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Diverse enteric pathogens, transmitted through human and animal feces, can cause gastroenteritis. Enteric viruses, such as human Aichi virus, specifically genotype A (AiV-A), are emerging pathogens that cause illnesses even at low doses and are spreading globally. This research developed a reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the 3CD junction and a reverse transcription colorimetric loop-mediated isothermal amplification (RT-cLAMP) duplex assay targeting junctions 2BC and 3CD of the AiV-A genome for rapid and sensitive detection of this virus in metropolitan and regional wastewater samples in Queensland, Australia. The performance of these assays was evaluated using control materials and by analyzing wastewater samples. In serially diluted control materials, RT-qPCR provided quantifiable data (mean 1.51 log 10 GC/2 μL of nucleic acid) down to a dilution of 1 × 10 -5  pg/μL. In comparison, the duplex RT-cLAMP assay detected down to 1 × 10 -4  pg/μL, indicating that its sensitivity was one order of magnitude less than that of RT-qPCR. Of the 38 wastewater samples from 38 metropolitan and regional wastewater treatment plants (WWTPs) in Queensland, Australia, 21 (55.3 %) tested positive by RT-qPCR with concentrations ranging from 3.60 to 6.23 log 10 GC/L. In contrast, only 15 (39.5 %) of 38 wastewater samples were positive using the duplex RT-cLAMP assay. The methods demonstrated substantial qualitative agreement (κ = 0.730), with a concordance of 86.5 %, demonstrating the reliability of RT-cLAMP for detecting AiV-A in wastewater samples. The duplex RT-cLAMP assay, despite demonstrating reduced detection sensitivity, has proven effective and holds promise as a supplementary approach, especially in settings with limited resources where rapid and affordable testing is crucial., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, a multiplex PCR method was developed for the detection of four diarrhea-associated viruses of canines, including canine bocavirus (CBoV), canine circovirus (CCV), torque teno canis virus (TTCV), and canine kobuvirus (CKV). Four pairs of compatible primers, one specific for each virus, were designed based on conserved sequences. After optimization of parameters such as primer concentration and annealing temperature in single and multiple amplifications, four specific fragments were amplified simultaneously with high sensitivity and specificity in one PCR reaction. The fragments amplified were 165 bp (CBoV), 345 bp (CCV), 506 bp (TTCV), and 666 bp (CKV) in length. The sensitivity of this one-step multiplex PCR is about 10 times lower than that of regular singleplex PCR. There was no cross-reaction with the canine pathogens canine parvovirus (CPV), canine distemper virus (CDV), or canine coronavirus (CCoV). Testing of canine fecal samples from China using the multiplex PCR assay revealed the presence of CBoV, CCV, TTCV, and CKV in 10.1%, 6.2%, 2.8%, and 1.7% of the samples, respectively. The results of multiplex PCR agreed with the singleplex PCR results with a coincidence rate of 100%. In addition, the complete genome sequences of the viruses in three CKV-positive samples were determined and found to be 95.7 - 96.6% identical to the reference strain US-PC0082 and genetically more distant from other animal kobuvirus. The multiplex PCR method established in this study is convenient, with high specificity and sensitivity, which will be helpful for the rapid differential diagnosis of CBoV, CCV, TTCV, and CKV infections., Competing Interests: Declarations Ethical approval All animal experiments conformed to the guidelines and regulatory requirements established by the Animal Care and Use Committee of Qingdao Agricultural University, Shandong Province, China. The owners’ consent was obtained to collect samples from diarrheic dogs. Conflict of interest All authors declare no conflict of interest., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Human enteric and some respiratory viruses are identified to be involved with acute gastroenteritis that can be shed in feces of infected persons into the environment. Understanding the abundance of these viruses in wastewater is significant when assessing the public health impacts associated with discharge of wastewater into the environment. This study aimed to investigate the prevalence and seasonal variation of human enteric adenovirus (HAdV), Aichi virus (AiV-1), bocavirus (HBoV), and respiratory syndrome coronavirus 2 virus (SARS-CoV-2) in wastewater as well as their prevalence among hospitalized children with acute gastroenteritis. The viruses were detected and quantified with real-time PCR. HAdV was the most detected virus in raw sewage (88.5%), treated sewage (28%), and stool gastroenteritis (74%), followed by HBoV (45.8% for raw sewage, 14.6% for treated sewage, and 55.3% for stool samples). The detection rate of AiV-1 was 59.4%, 19.8%, and 62.6% in raw sewage, treated sewage, and stool samples, respectively. The rate of SARS-CoV-2 detection in raw sewage, treated sewage, and stool samples was 33.3%, 7.3%, and 20.6%, respectively. The viral concentrations ranged between 4.50 × 10 1 and 8.75 × 10 7 GC/ml in raw sewage samples, 1.20 × 10 1 and 5.43 × 10 6 GC/ml in treated sewage samples, and 4.80 × 10 1 and 9.88 × 10 8 GC/gram in stool samples. The overall log means of virus reduction during the wastewater treatment process ranged from 1.68 log 10 (HAdV) to 3.31 log 10 (AiV-1). The peak prevalence of the four viruses in raw sewage samples occurred during the winter season. This study showed the high detection rates of the four targeted viruses in wastewater and demonstrated that virological surveillance of wastewater in local wastewater treatment plants is a suitable model for epidemiological monitoring of diarrheal and respiratory diseases in middle- and low-resource countries., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The whole-genome sequence (WGS) analysis of Aichivirus (AiV) identified in Korea was performed in this study. Using Sanger and Nanopore sequencing, the 8228-nucleotide-long genomic sequence of AiV (OQ121963) was determined and confirmed to belong to genotype A. The full-length genome of OQ121963 consisted of a 7296 nt open reading frame (ORF) that encodes a single polyprotein, and 5' UTR (676 nt) and 3' UTR (256 nt) at 5' and 3' ends, respectively. The ORF consisted of leader protein (L), structural protein P1 (VP0, VP1, and VP3), and nonstructural protein P2 (2A, 2B, and 2C) and P3 (3A, 3B, 3C, and 3D). The secondary structure analysis of the 5' UTR identified only stem-loop C (SL-C) and not SL-A and SL-B. The variable region of the AiV genome was analyzed by MegAlign Pro and reconfirmed by SimPlot analysis using 16 AiV whole genomes known to date. Among the entire regions, structural protein region P1 showed the lowest amino acid identity (96.07%) with reference sequence AB040749 (originated in Japan; genotype A), while the highest amino acid identity (98.26%) was confirmed in the 3D region among nonstructural protein region P2 and P3. Moreover, phylogenetic analysis of the WGS of OQ121963 showed the highest homology (96.96%) with JX564249 (originated in Taiwan; genotype A) and lowest homology (90.14%) with DQ028632 (originated in Brazil; genotype B). Therefore, the complete genome characterization of OQ121963 and phylogenetic analysis of the AiV conducted in this study provide useful information allowing to improve diagnostic tools and epidemiological studies of AiVs., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Clarifying the etiology of diarrhea cases of unknown cause is important in the fight against enteric infections. In this study, we aimed to investigate the role of canine kobuvirus (CaKoV), in cases of diarrhea of unknown origin in dogs. A total 121 swab samples from dogs with diarrhea were collected. Molecular analyses of the samples were performed. For this purpose, after the sequence reaction, a phylogenetic tree was created, and bioinformatics analyses were performed. The prevalence rate of CaKoV in the sampled population was determined as 16.5% (20/121). The presence of parvovirus and coronavirus, which are common viral agents in CaKoV-positive dogs, was determined as 35% (7/20) and 10% (2/20), respectively. The rate of dogs with only CaKoV detected was 65% (13/20). Phylogenetic analysis of CaKoV strains clustered together closely related to reference strains. There are very limited studies on the role of CaKoV in the etiology of diarrhea cases of unknown cause in dogs around the world. So far, only one study has been done on CaKoV in Turkey. In this report which includes molecular characterization and epidemiological data on CaKoV determined the importance of CaKoV in cases of diarrhea of unknown origin. More comprehensive studies are needed to better understand the pathogenesis, epidemiology, and biology of CaKoV and to determine effective strategies to combat it., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Recent epidemiological studies have discovered that a lot of cases of porcine epidemic diarrhea virus (PEDV) infection are frequently accompanied by porcine kobuvirus (PKV) infection, suggesting a potential relationship between the two viruses in the development of diarrhea. To investigate the impact of PKV on PEDV pathogenicity and the number of intestinal lymphocytes, piglets were infected with PKV or PEDV or co-infected with both viruses. Our findings demonstrate that co-infected piglets exhibit more severe symptoms, acute gastroenteritis, and higher PEDV replication compared to those infected with PEDV alone. Notably, PKV alone does not cause significant intestinal damage but enhances PEDV's pathogenicity and alters the number of intestinal lymphocytes. These results underscore the complexity of viral interactions in swine diseases and highlight the need for comprehensive diagnostic and treatment strategies addressing co-infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s).)

Calf diarrhea results in significant economic loss and is caused by a variety of pathogens, including enteric viruses. Many of these viruses, including bovine norovirus (BNoV), bovine torovirus (BToV), and bovine kobuvirus (BKoV), are recognized as the causative agents of diarrhea; however, they remain understudied as major pathogens. We developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) assay for rapid and simple detection of BNoV, BToV, and BKoV. Our method had high sensitivity and specificity, with detection limits of 1 × 10 2 copies/μL for BNoV, BToV, and BKoV, which is a lower detection limit than conventional RT-PCR for BNoV and BKoV and identical for BToV. We tested fecal samples from 167 diarrheic calves with our multiplex RT-qPCR method. Viral detection was superior to conventional RT-PCR methods in all samples. The diagnostic sensitivity of the multiplex RT-qPCR method (100%) is higher than that of the conventional RT-PCR methods (87%). Our assay can detect BNoV, BToV, and BKoV in calf feces rapidly and with high sensitivity and specificity., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Porcine epidemic diarrhea virus (PEDV) is a virus that can cause diarrhea in pigs, resulting in significant economic losses to the pig industry. The mutation of the virus and its co-infection with other enteroviruses leads to poor control of PEDV infection. In this study, we found that the diarrhea outbreak in a pig farm in Shandong Province was mainly caused by PEDV infection. Through high-throughput sequencing, we also detected one other diarrhea-related virus (porcine kobuvirus). In the phylogenetic analysis and molecular characterization of the detected PEDV S gene and PKV, it was found that the S gene of the PEDV strain detected in this study (named SD22-2) had more mutations than the CV777 strain. The highest homology between PKV (named SD/2022/China) detected in this study and other strains was only 89.66%. Based on polyprotein, we divided SD/2022/China strains into a new grouping (designated group 4) and detected recombination signals. In summary, SD22-2 detected in this study is a new PEDV variant strain, and SD/2022/China strain might be a novel PKV strain. We also found the co-infection of the new PEDV variant and the novel PKV isolated from piglets with diarrhea. Our data suggested the importance of continuous surveillance of PEDV and PKV., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Background: Metagenomic next-generation sequencing (mNGS) was used to assess patients with primary or secondary immune deficiencies (PIDs and SIDs) who presented with immunopathological conditions related to immunodysregulation., Methods: Thirty patients with PIDs or SIDs who presented with symptoms related to immunodysregulation and 59 asymptomatic patients with similar PIDs or SIDs were enrolled. mNGS was performed on organ biopsy. Specific Aichi virus (AiV) reverse-transcription polymerase chain reaction (RT-PCR) was used to confirm AiV infection and screen the other patients. In situ hybridization (ISH) assay was done on AiV-infected organs to identify infected cells. Virus genotype was determined by phylogenetic analysis., Results: AiV sequences were detected using mNGS in tissue samples of 5 patients and by RT-PCR in peripheral samples of another patient, all of whom presented with PID and long-lasting multiorgan involvement, including hepatitis, splenomegaly, and nephritis in 4 patients. CD8+ T-cell infiltration was a hallmark of the disease. RT-PCR detected intermittent low viral loads in urine and plasma from infected patients but not from uninfected patients. Viral detection stopped after immune reconstitution obtained by hematopoietic stem cell transplantation. ISH demonstrated the presence of AiV RNA in hepatocytes (n = 1) and spleen tissue (n = 2). AiV belonged to genotype A (n = 2) or B (n = 3)., Conclusions: The similarity of the clinical presentation, the detection of AiV in a subgroup of patients suffering from immunodysregulation, the absence of AiV in asymptomatic patients, the detection of viral genome in infected organs by ISH, and the reversibility of symptoms after treatment argue for AiV causality., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aichi virus C, a species in the genus Kobuvirus, causes diarrhea diseases in pigs and goats and pose health threat and economic loss for stock farming. A nearly complete genome sequence of caprine kobuvirus GCCDC14 was obtained from an anal swab of a black goat died from diarrhea collected in Hubei, China in 2019. Phylogenetic analyses suggested that GCCDC14 is a novel genotype of Aichi virus C, forming a sister branch to other caprine kobuviruses, with P1 and VP0 genes more closely related to porcine kobuviruses and VP3 in an independent branch. Compared to previous caprine kobuviruses, unique amino acid changes in the poly-l-proline type II helix structure of VP0 and VP1 were found, which may affect the cellular machinery of host and pathogenicity. This study indicates the presence of the kobuvirus with continuously evolving features and emphasizes the surveillance and genetic evolution investigation of kobuviruses for safety of husbandry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Aichivirus D (AiV-D) is a newly emerging Kobuvirus detected in bovine and sheep, and information is limited regarding its biological significance and prevalence. This study aimed to explore both the prevalence and characteristics of AiV-D in yaks. From May to August 2021, 117 fecal samples were collected from yaks with diarrhea in three provinces of China's Qinghai-Tibet Plateau, 15 of which were selected and pooled for metagenomic analysis. A high abundance of AiV-D sequences was obtained. Of the 117 diarrhea samples, 29 (24.8%) tested AiV-D-positive, including 33.3% (14/42) from Sichuan, 21.1% (8/38) from Qinghai, and 18.9% (7/37) from Tibet, respectively, suggesting a wide geographical distribution of the AiV-D in yaks in the Qinghai-Tibet Plateau. Furthermore, three AiV-D strains were successfully isolated using Vero cells. Significantly, the AiV-D strain could cause diarrhea, intestinal bleeding, and inflammation in yak calves via oral inoculation. The virus was distributed in the ileum, jejunum, duodenum, colon, cecum, and rectum. Based on phylogenetic analysis of the genome and capsid protein P1 (VP0, VP3, and VP1 genes), the yak AiV-D strains likely represent a novel genotype of AiV-D. On the whole, this study identified a novel genotype of AiV-D from yaks, which was successfully isolated, and confirmed that this virus is a diarrhea pathogen in yaks and has a wide geographical distribution in the Qinghai-Tibet Plateau. Our results expand the host range of AiV-D and the pathogen spectrum of yaks and have significant implications for diagnosing and controlling diarrhea in yaks. IMPORTANCE In this study, we identified and successfully isolated a novel genotype of AiV-D from yaks. Animal infection confirmed that this virus can cause diarrhea, intestinal bleeding, and inflammation in yak calves via oral inoculation. The virus was distributed in the ileum, jejunum, cecum, duodenum, colon, and rectum. All of these results have significant implications for diagnosing and controlling diarrhea in yaks. These novel AiV-D strains have a wide geographical distribution in yaks from the Qinghai-Tibet Plateau in China. In addition to expanding the host range of AiV-D and the pathogen spectrum of yaks, these findings can increase knowledge of the prevalence and diversity of AiV-D., Competing Interests: The authors declare no conflict of interest.

In this study, rectal samples collected from 60 stray dogs in dog shelters were screened for canine kobuvirus and other enteroviruses by quantitative real-time reverse transcription polymerase chain reaction. Canine kobuvirus was detected in 25% (15/60) of the samples. In the 15 positive samples, the coinfection rates of canine distemper virus, canine coronavirus, canine astrovirus, canine norovirus, and canine rotavirus were 26.67%, 20.00%, 73.33%, 0%, and 20.00%, respectively. Phylogenetic analysis based on partial VP1 sequences identified a novel canine kobuvirus that was a recombinant of canine and feline kobuvirus. Bayesian evolutionary analysis revealed that the rate of evolution of the VP1 gene of canine kobuvirus was 1.36 × 10 -4 substitutions per site per year (95% highest posterior density interval, 6.28 × 10 -7  - 4.30 × 10 -4 substitutions per site per year). Finally, the divergence time of VP1 was around 19.44 years ago (95% highest posterior density interval, 12.96-27.57 years)., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The newly identified porcine Kobuvirus (PKV) has raised concerns owing to its association with diarrheal symptom in pigs worldwide. The process involving the emergence and global spread of PKV remains largely unknown. Here, the origin, genetic diversity, and geographic distribution of PKV were determined based on the available PKV sequence information. PKV might be derived from the rabbit Kobuvirus and sheep were an important intermediate host. The most recent ancestor of PKV could be traced back to 1975. Two major clades are identified, PKVa and PKVb, and recombination events increase PKV genetic diversity. Cross-species transmission of PKV might be linked to interspecies conserved amino acids at 13-17 and 25-40 residue motifs of Kobuvirus VP1 proteins. Phylogeographic analysis showed that Spain was the most likely location of PKV origin, which then spread to pig-rearing countries in Asia, Africa, and Europe. Within China, the Hubei province was identified as a primary hub of PKV, transmitting to the east, southwest, and northeast regions of the country. Taken together, our findings have important implications for understanding the evolutionary origin, genetic recombination, and geographic distribution of PKV thereby facilitating the design of preventive and containment measures to combat PKV infection.

Porcine kobuvirus (PKV) infection is very common in both healthy pigs and diarrhea pigs throughout the world. However, there is no proof that it causes diarrhea, and little is known about its role in diarrhea. There are only a few reports concerning porcine kobuvirus separation at present, which makes investigating its invasion and pathogenesis mechanisms difficult. This study sequenced the entire genome of a porcine kobuvirus strain termed "Wuhan2020" after it was isolated from intestinal tissue samples of healthy piglets. The analysis results revealed that it shared the most resemblance with the WUH1 strain (89.5%) and belonged to the same evolutionary branch as the Hungarian strain S-1-SUN. The PKV was located using the in situ hybridization (ISH) approach, which revealed that it was colonized in intestinal villus epithelial cells and lymphocytes in the Peyer's patch. In general, we analyzed the genetic evolution of PKV, discovered PKV susceptible cells and determined PKV localization in the intestine of infected pigs, providing a reference for future research., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Porcine epidemic diarrhea virus (PEDV) frequently causes diarrhea outbreaks. However, whether newly discovered enteric viruses such as porcine kobuvirus (PKV) and porcine astroviruses (PAstVs) are also correlated with diarrhea is still unclear. Diarrhea outbreaks were reported in a PEDV-vaccinated pig farm in Xinjiang Uygur Autonomous Region of China from 2019 to 2020. PEDV was a common pathogen detected in fecal samples by routine RT-PCR assays. The PEDV positive fecal sample was used for pathogenic analysis due to the failure isolation of PEDV. The challenged neonatal piglets appeared watery diarrhea within one day post infection (dpi) and all died within 6 dpi. Histopathological and immunohistochemical examinations supported that PEDV is a major pathogen causing intestinal lesions. To further explore enteric viruses associated with neonatal piglet diarrhea, metagenomics sequencing was performed for the diarrheic piglets. Remarkably, PKV was the most abundant virus (58.33%) followed by PEDV (34.45%) and PAstVs (7.22%), which were also confirmed by real-time RT-PCR assays. Significant in vivo replications of PEDV and PKV could only be observed in challenged piglets whilst PAstVs maintained similar virus loads in both challenged and mock infected piglets. Overall, this study provides first pathogenic and metagenomic evidence that significant proliferations of PEDV and PKV are closely associated with severe diarrhea in neonatal piglets, while PAstVs likely play limited roles in neonatal piglet diarrhea., Competing Interests: Declaration of competing interest The authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Aichivirus C is an emerging virus in goats, but its biological significance remains unknown. In this study, 18 diarrheic and 16 non-diarrheic faecal samples of kids were collected from a farm with an on-going diarrheic outbreak in Sichuan Province, China in May 2021. Of these samples, 77.8% (14/18) of diarrheic samples were detected as Aichivirus C positive by RT-PCR, which was significantly higher than that of non-diarrheic faces (0%, p < .001); meanwhile, other common diarrhoea-causing pathogens in goats were not detected in diarrheic samples, except for two samples that were detected as caprine enterovirus positive, suggesting that Aichivirus C was associated with goat diarrhoea. Furthermore, five Aichivirus C strains were successfully isolated from positive samples using Vero cell lines and two isolates were further plaque-purified, named SWUN/F5/2021(10 -6.7 TCID 50 /0.1 mL) and SWUN/F6/2021(10 -7 TCID 50 /0.1 mL). Interestingly, Aichivirus C strain could cause systemic infection in experimental kids via oral administration, with the main clinical manifestation being severe watery diarrhoea. Histopathological changes observed in the duodenum and jejunum were characteristic, with shedding of mucosal epithelial cells. In addition, the virus was detected in tissues of diarrhoea kids naturally infected with Aichivirus C, exhibiting pathological changes similar to those of experimental infections. Overall, this study first isolated Aichivirus C and confirmed its pathogenicity in kids, with further study needed to better understand the virus pathogenicity. As Aichivirus C has been detected in South Korea, Italy and the USA and widely prevalent in southwest China, the results obtained here have significant implications for the diagnosis and control of diarrhoea in goats., (© 2022 Wiley-VCH GmbH.)

Kobuviruses are known to infect the gastrointestinal tract of different animal species. Since its discovery in 2003, bovine kobuvirus (BKV) has been identified in faecal samples from diarrhoeic cattle in many countries, but only recently in North America. Although its possible role as an agent of calf diarrhoea remains to be determined, evidence is mounting. Our study reports for the first time the detection of BKV in faecal samples from diarrhoeic calves raised in Quebec, Canada. BKV was more commonly identified than eight known and common enteric calf pathogens. Further sequence analysis revealed that Canada BKV strain 1,043,507 was more closely correlated with the US BKV IL35164 strain than other BKV strains with complete genome. Continued surveillance and genomic characterization are needed to monitor BKV in the cattle around the world., (© 2021 Wiley-VCH GmbH.)

Background: Murine Kobuvirus (MuKV) is a novel picornavirus of the genus Kobuvirus , and was first identified in the feces of murine rodents in the USA in 2011. There is limited information on the transmission route of MuKV. Thus, we conducted a study to investigate virus detection rates in fecal, serum, throat, and lung tissue samples from murine rodents., Results: A total of 413 fecal samples, 385 lung samples, 269 throat swab samples, and 183 serum samples were collected from 413 murine rodents ( Rattus norvegicus, Rattus tanezumi , and Rattus rattus ) captured in urban Shenzhen. Kobuviruses were detected via RT-PCR. Only fecal samples were positive, with prevalence rates of 34.9% in Rattus norvegicus and 29.4% in Rattus tanezumi . Phylogenetic analysis based on partial 3D and complete VP1 sequence regions indicated that all of the MuKV sequences obtained belonged to Aichivirus A , and were genetically closely related to other MuKVs reported in China, Hungary, and the USA. Twenty-eight full-length MuKV sequences were acquired. Phylogenetic analysis of two sequences randomly selected from the two species (SZ59 and SZ171) indicated that they shared very high nucleotide and amino acid identity with one another (94.0 and 99.3%, respectively), and comparison with human Kobuvirus revealed amino acid identity values of ~80%. Additionally, a sewage-derived sequence shared high similarity with the rat-derived sequences identified in this study, with respective nucleotide and amino acid identity values from 86.5 and 90.7% to 87.2 and 91.1%., Conclusion: The results of the current study provide evidence that murine Kobuvirus is transmitted via the fecal-oral route., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gao, He, Fu, Li, He and Chen.)

Kobuviruses are an unusual and poorly characterized genus within the picornavirus family and can cause gastrointestinal enteric disease in humans, livestock, and pets. The human kobuvirus Aichi virus (AiV) can cause severe gastroenteritis and deaths in children below the age of 5 years; however, this is a very rare occurrence. During the assembly of most picornaviruses (e.g., poliovirus, rhinovirus, and foot-and-mouth disease virus), the capsid precursor protein VP0 is cleaved into VP4 and VP2. However, kobuviruses retain an uncleaved VP0. From studies with other picornaviruses, it is known that VP4 performs the essential function of pore formation in membranes, which facilitates transfer of the viral genome across the endosomal membrane and into the cytoplasm for replication. Here, we employ genome exposure and membrane interaction assays to demonstrate that pH plays a critical role in AiV uncoating and membrane interactions. We demonstrate that incubation at low pH alters the exposure of hydrophobic residues within the capsid, enhances genome exposure, and enhances permeabilization of model membranes. Furthermore, using peptides we demonstrate that the N terminus of VP0 mediates membrane pore formation in model membranes, indicating that this plays an analogous function to VP4. IMPORTANCE To initiate infection, viruses must enter a host cell and deliver their genome into the appropriate location. The picornavirus family of small nonenveloped RNA viruses includes significant human and animal pathogens and is also a model to understand the process of cell entry. Most picornavirus capsids contain the internal protein VP4, generated from cleavage of a VP0 precursor. During entry, VP4 is released from the capsid. In enteroviruses this forms a membrane pore, which facilitates genome release into the cytoplasm. Due to high levels of sequence similarity, it is expected to play the same role for other picornaviruses. Some picornaviruses, such as Aichi virus, retain an intact VP0, and it is unknown how these viruses rearrange their capsids and induce membrane permeability in the absence of VP4. Here, we have used Aichi virus as a model VP0 virus to test for conservation of function between VP0 and VP4. This could enhance understanding of pore function and lead to development of novel therapeutic agents that block entry.

Many enteric viruses are found in pig farms around the world and can cause death of animals or important production losses for breeders. Among the wide spectrum of enteric viral species, porcine Sapelovirus (PSV), porcine Kobuvirus (PKoV) and porcine Astrovirus (PAstV) are frequently found in pig feces. In this study we investigated sixteen pig farms in Corsica, France, to evaluate the circulation of three enteric viruses (PKoV, PAstV-1 and PSV). In addition to the three viruses studied by RT-qPCR (908 pig feces samples), 26 stool samples were tested using the Next Generation Sequencing method (NGS). Our results showed viral RNA detection rates (i) of 62.0% [58.7-65.1] (n = 563/908) for PSV, (ii) of 44.8% [41.5-48.1] (n = 407/908) for PKoV and (iii) of 8.6% [6.8-10.6] (n = 78/908) for PAstV-1. Significant differences were observed for all three viruses according to age (P-value = 2.4e-13 for PAstV-1; 2.4e-12 for PKoV and 0.005 for PSV). The type of breeding was significantly associated with RNA detection only for PAstV-1 (P-value = 9.6e-6). Among the 26 samples tested with NGS method, consensus sequences corresponding to 10 different species of virus were detected. This study provides first insight on the presence of three common porcine enteric viruses in France. We also showed that they are frequently encountered in pigs born and bred in Corsica, which demonstrates endemic local circulation., Competing Interests: The authors have declared that no competing interests exist.

Calf diarrhea is one of the common diseases involved in the process of calf feeding. In this study, a sample of calf diarrhea that tested positive for bovine coronavirus and bovine astrovirus was subjected to high-throughput sequencing. The reassembly revealed the complete genomes of bovine norovirus, bovine astrovirus, bovine kobuvirus, and the S gene of bovine coronavirus. Phylogenetic analysis showed that the ORF2 region of bovine astrovirus had the lowest similarity with other strains and gathered in the Mamastrovirus unclassified genogroup, suggesting a new serotype/genotype could appear. Compared with the most closely related strain, there are six amino acid mutation sites in the S gene of bovine coronavirus, most of which are located in the S1 subunit region. The bovine norovirus identified in our study was BNoV-GIII 2, based on the VP1 sequences. The bovine kobuvirus is distributed in the Aichi virus B genus; the P1 gene shows as highly variable, while the 3D gene is highly conserved. These findings enriched our knowledge of the viruses in the role of calf diarrhea, and help to develop an effective strategy for disease prevention and control.

Recently, murine kobuvirus (MuKV), a novel member of the family Picornaviridae , was identified in faecal samples of Rattus norvegicus in China. The limited information on the circulation of MuKV in other murine rodent species prompted us to investigate its prevalence and conduct a genetic characterization of MuKV in Rattus losea , Rattus tanezumi and Rattus norvegicus in China. Between 2015 and 2017, 243 faecal samples of these three murine rodent species from three regions in southern China were screened for the presence of MuKV. The overall prevalence was 23.0% (56/243). Three complete MuKV polyprotein sequences were acquired, and the genome organization was determined. Phylogenetic analyses suggested that our sequences were closely related to Chinese strains and belong to the species Aichivirus A in the genus Kobuvirus . Additional studies are required to understand the true prevalence of MuKV in murine rodent populations in China.

Between 2016 and 2018, the prevalence of porcine kobuvirus (PKoV) and porcine astrovirus (PAstV) in Korean wild boars (n = 845) was 28.0% and 10.7%, respectively. Coinfection by both viruses was detected in 5.1% of boars. Phylogenetic analysis revealed that 134 PKoV isolates belonged to diverse lineages within the species Aichivirus C; however, one strain (WKoV16CN-8627) clustered with bovine kobuvirus (Aichivirus B). Forty-seven PAstVs belonged to lineage PAstV4, and only one strain (WAst17JN-10931) was a novel addition to lineage PAstV2. The two viruses were more prevalent in boars weighing ≤ 60 kg than in boars weighing > 61 kg., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Viruses are the primary cause of acute gastroenteritis in children all over the world. Understanding the emergence and genetic variation of these viruses may help to prevent infections. Aichivirus (AiV) is a member of the Kobuvirus genus, which currently contains six officially recognized species: Aichivirus A-F. The species AiV A contains six types including Aichivirus 1 (AiV 1) and eventually, three genotypes have been identified in the human AiV 1 (named A to C). The present study describes the identification and sequencing of the polyprotein gene of a human AiV 1 strain PAK419 via NGS in Pakistani children with acute gastroenteritis. Our study strain PAK419 was classified as AiV 1 genotype A, most commonly found in Japan and Europe, and closely related to non-Japanese and European strains on the phylogenetic tree. PAK419 showed 95-98 % nucleotide sequence identity with strains isolated from Ethiopia (ETH/2016/P4), Australia (FSS693) and China (Chshc7). On phylogenetic observation PAK419 formed a distinct cluster in the AiV 1 genotype A with the above mentioned and other human AiV strains detected around the world (Germany, Brazil, Japan, Thailand, Korea and Vietnam). The data clearly showed that Pakistani AiV strains and human strains identified from all over the world are distinct from Aichivirus strains found in bovine, swine, canine, feline, caprine, ferret, bat, and environmental samples. The distinguishing characteristics of the AiV genome showed a lower probability of inter-genotypic recombination events, which may support the lack of AiV serotypes. PAK419 also had a high content of C nucleotide (37.4 %), as found in previous studies, which could also restrict the possible genetic variation of AiV. This study demonstrate the power of NGS in uncovering unknown gastroenteric etiological agents circulating in the population., (Copyright © 2021. Published by Elsevier B.V.)

Bovine kobuvirus (BKV) is a single-stranded, positive sense, non-enveloped RNA virus in genus Kobuvirus of family Picornavirus. BKV was first identified in the culture media of HeLa cell containing calf serum in 2003. Since then, BKV has been detected in 13 countries of four different continents, suggesting widespread in the world. Herein, we review the detection and genomic characterization of BKV in 13 countries. All studies tested bovine faecal samples for BKV. These studies provide evidence that BKV might be a causative agent for neonatal calf diarrhoea. Therefore, further efforts including animal challenge study are urgently needed to unveil the pathogenicity of BKV., (© 2020 Wiley-VCH GmbH.)

Swine enteric viral infections are responsible for substantial economic losses in the pork industry worldwide. Porcine epidemic diarrhea (PEDV) is one of the main causative agents of diarrhea in lactating pigs, and reports of PEDV coinfection with other enteric viruses highlight the importance of viral interactions for disease presentation and outcomes. Using next-generation sequencing (NGS) and sequence analyses from samples taken from piglets with acute diarrhea, we explored the possible interactions between PEDV and other less reported pathogens. PEDV coinfection with porcine kobuvirus (PKV) was detected in 36.4% (27/74) of samples. Full genomes from porcine coronavirus and kobuvirus were obtained, as was a partial porcine sapovirus genome (PSaV). The phylogenetic results show the clustering of these strains corresponding to the geographical relationship. To our knowledge, this is the first full genome and isolation report for porcine kobuvirus in México, as well as the first phylogenetic analysis for porcine sapovirus in the country. The NGS approach provides a better perspective of circulating viruses and other pathogens in affected production units.

A duplex SYBR Green I-based real-time PCR assay was established for the simultaneous detection of canine kobuvirus (CaKoV) and canine astrovirus (CaAstV). This assay can easily distinguish the two viruses according to their different melting temperatures (Tm) of 80 °C for CaKoV and 86.5 °C for CaAstV; other canine enteroviruses used as controls showed no specific melting peaks. The detection limit of this assay was determined to be 10 1 copies/μL for both viruses. This method exhibited high repeatability and reproducibility, with a coefficient of variation less than 1.5 %. A total of 48 fecal samples were collected for clinical testing by real-time PCR and confirmed by sequencing. Real-time PCR assay showed a 10.4 % CaKoV-positive rate and a 4.2 % CaAstV-positive rate, and the positive rate of co-infection of the two viruses was 2.1 %, which was consistent with the sequencing results. This assay has many advantages over conventional PCR: it is rapid, sensitive, specific, and reliable for detecting these two viruses in one sample, and it can be used as a tool to detect CaKoV and CaAstV infection or co-infection in clinical settings., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Enteric viruses are the leading cause of diarrhea in children globally. Identifying viral agents and understanding their genetic diversity could help to develop effective preventive measures. This study aimed to determine the detection rate and genetic diversity of four enteric viruses in Gabonese children aged below five years. Stool samples from children <5 years with ( n = 177) and without ( n = 67) diarrhea were collected from April 2018 to November 2019. Norovirus, astrovirus, sapovirus, and aichivirus A were identified using PCR techniques followed by sequencing and phylogenetic analyses. At least one viral agent was identified in 23.2% and 14.9% of the symptomatic and asymptomatic participants, respectively. Norovirus (14.7%) and astrovirus (7.3%) were the most prevalent in children with diarrhea, whereas in the healthy group norovirus (9%) followed by the first reported aichivirus A in Gabon (6%) were predominant. The predominant norovirus genogroup was GII, consisting mostly of genotype GII.P31-GII.4 Sydney. Phylogenetic analysis of the 3CD region of the aichivirus A genome revealed the presence of two genotypes (A and C) in the study cohort. Astrovirus and sapovirus showed a high diversity, with five different astrovirus genotypes and four sapovirus genotypes, respectively. Our findings give new insights into the circulation and genetic diversity of enteric viruses in Gabonese children.

Introduction: Salivirus (SalV) represents an emerging problem in public health especially during the recent years. In this study, the Bayesian evolutionary history and the spread of the virus through the different countries have been reported., Methodology: a database of 81 sequences of SalV structural VP1 fragment were downloaded from GenBank, aligned and manually edited by Bioedit Software. ModelTest v. 3.7 software was used to estimate the simplest evolutionary model fitting the sequence dataset. A Maximum-Likelihood tree has been generated using MEGA-X to test the "clockliness" signal using TempEst 1.5.1. The Bayesian phylogenetic tree was built by BEAST. Homology modelling was performed by SWISS-Model and protein variability evaluated by ConSurf server., Results: the phylogenetic tree showed a clade of SalV A2 and three main clades of SalV A1, revealing several infections in humans in South Korea, India, Tunisia, China, Nigeria, Ethiopia and USA. The Bayesian maximum clade credibility tree and the time of the most common recent ancestor dated back the root of the tree to the year 1788 with the probable origin in USA. Selective pressure analysis revealed two positive selection sites, His at 100th and Leu at 116th positions that at the homology modelling resulted important to guarantee protein stability and variability. This could contribute to the development of new mutations modifying the clinical features of this evolving virus., Conclusions: Bayesian phylogenetic and phylodynamic represented a useful tool to follow the transmission dynamic of SalV and to prevent new epidemics worldwide., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2021 Silvia Angeletti, Domenico Benvenuto, Marta Fogolari, Cecilia De Flora, Giancarlo Ceccarelli, Ivana Maida, Riccardo Bazzardi, Silvia Spoto, Stefano Pascarella, Boban Mugosa, Massimo Ciccozzi.)

Aichivirus 1 (AiV-1) is an enteric virus that has been documented to be the causative agent of diarrhea in humans. It is transmitted by fecal-oral route, through person-to-person contact, consumption of contaminated food or water, or recreation of contaminated water. AiV-1 is highly prevalent in water samples and has been proposed as a potential indicator of fecal contamination in water reservoirs. This study aimed to investigate the prevalence and genetic diversity of AiV-1 in environmental water samples in Thailand. A total of 126 samples were collected monthly from November 2016 to July 2018 from various sources of environmental water including irrigation water, reservoir, river, and wastewater. The presence of AiV-1 was detected by RT-nested PCR of the 3CD region and further analyzed by phylogenetic analysis. The AiV-1 was detected in 28 out of 126 (22.2%) of tested samples. A high frequency of AiV-1 detection was in wastewater (52.4%). All 28 AiV-1 strains detected in this study belonged to the genotype B and were closely related to AiV strains detected previously in environmental waters and in humans worldwide. This study demonstrated, for the first time, the contamination of AiV-1 in various sources of water samples in Thailand and provided a better insight into the prevalence of AiV-1 in environmental waters and its potential risk of human health.

Caprine kobuvirus (CKoV), a member of the genus Kobuvirus, has only been identified in South Korea and Italy until now. In this study, 24 goat diarrheic fecal samples were collected from 3 farms in Sichuan province, China, and 87.5% (21/24) samples were detected as CKoV positive by RT-PCR. Meanwhile, full-length VP0, VP3, and VP1 genes were simultaneously cloned from 17 clinical samples. Phylogenetic analysis showed that all CKoV strains were most closely related to porcine kobuvirus based on amino acid (aa) sequences of VP0 and VP3 proteins, but CKoV strains were closely related to with Aichivirus B strains (ferret, bovine, and sheep kobuvirus) based on aa sequences of the VP1 protein. Interestingly, compared with known CKoV strains in the GenBank database, Chinese CKoV strains have unique amino acid changes in VP0 and VP1 proteins. Moreover, the first Chinese CKoV nearly complete genome was successfully obtained from a diarrheic fecal sample, named SWUN/F11/2019. Compared with the two known CKoV strains, five aa mutations (S60A, L252I, V267T, I, V 306 L, V331I) were found in the VP0 gene and 7 aa mutations (S57N, G, T243A, V244I, T, A248V, L, S251A, R252H, and M255L) were found in VP1 in the SWUN/F11/2019 genome. This was the first report of the detection and molecular characteristics of CKoV from goats in China, which could be helpful for improving the understanding of the prevalence and genetic evolution of CKoV., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Canine kobuvirus (CaKoV), a newly described virus, is the causative agent of gastroenteritis in dogs. In this study, 57 fecal samples from dogs with diarrhea in Anhui Province, eastern China, were collected. Among these, five samples were identified to be infected with CaKoV, by polymerase chain reaction targeting the CaKoV 3D gene. The five CaKoV strains were subjected to phylogenetic analysis. The sequences of VP1 from the five CaKoV strains were 93.6%-96.1% identical to each other and 91.75%-97.95% identical to other reported CaKoV VP1 sequences. In addition, the complete genome of one strain was successfully amplified and sequenced. The genome consisted of 8223 nucleotides and shared 94.6%-97.0% nucleotide and 93.1%-94.0% amino acid sequence identity with other CaKoV isolates. Phylogenetic analysis revealed that the CaKoV strain from Anhui Province was similar to other Chinese strains, and it was more closely related to feline and mouse kobuviruses than to sheep and bovine kobuviruses. Interestingly, all of the CaKoV-positive samples were coinfected with canine parvovirus. The finding of CaKoV infection in dogs with diarrhea and coinfection with canine parvovirus are a cause for concern and highlight the need for management and preventive measures.

Kobuvirus infections are common among humans, rodents, carnivores, pigs, and ruminants. We report herein the complete genome sequence of a novel caprine kobuvirus (MN604700) from diarrheic kids in Minnesota. Whole-genome sequencing revealed a kobuvirus genome of 8,139 nt with a single ORF region encoding a polyprotein of 2,480 amino acids. Further analysis revealed nt substitutions along the genome compared with that of the caprine kobuvirus reference strain, with 93% identity. Phylogenetic analysis indicated that the clade of the caprine kobuvirus was most closely related to porcine kobuviruses rather than bovine or ovine kobuviruses. Using primers designed from this genome, caprine kobuvirus was identified in the stools of other goats. Sanger sequencing of PCR products indicated 3D and VP1 gene nucleotides of this latter strain were 95% and 91% identical with those of MN604700, respectively. There were 35 and 101 nt substitutions in 3D and VP1 genes, respectively. Findings of kobuvirus over a 2-y period may indicate an endemic state, which needs further research. In addition, screening for kobuviruses over large geographic areas is needed to identify the evolutionary connections among different strains.

Porcine Kobuvirus (PKV) infection is very common in pigs throughout the world. Since it has never been investigated in Serbia, to contribute to the knowledge of Porcine Kobuvirus, its role, and distribution, we tested 200 samples from domestic pigs and wild boars. From domestic pigs, 10 fecal, 22 spleen and 68 serum samples, and 100 spleen samples from wild boars were tested. The virus prevalence determined by real-time RT-PCR in domestic pigs was 22% and in wild boars 6%. The phylogenetic analysis of 3D region revealed that Serbian strains are closest related to the Hungarian strain from wild boar from 2011. This is the first report on PKV in Serbia in domestic pigs and wild boars, implying its wide circulation. Although the infection could not be directly related to any clinical manifestation, the frequency of virus found in feces suggests viral affinity to the gastrointestinal tract. However, due to the rather ubiquitous presence of PKV, the clinical and pathological assessment have to be considered when PKV infection is diagnosed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

Monthly sampling was conducted at a drinking water treatment plant (DWTP) in Southern Louisiana, USA from March 2017 to February 2018 to determine the prevalence and reduction efficiency of pathogenic and indicator viruses. Water samples were collected from the DWTP at three different treatment stages (raw, secondary-treated, and chlorinated drinking water) and subjected to quantification of seven pathogenic viruses and three indicator viruses [pepper mild mottle virus (PMMoV), tobacco mosaic virus (TMV), and crAssphage] based on quantitative polymerase chain reaction. Among the seven pathogenic viruses tested, only Aichi virus 1 (AiV-1) (7/12, 58%) and noroviruses of genogroup II (NoVs-GII) (2/12, 17%) were detected in the raw water samples. CrAssphage had the highest positive ratio at 78% (28/36), and its concentrations were significantly higher than those of the other indicator viruses for all three water types (P < 0.05). The reduction ratios of AiV-1 (0.7 ± 0.5 log 10 ; n = 7) during the whole treatment process were the lowest among the tested viruses, followed by crAssphage (1.1 ± 1.9 log 10 ; n = 9), TMV (1.3 ± 0.9 log 10 ; n = 8), PMMoV (1.7 ± 0.8 log 10 ; n = 12), and NoVs-GII (3.1 ± 0.1 log 10 ; n = 2). Considering the high abundance and relatively low reduction, crAssphage was judged to be an appropriate process indicator during drinking water treatment. To the best of our knowledge, this is the first study to assess the reduction of crAssphage and TMV during drinking water treatment.

Background: Murine kobuviruses (MuKV) are newly recognized picornaviruses first detected in murine rodents in the USA in 2011. Little information on MuKV epidemiology in murine rodents is available. Therefore, we conducted a survey of the prevalence and genomic characteristics of rat kobuvirus in Guangdong, China., Results: Fecal samples from 223 rats (Rattus norvegicus) were collected from Guangdong and kobuviruses were detected in 12.6% (28) of samples. Phylogenetic analysis based on partial 3D and complete VP1 sequence regions showed that rat kobuvirus obtained in this study were genetically closely related to those of rat/mouse kobuvirus reported in other geographical areas. Two near full-length rat kobuvirus genomes (MM33, GZ85) were acquired and phylogenetic analysis of these revealed that they shared very high nucleotide/amino acids identity with one another (95.4%/99.4%) and a sewage-derived sequence (86.9%/93.5% and 87.5%/93.7%, respectively). Comparison with original Aichivirus A strains, such human kobuvirus, revealed amino acid identity values of approximately 80%., Conclusion: Our findings indicate that rat kobuvirus have distinctive genetic characteristics from other Aichivirus A viruses. Additionally, rat kobuvirus may spread via sewage.

The major enteric RNA viruses in pigs include porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (PRV-A), porcine kobuvirus (PKV), porcine sapovirus (PSaV) and porcine deltacoronavirus (PDCoV). For differential diagnosis, a multiplex RT-PCR method was established on the basis of the N genes of TGEV, PEDV and PDCoV, the VP7 gene of PRV-A, and the polyprotein genes of PKV and PSaV. This multiplex RT-PCR could specifically detect TGEV, PEDV, PDCoV, PRV-A, PKV and PSaV without cross-reaction to any other major viruses circulating in Chinese pig farms. The limit of detection of this method was as low as 10 0 -10 1  ng cDNA of each virus. A total of 398 swine faecal samples collected from nine provinces of China between October 2015 and April 2017 were analysed by this established multiplex RT-PCR. The results demonstrated that PDCoV (144/398), PSaV (114/398), PEDV (78/398) and PRV-A (70/398) were the main pathogens, but TGEV was not found in the pig herds in China. In addition, dual infections, for example, PDCoV + PSaV, PDCoV + PRV-A, PRA-V + PSaV and PEDV + PDCoV, and triple infections, for example, PDCoV + PRV-A + PSaV and PEDV + PDCoV + PKV, were found among the collected samples. The multiplex RT-PCR provided a valuable tool for the differential diagnosis of swine enteric viruses circulating in Chinese pig farms and will facilitate the prevention and control of swine diarrhoea in China., (© 2019 Blackwell Verlag GmbH.)

Picornaviruses infect a wide range of mammals including livestock such as cattle and swine. As with other picornavirus genera such as Aphthovirus, there is emerging evidence of a significant economic impact of livestock infections caused by members of the genera Enterovirus and Kobuvirus. While the human-infecting enteroviruses and kobuviruses have been intensively studied during the past decades in great detail, research on livestock-infecting viruses has been mostly limited to the genomic characterization of the viral strains identified worldwide. Here, we extend our previous studies of the structure and function of the complexes composed of the non-structural 3A proteins of human-infecting enteroviruses and kobuviruses and the host ACBD3 protein and present a structural and functional characterization of the complexes of the following livestock-infecting picornaviruses: bovine enteroviruses EV-E and EV-F, porcine enterovirus EV-G, and porcine kobuvirus AiV-C. We present a series of crystal structures of these complexes and demonstrate the role of these complexes in facilitation of viral replication.

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.

In this study, 96 diarrheic and 77 non-diarrheic fecal samples from dairy calves were collected from 14 dairy farms in 4 provinces to investigate the molecular prevalence and genomic characteristics of Bovine Kobuvirus (BKoV) in China. The results showed that the BKoV positive rate for the diarrheic feces (35.42%) was significantly higher than that for the non-diarrheic feces (11.69%, p < 0.001). Interestingly, three potential novel VP1 lineages were identified from 15 complete VP1 sequences, and a unique triple nucleotide insertion which can result in an aa insertion, was first observed in the 11/12 VP0 fragments with 660 bp long in this study, compared with known BKoV VP0 sequences. Moreover, the first Chinese BKoV genome was successfully obtained from a diarrheic fecal sample, named CHZ/CHINA. The open reading frame (ORF) of the genome from strain CHZ/China shares 87.4%-88.3% nucleotide (nt) and 93.7%-96.4% amino acid (aa) identity, compared with the three known genomes of BKoV. Interestingly, phylogenetic tree based on aa sequences of these genomes showed that CHZ/CHINA was clustered into an independent branch, suggesting the strain may represent a novel BKoV strain. The findings contribute to better understanding the molecular characteristics and evolution of BKoV., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The involvement of picornaviruses in calf diarrhoea was evaluated by the analysis of 127 faecal samples collected from diarrhoeic calves during 2014-2016. Virus detections were carried out by PCR using generic or specific primer pairs. One-third of the faecal samples (33.86%) were found to be positive for one or more of the studied viruses. Bovine kobuvirus was detected in 22.83%, bovine hungarovirus in 11.02%, while bovine enterovirus 1 in 5.51% of the samples. The sequences of the PCR products indicated the existence of novel variants in all the three virus species. When comparing the partial sequences, the nucleotide sequence identities between our newly detected viruses and those previously deposited to the GenBank ranged between 76 and 99%. Phylogenetic analyses revealed a novel lineage within the species Hunnivirus A. Our findings suggest that these viruses should be regarded as possible aetiological agents of calf diarrhoea. Based on the newly determined sequences, we designed and tested a new generic PCR primer set for the more reliable detection of bovine hungaroviruses. This is the first report on the molecular detection of the presence of bovine hungarovirus, bovine kobuvirus and bovine enterovirus 1 in the faecal samples of diarrhoeic calves in Turkey.

Background: In recent years, Aichi virus (AiV) has been involved in acute viral gastroenteritis outbreaks. However, the common pathogenesis of AiV releases more in subclinical infections underestimating the impact of AiV in human health., Objectives: The present study describes the presence and genetic diversity of AiV in patients with gastroenteritis in Northwestern Spain., Study Design: A total of 2667 stool samples, obtained between July 2010 and June 2011, from diarrheic outpatients were studied for detection and molecular characterization of AiV using PCR techniques followed by sequencing and phylogenetic analyses., Results: The virus was detected in 124 (5.0%) of the samples among all age groups. Coinfections were also detected, from the 124 positive samples, 72 (58.1%) were positive only for AiV, whereas mixed contaminations with Norovirus genogroup I or genogroup II, Sapovirus, or other enteric pathogens were detected in 52 (41.9%) samples. A total of 70 positive samples could be genotyped, being characterized as genotype A (58.6%) or B (41.4%). AiV was detected from August to April, being the highest number of AiV positive samples detected during autumn and winter seasons., Conclusions: This survey remarks the importance of emerging enteric viruses in patients who require medical assistance, and offers more information about the real importance of AiV as gastroenteritis agent., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The picornavirus Aichi virus (AiV) is a non-enveloped RNA virus that causes acute gastroenteritis symptoms, such as diarrhea, abdominal pain, nausea, vomiting, and fever. Antiviral host defense involves the fast response of type I interferon (IFN) and the secretion of inflammatory cytokines against pathogens. However, the intestinal inflammatory and antiviral response to AiV infection is poorly understood. This study evaluated the antiviral activity of intestinal epithelial cells (IECs), which form a single-cell layer separating the bowel wall from pathogens. Isolated primary mouse IECs were subjected to AiV infection and virion production, inducing the mRNA expression of type I/type III IFNs and inflammatory cytokines. The mechanism involved induced the expression of phospho-IFN regulatory factor 3 and mitochondrial antiviral-signaling protein of type I IFN signaling. These findings were also observed in AiV-infected human colon carcinoma cells. In summary, a viral productive and pathogenic infection of AiV in primary murine IECs is validated.

Background: Canine Kobuvirus (CaKoV) has been detected both in healthy and diarrheic dogs and in asymptomatic wild carnivores. In this study, we conducted a survey of CaKoV at small animal hospitals in Bangkok and vicinity of Thailand during September 2016 to September 2018., Results: Three hundred and seven rectal swab samples were collected from healthy dogs (n = 55) and dogs with gastroenteritis symptoms (n = 252). Of 307 swab samples tested by using one-step RT-PCR specific to 3D gene, we found CaKoV positivity at 17.59% (54/307). CaKoVs could be detected in both sick (19.44%) and healthy (9.09%) animals. In relation to age group, CaKoV could be frequently detected in younger dogs (25.45%). Our result showed no seasonal pattern of CaKoV infection in domestic dogs. In this study, we characterized CaKoVs by whole genome sequencing (n = 4) or 3D and VP1 gene sequencing (n = 8). Genetic and phylogenetic analyses showed that whole genomes of Thai CaKoVs were closely related to Chinese CaKoVs with highest 99.5% amino acid identity suggesting possible origin of CaKoVs in Thailand., Conclusions: In conclusion, this study was the first to report the detection and genetic characteristics of CaKoVs in domestic dogs in Thailand. CaKoVs could be detected in both sick and healthy dogs. The virus is frequently detected in younger dogs. Thai CaKoVs were genetically closely related and grouped with Chinese CaKoVs. Our result raises the concerns to vet practitioners that diarrhea in dogs due to canine Kobuvirus infection should not be ignored.

To rapidly distinguish Canine distemper virus (CDV), canine parvovirus (CPV), and canine kobuvirus (CaKoV) in practice, a one-step multiplex PCR/RT-PCR assay was developed, with detection limits of 10 2.1 TCID 50 for CDV, 10 1.9 TCID 50 for CPV and 10 3 copies for CaKoV. This method did not amplify nonspecific DNA or RNA from other canine viruses. Therefore, the assay provides a sensitive tool for the rapid clinical detection and epidemiological surveillance of CDV, CPV and CaKoV in dogs.

Canine kobuvirus (CaKV) is a member of the Picornaviridae family and the Kobuvirus genus. CaKV was first described in fecal samples from diarrheic dogs in the USA in 2011, with subsequent reports in the UK, Italy, South Korea, China, Tanzania, and Japan. CaKV is frequently identified in feces of animals with or without clinical signs of gastroenteritis. The present study investigated the presence of CaKV in fecal samples from 53 diarrheic dogs from Londrina, southern Brazil. Using a RT-PCR assay, CaKV RNA was identified in three dogs, resulting in an overall occurrence rate of 5.7%. In addition, coinfection with canine parvovirus subtype 2b was detected in all CaKV-positive diarrheic fecal samples. Using a phylogenetic analysis based on the VP1 gene sequence, the Brazilian CaKV field strains were found to be very similar to a previously identified CaKV strain from Brazil that was found in the tissue of a puppy and were also found to be clustered with other CaKV strains detected worldwide and other kobuvirus strains identified in mouse, feline, and human hosts.

Several viruses encounter various bacterial species within the host and in the environment. Despite these close encounters, the effects of bacteria on picornaviruses are not completely understood. Previous work determined that poliovirus (PV), an enteric virus, has enhanced virion stability when exposed to bacteria or bacterial surface polysaccharides such as lipopolysaccharide. Virion stabilization by bacteria may be important for interhost transmission, since a mutant PV with reduced bacterial binding had a fecal-oral transmission defect in mice. Therefore, we investigated whether bacteria broadly enhance stability of picornaviruses from three different genera: Enterovirus (PV and coxsackievirus B3 [CVB3]), Kobuvirus (Aichi virus), and Cardiovirus (mengovirus). Furthermore, to delineate strain-specific effects, we examined two strains of CVB3 and a PV mutant with enhanced thermal stability. We determined that specific bacterial strains enhance thermal stability of PV and CVB3, while mengovirus and Aichi virus are stable at high temperatures in the absence of bacteria. Additionally, we determined that bacteria or lipopolysaccharide can stabilize PV, CVB3, Aichi virus, and mengovirus during exposure to bleach. These effects are likely mediated through direct interactions with bacteria, since viruses bound to bacteria in a pulldown assay. Overall, this work reveals shared and distinct effects of bacteria on a panel of picornaviruses. IMPORTANCE Recent studies have shown that bacteria promote infection and stabilization of poliovirus particles, but the breadth of these effects on other members of the Picornaviridae family is unknown. Here, we compared the effects of bacteria on four distinct members of the Picornaviridae family. We found that bacteria reduced inactivation of all of the viruses during bleach treatment, but not all viral strains were stabilized by bacteria during heat treatment. Overall, our data provide insight into how bacteria play differential roles in picornavirus stability., (Copyright © 2019 Aguilera et al.)