In Vivo Expression And Antiviral Effects Of PRRSV Soluble Receptors Sn And CD163

Porcine reproductive and respiratory syndrome(PRRS)is highly contagious swine infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),which is characterized by late-term maternal reproductive failure,increased mortality of newborn piglets,and respiratory disease in growing pigs.PRRS was introduced into China in 1995 and has become the most important infectious disease in modern pig farms.Since 2006,the highly pathogenic PRRSV has brought great economic losses to pig industry.At present,PRRS is mainly controlled by immunization with attenuated live vaccine.Although the vaccine can induce fairly immune protection against homologous strain infection,the immunoprotection against heterologous stains is controversial with the risk of virulence-conversing and virus spread.PRRSV is one of the fastest-mutation RNA viruses with complex infection and immune evasion mechanisms.Therefore,the development of new PRRS vaccines faces great challenges,and development of new anti-PRRSV strategy is needed urgently.Porcine alveolar macrophage(PAM)is the main target cell of PRRSV with the involvement 2 major cellular receptors.Among them,CD163 is the core receptor using its 5-9 scavenger receptor cysteine-rich domains(SRCR5-9)as the virus-binding region.Sialoadhesin(Sn)is involved in the virus attachment and internalization with the first four immunoglobulin-like domains(Sn4D)as PRRSV-binding region.Our previous study showed that the recombinant adenoviruses(rAds)expressed Sn4D-Fc and SRCR59-Fc fusion proteins can act as the soluble receptors to block PRRSV infection of PAM.However,whether these rAds can express the encoded soluble receptors with sufficient anti-PRRSV effect in pigs remains to be tested.1.Expression,purification and activity of PRRSV soluble receptor-capturing proteinStreptococcus G protein(SPG)is a Ig-binding protein of multi-species and the Ig-binding site is located in its C2 domain.Elastin like polypeptide(ELP)is a novel recombinant protein purification tag with temperature-sensitive reversible phase-transition property.Therefore,ELP fusion proteins can be purified by inverse transition cycling(ITC).To establish the methods for purification and detection of soluble receptors Sn4.D-Fc and SRCR59-Fc,in this study the coding sequence for SPG C2 domain was cloned into ELP fusion expression vector pELP-C2,and transformed into BLR(DE3)E.coli.The expression of ELP-C2 fusion protein was induced with IPTG.The conditions for soluble fusion protein expression and purification were optimized.The results showed that ELP-C2 fusion was expressed as a soluble protein at 37℃ with a relatively low expression level.The expression level could be increased by lowering induction temperature and extending induction time,with the highest expression level for 30 h at 26 0C.The transition temperature of ELP-C2 fusion protein was 32℃ in the presence of 3M NaCl.The ELP-C2 fusion protein could be purified to higher than 90%purity after two rounds of ITC.Western-blotting analysis showed that the ELP-C2 fusion protein could bind to 5 mammalian IgG and chicken IgY.The successful expression and purification ELP-C2 fusion protein with Ig-binding activity laid the solid foundation for developing the method for purification and detection of Sn4D-Fc and SRCR59-Fc soluble receptors.2.Detection method establishment and in vivo expression of PRRSV soluble receptorsIn light of the fact that ELP-C2 fusion protein can bind to chicken IgY which is easily available,the conditions for Ig binding to and elution off ELP-C2 protein was optmized using chicken IgY.The results showed that chicken IgY could be efficiently captured from soluble egg protein by ELP-C2 protein under the optimal binding conditions at 16℃ with 125μg/mL ELP-C2 protein.The bound IgY could be efficiently eluted off the fusion protein under the optimal conditions with 0.1M glycine(pH 9.5)at 28℃ for 5min.To validate the usability of ELP-C2 protein for binding capture of Sn4D-Fc and SRCR59-Fc soluble receptors.PK-15 cells were transduced with rAd-Sn4D-Fc or rAd-SRCR59-Fc and the cell medium was collected 2 days after transduction.The protein binding assay showed that both Sn4D-Fc and SRCR59-Fc could be purified from the cell medium by ELP-C2 binding capture with a purity of 93.1%or 94.3%.The purified Sn4D-Fc was used as the antigen of sandwich ELISA with the mouse anti-porcine Sn immune serum as the coating antibody and biotin-labeled anti-Sn polyclonal antibody as the second antibody,with a detection sensitivity of 0.4ng/mL.Finally,piglets were injected with rAd-Sn4D-Fc and rAd-SRCR59-Fc,and the serum samples were collected for Sn4D-Fc detection using the established sandwich ELISA or SRCR59-Fc detection using commercial ELISA kit.The results showed that the two soluble receptors could be expressed and secreted into the circulation of rAd-injected piglets for 15 days.3.Generation of pig model for contagious PRRSV infectionTo mimic the natural PRRSV infection and to evaluate the challenge protective effect of the soluble receptors,three PRRSV-naive piglets were inoculated with artificially infected piglets of highly pathogenic PRRSV strain JXA1.Their clinical symptoms were observed daily for symptom scoring,and serum and fecal samples were collected regularly for PRRSV detection.The animals died of PRRS were examined pathologically and the PRRSV loads in different tissues were detected by quantitative RT-PCR.Results showed that the rectal temperature of the infected pigs rose obvious from day 4 after cohabitation with typical PRRS symptoms appeared from day 5,which were 2 and 3 days later than the artificial inoculation pig.Typical symptoms of PRRS appeared from day 7,and the symptom score was close to the artificial infected pig.From day 3 after cohabitation,viremia was detected in all piglets,which reached to and maintaining at a high level(8,6 log10/mL)on day 10.From day 5 after cohabitation,all fecal samples were positive for PRRSV,and the fecal virus emission was continued to a high level on day 7(3.9 log10/0.1g).Three contagious infected piglets died after twelfth and thirteenth days of cohabitation,which were 1-2 days later than the artificial inoculation pig.The virus load of main organs and gross or micropathological changes of contagious infected piglets are similar to the artificial inoculation pig.These data suggest that the pig model for contagious PRRSV infection was established and usable to evaluate the protective effect of soluble receptors against PRRSV challenge.4.Anti-PRRSV effect of the soluble receptors against JXA1 strainTo evaluate the protective effect of soluble receptors against PRRSV challenge,nine PRRSV-naive piglets were divided into three experimental groups(n=3)and injected muscularly with rAd-Sn4D-Fc(group Ⅰ),rAd-SRCR59-Fc(group Ⅱ)or rAd-Sn4D-Fc plus rAd-Sn4D-Fc(group Ⅲ).On day 5 after rAd injection,each group was housed with one piglet experimentally infected with PRRSV strain JXA1.Their clinical symptoms were observed,morbidity/mortality recorded,and blood samples and fecal samples were collected for PRRSV detection.The results showed that,compared to the challenge control group,the time of high fever and PRRS symptoms was delayed by 2,3,5 days and 3,3,4 days in groups,Ⅱ andⅢ,respectively.Compared to no death in group Ⅲ,the three piglets in group Ⅰ died at day 23,24 and 25,and one piglet in group Ⅱ died at day 23 with typical PRRS symptoms and lesions.Pigs in group Ⅰshowed minor interstitial pneumonia of lung than challenge group,while group Ⅱand Ⅲ did not show obvious gross or microscopic lesions.The appearance of viremia and fecal virus emission were delayed by 2,2,4 days and 2,2,5 days,respectively.The viral loads of organs in rAd groups were lower than challenge group,furthermore,group Ⅲ was the lowest,followed by group Ⅱ.These experiments demonstrated that PRRSV infection of pigs could be blocked partially by injection with rAd-Sn4D-Fc or rAd-SRCR59-Fc,or completely by injection with rAd-Sn4D-Fc plus rAd-SRCR59-Fc.These data confirmed the in vitro additive anti-PRRSV effect of soluble Sn and CD163 receptors.5.Anti-PRRSV effect of soluble receptors against different strainsTo confirm the in vivo anti-PRRSV effect of the two soluble receptors,six PRRSV-naive piglets were divided into two groups and injected with rAd-Sn4D-Fc + rAd-Sn4D-Fc.On day 5 after rAd injection,each group was housed with one piglet infected with PRRSV strain JS07 or SH1705,and the anti-viral effects were evaluated according to the above.Compared to the challenge group JS07 and SH1705,the time of high fever was delayed by 4 or 8 days,and clinical symptom was delayed by 2 days by rAd injection,with the symptom scores significantly lower(P<0.05)than JS07 from day 7 to 17 and SH1705 from day 7-20(P<0.05).Compared to one death in strain SH1705 and strain JS07 challenge group,all of the six rAd-injected piglets were survived from the challenge.The lungs of dead pigs showed typical interstitial pneumonia,while no obvious pathological changes in rAd-injected piglets after culling.The appearance of viremia and fecal virus emission were delayed by 4 days and 3 days,respectively.Viral loads of spleen,lung,and lymph node in rAd-injected pigs were significantly lower(P<0.05)than JS07 challenge group,which of spleen,lung,kidney and lymph node were significantly lower(P<0.05)than SH1705 challenge group.These data demonstrated that the rAd-delivered two soluble receptors could protect pigs from different strain PRRSV infection,and the two rAds could be further developed as novel vaccines for PRRS control.