Respiratory Syncytial Virus Interferon Antagonist NS1 Protein Suppresses and Skews the Human T Lymphocyte Response

We recently demonstrated that the respiratory syncytial virus (RSV) NS1 protein, an antagonist of host type I interferon (IFN-I) production and signaling, has a suppressive effect on the maturation of human dendritic cells (DC) that was only partly dependent on released IFN-I. Here we investigated whether NS1 affects the ability of DC to activate CD8+ and CD4+ T cells. Human DC were infected with RSV deletion mutants lacking the NS1 and/or NS2 genes and assayed for the ability to activate autologous T cells in vitro, which were analyzed by multi-color flow cytometry. Deletion of the NS1, but not NS2, protein resulted in three major effects: (i) an increased activation and proliferation of CD8+ T cells that express CD103, a tissue homing integrin that directs CD8+ T cells to mucosal epithelial cells of the respiratory tract and triggers cytolytic activity; (ii) an increased activation and proliferation of Th17 cells, which have recently been shown to have anti-viral effects and also indirectly attract neutrophils; and (iii) decreased activation of IL-4-producing CD4+ T cells - which are associated with enhanced RSV disease - and reduced proliferation of total CD4+ T cells. Except for total CD4+ T cell proliferation, none of the T cell effects appeared to be due to increased IFN-I signaling. In the infected DC, deletion of the NS1 and NS2 genes strongly up-regulated the expression of cytokines and other molecules involved in DC maturation. This was partly IFN-I-independent, and thus might account for the T cell effects. Taken together, these data demonstrate that the NS1 protein suppresses proliferation and activation of two of the protective cell populations (CD103+ CD8+ T cells and Th17 cells), and promotes proliferation and activation of Th2 cells that can enhance RSV disease

Low CCR7-Mediated Migration of Human Monocyte Derived Dendritic Cells in Response to Human Respiratory Syncytial Virus and Human Metapneumovirus

Human respiratory syncytial virus (HRSV) and, to a lesser extent, human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), can re-infect symptomatically throughout life without significant antigenic change, suggestive of incomplete or short-lived immunity. In contrast, re-infection by influenza A virus (IAV) largely depends on antigenic change, suggestive of more complete immunity. Antigen presentation by dendritic cells (DC) is critical in initiating the adaptive immune response. Antigen uptake by DC induces maturational changes that include decreased expression of the chemokine receptors CCR1, CCR2, and CCR5 that maintain DC residence in peripheral tissues, and increased expression of CCR7 that mediates the migration of antigen-bearing DC to lymphatic tissue. We stimulated human monocyte-derived DC (MDDC) with virus and found that, in contrast to HPIV3 and IAV, HMPV and HRSV did not efficiently decrease CCR1, 2, and 5 expression, and did not efficiently increase CCR7 expression. Consistent with the differences in CCR7 mRNA and protein expression, MDDC stimulated with HRSV or HMPV migrated less efficiently to the CCR7 ligand CCL19 than did IAV-stimulated MDDC. Using GFP-expressing recombinant virus, we showed that the subpopulation of MDDC that was robustly infected with HRSV was particularly inefficient in chemokine receptor modulation. HMPV- or HRSV-stimulated MDDC responded to secondary stimulation with bacterial lipopolysaccharide or with a cocktail of proinflammatory cytokines by increasing CCR7 and decreasing CCR1, 2 and 5 expression, and by more efficient migration to CCL19, suggesting that HMPV and HRSV suboptimally stimulate rather than irreversibly inhibit MDDC migration. This also suggests that the low concentration of proinflammatory cytokines released from HRSV- and HMPV-stimulated MDDC is partly responsible for the low CCR7-mediated migration. We propose that inefficient migration of HRSV- and HMPV-stimulated DC to lymphatic tissue contributes to reduced adaptive responses to these viruses

Effects of Human Respiratory Syncytial Virus, Metapneumovirus, Parainfluenza Virus 3 and Influenza Virus on CD4+ T Cell Activation by Dendritic Cells

BACKGROUND: Human respiratory syncytial virus (HRSV), and to a lesser extent human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), re-infect symptomatically throughout life without antigenic change, suggestive of incomplete immunity. One causative factor is thought to be viral interference with dendritic cell (DC)-mediated stimulation of CD4+ T cells. METHODOLOGY, PRINCIPAL FINDINGS: We infected human monocyte-derived DC with purified HRSV, HMPV, HPIV3, or influenza A virus (IAV) and compared their ability to induce activation and proliferation of autologous CD4+ T cells in vitro. IAV was included because symptomatic re-infection without antigenic change is less frequent, suggesting that immune protection is more complete and durable. We examined virus-specific memory responses and superantigen-induced responses by multiparameter flow cytometry. Live virus was more stimulatory than inactivated virus in inducing DC-mediated proliferation of virus-specific memory CD4+ T cells, suggesting a lack of strong suppression by live virus. There were trends of increasing proliferation in the order: HMPV<HRSV<HPIV3<IAV, and greater production of interferon-γ and tumor necrosis factor-α by proliferating cells in response to IAV, but differences were not significant. Exposure of DC to HRSV, HPIV3, or IAV reduced CD4+ T cell proliferation in response to secondary stimulus with superantigen, but the effect was transitory and greatest for IAV. T cell cytokine production was similar, with no evidence of Th2 or Th17 skewing. CONCLUSIONS, SIGNIFICANCE: Understanding the basis for the ability of HRSV in particular to symptomatically re-infect without significant antigenic change is of considerable interest. The present results show that these common respiratory viruses are similar in their ability to induce DC to activate CD4+ T cells. Thus, the results do not support the common model in which viral suppression of CD4+ T cell activation and proliferation by HRSV, HMPV, and HPIV3 is a major factor in the difference in re-infectability compared to IAV

Different Domains of the RNA Polymerase of Infectious Bursal Disease Virus Contribute to Virulence

BACKGROUND: Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. IBDV has a bi-segmented double-stranded RNA genome. Segments A and B encode the capsid, ribonucleoprotein and non-structural proteins, or the virus polymerase (RdRp), respectively. Since the late eighties, very virulent (vv) IBDV strains have emerged in Europe inducing up to 60% mortality. Although some progress has been made in understanding the molecular biology of IBDV, the molecular basis for the pathogenicity of vvIBDV is still not fully understood. METHODOLOGY, PRINCIPAL FINDINGS: Strain 88180 belongs to a lineage of pathogenic IBDV phylogenetically related to vvIBDV. By reverse genetics, we rescued a molecular clone (mc88180), as pathogenic as its parent strain. To study the molecular basis for 88180 pathogenicity, we constructed and characterized in vivo reassortant or mosaic recombinant viruses derived from the 88180 and the attenuated Cu-1 IBDV strains. The reassortant virus rescued from segments A of 88180 (A88) and B of Cu-1 (BCU1) was milder than mc88180 showing that segment B is involved in 88180 pathogenicity. Next, the exchange of different regions of BCU1 with their counterparts in B88 in association with A88 did not fully restore a virulence equivalent to mc88180. This demonstrated that several regions if not the whole B88 are essential for the in vivo pathogenicity of 88180. CONCLUSION, SIGNIFICANCE: The present results show that different domains of the RdRp, are essential for the in vivo pathogenicity of IBDV, independently of the replication efficiency of the mosaic viruses

Étude de l'épidémiologie moléculaire du segment B de l'avibinavirus de la bursite infectieuse aviaire et bases moléculaires de la virulence chez ce virus

La bursite infectieuse est une maladie du poulet (Gallus gallus) causée par un virus (noté IBDV) de la famille des Birnaviridae, genre Avibirnavirus. Certaines souches d'IBDV dites hypervirulentes (vvIBDV) induisent une mortalité supérieure à 30 %. Il n'y a pas de marqueur génétique de virulence pour les caractériser. Afin de comparer l'épidémiologie moléculaires des deux segments génomiques de l'IBDV, notés A et B, de courtes régions génomiques phylogénétiquement représentatives de chacun des segments ont éte identifiées, en comparant les phylogénies dérivées de séquences nucléotidiques complètes ou partielles déjà publiées. Ces régions correspondent au tiers moyen du gène VP2 (segment A) et aux deux tiers 5' du gène VP1 (segment B). Ces régions ont été amplifiées et séquencées chez 50 souches virales d'origines et de phénotypes variés. Leur étude phylogénétique suggère que les segments génomiques ont co-évolué chez 70 % des virus étudiés (dont les vvIBDV), mais que des phénomènes de réassortiment pourraient avoir affecté 13 % des souches virales. Un des virus potentiellement réassortant (segment A apparenté aux vvIBDV, segment B non vvIBDV), inoculé expérimentalement à des poulets sensibles, avait un pouvoir pathogène réduit sans que la réplication virale apparaisse modifiée. Les bases moléculaires de la pathogénicité de l'IBDV ont finalement été recherchées chez deux souches virales apparentées aux vvIBDV identifiées lors de l'étude phylogénétique, l'une issue d'une lignée particulière de virus pathogènes (88180), l'autre apparentée aux vvIBDV typiques mais dotée d'un pouvoir pathogène réduit (94432). Un système de génétique inverse a été développé pour chacune. Le pouvoir pathogène in vivo des clones moléculaires correspondants s'est avéré comparable à celui des virus parentaux. Onze virus génétiquement modifiés dérivés des clones moléculaires ont été construits et caractérisés in vivo. Le pouvoir pathogène de 88180 semble lié au segment A et à plusieurs régions du segment B, alors que l'atténuation de 94432 s'avère liée à trois mutations peptidiques codées par le segment A.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Compared pathogenicity of mc88180, A88BCU1 and five recombinant viruses with mosaic segment B in five-week-old SPF chickens (experiment <i>iii</i>).

<p>See in Table S1 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028064#pone-0028064-g001" target="_blank">Figure 1</a> the genetic make up of the viruses with mosaic segment B and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028064#pone.0028064.s003" target="_blank">Table S3</a> the summarized protocol of the animal experiment. On the first day of the experiment, SPF chickens were housed in eight groups of 20 chickens of comparable sex and weight. On the same day, each bird was inoculated by the intranasal route with a standardized dose of the relevant virus (10⁵ EID₅₀ by bird). A group was kept as a mock-inoculated control receiving PBS only. <b>A</b>) % cumulated mortality at 5 days post infection, <b>B</b>) BF to body weights ratio (‰) at day 21 (n = 10 chickens per group). The box plots show the median (horizontal line) flanked by the 2^nd and 3^rd quartile. The outer bars show the range of values. <b>C</b>) and <b>D</b>) Histological lesion scoring at day 4 and 21, respectively (n = 5 chickens per group). The outer bars show the range of values. <b>E</b>) VN titer at day 21 (in log₂, n = 8 to 10 chickens depending on the group). <b>F</b>) Virus titer at day 4 (in log₁₀ EID_50/gr, n = 5 chickens per group). Treatments sharing the same lowercase letter do not differ significantly according to the kruskal-Wallis test (BF to body weights ratio, lesion score, VN titer and virus titer) or according to the Chi-squared test (% mortality), at the p≤0.05 confidence level.</p

Mean individual symptomatic index between 1 to 10 day post inoculation in chickens inoculated with mosaic recombinant IBDV strains.

<p>The symptomatic index was calculated according a scale ranging from 0 to 3 with, with 0 meaning “lack of signs”, 1 meaning “typical IBD signs (ruffled feathers) conspicuous in quiet bird only, the bird stimulated by a sudden change in environment (light, noise, or vicinity of experiment observer) appears normal, motility is not reduced”; 2 meaning “typical IBD signs conspicuous even when bird is stimulated, dehydration is apparent, motility may be slightly reduced” and 3 standing for “typical severe IBD signs with prostration or death”. mc88180 induced significantly more morbidity than all other viruses from 3 to 9 DPI (p≤0.0001 to p≤0.003), whereas no significant difference in the severity of clinical signs was observed between the groups receiving the other viruses or mock-inoculated., A simplified representation of the chimeric segments B (based on the ones described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028064#pone-0028064-g001" target="_blank">Figure 1</a>) used in association with the segment A of 88180 to generate the indicated mosaic recombinant viruses is shown.</p