Identification Of Nonessential Sequences In NSP2Region Of An Attenuated Porcine Reproductive And Respiratory Syndrome Virus And Expression Of Foreign Genes In The Nonessential Regions

Porcine reproductive and respiratory syndrome (PRRSV), the etiological agent of porcinereproductive and respiratory syndrome (PRRS), is mainly characterized with either reproductive failurein pregnant sows, or respiratory tract distress particularly in sucking pigs. PRRS was first reported in theUnited States and then it was prevalent globally. In China, PRRS was first found and identified in1995and then the disease spread in all provinces. In2006, one so-called "high fever disease" broke out inswine herds in southern provinces of China, characterized by high fever, high morbidity and mortality,and caused enormous economic losses to swine industry. Subsequently, a variant PRRSV strain wasfound to be the etiological agent for this epidemic, and it was later named as high pathogenic (HP)PRRSV. So far, there were four different type of PRRSV strains prevalent in Chinese swine herds:Classical PRRSV and its derivative live vaccine virus, HP-PRRSV and its derivative live vaccine virus.This situation has made control or eradication of PRRS extremely difficult in China. Therefore, amarker vaccine is desirable for control of PRRS. It was reported that non-structural protein2(NSP2) isthe largest PRRSV replicative protein, having significantly difference in length among the differentPRRSV strains and containing nonessential region for viral replication. Therefore, the nonessentialregions in nsp2gene of an attenuated PRRSV (HuN4-F112) were firstly identified based on aninfectious cDNA clone, then the marker NDV NP49gene and the CSFV E2epitope were inserted intothe nonessential regions, respectively. The viable deletion or recombinant viruses was rescued from thefull-length cDNA infectious clones in vitro. And the marker recombinant viruses were evaluated inpiglets. These results that we got from this study could lay foundation in development of marker vaccineand using PRRSV NSP2as a viral vector.In the study, we first identified the nonessential regions of NSP2. A series of mutants with in-framedeletions in the nsp2coding region were engineered by the method of mutant PCR, based on theinfectious molecular clone of HuN4-F112vaccine strain. The deleted full-length cDNA clones wereassembled by cloning and splice of the gene fragments. The completely assembled full-length cDNAclones were confirmed by sequencing and enzyme digestion of Swa Ι. Capped RNAs were transcribedin vitro from the full-length cDNA clones of the viral genome and transfected into BHK-21cells byliposome to acquire the rescued virus. Then, the rescued recombinant viruses were passaged onMARC-145cells. The successfully rescued viruses were tested by RT-PCR, enzyme digestion, genomesequencing and IFA. The results showed that the rescued viruses could be distinguished from theparental and the deleted nsp2gene region with the mutant genetic marker (MluⅠenzyme site at14667ntof genome). IFA showed that the deletion mutants could react with the antibody of NSP4and the Nprotein. The mutants were also detected by viral growth characters (growth curve and plaque assay).The results demonstrated that the region between amino acids (aa)480to667of nsp2tolerateddeletions. Characterization of the mutants demonstrated that those with deletion in nsp2region did notaffect the viral growth on MARC-145cells, but led to earlier PRRSV replication increased. The regioncorresponding to amino acids (aa)480-667within nsp2of an attenuated PRRSV was identified to be nonessential for viral replication in MARC-145cell, and the results also suggested that the nonessentialregion might be an ideal insertion region for expressing foreign genes in PRRSV genome.And then, two rescued viruses with deletions in NSP2region (aa480-532or aa508-532) were usedas vectors to express the foreign marker genes. A molecular marker of immunodominant B-cell epitope(49amino acids at C-terminal) of nucleoprotein (NP) of Newcastle disease virus (NDV) and a majorneutralizing antigenic epitope(15amino acids) of E2gene of Classical swine fever virus(CSFV) wereinserted into the deleted NSP2regions of the two rescued viruses, respectively, using the method ofmutant PCR technology. The full-length cDNA clones with markers assembled and the recombinantviruses rescued as mentioned above. The four rescued viruses were confirmed by RT-PCR, enzymedigestion, and genome sequencing, and named as rHuN4-F112-Δ480-532+NP49,rHuN4-F112-Δ508-532+NP49, rHuN4-F112-Δ480-532+E2ep and rHuN4-F112-Δ508-532+E2ep,respectively. The results showed that the rescued viruses could be distinguished from the parental by themutant genetic marker (MluⅠenzyme site at14667nt of genome) and the insert in nsp2region. Theresults of IFA with antibodies against NDV and CSFV and sequencing of inserts showed that the insertforeign marker was stable and could be expressed during the virus serial passage. The results of plaqueassay and viral growth curve showed that the recovery viruses possessed similar characterses to those ofthe parental virus.Further, two recombinant marker viruses, rHuN4-F112-Δ480-532+NP49and rHuN4-F112-Δ508-532+NP49, were evaluated in piglets for protective efficiency against challenge by HP-PRRSV andantibody development to NDV NP49.20piglets were divided into4groups, five piglets for each group.Piglets of groups1-3were inoculated intramuscularly by rHuN4-F112-Δ508-532+NP49,rHuN4-F112-Δ480-532+NP49or HuN4-F112at dose of105TCID50, respectively. Piglets of groups4were inoculated intramuscularly by2mL DMEM (2%FBS) as mock control. On28dayspost-immunization (DPI), all animals were challenged with HP-PRRSV HuN4(3×104TCID50/pig).All the animals were euthanized28days post-challenge (DPC). The results showed that piglets in group2(innoculated with rHuN4-F112-Δ508-532+NP49) and group3(innoculated with rHuN4-F112) were100%protected (5/5), and piglets in group1(innoculated with rHuN4-F112-Δ480-532+NP49) were60%protected(3/5), while the piglets of group4were all dead. The results of antibody detection showedthat all piglets in group1,2and3developed antibodies to PRRSV, only piglets in group1and2developed antibodies to NP49, but absence of antibodies to deleted25aa until28days after challengewith HP-PRRSV. In conclusion, the recombinant PRRSV, rHuN4-F112-Δ508-532+NP49, can befurther developed as a marker vaccine and used for eradication of PRRS.