Strong evolutionary constraints against amino acid changes in the P2 subdomain of sapovirus GI.1 capsid protein VP1.

Sapovirus (SaV) is a nonenveloped RNA virus that causes acute gastroenteritis in humans. Although SaV is a clinically important pathogen in children, an effective vaccine is currently unavailable. The capsid protein VP1 of SaVs forms the outer shell of the virion and is highly diverse, as often seen in the virion-surface proteins of RNA viruses, creating an obstacle for vaccine development. We here report a unique phenomenon pertaining to the variation of SaV VP1. Phylogenetic and information entropy analyses using full-length VP1 sequences from a public database consistently showed that the amino acid sequences of the VP1 protein have been highly conserved over more than 40 years in the major epidemic genotype GI.1 but not in GI.2. Structural modeling showed that even the VP1 P2 subdomain, which is arranged on the outermost shell of the virion and presumably exposed to anti-SaV antibodies, remained highly homogeneous in GI.1 but not in GI.2. These results suggest strong evolutionary constraints against amino acid changes in the P2 subdomain of the SaV GI.1 capsid and illustrate a hitherto unappreciated mechanism, i.e., preservation of the VP1 P2 subdomain, involved in SaV survival. Our findings could have important implications for the development of an anti-SaV vaccine.

Detection and isolation of genotype 3 subtype b hepatitis E viruses from wild boars in Japan.

To conduct an epidemiological study of hepatitis E virus (HEV) in Japanese wild boars, we collected 179 serum and 162 fecal specimens from wild boars in eight Japanese prefectures; 39 of the serum samples (21.8%) were positive for anti-HEV IgG antibodies. RT-qPCR revealed HEV RNA in 11 serum samples (6.1%) and 5 fecal samples (3.1%). We obtained 412 bp of the viral genome sequences of ORF2 from five pairs of serum and fecal samples. All strains were subtype b in genotype 3 (HEV-3b) but separated into different clusters. We determined the entire genome sequence of HEV-3b strain WB0567 using a fecal specimen and isolated this strain by cell culture using PLC/PRF/5 cells. Eleven nucleotide mutations had occurred during virus replication. These results suggest that HEV-3b circulated uniformly among wild boars in Japan. Direct sequencing using a suspected animal's samples is indispensable for predicting original HEV nucleotide sequences.

Comprehensive full genome analysis of norovirus strains from eastern India, 2017-2021.

BACKGROUND: Worldwide, noroviruses are the leading cause of acute gastroenteritis (AGE) in people of all age groups. In India, norovirus rates between 1.4 to 44.4% have been reported. Only a very few complete norovirus genome sequences from India have been reported. OBJECTIVE: To perform full genome sequencing of noroviruses circulating in India during 2017-2021, identify circulating genotypes, assess evolution including detection of recombination events. METHODOLOGY: Forty-five archived norovirus-positive samples collected between October 2017 to July 2021 from patients with AGE from two hospitals in Kolkata, India were processed for full genome sequencing. Phylogenetic analysis, recombination breakpoint analysis and comprehensive mutation analysis were also performed. RESULTS: Full genome analysis of norovirus sequences revealed that strains belonging to genogroup (G)I were genotyped as GI.3[P13]. Among the different norovirus capsid-polymerase combinations, GII.3[P16], GII.4 Sydney[P16], GII.4 Sydney[P31], GII.13[P16], GII.16[P16] and GII.17 were identified. Phylogenetic analysis confirmed phylogenetic relatedness with previously reported norovirus strains and all viruses were analyzed by Simplot. GII[P16] viruses with multiple residue mutations within the non-structural region were detected among circulating GII.4 and GII.3 strains. Comprehensive mutation analysis and selection pressure analysis of GII[P16] viruses showed positive as well as negative selection sites. A GII.17 strain (NICED-BCH-11889) had an untypeable polymerase type, closely related to GII[P38]. CONCLUSION: This study highlights the circulation of diverse norovirus strains in eastern India. These findings are important for understanding norovirus epidemiology in India and may have implications for future vaccine development.

Emergence of Intergenogroup Reassortant G9P[4] Strains Following Rotavirus Vaccine Introduction in Ghana.

Rotavirus (RVA) is a leading cause of childhood gastroenteritis. RVA vaccines have reduced the global disease burden; however, the emergence of intergenogroup reassortant strains is a growing concern. During surveillance in Ghana, we observed the emergence of G9P[4] RVA strains in the fourth year after RVA vaccine introduction. To investigate whether Ghanaian G9P[4] strains also exhibited the DS-1-like backbone, as seen in reassortant G1/G3/G8/G9 strains found in other countries in recent years, this study determined the whole genome sequences of fifteen G9P[4] and two G2P[4] RVA strains detected during 2015-2016. The results reveal that the Ghanaian G9P[4] strains exhibited a double-reassortant genotype, with G9-VP7 and E6-NSP4 genes on a DS-1-like backbone (G9-P[4]-I2-R2-C2-M2-A2-N2-T2-E6-H2). Although they shared a common ancestor with G9P[4] DS-1-like strains from other countries, further intra-reassortment events were observed among the original G9P[4] and co-circulating strains in Ghana. In the post-vaccine era, there were significant changes in the distribution of RVA genotype constellations, with unique strains emerging, indicating an impact beyond natural cyclical fluctuations. However, reassortant strains may exhibit instability and have a limited duration of appearance. Current vaccines have shown efficacy against DS-1-like strains; however, ongoing surveillance in fully vaccinated children is crucial for addressing concerns about long-term effectiveness.

Prevalence and Characterization of Gastroenteritis Viruses among Hospitalized Children during a Pilot Rotavirus Vaccine Introduction in Vietnam.

Rotavirus (RV), norovirus (NoV), sapovirus (SaV), and human astrovirus (HAstV) are the most common viral causes of gastroenteritis in children worldwide. From 2016 to 2021, we conducted a cross-sectional descriptive study to determine the prevalence of these viruses in hospitalized children under five years old in Nam Dinh and Thua Thien Hue provinces in Vietnam during the pilot introduction of the RV vaccine, Rotavin-M1 (POLYVAC, Hanoi, Vietnam). We randomly selected 2317/6718 (34%) acute diarrheal samples from children <5 years of age enrolled at seven sentinel hospitals from December 2016 to May 2021; this period included one year surveillance pre-vaccination from December 2016 to November 2017. An ELISA kit (Premier Rotaclone®, Meridian Bioscience, Inc., Cincinnati, OH, USA) was used to detect RV, and two multiplex real-time RT-PCR assays were used for the detection of NoV, SaV and HAstV. The prevalence of RV (single infection) was reduced from 41.6% to 22.7% (p < 0.0001) between pre- and post-vaccination periods, while the single NoV infection prevalence more than doubled from 8.8% to 21.8% (p < 0.0001). The SaV and HAstV prevalences slightly increased from 1.9% to 3.4% (p = 0.03) and 2.1% to 3.3% (p = 0.09), respectively, during the same period. Viral co-infections decreased from 7.2% to 6.0% (p = 0.24), mainly due to a reduction in RV infection. Among the genotypeable samples, NoV GII.4, SaV GI.1, and HAstV-1 were the dominant types, representing 57.3%, 32.1%, and 55.0% among the individual viral groups, respectively. As the prevalence of RV decreases following the national RV vaccine introduction in Vietnam, other viral pathogens account for a larger proportion of the remaining diarrhea burden and require continuing close monitoring.

A household survey of intrafamily norovirus transmission.

Norovirus (NoV) is a leading cause of epidemic and sporadic gastroenteritis in people of all ages. Humans are the primary source of NoV and household contact is one of the risk factors for NoV transmission. However, the mechanisms underlying person-to-person NoV transmission are poorly understood. Here we conducted a survey to profile the frequency and characteristics of intrafamily NoV transmission. Stool samples were collected every week from three households between 2016 and 2020; the total number of samples was 1105. The detection of NoV and the genotyping were performed by reverse transcription-polymerase chain reaction targeting the capsid region and direct sequencing methods. NoV was detected in 3.4% of all samples. Eight NoV genotypes were identified. The most common genotype was GII.17, followed in order by GII.6, GI.6, GII.4, GI.3, and GI.2/GI.8/GI.9. Most NoV-positive samples were obtained from asymptomatic individuals. The highest number of NoV transmissions was found in household 3 (6 infections), followed by household 2 (2 infections), while household 1 had no NoV transmission, suggesting that asymptomatic NoV carriers play a major role in infection as NoV reservoirs in the households. Further clarification of the mode of infection will contribute to improved understanding and an appropriate prevention.

Low Replication Efficiency of a Japanese Rabbit Hepatitis E Virus Strain in the Human Hepatocarcinoma Cell Line PLC/PRF/5.

A Japanese rabbit hepatitis E virus (HEV) strain, JP-59, has been identified in a feral rabbit. When this virus was transmitted to a Japanese white rabbit, it caused persistent HEV infection. The JP-59 strain shares an <87.5% nucleotide sequence identity with other rabbit HEV strains. Herein, to isolate JP-59 by cell culture, we used a 10% stool suspension recovered from a JP-59-infected Japanese white rabbit and contained 1.1 × 107 copies/mL of the viral RNA and using it to infect a human hepatocarcinoma cell line, PLC/PRF/5. No sign of virus replication was observed. Although long-term virus replication was observed in PLC/PRF/5 cells inoculated with the concentrated and purified JP-59 containing a high titer of viral RNA (5.1 × 108 copies/mL), the viral RNA of JP-59c that was recovered from the cell culture supernatants was <7.1 × 104 copies/mL during the experiment. The JP-59c strain did not infect PLC/PRF/5 cells, but its intravenous inoculation caused persistent infection in rabbits. The nucleotide sequence analyses of the virus genomes demonstrated that a total of 18 nucleotide changes accompanying three amino acid mutations occurred in the strain JP-59c compared to the original strain JP-59. These results indicate that a high viral RNA titer was required for JP-59 to infect PLC/PRF/5 cells, but its replication capability was extremely low. In addition, the ability of rabbit HEVs to multiply in PLC/PRF/5 cells varied depending on the rabbit HEV strains. The investigations of cell lines that are broadly susceptible to rabbit HEV and that allow the efficient propagation of the virus are thus needed.

Distribution of Human Sapovirus Strain Genotypes over the Last Four Decades in Japan: a Global Perspective.

Sapovirus (SaV) infections are a public health problem because they cause acute gastroenteritis in humans of all ages, both sporadically and as outbreaks. However, only a limited amount of SaV sequence information, especially whole-genome sequences for all the SaV genotypes, is publicly available. Therefore, in this study, we determined the full/near-full-length genomic sequences of 138 SaVs from the 2001 to 2015 seasons in 13 prefectures across Japan. The genogroup GI was predominant (67%, n = 92), followed by genogroups GII (18%, n = 25), GIV (9%, n = 12), and GV (6%, n = 9). Within the GI genogroup, four different genotypes were identified: GI.1 (n = 44), GI.2 (n = 40), GI.3 (n = 7), and GI.5 (n = 1). We then compared these Japanese SaV sequences with 3,119 publicly available human SaV sequences collected from 49 countries over the last 46 years. The results indicated that GI.1, and GI.2 have been the predominant genotypes in Japan, as well as in other countries, over at least four decades. The 138 newly determined Japanese SaV sequences together with the currently available SaV sequences, could facilitate a better understanding of the evolutionary patterns of SaV genotypes.

Open Reading Frame 4 Is Not Essential in the Replication and Infection of Genotype 1 Hepatitis E Virus.

Genotype 1 hepatitis E virus (HEV-1), unlike other genotypes of HEV, has a unique small open reading frame known as ORF4 whose function is not yet known. ORF4 is located in an out-framed manner in the middle of ORF1, which encodes putative 90 to 158 amino acids depending on the strains. To explore the role of ORF4 in HEV-1 replication and infection, we cloned the complete genome of wild-type HEV-1 downstream of a T7 RNA polymerase promoter, and the following ORF4 mutant constructs were prepared: the first construct had TTG instead of the initiation codon ATG (A2836T), introducing an M→L mutation in ORF4 and a D→V mutation in ORF1. The second construct had ACG instead of the ATG codon (T2837C), introducing an M→T mutation in ORF4. The third construct had ACG instead of the second in-frame ATG codon (T2885C), introducing an M→T mutation in ORF4. The fourth construct contained two mutations (T2837C and T2885C) accompanying two M→T mutations in ORF4. For the latter three constructs, the accompanied mutations introduced in ORF1 were all synonymous changes. The capped entire genomic RNAs were generated by in vitro transcription and used to transfect PLC/PRF/5 cells. Three mRNAs containing synonymous mutations in ORF1, i.e., T2837CRNA, T2885CRNA, and T2837C/T2885CRNA, replicated normally in PLC/PRF/5 cells and generated infectious viruses that successfully infected Mongolian gerbils as the wild-type HEV-1 did. In contrast, the mutant RNA, i.e., A2836TRNA, accompanying an amino acid change (D937V) in ORF1 generated infectious viruses upon transfection, but they replicated slower than the wild-type HEV-1 and failed to infect Mongolian gerbils. No putative viral protein(s) derived from ORF4 were detected in the wild-type HEV-1- as well as the mutant virus-infected PLC/PRF/5 cells by Western blot analysis using a high-titer anti-HEV-1 IgG antibody. These results demonstrated that the ORF4-defective HEV-1s had the ability to replicate in the cultured cells, and that these defective viruses had the ability to infect Mongolian gerbils unless the overlapping ORF1 was accompanied by non-synonymous mutation(s), confirming that ORF4 is not essential in the replication and infection of HEV-1.

Complete genome analyses of G12P[8] rotavirus strains from hospitalized children in Surabaya, Indonesia, 2017-2018.

Rotavirus A (RVA) is a major viral cause of acute gastroenteritis (AGE) worldwide. G12 RVA strains have emerged globally since 2007. There has been no report of the whole genome sequences of G12 RVAs in Indonesia. We performed the complete genome analysis by the next-generation sequencing of five G12 strains from hospitalized children with AGE in Surabaya from 2017 to 2018. All five G12 strains were Wa-like strains (G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1) and were clustered into lineage-III of VP7 gene phylogenetic tree. STM430 sample was observed as a mixed-infection between G12 and G1 strains: G12/G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. A phylogenetic tree analysis revealed that all five Indonesian G12 strains (SOEP379, STM371, STM413, STM430, and STM433) were genetically close to each other in all 11 genome segments with 98.0%-100% nucleotide identities, except VP3 and NSP4 of STM430, suggesting that these strains have originated from a similar ancestral G12 RVA. The VP3 and NSP4 genome segments of STM430-G12P[8] were separated phylogenetically from those of the other four G12 strains, probably due to intra-genotype reassortment between the G12 and G1 Wa-like strains. The change from G12P[6] lineage-II in 2007 to G12P[8] lineage-III 2017-2018 suggests the evolution and diversity of G12 RVAs in Indonesia over the past approximately 10 years.

Characterization of a Human Sapovirus Genotype GII.3 Strain Generated by a Reverse Genetics System: VP2 Is a Minor Structural Protein of the Virion.

We devised a reverse genetics system to generate an infectious human sapovirus (HuSaV) GII.3 virus. Capped/uncapped full-length RNAs derived from HuSaV GII.3 AK11 strain generated by in vitro transcription were used to transfect HuTu80 human duodenum carcinoma cells; infectious viruses were recovered from the capped RNA-transfected cells and passaged in the cells. Genome-wide analyses indicated no nucleotide sequence change in the virus genomes in the cell-culture supernatants recovered from the transfection or those from the subsequent infection. No virus growth was detected in the uncapped RNA-transfected cells, suggesting that the 5'-cap structure is essential for the virus' generation and replication. Two types of virus particles were purified from the cell-culture supernatant. The complete particles were 39.2-nm-dia., at 1.350 g/cm3 density; the empty particles were 42.2-nm-dia. at 1.286 g/cm3. Two proteins (58-kDa p58 and 17-kDa p17) were detected from the purified particles; their molecular weight were similar to those of VP1 (~60-kDa) and VP2 (~16-kDa) of AK11 strain deduced from their amino acids (aa) sequences. Protein p58 interacted with HuSaV GII.3-VP1-specific antiserum, suggesting that p58 is HuSaV VP1. A total of 94 (57%) aa of p17 were identified by mass spectrometry; the sequences were identical to those of VP2, indicating that the p17 is the VP2 of AK11. Our new method produced infectious HuSaVs and demonstrated that VP2 is the minor protein of the virion, suggested to be involved in the HuSaV assembly.

Mongolia Gerbils Are Broadly Susceptible to Hepatitis E Virus.

Although cell culture systems for hepatitis E virus (HEV) have been established by using cell lines such as PLC/PRF/5 and A549, small-animal models for this virus are limited. Since Mongolia gerbils are susceptible to genotype 1, 3 and 4 HEV (HEV-1, HEV-3 and HEV4), we intraperitoneally inoculated Mongolia gerbils with HEV-5, HEV-7, HEV-8, rabbit HEV or rat HEV in addition to the above three genotypes to investigate the infectivity and to assess whether Mongolia gerbil is an appropriate animal model for HEV infection. The results indicated that (i) HEV-5 and rat HEV were effectively replicated in the Mongolia gerbils in the same manner as HEV-4: large amounts of the viral RNA were detected in the feces and livers, and high titers of the serum anti-HEV IgG antibodies were induced in all animals. The feces were shown to contain HEV that is infectious to naïve gerbils. Furthermore, HEV-4, HEV-5 and rat HEV were successfully transmitted to the gerbils by oral inoculation. (ii) Although the viral RNA and serum anti-HEV IgG antibodies were detected in all animals inoculated with HEV-1 and HEV-8, both titers were low. The viral RNA was detected in the feces collected from two of three HEV-3-inoculated, and one of three HEV-7-inoculated gerbils, but the titers were low. The serum antibody titers were also low. The viruses excreted into the feces of HEV-1-, HEV-3-, HEV-7- and HEV-8-inoculated gerbils failed to infect naïve Mongolia gerbils. (iii) No infection sign was observed in the rabbit HEV-inoculated gerbils. These results demonstrated that Mongolia gerbils are broadly susceptible to HEV, and their degree of sensitivity was dependent on the genotype. Mongolia gerbils were observed to be susceptible to not only HEVs belonging to HEV-A but also to rat HEV belonging to HEV-C1, and thus Mongolia gerbil could be useful as a small-animal model for cross-protection experiments between HEV-A and HEV-C1. Mongolia gerbils may also be useful for the evaluation of the efficacy of vaccines against HEV.

Experimental Cross-Species Transmission of Rat Hepatitis E Virus to Rhesus and Cynomolgus Monkeys.

Rat hepatitis E virus (rat HEV) was first identified in wild rats and was classified as the species Orthohepevirus C in the genera Orthohepevirus, which is genetically different from the genotypes HEV-1 to HEV-8, which are classified as the species Orthohepevirus A. Although recent reports suggest that rat HEV transmits to humans and causes hepatitis, the infectivity of rat HEV to non-human primates such as cynomolgus and rhesus monkeys remains controversial. To investigate whether rat HEV infects non-human primates, we inoculated one cynomolgus monkey and five rhesus monkeys with a V-105 strain of rat HEV via an intravenous injection. Although no significant elevation of alanine aminotransferase (ALT) was observed, rat HEV RNA was detected in fecal specimens, and seroconversion was observed in all six monkeys. The partial nucleotide sequences of the rat HEV recovered from the rat HEV-infected monkeys were identical to those of the V-105 strain, indicating that the infection was caused by the rat HEV. The rat HEV recovered from the cynomolgus and rhesus monkeys successfully infected both nude and Sprague-Dawley rats. The entire rat HEV genome recovered from nude rats was identical to that of the V-105 strain, suggesting that the rat HEV replicates in monkeys and infectious viruses were released into the fecal specimens. These results demonstrated that cynomolgus and rhesus monkeys are susceptible to rat HEV, and they indicate the possibility of a zoonotic infection of rat HEV. Cynomolgus and rhesus monkeys might be useful as animal models for vaccine development.

Development of New SNP Genotyping Assays to Discriminate the Omicron Variant of SARS-CoV-2.

The World Health Organization designated Omicron (B.1.1.529 lineage) of SARS-CoV-2 as a new variant of concern on November 26, 2021. The risk to public health conferred by the Omicron variant is still not completely clear, although its numerous gene mutations have raised concerns regarding its potential for increased transmissibility and immune escape. In this study, we describe the development of two single-nucleotide polymorphism genotyping assays targeting the G339D or T547K mutations of the spike protein to screen for the Omicron variant. A specificity test revealed that the two assays successfully discriminated the Omicron variant from the Delta and Alpha variants, each with a single nucleotide mismatch. In addition, a sensitivity test showed that the G339D and T547K assays detected at least 2.60 and 3.36 RNA copies of the Omicron variant, respectively, and 1.59 RNA copies of the Delta variant. These results demonstrate that both assays could be useful for detecting and discriminating the Omicron variant from other strains. In addition, because of the rapid and unpredictable evolution of SARS-CoV-2, combining our assays with previously developed assays for detecting other mutations may lead to a more accurate diagnostic system.

Generation of a Bactrian camel hepatitis E virus by a reverse genetics system.

Bactrian camel hepatitis E virus (HEV) is a novel HEV belonging to genotype 8 (HEV-8) in the Orthohepevirus A species of the genus Hepevirus in the family Hepeviridae. HEV-8 cross-transmits to cynomolgus monkeys and has a potential risk for zoonotic infection. Until now, neither a cell-culture system to grow the virus nor a reverse genetics system to generate the virus has been developed. To generate replication-competent HEV-8 and to establish a cell-culture system, we synthesized capped genomic HEV-8 RNAs by in vitro transcription and used them to transfect into PLC/PRF/5 cells. A HEV-8 strain, HEV-8M2, was recovered from the capped HEV-8 RNA-transfected cell-culture supernatants and subsequently passaged in the cells, demonstrating that PLC/PRF/5 cells were capable of supporting the replication of the HEV-8, and that a cell-culture system for HEV-8 was successfully established. In addition to PLC/PRF/5 cells, A549 and Caco-2 cells appeared to be competent for the replication, but HepG2 C3/A, Vero, Hela S3, HEp-2C, 293T and GL37 cells were incompetent. The HEV-8M2 strain was capable of infecting cynomolgus monkeys by an intravenous inoculation, indicating that HEV-8 was infectious and again carried a risk for zoonotic infection. In contrast, HEV-8 did not infect nude rats and BALB/c nude mice, suggesting that the reservoir of HEV-8 was limited. In addition, the replication of the HEV-8M2 strain was efficiently abrogated by ribavirin but not by favipiravir, suggesting that ribavirin is a drug candidate for therapeutic treatment of HEV-8-induced hepatitis. The infectious HEV-8 produced by a reverse genetics system would be useful to elucidate the mechanisms of HEV replication and the pathogenesis of type E hepatitis.

Isolation and characterization of mammalian orthoreovirus type 3 from a fecal sample from a wild boar in Japan.

Mammalian orthoreoviruses (MRVs) have been identified in various mammalian species, including humans, bats, and pigs. However, isolation and complete genome sequences of MRVs from wild boars have not yet been reported. In this study, we isolated, sequenced, and analyzed an MRV from a free-living wild boar in Japan using the porcine-sapelovirus-resistant cell line N1380. Complete and empty virus particles were obtained from the N1380 cell culture supernatants, and complete genome sequences were obtained from complete virus particles. Sequence analysis revealed that the isolated MRV, named TY-14, could be classified as MRV3 and had a close genetic relationship to an MRV2 isolate from a lion in a Japanese zoo (L2, L3, and M3 genes) and a human MRV2 isolate from Japan (S2 gene). Phylogenetic analysis showed that TY-14 clustered only with bat MRVs in the M1 phylogenetic tree but formed a cluster with several animal MRVs in the M2 and S3 phylogenetic trees and branched independently in the L1, S1, and S4 phylogenetic trees, suggesting a genetic relationship to viruses of unknown origin. Recombination events were identified in the M2 gene. These results suggest that TY-14 was generated by reassortment and recombination events involving MRVs circulating in Japan, viruses from bats, and other viruses of unknown origin.

Molecular epidemiology and genetic diversity of norovirus infection in children hospitalized with acute gastroenteritis in East Java, Indonesia in 2015-2019.

Noroviruses are recognized as a leading cause of outbreaks and sporadic cases of acute gastroenteritis (AGE) among individuals of all ages worldwide, especially in children <5 years old. We investigated the epidemiology of noroviruses among hospitalized children at two hospitals in East Java, Indonesia. Stool samples were collected from 966 children with AGE during September 2015-July 2019. All samples were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for the amplification of both the RNA-dependent RNA polymerase (RdRp) and the capsid genes of noroviruses. The genotypes were determined by phylogenetic analyses. In 2015-2019, noroviruses were detected in 12.3% (119/966) of the samples. Children <2 years old showed a significantly higher prevalence than those ≥2 years old (P = 0.01). NoV infections were observed throughout the year, with the highest prevalence in December. Based on our genetic analyses of RdRp, GII.[P31] (43.7%, 31/71) was the most prevalent RdRp genotype, followed by GII.[P16] (36.6%, 26/71). GII.[P31] was a dominant genotype in 2016 and 2018, whereas GII.[P16] was a dominant genotype in 2015 and 2017. Among the capsid genotypes, the most predominant norovirus genotype from 2015 to 2018 was GII.4 Sydney_2012 (33.6%, 40/119). The most prevalent genotype in each year was GII.13 in 2015, GII.4 Sydney_2012 in 2016 and 2018, and GII.3 in 2017. Based on the genetic analyses of RdRp and capsid sequences, the strains were clustered into 13 RdRp/capsid genotypes; 12 of them were discordant, e.g., GII.4 Sydney[P31], GII.3[P16], and GII.13[P16]. The predominant genotype in each year was GII.13[P16] in 2015, GII.4 Sydney[P31] in 2016, GII.3[P16] in 2017, and GII.4 Sydney[P31] in 2018. Our results demonstrate high detection rates and genetic diversity of norovirus GII genotypes in pediatric AGE samples from Indonesia. These findings strengthen the importance of the continuous molecular surveillance of emerging norovirus strains.

Sapoviruses detected from acute gastroenteritis outbreaks and hospitalized children in Taiwan.

BACKGROUND/PURPOSE: Sapoviruses (SaVs) become important pathogens causing both sporadic and outbreaks of acute gastroenteritis (AGE) after rotavirus vaccination era worldwide. SaVs were included in AGE screening items when norovirus and rotavirus were negative in Taiwan CDC since 2008. However, no complete SaV genome sequence of any genotype detected in Taiwan was determined. This study aimed to investigate SaVs infection and complete genome sequences detected in Taiwan. METHODS: This prospective survey, SaVs samples with untyped or weak PCR result were selected for testing the new design qRT-PCR assay from AGE hospitalized children during 2008-2011, 2016-2017 and AGE outbreak in 2012-2014. Those were genetically characterized using long RT-PCR with different primer combinations as well as primer independent deep sequencing and with 5' RACE and 3' terminal region targeting RT-PCR. RESULTS: Overall, 14 SaV-AGE hospitalized children and 4 SaV-AGE outbreaks were enrolled in this study. In addition to the AGE symptoms, 6 children also showed URI symptoms (cough, pharyngitis, rhinorrhea and nasal congestion). The detected 19 SaVs were classified as eight genotypes (GI.1, GI.2, GI.3, GII.2, GII.3, GII.5, GII.8, and GIV.1) and the complete genome sequence of representative strain for each genotype were determined except GI.3. The GII.3 was the most major genotype following GI.1 and GIV.1. CONCLUSION: Our result confirmed that SaV is one of the pathogens detected from Taiwanese AGE patients. Multiple SaV genotype strains would associate with AGE as similar to those detected in different countries/areas. The whole genome of SaV strains detected including rarely reported GII.8 was firstly determined.

Persistent infection with a rabbit hepatitis E virus created by a reverse genetics system.

Rabbit hepatitis E virus (HEV) is a novel zoonotic infectious agent. Although a cell culture system to grow the virus has been established, there is currently no reverse genetics system for generating the virus. In this study, capped genomic rabbit HEV RNAs generated by in vitro transcription were transfected into PLC/PRF/5 cells, and the recovered viruses were subsequently passaged in the cells. The cell culture supernatant was capable of infecting rabbits negative for anti-HEV antibody by intravenous and oral inoculation, indicating that rabbit HEV generated by the reverse genetics system is infectious. Genome-wide analyses indicated that no nucleotide sequence change occurred in the virus genomes that were recovered from the cell culture supernatant after transfection and passaged one time or in the virus genomes recovered from faecal specimens of the infected rabbits. Ribavirin, a broad-spectrum anti-viral inhibitor, efficiently abrogated virus replication ex vivo and transiently suppressed the virus growth in the virus-infected rabbits, suggesting that this reagent is a candidate for therapeutic treatment. In addition, transmission of rabbit HEV to rabbits caused persistent infection, suggesting that the virus-infected rabbit could be an animal model for virus-induced hepatitis. The infectious rabbit HEV produced by a reverse genetics system would be useful to elucidate the mechanisms of HEV replication and the pathogenesis of viral hepatitis.

Hepatitis E virus infection in 6-month-old pigs in Taiwan.

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E. Genotype 3 (G3) and 4 (G4) HEV have recently been identified in and isolated from swine as the main HEV genotypes worldwide. However, there is limited information on HEV infection status among pigs in Taiwan, especially pigs in the stage before transportation to the slaughterhouse. To determine the frequency of HEV infection among pigs in Taiwan, we detected and quantified HEV RNA contained in 295 fecal specimens collected from 6-month-old pigs bred in 30 pig farms located in 8 counties. We found that 25.1% (74/295) of the fecal specimens were positive for HEV RNA by a quantitative real-time reverse transcription-polymerase chain reaction, and the copy number ranged from 2.3 × 103 to 2.08 × 107 copies/g. Amplification of a 338 bp sequence in ORF2 was achieved in 16 of 74 HEV RNA-positive samples, and their nucleotide sequences were determined. Two HEV sequences appeared to belong to subtype 3a of G3 and the remaining 14 HEV sequences belonged to subtype 4b of G4 (G4b). The entire genome sequence of two G4b HEVs was obtained by next-generation sequence analyses, and the phylogenetic analyses indicated that unique G4b HEVs were circulating in pig farms in Taiwan. In the present study, we found that both G3 and G4 HEVs were circulating in Taiwanese pig farms and G4b was the predominant subtype. In addition, the relatively high detection frequency of HEV RNA in the 6-month-old pigs indicated that Taiwanese pigs just before transportation to the slaughterhouse are at risk of carrying HEVs, and thus thorough cooking or heating of pork meat or organs is needed before consumption in Taiwan and possibly in other countries as well.