Porcine Reproductive And Respiratory Syndrome Virus GP5 Research Articles

Mass Spectrometry-Based Proteomic Analysis of Potential Host Proteins Interacting with GP5 in PRRSV-Infected PAMs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus causing a large economic impact on the swine industry. The structural protein GP5 of PRRSV plays a pivotal role in its pathogenicity and immune evasion. Virus-host interactions play a crucial part in viral replication and immune escape. Therefore, understanding the interactions between GP5 and host proteins are significant for porcine reproductive and respiratory syndrome (PRRS) control. However, the interaction network between GP5 and host proteins in primary porcine alveolar macrophages (PAMs) has not been reported. In this study, 709 GP5-interacting host proteins were identified in primary PAMs by immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis revealed that these proteins were involved in multiple cellular processes, such as translation, protein transport, and protein stabilization. Subsequently, immunoprecipitation and immunofluorescence assay confirmed that GP5 could interact with antigen processing and presentation pathways related proteins. Finally, we found that GP5 may be a key protein that inhibits the antigen processing and presentation pathway during PRRSV infection. The novel host proteins identified in this study will be the candidates for studying the biological functions of GP5, which will provide new insights into PRRS prevention and vaccine development.

The glycoprotein 5 of porcine reproductive and respiratory syndrome virus stimulates mitochondrial ROS to facilitate viral replication.

Porcine reproductive and respiratory syndrome virus (PRRSV) presents a significant economic concern for the global swine industry due to its connection to serious production losses and increased mortality rates. There is currently no specific treatment for PRRSV. Previously, we had uncovered that PRRSV-activated lipophagy to facilitate viral replication. However, the precise mechanism that PRRSV used to trigger autophagy remained unclear. Here, we found that PRRSV GP5 enhanced mitochondrial Ca2+ uptake from ER by promoting ER-mitochondria contact, resulting in mROS release. Elevated mROS induced autophagy, which alleviated NLRP3 inflammasome activation for optimal viral replication. Our study shed light on a novel mechanism revealing how PRRSV exploits mROS to facilitate viral replication.

Comparison of heterologous prime-boost immunization strategies with DNA and recombinant vaccinia virus co-expressing GP3 and GP5 of European type porcine reproductive and respiratory syndrome virus in pigs

Vaccination is principally used to control and treat porcine reproductive and respiratory syndrome virus (PRRSV) infection. This study investigated immunogenicity and protective efficacy of heterologous prime-boost regimens in pigs, including recombinant DNA and vaccinia virus vectors coexpressing PRRSV European genotype (EU) isolate GP3 and GP5: group A, pVAX1-EU-GP3-GP5 prime and rddVTT-EU-GP3-GP5 boost; group B, rddVTT-EU-GP3-GP5 prime and pVAX1-EU-GP3-GP5 boost; group C, empty vector pVAX1; group D, E3L gene-deleted vaccinia virus E3L− VTT. Vaccine efficacy was tested in an EU-type PRRSV (Lelystad virus strain) challenge pig model based on evaluating PRRSV-specific antibody responses, neutralizing antibodies, cytokines, T lymphocyte proliferation, CD4+ and CD8+ T lymphocytes, clinical symptoms, viremia and tissue virus loads. Plasmid DNA was delivered as chitosan-DNA nanoparticles, and Quil A (Quillaja) was used to increase vaccine efficiency. All piglets were boosted 21 days post the initial inoculation (dpi) and then challenged 14 days later. At 14, 21, 28 and 35 dpi, groups A and B developed significantly higher PRRSV-specific antibody responses compared with control groups C and D. Two weeks after the boost, significant differences in neutralizing antibody and IFN-γ levels were observed between groups A, C, D and B. At 49 dpi, groups A and B had markedly increased peripheral blood CD3+CD4+ T cell levels. Following virus challenge, group A showed viremia, but organ virus loads were lower than those in other groups. Thus, a heterologous prime-boost vaccine regimen (rddVTT-EU-GP3-GP5 prime, pVAX1-EU-GP3-GP5 boost) can improve humoral- and cell-mediated immune responses to provide resistance to EU-type PRRSV infection in vivo.

Genetic variation and recombination analysis of the GP5 (GP5a) gene of PRRSV-2 strains in China from 1996 to 2022.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been prevalent in China for more than 25 years and remains one of the most significant pathogens threatening the pig industry. The high rate of mutation and frequent recombination of PRRSV have exacerbated its prevalence, particularly with the emergence of highly pathogenic PRRSV (HP-PRRSV) has significantly increased the pathogenicity of PRRSV, posing a serious threat to the development of Chinese pig farming. To monitor the genetic variation of PRRSV-2 in China, the GP5 sequences of 517 PRRSV-2 strains from 1996 to 2022 were analyzed and phylogenetic trees were constructed. Furthermore, a total of 60 PRRSV strains, originating from various lineages, were carefully chosen for nucleotide and amino acid homologies analysis. The results showed that the nucleotide homologies of the PRRSV GP5 gene ranged from 81.4 to 100.0%, and the amino acid homologies ranged from 78.1 to 100.0%. Similarly, the PRRSV GP5a gene showed 78.0 ~ 100.0% nucleotide homologies and 70.2 ~ 100.0% amino acid homologies. Amino acid sequence comparisons of GP5 and GP5a showed that some mutations, such as substitutions, deletions, and insertions, were found in several amino acid sites in GP5, these mutations were primarily found in the signal peptide region, two highly variable regions (HVRs), and near two T-cell antigenic sites, while the mutation sites of GP5a were mainly concentrated in the transmembrane and intramembrane regions. Phylogenetic analysis showed that the prevalent PRRSV-2 strains in China were divided into lineages 1, 3, 5, and 8. Among these, strains from lineage 8 and lineage 1 are currently the main prevalent strains, lineage 5 and lineage 8 have a closer genetic distance. Recombination analysis revealed that one recombination event occurred in 517 PRRSV-2 strains, this event involved recombination between lineage 8 and lineage 1. In conclusion, this analysis enhances our understanding of the prevalence and genetic variation of PRRSV-2 in China. These findings provide significant insights for the development of effective prevention and control strategies for PRRS and serve as a foundation for future research in this field.

Immunological evaluation of recombination PRRSV GP3 and GP5 DNA vaccines in vivo.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a threat to the health of pigs worldwide, but commercially available vaccines offer limited protection against PRRSV infection. It is necessary to develop a more effective DNA vaccine. The immunological effects of DNA vaccines with three adjuvants were examined in pigs (Susscrofa domestica) challenged with PRRSV. These DNA vaccines, which encoded PRRSV GP3 and GP5, were formulated with A1, A2, and A3. Serum specific and neutralizing antibodies, IL-4, IFN-γ, IL-2, IL-10, CD4+ and CD8+T-lymphocytes, health status, histopathology, and viral loads were determined. The results showed that the use of adjuvant A3 led to higher levels of neutralizing antibodies and a lower viral load in pigs compared to the other adjuvants. The neutralizing antibody titers of the pVAX-GP35+A1 and pVAX-GP35+A3 groups reached a peak of 1:19 at 35 dpi. The maximum concentration of IL-4 was 136.77 pg/mL in the pVAX-GP35+A3 group. At 35 dpi, the IFN-γ concentration in the pVAX-GP35+A1 group was 227.4 pg/mL. pVAX-GP35+A3 group shows the highest IL-2 and IL-10 expression to the peak of 597.6 pg/mL and 189.1 pg/mL, respectively. We found a formulation demonstrated beneficial immune outcomes. This study provides an alternative vaccine to protect pigs from PRRSV.

The Construction and Immunogenicity Analyses of Recombinant Pseudorabies Virus With NADC30-Like Porcine Reproductive and Respiratory Syndrome Virus-Like Particles Co-expression.

Porcine reproductive and respiratory syndrome (PRRS) and pseudorabies (PR) are highly infectious swine diseases and cause significant financial loss in China. The respiratory system and reproductive system are the main target systems. Previous studies showed that the existing PR virus (PRV) and PRRS virus (PRRSV) commercial vaccines could not provide complete protection against PRV variant strains and NADC30-like PRRSV strains in China. In this study, the PRV variant strain XJ and NADC30-like PRRSV strain CHSCDJY-2019 are used as the parent for constructing a recombinant pseudorabies virus (rPRV)-NC56 with gE/gI/TK gene deletion and co-expressing NADC30-like PRRSV GP5 and M protein. The rPRV-NC56 proliferated stably in BHK-21 cells, and it could stably express GP5 and M protein. Due to the introduction of the self-cleaving 2A peptide, GP5 and M protein were able to express independently and form virus-like particles (VLPs) of PRRSV in rPRV-NC56-infected BHK-21 cells. The rPRV-NC56 is safe for use in mice; it can colonize and express the target protein in mouse lungs for a long time. Vaccination with rPRV-NC56 induces PRV and NADC30-like PRRSV specific humoral and cellular immune responses in mice, and protects 100% of mice from virulent PRV XJ strain. Furthermore, the virus-neutralizing antibody (VNA) elicited by rPRV-NC56 showed significantly lower titer against SCNJ-2016 (HP-PRRSV) than that against CHSCDJY-2019 (NADC30-like PRRSV). Thus, rPRV-NC56 appears to be a promising candidate vaccine against NADC30-like PRRSV and PRV for the control and eradication of the variant PRV and NADC30-like PRRSV.

A swine arterivirus deubiquitinase stabilizes two major envelope proteins and promotes production of viral progeny.

Arteriviruses are enveloped positive-strand RNA viruses that assemble and egress using the host cell’s exocytic pathway. In previous studies, we demonstrated that most arteriviruses use a unique -2 ribosomal frameshifting mechanism to produce a C-terminally modified variant of their nonstructural protein 2 (nsp2). Like full-length nsp2, the N-terminal domain of this frameshift product, nsp2TF, contains a papain-like protease (PLP2) that has deubiquitinating (DUB) activity, in addition to its role in proteolytic processing of replicase polyproteins. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nsp2TF localizes to compartments of the exocytic pathway, specifically endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and Golgi complex. Here, we show that nsp2TF interacts with the two major viral envelope proteins, the GP5 glycoprotein and membrane (M) protein, which drive the key process of arterivirus assembly and budding. The PRRSV GP5 and M proteins were found to be poly-ubiquitinated, both in an expression system and in cells infected with an nsp2TF-deficient mutant virus. In contrast, ubiquitinated GP5 and M proteins did not accumulate in cells infected with the wild-type, nsp2TF-expressing virus. Further analysis implicated the DUB activity of the nsp2TF PLP2 domain in deconjugation of ubiquitin from GP5/M proteins, thus antagonizing proteasomal degradation of these key viral structural proteins. Our findings suggest that nsp2TF is targeted to the exocytic pathway to reduce proteasome-driven turnover of GP5/M proteins, thus promoting the formation of GP5-M dimers that are critical for arterivirus assembly.

Co-expression of self-cleaved multiple proteins derived from Porcine Reproductive and Respiratory Syndrome Virus by bi-cistronic and tri-cistronic DNA vaccines

Porcine Reproductive and Respiratory Syndrome caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) remains one of the important diseases in swine industry. A vaccine that is safe, effective and also elicit broad immune response against multiple antigens is desirable. In this study, we developed multi-cistronic DNA vaccines capable of co-expressing multiple structural proteins derived from PRRSV. To preserve the structure and function of each antigen protein, we employed self-cleaving 2A peptides to mediate separation of multiple proteins expressed by multi-cistronic genes. Six bi-cistronic genes encoding PRRSV GP5 and M proteins were generated, by which each construct contains different 2A sequences derived from Foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) either with or without furin cleavage site (Fu). Vectored by the mammalian expression plasmid pTH, all six bi-cistronic genes co-expressed the proteins GP5 and M at comparable level. Importantly, all six types of 2A sequences could mediate a complete self-cleavage of the GP5 and M. We next generated tri-cistronic DNA vaccines co-expressing the PRRSV proteins GP5, M and N. All homologous and heterologous combinations of P2A and F2A in tri-cistronic genes yielded a complete self-cleavage of the GP5, M and N proteins. Our study reports a success in co-expression of multiple PRRSV structural proteins in discrete form from a single vaccine and confirms feasibility of developing one single vaccine that provides broad immune responses against PRRSV.

Replacing the decoy epitope of PCV2 capsid protein with epitopes of GP3 and/or GP5 of PRRSV enhances the immunogenicity of bivalent vaccines in mice

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), and reproductive failure and causes economic losses in the domestic swine industry. The decoy epitope (169–180 amino acid (aa)) of the PCV2 capsid (Cap) protein is an immunodominant epitope and diverts the immune response away from protective epitopes. The mixed infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most common co-infections in the pig industry and shows more severe clinical symptoms. Linear B-cell antigenic epitopes of PRRSV GP3 epitope Ⅰ (61–72aa) and PRRSV GP5 epitope Ⅳ (187–200aa) efficiently elicited neutralizing antibodies against PRRSV. The recombinant baculovirus expressing the Cap protein (Bac-Cap) was modified by replacing the decoy epitope of the Cap protein with either the PRRSV GP3 epitope Ⅰ, the PRRSV GP5 epitope Ⅳ, or the PRRSV GP3 epitope Ⅰ- GP5 epitope Ⅳ to produce the recombinant baculoviruses Bac-Cap-GP3, Bac-Cap-GP5 and Bac-Cap-GP35. The four recombinant baculoviruses were successfully established and characterized as demonstrated with western blot analysis and immunofluorescence assay. Immunogenicities of the four recombinant baculoviruses in mice were tested in sera harvested at 21 and 42 days post-primary immunization. The titers of antibodies in the sera were determined by a PCV2-specific enzyme-linked immunosorbent assay (ELISA) and a serum neutralization assay. The serum IFN-γ levels were measured by indirect ELISA. The results showed that Bac-Cap-GP3, Bac-Cap-GP5, and Bac-Cap-GP35 elicited higher GP3/GP5 and Cap antibody titers than the Bac-Cap. Virus neutralization test also confirmed that the serum from the Bac-Cap-GP3 immunized mice had high levels of the both PCV2 and PRRSV neutralization antibodies. These findings collectively demonstrated that substituting the decoy epitope of the PCV2 capsid substituted with PRRSV epitopes could be developed into an effective vaccine against PCV2.

Construction and optimization of Lactobacillus plantarum expression system expressing glycoprotein 5 of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) causes serious reproductive failure and respiratory disease in pigs. Although numerous vaccines were developed against the virus, licensed vaccines showed limited efficacy. Here, we describe the construction and optimization of Lactobacillus plantarum expression system of PRRSV GP5 gene. The wild-type truncated GP5 or codon-optimized truncated GP5 was linked with endogenous signal peptide and target peptides (DCpep or Mpep) at 5′ and 3′ end of the gene, respectively. Then, the fragments were cloned into the L. plantarum expression plasmid pSIP411 and expressed under the induction of SppIP. As a result, PRRSV GP5 genes with optimized codons have higher expressions than that of the GP5 genes with wild-type codons, indicating codons optimization is an effective way to enhance the expression of an exogenous gene in L. plantarum. Further analysis showed that the codon-optimized GP5 with endogenous signal peptide can be effectively displayed on the surface of the L. plantarum, and the GP5 harboring target peptide Mpep displayed the highest antigenicity than the others. The highest production of PRRSV GP5 was obtained under the following conditions: L. plantarum harboring the plasmid pSIP-1320-O5MH are induced with 200 ng/mL SppIP at 33 °C for 7 h.

Interaction of porcine reproductive and respiratory syndrome virus major envelope proteins GP5 and M with the cellular protein Snapin

BackgroundPorcine reproductive and respiratory syndrome (PRRS) is characterized by abortions in pregnant sows and respiratory disease, particularly in young pigs. The causative agent is porcine reproductive and respiratory syndrome virus (PRRSV), a member of the arterivirus family. GP5 and M are the major envelope proteins encoded by PRRSV. To further characterize these two viral proteins, a yeast two-hybrid approach was utilized to identify interacting partners of PRRSV GP5 and M proteins. MethodsInteracting partners of PRRSV GP5 and M were identified using a porcine macrophage cDNA library yeast two-hybrid screen. Subsequently, the interactions between PRRSV GP5/M and the cellular protein Snapin were mapped using truncated versions of the GP5 and M proteins in a yeast two-hybrid assay to localize the interactions. The Snapin gene from the African green monkey kidney cell line MARC-145, which is permissive to PRRSV, was cloned and sequenced, and compared to porcine Snapin. Cellular Snapin expression was reduced in PRRSV-infected cells via Snapin-specific siRNA targeting. ResultsHere we show that the cellular Snap-Associated Protein (Snapin), an accessory protein of the SNARE membrane fusion network and also a member of the BLOC-1 complex, specifically interacts with GP5 and M. Inhibition of Snapin expression via siRNA targeting of Snapin results in the reduction of PRRSV replication. ConclusionsThe PRRSV GP5 and M proteins are known to form a heterodimeric complex which is important for viral structure and infectivity, and both PRRSV proteins can interact with cellular Snapin. Snapin knock-down suggests these interactions could be important in the PRRSV lifecycle. GP5 and M proteins may interact with Snapin to exploit its roles in intracellular transport and membrane fusion.

Construction and Detection of Recombinant Adeno-Associated Virus Co- Expressing PRRSV GP5, M Proteins and shRNA

Porcine reproductive and respiratory syndrome virus (PRRSV) is a severe threat to the swine industry and has caused heavy economic losses worldwide. The currently used inactivated and attenuated virus vaccines have several shortcomings, such as unsafety and low protection rate, so it is urgent to develop a new vaccine. To explore and develop a novel vaccine against PRRSV, a bi-functional recombinant adeno-associated virus, expressing ORF5 and ORF6 proteins as well as a short interfering RNA (shRNA) against ORF7, was constructed. The shRNA against ORF7 was inserted into the sequence forward of the U6 promoter of pAAV-U6-IRES-hrGFP, and ORF5 and ORF6 were cloned into the sequence after the CMV promoter. 293T cells that were co-transfected with this vector along with pAAV-RC and pHelper produced a recombinant adeno-associated virus. 293T cells transduced with this recombinant virus expressed GP5 and M proteins, and Marc145 cells transduced with this recombinant virus suppressed the replication of PRRSV. The infective titer of the reconstructed virus was 1.9 × 1010 v.gmL as measured by the dot blotting method, and GP5 and M proteins were detected by western blot. The successful construction of rAAV-shRNA-ORF5-6 paves the way for the development of novel bi-functional vaccines against PRRSV.

Porcine GPX1 enhances GP5-based DNA vaccination against porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) has been causing huge economic losses to the swine industry worldwide. Commercial PRRSV vaccines including killed and modified live ones are available. However the protective efficacy of these vaccines is incomplete. Thus, it is in urgent need to develop safer and more effective PRRSV vaccines. In this study, we constructed a recombinant plasmid co-expressing porcine glutathione peroxidase-1 (GPX1) and the envelope glycoprotein (GP5) encoding-gene of PRRSV (pcDNA3.1-GPX1-LSynORF5), and investigated the immune response induced following inoculation of mice and piglets. Significantly enhanced GP5-specific antibody, PRRSV-specific neutralizing antibody, IFN-γ level, as well as lymphocyte proliferation response, were induced in mice and pigs immunized with the DNA construct encoding GPX1 and GP5 compared with those inoculated with a construct encoding PRRSV GP5 only (pcDNA3.1-SynORF5). The enhanced cellular immune response in pigs induced by pcDNA3.1-GPX1-LSynORF5 was comparable to that induced by the attenuated virus vaccine JXA1-R, although the humoral immune response induced by the plasmid was much lower than the response induced by JXA1-R. Following the challenge with highly pathogenic PRRSV, less-severe clinical signs and rectal temperatures were observed in pigs immunized with the GPX1-GP5 construct compared with the control group. However, the viraemia of groups immunized with plasmid was more severe than that inoculated with JXA1-R, and it is likely that this could be attributed to the poor humoral response induced by the GPX1-GP5 construct. These results demonstrated that inclusion of GPX1 in a PRRSV DNA vaccine leads to an adjuvant effect, enhancing the humoral and cellular responses following vaccination.

Monkey Viperin Restricts Porcine Reproductive and Respiratory Syndrome Virus Replication.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen which causes huge economic damage globally in the swine industry. Current vaccination strategies provide only limited protection against PRRSV infection. Viperin is an interferon (IFN) stimulated protein that inhibits some virus infections via IFN-dependent or IFN-independent pathways. However, the role of viperin in PRRSV infection is not well understood. In this study, we cloned the full-length monkey viperin (mViperin) complementary DNA (cDNA) from IFN-α-treated African green monkey Marc-145 cells. It was found that the mViperin is up-regulated following PRRSV infection in Marc-145 cells along with elevated IRF-1 gene levels. IFN-α induced mViperin expression in a dose- and time-dependent manner and strongly inhibits PRRSV replication in Marc-145 cells. Overexpression of mViperin suppresses PRRSV replication by blocking the early steps of PRRSV entry and genome replication and translation but not inhibiting assembly and release. And mViperin co-localized with PRRSV GP5 and N protein, but only interacted with N protein in distinct cytoplasmic loci. Furthermore, it was found that the 13–16 amino acids of mViperin were essential for inhibiting PRRSV replication, by disrupting the distribution of mViperin protein from the granular distribution to a homogeneous distribution in the cytoplasm. These results could be helpful in the future development of novel antiviral therapies against PRRSV infection.

MYH9 is an Essential Factor for Porcine Reproductive and Respiratory Syndrome Virus Infection.

Porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is an important swine disease worldwide. PRRSV has a limited tropism for certain cells, which may at least in part be attributed to the expression of the necessary cellular molecules serving as the virus receptors or factors on host cells for virus binding or entry. However, these molecules conferring PRRSV infection have not been fully characterized. Here we show the identification of non-muscle myosin heavy chain 9 (MYH9) as an essential factor for PRRSV infection using the anti-idiotypic antibody specific to the PRRSV glycoprotein GP5. MYH9 physically interacts with the PRRSV GP5 protein via its C-terminal domain and confers susceptibility of cells to PRRSV infection. These findings indicate that MYH9 is an essential factor for PRRSV infection and provide new insights into PRRSV-host interactions and viral entry, potentially facilitating development of control strategies for this important swine disease.

Glycoprotein 5 of porcine reproductive and respiratory syndrome virus strain SD16 inhibits viral replication and causes G2/M cell cycle arrest, but does not induce cellular apoptosis in Marc-145 cells

Cell apoptosis is common after infection with porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV GP5 has been reported to induce cell apoptosis. To further understand the role of GP5 in PRRSV induced cell apoptosis, we established Marc-145 cell lines stably expressing full-length GP5, GP5Δ84-96 (aa 84-96 deletion), and GP5Δ97-119 (aa 97-119 deletion). Cell proliferation, cell cycle progression, cell apoptosis and virus replication in these cell lines were evaluated. Neither truncated nor full-length GP5 induced cell apoptosis in Marc-145 cells. However, GP5Δ97-119, but not full-length or GP5Δ84-96, induced a cell cycle arrest at the G2/M phase resulting in a reduction in the growth of Marc-145 cells. Additionally, GP5Δ84-96 inhibited the replication of PRRSV in Marc-145 cells through induction of IFN-β. These findings suggest that PRRSV GP5 is not responsible for inducing cell apoptosis in Marc-145 cells under these experimental conditions; however it has other important roles in virus/host cell biology.

Intracellular expression of an anti-idiotypic antibody single-chain variable fragment reduces porcine reproductive and respiratory syndrome virus infection in MARC-145 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome; it is one of the most economically important viral diseases affecting the swine industry worldwide. At present, neither live-attenuated nor inactivated PRRSV vaccines can provide sustainable disease control. Our previous studies have demonstrated that PRRSV infection can produce the auto-anti-idiotypic antibodies (aAb2s) specific to the idiotypic antibodies against PRRSV GP5, which plays an important role in the host immune responses to PRRSV infection. In the present study, a single-chain variable antibody fragment (scFv) from the monoclonal anti-idiotypic antibody specific for the idiotypic antibody against GP5 was expressed in MARC-145 cells and its effect on virus infection in vitro was evaluated. An scFv was constructed from the anti-idiotypic antibody (Mab2-5G2) and was named 5G2scFv. The lentiviral vector system was used as a vehicle to deliver 5G2scFv into MARC-145 cells. The effect of 5G2scFv expression in MARC-145 was analysed by determining the PRRSV N protein level and the virus titre in the supernatant. Virus attachment and the level of type I interferon (IFN) were determined to elucidate the mechanism of the scFv effect. 5G2scFv was delivered in MARC-145 cells using the lentiviral vector system as confirmed by the western blot and indirect immunofluorescence assays. The PRRSV challenge experiments demonstrated that expressed 5G2scFv in MARC-145 strongly reduced PRRSV infection and replication by inhibiting protein synthesis and progeny virus production. This effect was not due to the change of viability or virus binding, but increased IFN-α at messenger RNA and protein levels. The expression of the anti-idiotypic antibody 5G2scFv in MARC-145 cells has the interferential effect on PRRSV infection in the cells by induction of IFN-α, which provides a novel therapeutic approach for PRRSV infection.

Chimeric influenza-virus-like particles containing the porcine reproductive and respiratory syndrome virus GP5 protein and the influenza virus HA and M1 proteins.

Both porcine reproductive and respiratory syndrome and swine influenza are acute, highly contagious swine diseases. These diseases pose severe threats for the swine industry and cause heavy economic losses worldwide. In this study, we have developed a chimeric virus-like particle (VLP) vaccine candidate for porcine reproductive and respiratory syndrome virus (PRRSV) and H3N2 influenza virus and investigated its immunogenicity in mice. The HA and M1 proteins from the H3N2 influenza virus and the PRRSV GP5 protein fused to the cytoplasmic and transmembrane domains of the NA protein were both incorporated into the chimeric VLPs. Analysis of the immune responses showed that the chimeric VLPs elicited serum antibodies specific for both PRRSV GP5 and the H3N2 HA protein, and they stimulated cellular immune responses compared to the responses to equivalent amounts of inactivated viruses. Taken together, the results suggested that the chimeric VLP vaccine represents a potential strategy for the development of a safe and effective vaccine to control PRRSV and H3N2 influenza virus.

Enhanced immune responses in pigs by DNA vaccine coexpressing GP3 and GP5 of European type porcine reproductive and respiratory syndrome virus

The European (EU) type of porcine reproductive and respiratory syndrome virus (PRRSV) has recently emerged in China. In this study, three recombinant DNA vaccines, pVAX1-EU-ORF3-ORF5 (coexpressing EU type PRRSV GP3 and GP5), pVAX1-EU-ORF3 and pVAX1-EU-ORF5, were constructed and evaluated for their abilities to induce humoral and cellular responses as well as to protect piglets against homologous virus challenge. All piglets were given booster vaccinations at 21 days after the initial inoculation and then challenged 14 days later. Pigs inoculated with pVAX1-EU-ORF3-ORF5 developed significantly higher (P<0.05) PRRSV-specific antibody responses, neutralizing antibodies and levels of IL-4 and IL-10 than those given pVAX1-EU-ORF3, pVAX1-EU-ORF5 or pVAX1. Moreover, pigs immunized with pVAX1-EU-ORF3-ORF5 had markedly increased levels of IFN-γ and IL-2 in serum and T-lymphocytes (CD3+CD4+ and CD3+CD8+ T cells) in peripheral blood. Thus, EU-type PRRSV GP3 and GP5 proteins demonstrated good immunogenicity and reactogenicity and could induce cellular immunity in pigs. Following challenge with the Lelystad virus (LV) strain, piglets inoculated with pVAX1-EU-ORF3-ORF5 showed viremia and virus load distributed in organ tissues that were significantly lower (P<0.05) than those in the pVAX1-EU-ORF3 group and control group, and slightly lower than those in the pVAX1-EU-ORF5 group (P>0.05). As GP3 could enhance humoral- and cell-mediated immune responses to GP5, the results of this study suggested that these two proteins delivered by a vaccine can synergistically induce immunity against PRRSV.

English

Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease that has devastated the swine industry worldwide. Vaccination with live attenuated vaccine or inactivated vaccine is the main treatment to control PRRS. However, the disadvantages such as virulence resumption of the attenuated vaccine and low immunogenicity of the inactivated vaccine call for a more efficient and safer genetically engineered vaccine. In this study, the structural protein GP5 of the PRRS virus (PRRSV), one of the major protective antigens which stimulates a protective immune response was selected to develop a genetically engineered subunit vaccine. In order to promote the immune reaction of the host to GP5, heat shock protein 70 (Hsp70) was selected as immuno-adjuvant to enhance PRRSV GP5 immunogenicity. The Hsp70 gene was amplified by PCR from&nbsp;attenuated&nbsp;Mycobacterium bovis, and the PRRSV GP5 gene was amplified by RT-PCR from the total RNA of PRRSV SCQ strain which was isolated, identified and maintained by the Animal Biotechnological Center, Sichuan Agricultural University, China. The fusion expressing plasmid pET32-GP5-Hsp70 was constructed and expressed in&nbsp;Escherichia coli&nbsp;BL21. Ni2+-chelating resin was used to purify the His-tagged fusion protein expressed under optimized expressing conditions. The rabbit anti-GP5-Hsp70 fusion protein antibody was made, and Western blot assay verified the successful expression of the fusion protein, making it possible for further investigation whether Hsp70 could improve the immunogenicity of the PRRSV GP5 subunit vaccine, or evaluating the immunogenicity of the GP5-Hsp70 subunit vaccine. Key words:&nbsp;Porcine reproductive and respiratory syndrome virus (PRRSV) GP5 gene,&nbsp;Mycobacterium bovis&nbsp;Hsp70 gene, cloning, prokaryotic expression, identification.