Analysis of rabies virus pathogenicity and application to the vaccine by using reverse genetics

从缺失全部P基因的HEP-Flury株的cDNA反遗传得到了P基因缺失的狂犬病毒。利用补充缺陷基因功能的细胞系，通过挽救病毒来繁殖和扩增缺陷病毒。P基因缺陷（def-P）病毒在细胞系中复制其基因组并产生子代病毒，这些子代病毒组成部分表达P蛋白，尽管与亲本菌株相比，其生长速度略有减慢。相反，当def-P病毒感染不表达P蛋白的细胞时，在被感染的宿主中没有产生子代病毒。def-P病毒在成年和哺乳期小鼠中具有致病性，即使在颅内接种也是如此。在小鼠中接种def-P病毒可诱导高水平的病毒中和抗体，并对致死性狂犬病感染产生保护性免疫。这些结果证明了基因缺陷病毒作为一种新型狂犬病减毒活疫苗的潜在效用。为了生产发展中国家人民负担得起的抗狂犬病免疫球蛋白，用大肠杆菌表达的部分狂犬病病毒G蛋白免疫蛋鸡。免疫球蛋白（IgY）是从免疫母鸡产蛋的蛋黄中纯化出来的。结果表明，该抗体能特异性结合病毒粒子和感染狂犬病毒的细胞，并能中和狂犬病毒的感染性。此外，将该抗体接种到感染狂犬病毒的小鼠体内，可降低病毒引起的死亡率。针对大肠杆菌中表达的G蛋白部分的IgY可以作为目前可用的抗狂犬病人或马免疫球蛋白的可能替代品。

Rabies virus deficient in the P gene was generated by reverse genetics from cDNA of HEP-Flury strain lacking the entire P gene. The defective virus was propagated and amplified by rescue of virus, using a cell line that complemented the functions of the deficient gene. The P gene-deficient(def-P) virus replicated its genome and produced progeny viruses in the cell lines that constitutively expressed the P protein, although it grew at a slightly retarded rate compared to the parental strain. In contrast, no progeny virus was produced in the infected host when the def-P virus infected cells that did not express the P protein. The def-P virus was apathogenic in adult and suckling mice, even when inoculated intracranially. Inoculation of def-P virus in mice induced high levels of virus-neutralizing antibody and conferred protective immunity against a lethal rabies infection. These results demonstrate the potential utility of gene-deficient virus as a novel live attenuated rabies vaccine.In an attempt to produce anti-rabies immunoglobulin affordable for people living in developing countries, layer chickens were immunized with a part of the G protein of rabies virus expressed in Escherichia coli. Immunoglobulin (IgY) was purified from the yolks of eggs layed by the immunized hens. It was indicated that the antibody specifically bound to virions as well as cells infected with rabies virus and neutralized rabies virus infisctivity. Moreover, inoculation of the antibody into mice infected with rabies virus reduced the mortality caused by the virus. The IgY directed to the part of the G protein expressed in E.coli could be served as a possible alternative to currently available anti-rabies human or equine immunoglobulin.