| Hantaviruses (HV), which belong to the Hantavirus genus of the Bunyaviridae family, possess a three-segmented, single-stranded RNA genome of negative polarity. The viral RNA segments are designated as large (L), medium (M) and small (S), which encode the structural proteins, RNA polymerase, envelope glycoproteins (Gl and G2), and nucleocapsid protein (NP) respectively. Rodents are the natural carrier animals of hantaviruses. The viruses are transmitted to human through aerosolization of rodent excreta.In humans hantaviruses cause two main type of infections:hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). 100,000 cases of HFRS are documented annually in the world, and the mortality rate is 2%~10%. Among the HFRS cases, 90% occurred in China. HFRS is the most serious viral infectious disease except viral hepatitis in China.The most efficient prevention method is vaccine. Up to now, many kinds of vaccines have been developed, which has played a very important role in preventing and control HFRS. There are only two kinds of hantaviruses found in China: HTN and SEO. Due to the differences in clinical manifestation and antigenecity between the two types of viruses and two kinds of diseases caused by the two genotypes, bivalent vaccines needed to be developed.First isolated by Yang Weisong from a fetal liver whose mother suffered from HFRS in Xi'an in 1984, 84FLi strain was identified as a HTN virus by PRNT and partial gene analysis. Owing to the good adaptivity to Vero cells and stable multiplication with high liters, 84FLi strain was selected as the candidate vaccine of HTN by department of hemorrhagic fever and arboviruses, institute of Virology, Chinese Center of Diseases Control and Prevention. To characterize 84FLi strain more definitively and learn about its molecular characters, the complete genome structural information is needed.The cDNAs of L-. M and S segments were amplified by RT-PCR. The purified PCR products or the recombinant pMD18-T vector harboring the PCR products were sequenced and homology analysis was done using the sequences data from GenBank.It showed that the complete genome of strain 84FL1 was composed of L (6533bp), M (3616bp) and S (1688bp), coding 2151, 1135 and 429 amino acids respectively. The entire sequence composition was 3830A, 2050C, 2510G and 3447T, the GC and AT contents were 39.21% and 60.79% respectively. The Genbank accession numbers of the three segments are AY017064^ AF345636 and AF336826 respectively. Homology analysis showed that 84FLi was highly related to other Chinese Hantaan virus isolates. At the same time, 84FLi strain and other two Chinese isolates RG9 and Chen4 are in the same subtype.The M genome segment of 84FLi virus was amplified into two fragments by two pairs of primers with the method of RT-PCR, and the PCR products were cloned into pMD18-T vector. The full length M genome segment cDNA clone (pMD84M) was construced through digesting the two partial clones with Bgl II and Sal I and ligating together. In this study, baculovirus surface display system was first used to express glycoprotein of hantavirus. The Gl and G2 glycoprotein baculovirus expression vectors were construced by inserting the coding region of Gl and G2 glycoprotein of 84FLi strain into transfer vector pBACsurf-1 between the upstream gp64 signal sequence and downstream gp64 mature domain. Thus make it possible to further express the glycoprotein and study the affect of glycosilation to expression and its function in the future.
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