A nonstructural and antigenic glycoprotein is encoded by ORF3 of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus

Summary.  Open reading frame 3 (ORF3) of the genome of porcine reproductive and respiratory syndrome virus (PRRSV), Quebec strain IAF-Klop, was reverse-transcribed and cloned into the procaryotic expression vector pGEX-4T-1, then subcloned into the eucaryotic expression vector pAdCMV5 which was used as a shuttle vector to generate a replication-defective recombinant adenovirus. The procaryotic GST-ORF3 recombinant fusion protein was used to raise a monospecific antiserum in rabbits. By Western-immunoblotting with PRRSV-infected cell extracts, the ORF3 encoded protein had an estimated molecular mass (M_r) of 42 kDa, similar to that of the protein expressed by the adenovirus vector. Endoglycosidase F digestion showed that the ORF3 encoded protein occurs in an highly glycosylated form (GP₃) in the infected MARC-145 cells. Pulse-chase and radioimmunoprecipitation experiments revealed that the GP₃ protein was present in amounts equivalent to those of the N, M, and GP₅ proteins in the infected cells, whereas no GP₃ could be detected in purified virions. During the first 30 min of chase, the GP₃ undergoes a gradual downward shift of its apparent M_r, thought to result from trimming of the mannose-rich glycan structures. Tested convalescent pig sera that were found to be seropositive to PRRSV by indirect immunofluorescence reacted positively with the recombinant GST-ORF3 fusion protein by immunoblotting. Data indicated that the ORF3 protein of the Quebec reference strain of PRRSV is a highly glycosylated and antigenic protein, which is nonstructural. Received February 3, 1998 Accepted May 16, 1998     

Mutagenesis analysis of porcine reproductive and respiratory syndrome virus nonstructural protein 7

Nonstructural protein 7 (nsp7), which is flanked by nsp6 and nsp8, is one of the most conserved nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV). Nonstructural protein (nsp)-specific antibodies are produced in high titers in response to virus replication, especially against nsp1a, nsp1b, nsp2, and nsp7. However, many regional aspects of nsp7 are still veiled, such as its impact on viral replication and virulence or the immunological mechanism between virus and host. Based on the structure of the predicted nsp7 domain, we have constructed a series of large mutations and deletions. We ultimately demonstrated all mutations (nsp7, nsp7α/nspβ) and the majority of substitutions of nsp7 affected the PRRSV replicative cycle in some ways and were fatal for viral recovery, which indicates that these are significant to structure or function of the nsp7. What’s more, the mutant vOKXH-nsp7 (F40A) indeed caused some of the variation compared with the parental virus vOKXH-GD, which shortens the amount of time needed to reach its highest viral titer, and decreases the concentration of the highest viral titer, obstructing viral mRNA and protein synthesis. Consequently, these valuable results possibly provide the first direct evidence that the nsp7 is really a critical protein domain for the RNA synthesis and the translation of viral protein of PRRSV.     

Evolution of ORF5 of Spanish porcine reproductive and respiratory syndrome virus strains from 1991 to 2005

ORF5 sequences of porcine reproductive and respiratory syndrome virus (PRRSV) were analysed to determine genetic diversity, codon usage, positive and negative selection sites and potential changes in the predicted glycoprotein 5 (GP5). A hypothetical GP5 containing all selected sites was constructed to determine its characteristics. These sequences corresponded to isolates obtained 10 years apart (1991-1995, 18 strains) and a second set (n = 46) from 2000 to 2005. Similarity to Lelystad virus (LV) decreased from 95.5% in 1991-1995 to 89.5% in 2000-2005. Three highly variable regions were found in ORF5. Codon usage was different in both sets for leucine, glutamine, serine and proline. Thus, 2000-2005 sequences used codons more similar to those present in highly expressed pig genes compared to the 1991-1995 set. Twenty four sites of positive selection and 20 sites of negative selection were found in GP5, most of them in transmembrane regions. Additional glycosylation in N37 of GP5 was common in 2000-2005 but some sequences lack a glycosylation site in N46. The hypothetical GP5 was only 88.1% similar to LV and was less hydrophobic. Taking together these results suggest that PRRSV is still adapting to pig cells.     

T cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus

Summary.  The identification of antigens recognized by T cell responses has become fundamental for developing effective immunizations against viral infections. Lymphocyte proliferation and delayed-type hypersensitivity responses to porcine reproductive and respiratory syndrome virus (PRRSV) infection have been demonstrated. However, the polypeptide specificity of T cell responses to PRRSV is unknown. To identify the PRRSV polypeptides recognized by porcine lymphocytes two approaches were employed. First polypeptides of purified virions were separated by SDS-PAGE and particle suspensions obtained from nitrocellulose blots were used as antigens. Second, the polypeptides encoded by ORFs 2, 4, 5, 6, and 7 of the strain VR-2 332 were expressed as fusion proteins with a histidine tag in mammalian cells, using vaccinia virus as expression system. Significant antigen-specific proliferation responses to the matrix and envelope proteins from purified virions were obtained. This finding was supported by specific and dose-dependent proliferation responses to the recombinant polypeptides encoded by ORF2, 5 and 6 detected in virus-infected but not in control pigs. These results demonstrate that T-cell responses can be detected to individual PRRSV polypeptides. The greater response to the product of ORF6 than to the other PRRSV polypeptides indicates that the viral matrix polypeptide may have a major role in cellular immunity. Received April 8, 1998 Accepted July 28, 1998     

Suppression of porcine reproductive and respiratory syndrome virus replication in MARC-145 cells by shRNA targeting ORF1 region

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in swine producing area. The current vaccine strategies cannot provide complete protection against PRRSV. The objective of this study was to determine if specific short-hairpin RNA (shRNA) directed against different genomic regions of ORF1b of PRRSV could be utilized to inhibit virus replication in MARC-145 cells. Two shRNA expression vectors targeting ORF1b gene of PRRSV were constructed and delivered into MARC-145 cells, and then infected with PRRSV. The results showed that PRRSV-specific cytopathic effect (CPE) could be inhibited in the cells transfected with pSUPER-P2 and pSUPER-P3, and the virus titers in the cells transfected with pSUPER-P2 and pSUPER-P3 were lower than those control cells by approximately 100 fold. Moreover, the expression of ORF1 of PRRSV in the cells was reduced both at RNA and protein levels comparing to the controls. It indicated that vector-based shRNA targeting ORF1 region could effectively inhibit PRRSV replication in MARC-145 cells. Guanming Li and Juan Huang contributed equally to this work.     

Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2.

Infectious porcine circovirus type 2 (PCV2) was generated following transfection of a porcine retina cell line (VIDO R1) with cloned circovirus DNA. Expression of open reading frame 2 (ORF2) was detected at 24 h postinfection and onwards increasingly throughout the infection by Western blot analysis using ORF2 specific polyclonal antibody. Moreover, the ORF2 protein was also detected in purified PCV2 virus, indicating that ORF2 is a structural component of PCV2 viral capsid. Nuclear localization of PCV2 ORF2 was demonstrated by immunofluorescence assay in PCV2-infected cells. An analysis of the subcellular localization of a series of truncation mutants of ORF2 fused with the green fluorescent protein indicated that the nuclear localization signal of ORF2 was conferred by the N-terminal 41 amino acids. This domain was further analyzed through site-directed mutagenesis, suggesting that the presence of basic amino acid residues at positions 12 to 18 and 34 to 41 are important for the strict nuclear targeting of PCV2 ORF2.     

Antigenic structure analysis of glycosylated protein 3 of porcine reproductive and respiratory syndrome virus

The function of the glycosylated protein 3 (GP3), a porcine reproductive and respiratory syndrome virus (PRRSV) associated protein is poorly known. In the present study, the gene encoding GP3 (ORF3), lacking the highly hydrophobic domain in the N- and C-termini was expressed as GST-fusion proteins in E. coli. Monoclonal antibodies (MAbs) against GP3 were developed and used to probe a series of GP3 peptides using ELISA. After precise analysis by sequential deletion of the terminal amino acid residues from each peptide, the minimal epitopes recognized by the MAbs were localized to W(74)CRIGHDRCGED(85) and Y(67)EPGRSLW(74). The epitope sequences were well conserved among most of the North American-type isolates, with the exception of two amino acid mutations in both epitopes in a few of these isolates. Mutational analysis revealed that these mutants were not recognized by any of the five MAbs, indicating that genetic variation could lead to altered antigenicity. Eight out of nine peptide fragments, 58-72aa, 73-87aa, 88-101aa, 102-115aa, 50-65aa, 66-81aa, 80-95aa and 94-109aa were recognized by PRRSV-positive pig serum as determined by Western blot analysis. The results herein may elucidate partially the antigenic structure of GP3 and variations of PRRSV.     

Emergence of porcine reproductive and respiratory syndrome virus deletion mutants: correlation with the porcine antibody response to a hypervariable site in the ORF 3 structural glycoprotein

By using porcine immune sera to select a library of phage-displayed random peptides, we identified an antigenic sequence (RKASLSTS) in the C-terminus of the ORF 3 structural glycoprotein of European-type porcine reproductive and respiratory syndrome virus (PRRSV). Through the use of overlapping reading frames, the same PRRSV genetic locus codes for the ORF 3 "RKASLSTS" sequence, and a previously described ORF 4 epitope (Meulenberg, J. J. M., Van Nieuwstadt, A. P., Van Essen-Zandbergen, A., and Langeveld, J. P. M., 1997, J. Virol. 71, 6061-6067). Sequence analysis identified naturally occurring deletion mutants at this ORF 34 site. Phylogenetic analysis showed the presence of a highly accurate ORF 3 molecular clock, according to which deletion mutants and nondeleted viruses evolved at differing speeds. Furthermore, deletion mutants and nondeleted viruses evolved as separate lineages. These distinctions suggested that deletion mutants were a hitherto unrecognized subtype of European-type PRRSV. Currently, deletion mutants appear to be outcompeting nondeleted viruses in the field, highlighting the importance of the porcine antibody response against the minor structural glycoproteins of European-type PRRSV for viral evolution.     

Inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with 2-aminopurine

Summary.  Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to a group of RNA viruses that establish persistent infections. A proposed strategy for evading immunity during persistent PRRSV infection is by preventing the induction of IFN activity in pigs and/or by blocking the activation of antiviral proteins in permissive cells. IFN-γ mRNA expression was observed in the lymph nodes and lungs of pigs infected with wild-type PRRSV strain SDSU-23983. Pretreatment of MARC-145 cells with IFN-γ inhibited wild-type (SDSU-23983 P6) and culture-adapted (SDSU-23983 P136) PRRS viruses in a dose-dependent manner and at relatively low concentrations. The effect of IFN-γ on virus replication included reductions in the number of infected cells, virus yield, and RNA content in single cells. Virus replication was partially restored by the addition of 2-aminopurine (2-AP), an inhibitor of dsRNA inducible protein kinase (PKR). The addition of 2-AP also restored the viral RNA content per cell to near normal levels, suggesting that inhibition of viral RNA synthesis was through PKR. The principal difference between P6 and P136 isolates was the recovery of P136 replication with lower concentrations of 2-AP. Immunostaining with anti-PKR antibody showed a redistribution of PKR from the cytoplasm into nucleoli of infected cells. Received March 7, 2000 Accepted August 16, 2000     

Mechanisms of suppression of interferon production by porcine reproductive and respiratory syndrome virus

The recently emerged porcine reproductive and respiratory syndrome virus (PRRSV) leads to one of the most economically significant infectious diseases of swine worldwide. The virus modulates the host innate and adaptive immunity to escape its immune response to facilitate the infection. Interferons (INFs) are principal antiviral cytokines, which represent components of the innate immunity and are regarded as a bridge between the innate and adaptive immunity. Currently, accumulating evidence indicates that the virus has developed various strategies to counteract the IFN production. Here, various mechanisms utilized by the virus to antagonize the IFN induction are reviewed.     

Gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be one of the most important diseases facing swine industry today. Following PRRSV infection pigs develop both humoral and cell-mediated responses following PRRSV exposure; however, the relative importance in protection and clearance of the virus is not yet completely understood. Swine contain a large percentage of gammadelta T-lymphocytes in peripheral circulation capable of responding to various pathogens in both an innate and specific immune response. The objectives of this study were to determine whether gammadelta lymphocytes functionally respond to PRRSV upon initial exposure and re-exposure. Four month old PRRSV free gilts were intranasally inoculated with a field isolate MN-30100 then assessed at various time points post infection. On day 120, pigs were re-exposed with MN-30100 PRRSV strain and subsequently were bled on days 0, 7, and 14 post re-exposure. Lymphocyte subpopulations, antigen specific proliferation, and IFN-gamma production were evaluated throughout the study. Circulating gammadelta lymphocytes in PRRSV exposed animals expanded between days 14 to 70 (d14-d70, p = 0.016); following antigen stimulation, gammadelta lymphocyte proliferated by day 14 (d0-d14, p = 0.001) continuing through day 60. gammadelta lymphocytes produced IFN-gamma by day 14 pi continuing through day 50 (d0-d50, p = 0.004). Following re-exposure both gammadelta+ and CD4+ lymphocytes increased in IFN-gamma production. These results are not fully conclusive on the role of gammadelta lymphocytes against PRRSV; the data indicate that gammadelta lymphocytes specifically respond to PRRSV.     

Heterogeneity of swine lung macrophages inoculated by porcine reproductive and respiratory syndrome virus

The biological features of porcine alveolar macrophages (PAMs) and interstitial macrophages (IMs) were investigated, including morphology, nitric oxide (NO) secretion, cell viability and porcine reproductive and respiratory syndrome virus (PRRSV) mRNA expression post-inoculation with TJ-F10 or TJM-F92. Viability and NO secretion of PAMs and IMs were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Griess's assay, respectively. mRNA expression of PRRSV, inducible nitric oxide synthase (iNOS) and Arginase1 (Arg1) in PAMs and IMs were detected by quantitative real-time polymerase chain reaction technique. Our results show that PAMs were bigger and more granular than IMs and the Arg1/iNOS value was much higher in PAMs than in IMs. In addition, the vaccine strain TJM-F92 evoked higher NO production in PAMs and IMs compared with the wild type strain TJ-F10. In conclusion, our results indicate that the PAMs and IMs are heterogeneous in morphology, NO production and susceptibility to PRRSV.     

Porcine reproductive and respiratory syndrome virus infects mature porcine dendritic cells and up-regulates interleukin-10 production

Porcine reproductive and respiratory syndrome virus (PRRSV) infects mature dendritic cells (mDCs) derived from porcine monocytes and matured with lipopolysaccharide. The infection of mDCs induced apoptosis, reduced the expression of CD80/86 and major histocompatibility complex class II molecules, and increased the expression of interleukin-10, thus suggesting that such mDC modulation results in the impairment of T-cell activation.     

Induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication

Autophagy is an evolutionarily conserved lysosome-dependent degradation pathway that acts in the maintenance of cellular homeostasis and plays important functions in viral replication and pathogenesis. In this study, we investigated the role of autophagy in the replication of porcine reproductive and respiratory syndrome virus (PRRSV), an agent that has caused devastating losses in the international swine industry since the late 1980s. Using protein quantification and microscopy, we observed that PRRSV infection results in LC3-I/II conversion, an increased accumulation of punctate GFP-LC3-expressing cells, and a higher number of autophagosome-like double-membrane vesicles in the cytoplasm of host cells. Inhibition of autophagy using 3-methyladenine (3-MA) or small interfering RNAs targeting ATG7 and Beclin-1 led to a significant reduction in PRRSV titers and protein expression. Conversely, induction of autophagy by rapamycin resulted in increased viral replication. These results demonstrate that PRRSV infection induces autophagy which, in turn, enhances viral replication efficiency.     

Multiplex method for simultaneous serological detection of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

Porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) are major contributors to the porcine respiratory disease complex (PRDC). Routine serological diagnosis and surveillance play an important role in the prevention of PRDC, as it is a leading cause of economic losses to the swine industry. We herein describe an advanced microsphere-based immunoassay that permits the simultaneous detection of antibodies to PCV2 and PRRSV, thereby reducing the time and effort involved in testing. Recombinant PRRSV nucleoprotein antigen and the PCV2 capsid antigen were coupled to fluorophore-dyed beads with distinct spectral addresses. Weekly serum samples from 72 pigs that were experimentally exposed to either PCV2, PRRSV, or both PCV2 and PRRSV were used to validate the microbead assay (MBA) in comparison with the "gold standard" enzyme-linked immunosorbent assays. The kinetics of the PCV2- and PRRSV-specific antibody responses measured by the microbead assay were comparable to those of the standard assays; Spearman's rank correlations were 0.72 (P < 0.001) for PRRSV and 0.80 (P < 0.001) for PCV2. Diagnostic sensitivity and specificity were determined using field sera whose positive or negative status was determined by the standard tests. The diagnostic sensitivity and specificity were both 98% for PCV2 and were 91% and 93%, respectively, for PRRSV (kappa coefficients, 0.85 and 0.67 for PCV2 and PRRSV, respectively). Multiplexing did not interfere with assay performance or diagnostic sensitivity. Therefore, the described study demonstrates proof of concept for the development of more versatile and economical microbead array-based multiplex serological test panels for veterinary use.     

Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a devastating disease in swine. The presence and transmission of PRRSV by boar semen has been demonstrated by using a swine bioassay. In this assay, 4- to 8-week-old pigs were inoculated intraperitoneally with semen from PRRSV-infected boars. Seroconversion of these piglets indicated the presence of PRRSV in semen. Seroconversion in gilts has also been demonstrated following artificial insemination with semen from PRRSV-infected boars. These methods of detecting PRRSV in boar semen are time-consuming, laborious, and expensive. The objective of this study was to develop a reliable and sensitive PCR assay to directly detect PRRSV in boar semen. Primers from open reading frames 1b and 7 of the PRRSV genome were used in nested PCRs. Virus was detected at concentrations as low as 10 infectious virions per ml in PRRSV-spiked semen. Specificity was confirmed by using a nested PCR and a 32P-labeled oligonucleotide probe. The primers did not react with related arteriviruses or other swine viruses. The PCR assay showed good correlation with the swine bioassay, and both methods were superior to virus isolation. To consistently identify PRRSV in boar semen, the cell fraction was separated by centrifugation at 600 x g for 20 min, a lysis buffer without a reducing agent (2-mercaptoethanol) was used, and nondiluted and 1:20-diluted cell fractions were evaluated by PCR. PRRSV was not reliably detected in the seminal plasma fraction of boar semen.