The molecular genetics of feline coronaviruses: comparative sequence analysis of the ORF7a/7b transcription unit of different biotypes

AbstractFeline coronaviruses (FCoVs) have been subdivided into feline enteric coronaviruses (FECVs) and feline infectious peritonitis viruses (FIPVs) on the basis of pathogenic properties. Serologically, a distinction has been made between type I and II FCoVs, the latter of which more closely resemble canine coronavirus (CCV). To gain more insight into the genetic relationships between different FCoV biotypes, we determined the nucleotide sequences of the ORF7a/7b transcription unit of nine strains. The following observations were made: (i) The sequences are 87-100% identical. In this part of the genome, type I and II FCoVs are more closely related to each other than to CCV. To explain the genetic and antigenic differences between the spike genes of type I and II FCoVs, we postulate that type II FCoVs have arisen by an RNA recombination event between a type I FCoV and CCV. (ii) The avirulent "FECV" strains UCD and 79-1683 are more similar to virulent "FIPV" strains than to each other. Our findings thus support the notion that FECV and FIPV are not different species but merely virulence variants. (iii) In contrast to FECV 79-1683, FECV UCD contains an intact ORF7b, indicating that ORF7b deletions are not a universal distinguishing property of FECVs. (iv) ORF7b deletions readily occur in vitro, correlating with loss of virulence. By reverse transcription polymerase chain reaction analysis, we show that in naturally occurring FCoVs ORF7b is maintained Thus, ORF7b seems to provide a distinct selective advantage during natural infection

Feline coronavirus type II strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type I and canine coronavirus

Recent evidence suggests that the type II feline coronavirus (FCoV) strains 79-1146 and 79-1683 have arisen from a homologous RNA recombination event between FCoV type I and canine coronavirus (CCV). In both cases, the template switch apparently took place between the S and M genes, giving rise to recombinant viruses which encode a CCV-like S protein and the M, N, 7a, and 7b proteins of FCoV type I (K. Motowaka, T. Hoh- datsu, H. Hashimoto, and H. Koyama, Microbiol. Immunol. 40:425-433, 1996; H. Vennema, A. Poland, K. Floyd Hawkins, and N. C. Pedersen, Feline Pract. 23:40-44, 1995). In the present study, we have looked for additional FCoV-CCV recombination sites. Four regions in the pol gene were selected for comparative sequence analysis of the type II FCoV strains 79-1683 and 79-1146, the type I FCoV strains TN406 and UCD1, the CCV strain K378, and the TGEV strain Purdue. Our data show that the type II FCoVs have arisen from double recombination events: additional crossover sites were mapped in the ORF1ab frameshifting region of strain 79-1683 and in the 5' half of ORF1b of strain 79-1146

Detection of feline coronavirus RNA in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase PCR

A nested reverse transcriptase PCR (RT-nPCR) was developed for the detection of feline coronavirus (FCoV) RNA in the feces, tissues, and body fluids of infected cats. The RT-nPCR was targeted to the highly conserved 3'-untranslated region of the viral genome and will detect most, if not all, feline coronaviruses in the field. With the RT-nPCR, FCoV RNA was detected in plasma samples from experimentally infected cats as early as 2 days postinoculation. FCoV RNA was also detected in serum, plasma, or ascitic fluid samples from 14 of 18 cats (78%) with naturally occurring feline infectious peritonitis (FIP). The use of RT- PCR for FIP diagnosis is limited because of the occurrence of apparently healthy FCoV carriers. These asymptomatic cats shed the virus in the feces and, in a number of cases, also had detectable virus in the plasma. Because of the nature of FCoV infections, our RT-PCR assay with plasma or serum cannot be used to establish a definite diagnosis of FIP. However, this assay does provide a new means to identify asymptomatic FCoV carriers. As such, RT-nPCR will be of use to screen cats before their introduction into FCoV-free catteries. Moreover, this assay provides an important tool to study the epidemiology of FCoV

Placebo-controlled evaluation of a modified life virus vaccine against feline infectious peritonitis: safety and efficacy under field conditions

A modified live virus vaccine against feline infectious peritonitis (FIP) was evaluated in a double blind, placebo-controlled field trial in two high-risk populations. The vaccine was found to be safe and efficacious in one population of cats that had low antibody titre against feline coronavirus (FCoV) at the time of vaccination. Although clinically healthy at the time of vaccination, retrospectively some vaccinees that later came down with FIP were found to be RT-PCR positive for FCoV in plasma and showed changes in blood parameters consistent with early stage of FIP. It is concluded that vaccination can protect cats with no or low FCoV antibody titres and that in some cats vaccine failure was probably due to pre-existing infection

Persistence and evolution of feline coronavirus in a closed cat-breeding colony

AbstractFeline coronavirus (FCoV) persistence and evolution were studied in a closed cat-breeding facility with an endemic serotype I FCoV infection. Viral RNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in the feces and/or plasma of 36 of 42 cats (86%) tested. Of 5 cats, identified as FCoV shedders during the initial survey, 4 had detectable viral RNA in the feces when tested 111 days later. To determine whether this was due to continuous reinfection or to viral persistence, 2 cats were placed in strict isolation and virus shedding in the feces was monitored every 2–4 days. In 1 of the cats, virus shedding continued for up to 7 months. The other animal was sacrificed after 124 days of continuous virus shedding in order to identify the sites of viral replication. Viral mRNA was detected only in the ileum, colon, and rectum. Also in these tissues, FCoV-infected cells were identified by immunohistochemistry. These findings provide the first formal evidence that FCoV causes chronic enteric infections. To assess FCoV heterogeneity in the breeding facility and to study viral evolution during chronic infection, FCoV quasispecies sampled from individual cats were characterized by RT-PCR amplification of selected regions of the viral genome followed by sequence analysis. Phylogenetic comparison of nucleotides 7–146 of ORF7b to corresponding sequences obtained for independent European and American isolates indicated that the viruses in the breeding facility form a clade and are likely to have originated from a single founder infection. Comparative consensus sequence analysis of the more variable region formed by residues 79–478 of the S gene revealed that each cat harbored a distinct FCoV quasispecies. Moreover, FCoV appeared to be subject to immune selection during chronic infection. The combined data support a model in which the endemic infection is maintained by chronically infected carriers. Virtually every cat born to the breeding facility becomes infected, indicating that FCoV is spread very efficiently. FCoV-infected cats, however, appear to resist superinfection by closely related FCoVs