Molecular characterization of the FCoV-like canine coronavirus HLJ-071 in China.

BACKGROUND: According to the differences of antigen and genetic composition, canine coronavirus (CCoV) consists of two genotypes, CCoV-I and CCoV-II. Since 2004, CCoVs with point mutations or deletions of NSPs are contributing to the changes in tropism and virulence in dogs. RESULTS: In this study, we isolated a CCoV, designated HLJ-071, from a dead 5-week-old female Welsh Corgi with severe diarrhea and vomit. Sequence analysis suggested that HLJ-071 bearing a complete ORF3abc compared with classic CCoV isolates (1-71, K378 and S378). In addition, a variable region was located between S gene and ORF 3a gene, in which a deletion with 104 nts for HLJ-071 when compared with classic CCoV strains 1-71, S378 and K378. Phylogenetic analysis based on the S gene and complete sequences showed that HLJ-071 was closely related to FCoV II. Recombination analysis suggested that HLJ-071 originated from the recombination of FCoV 79-1683, FCoV DF2 and CCoV A76. Finally, according to cell tropism experiments, it suggested that HLJ-071 could replicate in canine macrophages/monocytes cells. CONCLUSION: The present study involved the isolation and genetic characterization of a variant CCoV strain and spike protein and ORF3abc of CCoV might play a key role in viral tropism, which could affect the replication in monocyte/macrophage cells. It will provide essential information for further understanding the evolution in China.

Molecular characterization of canine coronaviruses: an enteric and pantropic approach.

Canine coronavirus (CCoV) generally causes an infection with high morbidity and low mortality in dogs. In recent years, studies on coronaviruses have gained a momentum due to coronavirus outbreaks. Mutations in coronaviruses can result in deadly diseases in new hosts (such as SARS-CoV-2) or cause changes in organ-tissue affinity, as occurred with feline infectious peritonitis virus, exacerbating their pathogenesis. In recent studies on different types of CCoV, the pantropic strains characterized by hypervirulent and multi-systemic infections are believed to be emerging, in contrast to classical enteric coronavirus infections. In this study, we investigated emerging hypervirulent and multi-systemic CCoV strains using molecular and bioinformatic analysis, and examined differences between enteric and pantropic CCoV strains at the phylogenetic level. RT-PCR was performed with specific primers to identify the coronavirus M (membrane) and S (spike) genes, and samples were then subjected to DNA sequencing. In phylogenetic analysis, four out of 26 samples were classified as CCoV-1. The remaining 22 samples were all classified as CCoV-2a. In the CCoV-2a group, six samples were in branches close to enteric strains, and 16 samples were in the branches close to pantropic strains. Enteric and pantropic strains were compared by molecular genotyping of CCoV in dogs. Phylogenetic analysis of hypervirulent pantropic strains was carried out at the amino acid and nucleotide sequence levels. CCoV was found to be divergent from the original strain. This implies that some CCoV strains have become pantropic strains that cause multisystemic infections, and they should not be ruled out as the cause of severe diarrhea and multisystemic infections.

Nasal virome of dogs with respiratory infection signs include novel taupapillomaviruses.

Using viral metagenomics, we characterized the mammalian virome of nasal swabs from 57 dogs with unexplained signs of respiratory infection showing mostly negative results using the IDEXX Canine Respiratory Disease RealPCR™ Panel. We identified canine parainfluenza virus 5, canine respiratory coronavirus, carnivore bocaparvovirus 3, canine circovirus and canine papillomavirus 9. Novel canine taupapillomaviruses (CPV21-23) were also identified in 3 dogs and their complete genome sequenced showing L1 nucleotide identity ranging from 68.4 to 70.3% to their closest taupapillomavirus relative. Taupapillomavirus were the only mammalian viral nucleic acids detected in two affected dogs, while a third dog was coinfected with low levels of canine parainfluenza 5. A role for these taupapillomavirues in canine respiratory disease remains to be determined.

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.

New and emerging pathogens in canine infectious respiratory disease.

Canine infectious respiratory disease is a common, worldwide disease syndrome of multifactorial etiology. This review presents a summary of 6 viruses (canine respiratory coronavirus, canine pneumovirus, canine influenza virus, pantropic canine coronavirus, canine bocavirus, and canine hepacivirus) and 2 bacteria (Streptococcus zooepidemicus and Mycoplasma cynos) that have been associated with respiratory disease in dogs. For some pathogens a causal role is clear, whereas for others, ongoing research aims to uncover their pathogenesis and contribution to this complex syndrome. Etiology, clinical disease, pathogenesis, and epidemiology are described for each pathogen, with an emphasis on recent discoveries or novel findings.

Phylogeny of canine coronavirus (CCoV) from Brazilian dogs based on membrane protein partial sequences / Filogenia de Coronavírus canino (CCoV) de cães brasileiros com base em sequências parciais da proteína de membrana

Este artigo descreve a anteriormente desconhecida diversidade molecular de amostras brasileiras de Coronavírus canino (CCoV). Vinte e duas amostras foram submetidas à análise da sequência parcial do gene codificador da proteína de membrana, sendo 12 classificadas como CCoV Tipo II e 10 como CCoV Tipo I e uma possível sublinhagem tipicamente brasileira foi encontrada para o CCoV Tipo II.

Tropism and pathological findings associated with canine respiratory coronavirus (CRCoV).

Canine infectious respiratory disease (CIRD) occurs frequently in densely housed dog populations. One of the common pathogens involved is canine respiratory coronavirus (CRCoV), however little is known regarding its pathogenesis and the role it plays in the development of CIRD. The pathogenesis of five geographically unrelated canine respiratory coronavirus (CRCoV) isolates was investigated. Following experimental infection in dogs, all five CRCoV isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. The presence of CRCoV was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. Viral shedding was readily detected from the oropharynx up to 10 days post infection, but there was little or no evidence of rectal shedding. The successful re-isolation of CRCoV from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of CRCoV for respiratory tissues and fulfils the final requirement for Koch's postulates. By study day 14 dogs had seroconverted to CRCoV and the antibodies raised were neutralising against both homologous and heterologous strains of CRCoV in vitro, thus demonstrating antigenic homogeneity among CRCoV strains from the two continents. Defining the role that CRCoV and other agents play in CIRD is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. Here we have successfully developed a model for studying the pathogenicity and the role of CRCoV in CIRD.

Involvement of FOXO transcription factors, TRAIL-FasL/Fas, and sirtuin proteins family in canine coronavirus type II-induced apoptosis.

n our previous study, we have shown that canine coronavirus type II (CCoV-II) activates both extrinsic and intrinsic apoptotic pathway in a canine fibrosarcoma cell line (A-72 cells). Herein we investigated the role of Sirtuin and Forkhead box O (FOXO) families in this experimental model using Nortern Blot and Western Blot analysis. Our results demonstrated that mitochondrial SIRT3 and SIRT4 protein expression increased from 12 and 24 h post infection (p.i.) onwards, respectively, whereas the nuclear SIRT1 expression increased during the first 12 h p.i. followed by a decrease after 36 h p.i., reaching the same level of control at 48 h p.i. Sirtuins interact with/and regulate the activity of FOXO family proteins, and we herein observed that FOXO3A and FOXO1 expression increased significantly and stably from 12 h p.i. onwards. In addition, CCoV-II induces a remarkable increase in the expression of TNF-related apoptosis-inducing ligand (TRAIL), while we observed a slight up-regulation of FasL/Fas at 36 p.i. with a decrease of both proteins at the end of infection. Furthermore, we found that virus infection increased both bax translocation into mitochondria and decreased bcl-2 expression in cytosol in a time-dependent manner.These data suggest that FOXO transcription factors mediate pro-apoptotic effects of CCoV-II, in part due to activation of extrinsic apoptosis pathway, while some Sirtuin family members (such as SIRT3 and SIRT4) may be involved in intrinsic apoptotic pathway. Moreover, these results propose that TRAIL is an important mediator of cell death induced by CCoV-II during in vitro infection.

Basic science track. Entry and release of canine coronavirus from polarized epithelial cells.

Virus entry into and release from epithelial cells are polarized as a result of the distribution of the viral receptors. In order to establish the polarity of entry and release of CCoV from epithelial cells, the interactions of the virus with A72 and CrFK cells grown on permeable supports was evaluated, and the amount of infective virus in the apical and in the basolateral media was determined and compared. Infection of A72 cells after different times post seeding demonstrated that CCoV grow after infection from both apical and basolateral sides. In CrFK cells, CCoV was observed in both compartments only in the later phase of the infection. To establish the reciprocal binding of CCoV on plasma membrane, A72 cells on a permeable support were preincubated with a mAb specific for CCoV. Infection from the apical side was blocked by mAb applied to that side; in contrast, such treatment on the basolateral side had no effect on the infectious process. Similarly, the low levels of CCoV observed after basolateral exposure to virus was abolished following mAb treatment of that side. The identification of CCoV into the basolateral medium could play an important role in the viral pathogenesis.

An update on canine coronaviruses: viral evolution and pathobiology.

The emergence of human severe acute respiratory syndrome incited renewed interest in animal coronaviruses (CoVs) as potential agents of direct and indirect zoonoses. The reinforced epidemiological surveillance on CoVs has led to the identification of new viruses, genotypes, pathotypes and host variants in animals and humans. In dogs, a CoV associated with mild enteritis, canine coronavirus (CCoV), has been known since 1970s. CoV strains with different biological and genetic properties with respect to classical CCoV strains have been identified in dogs in the last few years, leading to a full reconsideration of the CoV-induced canine diseases. The genetic evolution of dog CoVs is paradigmatic of how CoVs evolve through accumulation of point mutations, insertions or deletions in the viral genome, that led to the emergence of new genotypes (CCoV type I), biotypes (pantropic CCoV) and host variants (canine respiratory coronavirus). This paper is a review of the current literature on the recent genetic evolution of CCoV and emergence of new CoVs in the dog. The significances of the newly acquired information for the canine health status and prophylaxis programmes are also discussed.

Canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex.

Infectious respiratory disease in dogs is a constant challenge because of the involvement of several pathogens and environmental factors. Canine respiratory coronavirus (CRCoV) is a new coronavirus of dogs, which is widespread in North America, Japan, and several European countries. CRCoV has been associated with respiratory disease, particularly in kenneled dog populations. The virus is genetically and antigenically distinct from enteric canine coronavirus; therefore, specific tests are required for diagnosis.

Comparison of the prevalence of enteric viruses in healthy dogs and those with acute haemorrhagic diarrhoea by electron microscopy.

OBJECTIVES: To evaluate prevalence of enteric viruses in healthy dogs and to compare it with prevalences in dogs with acute haemorrhagic diarrhoea. METHODS: Faecal samples were collected from 200 healthy dogs and examined by electron microscopy for presence of viral particles. Data were compared with viral prevalences that had been determined retrospectively by electron microscopy for 936 dogs with acute haemorrhagic diarrhoea. RESULTS: There were significantly more negative faecal samples among the healthy dogs (82.0 per cent) compared with 55.8 per cent in dogs with acute haemorrhagic diarrhoea (P<0.001). With a prevalence of 17.5 per cent, significantly more healthy dogs were shedding coronavirus compared with 11.6 per cent in dogs with acute haemorrhagic diarrhoea (P=0.034). Parvovirus was only detected in one healthy dog (0.5 per cent), thus with a prevalence that was significantly lower than 16.0 per cent detected in the dogs with acute haemorrhagic diarrhoea (P<0.001). Paramyxovirus was not found in any of the healthy dogs but was found in 9.3 per cent of dogs with acute haemorrhagic diarrhoea (P<0.001). CLINICAL SIGNIFICANCE: Results suggest that shedding of parvovirus and paramyxovirus is strongly associated with acute haemorrhagic diarrhoea. However, coronavirus seems to be even more prevalent among healthy dogs, raising the need for further studies to investigate the strain-associated pathogenicity of this virus.

Experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia.

A pantropic canine coronavirus (CCoV) strain (CB/05) has been recently associated to a fatal outbreak of systemic disease in young dogs. We report the clinical, virological and serological findings in dogs experimentally infected with strain CB/05. The dogs, three 2.5-month-old and two 6-month-old pups, were successfully infected, shedding viral RNA with their faeces for the entire observation period (21 days) and displaying systemic clinical signs resembling those observed during the course of natural infection. Leucopenia (acute lymphopenia) occurred in all infected dogs, with values dropping below 60% of the initial counts. Considering the severity of the CB/05-induced disease, two of the youngest pups were euthanized for ethical reasons at days 8-9 postinfection, whereas the other pups underwent a slow but progressive improvement of their clinical status with complete recovery. At postmortem examination, remarkable lesions were observed in the internal organs of the euthanized pups, that tested positive for CCoV by real-time RT-PCR and virus isolation on cell cultures. All pups seroconverted for CCoV, as shown by the high optical density values and antibody titres detected by ELISA and virusneutralisation tests, respectively. The present study confirms that strain CB/05 is highly pathogenic for dogs, being able to induce a severe disease (and in some cases the death) even in experimental conditions.

Canine coronavirus inactivation with physical and chemical agents.

Canine coronavirus (CCoV) is responsible for mild or moderate enteritis in puppies. The virus is highly contagious and avoiding contact with infected dogs and their excretions is the only way to ensure disease prevention. Since no studies have yet focused on the sensitivity of CCoV to chemical biocides the present investigation examined the efficiency of physical and chemical methods of viral inactivation. CCoV infectivity was stable at +56 degrees C for up to 30 min, but tended to decrease rapidly at +65 degrees C and +75 degrees C. Germicidal ultra-violet (UV-C) light exposure demonstrated no significant effects on virus inactivation for up to 3 days. CCoV was observed to be more stable at pH 6.0-6.5 while extreme acidic conditions inactivated the virus. Two tested aldehydes inactivated the virus but their action was temperature- and time-dependent. The methods for CCoV inactivation could be applied as animal models to study human coronavirus infection, reducing the risk of accidental exposure of researchers to pathogens during routine laboratory procedures.

Canine coronavirus highly pathogenic for dogs.

Canine coronavirus (CCoV) is usually responsible for mild, self-limiting infections restricted to the enteric tract. We report an outbreak of fatal disease in puppies caused by a pathogenic variant of CCoV that was isolated from organs with severe lesions.

Genetic evolution of canine coronavirus and recent advances in prophylaxis.

Since the first identification of the virus in 1971, the disease caused by canine coronavirus (CCoV) has not been adequately investigated and the role that the virus plays in canine enteric illness has still not been well established. In the last decade, as a consequence of the relatively high mutation frequency of RNA positive stranded viruses, CCoV has evolved and a new genotype has been identified in the faeces of infected dogs. The several studies carried out by different researchers have focused upon the epidemiological relevance of these viruses and, considering the wide diffusion of CCoV infections among dog populations, the author underlines the need for further investigation on the biology of CCoV and on the pathogenetic role of their infections.

Feline and canine coronaviruses are released from the basolateral side of polarized epithelial LLC-PK1 cells expressing the recombinant feline aminopeptidase-N cDNA.

In this study feline (FECV and FIPV) and canine (CCoV) coronavirus entry into and release from polarized porcine epithelial LLC-PK1 cells, stably expressing the recombinant feline aminopeptidase-N cDNA, were investigated. Virus entry appeared to occur preferentially through the apical membrane, similar to the entry of the related porcine coronavirus transmissible gastroenteritis virus (TGEV) into these cells. However, whereas TGEV is released apically, feline and canine coronaviruses were found to be released from the basolateral side of the epithelial cells. These observations indicate that local infections as caused by TGEV, FECV and CCoV do not strictly correlate with apical release, as suggested by earlier work.

Canine coronavirus infections in Japan: virological and epidemiological aspects.

Ten strains, eight field and two reference laboratory strains, of canine coronavirus (CCV) were comparatively examined with respect to antigenic relationships and pathogenic potential in dogs. With monoclonal antibodies and hyperimmune antisera to feline coronavirus and CCV, respectively, varying degrees of antigenic diversities were found among the strains by neutralization and immunofluorescence assays, but it was felt that they belong to one serotype. Specific-pathogen-free puppies experimentally inoculated with some CCV strains manifested clinical symptoms, but there was a difference in their virulence. In order to elucidate the prevalence of CCV infections in dogs in Japan, we tested for neutralizing antibodies to CCV in 467 field dogs, and found a prevalence of 44.1%. Moreover, by using nested reverse transcriptase-polymerase chain reaction on rectal swabs of 100 diarrheic dogs recently presented in veterinary clinics, evidence of CCV in 16% of these specimens was found. The results suggested that CCV infection is more widespread than expected in dogs, and that CCV is a significant etiologic factor in canine diarrhea also in Japan.

Differences in virus receptor for type I and type II feline infectious peritonitis virus.

Feline infectious peritonitis viruses (FIPVs) are classified into type I and type II serogroups. Here, we report that feline aminopeptidase N (APN), a cell-surface metalloprotease on the intestinal, lung and kidney epithelial cells, is a receptor for type II FIPV but not for type I FIPV. A monoclonal antibody (MAb) R-G-4, which blocks infection of Felis catus whole fetus (fcwf-4) cells by type II FIPV, was obtained by immunizing mice with fcwf-4 cells which are highly susceptible to FIPV. This MAb also blocked infection of fcwf-4 cells by type II feline enteric coronavirus (FECV), canine coronavirus (CCV), and transmissible gastroenteritis virus (TGEV). On the other hand, it did not block infection by type I FIPVs. MAb R-G-4 recognized a polypeptide of relative molecular mass 120-130 kDa in feline intestinal brush-border membrane (BBM) proteins. The polypeptide possessed aminopeptidase activity, and the first 15 N-terminal amino acid sequence was identical to that of the feline APN. Feline intestinal BBM proteins and the polypeptide reacted with MAb R-G-4 (feline APN) inhibited the infectivity of type II FIPV, type II FECV, CCV and TGEV to fcwf-4 cells, but did not inhibit the infectivity of type I FIPVs.