Isolation,Identification And Pathogenicity Analysis Of Tick-borne Parainfluenza Virus Type 5

As a vector of various zoonoses,ticks have a serious impact on human health and the sustainable and stable development of animal husbandry,causing increasing economic losses in animal husbandry worldwide.With the impact of human activities and animal trade,the spread of ticks has been further expanded,which may lead to the occurrence and prevalence of a larger range of tick-borne diseases.Therefore,in order to ensure the safety of human life and the healthy and stable development of animal husbandry,it is necessary to master the animal virus community carried by ticks,identify the known and unknown pathogens,isolate and identify the important pathogens indicated,and study the genetic variation and pathogenicity.Parainfluenza virus type 5(PIV5)was initially isolated from monkey kidney cells,with the passage of time more and more data suggest that PIV5 reports on animal pathogenic infection and host range increase gradually,infected host spectrum gradually from the domestic economic animals to wild animals and animal husbandry economy,forcing we gradually began to pay attention to it.At present,there has been no report of PIV5 infection by ticks.PIV5 is mainly transmitted by respiratory route and it is now known that PIV5 can lead to acute bronchitis in sick dogs clinically,and cause secondary infection and other symptoms.In addition,the PIV5 has also been found to cause respiratory diseases such as pulmonary dyspnea and interstitial pneumonia in calves,and PIV5 infection can also cause non-suppurative encephalitis and neurological symptoms in cattle,resulting in death of the infected animals.The disease has been widely paid attention to causing more and more serious clinical symptoms.Viral metagenomics,as a new method to study viruses,can analyze the samples directly enrichment virus nucleic acid mixtures of different sources,which has high accuracy and high flux,high sensitivity and low cost advantages,can be done at the same time traditional genomics such as sequence and annotation,and do not need to be done in tissue culture in vitro amplification and to develop.As a new method,when the clinical symptoms of the pathogen are not obvious,it can identify and find the pathogen,excavate the potential pathogen in the suspected sample to the greatest extent,and provide technical support for the discovery,isolation and identification of the pathogen.In this study,with next-generation sequencing and virus metagenomic analyses used to assess the viromes of Ixodes persulcatus.Firstiy,the ticks collected from the Greater Khingan Mountains,Heihe and Yichun regions of Heilongjiang province were sampled by flag method,with morphology preliminary identifition,the ticks were grinded processing by liquid nitrogen and diluted in PBS,through high-speed centrifugation and membrane filtration to remove impurity elements,after using the nuclease digestion,extraction of virus nucleic acid using random primer amplification,concentration and purification,the products were analyzed by high-throughput sequencing.The low-quality sequences in the original data were screened and filtered,and the obtained high-quality sequences were assembled.A total of 390,455,830 reads remained and then adaptor and quality trimmed them.The high quality reads were 385,965,588.Following compared and assembled revealed that 3,642,653 reads showed identity to annotated viral ORFs.It mainly includes virus sequences corresponding to Bunyavirida,Paramyxoviridae,Poxviridae,Baculoviridae,Ascoviridae,Siphoviridae,Nudiviridae,Herpesviridae,Polydnaviridae,Panoravirus,Reoviridae,Hypoviridae,Ortervirales and so on.In order to further verify the results of metagenomic sequencing,the virus was isolated and identified systematically by cell isolation and culture,PCR detection,electron microscope observation,immunofluorescence identification and full-length gene amplification.First,the tick treatment solution was inoculated into Vero cells.On the 5th day,there were obvious lesions,such as drawing,becoming round and shedding,and cell aggregation.Nucleic acids were extracted from the supernatant of diseased cells and the treatment solution of tick samples for RT-PCR amplification.The amplified fragment with a size of 213 bp was obtained,which was consistent with the expected result.The cell culture was subjected to repeated freeze-thaw and blind transmission for 3generations,after which the virus was harvested and inoculated with the virus venom of the fourth generation Vero cells,the TCID₅₀ of the isolate was calculated as 1×10^5.7 TCID₅₀/m L by Reed-Muench method.The diameter of the virions observed under electron microscopy was 50-60 nm,and the complete virions were spherical with capsule membrane,which was in line with the characteristics of PIV5 virions under electron microscopy,and specific fluorescence could be observed by immunofluorescence identification.The full-length genome sequence of the isolated strain was amplified by RT-PCR for sequencing analysis,and it was proved that there was PIV5 in the vector tick.It was named Tick/HLJ/2019and the login number was MW051776.Genetic analysis of the full-length gene sequence of the virus showed that the Tick/HLJ/2019 isolates were in the same evolutionary branch with the Chinese red panda ZJQ-221 isolates,the siberian tiger SR and HMZ isolates,the pangolin GD18 and Can isolates and the Korean dog 1168-1 isolates,and the genetic relationship was relatively close.Different from other PIV5reference strains from pigs,cattle,macaque kidney cells,human and horse,it is suggested that the virus may be transmissible among wild animals.Ferret animal model experimentally infected with Tick/HLJ/2019 virus by intranasal inoculation developed severe respiratory distress with pneumonia and neurologic tissue damage inflammation.The findings of this study confirm the presence of PIV5 in tick and indicate this arthropod as a possible natural reservoir.Further surveillance of the circulating transmission of PIV5 in ticks is necessary for tick-borne disease control and vaccine research in China.With high-throughput sequencing technology of virus metagenomic analyses was used to assess virus community in Ixodes persulcatus,and the tip of the important pathogenic PIV5 separation identification,genetic evolution and pathogenic analysis,in order to fully understand the spread of the disease,and make effective control strategy,to assess the potential risk to lay the foundation for animal and human health.