Whole genome sequencing and genome characterization of Aichivirus isolated from Korean adults.

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.

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.

Seasonal Prevalence and Detection of Enteric and Respiratory Viruses in Wastewater and Hospitalized Children with Acute Gastroenteritis.

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 × 101 and 8.75 × 107 GC/ml in raw sewage samples, 1.20 × 101 and 5.43 × 106 GC/ml in treated sewage samples, and 4.80 × 101 and 9.88 × 108 GC/gram in stool samples. The overall log means of virus reduction during the wastewater treatment process ranged from 1.68 log10 (HAdV) to 3.31 log10 (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.

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.

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 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).

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.

[Neurotropic bovine astrovirus-associated encephalitis: An underdiagnosed disease in South America?] / Encefalitis asociada a astrovirus bovino neurotrópico, ¿una enfermedad subdiagnosticada en Sudamérica?

We describe a case of neurotropic bovine astrovirus-associated encephalitis in a Jersey dairy cow from the department of San José, Uruguay. This represents the second case of this condition reported in the Southern Hemisphere. The cow was the only one affected in a herd of 70 cows, showing neurological signs with a 2-day clinical course, before dying spontaneously. Histopathological examination revealed lymphocytic, histiocytic, and plasmacytic meningoencephalitis with neuronal necrosis, without detectable inclusion bodies. Other infectious agents, including Rabies virus(Lyssavirus), Bovine alphaherpesvirus-1 and Bovine alphaherpesvirus-5(Varicellovirus), Bovine viral diarrhea virus(Pestivirus), West Nile virus(Flavivirus), Listeria monocytogenes, Histophilus somni and other bacteria, were not detected in the brain. We propose that given the recent discovery of neurotropic astroviruses in various mammalian species, including humans, cases of astrovirus encephalitis may have gone undetected in South America. We briefly discuss the differential pathologic diagnosis of infectious bovine encephalitis.

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.

Prevalence and Genetic Diversity of Aichi Virus 1 from Urban Wastewater in Senegal.

Aichi virus 1 (AiV-1) has been proposed as a causative agent of human gastroenteritis. In this study, raw, decanted, and treated wastewater samples from a wastewater treatment plant in an urban area of Dakar, Senegal, were collected. AiV-1 was detected in raw (70%, 14/20), decanted (68.4%, 13/19), and treated (59.3%, 16/27) samples, revealing a noticeable resistance of AiV-1 to chlorine-based treatment. Phylogenetic analysis revealed that all sequences clustered within genotype B. Our study presents the first report on the detection of AiV-1 in the environment of Dakar and constitutes indirect evidence of virus circulation in the population.

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.

Grape seed extract against Aichi virus infectivity in model foods and contact surfaces.

Aichi virus (AiV) is an enteric virus that affects humans and is prevalent in sewage waters. Effective strategies to control its spread need to be explored. This study evaluated grape seed extract (GSE) for: a) antiviral potential towards AiV infectivity at 37 °C and room temperature (RT); b) antiviral behavior in model foods (apple juice (AJ) and 2% fat milk) and also simulated gastric environments; and c) potential application as a wash solution on stainless steel surfaces. GSE at 0.5 mg/mL decreased AiV suspensions containing ~4.75 log PFU/mL to titer levels that were not detected after 30 s at both 37 °C and RT. Infectious AiV titers were not detected after 5 min treatment with 1 mg/mL GSE at 37 °C in AJ. GSE at 2 mg/mL and 4 mg/mL in 2% fat milk decreased AiV after 24 h by 1.18 and 1.57 log PFU/mL (4.75 log PFU/mL to 2.86 and 3.25 log PFU/mL), respectively. As a surface wash, GSE at 1 mg/mL after 30 s decreased AiV to undetectable levels under clean conditions. With organic load (mimicking unclean conditions), 2 and 4 mg/mL GSE reduced AiV after 5 min by 1.13 and 1.71 log PFU/mL, respectively. Overall, GSE seems to be a promising antiviral agent against AiV at low concentrations and short contact times.

KOBUVIRUS DETECTION IN THE CRITICALLY ENDANGERED PYGMY HOG (PORCULA SALVANIA), INDIA.

Pygmy hogs (Porcula salvania) are the smallest and rarest wild suid. It is categorized as a Critically Endangered species as per the Red List of the International Union for Conservation of Nature. This study reports the first detection of a single-stranded RNA virus species, Aichivirus C, belonging to the genus Kobuvirus (KobV) and the family Picornaviridae, in pygmy hogs. KobV species are identified as a cause of acute gastroenteritis among children in India. As of now, there exists no report on the detection of KobV in animals from India. We used a detection assay based on reverse transcription-polymerase chain reaction for KobV screening in pygmy hogs from a conservation center in India. The 3D polymerase gene-based molecular analysis revealed KobV presence in the Indian wild suid, pygmy hogs. Of the 15 samples tested, three were found positive for picornaviruses and were negative for rotavirus A, rotavirus C, astrovirus, picobirnavirus and caliciviruses. Nucleotide-based sequence analysis of the partial 3D polymerase gene revealed close identity with porcine KobV from the Czech Republic (JX232619, 90.6%-91.6%) and Hungary (NC_011829, 89.8%-91.6%), wherein one of the current study strains clustered with the Czech Republic JX232619 strain in the phylogenetic tree. Further investigation of the role of KobV in health and disease of pygmy hogs is warranted.

Bovine Kobuvirus in Calves with Diarrhea, United States.

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.

Kobuviruses carried by Rattus norvegicus in Guangdong, China.

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.

One-step multiplex TaqMan probe-based method for real-time PCR detection of four canine diarrhea viruses.

Viral canine diarrhea has high morbidity and mortality and is prevalent worldwide, resulting in severe economic and spiritual losses to pet owners. However, diarrhea pathogens have similar clinical symptoms and are difficult to diagnose clinically. Thus, fast and accurate diagnostic methods are of great significance for prevention and accurate treatment. In this study, we developed a one-step multiplex TaqMan probe-based real-time PCR for the differential diagnosis of four viruses causing canine diarrhea including, CPV (Canine Parvovirus), CCoV (Canine Coronavirus), CAstV (Canine Astrovirus), and CaKoV (Canine Kobuviruses). The limit of detection was up to 102 copies/µL and performed well with high sensitivity and specificity. This assay was optimized and used to identify possible antagonistic relationships between viruses. From this, artificial pre-experiments were performed for mixed infections, and a total of 82 canine diarrhea field samples were collected from different animal hospitals in Zhejiang, China to assess the method. The virus prevalence was significantly higher than what previously reported based on RT-PCR (Reverse Transcription-Polymerase Chain Reaction). Taken together, these results suggest that the method can be used as a preferred tool for monitoring laboratory epidemics, timely prevention, and effective monitoring of disease progression.

Structural basis for hijacking of the host ACBD3 protein by bovine and porcine enteroviruses and kobuviruses.

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.

Identification and full-genome sequencing of canine kobuvirus in canine fecal samples collected from Anhui Province, eastern China.

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.

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

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

Environmental monitoring of Aichi virus and human bocavirus in samples from wastewater treatment plant, drain, and River Nile in Egypt.

Wastewater plays a major role in water pollution causing transmission of several viral pathogens, including Aichi virus (AiV) and human bocavirus (HBoV), associated with gastrointestinal illness in humans. In this study, we investigated the presence of AiV and HBoV in aquatic, sludge, sediment matrices collected from Abu-Rawash wastewater treatment plant (WWTP), El-Rahawy drain, Rosetta branch of the River Nile in Egypt by conventional polymerase chain reaction (PCR). AiV RNA was detected in 16.6% (2/12), 8.3% (1/12), 8.3% (1/12), 22% (16/72), 12.5% (3/24), 4% (1/24), and 0/24 (0%) of untreated raw sewage, treated sewage, sewage sludge, drainage water, drain sediment, river water, and river sediment, respectively. On the other hand, HBoV DNA was detected in 41.6% (5/12), 25% (3/12), 16.6% (2/12), 48.6% (35/72), 29% (7/24), 3/24 (12.5%), 4% (1/24) of untreated raw sewage, treated raw sewage, sewage sludge, drainage water, drain sediment, river water, and river sediment, respectively. This study provides data on the presence of these viruses in various types of water samples that are valuable to environmental risk assessment. In addition, the current study demonstrates the importance of environmental monitoring as an additional tool to investigate the epidemiology of AiV and HBoV circulating in a given community.