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.

Diversity of human sapovirus genotypes detected in Japanese pediatric patients with acute gastroenteritis, 2014-2017.

Sapovirus (SaV) is one of the pathogens related to acute gastroenteritis (AGE) in adults and children worldwide. This study reported the diversity of SaV genotypes in children with AGE in Japan from July 2014 to June 2017. Of a total of 2259 stool samples tested by using reverse transcription-PCR method and further analyzed by nucleotide sequencing, 114 (5.0%) were positive for SaV and GI.1 (83.3%) was the most predominant genotype, followed by GII.1, GIV.1, GI.2, GI.3, and GII.3 genotypes. Monthly distribution analysis demonstrated two epidemic peaks from July to December 2015 and February to May 2017. However, no detection peak was observed in 2014 and 2016. Phylogenetic analysis of the complete VP1 nucleotide sequences of these GI.1 strains revealed two major clusters of GI.1 and each of which contained GI.1 strains of both 2015 and 2017. This study suggests that the continuous surveillance of SaV is needed to monitor high genetic diversity in Japanese children with AGE.

Evaluation of Immunochromatographic Tests for Detection of a Wide Variety of Group A Rotavirus Genotypes and Adenovirus.

BACKGROUND: Viral gastroenteritis is one of the most common illnesses in humans worldwide, and different viral agents have been shown to be associated with the disease. Among these, rotaviruses and adenoviruses are the responsible causative agents of acute gastroenteritis and causing numerous outbreaks. Therefore, a simple and rapid diagnostic tool, such as an immunochromatographic (IC) test, is required for rapid diagnosis, especially during an outbreak of these pathogens. METHODS: The efficiency of two commercial IC kits were evaluated for simultaneous detections of rotavirus and adenovirus in clinical stool specimens by a single test kit. RESULTS: The data demonstrated that both IC test kits could detect either adenovirus or rotavirus positive alone, as well as mixed infections of both viruses in a single stool specimen. In addition, a wide variety of rotavirus genotypes, including G1-P[8]-I1, G2-P[4]-I2, G3-P[8]-I2, G8-P[8]-I2, and G9-P[8]-I1 could be detected by both IC kits. The detection limit of the kits for the detection of rotavirus and adenovirus were comparable to those of real-time PCR at 105 copies/mL. CONCLUSIONS: These two IC test kits could be used as an alternative choice for rapid screening of rotavirus and adenovirus in the stool specimens, especially during the seasonal outbreak of acute gastroenteritis.

Molecular epidemiology of rotavirus gastroenteritis in Japan during 2014-2015: Characterization of re-emerging G2P[4] after rotavirus vaccine introduction.

Rotavirus vaccines have been available in Japan since 2011. This study conducted to monitor the trend of group A rotavirus (RVA) genotypes 3 years after vaccine introduction. A total of, 539 fecal samples were collected from children with acute gastroenteritis in six regions during July 2014-June 2015. Among them, 178 samples (33.0%) were positive for RVA. The most predominant genotype was G1P[8] (35.9%) followed by G2P[4] (26.4%), G9P[8] (21.3%), G3P[8] (4.5%), and G3P[9] (4.5%). The detection rate of G2P[4] was increased soon after vaccine introduction. Sequence analyses of VP7 and VP4 genes of the representative G2P[4] strains were found to be clustered in sub-lineage IVa of lineage IV. It is noteworthy that one amino acid substitution in the antigenic epitope (Q114P) of VP4 gene was found in representative G2P[4] strains of the current study. However, it is unclear whether the change in antigenic epitope is due to the effect of vaccination or due to natural variation, warranting further continuous monitoring of rotavirus evolution after vaccine introduction.

Evaluation of Immunochromatographic Test for Dual Detection of Noroviruses and Group A Rotaviruses in Stool Samples.

BACKGROUND: Viral enteric infections, such as noroviruses and rotaviruses are considered as the major causes of acute gastroenteritis in young children and elderly people all over the world. These viruses are responsible for numerous outbreaks of nonbacterial gastroenteritis in several settings such as hospitals, day care centers, nursing homes, and restaurants. Therefore, a rapid and sensitive diagnostic tool for virus detections could be helpful for disease control. METHODS: A new immunochromatographic test for the dual detection of noroviruses and group A rotaviruses was evaluated by using specimens that were known to be positive for noroviruses and rotaviruses, as well as other diarrheal viruses. RESULTS: The sensitivity of detections for noroviruses and group A rotaviruses were 89.5% and 100%, respectively, while the specificity was 100% for both viruses. This immunochromatographic test was reactive to several norovirus and group A rotavirus genotypes (NoV GII.2, GII.3, GII.4, GII.6, GII.7, GII.17 and RVA G1P[8], G2P[4], G3P[8], G8P[8], G9P[8]). CONCLUSIONS: The speed and ease of use of the immunochromatographic test makes this kit particularly attractive as an alternative method for the detections of noroviruses and group A rotaviruses in the primary care unit.

Detection and characterization of Aichi virus 1 in pediatric patients with diarrhea in Thailand.

Kobuvirus is a newly discovered virus that belongs to the Kobuvirus genus in Picornaviridae family, which comprised of three species including Aichivirus A, Aichivirus B, and Aichivirus C. The kobuvirus isolated from human has been classified as Aichi virus 1 and belongs to Aichivirus A species. The present study aimed to assess the epidemiology and to perform molecular characterization of Aichi virus 1 in children admitted to hospitals with acute gastroenteritis in Chiang Mai, Thailand. A total of 923 fecal specimens collected from January, 2011 to December, 2013 were screened for the presence of Aichi virus 1 by RT semi-nested PCR. Out of 923 fecal specimens tested, Aichi virus 1 was detected with the prevalence of 2.6% (24/923). Of these, 0.3% (3/923) was genotype A and 2.3% (21/923) were genotype B. It is interesting to note that the genotype A showed the nucleotide sequence closely related to the Aichi virus reference strain isolated from sewage in Tunisia, while genotype B was most closely related to other human Aichi virus B reference strains. The results suggest that Aichi virus 1 of both genotypes A and B are circulating in pediatric patients in Thailand. J. Med. Virol. 89:234-238, 2017. © 2016 Wiley Periodicals, Inc.

Multiplex RT-PCR for rapid detection of viruses commonly causing diarrhea in pediatric patients.

Multiplex RT-PCR method using five sets of panel primers was developed for the detection of diarrheal viruses, including rotavirus A, B, and C, adenovirus, astrovirus, norovirus GI and GII, sapovirus, Aichi virus, parechovirus, enterovirus, cosavirus, bocavirus, and Saffold virus. The sensitivity of the method was evaluated and tested with 751 fecal specimens collected from Japanese children with acute diarrhea. Several kinds of viruses were detected in 528 out of 751 (70.3%) fecal specimens. Mixed-infection with different viruses in clinical specimens could also be effectively detected. The method proved to be reliable with highly sensitive and specific and useful for routine diagnosis. J. Med. Virol. 89:818-824, 2017. © 2016 Wiley Periodicals, Inc.

Noroviruses and sapoviruses associated with acute gastroenteritis in pediatric patients in Thailand: increased detection of recombinant norovirus GII.P16/GII.13 strains.

Enteric caliciviruses, including noroviruses (NoVs) and sapoviruses (SaVs), are recognized as important etiologic agents of acute gastroenteritis (AGE) with considerable genetic diversity. In order to gain an overview of the molecular epidemiology of human NoVs and SaVs in children hospitalized with AGE in Chiang Mai, Thailand, a total of 889 fecal specimens were collected from 2012 to 2014 and screened for NoVs and SaVs. Out of 889 fecal specimens, 154 (17.3%) and 6 (0.7%) were positive for NoV GII isolates and SaV, respectively. Among the NoV GII, 10 different genotypes were identified with genotype GII.4 being predominant (103 strains), followed by GII.3 (17 strains), GII.13 (13 strains), GII.1 (7 strains), GII.6 (7 strains), GII.7 (2 strains), GII.17 (2 strains), and one each of GII.2, GII.15, and GII.21 genotypes. It was observed that four variants of NoV GII.4 (Den Haag 2006b, Apeldoorn 2007, New Orleans 2009, Sydney 2012) were detected from 2012 to 2014. Analysis of partial nucleotide sequences of RdRp and VP1 of the emerging NoV GII.13 strains (9 of 13 strains) revealed that they all were GII.P16/GII.13 recombinants. In addition, four different genotypes of SaV, GI.1 (2 strains), GII.1 (1 strain), GII.4 (2 strains), and GIV.1 (1 strain) were detected. The data revealed heterogeneity and a highly dynamic distribution of NoV and SaV genotypes circulating in children admitted to hospitals with AGE in Chiang Mai, Thailand, during the period of 2012 to 2014.

A 3-Month-Old Child with Acute Gastroenteritis with Enterovirus D68 Detected from Stool Specimen.

Enterovirus D68 (EV-D68) is known to be causative agent of mild to severe upper and lower respiratory illnesses in sporadic cases and outbreaks. We present a case report of a 3-month-old child with acute gastroenteritis who visited a pediatric clinic in Kyushu area in Japan in 2015. A stool sample collected from the patient was screened for diarrheal viruses by multiplex RT-PCR. The result showed that the sample was positive only for enterovirus, and EV-D68 clade B3 was identified by sequence analysis of the viral protein 1 gene. This study supports an association between EV-D68 infection and acute gastroenteritis.

Molecular Epidemiological Traits of Group A Rotaviruses in Japanese Children During Transitional Period of Rotavirus Vaccine Implementation, 2011 - 2014.

BACKGROUND: Group A rotavirus (RVA) vaccines have been introduced in Japan since 2011. To investigate the molecular epidemiological traits of RVA during the transitional period of rotavirus vaccine implementation in Japan, this study was undertaken by following up three-decade long surveillance conducted in the same regions. METHODS: RVA were screened and genotyped by RT-PCR from diarrheal samples collected from non-hospitalized patients in six localities (Hokkaido, Tokyo, Shizuoka, Osaka, Kyoto, and Saga) during 2011 - 2014. Selected samples were sequenced to elucidate the evolutionary trend. RESULTS: Among 1858 specimens, the detection rate of RVA declined to 4.0% in 2013 - 2014 from 17.9% in 2011 - 2012 and 22.1% in 2012 - 2013. G1P[8] was the most predominant genotype in the first two years accounting for more than half, and G9P[8] showed the highest detection rate as 35.0% in the last year. Interestingly, the proportional rate of G2 strains in the studied period increased from 0% to 25%. VP6 genotyping revealed that DS-1 like reassortant G1P[8] strains were detected all over Japan and their prevalence fluctuated greatly from 35.0% to 89.5%. Sequence analysis of VP6 showed that strains in the current strains were closely related but distinct from the original reference strains, namely Wa and DS-1. CONCLUSIONS: The detection rates of RVA, their GP combinations, prevalence of reassortant strains varied greatly after the introduction of rotavirus vaccines in Japan. Continuous monitoring is warranted to refine future vaccine strategy.

Multiplex PCR for the Detection of 10 Viruses Causing Encephalitis/Encephalopathy and its Application to Clinical Samples Collected from Japanese Children with Suspected Viral.

BACKGROUND: Acute encephalitis is a serious neurological condition having a high mortality rate and affecting both children and adults. This study aimed to develop a multiplex PCR method for the simultaneous screening of clinical samples for the presence of the 10 viruses presently considered as the major viral causes of acute encephalitis/ encephalopathy in Asia. METHODS: Using previously published primers that have been widely used to screen for herpes virus-6, influenza A virus, human parechovirus, herpes simplex viruses 1 and 2, Japanese encephalitis virus, group A rotavirus, enterovirus, adenovirus, and dengue virus in clinical samples, a single-tube multiplex PCR assay was developed and was tested for its sensitivity and specificity. The method was then applied to screen 57 clinical samples, consisting of 13 fecal samples, 5 throat swabs, 3 post-nasal swabs, 18 serum samples, and 18 cerebrospinal fluid (CSF) samples, collected from 18 hospitalized Japanese children with suspected viral encephalitis/encephalopathy for the target viruses, and the results were compared with those of a monoplex PCR method. RESULTS: Positive viral controls of the 10 viruses were correctly typed using this multiplex PCR method. The multiplex PCR method showed high specificity with no unspecific amplification to non-target viruses. The results of applying this PCR method for screening clinical samples showed that 6 fecal samples, 2 serum samples, and 1 CSF sample collected from 7 patients were positive for a virus, specifically group A rotavirus (4 patients, 22.2%), enterovirus (2 patients, 11.1%), or adenovirus (1 patient, 5.6%). In comparison with monoplex PCR, for group A rotavirus, enterovirus, and adenovirus, the sensitivity of this multiplex PCR method decreased for serum, cerebrospinal fluid, and throat swab samples. CONCLUSIONS: This newly developed multiplex PCR method is a simple, rapid diagnostic tool and can be used to screen clinical samples for viruses causing acute encephalitis/encephalopathy in children in Asian countries.

Epidemiology of gastroenteritis viruses in Japan: Prevalence, seasonality, and outbreak.

Acute gastroenteritis has been recognized as one of the most common diseases in humans and continues to be a major public health problem worldwide. Several groups of viruses have been reported as the causative agents of acute gastroenteritis, including rotavirus, norovirus, sapovirus, human astrovirus, adenovirus, and an increasing number of others which have been reported more recently. The epidemiology, prevalence, seasonality, and outbreaks of these viruses have been reviewed in a number of studies conducted in Japan over three decades. Rotavirus and norovirus were the two most common viruses detected almost equally in children under 5 years of age who were suffering from acute gastroenteritis. Like many other countries, the main rotavirus strains circulating in pediatric patients in Japan are G1P[8], G2P[4], G3P[8], and G9P[8]. Norovirus GII.4 was involved in most outbreaks in Japan and found to be associated with the emergence of new variants Sydney_2012. The classic human astrovirus, MLB, and VA clades astroviruses were also commonly found in pediatric patients with acute diarrhea. The sapovirus and adenovirus have been identified as the minor viral causative agents for acute gastroenteritis in Japan.

Multiple astrovirus MLB1, MLB2, VA2 clades, and classic human astrovirus in children with acute gastroenteritis in Japan.

This study investigated the genetic variability of astroviruses in children with acute gastroenteritis in Japan in 2012 to 2013. A total of 330 fecal specimens collected from children with diarrhea were tested for astroviruses using RT-PCR and nucleotide sequencing techniques. Among the tested specimens, 54 (16.4%) were positive for astrovirus. Three different phylogenetic clades were detected. MLB was the most predominant clade (35 samples), followed by classic astrovirus (17 samples), and VA astrovirus (2 samples). Two types of MLB were identified (MLB1 and MLB2), while classic astroviruses were HAstV1 and HAstV4. Two VA astroviruses were identified as VA2.

Cosavirus (family Picornaviridae) in pigs in Thailand and Japan.

Cosavirus is a recently established genus in the family Picornaviridae. The present study investigated the prevalence and genetic diversity of cosaviruses in stool samples collected from piglets and pigs with and without diarrhea in Thailand and Japan. It was observed that the cosavirus-positive rate in Thailand was higher than in Japan (55.4 % vs. 18.9 %). Phylogenetic analysis of a portion of the 5' untranslated region showed that porcine cosavirus strains clustered together in the same branch with members of the species Cosavirus A. These strains showed 97.0 to 100 % nucleotide sequence identity to each other. The virus concentration of cosavirus was very low compared with that detected in a infant with diarrhea. These results demonstrated that cosaviruses were circulating in the swine populations of both countries during the study term; however, it remains unclear whether the virus causes diarrhea in piglets and pigs.

Four-year study of viruses that cause diarrhea in Japanese pediatric outpatients.

Acute gastroenteritis continues to be a major public health problem worldwide. A wide variety of viruses associated with diarrhea disease is being reported continually. This study investigated the epidemiological situation of viruses that cause diarrhea in Japanese pediatric patients. This study enrolled a total of 2,381 fecal specimens collected between 2009 and 2013 from Japanese children with acute gastroenteritis. There is currently a 70.4% prevalence of viruses causing diarrhea among these Japanese pediatric outpatients. Norovirus was detected in 39.3% of the patients, whereas the prevalence of rotavirus, human parechovirus, enterovirus, adenovirus, sapovirus, astrovirus, and Aichi virus was 20.1, 6.6, 6.1, 5.6, 4.8, 2.3, and 0.1%, respectively. Co-infections were observed at the prevalence rates of 13.4 and 0.5% for double infections and triple infections, respectively. Mixed viral infections were found commonly in Japanese outpatients, and the norovirus seemed to play a major role in co-infections. Viral diarrhea cases were detected mostly in children younger than 3 years of age. The norovirus and rotavirus can be detected throughout the year, with a peak during the cold and dry seasons, whereas other common viruses are found during no specific season. Surveillance data revealed that a wide variety of viruses has caused diarrhea to circulate currently in Japanese pediatric outpatients, with very high detection rates; and norovirus and rotavirus are the most important pathogens. The data obtained from this study are valuable for compiling the overall picture of several viruses that causes diarrhea and associates with acute gastroenteritis in the Japanese pediatric population.

Rapid diagnostic tests apply for pediatric infections at outpatient clinic setting.

BACKGROUND: Early identification of the etiology of infection is beneficial. Most infections are treated as outpatients. However, facilities for rapid diagnosis are not available in clinic settings. METHODS: We applied Immunochromatography (IC) and Loop-mediated Isothermal Amplification (LAMP) methods to rapidly diagnose pathogens among 31 children with respiratory infection and 12 with gastroenteritis at a clinic in Saitama prefecture, Japan. Pathogens were then screened by multiplex conventional and real-time PCRs and bacterial culture. RESULTS: Respiratory pathogens were found in 64.5%. Despite the narrow spectrum, rapid tests identified pathogens in 28.6% of cases with a high agreement rate of 89.3% with PCR. Gastroenteritis pathogens were found in 66.7%. E. coli was positive in 3 cases and all were negative for verotoxin by LAMP. The agreement rate of IC and PCR assay was high, 100%. CONCLUSIONS: IC and LAMP are reliable and suitable methods in limited-resource settings for early pathogenic identification, which will help appropriate management, avoid unnecessary intervention, and cost saving.

Genetic analysis and homology modeling of capsid protein of norovirus GII.14.

In this study, a more detailed genetic characterization of the VP1 capsid protein of uncommon norovirus (NoV) GII.14 strains reported previously in Japan and China was performed using sequence analyses and homology modeling technique. The result of genetic comparison with the M7 prototype strain of GII.14 revealed that 10 amino acid mutations were observed at the same positions across the P2 and P1-2 subdomains in both Japanese and Chinese strains. By the homology modeling of the P domain, 7 out of these 10 mutations were predicted to be located on the surface-exposed P2 and P1-2 subdomains. All GII.14 strains had an altered RGD-like motif (RGT → KGT). While the Chinese strains contained 5 random amino acid changes in the S domain and the P2 subdomain, these changes were not detected in the Japanese strains. In addition, the histo-blood group antigen (HBGA)-binding interfaces remain identical to those of the previously determined GII.4 structure (VA387), suggesting the conservation of HBGA binding profile within the GII genogroup. Taken together, this report provides supportive structural data that antigenic drifts that occurred mostly in the P2 and P1-2 subdomains might be sufficient to generate new mutants, thus permitting the GII.14 virus to escape the host pre-existing immunity. These results also suggest the need for comparing the evolutionary profiles and structural models of rare NoV genotypes to an insight into NoV evolution.

Molecular characterization of norovirus variants and genetic diversity of noroviruses and sapoviruses in Thailand.

Norovirus (NoV) and Sapovirus (SaV) have been reported as a common cause of acute gastroenteritis worldwide. For a decade, surveillances of NoV and SaV have been conducted continually in Thailand. To monitor the epidemiological situation and to determine the genetic variation of NoV and SaV in Chiang Mai, Thailand, 567 samples collected from pediatric patients hospitalized with acute gastroenteritis were examined during 2007, and 2010-2011 by semi-nested RT-PCR and nucleotide sequencing methods. NoV was detected at 15.9%. Phylogenetic analysis revealed multiple NoV genotypes, GI/14 (1.1%), GII/1 (1.1%), GII/2 (1.1%), GII/3 (4.4%), GII/4 (65.6%), GII/6 (10.0%), GII/7 (2.2%), GII/12 (4.4%), GII/13 (3.3%), GII/16 (5.7%), and unclassified genotype (1.1%), circulating in this area. Among these, NoV GII/4 was the most prevalent genotype with a predominance of GII/4 2009 over other variants, 1996, 2006a, and 2006b. For SaV, the prevalence was 1.2% which was much lower than those of NoV and only SaV GI/1 was detected. This study highlights the epidemiology of NoV and SaV and genetic diversity of viruses circulating in pediatric patients hospitalized with acute gastroenteritis in Chiang Mai, Thailand.

Diverse genotypes of norovirus genogroup I and II contamination in environmental water in Thailand during the COVID-19 outbreak from 2020 to 2022.

Noroviruses (NoVs) are the most significant viral pathogens associated with waterborne and foodborne outbreaks of nonbacterial acute gastroenteritis in humans worldwide. This study aimed to investigate the prevalence and diversity of NoVs contaminated in the environmental water in Chiang Mai, Thailand. A total of 600 environmental water samples were collected from ten sampling sites in Chiang Mai from July 2020 to December 2022. The presence of NoV genogroups I (GI), GII, and GIV were examined using real-time RT-PCR assay. The genotype of the virus was determined by nucleotide sequencing and phylogenetic analysis. The results showed that NoV GI and GII were detected at 8.5% (51/600) and 11.7% (70/600) of the samples tested, respectively. However, NoV GIV was not detected in this study. NoV circulated throughout the year, with a higher detection rate during the winter season. Six NoV GI genotypes (GI.1-GI.6) and eight NoV GII genotypes (GII.2, GII.3, GII.7, GII.8, GII.10, GII.13, GII.17, and GII.21) were identified. Among 121 NoV strains detected, GII.17 was the most predominant genotype (24.8%, 30 strains), followed by GII.2 (21.5%, 26 strains), GI.3 (17.4%, 21 strains), and GI.4 (16.5%, 20 strains). Notably, NoV GII.3, GII.7, GII.8, and GII.10 were detected for the first time in water samples in this area. This study provides insight into the occurrence and seasonal pattern of NoV along with novel findings of NoV strains in environmental water in Thailand during the COVID-19 outbreak. Our findings emphasize the importance of further surveillance studies to monitor viral contamination in environmental water.

Emergence of norovirus GII/4 2006a and 2006b variants in hospitalized children with acute gastroenteritis in Thailand.

BACKGROUND: Norovirus (NoV) is recognized as a significant cause of acute gastroenteritis in infants and young children worldwide. This study investigated the prevalence of NoV infection in hospitalized children with gastroenteritis in Chiang Mai, Thailand in 2006. METHODS: A total of 156 fecal specimens were collected from children with diarrhea admitted to McCormick Hospital in 2006. All fecal specimens were examinedffor NoV by RT-PCR and the genotypes were identified by sequence analysis. RESULTS: A high prevalence of NoV infection was detected (20.5%, 32/156). NoV GII/4 was the most predominant genotype with a prevalence of 87.5% (28/32), while GII/3, GII/6, GII/12, and GII/15 were less common (3.1% each). Among GII/4 strains, 2006b variant (75%, 21/28) emerged as the leading strain and dominated over the Hunter'04-like variant, which was the most common strain in the previous season of 2005. In addition, the 2003, 2004, and 2006a variants were also detected. NoV infections were most commonly observed in the rainy season in Thailand. CONCLUSIONS: This study demonstrated the emergence of GII/4 2006b variants as the major pathogen causing acute gastroenteritis among infants and children at the age of less than 5 years old who admitted to hospital in Chiang Mai, Thailand in 2006. Additionally, other GII/4 variants of 2003, 2004, and 2006a were also reported.