Category: Scientific

Project leader: Cynthia Leifer
Sponsor: Cornell Feline Health Center Research Grants Program
Original Name: Development of Feline Chimeric Antigen Receptor-Expressing Natural Killer Cells (CAR-NK) Against a Feline Coronavirus Spike Protein
Project amount: $ 55,200
Project period: July 2021 to June 2022

DESCRIPTION (provided by the applicant):

Feline coronavirus (FCoV) causes a subclinical infection that can develop into FIP's lethal infectious peritonitis. It is caused by the feline infectious peritonitis virus (FIPV), which occurs when a mutation occurs in the spike of the FCoV virus. Despite more than three decades of virus research, there is still no approved treatment for FIP. This is partly due to the fact that FIPV type I causes most clinical infections, but is not very successful in cultures and there are few reagents available for its study, while type II grows well, has more reagents, but generally does not cause clinical infections. disease. The development of new immunotherapies requires significant preclinical study and validation, and due to limitations in the FIPV type I study, no immunotherapies have been developed for clinical FIP. We want to try a new immunotherapy using in vitro FIPV type II research, which could lead to the treatment of FIPV cats caused by FIPV type I. Immunotherapy, which has recently gained attention in the treatment of human cancers, are immune cells treated with a chimeric antigen receptor ( CAR). This approach has not yet been developed for acute viral infections such as FIPV, nor has it been used in cats. CAR consists of two main components: a single chain antibody fragment (ScFv) and a signal domain from the immune costimulatory receptor. We propose to use a FIPV-specific ScFv spike protein expressed in feline immune cells that could be administered to a feline to screen for and destroy FIPV-infected cells. Current human CAR therapies require the use of autologous T cells because allogeneic T cells kill host cells and lead to graft-versus-host disease.
NK cells (natural killers) are of increasing interest because NK cells do not induce graft-versus-host disease and can therefore be used in "conventional" therapies. However, little is known about feline NK cells. We hypothesize that genetically modified feline NK cells expressing CAR targeting the feline infectious peritonitis virus type II spike protein will recognize and kill FIPV-infected target cells in vitro.

To achieve our goals:

1. determine the cytotoxic activity of a subset of NK cells in comparison with NKT and T cells for the validation of feline NK cells as potential CAR carriers,
2. for cloning and sequencing, we will use currently available hybridoma spike anti-FIPV type II antibodies to construct a CAR directed against spike type II protein, and
3. in vitro we validate the NK cell assay and data demonstrating the effect of CAR-NK cells killing FIPV type II infected cells.

The completion of this study will provide data important for the development of CAR-NK therapy for FIPV type I, a completely new treatment for this devastating disease.

1.4.2021, Translation 8.5.2021
Stout, Alison E., André, Nicole M., and Whittaker, Gary R.
Original article: FELINE CORONAVIRUS AND FELINE INFECTIOUS PERITONITIS IN NONDOMESTIC FELID SPECIES

Abstract

Feline coronavirus (FCoV) is reported worldwide and is known to cause disease in both domestic and non-domesticated feline species. Although FCoV often leads to mild or subclinical disease, a small subset of cats succumb to the deadly systemic disease, feline infectious peritonitis (FIP). The outbreak of FIP in cheetahs (Acinonyx jubatus) in the zoo's collection highlighted the devastating effect of introducing FCoV into a group of animals. In addition to cheetahs, FIP has also been recorded in European wild cats (Felis silvestris), tigers (Panthera tigris), mountain lions (Puma concolor) and lions (Panthera leo). This article provides an overview of reported cases of FIP in non-domesticated cat species and highlights FCoV in non-domesticated cat populations.

Introduction

Infectious diseases pose a significant threat to wildlife conservation.⁷⁴ Soil fragmentation has the potential to allow higher populations to grow in natural habitats than usual, and urban sprawl may increase the interaction of wild cats with domestic cats.¹⁵ Recently, the COVID-19 pandemic also highlighted undelivered felines when the coronavirus SARS-CoV-2 was first detected in a tiger (Panthera tigris) at the Bronx Zoo in New York, NY, with subsequent cases diagnosed in other cats kept in captivity in the same zoo; ¹²² although the condition of these animals has improved since diagnosis, coronaviruses remain a concern for the health of cats in both domestic and non-domestic environments.

Feline coronavirus infections are currently widespread in the domestic cat population. ¹⁷ In most domestic cats, the disease is either asymptomatic or manifests as self-limiting mild diarrhea. ⁸³ However, a small subset of cats experience an "internal mutation" that leads to macrophage tropism⁴⁶, wherein these cats succumb to a systemic disease known as FIP. ^9,60,99 FCoV classified as alpha-coronavirus (species alpha-coronavirus-1) can be divided into two biotypes: benign feline enteric coronavirus or feline infectious peritonitis virus (FIPV).⁸² Furthermore, the virus can be classified according to serotype as feline coronavirus type I or type II.⁵² These viral types originally differed based on a different antibody response, but it has recently been proposed that they be considered different genetic strains based on their spike glycoproteins.⁶⁸ Type II viruses are much less common in natural infections, yet they are more easily isolated and propagated in cell culture systems, and have been characterized in more detail. The type II virus is considered to be derived from double homologous recombination between the native FCoV type and canine coronavirus.⁴⁶

The viral spike protein is the driving mechanism of cellular tropism and pathogenesis. The FCoV type II receptor is known to be feline aminopeptidase (fAPN), ¹¹⁴ while type I viruses have a different and as yet unknown receptor. ²¹ Another feature distinguishing between the two virus types is the fusion activation process, the type I virus contains two proteolytic activation points (at sites S1 / S2 and S29), while the type II virus has a single cleavage point S29.⁵² Although both serotypes are known to cause FIP, many questions remain as to the natural behavior of each and the host's response to infection.

Antibody responses to both type I and type II FCoV have been reported in captive cats in the United States, with reports specifically reporting a response to FCoV type I in cheetahs, African lions (Panthera leo) and Bengal tigers (Panthera tigris tigris). ), and the response against FCoV type II in cheetahs, jaguars (Panthera onca), African lions, Bengal tigers, lynxes (Lynx canadensis), snow leopards (Panthera uncia) and African leopards (Panthera pardus pardus).⁵⁶ In some cases, type II FCoV is considered to be more transmissible compared to type I virus in domestic cats, ¹²³ although due to the presence of both types of viruses in the study facility, this could not be confirmed. The situation in feral cats is unknown.

Clinically, there are two classic forms of the disease in cats, namely effusion or non-fusion ("wet" or "dry"). The wet form of FIP is characterized by effusion in the abdominal or thoracic cavity, or both, or in the pericardial cavities, and the dry form by the presence of pyogranulomatous lesions. However, the clinical symptoms associated with the FIPV biotype can be quite variable and non-specific. FCoV and FIP remained a major global challenge. Vaccination is not recommended, in part due to concerns about negative outcomes due to exacerbation of antibody-dependent infection in the presence of non-neutralizing antibodies.¹²⁵ Despite promising initial studies ^73,84 and other treatment options still do not have approved antiviral therapies. Prevention is difficult and pre-mortem diagnosis is challenging. Current PCR and serological testing methodologies do not distinguish between pathological forms of the virus and antibody titers are unpredictable. In feline populations, FCoV secretion is often observed in non-FIP positive animals. Likewise, antibody titers only provide evidence of previous exposure and a negative antibody test in domestic cats does not rule out the possibility of FCoV secretion.²⁹ Depending on the antibody assay, sensitivity and specificity may provide variable assistance in the diagnosis of FIP, as found by Felten and Hartmann.²⁸ Immunohistochemistry (IHC) currently remains the gold standard for the diagnosis of FIP.²⁸ Managing the virus can be problematic in the management of captive species, and limited reference ranges for blood analysis, high stress scenarios, and possible new presentations are uncertain.

FIP and FCoV in non-domesticated species

Cheetah (Acinonyx jubatus): In 1979 Horzinek and Osterhaus⁵¹ demonstrated a serological response to FCoV in cheetahs for the first time (Table 1). In 1982, it became clear that cheetahs could suffer from the same serious illness as FCoV-infected domestic cats when a FIP outbreak in captive-bred cheetahs at Winston Zoo, Oregon. ^27,88 The first individual (8.5 year old female) was recently introduced into the herd and had non-specific symptoms of the disease including lethargy, anorexia, dehydration, fever and jaundice with hematocrit 30%, hyperproteinemia (9.1 g / dl), leukocytosis and azotemia.⁸⁸ Ascites was present at post-mortem examination, along with fibrinous lesions adjacent to the abdominal organs and fluid in the thoracic cavity.⁸⁸ Petechization has been reported in several organs.⁸⁸ In the months that followed between this initial case and other cases of FIP, occasional diarrhea was observed in other cheetahs.²⁷ Pathology of tissue samples from other animals that died of FIP revealed multifocal necrotic lesions affecting the abdominal organs (liver, kidney, pancreas, spleen), lymph nodes, and thyroid gland. Facial erosions and ulcerative glossitis have also been observed.²⁷ Although it is not possible to rule out calicivirus, which also causes these ulcers, there are a number of recent cases with human patients with COVID-19 who have experienced tongue ulcers due to infection.⁹⁴ Longitudinal serological monitoring following the first case of cheetah FIP showed seroconversion, and by 1983 each cheetah in the park was serologically positive.^6,27,45 During this epidemic, almost 60% cheetahs succumbed to FIP.^25,45 Early characterization of the cheetah isolate involved inoculation of several cell lines, including feline Crandall-Rees kidney cells (CRFK), Felis catus-4 fetal cells (FCWF-4), and two kidney-derived cheetah cell lines (AjuKid234) or fibroblasts (AjuFib238).²⁴ CRFK cells showed similar properties after inoculation with cheetah isolates compared to the two FCoV type I strains, while FCWF-4 cells were less permissive to the isolate. Across cheetah cell lines, AjuKid234 cell transmissibility was low.²⁴

Other cases of FIP have also been reported in a litter of three caught, approximately 6-month-old wild cheetahs, co-infected with Toxoplasma gondii.¹¹⁹ Clinical pathology data revealed hypochromic anemia, neutrophilia, lymphopenia and eosinopenia with normal total protein but elevated alpha-2 globulin and gamma globulins.¹¹⁹ In addition to fibrinous peritonitis and lesions affecting the abdominal organs, pulmonary edema and gastric blood have also been reported.¹¹⁹ In this report, the authors point to a lack of diet; however, other predisposing factors may include relocation stress and genetic predisposition.¹¹⁹ In a pathological study of 31 dead cheetahs kept in captivity in North America, the FIP was considered the cause of death in two animals.⁷¹ Although FIP was undeniably considered a fatal disease in domestic cats, many hypotheses have been considered that have made cheetahs particularly susceptible to FIP, including genetic control of the immune response, especially the homogeneity of the majority histocompatibility complex (MHC). ^25,79 Analysis of the cheetah-infected virus showed deletion mutations in gene 7a, although interestingly, genes 7a and 7b were intact in cheetahs with FIP.⁵⁸ Since the first recorded case of cheetah FIP, the disease remains sporadic despite evidence of exposure. No further devastating losses have been observed since the initial outbreaks of the FIP epidemic in cheetahs, although research continues.

Following the outbreak of FIP in Oregon, FCoV monitoring revealed a common antibody response to the virus (Table 1). Initial tests have shown that faecal excretion and antibody titers are often inconsistent.⁴⁵ In a study of farmed and wild South African cheetahs, an overall seroprevalence of 57% (169/298) was observed compared to 36% (66/182) in those who secreted the viral antigen, as assessed by nested reverse transcription polymerase chain reaction (RT / nCR). .⁵⁷ In 49 animals that shed viral antigen, 46 showed an antibody response against FCoV type I, 45 showed an antibody response against FCoV type II, and three showed no antibody response.⁵⁷ Whether this high seroprevalence against both type I and II FCoV was caused by a number of circulating viruses, cross-reactivity, or other factors remains unknown, but this finding is unique. In South African cheetahs that were observed longitudinally (n¼48), almost a fifth (n¼10) excreted antigen at more than one time point, even after a negative test after a positive test.⁵⁷ In serological research, Munson et al. in Namibia⁷² were more seropositive males, with 5/14 (36%) juvenile males showing seroprevalence compared to 0/8 juvenile females and 13/37 (35%) adult males compared to 3/13 (23%) adult females.

Lifestyle and risk factors for wild and captive species may vary depending on the disease. In a study among captive cheetahs in the United States, three of the 22 healthy cheetahs shed viral antigen (RT / nPCR) (Table 2).⁵⁵ Of the three viral antigen shedding animals, one animal was positive for anti-FCoV type I antibodies and the other two were seronegative.⁵⁵ In another 19 animals, the antibody response was determined in 15 animals, of which 10 showed no antibodies, four animals were seropositive against type I virus and one animal was seropositive against both type I and type II viruses.⁵⁵ However, in another study on 10 cheetahs that did not shed the virus, the antibodies were common against type II virus (5/10) compared to type I (4/10) or both (1/10). Discrepancies in the response of specific antibodies may be due to changes in management strategies, seasonal fluctuations, or differences in sample handling. In addition, the differences between the antibody response and faecal excretion pose a challenge to the actual disease state of the animal, both in captive cheetahs and in wild cheetahs. To provide guidance on the testing strategy, faecal samples were collected from 25 cheetahs in captivity in the United States for 30 days.³⁸ Of these faecal samples, viral antigen was found in 13% (4/31 samples) fecal samples from one animal to 93% (26/28 samples) samples from one animal.³⁸

The role of persistent cheetah infections in the development of ulcerative colitis and other long-term consequences remains relatively unclear and represents a potential area for study.^96,112 In 1993 Munson⁷¹ described in two dead cheetahs with lesions characteristic of FIP also pathological changes in the villi of the small intestine in one animal (FIP did not consider the cause of death in even one cheetah). In a sample of nine cheetahs with necrotizing colitis and diarrhea that ruled out FCoV, six animals showed antibodies only to FCoV type I, two animals showed antibodies to both type I and type II, and one animal was not positive for antibodies of any serotype.⁵⁶ Furthermore, FCoV was diagnosed in cheetah pups showing acute hind limb paresis, but no apparent relationship to serology was demonstrated, and antibody titers varied from animal to animal during testing.^120,121 Feline leukoencephalomyelopathy (LFL) in large cats has been identified in many cheetahs and two other feline species in the United States. The disease is characterized by progressive vision loss, disorientation and difficulty eating.⁷ In the process of understanding the etiology of LFL, 14 animals were tested immunohistochemically for FCoV, but no animals in this small subgroup showed positive.⁷

European wild cat (Felis silvestris): In 1993, Watt and colleagues¹²⁴ reported an outbreak of FIP in European feral cats in a closed colony that had existed for almost 14 years and affected only males. Numerous clinical signs have been reported, along with multisystem organ involvement, including liver, kidney, intestine, brain, lung, heart, and lymphatic tissue.¹²⁴ Respiratory symptoms were noted in two cats.¹²⁴ It has not been established how cats became infected and it is considered unlikely that a stray cat could introduce the virus. Whether the source of the contamination could have been the nurse or the nurses at the zoo was not further investigated. It was also not determined whether there was long-term shedding of the virus. Interestingly, a previous study by McOrist et al.⁶⁴ was unable to identify European feral kittens as serologically positive for FCoV exposure by indirect immunofluorescence assay (IFA) performed on plasma and directed against the Wellcome virus strain (Table 3). Most recently, however, Heddergott et al.⁴⁴ reported 47.1% (16/34) seroprevalence in a sample of 34 European wild cats in Luxembourg and found that increasing age was a predictor of antibody status. Of the 16 seropositive cats, 75% were seropositive for at least one other common feline viral infection (feline leukemia virus, feline parvovirus, feline calicivirus, or feline herpesvirus).⁴⁴ Paradoxically, another group of European feral cats was seronegative in Portugal (0/26), although a third (5/15) of faecal samples were positive for viral antigen (Tables 2, 3).¹⁹ Differences in sampling and processing may explain some inconsistencies; Possible additional differences include interactions with domestic and other felines and the nature of FCoV infections.

American Puma (Puma concolor): A case of FIP has been reported in a young male American cougar, moving freely in California, who, in addition to enlarged lymph nodes, also had lesions in the small and large intestine, heart, lungs and brain. Interestingly, the kidneys appeared histologically normal, but viral RNA was evident.¹⁰⁸ This case report is unique in demonstrating FIP in a free-moving animal, especially in a species that often lives alone. The risk of further cases of FIP in free-ranging bombs remains unknown and other pathogens, such as the zoonotic disease Yersinia pseudotuberculosis, have been reported in bombs with pyogranulomatous liver lesions and peritonitis.³

According to serological research in California bombs, seroprevalence appears to fluctuate during the 18 years during which samples were taken.³³ In 1992, only three samples were taken, but one animal was positive, while in the next 3 years no animal was positive (n ¼46). By 1996, seroprevalence began to rise and peaked in 2000–2001, and after another period of decline, the highest seroprevalence reached almost 50% (n ¼ 29).³³ Longitudinal data provided by Foley et al.³³ are important because previous years do not predict future seroprevalence. An additional study in the Rocky Mountains demonstrates a high degree of correlation between antibody levels in females and their pups, even when the pups were 4 months of age or older at the time of sampling.⁴ In addition, statistical modeling suggests that increasing age and population location are risk factors for FCoV exposure.⁴ Other seroprevalences have been reported in the United States (Table 3).

Tiger (Panthera tigris): FIP was reported in an 8-year-old male tiger in captivity in Romania, which initially showed non-specific clinical signs.⁴⁹ The postmortem examination corresponded to FIP and revealed uveitis, abdominal discharge and, in addition to pericarditis, serous fluid in the pericardial space.⁴⁹ Macrophage staining for FIP was consistent with the diagnosis, with infected macrophages being identified in the lung and other tissues.⁴⁹ Amyloid deposits have also been reported.⁴⁹ There was an increase in serum amyloid A in cats with FIP, potentially similar to human patients with SARS-CoV-2,^39,69 Interestingly, amyloidosis is often observed in black-legged cats (Felis nigripes); thus, there may be other host factors that lead to the presence of amyloid plaques in the tiger.¹¹¹ Finally, in a tiger with FIP, liquefied necrosis was detected in the spleen, although this may not be specific for FIP infection.⁴⁹

In addition to the described case of FIP in tigers, one case of peritonitis in tigers was recorded in a survey of Italian zoos between 2004 and 2015; however, whether it was related to FCoV infection has not been established and is unlikely.¹⁰¹ A compilation of mortality data on captive-bred tigers in India identified peritonitis in addition to enteritis, gastritis, gastroenteritis, hepatitis and gastric ulcers.¹⁰⁷ Although no viral etiology has been reported, gastrointestinal diseases have been one of the most common causes of mortality, even during the monsoon season.¹⁰⁷

In Siberian or Amur tigers (Panthera tigris altaica), 43% animals (n-44) were positive for the presence of FCoV antibodies (Table 4).⁴² Neither sex nor age was associated with seroprevalence, although longitudinal monitoring led to three other tigers becoming seropositive.⁴² In one case report of a Siberian tiger that succumbed to a morbillivirus infection, IFA demonstrated the presence of anti-FCoV antibodies, although this was unlikely to have contributed to the animal's death.⁹⁰ In the case report of an captured Amur tiger, which was originally presented with anorexia and lethargy, the IFA was negative for FCoV; further diagnosis revealed bacterial pyothorax.¹⁰⁴ Virus shedding was observed in another Siberian tiger considered clinically healthy, but no antibody response.⁵⁶

Of the three Bengal tigers housed in the same institution, two showed antibody responses to both type I and type II FCoV, and the third showed anti-type II antibodies, although none of these animals secreted viral antigen.⁵⁶

Lion (Panthera leo): In 1970, Colby reported two cases of FIP in lions.¹³ Although this initial report did not provide any clinical details, the ocular form of FIP in a 15-year-old captive lion was recently described and confirmed to be blind.⁷⁵ The patient's clinical pathology, including anisocytosis and hypochromasia, was non-specific for FIP, although PCV (HCT) was lower than normal (31%) and total plasma protein was higher than normal (7.8 g / dl), values commonly found in domestic Histopathology revealed bilateral panuveitis and retinal detachment along with an infiltrate of lymphocytes, plasma cells and several macrophages.⁷⁵ In the anterior chamber, the exudate was characterized as eosinophilic and proteinaceous, and lesions were found in the brain.⁷⁵

In free-ranging lions in East Africa, 57% serum samples were evaluated as evidence of an antibody response by IFA (Table 3). ⁴⁷ FCoV secretion was previously observed in the captive African lion, but without an antibody response.⁵⁶ Similarly, two other lions in different institutions did not actively secrete the virus, but showed antibody responses.^56,57 Interestingly, one of these lions showed a low-level antibody response to both type I and type II FCoV.⁵⁶. In the case of a series of lions with encephalitis, one lion was tested for antibodies to FCoV, but appeared negative.¹¹⁶ In a study of inbred lions compared to an outbred population, the relationship between inbreed status and FCoV antibodies was not statistically significant.¹¹⁵ A longitudinal study of lions in the Serengeti showed a high degree of seroprevalence variability each year, especially in younger animals.⁸⁰ In addition, these data revealed possible effects on fertility and a possible link between the spread of the virus and higher population densities.⁸⁰ The second longitudinal study showed a high degree of seroprevalence; in most age groups more than 50% animals demonstrated seroprevalence and a trend of increasing seroprevalence with increasing age, with a few exceptions³⁴ (additional data from the study). In addition, FCoV seroprevalence may predict the occurrence of calicivirus or feline immunodeficiency virus (FIV).³⁴

Serval (Leptailurus serval): Juan-Salle´sa et al.⁵⁴ report two FIP cases in a group of four coexisting servers. Case 1, a two-year-old male, stopped entering the indoor area where the animals were routinely fed. It was not determined whether the cause was neurological behavioral changes or anorexia. Case 2 was an 8-year-old female with a melengesterol acetate implant, with non-specific clinical signs and mild abdominal distension.⁵⁴ Exploratory laparotomy revealed abdominal effusion and FIP-like lesions, and clinical pathology demonstrated leukocytosis and anemia. Although no extensive lesions were reported in Case 1, histology revealed fibrinopurulent ventriculitis; accumulation of neutrophils, fibrin and macrophages in the brain; and severe leptomeningitis. Focal pyogranulomatous lesions were present in the kidneys.⁵⁴ In case 2, in addition to pyogranulomatous necrotizing splenitis, pyogranulomatous lesions were also present in the mesentery, liver and gallbladder.⁵⁴

Feature: VanRensburg and Silkstone¹¹⁹ mention personal communication about FIP that has affected at least one red lynx. Of the 25 red lynx in the Golden Gate National Recreation Area near San Francisco, California, the only urban red lynx was serologically positive for FCoV (Table 5).⁹⁵ FCoV secretion was reported in two captive red lynx in the US Zoo, despite the absence of an antibody response.⁵⁶ Similarly, in the second US zoological collection, one healthy feature in captivity secreted FCoV in the absence of an antibody response, while the other trait did not secrete virus but showed a low-level antibody response against FCoV type II.⁵⁶

The Spanish lynx (Lynx pardinus) was once considered one of the most endangered wild carnivores, and as such, the risks of FCoV infection could be devastating. In one study of 22 Spanish traits evaluated by competitive enzyme-linked immunosorbent assay (ELISA) and 25 animals evaluated by IFA or rapid immunochromography, no animals showed signs of exposure or active FCoV infection.⁶⁷. However, further studies have shown a seroprevalence of approximately 26%, with no apparent relationship between FCoV and FeLV.⁶⁵ In a single sample of the Spanish lynx, 19/74 were positive for FCoV IFA exposure, although 0/68 animals shed virus in the faeces, as assessed by RT-PCR.⁶⁵ In two other lynx features for which the cause of death could not be determined, neither animal was found to be IHC positive for FCoV, despite pancreatitis and fibrinous serositis in one of these animals.¹⁰² The effect of FCoV on the development of other disease pathologies, including membrane glomerulonephritis and lymphoid cell depletion, has also been investigated, but no study has shown an apparent link to disease development.^53,86

Leopard (Panthera pardus): FIP has also been reported in leopards in Germany.¹¹⁷ No exclusion of FCoV was observed in three African leopards at two US zoos, but all three were serologically positive for type II FCoV.⁵⁶ Of the other two African leopards in South Africa, only one animal was seropositive and none shed the virus (Tables 2, 4).⁵⁷

Bengal wild cat (Prionailurus bengalensis): Between 2005 and 2006, 35 of a total of 1,453 rectal swab specimens obtained from Asian leopard cats in wildlife markets were positive for the new coronavirus, which found that the spike protein was phylogenetically similar to the spike protein of other alpha-coraviruses.¹⁶ However, this virus showed a phylogenetic relationship outside the group, suggesting that it separated at the beginning of the coronavirus evolutionary history, or probably represented an unidentified group of coronaviruses. Recent analysis suggests that the spike protein is related to a newly identified genus of deltacoronavirus.¹¹⁰ According to previously published results of an RT seminested PCR assay targeting viral polymerase, 8.8% (3/34) wild Bengal cats were positive for pancoronavirus, with a close phylogenetic relationship to FCoV.¹² In Iriomote cats, subspecies (Prionailurus bengalensis iriomotensis), most animals (14/17) tested by ELISA against canine coronaviruses showed the presence of antibody (Tables 2, 4).⁷⁰

Clouded Leopard (Neofelis nebulosa): In a survey of the causes of mortality of non-domesticated carnivores at Nandankanan Biological Park in Bhubaneswar, Orissa, India, peritonitis was thought to be the cause of death in one clouded leopard; ⁹² however, FCoV has not been studied.

Snow Leopard (Panthera uncia): FCoV secretion was observed in a healthy captive snow leopard, although no antibody response was evident.⁵⁶ In contrast, virus excretion was not observed in two snow leopards at different institutions, but were positive for antibodies to FCoV type II. ⁵⁶ In the case report of a 6-week-old captured bred male snow leopard with neurological disease, including cerebellar degeneration and encephalomyelopathy, FCoV was investigated as a potential cause, but was not identified.¹⁰⁹

Ocelot (Leopardus pardalis): Philoni et al.³⁰ reported one seropositive, caught, apparently healthy Amazonian ocelot, which was also serologically positive for feline herpesvirus¹, feline calicivirus, feline parvovirus and Bartonella henselae. This single observation may have been an anomaly and the result of other underlying medical conditions. Interestingly, however, the anti-B. henselae antibody response was associated with the anti-FCoV antibody in domestic cats.⁴⁰ It is not known whether a similar association exists for ocelots or other cats. Natural FCoV circulation in isolated ocelots nevertheless appears to be rare (Tables 2, 4).^31,35

Swamp cat (Felis chaus): In a survey of non-domesticated feline carnivores in Nandankanan Biological Park, peritonitis was thought to be the cause of death in one jungle cat; ⁹² however, FCoV has not been studied.

American Jaguar (Panthera onca): FIP was considered a differential diagnosis in a case report of a newborn jaguar with respiratory difficulties that progressed to ascites.³⁶ However, the confirmatory diagnosis did not take place and the pup recovered after extensive treatment.³⁶ In two apparently healthy jaguars in captivity, FCoV secretion was observed; in addition, one of these animals showed an antibody response to FCoV type II. (Tables 2, 4)⁵⁶

Caracal caracal: Four caracals showed an antibody response, although none animal excreted FCoV (Tables 2, 4).^56,57

Discussion

In addition to other infectious diseases, feline coronavirus challenges the effort to preserve non-domesticated felines.⁷⁴ Credible FIP records exist for seven non-domesticated species of felines and 15 species have shown seroprevalence. However, antigen release is observed relatively rarely. Screening and diagnostic testing are challenging and it is still difficult to determine the source of FCoV infection. In the cases reported by Watt et al.¹²⁴ and Juan-Salle et al., ⁵⁴ it was considered unlikely that the stray cat would be the source of the infection. Whether the virus was constantly circulating in these animals, or whether the virus was introduced in any other way, remains unknown. This leads to the need to always be vigilant with regard to biosecurity, especially in the case of endangered animals. The use of faeces to identify FCoV creates a strategy for easy sampling of both wild and captive animals; however, PCR from fecal contents may be inhibited.²⁰ In addition, treacherous onset in some cases of FIP often requires an investigation of the potential or exposure to FCoV infection. The spectrum of clinical manifestations, in addition to the "standard clinical picture", also includes dermatological lesions, ^10,93 myocarditis 22 and upper respiratory tract diseases.²

Reports of FIP outbreaks in non-domestic cats have shown that males are likely to be at higher risk than females, similar to those observed in domestic cats. FIP has only been found in European wild male cats, and through individual reports we know of FIP in mountain lion, lion and tiger, which only affected males. ^{49,75,108, 124} In domestic cats, males are also over-represented in FIP cases.⁷⁸ In addition, in humans, SARSCoV-2 infection led to more serious consequences in men compared to women.⁶⁶

While shelters and kennels are the optimal place for FCoV to spread, FCoV circulation in free-moving species can be improved by the solitary lifestyle of some of these species. The virus survives in the environment for a relatively short time, so that the transmission takes place mainly through direct or indirect interactions within the species or with other felines. In some cases, domestic cats can be a source of prey for larger non-domesticated felines. It is not known whether an infection could occur in this way, but it could be the cause of certain antibody responses or antigen secretion in the feces. Habitat changes, including fragmentation and habitat expansion, may, in addition to exposing domestic cats, lead to increased interactions within the same species in the wild.

Although antibody testing is useful for detecting previous exposure, even in non-domesticated species, a seronegative assay does not always indicate the absence of virus shedding.^38,56 This discrepancy poses a challenge to understanding the true levels of FCoV circulating in feral cat populations. Several testing strategies have demonstrated the ability to detect virus shedding with a high degree of reliability, including the feasible possibility of obtaining five samples in a row.³⁸ Taking into account critically endangered species, individuals must be adequately tested before being introduced to individuals of the same species, together with stress minimization, unnecessary anesthesia, etc. Sequential testing of faecal samples has been performed in captive animals, and similar strategies may be useful for understanding the presence or absence of FCoV in wild animal populations.

Acknowledgments: The authors greatly appreciate the members of the Whittaker Lab and the Cheetah Conservation Fund for their helpful discussion in preparing this manuscript. Jobs Dr. Stouta is supported by the National Institutes of Health comparative medicine training program (T32OD011000). The work in the author's laboratory (GRW) is partly funded by the Cornell Feline Health Center and the Winn Feline Foundation.

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02.2019, Translation 7.5.2021
Aydin Hakan, Yildirim Serkan
Original complete article: Investigation of the relationship between feline infectious peritonitis and retroviruses in cats

Abstract

Feline infectious peritonitis (FIP) infection is a highly pathogenic and fatal systemic inflammatory disease of cats. Although FIP infection is associated with the immune system, there are several predisposing factors for the disease. FIP has a high degree of interaction with other infectious agents. Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections play a predisposing role by reducing immunity to FIP. This study was performed with Turkish Van cats, a cat breed specific to the Turkish Lake Van region, between 2014 and 2015. Ascites and signs of organ damage in 15 Van cats were identified by pathological analysis at Van Yüzüncü Yıl University. Polymerase chain reaction (PCR) was used to examine the presence of FIV and FeLV in the homogenate of brain, lung, liver, kidney, spleen and intestinal samples with lesions. FIV was identified in 1 (6.7%) of 15 cats and all cat samples were positive for FeLV (100%). Phylogenetic analysis of the positive samples revealed that it was endogenous FeLV. In the FIV phylogenetic analysis, our study strain was found to belong to the FIV subgroup B. The fact that FIP-positive cats were also positive for FeLV has drawn attention to the pathogenesis of endogenous-FeLV in malignancies, which has not yet been understood. In particular, it was concluded that the prevalence of FIP infection can be reduced by paying attention to the vaccination of young cats with FIV and FeLV in environments with a high cat population.

Introduction

Feline infectious peritonitis (FIP) is a fatal disease caused by the feline infectious peritonitis virus (FIPV), which is the result of a coronavirus mutation (FCoV). While FCoV has an enteric form, FIPV is a pathogenic type with a systemic infection. The dramatic increase in the cat population after World War II and the subsequent placement of cats in shelters paved the way for the most important scenario of the FIP. FCoV is thought to spread in the cat population due to their mass concentration, leading to mutant FIPV ^{[1, 2]}. Although the disease is not common in older cats, FIP infection is one of the leading causes of death in young cats aged 3 to 24 months. There are two types of FIP: effusive (wet) and granulomatous (dry). While the wet form is the most common and takes the form of inflammatory exudate in body cavities, the granulomatous form attacks the organs ^{[1, 3]}. The hypothesis of FIP is based on the fact that viral replication in cats with FCoV occurs in enterocytes. However, another variant of FIPV that can replicate in macrophages due to a mutation in the FCoV genome has also been published. ^[4]. FIP is a slow, progressive disease and can last for weeks or months. However, retroviral opportunistic infections such as feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV), which cause immunosuppression, lead to the inevitable death by reduced immunity to FIP and accelerated disease. Although cats overcome FIP infection, secondary FeLV and FIV infection reactivates FIPV and dies in a short time. ^[4-6].

The Retroviridae family, which includes immune-suppressing viruses such as FIV and FeLV, has a linear, single-stranded, non-segmented, genome with a positive RNA polarity of 8-13 kb. Retroviruses that are morphologically enveloped and have icosahedral symmetry contain gag, pol and envelope gene regions ^{[7, 8]}.

The immune system plays an important role in the clinical manifestations of FIP (wet / granulomatous). In granulomatous cases (dry FIP), T-cell mediated cellular immunity and humoral immunity are key. In FeLV infections in particular, FeLV infection affects the clinic of FIP infection due to disruption of this immune system mechanism. ^[9]. In addition, cats with FIP infection in the form of a latent or limited infection may experience re-infection due to FeLV. This will again result in death due to the FIP [5]. FIV infection is one of the most important co-infection factors in FIP outbreaks in cats with FCoV. It is reported that FCoV can achieve a very high rate of replication in immunosuppressed cats due to FIV infection, and that this may be a predisposing factor for FCoV to FIPV mutation. In cats infected with FIV and FCoV, FIV causes a high degree of FCoV replication and may lead to an outbreak of FIP in a short time. ^[1]. These retroviruses, which disrupt the immune system, are the most important clinical indicators of FCoV mutation. ^[1]. In the case of FIP-induced immune suppression, the transmission of opportunistic pathogens such as FIV / FeLV may occur. ^{[10, 11]}. Although there are currently commercial vaccines against FIV and FeLV, there is no protective vaccination or effective treatment against FIP in Turkey. ^[12].

This study was conducted to determine the interactions between FIPV infection and retrovirus co-infection (FIV / FeLV) in Van cats, a cat breed originating in Turkey.

Materials and methods

The population for the study consisted of a total of 15 cats with suspected FIP, which were transported to Van Yüzüncü Yıl University between 2014 and 2015 for pathological analysis of the death of the Turkish Van cat breed. Refrigerated tissue samples from the brain, lungs, liver, kidneys, spleen and intestines were sent refrigerated to a virology laboratory. For histopathological examinations, tissue samples from all tissues were taken into 10% formalin solution. After 48 hours of fixation in formalin solution, the samples were washed with running tap water for 10 hours. After passing through alcohol (70 °, 80 °, 90 °, 96 ° and 100 °) and xylene, they were immersed in paraffin blocks. Four thick sections were taken from each block to prepare slides on a slide. For histopathological examination, they were stained with hematoxylin-eosin (HE) and examined by light microscopy.

Immunohistochemical examination

All sections taken into adhesion (poly-L-lysine) immunoperoxidase slides were passed through xylene and alcohol series for deparaffinization and dehydration. They were then washed for 5 minutes in distilled water, 5 minutes with phosphate buffer (PBS, pH 7.2) and the endogenous peroxidase was inactivated by standing in 3% H₂0₂ for 3 minutes. After washing for 5-10 minutes in PBS, they were incubated for 5 minutes with Protein Block, which is compatible with all primary and secondary antibodies, to prevent non-specific staining of the substrate. At the end of the incubation, the excess blocking solution remaining on the tissue sections was removed and drops of primary antibodies (VGEF, caspase 3, caspase 8, caspase 9, PCNA, neuronectin, TRPM 1, TRPM 2, α synuclein, apelin) and PBS in the control group were used without washing. In accordance with the primary antibody, they were left to stand overnight at +4°C. After washing twice for 5 min. with PBS, they were incubated for 10-30 min. at room temperature with biotinylated secondary antibody. Sections washed again with PBS were left to stand in streptavidin-peroxidase for 10-30 minutes, then washed in the same manner with PBS. After washing, 3-3 'diaminobenzidine (DAB) chromogen was added to the sections and the mixture was left to stand for 5-10 minutes. according to the uptake of the chromogen. After immersion in Mayer's hematoxylin for 1-2 minutes for staining, they were rinsed with running water. They were then passed through an alcohol and xylene series, covered with coverslips and examined with a light microscope (Leica DM 1000). The sections were scored as negative (-), mild (+), moderate (++) and severe (+++) positive according to their immunopositivity.

Investigation of retroviral infections in FIP positive cats

For FIV and FeLV DNA testing of FIP-positive cats, liver, spleen, kidney and brain tissue samples with lesions were taken according to pathological analysis. These tissue samples were combined to prepare a homogenate. Nucleic acid isolation was performed using a supernatant from this tissue homogenate using a commercial kit (GF-1 Nucleic Acid Extraction Kits, Vivantis, Malaysia). The resulting nucleic acid suspension was used as a template for PCR for FIV and FeLV analysis. For FIV DNA screening, nested-PCR was performed using primer pairs specific for the V3-V6 region of the env gene (Table 1). ^[13]. For FeLV detection, PCR was performed using primer pairs specific for the FeLV env gene region ^[14]. The primer sequences and product sizes are listed in Table 1.

Sequential and phylogenetic analysis

After gel electrophoresis, FIV / FeLV positive PCR products were subjected to sequence analysis. The raw data obtained after sequencing were confirmed by GenBank (NCBI National Center for Biotechnology Information / BLAST). Sequences of reference strains and studied samples were arranged by BioEdit program (version 7.0.5) ^[15]. Phylogenetic analysis was performed using the MEGA program (version 6.0) by the neighborhood method ^[16].

The results

Pathological findings

At necropsy, gray-white granulomatous lesions in the spleen, liver, kidneys, intestines, lungs and cerebral organs and serous atrophy were observed macroscopically in adipose tissue (Figure 1). In the effusion form, the mucous membranes were found to be strongly icteric and there was a discharge of 1-1.5 l in the abdominal cavity (Figure 2).

Microscopic examination revealed, especially in the non-fusible form, pyogranulomatous lesions, necrotic in the middle, spleen, liver, kidneys, intestines, lungs and brain tissues, surrounded by neutrophils, macrophages, lymphocytes and plasma cells (Figure 3). Vasculitis was found in the vessels in these organs (Figure 4). While these granules were generally observed near the serosa, some of them progressed to parenchymal tissue, especially in the kidneys.

Virological findings

FIV / FeLV DNA was examined by molecular methods in 15 Van cats with FIP diagnosed by pathological analysis. As a result of PCR analysis, FeLV DNA was detected in all 15 cats (100%). In the phylogenetic analysis of FeLV strains in our study and GenBank reference strains, FeLV strains formed two phylogenetic groups: exogenous and endogenous. Sheep and horse retroviruses have been used as a non-member group. In phylogeny, it was observed that all our study strains coalesced together with endogenous FeLV (enFeLV) (Figure 5).

PCR analysis identified FIV in 1 (6.7%) from 15 samples. Subsequently, a sequential reaction and phylogenetic analysis of the FIV strain was performed. Phylogenetic analysis of the FIV strain obtained from our study and reference strains from GenBank showed that the FIV strains were divided into 6 different subtypes from A to F. Our FIV strain was classified as FIV subtype B along with Turkish and Japanese strains (Figure 6).

Discussion

FIP is a systemic infection associated with the immune system caused by coronaviruses. This insidious and deadly infection significantly threatens domestic and street cats that share the same environment with humans, and even breeds of protected cats such as Van cats. Eliminating predisposing factors is the first combat strategy against FIP infection, for which there is not yet an effective method of prevention and treatment in Turkey. Of these factors, cleaning, hygiene and negative interaction in ordinary living spaces are important. Immunosuppressive diseases, such as FIV and FeLV, are the most important predisposing factors for the development of FIPV, a mutant form of coronavirus.

In our study, pathological analysis revealed FIP infection in cats of Van, a breed native to Turkey. Subsequently, the presence of FIV and FeLV was examined in tissue samples obtained from these cats.

FeLV and FIV are pathogenic infectious agents that cause immunosuppression mortality in domestic cats. While FIV is similar to human HIV infection, FeLV causes immunosuppressive, neoplastic, and hematopoietic disorders. ^[17]. Retroviral infections of cats are the most important trigger for FIP infection in cat homes and shelters. Successful results against retroviral infections have been achieved thanks to the aggressive policies of many Western countries, such as the eradication of infections through testing and vaccination. ^[18]. Although vaccines against FIV / FeLV infections exist in Turkey, an adequate and controlled vaccination program has not yet been established.

In our study, FeLV was detected in all cats with FIP. FeLV is divided into 6 groups: A, B, C, D, E and T. These types, called exogenous FeLV, are responsible for leukemia and tumor formation in cats. Endogenous FeLV (enFeLV) are FeLV sequences transfected into the genome of domestic cats. enFeLV proviruses are found in various organs and tissues and perform translation and transcription. However, due to the lack of essential parts of the viral genome, it cannot become an infectious viral particle. However, infection of a cat with exogenous FeLV (field strain) leads to recombination between the two (endogenous / exogenous) and transforms enFeLV into an infectious virus particle. However, it is still unclear whether enFeLV may be responsible for malignant formations in cats with lymphoma where exogenous strains are not detected. ^[19].

In our study, FeLV was detected in all tissue suspensions with pathological lesions collected from Van cats. Phylogenetic analysis showed that they were all enFeLV. Although this suggests that enFeLV may mutate and cause malignant formations, there is no information in the literature to support this hypothesis. There are several studies on coronavirus infection of cats in Turkey. In a study by Pratelli et al., FCoV seropositivity was determined in 62% cats living in environments such as shelters. ^[20]. In a study by Sahna et al. by molecular methods, the rate was identified by FCoV 54% ^[21]. A study in Turkey identified a rare ocular form of FIP infection in cats ^[22]. In a retrospective study of FCoV, FIV and FeLV in 169 sick cats in Turkey, seroprevalence ratios were determined to be 37%, 11% and 1% ^[23]. Similarly, in a study investigating FCoV, FIV and FeLV in chronic gastrointestinal, urinary, symptomatic and healthy cats in Turkey, viral substances were detected in 45.5%, 9.5% and 20.5%, respectively [24]. In our study, one cat was found to be co-infected with FIV and FeLV. In the phylogenetic analysis of the FIV strain identified in our study, it was found to be a subtype group B. In a previous study performed in cats in Turkey, subtype B was identified in parallel with our results. ^[25]. In a study of FIV and FeLV infections in cats, the incidence rates of FIV and FeLV were found to be 22.3% and 5.8%, respectively. ^[26]. Because our study was limited to a specific group, a relatively low FIV / FELV rate was achieved.

Conclusions

Eliminating predisposing factors is the first combat strategy against FIP infection, for which a method of prevention and treatment is not yet effective in Turkey. Although there are vaccines against FIV / FeLV infections that are predisposing factors for FIP, an adequate and controlled vaccination program has not yet been established. Due to the increased incidence of FCoV and predisposing factors, vaccination and awareness of cat owners is important in this regard in order to minimize the spread of FIPV. There is a need to establish a national eradication and control policy for agents such as FIPV and FeLV, which prepare the ground for FIPV infection in shelter cats and even in protected cat breeds.

Compliance with ethical standards

Acknowledgement

SY: For a time he worked at Van Yüzüncü Yıl University. At that time, cat samples were obtained. SY: provided cat samples and performed pathological analyzes, HA: performed virological analysis. SY and HA read the whole article and agreed.

Disclosure of conflicts of interest

The authors declare that they are not aware of the conflict of interest.

Declaration of ethical approval

This study was approved by the Ethics Committee of the Veterinary Faculty Unit of Van Yüzüncü Yıl University (Protocol No. 2015/03).

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29.1.2021, Translation 2.5.2021
Alison E. Stout DVM, Nicole André LVT, Joshua Zimmerberg MD PhD, Susan C. Baker PhD and Gary R. Whittaker PhD
Original article: Coronaviruses as a cause of vascular disease: a comparative medicine approach

Contact the author: e-mail: grw7@cornell.edu. 618 Tower Rd., Ithaca NY 14853 USA.

Abstract

COVID-19 caused by SARS-CoV-2 often manifests as a respiratory disease including cough, dyspnoea, fever and olfactory loss. However, other manifestations of the disease occur in many organ systems, at least in part due to vasculitis and endothelitis. COVID-19-associated multisystem inflammatory syndrome in children (MIS-C) has recently been identified as part of SARS-CoV-2 infection. In feline medicine, feline coronavirus is a common pathogen that can lead to a deadly disease called feline peritonitis (FIP). Like human COVID-19, the clinical manifestations of FIP are due in part to coronavirus-induced vasculitis, which can also lead to fatal multisystemic inflammatory syndrome in cats. Studies investigating how feline coronavirus infection can cause disseminated vasculitis in FIP cats will provide new information that may help people understand COVID-19. We strive for a comparative medicine approach to tackling coronavirus diseases.

The COVID-19 pandemic in 2019-2020 is caused by the recently discovered zoonotic beta-coronavirus classified as SARS-CoV-2. COVID-19 often manifests as a respiratory disease manifested by fever, cough, dyspnoea, olfactory loss, headache, and malaise. More severe disease may include acute respiratory distress syndrome (ARDS); however, the disease also appears to affect other organ systems, including the gastrointestinal, central nervous system (CNS), cardiovascular, renal, hepatic, endocrine, dermatological, hematological, and ophthalmic systems.¹⁷ These systemic symptoms are thought to be due to viral endothelial cell infection and endothelitis⁴⁹ leading to vasculitis.⁵ COVID-19 can be considered both a respiratory disease and a vascular disease, with widespread viral tropism and high transmissibility contributing to the current public health crisis and the differentiation of SARS-CoV-2 from SARS-CoV, despite the receptor (ACE2) common to both viruses.

After studying the history and pathogenesis of feline coronavirus, we see parallels between COVID-19 and feline infectious peritonitis (FIP) with a wide variety across human and feline coronavirus infections, as originally found in SARS.⁴¹ Feline coronavirus (FCoV) in cats is generally considered to be an initial gastrointestinal or rarely respiratory infection that results in FIP, a systemic vascular disease with high morbidity and mortality in some animals.⁴⁶ This feline disease, originally called chronic fibrinous peritonitis, was first described in 1963. ²²; shortly afterwards, the etiological agent (causative agent) was recognized as a coronavirus. This disease, now called feline infectious peritonitis (FIP), occurs worldwide, is almost always fatal, and mainly affects young cats (under 2 years of age).^28,38 The etiological agent responsible for most cases of FIP is feline coronavirus (FCoV), alpha coronavirus.²⁵ While FCoV transmission and infection are widespread in domestic cats, systemic disease develops in only a small subset of animals (Figure 1). Common to these various forms of the disease is the identification of virus-induced vasculitis.²⁷ This review highlights the similarities between COVID-19 and FIP and opens up opportunities for cooperation between the medical and veterinary communities in the fight against these devastating diseases.

Clinical and pathological profile of feline coronavirus infection: parallels with COVID-19

Like COVID-19 in humans, FIP is involved in many manifestations of the disease in cats (Figure 2), which are described as "variably manifested by multiple organ failure, seizures, generalized effusion, or shock."¹⁴ The disease is commonly classified in one of two clinical forms, effusive or "wet" (characterized by accumulation of protein effusion mainly in the peritoneal or pleural cavities) and non-fusive or "dry" (characterized by granulomatous lesions in many organs, including the brain).¹⁹ A “mixed form” is also considered, in the clinical spectrum of which granulomatous and pyogranulomatous lesions occurring in the serous part of the liver, kidneys and intestines are common. The spectrum of pathologies observed in FIP includes serositis, hepatitis, nephritis, uveitis and chorioretinitis.^38,55 Neuropathological manifestations of FIPV in cats include meningitis, meningoencephalitis, ependymitis, and choroidal plexitis with inflammation ranging from lymphoplasma to pyogranulomatous, and often including vasculitis and perivasculitis. Ophthalmic manifestations, including uveitis and chorioretinitis, are also common. Case reports point to associated heart lesions, including myocarditis and fibrinous epicarditis.^12,34 Dermatological lesions that are less commonly observed in FIP include erythema, nodules, and papules.^4,8,10 In some cases, early respiratory symptoms have been reported^8,54 and in some cats, immunostaining identified the virus in the lung parenchyma.²⁷ Our latest laboratory has confirmed the presence of the virus in the upper respiratory tract by immunostaining.¹

A similar feature in FIP outbreaks and patients with COVID-19 is extensive vascular involvement.^3,5,20 In one of the first reports recognizing vascular involvement in FIP cases, four types of lesions were described: “perivascular edema,” “vascular wall degeneration,” “endothelial proliferation,” and “adventitial and/or perivascular infiltration.”²⁰ In 1989, Boudreaux and colleagues described FIP lesions in several cats as "obliterating" vascular walls. Similarly, August concluded in 1984 that "FIP could be more accurately termed feline coronavirus vasculitis."³ As a naturally occurring disease in cats, FIP and FCoV infection provide an opportunity to understand the pathogenesis of SARS-CoV-2, especially with regard to the co-development of vascular manifestations.

Although observations of myocarditis, rhinitis or skin lesions in FIP are rare, they allow comparison with COVID-19^1,8,12,42. Frostbite-like lesions, a condition colloquially called "COVID-fingers", have been reported in several patients with COVID-19, although the direct association of these manifestations with COVID-19 remains open.^30,33 In cats, a similar condition is known as plasma cell subdermatitis and may also be associated with FCoV. Plasma cell pododermatitis manifests as paw inflammation along with the potential for color changes, ulcerations, and cracks.⁴⁰ In the case of feline subdermatitis, an association with an infectious disease has been suspected.¹¹ In addition to observing similar paw lesions in FCoV-secreting cats, our laboratory has previously identified subdermatitis in cats due to FIP. Although "COVID fingers" and subdermatitis in cats are rare consequences of infection, they further highlight the spectrum of similarities between COVID-19 and FIP.

One of the characteristic symptoms of COVID-19 is anosmia (loss of smell). Also, in most cases of FIP, the onset of anorexia is common.^38,53 Although a non-specific clinical symptom that can be attributed to many different causes, anorexia can be attributed to loss of odor perception - an extremely important sensation that drives appetite in cats. While anosmia is difficult to measure in cats, the relationship between olfactory bulb infection and possible spread to the brain by human and feline coronaviruses deserves further evaluation, especially with regard to the neurological manifestations of both diseases.^14,29

Immune component of FIP disease

Based on previous observations of complement and immunoglobulin circulation, including immune complexes, FIP is classically characterized as immune-mediated.^24,39 In particular, type III hypersensitivity and Arthus-like reactions lead to the development of immune complexes that can build up in the walls of blood vessels, leading to vasculitis. Despite attempts to classify FIP as a type III hypersensitivity reaction, further work by Kipar and colleagues has shown numerous differences.^26,27 Type III hypersensitivity has also been studied in patients with COVID-19.⁴³ The importance of FIP as a type IV hypersensitivity reaction with apparent perivascular lesions consisting of T-lymphocytes, granulocytes and a characteristic feature of FIP, macrophage infiltrates, was also evaluated.³⁶ Delayed-type hypersensitivity is observed in cats inoculated intradermally with FCoV, leading to perivascular cellular infiltrates of macrophages, neutrophils and lymphocytes in the absence of immune complexes.⁵² However, cellular extravasation associated with FIP lesions contributes to endothelial disruption and the inflammatory response that underlies FIP.27 Specifically, antigen (LFA) -1 associated with β2 integrin adhesion molecules and lymphocytes, β1 integrin very late antigen (VLA) -4 and β2 macrophage antigen (Mac-1) have been demonstrated to be increased in peripheral blood leukocytes isolated from cats with FIP.35 Integrins (LFA) -1 and (VLA) -4 play an important role in neutrophil and monocyte migration across the endothelium and are involved in the development of vasculitis in other disease processes,^9,47 while (Mac-1) may play an additional role in the development of thrombosis.⁵¹ The hypothetical role of endothelitis, which underlies COVID-19⁴⁹, parallels previous findings on FIP and the resulting acute vasculitis.

The interaction between vascular pathogenesis and hemodynamics has long been known in FIP.⁵³ In addition to peripheral pyogranulomatous lesions, phlebitis, endothelial edema with inflammatory infiltrates of tunica media, thrombophlebitis and thrombosis were observed in cats experimentally infected with FCoV, classical FIP.⁵³ Additionally, thrombocytopenia, anemia, jaundice, decreased clotting factors VII-XII, increased antithrombin III, and prolonged activated partial thromboplastin time, prothrombin time, and thrombin time are observed after experimental infection.^7,53 Thus, the end result for some cats with FIP is the development of disseminated intravascular coagulation.^7,53 This is in contrast to the course in human patients with JIS, who are often anticoagulated at some point in their hospital stay, and the rare development of DIC in humans is associated with a worse course in COVID-19.⁴⁸ The fibrin product D-dimer, a DIC marker, has been studied rarely in cats with FIP, but has been increased in two cases - as expected, observations in patients with COVID-19 have shown this.

The development of FIP in a subset of cats, although most FCoV-infected cats undergo only a subclinical phase, is thought to be the result of an initial immune response.³⁷ Strong cell-mediated immunity (CMI) together with humoral immunity is thought to lead to a subclinical course of the disease, while a lack of CMI leads to an effusive form of the disease and partial CMI leads to a dry form of the disease.³⁷ Similarly, with respect to the immune response, multisystem inflammatory syndrome in children (MIS-C), which may be a manifestation of SARS-CoV-2, has recently been described.¹⁶which may develop due to a delayed response to interferon or due to an aberrant IgG response promoting disease progression.⁴⁴ In addition to MIS-C, multisystem inflammatory syndrome in adults (MIS-A) has been described as a result of SARS-CoV-2 exposure.³²

Antibody-dependent exacerbation of infection

In the case of SARS-CoV-2, there were concerns about antibody-dependent enhancement (ADE) infection,¹³ largely based on experience with other human viruses, such as respiratory syncytial virus, influenza and dengue; despite the need for caution, formal evidence linking ADE to COVID-19 clinical outcomes remains insufficient.² In cats, it is noteworthy that the involvement of ADE in FCoV infection and the development of FIP with antibodies that would provide means for FCoV to gain macrophage entry has long been considered.²¹ So far, despite several trials, a viable FCoV / FIP vaccine is still not available.^45,50 In contrast to these FIP vaccine studies, preliminary immunization results for COVID-19, for example with stabilized spike immunogens, documented neutralizing, not potentiating, antibody responses.²³

Perspectives

COVID-19 is a complex and challenging disease in humans, and similarly, FIP is a complex disease in cats with many factors contributing to the outcome of the disease. By analogy with the persisting question in metastatic tumor research, "seed and soil", the availability of immunological, pathological and physiological samples and data on these similarly fatal inflammatory syndromes from two different coronaviruses in their own host species allow testing of structure / function hypotheses in venous vasculitis . In addition, due to the lack of enthusiasm for clinical biopsy of human patients with fulminant COVID-19 and the great difficulty in achieving good preservation of pathological specimens, it will take a very long time to learn more about the presence or absence of coronavirus in human patients at baseline and during their progression. inflammatory phase of COVID-19.

As a naturally occurring disease with many parallels with COVID-19, we think that FIP may help in future COVID-19 research. Both baseline vasculopathy in both FIP and COVID-19 underline the need to think of these diseases as multisystemic rather than organ-specific diseases. Although often considered a key enteric mode of transmission for FCoV, it is clear that the oropharyngus is a robust portal to host entry. Another factor to consider is that SARS-CoV-2 infects cats.15,18 Although not currently considered a serious disease in cats, it is not a robust animal model for COVID-19, The increased rate of SARS-CoV-2 infection in cats and possible FCoV co-infections raise, in addition to the possibility of viral recombination, concerns about the overlap of pathology and clinical manifestations of human and feline coronaviruses with surprisingly similar vascular disease profiles. However, the fact that SARS-CoV-2 naturally infects cats may offer a model for preclinical evaluation of SARS-CoV-2 vaccines. We believe that further studies are needed to investigate how coronaviruses can cause vascular disease in order to eliminate these devastating diseases, and FIP provides a platform for joint clinical trials bridging feline and human medicine.

Financing

AES is supported by the NIH Comparative Medicine Training Program T32OD011000. GRW is funded by NIH grants R01AI135270 and R21AI135373, SCB is funded by NIH grant R01 AI085089. FIP research in the author's laboratories is funded in part by the Winn Feline Foundation and the Cornell Feline Health Center.

Thanks

We thank Andrew Miller, Robert Goggs, Natalie Zatz and Beth Licitra for critical reading of the manuscript and helpful discussion.

Conflict of interests

The authors are not aware of any conflict of interest.

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