Rapid and sensitive identification of RNA from the emerging pathogen,coxsackievirus A6

Feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) are two important coronaviruses of domestic cat worldwide. Although FCoV is prevalent among cats; the fastidious nature of type I FCoV to grow on cell culture has limited further studies on tissue tropism and pathogenesis of FCoV. While several studies reported serological evidence for FCoV in Malaysia, neither the circulating FCoV isolated nor its biotypes determined. This study for the first time, describes the isolation and biotypes determination of type I and type II FCoV from naturally infected cats in Malaysia. Of the total number of cats sampled, 95% (40/42) were RT-PCR positive for FCoV. Inoculation of clinical samples into Crandell feline kidney cells (CrFK), and Feline catus whole fetus-4 cells (Fcwf-4), show cytopathic effect (CPE) characterized by syncytial cells formation and later cell detachment. Differentiation of FCoV biotypes using RT-PCR assay revealed that, 97.5% and 2.5% of local isolates were type I and type II FCoV, respectively. These isolates had high sequence homology and phylogenetic similarity with several FCoV isolates from Europe, South East Asia and USA. This study reported the successful isolation of local type I and type II FCoV evident with formation of cytopathic effects in two types of cell cultures namely the CrFK and Fcwf-4 , where the later cells being more permissive. However, the RT-PCR assay is more sensitive in detecting the antigen in suspected samples as compared to virus isolation in cell culture. The present study indicated that type I FCoV is more prevalent among cats in Malaysia.     

A reverse genetics approach to study feline infectious peritonitis

Feline infectious peritonitis (FIP) is a lethal immunopathological disease caused by feline coronaviruses (FCoVs). Here, we describe a reverse genetics approach to study FIP by assessing the pathogenicity of recombinant type I and type II and chimeric type I/type II FCoVs. All recombinant FCoVs established productive infection in cats, and recombinant type II FCoV (strain 79-1146) induced FIP. Virus sequence analyses from FIP-diseased cats revealed that the 3c gene stop codon of strain 79-1146 has changed to restore a full-length open reading frame (ORF).     

Sequence analysis of the nucleocapsid gene of feline coronaviruses circulating in Italy

Molecular analysis of the N genes of feline coronaviruses (FCoV) strains detected in naturally infected cats were carried out to investigate the genetic diversity among these viruses. Phylogeny showed a general clustering trend on the basis of geographic origin rather than on virulence characteristics. The analysis of the pattern of nucleotide substitutions disclosed "hot spots" sites which may represent immunological domains. In conclusion, our results demonstrate that the N gene does not carry mutations associated with the pathotypical switch FECV --> FIPV. During persistent infection, the individual qualitative immune response might address the accumulations of mutations in the N gene and the development of FIP.     

A serologic survey of wild felids from central west Saudi Arabia

Forty-five wildcats (Felis silvestris), 17 sand cats (Felis margarita), and 17 feral domestic cats were captured in central west Saudi Arabia, between May 1998 and April 2000, with the aim to assess their exposure to feline immunodeficiency virus/puma lentivirus (FIV/PLV), feline leukaemia virus (FeLV), feline herpesvirus (FHV-1), feline calicivirus (FCV), feline coronavirus (FCoV), and feline panleukopenia virus (FPLV). Serologic prevalence in wildcats, sand cats, and feral domestic cats were respectively: 6%, 0%, 8% for FIV/PLV; 3%, 8%, 0% for FeLV; 5%, 0%, 15% for FHV-1; 25%, 0%, 39% for FCV; 10%, 0%, 0% for FCoV; and 5%, 0%, 8% for FPLV. We recorded the first case of FeLV antigenemia in a wild sand cat. Positive results to FIV/PLV in wildcats and feral cats confirmed the occurrence of a feline lentivirus in the sampled population.     

Identification of a feline coronavirus type I strain from a cat with feline infectious peritonitis by RT-pCR and phylogenetic analysis

A case of feline infectious peritonitis (FIP) in an 11-month-old European shorthair cat is reported. The infected cat displayed loss of weight, respiratory distress, ascitis, anemia and died within 15 days after the first appearance of clinical signs. Lesions typical of a mixed form (effusive and non-effusive) of FIP were observed and by RT-PCR a feline coronavirus (FCoV) type I strain was detected in several tissues. The RT-PCR results were confirmed by sequence analysis of the amplified products. Phylogeny carried out on fragments of the M and S genes showed that the FCoV strain segregates with typical type I FCoVs.     

Development of Clinical Signs and Occurrence of Feline Corona Virus Antigen in Naturally Infected Barrier Reared Cats and Their Offspring

The onset and pattern of the clinical signs of feline corona virus (FCoV) infection in cats were studied in a setting behind an isolation barrier. Two FCoV-seropositive cats were the source of the infection, and 3 barrier reared cats–initially FCoV-seronegative–were the recipients. The first clinical sign in the recipients appeared 11 days after contact with the source of infection. After 2 years 1 male and 1 female of the recipients started to breed. Their offspring developed clinical signs of disease at an age of 4-5 weeks. A pattern of recurring upper respiratory tract signs and conjunctivitis at intervals of about 4 months was observed in both the recipients and their offspring, while CNS dependent signs and wasting remained or got worse, once developed. Once demonstrated, FCoV antigen persisted in membrana nictitans throughout the investigation, and was found in all cats but 4 (90%). The offspring died during 2 periods, around the first week of life (9/37), and at 3-5 months of age (5/25). For comparison 3 offspring were euthanised at an age of 1 day and 16 offspring at an age of 3-6 months. FCoV antigen was demonstrated in all organs investigated (100%) from offspring dying during the first period, and in 97% from those dying during the second period. For the offspring euthanised during the same 2 periods the corresponding findings were 95% and 85%. Offspring euthanized between 9 and 17 months (4 kittens) had antigen in 67% of all investigated organs. The incidence of FCoV antigen in almost every organ in the investigated newborn kittens suggests an intrauterine infection. The demonstration of FCoV antigen in all euthanised cats, suggests a persistant infection. Virus was cultivated from membrana nictitans, that was FCoV antigen positive in the M3 test.     

Determination of the cell tropism of serotype 1 feline infectious peritonitis virus using the spike affinity histochemistry in paraffin‐embedded tissues

Unlike for serotype II feline coronaviruses (FCoV II), the cellular receptor for serotype I FCoV (FCoV I), the most prevalent FCoV serotype, is unknown. To provide a platform for assessing the pattern by which FCoV I attaches to its host receptor(s), HEK293 cell lines that stably express the ectodomains of the spike (S) proteins derived from a FCoV I feline enteric coronavirus strain UU7 (FECV UU7) and a feline infectious peritonitis virus strain UU4 (FIPV UU4) were established. Using the recombinant S proteins as probes to perform S protein affinity histochemistry in paraffin‐embedded tissues, although no tissue or enteric binding of FECV UU7 S protein was detected, it was found that by immunohistochemistry that the tissue distribution of FIPV UU4 S protein‐bound cells correlated with that of FIPV antigen‐positive cells and lesions associated with FIP and that the affinity binding of FIPV UU4 S protein on macrophages was not affected by enzymatic removal of host cell‐surface sialic acid with neuraminidase. These findings suggest that a factor(s) other than sialic acid contribute(s) to the macrophage tropism of FIPV strain UU4. This approach allowed obtaining more information about both virus–host cell interactions and the biological characteristics of the unidentified cellular receptor for FCoV I.

     

Detection of Norovirus and Sapovirus from diarrheic dogs and cats in Japan

Norovirus (NoV) and sapovirus (SaV) are important causes of human diarrhea. In this study, between 2007 and 2014 fecal samples were collected from 97 dogs and 83 cats with diarrhea and examined to determine the prevalence of NoV and SaV infections in Japan. To detect caliciviruses, approximately 300 bases targeting the polymerase gene were amplified using RT‐PCR and subjected to phylogenetic and homology analyses. Specific PCR products were obtained from four canine and nine feline samples: two canine and one feline isolate were classified as NoV, two canine isolates as SaV and the remaining eight feline isolates as vesivirus (VeV). The three NoV isolates were classified into the same clade as that of known canine and feline NoVs; their homologies (75.9–92.3%) were higher than those with human genogroup IV (GIV) NoVs (59.1–65.9%). The homology of the feline NoV isolate with previously reported feline NoV isolates was particularly high (91.7–92.3%). Regarding SaV, the two canine isolates were classified into the same clade as known canine SaVs and their homologies (72.5–86.5%) were higher than those with other mammal SaVs (20.7–58.0%). The eight feline VeV isolates were assumed to be feline calicivirus. The present study is the first report of the presence of NoV‐ and SaV‐infected dogs and cats in Japan. The findings suggest there are species‐specific circulations of NoV and SaV among dogs and cats, in Japan.     

Peptides Corresponding to the Predicted Heptad Repeat 2 Domain of the Feline Coronavirus Spike Protein Are Potent Inhibitors of Viral Infection

Background

Feline infectious peritonitis (FIP) is a lethal immune-mediated disease caused by feline coronavirus (FCoV). Currently, no therapy with proven efficacy is available. In searching for agents that may prove clinically effective against FCoV infection, five analogous overlapping peptides were designed and synthesized based on the putative heptad repeat 2 (HR2) sequence of the spike protein of FCoV, and the antiviral efficacy was evaluated.

Methods

Plaque reduction assay and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay were performed in this study. Peptides were selected using a plaque reduction assay to inhibit Feline coronavirus infection.

Results

The results demonstrated that peptide (FP5) at concentrations below 20 μM inhibited viral replication by up to 97%. The peptide (FP5) exhibiting the most effective antiviral effect was further combined with a known anti-viral agent, human interferon-α (IFN-α), and a significant synergistic antiviral effect was observed.

Conclusion

Our data suggest that the synthetic peptide FP5 could serve as a valuable addition to the current FIP prevention methods.     

Genome organization and reverse genetic analysis of a type I feline coronavirus

In this study we report the complete sequence and genome organization of the serotype I feline coronavirus (FCoV) strain Black. Furthermore, a reverse genetic system was established for this FCoV strain by cloning a full-length cDNA copy into vaccinia virus. This clone served as basis for the generation of recombinant FCoV (recFCoV) that was shown to bear the same features in vitro as the parental FCoV. Using this system, accessory 3abc genes in the FCoV genome were replaced by green fluorescent protein (recFCoV-GFP) and Renilla luciferase genes (recFCoV-RL). In addition, we showed that feline CD14⁺ blood monocytes and dendritic cells can be easily detected after infection with recFCoV-GFP. Thus, our established reverse genetic system provides a suitable tool to study the molecular biology of serotype I FCoV.     

Emergence of Pathogenic Coronaviruses in Cats by Homologous Recombination between Feline and Canine Coronaviruses

Type II feline coronavirus (FCoV) emerged via double recombination between type I FCoV and type II canine coronavirus (CCoV). In this study, two type I FCoVs, three type II FCoVs and ten type II CCoVs were genetically compared. The results showed that three Japanese type II FCoVs, M91-267, KUK-H/L and Tokyo/cat/130627, also emerged by homologous recombination between type I FCoV and type II CCoV and their parent viruses were genetically different from one another. In addition, the 3′-terminal recombination sites of M91-267, KUK-H/L and Tokyo/cat/130627 were different from one another within the genes encoding membrane and spike proteins, and the 5′-terminal recombination sites were also located at different regions of ORF1. These results indicate that at least three Japanese type II FCoVs emerged independently. Sera from a cat experimentally infected with type I FCoV was unable to neutralize type II CCoV infection, indicating that cats persistently infected with type I FCoV may be superinfected with type II CCoV. Our previous study reported that few Japanese cats have antibody against type II FCoV. All of these observations suggest that type II FCoV emerged inside the cat body and is unable to readily spread among cats, indicating that these recombination events for emergence of pathogenic coronaviruses occur frequently.     

Viral infections in free-living populations of the European wildcat

While the importance of viral infections is well studied in domestic cats, only limited information is available on their occurence and prevalence in the European wildcat (Felis silvestris silvestris). The aim of this study was to determine the prevalence of antibodies to feline coronavirus (FCoV), calicivirus (FCV), herpesvirus (FHV), parvovirus (FPV), immunodeficiency virus (FIV), leukemia virus (FeLV), and FeLV antigenemia in 51 European wildcat sera. Samples were collected between 1996 and 1997 from wildcat populations in France, Switzerland, and Germany. Antibodies to FCoV were detected in two cats (4%) and FCoV RNA was detected in feces of one of these two cats. Antibodies to FCV, FHV and FPV were found at relatively low frequencies of 16%, 4%, and 2%, respectively. Antibodies to FIV were not detected. Although antigen and antibodies to FeLV were detected in 49%, and 75%, respectively, no evidence of FeLV-associated pathology was found. From the low prevalence of FCoV, FCV, FHV and FPV infections and from the fact that the European wildcats live solitarily, it was concluded that these viral infections do not spread readily within a population. Therefore, it may be assumed that release into the wild of European wildcats bred in captivity would not bring about a high risk of introducing of these viral infections to the free-ranging wildcats. As an exception, wildcats should be tested for absence of FIV infection before release if they were at risk to acquire this infection from domestic cats.     

Feline viruses in wildcats from Scotland

Few data are available on the prevalence of feline viruses in European wildcats (Felis silvestris). Previous surveys have indicated that wildcats may be infected with the common viruses of domestic cats, apart from feline immunodeficiency virus (FIV). In the present study, 50 wildcats trapped throughout Scotland (UK) between August 1992 and January 1997 were tested for evidence of viral infection. All were negative for FIV by several serological or virological methods. By contrast, 10% of the cats were positive for feline leukemia virus (FeLV) antigen and infectious virus was isolated from 13% of a smaller subset. Of the wildcats tested for respiratory viruses, 25% yielded feline calicivirus (FCV) and although no feline herpesvirus was isolated, 16% of the samples had neutralizing antibodies to this virus. Antibodies to feline coronavirus (FCoV) were found in 6% of samples. Feline foamy virus (FFV) was an incidental finding in 33% of samples tested. This study confirms that wildcats in Scotland are commonly infected with the major viruses of the domestic cat, except for FIV.     

Factors associated with pathogen seroprevalence and infection in Rocky Mountain cougars

Serological and genetic material collected over 15 years (1990-2004) from 207 cougars (Puma concolor) in four populations in the Rocky Mountains were examined for evidence of current or prior exposure to feline immunodeficiency virus (FIV), feline parvovirus (FPV), feline coronavirus (FCoV), feline calicivirus (FCV), canine distemper virus (CDV), feline herpesvirus (FHV), and Yersinia pestis. Serologic data were analyzed for annual variation in seroconversions to assess whether these pathogens are epidemic or endemic in cougars, and to determine whether family membership, age, sex, or location influence risk of exposure. FIV and FPV were clearly endemic in the studied populations, whereas exposure to FCoV, FCV, CDV, and Y. pestis was more sporadic. No evidence was found for FHV. Age was the most consistent predictor of increased exposure risk, often with no other important factors emerging. Evidence for transmission within family groups was limited to FIV and FCoV, whereas some indication for host sex affecting exposure probability was found for FIV and Y. pestis. Overall, cougar populations exhibited few differences in terms of pathogen presence and prevalence, suggesting the presence of similar risk factors throughout the study region.     

Serosurvey of viral infections in free-ranging Namibian cheetahs (Acinonyx jubatus)

Cheetahs (Acinonyx jubatus) in captivity have unusually high morbidity and mortality from infectious diseases, a trait that could be an outcome of population homogeneity or the immunomodulating effects of chronic stress. Free-ranging Namibian cheetahs share ancestry with captive cheetahs, but their susceptibility to infectious diseases has not been investigated. The largest remaining population of free-ranging cheetahs resides on Namibian farmlands, where they share habitat with domestic dogs and cats known to carry viruses that affect cheetah health. To assess the extent to which free-ranging cheetahs are exposed to feline and canine viruses, sera from 81 free-ranging cheetahs sampled between 1992 and 1998 were evaluated for antibodies against canine distemper virus (CDV), feline coronavirus (feline infectious peritonitis virus; FCoV/ FIPV), feline herpesvirus 1 (FHV1), feline panleukopenia virus (FPV), feline immunodeficiency virus (FIV), and feline calicivirus (FCV) and for feline leukemia virus (FeLV) antigens. Antibodies against CDV, FCoV/FIPV, FHV1, FPV, and FCV were detected in 24, 29, 12, 48, and 65% of the free-ranging population, respectively, although no evidence of viral disease was present in any animal at the time of sample collection. Neither FIV antibodies nor FeLV antigens were present in any free-ranging cheetah tested. Temporal variation in FCoV/FIPV seroprevalence during the study period suggested that this virus is not endemic in the free-ranging population. Antibodies against CDV were detected in cheetahs of all ages sampled between 1995 and 1998, suggesting the occurrence of an epidemic in Namibia during the time when CDV swept through other parts of sub-Saharan Africa. This evidence in free-ranging Namibian cheetahs of exposure to viruses that cause severe disease in captive cheetahs should direct future guidelines for translocations, including quarantine of seropositive cheetahs and preventing contact between cheetahs and domestic pets.     

First evidence of feline herpesvirus, calicivirus, parvovirus, and Ehrlichia exposure in Brazilian free-ranging felids

Serum samples from 18 pumas (Puma concolor), one ocelot (Leopardus pardalis), and two little spotted cats (Leopardus tigrinus) collected from free-ranging animals in Brazil between 1998 and 2004 were tested by indirect immunofluorescence (IFA) for antibodies to feline herpesvirus 1 (FHV 1), calicivirus (FCV), coronavirus (FCoV), parvo-virus (FPV), Ehrlichia canis, Anaplasma pha-gocytophilum, and Bartonella henselae. Serum samples also were tested, by Western blot and ELISA, for feline leukemia virus (FeLV) specific antibodies and antigen, respectively, by Western blot for antibodies to feline immunodeficiency virus (FIV), and by indirect ELISA for antibodies to puma lentivirus (PLV). Antibodies to FHV 1, FCV, FCoV, FPV, FeLV, FIV, PLV or related viruses, and to B. henselae were detected. Furthermore, high-titered antibodies to E. canis or a closely related agent were detected in a puma for the first time.     

Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor

Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further development for important coronaviruses in animals and humans.     

Serosurvey of free-ranging Amur tigers in the Russian Far East

Wild Amur tigers (Panthera tigris altaica, n=44) from the Russian Far East were tested for antibodies to feline leukemia virus, feline corona virus (FCoV), feline immunodeficiency virus, feline parvovirus (FPV), canine distemper virus (CDV), Toxoplasma gondii, and Bartonella henselae. Antibodies to FCoV, CDV, FPV, and T. gondii were detected in 43, 15, 68, and 42% of tigers, respectively. No differences were detected in antibody prevalence estimates between tigers captured as part of a research program and those captured to mitigate human-tiger conflicts. Domestic dogs (Canis familiaris) were tested as a potential source for CDV; 16% were vaccinated against CDV and 58% of unvaccinated dogs were antibody positive for CDV. A high percentage of tigers were exposed to potential pathogens that could affect the survival of this species. We recommend continued monitoring of wild tigers throughout Asia, development of standardized sampling and postmortem examination procedures, and additional research to better understand potential domestic and wild animal sources for these pathogens.