Genetic Studies Of Rotavirus Pathogenesis And Development Of Rotavirus Vaccines

以前，在涉及致菌性新生猪的半同源系统和强毒株猪轮状病毒株（SB-1A）和无毒毒株人轮状病毒株（DS-1）及其复制株的研究中，我们证明：（i）第三（VP 3）、第四（VP 4）、第九（VP 7），和第十（NSP 4）猪轮状病毒基因在轮状病毒感染仔猪的毒力中各自发挥重要的独立作用;和（ii）所有四种猪轮状病毒毒力相关基因都是诱导仔猪腹泻和轮状病毒脱落所必需的。这些观察结果表明，一个潜在的新的战略减毒野生型人轮状病毒的主要流行病学的重要性和其应用开发一个安全有效的疫苗。之前，我们开发了基于恒河猴（RRV）、牛（UK）和猪（Gottfried）的多价候选疫苗，旨在为流行病学重要性的VP 7（G）血清型1-4、8和9提供抗原覆盖。今年，为了研究鼠轮状病毒EB株的哪些基因参与同源小鼠模型中腹泻的诱导，我们在4-5日龄CD-1小鼠中连续传代细胞培养适应的无毒（非致腹泻）鼠轮状病毒EB株，直到病毒变得有毒性（致腹泻）。然后将该毒性病毒接种到原代非洲绿色猴肾（AGMK）细胞上并连续传代18次。在小鼠幼仔中显示无毒力的第18代细胞培养物传代病毒在CD-1小鼠中的后续连续传代期间再次变得有毒力。当在原代AGMK细胞中再次连续传代18次时，该毒力病毒在小鼠中变为无毒力。对本研究中产生的所选毒力和无毒力病毒的所有11个基因进行测序，结果显示，与无毒力病毒相比，毒力病毒具有（i）基因4（编码外衣壳VP 4）和基因10（编码病毒肠毒素NSP 4）中的不同突变，以及（ii）其他基因中无显著突变。此外，在小鼠中观察到强毒和无毒力病毒之间的病毒复制效率没有显著差异。我们产生了3种表达NSP 4蛋白的杆状病毒重组体，所述NSP 4蛋白来源于高小鼠传代的毒性EB病毒（样品A）、低小鼠传代的毒性EB病毒（样品B）或细胞培养传代的无毒力EB病毒（样品C）。 在小鼠幼仔中经口接种后，在样品A组中91.6%（11/12）、样品B组中12.5%（1/8）和样品C组中0%（0/12）中观察到腹泻的发展，表明NSP 4蛋白在该模型的发病机制中起重要作用。

Previously, in studies involving a semi-homologous system of gnotobiotic newborn pigs and a virulent porcine rotavirus strain (SB-1A) and an avirulent human rotavirus strain (DS-1) and their reassortants, we demonstrated that: (i) the third (VP3), fourth (VP4), ninth (VP7), and tenth (NSP4) porcine rotavirus gene each play an important independent role in the virulence of rotavirus infection in piglets; and (ii) all four of the porcine rotavirus virulence-associated genes are required for the induction of diarrhea and the shedding of rotavirus by piglets. These observations suggested a potential new strategy for attenuation of wild-type human rotaviruses of major epidemiologic importance and its application to the development of a safe and effective vaccine. Previously, we developed rhesus (RRV)-, bovine (UK)-, and porcine (Gottfried)-based multivalent vaccine candidates which were designed to provide antigenic coverage for VP7 (G) serotypes 1-4, 8 and 9 of epidemiologic importance. This year, in order to study which gene(s) of a murine rotavirus EB strain are involved in induction of diarrhea in a homologous mouse model, we passaged a cell culture-adapted avirulent (non-diarrheagenic) murine rotavirus EB strain serially in 4-5 day-old CD-1 mice until the virus became virulent (diarrheagenic). This virulent virus was then inoculated onto primary African green monkey kidney (AGMK) cells and passaged serially 18 times. The 18th cell culture-passaged virus that was shown to be avirulent in mouse pups became virulent again during subsequent serial passage in CD-1 mice. This virulent virus when passaged again serially 18 times in primary AGMK cells became avirulent in mice. Sequencing of all 11 genes of selected virulent and avirulent viruses generated in this study, revealed that virulent viruses when compared with avirulent viruses bore (i) distinct mutations in gene 4 (encoding outer capsid VP4) and in gene 10 (encoding viral enterotoxin NSP4) and (ii) no significant mutations in other genes. In addition, no significant difference in virus replication efficiency in mice was observed between virulent and avirulent viruses. We generated 3 baculovirus recombinants expressing the NSP4 protein derived from high mouse-passaged virulent EB virus (sample A), low mouse-passaged virulent EB virus (sample B), or cell culture-passaged avirulent EB virus (sample C). Upon oral inoculation in mouse pups, the development of diarrhea was observed in 91.6% (11 of 12) in sample A group; 12.5% (1 of 8) in sample B group; and 0% (0 of 12) in sample C group, indicating that the NSP4 protein played an important role in pathogenesis in this model.