Proteome analysis of differential protein expression in porcine alveolar macrophages regulated by porcine reproductive and respiratory syndrome virus nsp1β protein

Porcine reproductive and respiratory syndrome virus (PRRSV), an acute infectious disease agent in swine, causes enormous economic losses to the global swine industry. PRRSV nonstructural protein 1β (nsp1β) plays a critical role in viral subgenomic mRNA synthesis and host immune regulation. However, the global changes of cellular gene expression in natural target cells regulated by the nsp1β have not yet been identified. Here, isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography-tandem mass spectrometry were used to quantitatively identify cellular proteins in porcine alveolar macrophage (PAM) 3D4/21 cells transduced with recombinant lentivirus expressing PRRSV nsp1β that are differentially expressed compared with PAM 3D4/21 cells transduced with recombinant lentivirus expressing GFP. Of the 425 cellular proteins detected as differentially expressed, 186 were upregulated and 239 were downregulated. Based on the identities of the differentially expressed cellular proteins and the essential role of nsp1β in interferon (IFN) activation and inflammatory factor antagonism during PRRSV infection, we propose a potential mechanism in which nsp1β inhibits IFN induction and nuclear factor κB (NF-κB) signaling pathways. Our results suggest that mitochondrial antiviral signaling (MAVS) protein and translocases of outer membrane complex 70 (TOM70), involved in type I IFN induction, were downregulated, while protein phosphatase 1A (PPM1A), related to the inhibition of NF-κB pathway activation, was upregulated in nsp1β-overexpressed PAM 3D4/21 cells. These data provide valuable information for better understanding the potential biological function of nsp1β during PRRSV infection and the mechanism of virus escape from host immune surveillance of viral replication.     

Interleukin-1β expression by a recombinant porcine reproductive and respiratory syndrome virus

The cytokine interleukin-1 beta (IL-1β) is a potent inflammatory mediator in response to infection, and can be used as an immunological adjuvant. In this study, we constructed a recombinant porcine reproductive and respiratory syndrome virus (vP129/swIL1β) expressing swine IL-1β from the separate subgenomic mRNA inserted between the ORF1b and ORF2 genome region. MARC-145 cells infected with vP129/swIL1β secreted 1947 pg of IL-1β per 2 × 10⁵ cells at 36 h post-infection. In vitro growth kinetics analysis in MARC-145 cells showed that the vP129/swIL1β virus had a similar replication rate as that of parental virus. We further performed in vivo characterization of the vP129/swIL1β virus in a nursery pig disease model. The vP129/swIL1β infected pigs did not show visible clinical signs, while respiratory distress and lethargy were evident in pigs infected with the parental virus. The expression of various cytokines from peripheral blood mononuclear cells measured by fluorescent microsphere immunoassay showed that IL-1β, IL-4 and IFN-γ expression levels were up-regulated in pigs infected with vP129/swIL1β at 7 and 14 days post-infection. However, no detectable level of IL-1β was found in serum samples from pigs infected with either vP129/swIL1β or parental virus. In summary, this study demonstrated a recombinant PRRSV as a useful tool to study the role of different cytokines in disease progression and immune responses, which represents a new strategy for future therapeutic application and vaccine development.     

Molecular epidemiology of porcine reproductive and respiratory syndrome virus (PRRSV) in Québec

Porcine reproductive and respiratory syndrome virus (PRRSV) strains identified in samples from 226 field cases originating from Québec herds and submitted over a 4-year period (March 1998-July 2002) were studied. Sequencing of PRRSV strains was performed on the ORF5 gene amplified product and restriction fragment length polymorphism (RFLP) patterns for enzymes MluI, HincII and SacII were determined on these sequences. Twenty-four other PRRSV isolates including three vaccine strains were also included for comparison purposes and the total of 250 PRRSV strains were used in a phylogenetic analysis. Clinical and epidemiological data were collected through a questionnaire for each of the submitted field cases. About 75% of the cases were submitted during autumn and winter. Over 60% of the cases were submitted for reproductive problems, 33% for respiratory problems and 6% for increased PRRSV serological titers in the herd in absence of clinical signs. In 69% of the cases there was a PRRS vaccination program for the herd. However, only 26% of the animals from which samples were obtained had been vaccinated themselves. The genomic analysis of this large number of strains revealed a great variability of PRRSV ORF5 with 59% of amino acid positions being polymorphic. A total of 29 RFLP patterns were obtained. The main RFLP patterns obtained were 1-8-4 (28%), 1-4-4 (16%), 1-2-4 (9%) and 1-11-4 (9%). The global findings derived from the molecular analysis of 226 PRRSV strains suggest that PRRSV circulating in Québec represent a different sub-population of strains. Vaccine-like strains were identified in 10% of the cases. A phylogenetic tree enabled the identification of 44 groupings comprising two to 23 strains each. Of the 250 sequences analyzed, 183 (73%) could be included in one of these groupings. The data collected from the questionnaires were used to establish epidemiological links between strains within groupings. The main relationships between strains within a grouping were the introduction of infected animals (19%) and area spread (33%). In 40% of the cases from which an area spread was suspected, herds were located within 3 km from another. Aerosol transmission was suspected in several cases, more than half of which belonged to different owners. In 41 herds, more than one strain (2-8) were identified over a period varying from 3 months to 4 years. Data indicated that a PRRSV strain can persist in a herd up to 3.5 years displaying as little as 2% variation in ORF5 during this time. In 78% of the herds with multiple submissions genetically different strains were identified; often within 1 year of the original identification. These genetically distinct strains were often associated with a recrudescence of moderate to severe clinical signs. Coexistence on the same farm of different PRRSV strains was also observed.     

Porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-β production by inhibiting IRF3 activation in immortalized porcine alveolar macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) infection appears to elicit a weak innate immune response suppressing type 1 interferon (IFN) production. Recent studies have revealed that several nonstructural proteins encoded by the PRRSV genome independently antagonize the type 1 IFN system. The present study sought to identify the structural proteins that possess the immune evasion properties in immortalized porcine alveolar macrophages (PAM). Each structural protein gene was stably expressed in a porcine monocyte-derived macrophage cell line, PAM-pCD163, and tested for its potential to inhibit IFN-β induction. We then focused on the nucleocapsid (N) protein, which has a strong inhibitory effect on dsRNA-induced IFN-β production. Upon dsRNA stimulation, IFN-β production was shown to decrease proportionally with increasing levels of N expression. Furthermore, the PRRSV N protein was found to down-regulate IFN-dependent gene production by dsRNA. Taken together, these results indicate the ability of N to modulate the dsRNA-mediated IFN induction pathways. In addition, the N protein significantly interfered with dsRNA-induced phosphorylation and nuclear translocation of IRF3. Our data suggest that the PRRSV N protein is a responsible component, independent of other nonstructural elements, for evading the IFN response by antagonizing IRF3 activation.     

Conserved nucleotides in the terminus of the 3′ UTR region are important for the replication and infectivity of porcine reproductive and respiratory syndrome virus

The 3′ untranslated region (3′ UTR), including the poly (A) tail, reportedly plays an important role in arterivirus replication, but the roles of the cis-acting elements present in the 3′ UTR of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unknown. In the present study, PCR-based mutagenic analysis was conducted on the 3′ UTR of PRRSV infectious full-length cDNA clone pAPRRS to investigate the structure and function of the conserved terminal nucleotides between the poly (A) tail and the 3′ UTR region. Our findings indicated that the conservation of the primary sequence of the 3′ terminal nucleotides, rather than the surrounding secondary structure, was vital for viral replication and infectivity. Four nucleotides (nt) (5′-¹⁵⁵¹⁷AAUU¹⁵⁵²⁰-3′) at the 3′ proximal end of the 3′ UTR and the dinucleotide 5′-AU-3′ exerted an important regulatory effect on viral viability. Of the five 3′-terminal nucleotides of the 3′ UTR (5′-¹⁵⁵⁰³AACCA¹⁵⁵⁰⁷-3′), at least three, including the last dinucleotide (5′-CA-3′), were essential for maintaining viral infectivity. Taken together, the 3′-terminal conserved sequence plays a critical role in PRRSV replication and may function as a contact site for specific assembly of the replication complex.     

Molecular characterization and recombination analysis of porcine reproductive and respiratory syndrome virus emerged in southwestern China during 2012–2016

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen causing tremendous economic losses to the swine industry. To investigate the prevalence of PRRSV of genotype 2 (North American type, NA-type) in southwestern China, the Nsp2 hypervariable region (Nsp2 HV) and ORF5 of 61 PRRS viruses collected during 2012–2016 were sequenced and analyzed. All the virus detected clustered into the JXA1-like (52/61), VR-2332-like (7/61), and NADC30-like (2/61) sub-genotypes. Five deletions in Nsp2 HV were detected in addition to the typical 30aa discontinuous deletion in HP-PRRSV, and two of these five were not reported previously. Strikingly, two PRRS virus (SCnj16 and SCcd16) isolated in 2016 contained the classic HP-PRRSV molecular marker in the Nsp2-coding region, but belonged to the NADC30-like sub-genotype on the ORF5 gene. Further recombination and phylogenetic analysis on the two complete genomic sequences revealed that they may have originated from recombination events between the NADC30 and Chinese HP-PRRSV strains. The present study suggests that the endemic PRRSVs in the region have continuously evolved and new vaccine strategies are necessary for more efficient control of the virus.     

Antigenic and genetic variations of the 15 kD nucleocapsid protein of porcine reproductive and respiratory syndrome virus isolates

Summary.  The antigenic variability of the 15 kD nucleocapsid protein of porcine reproductive and respiratory syndrome (PRRS) virus was characterized with a panel of 24 monoclonal antibodies (MAbs) raised against the American PRRS virus isolate ISU-P. Five continuous epitopes designated EpORF7-A through E were revealed by the reactivity pattern of these MAbs with 67 American field isolates, two modified-live vaccine viruses, and the European Lelystad virus as determined by the indirect immnofluorescence assay and Western immunoblotting and confirmed by additivity and blocking enzyme-linked immunosorbent assays. The reactivity pattern of isolates in the IFA permitted their subdivision into 4 American antigenic groups which represented 84.1, 11.6, 2.9 and 1.4% of viruses tested. The antigenic variation among isolates was correlated to single, group specific nucleotide substitutions and mediated by a combination of at least 4 of the 5 epitopes. EpORF7-A was conserved in all American isolates and the Lelystad virus which constituted a separate antigenic group. Consequently, monoclonal antibodies specific for EpORF7-A may prove useful as the antigenic basis for a universal diagnostic test for the PRRS virus. EpORF7-C, D and E were only present in the American isolates tested. Received April 6, 1998 Accepted September 22, 1998     

Expression of oestrogen receptor α in the endometrium of porcine reproductive and respiratory syndrome virus-infected gilts

Porcine reproductive and respiratory syndrome (PRRS) virus infection often lead to infertility in gilts and sows. Nevertheless, the impact of PRRS virus on the endometrial function has not been fully elucidated. The present study aimed to determine the effect of PRRS virus infection on the expression of oestrogen receptor (ER) α in the endometrium of gilts. Uterine tissues from 54 gilts were classified into two groups according to PRRS virus detection using immunohistochemistry (26 positive and 28 negative samples). The reproductive status was classified as prepubertal, luteal and follicular phases. The expression of ERα in the epithelium, subepithelium and glandular tissue layers of the endometrium was determined by immunohistochemistry. ERα immunostaining was detected in 22.2, 13.5 and 33.6 % of the cells in the epithelial, subepithelial and glandular tissue layers, respectively. The ERα immunostaining in the glandular layer of the endometrium in follicular-phase gilts was higher than that in prepubertal gilts (46.1 and 15.2 %, respectively, P?=?0.011). The ERα immunostaining in the glandular layer of the endometrium was positively correlated with body weight (r?=?0.138, P?=?0.020) and average daily gain (r?=?0.169, P?=?0.005) of the gilts. The ERα immunostaining in all tissue layers of the endometrium with PRRS virus detection did not differ significantly compared to that without PRRS virus (P?>?0.05). However, in prepubertal gilts, the ERα immunostaining cells in the epithelial layer of PRRS virus negative (9.5 %) tended to be lower than PRRS virus positive (26.3 %, P?=?0.196) uterine tissues. This tendency indicates some impact of PRRS virus infection on the ERα expression in prepubertal gilts.     

Molecular analysis of the ORFs 3 to 7 of porcine reproductive and respiratory syndrome virus,Québec reference strain

Summary The cDNA sequence of the 3-terminal genomic region of the Québec IAF-exp91 strain of porcine reproductive and respiratory syndrome virus (PRRSV) was determined and compared to those of other reference strains from Europe (Lelystad virus) and US (ATCC VR2385, MN-1b). The sequence (2834 nucleotides) which encompassed ORFs 3 to 7 revealed extensive genomic variations between the Québec strain and Lelystad virus (LV), resulting from high number of base substitutions, additions and deletions. The ORFs 5, 3, and 7 seemed to be relatively the most variable; the predicted encoding products of the Québec and LV strains displayed only 52%, 54%, and 59% amino acid identities, respectively. Nevertheless, in vitro translation experiments of the structural genes (ORFs 5, 6, and 7) and radio-immunoprecipitation assays with extracellular virions gave results similar to those previously reported for LV. In contrast, close genomic relationships were demonstrated between Québec and US strains. Taking together, these results indicate that, although structurally similar, North American PRRSV strains belong to a genotype distinct from that of the LV, thus supporting previous findings that allowed to divide PRRSV isolates into two antigenic subgroups (U.S. and European).The nucleotide sequence data reported in this paper will appear in the EMBL and GenBank nucleotide sequence databases under accession number L40898.     

Endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp11 was essential for nsp11 to inhibit IFN-β induction

Previous studies have shown that PRRSV nsp11, which was an endoribonuclease, was an interferon antagonist, however, the mechanism that nsp11 inhibited IFN-β production was unclear. To explore whether the endoribonuclease was required for nsp11 to disrupt the IFN-β production, substitutions of the presumed catalytic histidine and lysine residues of nsp11 were introduced into plasmid pcDNA 3.1-FLAG. The results showed that mutation that inactivated endoribonuclease made nsp11 lose its ability to inhibit Poly(I:C)-induced IFN-β promoter activity. In conclusion, our present work indicated that the endoribonuclease activity of nsp11 was essential for nsp11 to inhibit the IFN-β induction.     

RNA interference inhibits yellow fever virus replication in vitro and in vivo

RNA interference (RNAi) is a process that is induced by double stranded RNA and involves the degradation of specific sequences of mRNA in the cytoplasm of the eukaryotic cells. It has been used as an antiviral tool against many viruses, including flaviviruses. The genus Flavivirus contains the most important arboviruses in the world, i.e., dengue (DENV) and yellow fever (YFV). In our study, we investigated the in vitro and in vivo effect of RNAi against YFV. Using stable cell lines that expressed RNAi against YFV, the cell lines were able to inhibit as much as 97% of the viral replication. Two constructions (one against NS1 and the other against E region of YFV genome) were able to protect the adult Balb/c mice against YFV challenge. The histopathologic analysis demonstrated an important protection of the central nervous system by RNAi after 10 days of viral challenge. Our data suggests that RNAi is a potential viable therapeutic weapon against yellow fever.

Evaluation of a DNA vaccine candidate co-expressing GP3 and GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) with interferon α/γ in immediate and long-lasting protection against HP-PRRSV challenge

Porcine reproductive and respiratory syndrome virus (PRRSV) has become one of the most economically important diseases to the global pork industry. Current vaccination strategies only provide a limited protective efficacy. In this study, a DNA vaccine, pVAX1^?-??-??-GP35, co-expressing GP3 and GP5 of PRRSV with interferon ??/?? was constructed, and its immediate and long-lasting protection against highly pathogenic PRRSV (HP-PRRSV) challenge were examined in pigs. For immediate protection, the results showed that pVAX1^?-??-??-GP35 could provide partially protective efficacy, which was similar to the pVAX1^?-??-?? (expressing interferon ??/??). For long-lasting protection, pigs inoculated with pVAX1^?-??-??-GP35 developed significantly higher PRRSV-specific antibody response, T cell proliferation, IFN-??, and IL-4, than those vaccinated with pVAX1^?-GP35 (expressing GP3 and GP5 of PRRSV). Following homologous challenge with HP-PRRSV strain SD-JN, pigs inoculated with pVAX1^?-??-??-GP35 showed almost no clinical signs, no lung lesions, and significantly lower viremia, as compared to those in pVAX1^?-GP35 group. It indicated that pVAX1^?-??-??-GP35 could induce enhanced immune responses and provide both immediate and long-lasting protection against HP-PRRSV challenge in pigs. The DNA vaccine pVAX1^?-??-??-GP35 might be an attractive candidate vaccine for the prevention and control of HP-PRRSV infections.     

Nonstructural protein 1α subunit-based inhibition of NF-κB activation and suppression of interferon-β production by porcine reproductive and respiratory syndrome virus

Induction of type I interferon (IFN-α/β) is an early antiviral response of the host, and porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to downregulate the IFN response during infection in cells and pigs. We report that the PRRSV nonstructural protein 1α (Nsp1α) subunit of Nsp1 is a nuclear-cytoplasmic protein distributed to the nucleus and contains a strong suppressive activity for IFN-β production that is mediated through the retinoic acid-inducible gene I (RIG-I) signaling pathway. Nsp1α suppressed the activation of nuclear factor (NF)-κB when stimulated with dsRNA or tumor necrosis factor (TNF)-α, and NF-κB suppression was RIG-I-dependent. The suppression of NF-κB activation was associated with the poor production of IFN-β during PRRSV infection. The C-terminal 14 amino acids of the Nsp1α subunit were critical in maintaining immunosuppressive activity of Nsp1α for both IFN-β and NF-κB, suggesting that the newly identified zinc finger configuration comprising of Met180 may be crucial for inhibitory activities. Nsp1α inhibited IκB phosphorylation and as a consequence NF-κB translocation to the nucleus was blocked, leading to the inhibition of NF-κB stimulated gene expression. Our results suggest that PRRSV Nsp1α is a multifunctional nuclear protein participating in the modulation of the host IFN system.     

Determination of 5′-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs

Summary.  We determined the untranslated 5′-leader sequence for three different isolates of porcine reproductive and respiratory syndrome virus (PRRSV): pathogenic European- and American-types, as well as an American-type vaccine strain. 5′-leader from European- and American-type PRRSV differed in length (220 and 190 nt, respectively), and exhibited only approximately 50% nucleotide homology. Nevertheless, highly conserved areas were identified in the leader of all 3 PRRSV isolates, which constitute candidate motifs for binding of protein(s) involved in viral replication. These comparative data provide a priori knowledge for mutational identification of virulence determinants in the 5′ nontranslated part of the PRRSV genome. Received October 12, 1998 Accepted January 8, 1999     

(?)-Epigallocatechin-3-gallate inhibits the replication cycle of hepatitis C virus

(-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea. In this study, we found that hepatitis C virus (HCV) infection was significantly suppressed by EGCG in an HCV cell culture (HCVcc) system using a JFH1-GFP chimeric virus, with a 50 % effective concentration (EC(50)) of 17.9 μM. The inhibitory activity of EGCG was confirmed by monitoring HCV RNA and protein expression levels in Huh7.5.1 cells infected with the JFH1 virus. Moreover, we demonstrated that the inhibitory mechanisms of EGCG were attributable to the suppression of both the HCV entry and RNA replication steps, although EGCG had little effect on translation directed by the viral internal ribosome entry site (IRES). Furthermore, HCV could be rapidly eliminated from cell cultures after two and five passages in the presence of 50 and 25 μM EGCG, respectively. These results indicate that EGCG is a potential candidate as a preventive and antiviral drug for HCV infection.