| The rapid development of high-throughput sequencing technologyrendered its application in the field of virology more widely in the last decade.High-throughput sequencing hasgreat advantages over Sanger sequencing,whether it is inemergency response ofsudden epidemics,discovery ofnovelviruses,or in large-scale molecular epidemiological investigations.SFTSV is a new tick-borne phlebovirus inbunyaviridae family,which was discoveredas the etiology of Severe fever with thrombocytopenia syndrome(SFTS)in China in 2009 andwas now prevalent in at least 20 provinces in China.Since 2012,cases of SFTSVhavealso been reported in Japan and South Korea.After the discovery of SFTSV,several novel viruses inphlebovirusgenus closely related to SFTSV,such as,Heartland Virus in the United States,was found or newlygenetic characterised.SFTSV,as a representative of the newly emerging group of viruses in the phlebovirus genus,has an unclear molecular evolutionary mechanism that needs to be fully studied.In the first part of the paper,whole genome sequencing of 72 SFTSV samples,including 42 serum samples and 30 samples of cell culture supernatant,was carried out by high-throughput sequencing.The average sequencing depth was more than 10000×.Bioinformatics analysis was performed on the newly abtained sequences and the data sets of all the SFTSV full-length genome sequences in the ViPR database.First,we clearly divided the six genotypes of SFTSV.Combined with its geographical distribution,we found that genotype F was the dominant epidemic genotype of Japan,South Korea and Zhejiang provine of Chinawhere the mortality rate of SFTS was high,Henan,Hubei,Anhui which were the three provines aroundDabie Mountain were the main areas of genotype A SFTSV epidemic,Shandong and Liaoning are the main regions of the epidemic of genotype B,and the genotypes are the most abundant in Jiangsu and Anhui provinces which were in the middle of the three above regions.A large number of reassortant strains and recombinant strainsof SFTSV were found in this study.Three were 23 reassortant SFTSV strains,accounting for 7.7%of the dataset,and 37 recombination events,making SFTSV the first reported negative-strand segmented RNA viruses with such high recombination frequency(L segment 5.1%,M segment 3.6%,S segment 0.8%).Three recombinant strains and one reassortment strain which host were non-human animals were reported for the first time.In this study,we found that the overall selection pressure of SFTSV came from negative selection,although 52 positive selection sites in the SFTSV genomewere identified.We also found a number of genotype-specific mutation sites,and proposed that two mutations(T501S and P662S)in Glycoprotein of genotype F might beassociated with the high pathogenicity of genotype F.Many co-mutation sites and their corresponding branches in the phylogenetic tree were identified as well.Finally,we used the BEAST software to analyze the origin of SFTSV in time and space.It was found that SFTSV originated in the early 18th century,and the most likely origin was from Zhejiang province(about 50%probability).Besides,genotype F is the most primitive genotype.Based on the above analysis,we have a comprehensive understanding of the genetic distribution differences,evolutionary driving forces and origin of SFTSV.This will provide the molecular fonudation for the prevention and control of SFTSV,and help the studies of other newly discovered tick-borne phlebovirus as reference data and research ideas.Zika virus(ZIKV)is a mosquito-borne flavivirus.If the virus cause fetal infectionthrough vertical transmission,it can lead to microcephaly and other birth defects.Since 2015,the disease had rapidly spread in Brazil and more than70 countries,and caused extensive concern worldwide.In February,2016,the first Zika inported case in China wasreported.Till now,24 cases of Zikavirus infection had been diagnosed in China.In the second part of this paper,we isolated and identified a Zikavirus from animported Zika case toChina in February,2016.The whole genome of the newly isolated vius strain ZKC2/2016 was sequenced by high-throughput sequencing and RACE PCR.The ZKC2/2016 strain was 10807bp in longth and belonged to the Asian lineage of ZIKV.RNA secondary structure prediction showed that its 3’-untranslated region(UTR)has multiple stem-loop structures.Phylogenetic analysis of E protein and NS5 protein revealed that nine ZIKV species in China present three small clusters in the Asian lineage.The molecular evolutionary analysis and epidemiological analysis showed that there were significant differences in epidemiological pathways and site-specific mutations among the three ZIKV clusters in China.Then,two different transmission sources of Zika virus to China were identified,namely,the ZIKV epidemic in the South Pacific Islands in 2013 and the epidemic in Central and South America in 2014-2015.In order to study the length of the genome of ZIKV,which was a disputableissue,we used multiple sequence alignment and RNA secondary structure prediction to compare the all the ZIKV genomes which were claimed to be a full-length genome in GenBank.We found that the major difference ofthe length of ZIKV genomse laid in the 5’ and 3’-UTR regions,and the genomes of less than 10790 bp were probably not complete ZIKV genomes due to lacking conserved secondary RNA structures in untranslated regions which were playing important roles in flavivirus replication.In summary,the high-throughput sequencing technology was successfully applied to obtain viral genome sequencesin this study.Molecular evolutionary analysis was conducted to study genome characteristics,phylogenetics,gene mutations and several other features of SFTSV and ZIKV,which provided important insights to understand and even predict the evolution of these viruses. |
