| Bovine adenovirus type 3(BAd V-3),a member of the mammalian genus Mastadenovirus in the family Adenoviridae,is a linear double-stranded DNA virus without envelope encapsulation,which can cause respiratory symptoms such as fever,pneumonia and bronchitis in calves and is one of the important pathogens leading to respiratory diseases in cattle.BAd V-3 infection has been widely prevalent in cattle herds in China,and there are fiber gene-deleted strains,showing a unique evolutionary trend.In order to further understand the distribution and prevalence of BAd V-3 in China,as well as the pathogenicity of the deletion strain,in this experiment,on the one hand,the etiology of BAd V-3 was investigated in respiratory and nasal swab samples from six provincial sick cattle in China,and the fiber deletion segment in the positive samples was typed,and on the other hand,the pathogenicity of the fiber deletion strain on BALB/c mice was studied.The following results were obtained:1.Etiological investigation results of BAd V-3 in 6 cattle farms in ChinaFrom October 2021 to January 2022,a total of 140 respiratory swab samples from sick cattle characterized by runny nose,cough,dyspnea and fever were collected from 9 largescale cattle farms in 6 provinces(Sichuan Province,Shanxi Province,Henan Province,Hebei Province,Jiangsu Province and Inner Mongolia Autonomous Region)in China.All samples were tested based on the TB Green Real-time PCR method using primers designed with conserved regions of BAd V-3 hexahedral(hexon)established in our laboratory.The results showed that the detection rate of BAd V-3 was 36.4%(51/140)and the field positive rate was 88.9%(8/9)in 140 nasal swab samples.Among them,the detection rates were as high as 82.9%(29/35)and 83.3%(10/12)in Sichuan and Henan provinces,20%(4/20)and 10.4%(5/48)in Jiangsu Province,Shanxi Province and Inner Mongolia Autonomous Region,respectively,and not detected(0/10)in Hebei Province.Among them,the detection rate of BAd V-3 was 26%(26/100)and the field positive rate was 85.7%(6/7)in 100 respiratory samples from adult beef cattle;the detection rate of BAd V-3 was 62.5%(25/40)and the field positive rate was 100%(2/2)in 40 respiratory samples from dairy cows,and the above results indicated that BAd V-3 was widespread in sampled farms.2.Typing of fiber missing segments and detection of some molecular characteristics in positive samplesPrevious studies have found that there are two types of BAd V-3 fiber gene: deletion type and traditional type.In order to determine which genotype is the dominant strain in China and understand its molecular characteristics,this study first designed a pair of specific detection primers for the 426 bp transaxial deletion segment to detect the fiber deletion segment in 51 positive samples from 140 samples in China.The results showed that the traditional type accounted for 80.4%(41/51)of the total positive rate and 77.8%(7/9)of the field positive rate,which were distributed in Sichuan Province,Shanxi Province,Henan Province,Jiangsu Province and Inner Mongolia Autonomous Region.The deletion type accounted for 19.6(10/51)of the total positive rate,33.3%(3/9)of the field positive rate,and was only detected in Sichuan and Henan provinces,with detection rates of: 24.1%(7/29)and 30%(3/10),respectively.Subsequently,some BAd V-3 positive samples were amplified for fiber gene sequence fragments,and the fiber fragments of the 14 cloned positive samples were compared with the fiber genes of BAd V-3 existing in Gen Bank and a phylogenetic tree was constructed,and the results showed that 11 positive samples clustered into a large independent branch with foreign strains,with a sequence length of 938 bp and no gene deletion phenomenon;3 strains had a sequence length of 695 bp,which was 243 bp(81 aa)missing compared with other strains and clustered into one branch with 8 Chinese strains.The results of fiber gene fragment amplification were consistent with those of primer examination of missing segments in the axial region.The above data indicate that the main epidemic in China is mainly the traditional type,and the fiber deletion strain is only endemic in some provinces of China.The results of this experiment enrich the molecular characteristics of BAd V-3 in China and provide guidance for understanding the unique genetic evolution of BAd V-3 in China.3.Pathogenicity of deletion strain BO/YB24/17/CH in BALB/c miceAlterations in the fiber axis length of the Fiber gene will affect the interaction between the virus and the receptor which in turn affects tissue hobby and pathogenicity.Therefore,in order to understand whether the deletion of this region causes an effect on the pathogenicity of BAd V-3,the pathogenicity of mice infected with the deletion strain BO/YB24/17/CH was studied in this experiment.Three-week-old SPF BALB/c female mice were artificially infected intranasally with 200 ul/mouse(105.3 TCID 50/m L)of virus.The clinical symptoms of mice were observed and recorded daily after inoculation,and the mice were dissected on days 1,3,5,7,9,11,13,and 15 after inoculation to collect heart,liver,spleen,lung,kidney,trachea,and blood for experiments.The results showed that 3 ～ 7 days after inoculation,the infected mice showed mild clinical symptoms such as drowsiness,unkempt coat,clumping,body tremor,and decreased weight gain rate,and the infected mice showed weight loss on the 3rd day after inoculation,which gradually returned to normal after reaching the minimum value on the 6th day.Gross necropsy lesions revealed congestion in the lungs of infected mice from day 3 after infection,scattered pulmonary hemorrhagic spots on day 9 after infection,and lobar atrophy in the lungs on day 15 after infection.Swelling and darkening were observed in the spleen on day 5 post-infection.Further histopathological observation revealed inflammatory cell infiltration,type II alveolar cell hyperplasia,and typical widening of the alveolar septa in the lungs of mice on day 3 after infection.On the 5th day,the spleen showed mild dilatation of nodules and blurred demarcation of red pulp and white pulp.Cilia damage and epithelial cell detachment were observed in tracheal infection13.Immunohistochemical results further confirmed that the above lesions were caused by virus infection in organs,and the virus mainly targeted the infected mouse tracheal mucosa and alveolar epithelial cells.Subsequently,the distribution of the virus in the organs of BALB/c mice was detected by q PCR and virus detection.The results showed that unlike the previously reported traditional strains,which replicated only in the lungs and trachea,the deletion strains could infect and replicate in the heart,liver,spleen,lungs,kidneys,trachea and even blood of BALB/c mice,and were continuously detected at 1 – 15 days after infection.The lungs decreased after peaking on day 3,and the heart,liver,spleen,and kidneys decreased after peaking on day 5.The above results indicate that the virus can cause viremia,has a wider range of tissue habits,and the duration of virus infection in vivo compared with the traditional type,which is closely related to the absence of fiber axis region.The results of this experiment further understand the pathogenicity of fiber gene deletion strain BO/YB24/17/CH in BALB/c mice,providing a reference for the prevention and control of BAd V-3 and the development of vaccines. |
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