RNAi Targeting PRRSV N Gene And Immunogenicity Of Viral Glycoprotein Without ADE Epitopes

Porcine reproductive and respiratory syndrome virus (PRRSV) is an envelopedsingle and positive-stranded RNA virus, belong the order of Nidovirales, familyArteriviridae, genus Arterivirus. Approximately, the size of PRRSV genome is15kbin length comprises at least10open reading frames. PRRSV is associated withporcine reproductive and respiratory syndrome (PRRS) is characterized byreproductive failure and severe trouble respiratory in piglets. Recognized in China in1996, PRRS was caused the serious economic losses in swine industry throughout thecountry.Furthermore, the PRRSV nucleocapsid protein (N), encoded by ORF7,constitutes about20–40%of the total protein of the virion and is essential in assemblyand disassembly of the virion. In addition, N protein is one of the most immunogenicof the virus and suitable candidate for the detection of virus-specific antibodies anddiagnosis of the PRRS disease. Also, N gene is directly involved in the virusreplication. Thus, the N gene plays a vital role during the life cycle of PRRSV. Inother hand, GP5is one of the major structural proteins encoded by PRRSV with aweight of24.5-26KDa. The GP5had been associated with the development ofneutralizing antibodies and protection. Moreover, it was shown that demonstrated thatthe primary neutralizing epitopes of both the European and North American PRRSVstrains are closely related and located in the same position of middle of the GP5gene.The GP5protein was shown important in the study of PRRSV pathogenicity,diagnosis and prevention. It is also the best candidate for developed geneticallyengineered vaccine against PRRSV. However, it was shown that the GP5ectodomainconserved a highly neutralizing epitope B (Aa37-45) and a high variability ofnon-neutralizing epitope A (Aa27-31). In other hand, the core of an epitope locates in the forestall seven amino acids of B epitope, make A epitope as a decoy epitope.When A/B epitopes concurrence, the neutralizing role of B epitope will be completelycovered by A epitope to reduce the neutralizing reaction. This phenomenon is calledantibody-dependent enhancement (ADE effect).Accordingly in this study, three theoretically effective interference target sites(71-91,144-164,218-238) were screened and targeting the nucleocapsid (N) gene ofPRRSV were designed. The three siRNA-expressing plasmids were constructed,respectively named p2.1-N71, p2.1-N144and p2.1-N218; then the application of theRNAi technique to inhibit the replication of PRRSV in MARC-145cell was explored.Experimental results demonstrated that these three siRNA-expressing plasmids couldeffectively and significantly inhibit the replication of PRRSV by93.2%,83.6%and89.2%in MARC-145cells, respectively. Among these three siRNA-expressingplasmids, p2.1-N71was found to be most effective, while p2.1-N144and p2.1-N218displayed relatively weak inhibition of virus replication. The results indicated thatsiRNA-expressing plasmids targeting the N gene of PRRSV could significantly inhibitPRRSV replication in MARC-145cells. Additionally, blasting the N gene of PRRSVJL07SW strain in NCBI revealed that there was100%nucleotide sequence homologywith the N gene of at least32different PRRSV strains which belong to NorthernAmerican type PRRSV. Therefore, we deduce that N71is a good target to suppressNorthern American PRRSV replication by RNAi technique.In addition, we use Protein in bioinformatics software DNAStar to predict GP5protein secondary structure. The results demonstrated that GP5protein belongs toβ-based protein folding, and potential advantages epitopes of GP5protein are the finalscreening:30-41,49-57,130-142,146-156,159-175and191-199amino acids. Basedon forecast results and the literature reports, we will be missing the27-31amino acidsof GP5protein (△aGP5), and in the absence of sites connected to the37-45aminoacids (bGP5) was amplified by PCR. Adenovirus pShuttle vectors were construsted,named pShuttle-△aGP5, pShuttle-GP5and pShuttle-bGP5, and a negative controlvector, named pShuttle-K. After four pShuttle vectors and adenovirus backboneplasmid pacAd5are linearized with PacⅠ, and transfected HEK293AD cellspackaging four adenoviruses, namely AD-△aGP5, AD-GP5, AD-bGP5, and AD-K. The results show that the correct packaging of the adenoviruses, F6-generationadenovirus when stability is still good, the size of the protein expressed about25KD,consistent with the expected results through the adenovirus stability, Western blot andindirect immunofluorescence.The N gene targeting PRRSV replication inhibition effect of a good p2.1-N71interference plasmid and adenovirus packaging AD-△aGP5, AD-GP5, AD-bGP5,AD-K and15-day-old piglets were immunized, after intranasal inoculation strainPRRSV JL07SW two week (TCID50of106.2ml-1)1mL/head, testing their level ofimmunity and immune cells induced humoral. Vaccination group and the controlgroup immunized piglets were detected antibodies by indirect ELISA and Serumdilution method after immunized0,7,14,21,28,35,42and56d. Total RNA wasextracted virus as a template for real-time quantitative PCR, detection of piglet serumviral titers. The results showed that except the negative control group, in each testgroup, antibody levels were significantly higher (P<0.01). Three adenovirusimmunohistochemical comparison, AD-bGP5immunohistochemistry inducedantibody levels produced slightly higher than other immune group, but the differencewas not significant (P>0.05). The results of identification of T lymphocyte subsetsshowed that the recombinant adenovirus immunity in pigs can significantly increasethe number of T lymphocyte subgroups, enhance the immune response cells.In summary, N genes and GP5gene in pigs to produce specific antibodies inducegood humoral and cellular immune response, effectively inhibit PRRSV infection maybe caused by structural gene ADE effects. This study PRRSV infection prevention andcontrol PRRS vaccine development and provide a theoretical basis and experimentaldata.