Epidemiological and phylogenetic analysis of canine kobuviruses in Tangshan, China.

Canine kobuviruses (CaKoV) have been found in healthy and diarrheic dogs as well as asymptomatic wild carnivores in various countries. In order to investigate the prevalence and evolution of CaKoV in Tangshan, China, 82 dog fecal samples from pet hospitals in Tangshan were subjected to RT-PCR targeting a segment of the 3D gene of CaKoV. Using this method, we identified CaKoV in 14 samples (17.07%, 14/82). Of the CaKoV-positive samples, 78.57% (11/14) and 50% (7/14) were positive for canine parvovirus and canine coronavirus, respectively. The nucleotide sequences of the 14 strains 96.6%-100% identical to each other and 77.6%-99.2% identical to representative sequences from the NCBI GenBank database. We also amplified the 14 VP1 gene sequences and found that they were 93.3%-99.6% identical to each other and 73.3%-97.8% identical to representative sequences from the NCBI GenBank database. Phylogenetic analysis revealed that the 14 CaKoV strains from Tangshan are closely related to those identified in China and Thailand and display less similarity to those found in Africa, the United States, and Europe. Our data suggest that CaKoV circulated in young pet dogs in Tangshan and displays a high co-infection rate with CCoV and CPV. However, the relationship between the three viruses and their roles in the host requires further investigation.

Detection and genetic characterization of kobuvirus in cats: The first molecular evidence from Northeast China.

Feline kobuvirus (FeKoV), a novel picornavirus of the genus kobuvirus, was initially identified in the feces of cats with diarrhea in South Korea in 2013. To date, there is only one report of the circulation of kobuvirus in cats in southern China. To investigate the presence and genetic variability of FeKoV in northeast China, 197 fecal samples were collected from 105 cats with obvious diarrhea and 92 asymptomatic cats in Shenyang, Jinzhou, Changchun, Jilin and Harbin regions, Northeast China, and viruses were detected by RT-PCR with universal primers targeting all kobuviruses. Kobuvirus was identified in 28 fecal samples with an overall prevalence of 14.2% (28/197) of which 20 samples were co-infected with feline parvovirus (FPV) and/or feline bocavirus (FBoV). Diarrhoeic cats had a higher kobuvirus prevalence (19.1%, 20/105) than asymptomatic cats (8.7%, 8/92). By genetic analysis based on partial 3D gene, all kobuvirus-positive samples were more closely related to previous FeKoV strains with high identities of 90.5%-97.8% and 96.6%-100% at the nucleotide and amino acid levels. Additionally, phylogenetic analysis based on the complete VP1 gene indicated that all FeKoV strains identified in this study were placed into a cluster, which separated from other reference strains previously reported, and three identical amino acid substitutions were present at the C-terminal of the VP1 protein for these FeKoV strains. Furthermore, two complete FeKoV polyprotein genomes were successfully obtained from two positive samples and designated 16JZ0605 and 17CC0811, respectively. The two strains shared 92.9%-94.9% nucleotide identities and 96.8%-98.4% amino acid identities to FeKoV prototype strains. Phylogenetic analysis indicated that FeKoVs were clustered according to their geographical regions, albeit with limited sequences support. This study provides the first molecular evidence that FeKoV circulates in cats in northeast China, and these FeKoVs exhibit genetic diversity and unique evolutionary trend.

Detection and genetic characterization of bovine kobuvirus from calves in Egypt.

Kobuviruses are small non-enveloped RNA viruses that probably cause diarrhea in cattle and swine. Since its discovery in 2003, few studies have addressed bovine kobuvirus (BKoV; a species of Aichivirus B) infections. BKoV has been reported in Europe, Asia, and South America, suggesting a worldwide distribution. To investigate the presence of BKoV in Egypt, 36 fecal specimens from diarrheic calves in two different Egyptian provinces (Cairo and Sharkia) were screened by RT-PCR and 24 (66.7%) were found positive for BKoV. RNA from one of the positive samples (BKoV/Egy-1/KY407744) was subjected to next-generation sequencing to determine the complete BKoV genome sequence. When compared to the only recorded BKoV genome sequence (BKoV/U-1/AB084788), the studied strain showed 94 amino acid (aa) substitutions through its entire polyprotein (2463 aa), one nucleotide (nt) insertion and one nt deletion in the 2B gene and 4-nt deletions in the UTRs (2 each). Additionally, five VP1 and seven 3D sequences were obtained from other samples by using RT-PCR and Sanger sequencing. A discrepancy in the phylogenetic topography of VP1 and 3D was observed, where the Egyptian VP1 sequences were classified as a distinct cluster within the proposed lineage 1 (genotype A), which also contained strains from the UK, Brazil, and Japan. While, the 3D sequences from Cairo were related to those of Chinese strains unlike Sharkia ones that were more closer to Korean  strains. To the best of our knowledge, this is the first detection and genomic characterization of BKoV in Egypt or indeed Africa.

Phylogenetic analysis of VP1 and RdRP genes of Brazilian aichivirus B strains involved in a diarrhea outbreak in dairy calves.

Aichivirus B has been reported worldwide in calves and adult cattle with and without diarrhea. The aim of this study was to describe the molecular characteristics of the RdRP and VP1 genes of aichivirus B strains identified as the most frequent etiologic agent in a neonatal diarrhea outbreak in a high-production Brazilian dairy cattle herd. Preliminary laboratory analysis ruled out important enteropathogens (Cryptosporidium spp; Eimeria spp., E. coli F5, and bovine coronavirus). Fecal samples from diarrheic (n = 24) and asymptomatic (n = 5) calves up to 30 days old were collected for virological analysis. RT-PCR assays were performed for the detection of aichivirus B RdRP and VP1 genes and for rotavirus A VP7 and VP4 genes in fecal samples. Asymptomatic calves (control group) were negative for both viruses. Aichivirus B and rotavirus A G10P[11] genotypes were found in 54.2% (13/24) and 25% (6/24) of the diarrheic fecal samples, respectively. Aichivirus B was only identified (83.3%, 10/12) in calves up to two weeks old. Phylogenetic analysis based on the RdRP gene grouped the Brazilian strains in a new branch within the aichivirus B group. Comparative analysis of the nucleotide sequence of the VP1 gene of Brazilian and Chinese aichivirus B strains allowed the strains identified in this study to be classified in the putative lineage 1. This is the first description of a high rate of aichivirus B detection in a diarrhea outbreak in dairy calves, and the first phylogenetic study of the VP1 gene of aichivirus B wild-type strains performed in South America.

Aichivirus in Children with Diarrhea in Northern Italy.

OBJECTIVE: Since its discovery, Aichivirus (AiV) A has been detected, with an incidence of 0.9-4.1%, primarily when studying outbreaks of diarrhea in children or young adults. In this paper, we report the first detection of AiV in Piedmont, Italy, in pediatric patients. METHODS: A total of 159 fecal specimens (from 96 males and 63 females) previously screened for rotaviruses, adenoviruses, noroviruses, human parechoviruses, saliviruses, and sapoviruses were collected from infants and children with acute gastroenteritis. RESULTS: The most commonly detected virus was norovirus GII (33.80%), fol lowed by rotavirus (21.30%), astrovirus (18.87%), boca virus (13.92%), sapovirus (10.90%), parechovirus (8%), norovirus GI (6.70%), adenovirus (1%), and salivirus (0.52%). Real-time polymerase chain reaction detected AiV A in 1 (0.62%) case subjects. AiV A was detected in monoinfection only in January. CONCLUSIONS: Our results indicate that AiV may be associated with a limited number of diarrhea cases in pediatric patients.

Large-scale screening and characterization of enteroviruses and kobuviruses infecting pigs in Vietnam.

A recent survey of pigs in Dong Thap province, Vietnam identified a high frequency of enterovirus species G (EV-G) infection (144/198; 72.7%). Amongst these was a plethora of EV-G types (EV-G1, EV-G6 and four new types EV-G8-EV-G11). To better characterize the genetic diversity of EV-G and investigate the possible existence of further circulating types, we performed a larger-scale study on 484 pig and 45 farm-bred boar faecal samples collected in 2012 and 2014, respectively. All samples from the previous and current studies were also screened for kobuviruses. The overall EV infection frequency remained extremely high (395/484; 81.6%), but with comparable detection rates and viral loads between healthy and diarrhoeic pigs; this contrasted with less frequent detection of EV-G in boars (4/45; 8.9%). EV was most frequently detected in pigs ≤ 14 weeks old (∼ 95%) and declined in older pigs. Infections with EV-G1 and EV-G6 were most frequent, whilst less commonly detected types included EV-G3, EV-G4 and EV-G8-EV-G11, and five new types (EV-G12-EV-G16). In contrast, kobuvirus infection frequency was significantly higher in diarrhoeic pigs (40.9 versus 27.6%; P = 0.01). Kobuviruses also showed contrasting epizootiologies and age associations; a higher prevalence was found in boars (42%) compared with domestic pigs (29%), with the highest infection frequency amongst pigs >52 weeks old. Although genetically diverse, all kobuviruses identified belonged to the species Aichivirus C. In summary, this study confirms infection with EV-G was endemic in Vietnamese domestic pigs and exhibits high genetic diversity and extensive inter-type recombination.

Genomic characterization of a US porcine kobuvirus strain.

Porcine kobuvirus has been detected from pig fecal samples in the USA, but there is still no information on the full-length genomes. In this study, we characterized the first complete genomic sequence of a US porcine kobuvirus strain OH/RV50/2011. The viral genome is 8123 nucleotides (nt) long, including a 576-nt 5'-untranslated region (UTR), a 7380-nt polyprotein encoding sequence, and a 167-nt 3'-UTR. A complete genome sequence alignment suggested that two types of porcine kobuviruses were found based on whether a 30-aa deletion existed in the 2B encoding region. Furthermore, several conserved motifs that can be used for the design of universal kobuvirus or porcine kobuvirus-specific primers were verified in non-structural protein genes. Phylogenetic analysis based on the complete genome sequence showed that RV50 was grouped with other porcine kobuviruses and more closely related to Chinese strains. Secondary structure analysis of the 5'-UTR showed that RV50 has three stem-loop domains in the first 108 nt and has a potential hepacivirus-/pestivirus-like type IV group-B-like internal ribosomal entry site, like the porcine kobuvirus prototype strain S-1. Codon usage analysis showed that the most preferred usage tends to be C or U at the end of a codon in a porcine kobuvirus genome. These results will be useful in understanding the evolution of porcine kobuviruses .

Molecular evolution of kobuviruses in cats.

Aichi virus, a causative agent of human gastroenteritis, is one of a number of animal viruses belonging to the genus Kobuvirus within the family Picornaviridae. The kobuvirus genome encodes several structural and nonstructural proteins; the capsid proteins encoded by the VP1 gene are key immunogenic factors. Here, we used the VP1 region to determine substitution rates and the time to the most recent common ancestor (TMRCA) by comparing feline kobuvirus (FKoVs) sequences with kobuvirus sequences isolated from members of other species. The substitution rate for FKoVs was 1.29 × 10(-2 )substitutions/site/year (s/s/y) and the TMRCA was 5.3 years.

Genetic characteristics of the complete feline kobuvirus genome.

We sequenced the complete genome of a feline kobuvirus and determined relationships with other kobuviruses. This kobuvirus has an 8,269-nucleotide-long RNA genome, excluding the poly(A) tail. The genome contains a 7,311-bp open reading frame (ORF) encoding a putative polyprotein precursor of 2,437 amino acids, a 717-bp 5'-untranslated region (UTR), and a 241-bp 3'-UTR. The L protein sequence was found to be the most variable region in the feline kobuvirus genome. Interestingly, the 5'-UTR B and C stem-loops were conserved as observed with other kobuviruses; however, a secondary structure corresponding to stem-loop A was not found in the full length 5'-UTR sequence. Phylogenetic tree analysis showed that kobuviruses can be divided into 3 main groups. The feline kobuvirus belongs to the Aichivirus A species containing Aichivirus, mouse kobuvirus, and canine kobuvirus.

Canine kobuvirus infections in Korean dogs.

To investigate canine kobuvirus (CaKoV) infection, fecal samples (n = 59) were collected from dogs with or without diarrhea (n = 21 and 38, respectively) in the Republic of Korea (ROK) in 2012. CaKoV infection was detected in four diarrheic samples (19.0 %) and five non-diarrheic samples (13.2 %). All CaKoV-positive dogs with diarrhea were found to be infected in mixed infections with canine distemper virus and canine parvovirus or canine adenovirus. There was no significant difference in the prevalence of CaKoV in dogs with and without diarrhea. By phylogenetic analysis based on partial 3D genes and complete genome sequences, the Korean isolates were found to be closely related to each other regardless of whether they were associated with diarrhea, and to the canine kobuviruses identified in the USA and UK. This study supports the conclusion that CaKoVs from different countries are not restricted geographically and belong to a single lineage.

Molecular detection and characterization of Aichivirus A in adult patients with diarrhea in Thailand.

Viral gastroenteritis is a common public health problem that causes morbidity and mortality worldwide. Recently, new viruses causing gastroenteritis have been identified. Among these, Aichivirus has also been proposed as a causative agent of gastroenteritis in human. Most studies have been conducted in infants and children, the information in adults is limited. Therefore, the purpose of this study was to investigate the epidemiology and molecular characterization of Aichivirus in adult patients with diarrhea. A total of 332 fecal specimens collected from January to December 2008 were screened for the presence of Aichivirus by reverse transcription-PCR (RT-PCR) method. Out of 332 fecal specimens tested, Aichivirus was detected with the prevalence of 0.9% (3/332). The data indicate that the prevalence of Aichivirus in adults was as low as those reported in children in Thailand. Phylogenetic analysis of the VP1 sequence revealed that one Aichivirus belonged to genotype A, while other two Aichiviruses were genotype B. In conclusion, this study provided the molecular epidemiological data of Aichivirus circulating in adult patients with diarrhea at low prevalence and the viruses were genetically variable as both genotypes A and B were found in this population.

Molecular and phylogenetic analysis of the porcine kobuvirus VP1 region using infected pigs from Sichuan Province, China.

BACKGROUND: Porcine kobuvirus (PKoV) is a member of the Kobuvirus genus within the Picornaviridae family. PKoV is distributed worldwide with high prevalence in clinically healthy pigs and those with diarrhea. METHODS: Fecal and intestinal samples (n = 163) from pig farms in Sichuan Province, China were obtained to determine the presence of PKoV using reverse transcription polymerase chain reaction assays. Specific primers were used for the amplification of the gene encoding the PKoV VP1 protein sequence. Sequence and phylogenetic analyses were conducted to clarify evolutionary relationships with other PKoV strains. RESULTS: Approximately 53% (87/163) of pigs tested positive for PKoV. PKoV was widespread in asymptomatic pigs and those with diarrhea. A high prevalence of PKoV was observed in pigs younger than 4 weeks and in pigs with diarrhea. Phylogenetic analysis of 36 PKoV VP1 protein sequences showed that Sichuan PKoV strains formed four distinct clusters. Two pigs with diarrhea were found to be co-infected with multiple PKoV strains. Sequence and phylogenetic analyses revealed diversity within the same host and between different hosts. Significant recombination breakpoints were observed between the CHN/SC/31-A1 and CHN/SC/31-A3 strains in the VP1 region, which were isolated from the same sample. CONCLUSION: PKoV was endemic in Sichuan Province regardless of whether pigs were healthy or suffering from diarrhea. Based on our statistical analyses, we suggest that PKoV was the likely causative agent of high-mortality diarrhea in China from 2010. For the first time, we provide evidence for the co-existence of multiple PKoV strains in one pig, and possible recombination events in the VP1 region. Our findings provide further insights into the molecular properties of PKoV, along with its epidemiology.

Sequence analysis of porcine kobuvirus VP1 region detected in pigs in Japan and Thailand.

Porcine kobuvirus is a new candidate species of the genus Kobuvirus in the family Picornaviridae, and information is still limited. The identification of porcine kobuvirus has been performed by the sequence analyses of the 3D region of the viruses. Therefore, the purpose of this study was to characterize the molecular properties of VP1 nucleotide sequences of the porcine kobuviruses isolated from porcine stool samples in Japan during 2009 and Thailand between 2006 and 2008. In addition, previous identification of a unique porcine kobuvirus; Japanese H023/2009/JP, which is a bovine kobuvirus-like strain based on sequence analysis of the 3D region, was also included in this study. All of the strains were amplified by the VP1-specific primer pair: the amplicons were subjected to direct sequencing and compared with the VP1 nucleotide sequences of reference strains. The VP1 sequences of strains from the GenBank database revealed high nucleotide sequence identity at 84.3-100%. On the other hand, the nucleotide identities among the 15 porcine kobuvirus strains analyzed in this study ranged from 78.8 to 99.8%. The results revealed that diversity of the strains in this study were higher than those of the strains in previous studies. Furthermore, it was found that the VP1 region of the bovine kobuvirus-like strain, H023/2009/JP, clustered with nine porcine kobuvirus strains that were isolated in Thailand and Japan. Since this strain was previously found to be closely related to bovine kobuviruses in the 3D gene region, it may be a natural recombinant.

Characterization of a canine homolog of human Aichivirus.

Many of our fatal "civilization" infectious diseases have arisen from domesticated animals. Although picornaviruses infect most mammals, infection of a companion animal is not known. Here we describe the identification and genomic characterization of the first canine picornavirus. Canine kobuvirus (CKoV), identified in stool samples from dogs with diarrhea, has a genomic organization typical of a picornavirus and encodes a 2,469-amino-acid polyprotein flanked by 5' and 3' untranslated regions. Comparative phylogenetic analysis using various structural and nonstructural proteins of CKoV confirmed it as the animal virus homolog most closely related to human Aichivirus (AiV). Bayesian Markov chain Monte Carlo analysis suggests a mean recent divergence time of CKoV and AiV within the past 20 to 50 years, well after the domestication of canines. The discovery of CKoV provides new insights into the origin and evolution of AiV and the species specificity and pathogenesis of kobuviruses.

Complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus, a member of a new species in the genus Kobuvirus, family Picornaviridae.

Kobuvirus is a new genus in the family Picornaviridae. Two species are currently known: Aichi virus (human kobuvirus) and Bovine kobuvirus (U-1). In this study, the complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus (Kobuvirus/swine/S-1-HUN/2007/Hungary, EU787450) were determined. The structure of the S-1-HUN genome, VPg-5'UTR-leader protein-structural proteins (VP0, VP3, VP1)-non-structural proteins (2A-2C, 3A-3D)-3'UTR-poly(A) tail, was found to be typical of picornavirus. The 8210-nucleotide (nt)-long RNA genome contains a large open reading frame (7467 nt) encoding a potential polyprotein precursor of 2488 amino acids (aa) that has 57/56% and 63/64% nt/aa identity with Aichi virus and U-1, respectively. The 5'UTR contains a hepacivirus/pestivirus-like internal ribosomal entry site (IRES type IV group-B-like) with conserved pseudoknot, II and IIIa-f domains. A tandem repeat (a 30-amino-acid-long motif) was detected in 2B. Thirty-nine (65%) of the 60 fecal samples from pigs under the age of 6 months at the tested farm were positive (the incidence was 90% under the age of 3 weeks). Porcine kobuvirus belongs to a potential new species-the third-in the genus Kobuvirus.