Molecular Evolutionary Analysis Of Hantaan Virus And Establishment Of Multiplex QRT-PCR Assays For Hemorrhagic Fever Viruses Detection

Viral hemorrhagic fevers(VHFs)are a group of natural epidemic diseases with fever,hemorrhage and shock as the main clinical features.They have the characteristics of acute onset,more serious clinical manifestations and high mortality.Varity of enveloped RNA viruses in the family Flaviviridae,Filoviridae,Bunyavirales,Hantaviridae,Arenaviridae,Nairoviridae,Phenuiviridae can cause VHFs.The clinical symptoms of diseases caused by these agents varies differently and most of the diseases have no specific therapies and effective vaccines.Therefore,the establishment of rapid and effective laboratory testing methods and getting more knowledge of viruses'molecular evolutionary mechanism would be of great significance to the early diagnosis of patients,epidemiological studies of diseases,and ultimately the prevention and control of VHFs.Hantaan virus(HTNV)in the family Hantavirida,order of Bunyavirales caused hemorrhagic fever with renal syndrome(HFRS),which has raised serious concerns in Eurasia,in particular in China,Russia and South Korea.Previous studies reported genetic diversity and phylogenetic features of HTNV in different parts of China,but the analyses from the holistic perspective are rare.In the first part of this study,we analyzed all available complete sequences derived from the S,M and L segments with bio-informatic tools.11 phylogenetic groups were defined and showed geographic clustering;51 significant amino acid variants sites were found and 21 of them located in immune epitopes;11 recombinant events and 8 reassortments with highly divergent sequences were found and analyzed.We found that sequences from Guizhou were highly genetic divergent,contributed to multiple lineages of the phylogenetic tree,also to the recombination and reassortment events.Bayesian stochastic search variable selection(BSSVS)analysis revealed that Heilongjiang,Shaanxi and Guizhou played important roles in HTNV evolution and migration,the virus may originate from Zhejiang Province in east part of China and the virus population size expanded from 1980s to 1990s.These findings revealed the original and evolution features of HTNV which will help to understand Hantavirus epidemic trends and better for disease control and prevention.In the second part of this study,we updated and optimized the existing Real-Time RT-PCR methods for hemorrhagic fever virus in our laboratory,then designed the primers and probes for New York virus,Andes virus,and Sin Nombre virus and Zika virus using all the whole genome sequences obtained from Genebank.These primers and probes were combined and the system was optimized to establish a PCR detection module containing 8 multiplex PCR assays,which can detect 25 hemorrhagic fever viruses.We evaluated the sensitivity and specificity of the multiplex qRT-PCR assay using in vitro transcribed RNAs.The results showed that all of the LODs of the multiplex qRT-PCR assay are below 30copies/?l and equivalent to that of single-channel detection.Also,no non-specific reaction was found among the primer-probe sets.A total of 2000 healthy human sera samples,6336 clinically or simulated positive samples,and 60 rat lung samples were used to verify the performance of detection assays.The results showed that all the total intra-group coefficients of variation of the simulated positive samples with different concentrations of each virus was less than 5%.The single-channel and multiple detection results of clinical samples and rat lung samples were consistent,and no false positives appeared in the serum of healthy people It indicated that the multiplex qRT-PCR assays established in this study has good detection ability,and is expected to be used in pathogens screening and epidemiological studies of patients with VHFsIn summary,through this study,we have deepened our understanding of HTNV genome characteristics,phylogenetic and evolutionary mechanisms,and established multiple qRT-PCR assay that can be applied to detect 25 common hemorrhagic fever viruses.These results provide basic information for our understanding of hemorrhagic fever virus,and also provide technical support for the diagnosis and epidemiological research of viral hemorrhagic fever disease.