谷口 孝喜

The live attenuated human rotavirus vaccine strain RIX4414 (Rotarix®) is used worldwide to prevent severe rotavirus-induced diarrhea in infants. This strain was attenuated through the cell culture passaging of its predecessor, human strain 89-12, which resulted in multiple genomic mutations. However, the specific molecular reasons underlying its attenuation have remained elusive, primarily due to the absence of a suitable reverse genetics system enabling precise genetic manipulations. Therefore, we first completed the sequencing of its genome and then developed a reverse genetics system for the authentic RIX4414 virus. Our experimental results demonstrate that the rescued recombinant RIX4414 virus exhibits biological characteristics similar to those of the parental RIX4414 virus, both in vitro and in vivo. This novel reverse genetics system provides a powerful tool for investigating the molecular basis of RIX4414 attenuation and may facilitate the rational design of safer and more effective human rotavirus vaccines.

Vaccine development for herpes simplex virus 2 (HSV-2) has been attempted, but no vaccines are yet available. A plasmid-based reverse genetics system for Rotavirus (RV), which can cause gastroenteritis, allows the generation of recombinant RV containing foreign genes. In this study, we sought to develop simian RV (SA11) as a vector to express HSV-2 glycoprotein D (gD2) and evaluated its immunogenicity in mice. We generated the recombinant SA11-gD2 virus (rSA11-gD2) and confirmed its ability to express gD2 in vitro. The virus was orally inoculated into suckling BALB/c mice and into 8-week-old mice. Serum IgG and IgA titers against RV and gD2 were measured by ELISA. In the 8-week-old mice inoculated with rSA11-gD2, significant increases in not only antibodies against RV but also IgG against gD2 were demonstrated. In the suckling mice, antibodies against RV were induced, but gD2 antibody was not detected. Diarrhea observed after the first inoculation of rSA11-gD2 in suckling mice was similar to that induced by the parent virus. A gD2 expressing simian RV recombinant, which was orally inoculated, induced IgG against gD2. This strategy holds possibility for genital herpes vaccine development.

Human rotavirus strains having the unconventional G3P[6] genotype have been sporadically detected in diarrheic patients in different parts of the world. However, the full genomes of only three human G3P[6] strains from Asian countries (China, Indonesia, and Vietnam) have been sequenced and characterized, and thus the exact origin and evolution of G3P[6] strains in Asia remain to be elucidated. Here, we sequenced and characterized the full genome of a G3P[6] strain (RVA/Human-wt/JPN/SO1199/2020/G3P[6]) found in a stool sample from a 3-month-old infant admitted with acute gastroenteritis in Japan. On full genomic analysis, strain SO1199 was revealed to have a unique Wa-like genogroup configuration: G3-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1. VP6 genotype I5 and NSP1 genotype A8 are commonly found in porcine rotavirus strains. Furthermore, phylogenetic analysis demonstrated that all 11 genes of strain SO1199 were closely related to those of porcine and/or porcine-like human rotaviruses and thus appeared to be of porcine origin. Thus, strain SO1199 was shown to possess a porcine-like genomic backbone and thus is likely to be the result of interspecies transmission of a porcine rotavirus strain. Of note is that all 11 genes of strain SO1199 were phylogenetically located in clusters, distinct from those of the previously identified porcine-like human G3P[6] strains from around the world including Asia, suggesting the occurrence of independent porcine-to-human zoonotic transmission events. To our knowledge, this is the first report on full genome-based characterization of a human G3P[6] strain that has emerged in Japan. Our findings revealed the diversity of unconventional human G3P[6] strains in Asia, and provide important insights into the origin and evolution of G3P[6] strains.

OBJECTIVES: Intestinal rotavirus (RV) vaccine replication and host immune response are suggested to be affected by several factors, including maternal antibodies, breastfeeding history, and gut microbiome, which are thought to be similar in pairs of twins. The aim of this study was to determine whether viral shedding from the fecal RV vaccine strain Rotarix® (RV1) and IgG and IgA responses to RV show similarity in pairs of twins. METHODS: Quantitative reverse transcription polymerase chain reaction specific to RV vaccine strain RV1 was used to monitor fecal RV1 viral shedding. RV IgG and IgA titers were measured using an in-house enzyme-linked immunosorbent assay. Fecal RV1 viral shedding and immune responses were compared between twins and singletons with mixed effects and fixed effects models. RESULTS: A total of 347 stool and 54 blood samples were collected from four pairs of twins and twelve singletons during the observation period. Although the kinetics of fecal RV1 viral shedding and immune responses differed among vaccinated individuals, they appeared to be similar within twin pairs. RV shedding after the first dose (P=0.049) and RV IgG titers during the entire observation period (P=0.015) had a significantly better fit in the fixed effect model that assumed that twins have the same response versus the model that assumed that twins have a different response. CONCLUSIONS: The similarity of RV vaccine viral replication in intestine and host immune responses in twin pairs was demonstrated using statistical analysis.

ロタウイルス(RV)は乳幼児嘔吐下痢症の病因ウイルスであり,小児において最も重要な下痢症ウイルスの一つとして知られている.RVは11本の分節2本鎖RNAをゲノムとして有する.11本のうち10本の遺伝子はモノシストロニックであるが,例外的にNSP5遺伝子はフレームの異なるORFにより2種類のタンパク(NSP5とNSP6)をコードする.培養細胞を用いて継代したRV株には,NSP6を部分的に欠損した株がごく少数報告されていることから,in vitroにおいてNSP6はRV増殖に必須でないことが示唆されていた.その一方で,自然界(便中)におけるNSP6欠損株の報告例はないため,in vivoにおけるNSP6の重要性もまた示唆されてきた.そこで今回,このNSP6の重要性を検討するために,リバースジェネティクス系を用いて,NSP6欠損ロタウイルスを作製し,培養細胞および乳のみマウスでの増殖能および病原性を解析した.本研究から得られたNSP6の意義について,培養細胞を用いたデータと乳のみマウスのデータをあわせて報告する.(著者抄録)

The emergence of unusual G9P[8]-E2 human rotaviruses in the Tokyo metropolitan area, Japan, in 2018 has been reported. During rotavirus strain surveillance in different regions of Japan (Mie, Okayama, and Chiba prefectures), G9P[8]-E2 strains were detected in children with diarrhea from all three prefectures. Here, we characterized the whole genome of seven representative G9P[8]-E2 strains. In the full-genome-based analysis, the seven study strains exhibited a unique genotype configuration with the NSP4 gene of genogroup 2 in a genogroup 1 genomic backbone: G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1. This genotype constellation was shared by the Tokyo G9P[8]-E2 strains. Phylogenetic analysis showed that all 11 genes, except NSP4, of the seven study strains appeared to have originated from co-circulating Wa-like G9P[8]-E1 strains. In contrast, NSP4 appeared to have originated from the co-circulating DS-1-like G2P[4]-E2 strains. Thus, G9P[8]-E2 strains appear to be derived through reassortment between G9P[8]-E1 and G2P[4]-E2 strains in Japan. Notably, the seven study G9P[8]-E2 strains and Tokyo G9P[8]-E2 strains were revealed to have 11-segment genomes almost indistinguishable from one another in their sequences (99.3-100%), indicating all these G9P[8]-E2 strains had a common origin. To our knowledge, this is the first description of the rapid spread of G9P[8]-E2 strains across a country.

Avian rotavirus A (RVA) is one of major enteric pathogens that cause diarrhoea in young avian individuals. Importantly, some of the avian RVA strains of G18P[17] genotype are naturally transmitted to and cause clinical diseases in mammalian species, indicating their potential risks to animal health. Although molecular information on the pathogenesis by avian RVA strains will be useful for estimating their risks, the absence of a reverse genetics (RG) system for these strains has hindered the elucidation of their pathogenic mechanisms. In this study, we aimed to establish an RG system for the avian G18P[17] prototype strain PO-13, which was isolated from a pigeon in Japan in 1983 and was experimentally shown to be pathogenic in suckling mice. Transfection with plasmids expressing 11 genomic RNA segments of the strain resulted in rescue of the infectious virus with an artificially introduced genetic marker on its genome, indicating that an RG system for the PO-13 strain was successfully established. The rescued recombinant strain rPO-13 had biological properties almost identical to those of its wild-type strain (wtPO-13). Notably, both rPO-13 and wtPO-13 induced diarrhoea in suckling mice with similar efficiencies. It was thus demonstrated that the RG system will be useful for elucidating the pathogenic mechanisms of the PO-13 strain at the molecular level. This is the first report of the establishment of an RG system for an avian RVA strain.

The group A rotavirus (RVA) genome comprising 11 double-stranded RNAs encodes six structural proteins (VP1-VP4, VP6, and VP7) and six non-structural proteins (NSP1-NSP6). Among these 12 rotaviral proteins, NSP6 has been less studied as to its function. We previously prepared a recombinant NSP6-deficient RVA derived from simian strain SA11-L2 by reverse genetics, and found that the NSP6-deficient virus grew well in cell culture, although its growth was less abundant than that of the parental SA11-L2 strain. In this study, we examined the potency of a recombinant RVA incapable of NSP6 expression to cause diarrhoea in suckling mice. The suckling mice infected with the NSP6-deficient virus apparently experienced diarrhoea, although the symptom was milder and the duration of diarrhoea was shorter than in the mice infected with the authentic SA11-L2 strain. Thus, together with the results obtained for cultured cells in the previous study, it can be concluded that NSP6 is not necessarily required for replication and pathogenicity in vitro and in vivo.

Rotavirus (RV) is a leading cause of gastroenteritis in children. In Japan, Rotarix (RV1; GlaxoSmithKline), which is a monovalent vaccine derived from human RV (G1P[8]), has been introduced since November 2011, and RotaTeq (RV5; MSD) which is an pentavalent, human-bovine mono-reassortant vaccine (G1, G2, G3, G4, and P1A[8]), has been introduced since July 2012. Long-term follow-up on vaccine efficacy and RV genotypical change should be carried out in order to control RV infection. The RV gastroenteritis (RVGE) outbreak occurred during the 2018/2019 season in Aichi prefecture, Japan. Therefore, the molecular epidemiology of RV among three different groups of RVGE, which were outpatients who received RV1, those who received RV5, and those without vaccination, was explored. Clinical features of RVGE patients were compared among the three patient groups. Children less than 15 years of age with gastroenteritis who visited any of seven pediatric practices between January and June 2019 were enrolled in the study. G, P, and E genotypes were determined by direct sequencing of reverse transcription-polymerase chain reaction products amplified from stool samples. Among 110 patients, there were 27, 28, and 55 in the RV1-vaccinated, RV5-vaccinated, and unvaccinated groups, respectively. The most frequent genotype was G8P[8] (92/110 patients, 83.6%). Genotype distributions did not significantly differ among the three patient groups (P = .125). Mean Vesikari score was significantly lower among RV1-vaccinated (7.1) and RV5-vaccinated patients (6.4) than among unvaccinated patients (10.2) (P < .001). Even in RVGE patients treated in an outpatient clinic, RV vaccine reduced the severity of the disease in this cohort.

In infants with immunodeficiency, rotavirus (RV) vaccines can be continuously excreted in stool. We analysed nosocomial infection with RV vaccine strain in immunodeficient paediatric patients. RV1 RNAs were detected in stool and serum samples from case A, who was vaccinated with RV1, and case B, who was not. PAGE analysis of serial stool samples of case A revealed several rearrangements of the RV genome. In case B, the only band pattern detected was the same as a rearrangement detected in case A at the same time. In summary, RV vaccination of infants with immunodeficiency poses a risk of nosocomial infections.

Human rotavirus strains having the unconventional G4P[6] genotype have been sporadically identified in diarrheic patients in different parts of the world. However, the whole genome of only one human G4P[6] strain from Africa (central Africa) has been sequenced and analyzed, and thus the exact origin and evolutionary pattern of African G4P[6] strains remain to be elucidated. In this study, we characterized the full genome of an African G4P[6] strain (RVA/Human-wt/KEN/KCH148/2019/G4P[6]) identified in a stool specimen from a diarrheic child in Kenya. Full genome analysis of strain KCH148 revealed a unique Wa-like genogroup constellation: G4-P[6]-I1-R1-C1-M1-A1-N1-T7-E1-H1. NSP3 genotype T7 is commonly found in porcine rotavirus strains. Furthermore, phylogenetic analysis showed that 10 of the 11 genes of strain KCH148 (VP7, VP4, VP6, VP1-VP3, NSP1, and NSP3-NSP5) appeared to be of porcine origin, the remaining NSP2 gene appearing to be of human origin. Therefore, strain KCH148 was found to have a porcine rotavirus backbone and thus is likely to be of porcine origin. Furthermore, strain KCH148 is assumed to have been derived through interspecies transmission and reassortment events involving porcine and human rotavirus strains. To our knowledge, this is the first report on full genome-based characterization of a human G4P[6] strain from east Africa. Our observations demonstrated the diversity of human G4P[6] strains in Africa, and provide important insights into the origin and evolutionary pattern of zoonotic G4P[6] strains on the African continent.

BACKGROUND: Acute gastroenteritis is the most common cause of illness and death in infants and young children worldwide. Rotaviruses (RVs) are the major viruses that cause acute gastroenteritis in young children, especially in developing countries in Asia and Africa. METHODS: The presence of rotavirus antigens in sera of four unvaccinated pediatric patients, aged between 4 and 6 years with severe diarrhea and dehydration, were detected by using three immunochromatographic (IC) kits. In addition, the presence of anti-rotavirus IgG, IgA, and IgM antibodies and their concentrations in patient sera were also determined by enzyme immunoassay (EIA). RESULTS: All three kits could detect rotavirus antigen in patient sera with different intensity of the test lines. When patient sera were pretreated with anti-VP6 rotavirus mouse monoclonal antibody prior to testing, the rotavirus positive test lines disappeared, suggesting that all patient sera contained VP6 protein antigen of rotavirus. Assessment of antibody concentration in these patient sera revealed that all patient sera contained IgG, IgA, and IgM antibodies against rotavirus antigen at different concentrations. CONCLUSIONS: The sensitivity of rotavirus protein detection in the patient sera of one IC kit brand was comparable to those of the EIA, suggesting this IC kit could be an alternative screening method for rapid diagnosis of rotavirus infection.

Human rotaviruses (HuRVAs) are highly important causes of acute gastroenteritis in infants and young children worldwide. A lack of reliable and reproducible reverse genetics systems for HuRVAs has limited a proper understanding of HuRVA biology and also the rational design of live-attenuated vaccines. Since the development of the first reverse genetics system for RVAs (partially plasmid-based reverse genetics system) in 2006, there have been many efforts with the goal of generating infectious recombinant HuRVAs entirely from cloned cDNAs. However, the establishment of a HuRVA reverse genetics system was very challenging until 2019. This review article provides an overview of the historical background of the recent development of long-awaited HuRVA reverse genetics systems, beginning with the generation of recombinant human-simian reassortant RVAs with the aid of a helper virus in 2006 and the generation of recombinant animal (simian) RVAs in a helper virus-free manner in 2017, and culminating in the generation of recombinant HuRVAs entirely from plasmid cDNAs in 2019. Notably, the original HuRVA reverse genetics system has already been optimized to increase the efficiency of virus generation. Although the application of HuRVA reverse genetics systems has only just been initiated, these technologies will help to answer HuRVA research questions regarding viral replication and pathogenicity that could not be addressed before, and to develop next-generation vaccines and intestine-specific rotaviral vectors.

The exact evolutionary patterns of human G4P[6] rotavirus strains remain to be elucidated. Such strains possess unique and strain-specific genotype constellations, raising the question of whether G4P[6] strains are primarily transmitted via independent interspecies transmission or human-to-human transmission after interspecies transmission. Two G4P[6] rotavirus strains were identified in fecal specimens from hospitalized patients with severe diarrhea in Thailand, namely, DU2014-259 (RVA/Human-wt/THA/DU2014-259/2014/G4P[6]) and PK2015-1-0001 (RVA/Human-wt/THA/PK2015-1-0001/2015/G4P[6]). Here, we analyzed the full genomes of the two human G4P[6] strains, which provided the opportunity to study and confirm their evolutionary origin. On whole genome analysis, both strains exhibited a unique Wa-like genotype constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The NSP1 genotype A8 is commonly found in porcine rotavirus strains. Furthermore, on phylogenetic analysis, each of the 11 genes of strains DU2014-259 and PK2015-1-0001 appeared to be of porcine origin. On the other hand, the two study strains consistently formed distinct clusters for nine of the 11 gene segments (VP4, VP6, VP1-VP3, and NSP2-NSP5), strongly indicating the occurrence of independent porcine-to-human interspecies transmission events. Our observations provide important insights into the origin of zoonotic G4P[6] strains, and into the dynamic interaction between porcine and human rotavirus strains.

With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.

Group A rotavirus is a leading cause of severe acute gastroenteritis worldwide. In this study, the first complete coding sequences of 11 RNA segments of human group A rotavirus G12P[8] in Japan were determined by an unbiased viral metagenomics. Its genomic constellation (VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes) was identified as G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. When performing the genetic analysis, we discovered an intergenotypic recombination event in the pig group A rotavirus G12P[8] strain BUW-14-A008. The novel recombination was found between two different genotypes G12 and G3 in the VP7 gene, and P[8] and P[13] in the VP4 gene.

An unusual rotavirus strain with the G3P[10] genotype (RVA/Human-wt/THA/MS2015-1-0001/2015/G3P[10]) was identified in a stool sample from a hospitalized child aged 11 months with severe gastroenteritis in Thailand. In the current study, we sequenced and characterized the full genome of strain MS2015-1-0001. On full-genomic analysis, strain MS2015-1-0001 exhibited the following genotype configuration: G3-P[10]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which is identical or closely related to those of bat and bat-like rotavirus strains (MYAS33-like). Furthermore, phylogenetic analysis revealed that all 11 genes of strain MS2015-1-0001 appeared to be of bat origin. Our findings provide evidence for bat-to-human interspecies transmission of rotaviruses and important insights into dynamic interactions between human and bat rotavirus strains.

Species A rotaviruses are a major cause of acute gastroenteritis in infants and young children worldwide. Reassortment is a common phenomenon due to the segmented nature of the rotavirus genome. The complete coding sequences of a species A rotavirus strain isolated from the feces of a child with acute gastroenteritis in Japan in 2018 were determined using an unbiased viral metagenomics approach. The genetic analysis revealed that the rotavirus strain had an unusual genomic constellation (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1), suggesting reassortment of a genotype 1 with a genotype 2 rotavirus, from which the NSP4-encoding gene was acquired.

Group A rotavirus (RVA) rarely causes severe complications such as encephalitis/encephalopathy. However, the pathophysiology of this specific complication remains unclear. Next-generation sequence analysis was used to compare the entire genome sequences of RVAs detected in patients with encephalitis/encephalopathy and gastroenteritis. This study enrolled eight patients with RVA encephalitis/encephalopathy and 10 with RVA gastroenteritis who were treated between February 2013 and July 2014. Viral RNAs were extracted from patients' stool, and whole-genome sequencing analysis was carried out to identify the specific gene mutations in RVA obtained from patients with severe neurological complications. Among the eight encephalitis/encephalopathy cases, six strains were DS-1-like G1P[8] and the remaining two were Wa-like G1P[8] (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1). Meanwhile, eight of the 10 viruses detected in rotavirus gastroenteritis patients were DS-1-like G1P[8], and the remaining two were Wa-like G1P[8]. These strains were further characterized by conducting phylogenetic analysis. No specific clustering was demonstrated in RVAs detected from encephalitis/encephalopathy patients. Although the DS-1-like G1P[8] strain was predominant in both groups, no specific molecular characteristics were detected in RVAs from patients with severe central nervous system complications.

Reverse genetics technology allows one to engineer replication-competent viruses from cloned cDNAs at will. Since the establishment of the initial reverse genetics system for species A rotaviruses (RVAs) requiring a helper virus in 2006, attempts have been successfully made to improve this technology. Efficient generation of replication-competent RVAs is now possible from just 11 T7-driven plasmids encoding an RVA genome when the quantity ratio of the two rescue T7-driven plasmids for the NSP2 and NSP5 segments is increased by 3-fold in relation to that of the other nine plasmids (11 plasmid-only system). Further, it is now possible to generate recombinant RVAs even with severely less efficient infectivity by using the 11 plasmid-only system, which has not been possible with the existing approaches. More importantly, the 11 plasmid-only system does not need any helper expression plasmid, and thus this simplest and robust system has a clear advantage over the existing systems in terms of safety. This 11 plasmid-only system should contribute to the development of safe next-generation vaccines and vaccine vectors.

Reassortment is an important mechanism in the evolution of group A rotaviruses (RVAs), yielding viruses with novel genetic and phenotypic traits. The classical methods for generating RVA reassortants with the desired genetic combinations are laborious and time-consuming because of the screening and selection processes required to isolate a desired reassortant. Taking advantage of a recently developed RVA reverse genetics system based on just 11 cloned cDNAs encoding the RVA genome (11 plasmid-only system), we prepared a panel of simian SA11-L2 virus-based single-gene reassortants, each containing 1 segment derived from human KU virus of the G1P[8] genotype. It was shown that there was no gene-specific restriction of the reassortment potential. In addition to these 11 single-gene reassortants, a triple-gene reassortant with KU-derived core-encoding VP1-3 gene segments with the SA11-L2 genetic background, which make up a virion composed of the KU-based core, and SA11-L2-based intermediate and outer layers, could also be prepared with the 11 plasmid-only system. Finally, for possible clinical application of this system, we generated a series of VP7 reassortants representing all the major human RVA G genotypes (G1-4, G9 and G12) efficiently. The preparation of each of these single-gene reassortants was achieved within just 2 weeks. Our results demonstrate that the 11 plasmid-only system allows the rapid and reliable generation of RVA single-gene reassortants, which will be useful for basic research and clinical applications.

A reverse genetics technology is an incredibly useful technique both for a proper understanding of different aspects of virus biology and for the generation of complementary DNA (cDNA)-derived infectious viruses, which can act as safe and effective vaccines and viral vectors. Rotaviruses (RVAs), especially human RVAs (HuRVAs), had been very refractory to this technology until very recently. Here, we describe the historical background of the development of a long-awaited HuRVA reverse genetics system, culminating in the generation of replicative HuRVAs entirely from cloned cDNAs.

The emergence and rapid spread of unusual DS-1-like intergenogroup reassortant rotaviruses having G1/3/8 genotypes have been recently reported from major parts of the world (Africa, Asia, Australia, Europe, and the Americas). During rotavirus surveillance in Thailand, three novel intergenogroup reassortant strains possessing the G9P[8] genotype (DBM2017-016, DBM2017-203, and DBM2018-291) were identified in three stool specimens from diarrheic children. In the present study, we determined and analyzed the full genomes of these three strains. On full-genomic analysis, all three strains were found to share a unique genotype constellation comprising both genogroup 1 and 2 genes: G9-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis demonstrated that each of the 11 genes of the three strains was closely related to that of emerging DS-1-like intergenogroup reassortant, human, and/or locally circulating human strains. Thus, the three strains were suggested to be multiple reassortants that had acquired the G9-VP7 genes from co-circulating Wa-like G9P[8] rotaviruses in the genetic background of DS-1-like intergenogroup reassortant (likely equine-like G3P[8]) strains. To our knowledge, this is the first description of emerging DS-1-like intergenogroup reassortant strains having the G9P[8] genotype. Our observations will add to the growing insights into the dynamic evolution of emerging DS-1-like intergenogroup reassortant rotaviruses through reassortment.

The emergence of unusual DS-1-like intergenogroup reassortant rotaviruses with a bovine-like G8 genotype (DS-1-like G8P[8] strains) has been reported in several Asian countries. During the rotavirus surveillance program in Japan in 2017, a DS-1-like G8P[8] strain (RVA/Human-wt/JPN/SO1162/2017/G8P[8]) was identified in 43 rotavirus-positive stool samples. Strain SO1162 was shown to have a unique genotype constellation, including genes from both genogroup 1 and 2: G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis revealed that the VP1 gene of strain SO1162 appeared to have originated from DS-1-like G1P[8] strains from Thailand and Vietnam, while the remaining 10 genes were closely related to those of previously reported DS-1-like G8P[8] strains. Thus, SO1162 was suggested to be a reassortant strain that acquired the VP1 gene from Southeast Asian DS-1-like G1P[8] strains on the genetic background of co-circulating DS-1-like G8P[8] strains. Our findings provide important insights into the evolutionary dynamics of emerging DS-1-like G8P[8] strains.

The emergence and rapid spread of novel DS-1-like intergenogroup reassortant rotaviruses having the equine-like G3 genotype (DS-1-like G3P[8] strains) have been recently reported from several countries. During rotavirus surveillance in Japan in 2015–2016, three DS-1-like G3P[8] strains were identified from children with severe diarrhea. In the present study, we sequenced and characterized the full genomes of these three strains. On full-genomic analysis, all three strains showed a unique genotype constellation including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis revealed that each of the 11 genes of the three strains was closely related to that of Japanese DS-1-like G1P[8] and/or Japanese equine-like G3P[4] human strains. Thus, the three study strains were suggested to be reassortants that acquired the G3-VP7 gene from equine G3 rotaviruses on the genetic background of DS-1-like G1P[8] strains. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G3P[8] strains.

Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PItransfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBPmediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs.

The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmidonly-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.

A monovalent rotavirus vaccine (RV1) was introduced into the National Immunization Program in Kenya in July 2014. We examined the impact of the vaccine on hospitalization for all-cause acute gastroenteritis (AGE) and rotavirus-specific AGE and strain distribution at a large referral hospital which serves a predominantly pen-urban population in Central Kenya. Data on rotavirus AGE and strain distribution were derived from ongoing hospital-based AGE surveillance. Hospital administrative data were used to compare trends in all-cause AGE. Pre-vaccine (July 2009-June 2014) and post-vaccine (July 2014-June 2016) periods were compared for changes in hospitalization for all-cause AGE and rotavirus AGE and strain distribution. Following the vaccine introduction, the proportion of children aged &lt; 5 years hospitalized for rotavirus declined by 30% (95% CI: 19-45%) in the first year and 64% (95% CI: 49-77%) in the second year. Reductions in rotavirus positivity were most pronounced among the vaccine-eligible group (&lt; 12 months) in the first year post-vaccination at 42% (95% CI: 28-56%). Greater reductions of 67% (95% CI: 51-79%) were seen in the second year in the 12-23 months age group. Similarly, hospitalizations for all-cause AGE among children &lt;5 years of age decreased by 31% (95% CI: 24-40%) in the first year and 58% (95% CI: 49-67%) in the second year of vaccine introduction. Seasonal peaks of rotavirus and all cause AGE were reduced substantially. There was an increased detection of G2P[4], G3P[6] and G3P[8], which coincided temporally with the timing of the vaccine introduction. Thus, introducing the rotavirus vaccine into the routine immunization program in Kenya has resulted in a notable decline in rotavirus and all-cause AGE hospitalizations in Central Kenya. This provides early evidence for public health policy makers in Kenya to support the sustained use of the rotavirus vaccine in routine immunizations. (C) 2017 Elsevier Ltd. All rights reserved.

In Japan, monovalent and pentavalent rotavirus (RV) vaccines were approved in 2011 and 2012, respectively. To monitor changes in the RV genotypes before and after vaccine introduction, we performed a prospective observational study among children (&lt; 5 years) with gastroenteritis who tested RV -positive on antigen rapid tests. Stool samples were collected from 3 different sites in Japan: Tsu City, Mie Prefecture; Kurashiki City, Okayama Prefecture; and Isumi City, Chiba Prefecture. RV genotypes were determined using reverse transcription-polymerase chain reaction. In Tsu City, G3P[8] was dominant (61.0-77.1%) before vaccine introduction, but decreased after introduction. Meanwhile, in an inverse proportion to the decrease in G3P[8], G1P[8] increased until the 2013/14 season, when a sudden predominance of G2P[4] (100%) occurred. A similar trend was observed in Kurashiki City in terms of the extent of reduction in G3P[8] and the emergence of G2P[4]. In Isumi City, G1P[8] was dominant (70.3%) before vaccine introduction, and G9P[8] became predominant (83.3%) in the 2013/14 season. To determine whether the genotype changes are attributable to vaccines or natural epidemiological changes, ongoing continuous monitoring of the RV genotypes is required.

This cross-sectional descriptive study aimed to investigate the incidence of rotavirus and enteric bacterial infections among children up to 5 years old with diarrhea living in suburban and rural areas of Kenya. Between August 2011 and December 2013, a total of 1,060 diarrheal fecal specimens were obtained from 722 children at Kiambu County Hospital (KCH), located in a suburban area, and from 338 children from Mbita District Hospital (MDH), located in a rural part of western Kenya. Of the 1,060 isolates, group A rotavirus was detected in 29.6% (214/722) and 11.2% (38/338) fecal specimens from KCH and MDH, respectively. Diarrheagenic Escherichia coli (DEC) was found to be the most frequently isolated bacterial pathogens in both study areas (32.8% at KCH and 44.1% at MDH). Two different mixed infection patterns (virus/bacteria and bacteria/bacteria) were observed among patients. A significantly higher infection rate of rotavirus (17.6%, p = 0.001) and DEC (10.5%, p = 0.007) were observed during the dry season. Our study found that in both suburban and rural settings in Kenya, rotavirus and DEC are the principal cause of pediatric diarrhea and exhibit higher incidence during the dry season.

A highly sensitive biosensor to detect norovirus in environment is desired to prevent the spread of infection. In this study, we investigated a design of surface plasmon resonance (SPR)-assisted fluoroimmunosensor to increase its sensitivity and performed detection of norovirus virus-like particles (VLPs). A quantum dot fluorescent dye was employed because of its large Stokes shift. The sensor design was optimized for the CdSe-ZnS-based quantum dots. The optimal design was applied to a simple SPR-assisted fluoroimmunosensor that uses a sensor chip equipped with a V-shaped trench. Excitation efficiency of the quantum dots, degree of electric field enhancement by SPR, and intensity of auto fluorescence of a substrate of the sensor chip were theoretically and experimentally evaluated to maximize the signal-to-noise ratio. As the result, an excitation wavelength of 390 nm was selected to excite SPR on an Al film of the sensor chip. The sandwich assay of norovirus VLPs was performed using the designed sensor. Minimum detectable concentration of 0.01 ng/mL, which corresponds to 100 virus-like particles included in the detection region of the V-trench, was demonstrated. (C) 2016 The Authors. Published by Elsevier B.V.

Between July 2009 and June 2014, a total of 1,546 fecal specimens were collected from children &lt;5 years of age with acute gastroenteritis admitted to Kiambu County Hospital, Central Kenya. The specimens were screened for group A rotavirus (RVA) using ELISA, and RVA-positive specimens were subjected to semi-nested RT-PCR to determine the G and P genotypes. RVA was detected in 429/1,546 (27.5%) fecal specimens. RVA infections occurred in all age groups &lt;59 months, with an early peak at 6-17 months. The infections persisted year-round with distinct seasonal peaks depending on the year. G1P[8] (28%) was the most predominant genotype, followed by G9P[8] (12%), G8P[4] (7%), G1P[4] (5%), G9P[4] (4%), and G12P[6] (3%). In the yearly change of G and P genotypes, a major shift from G9P[8] to G1P[8] was found in 2012. Phylogenetic analysis of the nucleotide sequences of the VP7 and VP4 genes of seven strains with unusual G8 or P[6] showed that the VP7 nucleotide sequences of G8 were clustered in lineage 6 in which African strains are included, and that there are at least two distinct VP4 nucleotide sequences of P[6] strains. These results represent basic data on RVA strains circulating in this region before vaccine introduction. (C) 2016 Wiley Periodicals, Inc.

Rotavirus gastroenteritis is an important cause of childhood morbidity and mortality in Kenya. In July 2014, Kenya introduced the rotavirus vaccine into her national immunization program. Although immunization coverage is crucial in assessing the real-world impact of this vaccine, variability in the vaccine coverage across the country is likely to occur. In view of this, we estimated the extent of coverage for the rotavirus vaccine at two socio-economically different sub-counties using the administrative data. The findings indicate disparities in vaccine coverage and access between the sub-counties and, thus, underscore the need to strengthen immunization systems to facilitate timely, accessible, and equitable vaccine delivery across the country. Both sub-counties recorded high vaccine dropout, suggestive of poor utilization of the vaccine. In this regard, increased social mobilization is needed to encourage vaccine compliance and to enhance tracking of vaccine defaulters. While efforts to improve the accuracy of the administrative coverage estimates are crucial, vaccination coverage surveys will be needed to verify the administrative coverage data and help identify specific factors relating to rotavirus vaccine coverage in the country.

An unusual rotavirus strain with the G9P[23] genotype (RVA/Human-wt/THA/KKL-117/2014/G9P[23]) was identified in a stool specimen from a 10-month-old child hospitalized with severe diarrhoea. In this study, we sequenced and characterized the complete genome of strain KKL-117. On full-genomic analysis, strain KKL-117 was found to have the following genotype constellation: G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1. The non-G/P genotype constellation of this strain (I5-R1-C1-M1-A8N1- T1-E1-H1) is commonly shared with rotavirus strains from pigs. Furthermore, phylogenetic analysis indicated that each of the 11 genes of strain KKL-117 appeared to be of porcine origin. Our observations provide important insights into the dynamic interactions between human and porcine rotavirus strains.

Of 2,754 stool specimens collected from children with acute gastroenteritis during 2013-2014 in Sukhothai and Phetchaboon provinces, Thailand, 666 (24.2%) were positive for rotavirus A (RVA) in polyacrylamide gel electrophoresis (PAGE). The G and P types of all RVA-positive specimens were determined by semi-nested RT-PCR. G1P[8] (56.5%) was most prevalent, followed by G2P[4] (22.1%). Unusual G8P[8] human RVAs (HuRVAs) were detected at a high frequency (20.0%). Interestingly, 171 of the 376 G1P[8] HuRVAs and all of the 133 G8P[8] HuRVAs showed a short RNA pattern in PAGE. Thus, it was shown that the properties of HuRVAs have been markedly unusual in recent years in Thailand. (C) 2016 Wiley Periodicals, Inc.