Emerging Variants of Canine Enteric Coronavirus Associated with Outbreaks of Gastroenteric Disease.

A 2022 canine gastroenteritis outbreak in the United Kingdom was associated with circulation of a new canine enteric coronavirus closely related to a 2020 variant with an additional spike gene recombination. The variants are unrelated to canine enteric coronavirus-like viruses associated with human disease but represent a model for coronavirus population adaptation.

Genomic analysis of canine pneumoviruses and canine respiratory coronavirus from New Zealand.

AIMS: To isolate canine respiratory coronavirus (CRCoV) and canine pneumovirus (CnPnV) in cell culture and to compare partial genomic sequences of CRCoV and CnPnV from New Zealand with those from other countries. METHODS: Oropharyngeal swab samples from dogs affected by canine infectious respiratory disease syndrome that were positive for CnPnV (n = 15) or CRCoV (n = 1) by virus-specific reverse transcriptase quantitative PCR (RT-qPCR) in a previous study comprised the starting material. Virus isolation was performed in HRT-18 cells for CRCoV and RAW 264.7 and Vero cells for CnPnV. The entire sequence of CnPnV G protein (1,266 nucleotides) and most (8,063/9,707 nucleotides) of the 3' region of CRCoV that codes for 10 structural and accessory proteins were amplified and sequenced. The sequences were analysed and compared with other sequences available in GenBank using standard molecular tools including phylogenetic analysis. RESULTS: Virus isolation was unsuccessful for both CRCoV and CnPnV. Pneumovirus G protein was amplified from 3/15 (20%) samples that were positive for CnPnV RNA by RT-qPCR. Two of these (NZ-048 and NZ-049) were 100% identical to each other, and 90.9% identical to the third one (NZ-007). Based on phylogenetic analysis of the G protein gene, CnPnV NZ-048 and NZ-049 clustered with sequences from the USA, Thailand and Italy in group A, and CnPnV NZ-007 clustered with sequences from the USA in group B. The characteristics of the predicted genes (length, position) and their putative protein products (size, predicted structure, presence of N- and O-glycosylation sites) of the New Zealand CRCoV sequence were consistent with those reported previously, except for the region located between open reading frame (ORF)3 (coding for S protein) and ORF6 (coding for E protein). The New Zealand virus was predicted to encode 5.9 kDa, 27 kDa and 12.7 kDa proteins, which differed from the putative coding capacity of this region reported for CRCoV from other countries. CONCLUSIONS: This report represents the first characterisation of partial genomic sequences of CRCoV and CnPnV from New Zealand. Our results suggest that the population of CnPnV circulating in New Zealand is not homogeneous, and that the viruses from two clades described overseas are also present here. Limited conclusions can be made based on only one CRCoV sequence, but the putative differences in the coding capacity of New Zealand CRCoV support the previously reported variability of this region. The reasons for such variability and its biological implications need to be further elucidated.

Detection and molecular characteristics of canine coronavirus in Chengdu city, Southwest China from 2020 to 2021.

Canine coronavirus (CCoV) is generally thought of as a mild, but highly contagious, enteritis of young dogs. This study was to investigate the molecular detection and characteristics of CCoV in Chengdu city, Southwest China. 218 canine fecal samples were collected from four animal hospitals and one animal shelter from 2020 to 2021. Fifty-nine CCoV-positive samples were detected by RT-PCR, including 40 CCoV-I, 25 CCoV-IIa, one CCoV-IIb and 10 untyped. To further analyze the genetic diversity of CCoV, we amplified ten complete spike (S) genes, including four CCoV-I and six CCoV-II strains. The amino acid sequence obtained in this study revealed 85.95% ± 12.55% homology with the reference strains. Moreover, in the N-terminal structural domain, there were two amino acid insertions (17QQ18) in two strains of CCoV-I and four amino acid insertions (95IGTN98) in CCoV-IIb strain. Interestingly, we identified that the S1/S2 cleavage site of the S protein of CCoV strains (SWU-SSX3 and SWU-SSX10) were consistent with feline coronavirus (FCoV). In the evolutionary tree, a strain of CCoV-I (SWU-SSX10) was found to be more closely related to FCoV, while SWU-SSX7 of CCoV-IIb was more closely related to coronavirus from the Chinese ferret badger. In addition, for the first time, recombination in a CCoV-IIb strain was found to occur between two subtypes occurring in the C domain of the S1 subunit, with a breakpoint starting at 2141 nt. The results enriched the epidemiological information of CCoV and provided an important reference for the prevention of CCoV in Chengdu city, Southwest China.

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.

Development of a nanoparticle-assisted PCR assay to distinguish canine coronaviruses I and II.

Nanoparticle-assisted PCR (nanoPCR) is a novel method for the simple, rapid, and specific detection of viruses. We developed a nanoPCR method to detect and differentiate canine coronavirus I (CCoV I) and II (CCoV II). Primer pairs were designed against the M gene conserved region of CCoV I and CCoV II, producing specific fragments of 239 bp (CCoV I) and 105 bp (CCoV II). We optimized the annealing temperature and primer concentrations for the CCoV nanoPCR assay and assessed its sensitivity and specificity. Under optimized nanoPCR reaction conditions, the detection limits were 6.47 × 101 copies/µL for CCoV I and 6.91 × 102 copies/µL for CCoV II. No fragments were amplified using other canine viruses as templates. The sensitivity of the nanoPCR assay was 100-fold higher than that of a conventional RT-PCR assay. Among 60 clinical samples collected from Beijing, China, the assay detected 12% positive for CCoV I and 48% positive for CCoV II. Our nanoPCR method is an effective method to rapidly detect CCoV I and CCoV II alone, or as a mixed infection, in dogs.

Detection and Molecular Characterization of Canine Alphacoronavirus in Free-Roaming Dogs, Bangladesh.

Canine coronavirus (CCoV) is widespread among the dog population and causes gastrointestinal disorders, and even fatal cases. As the zoonotic transmission of viruses from animals to humans has become a worldwide concern nowadays, it is necessary to screen free-roaming dogs for their common pathogens due to their frequent interaction with humans. We conducted a cross-sectional study to detect and characterize the known and novel Corona, Filo, Flavi, and Paramyxoviruses in free-roaming dogs in Bangladesh. Between 2009-10 and 2016-17, we collected swab samples from 69 dogs from four districts of Bangladesh, tested using RT-PCR and sequenced. None of the samples were positive for Filo, Flavi, and Paramyxoviruses. Only three samples (4.3%; 95% CI: 0.9-12.2) tested positive for Canine Coronavirus (CCoV). The CCoV strains identified were branched with strains of genotype CCoV-II with distinct distances. They are closely related to CCoVs from the UK, China, and other CoVs isolated from different species, which suggests genetic recombination and interspecies transmission of CCoVs. These findings indicate that CCoV is circulating in dogs of Bangladesh. Hence, we recommend future studies on epidemiology and genetic characterization with full-genome sequencing of emerging coronaviruses in companion animals in Bangladesh.

Etiology and genetic evolution of canine coronavirus circulating in five provinces of China, during 2018-2019.

As the outbreaks of COVID-19 in worldwide, coronavirus has once again caught the attention of people. Canine coronavirus is widespread among dog population, and sometimes causes even fatal cases. Here, to characterize the prevalence and evolution of current circulating canine coronavirus (CCoV) strains in China, we collected 213 fecal samples from diarrheic pet dogs between 2018 and 2019. Of the 213 samples, we found 51 (23.94%) were positive for CCoV. Co-infection with canine parvovirus (CPV), canine astrovirus (CaAstV), canine kobuvirus (CaKV), Torque teno canis virus (TTCaV) were ubiquitous existed. Mixed infection of different CCoV subtypes exists extensively. Considering the limited sequences data in recent years, we sequenced 7 nearly complete genomes and 10 complete spike gene. Phylogenetic analysis of spike gene revealed a new subtype CCoV-II Variant and CCoV-IIa was the most prevalent subtype currently circulating. Moreover, we identified strain B906_ZJ_2019 shared 93.24% nucleotide identifies with previous strain A76, and both of them clustered with CCoV-II Variant, which were not well clustered with the known subtypes. Recombination analysis of B906_ZJ_2019 indicated that strain B906_ZJ_2019 may a recombinant variant between CCoV-I and CCoV-II, which is consistent with strain A76. Furthermore, amino acid variations widely existed among current CCoV-IIa strains circulating in China and the classic CCoV-IIa strains, in spite of the unknown functions. In a word, we report a useful information as to the etiology and evolution of canine coronavirus in China based on the available sequences, which is urgent for the devise of future effective disease prevention and control strategies.

Multiplex PCR methods for detection of several viruses associated with canine respiratory and enteric diseases.

Viral respiratory and intestinal infections are the most common causes of canine viral illness. Infection with multiple pathogens occurs in many cases. Rapid diagnosis of these multiple infections is important for providing timely and effective treatment. To improve diagnosis, in this study, two new multiplex polymerase chain reactions (mPCRs) were developed for simultaneous detection of canine respiratory viruses (CRV) and canine enteric viruses (CEV) using two separate primer mixes. The viruses included canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine circovirus (CanineCV), canine coronavirus (CCoV) and canine parvovirus (CPV). The sensitivity of the mPCR results showed that the detection limit of both mPCR methods was 1×104 viral copies. Twenty nasal swabs (NS) and 20 anal swabs (AS) collected from dogs with symptoms of respiratory disease or enteric disease were evaluated using the novel mPCR methods as a clinical test. The mPCR protocols, when applied to these respiratory specimens and intestinal samples, could detect 7 viruses simultaneously, allowing rapid investigation of CRV (CAV-2, CDV, CIV and CPIV) and CEV (CAV-2, CanineCV, CCoV and CPV) status and prompt evaluation of coinfection. Our study provides an effective and accurate tool for rapid differential diagnosis and epidemiological surveillance in dogs.

A fast and simple one-step duplex PCR assay for canine distemper virus (CDV) and canine coronavirus (CCoV) detection.

The one-step polymerase chain reaction (one-step PCR) detection assay is an innovative PCR detection method, eliminating nucleic acid extraction steps, in which samples can be directly added to PCR reagents for testing. For simultaneous detection of CDV and CCoV, a sensitive and specific one-step duplex PCR (one-step dPCR) assay was developed with two pairs of primers that were designed based on H and M gene sequences of CDV and CCoV, respectively. The one-step dPCR with optimized detection conditions has high specificity and sensitivity; independent sequencing assays further verified these results.

Prevalence and genome characteristics of canine astrovirus in southwest China.

The aim of this study was to investigate canine astrovirus (CaAstV) infection in southwest China. We collected 107 faecal samples from domestic dogs with obvious diarrhoea. Forty-two diarrhoeic samples (39.3 %) were positive for CaAstV by RT-PCR, and 41/42 samples showed co-infection with canine coronavirus (CCoV), canine parvovirus-2 (CPV-2) and canine distemper virus (CDV). Phylogenetic analysis based on 26 CaAstV partial ORF1a and ORF1b sequences revealed that most CaAstV strains showed unique evolutionary features. Interestingly, putative recombination events were observed among four of the five complete ORF2 sequences cloned in this study, and three of the five complete ORF2 sequences formed a single unique group, suggesting that these strains could be a novel genotype. We successfully sequenced the complete genome of one CaAstV strain (designated 2017/44/CHN), which was 6628 nt in length. The features of this genome include putative recombination events in the ORF1a, ORF1b and ORF2 genes, while the ORF2 gene had a continuous insertion of 7 aa in region II compared with the other complete ORF2 sequences available in GenBank. Phylogenetic analysis showed that 2017/44/CHN formed a single group based on genome sequences, suggesting that this strain might be a novel genotype. The results of this study revealed that CaAstV circulates widely in diarrhoeic dogs in southwest China and exhibits unique evolutionary events. To the best of our knowledge, this is the first report of recombination events in CaAstV, and it contributes to further understanding of the genetic evolution of CaAstV.

A multiplex PCR method for the simultaneous detection of three viruses associated with canine viral enteric infections.

The aim of this study was to establish a multiplex PCR (mPCR) method that can simultaneously detect canine parvovirus (CPV-2), canine coronavirus (CCoV) and canine adenovirus (CAV), thereby eliminating the need to detect these pathogens individually. Based on conserved regions in the genomes of these three viruses, the VP2 gene of CPV-2, the endoribonuclease nsp15 gene of CCoV, and the 52K gene of CAV were selected for primer design. The specificity of the mPCR results showed no amplification of canine distemper virus (CDV), canine parainfluenza virus (CPIV), or pseudorabies virus (PRV), indicating that the method had good specificity. A sensitivity test showed that the detection limit of the mPCR method was 1 × 104 viral copies. A total of 63 rectal swabs from dogs with diarrheal symptoms were evaluated using mPCR and routine PCR. The ratio of positive samples to total samples for CPV-2, CCoV, and CAV was 55.6% (35/63) for mPCR and 55.6% (35/63) for routine PCR. Thirty-five positive samples were detected by both methods, for a coincidence ratio of 100%. This mPCR method can simultaneously detect CCoV (CCoV-II), CAV (CAV-1, CAV-2) and CPV-2 (CPV-2a, CPV-2b, CPV-2c), which are associated with viral enteritis, thereby providing an efficient, inexpensive, specific, and accurate new tool for clinical diagnosis and laboratory epidemiological investigations.

Molecular characterization of canine parvovirus and canine enteric coronavirus in diarrheic dogs on the island of St. Kitts: First report from the Caribbean region.

Although canine parvovirus (CPV) and canine enteric coronavirus (CCoV) are important enteric pathogens of dogs and have been studied extensively in different parts of the world, there are no reports on these viruses from the Caribbean region. During 2015-2016, a total of 104 diarrheic fecal samples were collected from puppies and adult dogs, with or without hemorrhagic gastroenteritis, on the Caribbean island of St. Kitts (KNA). By PCR, 25 (24%, n=104) samples tested positive for CPV. Based on analysis of the complete deduced VP2 amino acid sequences, 20 of the KNA CPV strains were assigned to new CPV-2a (also designated as CPV-2a-297A). On the other hand, the VP2 genes of the remaining 5 strains were partially characterized, or could not be sequenced. New CPV-2a was the predominant CPV variant in St. Kitts, contrasting the molecular epidemiology of CPV variants reported in most studies from nearby North and South American countries. By RT-PCR, CCoVs were detected in 5 samples (4.8%, n=104). Based on analysis of partial M-protein gene, the KNA CCoV strains were assigned to CCoV-I genotype, and were closely related to CCoV-I strains from Brazil. To our knowledge, this is the first report on detection and genetic diversity of CPV and CCoV in dogs from the Caribbean region, and underscores the importance of similar studies in the other Caribbean islands.

Discovery of a novel canine respiratory coronavirus support genetic recombination among betacoronavirus1.

Although canine respiratory coronavirus (CRCoV) is an important respiratory pathogen that is prevalent in many countries, only one complete genome sequence of CRCoV (South Korea strain K37) has been obtained to date. Genome-wide analyses and recombination have rarely been conducted, as small numbers of samples and limited genomic characterization have previously prevented further analyses. Herein, we report a unique CRCoV strain, denoted strain BJ232, derived from a CRCoV-positive dog with a mild respiratory infection. Phylogenetic analysis based on complete genome of all available coronaviruses consistently show that CRCoV BJ232 is most closely related to human coronavirus OC43 (HCoV-OC43) and BCoV, forming a separate clade that split off early from other Betacoronavirus 1. Based on the phylogenetic and SimPlot analysis we propose that CRCoV-K37 was derived from genetic recombination between CRCoV-BJ232 and BCoV. In detail, spike (S) gene of CRCoV-K37 clustered with CRCoV-BJ232. However orf1ab, membrane (M) and nucleocapsid (N) genes were more related to Bovine coronavirus (BCoV) than CRCoV-B232. Molecular epidemic analysis confirmed the prevalence of CRCoV-BJ232 lineage around the world for a long time. Recombinant events among Betacoronavirus 1 may have implications for CRCoV transmissibility. All these findings provide further information regarding the origin of CRCoV.

Characterization of pantropic canine coronavirus from Brazil.

Characterization of canine coronavirus (CCoV) strains currently in circulation is essential for understanding viral evolution. The aim of this study was to determine the presence of pantropic CCoV type IIa in tissue samples from five puppies that died in Southern Brazil as a result of severe gastroenteritis. Reverse-transcriptase PCR was used to generate amplicons for sequence analysis. Phylogenetic analysis of the CCoV-IIa strains indicated that they were similar to those found in other countries, suggesting a common ancestor of these Brazilian isolates. This is the first report of pantropic CCoV-II in puppies from Latin America and our findings highlight that CCoV should be included as a differential diagnosis when dogs present with clinical signs and lesions typically seen with canine parvovirus infection.

Genotypic characterization of canine coronaviruses associated with fatal canine neonatal enteritis in the United States.

Emerging canine coronavirus (CCoV) variants that are associated with systemic infections have been reported in the European Union; however, CCoV-associated disease in the United States is incompletely characterized. The purpose of this study was to correlate the clinicopathological findings and viral antigen distribution with the genotypic characteristics of CCoV in 11 puppies from nine premises in five states that were submitted for diagnostic investigation at Cornell University between 2008 and 2013. CCoV antigen was found in epithelial cells of small intestinal villi in all puppies and the colon in 2 of the 10 puppies where colon specimens were available. No evidence of systemic CCoV infection was found. Comparative sequence analyses of viral RNA extracted from intestinal tissues revealed CCoV-II genotype in 9 out of 11 puppies. Of the nine CCoV-IIs, five were subtyped as group IIa and one as IIb, while three CCoVs could not be subtyped. One of the CCoV-IIa variants was isolated in cell culture. Infection with CCoV alone was found in five puppies, of which two also had small intestinal intussusception. Concurrent infections with either parvovirus (n = 1), attaching-effacing Escherichia coli (n = 4), or protozoan parasites (n = 3) were found in the other six puppies. CCoV is an important differential diagnosis in outbreaks of severe enterocolitis among puppies between 4 days and 21 weeks of age that are housed at high population density. These findings will assist with the rapid laboratory diagnosis of enteritis in puppies and highlight the need for continued surveillance for CCoV variants and intestinal viral diseases of global significance.

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.

Nucleic acid-based differential diagnostic assays for feline coronavirus.

Feline coronavirus (FCoV) is a pleomorphic, enveloped, positive-sense single-stranded RNA virus. Owing to the differences in its genotype, FCoV belongs to a separate clade along with other viruses, such as transmissible gastroenteritis virus (TGEV) and canine coronavirus (CCoV), which can be isolated from cats. In this study, a PCR assay was developed to differentiate these coronaviruses concurrently. Multiplex differential RT-PCR was performed with primers based on the highly conserved coronavirus membrane protein. Three primer sets were designed: a primer pair (S1 and S2) that can bind to conserved sequences in all target coronaviruses, a CCoV-specific primer (S3), and a TGEV-specific primer (S4). Because of the high sequence homology among FCoV, CCoV, and TGEV, a nucleotide preceding the last pair of dissimilar nucleotides in S3 and S4 was substituted with an inosine to allow primer binding. This assay could detect and differentiate FCoV (n=7), CCoV (n=4), and TGEV (n=8) precisely and did not show any cross-reactivity with other pathogens. These results suggest that this molecular approach provides a rapid and reliable way to detect FCoV, especially in feline clinical specimens.

Detection and genetic characterization of Canine parvovirus and Canine coronavirus strains circulating in district of Tirana in Albania.

An epidemiological survey for Canine parvovirus 2 (CPV-2) and Canine coronavirus (CCoV) was conducted in Albania. A total of 57 fecal samples were collected from diarrheic dogs in the District of Tirana during 2011-2013. The molecular assays detected 53 and 31 CPV- and CCoV-positive specimens, respectively, with mixed CPV-CCoV infections diagnosed in 28 dogs. The most frequently detected CPV type was 2a, whereas IIa was the predominant CCoV subtype. A better comprehension of the CPV-CCoV epidemiology in eastern European countries will help to assess the most appropriate vaccination strategies to prevent disease due to infections with these widespread agents of acute gastroenteritis in the dog.

Infection of cats with atypical feline coronaviruses harbouring a truncated form of the canine type I non-structural ORF3 gene.

Feline and canine coronaviruses (FCoV and CCoV, respectively) are common pathogens of cats and dogs sometimes leading to lethal infections named feline infectious peritonitis (FIP) and canine pantropic coronavirus infection. FCoV and CCoV are each subdivided into two serotypes, FCoV-I/II and CCoV-I/II. A phylogenetic relationship is evident between, on one hand, CCoV-I/FCoV-I, and on the other hand, CCoV-II/FCoV-II, suggesting that interspecies transmission can occur. The aim of the present study was to evaluate the prevalence of coronavirus (CoV)-infected cats according to their contact with dogs and to genetically analyse the CoV strains infecting cats. From 2003 to 2009, we collected 88 faecal samples from healthy cats and 11 ascitic fluids from FIP cats. We investigated the possible contact with dog in the household and collected dogs samples if appropriate. Out of 99 cat samples, 26 were coronavirus positive, with six cats living with at least one dog, thus showing that contact with dogs does not appear as a predisposing factor for cats CoV infections. Molecular and phylogenetic analyses of FCoV strains were conducted using partial N and S sequences. Six divergent strains were identified with the N gene clustering with CCoV-I whereas the 3' end of S was related to FCoV-I. Further analysis on those six samples was attempted by researching the presence of the ORF3 gene, the latter being peculiar to CCoV-I to date. We succeeded to amplify the ORF3 gene in five samples out of six. Thus, our data strongly suggest the circulation of atypical FCoV strains harbouring the CCoV-I ORF3 gene among cats. Moreover, the ORF3 genes recovered from the feline strains exhibited shared deletions, never described before, suggesting that these deletions could be critical in the adaptation of these strains to the feline host.