Preliminary Study Of Small RNA High-throughput Sequencing For Tick-borne Virus Detection

Ticks are an important zoonotic parasite.Besides biting and sucking blood,ticks carry viruses,bacteria,rickettsia,protozoa and other pathogens which transmit many diseases.Tick-borne viruses are rich in species,complex in composition and widespread in ticks.With economic development and increased human activity,tick-borne viruses increasingly threaten human and livestock health.The establishment of an accurate,rapid and convenient detection method for tick-borne viruses is of great importance.With the rapid development of high-throughput sequencing technology,many studies have used macrogenomics to detect tick-borne pathogens.However,macrogenomics based on DNA sequencing cannot detect RNA viruses without DNA stage.For this reason,the method based on transcriptome sequencing is used to detect tick-borne virus,but this method obtains a large number of redundant host sequences.Small RNA high-throughput sequencing to detect pathogens is more sensitive than transcriptomic analysis.At present,it has been successfully used to detect the DNA fragments of African classical swine fever(ASFV)and tick-borne rickettsia.Furthermore,this method has more advantages in detecting RNA virus.Therfore,small RNA high-throughput sequencing was used to detect tick-borne virus in this study,which mainly included the followings:1)By integrating NCBI virus data,a local database of invertebrate transmitted virus was established,which laid a foundation for improving the efficiency and accuracy of small RNA highthroughput sequencing in tick-borne virus detection.2)Small RNA high-throughput sequencing was carried out on Amblyomma testudinarium collected from Yunnan ? Dermacentor nuttalli and Dermacentor niveus collected from Xinjiang respectively.The sequenced small RNA high-throughput raw data were processed for tick-borne virus detection.It was found that there were small RNA fragments of Mogiana tick virus and Stealth virus(AF191073)in Amblyomma testudinarium from Yunnan.The following viruses: Bluetongue virus,Stealth virus,Colorado tick fever virus,Epizootic hemorrhagic disease virus,African horsesickness virus,Equine encephalosis virus,Rift Valley fever virus,Vesicular stomatitis virus,Tehran virus,Uukuniemi virus were detected from the Dermacentor ticks collected in Xinjiang.3)PCR amplification and Sanger sequencing were used to verify the existence of Mogiana tick virus in Amblyomma testudinarium,which proved the feasibility of small RNA high-throughput sequencing method for tick-borne virus detection.Then,combining the results of Sanger sequencing and small RNA sequencing,the full-length genome data of Mogiana tick virus are obtained and 5? and 3?untranslated regions(UTRs)were precisely annotated at 1-bp resolution.It is confirmed for the first time internationally that using 5?end and 3?end small RNA annotation method can obtain complete fulllength sequences of virus genome.4)Based on PCR amplification and in-depth mining and analysis of small RNA sequences,it was confirmed that the Stealth virus(AF191073)reported on Genbank was misidentified,which should be part of the 23 s rDNA sequence of Ochrobactrum quorumnocens.Tick samples of Amblyomma testudinarium from Yunnan,Dermacentor niveus and Dermacentor nuttalli from Xinjiang detected Ochrobactrum quorumnocens.It is also proved that high-throughput sequencing of small RNA can detect not only viruses but also ribosomal RNA of bacteria.The studies showed that the method based on small RNA high-throughput sequencing can be used not only to detect tick viruses,but for many viruses.In addition to viruses,other microorganisms,including bacteria,can also be detected by this method.However,due to the limitation of the sequencing depth of this study,it needs to be further studied whether all viruses can be detected,or what kind of sequencing depth can be detected comprehensively.