Discovery And Epidemiology Of A Novel Segmented Flavivirus

With the help of high-throughput sequencing technology,researchers have found a number of new segmented flavivirus named as Jingmenvirus,including Jingmen tick virus,Mogiana tick virus,and Guaico Culex virus.The virus genome can be divided into four or five segments.Two of the segments that derived from NS2b-NS3 and NS5 segments of the classical flavivirus are important non-structural proteins with enzymatic functions,while the other segments express viral structural proteins derived from unknown sources.The virus can infect a wide range of hosts,including arthropods such as ticks and mosquitoes,animals such as cattle and monkey(Procolobus rufomitratus).Moreover,the virus is widely distributed in central and eastern areas of China,Brazil,the United States and Uganda.In this study,a new segmental flavivirus-Alongshan virus(ALSV)was isolated and identified,and epidemiological studies of ALSV on human and animals were carried out.Firstly,to isolate and identify the ALSV,blood samples collected from hospitalized tick-bite patients at the sentinel hospital in April 2017 in northeast China were collected and analyzed by high-throughput sequencing technology.Sequencing results showed that one patient's blood sample contained virus nucleotide sequences similar to Jingmen tick virus(JMTV)with a homology of 57.4-74.5%.Moreover,mild CPE occurred in Vero cell lines after inoculation of the patient's blood samples at 5-7 dpi.After concentration,the electron microscopy results revealed that these virions are enveloped spherical or nearly spherical particles,with a diameter of approximately 80-100 nm,indicating morphological similarity with flaviviruses.Furthermore,the whole genome sequence of ALSV was amplified by nest RT-PCR and RACE successfully with a total length of 11350 bp.The genome was divided into four segments with the length of 2995 bp,2806 bp,2811 bp and 2738 bp,respectively.Considering the first patient was identified in the Alongshan area of Inner Mongolia,the virus was named as Alongshan virus(ALSV).Secondly,to explore the function of the four segments of ALSV,bioinformatic analysis was performed.Homologous analysis of amino acid sequences was conducted with MegAlign software,revealing an amino acid homology of 23.7-80.9% between ALSV and JMTV.Proteomics analysis showed that ALSV had NSP1 and NSP2 non-structural proteins which derived from flavivirus NS5 and NS2b-NS3 fragments,the virus also had three predicted structural proteins named with VP1 envelope protein,VP2 nucleocapsid protein and VP3 membrane protein.Conserved domain analysis showed that there were 6 conserved Motifs in NSP1,and 9 conserved Motifs in NSP2.Phylogenetic analysis showed that ALSV,JMTV and the variants formed a single topological branch in the genus of flavivirus,pestivirus and hepatitis virus.Meanwhile,ALSV formed a small branch separately from JMTV,indicating that ALSV is a new segmented flavivirus.Thirdly,to investigate the epidemiological characteristics of ALSV infection in tick-bite patients in this area,a total of 86 cases of acute ALSV infection were detected by screening 374 tick-bite inpatient blood samples collected from April to August 2017 using nest PCR.The statistical analysis of positive patients showed that ALSV patients were mainly concentrated in the east of Inner Mongolia and the Greater Hinggan mountains in Heilongjiang.The proportion of male(73.3%)was significantly higher than female(26.7%)in patients,and most of them were field workers(97.7%)with a history of tick bites(95.3%).In general,headache(80.2%),fever(77.9%)and fatigue(59.3%)were the main symptoms of the patients,and the patients usually became symptomatic with a incubation period of 3-7 days and recovered after 10-14 days.Lastly,to investigate the epidemiology of ALSV in cattle,sheep and ticks,240 sheep and 240 bovine serum samples were collected in Hulunbuir,Inner Mongolia in May 2018.Indirect ELISA showed that the positive rate of ALSV ELISA antibody was 4.6-9.2% in sheep and cattle,and a prevalence of 1.7-4.2% with VNT,the studies suggested that there were past viral infections in cattle and sheep population.In the same time,ALSV RNA was detected by RT-qPCR,and the total positive rate of ALSV in cattle and sheep was 26.3-27.5%.Research revealed that there were ongoing viral infections in the animal population,and the cattle and sheep might be the reservoir hosts of ALSV in nature.Moreover,a total of 1105 ticks were collected in Heilongjiang and Inner Mongolia.ALSV was detected in Ixodes persulcatus,with positive rates of 3.7% in Heilongjiang and 6.5% in Inner Mongolia,respectively.These results revealed that I.persulcatus may be the vector of ALSV.Phylogeny analysis found that ALSV of each host source clustered together in a topological branch,which indicated that the ALSV from different host sources represent was the same epidemic virus.The virus formed a complete transmission circle in the epidemic source areas,which may be a new tick-borne zoonosis.In conclusion,a new segmented flavivirus was identified and isolated in humans and named as Alongshan virus.As the world's first segmental flavivirus found to infect humans,ALSV has important guiding significance for the prevention and control of tick-borne diseases and provides a new direction for the research of tick-borne diseases.