Melissa A. Kennedy

Deletions in the 7a ORF of feline coronavirus associated with an epidemic of feline infectious peritonitis

Abstract A population of Persian cats experienced an epidemic of feline infectious peritonitis (FIP) over 2 years. Twelve cases of FIP occurred in litters born during this period. Cats contracting FIP were all genetically related through the sire. Feline coronavirus (FCoV) genomic RNA was detected consistently in this study in biologic samples from adult cats, kittens suffering from FIP, and their siblings. Analysis of viral 7a/7b open reading frame (ORFs) were analyzed and revealed two distinct virus variants circulating in the population, one with an intact 7a ORF and one with two major deletions in the 7a ORF. The 7b ORFs were intact and similar among all virus isolates, although point mutations resulting in amino acid changes were present. The sire was determined to be infected with both variants, and was persistently virus-infected. We speculate the deletion variant arose from the non-deletion variant during viral replication in this population, possibly in the sire.

Detection of feline coronavirus in captive Felidae in the USA

Feline coronavirus (FCoV) is an important pathogen of domestic and nondomestic Felidae. Investigation into the prevalence of FCoV in exotic Felidae has relied primarily on serology. The usefulness of genetic detection of FCoV using reverse transcription and nested polymerase chain reaction (RT/nPCR) for viral screening was investigated. Seventy-five biologic samples, primarily feces, from captive felids from 11 institutions were tested using PCR. Serum samples collected from all but 12 of these animals were tested for antibodies to type I and type II FCoV by indirect immunofluorescence. Twenty-four animals were positive using RT/nPCR for virus. Twenty-nine animals were seropositive to type I and/or type II FCoV. From serologic data, infection with a virus antigenically related to FCoV type I occurred most commonly. Serology did not correlate with virus shedding because 13 animals were seronegative to FCoV type I and II but positive using RT/nPCR for virus. Conversely, 20 animals were seropositive but negative using RT/nPCR for FCoV. Some of the populations in which virus was detected had experienced health problems, including feline infectious peritonitis (FIP), necrotizing colitis, and mild enteritis. In addition to its role in FIP, this virus may play a role in gastrointestinal diseases of infected animals. This study demonstrates that FCoV is a significant infectious agent of captive felids because over half of the animals tested were positive by viral genetic detection, serology, or both. Dependence upon one method for detection of infection is unreliable.

Genetic Analysis of Feline Caliciviruses Associated with a Hemorrhagic-Like Disease

Feline calicivirus (FCV) is 1 of the most common causes of upper respiratory tract disease in cats. Other disease syndromes associated with FCV infection have been reported. Recently, calicivirus infection associated with a hemorrhagic-like disease leading to significant mortality in cats has been reported. The clinical signs are similar to those observed with the calicivirus of rabbit hemorrhagic disease. This study characterized 2 FCV isolates associated with hemorrhagic-like disease. Nucleotide sequencing of the complete genome has been done for these 2 isolates as well as for 4 additional isolates representing other disease syndromes. Previously reported sequence data for the entire genome of classical FCV (6 isolates) and a portion of the capsid gene for hemorrhagic-like FCV (3 isolates), isolated in different regions of United States were used in the genetic analysis. Sequence data were used to determine relationships among the isolates and any correlation with phenotype. Nucleotide sequence comparisons of the entire genome and individual open reading frames revealed high homology among all isolates. Data suggest that the virulence may have genetic determinants on the basis of phylogenetic clustering of the isolates associated with hemorrhagic-like disease.

SEROLOGIC SURVEY OF DOMESTIC FELIDS IN THE PETEN REGION OF GUATEMALA

Abstract Blood samples were analyzed from 30 domestic cats (Felis domesticus) from the Petén region of Guatemala to determine the seroprevalence of common pathogens that may pose a potential risk to native wild felids. Eight of the cats had been vaccinated previously; however, owners were unable to fully describe the type of vaccine and date of administration. In addition, blood samples were obtained from two captive margays (Leopardus wiedii). Samples were tested for antibodies to feline immunodeficiency virus, Dirofilaria immitis, feline panleukopenia virus, feline herpesvirus, feline coronavirus, canine distemper virus, and Toxoplasma gondii and for feline leukemia virus (FeLV) antigen. Fifty percent or more of the cats sampled were seropositive for feline herpesvirus (22 of 30), feline panleukopenia (15 of 30), and T. gondii (16 of 30). Five cats were positive for FeLV antigen. Both margays were seropositive for feline coronavirus and one was strongly seropositive to T. gondii. All animals were seronegative for D. immitis. This survey provides preliminary information about feline diseases endemic to the Petén region.

Correlation of genomic detection of feline coronavirus with various diagnostic assays for feline infectious peritonitis.

Feline infectious peritonitis (FIP) is a fatal disease of domestic and nondomestic felids caused by a feline coronavirus (FCV). It is a significant problem in catteries, multiple cat households, and shelters. 14,18 FIP can manifest as an effusive peritonitis and/or pleuritis, with a relatively short disease course ending in death. A protracted course with granulomatous lesions affecting multiple organs may occur, which also invariably progresses to death. 8,11,12 The FCVs are closely related and include two biotypes: those that are virulent and cause FIP and those that are avirulent. 7 The avirulent group, known as feline enteric coronaviruses (FECV), may be associated with mild enteric disease or subclinical infection in cats. 11,12 FCVs are also classified into serotypes 1 and 2 based on antigenicity. 7,12 Both serotypes contain virulent and avirulent biotypes, and FCVs within a serotype are indistinguishable in the laboratory. FCVs are ubiquitous, especially in environments such as catteries, where large numbers of cats are housed together and where the majority of cats are seropositive. 12 As a result, antemortem diagnosis for FIP is difficult because no detection assay sensitive enough and specific for the virus causing FIP is routinely available. 12 This problem is further compounded because consistent genetic differences between the virulent and avirulent biotypes of FCV have not been identified. 16 An assay has been developed using reverse transcription and a nested polymerase chain reaction (PCR) to detect FCV genomic material in a variety of biologic samples from cats. The genomic region targeted encompasses the 7a and 7b open reading frames (ORFs). This region, specifically that encoding the 7b ORF, may correlate with virulence.

Evaluation of antibodies against feline coronavirus 7b protein for diagnosis of feline infectious peritonitis in cats.

OBJECTIVE To determine whether expression of feline coronavirus (FCoV) 7b protein, as indicated by the presence of specific serum antibodies, consistently correlated with occurrence of feline infectious peritonitis (FIP) in cats. SAMPLE POPULATION 95 serum samples submitted for various diagnostic assays and 20 samples from specific-pathogen-free cats tested as negative control samples. PROCEDURES The 7b gene from a virulent strain of FCoV was cloned into a protein expression vector. The resultant recombinant protein was produced and used in antibody detection assays via western blot analysis of serum samples. Results were compared with those of an immunofluorescence assay (IFA) for FCoV-specific antibody and correlated with health status. RESULTS Healthy IFA-seronegative cats were seronegative for antibodies against the 7b protein. Some healthy cats with detectable FCoV-specific antibodies as determined via IFA were seronegative for antibodies against the 7b protein. Serum from cats with FIP had antibodies against the 7b protein, including cats with negative results via conventional IFA. However, some healthy cats, as well as cats with conditions other than FIP that were seropositive to FCoV via IFA, were also seropositive for the 7b protein. CONCLUSIONS AND CLINICAL RELEVANCE Expression of the 7b protein, as indicated by detection of antibodies against the protein, was found in most FCoV-infected cats. Seropositivity for this protein was not specific for the FCoV virulent biotype or a diagnosis of FIP.

Genetic relatedness and pathogenicity of equine herpesvirus 1 isolated from onager, zebra and gazelle.

Summary.Equine herpesvirus 1 was isolated from an onager in 1985, a zebra in 1986 and a Thomson’s gazelle in 1996 in USA. The genetic relatedness and pathogenicity of these three viruses were investigated based on the nucleotide sequences of the glycoprotein G (gG) gene, experimental infection in hamsters, and comparison with horse isolates. The gG gene sequences of EHV-1 from onager and zebra were identical. The gG gene sequences of the gazelle isolate showed 99.5% identity to those of onager and zebra isolates. The gG gene sequences of EHV-1 isolated from horses were 99.9–100% identical and 98, 98 and 97.8% similar to gG from onager, zebra and gazelle isolates, respectively. Hamsters inoculated with onager, zebra and gazelle isolates had severe weight loss, compared with hamsters inoculated with horse isolates. The histopathological findings were related to the virulence of each isolate. The results indicated that EHV-1 isolates from onager, zebra and gazelle differ from horse EHV-1 and are much more virulent in hamsters.

Serologic reactivity using conserved envelope epitopes in feline lentivirus-infected felids

An enzyme-linked immunosorbent assay (ELISA) based on synthetic peptides identical to lentivirus envelope protein amino acid sequences was used to study serologic reactivity of lentivirus-infected domestic cats and nondomestic felids. One feline immunodeficiency virus (FIV) peptide, P237, was consistently recognized by antibodies from FIV-infected cats, but 2 other FIV peptide antigens were not. The molecular basis for this serologic reactivity was examined. Lentivirus-infected nondomestic Felis species reacted intensely with a puma lentivirus (PLV) peptide corresponding to the conserved FIV peptide. However, lentivirus-infected Panthera species, from which a different lentivirus has been isolated, did not react with the PLV. FIV-infected domestic felids also did not have significant reactivity with the PLV peptide. The peptide ELISA is comparable in sensitivity and specificity to western blot analysis and a commercial enzyme immunoassay. Unlike the other assays, however, the peptide ELISA is inexpensive, requires a small amount of serum, enables the study of specific isotype reactivity, and discriminates between antibodies to FIV and those to PLV. Antibody tests based upon the FIV and the PLV peptides should be useful for detecting the possible introduction of FIV into exotic felids or of lentiviruses from nondomestic felids into the domestic cat population.

Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains

Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors’ laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10−1 TCID50). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

Complete genome sequence of an astrovirus identified in a domestic rabbit (Oryctolagus cuniculus) with gastroenteritis

A colony of domestic rabbits in Tennessee, USA, experienced a high-mortality (~90%) outbreak of enterocolitis. The clinical characteristics were one to six days of lethargy, bloating, and diarrhea, followed by death. Heavy intestinal coccidial load was a consistent finding as was mucoid enteropathy with cecal impaction. Preliminary analysis by electron microscopy revealed the presence of virus-like particles in the stool of one of the affected rabbits. Analysis using the Virochip, a viral detection microarray, suggested the presence of an astrovirus, and follow-up PCR and sequence determination revealed a previously uncharacterized member of that family. Metagenomic sequencing enabled the recovery of the complete viral genome, which contains the characteristic attributes of astrovirus genomes. Attempts to propagate the virus in tissue culture have yet to succeed. Although astroviruses cause gastroenteric disease in other mammals, the pathogenicity of this virus and the relationship to this outbreak remains to be determined. This study therefore defines a viral species and a potential rabbit pathogen.