The Isolation,Identification,and Pathogenicity Research On Pseudorabies Virus Strain GDHD-1

Pseudorabies(PR)is a highly contagious disease caused by pseudorabies virus(PRV).PR widely distributes across the world,PR is associated with a variety of disease such as reproductive failure,respiratory syndrome,neurological symptoms,diarrhea,stunted growth,and it causes huge economic losses to the pig industry.In recent years,it has continued to occur and become popular.Because of the antigenic variation and new PRV pandemic strain,the existing vaccine can't fully protect animals from infection.Therefore,it is necessary to develop a vaccine against the new PRV pandemic strains.In 2014,a large number of nuersery pigs died in a farm in Guangdong,and the initial symptoms pointed out the PRV infection.In this study,the PRV wild strain was isolated and identified,and its pathogenicity was studied,which contribute to developing a vaccine against new PRV strains infection.1.The isolation and identification of PRV strain from Guangdong provience.We collected materials of suspected PRV-infected pigs from the farm in Guangdong province to isolate the virus.The virus was identified by PCR,Vero cells infection,indirect immunofluorescence assay,and electron microscopy observation.gE specific products of wild strain PRV was amplified by PCR in brain tissue samples,which suggested that the pigs were infected by PRV wild virus.After 36h incubation with the supernatant of materials,Vero cells were showed typical cytopathic effect,such as rounding,swelling and gathering off,and gE specific product was also found by the PCR.Moreover,Vero cells which treated with the virus showed specific fluorescence signal in indirect immunofluorescence assay using positive PRV serum.Virus particles around 150nm were observed by transmission electron microscope.These virus particles are spherical,enveloped and covered by spikes,showing typical PRV particles characteristics.The titer of virus was 107TCID50/ml.The homology analysis of gE showed that the homology between 97.5%?99.9%with reference strains,which is close to the virulent strains isolated during 2013-2014.Our results indicated that pigs in the farm suffered PRV wild strain infection,designated strain GDHD-1.2.Pathogenicity of PRV strain GDHD-1.The pathogenicity of strain PRV strain GDHD-1 was analyzed in New Zealand white rabbits and piglets.Six 10-week-old healthy New Zealand white rabbits were divided intothree group:two rabbits were hypodermically injected 2ml 107 TCID50 virus suspension;two rabbits were hypodermically injected 2ml 106TCID50 virus suspension;and the last two were treated with 2ml sterile saline as control.Four rabbits infected GDHD-1 appeared itching and neurological symptoms after 24h,and all died within 48-72h.The rabbits in high-titer virus group died significantly faster than those in low-titer virus group.These results confirmed that PRV strain GDHD-1 is virulent to New Zealand white rabbits.Piglets were divided into three groups(5 pigs per group):the pigs in the first group were inoculated intranasally with 107 TCID50 virus 2ml,the pigs in second group were inoculated intranasally with 106TCID50 virus 2ml,and the pigs in the third group inoculated 2ml sterile saline as control.The results showed that GDHD-1 caused typical PRV symptoms in the first and second groups such as high fever,respiratory difficulty and neurological symptoms,and all pigs died within 10 days,what's more,pigs in high-titer virus group died significantly faster than those in low-titer virus group;while the pigs in control group showed normal.Piglets with maternal anti-PRV antibodies were divided into three group(5 pigs per group):The pigs in first group were inoculated intranasally with 107TCID5O virus 2ml,those in the second group were inoculated intranasally with 106TCID50 virus 2ml,and the pigs in the third group inoculated 2ml sterile saline as control.The result demonstrated that GDHD-1 caused typical PRV symptoms in the first and second groups such as high fever,respiratory difficulty and neurological symptoms,and all pigs died within 11 days.However,the pigs in control group showed normal.Additionally,8-day-old piglets were divided into three groups(5 pigs per group):the pigs in first group were immunized with 2ml domestic PRV attenuated vaccine;the pigs in second group were immunized with 2ml imported PRV attenuated vaccine;the pigs in the third group were inoculated with the same amount of sterile saline as control.After 21 days of immunization,the pigs in first and second groups were inoculated intranasally with 2ml virus suspension with titer 107TCID50,and the pigs in control group were inoculated 2ml sterile saline.Pigs in first group appeared symptoms of high fever,respiratory difficulty,and ataxia,and then all pigs in this group died within 7 days.The pigs in second group appeared moderate symptoms such as fever,depression,lethargy,and cough,but all pigs died within 10 days.The pigs in control group were normal.These date showed that PRV strain GDHD-1 was virulent to piglets,and neither maternal antibody nor commercially attenuated vaccine could protect piglets from its infection.These findings contribute to selecting the suitable PRV strain for developing a vaccine against the new PRV pandemic strains infection.