Studies On Genetic Variation Of VP1 Gene Of Foot And Mouse Disease Virus New Swine Strain

The picornavirus are highly susceptible to false replication during natural epidemics.The mutation rate of FMDV is very high,which increases the difficulty of prevention and control of foot-and-mouth disease.Through the development and implementation of the technology of"Clinical Evaluation of Immunization Effect of Swine FMD Vaccine",the level of FMDV immunized antibodies were improved.The rate of wild strain infection were greatly reduced by detecting of 3ABC anbibodies.The FMDV is spread only seen in sporadic outbreaks under immunity expression.Using new FMDV stains O/GZBY/2010 and A/GDMM/2013 of swine to infect with cells,suckling mice,and immunized or non-immunized piglets,the results were analyzed the genetic evolution of FMDV,using high-throughput sequencing and reverse genetics techniques to study FMDV variation under different conditions,provided theoretical support for studying the mechanism of interaction between FMDV and host,alerted the new epidemic situation of foot-and-mouth disease,and provided new ideas for the prevention and control of FMD.1.Formulation and implementation of“Clinical Evaluation of Immunization Effect of Swine FMD Vaccine”The“Clinical Evaluation of Immunization Effect of swine FMD Vaccine”technology was implemented in 208 demonstration counties in 34 experiment stations in 21 provinces to improve the level of FMD immune antibodies of swine in the country during 7 years.In 2017,the positive rate of O type FMD antibody of piglets was 93.93%with secondary immunization,which was 31.83%higher than the rate of pre-implementation,and the antibody positive rate after breeding immunization was97.62%,which was 23.82%higher than the rate of pre-implementation.The positive rate of type A FMDV antibodies were 94.67%of breeding pigs,87.96%of piglets,and 0.71%of 3ABC which was 3.69%lower than the 4.4%of 2016.The levels positive rate of immune antibodies of pigs and piglets had been greatly improved,exceeding the national standard of“The qualified rate of immune antibody is greater than or equal to 70%and is judged to be immunologically qualified..”With the increase of FMD immune antibodies,the wild-type infection rate gradually decreased,and the detection rate of FMDV infectious antibodies(3ABC)was significantly lower than that of 2016.In the process of FMDV genetic derivation,it shows diversity and abundant variation types.FMDV strains are much more than those isolated in the field.The continuous action of a single external factor will change the trend of FMDV mutation.After the overall immune antibody of the farm is improved,FMDV also exhibits a variant strain under immunological stress,and the new mutant strain infects the immune or immune blank animals.2.Variation of VP1 gene in O/GZBY/2010 and A/GDMM/2013 strains under immune and non-immune pressureThe challenge tests were performed with 82 piglets immunized and non-immunized with type O and type A FMDV.The FMD incidence of non-immune piglets was 20/52,and that of immunized piglets was 7/30.Type O FMDV is more susceptible to mutation than type A FMDV during the infection of this animal.Among 27 piglets infected with type O FMDV,there is no mutation of FMDV VP1 gene of 12 piglets(2-5 days of onset),containing non-immuned of 9 piglets and immuned of 3 piglets.Analysis of VP1 of type O FMDV of No.024 non-immuned piglet(tenth day of onset),amino acid had a change at S₁₄₀?L₁₄₀.Analysis of VP1 of type O FMDV of No.130 immuned piglet(sixth day of onset),amino acid had a change at S₁₄₀?P₁₄₀.Among 11 piglets(4-7 days of onset)infected with type A FMDV,there is no mutation of FMDV VP1 gene,containing non-immuned of 9piglets and immuned of 2 piglets.Analysis of VP1 of type A FMDV of No.3212non-immuned piglet(eighth day of onset),amino acid had a change at R₁₅₂?G₁₅₂.According to the results of the challenge test,VP1 gene of FMDV was not mutated in12 immunized and non-immunized piglets(2-5 days of onset)infected with type O FMDV and 11 immunized and non-immunized piglets(4-7 days of onset)infected with type A FMDV.FMDV VP1 genes of 3 piglets were mutated in four immunized and non-immunized piglets with a longer onset time.The RGD flanking sequence of FMDV VP1 gene was S₁₄₀?P₁₄₀ in No.130 piglets with immuned type O FMD antibody 1:32 before challenge test,which was consistent with the variation trend of O/SKR/4/2010 strain to O/SKR/GJ/2016 strain after the increase of immune antibody in Korea in 2011.From the results of the animal challenge test and epidemiological investigation,the mutation rate of FMDV in acute infection period was lower than that in chronic infection.The longer the piglets infected with FMD,VP1 gene of FMDV was more likely to mutate.3.VP1 gene mutation of FMDV in cells and suckling mice with different translation speedsA/GDMM/2013M strain was successfully rescued by reverse genetics techinique,and IRSE was replaced on the basis of A/GDMM/2013M strain to rescue A/GDMM/2013M1.A/GDMM/2013,A/GDMM/2013M and A/GDMM/2013M1strains were propagated in BHK-21 cell for 20th generation.Analysis of the VP1,the nucleotides of VP1 gene were not mutated.The 3 strains were diluted to 1 MOI(Multiplicity of infection,MOI)to infect PK-15 cells.At 4,8 and 12 h,the expression of FMDV VP1 protein and mRNA was lower than that of the other two strains.Suckling mice were infected with three strains of type A FMDV.The A/GDMM/2013M infected mice had the longest lesion time,and there were three residues to change under passaging condition.Other two strains with shorter onset time only had one residue mutation.A/GDMM/2013 strain has lower virus titer,slower translation speed,the longer onset time after infection with FMDV.VP1 gene is more likely to mutate.This is consistent with the results of the immune and non-immune piglet challenge tests.The virus has a longer onset time in the animal body,and the virus is more susceptible to mutation.This is consistent with the results of the immuned and non-immuned piglet challenge tests.Suckling mice infected with FMDV has a longer onset time to produce typical FMD symptoms.FMDV is more susceptible to mutation.4.Selecting different genes using high-throughput sequencing technology to analyze the interaction between FMD mutant plasmids,the relationship between strains and hosts12 piglets infected with and un-infected with O/GZBY/2010 strain were sequenced and analyzed using high-throughput sequencing technology to analysis transcriptome.The differentially expressed genes of CXCL10(C-X-C motif chemokine 10,CXCL10)and IFN-?(interferon-beta,IFN-?)were selected.By qRT-PCR analysis,it was found that the signal pathways of NF-kappa B and IFN-beta were activated,producing chemokines CXCL10 and cytokine IFN-?.RGD flanking sequence of type O FMDV VP1 gene of No.130 immunized piglets changed S₁₄₀?P₁₄₀ after challenge.The VP1 gene S₁₄₀?P₁₄₀ eukaryotic expression vector of O-type FMDV was constructed for cell transfection.The 3D structure of VP1 did not change significantly.The mutated O-type VP1 gene can promote the activation of NF-?B and IFN-?signaling pathways,and promote the high expression of chemokine CXCL10 and type I interferon IFN-?,making the host immune response and antiviral ability significant.Enhanced.Construction of type O FMDV VP1 gene S140?P140 plasmid and transfection,the mutant VP1 gene of type O can activate the NF-kappa B and IFN-beta signaling pathway,and promote the high expression of CXCL10 and IFN-?.The immune response and antiviral ability of the host were significantly enhanced.PK-15 cells were infected with the strains of A/GDMM/2013,A/GDMM/2013M and A/GDMM/2013M1,collected at 4 h,8 h and 12h,respectively.The CXCL10 and IFN-?produced by A/GDMM/2013M strains increased gradually,and produced by A/GDMM/2013M1 strains highest at 8 h and the lowest at12 h.At 8 h,the actived IFN-?produced by A/GDMM/2013M strain was significantly higher than A/GDMM/2013M1.With the prolongation of infection time of host cells by FMDV,the longer the inhibition of FMDV infection,the higher the expression of intracellular chemokines CXCL10 and IFN-?,FMDV strain is more susceptible to mutate.This study can broaden the understanding of FMDV mutation,and preliminarily clarifies the genetic evolution of porcine FMDV VP1 gene.More importantly,it reveals that FMDV have lower virulence,slower translation speed,longer onset,increase expression of chemokine CXCL10 and cytokines IFN-?,the more susceptible of FMDV VP1 to mutant.The mutation rate of FMDV is lower than that of chronic infection during the acute infection period.The FMDV genetic evolution law is analyzed to provide a mechanism for the study of FMDV-host interaction mechanism and early warning of foot-and-mouth disease.Theoretical support,while providing new ideas for the prevention and control of FMD.To analyze the genetic evolution of FMDV,it provided theoretical support for the study of interaction mechanism between FMDV and host,providing theoretical support for early warning of new outbreaks of foot-and-mouth disease,providing new ideas for the prevention and control of FMD.