The Regulatory Role Of Fusion Bursin In The Active Immunity Of Mice Against Pseudorabies Virus Variant Strains

Pseudorabies（PR）is a severe acute infectious disease caused by Pseudorabies virus（PRV）infection of animals,usually causing breathing difficulties,neurological symptoms and itching（except pigs）,etc.The symptoms and fatality rate are extremely high,which brings huge harm to the breeding industry and also threatens human public health security.Since 2011,a new round of swine PR outbreaks has occurred in china.The virus spreads very fast.The original classic vaccine Bartha-K61 can’t afford the effective protection,We have not yet developed specific drugs to prevent and treat the disease.Vaccine immunization is still the main means of prevention.Tripeptide Bursin（BS）is an active small molecule peptide with a structure of lysine-histidine-glycine（Lys-His-Gly-NH2）,extracted from avian bursa.A large number of studies have shown that BS has the function of promoting the growth and development of the body and enhancing the immune level of the body.The prokaryotic expression fusion BS developed by our research group is a fusion protein of tandem-expressed BS tetrapeptide and serum albumin-binding peptide,which is an analog of natural BS.The preliminary research of our research group shows that this substance can significantly improve the production performance of immunosuppressed animals.and immune properties,it is worth noting that the fusion BS has a significant antiviral effect,which is rarely reported in the existing BS-related literature.Therefore,this study used PK-15 cells and mice as models to explore the effect of fusion BS on PRV replication and its pathogenic effect in vitro and in vivo,so as to provide new insights for exploring the antiviral effect of fusion BS and clinical prevention and control of PR ideas.Antiviral effect of fusion BS in PRV on PK-15 cells1.Determination of TCID₅₀In this study,the PRV-MQ18 virus stock solution was blindly passed for 6 generations and purified for TCID₅₀ determination.The result was that the virus titer of PRV-MQ18 was 10^-8.52/0.1m L.2.Determination of optimal fusion BS incubation concentrationIn order to determine the optimal concentration of fusion BS on PK-15 cells cultured in vitro,five concentration gradients of 0μg/m L,1μg/m L,5μg/m L,10μg/m L,and 50μg/m L were set to treat cells for 24h,48h,After72h,the samples were collected with CCK8 kit for cell viability detection.The results showed that 5μg/m L of fused BS had less toxicity to PK-15cells,and 48h was the best.3.Exploring the optimal concentration of fusion BS on PRV-infected cellsThe PK-15 cells cultured in vitro were inoculated with 3.3×104TCID₅₀ of PRV,and the final concentrations of 1μg/m L,5μg/m L,10μg/m L of fused BS were added for 48h,and the samples were collected for q-PCR to detect the virus The expression level of g E gene,the results showed that:when the concentration of fusion BS was 5μg/m L,the expression of viral genes decreased by 31%（P<0.001）,and when the concentration of 1μg/m L,the expression of viral genes decreased by 27%（P<0.001）,and only decreased by 15%（P<0.05）when the concentration of 10μg/m L,so5μg/m L fusion BS was the optimal concentration.4.Exploring the titer of PRV infection under the optimal concentration of fusion BSCells were inoculated at concentrations of 3.3×10⁶ TCID₅₀,3.3×10⁵TCID₅₀,3.3×10⁴ TCID₅₀,and 3.3×10³ TCID₅₀,and fused BS with a final concentration of 5μg/m L was added for 48h.The samples were collected for q-PCR detection.The inhibitory rate of viral gene expression was 23%（P<0.01）at 3.3×10⁵ TCID₅₀,and 33%（P<0.01）at virus titer of 3.3×10⁴TCID₅₀.5.Explore the optimal virus titer and the optimal concentration of fusion BS for action timePRV-MQ18 with a viral titer of 3.3×10⁴ TCID₅₀ was inoculated into PK-15 cells,and fused BS with a final concentration of 5μg/m L was added for 24h,48h,and 72h,and the samples were collected for q-PCR detection.The results showed that,At 48h,the expression of g E gene in the fusion BS group decreased by 27%（P<0.001）,which was 19%lower than that in the fusion BS group at 24h（P<0.05）.The expression of g E gene was more inhibited,while at 72h,it is speculated that the difference is not obvious due to the excessive virulence of the virus,resulting in a large number of cell death.Therefore,the fusion BS with a final concentration of 5μg/m L acts on the cells,and the best antiviral infection effect can be obtained under the virus titer of 3.3×10⁴ TCID₅₀ for 48h.6.Antiviral effect of fusion BS on PRV under three modes of administrationBased on this result,three administration modes were designed.Group A:Incubate with 5μg/m L fused BS 6h before cell challenge,and continue to culture in 5μg/m L fused BS medium for 48h after PRV incubation is completed;Group B:at the same time of PRV incubation,5μg/m L fused BS solution was added,and then placed in 5μg/m L fused BS medium for 48h;Group C:routinely cultured for 6h after PRV incubation,and then replaced with fused BS medium containing 5μg/m L for 48h;a blank control group and a model control group were also set up,and the virus was incubated and the medium was replaced at the same time.Continue to cultivate for 48h;The results showed that compared with the infection group,the three administration modes All of them can effectively inhibit the replication of g E gene,and group A has the best effect,with an inhibition rate of 34%（P<0.001）,while group B is 25%（P<0.01）,and group C is only 20%（P<0.05）.The above results show that the fusion BS at the concentration of5μg/m L and the virus titer at 3.3×10⁴ TCID₅₀ for 48h on PK-15 cells can show a good ability to inhibit the replication effect of the virus gene.Therefore,the fusion BS may Can be a kind of inhibitor against PRV infection.Antiviral effect of fusion BS in mice1.Grouping and handling of experimental animalsSPF 6-8 week-old female BALB/c mice were divided into 5 groups after being adapted to feeding:group A was injected with soluble fusion BS purified concentrate in the legs on the 7th and 14th days;group B was injected subcutaneously in the legs on the 7th and 14th days.Bartha-K61live vaccine was injected;group C was simultaneously injected with fusion BS+Bartha-K61 live vaccine on the 7th and 14th days;model group D and blank E group were immunized with DMEM culture medium at the same time.2.Determination of LD₅₀Dilute the virus stock solution to 9 concentrations(10⁰ TCID₅₀～10⁸TCID₅₀),and infect 5 mice by gavage with PRV of each gradient.After feeding for 7 days,LD₅₀=10^-1.48/0.1m L.3.Mouse Mortality and Post-Challenge SurvivalAfter the completion of the second immunization and before the challenge,the mortality rate of mice in group B（vaccine group）reached75%,and the mortality rate of group C（fusion BS+vaccine group）was only 55%.8～10h in the evening.After challenge,the survival rate of mice in group A was 30%,group B was 20%,group C was 33%,and group D was 13%.The comparison found that the survival rate of mice in other groups was higher than that in group D model group.The above results preliminarily illustrate Fusion of BS can reduce the lethality after PRV infection.4.CD₄⁺/CD₈⁺detectionOn the 5th week after the second immunization（the 7th day after the challenge）,the anticoagulation of the mice in each group was detected by flow cytometry.The results showed that the ratio of serum CD₄⁺/CD₈⁺in the model group（group D）mice Significantly lower than the blank group（group E）（P<0.05）,the difference between group A and group B was not significant,while the CD₄⁺/CD₈⁺ratio of mice in group C was significantly higher than that in groups A,B,and D（P<0.05）,indicating fusion BS can enhance the immunity of mice,and the effect of mixed immunity with vaccine is better.5.Serum g B antibody detectionOn the 5th week,mice from each group were randomly selected for serum g B antibody detection after blood collection.The results showed that the serum g B antibody levels of mice in other groups were higher than those in group D（P<0.001）,and the antibody level of group C was slightly higher than that of group B,the effect was better;the level of g B antibody produced in group A was moderate（P<0.01）,and the ability of group D to produce g B antibody without the interference of fusion BS was poor（P<0.05）,indicating that fusion BS can promote the production of g B antibody.6.q-PCR to detect the level of virus replication between mouse tissuesThe results of q-PCR detection of sampling at 5W showed that the virus expression levels in each tissue of the mice in groups A,B,and C were lower than those in group D,and the virus expression levels between the tissues of group D and group C were all the same as those in group D.The most significant difference（P<0.001）;between group D and group A,the viral load in liver and lung tissue was significantly different（P<0.01）,and the difference between other tissues was significant（P<0.05）;group D and group B were significantly different（P<0.05）.Among the groups,the differences in the amount of virus in heart,liver and lung tissues were extremely significant（P<0.001）,and the differences among other tissues were significant（P<0.01）.Positive effect,inhibits the replication of PRV.7.HE experimentPathological section observation showed that the symptoms of groups A,B,C,and E were significantly relieved compared with group D（model group）.Among all the tissues,in the heart tissue,group C had the best immune effect,effectively reducing the degree of myocardial fiber rupture;In the liver tissue,the hepatocytes of the mice in group D swelled and became enlarged and vacuolar degeneration occurred,and the hepatic sinusoids disappeared.In spleen tissue,group D had obvious congestion,and the boundary between red and white pulp was blurred.After immunization,group C had the best effect and the mildest symptoms;in lung tissue,a large number of alveoli in group D were highly dilated,and the alveolar wall became thicker.Alveolar hemorrhage can be seen,lung lesions are significantly reduced after immunization,and group C has the best immune effect in each group;In renal tissue,the glomerular wall is thickened,and renal tubular necrosis can effectively relieve symptoms after immunization.In the brain tissue,the cerebral nerve cells of the mice in the D group were extensively degenerated and swollen,disintegrated and necrotic in a large amount,and the nerve cell lesions were greatly reduced after immunization,and the immunization effect of the C group was also the best.The above results show that the fusion BS can play a good role in mice and reduce the symptoms of virus infection in the body.ConclusionStudies have shown that the fusion BS has a better performance at the cellular level and animal level,it has a positive effect against viruses,it is also expected to become new type of antiviral drug.