有4个基因组和18个基因型的人类沙泊病毒(HuSaV)负责急性胃肠炎。为了理解它们的抗原性和病毒学差异,获得不同菌株的病毒库存至关重要。以前,我们利用人十二指肠来源的细胞系HuTu80和糖胆酸,共轭胆汁酸,复制和繁殖GI.1、GI.2和GII.3HuSaVs(H.Takagi等人。,美国A117:32078-32085,2020,https://10.1073/pnas.2007310117)。首先,我们研究了HuTu80传代数对HuSaV繁殖的影响。第二,我们证明牛磺胆酸盐相对于糖胆酸盐提高了粪便标本的初始复制成功率和病毒RNA水平.通过繁殖15个HuSaV基因型(GI.1-7,GII.1-5,-8和GV.1-2),并完成含有1.0×109至3.4×1011病毒基因组拷贝/mL的病毒原液的制备,我们发现所有菌株的复制都需要胆汁酸,GII.4显示对牛磺胆酸盐的严格要求。在连续传代的病毒培养物的放大过程中,推导出的病毒VP1序列与粪便中的原始序列相同或仅相差两个氨基酸。此外,我们从九种不同基因型的菌株中纯化病毒粒子,并将它们用作免疫原用于产生抗血清。使用兔和豚鼠抗血清对15种不同基因型中的每一种进行的酶联免疫吸附测定(ELISA)揭示了跨基因组繁殖病毒之间的不同抗原性以及基因型之间的差异。获得生物标记的病毒资源和确定关键培养条件对于深入了解HuSaV感染的常见机制将是有价值的。
目的:控制人类萨波病毒,导致所有年龄段的人急性胃肠炎,具有挑战性,因为它与类似人类诺如病毒引起的爆发有关。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的控制方法至关重要。在这项研究中,确定了在常规人十二指肠细胞系中人类萨波病毒繁殖的两个关键因素,分离出15个在遗传和抗原水平上不同的基因型菌株,并用于制备病毒原种。诺如病毒的病毒原液制备尚未成功,与沙波病毒属于同一家族。确保多种人道病毒株的病毒储备代表了建立可靠的实验系统的重大进展,该系统不依赖于有限的病毒阳性粪便材料。
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 × 109 to 3.4 × 1011 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.
OBJECTIVE: 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.