Isolation,Identification And Epidemiological Studies Of Tacheng Tick Virus 2

Recently,many novel pathogens have been discovered and characterized with the rapid development of meta-genomics technology and its application in insect-borne diseases.These pathogens have been of significant importance in public health for humans and animals.In 2005,Tacheng Tick Virus 2(TCTV2),a novel negative-sense single stranded RNA virus,was identified in ticks by meta-genomics.The virus belongs to the genus Phlebovirus,family Phenuiviridae,class Bunyavirale.However,rarely any epidemic data is available in humans and animals,in addition to pathological investigation.In the current study,TCTV2 was isolated from Asiatic Hyalomma by using SMMC cell culture in Xinjiang region.The morphology of TCTV2 has been studied with EM.Then,the genomic sequence of the L fragment and S fragment of TCTV2 were amplified by semi-nested RT-PCR.A highly specific ELISA method has been established with a recombinantly expressed N protein as antigen.With the established ELISA protocol,epidemic surveillance was carried out on the blood samples from herdsmen in some areas of Xinjiang.Moreover,infection status of cattle,sheep,and ticks was monitored by using semi-nested RT-PCR method.The presented study clarified the prevalence status of TCTV2 in humans,sheep and ticks,which have potential significance for monitoring,prevention,and early warning of the new tick-borne zoonotic disease.1.Obvious cytopathic changes were observed in SMMC cells that inoculated with tick grinded supernatant after three passages.TCTV2 infection was confirmed with PCR and sequencing that showed 99% identity to TCTV2 KM817744(S segment).The morphology of the TCTV2 particles was investigated under the electron microscopy.Spherical structure with the diameter of 90-100 nm,a typical characteristic of Bunya virus,was observed.Collectively,these results showed that the TCTV2 was successfully isolated.2.L and S segments of the TCTV2 were obtained with RT-PCR which followed by phylogenetic analysis.The sequencing results showed that the L and S segments of the virus contained 6659 bp and 2181 bp nucleotide,respectively.Phylogenetic analysis showed that the identity of L segment and S segment with the corresponding fragment of TC252(Genbank accession: KM817684 and KM817744)were 98.8% and 99.23%,respectively.This suggest that the newly isolated TCTV2 strain is probably a derivative of the original strain that found in Xinjiang in 2015.3.The N protein was recombinantly expressed in E.coli.Then,the Ig M and Ig G of herdsmen serum samples were detected by indirect ELISA using purified N protein as antigen.The overall infection rate of the TCTV2 in the studied population was 9.55% while 2.95% were in the early stage of infection,which suggest that the TCTV2 has a high infection rate in herdsmen.4.Semi-nested RT-PCR was used to investigate the epidemic status of TCTV2 in domestic animals and ticks.The result showed that the positive rate was 4.23% in sheep blood samples.In some areas of Xinjiang,the total infection rate of the TCTV2 reached 11.95%,and the Dermacentor marginatus collected from horse had an infection rate of 55%.Then,the infection rate of the Asiatic Hyalomma collected from camel body surface reached 19.04%.The result indicated that Dermacentor and Hyalomma are important vector of the TCTV2.The virus was present in host blood and may transmitted to other hosts by ticks.