Adaptive Evolution of Feline Coronavirus Genes Based on Selection Analysis.

PURPOSE: We investigated sequences of the feline coronaviruses (FCoV), which include feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV), from China and other countries to gain insight into the adaptive evolution of this virus. METHODS: Ascites samples from 31 cats with suspected FIP and feces samples from 8 healthy cats were screened for the presence of FCoV. Partial viral genome sequences, including parts of the nsp12-nsp14, S, N, and 7b genes, were obtained and aligned with additional sequences obtained from the GenBank database. Bayesian phylogenetic analysis was conducted, and the possibility of recombination within these sequences was assessed. Analysis of the levels of selection pressure experienced by these sequences was assessed using methods on both the PAML and Datamonkey platforms. RESULTS: Of the 31 cats investigated, two suspected FIP cats and one healthy cat tested positive for FCoV. Phylogenetic analysis showed that all of the sequences from mainland China cluster together with a few sequences from the Netherlands as a distinct clade when analyzed with FCoV sequences from other countries. Fewer than 3 recombination breakpoints were detected in the nsp12-nsp14, S, N, and 7b genes, suggesting that analyses for positive selection could be conducted. A total of 4, 12, 4, and 4 positively selected sites were detected in the nsp12-nsp14, S, N, and 7b genes, respectively, with the previously described site 245 of the S gene, which distinguishes FIPV from FECV, being a positive selection site. Conversely, 106, 168, 25, and 17 negative selection sites in the nsp12-14, S, N, and 7b genes, respectively, were identified. CONCLUSION: Our study provides evidence that the FCoV genes encoding replicative, entry, and virulence proteins potentially experienced adaptive evolution. A greater number of sites in each gene experienced negative rather than positive selection, which suggests that most of the protein sequence must be conservatively maintained for virus survival. A few of the sites showing evidence of positive selection might be associated with the more severe pathology of FIPV or help these viruses survive other harmful conditions.

Construction and Immunogenicity of Virus-Like Particles of Feline Parvovirus from the Tiger.

Feline panleukopenia, caused by feline parvovirus (FPV), is a highly infectious disease characterized by leucopenia and hemorrhagic gastroenteritis that severely affects the health of large wild Felidae. In this study, tiger FPV virus-like particles (VLPs) were developed using the baculovirus expression system. The VP2 gene from an infected Siberian tiger (Panthera tigris altaica) was used as the target gene. The key amino acids of this gene were the same as those of FPV, whereas the 101st amino acid was the same as that of canine parvovirus. Indirect immunofluorescence assay (IFA) results demonstrated that the VP2 protein was successfully expressed. SDS-PAGE and Western blotting (WB) results showed that the target protein band was present at approximately 65 kDa. Electron micrograph analyses indicated that the tiger FPV VLPs were successfully assembled and were morphologically similar to natural parvovirus particles. The hemagglutination (HA) titer of the tiger FPV VLPs was as high as 1:218. The necropsy and tissue sections at the cat injection site suggested that the tiger FPV VLPs vaccine was safe. Antibody production was induced in cats after subcutaneous immunization, with a >1:210 hemagglutination inhibition (HI) titer that persisted for at least 12 months. These results demonstrate that tiger FPV VLPs might provide a vaccine to prevent FPV-associated disease in the tiger.

Feline Panleukopenia: A Re-emergent Disease.

Feline panleukopenia (FPL) is caused by a Carnivore protoparvovirus infection. Feline parvovirus (FPV) causes most cases. When Canine parvovirus 2 (CPV-2) first emerged, it could not replicate in cats. All current CPV variants (CPV-2a-c) can infect cats to cause subclinical disease or FPL. Feline panleukopenia has re-emerged in Australia in shelter cats associated with failure to vaccinate. Parvoviruses can remain latent in mononuclear cells post-infection. Molecular methods such as polymerase chain reaction are used to determine the infecting strain. Current perspectives on causes, epidemiology, diagnosis, treatment, prognostic indicators, and management of outbreaks in shelters are reviewed.

Lymphoplasmacytic Meningoencephalitis and Neuronal Necrosis Associated With Parvoviral Infection in Cats.

Neurologic manifestations other than cerebellar hypoplasia are rarely associated with feline panleukopenia virus (FPV) infection in cats. Here the authors describe lymphoplasmacytic meningoencephalitis and neuronal necrosis in 2 cats autopsied after exhibiting ataxia and nystagmus. Gross changes consisted of cerebellar herniation through the foramen magnum, with flattening of cerebrocortical gyri and narrowing of sulci. Histologically, lymphoplasmacytic meningoencephalitis, extensive neuronal necrosis, and neuroaxonal degeneration with digestion chambers were present in the telencephalon and brain stem in both cats. Frozen brain tissue of both cats was positive for parvoviral antigen via fluorescent antibody testing, and formalin-fixed, paraffin-embedded tissue sections of brain were immunoreactive for parvovirus antigen and positive for parvoviral DNA on in situ hybridization. Frozen brain tissue from 1 case was positive for parvovirus NS1 and VP2 genes using conventional polymerase chain reaction, and subsequent DNA sequencing and phylogenetic analysis revealed that the viral strain was a FPV. Reverse transcription quantitative polymerase chain reaction on formalin-fixed, paraffin-embedded brain tissue revealed high levels of parvovirus in both cases, supporting an acute and active viral infection. Although rare, FPV infection should be considered in cases of lymphoplasmacytic meningoencephalitis and neuronal necrosis in cats.

Neuronal Vacuolization in Feline Panleukopenia Virus Infection.

Feline panleukopenia virus (FPV) infections are typically associated with anorexia, vomiting, diarrhea, neutropenia, and lymphopenia. In cases of late prenatal or early neonatal infections, cerebellar hypoplasia is reported in kittens. In addition, single cases of encephalitis are described. FPV replication was recently identified in neurons, although it is mainly found in cells with high mitotic activity. A female cat, 2 months old, was submitted to necropsy after it died with neurologic deficits. Besides typical FPV intestinal tract changes, multifocal, randomly distributed intracytoplasmic vacuoles within neurons of the thoracic spinal cord were found histologically. Next-generation sequencing identified FPV-specific sequences within the central nervous system. FPV antigen was detected within central nervous system cells, including the vacuolated neurons, via immunohistochemistry. In situ hybridization confirmed the presence of FPV DNA within the vacuolated neurons. Thus, FPV should be considered a cause for neuronal vacuolization in cats presenting with ataxia.

Feline Panleukopenia Virus Is Not Associated With Myocarditis or Endomyocardial Restrictive Cardiomyopathy in Cats.

Canine parvovirus-2 (CPV-2) is nearly indistinguishable from feline panleukopenia virus (FPV) and is a well-known cause of viral myocarditis in young puppies; however, it is not known whether either FPV or CPV-2 naturally infects feline cardiomyocytes and causes myocarditis. Endomyocarditis (EMC) and left ventricular endomyocardial fibrosis (LVEF), clinically known as "endomyocardial restrictive cardiomyopathy," are important feline heart diseases suspected to have an infectious etiology. A continuum is suggested with EMC representing the acute reaction to an unknown infectious agent and LVEF the chronic manifestation of repair. The purpose of this study was to determine (1) whether there is natural parvovirus infection of the feline myocardium and (2) whether parvoviral infection is associated with feline EMC and/or LVEF. In a retrospective study, polymerase chain reaction and sequencing for the parvovirus VP1/2 gene was performed on archived heart tissue from cats with endomyocardial disease and controls. Similar methods were used prospectively on myocardial tissues from shelter-source kittens. Although 8 of 36 (22%; 95% confidence interval [CI], 11%-40%) shelter kittens had parvoviral DNA in myocardial tissue, VP1/2 DNA was not detected in 33 adult cases or 34 controls (95% CI, 0% to ∼11%). These findings were confirmed by in situ hybridization: adult cats did not have detectable parvovirus DNA, although rare intranuclear signal was confirmed in 7 of 8 shelter-source kittens. In kittens, parvovirus was not significantly associated with myocarditis, and in situ hybridization signal did not colocalize with inflammation. Although infection of cardiomyocytes was demonstrated in kittens, these data do not support a role for parvovirus in EMC-LVEF.

Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus.

The cell cycle-associated neuronal death hypothesis, which has been proposed as a common mechanism for most neurodegenerative diseases, is notably supported by evidencing cell cycle effectors in neurons. However, in naturally occurring nervous system diseases, these markers are not expressed in neuron nuclei but in cytoplasmic compartments. In other respects, the Feline Panleukopenia Virus (FPV) is able to complete its cycle in mature brain neurons in the feline species. As a parvovirus, the FPV is strictly dependent on its host cell reaching the cell cycle S phase to start its multiplication. In this retrospective study on the whole brain of 12 cats with naturally-occurring, FPV-associated cerebellar atrophy, VP2 capsid protein expression was detected by immunostaining not only in some brain neuronal nuclei but also in neuronal cytoplasm in 2 cats, suggesting that viral mRNA translation was still occurring. In these cats, double immunostainings demonstrated the expression of cell cycle S phase markers cyclin A, cdk2 and PCNA in neuronal nuclei. Parvoviruses are able to maintain their host cells in S phase by triggering the DNA damage response. S139 phospho H2A1, a key player in the cell cycle arrest, was detected in some neuronal nuclei, supporting that infected neurons were also blocked into the S phase. PCR studies did not support a co-infection with an adeno or herpes virus. ERK1/2 nuclear accumulation was observed in some neurons suggesting that the ERK signaling pathway might be involved as a mechanism driving these neurons far into the cell cycle.

Feline panleukopaenia virus in captive non-domestic felids in South Africa.

An outbreak of feline panleukopaenia virus (FPLV) infection was diagnosed by pathology, electron microscopy and polymerase chain reaction (PCR) in vaccinated captive-bred subadult cheetahs in South Africa. Subsequent to this disease outbreak, 12 cases of FPLV diagnosed on histology were confirmed by PCR in captive African black-footed cat, caracal, cheetah, lion, ocelot and serval. Phylogenetic analyses of the viral capsid protein gene on PCR-positive samples, vaccine and National Center for Biotechnology Information (NCBI) reference strains identified a previously unknown strain of FPLV, present since at least 2006, that differs from both the inactivated and the modified live vaccine strains. A previously described South African strain from domestic cats and cheetahs was identified in a serval. Surveys of FPLV strains in South African felids are needed to determine the geographical and host species distribution of this virus. Since non-domestic species may be reservoirs of parvoviruses, and since these viruses readily change host specificity, the risks of FPLV transmission between captive-bred and free-ranging carnivores and domestic cats and dogs warrant further research.

Ultrasonographic diagnosis of a fibrinonecrotic colonic cast in a kitten with feline panleukopenia virus.

Ultrasonography of a cat with diarrhoea and vomiting revealed a multi-layered, discrete linear structure within the large intestine with retention of the intestinal layers which could potentially be confused with an intestinal intussusception. The structure was ultimately expelled from the large intestine during defecation, and confirmed as a fibrinonecrotic cast. The origin of the fibrinonecrotic cast was assumed to be an intestinal pseudo-membrane formed in enteritis caused by immune suppression due to the panleukopenia virus. To our knowledge, this is the first ultrasonographic description of a fibrinonecrotic cast and spontaneous passage of the colonic cast in the veterinary field.

Fatal infection with feline panleukopenia virus in two captive wild carnivores (Panthera tigris and Panthera leo).

Two cases of fatal infection caused by parvovirus in a white tiger (Panthera tigris) and an African lion (Panthera leo) at the Lisbon Zoo (Portugal) are described. Gross findings at necropsy were catharral enteritis in the tiger and severe hemorrhagic enteritis in the lion. Histopathologic examination revealed, in both animals, intestinal crypt necrosis and lymphocyte depletion in the germinal centers of the mesenteric lymph nodes. Bacteriologic examination was negative for common bacterial pathogens, including Salmonella. Amplification of the parvovirus VP2 complete gene was achieved in both cases and sequencing analysis identified these isolates as feline panleukopenia virus (FPLV). The nucleotide sequences obtained from these two viruses were genetically indistinguishable. The phylogenetic analysis of FPLV strains from domestic cats obtained in the Lisbon area revealed the circulation of FPLV strains highly similar to those isolated from the tiger and lion, which strongly suggests that stray cats may have been the source of infection.

[An update on viral diseases of the dog and cat]. / Recente ontwikkelingen op het gebied van virusziekten bij de hond en de kat.

In this review, recent developments in the field of viral diseases of the dog and the cat are discussed. In the dog, infection with the coronavirus type 2 is associated with respiratory signs, while infection of a highly pathogenic strain of the coronavirus type 1 has been identified as the cause of mortality in puppies. A new strain of the canine parvovirus is identified, from which the pathogenicity is not yet completely clarified. Infection with West Nile virus is associated with progressive neurological disease and subclinical infections in dogs. Infection with equine influenza A (H3N8) or a highly related influenza virus can cause severe respiratory disease and mortality in greyhounds and other dogs. Infection with avian influenza A (H5N1) can cause disease and mortality in cats and is mostly subclinical in dogs. A number of outbreaks of highly virulent strains of the calicivirus in cats have been described.

Parvovirus infection in domestic companion animals.

Parvovirus infects a wide variety of species. The rapid evolution, environmental resistance, high dose of viral shedding, and interspecies transmission have made some strains of parvovirus infection difficult to control within domestic animal populations. Some parvoviruses in companion animals, such as canine parvovirus (CPV) 1 and feline parvovirus, have demonstrated minimal evolution over time. In contrast, CPV 2 has shown wide adaptability with rapid evolution and frequent mutations. This article briefly discusses these three diseases, with emphasis on virus evolution and the challenges to protecting susceptible companion animal populations.

Concurrent infection of a cat with cowpox virus and feline parvovirus.

Concurrent infection with cowpox and feline parvovirus was diagnosed in a 5-month-old male European Short Hair cat. Microscopical examination of the facial skin, ears and foot pads revealed multifocal to coalescing, ulcerative to necrotizing dermatitis and panniculitis with ballooning epidermal degeneration and eosinophilic cytoplasmic inclusion bodies. Immunohistochemistry, polymerase chain reaction testing and virus isolation confirmed infection with a strain of cowpox virus similar to that isolated from a cat in Germany 5 years previously. Lymphoid tissues were depleted and there was catarrhal enteritis caused by feline parvovirus as confirmed by immunohistochemistry and in-situ hybridization. This co-infection did not result in a more severe and rapid course of the poxvirus-associated disease.

Acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein.

In feline coronavirus (FCoV) pathogenesis, the ability to infect macrophages is an essential virulence factor. Whereas the low-virulence feline enteric coronavirus (FECV) isolates primarily replicate in the epithelial cells of the enteric tract, highly virulent feline infectious peritonitis virus (FIPV) isolates have acquired the ability to replicate efficiently in macrophages, which allows rapid dissemination of the virulent virus throughout the body. FIPV 79-1146 and FECV 79-1683 are two genetically closely related representatives of the two pathotypes. Whereas FECV 79-1683 causes at the most a mild enteritis in young kittens, FIPV 79-1146 almost invariably induces a lethal peritonitis. The virulence phenotypes correlate with the abilities of these viruses to infect and replicate in macrophages, a feature of FIPV 79-1146 but not of FECV 79-1683. To identify the genetic determinants of the FIPV 79-1146 macrophage tropism, we exchanged regions of its genome with the corresponding parts of FECV 79-1683, after which the ability of the FIPV/FECV hybrid viruses to infect macrophages was tested. Thus, we established that the FIPV spike protein is the determinant for efficient macrophage infection. Interestingly, this property mapped to the C-terminal domain of the protein, implying that the difference in infection efficiency between the two viruses is not determined at the level of receptor usage, which we confirmed by showing that infection by both viruses was equally blocked by antibodies directed against the feline aminopeptidase N receptor. The implications of these findings are discussed.

Apoptosis in feline panleukopenia and canine parvovirus enteritis.

Tissue samples of cats and dogs with panleukopenia and parvovirus enteritis, respectively, were examined for the presence of viral antigen-positive cells and apoptotic cells by immunohistochemistry and by TUNEL assay (Terminal Transferase-Mediated dUTP Nick End Labelling). Compared to control animals, infected cats and dogs generally had more TUNEL-positive cells. Cell types positive for parvovirus antigen, for example digestive tract epithelial and mesenchymal cells, and lymphocytes and macrophages in lymphoid tissues were also positive for TUNEL signals. Occasionally, TUNEL signal and viral antigen were present in the same tissue areas, suggesting a direct viral trigger of apoptosis. More frequently, however, there was no complete overlap of antigen and TUNEL-positive areas. The results of this study indicate that apoptotic cell death contributes significantly to the widespread tissue damage of parvovirus infection in cats and dogs.

Feline panleukopenia virus revisited: molecular characteristics and pathological lesions associated with three recent isolates.

The low incidence of clinical signs or pathological lesions compatible with feline panleukopenia in cats has created the perception among practitioners that the disease has disappeared since the emergence of canine parvovirus type 2 in the late 1970s. Three parvoviruses that were recently isolated from a domestic cat and 2 cheetahs in cell culture or detected by means of the polymerase chain reaction were shown to be typical feline parvoviruses. Phylogenetic comparison with other FPV isolates did not reveal a particular African cluster.

Enterocolitis associated with dual infection by Clostridium piliforme and feline panleukopenia virus in three kittens.

Dual infection by Clostridium piliforme and feline panleukopenia virus (FPLV) was found in three kittens. In all cases, we found focal necrosis and desquamation of epithelial cells with occasional neutrophil infiltration in the large intestine. Large filamentous bacilli and spores were observed in the epithelium by using the Warthin-Starry method. Electron microscopy revealed the vegetative forms with characteristic peritrichous flagella and spore forms. Immunohistochemically, these bacilli showed a positive reaction with mouse antisera against the RT and MSK C. piliforme strains. Polymerase chain reaction (PCR) using cecum specimens demonstrated the 196-bp band specific to C. piliforme 16S rRNA. All three kittens were also diagnosed as FPLV-infected on the basis of the characteristic mucosal lesions, including intranuclear inclusions and PCR study for the FPLV genomic DNA. The PCR techniques are useful for confirming the C. piliforme and FPLV infection in spontaneous cases.

Bone-marrow changes in infectious diseases and lymphohaemopoietic neoplasias in dogs and cats--a retrospective study.

Bone-marrow changes in infectious diseases due to feline infectious peritonitis virus (FIPV), feline immunodeficiency virus (FIV), parvovirus (PV, canine and feline) and canine distemper virus (CDV), and in the lymphohaemopoietic neoplasias (LHNs) usually associated with feline leukaemia virus infection were studied in samples obtained from 204 cats and 82 dogs at necropsy. The study demonstrated (1) no changes, (2) non-specific reactive changes, and (3) disease-specific changes (similar to those occurring in extramedullary sites) in: 51.2, 48.8 and 9.7% of 41 cases of FIPV infection, respectively; 0, 100 and 0% of nine cases of FIV infection, respectively; 1.3, 0 and 92% of 75 cases of canine PV infection, respectively; 5.3, 3.9 and 84% of 76 cases of feline PV infection, respectively; 71.4, 28.6 and 0% of seven cases of CDV infection, respectively; and 35.9, 52.6 and 11.5% of 78 cases of LHN, respectively. The distribution of the disease-specific bone-marrow changes was either diffuse or focal; diffuse changes were frequently found in cases of feline and canine PV infection, and focal changes were found inconsistently in FIPV infections and feline LHN. To the extent that the bone marrow showed any changes in FIV and CDV infections, they were mostly reactive and not pathognomonic.

Panleukopenia-like syndrome of FeLV caused by co-infection with FeLV and feline panleukopenia virus.

To study the effect of interferon on feline leukemia virus (FeLV) infection, 30 specific pathogen free (SPF) cats were infected with the apathogenic FeLV A Glasgow. Unexpectedly, between 5 and 8 weeks after FeLV infection, all 19 cats with persistent FeLV infection but not the FeLV-negative cats died from a panleukopenia-like syndrome. No feline panleukopenia virus (FPLV) antigen was found in feces by latex agglutination, enzyme-linked immunosorbent assay (ELISA) or immunoelectron microscopy. No enteropathogenic bacteria were found. Histopathology revealed changes resembling those of FPLV infection such as destruction of crypts and pancytopenia of bone marrow. Neither clinical signs nor seroconversion to FPLV could be induced by transmitting intestinal extracts to two SPF cats. However, FPLV antigen was demonstrated by immunofluorescence assay in intestinal cryostat sections of diseased animals. FPLV could also be demonstrated in intestinal extracts by immunoelectron microscopy, by latex agglutination and ELISA after anti-FPLV antibodies were removed from immune-complexed FPLV by ultracentrifugation over a CsCl gradient at pH 2.0. From these experiments it was concluded that the panleukopenia-like syndrome of FeLV may not be caused by FeLV alone but at least in some cases by co-infection with FeLV and FPLV. In addition, some form of 'cooperation' between FeLV and FPLV must be postulated because neither virus alone induced symptoms.