Identification Of Tick-borne Encephalitis Virus And Studies On Their Biological Properties And Complete Genome Sequence Determination

In the experiment, tick-borne encephalitis virus(TBEV) MDJ-01 strain isolated in Mudanjing in 2001 was identified.and the biological characters and molecular biological characters were studied on,with Senzhang strain isolated in 1953 as a control.TBEV MDJ-01 strain and Senzhang strain were passed in sucking mouse by intracerebral inoculation. The sucking mouse died on different time.Senzhang strain had -higher MOI than MDJ-01 strain in BHK-21 cells.Physicochemical tests results showed that they belonged to acid-unlabile enveloped viruses with RNA. The sucking mouse brain were inoculated with MDJ-01 strain to make electron microscopic examination,results showed that the virus was a spheral particle with membran which had a diameter of about 40 nm.By indirect fluorescent antibody test MDJ-01 strain was identified with TBEV.A part of region encoding E protein was expanded by RT-PCR and sequenced.The nucleotide sequences of two strain viruses were compared with sequences in GenbankjSequence homology analyses revealed MDJ-01 strain and Senzhang strain had the highest homology with TBEV Oshima5-10, respectively,which were 95%,94%.MDJ-01 strain was identified with TBEV again.The complete nucleotide sequences of two strain viruses were determined.Ten pairs of primers were designed based on the nucleotide sequences of TBEV Sofjin-HO,Oshima5-10 by DNAStar software.The virus cDNA was expanded by RT-PCR in ten parts.sequenced by clones method.5' terminal and 3' terminal cDNA was expanded by RACE method.The nucleotide sequences and deduced amino acid sequences of two strain viruses were compared with the sequences in Genbank by DNAStar software. 1-10997 nt sequence of MDJ-01 strain and 1-10675 nt sequence of Senzhang strain were obtained,but 3' terminal about 200 nt sequence wasn't obtained. 5' UTR of MDJ-01 strain was 131nt,but 5' UTR of Senzhang strain was 129nt,the homology between two strain 5' UTR was 99.7%.Compared with Senzhang strain,MDJ-01 had four mutations,such as G19-A, C71-T, T75-C and two nucleotides inserted after 50 nt. The complete nucleotide sequences of the coding region of two strain viruses were 10245 nt, which encoded a polyprotein of 3414 amino acids. The homology of nucleotide sequences and deduced amino acide sequences were 98. 0%, 99. 3%, respectively, so we thought MDJ-01 strain mutated from Senzhangstrain. Compared with Senzhang strain, MDJ-01 strain had 24 amino acides mutated.We found 12 subtype-special amino acids which may be the cause for different characters from three subtypes. MDJ-01 strain and Senzhang strain lay at the Far Eastern subtype branch in the TBEV phylogenetic tree.MDJ-01 strain was compared with Senzhang strain in biological characters and complete genome sequence.All helped to ascertain the cause for tick-borne encephalitis erupted in china northern eastern in late years,to study on the complete cDNA of TBEV ,genome structure and function,diagnose,prevalence and therapy.the 12 subtype-special amino acids but 486 amino acids weren't reported, and were important to identify TBEV strain isolated newly with which subtype.