Study on the structure and function of viral proteins using artificial particles of human rotavirus

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1. Self-assembled, artificial and empty single-shelled particles (VP2/6) were prepared by using baculovirus expression system, and they were purified in a large quantity. VP2/6 particles accompanied with mucosal adjuvant (mLT-135, mLT-H44A, recombinant B subunit) were intranasally immunized in mice. Significant IgG and IgA responses in serum and stool were observed when mLT-135 was used as a mucosal adjuvant. In the inoculation with viruent murine strain EW for the immunized mice, a marked decrease of virus antigen in stools was observed in the case of mLT-135. Furthermore, a strong CTL response was also observed. Expression of mRNA for Th1 or Th2 cytokines was detected in the spleen cells from the immunized mice. Thus, artificial empty single-shelled particles combined with mLT-135 induced efficiently both of humoral and cellular immune responses in mice, suggesting the utility of the artificial particles and mucosal adjuvant as a vaccine for humans against rotavirus infection.2. We o … More bserved that human rotaviruses with G serotype other than G3 also induced diarrhea in suckling mice. This finding is useful for infection experiments using human rotavirus strains in mice.3. We carried out the following experiments for trying to establish reverse genetics of rotavirus.(1) RNA transcripts from VP4 or VP7 gene of strain SAT11-L2 which is less dependent on trypsin were transfected to MA 104 cells with the aid of a helper virus, human strain KU which is highly dependent on trypsin.(2) We prepared several constructs for transfection : T7 promoter-rotavirus VP4 or VP7 gene-rybozyme from type D hepatitis virus-T7 terminater. Truncated NSP1 genes which are not necessarily required for replication were connected to picornavirus IRES and GFP gene, and they were inserted in the above plasmid. The RNA transcripts were transfected into MA 104 cells infected with human strain KU.The screening assay for detecting the cells expressing GFP or producing infectious reassortant virus was developed. However, infectious artificial rotavirus has not been successfuly isolated so far. Less