The porcine reproductive and respiratory syndrome virus (PRRSV) can cause reproductive barriers in breeding pigs and respiratory symptoms in piglets. In this review, we summarize research progress of the infection and replication mechanisms of the PRRSV. We also review the virulence determinants of the PRRSV. All these fundamental studies are important for the control and elimination of the PRRSV.
Animals ; Porcine Reproductive and Respiratory Syndrome ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; pathogenicity ; physiology ; Swine ; Virulence ; Virus Replication

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a threat, causing economically significant impacts on the swine industry worldwide. Unfortunately, the traditional control strategies and conventional vaccines fail to provide sustainable disease control, in particular against genetically diverse strains, as they suffer from both antigenic heterogeneity and various immune evasion strategies of PRRSV. In this paper, latest research progress in immunology and immune evasion of PRRSVis summarized to provide a referenc for PRSSV prevention and control as well as the design of new vaccines.
Animals ; Immune Evasion ; Porcine Reproductive and Respiratory Syndrome ; immunology ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; immunology ; Swine ; Viral Proteins ; genetics ; immunology

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which causes great economic losses. At the moment, no effective neutralizing antibody is available for scientific research and treatment. Therefore, developing a method for screening the neutralizing monoclonal antibodies is of great significance for the prevention and treatment of PRRSV and the screening of antigen sites. Monoclonal antibodies have been widely used in the treatment and diagnosis of many human and animal diseases. Therefore, screening effective neutralizing antibodies for different pathogens is an urgent task. Among the methods for monoclonal antibody screening, B cell immortalization is an effective method to obtain neutralizing monoclonal antibody. Specifically, in this study, the bcl-6 and bcl-xl genes were connected by f2a and then the yielded product was ligated to a vector for retrovirus packaging. The swine lymphocytes immunized with PRRSV were infected the yielded mature viruses and cultured in the complete medium containing CD40L and IL21 cytokines. Then, CD21 was used as the marker to screen B cells with the magnetic bead method. Finally, monoclonal B cells were obtained and the secretion of antibodies was tested. The results showed that the plasmid, either being transfected alone or with the packaged plasmids, could be expressed, and that the packaged retrovirus could infect the cells. Moreover, the infected lymphocytes secreted antibodies, so did the screened B cells. Therefore, the method for screening monoclonal antibody against PRRSV was successfully established.
Animals ; Antibodies, Monoclonal ; Antibodies, Neutralizing ; Antibodies, Viral ; Humans ; Porcine Reproductive and Respiratory Syndrome/prevention & control* ; Porcine respiratory and reproductive syndrome virus/genetics* ; Swine

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*

Porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 7 (Nsp7) plays an important role in the induction of host humoral immune response and could serve as an ideal antigen for serological genotyping assay for PRRSV based on the significant difference in immunoreactivities of North American (NA) and European (EU) PRRSV Nsp7. In this study, Nsp7 of NA and EU PRRSVwas separately expressed and purified using prokaryotic expression system. The purified recombinant Nsp7 proteins reacted with serum antibodies against corresponding genotype PRRSV in Western blotting. However, nonspecific reaction of whole recombinant Nsp7 with antibodies against another genotype PRRSV was observed, indicating that whole NA PRRSV Nsp7 and EU PRRSV Nsp7 have similar antigenic epitopes and recombinant proteins could not be used for genotyping of antibodies against PRRSV. Based on the analysis of similar antigenic epitopes at the hydrophilic region of NA PRRSV Nsp7 and EU PRRSV Nsp7 by bioinformatics assessment, partial Nsp7 gene region deleted sequences encoding similar antigenic epitopes was constructed by fusion PCR. The recombinant truncated Nsp7 (NA-deltaNsp7 and EU-deltaNsp7, about 43 kDa) was expressed and the molecular weight was about 43 kDa. The results of Western blotting showed that NA-deltaNSP7 and EU-deltaNSP7 could be specifically recognized by positive serum to NA or EU PRRSV individually and nonspecific reaction was eliminated. This study provided a basis for further development of serological genotyping assay for North American and European genotype PRRSV infection.
Animals ; Genotype ; Porcine respiratory and reproductive syndrome virus ; classification ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; immunology ; Swine ; Viral Nonstructural Proteins ; biosynthesis ; immunology

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

In recent years, mass outbreaks of highly pathogenic (HP) porcine reproductive and respiratory syndrome virus (PRRSV) have spread all over the Chinese swine industry. Based on the first infectious cDNA clone of HP PRRSV strain pJX143 and that of an attenuated PRRSV, pAPRRS, constructed in our group, we constructed several chimeric clones with various substitutions of structural protein genes (ORF4-7) and 3' UTR between attenuated pAPRRS and virulent pJX143.Upon transfection of MA-104 cultured cells, all chimeric constructs pSX12, p5NX12, and p56N12 were rescued. The rescued viruses maintained the similar virological properties, based on the results of the growth curve of the rescued viruses. To test if the chimeric viruses can be used as a vaccine candidate, vSX12 and v56N12 vaccinated pigs were challenged with the HP PRRSV JX143 strain. As a result, the vSX12 vaccinated pigs were all seroconverted by 14-day-post vaccination, while v56N12 vaccinated pigs showed poor antibody response. Upon challenge, the vSX12-vaccinated group showed no signs of clinical PRRS syndrome, and virema period was shorten to 6 days post-challenge. Our results demonstrated that 1) vSX12 chimeric virus is a good vaccine candidate; 2) the virulence determinants of HP PRRSV probably located in coding regions other than ORF3-7 and 3' UTR, as our chimeric viruses were proved to be attenuated.
Animals ; Cloning, Molecular ; Open Reading Frames ; Porcine Reproductive and Respiratory Syndrome ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; Recombination, Genetic ; genetics ; Swine ; Vaccines, Attenuated ; immunology ; Viral Envelope Proteins ; Viral Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

In order to understand the prevalence and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) in China and to develop subunit vaccine against the epidemic lineage, the genetic evolution analysis of PRRSV strains isolated in China from 2001 to 2021 was performed. The representative strains of the dominant epidemic lineage were selected to optimize the membrane protein GP5 and M nucleotide sequences, which were used, with the interferon and the Fc region of immunoglobulin, to construct the eukaryotic expression plasmids pCDNA3.4-IFNα-GP5-Fc and pCDNA3.4-IFNα-M-Fc. Subsequently, the recombinant proteins IFNα-GP5-Fc and IFNα-M-Fc were expressed by HEK293T eukaryotic expression system. The two recombinant proteins were mixed with ISA206VG adjuvant to immunize weaned piglets. The humoral immunity level was evaluated by ELISA and neutralization test, and the cellular immunity level was detected by ELISPOT test. The results showed that the NADC30-like lineage was the main epidemic lineage in China in recent years, and the combination of IFNα-GP5-Fc and IFNα-M-Fc could induce high levels of antibody and cellular immunity in piglets. This study may facilitate the preparation of a safer and more effective new PRRSV subunit vaccine.
Humans ; Animals ; Swine ; Porcine respiratory and reproductive syndrome virus/genetics* ; Porcine Reproductive and Respiratory Syndrome/prevention & control* ; HEK293 Cells ; Viral Envelope Proteins/genetics* ; Antibodies, Viral ; Viral Vaccines/genetics* ; Recombinant Proteins ; Vaccines, Subunit

Despite global efforts to control porcine reproductive and respiratory syndrome virus (PRRSV) infection, the virus continues to cause economic problems in the swine industry worldwide. In this study, we attempted to generate and characterize a panel of stable BHK cell lines that constitutively express the nucleocapsid (N) protein of type 1 or type 2 PRRSV. The established BHK cell lines were found to react well with N-specific antibodies as well as the hyperimmune serum of pigs raised against each genotype of PRRSV. Taken together, the data implicate a potential usefulness for the newly generated stable cell lines as a diagnostic reagent for PRRSV serology.
Animals ; Antibodies, Viral/analysis/immunology ; Blotting, Western/veterinary ; Cell Line ; Cricetinae ; Female ; Genotype ; Nucleocapsid Proteins/genetics/*immunology ; Porcine Reproductive and Respiratory Syndrome/diagnosis/*immunology ; Porcine respiratory and reproductive syndrome virus/genetics/*immunology ; Swine ; Transfection/veterinary

To develop an attenuated vaccine against the highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) virus, the HP-PRRS virus strain TJ was attenuated by serial passages and plaque cloned every 5 to 10 passages in Marc-145 cells. Genetic variation and pathogenicity of HP-PRRSV strain TJ in the course of attenuation were analyzed. The results showed that the strain TJ sustained various sequence changes during the course of attenuation. Fifty-eight amino acids changes and a new continuous 120 amino acids deletion after the discontinuous 30 amino acids deletion (sites 481 and 533-561) occurred in strain TJ passages 140, and the position of 120 amino acids deletion was between 628 to 747 according to VR-2332. Animal test showed that the pathogenicity of strain TJ passages 20 was attenuated obviously, so we presume that genetic variation in nonstructural protein nsp2-nsp5, nsp7 and structural protein GP5 during the attenuation provides the molecular bases for the observed attenuated phenotype.
Amino Acid Sequence ; Animals ; Genetic Variation ; Molecular Sequence Data ; Porcine Reproductive and Respiratory Syndrome ; virology ; Porcine respiratory and reproductive syndrome virus ; classification ; genetics ; isolation & purification ; pathogenicity ; Sequence Deletion ; Serial Passage ; Swine ; Vaccines, Attenuated ; genetics ; isolation & purification ; Virulence