Screening Of MicroRNA Related To Neutralizing Antibody Production In Porcine Reproductive Respiratory Syndrome

Porcine reproductive and respiratory syndrome(PRRS),also called blue-ear pig disease,is a kind of acute contagious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),which is characterized as premature delivery,abortion,stillbirth,birth of mummified fetuses in pregnant sows and coughing,dyspnea,polypnea in piglets with a very high morbidity and mortality.PRRS spreads rapidly and brings huge economic loss to Chinese swine industry.PRRSV infection can lead to antibody-dependent enhancement(ADE)and neutralizing antibody delaying,therefore,the immue antibody of PRRSV vaccine generated plays a certain role in the protective immunity of PRRS instead of completely protective effect.Which implies that the level of immune antibody of PRRSV vaccine is not completely correlated with the immunity protection.Hence,the immune antibody level of PRRSV vaccine is not the single standard for measuring the ability of anti-PRRSV infection.The neutralizing antibody against PRRSV has a protective function,howerver,the neutralizing test which is the primary testing method of the neutralizing antibody against PRRSV takes time and energy and the other testing methods are expensive.Therefore,it is necessary to screen the immune molecules related with the generation of neutralizing antibody to determine the molecules of PRRSV immune protection,and provide theoretical basis for assessing the immune protection effect of PRRSV vaccine.Mi RNAs is a type of small non-coding single-stranded ribonucleic acid(RNA)molecule.Mature mi RNAs are complementary with 3’-end non-coding region of the target genes in cells More and more evidences have indicated that mi RNAs are indispensable regulatory factors in the national/acquired immunity of the body.In the present study,five clean female piglets with negative PRRSV antigen-antibody,negative classical swine fever virus(CSFV)antigen-antibody,negative pseudorabies virus(PRV)antigen-antibody and negative porcine circovirus(PCV)antigen-antibody were screened out.The laboratory animals were fed separately in a sterile hog house and all the feed and tools were up to specifications.The hog house could be entered only after disinfection.On day 1 of the test,the laboratory animals were injected with CH-1R strain of attenuated vaccine on the neck muscles,2 portions/piglet.Blood samples were collected before inoculation and on day 14,day 21,day 28,day 35,day 42,day 49,day 56,day 63,day70 and day 77 after inoculation,which were tested for the generation of immune antibody and neutralizing antibody via enzyme-linked immunosorbent assay(ELISA)and serum neutralization test.Moreover,real-time polymerase chain reaction(PCR)was adopted to detect the transcriptional level of mi RNAS related to cellular immunity and adhesionmolecules correlated with generation of antibodies.The mean values of ELISA of the five piglets before inoculation and 2-11 weeks after inoculation were 0.20,0.54,1.43,2.46,2.25,1.54,1.71,2.11,2.51,2.80 and 2.14,indicating that antibodies were generated after inoculation of CH-1R strain of attenuated vaccine.The antibody values gradually increased at 2-4 weeks,decreased slightly at 4-6 weeks,increased gradually at 7-10 weeks and decreased again at 11 weeks,indicating that the antibody value of immune serum presented a gradient-like change and showed a growth and decline rule of the antibody.The detection results of the adhesion molecules related to generation of antibodies showed that the transcriptional level of IL-4,CD19 and CCR4 increased at 0-6weeks,decreased at 7-10 weeks and increased at 11 weeks except IL-4.The transcriptional level of adhesion molecule CXCR5 decreased at 0-7 weeks and increased at 8-11 weeks.The detection results of the neutralizing antibody revealed that the neutralizing antibody was not found in all laboratory animals at 0-4 weeks and was generated from 5 weeks,the valence of which gradually increased,reaching a peak value at 8-9 weeks(1:32)and decreasing at 10-11weeks(1:16).The detection results of mi RNAs showed that the transcriptional level of mi R-26 a and mi R-155 increased gradually at 2-8 weeks,significantly higher than that of let-7f and mi R-181 a,reaching a peak value at 8 week and gradually decreasing from 9-11 weeks.In summary,mi R-155,mi R-26 a have the same changing trend with neutralizing antibody,which can be used as an immune factor to evaluate the neutralization antibody production of PRRSV.It provides a more convenient evaluation marker for the monitoring of immune protective antibodies in PRRS,and has important clinical significance for the surveillance of neutralizing antibodies in PRRS.