Establishment Of EHV-1 XJ2015 Strain Disease And Preliminary Exploration Of Inactivated Vaccine

Equine herpesvirus-1（EHV-1）and equine herpesvirus-4（EHV-4）are pathogens of equine rhinopneumonitis（ER）,both of which cause respiratory diseases in Equine animals.However,in addition to cause respiratory diseases in horses,EHV-1 can also cause abortion,stillbirth and deaths of newborn foals in pregnant horses.It is a pathogen causing different clinical syndromes in horses,causing serious economic losses to the equine industry.At present,the most effective means to prevent the disease is still vaccination,and there is no commercial vaccine in China.Therefore,it is of great practical significance to develop a safe and effective inactivated EHV-1 vaccine.1.The purified virus of EHV-1 XJ2015 liquid was diluted by 10 times,and the virus liquids of 10⁶,10⁵,10⁴and 10³TCID₅₀/m L were used to infect each group of test horses through the nasal cavity.The clinical symptoms,nasal detoxification amount,and viral blood were compared and analyzed.EHV-1 XJ2015strain equine disease model was established.A clinical scoring table was established,and by setting the clinical symptom score of infected horses for 2 consecutive days during the test period to be the score>1,or the total score>3,the disease was determined to be onset,and the half of the infection amount of the EHV-1 XJ2015 strain to the horses was calculated.The results showed that the infected horses showed fever,body temperature over 38.8℃,loss of appetite,mucopurulent nasal fluid,and swollen mandibular lymph nodes.In severe cases,clinical symptoms such as difficulty in getting up,procrastination,and repeated getting up and down occurred,the incidence rate It is positively correlated with the infectious dose,and its ID₅₀is 10^4.33TCID₅₀/m L;the virus is abundantly enriched in the lungs and brain tissues of horses,there are obvious pathological changes such as interstitial pneumonia and neuronal edema.2.In this study,EHV-1 XJ2015 strain was used as the seed virus to inoculate RK-13 cells for virus amplification,the virus by saturated ammonium sulfate precipitation concentrated virus,and the virus was purified by sucrose density gradient centrifugation.The titer of 1 L was 10^9.66TCID₅₀/m L virus.After concentration and purification of the original solution,2 m L of the purified virus with a titer of 10^12.19TCID₅₀/m L can be obtained,the recovery rate is 67.77%,and the removal rate of contaminants is 97.25%.The virus solution can be completely inactivated by 0.02%β-BPL for 24 h.After hydrolyzing at 37℃for2 h,the A,GEL-02,and ISA35-VG adjuvants are mixed with the inactivated virus under low shear force.Prepared into three different adjuvant EHV-1 inactivated vaccines.3.The virus titer of 10⁸,10⁷,10⁶TCID₅₀/m L inactivated virus through the neck muscle injection immune group test horses,42 d after two immune attack by 100 ID₅₀poison protection test,through clinical symptoms,nasal fluid and content of the virus in the blood and the duration of the evaluation of the inactivated vaccine immune effect,and determine the best protection and immune dose.The results showed that 10⁸TCID₅₀/m L and 10⁷TCID₅₀/m L inactivated virus immunized horses did not develop disease,the protection rate was 100%,10⁶TCID₅₀/m L inactivated virus immunized horses protection rate was 50%.The 10⁸TCID₅₀/m L immunization group had the best protective effect with less virus content in nasal fluid and blood and short duration.The optimal immune dose was combined with A,GEL-02 and ISA-35VG adjuvants to prepare the vaccine,and the optimal adjuvants were screened through the equine immune test.The results showed that the antibody level of the adjuvant A vaccine group was the highest,and the average neutralizing antibody was 1:350 after two immunization,which was significantly different from that of the Ag control group（P<0.01）,and significantly different from that of the Ag+GEL-02 and Ag+ISA-35Vg groups（P<0.05）.It shows that the inactivated vaccine prepared by A adjuvant has the highest level of humoral immune response,so it is selected as the best adjuvant.In summary,this experiment successfully established the EHV-1 XJ2015 strain horse disease model,and evaluated the vaccine immune efficacy through the challenge protection test.After the optimal immunization dose and A adjuvant are used to immunize the horse,the immune effect is the best.Laid a theoretical foundation for the later production of commercial vaccines.