History of Feline infectious Peritonitis 1963-2022 – First description to Successful Treatment

Niels C. Pedersen
Center for Companion Animal Health, School of Veterinary Medicine, University of California, 944 Garrod Drive, Davis, CA, 95616, USA
Original article: History of Feline infectious Peritonitis 1963-2022 – First description to Successful Treatment
17.4.2022

Abstract

This article discusses the development of knowledge about feline infectious peritonitis (FIP) from its recognition in 1963 to the present and has been prepared to inform veterinarians, cat rescuers and carers, shelter staff and cat lovers. The causative agent of the feline coronavirus and its relationship to the ubiquitous and minimally pathogenic feline intestinal coronavirus, epizootology, pathogenesis, pathology, clinical signs and diagnostics are briefly mentioned. The main emphasis is placed on the risk factors influencing the incidence of FIP and the role of modern antivirals in successful treatment.

Introduction

Figure 1. Photo of the author and Dr. Jean Holzworthová (1915-2007) from 1991. Dr. Holzworth was the best feline veterinarian the author knew and was responsible for the first report of FIP as a specific disease. She spent her entire career at Angell Memorial Animal Hospital in Boston.

Feline infectious peritonitis (FIP) was described as a specific disease in 1963 by veterinarians at Angell Memorial Animal Hospital in Boston (Holzworth 1963) (Fig. 1). Pathology records from this institution and Ohio State University failed to identify earlier cases (Wolfe and Griesemer 1966), although identical cases were soon recognized worldwide. The initial pathological descriptions were of diffuse inflammation of the tissues lining the abdominal cavity and abdominal organs with extensive effusion of inflammatory fluid, after which the disease was eventually named (Wolfe and Griesemer 1966, 1971) (Figs. 2, 3). A second and less common clinical form of FIP, which presents with less diffuse and more widespread granulomatous lesions involving organ parenchyma, was first described in 1972 (Montali and Strandberg 1972) (Figs. 3,4). The presence of inflammatory effusions in the body cavity in the common form and the absence of effusions in the less common form led to the names wet (effusion, non-parenchymatous) and dry (non-effusion, parenchymatous) FIP.

The prevalence of FIP appears to have increased during the panzootic disease caused by feline leukemia virus (FeLV) in the 1960s–1980s, when many cases of FIP were found to be associated with FeLV (Cotter et al., 1973; Pedersen 1976a). Subsequent management of FeLV infection in owned cats through rapid testing and vaccination resulted in an increase in FIP cases. However, recent interest in breeding/rescue along with effective treatment has led to increased awareness of the disease and its diagnosis.

Figure 2. Gross necroptic appearance of the abdominal cavity of a cat with acute onset wet FIP. The abdomen is filled with several hundred ml of yellow viscous fluid, the omentum is reddened, edematous and contracted, and fibrin deposits (arrows) are visible on the surface of the spleen and the edges of the liver. A fiber of fibrin can be seen on the spleen
Figure 3. Appearance of an open abdomen at autopsy of a cat that died of a chronic form of effusive FIP. The abdomen is filled with a viscous, yellow-colored exudate, and the omentum is thickened and contracted. The main lesions are in the liver with numerous plaque-like structures (pyogranulomas) on the cover. More circumscribed lesions (granulomas), also oriented on the serous surface, look more fleshy and are elevated above the surface. These lesions also involve the underlying liver parenchyma and are more typical of dry FIP. This is an example of a case of FIP that transitions between the wet and dry forms (arrow).
Figure 4A – Gross section of kidneys of two cats with dry form of FIP. The lesions are superficial and extend into the underlying parenchyma.
Figure 4B - lesions of the dry form of FIP in organs such as the kidneys, cecum, colon and intestinal lymph nodes (Fig. 5) were grossly confused with renal lymphoma.
Figure 5. Gross enlargement of ileo-cecal-colic lymph nodes in a cat with dry FIP.

Etiological factor

The first attempts did not allow identifying the causative agent of FIP, but confirmed its infectious nature (Wolfe and Griesemer 1966). A viral etiology was established in 1968 using ultrafiltrates of infectious material (Zook et al., 1968). The causative virus was subsequently identified as a coronavirus (Ward 1970), which is closely related to enteric coronaviruses of dogs and pigs (Pedersen et al., 1978).

Confusion arose when feline enteric coronavirus (FECV) was isolated from the feces of healthy cats and proved to be indistinguishable from feline infectious peritonitis virus (FIPV) (Pedersen et al., 1981). Unlike FIPV, which readily induced FIP in laboratory cats, experimental infections with FECV were largely asymptomatic. The relationship between the two viruses became clear when FIPVs were found to be FECV mutants that arise in the body of every cat with FIP (Vennema et al., 1995; Poland et al., 1996).

FECV is ubiquitous in feline populations worldwide and is first shed in faeces from approximately 9–10 weeks of age, coinciding with the loss of maternal immunity (Pedersen et al., 2008 ;). The infection takes place via the faecal-oral route and targets the intestinal epithelium, and the primary signs of enteritis are mild or inconspicuous (Pedersen et al., 2008; Vogel et al., 2010). Subsequent faecal excretion occurs from the colon and usually stops after several weeks or months (Herrewegh et al., 1997; Pedersen et al., 2008; Vogel et al., 2010). Immunity is short-lived and repeated infections are common (Pedersen et al., 2008; Pearson et al., 2016). Over time, stronger immunity eventually develops and cats older than 3 years are less likely to shed the infection in their faeces (Addie et al., 2003). FECV is constantly subject to genetic drift into locally and regionally identifiable clades (Herrewegh et al., 1997; Pedersen et al., 2009).

FECV and FIPV are classified as biotypes of the feline coronavirus (FCoV) subspecies. The genomes of FECV and FIPV biotypes are related at >98 %, but with unique host cell tropism and pathogenicity (Chang et al., 2012; Pedersen et al., 2009). FECVs infect the mature intestinal epithelium, whereas FIPVs lose intestinal tropism and acquire the ability to replicate in monocytes/macrophages. The published names FECV or FIPV will be used here when discussing aspects of the disease specific to each biotype, while the term FCoV will be used when discussing features common to both biotypes.

Three types of mutations are involved in the biotype change of FECV to FIPV. The first type, which is unique to each cat with FIP (Poland et al., 1996), consists of an accumulation of missense and nonsense mutations in the c-terminus of the auxiliary 3c gene, often resulting in truncated 3c gene products (Pedersen et al., 2012 ; Vennema et al., 1995). The second type of mutation consists of two specific single nucleotide polymorphisms in the fusion peptide of the S gene, one or the other form being common to >95 % FIPV and absent in FECV (Chang et al., 2012). A third type of mutation, unique to each FIPV isolate and not found in FECV, occurs in and around the furin cleavage motif between the receptor binding domain (S1) and the fusion domain (S2) of the spike gene (S) (Licitra et al., 2013). These mutations have different effects on furin cleavage activity. Together and in an as yet undetermined manner, they are responsible for the shift of the tropism of the host cell from the enterocyte to the macrophage and for the profound change in the form of the disease.

FCoV, and therefore FECV and FIPV, exist in two serotypes identified by antibodies against the viral neutralizing epitope on the S gene (Herrewegh et al., 1998; Terada et al., 2014). Serotype I FCoVs are identified in cat sera and are prevalent in most countries. Serotype II FCoVs result from recombination with the S part of the canine coronavirus gene (Herrewegh et al., 1998; Terada et al., 2014) and are identified by canine coronavirus antibodies. Serotype II FIPVs are easily cultured in tissue culture, whereas serotype I FIPVs are difficult to adapt to growth in vitro. Serotype I and II FECVs were not grown in conventional cell cultures (Tekes et al., 2020).

FIPVs are found exclusively in activated monocytes and macrophages in affected tissues and effusions and are not secreted into the environment. Therefore, cat-to-cat (horizontal) transmission of FIPV is not the main mode of spread. Rather, FIP follows the pattern of an underlying enzootic FECV infection, with sporadic cases and occasional small outbreaks of disease (Foley et al., 1997). These clusters of cases can be mistaken for epizootics. The only report of an epizootic occurrence of FIP was associated with a single serotype II virus that appeared to develop in a shelter housing both dogs and cats (Wang et al., 2013). Horizontal transmission in this case followed an epizootic rather than an enzootic disease model, with infection spreading rapidly to cats of all ages and in close contact with the index case (Wang et al., 2013).

The low incidence of FIP cases in the population suggests that FIPV mutations arise infrequently. However, studies involving FECV infection in immunocompromised cats infected with FIV and FeLV suggest that FIP mutants may be common but only cause disease under certain circumstances. Nineteen cats infected with feline immunodeficiency virus (FIV) for 6 years and a control group of 20 littermates not infected with FIV were orally challenged with FECV (Poland et al., 1996). Cats in both groups remained asymptomatic for two months when two cats in the FIV-infected group developed FIP. In a second study, 26 young cats with enzootic FECV infection from a breeding colony with no history of FIP were contact-exposed to FeLV carriers (Pedersen et al., 1977). Two kittens in the group subsequently developed FIP 2–10 weeks after becoming FeLV viremic. The question remains, how long can FIPV viruses survive in the body before they are eliminated? According to one of the theories, they persist in the body for a certain time and become pathological only if immunity against them is impaired (Healey et al., 2022). This theory is supported by the way immunity to FeLV develops. Most cats resist FeLV by kitten age and develop robust and permanent immunity, but this occurs within a few weeks, during which the virus persists in a subclinical or latent state (Pedersen et al., 1982; Rojko et al., 1982). . Methylprednisolone given during this period, but not after, will abolish developing immunity and lead to a state of persistent viremia.

Epizootology

Epizootiology is the study of the occurrence, spread and possible control of animal diseases and the influence of environmental, host and agent factors. FIP is considered one of the most important infectious causes of death in cats, although there are no precise data on prevalence. It is estimated that 0.3–1.4 % deaths of cats presented to veterinary institutions are related to FIP (Rohrbach et al., 2001; Pesteanu-Somogyi et al., 2006; Riemer et al., 2016) and in some shelters and breeding stations up to 3.6–7.8 % (Cave et al., 2002). FIP is also described as an environmental disease with a higher incidence of multiple cats. Three-quarters of the FIP cases in the currently ongoing treatment study came from the field through foster carers/rescues and cat shelters, 14 % from kennels, and only 11 % from households.1

Studies based on cases observed in academic institutions have demonstrated the influence of age and gender on the incidence of FIP (Rohrbach et al., 2001; Pesteanu-Somogyi et al., 2006; Pedersen 1976a; Worthing et al., 2012; Riemer et al., 2016) . Three-quarters of the cases in these cohorts occurred in cats younger than 3 years of age, and few cases occurred after 7 years of age. This was also confirmed by a current and ongoing field study from the Czech Republic and Slovakia, in which it was found that more than 80 % cases of FIP occurred in cats under 3 years of age and only 5 % in cats older than 7 years (Fig. 6) .1 Earlier institutional studies differed on the effect of sex, but indications were that male cats were slightly more susceptible to FIP than female cats. This was also confirmed by current data from the field, which show a ratio of males to females of 1.3:1.1. It is unclear whether castration affects the incidence of FIP, with some reports suggesting that it may increase susceptibility (Riemer et al., 2016), while others do not report such a clear effect.1

Figure 6. Age of more than 607 cats from the Czech Republic and Slovakia at the time of diagnosis and treatment of FIP.1 Thirty percent of infections were in cats six months of age or younger, 50 % at one year of age, and 85 % at three years of age or younger.

Other environmental and viral risk factors have been implicated in the increased incidence of FIP, but their significance requires knowledge of disease occurrence in their absence. A possible baseline may have been provided by a study of enzootic FECV infection, which had been unrecognized for many years in a well-managed specific pathogen-free breeding colony (Hickman et al., 1995). This colony was kept in strict quarantine free of other infections and the standard of nutrition and husbandry was high. This colony produced hundreds of kittens each year before the first case of FIP was diagnosed. Such observations suggest that FIP may be a rare phenomenon in the absence of risk factors.

The importance of moving to a new home as a risk factor for FIP is only now being appreciated. Breeders, many of whom have not experienced any cases of FIP in their litters, are most concerned about the announcement that one of their kittens has developed FIP shortly after going to a new home. A recent study found that more than half of cats with FIP had experienced a change in environment, shelter or capture in the weeks before the illness.1 Cats are known to hide outward signs of stress, even when suffering from serious internal disease consequences. Even simple procedures such as changing the cage suppress immunity and reactivate latent herpes virus shedding and disease symptoms in cats (Gaskell and Povey, 1977). Stressful situations, even those that seem minor, can cause a decrease in lymphocyte levels and “sickness behavior” (Stella et al., 2013).

Differences in the genetic make-up of enzootic FCoV strains may also contribute to the prevalence of FIP in the population. Serotype II FIPVs are thought to be more virulent than serotype I and more likely to be transmitted from cat to cat (Lin et al., 2009; Wang et al., 2013). It is also possible that certain FECV clades are more susceptible to mutation to FIPV, which should be studied. The author also observed a disproportionately high proportion of cats with neurologic FIP in some regions, suggesting that genetic determinants in certain FCoV strains may be more neurotropic.

Immunodeficiencies associated with retroviruses are associated with susceptibility to FIP. Up to half of FIP cases during the peak of FeLV panzootic disease were persistently infected with FeLV (Cotter et al., 1973; Pedersen 1976a; Hardy 1981). FeLV infection causes suppression of T-cell immunity, which may inhibit the protective immune response to FIP. The importance of FeLV infection for the incidence of FIP has declined significantly since the 1980s, when carrier elimination and vaccination pushed FeLV back into the wild, where exposures are less severe and immunity is the usual outcome. Chronic feline immunodeficiency virus (FIV) infection has also been shown to be a risk factor for FIP in FECV-infected cats under experimental conditions (Poland et al., 1996). In one recent field study, FeLV infection was recognized in 2 % and FIV in 1 % cats treated for FIP.1

The incidence of FIP in purebred cats is reported to be higher than in random breeding cats, with some breeds appearing to be more susceptible than others (Pesteanu-Somogyi et al., 2006; Worthing et al., Genetic predisposition to FIP has been investigated in several Persian cat breeds and is estimated to account for half the risk of the disease (Foley et al., 1997). Some breeds, such as the Birman, are more susceptible to developing dry than wet FIP (Golovko et al., 2013). Attempts to identify specific genes associated with susceptibility for FIP in Burmese cats included several immune-related genes, but none reached the desired significance (Golovko et al., 2013).The largest study of genetic susceptibility to FIP showed that it is extremely polymorphic and reported consanguinity as a major risk factor. breeding (Pedersen et al., 2016).Specific polymorphisms in several genes have also been associated with high levels of FECV shedding among several breeding cat breeds (Bubeniko and et al., 2020).

In females, FIP, usually the wet form, may develop during pregnancy or in the perinatal period. This phenomenon resembles the suppression of immunity in pregnant women and the predisposition to certain infections (Mor and Cardenas 2010). It is not clear whether subclinical FIP is activated by pregnancy or by increased susceptibility to new infection. Maternal infection early in pregnancy results in fetal death and resorption, while later infections often result in abortion (Fig. 7). Kittens can be born healthy, but develop disease in the perinatal period and die. Some babies are born uninfected thanks to the effectiveness of the placental barrier between mother and fetus or thanks to the help of antiviral treatment (Fig. 8).

Figure 7. Aborted kittens from a dam that developed wet FIP late in pregnancy. Miscarriage was the first symptom of FIP, quickly followed by the classic symptoms of abdominal wet FIP. The mother was successfully cured of FIP with the antiviral GS-441524.
Figure 8. This mother developed symptoms of wet abdominal FIP 3 weeks after the onset of pregnancy and was successfully treated with GS-441524. Subsequently, she gave birth to a litter of four kittens by caesarean section, one of which died and three survived and grew up healthy. Treatment was given during the remaining 6 weeks of gestation and continued for 6 weeks during which the kittens were successfully nursed. GS-441524 had no apparent side effects on the mother or the kittens.

A possible increase in the number of cases of FIP was observed in cats older than 10 years in studies conducted 50 years ago (Pedersen 1976a). Slightly more than 3 % cases of FIP in a recent study occurred in cats 10 years of age and older and 1.5 % in cats 12 years of age and older (Fig. 6).1 The occurrence of FIP in the elderly often involves two different scenarios. The first scenario also involves exposure to FECV faecal excretion, but in a unique way. It is common for old cats to mate as kittens and live together in relative isolation unexposed to FECV for many years. One cat in the pair dies, is left alone, and a much younger companion obtained from a rescue organization, shelter, or kennel is brought into the household that has a high probability of excreting FECV. Older cats are also susceptible to the same FIP risk factors as younger cats, as well as other factors associated with aging. The first of these is the impact of aging on the immune system, with the most consequential being the deterioration of cellular immune function (Day 2010). Other risk factors associated with old cats include the debilitating and potentially immunosuppressive effects of diseases such as cancer and chronic diseases of the kidneys, liver, oral cavity and intestines. Some diseases in old cats can be mistaken for FIP or complicate the treatment of FIP if they are present at the same time.

Other risk factors that need further investigation include loss of maternal systemic immunity by separation at birth, early weaning and loss of lactogenic immunity, malnutrition, common kitten infectious diseases, early neutering, vaccination, congenital heart defects, and even a shelter fire (Drechsler et al.), 2011; Healey et al., 2022; Pedersen 2009, Pedersen et al. 2019).1 However, the most important positive risk factor remains the presence of FECV in the population (Addie et al., 1995). The prevalence of FIP in several Persian cat breeds was also related in one study to the proportion of cats that shed FECV at a given time and to the proportion of these cats that are chronic shedders (Foley et al., 1997). The importance of exposure to FECV supports the need to find ways to either prevent infection or reduce its severity. One of the first steps is a better understanding of FECV immunity (Pearson et al., 2019).

Pathogenesis

The first interface between FECV and the immune system is the lymphatic tissues of the intestine (Malbon et al., 2019, 2020). Although the downstream events leading to FIP are not fully understood, it is possible to speculate based on what is already known about FECV and FIPV infections, other macrophage-tropic infections, and viral immunity in general. During intestinal infection, FECV particles and proteins reach the local lymphatic tissues and are processed by phagocytic cells first into peptides and finally into amino acids. Some of these peptides will be recognized as foreign when arrayed on the cell surface, triggering innate (innate or non-specific) and adaptive (acquired or specific) immune responses (Pearson et al., 2016). FECVs also mutate to FIPV at the same time and in the same cell type. Some of these mutations will allow the virus to replicate in these or closely related cells of a specific monocyte/macrophage lineage.

The host cell for FIPV appears to be a specific class of activated monocytes found around venules on the surface of intestinal and thoracic organs, mesentery, omentum, uveal tract, meninges, choroid and ependyma of the brain and spinal cord, and freely in effusions. These cells belong to the activated (M1) class (Watanabe et al., 2018) and resemble a subpopulation of small peritoneal macrophages described in mice (Cassado et al., 2015). This type of cell arises from circulating bone marrow-derived monocytes that are rapidly mobilized from the blood in response to infectious or inflammatory stimuli. A similar-looking population of activated monocytes has been described around blood vessels in the retina affected by FIP (Ziolkowska et al., 2017). These cells stained for calprotectin, indicating their blood origin. Although FIPV infection occurs initially in smaller activated monocytes, viral replication is most intense in large, vacuolated, terminally differentiated macrophages (Watanabe et al., 2018). The virus released from these cells rapidly infects activated monocytes produced in the bone marrow and drawn to the site from the bloodstream.

The cellular receptor used by FECVs to infect intestinal epithelial cells has not yet been determined. The cellular receptor that FIPVs use to infect activated monocytes is also unknown. RNAs for conventional coronavirus receptors such as aminopeptidase N (APN), angiotensin converting enzyme 2 (ACE2) and CD209L (L-SIGN) were not upregulated in infected peritoneal cells of cats with experimental FIP, and CD209 (DC-SIGN) was significantly underexpressed (Watanabe et al., 2018). An alternative route of infection of activated monocytes may involve immune complexation of the virus and entry into cells by phagocytosis (Dewerchin et al., 2008, 2014; Van Hamme et al., 2008). Activated monocytes in lesions stain strongly positive for FIPV antigen, IgG and complement (Pedersen, 2009) and mRNA for FcγRIIIA (CD16A/ADCC receptor) is markedly increased in infected cells (Watanabe et al., 2018), supporting infection through immune complexation and alternative receptors related to phagocytosis.

Macrophage pathogens are intracellular and elimination of infected cells occurs through lymphocyte-mediated killing. The first line of defense is non-specific lymphocytes, and if they fail, an adaptive immune response to FIPV follows through specific T-lymphocytes. If infected activated monocytes and macrophages fail to be contained and eliminated, they may disseminate locally in the abdominal cavity, possibly from lymph nodes in the lower intestinal region and the site of FECV replication. Spread locally and to distant sites via the bloodstream is by infected monocyte cells (Kipar et al., 2005).

FIP occurs in two basic forms, wet (effusive, nonparenchymatous) (Figures 2 and 3) or dry (noneffusive, parenchymatous) (Figures 4 and 5), with wet FIP accounting for 80 % cases.1 The term "wet" refers to a characteristic fluid discharge in the abdomen or chest (Wolfe and Griesemer 1966, 1971). Wet FIP lesions are dominated by inflammation reminiscent of immediate or Arthus-type hypersensitivity (Pedersen and Boyle, 1980), whereas dry FIP lesions resemble delayed-type hypersensitivity reactions (Montali and Strandberg 1972; Pedersen 2009). The wet and dry forms of FIP therefore reflect competing influences of antibody and cell-mediated immunity and associated cytokine pathways (Malbon et al., 2020, Pedersen 2009). Immunity to FIPV-infected cells, which is the norm, is thought to involve strong cell-mediated responses (Kamal et al. 2019). Dry FIP is thought to occur when cell-mediated immunity is partially effective in suppressing infection, and wet FIP when cellular immunity is ineffective and humoral immune responses predominate.

FIP is considered unique among macrophage infections because it is viral, but the dry form shares many clinical and pathogenic features with feline diseases caused by systemic mycobacterial (Gunn-Moore et al., 2012) and fungal infections (Lloret et al., 2013). . Similarities in pathogenesis also exist between wet FIP and antibody-enhanced viral infections such as dengue fever and dengue hemorrhagic shock syndrome (Pedersen and Boyle 1980; Rothman et al., 1999; Weiss and Scott 1981).

Host responses are thought to solely determine the outcome of FIPV infection and the resulting forms of disease. However, macrophage-tropic pathogens have evolved their own unique defense mechanisms against the host (Leseigneur et al., 2020). One of the mechanisms is the delay of programmed cell death (apoptosis). Delayed apoptosis allows sustained microbial replication and eventual release of more infectious agents, as has also been described in FIPV-infected macrophages (Watanabe et al., 2018). FIPV can also control the recognition and killing of infected activated monocytes by specific or non-specific T-cells. The cell surface targets for T-cells that kill infected cells are likely FIPV proteins (antigens) expressed on major histocompatibility complex class I (MHC-I) receptors. However, surface expression of viral antigens by MHC-I receptors was not detected on FIPV-positive cells collected from FIP tissues or effusions (Cornelissen et al., 2007). DC-Sign has been proposed as a receptor for FIPV (Regan and Whitaker, 2008), but RNA for DC-Sign is markedly underexpressed by infected peritoneal cells, whereas RNA for Fc (MHC-II) receptors is markedly overexpressed and RNA for MHC -I is reduced (Watanabe et al., 2018). This suggests that the normal mode of infection of host cells may be altered by FIPV to favor infection by phagocytosis instead of binding to specific viral receptors on the cell surface, fusion with the cell membrane, and internalization.

Pathology

Detailed descriptions of the gross and microscopic lesions in the wet form of FIP were first described by Wolfe and Griesemer (1966, 1971). The disease is characterized by vasculitis involving venules in the tissues lining the abdominal or thoracic cavity, organ surfaces, and supporting tissues such as the mesentery, omentum, and mediastinum. The inflammatory process leads to effusions in the abdominal or chest cavity up to a volume of one liter or more (Fig. 2, 3). The underlying lesion is a pyogranuloma, which consists of a focal accumulation of activated monocytic cells in various stages of differentiation, interspersed with non-degenerate neutrophils and sparse numbers of lymphocytes. Pyogranulomas are superficially oriented and appear grossly and microscopically as single and coalescent plaques (Fig. 2).

FIPV antigen is immunohistochemically (IHC) observed only in activated monocytes in lesions and effusions (Litster et al., 2013). Large vacuolated terminally differentiated macrophages are particularly rich in virus (Watanabe et al., 2018), reminiscent of the lepromatous form of leprosy (deSousa et al., 2017). Lymph nodes located near the sites of inflammation are hyperplastic and enlarged.

The relationship between dry and wet FIP was first described in 1972 in a report of cases of unknown etiology with similar pathology (Montali and Strandberg 1972). As the authors state, "this pathological syndrome was characterized by granulomatous inflammation in various organs, but mainly affected the kidneys, visceral lymph nodes, lungs, liver, eyes and leptomeninges". Tissue extracts of these lesions induced wet FIP in laboratory cats, confirming that wet and dry FIP are caused by the same agent.

The gross and microscopic pathology of dry FIP resembles that of other macrophage-tropic infections such as feline systemic blastomycosis, histoplasmosis, coccidioidomycosis (Lloret et al., 2013), tuberculosis and leprosy (Gunn-Moore et al., 2012). Lesions of dry FIP mainly involve the abdominal organs (Figs. 5, 6) and are rare in the thoracic cavity (Montali and Strandberg 1972; Pedersen 2009). Lesions are less widespread and focal than in wet FIP, with a tendency to extend from the serous surfaces into the parenchyma of the underlying organs (Figs. 5, 6). The target of the host immune response are small aggregates of infected monocytic cells associated with venules, similar to pyogranulomas in wet FIP, but surrounded by dense accumulations of lymphocytes and plasma cells and variable fibrosis. The florid hyperemia, edema, and microhemorrhage associated with wet FIP are mostly absent, therefore significant effusions in the body cavities are absent. The host response to foci of infection gives the lesions a gross tumor-like appearance (Figs. 5, 6). Infected activated monocytes in the central focus of infection are less dense and contain lower levels of virus than in the wet form (Pedersen 2009;), a feature of the tuberculoid form of leprosy (de Sousa et al., 2017). Lesions in some places, for example on the wall of the large intestine, can cause a dense surrounding zone of fibrosis, which resembles classic tuberculosis granulomas. Transitional forms also exist between wet and dry forms in a small number of cases and are mostly recognizable at autopsy (Fig. 3).

Ocular and neurological FIP are classified as forms of dry FIP (Montali and Strandberg 1972). However, pathology in the uveal tract and retina and in the ependyma and meninges of the brain and spinal cord is intermediate between wet and dry FIP (Fankhauser and Fatzer 1977; Peiffer and Wilcock 1991). This can be explained by the effect of the blood-ocular and blood-brain barrier in protecting these areas from systemic immune reactions.

Clinical characteristics of FIP

The five most common symptoms in cats with FIP, regardless of clinical form and frequency of occurrence, are lethargy, loss of appetite, enlarged abdominal lymph nodes, weight loss, fever, and deteriorating coat.1 These symptoms can appear quickly, within a week, or they can exist for many weeks or even months before a diagnosis is made. The course of the disease tends to be more rapid in cats with wet FIP than with dry FIP, and growth retardation is common in young cats, especially those with more chronic disease. 20 % cats with fever as the main symptom are eventually diagnosed with FIP (Spencer et al., 2017).

The wet form of FIP occurs in approximately 80 % cases, more often in younger cats, and tends to be more severe and more rapidly progressive than the dry form. Abdominal effusion (ascites) is four times more common than pleural effusion, with abdominal distension (Fig. 9) and dyspnea being common symptoms. Pyrexia and jaundice are more common symptoms in cats with wet than dry FIP (Tasker, 2018).

Figure 9.  Adult longhair cat with chronic abdominal wet FIP. The cat was in acceptable health except for mild weight loss, lethargy, poor coat quality, and occasional low-grade fever. Abdominal distension was not noted for some time, and the abdominal fluid contained relatively low protein and white blood cell counts.
Figure 9. A young cat who presented with rapid onset of high fever, loss of appetite, abdominal distension and abdominal fluid with a high protein and white blood cell content.

Most cats with dry FIP present with disease symptoms limited to the abdomen and/or chest. The most common clinical signs of dry FIP are palpable or ultrasound-identifiable masses in the kidney (Fig. 4), cecum, colon, liver, and associated lymph nodes (Fig. 5). Lesions of dry FIP usually spare the thoracic cavity and rarely occur in the skin, nasal passages, pericardium, and testes as part of a wider systemic disease.

Neurological and ocular disease are the sole or secondary features of 10 % of all FIP cases and are 10 times more often associated with dry than wet FIP (Pedersen 2009). The neurological and ocular forms of FIP have been classified as forms of dry FIP, but it may be more appropriate to classify them as distinct forms of FIP resulting from the modifying effects of the blood-ocular and blood-brain barriers behind which they occur. These barriers have a strong impact on the nature of eye and central nervous system (CNS) disease and response to antiviral therapy.

Clinical signs of neurologic FIP involve both the brain and spinal cord and include posterior weakness and ataxia, generalized incoordination, seizures, mental dullness, anisocoria, and varying degrees of fecal and/or urinary incontinence (Foley et al., 1998; Dickinson et al., 2020) ( Fig. 10). Extreme intracranial pressure can lead to sudden herniation of the cerebellum and brainstem into the spinal canal and spinal shock syndrome. Prodromal symptoms include compulsive wall or floor licking, litter eating, involuntary muscle twitching, and reluctance or inability to jump to high places. Eye involvement may precede or accompany neurological disease. Neurological FIP is a common phenomenon with antiviral therapy, either occurring during treatment of non-CNS forms of FIP or as a manifestation of disease relapse after treatment cessation (Pedersen et al., 2018, 2019; Dickinson et al., 2020).

Figure 10. A young cat with dry FIP and neurological impairment. The cat is lethargic, emaciated and with poor fur. The fur in the perineal area is wet and stained from urinary incontinence.
Figure 11. This cat's right iris staining was the first sign of FIP-associated uveitis. There is a slight haze in the anterior chamber, and there are fibrin deposits rich in red blood cells on the inside of the cornea. The pupils are also unequal (anisocoria).
Figure 12. A young cat with ocular FIP presenting as anterior uveitis in the right eye with secondary glaucoma causing globe enlargement. The iris has changed color due to inflammation, the vessels at the base of the iris are congested, and there is turbidity of aqueous humor and inflammatory products on the back of the cornea. Intraocular pressure is usually low in uncomplicated uveitis but elevated in cats with glaucoma.
Figure 13. This young cat had anterior uveitis, but her FIP therapy with GS-441524 was delayed, allowing glaucoma to develop in both eyes. Treatment cleared the underlying uveitis and greatly improved external health, but secondary glaucoma and blindness persisted.

Eye involvement is usually obvious and is confirmed by ophthalmoscopic examination of the anterior and posterior chambers. Ocular FIP affects the iris, ciliary bodies, retina, and optic disc to varying degrees (Peiffer and Wilcock, 1991; Ziółkowska et al., 2017; Andrew, 2000). The earliest symptom is often a unilateral change in the color of the iris (Fig. 11). The anterior chamber may appear cloudy and may show high protein levels and water turbidity on refraction. Inflammatory products in the form of activated macrophages, red blood cells, fibrin markers and small blood clots are washed into the anterior chamber. This material often adheres to the back of the cornea as keratic precipitates (Fig. 12). The disease can also affect the retina in tapetal and non-tapetal areas and lead to retinal detachment. Intraocular pressure is usually low, except in cases complicated by involvement of the ciliary body and glaucoma (Fig. 12, 13).

FIP Diagnosis

Signaling, environmental history, clinical signs, and physical examination findings often point to FIP (Tasker, 2018). A thorough physical examination should include body weight and temperature, coat and body condition, manual palpation of the abdomen and abdominal organs, gross assessment of cardiac and pulmonary function, and a cursory examination of the eyes and neurological system. Strong suspicion of an effusion in the abdominal or thoracic cavity may warrant confirmatory aspiration and even in-house fluid analysis as part of the initial examination.

Abnormalities in the complete blood count (CBC) and basic serum biochemical panel are important factors in the diagnosis of FIP (Tasker, 2018; Felten and Hartmann, 2019) and monitoring of antiviral therapy (Pedersen et al., 2018, 2019; Jones et al., 2021). ; Krentz et al., 2021) (Fig. 14). Total leukocyte counts are most likely high in cats with wet FIP, but low counts can occur with severe inflammation. A high leukocyte count is often associated with neutrophilia, lymphopenia, and eosinopenia. Mild to moderate non-regenerative anemia is also frequently seen in both wet and dry FIP. Total protein is usually elevated due to elevated globulin levels, while albumin values tend to be low (Fig. 14). This results in an A:G ratio that is often lower than 0.5-0.6 and is considered one of the most consistent indicators of FIP. However, a low A:G ratio can occur in situations where both albumin and globulin are within the reference range or in other diseases. Therefore, the A:G ratio should not be the only FIP indicator and should always be evaluated in the context of other FIP indicators (Tasker, 2018; Felten and Hartmann, 2019). Serum protein values obtained from most serum chemistry panels are usually adequate. Serum protein electrophoresis can provide additional information, especially if protein values from serum chemistry are questionable (Stranieri et al., 2017).

Figure 14. Complete blood count (CBC) (a) of a young cat with acute wet abdominal FIP. Although the leukocyte count was not elevated, relative but not absolute neutrophilia, relative and absolute lymphopenia, relative and absolute eosinopenia, and unresponsive anemia were noted, indicated by low red blood cells, hematocrit, and hemoglobin with a normal reticulocyte count.
Figure 14. Serum biochemical examination (b) of a young cat with acute wet abdominal FIP. Relevant values in the serum chemistry panel were elevated total protein, low albumin, high globulin, low albumin/globulin (A:G) ratio, and elevated total and direct bilirubin. Liver enzymes were normal except for mildly elevated AST and BUN and creatinine were normal, indicating the absence of significant liver or kidney disease. Globulin values are not always given, but a reasonable estimate can be calculated by subtracting the albumin level from the total protein.

Overreliance on CBC and serum biochemistry abnormalities can lead to diagnostic uncertainty when absent, despite the fact that no test value is consistently abnormal in all cases of FIP (Tasker, 2018)1. The biggest differences are between the clinical form of the disease, with leukocytosis and lymphopenia being more common in cats with wet than with dry FIP (Riemer et al., 2016). Hyperbilirubinemia is common in cats with FIP, but especially in cats with wet FIP (Tasker, 2018). The author also found that many cats with primary neurological FIP show minor or no blood abnormalities. Blood test values for FIP also vary from study to study (Tasker, 2018).

A complete analysis of the effusion is important to diagnose wet FIP and to rule out other potential causes of fluid accumulation (Dempsey and Ewing, 2011). It includes color (clear or yellow), viscosity (thin or viscous), presence of precipitates, ability to form a partial clot on standing, protein content, leukocyte count, and differential. The nature of the fluid may vary depending on the duration of the disease and its severity. Effusions in cats with more severe disease usually have protein values close to serum values, are more viscous, contain more leukocytes, are more yellow in color, and have a greater ability to form partial clots on standing. Chronic effusions tend to be less inflammatory in nature, with lower protein and leukocyte counts, less viscous and clearer. These values can be determined on the spot in most clinics. The clotting factor is determined by comparing the fluid collected in the serum and in the anticoagulant tubes after standing. Color and viscosity can be approximated and protein levels can be estimated using a handheld refractometer to determine total solids. Cells are pelleted from the fluid and analyzed on a fast-stained slide using light microscopy, and the leukocyte count and differential are estimated. Cells include nonseptic neutrophils, small and medium-sized mononuclear cells, and large vacuolated macrophages (Fig. 15). It is important to note that effusions can occur in a variety of conditions, such as heart failure, cancer, hypoproteinemia, and bacterial infections. Effusions in these other diseases usually have different identifying features.

Figure 15. Stained smear of peritoneal cells centrifuged from the abdominal fluid of a cat with wet FIP and examined on a fast-stained slide by light microscopy. The predominant cells are large strongly vacuolated macrophages, smaller differentiated activated monocytes and neutrophils. The greatest concentration of viral particles is in the intracytoplasmic vacuoles of macrophages (arrows).
Figure 16. Positive result of the Rivalt test. A small sample of abdominal or thoracic fluid is carefully dropped into a small beaker filled with diluted acetic acid (8 ml of distilled water and 1 drop of concentrated acetic acid). Inflammatory proteins almost immediately precipitate and sink to the bottom (positive). Less inflammatory fluids will form diffuse precipitates (questionable) or diffuse freely in solution (negative).

A positive Rivalt test on abdominal or chest fluid is often used to diagnose FIP as a cause of effusion, and a negative test tends to rule it out (Fischer et al., 2010) (Fig. 16). However, the test may be positive in inflammatory effusions of another cause and negative in some cats with FIP. Therefore, Rivalt's test is most helpful in combination with other clinical findings of FIP and should not replace a thorough fluid analysis (Felten and Hartmann, 2019).

Serum total and direct bilirubin levels are often elevated, especially in cats with wet FIP (Fig. 14), and may be associated with jaundice and bilirubinuria. Hyperbilirubinemia in FIP is not caused by liver disease (Tasker, 2018), but rather by vasculitis, microhemorrhage, hemolysis, and destruction of damaged red blood cells by macrophages locally and in the liver. The released hemoglobin is finally metabolized to bilirubin, which is then conjugated in the hepatocytes and excreted in the urine. Glucuronidation is essential for bilirubin excretion, and genetic disorders affecting glucuronidation in humans prevent its excretion (Kalakonda et al., 2021). Cats as a species are deficient in the enzymes required for glucuronidation, making it difficult to excrete substances such as bilirubin (Court and Greenblatt 2000).

Although FIP can affect the kidneys and liver, it is not severe enough to cause significant loss of kidney or liver function. However, serum tests for blood urea nitrogen (BUN) and creatinine as indicators of kidney disease and alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma glutamyltransferase (GGT) as indicators of liver disease are often mildly elevated in cats with FIP, especially with a more acute and serious disease (Fig. 14). Therefore, slightly abnormal test values should not be interpreted excessively if other clinical signs of liver or kidney disease are not present, while their significant increase should point to the possibility of concurrent and possibly predisposing diseases of these organs.

Serum can also be tested for other markers of systemic inflammation, such as increased levels of alpha-1-acid glycoprotein (AGP) (Paltrinieri et al., 2007) and feline serum amyloid A (fSAA) (Yuki et al., 2020). They may also prove useful in monitoring response to antiviral therapy (Krentz et al., 2021).

Radiography can be helpful in identifying chest and abdominal effusions. Abdominal ultrasound can reveal a smaller amount of effusion, identify enlarged mesenteric and ileo-cecal-colic lymph nodes, thickening of the colonic wall and lesions in organs such as the kidneys, liver and spleen (Lewis and O'Brien 2010). It may also be useful in examining the chest for lesions and assisting with needle aspiration or biopsy.

Antibody titers against FCoV have decreased since the first report nearly 50 years ago (Pedersen 1976b). The reference antibody test uses indirect fluorescent antibody staining (IFA) IFA titers ≥ 1:3200 in FIP cats are higher than most FECV-exposed cats (1:25–1:400). Newer tests often use ELISA procedures for rapid in-house or laboratory testing, but are qualitative rather than quantitative. IFA antibody titers decrease during successful antiviral treatment in many cats, but remain high in others (Dickinson et al., 2020; Krentz et al., 2021). Sequential titers can show a gradual increase in titers during the development of FIP (Pedersen et al., 1977), but previous serum samples are rarely available for comparison. Like most tests, FCoV antibody levels should not be used as the sole criterion to diagnose or rule out FIP (Felten and Hartmann, 2019) or to assess treatment success (Krentz et al., 2021).

Reverse transcriptase polymerase chain reaction (RT-PCR) is the primary means of identifying FCoV RNA in inflammatory effusions, fluids, or affected tissues (Felten and Hartmann, 2019). Accessory gene 7b RNA is present at the highest levels in tissues, fluids or exudates infected with FECV or FIPV, making it the most sensitive target for detecting low levels of virus (Gut et al., 1999). RT-PCR for FIPV S gene mutations is often used in samples that are positive for 7b RNA to be specific for FIPV (Felten et al., 2017). Other studies suggest that RT-PCR assays for FIPV-specific S gene mutations have similar specificity for FIP, but at the cost of a significant loss of sensitivity (Barker et al., 2017). A decrease in sensitivity is associated with an increase in the number of false negative results. False-negative RT-PCR tests also occur in samples that do not contain sufficient numbers of infected macrophages or in cats with very low levels of virus. False-negative results are especially common when testing whole blood.

Immunohistochemistry (IHC) detects feline coronavirus nucleocapsid protein in formalin-fixed tissues with high sensitivity and specificity, but is not as popular as RT-PCR (Litster et al., 2013; Ziółkowska et al., 2019). Specimens for IHC must contain intact infected macrophages (Fig. 17), which requires careful separation of cells from effusions and mounting them on slides, or formalin-fixed, paraffin-embedded diseased tissues that show lesions compatible with FIP. The coronavirus antigen in macrophages within a typical FIP lesion or fluid is seen only in FIP, giving IHC a high level of specificity.

Figure 17. Histological section from the thickened colon of a cat with the intestinal form of FIP. The thickened wall contained foci of macrophages (square area), which stained positive (brown-red) for FIPV nucleocapsid protein by immunoperoxidase.

A thorough ophthalmological examination is necessary to diagnose the characteristic changes of FIP (Pfeiffer and Wilcock 1991; Andrew, 2000). A sample of aqueous humor from the anterior chamber of an inflamed eye may also be useful for cytology, PCR and IHC.

Neurological FIP is often diagnosed using contrast-enhanced magnetic resonance imaging (MRI) and is often associated with cerebrospinal fluid (CSF) analysis (Crawford et al., 2017; Tasker, 2018; Dickinson et al., 2020). However, these are expensive procedures that are not always available and carry a certain risk for the cat. MRI lesions include obstructive hydrocephalus, syringomyelia, and herniation of the foramen magnum with contrast enhancement of the meninges of the brain and spinal cord and ependyma of the third ventricle, mesencephalic aqueduct, and brainstem. CSF shows an increased number of proteins and cells (neutrophils, lymphocytes, monocytes/macrophages) and, if present, can be reliable material for PCR or IHC examination.

Neurologic and/or ocular forms of FIP are often confused with systemic feline toxoplasmosis, and many cats with FIP are empirically treated for toxoplasmosis before a diagnosis of FIP is made. Fortunately, the availability of effective treatment for FIP has curtailed this practice. Systemic toxoplasmosis is much less prevalent than FIP, and fewer than 1 % cats with FIP were serologically positive in one field study.1 Therefore, testing or treatment for toxoplasmosis should only be considered once FIP has been adequately diagnosed.

Antiviral treatment as a diagnostic tool

Figure 18. A cat with FIP at the start of treatment with GS-441524 (a) and after 1 week (b). The answer is quick, the fever will disappear within 24-48 hours and the general state of health will improve significantly within 1-2 weeks. This type of response is often used to confirm a diagnosis of FIP.

Situations commonly occur where clinical findings point to FIP but doubts remain. Then there is a choice of performing several diagnostic tests, which may not lead to a more definitive diagnosis. An alternative diagnostic approach is treatment with a suitable antiviral for 1-2 weeks in the correct dose for the suspected form of FIP.2 Treatment often produces clinical improvement in as little as 24-48 hours and this rapidly progresses over the next 2 weeks and the total duration of treatment (Fig. 18). No response to test treatment and/or deterioration in health would indicate the need for further investigation of the cause(s) of ill health.

FIP Treatment

Before 2017, there was no cure for FIP, and treatment was mainly aimed at alleviating the symptoms of the disease (Izes et al., 2020). Such supportive treatment was aimed at maintaining good nutrition, controlling inflammation (corticosteroids), changing immune responses (interferons, cyclophosphamide, chlorambucil) and inhibiting key cytokine responses (pentoxifylline and other TNF-alpha inhibitors). Nutritional supplements that were supposed to help specific organ functions were also commonly used, such as one (Polyprenyl Immunostimulant) that was supposed to improve immunity and prolong survival in cats with dry but not wet FIP (Legendre et al., 2017). The effect of good supportive care on survival could not be determined because most cats were euthanized after diagnosis or within days or weeks. The survival rate for even the mildest forms of dry FIP and the most permanent treatment in one study was only 13 % at 200 days and 6 % at 300 days (Legendre et al., 2017).

Many commercially available drugs and compounds inhibit FIPV infection or replication in vitro, some of which are drugs known to inhibit specific HIV or hepatitis C proteins, while others work by inhibiting normal cellular processes that the virus usurps for its own life cycle (Hsieh et al., 2010; Izes et al., 2020; Delaplace et al., 2021). These various drugs and agents include cyclosporine and related immunophilins, several nucleoside and protease inhibitors, vioporin inhibitors, pyridine N-oxide derivatives, chloroquine and related compounds, ivermectin, several plant lectins, ubiquitin inhibitors, itraconazole, and several antibiotics. However, the concentrations required to inhibit viral replication in vitro often approach toxic values for cells. It has also been difficult to transfer favorable in vitro conclusions to animals, and studies in sick cats have rarely followed. Ribavarin inhibits FIPV replication in vitro, but was not effective as a treatment for experimental FIP (Weiss et al., 1993). The efficacy of chloroquine was tested in laboratory cats infected with FIPV, but clinical outcomes in treated cats were only slightly better than untreated ones and hepatotoxicity was demonstrated (Takano et al., 2013). A 3-month-old kitten with chest wet FIP treated with itraconazole and prednisolone developed neurological FIP and was euthanized after 38 days of treatment (Kameshima et al., 2020). Mefloquine also inhibited FIPV replication at low concentrations in cultured feline cells without cytotoxic effects, and preliminary pharmacokinetic studies in cats appeared favorable (Yu et al., 2020), but evidence of its safety and efficacy in clinical trials in cats with FIP has yet to be established. published.

A breakthrough in the treatment of FIP occurred in 2016-2019 when antiviral drugs were reported that target specific FIPV proteins essential for replication. The first of these drugs was GC376, a major protease inhibitor (Mpro ) FIPV (Kim et al., 2016; Pedersen et al., 2018). Protease inhibitors prevent the formation of individual viral proteins by inhibiting their cleavage from polyprotein precursors. GC376 was able to cure all experimentally infected cats and 7 of 21 cats with naturally occurring wet and dry FIP, but was less effective for cats with ocular or neurological signs (Pedersen et al., 2018). The second of these drugs was GS-441514, the active part of the prodrug remdesivir (Gilead Sciences; Murphy et al., 2018; Pedersen et al., 2019). GS-441524 is an adenosine nucleoside analog that blocks FIPV replication by inserting a nonsense adenosine into the developing viral RNA. GS-441524 was also able to cure all experimentally infected cats (Murphy et al., 2018) and 25/31 cats with naturally occurring wet and dry FIP (Pedersen et al., 2019). It has also been shown to be effective at higher doses in several cats with ocular and neurological FIP (Pedersen et al., 2019) and is now the drug of first choice for cats with neurological FIP (Dickinson et al., 2020). GS-441524 has cured thousands of FIP cats from around the world over the past three years, with an overall cure rate of just over 90 % (Jones et al., 2021).1

Although the ability of GC376 and GS-441524 to treat cats has been known for several years, neither is currently legally available in most countries. The rights to GC376 have been purchased by Anivive, but it has not yet been launched.3 Potential conflicts with the development of remdesivir for the treatment of COVID-19 in humans led Gilead Sciences to withhold rights to GS-441524 for animal use, prompting the creation of an unapproved source for GS-441524 from China (Jones et al, 2021).1,2,4 Remdesivir is rapidly metabolized in the body to GS-441524 and has been approved for the treatment of FIP in some countries.2 GS-441524 can also be administered orally in higher doses and is currently commonly used in practice (Krentz et al., 2021).1

The efficacy of drugs such as GC376 and GS-441524 on FIP cats, the use of which preceded the COVID-19 pandemic, has been recognized by researchers investigating related SARS-CoV 2 inhibitors (Yan et al., 2020; Vuong et al., 2021). Remdesivir, an injectable drug called glaucoma (Gilead), has been used worldwide to reduce mortality from COVID-19 (Beigel et al., 2020). GC373, the active form of the prodrug GC376, has undergone simple modifications to increase efficacy and oral bioavailability (Vuong et al., 2021). The GC373-related drug, nirmatrelvir, has been successfully tested against early COVID-19 infections and has been approved for the treatment of early COVID-19 and marketed as paxlovid (Pfizer). Paxlovid consists of two medicines, nirmatrevir and the HIV protease inhibitor ritonavir. Ritonavir is not a significant inhibitor of SARS-CoV 2, but is reported to prolong the half-life of Mprowhen used in combination (Vuong et al., 2020). Nirmatrelvir and paxlovid have not been tested in cats with FIP at present, but based on experience with the closely related drug GC376, oral treatment of some forms of FIP may be important in the future.

Two other nucleoside analogs, EIDD-1931 and EIDD-2801 (Painter et al., 2021), have been investigated for the treatment of multiple RNA virus infections in humans and animals. EIDD-1931 is the experimental designation for beta-D-N4-hydroxycytidine, a compound widely studied since the 1970's. Beta-D-N4-hydroxycytidine is metabolized to a ribonucleoside analog, which is incorporated into RNA instead of cytidine and leads to fatal mutations in the viral RNA strand. The compound is an inhibitor of a wide variety of human and animal RNA viruses, including all known coronaviruses. EIDD-1931 was modified to increase oral absorption and was termed EIDD-2801 (molnupiravir) (Painter et al., 2021). Molnupiravir is deesterified in the body to its active ingredient, beta-D-N4-hydroxycytidine. Therefore, EIDD-1931 and molnupiravir are analogous to GS-441524 and remdesivir. Molnupiravir is marketed for the home treatment of primary COVID-19 under the names Lagevrio (Merck, USA) or Molnulup (Lupine, India).

Both EIDD-1931 and EIDD-2801 have been shown to be effective in inhibiting FIPV in tissue culture (Cook et al., 2021), and EIDD-2801 is currently used to treat some cases of FIP in the field.5,7 The effective concentration of 50 % (EC50) for EIDD-1931 against FIPV is 0.09 µM, EIDD-2801 0.4 µM and GS-441524 0.66 µM (Cook et al., 2021). The percentage cytotoxicity at 100 μM for these compounds is 2.8, 3.8 and 0.0. Thus, EIDD-1931 and -2801 are slightly more inhibitory to viruses, but more cytotoxic than GS-441524. Resistance to GS-441524 has been reported in some cases of FIP (Pedersen et al., 2019) and to remdesivir in patients with COVID-19 (Painter et al., 2021), but these isolates remain sensitive to molnupiravir (Sheahan et al., 2020). This may prove useful in combating resistance to GS-441524 in cats and humans and in developing multidrug therapy to prevent the development of resistance.

What will full approval of medicines like molnupiravir and paxlovid mean for cats? Full human approval should allow veterinarians in most countries to legally procure medicinal products authorized for human consumption for direct use in animals, provided that the guidelines for use in non-food producing animals are followed.6 This requires a reformulation of a medicine made for humans and purchased at a price for humans. Hopefully, antivirals similar or identical to those approved for humans will be licensed exclusively for animals and sold at a much lower price, but this is likely to take years.

Commercial and policy issues that limit the current use of antivirals such as GS-441524 in animal diseases such as FIP are for current cat owners and feline support groups who have already bypassed the current drug approval system and its emphasis on overriding human needs, irrelevant (Jones et al., 2021; Krentz et al., 2021). Advocates of FIP treatment are currently found around the world and often associate under the expanded FIP Warrior brand. Members of these groups often act as intermediaries between owners, veterinarians and antiviral suppliers and often provide advice to those who are unable to obtain veterinary treatment assistance. Some of these groups, such as FIP Warriors Czech Republic / Slovakia7, have placed their experience with FIP treatment on the Internet, where they provide much-needed information about current antiviral treatment.

Current situation of FIP treatment

The current drug of choice for the treatment of FIP is the adenosine nucleoside analog GS-441524, which was first published in the scientific literature under experimental conditions (Murphy et al., 2018) and later against naturally occurring disease (Pedersen et al., 2019). Although initial experimental and field studies of GS-441524 were conducted in collaboration between researchers at Gilead Sciences and the University of California, Davis, the relationship between Remdesivir and GS-441524 and the onset of the COVID-19 pandemic in 2019 led Gilead Sciences to eventually did not grant rights to use GS-441524 to animals on the grounds that it could interfere with the development of Remdesivir for human use.4 Objections to this decision have been raised directly by the company and in several internet forums.4 Subsequent pressure from cat owners, cat rescue groups and cat lovers, along with opportunistic Chinese drug manufacturers, quickly created an alternative unapproved source of GS-441524, its market and treatment network.4  This network has largely bypassed veterinarians, most of whom have decided to wait for the drug to be legalized (Jones et al., 2021). The result of this relationship was an almost seamless transition of FIP treatment with GS-441524 from the laboratory to a rapidly expanding worldwide network of groups, under the umbrella of FIP Warriors (Jones et al., 2021).4,7 

The sale and use of GS-441524 in practice for the treatment of FIP began almost immediately with the first publication of the results of field trials (Pedersen et al., 2019) (Fig. 19).

Figure 19.  Graph of the monthly development of treatment of cats from the Czech Republic and Slovakia since August 2019. This graph comes from the FIP Warrior CZ / SK website.1 These figures reflect the experiences of other FIP Warrior groups around the world. Since 2019, when the first field study GS-441524 was published (Pedersen et al. 2019), thousands of cats have been successfully treated for FIP worldwide. The winter peaks of the disease reflect a late spring and summer increase in the number of kittens born and a high incidence of FIP, which usually begins at 3 to 6 months of age (Fig. 6). This chart is from the FIP Warrior CZ / SK website.1
Figure 20. The main participants in the GS-441524 treatment. This chart is from the FIP Warriors CZ / SK website.1

The fact that GS-441524 is not legally approved for use in animals has prevented many veterinarians from recognizing or participating in this treatment. Only 25 % cats in the CZ / SK treated group received veterinary support during treatment (Fig. 20), although more veterinarians may have been involved in the diagnosis of the disease. Interestingly, this number was higher than the 8.7 % treated cats in the United States that received veterinary care (Jones et al., 2021). However, participants in CZ / SK studies and similar groups around the world are not without medical experience, as many of them are engaged in temporary care / rescue and have had considerable direct and indirect veterinary experience with cat diseases and their treatment and castration programs.

From the first laboratory studies and research of Chinese manufacturers, it was known that GS-441524 can be absorbed orally, although with less efficiency (Kim et al. 2016).9 The first sellers of GS-441524 further investigated this fact and found that effective blood levels could be achieved by increasing the amount administered orally compared to injection.8 Supplements have often been added to GS-441524 oral capsules or tablets, claiming that they increase absorption or have additive therapeutic benefits (Krentz et al., 2011). Most major retailers of GS-441524 now offer oral versions, and oral therapy is becoming increasingly popular either as a single treatment or in combination with GS-441524 (Figure 21). The success of GS-441524 oral therapy did not differ significantly from GS-441524 injection therapy (Figure 22).

Figure 21. Comparison of the use of oral (tablets or capsules) and injectable (subcutaneous) forms of GS-441524 for the treatment of FIP in cats from the Czech Republic and Slovakia. This chart is from the FIP Warriors CZ / SK website.1
Figure 22. There is no significant difference in treatment success with oral administration of GS-441524 compared to subcutaneously administered GS, but the actual amount (mg) of drug administered orally in each dose is up to twice the amount contained in the same dose of GS injection. This chart is from the FIP Warriors CZ / SK.1 website

The recommended dosing schedule for GS-441524 based on published data from field studies (Pedersen et al., 2019) was 4 mg / kg, subcutaneous (SC), daily (q24h), ie 4 mg / kg, SC, q24h. This recommended starting dose for cats with wet or dry FIP without ocular or neurological symptoms tended to increase to 6 mg / kg SC q24h over time (Fig. 23). 8 mg / kg SC q24h is the current recommended dose for cats with ocular symptoms and 10 or 12 mg / kg SC q24h for cats with neurological symptoms.

Figure 23. Daily dose of GS-441524, which was used to treat FIP in cats from the Czech Republic and Slovakia. The usual starting dose was 6 mg / day, with some cats requiring higher doses based on response to treatment, form of the disease and recurrence after treatment appeared to be successful. GS-441524 oral formulations are usually labeled to match the dosage used for the injectable drug, but contain up to twice the labeled amount. This chart is from the FIP Warrior CZ / SK website.1

The optimal duration of treatment, as determined in the initial clinical study, is 84 days (Pedersen et al., 2019). In some cases of acute wet FIP in younger cats, healing has been achieved in 6-8 weeks, but some cats need more than 84 days. As shown in Figure 24.72 % cats were treated for 81-90 days, 19 % longer and only 9 % were treated shorter. Unfortunately, there is no simple and accurate test to determine the moment of cure, and the decision to stop treatment is based on a complete return to health and normal blood test values. Cats treated for much longer than 100 days were usually those requiring a GS dose higher than 12 mg / kg per day by injection or equivalent oral dose, cats that relapsed during the 12-week post-treatment observation period, cats with neurological disease or cats that have become resistant to GS-441524.   

Figure 24. Duration of treatment with GS-141524 in 352 cats successfully treated for all forms of FIP. This chart is from the FIP Warriors CZ / SK website.1
Figure 25. Initial treatment was successful in 88.1 % cats and 6.2 % cats died or were sacrificed either due to insufficient response to treatment, financial reasons or treatment side effects. Another 5.7 % cats relapsed after initial treatment and approximately the same number of cats either recovered or died after further treatment. This chart is from the FIP Warriors CZ / SK.1 website

The treatment success rate for all forms of FIP in cats from the Czech Republic and Slovakia is 88.1 % in the first treatment, but when cats that relapsed after the first treatment and recovered after the second treatment (3.1 %) were included, the overall success rate was more as 91 % (Fig. 25). This cure rate is identical to the cure rate of other groups of FIP fighters (Jones et al., 2021). Treatment success did not differ between cats with wet or dry FIP and without ocular or neurological impairment (Fig. 26). However, the cure rate in cats with ocular and neurological impairments was lower, at 80 % compared to 92 % in all other forms of FIP (Fig. 26).

Figure 26.  Cure rate of cats with wet or dry FIP without ocular or neurological symptoms and cats with ocular or neurological disease as the main feature of their disease. This chart is from the FIP Warriors CZ / SK website.1
Figure 27.  The condition of the cats one year after the successful completion of treatment with GS-441524. This chart is from the FIP Warriors CZ / SK website.1

Cats that have been successfully treated for FIP have been followed for 4 to 5 years, including cases reported in the first field studies. There have been no recurrences or recurrent cases of FIP in this group of first field trials. Data on annual survival are available from a much larger population of the CZ / SK study, which shows that 90.5 % cats are still healthy one year after the end of treatment (Fig. 27). Only 1.3 % of these cats died from causes other than FIP and 8.2 % cohort is currently in an unknown medical condition. The low proportion of cats that died of unknown causes within a year of treatment and their positive response to treatment suggest that FIP has been diagnosed correctly.

EIDD-2801 (molnupiravir) is currently being used in the field for the main treatment and for the treatment of GS-441524-resistant cats.5,7,9 EIDD-1931, the active form of EIDD-2081, needs to be further researched because it is no longer covered by patent protection and is thus easily approved for use in animals if it is found to be truly safe and effective.5 Nirmatrelvir, an oral form of GC373 and a closely related GC376, still needs to be studied for the treatment of FIP.

Acknowledgement

I am indebted to Ladislav Mihok and his collaborator from "FIP Warriors Czech Republic / Slovakia" for allowing me to share data from their website. This website contains the most important, comprehensive and organized collection of data on FIP antiviral treatment today. The website also contains useful information and advice on starting, conducting and monitoring current treatment. The collection of cats and their data is continuously and regularly updated and at the time of writing this article included more than 600 cats with FIP.

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Footnotes

  1. FIP Treatment - Czechia / Slovakia. Basic data, 2022. https://docs.google.com/spreadsheets/d/e/2PACX-1vRAnj_FV_fteWIW1HXsROLuJ7YY1-i_Sf81BCmM9JT9LbCT2mcnwD1rL9IBsLCTB1U59CcnalOGjFqq/pubhtml?gid=1340189982&single=true  (Accessed 4 April2022).
  2. Hughes D, Howard G, Malik R, 2021. Treatment of FIP in cats with Remdesivir. Clinical review, 2021. The Veterinarian. https://www.turramurravet.com.au/wp-content/uploads/2021/07/FIP-Article_The-Veterinarian.pdf (Accessed 5 March 2022).
  3. Anonymous. Thanks to Cats, One Promising Coronavirus Treatment is Already in Development-The GC376 story. 2021,  https://anivive.com/coronavirus (Accessed 4 April 2022)
  4. Zhang S (2020) A Much-Hyped COVID-19 Drug Is Almost Identical to a Black-Market Cat Cure. The Atlantic. https://www.theatlantic.com/science/archive/2020/05/remdesivir-cats/611341/ (Accessed 4 April 2022).
  5. Pedersen NC, 2021. The long history of Beta-d-N4-hydroxycytidine and its modern application to the treatment of Covid-19 in people and FIP in cats. https://ccah.vetmed.ucdavis.edu/sites/g/files/dgvnsk4586/files/inline-files/Molnuparivir%20as%20a%20third%20antiviral%20drug%20for%20treatment%20of%20FIP%20v13_1.pdf  (Accessed 4 April 2022).
  6. American Veterinary Medical Association. Guidelines for veterinary prescription drugs. 2022 https://www.avma.org/resources-tools/avma-policies/guidelines-veterinary-prescription-drugs (Accessed 4 April 2022).
  7. FIP Warriors CZ / SK. https://www.fipwarriors.eu/en/ (accessed 15 April 2022).
  8. Pedersen NC, Jacque N, 2021. Treatment with oral formulations of GS-441524. https://sockfip.org/2021-treatment-with-oral-formulations-of-gs-441524/  (Accessed 11 December 2021).
  9. Pedersen NC, Jacque N. 2021. Alternative treatments for cats with FIP and natural or acquired resistance to GS-441524. https://ccah.vetmed.ucdavis.edu/sites/g/files/dgvnsk4586/files/inline-files/Approaches-to-drug-resistance-in-cats-treated-with-GS-441524-for-FIP-v3.pdf (Accessed 16 April 2022).
Read "History of feline infectious peritonitis 1963-2022 - from the first mention to successful treatment"

The history of Save Our Cats and Kittens over four decades and where we go from here

Niels C. Pedersen, DVM, PhD
December 2021
Original article: The history of Save Our Cats and Kittens over four decades and where we go from here

Niels C. Pedersen

Those who have followed my career know that I have many interests in addition to infectious diseases of cats. However, I am best known for feline medicine and diseases that plague multi-cat environments. This interest in infectious diseases started in 1965 as a second-year veterinary student but evolved after I joined the faculty of the UC Davis School of Veterinary Medicine in 1972. My first appointment was to help win President Nixon’s war on cancer. This war emphasized potential viral causes of cancer, in particular retroviruses and human leukemias. This was my entry back into the world of feline leukemia virus (FeLV). Of course, my interest was more on FeLV infection as it applied to cats than any application to human cancers. It became rapidly apparent that FeLV infection was a serious panzootic (pandemic) of cats that had unknowingly spread from feral to pet cats in the preceding decades and would account for one-third of mortality in cats in the 1960s and 70s. Cat lovers quickly mobilized once the virus was discovered and started raising money to support FeLV research. The original SOCK was created by a group of amazing cat lovers led by Vince, Connie and Dorothy Campanile and friends. SOCK it to leukemia became the rallying cry of the group and I was privileged to join forces with them from their beginning to end. Thereafter, donations from cat lovers and not federal research funds provided the bulk of our research into FeLV infection at UC Davis. This research led to an understanding of how FeLV became a pandemic of pet cats, how it caused a wide range of diseases, and how it could be controlled. FeLV infection of pet cats was brought under control in the 1970’s and 1980’s through rapid diagnostic tests and vaccination. The conquest of FeLV infection was one of the highlights of veterinary research of the period, and perhaps one of the most important contributions of modern feline medicine in the 20th century. SOCK it to leukemia had ultimately worked itself out of existence with over $1M dollars raised towards the ultimate conquest of FeLV infection. FeLV infection still exists in nature, where it remains a problem for a small number of younger cats coming into foster/rescues and shelters from the field.

During this same period, another highly fatal disease was rearing its head. Feline infectious peritonitis (FIP) was first reported in 1963 by veterinarians from the Angell Memorial Animal Hospital in Boston. It was later found to be closely linked to FeLV infection and the hope was that it would largely disappear with control of FeLV. This did not prove true and FIP soon replaced FeLV as a major infectious cause of deaths in cats up to this time. As a result, the torch was passed from SOCK it to leukemia to SOCK it to FIP. This was also a natural progression for my research. FIP was my first “love” from the time I helped research the first cases of FIP at UC Davis as a veterinary student in 1965. My interest in FIP only took second stage for a brief period in the 1980s with my work on HIV/AIDS and subsequent discovery of feline immunodeficiency virus (FIV). FIP has been my major research interest for the last three decades.

I am pleased to have had the support of SOCK FIP over these later years. One of our greatest discoveries at UC Davis was how an innocuous and ubiquitous feline enteric coronavirus (FECV) ends up causing such a highly fatal disease as FIP. Our theory that the virus of FIP arose as an internal mutation of FECV was first met with great skepticism but is now universally accepted. The internal mutation theory has led to a much better understanding of the conditions under which FIP occurs and how the FIP virus causes disease. Unfortunately, no one, including us, was able to find a successful vaccine for FIP. This failure led to my interest in curing rather than preventing FIP using modern antiviral drugs, which I became familiar with during the HIV/AIDS pandemic. The capstone of my almost 50-year experience with FIP was the discovery of two antiviral drugs that could cure FIP. Thousands of cats from round the world have been cured of FIP with antiviral drugs researched at UC Davis over the last 3 years. Our discoveries at UC Davis could have been impossible without the significant long-term financial and moral support of SOCK FIP and cat owners who have donated money.

The discovery of a cure for FIP has once again brought SOCK FIP to a logical ending, just as the conquest of FeLV infection ended the need for the original SOCK. Although I am retired, I continue to work with cat owners and caregivers on how to use antiviral drugs to treat FIP and will maintain my relationship with SOCK FIP as a consultant on FIP treatment and a lifelong member. Admittedly, there is still research to be done with FIP, mainly in the areas of disease prevention. Hopefully, others will take up this and other areas of FIP research. The question now is how SOCK can best improve the health of our cats and kittens. SOCK FIP is in the process of evaluating a broader mission than just FIP. This mission may or may not involve fund raising for research and could be more informational. We welcome suggestions on how the long history of SOCK’s can be used to improve the health of our cats and kittens. Read "Four Decades Save Our Cats and Kittens and What's Next"

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

ALVES, AE et al. Leucogram and serum acute phase protein concentrations in queens submitted to conventional or videolaparoscopic ovariectomy. Arquivo Brasileiro de Medicina Veterina- ria e Zootecnia, v.62, n.1, p.86-91, 2010. Available from:. Accessed: Oct. 10, 2018. doi: 10.1590 / S0102-09352010000100012.

BAUMANN, H. & GAULDIE, J. The acute phase response.
Immunol Today, v.15, n.2, p.74-80, 1994. Available from:
https://doi.org/10.1016/0167-5699(94)90137-6. Accessed: Aug. 21, 2018. doi: 10.1016 / 0167-5699 (94) 90137-6.

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"

FIP treatment with subcutaneous remdesivir followed by GS-441524 oral tablets

Richard Malik DVSc PhD FACVS FASM Center for Veterinary Education, University of Sydney
Original article: Treatment of FIP in cats with subcutaneous remdesivir followed by oral GS-441524 tablets

Translator's note: The article contains information about the real content of GS-441524 in tablets. However, this content may not correspond to the "equivalent" amount of GS-441524 in tablets from other manufacturers, where the actual content of GS-441524 is always slightly higher due to the known reduced oral bioavailability of the drug. Therefore, it is not possible to easily and unambiguously compare the recommended dosage of GS-441524 from BOVA in Australia and in our country.

Introduction

Infectious feline peritonitis (FIP) is an infectious disease, especially of young cats. It occurs when a feline enteric coronavirus that multiplies in the gut undergoes a critical mutation that changes its tissue tropism from enterocytes to macrophages. The FIP virus then circulates in the body in macrophages - this is the ultimate mechanism of the Trojan horse. This leads to disseminated infection and the development of fibrinoid necrotizing vasculitis and serositis due to the deposition of immune complexes consisting of feline antibodies and FIP viral antigens.

In general, there are two forms of FIP - effusive ("wet") FIP and non-effusive ("dry") FIP. The disease process itself can occur in the abdomen, thoracic cavity, pericardium, eyes or central nervous system. Combinations of dry and wet FIP with various tissues are not uncommon.

Until recently, the diagnosis of feline infectious peritonitis (FIP) was a death judgment for a feline patient. In recent years, however, this vision has been turned upside down as a result of the pioneering work of Professor Niels C. Pedersen and colleagues at UC Davis.

Over the last 12 months, many veterinarians in Australia have also successfully managed many cases of FIP using remdesivir and GS-441524.

Omega-interferon (Virbagen) and polyprenyl immunostimulant (PPI) were the first drugs used to treat FIP, and both had some effects in some patients. Omega interferon has been useful in cases of effusive ("wet") FIP, often combined with low-dose prednisolone according to the Ishid protocol, while PPI, pioneered by Al Legendre, has been more useful in cases of non-fusible FIP. In some cases, both drugs were used at the same time. The problem was that both forms of therapy were often expensive, especially when both drugs were used, so that although patients improved and could have transient clinical remissions during treatment, permanent clinical cures were rare. As a result, most veterinarians still considered the diagnosis of FIP a prelude to euthanasia.

That all changed a few years ago thanks to the culmination of FIP's lifelong research. Niels Pedersen. Niels is an amazing North American veterinarian of Danish descent. He grew up on a chicken farm and originally wanted to be a clinician for large animals, but with great foresight he decided on a scientific career. Shortly after graduating, he traveled to Canberry to the John Curtin School of Medical Research at ANU, where in the late 1960s he received a PhD in kidney transplant rejection immunology from Professor Bede Morris, using sheep as an experimental model to study lymphocyte kinetics.

When Niels returned to UC Davis, he focused on studying infections and immunity. Although he has contributed to a large number of topics in internal medicine and the genomics of dogs and cats, FIP has become his favorite disease due to its commonness and current complexity. His studies date from the 1980s, when he specialized in diagnostics, virology and pathogenesis, to the present, with an increasing focus on therapy.

Niels, in collaboration with colleagues from Kansas State University, has shown that a purposefully designed protease inhibitor GC-376 could prevent and cure experimentally induced FIP in laboratory cats.1,2 Field clinical trials with cats with naturally occurring disease have been disappointing, especially when cats have had an ocular form of FIP or CNS disease. He did not give up, so he switched to another drug - GS-4415243,4 - a nucleoside analogue developed by the North American pharmaceutical company Gilead. This molecule has been shown to be much more effective than GC-376 in the treatment of FIP, both in experimental infections and in spontaneous cases of FIP. Starting with pharmacokinetics and dose escalation studies using a wide range of clinical cases, Niels and colleagues found that the required dose depended on whether the patient had dry or wet FIP and whether the eye or central nervous system (CNS) was affected.5

Surprisingly, Gilead, the manufacturer who developed GS441524, has not yet shown interest in developing this molecule for the treatment of cats. To fill the gap for effective FIP therapy around the world, various laboratories in China and Eastern Europe have begun producing GS-441524 and selling it on the black market.

The wide availability of the GS-441524, often of high quality and initially very high price, provided dedicated owners with a way to save their cats with FIP. Studies by clinical pathologist Samantha Evans of Ohio State University have indicated a cure rate of approximately 80 % in the field. Until recently, the procurement of the drug was complicated and full of problems, which at some level were circumvented by various "FIP Warriors" groups on Facebook. Unfortunately for Australian cat lovers, APVMA and Vet Boards finally understood what was going on and the Border Force made it much more difficult to obtain GS-441524 and its safe import for veterinary use. Regulatory and Veterinary Committees' warnings against prosecutors were directed against veterinarians who allowed cats with FIP to be treated with black market drugs.

Ironically, the COVID 19 pandemic provided a new solution to this problem. Gilead developed remdesivir (GS-5734) as a drug for the treatment of hepatitis C, Ebola and human coronavirus disease. Remdesivir is a prodrug of GS-441524, which contains an additional chemical side chain (including a phosphate group) to improve intracellular penetration (Figure 1B). Remdesivir (as a product of Veklura) obtained a temporary marketing authorization (for two years) from TGA in July 2020 for the treatment of SARS-CoV-2 infections in human patients with COVID-19. This registration process would normally take several years, but the severity of the pandemic has accelerated this process, taking into account preliminary data from clinical trials. As remdesivir became a licensed human drug and Gilead licensed production worldwide, it meant more access to quality raw material. This circumvented the problems with the use of the drug purchased on the black market, as well as the problems of unknown purity and consistency of the product over time.

In 2020, the veterinary compounding company BOVA Australia provided reliable supplies of remdesivir in a suitable format for IV and subcutaneous application. Studies in Australia have determined that the shelf life after reconstitution exceeds 12 days and have confirmed in vitro efficacy against coronaviruses in tissue cultures. The analytical purity of the drug is regularly checked by HPLC. Over the past year, veterinarians in every Australian state have used remdesivir to treat cats with FIP. There have been a number of effusive and non-fusive cases, including some cats with ocular disorders (uveitis) and others with multifocal CNS disease. Based on treatment of approximately 500 cats treated between October 2020 and November 2021, remdesivir has been shown to be highly effective in managing FIP infections. It allows for a slightly simpler subcutaneous administration and the injection appears to be slightly less painful compared to GS-441524 and does not cause the local injection site reactions observed with GS-441524 injection. Remdesivir was originally used exclusively in Australia, although it has also been available in the UK from BOVA UK for the last 2 months.

The molecular weight of remdesivir is 603 g / mol, while the molecular weight of GS-441524 is 291 g / mol. This could suggest that treatment of cats with remdesivir requires approximately twice the dose of GS-441524, although this does not take into account the possible improvement in intracellular penetration of remdesivir into certain tissues compared to GS-441524. The proposed dose of remdesivir in human patients with COVID19 is 200 mg intravenously (IV), followed by 100 mg IV daily. For a 70 kg human patient, this represents a daily dose of 1.3 mg / kg, so using allometric scaling, a dose of 5-10 mg / kg per day was considered correct for a cat. However, our experience with the first 500 cases was that many cats eventually needed a higher dose of remdesivir for permanent cure, so we adjusted our recommended dosage upwards (see below). Remdesivir provides BOVA as a sterile 10 mg / ml solution ready for use in a 10 ml vial.

Figure 1. (A) BOVA Remdesivir reconstituted and ready for treatment. After reconstitution, the contents of the vial are stable for at least 120 days at 5 ° C - and the vial is usually consumed in 3-7 days. It is best to store the vial in the refrigerator. (B) The pathway that remdesivir travels intracellularly to activate as GS-441524.

At present, Australia and the United Kingdom are the only countries where remdesivir is readily available by prescription for veterinary use. However, veterinarians in India, New Zealand, South Africa and parts of Europe have also started using human medicine suppliers to access the medicine.

Diagnosis

Figure 2: Amazingly comprehensive and practical overview of FIP diagnostics by Severine Tasker.

A complete differential diagnosis of FIP is beyond the scope of this article, but readers are strongly encouraged to read the excellent article by Séverine Tasker in the Journal of Feline Medicine & Surgery. 6

Although FIP can occur in cats of any age, most cases occur in kittens and cats less than 3 years of age. Persistent and often high fever that does not respond to antibiotic therapy (and often NSAIDs) is a common finding, as is increased plasma total protein levels due to elevated globulin concentrations (diffuse gammopathy in serum electrophoresis). In effusive or "wet" FIP, the albumin to globulin ratio may drop to <0.45. Acute phase reactants such as serum amyloid A and α1-acid glycoprotein tend to be markedly elevated. Many cats with FIP also exhibit secondary immune-mediated hemolytic anemia, increased AST and ALT activities, and jaundice.

Diagnostic imaging is crucial for early diagnosis, which has been greatly facilitated by the introduction of digital radiology and the widespread availability of diagnostic ultrasound in small animal practice. Pleural effusion is readily recognizable from chest X-rays, while abdominal effusion is best detected by ultrasound (Figure 3), especially if high frequency probes are available. It is worth noting that in some cases, the fluid pockets may be focal and localized. Often there is some fluid around the kidney under the kidney sheath, kittens may have scrotal edema, while in rare cases the discharge is limited to the pericardial sac. But the key is - to look for (i) effusion in any body cavity, (ii) granulomas in the kidneys, liver or lungs, (iii) enlarged intra-abdominal and mesenteric lymph nodes (Figure 5) or marked thickening of the iliac-ecological area (f focal FIP ’) ( Figure 5). Chest X-rays after drainage of pleural effusion may show changes corresponding to viral pneumonia.

Figure 2: (A) Ultrasound of the abdomen showing abundant highly echogenic fluid (fibrin fibers) in cats with high protein ascites due to effusive FIP. (B) The fusion contains a viscous yellow to straw-colored liquid. (C) An X-ray of the abdomen with the appearance of cut glass indicating fluid in the abdomen.

If you see an effusion - puncture - because fluid is the best diagnostic sample.

Figure 3: Marked mesenteric lymphadenomegaly in a cat with dry FIP.

A fluid with a high protein content, often yellow to straw in color, is characteristic (Figure 3B). If you see granuloma in the organ or if the lymph nodes are clearly enlarged - do FNA (thin needle aspiration biopsy), apply a smear, use RapidDiff staining and look for neutrophils and macrophages (pyogranulomatous inflammation) without visible infectious agents (Figure 4). The two diseases most commonly confused with FIP in adult cats are lymphoma and some types of lymphocytic cholangitis (associated with high protein ascites).

Figure 4: RapidDiff stained aspirate with a thin needle from the mesenteric lymph nodes of a 4-year-old oriental cat with dry FIP. Distinctive macrophages are the key to cytological diagnosis. Photo courtesy of Trish Martin.

Of course, effusive disease is much easier to diagnose because ascitic, pericardial or pleural fluid provides a suitable sample that can be examined cytologically, by fluid analysis and immunofluorescence (IFA) for FIP antigen, or reverse transcriptase PCR to detect FIP nucleic acid. IFA is performed at VPDS, B14, University of Sydney (via Vetnostics, QML, ASAP, VetPath, Gribbles or IDEXX). However, it is usually the cheapest way to send the sample directly to the university laboratory.

Dry FIP is more problematic because it usually requires a thin-needle aspiration biopsy of pyogranulomatous lesions in the liver, kidneys, or abdominal lymph nodes. Occasionally, cases of wet FIP may show fluid samples that are negative for IFA and / or PCR testing, but the patient is still likely to have FIP, which is reflected in a favorable response to remdesivir or GS-441524 treatment.

Treatment

Since October 2020, we have been treating cats with FIP with remdesivir (IV and SCI) and more recently with GS-441524 (oral), so our protocols are constantly evolving with experience. About 500 cats have been treated so far. We try to avoid being too prescriptive in our recommendations, as we suspect that there is no one-size-fits-all protocol and that each case presents unique circumstances, including patient size, whether the cat is still "happy" and reasonably , or is depressed and dehydrated. An important factor is the emotional and financial commitment of the owner. A key feature that needs to be mentioned is that both drugs are very safe, even in sick cats and kittens.

Note that the following recommended doses are higher than those originally recommended a year ago. Although lower doses worked in many patients, we found that this was often the wrong economic consideration, as disease recurrence at the end of treatment and the development of viral resistance during treatment appear to be related to insufficient initial dosing. So we have learned to be more aggressive from the beginning, which is cheaper in the long run (ie 2nd therapy is not required)

Our greatest experience is with remdesivir. This drug is expensive and the owner has to commit to a costly treatment process that takes 3 months. For most clients, this represents an emotional and financial burden. My view is that in many cases it is better to spend money on antiviral therapy as such than on extensive diagnostics and monitoring.

Figure 5: Significant thickening of the ilico-ecological area of the Devon Rex cat with the so-called "focal FIP", a common form of non-fusive FIP. Photo courtesy of Penny Tisdall.

One of the approaches in newly diagnosed cats with severe disease is hospitalization of cats during the first 3-4 days of treatment. Patients begin treatment with remdesivir when receiving IV fluid therapy (typically 2-4 ml / kg / hr; first day Hartmann's solution or Plasmalyte followed by 0.45 % NaCl and 2.5 % dextrose containing 20 mmol KCl / l). On the 1st day of hospitalization, remdesivir is administered in a high dose intravenously (10-15 mg / kg diluted in 10 ml with saline and is given SLOWLY for 20-30 minutes or longer, manually or by infusion pump; in human patients, administration lasts 2 hours. ) to achieve an increased starting dose of drug distribution volume. This achieves fast antiviral efficacy. In cases with CNS disease, we recommend a daily IV dose of 20 mg / kg. Many cats may appear slightly depressed several hours after IV remdesivir infusion. In human patients, remdesivir may cause infusion-related reactions, including low blood pressure, nausea, vomiting, sweating or chills, but we have not observed these events in our feline patients.

The advantage of starting treatment intravenously is that dehydration, if present, is corrected and you have IV access if you need to take other medicines (eg anticonvulsants, corticosteroids). Importantly, once an IV catheter is inserted, daily injections of remdesivir do not cause any pain or discomfort. However, if the cat eats and is diagnosed in the early stages of the disease, then IV therapy is not required and the same doses can be given subcutaneously, saving a lot of money.

FIP cats treated with remdesivir typically improve significantly during the first 2-3 days. However, we found that cases of effusion, and especially those that resulted in pleural effusion prior to treatment, should be closely monitored, as the combination of the antiviral effect of remdesivir and a higher than maintenance dose of crystalloids may lead to transient worsening of pleural effusion. This requires drainage twice a day using a 19G butterfly needle (1.1 mm - cream color) and a 3-way stopcock (ideally using an ultrasonic guide to find the best place to insert the needle). These "secondary" pleural effusions can be fatal if not detected in time and appear to occur in approximately 1 in 10 cases of effusive FIPs treated with remdesivir.

Another problem that occasionally occurs at this time is the development of neurological symptoms, including seizures. Our view is that this is not the effect of the drug as such, but rather the unmasking of the subclinical CNS FIP. Such cats require careful monitoring, while the development of seizures requires the use of anticonvulsant drugs such as midazolam (0.3 mg / kg IV), alfaxane or propofol (administered IV to be effective), followed by levetiracetam (Keppra) (10- 20 mg / kg, PO every 8 hours). Phenobarbitone is a reliable anticonvulsant, but it tends to increase the metabolism of many drugs, and levetiracetam is probably safer until we better understand the pharmacokinetics and metabolism of remdesivir and GS-441524. Some doctors also administer dexamethasone or prednisolone as a single treatment to relieve CNS inflammation.

Although advocating initial IV therapy for the most severe cases of FIP, cats and kittens that are still "happy" and eating do not require IV therapy at first and may instead begin subcutaneous injections at 10-12 mg / kg / day (20 mg / kg in CNS diseases). This is, of course, much cheaper because cats or kittens do not have to be placed in an infusion pump and hospitalized in a stressful environment. For clients who have financial limits, this may be a more appropriate way to start therapy. Some skilled colleagues, such as Jim Euclid, have developed a hybrid approach where kittens receive subcutaneous fluids daily as a bolus with injected remdesivir.

The cats were then given continuous subcutaneous injections of remdesivir. It originally took 84 days, and such cases accounted for most of the cases we have dealt with so far. Recently, we have been using aggressive IV / SCI remdesivir for initial therapy, and then cats are switching to oral GS-441524 for 10 weeks of consolidation therapy.

After the initial use of lower doses, which were not successful in every patient, we now use the following treatment protocols:

  • for cats with wet FIP: 10-12 mg / kg once daily (SID) for 2 weeks
  • for cats with severe eye impairment: 15 mg / kg SID by subcutaneous injection (SCI) for 2 weeks; Cats with severe uveitis should also be given topical corticosteroids (Before Forte or Maxidex) for 2-3 days (no longer!) and atropine eye ointment.
  • for cats with neurological FIP with CNS symptoms: administer 20 mg / kg SID SCI for 2-4 weeks. 5

It is important that owners are properly instructed on how to optimally administer daily injections. Cats will perceive the injection as less painful if the remdesivir solution in the syringe is allowed to warm to room temperature instead of being refrigerated. In addition, if you teach them simple tasks such as using a new needle when injecting (ie use a needle other than the one used to draw the medicine from the vial) and using 21G (0.8mm - green) or 23G diameter needles (0.6mm - blue), injections will be more tolerable. Although 21G needles are larger, some cats may have the advantage of injecting faster. Alternatively, for simplicity, veterinarians can prepare injections for the whole week, which they will keep in the refrigerator, and will give a new injection every day.

For cats that continue to perceive SC injections as painful, we used gabapentin orally (50 to 100 mg per cat) and / or transmucosally or SC administered buprenorphine 30-60 minutes before sedation / analgesia injection. The area to be injected can also be trimmed so that a topical EMLA cream can be applied 30 minutes before the injection. BOVA produces a faster-acting local anesthetic gel that may be useful in some patients. In exceptional cases, we inserted a cephalic catheter every 4-5 days so that owners could administer IV therapy instead of SC injections. Injection site reactions reported with GS-441524 injected abroad do not appear to occur with remdesivir.

After 2-4 weeks of taking remdesivir and after the abdominal fluid has disappeared and the ocular and CNS symptoms have improved or disappeared, we are now proposing a switch to GS-441524 tablets. This is done for 3 reasons: (i) it reduces costs (ii) eliminates the pain problem of SC injections (iii) in some patients it is more effective. Remdesivir injections are probably more reliable than oral GS-441524, and in the worst cases, you might choose to give them for 4 weeks, but for most cats, 2 weeks and comfort and lower oral formulation costs outperform everything else.

The use of GS-441524 tablets is relatively new in Australia, but is widely used overseas. The recommended oral dose of GS441524 is usually the same as the SCI / IV remdesivir dose: wet cases of FIP receive 10-12 mg / kg PO SID, ocular cases 15 mg / kg PO SID and CNS cases 20 mg / kg (or higher). GS-441524 is more economical and even safer than remdesivir. In CNS cases where high doses are administered, it is probably best to administer 10 mg / kg PO every 12 hours (BID) to circumvent the "ceiling" effect referred to in the limited absorption of high doses.

Figure 6. Focal dry FIP with pyogranulomatous inflammation of intra-abdominal lymph nodes. Instead of exploratory laparotomy, lymph node biopsy, histology, and immunohistology, 3 days of remdesivir IV treatment may be more cost-effective if FIP is highly suspected. Enlarged lymph node FNA is probably an ideal diagnostic option for physicians with this set of skills.

Why are the dosages about the same? At mg / kg, GS441524 has twice as many active molecules as remdesivir (due to the difference in their molecular weight), but the bioavailability of GS-441524 is only 50 % (only half of what is given is absorbed, and this is affected by feeding and also the effect of the ceiling dose) - so these two factors cancel each other out.

We recommend that GS-441524 tablets be given with a small treat to mask the tablet, with the main meal being served 1 hour later. The tablets provided by BOVA are 50 mg tuna-flavored tablets, with four score lines, so they can be divided into halves or even quarters.

In situations where owners cannot afford full treatment, we use mefloquine (Lariam; 5 mg / kg orally once daily in capsules or 62.5 mg twice a week) after initial treatment with remdesivir / GS-441524.

Phillip McDonagh, Jacqui Norris, Merran Govendir and colleagues at the Sydney School of Veterinary Science have shown that mefloquine has an antiviral effect. 7 This is probably due to the fact that mefloquine usurps the biochemical intracellular pathways used by the FIP virus, a mechanism that has recently been demonstrated with clofazimine. 8 (anti-leprosy medicine), and several other medicines. In several cats, where owners could not afford a complete treatment with remdesivir, mefloquine proved to be effective in reaching the limit of clinical cure.

The main advantage of buying remdesivir and GS-441524 from BOVA for the treatment of FIP cases is that the products we use are subject to quality control. It's just a prescription with the client's name and address, the patient's name and the dose to be given, and the compounder can usually deliver the vials or tablets to any veterinarian in Australia within 24-48 hours.

At present, the price is 100 mg of vials of remdesivir 250$ plus GST and postage (the total price is usually about 280$). GS-441524 is sold in packs of 10 tablets for 600$ plus shipping and handling. By purchasing more vials and tablets at the same time, of course, postage and handling fees will be reduced. We believe that most owners will feel much more comfortable getting a product from a well-known Australian company than sending money overseas and hoping that drugs of unknown quality on the black market will reach Australia safely without being detained by customs.

There is no reason why a well-motivated veterinarian would not be able to handle these cases in his own practice. This is often more convenient for the owner, especially if they struggle with daily injections and need a practice near them.

Figure 7: Gs-441524 tablets from BOVA Australia. They are tuna flavored. They can be divided into halves or even quarters. MUCH EASIER than injections for most cats. Less stress and less cost.

Veterinarians who wish to explore this option or have general questions about FIP case management may email Sally Coggins (dr.sallyc@gmail.com), Richard Malik (richard.malik@sydney.edu.au), David Hughes (concordvets@concordvets.com.au), Grette Howard (drgretta@gmail.com) or Professor Jacqui Norris (jacqui.norris@sydney.edu.au), for advice on diagnosis or treatment. Many Australian veterinarians interested in FIP have gained considerable expertise in the management of these cases. For example, Andrew Spanner in Adelaide treated more than 20 cases with excellent results. Thus, there are already many feline medicine physicians and internal medicine specialists with experience in the treatment of FIP, and so veterinarians who are hesitant to treat their own cases have the opportunity to recommend these specialists to their clients.

Physicians who accept FIP cases from GPs include: QLD Rhett Marshall, Marcus Gunew, Alison Jukes, Rachel Korman; NSW Katherine Briscoe, Michael Linton, Randolph Baral, Melissa Catt; VIC - Carolyn O'Brien, Keshuan Chow, Amy Lingard; WA-Martine Van Boeijen and Murdoch University Veterinary Hospital; TAS Moira van Dorsselaer.

All of these doctors (and probably even more we don't know about) are happy to accept cases for diagnosis and therapy. Everyone is probably happy to discuss case management with you.

Figure 8: Bengal kitten with CNS and ocular FIP (A: before) and (B: after) after Remdesivira. This cat also had pulmonary granulomas.

Sally Coggins, working with Lara Boland, Emily Pritchard, Associate Professor Mary Thompson and Professor Jacqui Norris at the Sydney School of Veterinary Science, is interested in treating cases with comprehensive diagnosis and free monitoring. It will be part of Sally's doctoral program, so you will help her advance in her studies by sending her cases. We hope that through these studies, we will get a better idea of how quickly cats respond and when exactly treatment can be safely stopped. Owners will only have to pay for remdesivir and GS-441524 for therapy. This group is also interested in treating cases with interferon-omega and mefloquine.

In most cases, FIP is doing very well with GS-441524 or remdesivir. Niels Pedersen has gathered an amazing resource for veterinarians interested in FIP case management - https://sockfip.org/dr - pedersen - research / . The site also provides some recommendations on how to monitor cats during treatment. I'm not very protocol-oriented, so the key things for me to keep track of are appetite, attitude, activity levels, and changes in body weight and fitness over time. Most physicians like to monitor serum hematology and biochemistry every month to ensure that all measurable abnormalities improve, although this can be stressful for the patient and increase treatment costs. The trade-off is taking a few drops of blood to monitor PCV, total plasma protein (TPP) using refractometry, and plasma color to determine if anemia is improving, jaundice is subsiding, and gamma globulin levels are lowering, resulting in lower TPP.

Do not worry about transient increases in globulin levels at the start of treatment; when high protein effusions are absorbed, a lot of immunoglobulins enter the patient's plasma. This phenomenon may be common until the 8th week of treatment, but disappears by the 12th week.

Figure 9: Transverse plane MRI image in contrast to T1 weighting. Note: dilatation of the lateral ventricles with very slight emphasis on the ependymal lining (orange arrows). Image courtesy of Christine Thomas.

And what about a kitten with multifocal CNS disease, where FIP CNS is the most likely cause of clinical symptoms? The traditional approach is serology (to rule out cryptococcosis and toxoplasmosis), a good history and thiamine test to rule out vitamin B1 deficiency, followed by MRI scans (Figure 9) and CSF collection for fluid analysis and multiplex neuro-qPCR analysis). This approach is very expensive and there is also a certain risk of anesthesia and especially CSF collection. We found that a 3-5 day intravenous or sc. Remdesivir therapy can be used as a therapeutic test in cats with probable CNS FIP and is a cost-effective alternative to complete diagnostic processing, which can cost 3-5000$ or more.

Similarly, if exploratory laparotomy, abnormal tissue biopsy, histology, and immunohistochemistry for FIP antigen are used to diagnose dry intra-abdominal FIP versus 3-5 days of treatment with remdesivir or GS-441524, a drug test may be considered, which is a better choice from in terms of patient well-being and reduced costs. Most cats with non-fusive FIP experience rapid improvement with antiviral therapy, with normalized fever, improved appetite, and better overall attitude within 2 to 3 days. If the patient does not respond to antiviral therapy, then exploratory laparotomy and representative organ biopsy are reasonable, as the main differential diagnoses are lymphoma and lymphocytic cholangitis.

This is a matter of personal approach for each doctor. FNA for cytological and sometimes immunohistochemical examination or PCR is a convincing non-invasive option where this expertise is available, but sometimes it does not give a definitive answer. Some veterinarians insist on tissue diagnosis and positive immunohistology or PCR in each patient, while others would like to "treat treatable" with a 3-5-day remdesivir / GS-441524 application and proceed to exploratory laparotomy only when there is no clear response to therapy.

It is incredibly satisfying to see the transformation of cats and kittens, which are not well, into normal and happy cats. It's really something that will lift your spirits as a doctor. It's good science and good veterinary medicine!

Conclusions

In the past, the diagnosis of FIP was an intellectual exercise so that we could end the suffering of a cat or kitten with the certainty of an accurate diagnosis. Now, thanks to FIP's lifelong study, Dr. Niels Pedersen, we are able to successfully treat perhaps 80 % or more cats with FIP if the client has sufficient funds. It is too early to predict whether or how many will be repeated later.

There is a need for intensive study in diagnosis and case management, but with the necessary effort, a good veterinarian should be able to work with a determined owner to achieve a clinical cure. The most important thing is not to put too many obstacles in the way of the dedicated owner and support him during the 12-week marathon treatment course by helping him find the best way to treat his patient. This may include sedative / analgesic treatment to help the cat improve controllability and prevent discomfort when the client brings their cat to the clinic daily for remdesivir injections or switching to GS-441524 tablets when the stress from the injections is too great for the owner. It is important to go a long way and a payment plan can be provided that will allow determined clients to improve the affordability of treatment.

Finally, the impact of COVID-19 on coronavirus research has been indeed profound, and several very promising drugs are under development, such as molnupiravir from Merck and another oral drug from Pfizer.

OVERALL SUMMARY

2-step approach to therapy

Phase 1 - INDUCTION

IV / SC injections of Remdesivir

  • For cats with wet FIP: 10-12 mg / kg remdesivir by subcutaneous injection (SCI) once daily (SID) for 2 weeks
  • For cats with eye: 15 mg / kg SID remdesivir SCI for 2 weeks
  • For cats with neurological symptoms of FIP and CNS: remdesivir 20 mg / kg SID for 2 weeks

Phase 2 - CONSOLIDATION

Switch to GS-441524 tablets after 2 weeks of remdesivir injection

  • For cats with wet FIP: 10-12 mg / kg GS-441524 oral SID for 10 weeks
  • For cats with eye impairment: 15 mg / kg SID GS-441524 oral SID for 10 weeks
  • For cats with neurological symptoms of FIP and CNS: GS-441524 10 mg / kg oral BID (20 mg / kg / day) for 10 weeks

References

  1. Kim, Y .; Liu, H .; Galasiti Kankanamalage, AC; Weerasekara, S .; Hua, DH; Groutas, WC; Chang, KO; Pedersen, NC Reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor. PLoS Pathog. 2016, 12, e1005531.
  2. 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, 378–392.
  3. 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.
  4. Pedersen, NC; Perron, M .; Bannasch, M .; Montgomery, E .; Murakami,
    E .; Liepnieks, M .; Liu, H. Efficacy, and safety of the nucleoside analog GS441524 for treatment of cats with naturally occurring feline infectious peritonitis. J. Feline Med. Surg. 2019, 21, 271–281.
  5. Dickinson PJ, Bannasch M, Thomasy SM, et al. Antiviral treatment using the adenosine nucleoside analogue GS-441524 in cats with clinically diagnosed neurological feline infectious peritonitis. Journal of Veterinary Internal Medicine. 2020. doi: 10.1111 / jvim.15780.
  6. Tasker S. Diagnosis of feline infectious peritonitis: Update on evidence supporting available tests. Journal of Feline Medicine and Surgery.
    2018; 20 (3): 228-243. doi: 10.1177 / 1098612X18758592
  7. McDonagh, P .; Sheehy, PA; Norris, JM Identification, and characterization of small molecule inhibitors of feline coronavirus replication. Vet. Microbiol. 2014, 174, 438–447.
  8. Yuan, S., Yin, X., Meng, X. et al. Clofazimine broadly inhibits coronaviruses including SARS-CoV-2. Nature (2021).
    https://doi.org/10.1038/s41586-021-03431-4
  9. https://sockfip.org/ - THE BEST resource on the internet or anywhere for FIP.
Figure 10: Two cats with dry FIP after successful therapy. As an avid young veterinarian wrote to me not so long ago - "that's why I did science!"

COSTS:

2 kg kitten with wet FIP
4 × 100 mg remdesivir vials - 1000$
35 × 50 mg tablets GS-441524 - 2100$
Manipulation and GST - 30$ plus 310$ = 340$
A total of 3440$, approximately 290$ per week for 12 weeks

4 kg cat with dry FIP
7 × 100 mg remdesivir vials - 1750$
70 × 50 mg tablets GS-441524 - 4200$
Handling and GST 30$ plus 600$
A total of 6550$, about 545$ per week for 12 weeks

Figure 11: Two siblings who developed FIP and were successfully treated with remdesivir and GS441524.
Read "FIP treatment with subcutaneous remdesivir followed by oral tablets GS-441524"

Serum protein electrophoresis

Kristiina Ruotsalo, DVM, DVSc, ACVP & Margo S. Tant, BSc, DVM, DVSc
Original article: Serum Protein Electrophoresis - General

What are serum proteins?

Serum is the liquid part of the blood from which red blood cells, white blood cells and blood clotting factors have been removed. The serum contains a large amount of protein, which performs various functions. These functions include providing cell nutrition, protecting against infections, acting in inflammation, and acting as hormones or enzymes.

What is serum protein electrophoresis?

Protein electrophoresis is a specialized test that analyzes specific groups of proteins in the blood serum and measures the proportion of each group of proteins. Individual proteins have characteristic sizes and electric charges. Electrophoresis divides serum proteins into broad groups based on their size and electrical charge. The results of the analysis are shown in a special graph and a pattern of different proteins is used to diagnose specific diseases, including some types of cancer.

What proteins does the test measure?

"Globulin levels - tend to rise in diseases."

There are many different proteins in the blood, but protein electrophoresis focuses on only two classes of proteins, called albumin and globulin. There is only one type of albumin and it is found in the blood at relatively constant levels; it is a versatile protein with a number of important roles, including the transport of substances in the body. In contrast, there are many types of globulins, each with a specific function. Globulin levels are more variable than albumin and tend to increase in disease.

When a blood sample is analyzed by routine methods, albumin and total globulin levels are measured. Protein electrophoresis goes further and divides the total globulin into its individual parts, called globulin fractions, which are then measured individually. By analyzing the types and amounts of different proteins in your blood, it is often possible to determine the nature of your pet's disease.

Typically, globulins are divided into the following fractions: α1 (Alpha 1), α2 (Alpha 2), β1 (Beta 1), β2 (Beta 2), γ (Gamma)

Why are globulins important?

Globulins play an important role in the body's defense system; some are "first rescuers," like firefighters, and quickly appear in the bloodstream after any tissue injury. Others, called antibodies, are produced by lymphoid cells, a type of white blood cell, and appear in the bloodstream more slowly after injury. Antibodies are essential for the body's ability to defend itself against bacteria and other disease-causing organisms.

High total blood globulin levels in most cases indicate underlying inflammation or infectious disease, but sometimes indicate the presence of cancer, especially affecting lymphoid cells. When determining the type and distribution of globulins, protein electrophoresis can help us decide what kind of disease it may be.

How does the test work?

Belch electrophoresis is like sorting a bowl of mixed colored beads into separate groups according to color and size and then counting how many beads are in each group. The test is based on the fact that albumin and different globulins have different sizes and that each type of protein carries a different electrical charge than static electricity. The serum sample is prepared and placed on a special grid. When an electric current is applied, different proteins migrate across the lattice at different rates, causing them to divide into groups according to size and electric charge. For example, albumin is a relatively small molecule and carries a lot of "static electricity"; it travels the farthest and fastest of all proteins and is always the first to appear on the chart. Globulins are generally larger and move more slowly, and antibodies, which are the largest of globulins and have the least "static electricity", move very slowly and are the last to be shown on the graph.

"… Each type of protein carries a different electric charge"

Once the proteins are divided into their groups, it is possible to measure the amount of each protein and display the results in a graph. The shape of the graph helps us to understand the underlying disease.

When should protein electrophoresis be done?

"Protein electrophoresis is recommended whenever total globulin levels are elevated and the cause is unknown."

Protein electrophoresis is recommended whenever total globulin levels are elevated and the cause is unknown. The higher the level of total globulins, the more suitable it is to perform protein electrophoresis. Globulins usually grow when there is inflammation, tissue injury or infectious disease. More importantly, however, globulin levels can be very high in some types of lymphoid cell cancers. When preliminary blood tests indicate that total globulin levels are elevated, protein electrophoresis should be performed to try to determine if the underlying disease is inflammatory or neoplastic.

How is the graph (electrophoretogram) interpreted?

The most important thing in interpreting the electrophoresis graph is whether the globulin is increased due to the growth of many different globulins or due to the growth of only one type of globulin. When many different globulins are elevated, we speak of a polyclonal increase (poly = many; clonal = type); when only one type of globulin is responsible for the increase, we speak of a monoclonal increase (mono = one; clonal = type). Inflammation is typically polyclonal, while lymphoid neoplasia is more likely to be monoclonal. Unfortunately, there is some overlap between the two general classifications.

Do the results always provide a definitive diagnosis?

No, but some serious diseases, both inflammatory and neoplastic, form a characteristic pattern on the electrophoresis chart that can quickly lead to a definitive diagnosis. In many inflammatory conditions, protein electrophoresis can provide valuable information about the severity of inflammation, where it may be located, and what it may cause.

Examples of electrophoretogram

Panel A: Normal agarose gel electrophoretogram in dogs. The highest peak on the left is albumin, followed by α1 (2 peaks), α2 (2 peaks), β1 (2 peaks, β1a and β1b), β2 and γ (last flat peak).
Panel B: Serum from a cat with a virus infection feline infectious peritonitis (FIPV). Visible increase α2 globulins (arrow), indicating acute phase reactant response, and polyclonal gammopathy (arrow in the γ region). These results are typical, but not specific, for FIPV infection (they can be observed in other inflammatory conditions).
Panel C: Serum from a dog with multiple myeloma. There is a high narrow peak in the γ region, which indicates monoclonal gammopathy (arrow). Albumin concentrations are also reduced (compared to a normal dog in panel A).

Co-authors: Kristiina Ruotsalo, DVM, DVSc, ACVP & Margo S. Tant, BSc, DVM, DVSc Read "Serum Protein Electrophoresis"

Feline Infectious Peritonitis (FIP): Hope for cats on the horizon

Sam Taylor, BVetMed (Hons), CertSAM, MANZCVS, DipECVIM-CA, FRCVS and Emi Barker BSc (Hons), BVSc (Hons), PhD, DipECVIM-CA, MRCVS summarize ideas about the manifestations and diagnosis of this disease and represent a new era of treatment .

Emi BarkerSamantha Taylor, VetTimes Volume 51, Issue 32, Pages 16-19 | August 31, 2021
Original article: Feline infectious peritonitis: hope on the horizon for cats

Figure 1. "Classic" Cat with a "classic" FIP with a large abdominal effusion. Image: Feline Center, Langford Vets, University of Bristol

FIP is caused by virulent mutations in feline coronavirus (FCoV), which transform it from a mild and enteric infection to a serious systemic disease.

Like other coronaviruses, FCoV is a large enveloped RNA virus - this is important when considering immune system avoidance, environmental survival, detection, treatment and prevention. FIP has a high mortality rate and, until recently, treatments were relatively ineffective.

This article summarizes current views on the manifestations and diagnosis of this disease. It also represents a new era of FIP treatment in the context of the recent availability of legal medicines in the UK.

What causes FIP?

FCoV is an alpha-coronavirus that infects domestic cats and other cats. It is from the same genus as canine enteric coronavirus and swine gastroenteritis virus. FCoV cannot infect humans and is only distantly related to SARS-CoV-2, beta-coronavirus, and pathogen COVID-19.

FCoV, as a feline enteric coronavirus (FECV) biotype, is commonly detected in faeces - especially in cats living in multi-cat households. The infection typically spreads via the fecal-oral route when kittens or young cats are in contact with excreting cats. The chances of survival of this enveloped virus in the environment are generally poor unless the virus is trapped in feces and is sensitive to most disinfectants.

In some cats, and at some point after the initial infection - between viral replication in enterocytes and efficient replication in macrophages and monocytes - the less virulent FECV mutates into a virulent form associated with FIP - that is, the FIP virus biotype (FIPV). Certain mutations associated with this transition have been found in the spike protein gene, although none of them are yet pathognomonic for FIP.

The high frequency of genomic mutations - a hallmark of RNA viruses - can also facilitate the avoidance of the host immune response and drive tissue tropism, leading to various manifestations of the disease. Natural direct transmission of FIPV between cats is considered rare, and it is generally believed that FIPV - and subsequently FIP - results from a new mutation in the FCoV of an individually infected cat.

Figure 2. Mild jaundice and pallor in cats with FIP.

One of the many complexities of FCoV and FIP is that the infection manifests itself in many different ways depending on viral factors such as strain and dose, but also on the cat's immune response and genetic factors.

A strong cell-mediated immune (CMI) response to FCoV appears to provide protection against FIP. In contrast, cats with a predominantly antibody-mediated response with a weak CMI response typically succumb to the effusive "wet" form of the disease due to immune-mediated vasculitis, while cats with moderate CMI develop tissue granulomas typical of the non-fusive "dry" form of FIP.

It is important to keep in mind that the effusive and non-fusive forms of FIP can significantly overlap, leading to a wide range of symptoms; short episodes of the effusive form of FIP may occur rather than the predominantly non-fusive form, and conversely, effusions may form in the terminal stages of the non-fusive form of FIP. In addition, many exuded cats have tissue granulomas.

Clinical signs

Classic presentation of a young cat with protein-rich ascites (Figure 1) may offer easier diagnostics; however, other cats may be more of a diagnostic challenge. Common non-specific symptoms include lethargy, anorexia and weight loss.

Affected cats may be febrile with a moderate fever, typically less than 40 ° C, which often fluctuates and responds poorly to NSAIDs (non-steroidal anti-inflammatory drugs or non-steroidal anti-inflammatory drugs) or antimicrobials, and jaundice, if present, is usually mild (Figure 2).

Figure 3. Hyphema, uveitis and hypopyon in cats with ocular FIP.

High protein viscous effusions are formed in approximately 80% cats with FIP - most (approximately 85%) involve the abdominal cavity, while fewer cases show thoracic effusion (approximately 20%).

Pericardial effusions are occasionally observed, although rarely causing tamponade, and very rarely, scrotal effusions occur in uncastrated cats.

Pyogranulomatous lesions can occur in any tissue, and while they commonly involve the abdominal organs (e.g., mesenteric lymph nodes and kidneys), the disease may be limited to other organs such as the eyes, brain, or spinal cord.

Ocular symptoms include uveitis, ceramic clots, hypopyon, hyphaemia (Figure 3) and retinitis. Neurological symptoms include ataxia, seizures, nystagmus, hyperesthesia, and behavioral / mental changes.

Diagnosis

Although one abnormality does not in itself determine the diagnosis of FIP, nor does their absence rule out the diagnosis, the veterinarian may base the suspicion on FIP with the following findings, bearing in mind signaling, clinical picture, clinical pathology, and imaging results if no alternative diagnosis is present. , more likely explanation:

Figure 4. MRI scan of a cat with FIP showing obstructive hydrocephalus and increased contrast of the meninges.
  • Clinical examination - may reveal fever, jaundice, abdominal distension (ascites; organomegaly), chorioretinitis, ataxia, cranial nerve deficits.
  • Blood analysis - lymphopenia, non-regenerative anemia, microcytosis, neutrophilia, hyperglobulinemia, low albumin / globulin ratio (A: G; classically less than 0.4), hyperbilirubinemia, high α-1 acid glycoprotein (often markedly elevated, more than 1.5 mg / ml).
  • Diagnostic imaging - effusions, abdominal lesions, CNS abnormalities consistent with meningeal thickening and / or obstructive hydrocephalus (Figure 4).
  • Efusion analysis - non-aseptic pyogranulomatous inflammation with a relatively low cell number (total number of nuclear cells higher than 5 × 109/ l; neutrophils and macrophages) with high protein concentrations (often higher than 35 g / l) and low A: G (Figure 5).
  • Molecular diagnostics - positive results of FCoV RNA RT-PCR in fluids (eg effusions, aqueous humor, CSF; Note: false positive and negative results are possible in whole blood) or aspirates with a thin needle (FNA) of the affected organ (eg kidneys, liver, mesenteric lymph nodes); the higher the viral load, the more evidence of FIP. Note: RT-PCR cannot confirm the diagnosis of FIP.

High levels of FCoV antibodies indicate only previous FCoV infection and do not indicate a diagnosis of FIP

The definitive diagnosis of FIP is confirmed by positive immunohistochemical staining for coronavirus antigen in macrophages associated with pathological changes in FIP in formalin-fixed tissue samples. However, sampling for histopathology and immunohistochemical staining requires invasive procedures, which may be contraindicated in a sick cat.

Figure 5. Sweat analysis is useful in diagnosing FIP; where possible, take a fluid sample.

Alternatively, the presence of antigen-positive coronavirus cells in cytological specimens (effusion, aqueous humor, cytospin CSF preparations or FNA from any abnormal organs - such as mesenteric lymph node) showing pyogranulomatous changes is very helpful in diagnosis and allows less invasive specimen acquisition.

Some researchers have used cell pellets prepared from centrifuged effusion samples to improve the sensitivity of immunohistochemical staining (Tasker et al, 2021).

However, it is important to diagnose FIP as reliably as possible before treatment, as there are many other differential diagnoses for these clinical symptoms - including neoplasia (especially lymphoma), other infectious diseases (pyothorax, toxoplasmosis, mycobacteriosis, fungal infections)) and primary immune-mediated disease (idiopathic disease). , uveitis, lymphocyte cholangitis). On the Figure 6 is a graph indicating possible diagnostic pathways.

Minimally invasive sampling may include abdominal or thoracic effusions and FNA abnormal organs.

More detailed diagnostic flowcharts for the diagnosis of FIP are available on the website of the European Advisory Committee on Feline Diseases (www.abcdcatsvets.org/feline-infectious-peritonitis).

Figure 6. Access to FIP diagnostics

FIP treatment with antiviral drugs

In recent years, publications have focused on antiviral drugs (GS-441524, a nucleoside analog that inhibits viral RNA polymerase, and GC376, a viral protease inhibitor) with the potential to cure experimentally induced cats (Kim et al, 2016; Murphy et al, 2018) and naturally obtained (Pedersen et al, 2018; 2019; Dickinson et al, 2020) FIP.

Unfortunately, until recently (see below), legal formulations of these drugs were not commercially available, although some owners obtained and administered illegal formulations to their cats of unknown origin and at great expense.

Remdesivir, a prodrug of GS-441524, is an antiviral drug with a broad spectrum of activity against RNA viruses. It was originally developed to treat hepatitis C virus and Ebola virus in humans. Its development was then significantly accelerated due to the worldwide treatment of SARS-CoV-2.

In Australia, remdesivir has been legally available to veterinarians for several months as a "special" formulation that allows veterinarians to gain experience with this drug in the treatment of cats and kittens with FIP, where it has shown promising results. Unlike GS-441524, remdesivir has low oral bioavailability and is administered by intravenous infusion to human patients.

In the UK, remdesivir is legally available through Gilead Sciences, a patent-pending company that manufactures a medicinal product for human use. The currently available formulation is Veklury, a powder for reconstitution with water for solutions for injection to a final remdesivir concentration of 5 mg / ml. After reconstitution, it should be cooled and consumed within 24 hours.

Figure 7. Vials with reformulated remdesivir will be available to treat FIP, legally, in the UK from August 2021.

In cats, SC is usually administered unofficially, although some cats may benefit from initial IV administration. From August 2021, remdesivir will also be available from specialty drug manufacturers as a veterinary ál special ’(Figure 7) in the United Kingdom. The reformulated remdesivir will be supplied in vials containing 100 mg remdesivir, at a concentration of 10 mg / ml, allowing for smaller injection volumes, with a shelf life of at least three months when properly stored.

The experience of our colleagues from Australia (Malik, personal communication) allows us to design benefits according to the above plan. The authors emphasize the need to diagnose FIP before using this drug to ensure its proper use, while acknowledging that the diagnosis of FIP can be anticipated due to clinical or financial diagnostic limitations.

Costs, prolonged nature of the treatment cycle (recommended at least 12 weeks), potential discomfort with SC injections and risk of relapse should be discussed with cat owners before initiating therapy.

Owners may be instructed to give daily injections to their cat, but must be thoroughly trained to avoid unintentional self-administration, incorrect technique that may harm the cat, and to minimize the risk of the cat reacting to injections leading to bite or scratch injuries. This healing process requires committed owners and is an emotional as well as a significant financial commitment.

Depending on the clinical condition of the cat, supportive therapy is still needed (eg IV or SC fluids, antiemetic stimulation and appetite, analgesia, tube nutritional support, sepsis antimicrobials). Cats with uveitis may require topical treatment with corticosteroids and cats with neurological symptoms may need anti-seizure medication.

Figure 8. Bengal kitten with ocular and neurological FIP before (left) and after (right) remdesivir treatment.

Although systemic use of corticosteroids is not generally recommended concomitantly with the use of antivirals, short-term administration of corticosteroids may be considered in cats with a strong suspicion of secondary immune-mediated disease due to FIP (eg immune-mediated haemolytic anemia).

The success rate of treatment is high - 80% to 95% (Malik, personal communication; Figure 8) - and therefore we have reason to be optimistic when discussing treatment with clients, even though we are aware of the commitment and the associated costs and potential for relapse.

Increasing the success of treatment

The treatment is long and remdesivir can be painful when injected. Once opened, the medicine should be stored in the refrigerator and should be warmed to room temperature before injection. Needle size can affect discomfort; for some patients, a faster injection of a larger diameter "green" needle (21G) may be more advantageous, while a smaller diameter orange "orange" (25G) needle may be more advantageous. A new needle should be used for each injection.

Some cats will need to go to the clinic every day for an injection. Gabapentin or trazodone (both 50 mg to 100 mg orally per cat), given two hours before the deadline to reduce anxiety and pain, may benefit some cats. Other cats may need a dose of buprenorphine (0.02 mg / kg to 0.03 mg / kg transmucosally or IM in a clinic before treatment).

To reduce discomfort, the injection site can be trimmed and EMLA topical anesthetic cream applied 45 to 60 minutes before injection. Injection tolerance appears to vary between cats.

Remdesivir in the treatment of FIP


Dosage

  • FIP s exudates (ie ascites and / or pleural effusion), but without any ocular or neurological impairment: 7 mg / kg until 8mg / kg once a day.
  • FIP s ocular symptoms (that is, uveitis or other eye disorders, but without neurological impairment): 10 mg / kg once a day.
  • FIP s neurological symptoms: 12 mg / kg until 15mg / kg once a day.

Method of administration

  • Most cases: SC injection into the loose skin of the interscapular area.
  • Very severe cases: 10 mg / kg may initially be given by intravenous infusion (ie diluted in 10 ml saline and given slowly over 10 to 20 minutes) to achieve a rapid antiviral effect; This can be done after three to four days of SC injection as soon as the cat starts eating and its health improves. Note: After intravenous administration, some cats may experience depression for several hours.

Duration of treatment

  • Treatment of at least 84 days (ie 12 weeks) should be considered. This time is based on a clinical study with GS-441524 and the unofficial use of remdesivir to minimize the likelihood of FIP relapse.
  • After 84 days, treatment should only be discontinued if the patient is clinically OK and abnormal laboratory parameters have returned to normal.
  • If the response to treatment is only partial or unsatisfactory, a prolongation of treatment may be necessary.

Monitoring

  • In the first days, closely monitor for clinical signs:
    - Improvement should be rapid, within a few days, with weight gain and improvement in clinical symptoms.
    - Consider verifying the diagnosis if no improvement is seen (with regard to the following symptoms):
    Efusion (especially pleural) may worsen for one to two days at the start of treatment and may require therapeutic thoracocentesis or abdominocentesis. This appears to occur most frequently after IV treatment. Consider ultrasound monitoring once or twice daily.
    Neurological symptoms may initially appear or worsen in the first days of treatment. This may include the development of seizures that may require medical attention (eg levetiracetam 20 mg / kg to 30 mg / kg every eight hours).
  • The weight should be checked regularly and the doses adjusted accordingly.
  • Monitor PCV, total proteins (albumin and globulin), bilirubin and other abnormal parameters until they return to normal. The frequency of monitoring varies between veterinarians; a monthly assessment of biochemistry and hematology is usually performed, but should be adapted to the client's finances and the cat's response and behavior.
  • Serum globulins may increase initially, but any hyperglobulinemia usually resolves by week 12.
  • Remdesivir is reported to cause reno / hepatotoxicity in humans, but these have only been observed at higher doses by our Australian colleagues, who resigned when the dose was reduced. * Higher doses may be required depending on the response. Using lower doses to reduce costs may increase the likelihood of treatment failure. Please note that these dosing recommendations may change depending on the growing amount of data and clinical experience of veterinarians using this medicine. It is recommended to consult an expert in cats or internal medicine to discuss the individual case and the appropriate dosage.

Further treatment

The researchers are looking at the beneficial effects of immunostimulants and / or other antiviral medicines, such as interferons or mefloquine, once remdesivir treatment is stopped or if the injections are considered too painful. Most studies published to date have focused on the use of antiviral drugs alone.

Thanks

The authors would like to thank their Australian colleagues David Hughes, Rebecca Brady and Richard Malik for sharing their experiences with remedivirus treatment in cats with FIP. Thanks also to Séverine Tasker and Professor Gunn-Moore for comments on the article.

Do you need advice on FIP treatment?

Stephanie Sorrell and Danièlle Gunn-Moore of the University of Edinburgh will recruit cases to monitor the UK's response to the remdesivir, with more information coming soon.

If advice is needed in the meantime on the diagnosis and treatment of a suspected FIP case, send an e-mail to fipadvice@gmail.com

References

  • Dickinson PJ, Bannasch M, Thomasy SM, Murthy VD, Vernau KM, Liepnieks M, Montgomery E, Knickelbein KE, Murphy B and Pedersen NC (2020). Antiviral treatment using the adenosine nucleoside analogue GS-441524 in cats with clinically diagnosed neurological feline infectious peritonitis, Journal of Veterinary Internal Medicine 34(4): 1,587-1,593.
  • Kim Y, Liu H, Galasiti Kankanamalage AC, Sahani Weerasekara S, Hua DH, WC Groutas, Chang K and Pedersen NC (2016). Reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor, PLOS Pathogens 12(3): e1005531.
  • Murphy BG, Perron M, Murakami E, Bauer K, Park Y, Eckstrand C, Liepnieks M and Pedersen NC (2018). The nucleoside analog GS-441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies, Veterinary Microbiology 219: 226-233.
  • Pedersen NC, Kim Y, Liu H, Galasiti Kankanamalage AC, Eckstrand C, Groutas WC, Bannasch M, Meadows JM and Chang KO (2018). Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis, Journal of Feline Medicine and Surgery 20(4): 378-392.
  • Pedersen NC, Perron M, Bannasch M, Montgomery E, Murakami E, Liepnieks M and Liu H (2019). Efficacy, and safety of the nucleoside analog GS-441524 for treatment of cats with naturally occurring feline infectious peritonitis, Journal of Feline Medicine and Surgery 21(4): 271-281.
  • Tasker S and members of the European Advisory Board for Cat Diseases (2021). Feline infectious peritonitis guidelines, www.abcdcatsvets.org/feline-infectious-peritonitis
Read "Feline Infectious Peritonitis (FIP): Hope for Cats on the Horizon"

Combination therapy with fluoxetine and the nucleoside analogue GS-441524 shows synergistic antiviral effects against various SARS-CoV-2 variants in vitro

Complete original article: Combination Therapy with Fluoxetine and the Nucleoside Analog GS-441524 Exerts Synergistic Antiviral Effects against Different SARS-CoV-2 Variants In Vitro81 /

Authors: Linda Brunotte, Shuyu Zheng, Angeles Mecate-Zambrano, Jing Tang, Stephan Ludwig, Ursula Rescher, and Sebastian Schloer

Abstract

The ongoing SARS-CoV-2 pandemic requires effective and safe antiviral treatment strategies. Drug repurposing is a fast and inexpensive approach to developing new treatment options. The direct antiviral agent remdesivir was found to have antiviral activity against SARS-CoV-2. While remdesivir has only a very short half-life and bioactivation, which depends on prodrug activating enzymes, its plasma metabolite GS-441524 can be activated by various kinases, including adenosine kinase (ADK), which is moderately expressed in all tissues. The pharmacokinetics of GS-441524 suggest that it is a suitable antiviral drug that can be administered to patients with COVID-19. In this work, we analyzed the antiviral properties of combination therapy with the metabolite remdesivir GS-441524 and the antidepressant fluoxetine in a polarized Calu-3 cell culture model against SARS-CoV-2. Combination therapy with GS-441524 and fluoxetine was well tolerated and showed synergistic antiviral effects against the three circulating SARS-CoV-2 variants in vitro in commonly used drug interaction reference models. Thus, combination treatment with GS-441524 virus-targeted and host-targeted fluoxetine could offer a suitable therapeutic option for the treatment of SARS-CoV-2 infections.

Translator's note: Fluoxetine is produced under various trade names, of which Prozac is probably the best known.

… You can read the rest in complete article in english. Read "Combination therapy with fluoxetine and nucleoside analogue GS-441524 shows synergistic antiviral effects against different variants of SARS-CoV-2 in vitro"

SARS-CoV-2 study: The second possible potent mechanism of remdesivir has been identified

Original article: SARS-CoV-2 Research: Second possible effective mechanism of remdesivir discovered

Remdesivir suppresses host cell defense. This knowledge may be important for the development of drugs to fight various viruses. Photo: Bernard Chantal / shutterstock

When a cell is infected, SARS-CoV-2 not only causes the host cell to produce new viral particles. The virus also suppresses host cell defense mechanisms. The nsP3 viral protein plays a central role in this. Researchers at Goethe University, in collaboration with the Paul Paul Scherer Institute in Switzerland, found through structural analyzes that the degradation product of the virostatic agent remdesivir binds to nsP3. This points to another previously unknown potent mechanism of remdesivirus, which may be important for the development of new drugs to combat SARS-CoV-2 and other RNA viruses.

The virostatic agent remdesivir was developed to disrupt an important step in the propagation of RNA viruses, including SARS-CoV-2: the reproduction of the virus's own genetic material. This is represented by the RNA matrix by which the host cell directly produces viral proteins. However, to accelerate the production of their own proteins, RNA viruses also copy the RNA matrices themselves. For this purpose, they use their own specific protein (RNA polymerase), which is blocked by remdesvirus. More specifically, it is not the remdesivir itself that does, but rather the substance that is synthesized from the remdesivir in five steps as the remdesivir penetrates the cell.

In the second of these five steps, a product is formed from remdesivir, a substance with a somewhat impractical name, GS-441524 (in scientific terminology: a metabolite of remdesivir). GS-441524 is also a virostatic agent. As researchers in a group led by Professor Stefan Knapp of the Institute for Pharmaceutical Chemistry at Goethe University in Frankfurt found out, GS-441524 targets a SARS-CoV-2 protein called nsP3. nsP3 is a multifunctional protein whose tasks include suppressing the host cell defense response. The host cell is not helpless in the face of the virus because, among other things, it activates the inflammatory mechanisms by which it calls the endogenous immune system of the cell. However, the nsP3 protein helps the virus to suppress this call for help.

Professor Stefan Knapp explains: “GS-441525 inhibits the activities of the nsP3 domain, which is important for virus reproduction and which communicates with human cellular defense systems. Our structural analysis shows how this inhibition works, which allows us to lay an important foundation for the development of new and more effective antiviral drugs - effective not only against SARS-CoV-2. The target structure of GS-441524 is very similar in other coronaviruses, such as SARS-CoV and MERS-CoV, as well as in a series of alphaviruses, such as chikungunya virus. Therefore, the development of such drugs could help prepare for future viral pandemics. "

Translator's note: The nsP3 protein also contains the feline coronavirus FCoV / FIPV.

Publication: Xiaomin Ni, Martin Schröder, Vincent Olieric, May E. Sharpe, Victor Hernandez-Olmos, Ewgenij Proschak, Daniel Merk, Stefan Knapp, Apirat Chaikuad: Structural Insights into Plasticity and Discovery of Remdesivir Metabolite GS-441524 Binding in SARS-CoV ‑ 2 Macrodomain. ACS Med. Chem. Lett. 2021, 12, 603-609 https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00684 Read "SARS-CoV-2 research: The second possible effective mechanism of remdesivir has been discovered"

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