Molecular Epidemiological And Serological Research On Bat-borne Hantaviruses In Southern China

As important zoonotic pathogens,hantaviruses cause two major life-threatening diseases in human:Hemorrhagic fever with renal syndrome?HFRS?in Eurasia and Hantavirus cardiopulmonary syndrome?HCPS?in the Americas.Our country is most seriously affected by HFRS,among the 10,000-15,000 cases worldwide every year,more than 90%were from China.Hantaviruses?family Hantaviridae,order Bunyavirales?are a group of spherical or pleomorphic viruses with the diameter of 80-120 nm,containing three single-stranded RNA genomic segments designated as small?S?,medium?M?and large?L?with the total length of 11.8 kb.In the 10^th report of ICTV,41 species were contained in the genus Orthohantavirus.Rodents had been considered the only reservoir of hantavirus,yet 9 novel hantaviruses have been identified in bats worldwide since 2012 with their hosts of 6families from Africa,Asia and Europe respectively.These bat-borne hantaviruses showed abundant genetic diversity and ecology,raising the attention of science on whether bats are natural reservoirs of hantaviruses.However,there are a lot of defect and deficiency in the study of bat-borne hantaviruses:insuffiency of whole genomic sequence,failure on virus isolation,and no systematic investigation,leading to the poor understanding of bat-borne hantaviruses on their genetic diversity,pathogenicity and the cross-reactivity.Therefore,further research is urgently needed.Herein,we first applied the viral metagenomics platform based on high-throughput sequencing to research the genetic diversity of bat-borne hantaviruses in Southern China.During 2012 to 2016,1,451 bats were captured in Yunnan,Guangdong,Fujian and Zhejiang provinces and Guangxi Zhuang Autonomous Region,comprising22 species in 9 genera of 5 families.Through high-throughput sequencing,413 reads annotated to hantavirus were obtained.Then by RT-PCR,10 strains of hantaviruses were detected:a novel hantavirus were identified in the lung tissue of a black-bearded tomb bat from Laibin city and Baise city in Guangxi separately with the positive rate of 3.13%?1/32?and 2.56%?1/39?,designated Laibin virus?LAIV?strain BT20 and BT33;variants Xuan son virus?XSV?were detected in the lung tissue of Pomona roundleaf bats in Pu'er city in Yunnan Province and Laibin city,with the positive rate of 9.1%?5/55?and 7.5%?3/40?respectively.The whole genomic sequence of five representative strains?2 LAIVs and 3 XSVs?were successfully obtained,standing for the first obtain of complete genomic sequences of bat-borne hantavirus worldwide.Phylogenetic and homological analysis demonstrated LAIV were circulating in black-bearded tomb bats in Guangxi Province and XSV in Hipposideros pomona bats in Yunnan and Vietnam,forming the individual evolutionary group and transmission circle in Guangxi-Yunnan-Vietnam area.To figure out the distribution and host characteristics of bat-borne hantaviruses,serological investigation on LAIV,XSV and rodent-borne SEOV was further conducted.First,the prokaryotic expression vector of pET28a?+?-rNP for LAIV-BT33,XSV-AR18and SEOV-GuangzhouRn36 were constructed respectively,then the three recombinant nucleocapsid proteins?rNPs?were induced by IPTG,and purified through affinity chromatography of Nickel(Ni²⁺)column.To study the cross-reactivity among them,hyperimmune mouse sera were prepared by injecting the young mice with prokaryotic expressed rNPs,and rNPs were expressed in 293T and BHK cell lines,through the reaction among the rNPs and hyperimmune sera,cross-reactions were observed.Then indirect ELISA were established using prokaryotic expressed rNPs as the antigens.Totally,709 bat sera samples were detected by ELISA,collected from 13 sites in Yunnan,Fujian,Zhejiang provinces and Guangxi Zhuang Autonomous Region,comprising 16 bat species in 8 genera in 5 families.And 88 sera were furtherly tested by Western blotting,cut-off OD₄₉₂ values were settled based on the highest coincidence rate between Western blotting and ELISA?0.10 for LAIV and XSV,and 0.11 for SEOV?.Based on the standard of ELISA,131 of 709 bat sera?18.5%?were antibody positive to hantavirus?any of the three viruses?,of which 90?12.7%?were positive to LAIV,93?13.1%?to XSV and 93?13.1%?to SEOV.Furtherly,fluorescent antibody virus neutralization test?FAVNT?were performed to detect antibodies in 48 sera using SEOV cultured by Vero E6 cells,results showed that 9 sera were positive,conforming the existence of neutralized antibodies.To further explore the threat of bat-borne hantavirus spreading across species,human sera were collected from Honghe Yi Autonomous Prefecture?n=99?and Wenshan Zhuang and Miao Autonomous Prefecture in Yunnan Province?n=50?.Only one serum sampled from Honghe was positive by ELISA,both to XSV and SEOV-rNP,with the positive rate of 0.7%?1/149?.WB also showed its reactivity to both XSV and SEOV-rNP,and neutralized antibody with the titer of 1:34 was observed in FAVNT.In this study,we had conducted the research on molecular epidemiology and serology of bat-borne hantaviruses in Southern China.Through viral metagenomics,genomic sequencing and genetic evolution analysis,we obtained the molecular epidemic data such as genetic diversity,positive rate and geographic distribution of bat-borne hantavirus.Results of seroepidemiology and cross-reactivity demonstrated the wide hantavirus infection in bats in Southern China,furtherly confirmed that bats were natural hosts of hantaviruses.In addition,we found the area of Guangxi-Yunnan-Vietnam might be an original emerging area of bat-borne hantaviruses.This study provide important data for the origin-tracing,monitoring and predicting of hantavirus-caused disease,however,it will take further work to study whether bat-borne hantaviruses infect human or evolve into human pathogens.