| Pseudorabies(PR)is a porcine infectious disease caused by pseudorabies virus(PRV),which is characterized by neural symptoms in the piglets and miscarriage in pregnant sows.The disease has caused huge economic losses in pig industry.Since the 1990 s,most pig herds in China have been inoculated with vaccines,so PR had been effectively controlled.However,since the second half of 2011 in China,many provinces continued to outbreak PR in many pig farms vaccinated with Bartha-K61.Three strains of PRV were isolated in pig farms vaccinated with Bartha-K61 in Hebei and Shaanxi provinces,and designated them as HB-SH1-2015,HB-SH2-2015,SX-HZ-2015.Here,we studied cell propagation cultivation,PRV infection TCID50 determination,the rabbit test,the determination of viral load in rabbit brain tissue and lung tissue,gE gene nucleotide sequence analysis on three PRV strains,and obtained the following results.(1)PK-15 cells infection with the three PRV strains both appeared obvious lesions(CPE)at 24 h.Some cells appeared rounded and scattered in the foci at 24 h.Cells appeared rounded and aggregated at 48 h.Cells are broken and dead at 72 h.The cells did not appear obvious differences between different strains.Virus titers in HB-SH1-2015 is 10-11.2857TCID50/100 ?L,HB-SH2-2015 is 10-13.9027TCID50/100 ?L,and SX-HZ-2015 is 10-12.2877TCID50/100 ?L.(2)The rabbit infection test demonstrated that three PRV strains made rabbits a typical pseudo rabies symptoms,namely,spirit depressed,loss of appetite,body temperature,and part of rabbits eat hair phenomenon.After 10 days,rabbits appeared burnout,opisthotonos,cramps,and all rabbits died off.Rabbit pathologic autopsy results demonstrated that the three group rabbits showed the typical PRV lesions,hepatomegaly,edge necrosis,congestion,edema of the lungs,and congestion enlargement of the spleen,with necrosis foci.Real-time PCR ananlysis of infected rabbit brain tissue and lung tissue showed that viral load in brain tissue of HB-SH1-2015 strain and HB-SH2-2015 strain were 7.5×105 copies/g and 6.9×105 copies/g,respectively.There was no significant difference(P > 0.05)between them.However,viral load in SX-HZ-2015 strain was 1.2×106 copies/g,significantly higher than that two strain(P < 0.05)in Hebei.Viral loads of HB-SH1-2015 and HB-SH2-2015 strain in lung tissue were 3.8×1010 copies/g and 3.4×1010 copies/g,respectively,no significant difference(P > 0.05).Viral loads of SX-HZ-2015 strain in lung tissue was 7.6×1010 copies/g,significantly higher than that two strain(P < 0.01)in hebei.These data indicated that infection of rabbit brain tissue and lung tissue of SX-HZ-2015 strains were significantly stronger than that of HB-SH1-2015 strain and HB-SH2-2015 strain.(3)According to PRV gE gene nucleotide sequences analysis,the gE gene nucleotide homology was 99.9% between HB-SH1-2015 isolates and HB-SH2-2015 isolates,and the SX-HZ-2015 isolates was 99.3% homology with the other two strains.For gE gene mutations analysis,SX-HZ-2015 isolates demonstrated the lack of some sites and mutation compared to HB-SH1-2015 isolates and HB-SH2-2015 isolates.The gE gene nucleotide homology of HB-SH1-2015 isolates and HB-SH2-2015 strains was as high as 99.0% ~ 99.0% with domestic strains separated after 2012.However,the homology of SX-HZ-2015 strain is relatively low(98.3% ~ 99.0%)with the domestic popular PRV strain.Phylogenetic tree showed that the three isolates with domestic most of the PRV epidemic strains belong to the same branch,suggesting that three isolates have no obvious mutation at the genetic level,all belonging to the domestic common PRV strains. |
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