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Canine kobuvirus (CaKoV) is a pathogen associated with canine gastrointestinal disease (GID). This study examined 327 rectal swabs (RS), including 113 from Vietnam (46 healthy, 67 with GID) and 214 from Thailand (107 healthy and 107 with GID). CaKoV was detected in both countries, with prevalences of 28.3% (33/113) in Vietnam and 7.9% (17/214) in Thailand. Additionally, CaKoV was found in both dogs with diarrhea and healthy dogs. CaKoV was mainly found in puppies under six months of age (30.8%). Co-detection with other canine viruses were also observed. The complete coding sequence (CDS) of nine Vietnamese and four Thai CaKoV strains were characterized. Phylogenetic analysis revealed a close genetic relationship between Vietnamese and Thai CaKoV strains, which were related to the Chinese strains. CDS analysis indicated a distinct lineage for two Vietnamese CaKoV strains. Selective pressure analysis on the viral capsid (VP1) region showed negative selection, with potential positive selection sites on B-cell epitopes. This study, the first of its kind in Vietnam, provides insights into CaKoV prevalence in dogs of different ages and healthy statuses, updates CaKoV occurrence in Thailand, and sheds light on its molecular characteristics and immune evasion strategies.

Little is known about the epidemiology of Aichi virus, which is a new member of the family Picornaviridae, in the genus Kobuvirus. We report here on seroprevalence in France. Sera were screened using an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G. Of 972 sera tested, seroprevalence ranged from 25% for the 7-month-to-9-year-old age group to about 85% for the 30-to-39-year-old age group and older age groups. Our ELISA correlated well with the microneutralization technique. This study shows that Aichi virus is quite frequent in France and that seroconversion occurs before the age of 40.

Enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. The capacities of nanopore sequencing for viral diagnostics were investigated here. First, cell culture-grown porcine epidemic diarrhea virus and rotavirus A were pooled and sequenced on a MinION. Reads were already detected at 7 seconds after start of sequencing, resulting in high sequencing depths (19.2 to 103.5X) after 3 h. Next, diarrheic feces of a one-week-old piglet was analyzed. Almost all reads (99%) belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides , Escherichia and Enterococcus phages. Moreover, porcine kobuvirus was discovered in the feces for the first time in Belgium. Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding (10 6.42 –10 7.01 copies/swab) and diarrheic signs was not observed during a follow-up study. Retrospective analysis showed the widespread (n = 25, 56.8% positive) of genetically moderately related kobuviruses among Belgian diarrheic piglets. MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted. The true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded.

We detected bovine kobuvirus (BKV) in calves with diarrhea in the United States. The strain identified is related genetically to BKVs detected in other countries. Histopathologic findings also confirmed viral infection in 2 BKV cases. Our data show BKV is a potential causative agent for diarrhea in calves.

Viral calf diarrhea poses a significant challenge to the cattle industry worldwide due to its high morbidity and mortality rates, leading to substantial economic losses. The clinical symptoms associated with various diarrhea pathogens often overlap, complicating accurate diagnosis; thus, there is an urgent need for rapid and precise diagnostic methods to improve prevention and treatment efforts. In this study, we developed a one-step multiplex reverse-transcription quantitative real-time polymerase chain reaction (mRT-qPCR) that enables the simultaneous detection of three key viral pathogens responsible for calf diarrhea: bovine kobuvirus (BKoV), bovine astrovirus (BoAstV), and bovine torovirus (BToV). However, development of accurate and rapid methods to distinguish these three viruses is helpful for the early detection, disease surveillance, and control of viral calf diarrhea. Specific primers and minor groove binder (MGB)-based probes were designed targeting the 3D region of BKoV, ORF1 region of BoAstV, and N region of BToV. The sensitivity, specificity, and reproducibility ability were evaluated for the mRT-qPCR. Further, 80 bovine fecal samples were subjected to the mRT-qPCR, and the results were verified using conventional reverse-transcription PCR (RT-PCR) or PCR methods and sequencing methods. This novel method demonstrated high sensitivity and specificity,achieving a detection limit of 24 copies/mL for each pathogen. Furthermore, the assay exhibited excellent reproducibility, with coefficients of variation below 1.5%, a strong linear correlation (R > 0.996), and an amplification efficiency between 90% and 110%. Validation with 80 clinical samples from both diarrheic and non-diarrheic cattle across four farms in Shanghai showed a high degree of concordance with RT-PCR, with positive detection rates for BKoV, BoAstV, and BToV at 28.75%, 8.75%, and 3.75%, respectively, highlighting the predominance of BKoV and BoAstV. Notably, this study represents the first identification of BKoV, BoAstV, and BToV in the Shanghai region. The mRT-qPCR is a robust, rapid, and simple tool for identifying viral pathogens associated with calf diarrhea, facilitating the development of effective prevention and control measures that are vital for the future sustainability of the cattle industry.

Aichi virus (AiV) is an emerging single-stranded, positive-sense, non-enveloped RNA virus in the Picornaviridae that causes acute gastroenteritis in humans. The first case of AiV infection in Taiwan was diagnosed in a human neonate with enterovirus-associated symptoms; the virus was successfully isolated and propagated. To establish a method to detect AiV, we analyzed the antigen epitope and generated a polyclonal antibody against AiV viral protein 1 (VP1). This peptide-purified anti-AiV VP1 antibody showed high specificity against AiV VP1 without cross-reaction to nine other tested strains of Picornaviruses. The anti-AiV VP1 antibody was used in immunofluorescence analysis, immunoblotting, and enzyme-linked immunosorbent assay to elucidate the cell tropism and replication kinetics of AiV. Use of the anti-AiV VP1 antibody also revealed AiV infection restriction with interferon type I and polyI/C antiviral treatment. The AiV infection and detection system may provide an in vitro platform for AiV virology study.

We have determined the H-2 class II allele-specific amino acid motif of the agretope (the site of contact between the peptide antigen and the major histocompatibility complex) for a synthetic peptide composed of residues 43-58 of pigeon cytochrome c (p43-58). Residues 46 and 54 functioned as the agretope, and residues 50 and 52 functioned as the epitope (the site for contact between the peptide antigen and the T-cell antigen receptor). In general, agretopes and epitopes function independently. Thus, substitution of amino acids in the epitope does not significantly affect binding of the peptide antigen to a class II molecule. On the basis of these findings, synthetic peptide vaccines against influenza Aichi (H3N2) virus were prepared by introducing seven residues of the influenza virus hemagglutinin into the frame component residues 43-46 and 54-58 of p43-58 analogues including the agretopes for Akor Abpreviously determined on the p43-58 segment. These peptide vaccines induced both helper T-cell responses and production of antibodies that were specific for influenza Aichi hemagglutinin but not for the major histocompatibility complex binding frame in mice bearing Akor Ab. The antibodies produced neutralize the infectivity of influenza Aichi in vitro. The present findings should provide a basis for preparing potent peptide vaccines that function without producing side effects.

The present experiments were designed to assess the immunogenic properties of an inactivated influenza-virus vaccine made from a recombinant virus (X-31) that possesses the surface antigens of Aichi virus and the growth capacity in eggs of A0/PR8 virus. Antibody response in mice and rabbits was tested by assay for titers of hemagglutination inhibition, plaque inhibition, plaque-size reduction, and neuraminidase inhibition following immunization with graded doses of the recombinant vaccine and a standard Aichi virus vaccine of equivalent antigenic content, as determined by the chick-cell-agglutination test. The X-31 vaccine was slightly more effective in eliciting antibody to hemagglutinin in both rabbits and mice and was also more effective than Aichi vaccine in increasing the resistance of mice to the initiation of infection with Aichi virus. Similar titers of antibody to neuraminidase were elicited by the two vaccines, and both were more potent in stimulating antibody to hemagglutinin than to neuraminidase. The results indicate that the recombinant vaccine was at least as effective as a standard vaccine in its immunogenic properties. In addition, the results of these experiments raise questions regarding the accuracy of the chick-cell-agglutination test as a method for assaying immunogenic potency.