Detection and Molecular Characterization of Kobuviruses: An Agent of Canine Viral Diarrhea.

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.

Porcine kobuvirus enhances porcine epidemic diarrhea virus pathogenicity and alters the number of intestinal lymphocytes in piglets.

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.

Evolutionary dynamics of canine kobuvirus in Vietnam and Thailand reveal the evidence of viral ability to evade host immunity.

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.

A novel multiplex RT-qPCR assay for simultaneous detection of bovine norovirus, torovirus, and kobuvirus in fecal samples from diarrheic calves.

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 × 102 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.

Phylogenetic analysis and molecular characterization of the co-infection of the new variant of the porcine epidemic diarrhea virus and the novel porcine kobuvirus isolated from piglets with diarrhea.

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.

A black goat-derived novel genotype of Aichi virus C blurs the boundary between caprine and porcine kobuviruses.

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.

Chronic Aichi Virus Infection As a Cause of Long-Lasting Multiorgan Involvement in Patients With Primary Immune Deficiencies.

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.

Isolation and Characteristics of a Novel Aichivirus D from Yak.

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.

Detection and genetic characterization of canine kobuvirus from stray dogs in Shanghai, China.

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).

Evolutionary Origin, Genetic Recombination, and Phylogeography of Porcine Kobuvirus.

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.

Complete genomic sequence analysis and intestinal tissue localization of a porcine Kobuvirus variant in China.

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.

Pathogenic and metagenomic evaluations reveal the correlations of porcine epidemic diarrhea virus, porcine kobuvirus and porcine astroviruses with neonatal piglet diarrhea.

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.

Epidemiological Evidence for Fecal-Oral Transmission of Murine Kobuvirus.

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.

Aichivirus C isolate is a diarrhoea-causing pathogen in goats.

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 TCID50 /0.1 mL) and SWUN/F6/2021(10-7 TCID50 /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.

Membrane Interactions and Uncoating of Aichi Virus, a Picornavirus That Lacks a VP4.

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.

Detection and genome characterization of bovine kobuvirus (BKV) in faecal samples from diarrhoeic calves in Quebec, Canada.

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.

Next-Generation Sequencing Reveals Four Novel Viruses Associated with Calf Diarrhea.

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.

Epidemiology and genetic characteristics of murine kobuvirus from faecal samples of Rattus losea, Rattus tanezumi and Rattus norvegicus in southern China.

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.

Phylogenetic analysis of kobuviruses and astroviruses from Korean wild boars: 2016-2018.

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.

Whole genome analysis of Aichivirus A, isolated from a child, suffering from gastroenteritis, in Pakistan.

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.