Discovery And Confirmation Of Several New Tick-borne Pathogens In Parts Of Sichuan And Gansu Provinces Of China

Objectives(1)To discover new tick-borne pathogens that may existed in parts of Gansu and Sichuan provinces.(2)To understand the characteristics of representative new tick-borne pathogens.Methods(1)Tick collection,identification,preservation and transportation.Free ticks and parasitic ticks were collected by flagging and body surface collection methods respectively in Wuwei City of Gansu Province in northwest China and Wolong Giant Panda Reserve of Sichuan Province in southwest China.Ticks were distinguished by morphological identification.And the collected ticks were stored in porous centrifuge tubes with moistened filter paper and gauze,and then kept in an artificial climate box.Ice bags were used to keep low temperature during transportation of ticks.(2)High-throughput sequencing and analysis.The DNA/RNA extracted from tick samples is first tested for purity,concentration and integrity,followed by a library and sequencing.Quality control and remove hosts’ genome for the acquired data were constructed through bioinformatics software,which is helpful to discover pathogens.(3)PCR and q PCR detection.Total nucleic acid extracted from ticks slurry and culture was detected by using specific primers through conventional/nested PCR or one-step touchdown PCR or realtime fluorescent quantitative PCR methods.(4)Pathogen isolation and identification.Ticks’ slurry were inoculated on different cells(BHK21 、Vero81 、HUVEC 、CTVM23、IDE8),and then placed in the incubator.To ensure the growth of pathogens,we need to discard culture supernatant and add new culture medium every week.After 4weeks,DNA was extracted from the culture and use real-time fluorescence quantitative PCR to detect Ct value of Rickettsia sca1 gene.(5)Phylogenetic analysis.The representative reference sequences was selected on the basis of blast comparison results of pathogen sequences obtained.After comparing the reference sequences with the obtained sequences,the phylogenetic tree was constructs by maximum likelihood method and 1000 bootstrap value of MEGA＿11.0.13.Results(1)Collection of tick samples.The parasitic ticks of giant pandas and goats and free ticks were collected in Wolong Nature Reserve,Sichuan Province.A total of 19 adult ticks were collected,including three species of Ixodes acutitarsus,Haemaphysalis longicornis and Ixodes ovatus,of which three female ticks laid eggs.One hundred and thirty-four ticks of 6 species were collected from 9 sampling sites and 3 habitats(grassland,plain and desert)in Wuwei area,Gansu Province,all of which were adult ticks,including Dermacentor nuttalli,Dermacentor silvarum,Haemaphysalis kolonini,Haemaphysalis qinghaiensis,Hyalomma asiaticum and Hyalomma scupense.(2)High-throughput sequencing results.Fifteen RNA libraries were constructed from tick samples in Sichuan.After processing,165.15 GB of data and 110846 valid reads were finally obtained.After being processed by bioinformatics software,viruses belonging to 119 families and 538 genera were found.Through further sequence assembly,4 complete sequences similar to the Jingmen tick virus were discovered in 4 samples,with a total of 16 segments.Twenty-two RNA libraries were constructed using tick samples from Gansu Province.After processing the data,the effective data were 201.01 G data and1340100376 reads.After being processed by bioinformatics software,viruses belonging to 618 genera and 121 families were detected.Through further sequence assembly,14 virus sequences were obtained,which are similar to Bole tick virus 1,Bole tick virus 2,Bole tick virus 3,Changping tick virus 1 and Taishun tick virus.(3)Results of PCR and q PCR detection.An unknown Rickettsia and Rickettsia raoultii were detected in Haemaphysalis qinghaiensis and Dermacentor silvarum respectively in the tick slurry of Gansu Province.The unknown Rickettsia was the most similar to Candidatus Rickettsia thierseensis,with 84.09 % identity.(4)Pathogen isolation and culture results: After 4 weeks of inoculation and culture of tick slurry,Rickettsia sca1 gene was detected to be positive in the culture of Dermacentor silvarum,but it failed to achieve stable passage and proliferation after 3months of culture,and the Ct value remained around 30.(5)Phylogenetic analysis results.A potential new JMTV virus of Jingmen virus group was discovered in four tick samples in Sichuan,which was named as Sichuan tick virus(SCTV).Among the 14 full virus sequences in Gansu Province,one virus is an unknown flaviviridae virus,which has highest identity(84.87%)with Dermacentor reticulatus pestivirus-like virus 1.It is a potential new tick-borne virus,and is temporarily named "Gansu tick virus".An unknown Rickettsia was found in Haemaphysalis qinghaiensis in Gansu Province,which is the closest to Candidatus Rickettsia thiersensis,with 84.09% identity.It is suspected to be newly rickettsia,and is temporarily named "Rickettsia gansuensis",which is a new record of rickettsia in Gansu Province.Conclusion(1)Jingmen tick virus-like viruses and other tick-borne pathogens pose threat to wildlife in the Giant Panda Reserve of Sichuan Province.It is necessary to further expand the pathogen investigation of host itself and carry out risk assessment.(2)In Wuwei area of Gansu Province,China,there are various habitats and abundant tick-borne pathogens.It is worthwhile to further study the potential risks of tick-borne new viruses and new rickettsia.