Study On The Genomic Characteristics And Molecular Evolution Of Banna Virus (bav)

Banna Virus (BAV) is the prototype species of genus Seadornavirus within the family Reoviridae. BAV was initially isolated from patients with encephalitis and unknown fever in Xishuangbanna, Yunnan Province of China in 1987. Since then, BAV isolates have been obtained from pigs, bovines, ticks, and from mosquitoes including 3 genera and 10 species in China, Indonesia, and Vietnam, which are located in north temperate zone, subtropical zone and tropical zone, between the equator to northern latitude 42°. Since BAVs have been isolated from patient specimens and antibodies against BAV can be detected in patient serum, BAV is identified as an important emerging pathogen of human fever and viral encephalitis, and arouses great attention in disease prevention and control among the virologists and experts home and abroad.On the basis of sequencing the BAV complete genomes in a large scale and systematically analyzing on the characteristics of complete genome, a comprehensive analysis were conducted in this study, including phylogenetic analysis, time of most recent ancestor, population dynamics and the migration routes and dynamic. This study on BAV is first reported, and the results of this study will play a significant role in explaining the molecular evolution and origin of BAV as well as providing a theoretical basis to formulate strategies on disease prevention and control caused by BAV. The results of this study are as follows:1. Whole genome sequencing of Banna virusIn this study, the whole genome sequences of 20 BAV isolates in China from 1995 to 2007 were completed. These BAV isolates were isolated from mosquitoes including 3 genera and 10 species collected in Yunnan Province, Shanxi Province, Gansu and the isolation places cover subtropical and north temperate zone in China. The statistics of sequencing quality shows that the average quality value is 30.34, and the accuracy reach to 98.9%, which indicates that the quality of the sequence is highly reliable. Two sets of self-designed BAV whole genome amplifying primers with good specificity and running stability were used in the process of sequencing which has greatly improved the efficiency of sequencing.2. Genomic characteristics of Banna virusThe length of BAV's whole genome is 21,000 bp, which is composed of twelve segmented double-chained RNA with each segment's size distribution from 759bp to 3747 bp. The virus genome is abundant of purine and pyrimidine nucleotides (A and T accounts for 58.19%-65.39%) and every segment has a compact size with few redundant sequences. The individual gene segment of BAV is of monocistron structure including a merely open reading frame according to respective viral proteins from VP1 to VP12. The consequence analysis suggests that there are lots of highly conserved areas in BAV genome, which are the vital activity locuses involving sustaining viral functions and served as ideal targets for molecular diagnosis. The homology analysis of BAV whole genome sequence indicates homology of amino acids within individual segment is higher than nucleotides' manifesting most of the mutational sites distributing in the third position of the codon without change on amino acids coding. There is a significant detergency among homology of the first to twelve segments of BAV, of which the 12th segment shows a best fidelity (>84.8%,μ=92.5%) and the 9th varies most (>37.8%,μ=81.53%). It could be detected there are base group's deficiency, insertion, reconstruction, reassortment and multiplication within each BAV segment, prompting the genome structure of each segment of BAV is not very rigorous but dynamic and admitting losing and acquiring genetic material, which is supposed to be the impetus of BAV adaptive evolution. The phyletic evolution analysis of the complete viral genome shows, BAV could be divided into two genotypes, type A and type B, and type A could be subdivided into subtype A1 (isolated from north areas) and subtype A2 (isolated from south areas), embodying distinctive regional distribution characteristics. Subtype A1 and Subtype A2 contains type typical amino acids sites, which shows a genetic distinction during the BAV evolution and used to be a major molecular motif on developing BAV pathogenicity research. The evolution analysis results of each viral segment displays the 12th gene segment presents conserved structure, sufficient information louses for genotyping, and moreover it shows a similar consequent gene evolutionary tree compared to viral complete genome analysis conclusion, all the reasons above convinced that the 12th could be served as the molecular basis (part of the research results of this section were published on EID,2010) in virtual genotyping. Proceeding a further study, the evolution dendritic diagram of each individual BAV genetic segment presents in perfect consistency showing each individual gene has a different distinctions in evolvement3. Analysis of population dynamics and evolutionary historyIn this study, the complete genome sequence data from twenty strains of BAV detected in this subject and also four strains downloaded through GenBank were collected to build a BAV alignment whole genome except redundant sequence data sets, aiming for launching a BAV viral genome space dynamics research. Software BEAST, PAUP, Phylip and MigraPhyla have been used to carry out the virus selection pressure, molecular traceability, population evolution dynamics and geographical migration analysis. The results shows that BAV underwent purification selection pressure primarily in nature, in other words, most of the harmful mutations were eliminated by that pressure. The average rates of replaced basic group distributed between 10-3 to 10-4 magnitude, which was equivalent to the rates of other viruses of reovirus, such as blue tone virus, rotavirus etc. BAV each individual gene segment manifested different basic group replacing rate under distinct selection pressure, of which the 9th segment shows the fastest trend (1.345*10-2/nt/year) while the 12th segment shows the slowest (4.33*10-4/nt/year). The calculated consequence from Basic group replacement model shows interchange between pyrimidine (C converted to T) turns up most commonly, and then is the purine (A converted to G). In a sequence, the ratio of transition is higher than transversion. Except for the 11th gsegment optimum substitution modelis HKY, others are GTR. BEAST software were used to construct a analysis on molecular traceability and population evolution dynamicse, and the results show the time of most recent ancestor of BAV is -315 years (95%HPD:63-619), among which the ancestors of genotype A and genotype B emerged in 92 years ago (95%HPD:21-192) and 164 years ago (95%HPD:36-327) respectively from now on. Inferring from the present known BAV strains'isolation age information, the TMRCA of subgenotype A2 was the oldest. Population dynamic analysis shows the diversity of BAV group was under slowly decline situation since 1980, then appeared to a distinct decrease during 2000 to 2005, around 2005 the trend falls to the bottom level, and at present this situation has sustained between 101 to 102 magnitude. Migration dynamics shows that Yunnan Province, Gansu Province, Liaoning Province are the three primary diffusing zones of BAV. Tracing back to the BAV migration routes, it is suggested a highly similarity to our country's migratory birds flying routs, which manifests migratory birds have preformed a significant role in diffusing BAV.4. SummaryBanna Virus (BAV) is the prototype species of genus Seadornavirus within the family Reoviridae.This subject was the first report of the whole genome of BAV. We acquire primary structure panorama of BAV species group genome, accurately locate the highly conserved region within BAV genome, verify the type-special molecular motif, find out the impetus of BAV evolution and interpret the dynamics yielded from viral gene reassortment, all of these work has achieved effective genome molecular data excavation and analysis for developing BAV etiology research thoroughly. Meanwhile, develop a systemic bioinformatics research under the assistance of the international most advanced analysis methods, which clarified the biology significance involved in the genome data, confirmed the relationships between BAV molecular genotyping classification and virus strains, reviewed BAV's molecular origination time as well as population evolution trends and reconstructed BAV's migration routes. The final purpose generated from this subject is to apply the research achievements to BAV relative diseases control work. The results from BAV homology analysis, locations of the gene conserved segment and viral genotyping classification created a foundation towards exploitation of BAV molecular diagnosis reagents; the consequence acquired from calculation of BAV different segment's basic group replacement rates served as a important evidence for establishing different BAV epidemiological surveys on diverse time scales; the results of the BAV origination, population dynamic alteration, especially the data derived from migration routes and dynamics analysis also offered valuable reference materials to formulate the BAV prevention and control strategy.