Genetic Characterization Of Hantavirus In Key Epidemic Areas Of Hemorrhagic Fever With Renal Syndrom In China

Epidemic hemorrhagic fever(EHF),also named hemorrhagic fever with renal syndrome,is a zoonoses caused by Hantavirus(HVs)with a clinic signs of fever,bleeding and kidney injury.Hantaviruses belong to the Orthohantavirus genus of the Hantaviridae family and are tripartite negative-sensed RNA virus,which contain a tripartite negative-sensed RNA genome consisting of following three segments:the small-sized(S)segment encodes the viral nucleocapsid protein(NP),the medium-sized(M)segment encodes two viral glycoproteins(GPs,Gnand Gc),the large-sized(L)segment encodes a viral RNA-dependent RNA polymerase.Rodents are the main natural reservoir.Virus infection of host are mostly persisted for a long time and without any clinical signs of illness.Hosts carry viruses for a long time and keep releasing virus into environment through the urine,saliva and feces.Hantaviruses can be transsimited from host to human by eating contaminated foods or inhalation of contaminated aerosol or bitten by rodents,and causing serious diseases such as hemorrhagic fever with renal syndrome(HFRS)and Hantavirus cardiopulmonary syndrome(HPS).China is currently the country with the most serious HFRS epidemic in the world.The HFRS is widely spread(Cases have been reported in all provinces,municipalities and autonomous regions except Qinghai province)and there are a lot of cases and the mortality rate of this illness is high.It is an important zoonotic infectious disease that seriously threatens people’s lives.Epidemic surveillance and epidemiological studies are important means for disease prevention and control.The comparative study of Hantavirus prevalence in human population and mouse population is very important for the prediction of human epidemic situation and the evaluation of the protective efficacy of existing vaccines.To understand the epidemic characteristics of Hantavirus in the key epidemic area of HFRS in China and provide evidences for the optimization of the detection procedures for Hantavirus surveillance in rodents.Human and mouse samples were collected from key epidemic areas of epidemic hemorrhagic fever during 2018 spring in China(Hebei,Liaoning,Heilongjiang,Jiangxi,etc.).45 positive mouse lungs and 12 IgM positive human acute phase serums(the interval between onset date and sampling date is 6 days)were selected for virus isolation,and mouse lungs which were not successfully isolated virus were used to amplify the whole genome segment of hantavirus by PCR,All virus strain were sequenced trough the method of first generation sequencing and second generation sequencing,phylogenetic and homology of all virus gene sequences were analysis in this study.Meanwhile 392 mouse lung samples and mouse blood samples were used for comparision of three main detection methods of nucleic acide,antigen and antibody of Hantavirus.Results:a total of two strains of virus(LNTL001 and LNTL003)were isolated from rat lung samples in Liaoning province,and one strain(HBCD041 and HLJNH013)were isolated from rat lung samples in Hebei province and Heilongjiang province respectively.The whole genome of 16 strains of Hantavirus were obtained from the mouse lung samples from hebei,liaoning and heilongjiang by segmentation-amplification of the whole genome of Hantavirus,and the Gn and Gc gene partial segments of 5 strains of Hantavirus were obtained from the lung samples from Jiangxi province.The S,M and L sequences of the genome of 22 virus strains nd the Gn and Gc gene sequences of 5 virus strains were obtained by first or second generation sequencing.The phylogenetic analysis and homology analysis show that the genetic distance of all virus strains from hebei,Liaoning and Heilongjiang provinces were closed to the strains of 80-39,L99and Z37,and far away from the virus strains of 76-118,84Fli and Z10,and located at the same clade of S3 subtypes of SEOV.The homology between 4 isolations strains were 97%～99%,and the homology between 18 viruse strians which were directed sequenced from mouse lung strains were 97%～99%.The homology between virus strains from same province were 99%,while the homology between virus strains from differente province were 97%.The isolations the homology between 4 isolations strains and directed amplified virus strains were 99%.There are different several amino acides between this 22 virus and vaccine strains and the difference between L99 and new virus strains are more huger than Z37.5 virus strains directed amplified from mouse lung from Jiangxi province were validated by gene type specific RT-PCR:1(AYN20)were SEOV and 4(AYW10,AYW45,AYW50,GAW50)were HTNV,homology among 5 virus and all known S3 virus strains are lower than 93.6%,and form a new genetic branche independently which can be hypothesized to be a new hantavirus subtype.Beside,the strain of AYN20 and GAW50 have the possibility of gene recombination.The results of second-generation sequencing of four isolates were 99%consistent with those of first-generation sequencing,indicating that the first-generation sequencing and the second-generation sequencing results were in good consistent.In practice,one of them can be selected to the virus genome sequencing according to the sample quality and laboratory conditions.The results of this comparison study of 3 detection method of nucleic acide,antigen and antibody show that 46 antibody-positive mouse blood samples were detected by ELISA and thepositive rate were 12.53%,28 RNA-positive mouse lung samples were detected by RT-PCR,with a positive rate of 7.63%;And 24 were detected N protein antigen positive by IFA,and the positive rate was 2.3%.All IFA detection positive samples(100%,24/24)and most RT-PCR detection positive samples(89.3%,25/28)were with detected antibodies by the ELISA.The results showed that there was no significant differences between RT-PCR and IFA test results(χa2 = 0.64,P>0.05),with a Kappa coefficient of 0.71,and good consistency(Z=13.66,P<0.05),which is in accordance with the characteristics of Hantavirus infection and replication in rodents.The related detection based on the results of ELISA can significantly narrow the range of RT-PCR and IFA for detecting viral RNA or antigen(χb2 = 12.04,χc2 = 20.05,P<0.05)The results showed that most samples with detected viral RNA or antigens were with detectable antibodies,and the samples positive for antibody detection by ELISA basically contain the samples positive for antigen and RNA,and,which provides a robust evidence for the optimization of the detection procedures for hantavirus surveillance in rodents.In a conclusion,this research successfully isolate four virus strains from mouse lung samples from hebei,Heilongjiang and liaoning province,and all 27 virus strains were obtained through virus isolation and direct amplification,whole genome of 22 virus and Gn gene and Gc gene partial segment were sequenced by the method of first-generation sequencing and second-generation sequencing.The result of phylogenetic analysis show that Hantavirus had less variation in host animals in hebei,heilongjiang and liaoning,while Hantavirus had more variation in host animals in jiangxi province.The genetic characteristics of Hantavirus among hosts in the 4 key provinces of epidemic haemorrhagic fever were analyzed by genetic evolution.Three method to detect Hantavirus comparative study confirmed that the host animal tissue samples detected Hantavirus RNA and(or)structural protein antigen of specimens,the corresponding specific antibodies can be detected virus in blood samples,and vice versa,specimen contains the basic detection antibody can be detected with the antigen and RNA samples.In the HFRS host field epidemiological investigation and study,the antibody of Hantavirus can be firstly screened antibody,than validate the antigen and nucleic acide.This conclusion provides a robust evidence for the optimization of the detection procedures for Hantavirus surveillance in rodents.