Genome Sequencing And Analysis Of Rabies Street Virus DRV-AH08with Research On The Immunity Of Glycoprotein

Rabies, an ancient zoonotic disease, continues to present a global public health threat and is responsible for more than55,000human fatalities each year around the globe. The hazard and prevalence of rabies are different in different regions.China is currently in the third epidemic with2000-3000deaths every year.As a negative strand RNA virus, lyssaviruses possess a lot of variations. The International Committee on Taxonomy of Viruses （ICTV） divided the lyssaviruses into12genotypes in2012, including RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, ABLV, ARAV, KHUV, IRKV, WCBV and HIBV. RABV is most prevalent genotype in China.Glycoprotein plays a major role in virulence of rabies virus. Previous studies have demonstrated that different expression level of glycoprotein between attenuated and wild type rabies virus leads to difference in pathogenesis.In this present study, rabies virus isolated from a dog in2008in Anhui province, China, was sequenced and analyzed. Its glycoprotein was expressed via baculoviruses and its level of expression in mammalian and insect cells, its immunogenicity, and the protection against challenge were compared with that from a vaccine rabies virus strain （ERA）.Here is the summary of our findings:1Classification and genome sequencing of street rabies virus strain DRV-AH08Sequencing the entire genome showed that DRV-AH08shares close relationship with Henan₅q6, belonging to RABV. The genome sequence has been submitted to GeneBank （HQ450385）.2Genome structure and phylogenetic analysis of DRV-AH08The complete genome of DRV-AH08is11,924nucleotides in length, similar to other street viruses published to date. The coding sequences are as follows:1353nt for N,891nt for P,609nt for M,1575nt for G, and6384nt for L.Phylogenetic analysis revealed that indeed there are three clades of RABV around the globe, DRV-AH08could be grouped into clade â… . And clade â…  and â…¡ viruses share the same ancestor from clade â…¢, clade â…¡ RABVs exhibit closer relationship with the clade â…¢ than clade â…  viruses as shown by the deduced amino acids of the G. Molecular clock analysis estimates that clade â…  and â…¡ viruses diverged from clade â…¢ from939-1536years ago and these two clades diverted from195～670years ago.3Genome-wide amino acids analysis of China-I （currently dominant in China） and D11linage （dominant in Mexico）.To investigate whether these distinct environments could lead to different selective pressures, which resulting in mutations at the amino acid level, we chose two linages: China-I, which has emerged to become the dominant lineage in the current epidemic in China; D11Mexico lineage, associated with the West USA-Mexico border clade. Amino acid analysis revealed17amino acid mutations in the N, G and L proteins. Some of the mutations might be associated with virus replication, virulence and genes functions.4Comparison of the expression level of G protein between DRV-AH08and ERA.Eight recombinant baculoviruses were constructed to express the G protein of DRV-AH08and/or ERA:BAC-E1, BAC-S1, BAC-E2, BAC-S2, BAC-E1-E2, BAC-S1-S2, BAC-E1-S2, BAC-S1-E2. The results of cell transduction experiments indicated that the expression level of ERA glycoprotein was higher than that of DRV-AH08glycoprotein both in Sf9and BHK cells. Higher expression level of ERA G resulted in more incorporation of the G into baculovius virions when compared with DRV-AH08G.5Comparison of the immunogenicity of the G between DRV-AH08and ERA.Higher neutralizing antibody level was detected in mice immunized with1×108PFU baculoviruses expressing ERA G protein after primary or booster immunization （i.m） than those immunized with the G from DRV-AH08. After challenge with CVS-24, more mice immunized with recombinant baculoviruses expressing ERA G were protected than those immunized with recombinant baculoviruses expressing the G from DRV-AH08: BAC-E1-E2,100%, BAC-E1-S2,90%, BAC-S1-E2,80%,BAC-S1-S2,66.7%, BAC-E1,60%, BAC-E2,40%, BAC-S2,10%, BAC-S1,0%. Next, mice were immunized with vaccines expressing equal G protein:1×108PFU of BAC-E1or2×108PFU of BAC-S1. Yet, there was no VNA after the primary immunization and only low level after the boost immunization with the virus expressing the G from DRV-AH08. No protection was observed in mice immunized with virus expressing the G from DRV-AH08.These results suggested that the immunogenicity of ERA G protein is superior to DRV-AH08G. In addition, recombinant BAC-E1-E2expressing double G protein significantly enhanced the immunogenicity and protected all immunized mice against the challenge, indicating that BAC-E1-E2could be developed as a rabies vaccine.