Tomoichiro Oka

Human sapoviruses (HuSaVs), which cause acute gastroenteritis, are highly diverse. Fifteen HuSaV genotype strains (GI.1-7, GII.1-5, -8, and GV.1-2) were efficiently propagated in the human duodenum-derived cell line HuTu80 when supplemented with conjugated bile acid (T. Oka, T.-C. Li, K. Yonemitsu, Y. Ami, et al., J Virol 98:e00639-24, 2024, https://doi.org/10.1128/jvi.00639-24 ). However, the host cellular factors involved in HuSaV infection and propagation remain unidentified. Using a knockout approach, we newly identified and confirmed that CD36 is a critical cellular protein for the propagation of all 15 HuSaV genotypes tested in HuTu80 cells. When the CD36 gene was re-expressed in CD36 gene knockout HuTu80 cells, all HuSaV genotype strains, except for GI.6 and GII.3, recovered their viral propagation ability. To further demonstrate that human CD36 is critical for HuSaV propagation, we expressed human CD36 in HuSaV-insensitive non-human origin cells. Of the 15 HuSaV genotype strains tested, 10 from GI, GII, and GV (GI.3, -5, -6, GII.1-3, -5, -8, GV.1, and GV.2) were propagated in Chinese Hamster Ovary-K1 cells, and six from GII (GII.1-5, -8) were propagated in Vero cells. Although there were differences in the propagation ability between the genotypes tested, at least one of the three cell lines engineered to express the human CD36 gene acquired HuSaV propagation potential, confirming that human CD36 is an essential cellular factor for HuSaV propagation. IMPORTANCE: The host cellular factor(s) involved in the infection and propagation of sapovirus, which causes acute gastroenteritis in humans, remain unclear. By using a loss-of-gene function approach with a potential physiological infection site derived from a human cell line clone, which caused a marked cytopathic effect related to human sapovirus (HuSaV) propagation, we identified that CD36 was essential for the propagation of all 15 genotype strains of HuSaV tested. We confirmed different effects on distinct genotypes of HuSaV propagation by the re-expression of human CD36 in HuTu80 cells and human CD36 introduced into two non-human HuSaV-insensitive cells. This finding is an important step toward understanding common and genogroup/genotype-specific HuSaV propagation mechanisms, and the development of methods to control this highly contagious virus, including research on the development of anti-viral drugs and the establishment of HuSaV-susceptible animal models in the future.

Sapovirus (SaV) is one of the major viruses causing acute gastroenteritis. Of the 1981 fecal specimens collected through sentinel pediatric acute gastroenteritis pathogen surveillance in Mie Prefecture, Japan (2010–2022), 236 were positive for SaV, according to PCR screening. Whole or near‐whole genome sequences were determined for 158 strains by next‐generation sequencing. Genotype GI.1 was the most common of the nine SaV genotypes detected, followed by GII.3 and GII.1. Phylogenetic analysis showed that SaVs of these three genotypes separated into three different clusters depending on the year of detection, suggesting continuous genetic changes in the same genotype. Coinfections involving different SaV genotypes, as well as reinfections with SaV in the same individual, were observed in this study. The main clinical manifestations were diarrhea (68.4%) and vomiting (61.6%), with an increased rate of emesis, particularly in patients over 3 years of age. In addition, 18.1% of the children had fever. This study clarified the prevalence of viral genotypes as well as clinical findings of SaV‐positive gastroenteritis in children, and revealed trends by age.

There are four genogroups and 18 genotypes of human sapoviruses (HuSaVs) responsible for acute gastroenteritis. To comprehend their antigenic and virological differences, it is crucial to obtain viral stocks of the different strains. Previously, we utilized the human duodenum-derived cell line HuTu80, and glycocholate, a conjugated bile acid, to replicate and propagate GI.1, GI.2, and GII.3 HuSaVs (H. Takagi et al., Proc Natl Acad Sci U S A 117:32078–32085, 2020, https://10.1073/pnas.2007310117 ). First, we investigated the impact of HuTu80 passage number on HuSaV propagation. Second, we demonstrated that taurocholate improved the initial replication success rate and viral RNA levels in fecal specimens relative to glycocholate. By propagating 15 HuSaV genotypes (GI.1–7, GII.1–5, −8, and GV.1–2) and accomplishing preparation of viral stocks containing 1.0 × 10 ⁹ to 3.4 × 10 ¹¹ viral genomic copies/mL, we found that all strains required bile acids for replication, with GII.4 showing strict requirements for taurocholate. The deduced VP1 sequences of the viruses during the scale-up of serial passaged virus cultures were either identical or differed by only two amino acids from the original sequences in feces. In addition, we purified virions from nine strains of different genotypes and used them as immunogens for antiserum production. Enzyme-linked immunosorbent assays (ELISAs) using rabbit and guinea pig antisera for each of the 15 strains of different genotypes revealed distinct antigenicity among the propagating viruses across genogroups and differences between genotypes. Acquisition of biobanked viral resources and determination of key culture conditions will be valuable to gain insights into the common mechanisms of HuSaV infection. IMPORTANCE: The control of human sapovirus, which causes acute gastroenteritis in individuals of all ages, is challenging because of its association with outbreaks similar to those caused by human norovirus. The establishment of conditions for efficient viral propagation of various viral strains is essential for understanding the infection mechanism and identifying potential control methods. In this study, two critical factors for human sapovirus propagation in a conventional human duodenal cell line were identified, and 15 strains of different genotypes that differed at the genetic and antigenic levels were isolated and used to prepare virus stocks. The preparation of virus stocks has not been successful for noroviruses, which belong to the same family as sapoviruses. Securing virus stocks of multiple human sapovirus strains represents a significant advance toward establishing a reliable experimental system that does not depend on limited virus-positive fecal material.

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.

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 ®, 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.

Sapovirus (SaV) infections are a public health problem because they cause acute gastroenteritis in humans of all ages both as outbreaks and in sporadic cases. However, publicly-available SaV sequence information, especially whole genome sequences for all the SaV genotypes, is still limited. Therefore, in this study, we determined the full/near-full-length genomic sequences of 138 SaVs from the 2001-2015 seasons in 13 prefectures across Japan. The GI genogroup was predominant (67%, n = 92), followed by GII (18%, n = 25), GIV (9%, n = 12), and GV (6%, n = 9). Within the GI genogroup, four different genotypes were identified, as follows: 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 a total of 3119 public human SaV sequences 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. Together with public SaV sequences, the 138 newly determined Japanese SaV sequences could facilitate a better understanding of the evolutionary patterns of SaV genotypes.

Human parechovirus (PeV-A) is an RNA virus that belongs to the family Picornaviridae and it is currently classified into 19 genotypes. PeV-As usually cause mild illness in children and adults. Among the genotypes, PeV-A3 can cause severe diseases in neonates and young infants, resulting in neurological sequelae and death. In this study, we identify the human myeloid-associated differentiation marker (MYADM) as an essential host factor for the entry of six PeV-As (PeV-A1 to PeV-A6), including PeV-A3. The infection of six PeV-As (PeV-A1 to PeV-A6) to human cells is abolished by knocking out the expression of MYADM. Hamster BHK-21 cells are resistant to PeV-A infection, but the expression of human MYADM in BHK-21 confers PeV-A infection and viral production. Furthermore, VP0 capsid protein of PeV-A3 interacts with one extracellular domain of human MYADM on the cell membrane of BHK-21. The identification of MYADM as an essential entry factor for PeV-As infection is expected to advance our understanding of the pathogenesis of PeV-As.

ヒトサポウイルス（Human sapovirus; HuSaV）の発見から約40年以上を経て，ヒト十二指腸腫瘍由来細胞HuTu80とsupplementとして胆汁酸を加えることにより，HuSaV培養系を確立するに至った．一般的な細胞株に加え，クラシカル培地の使用や培養スケールアップも容易であるなど汎用的な手法となったが，ここに至るに十分なウイルス力価を得るための培養日数や供試サンプル選定のためのウイルス遺伝子保存性確認，HuTu80細胞の継代方法などが重要であった．現在15種のgenotypeについて増殖・ストック作製に成功しており，研究のためのリソースとして安定的確保に至った．これに続き精製抗原による抗血清の作成やその解析，不活性化条件評価を進められるようになった．本稿では培養細胞や胆汁酸の選択経緯や発表後のトピックを紹介するとともに，細胞変性の顕在化や糞便由来HuSaVのUV-C低感受性に関する解明など，今後の課題も提起する．

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.