Molecular Evolution of GII.P31/GII.4_Sydney_2012 Norovirus over a Decade in a Clinic in Japan.

Norovirus (NoV) genogroup II, polymerase type P31, capsid genotype 4, Sydney_2012 variant (GII.P31/GII.4_Sydney_2012) has been circulating at high levels for over a decade, raising the question of whether this strain is undergoing molecular alterations without demonstrating a substantial phylogenetic difference. Here, we applied next-generation sequencing to learn more about the genetic diversity of 14 GII.P31/GII.4_Sydney_2012 strains that caused epidemics in a specific region of Japan, with 12 from Kyoto and 2 from Shizuoka, between 2012 and 2022, with an emphasis on amino acid (aa) differences in all three ORFs. We found numerous notable aa alterations in antigenic locations in the capsid region (ORF2) as well as in other ORFs. In all three ORFs, earlier strains (2013-2016) remained phylogenetically distinct from later strains (2019-2022). This research is expected to shed light on the evolutionary properties of dominating GII.P31/GII.4_Sydney_2012 strains, which could provide useful information for viral diarrhea prevention and treatment.

Remarkable increase in the detection and molecular characterization of adenovirus F41 in children with acute gastroenteritis in Japan, 2017-2023.

Human adenovirus (HAdV) is one of the causative viruses of acute gastroenteritis (AGE) in children worldwide. Species F is known to be enteric adenovirus (genotypes 40 and 41) detected in stool samples. In Japan, we conducted an epidemiological study and molecular characterization of HAdV before and after the COVID-19 pandemic from 2017 to 2023. Among 821 patients, HAdV was detected in 118 AGE cases (14.4%). During a period of 6 years, the HAdV detection rates for each year were relatively low at 3.7% and 0%, in 2017-2018, and 2020-2021, respectively. However, the detection rate increased to remarkably high rates, ranging from 13.3% to 27.3% in the other 4-year periods. Of these HAdV-positive strains, 83.1% were F41 genotypes and 16.9% were other genotypes (A31, B3, C1, C2/C6, and C5). Phylogenetic analyses of the nucleotide and deduced amino acid sequences of the full-length hexon gene demonstrated that HAdV-F41 strains were comprised of three clades, and each clade was distributed across the study period from 2017 to 2023. Analysis of deduced amino acid sequences of the hexon gene of the representative HAdV-F41 strains from each clade revealed numerous amino acid substitutions across hypervariable regions (HVRs) from HVR-1 to HVR-7, two insertions in HVR-1 and HVR-7, and two deletions in HVR-1 and HVR-2 of the hexon gene compared to those of the prototype strain, particularly, those of clade 3 HAdV-F41 strains. The findings suggested that the HAdV-F41 of each clade was stable, conserved, and co-circulated for over two decades in Japan.

Evaluation of a novel triplex immunochromatographic test for rapid simultaneous detection of norovirus, rotavirus, and adenovirus on a single strip test.

BACKGROUND: Acute gastroenteritis is one of the major causes of morbidity and mortality in young children worldwide. Among these, rotavirus, norovirus, and adenovirus have been reported as the primary viral pathogens associated with the disease. Rapid diagnosis of viral pathogens is crucial when diarrhea outbreaks occur to ensure the timely administration of appropriate treatment and control measures. METHODS: We evaluated three immunochromatographic test kits designed for the detection of norovirus, rotavirus, and adenovirus in 71 stool specimens collected from children with diarrhea who visited clinics in Japan. The first kit is a triplex immunochromatographic test kit designed for simultaneous detections of norovirus, rotavirus, and adenovirus on a single strip (this kit was referred to as IC-A). The other two immunochromatographic test kits are a dual detection kit for rotavirus and adenovirus, and a single detection kit for norovirus (IC-B). The RT-PCR/PCR was used as the gold standard method. RESULTS: The results revealed that both IC-A and IC-B kits exhibited the same level of sensitivity of detection for rotavirus (72.7%) and adenovirus (22.7%), although the detection rate was lower than that of the RT-PCR/PCR method. However, there was a slight difference in the sensitivity of detection for norovirus between IC-A and IC-B, at 86.7% and 93.3%, respectively. The sensitivity of detection for adenovirus of both kits was relatively lower than those of RT-PCR method. This could be due to low viral load of adenovirus in clinical specimens below the detection limit of IC-A and IC-B kits. However, both immunochromatographic test kits (IC-A and IC-B) exhibited 100% specificity for norovirus, rotavirus, and adenovirus. CONCLUSIONS: The triplex immunochromatographic test kit (IC-A) designed for simultaneous detection of norovirus, rotavirus, and adenovirus has been proved to be more practical and convenient than the use of single or dual detection kits with more or less the same sensitivity and specificity of detections.

Genetic recombination and genotype diversity of norovirus GI in children with acute gastroenteritis in Thailand, 2015-2021.

BACKGROUND: Human norovirus is a predominant etiological agent responsible for acute gastroenteritis across all age groups. Recently, norovirus recombinant strains have been reported as the cause of norovirus outbreaks in several settings and the strains that cause outbreaks mostly belong to the norovirus GII. However, yet, the norovirus GI recombinant strains have never been reported previously in Thailand. The aims of this study were to investigate the genetic recombination and genotype diversity of norovirus GI strains in children hospitalized with acute gastroenteritis in Chiang Mai, Thailand during a period of seven years from 2015 to 2021. METHODS: A total of 2829 stool specimens were screened for norovirus GI by real-time PCR, and the polymerase and capsid genes were sequenced and analyzed. RESULTS: Of 2829 specimens tested, 12 (0.4%) were positive for norovirus GI. Of these, 7 out of 12 (58.3%) strains were identified as norovirus GI recombinant strains. Among 7 norovirus GI recombinant strains, 3, 3, and 1 were identified as GI.3[P13], GI.5[P4], and GI.6[P11], respectively. The remaining five strains were identified as non-recombinant strains of the GI.4[P4], GI.5[P5], and GI.6[P6] genotypes. CONCLUSIONS: The findings highlight the genetic diversity and multiple intergenotype recombinant strains of norovirus GI circulating in children with acute gastroenteritis in Chiang Mai, Thailand from 2015 to 2021. The detection of multiple intergenotype norovirus GI recombinant strains further underscore the complexity of norovirus GI strains circulating in this region.

Hepatitis B virus genotypes associated with pregnant women in Northern Thailand.

BACKGROUND: Mother-to-child transmission of hepatitis B virus (HBV) is the major route of transmission causing persistent infection. The prevalence of HBV infection and HBV genotypes found in different geographical areas varies from country to country. Therefore, this study was conducted to identify the HBV genotypes in HBV-infected pregnant women in Northern Thailand. METHODS: Stored blood samples that were collected from 145 HBsAg-positive pregnant women who gave birth at Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand from 2017 to 2020 were analyzed. The partial nucleotide sequence of the S gene of HBV was amplified by nested PCR and sequenced. All sequences were analyzed phylogenetically together with the reference strains to define the HBV genotypes. RESULTS: A total of 31 blood samples from 145 HBsAg-positive pregnant women were positive for HBV by nested PCR. The detected HBV strains were identified as presumptive subgenotypes C1 (77.4%; 24/31), B9 (9.7%; 3/31), C2 (3.2%; 1/31), B2 (3.2%; 1/31), B4 (3.2%; 1/31), and presumptive B4/C2 recombinant subgenotype (3.2%; 1/31). CONCLUSIONS: The findings revealed that presumptive subgenotype C1 was the most common subgenotype circulating in pregnant women in Northern Thailand and accounted for 77.4% of cases, followed by presumptive subgenotypes B9, C2, B2, and B4. Furthermore, this study reported, for the first time in Thailand, the HBV genotypes and presumptive subgenotypes, particularly subgenotype B9 circulating in pregnant women.

Distribution and molecular characterization of saffold virus and human cosavirus in children admitted to hospitals with acute gastroenteritis in Thailand, 2017-2022.

Saffold virus (SAFV) and human cosavirus (HCoSV) are emerging viruses of the Picornaviridae family. They have been shown to associate with gastrointestinal infection and more recently these viruses have also been demonstrated to associate with other clinical infections such as the respiratory tract, cardiovascular system, and the cerebral ventricular system. In this study, 2459 stool specimens collected from pediatric patients admitted to hospitals with acute gastroenteritis from January 2017 to December 2022, were screened for SAFV and HCoSV utilizing reverse transcription-polymerase chain reaction. Positive samples were then characterized into genotypes via nucleotide sequencing and bioinformatic analysis. Of the 2459 samples, 21 and 39 were positive for SAFV (0.9%) and HCoSV (1.6%), respectively. Three genotypes of SAFV were identified-SAFV-1 (38%), SAFV-2 (24%), and SAFV-3 (38%). Two genetic groups of HCoSV were identified-HCoSV-C (97%) and HCoSV-A (3%), demonstrating a large increase of HCoSV-C as compared to those reported previously from the same geographical region in Thailand. This study provides the prevalence of SAFV and HCoSV genotypes in Chiang Mai, Thailand during a period of 6 years from 2017 to 2022.

Molecular detection and characterization of SARS-CoV-2 in wastewater in Thailand during 2020-2022.

BACKGROUND: SARS-CoV-2 has been detected in feces of infected individuals and in wastewater in many countries, which indicates that wastewater may be used to monitor contamination of the virus in community. However, information about the presence of SARS-CoV-2 in different types of environmental water and their genetic characterization are still limited. The purpose of this study was to investigate the presence of SARS-CoV-2 contaminating in environmental water in Thailand. METHODS: We collected 600 water samples from 10 different sampling sites in Chiang Mai city, Thailand twice a month from July 2020 to December 2022. The SARS-CoV-2 RNA was detected by real-time RT-PCR and further amplified for ORF1a and S genes to investigate their genetic relationship to the reference strains by phylogenetic analysis. RESULTS: SARS-CoV-2 was detected at 0.17% in the wastewater sample collected in the vicinity of fresh market where the outbreak of COVID-19 cases were simultaneously reported. The detected SARS-CoV-2 strain (W323/21) had nucleotide and amino acid sequences identical to SARS-CoV-2 Delta variant. Amino acid sequence alignment of spike protein revealed that the W323/21 strain carried a mutation of D950N as it was demonstrated in Delta variant reference strains. CONCLUSIONS: The findings indicated that SARS-CoV-2 Delta variant was detected in wastewater in Chiang Mai, Thailand during the outbreak of COVID-19, suggesting a circulation of the virus in environmental water and in the community during the outbreak.

The Emergence and Widespread Circulation of Enteric Viruses Throughout the COVID-19 Pandemic: A Wastewater-Based Evidence.

Growing evidence shed light on the importance of wastewater-based epidemiology (WBE) during the pandemic, when the patients rarely visited the clinics despite the fact that the infections were still prevalent in the community as before. The abundance of infections in the community poses a constant threat of the emergence of new epidemic strains. Herein, we investigated enteric viruses in raw sewage water (SW) from Japan's Tohoku region and compared them to those from the Kansai region to better understand the circulating strains and their distribution across communities during the COVID-19 pandemic. Raw SW was collected between 2019 and 2022, concentrated by polyethylene-glycol-precipitation method, and investigated for major AGE viruses by RT-PCR. Sequence-based analyses were used to assess genotypes and evolutionary relationships. The most commonly detected enteric virus was rotavirus A (RVA) at 63.8%, followed by astrovirus (AstV) at 61.1%, norovirus (NoV) GII and adenovirus (AdV) at 33.3%, sapovirus (SV) at 25.0%, enterovirus (EV) at 19.4%, and NoV GI at 13.9%. The highest prevalence (46.0%) was found in the spring. Importantly, enteric viruses did not decline during the pandemic. Rather, several strains like NoV GII.2, DS-1-like human G3 (equine) RVA, MLB1 AstV, and different F41 HAdV emerged throughout the pandemic and spread widely over the Tohoku and Kansai regions. Tohoku's detection rate remained lower than that of the Kansai area (36 vs 58%). This study provides evidence for the emergence and spread of enteric viruses during the pandemic.

Possible Association between Genetic Diversity of Hepatitis B Virus and Its Effect on the Detection Rate of Hepatitis B Virus DNA in the Placenta and Fetus.

Background: The prevalence of HBV infection and HBV genotypes varies from country to country, and the role of HBV genotypes in the presence of HBV in the placenta and fetus has never been explored. This study was conducted to (1) identify HBV genotypes, and their frequencies, that infected Northern Thai pregnant women; (2) evaluate the association between HBV genotypes and the detection rate of HBV DNA in the placenta and fetus; (3) evaluate the association between specific mutations of the HBV genome and HBV DNA detection in placental tissue; and (4) identify the mutation of the HBV genome that might occur between maternal blood, placenta, and cord blood. Methods: Stored samples of the maternal blood, placental tissue, and cord blood that were collected from 145 HBsAg-positive pregnant Thai women were analyzed to identify HBV DNA. Results: Approximately 25% of infected mothers had fetal HBV DNA detection, including cases with concomitant HBV DNA detection in the placenta (77.3%). A total of 11.7% of cases with placental detection had no HBV DNA detection in the maternal blood, indicating that the placenta could be a site of HBV accumulation. Of the 31 HBV-positive blood samples detected by nested PCR, the detected strains were subgenotype C1 (77.4%), subgenotype B9 (9.7%), and subgenotype C2, B2, B4, and recombinant B4/C2 (3.2% for each). Genotype B had a trend in increased risk of placental HBV DNA detection compared to genotype C, with a relative risk of 1.40 (95% CI: 1.07-1.84). No specific point mutation had a significant effect on HBV DNA detection in placental tissue. Mutation of C454T tended to enhance HBV DNA detection in placental tissue, whereas T400A tended to have a lower detection rate. No mutation was detected in different sample types collected from the same cases. Conclusions: HBV DNA detection in the fetus was identified in approximately 25% of HBV-positive mothers, associated with the presence of HBV in the placenta in most cases. The placenta could possibly be a site of HBV accumulation. Subgenotype C1 was the most common subgenotype, followed by subgenotype B9. HBV genotype B possibly had a higher trend in intrauterine detection than HBV genotype C. Mutation is unlikely to occur during intrauterine exposure.

Diverse genotypes of human enteric and non-enteric adenoviruses circulating in children hospitalized with acute gastroenteritis in Thailand, from 2018 to 2021.

Human adenovirus (HAdV) is a common viral pathogen that causes diarrhea in children worldwide. The aim of this study was to investigate the prevalence and genotype diversity of HAdV strains circulating in children admitted to the hospitals with acute gastroenteritis (AGE) in Chiang Mai, Thailand, from 2018 to 2021. A total of 1,790 stool samples were screened for HAdV by PCR method, and 80 (4.5%) were positive for HAdV. Of these, children under 5 years of age accounted for 90.0% of HAdV-positive cases with the highest infection rate at the age group of 48-60 months old. The infection rate was not significantly different between boys and girls. The HAdV infection was detected sporadically throughout the year without a discrete seasonal pattern. Five species of both enteric and non-enteric HAdVs (A, B, C, E, and F) with 10 different genotypes, including HAdV-F41 (25.0%), HAdV-B3 (17.5%), HAdV-F40 (16.3%), HAdV-C1 (15.0%), HAdV-C5 (7.5%), HAdV-C2 (6.3%), HAdV-B7 (5.0%), HAdV-A12 (3.8%), HAdV-E4 (2.5%), and HAdV-B11 (1.3%), were detected in this study. In conclusion, our study reported the prevalence and seasonality of HAdV infection with a wide variety of HAdV genotypes circulating in children hospitalized with AGE during a period of 2018-2021 in Chiang Mai, Thailand. IMPORTANCE In the present study, the prevalence of human adenovirus (HAdV) infection in children with acute gastroenteritis (AGE) in Chiang Mai, Thailand, from 2018 to 2021 was detected at 4.5%. Diverse species and genotypes of HAdVs (HAdV-A12, HAdV-B3, HAdV-B7, HAdV-B11, HAdV-C1, HAdV-C2, HAdV-C5, HAdV-E4, HAdV-F40, and HAdV-F41) had been identified. The highest infection rate was found in children aged 48-60 months old. The HAdV infection was detected sporadically throughout the year. These findings imply that a wide variety of HAdV genotypes circulate in pediatric patients with AGE in Chiang Mai, Thailand.

Sapovirus infections in Japan before and after the emergence of the COVID-19 pandemic: An alarming update.

An increasing trend of sapovirus (SaV) infections in Japanese children during 2009-2019, particularly after the introduction of the voluntary rotavirus (RV)-vaccination program has been observed. Herein, we investigated the epidemiological situation of SaV infections from 2019 to 2022 when people adopted a precautionary lifestyle due to the emergence of the COVID-19 pandemic, and RV vaccines had been implemented as routine vaccines. Stool samples were collected from children who attended outpatient clinics with acute gastroenteritis and analyzed by reverse transcriptase-polymerase chain reaction to determine viral etiology. Among 961 stool samples, 80 (8.3%) were positive for SaV: 2019-2020 (6.5%), 2020-2021 (0%), and 2021-2022 (12.8%). The trend of SaV infection in Japanese children yet remained upward with statistical significance (p = 0.000). The major genotype was GI.1 (75%) which caused a large outbreak in Kyoto between December 2021 and February 2022. Phylogenetic, gene sequence and deduced amino acid sequence analyses suggested that these GI.1 strains detected in the outbreak and other places during 2021-2022 or 2019-2020 remained genetically identical and widely spread. This study reveals that SaV infection is increasing among Japanese children which is a grave concern and demands immediate attention to be paid before SaV attains a serious public health problem.

Genetic diversity and declining norovirus prevalence in infants and children during Japan's COVID-19 pandemic: a three-year molecular surveillance.

Noroviruses (NoVs) are a global concern, causing widespread outbreaks and sporadic acute gastroenteritis (AGE) cases across all age groups. Recent research has shed light on the emergence of novel recombinant strains of NoV in various countries. To delve deeper into this phenomenon, we extensively analyzed 1,175 stool samples collected from Japanese infants and children with AGE from six different prefectures in Japan over three years, from July 2018 to June 2021. Our investigation aimed to determine the prevalence and genetic characteristics of NoV associated with sporadic AGE while exploring the possibility of detecting NoV recombination events. Among the analyzed samples, we identified 355 cases positive for NoV, 11 cases attributed to GI genotypes, and 344 associated with GII genotypes. Notably, we discovered four distinct GI genotypes (GI.2, GI.3, GI.4, and GI.6) and seven diverse GII genotypes (GII.2, GII.3, GII.4, GII.6, GII.7, GII.14, and GII.17). The predominant genotypes were GII.4 (56.4%; 194 out of 344), followed by GII.2 and GII.3. Through dual genotyping based on sequencing of the ORF1/ORF2 junction region, we identified a total of 14 different RdRp/capsid genotypes. Of particular interest were the prevalent recombinant genotypes GII.4[P31] and GII.2[P16]. Notably, our study revealed a decrease in the number of children infected with NoV during and after the COVID-19 pandemic. These findings underscore the importance of continuous NoV surveillance efforts.

Predominance of ON1 and BA9 genotypes of human respiratory syncytial virus in children with acute respiratory infection in Chiang Mai, Thailand, 2020-2021.

BACKGROUND: Human respiratory syncytial virus (hRSV) is an important cause of acute respiratory infection, especially in children. Few studies have investigated molecular epidemiology of hRSV infection in Thailand. The aims of this study were to investigate the prevalence and genotype diversity of hRSV in children with acute respiratory infection (ARI) in Thailand. METHODS: A total of 383 nasopharyngeal swabs collected from children with ARI from October 2020 to September 2021 were screened for hRSV and nucleotide sequences of the hypervariable region 2 (HVR2) of G gene of the detected hRSV were analysed. RESULTS: Of 383 nasopharyngeal swabs, 104 (27.2 %) were positive for hRSV, of which 51 (49.0 %), 43 (41.3 %), and 10 (9.6 %) were hRSV-A, hRSV-B, and untypeable strains, respectively. All hRSV-A and hRSV-B were ON1 genotype and BA9 genotype, respectively. Most of the hRSV strains were detected in the cool months, November 2020 to February 2021. Phylogenetic analysis of the HVR2 sequence of G gene revealed three clusters of hRSV-A (ON1 genotype) and two clusters of hRSV-B (BA9 genotype). The hRSV-A strains in cluster 1 and 3 were closely related to the hRSV-A reference strains reported previously from other regions of Thailand whereas those in cluster 2 were closely related to the hRSV-A reference strains reported previously from Europe and Africa. For the hRSV-B strains, both clusters 1 and 2 were closely related to the hRSV-B reference strains reported previously from Europe, Australia, and Taiwan. The predicted N- and O-linked glycosylation sites were found along the length of HVR2 of G protein, mostly in the hRSV-B strains. CONCLUSIONS: The ON1 and BA9 were the only two hRSV genotypes that were co-predominant and solely detected in this study. The findings indicated that the ON1 and BA9 are the only two hRSV genotypes currently circulating in children with ARI in northern Thailand.

Genetic Diversity of Rotaviruses Circulating in Pediatric Patients and Domestic Animals in Thailand.

Rotavirus A is a highly contagious virus that causes acute gastroenteritis in humans and a wide variety of animals. In this review, we summarized the information on rotavirus described in the studies in the last decade (2008 to 2021) in Thailand, including the prevalence, seasonality, genetic diversity, and interspecies transmission. The overall prevalence of rotavirus infection in humans ranged from 15-33%. Rotavirus infection was detected throughout the year and most frequently in the dry and cold months, typically in March. The diversity of rotavirus genotypes varied year to year and from region to region. From 2008 to 2016, rotavirus G1P[8] was detected as the most predominant genotype in Thailand. After 2016, G1P[8] decreased significantly and other genotypes including G3P[8], G8P[8], and G9P[8] were increasingly detected from 2016 to 2020. Several uncommon rotavirus strains such as G1P[6], G4P[6], and G3P[10] have also been occasionally detected. In addition, most studies on rotavirus A infection in animals in Thailand from 2011 to 2021 reported the detection of rotavirus A in piglets and canine species. It was reported that rotavirus could cross the host species barrier between humans and animals through interspecies transmission and genetic reassortment mechanisms. The surveillance of rotavirus infection is crucial to identify the trend of rotavirus infection and the emergence of novel rotavirus genotypes in this country. The data provide information on rotavirus infection and the diversity of rotavirus genotypes circulating in the pre-vaccination period, and the data will be useful for the evaluation of the effectiveness of rotavirus vaccine implementation in Thailand.

Outbreak of human astroviruses 1 and Melbourne 2 in acute gastroenteritis pediatric patients in Japan during the COVID-19 pandemic, 2021.

BACKGROUND: Human astrovirus (HAstV) infection is one of the leading causes of acute gastroenteritis in young children. The present study reports the outbreak of HAstV in children with acute gastroenteritis in Kyoto, Japan, during the COVID-19 pandemic, 2021. METHODS: A total of 61 stool samples were collected from children with acute gastroenteritis who visited a pediatric outpatient clinic in Maizuru city, Kyoto, Japan from July to October, 2021. HAstV was screened by RT-PCR, and the genotypes were identified by nucleotide sequence analysis. RESULTS: Of 61 cases of acute gastroenteritis, 20 were mono-infected with HAstV alone. In addition, mixed infection of HAstV and NoV, and HAstV and RVA were also detected in 15 and 1 cases, respectively. Of 36 HAstV strains detected in this outbreak, 29 and 7 were HAstV1 and MLB2 genotypes, respectively. All HAstV1 strains were closely related to the HAstV1 reported from Thailand and Japan in 2021 and all of them belonged to subgenotype HAstV1a. Among MLB2, they were most closely related to the MLB2 strains reported from China in 2016 and 2018. CONCLUSIONS: After the kindergartens and schools were re-opened at the middle of 2021 in Japan, an outbreak of HAstV was reported. Control measures against the COVID-19 pandemics might affect the spread of diarrheal virus infection. Here we report the outbreak of HAstV1 and MLB2 in Kyoto, Japan, during COVID-19 pandemic in 2021.

Molecular Epidemiology of Classic, MLB, and VA Astroviruses in Children with Acute Gastroenteritis, 2014-2021: Emergence of MLB3 Strain in Japan.

Human astroviruses (HAstVs) are important causative pathogens of acute gastroenteritis (AGE) in children worldwide. MLB and VA HAstVs, which are genetically distinct from the previously known classic HAstVs, have been detected since 2008. To investigate the role of HAstVs in AGE, we conducted molecular detection and characterization of HAstVs circulating in children with AGE in Japan from 2014 to 2021. Out of 2,841 stool samples, HAstVs were detected in 130 (4.6%). MLB1 was the predominant genotype detected (45.4%), followed by HAstV1 (39.2%), MLB2 (7.4%), VA2 (3.1%), HAstV3 (2.3%), HAstV4, HAstV5, and MLB3 (0.8% each). The results demonstrated that HAstV infection in pediatric patients in Japan was dominated by the two major genotypes MLB1 and HAstV1, with a small proportion of other genotypes. The overall infection rates of MLB and VA HAstVs were higher than those of classic HAstVs. The HAstV1 strains detected in this study belonged solely to lineage 1a. The rare MLB3 genotype was detected for the first time in Japan. All three HAstV3 strains belonged to lineage 3c based on the ORF2 nucleotide sequence and were shown to be recombinant strains. IMPORTANCE HAstVs are one of the pathogens of viral AGE and are considered the third most common viral agents of AGE after rotavirus and norovirus. HAstVs are also suspected to be the causative agents of encephalitis or meningitis in immunocompromised patients and elderly persons. However, little is known about the epidemiology of HAstVs in Japan, especially that of MLBs and VA HAstVs. This study demonstrated epidemiological features and molecular characterization of human astroviruses encompassing a 7-year study period in Japan. This study highlights the genetic diversity of HAstV circulating in pediatric patients with acute AGE in Japan.

Genotype Diversity of Enteric Viruses in Wastewater Amid the COVID-19 Pandemic.

Viruses remain the leading cause of acute gastroenteritis (AGE) worldwide. Recently, we reported the abundance of AGE viruses in raw sewage water (SW) during the COVID-19 pandemic, when viral AGE patients decreased dramatically in clinics. Since clinical samples were not reflecting the actual state, it remained important to determine the circulating strains in the SW for preparedness against impending outbreaks. Raw SW was collected from a sewage treatment plant in Japan from August 2018 to March 2022, concentrated by polyethylene-glycol-precipitation method, and investigated for major gastroenteritis viruses by RT-PCR. Genotypes and evolutionary relationships were evaluated through sequence-based analyses. Major AGE viruses like rotavirus A (RVA), norovirus (NoV) GI and GII, and astrovirus (AstV) increased sharply (10-20%) in SW during the COVID-19 pandemic, though some AGE viruses like sapovirus (SV), adenovirus (AdV), and enterovirus (EV) decreased slightly (3-10%). The prevalence remained top in the winter. Importantly, several strains, including G1 and G3 of RVA, GI.1 and GII.2 of NoV, GI.1 of SV, MLB1 of AstV, and F41 of AdV, either emerged or increased amid the pandemic, suggesting that the normal phenomenon of genotype changing remained active over this time. This study crucially presents the molecular characteristics of circulating AGE viruses, explaining the importance of SW investigation during the pandemic when a clinical investigation may not produce the complete scenario.

Triple Intergenotype Recombination of Human Astrovirus 5, Human Astrovirus 8, and Human Astrovirus 1 in the Open Reading Frame 1a, Open Reading Frame 1b, and Open Reading Frame 2 Regions of the Human Astrovirus Genome.

Human astrovirus (HAstV) strains exhibit high levels of genetic diversity, and many recombinant strains with different recombination patterns have been reported. The aims of the present study were to investigate the emergence of HAstV recombinant strains and to characterize the recombination patterns of the strains detected in pediatric patients admitted to the hospital with acute gastroenteritis in Chiang Mai, Thailand. A total of 92 archival HAstV strains detected in 2011 to 2020 were characterized regarding their open reading frame 1a (ORF1a) genotypes in comparison with their ORF1b genotypes to identify recombinant strains. The recombination breakpoints of the putative recombinant strains were determined by whole-genome sequencing and were analyzed by SimPlot and RDP software. Three HAstV strains (CMH-N178-12, CMH-S059-15, and CMH-S062-15) were found to be recombinant strains of three different HAstV genotypes, i.e., HAstV5, HAstV8, and HAstV1 within the ORF1a, ORF1b, and ORF2 regions, respectively. The CMH-N178-12 strain displayed recombination breakpoints at nucleotide positions 2681 and 4357 of ORF1a and ORF1b, respectively, whereas the other two recombinant strains, CMH-S059-15 and CMH-S062-15, displayed recombination breakpoints at nucleotide positions 2612 and 4357 of ORF1a and ORF1b, respectively. This is the first study to reveal nearly full-length genome sequences of HAstV recombinant strains with a novel recombination pattern of ORF1a-ORF1b-ORF2 genotypes. This finding may be useful as a guideline for identifying other recombinant HAstV strains in other geographical regions and may provide a better understanding of their genetic diversity, as well as basic knowledge regarding virus evolution. IMPORTANCE Recombination is one of the mechanisms that plays a crucial role in the genetic diversity and evolution of HAstV. We wished to investigate the emergence of HAstV recombinant strains and to analyze the whole-genome sequences of the putative HAstV recombinant strains detected in pediatric patients with acute gastroenteritis in 2011 to 2020. We reported 3 novel intergenotype recombinant strains of HAstV5-HAstV8-HAstV1 at the ORF1a-ORF1b-ORF2 regions of the HAstV genome. The hot spots of recombination occur frequently near the ORF1a-ORF1b and ORF1b-ORF2 junctions of the HAstV genome. The findings indicate that intergenotype recombination of HAstV occurs frequently in nature. The emergence of a novel recombinant strain allows the new virus to adapt and successfully escape from the host immune system, eventually emerging as the predominant genotype to infect human populations that lack herd immunity against novel recombinant strains. The virus may cause an outbreak and needs to be monitored continually.

Detection of Six Different Human Enteric Viruses Contaminating Environmental Water in Chiang Mai, Thailand.

A 2-year surveillance study into enteric viruses contaminating environmental water samples was conducted in the city of Chang Mai, Thailand. The aim of the study was to investigate the prevalence of six different human enteric viruses, specifically, adenovirus (AdV), astrovirus (AstV), enteroviruses (EVs), human parechovirus (HPeV), rotavirus (RV), and saffold virus (SAFV), contaminating several types of environmental water using PCR and reverse transcription-PCR (RT-PCR) methods. All targeted viruses were detected with different levels of prevalence. The levels ranged from 0.8 to 4.8% (AdV, 0.8%; AstV, 4.8%; EV, 0.8%; HPeV, 3.2%; RV, 0.8%; SAFV, 3.2%). A wide variety of human enteric virus genotypes, including AdV-41, AstV-MLB1, coxsackievirus A, HPeV1, 5, and 6, RV G4[P8], and SAFV-2 and 3 were detected. The overall picture of the 13 human enteric viruses that were detected in environmental water in Chiang Mai, Thailand, is also summarized in this study. The data and the findings of this study will provide a better understanding of the viral dynamics in environmental water. The detection of these viruses in environmental water indicates there is the potential for human infection from this source. IMPORTANCE Human enteric viruses are a major cause of gastrointestinal illness, and these viruses can be introduced into environmental water through various routes. Viral contamination in water could play a significant role in human health. This study demonstrated the prevalence of six different enteric viruses, adenovirus, astrovirus, enteroviruses, human parechovirus, rotavirus, and saffold virus, contaminating environmental water. We also analyzed the overall prevalence of other enteric viruses that were in this area, and the findings revealed a wide diversity of the enteric viruses contaminating environmental water. The data provide a better understanding of the epidemiologic importance of viral contamination of the water and highlight the need for better management of wastewater disposal and effective environmental water treatment to prevent the human population from infection.