Development of the system for the formation of infectious particles of rotavirus with the aid of genetic engineering

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At the final stage of the research project, we succeeded in the development of the system of reverse genetics of rotavirus, although we have to employ a helper virus for the system. The methodology for the development is the following. 1) We prepared a plasmid (termed as pT7/VP4-SA11-L2) which the VP4 gene from a trypsin-independent simian rotavirus SA11-L2 clone with the ability of efficient multiplicity in cultured cell was inserted. 2) Vaccinia virus expressing T7 RNA polymerase was infected into COST cells. 3) A plasmid pT7/VP4(SA11-L2) was transfected. 4) A human rotavirus strain KU as a helper virus was infected into COST cells, and the cells were cultured for two days. 5) The culture fluid from the virus-infected cells was infected to MA-104 cells, and the cells were maintained for two days in the presence of a neutralizing monoclonal antibody YO-2C2, which inhibits specifically the growth of a helper virus KU. 6) RNA pattern of the RNA extracted from the culture fluid was confirmed by polyacrylamide gel electrophoresis.By these procedures, we succeeded in the preparation of infectious rotavirus particles which the VP4 gene was artificially inserted. Next, we prepared infectious rotaviruses with artificially mutated VP4 gene. The mutated VP4 genes are the one (pT7/VP4(SA11-L2)-Δ PstI) which a PstI restriction site on the gene was removed, and the other (pT7/VP4(SA11-L2)-Δ HaeII) whose a HaeII restriction site was removed by mutagenesis.Thus, we first developed the reverse genetics system for rotavirus. We try to prepare the infectious rotavirus with the mutated VP7 gene or NSP1 gene, and to prepare the infectious rotavirus with the aid of only plasmid DNAs in near future.