The Effects Of CD163Molecule In ADE Of PRRSV Infection

Porcine reproductive and respiratory syndrome (prrs) is Class B infectious diseases caused by porcine reproductive and respiratory syndrome virus(PRRSV) which is characterized by miscarriage, stillbirth in pregnant sows and respiratory problems of piglets, and also causes immune-suppression and persistent infection of pigs. PRRSV is a single-stranded RNA virus, which is provided with variability, antibody-dependent enhancement (ADE). To study ADE mechanism of PRRSV infection has important significance for prevention of PRRS and research on new vaccines.PRRSV has strict tropism for cell and is able to infect monocyte-macrophages in vitro and vivo, while receptors play a decisive role in mediating virus invasion of the host.Currently there is a consensus on the process of PRRSV infection PAM:first PRRSV particles adhere to the cell surface by receptor HS, causing Sn N terminal sialic acid binding site to combine with GP5/M complex of PRRSV, activating the Sn-induced signal path to induce endocytosis, following releasing PRRSV genome mRNA into the cytoplasm of cells by CD163molecule, then completing the process of proliferation. Studies show that SRCR5domain of CD163molecule may interact with GP2and GP4protein of the viral particles,however, it is not very clear that the effects of CD163molecule on the mechanism of in PRRSV entering the host cells, and it has not been reported whether the CD163molecule plays a role in PRRSV-ADE. In this test we cloned and expressed by prokaryote the the4extracellular regions of CD163molecule domains, immunizing mice to get positive sera, and prepared and purified the corresponding specific polyclonal antibodies. By studying whether these antibodies are able to block PRRSV and immune complexes infection, we identified the role of CD163molecule in PRRSV invasion hosts process, and explored the role of CD163in PRRSV-ADE.Scavenger receptor CD163protein, known as M130, is130KDa, which belongs to I type glycosylated protein, rich in cysteine. With the software prediction analysis we found porcine CD163molecule in PAM comprising the extracellular, transmembrane and cytoplasmic tail region, and the extracellular region containing a signal peptide (1-40AA) and9cysteine-rich (SRCR) domains, and the position of1-9SRCRs domains of CD163were respectively54AA-151AA,161AA-258AA,268AA-365AA,375AA-472AA,480AA-577AA,585AA-682AA,721AA-818AA,828AA-925AA and931AA-1028AA. In this test, CD163molecule was divided into four fragments to be cloned and expressed by prokaryote, namely CD163-P1(SRCR1-2), CD163-P2(SRCR3-4), CD163-P3(SRCR5-6) and CD163-P4(SRCR7-9). We prepared the polyclonal antiserum by immunizing the mice with the four recombinant proteins. The two-step method of ammonium sulfate precipitation method and the DE-52ion exchange chromatography technology was used to extract and purify to obtain four specific antibodies of high purity. With PBS lavage method, primary porcine alveolar macrophage cells were washed and plated out, when four kinds of purified anti-CD163antibodies and mixture antibody of the four antibodies were incubated1h to block PAM cells, which were treated with Hn-1/06PRRSV and diluted4times PRRSV-IgG immune complexes24h and48h, setting the negative control and blank control groups. We detected the PRRSV mRNA levels of infected cells with established PRRSV SYBR Green I real-time PCR method, and used the GraphPad Prism5software to handle with the datas that we got.In experiment one, we collected PAM from healthy piglets first, extracted cell total RNA, referred to GenBank:EU016226.1to design four pairs of specific primers, and amplified four gene fragments of CD163by RT-PCR, which were respectively698bp,721bp,609bp and988bp. Then we subcloned these fragments into the plasmid vector PET-32a, and successfully built the recombinant plasmids PET-CD163-PI, PET-CD163-P2, PET-CD163-P3and PET-CD163-P4. We transformed the recombinant plasmids into the expression strain BL21, and successfully expressed protein by IPTG inducing. The expressed proteins approximately were44.2kDa,45kDa,41.4kDa and52.0kDa, which consistented with the predicted fusion protein. By optimizing the time and theconcentration of IPTG inducing, we got a large number of expressed recombinant proteins CD163-P1, CD163-P2, CD163-P3and CD163-P4, and purified recombinant proteins by Urea. We immunized the mice with the purified recombinant proteins, and prepared four kinds of polyclonal antiserum, used indirect ELISA method to detect positive antibody titer, which were respectively1:10240,1:12800,1:25600and1:12800. Antibodies were extracted by ammonium sulfate precipitation method, and purified with the DE-52ion-exchange chromatography technology, before concentration of antibodies were determined by UV spectrophotometer.In experiment two, we used four preparated antibodies, and the mixture of four antibodies from the experiment one to block PAM1h, before PRRSV infected24h and48h, and we detected PRRSV mRNA levels of infected cells with established PRRSV SYBR Green I real-time PCR method, and used the GraphPad Prism5software to handle with the datas we got. The results showed that it was not obvious for the difference of PRRSV mRNA levels between mouse anti-pig CD163-P1、CD163-P2and CD163-P4control groups with mouse negative-IgG group at24h and48h, while the PRRSV mRNA levels for mouse anti-pig CD163-P3and CD163-H are lower than mouse negative-IgG group, and PRRSV mRNA levels for mouse anti-pig antibodies CD163-P3blocked group is0.78times as the mouse IgG-negative control group (P<0.01) at24h and is0.85times (P<0.05) at48h. It showed that the antibodies prepared by this method could block PRRSV infection PAM, and suggested that CD163-P3(SRCR5and SRCR6domains) could be involved in the process of PRRSV infected PAM cells while the other fragments of CD163did not affect PRRSV to invade host cells, which was consistent with the main function of SRCR5domain of CD163. It also showed that the antibodies that prepared、extracted and purified by the method had immunobiologic activities, which provided references for researching the mechanism of PRRSV infection and PRRSV-ADE.In experiment three, PAMs were blocked with four preparated antibodies, the mixture of four antibodies from the experiment one, and were treated with four times diluting immune complexes for24h and48h, detecting PRRSV mRNA levels of infected cells with established PRRSV SYBR Green I real-time PCR method, at the same time setting up PRRSV and immune complexes unblocked group, and using the GraphPad Prism5software to handle with the datas we got. In the result, after PAM were treated with immune complexes for24h and48h, the PRRSV mRNA level of mouse anti-pig CD163-P1and CD163-P2antibody groups was not significant difference compared with the corresponding mouse IgG negative control groups. The PRRSV mRNA level of mouse anti-pig CD163-P3antibody groups was significantly lower than the average control group, and the PRRSV mRNA level of mouse anti-pig CD163-P3antibody groups was0.87times as mouse IgG negative control group at24h, while it was0.75times (P<0.01) at48h; the PRRSV mRNA copies of the mouse anti-porcine CD163-P4groups were lower than mouse negative IgG control group, but the difference was not significant; the PRRSV mRNA copies of mouse anti-pig CD163-H antibody groups were significantly lower than mouse negative IgG control group, the mRNA level of PRRSV at24h was0.88times (P<0.05) as the mouse negative IgG control group, while the mRNA level of PRRSV was0.83times (P <0.05)as the control group at48h, showing that CD163-P3mouse anti-pig antibody can affect the PRRSV replication in the cell proliferation, indicating the receptor CD163-P3may be involved in the proliferation of immune complexes in cells. We speculated that the immune complexes invade the host cells by PRRSV protein GP2a and GP4interacting with CD163molecule, change the structure of PRRSV, promote to release the mRNA of PRRSV in the cytoplasm, then completed the proliferation of PRRSV, in which CD163-P3(SRCR5-6) plays a major role, and CD163-P4(SRCR7-9) may also have function slightly. In this study, a preliminary study was explored to the role of receptor CD163extracellular domains in PRRSV-ADE. It provided a reference for the study of the mechanism of ADE and the development of an effective vaccine, and was important for prevention and treatment of viral diseases.