Clinical and molecular links between COVID-19 and feline infectious peritonitis (FIP)

Arjun N. Sweet, Nicole M. André, Alison E. Stout, Beth N. Licitra and Gary R. Whittaker
Julia A. Beatty, Academic Editor and Séverine Tasker, Academic Editor

Original article: Clinical and Molecular Relationships between COVID-19 and Feline Infectious Peritonitis (FIP)

Abstract

The emergence of severe acute respiratory syndrome 2 (SARS-CoV-2) has led the medical and scientific community to address questions regarding the pathogenesis and clinical presentation of COVID-19; however, relevant clinical models other than humans are still lacking. In cats, the ubiquitous coronavirus, described as feline coronavirus (FCoV), can manifest as feline infectious peritonitis (FIP), a leading cause of mortality in young cats characterized by severe systemic inflammation. The diverse extrapulmonary symptoms of FIP and the rapidly progressive course of the disease, together with the proximate etiologic agent, represent a degree of overlap with COVID-19. This article reviews the molecular and clinical relationships between FIP and COVID-19. Although there are key differences between the two syndromes, these similarities encourage further investigation of feline coronaviruses as a naturally occurring clinical model for human coronavirus disease.

Keywords: feline infectious peritonitis, SARS-CoV-2, COVID-19, cats

1. Introduction

In the 1960s, feline infectious peritonitis (FIP) was described as a disease in domestic cats and was subsequently found to be of viral etiology, specifically feline coronavirus (FCoV). [1,2]. In most cats, infection with FCoV results in mild to inconspicuous clinical signs, but a small proportion of cats develop severe disease and succumb to the systemic form of the disease known as FIP [3]. In the years since the discovery of FCoV, many features of FCoV have remained misunderstood. Similarly, the COVID-19 pandemic, caused by the emergence of SARS-CoV-2, has raised many equally challenging questions regarding pathogenesis, transmissibility, and treatment. The widespread transmission of FCoV/SARS-CoV-2 and the insidious onset of severe symptoms in both FIP and COVID-19 limit the ability to detect the disease early—what may begin as mild or even mild clinical signs or symptoms can quickly lead to systemic disease [3,4]. We believe that FIP may represent a valuable, naturally occurring extrapulmonary model of COVID-19.

Both FCoV and SARS-CoV-2 belong to the family Coronaviridae [4,5], although to different genera (Figure 1). FCoV, together with similar animal coronaviruses such as canine coronavirus (CCoV) and porcine gastroenteritis virus (TGEV), belong to the genus alpha-coronaviruses. Community respiratory (CAR) human coronaviruses 229E and NL63 are also included in the genus Alphacoronaviruses. [6], the latter being associated with the common cold, hail and possibly Kawasaki disease in children [7]. In contrast, SARS-CoV-2, together with SARS-CoV (the causative agent of the severe acute respiratory syndrome outbreak in 2002-2003) and the Middle East respiratory syndrome coronavirus (MERS-CoV) belong to the genus betacoronaviruses [8], wherein SARS-CoV-2 and SARS-CoV belong to line B (sarbecovirus) and MERS-CoV belong to line C (merbecovirus). Less related beta coronaviruses include human coronavirus CAR OC43 (associated with the common cold), mouse hepatitis virus (MHV), and bovine coronavirus, which is associated with pneumonia and diarrhea in cattle; these viruses are in line A (embekovirus).

Figure 1
Phylogenetic tree of spike proteins of selected coronaviruses. The phylogenetic tree of maximum likelihood was constructed using the MEGAX program (100 bootstraps) from multiple alignment of spike protein sequences. Tip amino acid sequences were obtained from GenBank NCBI. Relevant numbers are: transmissible gastroenteritis virus / TGEV (P07946), severe acute respiratory syndrome coronavirus 2 / SARS-CoV-2 (YP_009724390.1), middle eastern respiratory syndrome / MERS-CoV coronavirus (AFS88 /36) 1. -1 (ACN89742), severe acute respiratory syndrome coronavirus / SARS-CoV (AAT74874.1), feline coronavirus / FCoV-Black (EU186072.1), bovine coronavirus / BCoV (P15777), canine coronavirus / CCo37.14) , human coronavirus / HCoV-OC43 (NC_006213.1), HCoV-229E (NC_002645.1) and HCoV-229E (NC_002645.1).

FCoV can be classified in two ways, the first of which relates to the form of the disease. Feline enteric coronavirus (FECV) is thought to cause a mild gastrointestinal form of the disease, while feline infectious peritonitis virus (FIPV) is associated with a fatal systemic infection known as FIP. [3]. FIPV differs from FECV in its ability to infect and replicate efficiently in monocytes and macrophages [9], causing systemic inflammation. FIPV is associated with a spectrum of clinical sequelae. At one end of the spectrum is effusive or “wet” FIP, which progresses rapidly and involves the accumulation of a highly proteinaceous exudate in the abdominal and/or thoracic cavity. At the other end of the spectrum is non-effusive or “dry” FIP, which can affect many organ systems but is usually characterized by neurological and ocular symptoms. Non-effusive FIP generally has a longer course of disease and is less common than its effusive counterpart. FCoV can also be divided into two serotypes – type I or type II – based on major differences in the spike protein of the virus that affect receptor binding and antibody response [10]. The receptor for FCoV type II is feline aminopeptidase N (fAPN) [11], while the receptor for type I viruses is not identified. Type I FCoV accounts for the vast majority of FIP cases [12].

The classification of SARS-CoV-2 virus into different variants based on genetic mutations is still ongoing as the virus continues to evolve. Viral lines that show the potential for increased transmissibility, treatment resistance, vaccine resistance, or increased morbidity and mortality have been identified as VOCs. The spectrum of diseases associated with COVID-19 is broad, ranging from asymptomatic and mild infections to acute respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), and multiorgan failure and death. Systemic inflammation in SARS-CoV-2 is not associated with macrophages and monocytes (as in FIP), but is responsible for a wide range of extrapulmonary symptoms. The SARS-CoV-2 receptor, angiotensin converting enzyme-2 (ACE-2), which plays an important role in the renin-angiotensin system and the development of pro-inflammatory status, appears to be involved. [13]. Multisystem inflammatory syndrome (MIS) in children and adults, as well as the post-acute course of SARS-CoV-2 infection (PASC), also known as "long COVID", are potential consequences of infection with COVID-19.

2. Transfer

As a group, coronaviruses are known for their ability to cause both respiratory and intestinal diseases and are usually transmitted in one or both ways. While FCoV is considered faecal-oral and SARS-CoV-2 is primarily respiratory, patients with COVID-19 may excrete the infectious virus in the faeces. [14], often for a long time, and FCoV can easily become infected by the oronasal route, which is a common method of experimentally vaccinating cats [15].

In most cases, FCoV infection is self-limiting, and although the virus can be detected systemically, replication outside the intestinal epithelium is weak. This form of the virus, referred to as FECV, is easily transmitted by the fecal-oral / oronasal route, with common anchors and swallowing of virus particles during purification being common sources of infection. The current understanding of FIP development involves internal mutation: in a small subset of FECV cases, a complex combination of host and viral factors leads to mutation (s) that allow efficient replication in macrophages and monocytes. [16]. These lethal variants are classified as FIPV and are associated with systemic inflammation, organ failure, and death. FIPV is generally considered non-transmissible because factors that increase its tropism on troprophages appear to limit its faecal-oral spread. [17]. FIP outbreaks were recorded in kennels and shelters. In these situations, congestion stress and high levels of virus in the environment may promote the conversion of FECV to FIPV. There is evidence that some FCoV strains may be more susceptible to this rebirth than others [18,19].

SARS-CoV-2 virus infection is primarily targeted at the respiratory epithelium, but as with FCoV, the virus may appear systemically without appropriate symptoms of infection. [20,21]. Asymptomatic individuals are a well-documented source of SARS-CoV-2 [22,23,24] and delivery includes inhalation of aerosols as well as contact with droplets [25]. The incubation times of SARS-CoV-2 and FECV range from 2 to 14 days [26]. The incubation time of FIP is highly variable, influenced by the time to internal mutation and the individual's immune response. The onset of FIP can occur several weeks to months after the initial infection [27,28,29,30]. Multisystem inflammatory syndrome in children (MIS-C), a severe manifestation of SARS-CoV-2, is also delayed by an initial infection with a median onset of 4 weeks. No viral factors have been associated with the development of MIS-C, but an immune-mediated component is thought.

Vertical transmission of FIP via the placenta or milk is considered rare. In the first experimental study in which a suckling cat was infected, one in four kittens succumbed to FIP [28]. Maternal antibodies appear to be effective in preventing transmission until approximately six weeks of age, when antibody levels decrease and kittens are susceptible to fecal-oral transmission. [31]. However, this maternally acquired immunity can be overcome early in life by high levels of exposure to FCoV – a Swiss study showed that kittens in large herds show infection as early as two weeks of age [32,33]. Vertical transmission poses a risk of SARS-CoV-2 infection. Placental transmission is rare but has been documented in fetuses of SARS-CoV-2 infected mothers. [34,35,36], as evidenced by virus detection in amniotic fluid, neonatal blood, umbilical cord blood and placental tissue. Transmission cases have been documented in both early and late pregnancies, but infection of the neonate with SARS-CoV-2 may not always occur in the uterus. Infection can also occur during childbirth or in close contact with the mother. The neonatal outcomes of COVID-19 infected mothers remain under study, and it is difficult to distinguish between the effects of SARS-CoV-2 infection and maternal comorbidities. Nevertheless, neonatal infection does not appear to be without sequelae, with one analysis showing that approximately 50 % infected neonates exhibited COVID-19-related clinical symptoms, including fever and respiratory and gastrointestinal symptoms. [37].

3. General clinical presentation

Clinical signs associated with both FIP and COVID-19 include fever, diarrhea, depression, weakness, anorexia, and dyspnoea. [1]. Typical manifestations of COVID-19 commonly include non-specific symptoms including fever, dry cough, fatigue, dyspnea, and myalgia [38]. Anosmia (loss of smell) and ageusia (loss of appetite) have also been frequently reported in COVID-19 and are more specific symptomatic indicators of the disease. [39]. Pneumonia, acute respiratory distress syndrome (ARDS) and sepsis may occur. Men appear to be at higher risk of developing more severe manifestations of COVID-19 [40,41], with several small studies confirming the same relationship between males and the development of FIP in cats [42,43].

The classic manifestation of FIP is the formation of effusion in the abdomen and / or thoracic cavity; although this manifestation has also been reported in COVID-19 [44], is very rare. In addition, FIP is manifested in various bodily systems that are similar to the extrapulmonary manifestations of COVID-19 (Figure 2 and Figure 3). The most similar feature of both diseases is endothelial dysfunction. Vasculitis is a hallmark of FIP pathology [45,46] with lesions characterized by perivascular edema and infiltration, vascular wall degeneration and endothelial proliferation [47]. In the case of COVID-19, extrapulmonary symptoms are thought to be caused by virus-mediated endothelitis, which leads to vasculitis, especially in veins with minor arterioles [48,49]. In the following sections, we will describe these extrapulmonary symptoms and point out key similarities and differences.

Figure 2
Summary of systemic clinical signs and pathological conditions associated with FIP. FIP is known to be a systemic infection with a variety of manifestations. Summarized possible systemic clinical signs associated with FIP are summarized, which include organ systems that are also affected by COVID-19. The most common symptoms of FIP are highlighted in red.

Figure 3
Summary of systemic clinical signs, symptoms, and pathologies associated with COVID-19. Respiratory symptoms of COVID-19 are the main manifestation of the disease. However, SARS-CoV-2 infection in humans can also result in a variety of extrapulmonary symptoms. Summarized are the systemic clinical signs and symptoms associated with COVID-19, which include organ systems that are also affected by FIP. The most common symptoms of COVID-19 are highlighted in red. ARDS stands for Acute Respiratory Distress Syndrome.

4. Biomarkers

Inflammatory biomarkers are important as prognostic markers in COVID-19 and as a means of differentiating FIP from other diseases. In FIP, IL-6 expression appears to be increased in the ascitic fluid of FIP-infected cats, presumably through increased expression in the heart and liver. [50,51]. Other acute phase proteins are also elevated in FIP infection. Alpha-1-acid glycoprotein (AGP) has been studied as a diagnostic marker of FIP, but may be elevated in other conditions, thereby limiting its specificity. [52,53]. Serum amyloid A (SAA) is another acute phase protein that appears to distinguish between FIPV and FECV infection, with FIPV-infected cats showing higher levels of SAA compared to FECV-infected cats and control cats without SPF. [54], but has limited utility in distinguishing FIP from other effusive conditions [55].

As with FCoV, subjects with severe COVID-19 have higher SAA levels compared to subjects with milder COVID-19. [56]. Higher SAA levels are also reported in patients who died of COVID-19 compared with those who survived [57]. C-reactive protein (CRP) is another marker that has been shown to be a promising biomarker in both FCoV and SARS-CoV-2 infections. Liver CRP synthesis is induced by IL-6 expression in response to inflammation [58] and is increased in FIP cases [59]. Elevated CRP levels in the early stages of COVID-19 are associated with a more severe course of the disease and higher mortality [60,61,62], which led to the recommendation to use it as a prognostic indicator in risk assessment in patients hospitalized for COVID-19. In contrast, one meta-survey found that IL-6 levels were elevated, but at least one order of magnitude lower in patients with COVID-19 than in patients with ARDS and non-COVID-19-related sepsis, suggesting a different mechanism of immune dysregulation. [63].

The D-dimer, although not specific for COVID-19 or FIP, is another interesting biomarker. D-dimer is released upon fibrin breakdown and is used as a clinical tool to eliminate thromboembolism [64]. Thrombotic events have often been documented in COVID-19 in several organ systems [65,66] and elevated D-dimer levels are associated with higher morbidity and mortality [67,68]. Similarly, thrombotic events can occur in FIP, and high levels of D-dimers, along with other symptoms of disseminated intravascular coagulation (DIC), can be observed in the final stages of FIP in both natural and experimental infections. [69,70].

5. Pathophysiology

5.1. Neurological

FIP is one of the major infectious neurological diseases in cats and the symptoms associated with central nervous system (CNS) infection are well documented. [71]. CNS symptoms are reported in approximately 40 % cases of dry FIP and may manifest as nystagmus, torticollis, ataxia, paralysis, altered behavior, altered mentoring, and seizures [72]. The wide range of symptoms supports the conclusion that the infection is not limited to a specific part of the CNS [73]. CNS infection is restricted to the monocyte and macrophage lines and leads to pyogranulomatous and lymphoplasmacytic inflammation, which usually affects leptomening, choroidal plexus and periventricular parenchyma. [74].

Documentation of neurological symptoms associated with SARS-CoV-2 CNS infection is limited compared to other coronaviruses [75]. The symptoms observed range from headache and confusion to seizures and acute cerebrovascular events. [76]. Virus detection in the brain is rare, suggesting that symptoms may not be directly related to CNS infection. Viral particles have been observed in neural capillary endothelial cells and a subset of cranial nerves, although such detection does not correlate with the severity of neurological symptoms. [77]. There is often no clear evidence of direct infection. Instead, inflammatory mediators, such as activated microglia, have been reported to contribute to microvascular damage and disease. [78,79].

Further comparison of the neuroinflammatory properties of SARS-CoV-2 and FCoV may provide new insights into the neurological manifestations of COVID-19. Further understanding of the neurological symptoms associated with SARS-CoV-2 is necessary to understand the progression of COVID-19 and the extent of CNS infection.

5.2. Ophthalmological

Ocular manifestations of FIP are more common in the dry form of the disease [80]. Mydriasis, iritis, retinal detachment, conjunctivitis, hyphema and keratic precipitates have been observed [81]. The most common ocular manifestation of FIP is uveitis, which can affect both the anterior and posterior uvea [80]. Viral antigen can also be detected in epithelial cells of the nitrating membrane, but viral antigen detection does not distinguish between FECV and FIPV [82].

Ocular manifestations of COVID-19 include conjunctivitis, chemosis, epiphora, conjunctival hyperaemia, and increased tear production [83]. Uveitis—a common ocular presentation of FIP—has also been observed in SARS-CoV-2 infection [84,85]. Tear fluid virus detection has led to concerns about ocular transmission in the first months of the COVID-19 pandemic [83,86]. SARS-CoV-2 RNA was detected in tear secretions and was isolated from ocular secretions, supporting the possibility of ophthalmic transmission [87,88]. Interestingly, in the above case study in China, out of 12 patients with ophthalmic symptoms, only 2 patients returned positive conjunctival tests, suggesting limited sensitivity in detecting virus from conjunctival specimens. [83].

5.3. Cardiovascular

Pericardial effusion is a less common manifestation of FIP, but is well documented in the literature [26,89,90,91]. FCoV was detected in the pericardium of cats with recurrent pericardial effusion, which later developed neurological symptoms [92]. Direct FCoV infection of the heart was documented in a 2019 case study of FIP-associated myocarditis with severe left ventricular hypertrophy and atrial enlargement. [93]. Immunohistochemistry (IHC) revealed the presence of FCoV-infected macrophages and associated pyogranulomatous lesions. [26]. Interestingly, severe SARS-CoV-2 infection with evidence of viral replication in the heart and lungs has recently been documented in cats with pre-existing hypertrophic cardiomyopathy (HCM). [94].

Unlike FIP, heart damage associated with SARS-CoV-2 infection appears to be much more prevalent. A study of 187 patients found that 27.8 % cases of COVID-19 showed evidence of myocardial damage, as evidenced by elevated cardiac troponin (TnT) levels. [95]. High levels of TnT were in turn associated with higher mortality. In a retrospective multicenter study of 68 patients with COVID-19, 27 deaths were attributable to myocardial damage and / or circulatory failure as one of the leading causes of mortality, with elevated C-reactive protein and IL-6 levels associated with higher mortality [96]. The increase in such inflammatory biomarkers in the blood suggests that the rapid inflammatory nature of COVID-19 may have a particularly detrimental effect on heart function. Diffuse edema as well as increased wall thickness and hypokinesis have been reported with COVID-19 infection. [97]. Cardiac tamponade was also observed in patients with COVID-19, with SARS-CoV-2 levels detectable in pericardial fluid. [98]. In contrast to FIP, in which direct invasion of FCoV-infected macrophages into the myocardium was observed in myocarditis, myocardial infection with SARS-CoV-2 virus is not clearly associated with mononuclear cell infiltration or myocarditis. [99]. This leads to considerations of multiple systemic factors in adverse cardiac outcomes – particularly dysregulation of inflammatory cytokines. The impact of SARS-CoV-2 infection on the cardiovascular system is an important element in our increasing understanding of the morbidity and mortality associated with COVID-19.

5.4. Gastroenterological

FCoV is excreted in feces and transmitted by the oronasal route. Initial FCoV infection targets the intestinal tract – infection may be subclinical or cats may develop diarrhea and, less commonly, vomiting. The primary infection lasts for several months and the virus can be shed for months to years [100,101]. Colonic epithelial cells appear to serve as a reservoir for persistent infection and excretion. [21]. The symptoms tend to be mild and spontaneous and only a small proportion of the animals go into the FIP stage. Fibrinous serositis and pyogranulomatous lesions with vasculitis are classic FIP lesions and can be found in the small and large intestines of affected cats. [102]. FIP can cause solitary mass lesions in the intestinal wall, although this is considered a rare presentation (26/156 cats in one study) [103]. These are usually found in the colon or ileocecal junction and have a pyogranulomatous character.

Gastroenterological symptoms are often reported with COVID-19 infection. ACE2, the cellular receptor for SARS-CoV-2, is widely expressed in glandular cells of the gastric, duodenal and rectal epithelium. Viral RNA and nucleocapsid were detected in these tissues [104], which supports their suitability for SARS-CoV-2 replication. Gastrointestinal (GI) symptoms range from general anorexia to diarrhea, nausea, vomiting and abdominal pain [105,106]. Excluding a less specific anorexia symptom, multiple meta-analyzes estimate the prevalence of GI symptoms in patients with COVID-19 at approximately 10 % to 20 %, with diarrhea being the most commonly reported symptom. [106,107,108]. Interestingly, GI symptoms in COVID-19 were observed without accompanying respiratory symptoms [105].

Faecal virus excretion is a major concern for COVID-19, as SARS-CoV-2 RNA may continue to be present in faeces even after reaching undetectable levels in upper airway samples. [109]. Although the detection of viral RNA in faeces alone does not necessarily indicate the presence of infectious virions, viable viral particles have been detected in faeces. [110]. The viral antigen persists in the cells of the gastrointestinal tract as well as in the convalescent phase, up to 6 months after healing [20]. In one case study, persistent colonic infection was associated with persistent gastrointestinal symptoms in a case of "long COVID" [111], which draws a parallel to the role of the colon epithelium as a reservoir for FCoV.

5.5. Dermatological

Dermatological changes have been reported with both SARS-CoV-2 and FIPV infections. Although papular skin lesions are rare, they are the primary dermatological manifestation of FIP, with several available case reports documenting papules. [81,112,113,114]. On histological examination, pyogranulomatous dermatitis, phlebitis, periflebitis, vasculitis and necrosis were reported in several FIP cases. [81,112,113,114,115].

The first report of dermatological manifestations associated with COVID-19 was recorded at Lecco Hospital in Lombardy, Italy [116]. In this study, 18/88 patients (20.4 %) developed a skin disorder, with 8/18 patients observed at the onset of the disease and 10/18 after hospitalization [116]. Clinical signs included erythematous rash (14/18 patients), diffuse urticaria (3/18 patients) and smallpox-like vesicles (1/18 patients) [116]. Lesions were observed mainly on the trunk (torsion) and pruritus was mild or absent [116]. The continuation of the pandemic brought better characteristics of the first observed dermatological symptoms, as well as the identification of rarer presentations. The most common dermatological manifestation of COVID-19 appears to be rash, often characterized by maculopapular lesions. [117,118]. Another predominant dermatological symptom also appears to be urticaria [118,119]. Importantly, neither rash nor urticaria is specific for COVID-19, which limits their positive predictive value. Varicella-like rash has been observed with SARS-CoV-2 infection and may be more specific due to its low prevalence in viral diseases. In particular, with missing lesions in the oral cavity and pruritus observed in COVID-19-associated rash, together with a previous history of varicella infection, the specificity of this presentation is reinforced. [118].

5.6. Teriogenological

Orchiditis and periorchitis with fibrinopurulent or granulomatous infiltrates as well as hypoplastic testes have been observed in several cases of FIP. [1,26,120]. Inflammatory mediators from the tuna surrounding the testicles caused enlargement of the testicles in cats with FIP [26,120]. In effusive FIP, enlargement of the spinal cord due to edema and peritonitis of tuna was observed [16]. Despite the apparent pathology of the male reproductive system in cats, FCoV has not been detected in sperm, which reduces the likelihood of sexual transmission. [121]. The pathology of the female reproductive system in FIP is less documented in the literature, but macroscopic lesions present in the ovaries of FIPV-infected cats have been observed. The surrounding uterine and ovarian vessels of these cats were surrounded by lymphocytes, macrophages, plasma cells and neutrophils. [122].

As with FIP, the pathology of COVID-19 is manifested in the male reproductive system. One study examining the testes of 12 COVID-19 patients found edema as well as lymphocyte and histiocytic infiltration, consistent with viral orchitis. [123]. These samples were also characterized by damage to the seminiferous tubules with a significant effect on Sertoli cells, as well as a reduced number of Leydig cells. In a separate study, germ cell damage was more pronounced despite similar Sertoli cell counts between SARS-CoV-2-infected individuals and uninfected controls, which represents a more direct relationship between infection and fertility. [124]. The extent to which SARS-CoV-2 may persist in the male reproductive tract is still being investigated. Although SARS-CoV-2 has been detected in human semen, it is questionable whether this is a true testicular infection or a consequence of a disrupted blood-epididymal / deferent barrier. [125,126].

Our knowledge of COVID-19 in the female reproductive system is still limited by the amount of literature and sample size of existing studies. Nevertheless, an understanding of the extent of SARS-CoV-2 in the female reproductive tract is essential to recognize any adverse effects on fertility. ACE2 is expressed in the ovaries, oocytes, and uterus, but limited coexpression of proteases such as TMPRSS2 and cathepsins L and B with ACE2 raises questions about the likelihood of ovarian / uterine infection. [127,128]. While in one study of 35 women diagnosed with COVID-19, SARS-CoV-2 was not detected in vaginal fluid or in exfoliated cervical cells, in a case study from Italy, SARS-CoV-2 was detected in vaginal fluid by RT-PCR ( Ct 37.2 on day 7 and Ct 32.9 on day 20 from the onset of symptoms), suggesting that infection of the female reproductive system is possible [129,130].

5.7. Immunological response

FIP is classically characterized as an immune-mediated disease based on early observations of complement and immunoglobulin circulation, even in the form of immune complexes. [131]. Components of type III and IV immune responses have been described [132]. Vasculitis and vasculitis-like lesions are thought to play a role in COVID-19 systemic complications that cannot be explained by direct organ infection, such as microthrombosis in the brain, kidney, spleen and liver. [133]. One type III hypersensitivity report has been identified in the COVID-19 literature [134]; however, immune complexes do not appear to play an important role in COVID-19 pathology. The mechanism of viral clearance and the inflammatory effects of the immune response are important areas of study for both FIP and COVID-19. Previous work investigating SARS-CoV has demonstrated the need for CD4 + T cells for virus clearance [135,136]. T-cell depletion was a recognized consequence of FCoV and was observed to be associated with more severe cases of COVID-19 [137,138,139]. In addition, FIP reduces both regulatory T cells and NK cells in the blood, mesenteric lymph nodes and spleen. [140]. High levels of IL-6 have previously been demonstrated in FIP ascites [50], and similarly, elevated levels of IL-6 appear to be related to disease severity and outcome in patients with COVID-19. [141]. The cytokine storm, characterized by overexpression of inflammatory cytokines, has been implicated in the pathogenesis of both infections. In FIP, this pathology is associated with monocyte and macrophage activation, while in COVID-19, the association with macrophages and monocytes is less clear. [142]. When considering the balance between cell-mediated immunity and humoral immunity, early reports suggested a link to strong humoral immunity leading to FIP [143]. However, humoral immunity may play a more beneficial role in patients with COVID-19 [144], especially given the potential clinical benefit of convalescent plasma / serum [145].

During the development of the SARS-CoV-2 vaccine, the antibody-dependent infection enhancement process (ADE), in which virus-antibody complexes enhance infection, was particularly important. FIPV has been shown to exhibit ADE in the presence of anti-FIPV antibodies [146]. This increase in infection appears to be serotype specific, with passive immunization of cats against FIPV type I or type II leading to ADE only after challenge with the same serotype for which the immunization was performed. [147]. As a result, ADE is a major challenge for the development of FIP vaccines. In diseases caused by human coronaviruses, ADE has yet to be fully understood. SARS-CoV has been found to have higher concentrations of anti-spike antibodies having a higher neutralizing effect, while more dilute concentrations are thought to contribute to ADE in vitro. [148]. In SARS-CoV-2, ADE was observed in monocyte lines but was not related to the regulation of pro-inflammatory cytokines [149]. Spike protein sequence modeling has identified possible ADE mechanisms that involve interaction with Fc receptors on monocytes and adipocytes [150]. If ADE played a role in SARS-CoV-2, the most likely mechanism would be overactivation of the immune cascade through Fc-mediated innate immune cell activation. [151,152]. There is currently insufficient evidence to suggest an ADE with the pathogenesis of SARS-CoV-2 and further research is needed to assess the true extent of the risk.

6. Molecular similarities between FCoV and SARS-CoV-2 spike proteins

Spike protein is a major factor in tissue and cell tropism and binds the cell receptor [153]. It is now well known that SARS-CoV-2 binds angiotensin converting enzyme-2 (ACE-2) as a primary receptor, a common feature of SARS-CoV. There are other binding partners for SARS-CoV-2, including heparan sulfate as a non-specific binding and neuropilin-1 (NRP-1), which may cause viral tropism for the olfactory and central nervous systems. [154,155]. In contrast, most alpha-virus viruses, including FCoV type II, use aminopeptidase (APN) virus entry. [9,153,156]. The receptor for FCoV type I has yet to be elucidated. The spike protein also mediates membrane fusion, which is activated by a complex process controlled by host cell proteases. [153]. While type I FCoV has two protease cleavage activation sites, designated S1 / S2 and S2 ′, type II FCoV has only one cleavage activation site (S2 ′). [10]. In comparison, SARS-CoV-2 is similar to FCoV-1 (and currently unique to SARS-related viruses) in that it has two identified cleavage sites (S1 / S2 and S2 ′), the first of which, the furin cleavage site or FCS is considered a significant factor in pandemic spread [157,158,159]. In both cases, the presence of S1 / S2 cleavage sites distinguishes FCoV-1 and SARS-CoV-2 from their close relatives. The importance of the cleavage activation site appears to be directly related to the proteases required for viral infection, and thus to another component of tissue tropism. In FCoV type I, the transition from FECV to macrophage-tropical FIPV was first demonstrated by amino acid substitutions at the S1 / S2 cleavage site in FIP-confirmed pathological specimens that are thought to reduce proteolytic priming of furin by similar proteases prior to S2-mediated fusion process activation. [72,160,161]. In SARS-CoV-2, TMPRSS-2 or other trypsin-like related proteases are a major activator of fusion and entry at S2 ′ [162] (Table 1), wherein the furin-like proteases prim the tip and S1 / S2 [163] and in particular have been shown to be rapidly regulated after adaptation to Vero E6 cells in culture and possibly also in extrapulmonary human tissues [164]. Thus, there appear to be remarkable similarities in host cell adaptation between the two viruses.

VirusGroupReceptorConsensus sequence S1 / S2 in circulating virusesConsensus sequence S2 ′ in circulating viruses
SARS-CoV-2Beta-coronavirusACE2SPRRAR | S
(* SHRRAR | S and SRRRAR | S)
SKPSKR | S
FCoV-1Alphacoronavirus (“clade A”)unknownSRRSRR | S (in FECV; mutated in FIPV)KR | S
FCoV-2Alphacoronavirus (“clade B”)APNabsentYRKR | S
Table 1
Summary of SARS-CoV-2 and two FCoV serotypes. The coronavirus spike glycoprotein, mediated by proteolytic cleavage, is a major driver of cell receptor binding and membrane fusion. The Taxonomic classification, host receptor, and amino acid sequences of the proteolytic cleavage site S1 / S2 and S2 ′ are summarized below.
*, Replaced in common variants.

7. Prevention and treatment: From social withdrawal to vaccines

Until now, the role of public health / public health measures has been a major driver in mitigating both the spread of FCoV and SARS-CoV-2. [3,31,165,166]. In this regard, many measures have been introduced for the affected population to reduce social distance, including orders to stay at home, the closure of unnecessary establishments and restrictions on public assemblies. [167]. Although not referred to as social distances, similar methods are often introduced or recommended in cat populations. [3]. Dreschler et al. summarize the methods that have been recommended in cat populations, especially in a multi-cat environment, including reducing the number of cats per room, frequent cage cleaning, and grouping cats according to excretion and / or serological status. [168]. Dreschler argues that quarantining cats exposed to FCoV / FIPV to limit the spread of FCoV in the population is neither effective nor beneficial given the likelihood of widespread FCoV infection in a multi-cat environment as well as the months required for development (and uncertainty in development) FIP. In contrast, quarantine people exposed to SARS-CoV-2 has the potential to reduce the spread of the disease and mortality [169]. Regardless of the extent of grouping or separation, the social difficulties caused by separation must be carefully considered for both cats and humans. In the case of cats, especially in connection with the premature weaning of their mothers, special attention must be paid in the weaning process to ensuring adequate socialization of the kittens. Similarly, in the case of COVID-19, the process of quarantine and / or isolation can be psychologically burdensome for individuals. A thorough cost-benefit analysis must often be carried out to compare the benefits of quarantine and isolation for public health with the negative psychological burden on the persons concerned in order to avoid unnecessary / ineffective quarantine. Where appropriate, justification as well as support to improve well-being should be provided [170].

Although the FIP vaccine is commercially available (Primucell), the benefit of FIP vaccination is still low. Primucell is an intranasal vaccine that uses an attenuated FIPV serotype 2 isolate. Although the FIP vaccine is commercially available (Primucell), the benefit of FIP vaccination is still low. Primucell is an intranasal vaccine that uses an attenuated FIPV serotype 2 isolate (FIPV-DF2), given in two doses 3 to 4 weeks apart to cats at least 16 weeks old. [171]. In a placebo-controlled experimental study in 138 cats, vaccinated cats did not show a significantly lower incidence of FIP compared to controls during the 12-month study period. After adjusting for FCoV titers, cats with lower antibody titers (100 or less) had a significantly lower incidence of FIP at the time of the first vaccination compared to cats with higher titers (400 or more). [172]. However, due to the high prevalence of FCoV, especially in multi-cat environments, attempts to alleviate FIP by vaccinating cats that are FCoV-naive at least 16 weeks of age may not be feasible due to the high potential for FCoV infection during the 16 weeks prior to vaccination. As a result, the American Association of Animal Hospitals and the American Association of General Practitioners for Cats do not recommend FIP vaccination. [173].

ADE remains a major concern of FIP vaccines. Several studies have attempted to reduce the incidence of FIP in experimentally infected cats with recombinant and other experimental vaccines, but ADEs have been reported repeatedly. In one placebo-controlled study in which purebred British Shorthairs and Specific Pathogen Free (SPF) Domestic Shorthairs were vaccinated with one of two recombinant FIPV type 2 (FIPV-DF2) vaccines, both candidate vaccines showed significantly reduced to no protection against challenge FIPV in non-SPF cats - with the majority of non-SPF animals showing ADE [174]. In a separate study, immunization of kittens with a vaccine virus recombined with the FIPV spike glycoprotein gene significantly shortened survival time after FIPV challenge compared to kittens immunized with wild-type vaccine virus. Importantly, low levels of neutralizing antibodies were observed in the group immunized against FIPV [175]. Concerns about ADE after FIPV immunization remain a challenging challenge in FIP prevention.

Unlike the FIP vaccination, the COVID-19 vaccines have played a more significant role in reducing the spread of the infection. Several types of vaccines have been produced that have demonstrated safety and efficacy in preventing symptomatic infection, severe disease, and death from COVID-19—including, but not limited to, mRNA vaccines (Pfizer/BioNTech and Moderna), viral vector vaccines (Janssen, AstraZeneca), and inactivated virus vaccines (Bharat Biotech, Sinovac) [176,177,178,179,180,181]. The first two vaccine platforms use the SARS-CoV-2 spike glycoprotein as the immunogen, while inactivated virus vaccines have the potential to elicit an immune response to viral components other than the spike glycoprotein. Despite the favorable safety profile of COVID-19 vaccines, adverse reactions occurred after vaccination, some of which were mediated by antibodies in analogy to ADE concerns with FIP vaccines. Thrombosis has been a documented problem, especially with AstraZeneca and Janssen vaccines. Although the exact mechanisms are being investigated, the inflammatory response is currently thought to lead to increased levels of platelet-activating antibodies, which bind to platelet factor 4 and lead to a hypercoagulable state. [182,183]. In contrast to the higher incidence of ADE in experimental FIP vaccines, the incidence of thrombotic events after administration of COVID-19 is low. [184].

In addition to the primary endpoints of vaccine studies, which focused on the prevention of symptomatic infection, serious illness, and death from COVID-19, many phase 3 vaccine studies did not address observations to assess the degree of prevention of asymptomatic infection. Beneficial efficacy against asymptomatic infection is important from a public health perspective, especially given that asymptomatic individuals can transmit COVID-19 and that routine monitoring testing is resource intensive and difficult to coordinate on a large scale. [22]. An important contribution towards this field is the real-world studies investigating the efficacy of the vaccine, which show a reduced risk of SARS-CoV-2 infection, as well as a reduced viral load in vaccine "breakthrough" infections. [185,186,187,188]. Such evidence supports the use of SARS-CoV-2 vaccines as a protective measure not only against severe COVID-19, but also as a crucial contribution in the management of the disease.

8. Clinical care and therapeutic options

In 1963, when the first clinical cases of FIP were described (before understanding the viral etiology), it was found that antibiotic treatment was often tried, but it clearly did not bring any benefit. [189]. Since this first report and without an effective vaccine, a number of therapies have been tried in cats with FIP. Ribavirin, a nucleoside analogue, has previously provided promising results against FCoV in an in vitro study [190], however, when administered to cats as an experimental treatment, led to poorer results in some cases [191]. Similarly, at the onset of the COVID-19 pandemic, ribavirin was used in several doses and in combination with other drugs. [192] and a study protocol was designed to examine the benefit in human patients [193]. However, another direct-acting antiviral drug (DAA) (remdesivir), a nucleoside analogue that acts as a chain terminator and raises minor toxicity concerns compared to ribavirin, is rapidly gaining prominence in the treatment of hospitalized patients with COVID-19. . Despite initial enthusiasm, remdesivir has not been shown to be effective in patients with such diseases in robust clinical trials; however, several reports have demonstrated the clinical benefit of the related nucleoside analog GS-441524 in the treatment of cats with FIP, including effusive, non-fusive, and neurological forms of the disease. [194,195,196,197]. At the time of writing, studies on the effectiveness of remdesivir in the treatment of FIP are being conducted in Australia and the United Kingdom. Interestingly, remdesivir is a prodrug form of GS-441524 [195]. Two orally available DAAs have recently entered clinical trials for COVID-19 and are currently awaiting FDA approval; molnupiravir (MK-4482 / EIDD-2801), a modified form of ribavirin, and Paxlovid (a protease inhibitor PF-07321332 in combination with ritonavir, which improves the half-life of PF-07321332) targeting the major protease (Mpro). It is noteworthy that the active substance Paxlovid is related to GC-376 and has previously been shown to be effective in a FIP clinical study. [196]. It will be very interesting to follow the development, FDA approval and use of these DAAs in relation to the relevant diseases caused by SARS-CoV-2 and FCoV.

Due to the inflammatory nature of both FIP and COVID-19, treatment often focuses on controlling the immune response. Although cats with FIP are often given glucocorticoids in an effort to alleviate the inflammatory symptoms of the disease, the clinical benefit is negligible. [198]. The use of corticosteroids in patients with COVID-19 does not appear to be insignificant, with some studies showing negative profiles [199]. However, their administration may be beneficial in severe cases of COVID-19 through the observed reduction in mortality [200,201]. Cyclosporine, an immunosuppressive drug that is often used to prevent organ rejection in transplant patients and to treat some autoimmune diseases, has been studied in both FIP and SARS-CoV-2. An in vitro study with cyclosporin A (CsA) using FCoV type II virus showed a reduction in virus replication [202], treatment of a 14-year-old cat CsA after unsuccessful IFN treatment resulted in clinical improvement, reduction in viral load and survival of more than 260 days [203]. Although there are currently no controlled studies on the use of CsA in the treatment of patients with COVID-19, in addition to safety concerns, potential mechanisms of action have been suggested. [204,205,206]. In addition, the cyclosporin A analogue, alisporivir, has shown in vitro effects on virus replication [207], similar to evidence that cyclophilin A blockade inhibits the replication of other coronaviruses [208].

Many antibiotics have been prescribed for both FIP and COVID-19, but not for their antimicrobial properties, but rather for their anti-inflammatory effects. [198]. For example, doxycycline may have helped prolong survival in cats with FIP [209]. Whether doxycycline would be of benefit to patients with COVID-19 is not currently known, but has been suggested as a possible part of the treatment of the disease. [210].

Interferons have also been studied in the treatment of FIP without a clear link to clinical improvement [211]. In human patients with COVID-19, combination therapy with interferon-β-1b with lopinavir, ritonavir and ribavirin compared with lopinavir and ritonavir alone was associated with a reduced duration of virus excretion and improved clinical outcomes in mild to moderate cases. [212].

Monoclonal antibodies targeting components of the immune response have the potential to reduce inflammatory cytokine levels. A small study in cats experimentally infected with FIPV-1146 demonstrated the benefit of anti-TNF-α in managing the disease [213]. Tocilizumab, an anti-IL-6 monoclonal antibody, was administered to patients with COVID-19 [214]. Due to the different clinical results reported, further research is needed with Tocilizumab [215,216].

The transfer of knowledge between species will undoubtedly affect cats as well as humans and even other species. Although many compounds are effective when studied in vitro, their use in vivo can lead to different results, including toxicity. In addition, the fact that a compound may show promising results in one species does not mean that the same effect will be observed in other species, especially when comparing similar but different viruses and virus-induced diseases.

9. MIS-C and PASC

In April 2020, the UK's National Health Service issued an alert about an increased incidence of multisystem inflammatory syndrome in children - many of whom tested positive for COVID-19 [217]. As the pandemic progressed, studies from other countries examining this inflammatory condition provided more detail towards a clinical understanding of what is now referred to as MIS-C, a rare presentation of COVID-19 in pediatric patients. MIS-C includes several organ systems. Cardiovascular dysregulation in MIS-C is often observed in the form of ventricular dysfunction, pericardial effusion and coronary artery aneurysms [218,219]. Gastrointestinal symptoms mimic appendicitis and include abdominal pain, vomiting, and diarrhea. Terminal ileitis is a common finding on imaging tests [220]. Many patients also experience neurocognitive symptoms, including headache and confusion. More serious neurological complications, including encephalopathy and stroke, are less common [218,221].

One area of significant clinical overlap between FIP and COVID-19 is the rare inflammatory manifestation of SARS-CoV-2 infection – multisystem inflammatory syndrome in children (MIS-C). MIS-C is seen in the pediatric population, just as FIP commonly affects young cats [43]. Like FIP, MIS-C has a systemic presentation involving multiple organ systems—including gastrointestinal, cardiovascular, and hematologic abnormalities [222]. As with the wet form of FIP, pleural effusions and ascites occur in MIS-C. [223]. Both syndromes also show overlap in vascular pathology. FIP shows granulomatous vasculitis, which overlaps with Kawasaki vascular syndrome observed in MIS-C [224]. MIS-C is thought to be a post-infectious disease associated with a previous SARS-CoV-2 infection [223,225]. FIP also has a delayed onset after the first exposure to FCoV and occurs only in a small subset of cases. Although cats with FIP can still shed FCoV in their feces, the mutations associated with the biotype switch from FECV to FIPV are thought not to be transmissible—supporting some degree of similarity in the limited infectious range of both FIP and MIS-C.

Recently, post-acute sequelae of COVID-19 (PASC) has been defined, which includes memory loss, gastrointestinal distress, fatigue, anosmia, dyspnea, etc. and is more often referred to as "long-term COVID". Along with MIS-C, PASC is a very active subject of research, which has been summarized by others [226], and together represent an excellent starting point for the use of feline medicine as a model of coronavirus-induced pathogenesis, possibly in an unexpected way [224].

10. SARS-CoV-2 infection in cats

Cats have now become widespread hosts for SARS-CoV-2 infections, in part due to the relative similarity of human and feline ACE2 receptors. Following cases reported in Hong Kong and Belgium in March 2020, the most notable early natural infection occurred at the Bronx Zoo in New York City, USA. In April, four tigers and three lions showed mild respiratory symptoms from their breeders, and SARS-CoV-2 was detected by PCR and sequencing. [227]. Consequently, infection of both domesticated and non-domesticated cats has become relatively common in cases where owners and caregivers are positive for SARS-CoV-2. From a clinical point of view, SARS-CoV-2 infection in cats is considered to be predominantly asymptomatic, with some animals showing mild respiratory symptoms. [228,229,230]. In general, severe respiratory symptoms do not appear to occur in cats, although severe respiratory distress may in some cases be related to the underlying hypertrophic cardiomyopathy (HCM) of cats. [94]. An increased incidence of myocarditis in dogs and cats has also been reported in the United Kingdom, associated with a sharp increase in variant B.1.1.7 (Alpha). [231]. There is a clear need for further studies in this area, as well as possible links between coronavirus infections in cats and multisystem inflammatory syndrome in children (MIS-C), which, as mentioned above, is a rare manifestation of COVID-19.

Laboratory animal studies have also been key to understanding SARS-CoV-2 infection in cats, which are very susceptible to infection by oronasal challenge. Experimentally infected cats showed mild respiratory symptoms or asymptomatic infection, virus shedding, virus-to-cat virus transmission, and a strong neutralizing antibody response. Recent studies have shown that long-term immunity exists after re-infection of cats, but cats may develop long-term consequences, including persistence of inflammation and other lung lesions. [232]. Overall, as with SARS-CoV in 2003, cats in particular can be an important source of information on the pathogenesis and immune responses elicited by SARS-CoV-2.

Thanks

Thanks to Annette Choi for helping with Figure 1 and all Whittaker Lab members for helpful discussions during the preparation of this manuscript.

Author shares

All authors have contributed to this article. All authors have read and agreed to the published version of the manuscript.

Financing

The work in the author's laboratory is partly funded by research grants from the National Institutes of Health, the EveryCat Foundation and the Cornell Feline Health Center. AES was supported by the NIH Comparative Medicine Training Program T32OD011000. Studies on FIP are also supported by the Michael Zemsky Fund for Cat Diseases.

Conflict of interests

The authors do not indicate any conflict of interest.

Footnotes

Publisher Note: The MDPI remains neutral in terms of jurisdictional claims in published maps and institutional affiliation.

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Read "Clinical and molecular links between COVID-19 and feline infectious peritonitis (FIP)"

Miscellaneous questions frequently arising during antiviral drug treatment for FIP and aftercare

23.3.2022
NC. Pedersen, DVM PhD
Original article: Miscellaneous questions frequently arising during antiviral drug treatment for FIP and aftercare

Several issues often arise during FIP treatment. Before addressing these issues, it is important to mention the FIP treatment itself. Only antivirals that target specific viral proteins and inhibit FIP replication have been shown to have therapeutic effects. Currently, these include nucleoside analogs and RNA replication inhibitors GS-441524 (and a related prodrug Remdesivir), Molnupiravir (EIDD-2801) and the viral protease inhibitor GC376. Proper administration of these drugs has resulted in the cure of all forms of FIP in more than 90 % with minimal side effects. Most treatments are completed without complications. However, certain issues that are the subject of this article often arise.  

I pointed out the problems associated with unwanted sexual behavior in intact females and males treated with specific antivirals. The questions often come from countries where castration is either postponed or not common practice. They fear that the stress of castration and vaccines may affect the outcome of antiviral treatment. I believe that such concerns are exaggerated. If a cat is in treatment and in remission or is considered cured, it is okay to sterilize or neuter it, but preferably in the least stressful way possible. Cats can be neutered and sterilized quickly and efficiently and returned to their homes on the same day (castration) or within one day (sterilization) with minimal preoperative, operative and postoperative drug treatment and restrictions (eg cages, E-collars). Such operations will be less stressful for cats (and owners, which will then be reflected in their cats) than their sexual behavior. 

I am also not in favor of hormonal treatment to prevent unwanted sexual behavior in males or females, and I feel that effective castration and sterilization will be less stressful in the long run than such preventive measures. Therefore, if it is necessary to permanently change this behavior, surgical castration is more appropriate than chemical.  

Some owners seem to want to keep cured cats intact so that they can be used for breeding later. We know that FIP has both genetic and environmental components, which has led to the recommendation that purebred cats that breed FIP kittens should not be used for breeding. This should be even more true for cats that have been cured of the FIP.  

As far as vaccines are concerned, many already know that I am not a big fan of adult vaccines and the first annual booster vaccines because I feel that immunity is long-lasting. I also think that rabies vaccines cannot be used routinely in cats, whether in terms of public health or cats. Nevertheless, I accept that these views are not generally accepted and that the laws in several states require rabies to be vaccinated against rabbits, in some vaccination is not required and in others it is recommended but not required. I have not noticed the consequences of routine vaccinations in any of our cured cats. However, it is not something I would recommend for cats undergoing treatment. The immune system of these cats is responsible for other things than responding to vaccines.  

What are the indications for drugs other than specific antivirals for the treatment of FIP? During the initial illness, supportive (symptomatic) treatment may be required to keep the cats alive long enough for the antivirals to take effect. Drugs often used in this early stage usually include antibiotics (doxycycline / clindamycin), analgesics (opioids, gabapentin), anti-inflammatory drugs (corticosteroids, NSAIDS), immunostimulants (interferons, non-specific immunostimulants), and drugs. I have tried to avoid excessive use of these drugs except for temporary use and only if it is strongly justified, especially in severely ill cats during the first days. The most important goal of FIP treatment is to stop the replication of the virus in macrophages, which immediately stops the production of the numerous inflammatory and immunosuppressive cytokines that cause the symptoms of FIP. Although some drugs, such as corticosteroids (prednisolone) or NSAIDs (meloxicam), may inhibit inflammatory cytokines and cause clinical improvement, they are not curative. They can also mask the beneficial effects of GS treatment, which are often monitored to assess the effect and course of treatment. The response to antiviral treatment is also used for diagnostic purposes. The only drugs that completely suppress these harmful cytokines and cure FIP are antivirals such as GS-441524, molnupiravir or GC376, and related compounds. These antivirals cause a dramatic improvement in fever, activity, appetite, etc. within 24-48 hours. This improvement will be much greater than any improvement made with other drugs. Therefore, if the use of other drugs is not warranted, they should be discontinued as soon as the symptoms of FIP have steadily improved. 

I also do not believe in many supplements that are said to treat or prevent problems with the liver, kidneys, immune system or other organs. These supplements are expensive and have not been shown to be effective in what they claim. B12 injections only treat B12 deficiency, which is rare, and not anemia in FIP. The same goes for other vitamins. This also applies to a wide range of nutritional supplements and special diets for cats of many types. There is no essential ingredient in any of these supplements that could be provided by well-tested commercial cat food brands. There is also a possibility that some supplements interfere with the absorption of oral antivirals.  

How should cats be monitored after treatment and during the post-treatment observation period? From a technical point of view, no further blood tests are needed, especially if routine health assessments such as weight, appetite and temperature are continued during this period. Blood tests during this period do not change the outcome and can only increase the cost of treatment and increase the owner's stress. However, it is common for successfully treated cats to routinely test for blood during a 12-week post-treatment observation, usually every 4 weeks, but sometimes more frequently. In some cases, routine blood testing is continued for 12 weeks after treatment, even out of fear of possible relapse or recurrence. Relapses or new infections after a 12-week observation period are rare and are preceded by external signs of the disease, such as weight loss, lethargy, anorexia, poor coat and fever, which would be the best indicators for a blood test. Blood test panels also contain many values, and it is not uncommon for one or more values to be abnormal in healthy cats. Care must be taken not to over-interpret such abnormalities and to lead to excessive concern or additional testing in order to determine their significance. For example, a mild to moderate increase in one in three liver enzymes in a healthy cat is much less important than in another cat with symptoms of the disease. Read "Various issues common during FIP antiviral treatment and aftercare"

FIP diagnostics overview

Original article: A review on the diagnosis of feline infectious peritonitis
3.3.2022

Jehanzeb Yousufa | Riyaz Ahmed Bhatb | Shahid Hussain Darb | Alisa Shafib | Snober Irshadc | Mohammad Iqbal Yatoob | Jalal Udin Parrahb | Amatul Muheeb | Abdul Qayoom Mirb

Abstract: Feline infectious peritonitis, or simply FIP, is a coronavirus viral disease in cats, usually less than three years old. It is manifested by an extreme inflammatory reaction in the tissues in the abdomen, kidneys and brain. This review article discusses the various diagnostic tests and their benefits in diagnosing cases of suspected FIP for definitive diagnosis. This review can help compare different diagnostic parameters and also raise awareness of their advantages and disadvantages.

Keywords: acute phase proteins, coronavirus, feline infectious peritonitis, Rivalt's test.

1. Introduction

Feline infectious peritonitis (FIP) is a well-known and widespread coronavirus (CoV) -induced systemic disease in cats, characterized by fibrinous-granulomatous serositis with protein-rich exudates in the body cavities, granulomatosis-announcing phlebitis in the gallbladder and periphels Scott 1981; Kipar et al. 2005). Feline CoV (FCoV) spreads via the faecal-oral route and is primarily infected with enterocytes (Pedersen 1995), but subsequently spreads systemically via monocyte viremia (Meli et al 2004; Kipar et al 2005). The increased ability of the virus to replicate has been shown to be a key feature in the development of FIP, and FIP is also thought to be caused by mutations in common feline enteric coronavirus (FECV), which occurs in cats worldwide and is not a serious infection (Pedersen et al 2009; Healey et al 2022). Approximately 10 % infected cats have mutations that result in feline infectious peritonitis. In large multi-cat situations, FECV is excreted in the faeces of most seemingly healthy cats and the transmission occurs through direct contact with faeces or contaminated litter and other fomits (Pedersen et al 2004). Kittens become infected at approximately 9 weeks of age (Pedersen et al. 2008). The time between the onset of clinical signs and death also varies, but younger cats and cats with effusive disease have a shorter course of disease than older cats and cats with non-effusive disease (Pedersen 2014). Even with severe FIP, some cats can live for months. in multi-feline situations, feline enteric coronavirus (FECV) is extremely common and highly contagious. Almost all cats that come into contact with FECV from secreting cats become ill, but on the other hand, the infection is usually asymptomatic or causes only mild temporary diarrhea (Pedersen et al. 2008; Vogel et al. 2010; Ermakov et al. 2021). On the other hand, feline infectious peritonitis virus (FIPV) is not transmitted via the faecal-oral route, but originates from avirulent FECV in a small percentage of infected cats and causes feline infectious peritonitis (FIP) (Pedersen et al 1981; Vennema et al. 1998). Anorexia, lethargy, weight loss, pyrexia, ocular and neurological symptoms such as gait abnormalities or inappropriate mentoring are non-specific (Giori et al 2011; Kipar et al 2014). The infection takes two forms: "wet" and "dry". The dry form causes inflammatory changes around the vessels, seizures, ataxia and excessive thirst, while the wet form leads to enlargement of the abdomen due to excessive accumulation of fluid in the abdominal cavity. Specificity is always the most important diagnostic value to consider in order to avoid misdiagnosis of FIP in unaffected cats.

2. Diagnostic tests for feline infectious peritonitis

The diagnosis takes into account the age, origin, clinical signs and physical examination of the cat. Abdominal distension with ascites, dyspnoea with pleural effusion, jaundice, hyperbilirubinuria, marked masses of the kidneys and / or mesenteric lymph nodes, uveitis and various neurological symptoms are common in cats with effusive (wet) or non-fusive (dry) forms of FIP. and / or spinal cord. Ocular changes often occur in cats with FIP, with the most common ocular disorders being retinal changes. Retinal cuff cuffs may occur, which appear as blurred gray lines on either side of the vessels. Occasionally, granulomatous changes in the retina occur. FIPV infection has been found to be associated with T-cell depletion by apoptosis, although the virus cannot infect CD4 + and CD8 + T-cells (Haagmans et al. 1996; De Groot et al. 2005). Due to the high mortality rate, many veterinarians and pet owners are cautious about a diagnosis based on "reasonable assurance". The challenge is to decide whether the test increases the likelihood that clinical symptoms are caused by FIP (indirect tests) or offers a definitive diagnosis (direct tests). It is important to note that the sensitivity and specificity of any indirect test will vary depending on the likelihood that the cat is infected due to other factors. This means that a positive predictive value of the test, such as complete blood count (CBC) or albumin: globulin (A: G) ratio, to predict FIP will be much higher in cats with FIP-like signaling than in cats with non-FIP signaling. It should be noted that the results of other indirect tests are only estimates and the results of additional indirect tests have the potential to confuse and support the diagnostic process.

3. Diagnostic tests

The problem with FIP diagnostics is that non-invasive tests are not reliable enough. In general, effusion tests have a significantly higher predictive value than blood tests (Stranieri et al. 2018; Hartmann et al. 2003). As a result, the identification of FIP ante mortem in cats without significant effusion is particularly difficult. The most useful ante-mortem indicators are positive anti-Corona (IgG) antibody titers in cerebrospinal fluid (CSF), high total serum protein, and MRI changes such as periventricular contrast enhancement, ventricular dilatation, and hydrocephalus. However, monoclonal antibodies from affected tissues and coronavirus-specific polymerase chain reaction (PCR) are valuable in post mortem evaluations (Foley et al. 1998). As a clear diagnosis cannot be made on the basis of symptoms, medical history and clinical and laboratory indicators alone, these factors should always be considered as a whole, sometimes in combination with other factors such as molecular or even more invasive diagnostic procedures.

3.1. Analysis of effusion samples

If FIP with effusion is suspected, the effusion sample can be incredibly helpful in making a diagnosis and then in hematological findings, so obtaining effusion samples should always be a top priority. In the case of ascites, the sample can be obtained by ultrasound-guided thin-needle aspiration or the "flying cat" technique. To identify small amounts of fluid in the chest and abdomen, ultrasonography provides useful assistance in locating effusion bags in the abdomen, while evidence of pericardial effusions can be obtained through suppressed cardiac echoes and electrocardiographic changes. Ultrasonography should be used repeatedly to identify any small volume effluents, and ultrasonography may also be used to guide the sampling of small bags of fluid. In cats with pericardial effusions, auscultation of the heart detects muffled sounds and the ECG reveals typical changes.
FIP effusions are often clear, viscous / sticky, straw yellow and rich in protein (cytology often describes a dense eosinophilic protein background) with a total protein concentration> 35 g / l (> 50 % globulins). Chlootic effusions are rarely described. FIP effusions are often pyogranulomatous in nature with macrophages, non-degenerate neutrophils and a relatively small number of lymphocytes. As a result, effusions are often referred to as modified transudates based on cell number (<5 × 109 cells / L), but exudates based on protein concentration (greater than 35 g / L). Typical FIP effusions have a low A: G ratio (see above) and an increased AGP content, which is similar to the serum content. A recent study (Hazuchova et al. 2017) found that AGP concentrations in effusions (> 1.55 mg / ml) are more useful (sensitivity and specificity 93 %) in distinguishing FIP cases from cases without FIP than serum AGP levels or other APPs.
The rival test is a simple test that can be used to distinguish transudate from exudate in a effusion sample (Barker and Tasker 2020). Positive results simply suggest that the effluent is exudate and not specific to FIP; positive transudate results have been documented in situations other than FIP (eg, bacterial / septic peritonitis and lymphoma) (Fischer et al. 2012).

3.2. Serum biochemistry

Although the changes in blood biochemistry observed in FIP cases are variable and often non-specific, there are several key anomalies that need to be addressed in order to confirm the diagnosis of FIP.

3.2.1. Acute phase proteins

Many inflammatory and non-inflammatory diseases produce acute phase proteins (APPs) in the liver in response to cytokines released by macrophages and monocytes (particularly interleukins 1 and 6 and tumor necrosis factor α).
AGP is an abbreviation for α1-acid glycoprotein and its examination may help in the diagnosis of FIP. Although the increase in AGP levels (> 0.48 mg / ml) is not specific for FIP, patients with FIP often have significantly high AGP levels (> 1.5 mg / ml). As a result, the magnitude of the increase may be valuable in helping to diagnose FIP, with higher levels more effectively increasing the suspicion index (Giori et al 2011; Hazuchova et al 2017).

3.2.2. Hyperglobulinemia

In 89% cases, hyperglobulinemia is present; often in association with hypoalbuminemia or low-normal serum albumin levels (observed in 64.5 % cases) (Riemer et al. 2016). Hyperproteinemia may not always occur due to the existence of hypoalbuminemia. The albumin: globulin (A: G) ratio is low in hyperglobulinemia and hypoalbuminemia (low-normal albumin concentration) and this parameter can be used to assess the likelihood of FIP in a particular case.

3.2.3. Hyperbilirubinemia

Hyperbilirubinemia occurs in 21-63 % cases of FIP and is more common in effusive FIP, where alanine aminotransferase (ALT), alkaline phosphatase (ALP) and γ-glutamyltransferase enzymes are commonly high (although these may be slightly increased in FIP cases). FIP is rarely associated with hyperbilirubinemia due to immune-mediated hemolytic anemia (IMHA) (Norris et al. 2012) and cats are often not severely anemic. In the absence of high liver enzyme activity or severe anemia, the presence of hyperbilirubinaemia should suggest FIP (note that sepsis and pancreatitis may cause hyperbilirubinaemia without increased liver enzyme activity). Based on a stepwise evaluation of cats with FIP, it was documented that hyperbilirubinemia was more typically recognized in cats just before death or euthanasia than in the first presentation (Harvey et al. 1996). In addition, higher bilirubin levels were observed in cats just before death or euthanasia than in the first presentation.

3.3. Hematology

In FIP, hematological changes are non-specific; however, there are several abnormalities that need to be checked to confirm the diagnosis. Lymphopenia is the most common change (55 - 77%) in cases, with a recent study (Riemer et al. 2016) revealing lymphopenia in only 49.5 % cases of FIP, with neutrophilia (39 - 57 %), left-sided and mild to severe normocytic, normochromic anemia (37-54 %) (Riemer et al. 2016; Norris et al. 2012). Recently, an association between FIP and microcytosis (with or without anemia) has been discovered. FIP can cause severe IMHA with concomitant regenerative anemia; however, this is an unusual phenomenon.

3.4. Serology

ELISAs, indirect immunofluorescence antibody assays, and rapid immunocompromination assays are the most common assays for anti-FCoV antibodies in serum (Addie et al. 2015). In most studies, cells infected with CoV pigs or cats are used as substrate and titers are measured in multiples of serum dilutions. A positive anti-FCoV antibody test means that the cat has been infected with FCoV and has seroconverted (which lasts 2-3 weeks after infection). The tests are therefore of limited clinical importance. Breed-dependent differences in anti-FCoV antibody titers were found, which could indicate differences in the breed's response to FCoV infection (Meli et al. 2013).
Although cats with FIP had higher anti-FCoV antibody titers than cats without FIP, no difference was found between the median anti-FCoV antibody titers in healthy cats and cats with suspected FIP. As a result, the titer in one animal is only marginally useful in identifying cats with FIP (Bell et al. 2006). Many clinically healthy cats (especially those in multi-cat households) have positive and often very high anti-FCoV antibody titers, while 10 % cats with FIP are seronegative, which could be due to virus binding to the antibody and its inaccessibility for serological testing. which also points to problems with interpretation (Meli et al 2013). A negative FCoV antibody test if dry FIP is suspected may be more effective at excluding FIP (Addie et al. 2009). Nevertheless, negative results have been observed in neurological FIP situations (Negrin et al 2007). As a result, doctors disagree on whether to perform a serological test in suspected cases, even though a positive result almost always means FCoV exposure.

3.5. Current trends in diagnostics

The use of anti-coronavirus antibody testing in cerebrospinal fluid (CSF) for diagnosis in cases involving the central nervous system is another breakthrough in which IgG is detected in the CSF. However, in most cases, the antibody was detected only in cats with high serum IgG titers (Boettcher et al. 2007)
An important difference between feline coronavirus and FIP infection is the behavior of the NSP3c gene. The infected tissue isolates from the second case were found to have a disrupted gene 3c, while in the first case the gene was intact. Mutation of the S1 / S2 locus and modulation of the furin recognition site, which is normally present in the S-gene of the enteric coronavirus (Levy and Hutsell 2019), is also a crucial contributing factor.
The diagnostic utility of cerebrospinal fluid immunocytochemistry is also used to diagnose FIP manifested by severe central nervous system involvement. Immunocytochemical staining (ICC) of feline coronavirus antibodies in cerebrospinal fluid macrophages is a highly sensitive test, especially for the diagnosis of ante mortem with a sensitivity of 85 % and a specificity of 83.3 % (Gruendl et al 2017).

4. Conclusions

In cats with suspected FIP, anamnesis, clinical signs and clinicopathological examinations should be correlated. The dry form is more difficult to diagnose than the wet form. In the wet form, laboratory analysis of the fluid can be performed, such as the Rivalt's test. If the test is negative, the probability of FIP is small, but if the test is positive, additional diagnostic tests should follow to confirm FIP. In FIP, the A: G ratio is low because hyperglobulinemia and hypoalbuminemia (low normal albumin concentration) are present, and this parameter can be used to assess the likelihood of FIP in a particular case. Patients with FIP often have significantly high levels of AGP (α1-acid glycoprotein). When distinguishing FIP cases from non-FIP cases, AGP concentrations in effusions (> 1.55 mg / ml) have a sensitivity and specificity of 93 %.

Conflict of interests

The authors declare that they do not have a conflict of interest.

Financing

No financial support was provided from any institute or other source.

5. Literature

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Outbreak of feline infectious peritonitis in a shelter in Taiwan: epidemiological and molecular evidence of horizontal transmission of a new type II feline coronavirus

Ying-Ting Wang,1 Bi-Ling Su,2 Li-En Hsieh,1 and Ling-Ling Chueh1
Original article: An outbreak of feline infectious peritonitis in a Taiwanese shelter: epidemiologic and molecular evidence for horizontal transmission of a novel type II feline coronavirus
Czech translation partially taken from: Results Confirmation of the FIP outbreak in the cat shelter - Sevaron
13.7.2013

Abstract

Infectious feline peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV) infection. FCoV can be divided into serotypes I and II. The virus that causes FIP (FIPV) is said to occur sporadically and does not often spread from one cat to another. An outbreak in one animal shelter in Taiwan was recently confirmed. FCoV from all cats in this shelter was analyzed to determine the epidemiology of this outbreak. Thirteen of the 46 (28,2%) cats with typical FIP symptoms were identified. Of these, FIP was confirmed in seven cats by necropsy or histopathological examination. Despite the fact that in this environment with more cats, more than one FCoV was identified, eight cats with symptoms of FIP were reliably found to be infected with FCoV type II. Sequence analysis revealed that FIPV type II, found from feline faeces, body effusions and granulomatous tissue homogenate from cats that underwent FIP, contained identical recombination in all cases. WITH gene. Two cats that succumbed to FIP were found to have an identical nonsense mutation in 3c gene. The excretion of this type II virus in faeces of the effusive form of FIP can be detected up to six days before the animal dies. In general, our data demonstrate that horizontal transmission of FIPV is possible and that FIP cats may pose a potential risk to other cats living in the same environment.

Introduction

Infectious feline peritonitis (FIP) is a fatal disease of cats caused by feline coronavirus (FCoV) infection. FCoV is an enveloped RNA virus that belongs to the species Alphacoronavirus, family Coronaviridae and in order Nidovirales. The size of the FCoV genome is approximately 28.9 kb, including the nonstructural replication gene; four structural genes that encode spike (S), envelope, membrane, and nucleocapsid proteins; and five helper / nonstructural genes 3abca 7ab[1].

Feline coronaviruses cause mild, invisible, and transient bowel infections and are ubiquitous among cat populations worldwide [2]. They occur in two serotypes, I and II [man]3]. Type I FCoV predominates here, while type II virus represents only 2-30% infections [48]. Following the accumulation of genetic evidence, it is apparent that FCoV type II was formed by two homologous recombinations between FCoV type I and canine coronavirus CoV (CCoV) [9,10]. Both serotypes can mutate in the host, lead to macrophage tropism and a systemic disease called infectious feline peritonitis [cat]2,11,12]. Due to poor virus shedding in FIP studies in cats, mutant FIP viruses (FIP-inducing FCoV, FIPV) appear to be contained only in diseased tissues and are not naturally transmitted in cat-to-cat contact [2,11,13,14].

In this article, we report an epizootic FIP in a shelter in Taiwan that was caused by a new Type II FCoV. Epidemiological and molecular examination of isolates from various healthy and sick cats from this shelter strongly suggests that the virus was introduced by moving kittens from another shelter with subsequent horizontal spread to adult cats with which the new kittens shared the shelter.

Materials and methods

Animals and sampling

A total of 46 cats from a private shelter were included in this study, which ran from September 2011 to August 2012. This shelter houses adult cats and from time to time a few kittens. All the cats were either strayed or rescued, and some of them were obtained from the homes of various private rescue stations where the rescued cats were temporarily housed. Before the onset of the disease, all cats lived together in an indoor environment without cages, sharing food, drink and toilets. Some cats were siblings, others were not related to them (Table 1).

Table 1
Information on all cats from this shelter in which FIP was suspected and in which the disease was confirmed

Hot girlAge 1 Date of admission to the shelter Date of onset of fever Date of death Clinical findings Necropsy findings Effusive / non - fusive
13mJune 16, 2011August 17, 2011September 1, 2011Fever, anorexia, ascites, neurological symptoms  
2a4mAugust 6, 2011ON THE 2September 21, 2011Clinical signs are not available  
3b3mJuly 11, 2011August 18, 2011September 25, 2011Fever, anorexia, weight loss, neurological symptoms  
42.5mJun. 08, 2011August 16, 2011September 28, 2011Fever, ascites, neurological symptoms  
5a4mAugust 6, 2011August 15, 2011October 20, 2011Fever, pleural effusion, diarrhea  
67mApril 24, 2011ON THEOctober 22, 2011Anorexia, weight loss, neurological symptoms  
73y6mResidentON THEOctober 27, 2011 Ascites, jaundice, granulomatous lesions in the kidney, fibrinous peritonitisEffusive
86mJuly 11, 2011ON THEDecember 14, 2011 Granulomatous changes in the kidneys, liver, lungs, brain and eyes Non-fusible
92yResidentON THEDecember 28, 2011 Ascites, pleural effusion and pericardial effusion, granulomatous changes in the kidneys, liver and intestine.Effusive / non - fusive
10 b3mJuly 11, 2011ON THENovember 5, 2011 Granulomatous changes in the kidneys, liver and omentum Non-fusible
11 c1y6mResidentON THEFebruary 14, 2012 Ascites and pleural effusion, jaundice, fibrinous peritonitis, granulomatous changes in the kidneys, liver, lungs and spleen.Effusive / non - fusive
12 c1y6mResidentON THEMarch 19, 2012 Jaundice, fibrinous peritonitis, granulomatous changes in the thoracic and abdominal walls, kidneys, liver, lungs, spleen omenta and eyes.Effusive / non - fusive
131y7mResidentON THEApril 13, 2012 Jaundice, enlargement of the liver and mesenteric lymph nodes, granulomatous changes in the kidneys and lungs.Non-fusible

1 Age of cats at the time of clinical signs of FIP.
2 Not available. 
a, b, c : siblings.

Faeces or rectal samples were taken from all asymptomatic cats at least once to monitor for FCoV. Body swabs, blood samples, swab specimens, including rectal, nasal, oral and conjunctival specimens, were taken as standard from cats that already showed signs of the disease or were suspected of having FIP. In addition to supportive care, cats with suspected FIP were treated with prednisolone (Prelon®, YF Chemical Corp., New Taipei City, Taiwan), benazepril (Cibacen®, Novartis, Barbera del Valles, Spain) and recombinant human interferon alpha (Roferon®-A). , Roche, Basel, Switzerland). Cats that succumbed to the disease were necropsied for pathological confirmation. During necropsy, body exudates were first removed with a needle and syringe, followed by swabs, blood, urine and granulomatous lesions on the internal organs. All samples were frozen at -20 ° C until use. All samples were tested for FCoV nested reverse transcription polymerase chain reaction (RT-nPCR) [man]15]. Samples with positive results were subsequently subjected to further analysis.

Sample preparation and reverse transcription

Swab samples were suspended in 1 ml of water treated with 0.1% diethyl pyrocarbonate (DEPC). Stool samples were suspended with 9x treated water 0.1% DEPC by vortexing. The suspension was centrifuged and the supernatant was transferred to a new tube. About 0.5 g of tissue was frozen and then crushed with a mortar and pestle in the presence of 2 ml of Trizol [16]. Total RNA was extracted from 300 μl of swab suspension, whole blood, faeces suspension, tissue homogenate and body effusion using Trizol. Twenty-one microliters of isolated RNA was reverse transcribed with specific primer N1 (5′-gctacaattgtatcctcaac-3 ′) or P211 [15] with Moloney mouse leukemia reverse transcription (Invitrogen, CA, USA). The reaction was incubated at 37 ° C for 60 min, at 72 ° C for 15 min and finally at 94 ° C for 5 min.

FCoV type determination by nested PCR

Nested PCR was performed for FCoV typing according to the procedures of Addie et al. [5] with a slight modification. After reverse transcription, 5 μl of complementary DNA was added to 25 μl of PCR mix (Invitrogen, CA, USA) according to the manufacturer's instructions for the following primer sets: S1 and Iffs to determine FCoV type I and S1 and Icfs to determine FCoV type II. Nested PCR was performed on 2 μl of the first PCR product using nested primers. The expected size of the second PCR achieved for type I and type II FCoV was 360 and 218 bp. RT-nPCR products were electrophoresed and then the target DNA fragments were purified (Geneaid Biotech, Ltd, Taipei) and sequenced (Mission Biotech, Taipei, Taiwan) - from both orientations.

Gene amplification, sequencing and analysis 3a and 3c  from FCoV type II

For amplification 3a of the FCoV type II gene from FIP cats, a set of specific primers was designed that is able to amplify from WITH type II gene to gen 3a. Complementary DNA, amplified with a primer set, targeted the 3 'end WITH FCoV type II gene (Icfs) and 5 ′ end 3a FCoVe gene (3aR2: 5′-caccaaaacctatacacacaag-3 ′). The temperature cycle was as follows: 5 minutes preheating at 94 ° C; 35 cycles of denaturation at 94 ° C for 20 s, annealing at 50 ° C for 20 s and extension at 72 ° C for 30 s; and final extension at 72 ° C for 5 minutes. This was followed by a second series of amplification using primers nIcfs and 3aR2; the expected product size was about 600 bp. Amplicons were electrophoresed, purified, and sequenced from both orientations to confirm nucleotide sequences.

For amplification 3c of the FCoV type II gene from FIP cats, a set of specific primers was designed that is able to amplify from WITH type II gene to gen 3c. Complementary DNA was amplified with forward primer (Icfs) and reverse primer (E68R: 5′-aatatcaatataattatctgctgga-3 ′ and N21R: 5′-gttcatctccccagttgacg-3 ′, respectively). The temperature cycle was as follows: 5 minutes preheating at 94 ° C; 40 cycles of denaturation at 94 ° C for 30 s, annealing at 46 ° C for 30 s and extension at 72 ° C for 90 s; and final extension at 72 ° C for 7 minutes. Following a second series of amplification using primers nIcfs and E68R, the products were electrophoresed, purified and sequenced from both orientations to confirm nucleotide sequences.

Phylogenetic analysis and recombinant analysis of FCoV type II

Several sequence alignments were performed using ClustalW 2.0 with manual editing in EditSeq (DNASTAR, Madison, USA). Phylogenetic analyzes were performed using MegAlign, version 7.2.1 (DNASTAR, Madison, USA). Bootscan and similar graphs were compiled using SimPlot 3.5.1 software (SCRoftware, Baltimore, USA).

The results

Confirmation of the FIP outbreak in the cat shelter

The shelter has been operating for three and a half years. Prior to August 2011, there were no records of FIP. The kittens (cats 1, 3, 4, 8 and 10) were moved to this shelter between June and July 2011. After arrival, these kittens played together and lived together with adult cats that lived here before. Prior to the outbreak, the kittens were individually taken to a veterinarian for vaccination and adoption visits. Fever was first detected in four kittens (cats 1, 3, 4, 5) within a few days (from 15 to 18 August) (Table 1). Clinical symptoms, e.g. fever, anorexia, neurological symptoms, shortness of breath and enlargement of the abdomen were observed over the next two months and the kittens gradually died between 1 September and 22 October (Table 1). Shelters from the shelter asked for our help on September 27. All cats housed in the shelter for a long time were immediately examined for FCoV using the RT-nPCR method. All FCoV-positive cats were isolated and kept separately. Nevertheless, starting in September, adult cats with FIP (cats 7-13) showed clinical signs similar to kittens, and all of these cats later died.

Six kittens (cats 1-6) with body effusions or neurological symptoms that succumbed in the first two months were not confirmed for necropsy (Table 1). Cat 1 was once brought to our teaching hospital and ascites (free fluid in the abdominal cavity) was taken from her. In cats 7-13, typical symptoms were found, namely ascites or pleural effusions in the body cavity (effusive FIP) and granulomatous lesions in some organs, especially in the kidneys, nuclei, lungs, omentum (forecourt) and eyes (non-effusive FIP). In cats 9, 11 and 12, necropsy showed a mixed form of the disease (Table 2) 1).

A total of 13 of the 46 cats (28.3%) died between September 2011 and April 2012 at FIP. At this time, 33 cats (71.7%) appeared to be clinically healthy and 26 of these asymptomatic cats (78.7%) were positive at least once for FCoV - detected from faeces using the RT-nPCR method. The other seven of these asymptomatic cats were negative for FCoV (Table 2) 2).

Table 2
Detection of the occurrence and type of FCoV from faeces samples in healthy cats from the same shelter

Hot girl
FCoV
Type
Oct. 2011Feb. 2012Jun. 2012 Jul. 2012
14+++++++untypable
15+ untypable
16++ untypable
17+++++++I
18++++++++I
19 +untypable
20 +untypable
21  
22++++ untypable
23++++I
24+ untypable
25++++++I
26 +untypable
27++++++I
28+++++++I
29  
30++++I
31   
32+++I
33 ++untypable
34++   I
35 ++untypable
36+++++I
37    
38  + untypable
39 ++++I
40 +untypable
41 ++untypable
42  +untypable
43    
44    
45    
46  + untypable

++: FCoV detected in the first round of PCR.
+: FCoV detected only in nested PCR.

FIPV type II was found in all cats that succumbed to FIP

In order to further investigate the relationship between these seven histopathologically confirmed FIP cats, the amplified DNA was typed, sequenced and analyzed. FIPV type II was detected in all eight animals that succumbed to FIP, from swabs, faeces, urine, body effusions, cerebrospinal fluid, and tissue homogenates (Table 3). Type II viruses that cause FIP have been found not only in diseased tissue but also in faeces samples (cats 7, 11, 12 and 13), nasal / oral / conjunctival swab samples (cats 7, 8, 9, 11 and 12). ) and in urine collected by cystocentesis (cat 11) (Table 3). Although no necropsy was performed, ascites from cat 1 - the first cat to die in the shelter at FIP - were available for analysis. This cat was confirmed to be infected with type II virus. In healthy animals, only type I or FCoV was detected from faeces samples without type determination (Table 2) 2). Cats 8, 9 and 13 were infected with both types of FCoV (Table 2) 3). Although it has been found that in this environment with many cats there is more than one type of FCoV, ie. type I, II or non-typed viruses, FCoV type II infection was found in all eight FIP cats, whereas this was not the case in healthy animals (Tables 2 and33).

Table 3
Characteristics 3c FCoV genes obtained from different samples of FIP cats

Hot girlFCoV genotypeWITH instead of gene crossingIntegrity 3c geneb
NIGHTR / FUA / PCSFLiLuKiBrSpIntR / FA / PLiLuKiBrSp
1   II       4250and intact     
7IIII II IIIIIIIIII 4250intact intactintactintact intact
8III   +IIII+        
9III II +II IIII+4250 G210 *   G210 *G210 *
10     ++IIII II4250    intact  
11IIIIIIII+IIIIII + 4250   E47 *   
12IIII IIII+IIII II+4250G210 *G210 *     
13 I / II  +++IIII+ 4250     Q218 * 

NIGHT, nose / mouth / conjunctival swabs; R / F, rectal swabs or stool samples; A / P, ascites or pleural effusion; CSF, cerebrospinal fluid; Li, liver; Lu, lungs; Ki, kidney; Br, brain; Sp, spleen; Int, gut.
+: FCoV positive, but virus type cannot be determined. -: FCoV negative.
a: FCoV / NTU2 / R / 2003; GenBank: DQ160294.
b: E47 *, G210 * and Q218 *: truncated 3c proteins with premature stop codons at amino acids 47, 210 and 218 were found.

FIPV type II of the same origin was found in cats that succumbed to FIP

To further investigate the relationship of these disease-causing type II viruses, which were isolated from cats that succumbed to FIP, sets of specific primers capable of specifically amplifying from the 3 'end were used to analyze viral sequences. WITH the type II gene has a subsequent gene. The identity of the 620 bp amplicons derived from the seven FIPV type II was approximately 98.7% to 99.8%. Phylogenetic analysis found that the type II FCoVs derived from the outbreak described above were all grouped into a separate cluster, which differs from the other four type II FCoVs currently available at GenBank, i. FIPV 79-1146 (GenBank: {“Type”: ”entrez-nucleotide”, “attrs”: {“text”: ”DQ010921 ″,” term_id ”:” 63098796 ″}} DQ010921), FCoV 79-1683 (GenBank: {“Type”: ”entrez-nucleotide”, “attrs”: {“text”: ”JN634064 ″,” term_id ”:” 384038902 ″}} JN634064), FCoV DF-2 (GenBank: {“Type”: ”entrez-nucleotide”, “attrs”: {“text”: ”DQ286389 ″,” term_id ”:” 87242672 ″}} DQ286389) and FCoV NTU156 (GenBank: {“Type”: ”entrez-nucleotide”, “attrs”: {“text”: ”GQ152141 ″,” term_id ”:” 240015188 ″}} GQ152141) (data not shown).

Recombination at the 3 ′ end WITH of the putative recombination site at nucleotide 4250 was determined in all FCoV type II animals obtained from body effusions and tissue homogenates in cats 1, 7, 9, 10, 11, 12 and 13 (Additional set 1) (Table 3). Sequences above this site show greater similarity to CCoV, whereas sequences beyond this site were more similar to type I FCoV (Fig. 1). 1). Indeed, these findings suggest that FCoV type II, found in all FIP cats, has a common origin.

Figure 1
FIPV recombination from cats 1, 7, 9, 10, 11, 12 and 13 on the S gene. Alignment of the 3 ′ end of the S gene with subsequent FCoV genes isolated from seven FIP cats with FCoV type I and CCoV. The light and dark shaded regions include greater similarity to CCoV and FCoV type I. The predicted recombination event occurred at nucleotide 4250 based on comparison to FCoV NTU2 and is indicated by an arrow. Sequences were obtained from FIPV found in individual samples and tissues and are summarized. NIGHT: swabs from the nose / mouth / conjunctiva; RS: rectal swabs; As: ascites; PE: pleural effusion; Li: liver; Lu: lungs; Ki: kidneys; Br: brain; Sp: spleen; dbd: days before death. GenBank accession number: FCoV C1Je (GenBank: DQ848678), FCoV Black (GenBank: EU186072), FCoV NTU2 (GenBank: DQ160294) and CCoV NTU336 (GenBank: GQ477367).

Identical nonsensical mutation on 3c The gene was found in two cats that succumbed to FIP

In order to further analyze the relationship of these FIPVs, they were 3c genes, a proposed virulence-associated FIP, are amplified from the disease-causing FCoV type II. Mutated 3c genes with identical premature stop codon at nucleotides 628-630 (amino acids 210, G210 *) were found in two FIP cats, cat 9 (ascites, spleen and brain) and 12 (ascites and rectal swabs from the day the cat died and four days previously) (Fig. 2A). It is worth noting that FIPV, obtained from cat 12, showed the same nonsense mutation as the virus in its ascites. Intact 3c the genes were discovered in cats 1, 7 and 10, which had previously succumbed to FIP. Two other clear / different nonsense mutations were found in cats 11 (E47 *) and 13 (Q218 *) (Fig. 1). 2AB, Table 3).

Figure 2:
Alignment of complete FIPV 3c genes from cats 1, 7, 9, 10, 11, 12 and 13. (A) The full length 3c genes analyzed in this study were aligned with FCoV type I, FCoV NTU2. The sequences were obtained from FIPV found in individual samples and tissues and are listed together. The box represents the identified premature stop codons. (B) The diagram shows the location of premature stop codons (PT) of gene 3c from different samples from different FIP cats.

FIPV type II excretion can be detected in the terminal phase in FIP cats

The occurrence of FCoV was continuously analyzed to elucidate a possible route of FIPV secretion and transmission. Disease-associated FCoV type II was found to be excreted by the nasal / oral / conjunctival route and faeces (Table 4). Faecal and nasal / oral / conjunctival type II shedding can be detected from day 6 (cat 11) and from day 4 (cat 12) before death. Viremia can be detected during the terminal stage in cats suffering from FIP up to 18 days before death, and concomitant faecal excretion was detected in one cat (cat 12) (Table 4).

Table 4
Excretion and serotypes of feline coronavirus detected in FIP cats in a cat shelter

Hot girlSampleDays before death
−80−66−60−57−50−43−36−29−25−23−20−18−14−12−8−6−40*
9Feces I            I  II
 NIGHT tampons                 II
 Viremie              II  +
 Efuze              IIII II
11Feces            II II
 NIGHT tampons               II
 Viremie             
 Efuze     +           II
12Feces+ +  IIII
 NIGHT tampons      IIII
 Viremie  II++   
 EfuzeII                II

+: FCoV positive; -: FCoV negative.
I, II: FCoV type I or type II.
*: Samples were taken immediately before euthanasia, except for cat 12, which were sampled after death.

Discussion

The possibility of horizontal transmission is generally questioned in FIP because (i) the occurrence of FIP is sporadic and it is common for only one of them to develop FIP in an environment with a large number of cats [2]; (ii) internal mutation theory, which describes that FIPV is a mutant generated from enteric FCoV in one cat [12,17]; (iii) there is insufficient evidence that the mutant FIPV is eliminated from FIP cats; and (iv) mutations 3c gene is unique for every FIP cat [man]11,13,18]. The current belief is that cats that have succumbed to FIP do not excrete and pass FIPV to other cats [11,13,14,1820]. Our data indicate that this outbreak of FIP was caused by viruses of the same origin. First, all cats that died of FIP had a type II infection, and recombination of these seven type II viruses was located at the same site. Recombination of type II viruses currently available in the genetic bank, i.e. FIPV 79-1146, FCoV 79-1683 and FCoV NTU156, were all unique, specific and occurred independently [9,10]. Second, FIPV, found in three kittens that died within the first two months after the onset of fever, had an intact 3c gene, whereas viruses from cats that survived longer (died four to eight months later) all contained a nonsensical mutation, i. G210 * (cats 9 and 12), E47 * (cat 11) and Q218 * (cat 13). Because the three nonsense mutations found in FIPV in these animals were all located at different sites, the viruses that originally infected these cats should be intact. 3c gene - similar to the virus found in kittens that died earlier. Following infection, local mutations occurred during virus replication in individual cats, resulting in FIPV with 3c a gene that carries meaningless mutations in different places. The finding that viruses, which were identified not only in tissues but also in faecal samples in two cats (cats 9 and 12), had an identical mutation in 3c gene, further confirmed that there was a horizontal transfer (Table 2) 3). Taken together, all of these findings demonstrated that highly virulent FIPV spread horizontally from one animal to another.

This is the first report of an FIPV type II outbreak with evidence of horizontal disease-causing FCoV transmission. The FIP broke out after five kittens (cats 1, 3, 4, 8 and 10) entered this shelter between June and July 2011. Because causative type II viruses with a specific genetic marker in the S gene have been confirmed as feline and canine coronavirus recombination, and some of the kittens that died earlier were found to have lived together or next to dogs between rescue and transport to the shelter. of these kittens may have been the source of this type II virus. Dogs and especially young dogs often shed large amounts of canine coronavirus in their faeces in shelters, and recombination between feline-canine and canine-feline coronavirus is already well documented [man]2123]. In addition, type II causative viruses have been detected in a number of excreta and secretions in cats that have died of FIP (Table 3), demonstrating that it is possible to spread between cats.

Although immediately after the first examination of all animals from this FCoV shelter, FCoV-secreting cats were housed in separate cages and transmission subsequently ceased, mortality at the onset of the disease was high (28%, 13/46). The results of three studies that looked at the outbreak of FIP have been reported earlier. The results of a four-year study conducted at a nearby cat kennel showed an average mortality of 17.3% [24]; the mortality rate from a ten-year study conducted at a nearby kennel was 29.4% (5/17) [25]. Another epidemic study conducted in seven kennels / shelters revealed >10% mortality [20]. The high incidence of FIP in these closed breeding stations could be influenced by genetically predisposed breeding animals. In our study, only a few FIP cats in this shelter were siblings and the other cats were not genetically related. Our study shows that even without the influence of genetic predisposing factors, FIP mortality can be high in a confined environment with a large number of cats if the spread of FCoV, which causes the disease, remains undetected.

In this environment with a large number of cats, three FIP cats were infected not only with FCoV type II, but also co-infected with FCoV type I (Table 3). Type I FCoV was found only in faecal samples, while type II FCoV was found in various samples, including body effusions, granulomatous tissue homogenates, and cerebrospinal fluid. This finding indicates that FCoV type II was a major cause of FIP in these doubly infected animals. This finding is consistent with our previous finding that FCoV type II infection is significantly associated with FIP [4].

The presence of FCoV in whole blood in the terminal phase has been identified previously [26,27]; however, to our knowledge, the presence of FIPV in faeces prior to the final stage of the disease was not published anywhere until our study. The excretion of this type II virus in faeces and by the nasal / oral / conjunctival route can be detected in the effusive form of FIP up to six days before the death of the animal. Another experimental study of the infection showed that inoculated viruses could not be detected until about two weeks after inoculation, before clinical signs of the disease developed [14]. In summary, FIPV transmission could occur at the beginning, before the manifestations of the disease and in the terminal phase. When the disease broke out in our case, all the cats were initially placed together in an open room. After seven cats gradually succumbed to the disease, all FCoV-positive cats were housed separately in cages and kept separately. Isolation probably inhibited disease transmission. This outbreak of disease, which killed 13 cats, allowed us to make it clear that FIPV can be transmitted horizontally and to show that the isolation of sick cats should be taken into account in an environment where more cats are present.

Competitive interests

The authors claim that they have no competitive interests.

Contributions and contributions of authors

YTW performed sampling and preparation, FCoV detection, type determination, amplification 3c gene and other analyzes and compiled a manuscript. The BLS supervised the sampling and treatment of all FIP animals and contributed to the compilation of the manuscript. LEH participated in the amplification 3c gene, genetic analysis and manuscript preparation. The LLC devised the study, participated in the design of the study, coordinated and participated in the preparation of the manuscript. All authors read and approved the final version of the manuscript.

Additional material

Additional file 1:

FIPV recombination site analysis in cats 1, 7, 9, 10, 11, 12 and 13 at WITH gene. Analysis of the plot similarity using the Kimur (two-parameter) distance model, the model of adjacent interconnected trees, and 100 replicates of the bootstrap showed that recombination had occurred and the putative crossing point is indicated by an arrow. 

Thanks

The authors would like to thank the caregivers in the mentioned cat shelter, without whose help this study would not have been possible.

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  17. Poland AM, Vennema H, Foley JE, Pedersen NC. Two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus. J Clin Microbiol. 1996; 44: 3180–3184. [PMC free article] [PubMed]
  18. Pedersen NC, Liu H, Scarlett J, Leutenegger CM, Golovko L, Kennedy H, Kamal FM. Feline infectious peritonitis: role of the feline coronavirus 3c gene in intestinal tropism and pathogenicity based upon isolates from resident and adopted shelter cats. Virus Res. 2012; 44: 17–28. doi: 10.1016 / j.virusres.2011.12.020. [PubMed] [Cross Ref]
  19. Foley JE, Poland A, Carlson J, Pedersen NC. Patterns of feline coronavirus infection and fecal shedding from cats in multiple-cat environments. J Am Vet Med Assoc. 1997; 44: 1307–1312. [PubMed]
  20. Foley JE, Poland A, Carlson J, Pedersen NC. Risk factors for feline infectious peritonitis among cats in multiple-cat environments with endemic feline enteric coronavirus. J Am Vet Med Assoc. 1997; 44: 1313–1318. [PubMed]
  21. Stavisky J, Pinchbeck G, Gaskell RM, Dawson S, German AJ, Radford AD. Cross sectional and longitudinal surveys of canine enteric coronavirus infection in kennelled dogs: a molecular marker for biosecurity. Infect Genet Evol. 2012; 44: 1419–1426. doi: 10.1016 / j.meegid.2012.04.010. [PubMed] [Cross Ref]
  22. Decaro N, Mari V, Elia G, Addie DD, Camero M, Lucente MS, Martella V, Buonavoglia C. Recombinant canine coronaviruses in dogs, Europe. Emerg Infect Dis. 2010; 44: 41–47. doi: 10.3201 / eid1601.090726. [PMC free article] [PubMed] [Cross Ref]
  23. Decaro N, Buonavoglia C. An update on canine coronaviruses: viral evolution and pathobiology. Vet Microbiol. 2008; 44: 221–234. doi: 10.1016 / j.vetmic.2008.06.007. [PubMed] [Cross Ref]
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  25. Watt NJ, MacIntyre NJ, McOrist S. An extended outbreak of infectious peritonitis in a closed colony of European wildcats (Felis silvestris) J Comp Pathol. 1993; 44: 73–79. doi: 10.1016 / S0021-9975 (08) 80229-0. [PubMed] [Cross Ref]
  26. de Groot-Mijnes JD, van Dun JM, van der Most RG, de Groot RJ. Natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease. J Virol. 2005; 44: 1036–1044. doi: 10.1128 / JVI.79.2.1036-1044.2005. [PMC free article] [PubMed] [Cross Ref]
  27. Tsai HY, Chueh LL, Lin CN, Su BL. Clinicopathological findings and disease staging of feline infectious peritonitis: 51 cases from 2003 to 2009 in Taiwan. J Feline Med Surg. 2011; 44: 74–80. doi: 10.1016 / j.jfms.2010.09.014. [PubMed] [Cross Ref]
Read "Outbreaks of feline infectious peritonitis in a shelter in Taiwan: epidemiological and molecular evidence of horizontal transmission of a new type II feline coronavirus"

Origin of abdominal or thoracic effusions in cats with wet FIP and causes of their persistence during treatment

Niels C. Pedersen, DVM, PhD
Pet Health Center
University of California, Davis
24.9.2021

Original article: Origin of abdominal or thoracic effusions in cats with wet FIP and reasons for their persistence during treatment


Origin of FIP exudates. Sweat in wet FIP comes from small vessels (venules) that line the surface of the abdominal and thoracic organs (visceral) and walls (parietal), mesentery / mediastinum, and omentum. The spaces around these vessels contain a specific type of macrophages that come from monocyte progenitors that constantly recirculate between the bloodstream, the interstitial spaces around the venules, the afferent lymph, the regional lymph nodes, and back into the bloodstream. Other sites of this recirculation are located in the meninges, brain ependyma, and uveal eye tract. A small proportion of these monocytes develop into immature macrophages (monocyte / macrophage) and eventually into resident macrophages. Macrophages are constantly looking for infections.

FIPV is caused by a mutation in feline enteric coronavirus (FECV) present in lymphoid tissues and lymph nodes in the lower intestine. The mutation changes FECV cell tropism from enterocytes to peritoneal-type macrophages. Monocytes / macrophages appear to be the first cell type to be infected. This infection causes more monocytes to leave the bloodstream and begin to turn into macrophages, which continue the cycle of infection. [2]. Monocytes / macrophages do not undergo programmed cell death as usually expected, but continue to mature into large virus-loaded macrophages. These large macrophages eventually undergo programmed cell death (apoptosis) and release large amounts of virus, which then infects new monocytes / macrophages. [1]. Infected monocytes / macrophages and macrophages produce several substances (cytokines) that mediate the intensity of inflammation (disease) and immunity (resistance). [1,2].

Inflammation associated with FIP leads to three types of changes in the venules. The first is loss of vascular wall integrity, micro-bleeding, and leakage of plasma protein rich in activated complement clotting and activation factors and other inflammatory proteins. The second type of damage involves thrombosis and blocking blood flow. The third injury occurs in more chronic cases and involves fibrosis (scarring) around the blood vessels. Variations in these three events determine the amount and composition of exudates according to the four Starling forces that determine the movement of fluids between the bloodstream and interstitial spaces. [3].

The classic effusion in wet FIP is mainly due to acute damage to the vessel walls and leakage of plasma into the interstitial spaces and finally into the body cavities. Protein that escapes into the interstitial spaces attracts additional fluids, which can be exacerbated by blocking venous blood flow and increasing capillary pressure. This type of effusion, known as exudate, also contains high levels of protein, which is involved in inflammation, immune responses and blood clotting.

This fluid also contains a large number of neutrophils, macrophages / monocytes, macrophages, eosinophils and a lower number of lymphocytes and red blood cells. This classic type of fluid has the consistency of egg white and forms weak clots containing a high amount of bilirubin. Bilirubin does not originate from liver disease, but rather from the destruction of red blood cells that escape into interstitial tissue cells and are taken up by monocytes / macrophages and macrophages. Red blood cells break down and hemoglobin is broken down into heme and globin. Globin is further metabolized to biliverdin (greenish color) and finally to bilirubin (yellowish color), which is then excreted by the liver. However, cats lack the enzymes used for conjugation and are therefore ineffective in removing bilirubin from the body. [4]. This leads to the accumulation of bilirubin in the bloodstream and gives the effusion a yellow tinge. The darker the yellow tint, the more bilirubin is in the effusion, the more severe the initiating inflammatory response and the more severe the resulting bilirubinemia, bilirubinuria and jaundice.

The opposite extreme of the classic and more acute effusion in FIP are effusions arising mainly from chronic infections and blockage of venous blood flow and consequent increase in capillary pressure. High capillary pressure results in effusion that is more distant to interstitial fluid than plasma, has a lower protein content, is watery rather than sticky, clear or slightly yellow in color, is not prone to clotting, and has a lower number of acute inflammatory cells such as neutrophils. There are also FIP effusions that are among these extremes, depending on the relative degree of acute inflammation and chronic fibrosis. These transient types of fluids are commonly referred to in the veterinary literature as modified transudate, but this is a misnomer. The modified transudate begins as a transudate and changes as it persists and causes mild inflammation. Low protein and cell effusions in FIP arise as exudates and not as transudates and do not conform to this description. The more correct term is "modified exudate" or "variant exudate outflow".

How long do sweats usually last in cats treated with GS-441524 or GC376? The presence of abdominal effusions often leads to a large dilation of the abdomen and is confirmed by palpation, hollow needle aspiration, X-ray or ultrasound. Cats with thoracic effusions are most often presented with severe shortness of breath and are confirmed by radiological examination and aspiration. Chest effusions are almost always removed to relieve shortness of breath and recur slowly compared to abdominal effusions. Therefore, abdominal effusions are usually not removed unless they are massive and do not interfere with respiration, as they are quickly replaced. Repeated drainage of abdominal effusions can also deplete proteins and cause harmful changes in fluid and electrolyte balance in severely ill cats.

Chest effusions disappear faster with GS-441524 treatment, with improved breathing within 24-72 hours and usually disappearing in less than 7 days. Abdominal effusions usually decrease significantly within 7-14 days and disappear within 21-28 days. The detection of exudates that persist after this time depends on their amount and method of detection. Small amounts of persistent fluid can only be detected by ultrasound.

Persistence of exudates during or after antiviral treatment. There are three basic reasons for the persistence of exudates. The first is the persistence of the infection and the resulting inflammation at a certain level, which can be caused by inappropriate treatment, poor medication or drug resistance. Inadequate treatment may be the result of incorrect dosing of the wrong drug or the acquisition of virus resistance to the drug. The second reason for fluid persistence is chronic venous damage and increased capillary pressure. This may be due to a low-grade infection or residual fibrosis from an infection that has been removed. The third reason for persistence is the existence of other diseases, which can also manifest as exudates. These include congenital heart disease, in particular cardiomyopathy, chronic liver disease (acquired or congenital), hypoproteinemia (acquired or congenital) and cancer. Congenital diseases causing effusions are more common in young cats, while acquired causes and cancer are more commonly diagnosed in older cats.

Diagnosis and treatment of persistent effusions. A thorough examination of the fluid, as described above, is a prerequisite for diagnosis and treatment. If the fluid is inflammatory or semi-inflammatory and the cell pellet is positive by PCR or IHC, the reason for the persistence of the infection must be determined. Was the antiviral treatment performed correctly, was the antiviral drug active and its concentration correct, was there evidence of acquired drug resistance? If the fluid is inflammatory and PCR and IHC are negative, what other diseases are possible? Low protein and non-inflammatory fluids that are negative for PCR and IHC indicate a diagnosis of residual small vessel fibrosis and / or other contributing causes such as heart disease, chronic liver disease, hypoproteinemia (bowel disease or kidneys). Some of the disorders causing this type of effusion may require an exploratory laparotomy with a thorough examination of the abdominal organs and a selective biopsy to determine the origin of the fluid. The treatment of persistent effusions will vary greatly depending on the end cause. Persistent effusions caused by residual small vessel fibrosis in cats cured of the infection often resolve after many weeks or months. Persistent discharges caused in whole or in part by other diseases require treatment for these diseases.

Identification and characteristics of persistent effusions. The presence of fluid after 4 weeks of GS treatment is unpleasant and is usually detected in several ways depending on the amount of fluid and its location. Large amounts of fluid are usually determined by the degree of abdominal dilation, palpation, X-ray and abdominal aspiration, while smaller amounts of fluid are best detected by ultrasound. Persistent pleural effusion is usually detected by X-rays or ultrasound. Overall, ultrasound is the most accurate means of detecting and semiquantitatively determining thoracic and abdominal effusions. Ultrasound can also be used in combination with thin needle aspiration to collect small and localized amounts of fluid.

The second step in examining persistent effusions is to analyze them based on color, protein content, white and red blood cell counts, and the types of white blood cells present. Fluids generated primarily by inflammation will have protein levels close to or equal to plasma and a large number of white blood cells (neutrophils, lymphocytes, monocytes / macrophages and large vacuolated macrophages). Fluids produced by increased capillary pressure are more similar to interstitial fluid with proteins closer to 2.0 g / dl and cell counts <200. The Rivalt test is often used to diagnose FIP-related effusions. However, this is not a specific test for FIP, but rather for inflammatory effusions. It is usually positive for FIP effusions that are high in protein and cells, but is often negative for very low protein and cell effusions. The effluents that are between these two types of effusions will be tested either positively or negatively, depending on where they are in the spectrum.

The third step is the analysis of exudates for the presence of FIP virus. This usually requires 5 to 25 ml or more of fluid. For fluids with a higher protein and cell count, a smaller amount may suffice, while for fluids with a low protein and cell count, a larger amount is required. The freshly collected sample should be centrifuged and the cell pellet analyzed for the presence of viral RNA by PCR or cytocentrifuged for immunohistochemistry (IHC). The PCR test should be for FIPV 7b RNA and not for specific FIPV mutations, as the mutation test does not have sufficient sensitivity and does not provide any diagnostic benefits [5]. Samples that are positive by PCR or IHC provide definitive evidence of FIP. However, up to 30 % samples from known cases of FIP may have a false negative test either due to an inappropriate sample and its preparation, or because the RNA level of the FIP virus is below the level of detection. It is also true that the less inflammatory the fluid, the lower the virus levels. Therefore, effusions with lower protein and white blood cell levels are more likely to be tested negative because viral RNA is below the detection limit of the test.

References

[1] Watanabe R, Eckstrand C, Liu H, Pedersen NC. Characterization of peritoneal cells from cats with experimentally-induced feline infectious peritonitis (FIP) using RNA-seq. Vet Res. 2018 49 (1): 81. doi: 10.1186 / s13567-018-0578-y.

[2]. Kipar A, Meli ML, Failing K, Euler T, Gomes-Keller MA, Schwartz D, Lutz H, Reinacher M. Natural feline coronavirus infection: differences in cytokine patterns in association with the outcome of infection. Vet Immunol Immunopathol. 2006 Aug 15; 112 (3-4): 141-55. doi: 10.1016 / j.vetimm.2006.02.004. Epub

[3] Brandis K. Starling's Hypothesis, LibreTexts. https://med.libretexts.org/Bookshelves/Anatomy_and_Physiology/Book%3A_Fluid_Physiology _(Brandis)/04%3A_Capillary_Fluid_Dynamics/4.02%3A_Starling%27s_Hypothesis

[4]. Court MH. Feline drug metabolism and disposition: pharmacokinetic evidence for species differences and molecular mechanisms. Vet Clin North Am Small Anim Pract. 2013; 43 (5): 10391054. doi: 10.1016 / j.cvsm.2013.05.002

[5]. Barker, EN, Stranieri, A, Helps, CR. Limitations of using feline coronavirus spike protein gene mutations to diagnose feline infectious peritonitis. Vet Res 2017; 48: 60. Read "Origin of abdominal or thoracic effusions in cats with wet FIP and causes of their persistence during treatment"

Acute phase proteins in cats

April 2019
Rita Mourão Rosa, Lisa Alexandra Pereira Mestrinho
Original article: Acute phase proteins in cats

ABSTRACT: Acute phase proteins (APPs) are proteins synthesized and released mainly by hepatocytes during cell damage or invasion of microorganisms. This article provides an overview of the use of APP in cat diseases, identifies their usefulness in the clinical setting, and analyzes 55 published papers. Serum amyloid A, alpha-1 acid glycoprotein and haptoglobin are indicators that the authors consider useful in monitoring the acute inflammatory response in cats. Although APP measurement is still not routinely used in veterinary medicine, along with clinical signs and other blood parameters, they are clinically of interest and useful in diseases such as feline infectious peritonitis, pancreatitis, renal failure, retroviral and calicivirus infections. Although there are commercially available kits for measuring feline APPs, standardization of tests for technical simplicity, greater species specificity, and less associated costs will allow for routine use in feline practice, as is the case in the human field.
keywords: inflammation, acute phase proteins, cat.

Introduction

Acute phase response (APR) is an early non-specific systemic innate immune response to a local or systemic stimulus that helps treat and restore homeostasis and minimize tissue damage when an organism is affected by trauma, infection, stress, surgery, neoplasia, or inflammation (GRUYS et al. , 2005; CRAY et al., 2009; ECKERSALL AND BELL, 2010). In this reaction, we observe several different systemic effects: fever, leukocytosis, hormonal changes - mainly cortisol and thyroxine concentrations, with secondary catabolic status and serum muscle, iron and zinc depletion (CERÓN et al. 2005, JAVARD et al. 2017).
Cytokines IL-1β, TNF-α, and especially IL-6, and approximately 90 minutes after injury, increase protein synthesis in hepatocytes, lymph nodes, tonsils, and spleen, as well as blood leukocytes. These newly formed proteins are called acute phase proteins (APPs) (TIZARD, 2013b).

Acute-phase proteins

APP concentrations may increase (APP positive) or decrease (APP negative) in response to inflammation (PALTRINIERI et al., 2008) (JOHNSTON & TOBIAS, 2018). They can activate leukocytosis and complement, cause protease inhibition, lead to blood clotting and opsonization - a defense mechanism that leads to the elimination of infectious agents, tissue regeneration and restoration of health (CRAY et al., 2009). APP can have two functions, pro- and / or anti-inflammatory, which must be fine-tuned to promote homeostasis (HOCHEPIED et al., 2003).

According to the size and duration of the reaction following the stimulus, three main groups of APP are distinguished (MURATA et al., 2004; PETERSEN et al., 2004; CERÓN et al.). Positive APP can be divided into two groups: the first group includes APP with an increase of 10 up to 1000-fold in humans or 10- to 100-fold in domestic animals in the presence of inflammation - e.g. c-reactive protein (CRP) and serum amyloid A (SAA). The second group are APPs, which increase 2 to 10-fold in an inflammatory response - e.g. haptoglobin and alpha-globulins. The last group included negative APP, in which the concentration decreases in response to inflammation - e.g. albumin (KANN et al., 2012).

Acute phase positive proteins


Positive APPs are glycoproteins whose serum concentrations, when stimulated by pro-inflammatory cytokines, increase by 25 % during the disease process and are released into the bloodstream. These concentrations can be measured and used in diagnosis, prognosis, monitoring of response to treatment, as well as general health screening. They can also be considered as quantitative biomarkers of the disease, highly sensitive to inflammation but not very specific, as an increase in APP can also occur in non-inflammatory diseases (CERÓN et al., 2005; ECKERSALL and BELL, 2010).

Positive APPs respond to cytokines differently, and these groups fall into two main classes. Type 1 APP, which includes AGP, complement component 3, SAA, CRP, haptoglobin and hemopexin, is regulated by IL-1, IL-6 and TNF-α as well as glucocorticoids. Type 2, which includes three fibrinogen chains (α-, β- and γ-fibrinogen) and various inhibitory proteases, is regulated by cytokines IL-6 and glucocorticoids (BAUMANN et al., 1990; BAUMANN & GAULDIE, 1994).

In cats, APP SAA or alpha-1-acid glycoprotein (AGP) is the most important. Blood SAA levels may indicate inflammatory conditions such as feline infectious peritonitis (FIP) and other infectious diseases such as calicivirus infection, chlamydia, leukemia and infectious immunodeficiency, as they increase 10- to 50-fold (TIZARD, 2013b). SAA can also be increased in other diseases, such as diabetes mellitus and cancer. Haptoglobin usually increases 2- to 10-fold and is particularly high in FIP (TIZARD, 2013b). Table 1 summarizes the individual positive APPs in the context of feline disease.

Acute phase negative proteins

The most significant negative APP is albumin, whose blood concentration decreases during APR due to amino acid aberrations towards the synthesis of positive APPs (CRAY et al., 2009; PALTRINIERI, 2007a). Other negative APPs are transferrin, transthyretin, retinol ligand, and cortisol binding protein, proteins involved in vitamin and hormone transport (JAIN et al., 2011).

Acute phase proteins in cat disease

Unlike cytokines, which are small in size and rapidly filtered by the kidney, acute phase proteins have a higher molecular weight (greater than 45 kDa) and consequently remain in plasma for longer (SALGADO et al., 2011).

APP levels can only indicate inflammation, and consequently their concentrations can help diagnose and monitor the disease. APP can help detect subclinical inflammation, distinguish acute from chronic disease, and predict its course (VILHENA et al, 2018; JAVARD et al., 2017). Because APRs begin before specific immunological changes occur, they can be used as an early marker of disease before leukogram changes occur, with their magnitude related to disease severity (PETERSEN et al., 2004; CÉRON et al., 2005; VILHENA et al., 2005). , 2018). For this reason, disease monitoring can be considered one of the most interesting and promising applications of APP.

APP levels along with clinical signs and blood tests have been evaluated in a variety of animal diseases (ie, FIP, canine inflammatory disease, leishmaniasis, ehrlichiosis, and canine pyometra) and have been shown to be useful in diagnosis, response to treatment, and prognosis (ECKERSALL et al. ), 2001; MARTINEZ-SUBIELA et al., 2005; SHIMADA et al., 2002; JERGENS et al., 2003; GIORDANO et al., 2004; PETERSEN et al., 2004; DABROWSKI et al., 2009; VILHENA et al., 2018).

To obtain complete information on APR, one major and one moderate positive as well as one negative APP should be evaluated simultaneously (CERÓN et al., 2008). High concentrations of major APP are usually associated with infectious diseases, usually systemic bacterial infection or immune-mediated disease (CERÓN et al., 2008; TROÌA et al., 2017). Although APPs should be analyzed along with white blood cell and neutrophil counts, they are most sensitive in the early detection of inflammation and infection (CERÓN et al., 2008; ALVES et al., 2010). However, the specificity of these proteins is low in determining the cause of the process, and also increases in physiological conditions such as pregnancy (PALTRINIERI et al., 2008).

APPThe disease
SAAFIP
Induced inflammation and surgery
Various diseases (pancreatitis, renal failure, FLUTD, tumors, diabetes mellitus; kidney disease, injury, etc.)
Sepsis
FeLV; hemotropic mycoplasma infections
Hepatozoonfelis and Babesia vogeli infection
Dirofilariaimmitis
FIV cats treated with recombinant feline interferon
AGPChlamydophila psittaci infection;
Pancreatitis and pancreatic tumors
FIP
Lymphoma and other tumors
Induced inflammation and surgery
FIV cats treated with recombinant feline interferon
Abscesses, pyothorax, adipose tissue necrosis
Various diseases (FLUTD, tumors, diabetes mellitus, kidney diseases, injuries, etc.)
HaptoglobinFIP
Induced inflammation and surgery
Abscesses, pyothorax, adipose tissue necrosis
Various diseases (FLUTD, tumors, diabetes mellitus, kidney diseases, injuries, etc.)
Hepatozoonfelis and Babesia vogeli infection
FeLV, hemotropic mycoplasmas
Dirofilariaimmitis
CRPFIV cats treated with recombinant feline interferon
Induced inflammation and surgery
Table 1 - Acute phase proteins studied for feline diseases.
Legend: Serum amyloid A (SAA), α1-acid glycoprotein (AGP), systemic inflammatory response syndrome (SIRS), feline lower urinary tract disease (FLUTD), feline infectious peritonitis (FIP), feline leukemia virus (FeLV), immunodeficiency virus cats (FIV); feline calicivirus (FCV).

Figure 1 shows the expected behavior of acute phase positive proteins based on revised studies. AGP, SAA and haptoglobin have been identified as useful indicators for monitoring the acute inflammatory response in cats (WINKEL et al., 2015; PALTRINIERI et al., 2007a, b; KAJIKAWA et al., 1999). APPs in cats were first identified after comparative measurements in the serum of clinically normal and diseased animals, in experimentally induced inflammation studies, and in postoperative studies (KAJIKAWA et al., 1999). The concentration of SAA reportedly increased first, followed by an increase in AGP and haptoglobin, in contrast to a less pronounced increase in CRP (KAJIKAWA et al., 1999). One study showed that CRP behaves similarly to SAA and AGP in cat inflammation (LEAL et al., 2014).

Serum Amyloid A

SAA is one of the major APPs in several species, important in both humans and cats (KAJIKAWA et al., 1999). It modulates the immune response by attracting inflammatory cells to tissues and leading to the production of multiple inflammatory cytokines (GRUYS et al., 2005; TIZARD, 2013a). Its concentration can increase more than 1,000 times in an inflammatory condition, which we then understand as inflammation (TAMAMOTO et al., 2013). However, such an increase can be observed in both non-inflammatory and inflammatory diseases and neoplasms (TAMAMOTO et al., 2013). According to a study in cats that underwent surgery, SAA levels begin to increase approximately 3 to 6 hours, peaking 21 to 24 hours after surgery (SASAKI et al., 2003).

Figure 1 - Idealized behavior of acute phase proteins in cats after inflammatory stimuli. The values representing the changes cannot be considered absolute. Increase in serum amyloid A (SAA) 3 to 6 h after challenge, peak at 21 to 24 h, peak size 10 to 50 times its basal plasma concentration. Alpha 1 acid glycoprotein (AGP) increases 8 h after challenge, peak at 36 h, size at peak time 2 to 10 times its baseline plasma concentration. Haptoglobulin (Hp) increase 24 h after challenge, peak 36 to 48 h, peak size 2 to 10 times its basal plasma concentration. C-reactive protein (CRP) increased 8 h after challenge, peak at 36 h, peak size 1.5 times its basal values.

Alpha 1-acid glycoprotein

Alpha 1-acid glycoprotein (AGP) is an acute phase-reactive protein found in the serum mucoid portion of serum (SELTING et al., 2000; WINKEL et al., 2015). Like most positive APPs, AGP is a glycoprotein synthesized predominantly by hepatocytes in APR and released into the bloodstream (CÉRON et al., 2005).

AGP can be used to monitor early interferon treatment in cats infected with feline immunodeficiency virus (FIV) (GIL et al., 2014). AGP as well as haptoglobin (Hp) are increased in anemic cats suffering from pyothorax, abscesses or fat necrosis (OTTENJANN et al., 2006).

Changes in AGP in feline neoplasia do not appear to be consistent across studies. Some of them do not describe any changes in cats with lymphoma (CORREA et al., 2001). Others point to an increase in both AGP and SAA in cats with sarcomas, carcinomas, or other round cell tumors (SELTING et al., 2000; TAMAMOTO et al., 2013; MEACHEN et al., 2015; HAZUCHOVA et al., 2017).

AGP is important as an indicator test for FIP, which is used specifically in Europe (CECILIANI et al., 2004). GIORI et al. examined the specificity and sensitivity of several tests in 12 cats, with 33.33 % cats being FIP negative based on histopathology and immunohistochemistry and 66.66 % cats being FIP positive confirmed by histopathology and immunohistochemistry. This author concludes that immunohistochemistry must always be performed to confirm FIP, but high concentrations of AGP can help support the diagnosis of FIP if immunohistochemistry cannot be performed and histopathology is not convincing.

Haptoglobin

Haptoglobin (Hp) is one of the most important acute phase proteins in cattle, sheep, goats, horses and cats (TIZARD, 2013a), synthesized mainly by hepatocytes but also by other tissues such as skin, lungs and kidneys (JAIN et al, 2011 ). Hp binds to iron molecules and makes them inaccessible to invasive bacteria, thereby inhibiting bacterial proliferation and invasion. Subsequently, it also binds to free hemoglobin, thus preventing its oxidation with lipids and proteins (TIZARD, 2013a), which justifies a reduction in Hp in case of hemolysis.

In cats, Hp usually increases 2- to 10-fold in inflammatory conditions, and is particularly high in FIP (TIZARD, 2013a). However, both Hp and SAA did not provide sufficient support to distinguish FIP from other causes of effusion compared to AGP (HAZUCHOVÁ et al., 2017).

Measurement APP

The serum is composed of a large number of individual proteins in which the detection of changes in its fractions can provide important diagnostic information (ECKERSALL, 2008).

Ideally, measurement of all serum proteins should be available so that they can be used as a diagnostic tool in relation to inflammatory diseases.
Currently, APPs (Table 2) can be determined by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, nephelometry, immunoturbidimetry (IT), Western blot, and messenger ribonucleic acid (mRNA) analysis (CÉRON et al., 2005; PALTRINIERI et al., 2008; SCHREIBER et al., 1989). Although some human APP tests have been automated for veterinary medicine, species-specific tests are still limited. Cross-species differences in APP and the limited availability of cross-reactive agents have so far contributed to the low routine level of APP determination in veterinary laboratories, especially in cats. Regardless, the technology is evolving and routine monitoring of clinically relevant APPs in cats can be expected in the near future.

Conclusion

Acute phase proteins in cats are biomarkers suitable for monitoring inflammation, along with other clinical and laboratory findings that are useful in diagnosing subclinical changes, monitoring the development and effect of the disease in the body, as well as in evaluating the response to treatment.

In cats, SAA APP, which is most pronounced in response to inflammation, is followed by AGP and haptoglobin, in contrast to CRP, which is used in other species.

Although there are commercially available kits for determining feline APPs, standardization of tests for technical simplicity, higher species specificity with lower associated costs will allow routine use in feline practice, as is done in human medicine.

AnalyzesProsCons
Radioimmunoassay24 to 48 hours to obtain results, specific operator skills required
ELISACommercially available species-specific kitsLack of automation, expensive, some "between-run" inaccuracy
Immunoturbidimetry30 minutes to obtain results, customizable with a biochemical analyzer
Western BlotLong time for immunoblot processing
Nephelometric immunoassaysThey depend on the cross-reactivity of the increased antiserum
Table 2 - Advantages and disadvantages of possible APP measurement techniques.

Appendix: APP and their position in the electrophoretogram

Although there are tests directly for a specific APP, it is useful to know in which region the electrophoretograms are located.

Electrophoretogram demonstration (Serum protein electrophoresis output)
Serum proteinElectrophoretic region
α1-acid glycoproteinα1 (alpha-1)
Serum Amyloid Aα (alpha)
Haptoglobinα2 (alpha-2)
Ceruloplasmin α2 (alpha-2)
Transferrinβ1 (beta-1)
C-reactive proteinγ (gamma)
Position of serum proteins in electrophoretogram

References

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BAUMANN, H. & GAULDIE, J. The acute phase response.
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BAUMANN, H. et al. Distinct regulation of the interleukin-1 and interleukin-6 response elements of the rat haptoglobin gene in rat and human hepatoma cells. Molecular and Cellular Biology, v.10, n.11, p.5967–5976, 1990. Available from: Accessed: Aug. 21, 2018. doi: 10.1128 / MCB.10.11.5967.

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Alternative treatments for cats with FIP and natural or acquired resistance to GS-441524

Niels C. Pedersen, Nicole Jacque, 3.11. 2021
Original article: Alternative treatments for cats with FIP and natural or acquired resistance to GS-441524

Abbreviations:
SC - subcutaneous
IV - intravenous
IM - to the muscle
PO - per os - orally
SID - once a day
BID - 2x this
q24h - once every 24 hours
q12h - once in 12 hours

Introduction

Antiviral resistance is well documented in diseases such as HIV / AIDS and hepatitis C. In some cases, this resistance is present in the infecting virus, but is more often due to long-term drug exposure. Resistance to GC376 [1] and GS-441524 [2] has also been documented in cats with naturally acquired FIP. Resistance develops based on mutations in regions of the viral genome that contain targets for the antiviral drug. For example, several amino acid changes (N25S, A252S or K260N) were detected in the GIP376-resistant FIPV isolate (3CLpro). [3]. A change in N25S in 3CLpro was found to cause a 1.68-fold increase in 50 % GC376 inhibitory concentration in tissue cultures [3]. Resistance to GC376, although recognized in initial field trials, has not yet been described. GC376 is less popular in the treatment of FIP and is not recommended for cats with ocular or neurological FIP. [1].

Natural resistance to GS-441524 was observed in one of 31 cats treated for naturally occurring FIP [2]. One of the 31 cats in the original GS-441524 field study also appeared to be resistant, as viral RNA levels did not decrease throughout the treatment period and the symptoms of the disease did not abate. Although this virus has not been studied, resistance to GS-5734 (Remdesivir), a prodrug of GS-441524, has been established in tissue culture by amino acid mutations in RNA polymerase and corrective exonuclease. [4].

Resistance to GS-441524 has been confirmed in a number of cats that have been treated for FIP with GS-441524 in the last 3 years, especially among cats with neurological FIP [5]. Resistance to GS441524 is usually partial and higher doses often cure the infection or significantly reduce the symptoms of the disease during treatment. Interestingly, resistance to GS-441524 has also been found in patients with Covid19 treated with Remdesivir [12]. An immunocompromised patient developed a prolonged course of SARS-CoV-2 infection. Remdesivirus treatment initially alleviated symptoms and significantly reduced virus levels, but the disease returned with a large increase in virus replication. Whole genome sequencing identified an E802D mutation in nsp12 RNA-dependent RNA polymerase that was not present in pre-treatment samples and caused a 6-fold increase in resistance.

Although the history of molnupiravir and its recent use in the treatment of FIP has been described [6], there are currently no studies documenting natural or acquired resistance to molnupiravir. Molnupiravir has been shown to function as an RNA mutagen causing several defects in the viral genome [7]while remdesivir / GS-441524 is a non-binding RNA chain terminator [8], which suggests that its resistance profile will be different.

Overcoming resistance to GS-441524

Drug resistance can only be overcome in two ways: 1) by gradually increasing the dose of the antiviral to achieve drug levels in body fluids that exceed the resistance level, or 2) by using another antiviral that has a different mechanism of resistance, either alone or in combination. So far, the first option has been chosen, which has proved effective in many cases. However, resistance to GS-441524 may be complete or so high that increasing the dose is no longer effective. In such cases, the second option is increasingly used. Currently available alternatives to GS-441524, although still from an unapproved market, are GC376 and molnupiravir.

Antiviral drug treatment regimens for resistance to GS-441524

GC376 / GS-441524


The combined GS / GC regimen has been shown to be effective in cats treated with GS-441524 at doses up to 40 mg / kg without cure due to resistance to GS-441524. It is better to intervene as soon as resistance to GS-441524 is detected, which will allow the cat to be cured sooner and at lesser cost to the owner.

Rainman is the current supplier of GC376, which comes in 4 ml vials at a concentration of 53 mg / ml.

GS / GC dosage: The dose of GS (SC or PO equivalent) in combination antiviral therapy is the same as the dose needed to adequately control the symptoms of the disease. This is usually the last dose used before the end of treatment and relapse. To this dose of GS-441524, GC376 is added at a dose of 20 mg / kg SC q24h regardless of the form of FIP. This is sufficient for most cats, including many cats with neuro FIP, but some will need higher doses. If remission of clinical signs is not achieved or blood tests are of concern, the dose of GC376 is increased by 10 mg / kg up to 50 mg / kg SC q24h.

Duration of treatment: An eight-week combination GC / GS treatment is recommended, which is added to previous GS monotherapy. Some cats were cured at 6 weeks of combination therapy, but relapse is more likely than at 8 weeks.

Side effects: Most cats have no serious side effects. However, about one in five cats may experience nausea or discomfort at the beginning of treatment and sometimes longer. These side effects do not appear to be dose dependent and can be treated with anti-nausea drugs such as Cerenia, Ondansetron or Famotidine. Ondansetron appears to have performed better in some cats.

Molnupiravir

Molnupiravir has been reported to be effective in monotherapy in cats with FIP by at least one Chinese retailer GS-441524 [9], but there are no reports of its use in cats with resistance to GS-441524. However, resistance to GS-441524 is unlikely to spread to molnupiravir. The fact that it has been found to be effective as an oral medicine also makes it attractive for treatment alone, as many cats resistant to GS-441524 have suffered from injections for a very long time.

A field study of molnupiravir reportedly consisted of 286 cats with various forms of naturally occurring FIP, which were examined in pet clinics in the United States, the United Kingdom, Italy, Germany, France, Japan, Romania, Turkey and China. Among the 286 cats that participated in the trial, no deaths occurred, including seven cats with ocular (n = 2) and neurological (n = 5) FIP. Twenty-eight of these cats were cured after 4-6 weeks of treatment and 258 after 8 weeks. All treated cats remained healthy 3-5 months later, a period during which cats that were not successfully cured would be expected to relapse. These data provide convincing evidence of the safety and efficacy of molnupiravir in cats with various forms of FIP. However, we hope that this field study will be written in the form of a manuscript, submitted for review and published. Nevertheless, it is now sold to cat owners with FIP. At least one other major retailer of GS-441524 is also interested in using molnupiravir for FIP, indicating a demand for further treatment of cats with FIP antivirals.

Molnupiravir dosage: The safe and effective dosing of molnupiravir in cats with FIP has not been established based on closely controlled and monitored field studies such as those performed for GC376 [1] and GS-441524. [2]. However, at least one seller from China in his flyer for a product called Hero-2801 [9] provided some pharmacokinetic and field trials of Molnuparivir in cats with naturally occurring FIP. This information does not clearly state the amount of molnupiravir in one of their “50 mg tablets” and the actual dosing interval (q12h or q24h?). The dose used in this study also appeared to be too high. Fortunately, the estimated starting dose of molnupiravir in cats with FIP can be obtained from published studies on EIDD-1931 and EIDD-2801. [15] in vitro on cell cultures and laboratory and field studies GS-441524 [14,18]. Molnupiravir (EIDD-2801) has an EC50 of 0.4 μM / μl against FIPV in cell culture, while the EC50 of GS-441524 is approximately 1.0 μM / μl. [18]. Both have a similar oral absorption of approximately 40-50 %, so an effective subcutaneous (SC) dose of molnupiravir would be approximately half the recommended starting dose of 4 mg / kg SC q24h for GS441524. [14] or 2 mg / kg SC q24h. The oral (PO) dose would be doubled to account for less effective oral absorption per 4 mg / kg PO q24h dose. The estimated initial effective oral dose of molnupiravir in cats with FIP can also be calculated from the available Covid-19 treatment data. Patients treated with Covid-19 are given 200 mg of molnupiravir PO q12h for 5 days. This dose was, of course, calculated from a pharmacokinetic study performed in humans, and if the average person weighs 60-80 kg (70 kg), the effective inhibitory dose is 3,03.0 mg / kg PO q12h. The cat has a basal metabolic rate 1.5-fold higher than humans, and assuming the same oral absorption in both humans and cats, the minimum dose for cats according to this calculation would be 4.5 mg / kg PO q12h in neocular and non-neurological forms of FIP. If molnupiravir crosses the blood-brain and blood-brain barriers with the same efficacy as GS-441524 [3,18], the dose should be increased to 1,51.5 and 2,02.0-fold to ensure adequate penetration into aqueous humor and cerebrospinal fluid for ocular cats (88 mg / kg PO, q12 h), respectively. neurological FIP (~ 10 mg / kg PO, q12 h). These doses are comparable to those used in ferrets, where 7 mg / kg q12h maintains sterilizing blood levels of the influenza virus drug (1.86 μM) for 24 hours. [10]. Doses in ferrets of 128 mg / kg PO q12h caused almost toxic blood levels, while a dose of 20 mg / kg PO q12h caused only slightly higher blood levels. [10].

Molnupiravir / GC376 or Molnupiravir / GS-441524

Combinations of molnupiravir with GC376 or GS-441524 will be used more and more frequently, not only to synergy or complement their individual antiviral effects, but also as a way to prevent drug resistance. Medicinal cocktails have been very effective in preventing drug resistance in HIV / AIDS patients [11]. However, there is currently insufficient evidence on the safety and efficacy of the combination of molnupiravir with GC376 or GS-441524 as initial treatment for FIP.

Case studies


Rocky - DSH MN Neuro FIP


A 9-month-old neutered domestic shorthair cat obtained as a rescue kitten had several weeks of seizures with increasing frequency, ataxia and progressive paresis. The blood tests were unremarkable. FIP treatment was started at a dose of 15 mg / kg BID GS-441524, which decreased to SID for about a week. The cat showed improvement, seizures stopped, and mobility increased within 24 hours of starting treatment. Within 5 days of treatment, the cat was able to move again. However, approximately 2 weeks after the start of treatment, the cat experienced loss of vision, decreased mobility, recovery of seizures and difficulty swallowing. Dose adjustments of levetiracetam and prednisolone were made, as well as a change in the composition of GS-441524, followed by a temporary improvement in motility and swallowing and a reduction in seizures, but overall the cat's condition worsened. The dose of GS-441524 was gradually increased to 25 mg / kg, with little or no improvement. At this point, GS was taken orally at a dose of 25 mg / kg (estimated to be approximately 12.5 mg / kg) and within 3 days, the cat began to move, improved vision, and stopped seizures with increased energy and appetite. Improvement in cats continued for approximately 4 weeks with oral administration of GS-441524, then stopped for approximately 3 weeks before rapidly progressing paresis. Oral doses up to 30 mg / kg SC equivalent have been tested but have no effect. GS-441524 was then injected at a dose of 20 mg / kg and the cat was able to move again within 4 days with good appetite and energy. After 2 weeks, a dose of GC376 20 mg / kg BID was added to the dosing regimen. The cat terminated 6 weeks of the GS441524 and GC376 combination therapy and then discontinued the treatment. Although the cat has certain permanent neurological deficits, its condition is stable, it has good mobility, appetite and activity for 9 months after the end of antiviral treatment.

Rocky's video: https://www.youtube.com/watch?v=RXB_NnfcMOY

Bucky - DSH MN Neuro / Eyepiece FIP


A four-month-old neutered domestic shorthair cat obtained as a rescue kitten was presented with a monthly history of lethargy and a progressive history of ataxia, hind limb paresis, spades, uveitis, anisocoria, and urinary and stool incontinence. Blood tests were mostly uncommon, with the exception of mild hyperglobulinemia. The A / G ratio was 0.6. The cat was treated with 10 mg / kg GS-441524 SC SID for 3 weeks. Activity, mentation and uveitis improved within 72 hours of starting treatment. During the first 2 weeks, a slow improvement in mobility and eye symptoms was observed, but then a plateau was reached. After 3 weeks, the dose of GS-441524 was increased to 15 mg / kg GS-441524 SC SID due to persistent neurological and ocular deficits. In addition, enlargement of the left eye due to glaucoma was noted at this time and the eye continued to swell until it was removed at week 8 of treatment.
Due to persistent weakness / lack of pelvic coordination and increasing lethargy, dose GS-441524 was increased to 20 mg / kg SC SID [or equivalent oral dose] at week 9 and 20 mg / kg SC BID was added to the regimen a few days later. GC376. Significantly increased activity and willingness to jump on elevated surfaces occurred within 48 hours of starting GS376 treatment. The combination treatment of GS-441524 and GC376 was maintained for 8 weeks. The cat has residual incontinence problems after treatment, but is otherwise clinically normal 6 months after treatment.

Boris - Maine Coon MI wet eye FIP


The five-month-old intact (uncastrated) Maine Coon cat, obtained from the breeder, had lethargy, anorexia, abdominal ascites, cough, anemia and neutrophilia. No biochemical analysis was performed to establish the diagnosis. The cat was treated with 6 mg / kg GS-441524 SC SID for 8 weeks. After six weeks of treatment, X-rays revealed nodules in the lungs, and after 8 weeks, hyperglobulinemia persisted. The GS-441524 dose was then increased to 8 mg / kg SC SID for 4 weeks. There was little improvement in blood tests and X-rays and the dose of GS-441524 was increased to 12 mg / kg SC SID over 4 weeks, followed by an increase to 17 mg / kg over 11 weeks, 25 mg / kg over 4 weeks and 30 mg / kg for 4 weeks. After 25 weeks of treatment, ultrasound revealed pleural abnormalities on the left side and X-rays showed no improvement in the pulmonary nodules. In addition, uveitis and retinal detachment have been reported in the right eye. Pulmonary aspirates that showed FIP-compliant inflammation were collected. After 33 weeks of treatment, 20 mg / kg SC BID GC376 was added to the regimen and the combined treatment of GS-441524 and GC376 was continued for 12 weeks. Increased activity was noted over several days. Over the course of 5 weeks, the weight gain accelerated, the cough subsided and the energy level increased. Blood tests showed an improvement in the A / G ratio, and chest X-rays showed a reduction in the lungs. After 84 days of combination antiviral therapy, the A / G ratio was 0.85 and the cat appeared clinically normal. The cat is currently 3 months after treatment.

References

  1. Pedersen NC, Kim Y, Liu H, Galasiti Kankanamalage AC, Eckstrand C, Groutas WC, Bannasch M, Meadows JM, Chang KO. Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis. J Feline Med Surg. 2018; 20 (4): 378-392.
  2. Pedersen NC, Perron M, Bannasch M, Montgomery E, Murakami E, Liepnieks M, Liu H. efficacy and safety of the nucleoside analog GS-441524 for the treatment of cats with naturally occurring feline infectious peritonitis. J Feline Med Surg. 2019; 21 (4): 271-281.
  3. Perera KD, Rathnayake AD, Liu H, et al. Characterization of amino acid substitutions in feline coronavirus 3C-like protease from a cat with feline infectious peritonitis treated with a protease inhibitor. J. Vet Microbiol. 2019; 237: 108398. doi: 10.1016 / j.vetmic.2019.108398
  4. Agostini ML, Andres EL, Sims AC, et al. Coronavirus susceptibility to the antiviral remdesivir (GS5734) is mediated by the viral polymerase and the proofreading exoribonuclease. MBio 2018; 9. DOI: 10.1128 / mBio.00221-18.
  5. Pedersen NC. 2021. The neurological form of FIP and GS-441524 treatment.
    https://sockfip.org/the-neurological-form-of-fip-and-gs-441524-treatment/
  6. Pedersen NC. The long history of beta-d-n4-hyroxycytidine and its modern application to treatment of covid019 in people and FIP in cats. https://sockfip.org/the-long-history-of-beta-d-n4-hydroxycytidineand-its-modern-application-to-treatment-of-covid-19-in-people-and-fip-in- cats /.
  7. Agostini, ML et al. Small-molecule antiviral beta-dN (4) -hydroxycytidine inhibits a proofreading-intact coronavirus with a high genetic barrier to resistance. J. Virol. 2019; 93, e01348.
  8. Warren, TK et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature 2016; 531, 381–385.
  9. FIP Warriors CZ / SK - EIDD-2801 (Molnupiravir) https://www.fipwarriors.eu/en/eidd-2801-molnupiravir/
  10. Toots M, Yoon JJ, Cox RM, Hart M, Sticher ZM, Makhsous N, Plesker R, Barrena AH, Reddy PG, Mitchell DG, Shean RC, Bluemling GR, Kolykhalov AA, Greninger AL, Natchus MG, Painter GR, Plemper RK . Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia. Sci Transl Med. 2019; 11 (515): eaax5866.
  11. Zdanowicz MM. The pharmacology of HIV drug resistance. Am J Pharm Educ. 2006; 70 (5): 100.doi: 10.5688 / aj7005100
  12. Gandhi, S, Klein J, Robertson A, et al. De novo emergence of a remdesivir resistance mutation during treatment of persistent SARS-CoV-2 infection in an immunocompromised patient: A case report. medRxiv, 2021.11.08.21266069AID
Read "Alternative treatment of cats with FIP and natural or acquired resistance to GS-441524"

The long history of Beta-d-N4-hydroxycytidine and its modern application to treatment of Covid-19 in people and FIP in cats.

Niels C. Pedersen DVM, PhD
Original article: The long history of Beta-d-N4-hydroxycytidine and its modern application to treatment of Covid-19 in people and FIP in cats.

Beta-d-N4-hydroxycytidine is a small molecule (nucleoside) that was studied in the late 1970s in the former Soviet Union as part of biological weapons research [2]. The weaponization of diseases such as smallpox was a worldwide threat, but the danger of using the smallpox virus for this purpose was too great. Smallpox was eradicated from the world, virtually all stocks were destroyed and further research was banned. This led the US and the Soviet Union to research other RNA viruses as biological weapons and antivirals to defend against them. The Venezuelan equine encephalomyelitis virus (VEEV) was one of the first viruses to be seriously considered for use as a biological weapon. [3]. VEEV is transmitted to humans by mosquito bites and causes high fever, headaches and encephalitis with swelling that can be fatal. Beta-d-N4-hydroxycytidine has been found to inhibit not only VEEV replication but also a wide range of alphaviruses, including Ebola, chikungunya, influenza virus, norovirus, bovine diarrhea virus, hepatitis C virus and respiratory syncytial virus. [3-8]. The first reports of an inhibitory effect of beta-d-N4-hydroxycytidine on human coronavirus NL63 date back to 2006 [9]. Recent studies have confirmed its inhibitory effect on a wide range of human and animal coronaviruses [8].

An important part of the recent history of beta-d-N4-hydroxycytidine is associated with the Emory Institute for Drug Development (EIDD) [1], where he received the experimental designation EIDD-1931. The US government has provided significant financial support for the study of antivirals against alphaviruses in institutions such as Emory since 2004. [10]. In 2014, the Defense Threat Reduction Agency provided institutional support to find an antiviral compound against VEEV and other alpha-coronaviruses. "N4-Hydroxycytidine and its derivatives and antiviral uses" were included in U.S. Patent Application 2016/106050 A1 of 2016 [11]. Additional funding in 2019 was provided by the National Institute of Allergies and Infections for fellowship of the esterified beta-d-N4-hydroxycytidine precursor (EIDD-2801) for the treatment of influenza. [10]. The stated purpose of the chemical changes of EIDD-2801 was to increase its oral bioavailability, which would ultimately allow beta-d-N4-hydroxycytidine to be administered as pills and not as injections. In 2019/2020, the focus of research changed from influenza to SARS-CoV-2 [2]. The commercialization of EIDD-2801 was entrusted to Emory's Ridgeway Biotherapeutics subsidiary, which subsequently worked with Merck on a lengthy and costly FDA approval process. The current version of EIDD-2081 for field testing was named Molnupiravir.

Beta-d-N4-hydroxycytidine, the active substance in Molnupiravir, exists in two forms as tautomers. In one form, it acts as a cytidine with a single bond between the carbon and the N-OH group. In its other form, which mimics uridine, it has an oxime with a double bond between the carbon and the N-OH group. In the presence of beta-d-N4-hydroxycytidine, viral RNA-dependent RNA polymerase reads it as uridine instead of cytidine and inserts adenosine instead of guanosine. Switching between forms causes inconsistencies during transcription, which results in numerous mutations in the viral genome and a cessation of viral replication. [8].

Merck's commitment to conditional and full FDA approval of Molnuparivir continues. In its statement, Merck stated: [12] "In anticipation of the results of the MOVe-OUT program, Merck manufactures Molnupiravir at its own risk. Merck expects to produce 10 million therapeutic doses by the end of 2021, with more expected to be produced in 2022. Merck is committed to providing timely access to Molnupiravir worldwide, if authorized or approved, and plans to introduce access to tiered prices based on World Bank admission criteria that reflect countries' relative ability to fund their pandemic health response. As part of its commitment to extend the global approach, Merck has previously announced that it has entered into non-exclusive voluntary licensing agreements for Molnupiravir with established generic manufacturers to accelerate the availability of Molnupiravir in more than 100 low and middle income countries (LMICs) following approval or emergency approval by local regulatory agencies. . " This "generosity" is unlikely to apply to use in animals.

Drugs to inhibit the current Covid-19 pandemic have been the subject of accelerated field trials in the last two years, and one of them, Remdesivir, has been approved for use in hospitalized patients in record time. Last year, Molnupiravir was submitted for conditional approval as an oral medicinal product for home treatment of the infection at an early stage. [12]. However, anti-coronavirus compounds have been developed previously for another common and serious feline disease, feline infectious peritonitis (FIP). These drugs include a protease inhibitor (GC376) [13] and an RNA-dependent RNA polymerase inhibitor (GS-441524), which is an active ingredient of Remdesivir [14]. The success of antiviral drugs in the treatment of FIP prompted a recent study by EIDD-1931 and EIDD-2801 for their ability to inhibit FIPV in tissue cultures. [15]. The effective EC50 concentrations for EIDD-1931 against FIPV are 0.09 μM, EIDD-2801 0.4 μM and GS441524 0.66 μM [15]. The percentage of cytotoxicity at 100 μM is 2.8, 3.8 and 0, respectively. Therefore, EIDD-1931 and EIDD-2801 are slightly more effective at inhibiting viruses, but also more cytotoxic than GS-441524. These laboratory studies suggest that EIDD-1931 and EIDD-2801 are excellent candidates for the treatment of FIP.

Although EIDD-1931 and EIDD-2801 are a great promise for the treatment of FIP, there are several obstacles that will make the legal use of these compounds unlikely in the near future. GS-441524, the active form of Remdesivir and patented by Gilead Sciences, was investigated for use in cats with FIP shortly before the Covid-19 pandemic. FIP research [14] therefore stimulated the potential use of Remdesivir against Ebola and not SARS-like coronavirus [14]. Although these studies were conducted in collaboration with scientists from Gilead Sciences, the company refused to grant GS-441524 rights to treatment in animals as soon as it became clear that there was a much larger market for Covid-19 in humans. [16]. Similarly, my attempts over the past 2-3 years at Emory, Ridgeback Biotherapeutics, and Merck Veterinary Division to investigate EIDD-1931 and EIDD2801 for the treatment of FIP in cats have either remained unanswered or rejected, no doubt for similar reasons why Gilead refused to grant rights for GS-441524. However, the great worldwide need for FIP treatment quickly supported the unapproved market for GS-441524 from China. The same need to treat FIP has recently aroused interest in Molnupiravir, also from China.

Situation with EIDD-1931 vs. EIDD-2801 / Molnupiravir and GS-441524 vs. Remdesivir raises the question of why some medicines are being converted to prodrugs for marketing purposes [17]. Remdesivir was reportedly esterified to increase antiviral activity, although studies in cats showed that GS-441524 and Remdesivir had similar viral inhibitory activity in tissue culture. [18]. However, Remdesivir was found to be poorly absorbed by the oral route and was therefore conditionally approved for injectable use only. EIDD-2801 was designed to increase the oral absorption of EIDD-1931, although previous research has shown that EIDD-1931 is well absorbed orally without esterification. [6]. The motives for the commercialization of Remdesivir instead of GS-441524 for human use have been scientifically questioned, as it appears to be better in several ways without further modification. [17]. Why EIDD-2801 was chosen for commercialization, when EIDD-1931 would be cheaper, 4 times more effective against viruses and one third less toxic than EIDD-2801 [15]? The strength of patent rights and the longevity of patents may be more important factors in these decisions. [16,17,19].

One of the problems in the treatment of FIP in cats is the blood-eye and blood-brain barriers, which become very important when the disease affects the eyes and / or the brain. [13, 14, 20]. This problem has been largely overcome in the treatment of ocular and neurological forms of FIP with GS-441524 by gradually increasing the dose to increase blood levels and thus drug concentrations in the ventricular fluid and / or brain. [20]. GC376, one of the most effective antivirals against FIP virus in culture [17], is not effective against ocular and neurological FIP due to the inability to get enough drug to these sites, even if the dose is increased several times[14]. Fortunately, it appears that EIDD-1931 can reach effective levels in the brain, as indicated by studies in horses with VEEV infection. [3]. Drug resistance is another problem that now occurs in some cats treated with GS-441524, especially in individuals with the neurological form of FIP. Long treatment procedures and difficulties in transporting enough drug to the brain support the development of drug resistance.

The short-term and long-term toxic effects of the drug candidate on the test person or animal are crucial. GS-441524 showed lower toxicity in cell cultures than GC376, EIDD-1931 and EIDD-2801 [15]. Most important, however, is the toxicity that manifests itself in vivo. GC376 is one of the drugs with the highest coronavirus inhibitory effect [15], but slows the development of adult teeth when given to young kittens [13]. No serious toxicity was observed during nearly three years of field use of GS-441524, reflecting the complete absence of cytotoxic effects in vitro at concentrations up to 400 µM. [18]. However, EIDD-1931 and EIDD-2801 show significant cytotoxicity at 100 μM [15]. Therefore, the ability of EIDD-1931 to make fatal mutations in RNA has been raising a number of questions for some time. [8, 21, 22]. This was the main reason why the application for the treatment of diseases was still delayed. However, the current recommended duration of treatment with Covid-19 Molnupiravir is only 5 days at the initial stage of treatment. [10]. However, the recommended duration of FIP treatment with GS-441524 is 12 weeks [14], which represents a much longer time for the manifestation of toxicity. Therefore, close observation of cats during treatment with EIDD-1931 or EIDD-2801, whether short-term or long-term, will be important.

All existing antiviral drugs have led to the development of drug resistance through mutations in the viral genome. Although Remdesivir appears to be less susceptible to such mutations compared to drugs used in viral diseases such as HIV / AIDS, resistance is well documented. [23-25]. Resistance to GS-441524 in cats treated for FIP was observed at a higher frequency, especially in cats with neurological FIP, where it is more difficult to deliver sufficient drug to the brain [13, 14, 20]. Resistance to GS-441524 in cats is also likely to be a major problem, as cats with FIP are often treated for 12 weeks or longer, while Remdesivir (and Molnupiravir) are recommended for only five days during the initial viremic stage of Covid-19. [16]. The problem of drug resistance in HIV / AIDS treatment is effectively addressed by using a cocktail of different drugs simultaneously with different resistance profiles. Mutants resistant to one drug will immediately inhibit other drugs, thus preventing their positive selection during treatment. Inhibition of resistance is particularly strong when the two drugs attack different proteins involved in virus replication. For example, GC376 is a protease inhibitor [13], while GS-441524 acts on an RNA-dependent RNA polymerase [18]. However, GC376 is not as well absorbed across the blood-brain barrier. Although the necessary research has not yet been performed, there appears to be no cross-resistance between GS-441524 and Molnupiravir and is as effective as GS-441524 in crossing the blood-brain barrier. [3]. This makes Molnupiravir (or 5-hydroxycytidine) an important contribution to the future treatment of FIP.

As expected, Molnupiravir has recently been tested on cats with FIP by at least one Chinese retailer, GS-441524, and preliminary results are available on the FIP Warriors website. [26]. Field trials included 286 cats with various forms of naturally occurring FIP observed at pet clinics in the United States, the United Kingdom, Italy, Germany, France, Japan, Romania, Turkey, and China. The 286 cats that participated in the study, including seven cats with ocular (n = 2) and neurological (n = 5) FIP, did not die. Twenty-eight of these cats were cured after 4-6 weeks of treatment and 258 after 8 weeks. All treated cats were healthy after 3-5 months, a period during which relapses would be expected to relapse unsuccessfully. These data provide convincing evidence of the safety and efficacy of Molnupiravir in cats with various forms of FIP. However, we hope that this field study will be written in manuscript form, submitted for review and published. Either way, Molnupiravir is already marketed to owners of cats with FIP. At least one other major retailer of GS-441524 is also interested in using Molnupiravir for FIP, indicating a demand for additional antiviral drugs for cats with FIP.

Safe and effective dosing for Molnupiravir in cats with FIP has not been published. However, at least one vendor from China provided certain pharmacokinetic and field test data for Molnuparivir in cats with naturally occurring FIP in a leaflet for the product Hero-2081. [26]. However, this information does not clearly indicate the amount of Molnupiravir in one of their "50 mg tablets" and the actual dosing interval (q12h or q24h?). Fortunately, the estimated starting dose of molnupiravir for cats with FIP can be obtained from published in vitro cell culture studies of EIDD-1931 and EIDD-2801. [15] and laboratory and field studies GS-441524 [14,18]. Molnupiravir (EIDD-2801) has an EC50 of 0.4 μM / μl against FIPV in cell culture, while the EC50 of GS-441524 is about 1.0 μM / μl. [18]. Both have a similar oral absorption of about 40-50 %, so the effective subcutaneous (SC) dose for Molnupiravir would be approximately half the recommended 4 mg / kg SC every 24 hours of the initial dose for GS441524. [14] or 2 mg / kg SC q24h. The per-os (PO) dose would be doubled to account for less effective oral absorption at a dose of 4 mg / kg PO every 24 hours. The estimated initial oral dose of molnupiravir for cats with FIP can also be calculated from the available Covid-19 treatment data. Patients treated for Covid-19 are given 200 mg of molnupiravir PO q12h for 5 days. This dose was evidently calculated from a pharmacokinetic study performed in humans, and if the average person weighs 60-80 kg (70 kg), the effective inhibitory dose is 3,03.0 mg / kg PO q12h. The cat has a basal metabolic ratio 1.5 times higher than humans, and assuming the same oral absorption in both humans and cats, the minimum dose for cats according to this calculation would be 4.5 mg / kg PO every 12 hours. Assuming that molnupiravir crosses the blood-brain barrier and the blood-brain barrier as efficiently as GS-441524 [3,18], the dose would be increased ~ 1.5 and ~ 2.0-fold to allow adequate penetration into the aqueous humor and cerebrospinal fluid for cats with ocular (~ 8 mg / kg PO, q12 h) or neurological FIP (~ 10 mg / kg PO, q12h). The treatment will last 10-12 weeks and the monitoring of the response to treatment will be identical to GS-441524 [14, 20]. These recommendations are based on published data assumptions and further experience with Molnupiravir will be required in this area. Molnupiravir is unlikely to be safer and more effective than GS-441524 in the treatment of FIP, but a third antiviral drug may be particularly useful in preventing resistance to GS-441524 (as a cocktail of antivirals with different resistance profiles) or in treating cats that no longer respond. good on GS-441524. It is largely unknown whether Molnupiravir will be without long-term toxic effects, as the active substance N4-hydroxycytidine is an extremely potent mutagen. [21] and the duration of FIP treatment is much longer than with Covid-19 and there is a likelihood of major side effects.

It is a pity that EIDD-1931 (N4-hydroxycytidine), the active substance in Molnupiravir, has not received much attention in the treatment of FIP cats than Molnupiravir. EIDD-1931 has a 4-fold greater inhibitory effect against the virus than Molnupiravir (EC50 0.09 vs. 0.4 μM) and the percentage of cytotoxicity is slightly lower (2.3% vs. 3.8% at 100 μM) [15]. N4-hydroxycytidine is also efficiently absorbed orally [3], which was downplayed in the development of EIDD-2801 (Molnupiravir). This scenario is identical to the GS-441524 vs. Remdesivir, the second of which, Remdesivir, was chosen for commercialization, although current research suggests that GS-441524 would be the best candidate.[17].

References

  1. Painter GR, Natchus MG, Cohen O, Holman W, Painter WP. Developing a direct acting, orally available antiviral agent in a pandemic: the evolution of molnupiravir as a potential treatment for COVID-19 [published online ahead of print, 2021 Jun 18]. Curr Opin Virol. 2021; 50: 17-22. doi: 10.1016 / j.coviro.2021.06.003
  2. Halford B. An emerging antiviral takes aim at COVID-19. c & en topics. 2020. https://cen.acs.org/pharmaceuticals/drug-development/emerging-antiviral-takes-aim-COVID19/98/web/2020/05.
  3. Painter GR, Richard A. Bowend RA, Bluemling GR et al. The prophylactic and therapeutic activity of a broadly active ribonucleoside analog in a murine model of intranasal Venezuelan equine encephalitis virus infection. Antiviral Res. 2019, 171: 104597
  4. Costantini, VP, Whitaker, T., Barclay, L., Lee, D., McBrayer, TR, Schinazi, RF, Vinje, J., 2012. Antiviral activity of nucleoside analogues against norovirus. Antivir. Ther.17 (6), 981–991. https://doi.org/10.3851/imp2229.
  5. Ehteshami, M., Tao, S., Zandi, K., Hsiao, HM, Jiang, Y., Hammond, E., Amblard, F., Russell, OO, Merits, A., Schinazi, RF, 2017. Characterization of beta-dN (4) -hydroxycytidine as a novel inhibitor of chikungunya virus. Antimicrob. Agents Chemother. 61 (4) e02395-02316. https://doi.org/10.1128/aac.02395-16.
  6. Stuyver, LJ, Whitaker, T., McBrayer, TR, Hernandez-Santiago, BI, Lostia, S., Tharnish, PM, Ramesh, M., Chu, CK, Jordan, R., Shi, J., Rachakonda, S ., Watanabe, KA, Otto, MJ, Schinazi, RF, 2003. Ribonucleoside analogue that blocks replication of bovine viral diarrhea and hepatitis C viruses in culture. Antimicrob. Agents Chemother. 47 (1), 244–254.
  7. Yoon J., Toots M, Lee S, Lee ME, et al., 2018. Orally efficacious broad-spectrum ribonucleoside analog inhibitor of influenza and respiratory syncytial viruses. Antimicrob. Agents Chemother. 2018, 62 (8): https://doi.org/10.1128/aac.00766-18
  8. Urakova N, Kuznetsova V, Crossman DK, Sokratian A, Guthrie DB, Kolykhalov AA, et al. β-d-N4Hydroxycytidine is a potent anti-alphavirus compound that induces a high level of mutations in the viral genome. J Virol. 2018, 92: e01965 – e01917. doi: 10.1128 / JVI.01965-17.
  9. Pyrc, K., Bosch, BJ, Berkhout, B., Jebbink, MF, Dijkman, R., Rottier, P., van der Hoek, L., 2006. Inhibition of human coronavirus NL63 infection at early stages of the replication cycle . Antimicrob. Agents Chemother. 2006, 50 (6): 2000–2008. https://doi.org/10.1128/aac.01598-05.
  10. Whitfill T. A likely new treatment for Covid-19 was made possible by government-funded innovation. STAT +. https://www.statnews.com/2021/10/05/government-funding-backed-molnupiravir-possible-newcovid-19-treatment/.
  11. Painter, GR, Guthrie, DB, Bluemling, G., Natchus, MG N4-Hydroxycytidine and Derivatives and Antiviral Uses Related Thereto. US Patent Application, 2016, 2016/106050 A1.
  12. Merck news release, October 1, 2021. https://www.merck.com/news/merck-and-ridgebacksinvestigational-oral-antiviral-molnupiravir-reduced-the-risk-of-hospitalization-or-death-by-approximately -50percent-compared-to-placebo-for-patients-with-mild-or-moderate /.
  13. Pedersen NC, Kim Y, Liu H, Galasiti Kankanamalage AC, Eckstrand C, Groutas WC, Bannasch M, Meadows JM, Chang KO. Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis. J Feline Med Surg. 2018, 20 (4): 378-392.
  14. Pedersen NC, Perron M, Bannasch M, Montgomery E, Murakami E, Liepnieks M, Liu H. efficacy and safety of the nucleoside analog GS-441524 for the treatment of cats with naturally occurring feline infectious peritonitis. J Feline Med Surg. 2019, 21 (4): 271-281.
  15. Cook SE, Vogel H and D. Castillo D. A rational approach to identifying effective combined anticoronaviral therapies against feline coronavirus. 2021. bioRxiv 2020.07.09.195016; doi: https://doi.org/10.1101/2020.07.09.195016
  16. Zhang S. A Much-Hyped COVID-19 Drug Is Almost Identical to a Black-Market Cat Cure. May 8, 2020 Shutterstock / The Atlantic, https://www.theatlantic.com/science/archive/2020/05/remdesivir-cats/611341/.
  17. Yan VC, Muller FL. Advantages of the Parent Nucleoside GS-441524 over Remdesivir for Covid-19 Treatment. ACS Medicinal Chemistry Letters. 2020, 11 (7): 1361-1366 DOI: 10.1021 / acsmedchemlett.0c00316
  18. Murphy BG, Perron M, Murakami E, Bauer K, Park Y, Eckstrand C, Liepnieks M, Pedersen NC. The nucleoside analog GS-441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies.Vet Microbiol. 2018, 219: 226-233.
  19. Common Dreams. Public citizen. Press release, August 4, 2020, https://www.commondreams.org/newswire/2020/08/04/public-citizen-scientists-gilead-and-federalscientists-have-neglected
  20. Dickinson PJ. Coronavirus Infection of the Central Nervous System: Animal Models in the Time of Covid-Front. Vet. Sci. 2020, 23: https://doi.org/10.3389/fvets.2020.584673
  21. Zhou S, Hill CS, Sarkar S, et al., Β-d-N4-hydroxycytidine Inhibits SARS-CoV-2 through lethal mutagenesis but Is also mutagenic to mammalian cells. J Infect Dis. 2021, 224: 415–419, https://doi.org/10.1093/infdis/jiab247.
  22. Cohen J, Piller C. Emails offer look into whistleblower charges of cronyism behind potential COVID-19 drug. ScienceInsider-Health. 2020, https://www.science.org/news/2020/05/emails-offer-look-whistleblowercharges-cronyism-behind-potential-covid-19-drug.
  23. Agostini ML, Andres EL, Sims AC, et al. Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease. MBio 2018; 9. DOI:
    10.1128 / mBio.00221-18.
  24. Szemiel AM, Merits A, Orton RJ, In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2. Plos Path, 2021, https: //doi.org/10.1371/journal.ppat.1009929.
  25. Martinot M, Jary A, Fafi-Kremer S, et al., Emerging RNA-Dependent RNA Polymerase Mutation in a Remdesivir-Treated B-cell Immunodeficient Patient With Protracted Coronavirus Disease 2019, Clinical Infectious Diseases, 2020 ;, ciaa1474, https: / /doi.org/10.1093/cid/ciaa1474
  26. FIP Warriors CZ / SK - EIDD-2801 (Molnupiravir) https://www.fipwarriors.eu/en/eidd-2801-molnupiravir/
Read "A long history of Beta-d-N4-hydroxycytidine and its modern application for the treatment of Covid-19 in humans and FIP in cats."

SUMMARY OF FIP TREATMENT USING GS-441524 (Dr. Pedersen)

Original article: SUMMARY OF GS-441524 TREATMENT FOR FIP
Niels C. Pedersen, DVM PhD, Professor Emeritus,
Pet Health Center, School of Veterinary Medicine, UC Davis

We use the same criteria to monitor treatment as described in clinical study published in JFMS (Journal of Feline Medicine and Surgery). Owners should monitor temperature, weight, activity, appetite, and clinical signs of the original disease at daily or weekly intervals. Blood tests - hematology and biochemistry (including serum protein values - total protein, albumin, globulin, A: G ratio) at the beginning of treatment and then every 4 weeks. It is always useful to update these values along with the weight in the form of a graph. The aim is to have a healthy, sensitive and active cat at the end of 12 weeks of treatment and with normal blood test values, especially in terms of hematocrit, total protein, globulin, albumin and A: G ratios. Significant weight gain is also a good sign, and some young or particularly emaciated cats can more than double their weight during treatment. This is, of course, an idealized treatment, and it should be appreciated that upward adjustments may be required if the response is slow or if complications such as ocular or neurological impairment occur during treatment.

Supportive (symptomatic) care may be required to stabilize cats that are critically ill at the time of diagnosis or during the first days of treatment with GS-441524 (GS). Abdominal effusion should not be aspirated unless it compresses the chest and interferes with respiration, as it is quickly replaced at the expense of the rest of the body. However, thoracic effusions are usually associated with varying degrees of dyspnoea and should be eliminated. Chest effusions return much more slowly. Symptomatic care also often includes fluids and electrolytes to suppress dehydration, antibiotics suspected of secondary bacterial infection and anti-inflammatory drugs (usually systemic corticosteroids), and rarely blood transfusions. Some cats with eye problems may also need topical medications to suppress severe inflammation and increased intraocular pressure (glaucoma).

Corticosteroids such as prednisolone should only be used during the first days of GS treatment and should be discontinued when there is a rapid improvement in health. Long-term use of corticosteroids with GS is strongly discouraged as it may mask the signs of improvement caused by GS, especially in cats with neurological FIP, has no therapeutic power and may interfere with the development of a protective immune response to FIP. It is possible that this immune response plays a major role in the final cure. If cats are on chronic steroid therapy, no dose reduction is required as there is no evidence that cats experience severe adrenal atrophy, which occurs in humans during long-term steroid therapy. Many owners, GS treatment consultants and veterinarians will use various promoted supplements to improve liver, kidney or immune system health, as well as vitamins such as B12. These substances do not have proven effectiveness and I consider them a waste of money.

Treatment with GS, which is the most common, can also be complicated by ulcers / lesions at the injection site. Treatment is difficult for both owners and cats because injections can be painful. In some cats, especially those with neurological impairments, there is a problem with the development of partial drug resistance, which requires an increase in dose. The response to treatment is usually within 24-72 hours and most cats return to normal or approach normal within 2-4 weeks, which is a good sign. We anticipate that the success rate of FIP treatment with GS-441424 is greater than 80%, given treatment failure due to misdiagnosis of FIP, inappropriate dosing, health complications, and drug resistance. Young cats are easier to treat and have a higher cure rate than cats older than 7 years. Cats with wet or dry FIP, with uncomplicated neurological or ocular symptoms, are easier to treat than cats with neurological FIP.

The starting dose for cats with wet or dry FIP without signs of ocular or neurological disease is 4-6 mg / kg daily for 12 weeks, with younger cats and wet FIP tending toward the lower limit and dry cases to the upper limit. Cats with ocular lesions and no neurological symptoms start with a dose of 8 mg / kg daily for 12 weeks. Cats with neurological symptoms start at a daily dose of 10 mg / kg for 12 weeks. If cats with wet or dry FIP initially show ocular or neurological symptoms, they switch to appropriate ocular or neurological doses. There is an oral form of GS available from at least two sources in China (Spark, Mutian), but I do not use it, so I do not know a comparable dosage. However, I do not recommend this if the injection dose rises above 10 mg / kg per day, as the effectiveness of oral absorption decreases at these high doses.

I recommend adjusting the dosage by weekly weight control. The weight gain of many of these cats can be huge, either because they are so skinny at first or they grow, or both. If weight loss occurs at the beginning of treatment, I remain at the original dose and do not reduce it. Failure to gain weight during treatment is considered a bad sign. We do not increase the dose unless there are serious reasons for this, such as worsening or improved blood test results, slow improvement, poor activity, restoration of the original clinical symptoms, or a change in the form of the disease, including ocular or neurological symptoms. This is where common sense comes in, because you don't want to get stuck on one blood level, which is not quite common, but does not affect the overall health of the cat. For example, globulin may still be a little high, but other important blood test values and health are very good. If there is a good reason to increase the dose, it should always be from +2 to +5 mg / kg per day and for at least 4 weeks. If these 4 weeks cause a prolongation of the 12-week duration of treatment, it is because of this dose adjustment. A positive response to any dose increase can be expected, and if you do not see an improvement, it means that the dose is still not high enough, drug resistance is developing, you have a bad GS brand, the cat does not have FIP, or there are other diseases that affect treatment.

One of the most difficult decisions is determining when to stop treatment. Although some cats, often younger with wet FIP, can be cured as early as 8 weeks, and possibly earlier, the usual duration of treatment is 12 weeks. Some cats may even require a dose adjustment and longer treatment periods. Critical blood levels such as hematocrit, total protein, albumin and globulin levels, and total white blood cell and absolute lymphocyte counts usually return to normal in treated cats after 8-10 weeks, when there is often an unexpected increase in activity levels. It is assumed, but there is no evidence yet, that after 8-10 weeks, the cat will develop its own immunity to infection. This is a situation that occurs in the treatment of hepatitis C in humans, which is also a chronic infection caused by the RNA virus, which often requires up to 12 or more weeks of antiviral treatment.

Unfortunately, there is no simple test to determine when a cure has taken place, and the fear of relapse often leads owners, treatment advisers and veterinarians to extend treatment beyond 84 days. Fear of relapses will also make people involved in the decision-making process too cautious about a single blood value that is slightly abnormal (eg, slightly high globulin or slightly low A: G ratio), or final ultrasound results suggesting suspected enlarged lymphatics. nodules, small amounts of fluid in the abdomen, or vague irregularities in organs such as the kidneys, spleen, pancreas, or intestines. It should be borne in mind that although most animals fall within the normal range of blood values, they are otherwise bell-shaped curves, and that there are a few exceptional patients who will have values at the edge of these curves. The ultrasound diagnosis must take into account the degree of pathology that may occur in the abdominal cavity affected by FIP, such as scars or some consequences in the form of organ changes in successfully treated cats. In situations where such questions arise, it is better to look more closely at the overall picture, and not just at one small part. The most important outcome of treatment is a return to normal health, which has two components - external health symptoms and internal health symptoms. External signs of health include a return to normal activity levels, an appetite, adequate weight gain or growth, and coat quality. The latter are often one of the best measures of health for a cat. Internal health symptoms are manifested by the return of certain critical values to normal based on periodic monitoring of complete blood counts and biochemistry. The most important values in the blood count are the hematocrit and the relative and absolute total number of white blood cells, neutrophils and lymphocytes. The most important values in biochemistry (or serum electrophoresis) are total protein, globulin, albumin and A: G ratio. Bilirubin is often elevated in cats by effusive FIP and may be useful in monitoring the severity and duration of inflammation. There are many other values in hematology and biochemistry panels, and it is not uncommon for some of them to be slightly higher or lower than normal, and it is better to ignore these values unless they are significantly elevated and associated with clinical symptoms - such as high urea and creatinine, which are also associated with increased water consumption, excessive urination, and abnormalities in urine analysis. The number of platelets in cats is notoriously low due to the trauma of blood collection and platelet aggregation, and should always be verified by a manual blood smear test. The final decision to discontinue or extend treatment when you encounter unclear doubts about different test procedures should always be based on external health manifestations more than on any single test result.

Different FIP groups have come up with different modifications of FIP treatment. Some groups will treat with an extremely high dose of GS from the beginning instead of increasing the dose only when indicated, or increase their GS dose in the last two weeks, or postpone treatment with a higher dose of GS in the hope of shortening the next two weeks. duration of treatment or reduce the likelihood of relapse. Some advocate the use of interferon omega or non-specific immunostimulants to further stimulate the immune system, and some use various other modifications. There is no evidence that modification of the extra high dose treatment will improve the cure rate. Similarly, interferon omega and non-specific immunostimulants have no demonstrated beneficial effects in FIP when administered as a single treatment or as adjuncts to GS. The practice of adding another antiviral drug, the viral protease inhibitor GC376, to the treatment of GS in cats that develop resistance to GS is also emerging, but this possibility still requires research. Finally, it is common for owners, treatment groups and veterinarians to add many supplements, tonics or injections (eg B12) to increase blood levels or to prevent liver or kidney disease. Such supplements are rarely needed in cats with pure FIP.

FIP relapses during the 12-week post-treatment observation period occur, and there is no simple blood test to predict whether a cure has occurred or is possible. Relapses usually involve infections that have entered the central nervous system (brain, spine, eyes) during treatment with wet or dry FIP, which has not been accompanied by neurological or ocular symptoms. The dose of GS-441524 used to treat these forms of FIP is often insufficient to effectively overcome the blood-brain or blood-eye barrier. The blood-brain barrier is more inaccessible than the blood-eye barrier, which explains why eye lesions are easier to treat than brain or spinal infections. Relapses that occur in the post-treatment period and that involve the eyes, brain or spine are usually treated for at least 8 weeks at an initial daily dose at least 5 mg / kg higher than the dose used during the primary treatment (eg 10, 12, 15 mg / kg daily). It is recommended that GS oral formulations not be used if the dose exceeds 10 mg / kg daily for injection, as intestinal absorption efficiency is reduced at high oral concentrations. Cats that cannot be cured of the infection at doses up to 15 mg / kg per day are likely to develop varying degrees of resistance to GS-441524. Partial resistance may allow the symptoms of the disease to be kept under control but not cured, while general resistance manifests itself in varying severity of clinical symptoms during treatment.

At the time of diagnosis, there may be resistance to GS-441524, but this is unusual. Rather, it occurs during treatment, and is often partial at first, leading to the need for higher dosing. In some cats, it may become complete. Resistance is a major problem in cats with neurological disease, especially those that have neurological symptoms or develop a brain infection during treatment, or during relapse after treatment has appeared to be successful. Many cats with partial drug resistance can be treated for signs of the disease, but relapse occurs as soon as treatment is stopped. The cats have been "treated" at the FIP for more than a year without healing, but eventually the resistance worsens or the owner runs out of money.

GS-441524 treatment shows no or minimal systemic side effects. It may cause mild kidney damage in some cats, but should not lead to kidney failure. Systemic vasculitis-type drug reactions have been observed in several cats and can be confused with injection site reactions. However, these drug reactions are in non-injectable areas and often go away on their own or respond well to short-term low-dose steroids. The main side effect of GS treatment is pain at the injection sites, which varies from cat to cat and according to the abilities of the person giving the injections (usually the owner). Injection site ulcers / lesions are a problem in some cats and usually occur when the injection site does not rotate (do not stay between the shoulders) and is not administered to the muscular and nervous layers under the skin. I recommend choosing places starting one inch behind the shoulder blades, down from the back to 1 to 2 inches in front of the tail and one third to half way down to the chest and abdomen. Many people use gabapentin before injections to relieve pain. The ulcers at the injection site are cleared of surrounding hair and gently cleaned 4 or more times a day with sterile cotton swabs soaked in dilute 1: 5 household hydrogen peroxide solution. They usually do not require any more complicated treatment and will heal in about 2 weeks.

We hope that the legal form GS-441524 will be available soon. The drug, called Remdesivir, is the greatest hope today, as Remdesivir breaks down into GS immediately when given intravenously to humans, mice, primates and cats. Remdesivir has received full US FDA approval, and similar approval is likely to follow in other countries. If so, it can be prescribed by any licensed human doctor and veterinarians. However, the use of Remdesivir in the United States is still limited to a specific subset of patients with Covid-19 and only under controlled conditions and with ongoing data collection. Until all restrictions are lifted, it will not be easily accessible for human use. I have no experience treating cats with Remdesivir instead of GS-441524. However, groups in Australia and some Asian countries are starting to use Remdesivir and are reporting the same results as GS-441524. The molar basis of Remdesivir is theoretically the same as GS-441524. GS-441524 free base has a molecular weight of 291.3 g / M, while Remdesivir has 602.6 g / M. Therefore, twice as much Remdesivir (602.6 / 291.3 = 2.07) would be needed to obtain 1 mg of GS-441524. The solvent for Remdesivir differs significantly from the solvent used for GS-441524 and is intended for IV use in humans. It is not known how diluted Remdesivir will behave when administered subcutaneously for 12 weeks or more. Mild signs of hepatotoxicity and nephrotoxicity have been observed with Remdesivir in humans. GS-441524 causes mild and non-progressive renal toxicity in cats, but without apparent hepatic toxicity. It is not clear whether the renal toxicity observed in humans receiving Remdesivir is due to its active ingredient (ie GS-441524) or to chemical agents designed to increase antiviral activity. Anivive is seeking GC376 approval for cats (and humans), but it will take another two or more years. GC376 is a viral protease inhibitor and acts differently from GS-441524, which inhibits early-stage viral RNA replication. Therefore, it is unlikely to have a significant synergistic viral inhibitory effect, but will be much more important in inhibiting drug resistance when used in combination therapy (such as combination antiviral therapy for HIV / AIDS).

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FIP treatment with oral forms GS-441524

Niels C. Pedersen, Nicole Jacque,
3.10.2021
Original article: FIP treatment with oral formulations of GS-441524

Introduction

Initial field testing of GS-441524 for FIP treatment involved subcutaneous administration. This route of administration was based on previous pharmacokinetic (PK) studies performed in laboratory cats. The intravenous and subcutaneous routes of injection yielded similarly high blood levels, which were maintained at virus-inhibiting concentrations for more than 24 hours. Oral administration has been found to lead to blood levels that peak after 2 hours, but reach only about 40 % peak levels of subcutaneous and intravenous administration (Pedersen NC, unpublished data, 2018). However, dogs that have a longer intestinal tract developed for omnivores can absorb up to 85 % GS441524 orally. [1, 5]. Dogs are often used as a surrogate for humans in oral absorption studies, so oral absorption in humans is also likely to be higher than in cats. 

Chinese suppliers of GS-441524 copied the diluent, drug concentration, and subcutaneous route used in the original published field study. Mutian was the first company to offer GS441524 on an unapproved market. Mutian was also the first company to investigate and offer an oral formulation. Mutian researchers found that effective blood levels of GS-441524 could only be achieved by increasing the concentration of the drug in their oral preparations. Other companies (eg Aura, Lucky) subsequently offered their own versions of the orally administered drug GS-441524. However, as of September 2021, Mutian no longer lists GS oral preparations (in any form) on its website. Aura, Lucky and Capella are currently the most widely used oral forms of GS441524 in the United States.

Current brands of capsules / tablets are sold as nutritional supplements and their labels list several common harmless chemical compounds and medicinal herbs, with GS-441524 not being listed at all. This is probably so that manufacturers avoid customs controls. Regardless of the list of ingredients, GS-441524 is the active ingredient in all oral products. The exact concentration of GS-441524 in the various oral preparations is kept secret by the vendors, but it is clearly higher (1.5-2-fold?) Than would be required if the drug was administered subcutaneously. 

Initially, we were critical of the oral route for two reasons. First, oral forms were more wasted by what was initially a rare and expensive resource. Second, published research on oral absorption of nucleosides (GS-441524 is a nucleoside) documents a concentration limit or ceiling for oral absorption [2-5]. Results with nucleoside-related EIDD-1931 showed a decrease in bioavailability from 56 to 36 % with increasing oral dose [6]. This limitation would theoretically make it difficult to achieve the extremely high blood levels needed to treat some forms of FIP (e.g., neurological) and / or to overcome the problem of acquired drug resistance. Oral bioavailability can also be significantly reduced by certain substances in the diet, and cat owners are known to use a large number of dietary supplements, some of which could adversely affect treatment. 

More and more owners and veterinarians appear to be using GS-441524 oral therapy for some or all of their treatment. The cost of GS-441524 oral products has been steadily declining and improving over the last two years. The problem of injection site reactions together with the more effective oral preparations GS-441524 have stimulated oral treatment and more and more cats are being treated with oral drugs either partially or completely. 

Composition and labeling

Most established oral preparations are small tablets that are easier to administer than larger capsules. Newer formulations, such as Sweeper, offer a soluble film form of GS-441524 to avoid "pill" difficulties in some cats. 

The actual amount of GS-441524 in the tablet / capsule and the recommended dosage of the oral medication will vary considerably depending on the form of the FIP, the vendor and the experience of the owner and the FIP treatment groups. Therefore, the actual amount (mg) of GS-441524 in a tablet or capsule is usually not reported. Instead of the actual amount of GS-441524 in a tablet or capsule, the seller's dosage is often based on the number of tablets needed per kg of weight, e.g. 1 tablet / kg orally (P0) every (q) 24 hours (h) for cats with wet or dry FIP and without ocular or neurological impairment. The amount of GS-41524 in one such tablet administered after 24 hours corresponds to a dose of 4-6 mg / kg SC after 24 hours, but the actual amount of GS in one tablet can be doubled as in 1 ml of injectable GS to compensate for the reduced bioavailability when administered oral route. 

In addition, one supplier (Aura / Spark) has tablets labeled for q12h administration and another for q24h dosing. 1 tablet / kg after 12 hours contains half the amount of GS-441524 (probably 4-6 mg) as 1 tablet / kg after 24 hours (probably 10 mg) - the reason is that dosing after 12 hours prevents a decrease in blood concentration 24 hours ago . However, effective blood levels after a single dose of PO or SC are maintained for 24 hours or longer in both cases. At doses corresponding to 10-15 mg / kg SC q24h, a further advantage of q8h or q12h over q24h may be an advantage, as it can help bypass the absorption ceiling. Therefore, in cats with doses corresponding to 10-15 mg / kg SC q24h or higher, a dose division of q8h or q12h is often used.

Dosage

The recommended starting dose of GS-441524 for cats with wet or dry FIP and without neurological or ocular symptoms is 4-6 mg / kg SC q24h. The injection dose for cats with ocular disease is 8 mg / kg SC q24h and for cats with neurological disease 10 mg / kg SC q24h. If a cat is started on wet FIP and then develops eye disease, the dose is immediately increased to 8 mg / kg SC q24h and if neurological symptoms develop, it is increased to 10 mg / kg SC q24h. Failure to treat FIP at doses higher than 15 mg / kg SC q24h indicates drug resistance. Doses of PO corresponding to 4-6, 8 and 10 mg / kg SC q24h are 10, 16 and 20 mg / kg PO q24h. (Note: some oral preparations are designated as SC equivalents, but in fact contain up to twice the reported mg GS) The recommended duration of treatment is 12 weeks, with dose increases if considered necessary. However, it is known that some cats can be cured in 6 weeks with any form of GS-441524, several in 8-10 weeks and almost all in 12 weeks. Young cats with abdominal wet FIP tend to respond the fastest, cats with dry FIP slower and cats with neurological FIP the slowest. Therefore, it is a "universal" recommendation to treat any cat with FIP, regardless of form, for at least 12 weeks. The daily dose in the form of PO can be divided into q12h, which may be advantageous in higher dose treatment to avoid an absorption ceiling. SC and PO treatment can be alternated q12h to avoid large injection volumes.

Oral GS dosing is less accurate than for injections. Tablets are difficult to separate because they tend to break, so halving is often the best thing to do. If the calculated dose after use falls within the indicated doses in the tablets, it is always recommended to round up to the nearest half of the tablet. 

Serving

All oral brands have similar instructions for the administration of capsules or tablets. Half an hour of fasting before and after administration is generally recommended. A small amount of treats can encourage cats to take the tablets, and many cats consume them when they are placed on a plate wrapped in treats (e.g., Churu). 

Costs

The price of oral GS has dropped significantly over the last year. Nevertheless, the relative price of the oral GS-441524 is 20-40 % higher (depending on the supplier) than its injectable version.  

Factors affecting oral and injectable administration 

Cats currently experiencing vomiting / regurgitation and diarrhea are generally considered unsuitable candidates for oral treatment with GS-441524. Therefore, cats with severe gastrointestinal disease are often injected at least until these problems are resolved. Most people, especially in the past, have started injecting GS-441524. The injection form is cheaper and the dosage is more precisely controlled. Absorption of GS-441524 is also more reliable by the subcutaneous route than by the oral route, which is often a critical factor in the initial treatment of cats that are initially seriously ill and unstable. Whether a cat will continue to inject GS-441524 is often conditioned by the owner's ability to inject as efficiently as possible, the cat's willingness to adapt to the pain of the injection, and the occurrence of injection wounds (lesions) at the injection site. Oral medications are often a welcome relief for both the owner and the feline patient in such situations. Some substances administered orally may interfere with the absorption of GS-441524. Therefore, you should avoid the inclusion of other medications and nutritional supplements unless they are necessary for the treatment of FIP. 

Comparison of the success of treatment with injection and oral GS-441524  

Assuming that the dosage is accurately calculated and properly adjusted, the success rate of the oral drug GS-441524 currently reflects the success rate of the injectable drugs. Nevertheless, differences in responses between oral and injectable GS-441524 have been reported. A small number of cats did not respond well to oral GS-441524 as initial treatment or led to relapses during injection replacement. Alternatively, switching cats to oral GS-441524 at the equivalent dose resulted in resolution of the disease, which did not respond well to injections. It is difficult to attribute these dramatic differences in formulation response as GS-441524 administered subcutaneously or orally enters the bloodstream and eventually the tissues. This is more likely to be due to the fact that the brands of GS-441524 injectable or oral medicine used prior to such a change were not good or that there were problems with absorption or administration. Indeed, there have been many cases where switching to another oral or injectable brand immediately resulted in improved response. 

It was originally thought that only the injectable form of GS-441524 could achieve the extremely high levels of blood and cerebrospinal fluid needed to effectively treat neurological disease, especially in situations where the virus developed varying degrees of drug resistance. However, oral markers such as Aura / Lucky have been effective in many cats with neurological FIP. This also applied to some cats that did not respond to the extremely high doses of GS441524 injection. More and more cats with neurological FIP are being treated exclusively with the oral form of GS. This is due either to greater experience with oral treatment in severe cases of FIP, or probably to higher quality oral products. 

An overview of currently available oral form brands GS-441524 

Note: The GS label and content reflect information provided by suppliers and have not been independently verified. 

Mutian - This is the original and most famous brand of the oral form GS-441524. It has been sold in several different forms, including several tablets and capsules. At the beginning of 2021, Mutian switched to the form of tablets, designated as 200 mg or 50 mg "Mutian" or "Xraphconn" - these deliver an equivalent SC dose of 10, respectively. 2.5 mg GS-441524. The tablets are significantly larger (8.5 mm diameter) than tablets from other suppliers. Recently, a new capsule formulation is rarely available. As of September 2021, Mutian's website no longer offers the option of PO. For all oral forms of Mutian, the supplier states the dosage: 100 mg / kg “Mutian” for wet / dry FIP, 150 mg / kg Mutian for ocular FIP and 200 mg / kg for neurological FIP. 

Aura / Spark - Aura is a long-established brand and is sold in tablets that are given every 12 or 24 hours. They are sold in versions q12h and q24h, but there is no difference in composition (ie extended release, etc.) between the two versions. The actual amount of GS-441524 in each tablet is not reported, but the label and effective dose are as follows:

MarkingInjectable equivalentDosage instructions
Aura 12h-1kgapprox. 2.5 mg / kgWet / dry: 1 tablet per kg twice a day
Ocular: 1.5 tablets per kg twice a day
Neurological: 2 tablets per kg twice a day  
Aura 24h – 1 kgapprox. 5 mg / kgWet / dry: 1 tablet per kg per day
Ocular: 1.5 tablets per kg per day
Neurological: 2 tablets per kg per day         
Aura 12h – 3 kgapprox. 7.5 mg / kgWet / dry: 1 tablet per 3 kg twice a day
Ocular: 1.5 tablets per 3 kg twice a day
Neurological: 2 tablets per 3 kg twice a day         
Aura 24h – 2 kgapprox. 10 mg / kgWet / dry: 1 tablet per 2 kg twice a day
Ocular: 1.5 tablets per 2 kg twice a day
Neurological: 2 tablets per 2 kg twice a day         

The equivalent oral dose for> 10 mg / kg daily GS injection is increased proportionately. The tablets can be combined regardless of the 12 / 24h label using an effective injection dose - for example, a 2.5 kg cat with a wet FIP could take one tablet 24h - 2 kg and one tablet 12h - 1 kg per day.

Lucky - Lucky tablets are designated 24h - 1 kg (equivalent dose 5-6 mg / kg SC) or 24h - 2 kg (equivalent dose approximately 10-12 mg / kg SC) and are said to have the same composition as comparable Aura tablets, although they have a different Face. For FIP without ocular or neurological symptoms, you should give one 1 kg tablet daily per kg cat weight or one 2 kg tablet for every 2 kg, rounded to the nearest half tablet. Multiply the number of tablets per day by 1.5 for ocular or 2 for neurological forms. 

MarkingInjectable equivalentWet / dry FIP dosing instructions (dosing doubles for neuro / ocular FIP)
Lucky 24h - 1 kgapprox. 5-6 mg1 tablet per kg per day
Lucky 24h - 2 kgapprox. 10-12 mg1 tablet per 2 kg per day

Capella - Capella produces two tablet sizes, 1 kg (dose 5-6 mg SC equivalent) and 2 kg (dose 10-12 mg SC equivalent). For FIP without ocular or neurological symptoms, you should give one 1 kg tablet daily per kg cat weight or one 2 kg tablet for every 2 kg and round up to the nearest half tablet. Multiply the number of tablets per day by 1.5 for ocular or 2 for neurological forms. 

Kitty Care - This is another low-cost brand that now offers both injectable and oral GS-441524. Each tablet is assumed to contain the equivalent of a 6 mg SC dose of GS-441524. 

Hero 16 -It is a well-known brand, which is supplied in easy-to-apply and divisible tablets intended for administration in a dose of one tablet per 2 kg body weight, such as Capella 2 kg tablets. Each tablet probably contains 16 mg of GS-441524. 

Rainman - This brand is popular in China and seems to have a good reputation in the countries where it is used. It is sold in 1 kg and 2 kg tablets, which are believed to contain the equivalent of 5-6 mg and 10-12 mg SC GS-441524. 

Mary - Mary is sold in capsules that probably contain the equivalent of 6 mg SC GS-441524

Additional brands- Panda, Pany, Sweeper, Sweeper movie

Reference studies on GI uptake of nucleosides similar to GS-441524 and GS-441524

  1. Thomas L. A precursor to remdesivir shows therapeutic potential for COVID-19. https://www.news-medical.net/news/20210209/A-precursor-to-remdesivir-showstherapeuticpotential-for-COVID-19.aspx.
  2. Painter GR, Bowen RA, Bluemling GR, et al. The prophylactic and therapeutic activity of a broadly active ribonucleoside analog in a murine model of intranasal venezuelan equine encephalitis virus infection. Antiviral Res. 2019; 171: 104597. doi: 10.1016 / j.antiviral.2019.104597
    After oral administration EIDD-1931 is quickly absorbed as evidenced by plasma T-max-values ranging between 0.5 and 1.0 h.Exposures are high (C-ma-xvalues range between 30 and 40μM) and are dose dependent, but significantly less than dose proportional. The observation of decreasing bioavailability with increasing dose may indicate capacity limited absorption, a phenomenon that has been reported for other nucleosides (de Miranda et al., 1981). EIDD-1931, like most endogenous nucleosides and xenobiotic nucleoside analogs, is a highly polar, hydrophilic molecule (cLog P = −2.2) and therefore likely to require functional transporters to cross cell membranes. This dependence would explain the capacity limited uptake seen in the pharmacokinetic studies done using the CD-1 mice. Earlier reports also indicated that nucleoside uptake into mouse intestinal epithelial cells is primarily mediated by sodium dependent concentrative nucleoside transporters (Cass et al., 1999; Vijayalakshmi and Belt, 1988).
  3. Cass, CE, Young, JD, Baldwin, SA, Cabrita, MA, Graham, KA, Griffiths, M., Jennings, LL, Mackey, JR, Ng, AM, Ritzel, MW, Vickers, MF, Yao, SY, 1999 .Nucleoside transporters of mammalian cells. Pharm. Biotechnol. 12313–12352
  4. de Miranda, P., Krasny, HC, Page, DA, Elion, GB, 1981. The disposition of acyclovir indifferent species. J. Pharmacol. Exp. Ther. 219 (2), 309–315
  5. Vijayalakshmi, D., Belt, JA, 1988. Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities. Biol. Chem. 263 (36), 19419–19423.
  6. Yan VC, Khadka S, Arthur K, Ackroyd JJ, Georgiou DK, Muller FL. Pharmacokinetics of Orally Administered GS-441524 in Dogs. bioRxiv, doi: https://doi.org/10.1101/2021.02.04.429674
  7. FIP Warriors CZ / SK, https://www.fipwarriors.eu/, https://www.facebook.com/groups/fipczsk
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