As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.
Swine ; Animals ; Induced Pluripotent Stem Cells/metabolism* ; Receptors, Cell Surface/genetics* ; Antigens, CD/metabolism* ; Porcine respiratory and reproductive syndrome virus/genetics*

The cDNA fragment encoding the truncated GP5 and the full-length M protein of Porcine Reproductive and Respiratory Syndrone Virus (PRRSV) were orderly fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pKG-56. After transformed into E. coli BL21 (DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-GP5-M fusion protein was expressed in high level. Western-blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against PRRSV. The fusion protein was further purified and used as an antigen to establish a novel PRRSV ELISA diagnose assay (P56-ELISA). Comparison between P56-ELISA and the abroad kit IDEXX-ELISA showed the two methods had 94.1 percent agreement by detecting 205 serum samples, indicating that the indirect P56-ELISA was specific and sensitive. The correlation between virus neutralization antibody of the infected pigs (not convalescent pigs) and antibody response to the fusion protein GP5-M was further studied. The regression function analysis suggested that there was no significant correlation between ELISA antibody response (OD630 nm) to the fusion protein GP5-M in clinical serum and their specific neutralizing titers.
Animals ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Glutathione Transferase ; metabolism ; Open Reading Frames ; Porcine Reproductive and Respiratory Syndrome ; diagnosis ; immunology ; Porcine respiratory and reproductive syndrome virus ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Swine ; Viral Envelope Proteins ; biosynthesis ; genetics

This study aims to express pig nuclear transcription factor-kappaB (NF-kappaB) p65/p50 fusion protein in E. coli Rosetta, and study its impacts on PRRSV proliferation in vitro. The p65 ORF and mature p50 encoding gene were amplified by RT-PCR, the products were cloned into the pET-21a(+) vector, then transformed into Escherichia coli Rosetta, recombinant fusion protein was expressed by IPTG induction, the expressed product was identified by SDS-PAGE and Western-Blot. The purified and re-folded p65/p50 was added to the 2% FBS DMEM, and the cytotoxicity on Marc145 was observed to select the optimum concentration. The effects of optimum concentration of p65/p50 on PRRSV proliferation activity were investigated by detecting PRRSV infection phase in the culture supernatant using real-time FQ-PCR method and drawing PRRSV one-step growth curve. The results showed the p65/p50-pET21a(+) prokaryotic expression vector were successfully constructed , recombinant p50 and p65 fusion protein was expressed abundantly in the form of inclusion body with molecular weight of 70kD, Western-Blot results showed that the rabbit anti-human p50 polyclonal serum, rabbit anti-human p65 purified antibody could bind specifically to p50 and p65 respectively. The optimum concentration of p65/p50 was 0.4 microg/mL. The real-time FQ-PCR results indicated that NF-kappaB p65/p50 could promote CPE appearance and PRRSV proliferation before CPE appeared, and suppress PRRSV proliferation after CPE appeared, and lower the virus titer levels significantly(P < 0.05). These results will provide some new insight of the pathogenic mechanism and treatment strategies of PRRS.
Animals ; Cell Line ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; NF-kappa B p50 Subunit ; genetics ; metabolism ; Porcine Reproductive and Respiratory Syndrome ; metabolism ; Porcine respiratory and reproductive syndrome virus ; genetics ; physiology ; Recombinant Fusion Proteins ; genetics ; metabolism ; Swine ; Transcription Factor RelA ; genetics ; metabolism ; Virus Replication

The objective of this study was to investigate the function of vimentin in PRRSV infection. Vimentin gene from Marc-145 cells was amplified by RT-PCR, cloned into pET-28a vector and expressed in Escherichia coli BL21(DE3). The expressed vimentin was confirmed by Western blot and purified which was used to immunize BALB/c mice for the production of antibodies. Vimentin and antibodies were tested for blocking PRRSV infection of Marc-145 cells. The binding of vimentin to PRRSV N and GP5 proteins were tested by the ELISA. The results showed that vimentin gene was amplified successfully and expressed as identified by SDS-PAGE and Western blot. Mouse anti-vimentin antibodies were produced with the titer of 10(5). PRRSV infection of Marc-145 cells was blocked partially by vimentin while blocked completely by the antibobies. In addition, vimentin was bound N protein, but not GP5. These results provide additional information on PRRSV entry into Marc-145 cells.
Animals ; Antibodies ; immunology ; metabolism ; Cell Line ; Escherichia coli ; genetics ; metabolism ; Female ; Genetic Vectors ; genetics ; Mice ; Mice, Inbred BALB C ; Porcine Reproductive and Respiratory Syndrome ; genetics ; metabolism ; virology ; Porcine respiratory and reproductive syndrome virus ; physiology ; Protein Binding ; physiology ; Recombinant Proteins ; genetics ; immunology ; isolation & purification ; metabolism ; Swine ; Vimentin ; genetics ; immunology ; isolation & purification ; metabolism ; Viral Proteins ; metabolism

Modified ORF5 (MORF5) and ORF6 gene of PRRSV were cloned into two multiple cloning sites of MVA transfer vector pLR-gpt to construct the recombinant plasmid pLR-MORF5/ORF6. Homologous recombination between pLR-MORF5/ORF6 and the wtMVA on BHK-21 cell line was mediated with liposome by infecting the cell with 0.01 MOI wtMVA two hours before transfecting the recombinant plasmid into the cell. When the cytopathic effect (CPE) was obvious, virus was collected from the cell plate and the recombinant virus was selected with drug selecting medium (2% MXHAT). After 12 cycles of selection, rMVA with a selection marker Eco gpt was obtained and named as rMVAgpt-MGP5/M. By infecting BHK-Cre expressing Cre recombinant enzyme, the Eco gpt marker in rMVAgpt-MGP5/M was deleted and this rMVA was named as rMVA-MGP5/M. The insertion of MORF5 and ORF6 into the MVA genome was confirmed with PCR analysis and the expression of MGP5 and M protein was identified with Western blot and IFA. Through biological study on the recombinant MVA, no obvious difference was observed between rMVA-MGP5/M and the wtMVA regarding to the CPE and growth curve. The recombinant MVA constructed in this study could coexpress the modified GP5 and M protein and the expressed product had good immunocompetence. Furthermore, the insertion of the MORF5 and ORF6 into MVA genome had no obvious effect on the replication and biological characteristics of this virus.
Animals ; Cell Line ; Genetic Vectors ; genetics ; Porcine Reproductive and Respiratory Syndrome ; prevention & control ; Porcine respiratory and reproductive syndrome virus ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Recombination, Genetic ; Swine ; Transfection ; Vaccines, DNA ; genetics ; immunology ; Vaccinia virus ; classification ; genetics ; metabolism ; Viral Envelope Proteins ; biosynthesis ; genetics ; Viral Matrix Proteins ; biosynthesis ; genetics

The highly virulent PRRSV isolate strain HN-1/06 was cultivated on Marc-145. To study the viral entry mechanisms, the GP5 gene of PRRSV isolate was amplified by RT-PCR and cloned into pcDNA3.0 to generate the expressing plasmid pcDNA-GP5. pcDNA-GP5 was transfected into 293T by the calcium phosphate precipitation method. Analysis of flow cytometry confirmed that the GP5 proteins were expressed in surface of the 293T cells. Then 293T cells were transfected with pcDNA-GP5, pHIT60 and pHIT111 plasmids to generate pseudotyping virus. The pseudotyping virus supernatant was harvested 48 hours post-transfection and was detected by Western blotting and infection assay. Western blotting indicated that the GP5 glycoproteins were incorporated into the retroviral pseudotyped virus. Infection assay showed that the pseudotyped virus infected 293T and Mark-145 cell. The pseudotyped virus could be used to further study infectious mechanism of PRRSV.
Animals ; Cell Line ; Cloning, Molecular ; Endothelial Cells ; cytology ; metabolism ; virology ; Leukemia Virus, Murine ; genetics ; metabolism ; Mice ; Porcine respiratory and reproductive syndrome virus ; chemistry ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Swine ; Transfection ; Viral Envelope Proteins ; biosynthesis ; genetics ; Virion ; genetics ; metabolism

The purpose of this study was to investigate the effects of porcine interleukin (IL)-2 and IL-4 genes on enhancing the immunogenicity of a porcine reproductive and respiratory syndrome virus ORF5 DNA vaccine in piglets. Eukaryotic expression plasmids pcDNA-ORF5, pcDNA-IL-2, and pcDNA-IL-4 were constructed and then expressed in Marc-145 cells. The effects of these genes were detected using an indirect immunofluorescent assay and reverse transcription polymerase chain reaction (RT-PCR). Characteristic fluorescence was observed at different times after pcDNA-ORF5 was expressed in the Marc-145 cells, and PCR products corresponding to ORF5, IL-2, and IL-4 genes were detected at 48 h. Based on these data, healthy piglets were injected intramuscularly with different combinations of the purified plasmids: pcDNA-ORF5 alone, pcDNA-ORF5 + pcDNA-IL-2, pcDNA-ORF5 + pcDNA-IL-4, and pcDNA-ORF5 + pcDNAIL-4 + pcDNA-IL-2. The ensuing humoral immune responses, percentages of CD4+ and CD8+ T lymphocytes, proliferation indices, and interferon-gamma expression were analyzed. Results revealed that the piglets co-immunized with pcDNA-ORF5 + pcDNA-IL-4 + pcDNA-IL-2 plasmids developed significantly higher antibody titers and neutralizing antibody levels, had significantly increased levels of specific T lymphocyte proliferation, elevated percentages of CD4+ and CD8+ T lymphocytes, and significantly higher IFN-gamma production than the other inoculated pigs (p < 0.05).
Animals ; Cell Line ; Escherichia coli/genetics ; Haplorhini ; Immunity, Cellular ; Interleukin-2/genetics/*metabolism ; Interleukin-4/genetics/*metabolism ; Neutralization Tests/veterinary ; Plasmids ; Porcine Reproductive and Respiratory Syndrome/*prevention & control ; Porcine respiratory and reproductive syndrome virus/*immunology ; Recombinant Proteins/genetics/metabolism ; Swine ; Vaccines, DNA/immunology ; Viral Envelope Proteins/*genetics/metabolism ; Viral Vaccines/*immunology

Porcine circovirus 2 (PCV2) has been implicated as the etiological agent of postweaning multisystemic wasting syndrome (PMWS). Co-infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) can result in severe economic losses to the swine industry. In this study, we constructed the recombinant adenovirus rAd-Cap-GP5 expressing Cap of PCV2 and GP5 of PRRSV. And the expression of Cap and GP5 protein in the HEK-293 cells inoculated with rAd-Cap-GP5 were confirmed by immunoperoxidase monolayer assay (IPMA), indirect immunofluorescence assay (IFA) and Western blotting, respectively. The immunogenicity of recombinant adenoviruses rAd-Cap-GP5 was examined in mice by vaccination with the recombinant adenovirus. The results showed that the mice could produce anti-PCV2 and PRRSV antibodies detected by indirect ELISA and virus neutralization assay. It indicated that rAd-Cap-GP5 could provide humoral immunity responses in mice. The recombinant adenovirus rAd-Cap-GP5 might be an attractive candidate vaccine for preventing the disease associated with PCV2 and PRRSV infection.
Adenoviridae ; genetics ; metabolism ; Animals ; Capsid Proteins ; biosynthesis ; genetics ; immunology ; Cell Line ; Circoviridae Infections ; prevention & control ; virology ; Circovirus ; genetics ; metabolism ; Immunization ; Mice ; Porcine Reproductive and Respiratory Syndrome ; prevention & control ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Transfection ; Viral Envelope Proteins ; biosynthesis ; genetics ; immunology

RNA interference (RNAi) is a powerful tool in gene function research. In order to investigate the role of GP2, GP3 and GP4 of porcine reproductive and respiratory syndrome virus (PRRSV) in the viral replication, small interference RNAs (siRNAs) directed to ORF2, ORF3 and ORF4 were designed and 12 short hairpin RNA (shRNA) expression vectors were constructed (designed as 21,22,23,24,31,32,33,34,41,42,43 and 44). Cells treated with shRNA expression vectors were infected by PRRSV. The effective shRNA expression vectors were selected by fluorescent quantatitive PCR (FQ-PCR). The virus titer of supernatant of the cells treated with effective shRNA expression vectors (23,24,31,34 and 41) were reduced by 184 to 4.65 folds compared with that of controls.
Genetic Vectors ; Open Reading Frames ; Porcine respiratory and reproductive syndrome virus ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; metabolism ; RNA, Viral ; chemistry ; metabolism ; Transduction, Genetic ; Viral Proteins ; genetics ; Virus Replication ; genetics

A porcine reproductive and respiratory syndrome virus (PRRSV) was obtained from clinic samples. Genes 5 and 6 encoding for the viral glycoprotein 5 and a membrane protein of the PRRSV designated as HH08 were amplified by reverse transcription-PCR. These sequences were compared with reference sequences derived from different geographical locations. The results indicated that the virus belongs to the North American type rather than European. Comparative analyses of the genetic diversity between the PRRSV isolate HH08 and other Chinese as well as foreign reference strains of PRRSV were discussed based on the sequence comparison and the topology of phylogenetic trees constructed in this study.
Animals ; Base Sequence ; China/epidemiology ; Gene Expression Regulation, Viral/physiology ; Genetic Variation ; Molecular Sequence Data ; *Phylogeny ; Porcine Reproductive and Respiratory Syndrome/epidemiology/virology ; Porcine respiratory and reproductive syndrome virus/*genetics/*metabolism ; Sequence Alignment ; Swine ; Viral Envelope Proteins/genetics/*metabolism ; Viral Matrix Proteins/genetics/*metabolism