Molecular Epidemiology Of Tick-borne Virus In Heilongjiang And Jilin Provinces

Ticks are a large group of ectoparasites that are exclusively specialized obligate hematophages and can transmit viruses,bacteria,spirochetes and rickettsia.So far,more than 80 tick-borne viruses have been identified.In recent years,tick-borne diseases has increasingly posed a great threat to human health and the development of animal husbandry.In this study,we collected 2 402 ticks from 9 sites in Heilongjiang(Jiamusi,Hegang and Yichun)and Jilin provinces(Changchun and Jiaohe)by drag-flag method and direct picking from infested domestic animals during 2018-2019.According to the morphological and 16 S r RNA identification,these ticks were divided into Ixodidae Dermacentor,Haemaphysalis and Ixodes,including 60.3% Dermacentor silvarum(1 449/2 402),14.1% Haemaphysalis japonica(339/2 402),8.0% Haemaphysalis concinna(192/2 402)and 17.6% Ixodes persulcatus(422/2 402).D.silvarum was the dominant tick species in these samples.Viral metagenomic results showed that a total of 3 866 032 reads were annotated to vertebrate viruses,belonging to 15 viral families : Retroviridae,Geminiviridae,Reoviridae,Picobirnaviridae,Endornaviridae,Adenoviridae,Circoviridae,Parvoviridae,Flaviviridae,Nairoviridae,Peribunyaviridae,Phenuiviridae,Picornaviridae,Rhabdoviridae and unclassified virus,as well as plant viral families,Partitiviridae and Luteoviridae.According to viral metagenomics results,RT-PCR and semi-nested PCR methods were established to detect tick-borne viruses.Sequence amplification and phylogenetic analysis of 11 selected viruses were performed based on high-throughput sequencing and sanger sequencing.They were found in ticks in Heilongjiang and Jilin provinces,including nine RNA viruses: Alongshan virus(ALSV),Ngoye virus(NGOV),Tick-borne encephalitis virus(TBEV),TCV-1,JLTV,Beiji nairovirus(BJNV),Pustyn Virus(PV),Heilongjiang Tick Virus 1(HLJTV 1),Jilin Tick Virus(JLTV),Jilin Luteovirus 2(JLLV2),Jilin partiti-like virus 1(JLPV 1),and two DNA viruses(bovine parvovirus-2(BPV2)and tick circovirus(TCV)).In this study,three species in flaviviruses which threaten to human and animal health were selected for molecular epidemiological study.NGOV nucleic acid sequence was detected in H.japonicain in Jilin province for the first time,and 916 nt of NS5 gene was amplified,which shares 78.82%-68.79% nucleotide homology with the reference and 77.42%-58.22% homology in amino acid.Phylogenetic analysis showed that Jiutai V-JL strain forms a separate branch with Senegal NGOV strain,which confirms that Jiutai V-JL strain belongs to Flavivirus,and it is closely related to tick-borne encephalitis virus and Louping-ill virus,but it is further related to the mosquito borne arbovirus.The closest viral sequence to the Ngoye virus found in China indicated the presence of the new Flavivirus.ALSV NS3-Like gene fragment(1 531 nt long)was amplified in Ixodes persulcatus in Yichun,Heilongjiang province,and share 93.08%-90.20% and 99.21%-98.43% nucleotide and amino acid homologies with reference sequence,respectively.Phylogenetic analysis showed that two ALSV strains obtained in this study formed an independent evolutionary branch,which was clustered with Heilongjiang strain ALSV H3,but it is f urther related to other flaviviruses.The whole TBEV genome was amplified in Jiaohe,Jilin province,the nucleotide and amino acid homology were 95.53%-93.86% and 99.15%-94.96% with epidemic and vaccine strain,respectively.Amino acid variation was mainly concentrated on the non-structural protein NS5.Phylogenetic analysis showed that the strain in this study and the far eastern subtype epidemic strains of tick-borne encephalitis were in the same evolutionary branch,which indicated that they have closer relationship,but the strain formed a separate branch within the far eastern subtype evolution branch.In this study,epidemiological and molecular study of several viruses were carried out on ti cks sampled in some areas of Heilongjiang and Jilin provinces.A new flavivirus was discovered in present study.The diversity of tick-borne virus species and its molecular genetic and evolutionary characteristics were identified,which provided scientific data for the prevention and control measures of tick-borne virus in the regions,and provided an early warning for the emergence of new tick-borne virus.The present study also indicates important social and public health significance.