| Porcine reproductive and respiratory syndrome is an infectious disease that can cause reproductive disorders in sows and respiratory disorders in piglets.Its pathogen is porcine reproductive and respiratory syndrome virus.According to retrospective experiments,positive serum was detected in the Canadian pig herd as early as 1987,and then reported in the southern United States.It spread to all parts of the world,causing huge economic losses to the pig industry and continuing to seriously threaten pig production.In 2012,the NADC30-like strain was reported for the first time in Henan,and its nucleotide and amino acid homology was similar to that of the American NADC30 strain.At present,the common commercial vaccines on the market have unsatisfactory immune protection effects on the NADC30-like strain.The PRRS caused by the NADC30-like strain in the field still poses a certain threat to the pig industry.Therefore,in order to provide applicable strains for the development of vaccines against NADC30-like strains,this experiment studies the in vitro passage changes of HENXX-9 and HENXX-8 strains,analyzes the genetic evolution rules between the parental strains and the passaged strains during the passage,and explores the differences The immune response changes after immunized mice with substitute strains and infected with Marc-145 cells in vitro.1.In vitro passage of NADC30-like HENXX-9 strain and HENXX-8 strainThe NADC30-like HENXX-9 strain and HENXX-8 strain were passed to the 80th generation in Marc-145 cells.As the number of passages increased,the time for the two strains to appear lesions on Marc-145 cells was shortened from 48h/36h to 24h.Compared with the HENXX-9 strain,the virus titer of the HENXX-9-F30 strain was 106.72TCID50/mL,no significant difference(P>0.001),and the HENXX-9-F80 virus titer was 107.11TCID50/mL,showing a significant increase(P<0.001);Compared with the HENXX-8 strain,the virus titer of the HENXX-8-F30 strain was 106.56TCID50/mL,with no significant difference(P>0.001),and the virus titer of the HENXX-8-F80 strain was 107.23TCID50/mL showed a very significant increase(P<0.0005).Therefore,as the number of passages increased,the virus titer of the two strains gradually increased,and their ability to adapt to Marc-145 cells was significantly enhanced.The virus titer of the HENXX-8-F80 strain was higher than that of the HENXX-9-F80 strain.2.Analysis of genetic variation of NADC30-like HENXX-9 strain and HENXX-8 strain during the passageIn order to study the genetic evolution of NADC30-like strains in the passage process,firstly compare and analyze the parental strains of HENXX-9 strain and HENXX-8 strain,the 10th generation(F10),the 20th generation(F20),and the 30th generation(F30).40th generation(F40),50th generation(F50),60th generation(F60),70th generation(F70)and 80th generation(F80)complete gene sequence and amino acid sequence of each protein,and then HENXX-9/HENXX9-F80 strain and HENXX-8/HENXX-8-F80 strain are compared with the reference virulent strain/attenuated strain of each protein and amino acid,and finally the above-mentioned strains and the epidemic strains in China in recent years are inherited Evolutionary analysis.The results showed that:(1)During the passage,HENXX-9 strain had 59 nucleotide variations,resulting in 31 amino acid variations;HENXX-8 strain had 108 nucleotide variations,corresponding to 31 amino acid variations.The amino acid at position 59 of the Nsp11 protein of HENXX-9 strain has repeated mutations of 59E?59K?59E,that is,the amino acid at position 59 of HENXX-9-F20?F60 is K59,and the amino acid at position 59 of HENXX-9-F70?F80 is E59.(2)HENXX-9 strain has 3 amino acid variants D83Ynsp2,D9NE,S132NGp2,and HENXX-8 strain has 6 amino acid variants R459KNsp2,G68EGP3,G73EGP3,I124VGP4,P15LGP4,G80CGP5,which are speculated to be related to weakening of virulence and immune characteristics.(3)HENXX-9/HENXX-9-F80 and the classic strains CH-1a,BJ,etc are in a large branch,belonging to Lineage 8,implying that it is related to the use of attenuated vaccines;HENXX-8/HENXX-8-F80 strains are related to The NADC30 strain belongs to a large branch and belongs to Lineage 1,which is a strain that has been widespread in China in recent years.3.Experiments on immunizing mice with NADC30-like HENXX-9 strains and HENXX-8 strains and the passage virus strainsIn order to explore the differences in immune response caused by different generation strains,this study immunized mice with different generation strains and JXA1-R vaccine strains,and evaluated the contents of IgG and neutralizing antibodies,cytokines IL-6 and IFN-? in the mouse serum Variety.(1)The results of IgG antibody showed that:from the point of view of antibody production time and antibody level,the antibody level in the serum of HEXX-9-F80 group mice reached the maximum at 14 days after immunization,which was earlier than other immunization groups;from the point of view of antibody duration,the serum antibody level of mice in the HENXX-8-F80 group continued to rise from 7 to 35 days after the first immunization.(2)The serum IL-6 content of mice in each group was higher than that of the control group,reaching the highest value 7 days after the first immunization.The prolonged immunization time reduced the IL-6 content in the serum of mice in the HNEXX-8-F80 group The results of IFN-? showed that the change trend of serum IFN-y in HENXX-9-F80 group,HENXX-8-F80 group and HENXX-9-F30 group was the same.The serum IFN-y in HENXX-9-F30 group was the same.The highest content and the largest increase after the second immunization.(3)Neutralizing antibodies were detected in the serum of mice in the JXA1-R vaccine group at 28 days after the first immunization.The neutralizing antibodies in the serum of each group of mice were detected only after 35 days after the first immunization.42 days after the first immunization,the level of neutralizing antibody was 1:11.6,and the level of neutralizing antibody in the HENXX-8-F80 group was 1:10.6.The difference was not significant(P>0.05),which was lower than the JXAI-R vaccine group.The level of neutralizing antibody in the serum of mice immunized for 42 days for the first time in each group reflected that the level of neutralizing antibody increased with the passage of passages.4.The effect of NADC30-like HENXX-9 strain and HENXX-8 strain and the passage virus strain on the expression level of related factors in Marc-145 cellsIn order to explore the cytokine changes caused by different generation strains of Marc-145 in vitro,this experiment used different generation strains and JXA1-R vaccine strain to infect Marc145 cells,and detect IL-6,IL-10 and TNF-? expression level at different time points.The results showed that(1)IL-6:HENXX-8 strain and its passaged strains caused a higher level of IL-6 expression than HENXX-9 strain and its passaged strains.It is speculated that HENXX-8-F80 strain has a stronger ability to cause inflammation than HENXX-9-F80 strain.(2)TNF-?:After infection at 24h,36h and 48h,the TNF-? content of HENXX-9-F80 strain was lower than that of HENXX-9 and HENXX-9-F30 groups,and the expression level of TNF-? in HENXX-8-F80 group Lower than HENXX-8 and HENXX-8-F30 group,HENXX-9-F80 strain TNF-? expression level is higher than HENXX-8-F80 group,it is speculated that HENXX-9-F80 has stronger immune regulation ability after being infected with Marc-145 cells HENXX-8-F80 strain.(3)IL-10:After 36h and 48h of infection,the IL-10 mRNA transcription level of the HENXX-9 group and the HENXX-8 group was higher than the passage strain group,and the IL-10 mRNA transcription level of the HENXX9-F80 group was higher In the HENXX-8-F80 group,it shows that with the increase of the passage number,the immunosuppression of the passaged strain on Marc-145 cells is weakened,and the immunosuppression caused by the HENXX-9-F80 strain is stronger than that of the HENXX-8-F80 strain.In summary,different generations of strains can cause immune responses,but to different degrees.For HENXX-9 and HENXX-8 in vitro passage changes and genetic evolution law analysis and comprehensive analysis of neutralizing antibody levels,HENXX-8-F80 is a selectable and applicable strain. |
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