Identification Of Bat Adenovirus And Cloning And Expression Of Hexon Protein

ObjectiveBats have been recognized as important viral hosts in recent years.This species have always brought great hidden dangers to human being because of their complex feeding habits,long distance flight,and close contact with humans and livestock,.The coastal climate in southeast China is warm,humid and fits an ideal habitat for many bats.It is also the natural epidemic focus of many diseases.Adenovirus is a kind of uncoated icosahedral virus with a size of about 70-90 nm and a linear double-stranded DNA genome of 26 to 46 kb.Adenoviruses can always makevertebrates infected,including mammals,birds,amphibians,reptiles and fish.Human diseases caused by adenovirus include acute respiratory diseases,epidemic keratoconjunctivitis,acute hemorrhagic cystitis,hepatitis,myocarditis and gastroenteritis.In order to uncover the pathogenic mechanism of bad adenovirus,the adenovirus carried by bats in the southeast coastal area was studied.Firstly,the positive rate of adenovirus carried by bats in this area was investigated.Then the whole sequence was isolated by virus isolation and its pathogenicity was studied.Methods1.Bat sample collectionA variety of adult bat anal swabs and throat swabs are collected from caves and tank tunnels in various areas of the southeast coast（Daishan,Dinghai,Taohuadao,Xiamen,Changle,etc.）,which are stored in liquid nitrogen tanks and brought back to the laboratory.Sample processing,taking a portion of the sample to extract nucleic acids.For the remaining samples collected,place them at-80 degrees.2.Primers design and sample population identificationBased on the bat adenovirus sequence already reported by NCBI,multiple specific primers were designed and PCR was performed on the collected sample nucleic acids.At the same time,the mitochondrial pigment B primer was used to identify the sample.The target fragment of the PCR positive sample was subjected to tapping recovery and purification,and the purified product was sent to Shanghai Shenggong Biological Co.,Ltd.for sequencing,and the sequencing result was subjected to BLAST alignment in the NCBI database.3.Virus separationThe homogenate prepared from the adenovirus positive sample was inoculated onto Vero E6 cells in a 6-well plate.The 6-well plates were incubated for 2 hours at37°C and 5%CO₂ to adsorb the virus,while gently shaking the 6-well plate every half an hour to aid adsorption.The cells were cultured in fresh medium（DMEM）,supplemented with 2%fetal bovine serum（FBS）,and cultured at 37°C,5%CO₂ for 2weeks or lesions（CPE）.The supernatant of the cell culture was blindly passed 3 times to monitor the CPE.They were inoculated onto Vero E6 and other cell lines according to the methods described above.4.Adenovirus suckling mice challenge experimentThe isolated and purified adenovirus was injected into the brain tissue of Kunming strain mice born for 3 days,a group of 12,each injection of 100 microliters,another set of two control groups,one group did not do any treatment,one group of injections 100microliters of DMEM.After 2 weeks of feeding under the same conditions,the brain,liver,kidney,lung and intestinal organs were taken and divided into three,one for nucleic acid extraction and virus identification;one was placed in 4%paraformaldehyde.In the subsequent immunohistochemistry experiments and HE staining experiments;one part was placed in glutaraldehyde solution for subsequent electron microscopic observation.Result1.Positive rate statisticsIn 2015 to 2019,a total of 552 bat samples were collected from the southeastern coastal areas of Zhou-shan,Dinghai,Taohua Island,Changle,Xiamen,etc.,36 batsamples were identified to carrry adenovirus.The total positive rate was 6.5%.The results of the population identification showed that 18 positive samples were from the phoenix head bat.2.Virus separationOn the 7th day of vaccination positive sample（second pass of blind transmission）,3 cell holes appeared in the 6-well plate（CPE）,and the PCR test was positive for adenovirus.After the blind passage,the CPE was more obvious,and the PCR positive was also more.Obviously,adenoviral particles with distinct characteristics can be seen under an electron microscope.The obtained adenovirus solution was purified by plaque purification,and the purified adenovirus was stored in a-80℃refrigerator for subsequent in vivo experiments and deep sequencing.3.Suckling mice challenge experimentThree groups of Kunming strains were sacrificed for 2 weeks under the same conditions,and the brain,liver,kidney,intestine and lung tissues were taken and divided into three.Adenovirus was detected in the intestinal tissues of 12 experimental mice.Both groups were negative.ConclusionsAdenovirus has a certain carrying rate in bats in the southeastern coastal areas,and there is a certain probability of cross-species transmission and pathogenicity.It is of great significance to the epidemiology and statistical study of bat-borne adenovirus in the southeast coastal areas.