Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
Research Center for Biosafety, Laboratory Animal and Pathogen Bank, National Institute of Infectious Diseases, Tokyo, Japan.
J Virol. 2024 Sep 17;98(9):e0063924. doi: 10.1128/jvi.00639-24. Epub 2024 Aug 12.
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 to 3.4 × 10 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.
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.
引起急性胃肠炎的人类杯状病毒（HuSaV）有 4 个基因群和 18 种基因型。为了了解它们的抗原和病毒学差异，获得不同毒株的病毒株至关重要。此前，我们利用人十二指肠细胞系 HuTu80 和甘氨胆酸，复制和扩增 GI.1、GI.2 和 GII.3 HuSaV（H. Takagi 等人，Proc Natl Acad Sci U S A 117：32078-32085，2020，https://10.1073/pnas.2007310117）。首先，我们研究了 HuTu80 传代数对 HuSaV 繁殖的影响。其次，我们证明与甘氨胆酸相比，牛磺胆酸可提高初始复制成功率和粪便标本中的病毒 RNA 水平。通过繁殖 15 种 HuSaV 基因型（GI.1-7、GII.1-5、-8 和 GV.1-2）并完成含 1.0×10 至 3.4×10 病毒基因组拷贝/ml 的病毒株制备，我们发现所有毒株的复制均需要胆盐，而 GII.4 对牛磺胆酸的要求严格。在连续传代病毒培养中扩大规模时，病毒培养物中病毒的推导 VP1 序列与粪便中原序列相同或仅相差两个氨基酸。此外，我们从不同基因型的 9 种毒株中纯化了病毒粒子，并将其用作抗血清产生的免疫原。使用针对不同基因型的 15 种不同毒株的兔和豚鼠抗血清进行酶联免疫吸附测定（ELISA）显示，在基因群之间，以及在基因型之间，增殖病毒之间具有不同的抗原性。获得生物库病毒资源并确定关键培养条件对于深入了解 HuSaV 感染的共同机制将非常有价值。
由于其与类似于人类诺如病毒引起的暴发有关，因此控制引起急性胃肠炎的人类杯状病毒（HuSaV）具有挑战性。建立各种病毒株的有效病毒繁殖条件对于了解感染机制和确定潜在控制方法至关重要。在这项研究中，确定了在常规人十二指肠细胞系中繁殖人类杯状病毒的两个关键因素，并分离并制备了 15 种不同基因型的病毒株，这些病毒株在遗传和抗原水平上均存在差异。尚未成功制备属于杯状病毒科的诺如病毒的病毒株。确保多种人类杯状病毒株的病毒株储备是朝着建立不依赖有限的病毒阳性粪便材料的可靠实验系统迈出的重要一步。
