Interleukin-4 suppresses the expression of macrophage NADPH oxidase heavy chain subunit (gp91-phox)

AbstractThe production of superoxide anion by NADPH oxidase is a principal nonspecific bactericidal activity of macrophages and neutrophils in host defense. However, exuberant production of superoxide anion also damages host tissues. Cloning and DNA sequencing of the 91 kDa subunit (gp91-phox) open reading frame indicated a high degree of sequence conservation, greater than 90% in nucleotide and amino acid sequences, between the porcine and human cDNAs. We show in pigs that interleukin-4 (IL-4), a T lymphocyte cytokine which plays a major role in mediating antibody responses to pathogens, suppresses superoxide anion production in macrophages by specifically reducing the level of mRNA encoding gp91-phox. Messenger RNA levels are suppressed approx. 70% within 4 h and persist for 24 h without any change in the rate of mRNA turnover. Nuclear run-on analysis showed that IL-4 did not alter the rate of gp91-phox gene transcription under conditions in which IL-1β transcription was inhibited. These results indicate that IL-4 suppresses the inflammatory response of macrophages by mechanisms that include post-transcriptional regulation of the 91 kDa catalytic subunit of NADPH oxidase, and transcriptional regulation of inflammatory cytokine expression

Age-dependent resistance to Porcine reproductive and respiratory syndrome virus replication in swine

<p>Abstract</p> <p>Background</p> <p>Porcine reproductive and respiratory syndrome virus (PRRSV) causes a prolonged, economically devastating infection in pigs, and immune resistance to infection appears variable. Since the porcine adaptive immune system is not fully competent at birth, we hypothesized that age influences the dynamics of PRRSV infection. Thus, young piglets, growing 16-20-week-old finisher pigs, and mature third parity sows were infected with virulent or attenuated PRRSV, and the dynamics of viral infection, disease, and immune response were monitored over time.</p> <p>Results</p> <p>Virulent PRRSV infection and disease were markedly more severe and prolonged in young piglets than in finishers or sows. Attenuated PRRSV in piglets also produced a prolonged viremia that was delayed and reduced in magnitude, and in finishers and sows, about half the animals showed no viremia. Despite marked differences in infection, antibody responses were observed in all animals irrespective of age, with older pigs tending to seroconvert sooner and achieve higher antibody levels than 3-week-old animals. Interferon γ (IFN γ) secreting peripheral blood mononuclear cells were more abundant in sows but not specifically increased by PRRSV infection in any age group, and interleukin-10 (IL-10) levels in blood were not correlated with PRRSV infection status.</p> <p>Conclusion</p> <p>These findings show that animal age, perhaps due to increased innate immune resistance, strongly influences the outcome of acute PRRSV infection, whereas an antibody response is triggered at a low threshold of infection that is independent of age. Prolonged infection was not due to IL-10-mediated immunosuppression, and PRRSV did not elicit a specific IFN γ response, especially in non-adult animals. Equivalent antibody responses were elicited in response to virulent and attenuated viruses, indicating that the antigenic mass necessary for an immune response is produced at a low level of infection, and is not predicted by viremic status. Thus, viral replication was occurring in lung or lymphoid tissues even though viremia was not always observed.</p

Impact of genotype 1 and 2 of porcine reproductive and respiratory syndrome viruses on interferon-α responses by plasmacytoid dendritic cells

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) infections are characterized by prolonged viremia and viral shedding consistent with incomplete immunity. Type I interferons (IFN) are essential for mounting efficient antiviral innate and adaptive immune responses, but in a recent study, North American PRRSV genotype 2 isolates did not induce, or even strongly inhibited, IFN-α in plasmacytoid dendritic cells (pDC), representing "professional IFN-α-producing cells". Since inhibition of IFN-α expression might initiate PRRSV pathogenesis, we further characterized PRRSV effects and host modifying factors on IFN-α responses of pDC. Surprisingly, a variety of type 1 and type 2 PRRSV directly stimulated IFN-α secretion by pDC. The effect did not require live virus and was mediated through the TLR7 pathway. Furthermore, both IFN-γ and IL-4 significantly enhanced the pDC production of IFN-α in response to PRRSV exposure. PRRSV inhibition of IFN-α responses from enriched pDC stimulated by CpG oligodeoxynucleotides was weak or absent. VR-2332, the prototype genotype 2 PRRSV, only suppressed the responses by 34%, and the highest level of suppression (51%) was induced by a Chinese highly pathogenic PRRSV isolate. Taken together, these findings demonstrate that pDC respond to PRRSV and suggest that suppressive activities on pDC, if any, are moderate and strain-dependent. Thus, pDC may be a source of systemic IFN-α responses reported in PRRSV-infected animals, further contributing to the puzzling immunopathogenesis of PRRS

Evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions

<p>Abstract</p> <p>Background</p> <p>Porcine reproductive and respiratory syndrome virus (PRRSV) causes chronic, economically devastating disease in pigs of all ages. Frequent mutations in the viral genome result in viruses with immune escape mutants. Irrespective of regular vaccination, control of PRRSV remains a challenge to swine farmers. In PRRSV-infected pigs, innate cytokine IFN-α is inhibited and the adaptive arm of the immunity is delayed. To elucidate both cellular and innate cytokine responses at very early stages of PRRSV infection, seven weeks old pigs maintained on a commercial pig farm were infected and analyzed.</p> <p>Results</p> <p>One pig in a pen containing 25 pigs was PRRSV infected and responses from this pig and one penmate were assessed two days later. All the infected and a few of the contact neighbor pigs were viremic. At day 2 post-infection, approximately 50% of viremic pigs had greater than 50% reduction in NK cell-mediated cytotoxicity, and nearly a 1-fold increase in IFN-α production was detected in blood of a few pigs. Enhanced secretion of IL-4 (in ~90%), IL-12 (in ~40%), and IL-10 (in ~20%) (but not IFN-γ) in PRRSV infected pigs was observed. In addition, reduced frequency of myeloid cells, CD4^-CD8⁺T cells, and CD4⁺CD8⁺T cells and upregulated frequency of lymphocytes bearing natural T regulatory cell phenotype were detected in viremic pigs. Interestingly, all viremic contact pigs also had comparable immune cell modulations.</p> <p>Conclusion</p> <p>Replicating PRRSV in both infected and contact pigs was found to be responsible for rapid modulation in NK cell-meditated cytotoxicity and alteration in the production of important immune cytokines. PRRSV-induced immunological changes observed simultaneously at both cellular and cytokine levels early post-infection appear to be responsible for the delay in generation of adaptive immunity. As the study was performed in pigs maintained under commercial environmental conditions, this study has practical implications in design of protective vaccines.</p

Temporal dynamics of co-circulating lineages of porcine reproductive and respiratory syndrome virus

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the most important endemic pathogen in the U.S. swine industry. Despite control efforts involving improved biosecurity and different vaccination protocols, the virus continues to circulate and evolve. One of the foremost challenges in its control is high levels of genetic and antigenic diversity. Here, we quantify the co-circulation, emergence and sequential turnover of multiple PRRSV lineages in a single swine-producing region in the United States over a span of 9 years (2009–2017). By classifying over 4,000 PRRSV sequences (open-reading frame 5) into phylogenetic lineages and sub-lineages, we document the ongoing diversification and temporal dynamics of the PRRSV population, including the rapid emergence of a novel sub-lineage that appeared to be absent globally pre-2008. In addition, lineage 9 was the most prevalent lineage from 2009 to 2010, but its occurrence fell to 0.5% of all sequences identified per year after 2014, coinciding with the emergence or re-emergence of lineage 1 as the dominant lineage. The sequential dominance of different lineages, as well as three different sub-lineages within lineage 1, is consistent with the immune-mediated selection hypothesis for the sequential turnover in the dominant lineage. As host populations build immunity through natural infection or vaccination toward the most common variant, this dominant (sub-) lineage may be replaced by an emerging variant to which the population is more susceptible. An analysis of patterns of non- synonymous and synonymous mutations revealed evidence of positive selection on immunologically important regions of the genome, further supporting the potential that immune-mediated selection shapes the evolutionary and epidemiological dynamics for this virus. This has important implications for patterns of emergence and re-emergence of genetic variants of PRRSV that have negative impacts on the swine industry. Constant surveillance on PRRSV occurrence is crucial to a better understanding of the epidemiological and evolutionary dynamics of co-circulating viral lineages. Further studies utilizing whole genome sequencing and exploring the extent of cross-immunity between heterologous PRRS viruses could shed further light on PRRSV immunological response and aid in developing strategies that might be able to diminish disease impact

Full genome sequence analysis of a wild, non-MLV-related type 2 Hungarian PRRSV variant isolated in Europe

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widespread pathogen of pigs causing significant economic losses to the swine industry. The expanding diversity of PRRSV strains makes the diagnosis, control and eradication of the disease more and more difficult. In the present study, the authors report the full genome sequencing of a Type 2 PRRSV strain isolated from piglet carcasses in Hungary. Next generation sequencing was used to determine the complete genome sequence of the isolate (PRRSV-2/Hungary/102/2012). Recombination analysis performed with the available full-length genome sequences showed no evidence of such event with other known PRRSV. Unique deletions and an insertion were found in the nsp2 region of PRRSV-2/Hungary/102/2012 when it was compared to the highly virulent VR2332 and JXA-1 prototype strains. A majority of amino acid alterations in GP4 and GP5 of the virus were in the known antigenic regions suggesting an important role for immunological pressure in PRRSV-2/Hungary/102/2012 evolution. Phylogenetic analysis revealed that it belongs to lineage 1 or 2 of Type 2 PRRSV. Considering the lack of related PRRSV in Europe, except for a partial sequence from Slovakia, the ancestor of PRRSV-2/Hungary/102/2012 was most probably transported from North-America. It is the first documented type 2 PRRSV isolated in Europe that is not related to the Ingelvac MLV

Longitudinal Surveillance of Porcine Rotavirus B Strains from the United States and Canada and In Silico Identification of Antigenically Important Sites

Citation: Shepherd, F.K.; Murtaugh, M.P.; Chen, F.; Culhane, M.R.; Marthaler, D.G. Longitudinal Surveillance of Porcine Rotavirus B Strains from the United States and Canada and In Silico Identification of Antigenically Important Sites. Pathogens 2017, 6, 64.Rotavirus B (RVB) is an important swine pathogen, but control and prevention strategies are limited without an available vaccine. To develop a subunit RVB vaccine with maximal effect, we characterized the amino acid sequence variability and predicted antigenicity of RVB viral protein 7 (VP7), a major neutralizing antibody target, from clinically infected pigs in the United States and Canada. We identified genotype-specific antigenic sites that may be antibody neutralization targets. While some antigenic sites had high amino acid functional group diversity, nine antigenic sites were completely conserved. Analysis of nucleotide substitution rates at amino acid sites (dN/dS) suggested that negative selection appeared to be playing a larger role in the evolution of the identified antigenic sites when compared to positive selection, and was identified in six of the nine conserved antigenic sites. These results identified important characteristics of RVB VP7 variability and evolution and suggest antigenic residues on RVB VP7 that are negatively selected and highly conserved may be good candidate regions to include in a subunit vaccine design due to their tendency to remain stable

Genomic and evolutionary inferences between American and global strains of porcine epidemic diarrhea virus

AbstractPorcine epidemic diarrhea virus (PEDV) has caused severe economic losses both recently in the United States (US) and historically throughout Europe and Asia. Traditionally, analysis of the spike gene has been used to determine phylogenetic relationships between PEDV strains. We determined the complete genomes of 93 PEDV field samples from US swine and analyzed the data in conjunction with complete genome sequences available from GenBank (n=126) to determine the most variable genomic areas. Our results indicate high levels of variation within the ORF1 and spike regions while the C-terminal domains of structural genes were highly conserved. Analysis of the Receptor Binding Domains in the spike gene revealed a limited number of amino acid substitutions in US strains compared to Asian strains. Phylogenetic analysis of the complete genome sequence data revealed high rates of recombination, resulting in differing evolutionary patterns in phylogenies inferred for the spike region versus whole genomes. These finding suggest that significant genetic events outside of the spike region have contributed to the evolution of PEDV

Establishment of Systems to Enable Isolation of Porcine Monoclonal Antibodies Broadly Neutralizing the Porcine Reproductive and Respiratory Syndrome Virus

The rapid evolution of porcine reproductive and respiratory syndrome viruses (PRRSV) poses a major challenge to effective disease control since available vaccines show variable efficacy against divergent strains. Knowledge of the antigenic targets of virus-neutralizing antibodies that confer protection against heterologous PRRSV strains would be a catalyst for the development of next-generation vaccines. Key to discovering these epitopes is the isolation of neutralizing monoclonal antibodies (mAbs) from immune pigs. To address this need, we sought to establish systems to enable the isolation of PRRSV neutralizing porcine mAbs. We experimentally produced a cohort of immune pigs by sequential challenge infection with four heterologous PRRSV strains spanning PRRSV-1 subtypes and PRRSV species. Whilst priming with PRRSV-1 subtype 1 did not confer full protection against a subsequent infection with a PRRSV-1 subtype 3 strain, animals were protected against a subsequent PRRSV-2 infection. The infection protocol resulted in high serum neutralizing antibody titers against PRRSV-1 Olot/91 and significant neutralization of heterologous PRRSV-1/-2 strains. Enriched memory B cells isolated at the termination of the study were genetically programmed by transduction with a retroviral vector expressing the Bcl-6 transcription factor and the anti-apoptotic Bcl-xL protein, a technology we demonstrated efficiently converts porcine memory B cells into proliferating antibody-secreting cells. Pools of transduced memory B cells were cultured and supernatants containing PRRSV-specific antibodies identified by flow cytometric staining of infected MARC-145 cells and in vitro neutralization of PRRSV-1. Collectively, these data suggest that this experimental system may be further exploited to produce a panel of PRRSV-specific mAbs, which will contribute both to our understanding of the antibody response to PRRSV and allow epitopes to be resolved that may ultimately guide the design of immunogens to induce cross-protective immunity