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17 p.-8 fig., SARS-CoV-2 vaccines currently in use have contributed to controlling the COVID-19 pandemic. Notwithstanding, the high mutation rate, fundamentally in the spike glycoprotein (S), is causing the emergence of new variants. Solely utilizing this antigen is a drawback that may reduce the efficacy of these vaccines. Herein we present a DNA vaccine candidate that contains the genes encoding the S and the nucleocapsid (N) proteins implemented into the non-replicative mammalian expression plasmid vector, pPAL. This plasmid lacks antibiotic resistance genes and contains an alternative selectable marker for production. The S gene sequence was modified to avoid furin cleavage (Sfs). Potent humoral and cellular immune responses were observed in C57BL/6J mice vaccinated with pPAL-Sfs + pPAL-N following a prime/boost regimen by the intramuscular route applying in vivo electroporation. The immunogen fully protected K18-hACE2 mice against a lethal dose (105 PFU) of SARS-CoV-2. Viral replication was completely controlled in the lungs, brain, and heart of vaccinated mice. Therefore, pPAL-Sfs + pPAL-N is a promising DNA vaccine candidate for protection from COVID-19., This work was funded by PTI-Salud Global (CSIC), Center for Technological and Industrial Development (CDTI), REACT-ANTICIPA-UCM (Comunidad de Madrid), and European Research Council (Advanced Grant VERDI, ERC2015AdG grant number 694160).

Viruses have evolved numerous strategies to impair immunity so that they can replicate more efficiently. Among those, the immunosuppressive effects of morbillivirus infection can be particularly problematic, as they allow secondary infections to take hold in the host, worsening disease prognosis. In the present work, we hypothesized that the highly contagious morbillivirus peste des petits ruminants virus (PPRV) could target monocytes and dendritic cells (DC) to contribute to the immunosuppressive effects produced by the infection. Monocytes isolated from healthy sheep, a natural host of the disease, were able be infected by PPRV and this impaired the differentiation and phagocytic ability of immature monocyte-derived DC (MoDC). We also assessed PPRV capacity to infect differentiated MoDC. Ovine MoDC could be productively infected by PPRV, and this drastically reduced MoDC capacity to activate allogeneic T cell responses. Transcriptomic analysis of infected MoDC indicated that several tolerogenic DC signature genes were upregulated upon PPRV infection. Furthermore, PPRV-infected MoDC could impair the proliferative response of autologous CD4+ and CD8+ T cell to the mitogen concanavalin A (ConA), which indicated that DC targeting by the virus could promote immunosuppression. These results shed new light on the mechanisms employed by morbillivirus to suppress the host immune responses. IMPORTANCE Morbilliviruses pose a threat to global health given their high infectivity. The morbillivirus peste des petits ruminants virus (PPRV) severely affects small-ruminant-productivity and leads to important economic losses in communities that rely on these animals for subsistence. PPRV produces in the infected host a period of severe immunosuppression that opportunistic pathogens exploit, which worsens the course of the infection. The mechanisms of PPRV immunosuppression are not fully understood. In the present work, we demonstrate that PPRV can infect professional antigen-presenting cells called dendritic cells (DC) and disrupt their capacity to elicit an immune response. PPRV infection promoted a DC activation profile that favored the induction of tolerance instead of the activation of an antiviral immune response. These results shed new light on the mechanisms employed by morbilliviruses to suppress the immune responses.

The tumour necrosis factor superfamily OX40L and CD70 and their receptors are costimulatory signalling axes critical for adequate T and B cell activation in humans and mice. In this work we inoculated groups of sheep with human recombinant adenovirus type 5 (Ad) expressingOvis aries(Oa)OX40L orOaCD70 or a control adenoviral vector to determine whether they could improve the immune response to the model antigen OVA. PBMCs and serum samples were obtained for analysis of the adaptive immune response to OVA at days 0, 15, 30 and 90 post-inoculation (pi). Recall responses to OVA were assessed at day 7 and 30 after the second antigen inoculation (pb) at day 90. Administration of these immunomodulatory molecules did not induce unspecific PBMC stimulation. WhileOaOX40L administration mainly increased TNF-α and IL-4 in PBMC at day 15 pi concomitantly with a slight increase in antibody titer and the number of IFN-γ producing cells, we detected greater effects on adaptive immunity afterOaCD70 administration. AdOaCD70 inoculation improved antibody titers to OVA at days 30 and 90 pi, and increased anti-OVA-specific IgG-secreting B cell counts when compared to control. Moreover, higher IFN-γ production was detected on days 7 pi, 7 pb and 30 pb in PBMCs from this group. Phenotypic analysis of T cell activation showed an increase in effector CD8+T cells (CD8+CD62L-CD27-) at day 15 pi in AdOaCD70 group, concurrent with a decrease in early activated cells (CD8+CD62L-CD27+). Moreover, recall anti-OVA CD8+T cell responses were increased at 7 pb in the AdOaCD70 group. AdOaCD70 administration could therefore promote CD8+T cell effector differentiation and long-term activity. In this work we characterized thein vivoadjuvant potential on the humoral and cellular immune response ofOaOX40L andOaCD70 delivered by non-replicative adenovirus vectors using the model antigen OVA. We present data highlighting the potency of these molecules as veterinary vaccine adjuvant.

Centro de Investigación en Sanidad Animal (CISA), The toll-like receptor (TLR) family comprises both cell-surface and intracellular receptors that recognize different types of pathogen-associated molecular patterns (PAMPs) leading to the production of pro-inflammatory cytokines and subsequent development of adaptive immunity. TLR2 is a cell-surface receptor initially thought to act as a bacterial sentinel but also shown to recognize a number of viral glycoproteins. In this study, we sought to characterize the role of TLR2 in the activation of the immune response by peste des petits ruminants virus (PPRV), a morbillivirus of the Paramixoviridae family that causes an acute, highly contagious disease in goats and sheep. Using human embryonic kidney (HEK) 293 cells stably expressing human (h)TLR2 but lacking any other TLR, we found that PPRV induces IL-8 production in a dose-dependent manner. That activation is only observed in cells expressing hTLR2 and is greatly reduced when the receptor is blocked by pretreatment with specific antibody. We identified hemagglutinin (H) as the viral protein responsible of TLR2 activation by performing the same assays with purified recombinant mammalian-expressed H protein. Exogenous addition of recombinant H protein to cell culture induces high levels of interleukin (IL)-8 only in TLR2-expressing cells. Moreover, H engagement on TLR2 in the monocytic cell line THP-1 activates extracellular-signal-regulated kinase (ERK) signaling. Stimulation of primary ovine dendritic cells with either inactivated PPRV or purified recombinant H protein results in transcription of pro-inflammatory cytokines and the secretion of the Th1-polarizing cytokine IL-12. The role of these host immune mechanisms in the control of PPR is discussed., This work was funded by grants AGL2015-64290R and RTI2018-094616-B-100 from the Ministerio de Ciencia (Spain); grant VetBioNet INFRAIA-731014 from the EU-H2020 and grant S2018/BAA-4370-PLATESA2 from Comunidad de Madrid (Fondo Europeo de Desarrollo Regional, FEDER). MA was funded by an FPI grant (BES-2013-066406). NS was the holder of a fellowship from OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agricultural Systems., 15 Pág.

17 Pág. Centro de Investigación en Sanidad Animal (CISA), Viral infections have long provided a platform to understand the workings of immunity. For instance, great strides towards defining basic immunology concepts, such as MHC restriction of antigen presentation or T-cell memory development and maintenance, have been achieved thanks to the study of lymphocytic choriomeningitis virus (LCMV) infections. These studies have also shaped our understanding of antiviral immunity, and in particular T-cell responses. In the present review, we discuss how bluetongue virus (BTV), an economically important arbovirus from the Reoviridae family that affects ruminants, affects adaptive immunity in the natural hosts. During the initial stages of infection, BTV triggers leucopenia in the hosts. The host then mounts an adaptive immune response that controls the disease. In this work, we discuss how BTV triggers CD8+ T-cell expansion and neutralizing antibody responses, yet in some individuals viremia remains detectable after these adaptive immune mechanisms are active. We present some unpublished data showing that BTV infection also affects other T cell populations such as CD4+ T-cells or γδ T-cells, as well as B-cell numbers in the periphery. This review also discusses how BTV evades these adaptive immune mechanisms so that it can be transmitted back to the arthropod host. Understanding the interaction of BTV with immunity could ultimately define the correlates of protection with immune mechanisms that would improve our knowledge of ruminant immunology., This work was funded by grants RTI2018-094616-B-100 from the Ministerio de Ciencia (Spain) and S2018/BAA-4370-PLATESA2 from Comunidad de Madrid (Fondo Europeo de Desarrollo Regional, FEDER). MA was funded by an FPI grant (BES-2013-066406). ALL was funded by an FPI grant (INIA).

14 Pág. Centro de Investigación en Sanidad Animal (CISA), The Morbillivirus peste des petits ruminants virus (PPRV) is the causal agent of a highly contagious disease that mostly affects sheep and goats and produces considerable losses in developing countries. Current PPRV control strategies rely on live-attenuated vaccines, which are not ideal, as they cannot differentiate infected from vaccinated animals (DIVA). Recombinant vector-based vaccines expressing viral subunits can provide an alternative to conventional vaccines, as they can be easily paired with DIVA diagnostic tools. In the present work, we used the bovine herpesvirus-4-based vector (BoHV-4-A) to deliver PPRV hemagglutinin H antigen (BoHV-4-A-PPRV-H-ΔTK). Vaccination with BoHV-4-A-PPRV-H-ΔTK protected sheep from virulent PPRV challenge and prevented virus shedding. Protection correlated with anti-PPRV IgGs, neutralizing antibodies and IFN-γ-producing cells induced by the vaccine. Detection of antibodies exclusively against H-PPRV in animal sera and not against other PPRV viral proteins such as F or N could serve as a DIVA diagnostic test when using BoHV-4-A-PPRV-H-ΔTK as vaccine. Our data indicate that BoHV-4-A-PPRV-H-ΔTK could be a promising new approach for PPRV eradication programs., This work was funded mainly by a TNA (Translational Access Activities) from VetBioNet INFRAIA-731014 from the EU-H2020. The study also received the following grants and funding: AGL2015-64290R and RTI2018-094616-B-100 from the Ministerio de Ciencia (Spain), grant S2018/BAA-4370-PLATESA2 from Comunidad de Madrid (Fondo Europeo de Desarrollo Regional, FEDER), and internal funding from Parma University.

The mammalian interferon (IFN) signaling pathway is a primary component of the innate antiviral response, and viral pathogens have evolved multiple mechanisms to antagonize this pathway and to facilitate infection. Bluetongue virus (BTV), an orbivirus of the Reoviridae family, is transmitted by midges to ruminants and causes a disease that produces important economic losses and restriction to animal trade and is of compulsory notification to the World Organization for Animal Health (OIE). Here, we show that BTV interferes with IFN-I and IFN-II responses in two ways, by blocking STAT1 phosphorylation and by degrading STAT2. BTV-NS3 protein, which is involved in virion egress, interacts with STAT2, and induces its degradation by an autophagy-dependent mechanism. This STAT2 degradative process requires the recruitment of an E3-Ub-ligase to NS3 as well as NS3 K63 polyubiquitination. Taken together, our study identifies a new mechanism by which a virus degrades STAT2 for IFN signaling blockade, highlighting the diversity of mechanisms employed by viruses to subvert the IFN response.

The anti-inflammatory master regulator IL-10 is critical to protect the host from tissue damage during acute phases of immune responses. This regulatory mechanism, central to T cell homeostasis, can be hijacked by viruses to evade immunity. IL-10 can be produced by virtually all immune cells, and it can also modulate the function of these cells. Understanding the effects of this multifunctional cytokine is therefore a complex task. In the present review we discuss the factors driving IL-10 production and the cellular sources of the cytokine during antiviral immune responses. We particularly focus on the IL-10 regulatory mechanisms that impact antiviral immune responses and how viruses can use this central regulatory pathway to evade immunity and establish chronic/latent infections.

Soluble cytokine decoy receptors are potent immune modulatory reagents with therapeutic applications. Some virus-encoded secreted cytokine receptors interact with glycosaminoglycans expressed at the cell surface, but the biological significance of this activity in vivo is poorly understood. Here, we show the type I interferon binding protein (IFNα/βBP) encoded by vaccinia and ectromelia viruses requires of this cell binding activity to confer full virulence to these viruses and to retain immunomodulatory activity. Expression of a variant form of the IFNα/βBP that inhibits IFN activity, but does not interact with cell surface glycosaminoglycans, results in highly attenuated viruses with a virulence similar to that of the IFNα/βBP deletion mutant viruses. Transcriptomics analysis and infection of IFN receptor-deficient mice confirmed that the control of IFN activity is the main function of the IFNα/βBP in vivo. We propose that retention of secreted cytokine receptors at the cell surface may largely enhance their immunomodulatory activity., Ministry of Economy and Competitiveness and European Union (European Regional Development’s Funds, FEDER) (grant SAF2015-67485-R) and by European Sequencing and Genotyping Infrastructure

The adaptive immune system utilizes multiple effector mechanisms to clear viral infections. Among those antibody-dependent cell-mediated cytotoxicity (ADCC) can help recognize and clear virus-infected cells. In the present work we evaluated ADCC contribution to immunity in two economically important viral diseases that affect ruminants: bluetongue and peste des petits ruminants. Immune sera obtained from sheep experimentally infected with bluetongue virus (BTV) serotype 8 or peste des petits ruminant virus (PPRV) IC'89 were used for this study. PPRV immune sera could bind to the surface of PPRV-infected ovine B cells while BTV immune sera was unable to bind to the surface of BTV-infected sheep cells but could recognize intracellular BTV antigens. BTV and PPRV immune serum ADCC potency was established using an ovine autologous cytotoxicity assay that employed an NK cell-enriched fraction as effector cells and a virus-infected B cell-enriched fraction as target cells. In this system, immune sera triggered ADCC against PPRV-infected cells, but not against BTV-infected cells. PPRV immune sera could recognize PPRV fusion and hemagglutinin proteins on the surface of transfected cells, and enhanced lysis of these cells in ADCC assays. This indicated that these viral antigens are natural ADCC targets during PPRV infection. The present work describes a novel effector immune mechanism against PPRV in the natural host that could contribute to virus clearance highlighting the importance of studying protective immune mechanisms to improve current vaccines by invoking all effector arms of immunity.

Peste des petits ruminants virus (PPRV) causes an economically important disease that limits productivity in small domestic ruminants and often affects the livestock of the poorest populations in developing countries. Animals that survive PPRV develop strong cellular and humoral responses, which are probably necessary for protection. Vaccination should thus aim at mimicking these natural responses. Immunization strategies against this morbillivirus using recombinant adenoviruses expressing PPRV-F or -H proteins can protect PPRV-challenged animals and permit differentiation of infected from vaccinated animals. Little is known of the T cell repertoire these recombinant vaccines induce. In the present work, we identified several CD4+ and CD8+ T cell epitopes in sheep infected with PPRV. We also show that recombinant adenovirus vaccination induced T cell responses to the same epitopes, and led to memory T cell differentiation. T cells primed by these recombinant adenovirus vaccines expanded after PPRV challenge and probably contributed to protection. These data validate the use of recombinant adenovirus expressing PPRV genes as DIVA strategies to control this highly contagious disease. Electronic supplementary material The online version of this article (10.1186/s13567-017-0482-x) contains supplementary material, which is available to authorized users.

Arboviruses cause acute diseases that increasingly affect global health. We used bluetongue virus (BTV) and its natural sheep host to reveal a previously uncharacterized mechanism used by an arbovirus to manipulate host immunity. Our study shows that BTV, similarly to other antigens delivered through the skin, is transported rapidly via the lymph to the peripheral lymph nodes. Here, BTV infects and disrupts follicular dendritic cells, hindering B-cell division in germinal centers, which results in a delayed production of high affinity and virus neutralizing antibodies. Moreover, the humoral immune response to a second antigen is also hampered in BTV-infected animals. Thus, an arbovirus can evade the host antiviral response by inducing an acute immunosuppression. Although transient, this immunosuppression occurs at the critical early stages of infection when a delayed host humoral immune response likely affects virus systemic dissemination and the clinical outcome of disease.

Ovine interferon tau (IFN-τ) is a unique type I interferon with low toxicity and a broad host range in vivo . We report the generation of a nonreplicative recombinant adenovirus expressing biologically active IFN-τ. Using the B6.A2G-Mx1 mouse model, we showed that single-dose intranasal administration of recombinant Ad5-IFN-τ can effectively prevent lethality and disease induced by highly virulent hv-PR8 influenza virus by activating the interferon response and preventing viral replication.

Long-term infections with viruses permit the generation of variants that evolve specific growth advantages in certain tissues and may show altered disease potentials. The selection of such variants is influenced by the host tissue and often involves virus-receptor interactions. Here we report studies of receptor usage by several lymphocytic choriomeningitis virus (LCMV) isolates that expressed different disease patterns. Consistent with our previous studies, we found that, with one exception, multiple LCMV variants that cause suppression of immune responses bound with high affinity to their cellular receptor alpha-dystroglycan (alpha-DG) and were dependent on alpha-DG for entry and infection. The exception also bound strongly to alpha-DG but was not dependent on alpha-DG for entry and infection. In contrast, those variants of LCMV that do not suppress the immune response either displayed low or no binding affinity for alpha-DG and used alternative receptors in addition to or instead of alpha-DG for entry and infection. For all alpha-DG binding variants, alpha-DG represents the preferred receptor in DG-expressing cells, as soluble alpha-DG blocked their infection of DG-deficient cells, indicating that binding of alpha-DG to the viral glycoprotein (GP) at the virion surface interferes with the GP's interaction with the alternative receptor. Biochemical characterization of the alternative receptor(s) for LCMV indicated that they are either protein(s) or protein-bound entities.

DCs play a pivotal role in bringing forth innate and adaptive immune responses. Viruses can specifically target DCs, rendering them ineffective in stimulating T cells, which can ultimately lead to immunosuppression. In the present study we have identified several potential mechanisms by which lymphocytic choriomeningitis virus (LCMV) induces immunosuppression in its natural murine host. The immunosuppressive LCMV variant clone 13 (Cl 13) infects DCs and interferes with their maturation and antigen-presenting capacity as evidenced by a significant reduction in the surface expression of MHC class I, MHC class II, CD40, CD80, and CD86 molecules. Additionally, Cl 13 infects hematopoietic progenitor cells both in vivo and in vitro, impairing their development. One mechanism by which hematopoietic progenitors are developmentally impaired is through the Cl 13-induced production of IFN-alpha and IFN-beta (IFN-alpha/beta). Mice deficient in the receptor for IFN-alpha/beta show a normal differentiation of progenitors into DCs despite viral infection. Thus, a virus can evolve a strategy to boost its survival by preventing the maturation of DCs from infected progenitor cells and by reducing the expression of antigen-presenting and costimulatory molecules on developed DCs.

Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivatedvaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8+- and CD4+-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5- BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection. © 2015 Martín et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Bluetongue virus (BTV) is a non-enveloped dsRNA virus that causes a haemorrhagic disease mainly in sheep. It is an economically important Orbivirus of the Reoviridae family. In order to estimate the importance of T cell responses during BTV infection, it is essential to identify the epitopes targeted by the immune system. In the present work, we selected potential T cell epitopes (3 MHC-class II-binding and 8 MHC-class I binding peptides) for the C57BL/6 mouse strain from the BTV-8 non-structural protein NS1, using H2b-binding predictive algorithms. Peptide binding assays confirmed all MHC-class I predicted peptides bound MHC-class I molecules. The immunogenicity of these 11 predicted peptides was then determined using splenocytes from BTV-8-inoculated C57BL/6 mice. Four MHC-class I binding peptides elicited specific IFN-γ production and generated cytotoxic T lymphocytes (CTL) in BTV-8 infected mice. CTL specific for 2 of these peptides were also able to recognise target cells infected with different BTV serotypes. Similarly, using a combination of IFN-γ ELISPOT, intracellular cytokine staining and proliferation assays, two MHC-class II peptides were identified as CD4+ T cell epitopes in BTV-8 infected mice. Importantly, two peptides were also consistently immunogenic in sheep infected with BTV-8 using IFN-γ ELISPOT assays. Both of these peptides stimulated CD4+ T cells that cross-reacted with other BTV serotypes. The characterisation of these T cell epitopes can help develop vaccines protecting against a broad spectrum of BTV serotypes and differentiate infected from vaccinated animals., This work was funded by grants AGL2009-07353, RyC-2010-06516 and AGL2011-25025 from Ministerio de Economía y Competitividad (Spain) and 228394-NADIR Integrating Activities 7th EU program.

alpha-Dystroglycan (DG) has been identified as the cellular receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus (LFV). This subunit of DG is a highly versatile cell surface molecule that provides a molecular link between the extracellular matrix (ECM) and a beta-DG transmembrane component, which interacts with the actin-based cytoskeleton. In addition, DG exhibits a complex pattern of interaction with a wide variety of ECM and cellular proteins. In the present study, we characterized the binding of LCMV to alpha-DG and addressed the role of alpha-DG-associated host-derived proteins in virus infection. We found that the COOH-terminal region of alpha-DG's first globular domain and the NH2-terminal region of the mucin-related structures of alpha-DG together form the binding site for LCMV. The virus-alpha-DG binding unlike ECM alpha-DG interactions was not dependent on divalent cations. Despite such differences in binding, LCMV and laminin-1 use, in part, an overlapping binding site on alpha-DG, and the ability of an LCMV isolate to compete with laminin-1 for receptor binding is determined by its binding affinity to alpha-DG. This competition of the virus with ECM molecules for receptor binding likely explains the recently found correlation between the affinity of LCMV binding to alpha-DG, tissue tropism, and pathological potential. LCMV strains and variants with high binding affinity to alpha-DG but not low affinity binders are able to infect CD11c+ dendritic cells, which express alpha-DG at their surface. Infection followed by dysfunction of these antigen-presenting cells contributes to immunosuppression and persistent viral infection in vivo.

Among cells of the immune system, CD11c(+) and DEC-205(+) splenic dendritic cells primarily express the cellular receptor (alpha-dystroglycan [alpha-DG]) for lymphocytic choriomeningitis virus (LCMV). By selection, strains and variants of LCMV that bind alpha-DG with high affinity are associated with virus replication in the white pulp, show preferential replication in a majority of CD11c(+) and DEC-205(+) cells, cause immunosuppression, and establish a persistent infection. In contrast, viral strains and variants that bind with low affinity to alpha-DG are associated with viral replication in the red pulp, display minimal replication in CD11c(+) and DEC-205(+) cells, and generate a robust anti-LCMV cytotoxic T lymphocyte response that clears the virus infection. Differences in binding affinities can be mapped to a single amino acid change in the viral glycoprotein 1 ligand that binds to alpha-DG. These findings indicate that receptor-virus interaction on dendritic cells in vivo can be an essential step in the initiation of virus-induced immunosuppression and viral persistence.

Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-143→Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin α v β 3 . In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type.

Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants caused by the Morbillivirus peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenoviruses serotype 5 (Ad5) expressing either the highly immunogenic fusion protein (F) or hemagglutinin protein (H) from PPRV were used to vaccinate sheep by intramuscular inoculation. Both recombinant adenovirus vaccines elicited PPRV-specific B- and T-cell responses. Thus, neutralizing antibodies were detected in sera from immunized sheep. In addition, we detected a significant antigen specific T-cell response in vaccinated sheep against two different PPRV strains, indicating that the vaccine induced heterologous T cell responses. Importantly, no clinical signs and undetectable virus shedding were observed after virulent PPRV challenge in vaccinated sheep. These vaccines also overcame the T cell immunosuppression induced by PPRV in control animals. The results indicate that these adenovirus constructs could be a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines. © 2014 Rojas et al.

Peste des petits ruminants is a highly contagious disease of small ruminants caused by a Morbillivirus, peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenovirus serotype 5 (Ad5) containing the highly immunogenic fusion protein (F) and hemaglutinine protein (H) genes from PPRV were constructed. HEK293A cells infected with either virus (Ad5-PPRV-F or -H) express F and H proteins respectively. These viruses were used to vaccinate mice by intramuscular inoculation. Both viruses elicited PPRV-specific B- and T-cell responses. Thus, after two immunizations, sera from immunized mice elicited neutralizing antibody response, indicating that this approach has the potential to confer protective immunity. In addition, we detected a significant antigen specific CD4+ and CD8+ T-cell response in mice vaccinated with either virus. These results indicate that these adenovirus constructs offer a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines. © 2013 Elsevier Ltd.

Hematopoietic stem cells (HSCs) give rise to progenitors with potential to produce multiple cell types, including dendritic cells (DCs). DCs are the principal antigen-presenting cells and represent the crucial link between innate and adaptive immune responses. Bluetongue virus (BTV), an economically important Orbivirus of the Reoviridae family, causes a hemorrhagic disease mainly in sheep and occasionally in other species of ruminants. BTV is transmitted between its mammalian hosts by certain species of biting midges ( Culicoides spp.) and is a potent alpha interferon (IFN-α) inducer. In the present report, we show that BTV infects cells of hematopoietic origin but not HSCs in immunocompetent sheep. However, BTV infects HSCs in the absence of type I IFN (IFN-I) signaling in vitro and in vivo . Infection of HSCs in vitro results in cellular death by apoptosis. Furthermore, BTV infects bone marrow-derived DCs (BM-DCs), interfering with their development to mature DCs in the absence of type I IFN signaling. Costimulatory molecules CD80 and CD86 and costimulatory molecules CD40 and major histocompatibility complex class II (MHC-II) are affected by BTV infection, suggesting that BTV interferes with DC antigen-presenting capacity. In vivo , different DC populations are also affected during the course of infection, probably as a result of a direct effect of BTV replication in DCs and the production of infectious virus. These new findings suggest that BTV infection of HSCs and DCs can impair the immune response, leading to persistence or animal death, and that this relies on IFN-I.

Arenaviruses include several important human pathogens, and there are very limited options of preventive or therapeutic interventions to combat these viruses. An off-label use of the purine nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is the only antiviral treatment currently available for arenavirus infections. However, the ribavirin antiviral mechanism action against arenaviruses remains unknown. Here we document that ribavirin is mutagenic for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) in cell culture. The mutagenic activity of ribavirin on LCMV was observed under singleand multiple-passage regimes and could not be accounted for by a decrease of the intracellular GTP pool promoted by ribavirin-mediated inhibition of inosine monophosphate dehydrogenase (IMPDH). Our findings suggest that the antiviral activity of ribavirin on arenaviruses might be exerted, at least partially, by lethal mutagenesis. Implications for antiarenavirus therapy are discussed. © 2011, American Society for Microbiology., BFU2008-02816/BMC from the Ministerio de Ciencia e Innovación (MICINN) and Fundación R. Areces. The Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) is funded by Instituto de Salud Carlos III

Although many chemokine genes have been identified in rainbow trout (Oncorhynchus mykiss) as in other teleost species, almost no studies focused on their biological role have been conducted, despite the fact that no clear inferences as to their functions can be made based on their low similarity to mammalian counterparts. In the current work, we have studied the regulation of mRNA transcription and protein expression of CK12, a rainbow trout CC chemokine previously catalogued within the CCL19/21/25 phylogenetic group. Our studies revealed that CK12 is strongly expressed both at mRNA and protein level in mucosal tissues. Mature lymphocyte populations also express CK12 both at mRNA and protein levels. Concerning its biological activity, a significant chemotatic activity towards purified recombinant CK12 in unfractionated leukocyte populations was observed in the spleen, but not in head kidney or blood. Consequently, a binding assay revealed that the number of leukocytes capable of binding CK12 was much more elevated in spleen populations than in leukocyte populations from other organs. This binding capacity was only observed in small lymphocytes that should account for resident inactivated lymphocytes, in contrast to mature lymphocytes that were responsible for CK12 production. Around 36% of these small lymphocytes were IgM+ cells, of which 40% had a CK12 binding capacity. On the other hand, 10% of thymocytes were also capable of CK12 binding, suggesting that both T and B immature lymphocytes are recruited by CK12. This work constitutes the first description of a mucosal-associated chemokine in fish in which important aspects of its regulation and functionality are revealed. © 2011 Elsevier Ltd.

Bluetongue virus (BTV), an economically important orbivirus of the Reoviridae family, is a non-enveloped, dsRNA virus that causes a haemorrhagic disease mainly in sheep, but little is known of the cellular immunity elicited against BTV. We observed that vaccination of interferon type I-deficient mice (IFNAR (-/-)), or inoculation of the wild type C57BL/6 strain with BTV-8, induced a strong T cell response. Therefore, we proceeded to identify some of the T cell epitopes targeted by the immune system. We selected, using H-2 b-binding predictive algorithms, 3 major histocompatibility complex (MHC)-class II-binding peptides and 7 MHC-class I binding peptides from the BTV-8 core protein VP7, as potential T cell epitopes. Peptide binding assays confirmed that all 7 MHC-class I predicted peptides bound MHC-class I molecules. Three MHC-class I and 2 MHC-class II binding peptide consistently elicited peptide-specific IFN-γ production (as measured by ELISPOT assays) in splenocytes from C57BL/6 BTV-8-inoculated mice and IFNAR (-/-)-vaccinated mice. The functionality of these T cells was confirmed by proliferation and cytotoxicity assays. Flow cytometry analysis demonstrated that CD8 + T cells responded to MHC-class I binding peptides and CD4 + T cells to MHC-class II binding peptides. Importantly, these 5 epitopes were also able to induced IFN-γ production in sheep inoculated with BTV-8. Taken together, these data demonstrate the activation of BTV-specific T cells during infection and vaccination. The characterisation of these novel T cell epitopes may also provide an opportunity to develop DIVA-compliant vaccination approach to BTV encompassing a broad-spectrum of serotypes. © 2011 Elsevier Ltd.

Background: New vaccine designs are needed to control diseases associated with antigenically variable RNA viruses. Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that inflicts severe economic losses. Although the current whole-virus chemically inactivated vaccine has proven effective, it has led to new outbreaks of FMD because of incomplete inactivation of the virus or the escape of infectious virus from vaccine production premises. We have previously shown that serial passages of FMD virus (FMDV) C-S8c1 at high multiplicity of infection in cell culture resulted in virus populations consisting of defective genomes that are infectious by complementation (termed C-S8p260). Principal Finding: Here we evaluate the immunogenicity of C-S8p260, first in a mouse model system to establish a proof of principle, and second, in swine, the natural host of FMDV C-S8c1. Mice were completely protected against a lethal challenge with FMDV C-S8c1, after vaccination with a single dose of C-S8p260. Pigs immunized with different C-S8p260 doses and challenged with FMDV C-S8c1 either did not develop any clinical signs or showed delayed and mild disease symptoms. C-S8p260 induced high titers of both FMDV-specific, neutralizing antibodies and activated FMDV-specific T cells in swine, that correlated with solid protection against FMDV. Conclusions: The defective virus-based vaccine did not produce detectable levels of transmissible FMDV. Therefore, a segmented, replication-competent form of a virus, such as FMDV C-S8p260, can provide the basis of a new generation of attenuated antiviral vaccines with two safety barriers. The design can be extended to any viral pathogen that encodes trans-acting gene products, allowing complementation between replication-competent, defective forms. © 2010 Rodríguez-Calvo et al., Ministerio de Ciencia e Innovación, Spain, and European Union, Network of Excellence, EPIZONE (Contract # FOOD-CT-2006-016236). CIBERehd (Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Work at Centro de Biología Molecular ‘‘Severo Ochoa’’ (CISC-UAM) was supported by an institutional grant from Fundación Ramón Areces

The Arenaviridae family currently comprises 22 viral species, each of them associated with a rodent species. This viral family is important both as tractable experimental model systems to study acute and persistent infections and as clinically important human pathogens. Arenaviruses are enveloped viruses with a bi-segmented negative-strand RNA genome. The interaction with the cellular receptor and subsequent entry into the host cell differs between Old World and New World arenavirus that use α-dystoglycan or human transferring receptor 1, respectively, as main receptors. The recent development of reverse genetic systems for several arenaviruses has facilitated progress in understanding the molecular biology and cell biology of this viral family, as well as opening new approaches for the development of novel strategies to combat human pathogenic arenaviruses. On the other hand, increased availability of genetic data has allowed more detailed studies on the phylogeny and evolution of arenaviruses. As with other riboviruses, arenaviruses exist as viral quasispecies, which allow virus adaptation to rapidly changing environments. The large number of different arenavirus host reservoirs and great genetic diversity among virus species provide the bases for the emergence of new arenaviruses potentially pathogenic for humans. © 2009 Elsevier B.V. All rights reserved.

Bluetongue (BT) is a noncontagious, insect-transmitted disease of ruminants caused by the bluetongue virus (BTV). A laboratory animal model would greatly facilitate the studies of pathogenesis, immune response and vaccination against BTV. Herein, we show that adult mice deficient in type I IFN receptor (IFNAR(-/-)) are highly susceptible to BTV-4 and BTV-8 infection when the virus is administered intravenously. Disease was characterized by ocular discharges and apathy, starting at 48 hours post-infection and quickly leading to animal death within 60 hours of inoculation. Infectious virus was recovered from the spleen, lung, thymus, and lymph nodes indicating a systemic infection. In addition, a lymphoid depletion in spleen, and severe pneumonia were observed in the infected mice. Furthermore, IFNAR(-/-) adult mice immunized with a BTV-4 inactivated vaccine showed the induction of neutralizing antibodies against BTV-4 and complete protection against challenge with a lethal dose of this virus. The data indicate that this mouse model may facilitate the study of BTV pathogenesis, and the development of new effective vaccines for BTV. © 2009 Calvo-Pinilla et al.

Recent recombinant DNA technology has provided novel approaches to develop marker and safe vaccines against bluetongue virus (BTV). To develop new vaccination strategies against BTV infection we have engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA) expressing VP2, VP5 and VP7 proteins from BTV-4. IFNAR(-/-) mice inoculated with DNA/rMVA-VP2, -VP5, -VP7 in an heterologous prime boost vaccination strategy generated significant levels of neutralizing antibodies against BTV-4 and they were completely protected against BTV-4 challenge. Interestingly, VP2 and VP7 proteins expressed in the DNA/rMVA vaccines induced a specific BTV T-cell response that might contribute to the protection of IFNAR(-/-) mice against challenge with BTV-4. In addition, antibodies against VP2, VP5, and VP7, but not NS3 were detected in the sera of DNA/rMVA-VP2, -VP5, -VP7 immunized mice confirming the DIVA (differentiating infected from vaccinated animals) properties of this vaccine. Overall, our results show that the heterologous prime boost vaccination with DNA/rMVA expressing VP2, VP5, and VP7 proteins protects against BTV-4 infection. © 2009 Elsevier Ltd. All rights reserved.

Foot-and-mouth disease virus (FMDV) is one of the most contagious animal viruses, causing a devastating disease in clovenhoofed animals with enormous economic consequences. Identification of the different parameters involved in the immune response elicited against FMDV remains unclear, and it is fundamental the understanding of such parameters before effective control measures can be put in place. In the present study, we show that interleukin-10 (IL-10) production by dendritic cells (DCs) is drastically increased during acute infection with FMDV in swine. In vitro blockade of IL-10 with a neutralizing antibody against porcine IL-10 restores T cell activation by DCs. Additionally, we describe that FMDV infects DC precursors and interferes with DC maturation and antigen presentation capacity. Thus, we propose a new mechanism of virus immunity in which a non-persistent virus, FMDV, induces immunosuppression by an increment in the production of IL- 10, which in turn, reduces T cell function. This reduction of T cell activity may result in a more potent induction of neutralizing antibody responses, clearing the viral infection., This research was supported by grant AGL2004-0049, AGL2007-61374 and CSD2006-07 from Ministerio de Educacion y Ciencia, Spain and EU, Netwok of Excellence, EPIZONE (Contract # FOOD-CT-2006-016236). F.D.-S. was a fellowship of INIA. T.R. was supported by a contract from Comunidad Auto´noma de Madrid. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

The characterization of virulence determinants of pathogenic agents is of utmost relevance for the design of disease control strategies. So far, two classes of virulence determinants have been characterized for viral populations: those imprinted in the nucleotide sequence of some specific genomic regions and those that depend on the complexity of the viral population as such. Here we provide evidence of a virulence determinant that depends neither on a genomic sequence nor on detectable differences in population complexity. Foot-and-mouth disease virus is lethal for C57BL/6 mice showing the highest viral load in pancreas. Virus isolated from pancreas after one passage in mice showed an attenuated phenotype, with no lethality even at the highest dose tested. By contrast, virus from sera of the same mice displayed a virulence similar to that of the parental wild-type clone and virus isolated from spleen displayed an intermediate phenotype. However, viral populations from pancreas, spleen, and serum showed indistinguishable consensus genomic nucleotide sequences and mutant spectrum complexities, as quantified according to the mutation frequencies of both entire genomic nucleotide sequences of biological clones. The results show that the populations with differing virulences cannot be distinguished either by the consensus sequence or by the average complexity of the mutant spectrum. Differential harvesting of virus generated by cell transfection of RNA from serum and pancreas failed to reveal genetic differences between subpopulations endowed with differing virulences. In addition to providing evidence of hidden virulence determinants, this study underlines the capacity of a clone of an RNA virus to rapidly diversify phenotypically in vivo. Copyright © 2008, American Society for Microbiology. All Rights Reserved., This research was supported by grants AGL2004-0049 and BFU2005-00863 from Ministerio de Educación y Ciencia, Spain; by the EU, Network of Excellence, EPIZONE (contract no. FOOD-CT-2006-016236); and by Fundación Ramón Areces. CIBERehd is funded by the Instituto de Salud Carlos III. M.S.-R. was supported by an FPU fellowship from Ministerio de Educación y Ciencia. F.D.-S.S. was supported by a fellowship from INIA (Spain).

In the current work, we have demonstrated that both rainbow trout (Oncorhynchus mykiss) interleukin 8 (IL-8), a CXC chemokine, and CK-6, a CC chemokine, are able of efficiently attracting RTS11, a rainbow trout established macrophage-monocyte-like cell line. Interestingly, two sub-populations of non-adherent cells are distinguishable by flow cytometry that could be identified as immature monocyte- and mature macrophage-like populations, respectively, and the two chemokines studied exert their effects on different populations. Although IL-8 specifically attracts the monocyte-like sub-population, CK-6 specifically attracts the macrophage-like cell sub-population. We have also determined the effects of both of these chemokines on RTS11 phagocytosis, respiratory burst and the expression of other immune-related genes. We found that IL-8 inhibited the phagocytosis capacity of RTS11 cells belonging to the macrophage-like profile. No effect was observed, however, on the respiratory burst, immune function that has been considerably affected throughout the establishment of the cell culture. Concerning the effect that IL-8 and CK-6 have on the expression of other immune genes, we found that IL-8 significantly induced the levels of expression of CK-6, IL-8, pro-inflammatory cytokines such as IL-1β and tumour necrosis factor α (TNF-α) of RTS11 cells. On the other hand, CK-6 induced the levels of expression of IL-8, iNOS and the integrin CD-18, while it had very faint effect on pro-inflammatory cytokines. These results constitute one of the very few studies in which the effect of IL-8, a CXC chemokine, on monocyte-like cells is described. Moreover, it demonstrates that different monocyte-macrophage sub-populations have different reactivity to different chemokines. © 2008 Elsevier Ltd. All rights reserved.

Transmissible spongiform encephalopaties (TSEs) or prion diseases develop as central nervous system (CNS) disorders characterized by extremely long incubation periods. Although TSEs do not go along with inflammatory infiltrates and/or antibody production against the prion protein (PrPSc), the immune system plays an important role in pathogenesis as long as different lymphoid organs (Peyer's patches, lymph nodes and spleen) may facilitate the accumulation and further spread of prions after peripheral exposure. In this work we investigated the changes in lymphoid and dendritic cell (DC) populations as well as the implications of different cytokines during disease progression after experimental oral inoculation of prions in a transgenic mouse model. At different days post-inoculation (dpi), T and B lymphocytes and DC populations from lymphoid organs, blood and brain were analyzed by flow cytometry and immunohistochemistry. Besides time related variations in lymphoid cell numbers due to the aging of the animals significant changes related with the infection were found in mesenteric lymph nodes, peripheral blood leukocytes (PBLs) as well as in spleen, affecting the CD4/CD8 ratio. In contrast, little or no variation was detected in Peyer's Patches or in thymus either associated with aging or the infection status. At individual time points significant differences between infected and control mice were seen in the CD8, CD4 and DC populations, with less evidence of differences in the B cell compartment. Finally, a pro-inflammatory phenotype occurred at early times in the spleen, where the levels of lymphotoxin-β mRNA were found augmented with respect to controls. Altogether, these results suggest that normal regulation of lymphocyte populations becomes altered along the progression of a prion infection. © 2007 Elsevier B.V. All rights reserved.

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious vesicular disease of cloven-hoofed animals. In the present study we use FMDV serotype C infection of swine to determine, by analytical techniques, the direct ex vivo visualization of virus-infected immune cells during the first 17 days of infection. We report, for the first time, that FMDV C-S8c1 can infect T and B cells at short periods of time postinoculation, corresponding with the peak of the viremia. There is a significant lymphopenia that involves CD3 + CD4 − CD8 +/− , CD3 + CD4 − CD8 + Tc, and CD3 + CD4 + CD8 + memory Th but not CD3 + CD4 + CD8 − naïve Th lymphocytes. In addition, a profound depletion of the vast majority of peripheral T cells in lymph nodes and spleen is observed. This selective depletion of T cells is not due mainly to in situ death via apoptosis as visualized by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique. Thus, early infection of T cells by FMDV may be the main cause of the observed T-cell depletion. Importantly, this lack of T cells is reflected in a reduced response to mitogen activation, which in many cases is totally eliminated. These data suggest a mechanism by which the virus causes a transient immunosuppression, subvert the immune systems, and spreads. These results have important implications for our understanding of early events in the development of a robust immune response against FMDV.

Objectives: Here we address the capacity of bone marrow-derived cells (BMDCs) to trans-differentiate into mature myocytes under the physiological stimulus of exercise training. Methods: For this purpose, we have transplanted bone marrow from mice ubiquitously expressing enhanced green fluorescence protein (eGFP) into host mice that have been subjected to a prolonged program of exercise. Results: In all successful bone marrow reconstitutions (greater than 80%), we observed rare but consistent events of bone marrow-derived cardiomyocytes, the frequency of which was unchanged upon exercise training. We have further determined whether these recruited myocytes are a product of trans-differentiation or fusion by the use of a genetic system that distinguishes cell fusion from trans-differentiation in a single-cell assay. Conclusions: We concluded that both in the unchallenged mouse and in the trained specimens, fusion is the most prominent mechanism by which bone marrow-derived cells are observed in the myocyte compartment.

Foot-and-mouth disease virus (FMDV) is a picornavirus that causes an acute vesicular disease of cloven-hoofed animals. This virus continues to be threat to livestock worldwide with outbreaks causing severe economic losses. However, very little is known about FMDV pathogenesis, partially due to the inconveniences of working with cattle and swine, the main natural hosts of the virus. Here we demonstrate that C57BL/6 and BALB/C adult mice are highly susceptible to FMDV infection when the virus is administered subcutaneously or intraperitoneally. The first clinical signs are ruffled fur, apathy, humped posture, and wasting, which are followed by neurological signs such as hind-limb paralysis. Within 2-3 days of disease onset, the animals die. Virus is found in all major organs, indicating a systemic infection. Mice developed microvesicles near the basal layer of the epithelium, event that precedes the vesiculation characteristics of FMD. In addition, a lymphoid depletion in spleen and thymus and severe lymphopenia is observed in the infected mice. When these mice were immunized with conventional inactivated FMDV vaccine, they were protected (100% of vaccinated animals) against challenge with a lethal dose of FMDV. The data indicate that this mouse model may facilitate the study of FMDV pathogenesis, and the development of new effective vaccines for FMD. © 2004 Elsevier Inc. All rights reserved.

International audience; RNA viruses evolve as complex distributions of mutants termed viral quasispecies. For this reason it is relevant to explore those environmental parameters that favour the selective advantage of some viral subpopulations over others. In the present study we provide direct evidence that the relative fitness of two competing viral subpopulations may depend on the multiplicity of infection (m.o.i.). Two closely related subpopulations of foot-and-mouth disease virus (FMDV) of serotype C, which differed in their history of cytolytic passages in BHK-21 cells, were subjected to growth-competition experiments in BHK-21 cells. One of the populations, termed S, was found to have a selective advantage over the other population, termed L, only when the competition passages were carried out at low m.o.i. In contrast, both populations, L and S, coexisted during serial passages carried out at high m.o.i. No differences between S and L were detected in assays of inhibition of infectivity by synthetic peptides, in cell binding-competition experiments, or in virulence for BHK-21 cells. However, FMDV S displayed increased heparin binding compared with L, and L higher virulence for Chinese hamster ovary (CHO) cells than S. These results with FMDV suggest that small differences in the interaction of the virus with the host cell may contribute to an m.o.i.-dependent selective advantage of one viral subpopulation over a closely related subpopulation. Therefore, different viral mutants from quasispecies replicating in vivo may be selected depending on the number of variant viruses relative to the number of susceptible cells.

International audience; The function of a loop exposed on the aphthovirus capsid (the G-H loop of protein VP1) has been explored by combining genetic and structural studies with viral mutants. The loop displays a dual function of receptor recognition and interaction with neutralizing antibodies. Remarkably, some amino acid residues play a critical role in both such disparate functions. Therefore residues subjected to antibody pressure for variation may nevertheless maintain a role in receptor recognition for which invariance is a requirement. Evolution of FMDV in cell culture may relax the requirements at this site and allow further increase of antigenic diversification. Essential residues at one stage of virus evolution may become dispensable at another not very distant point in the evolutionary landscape. Implications for FMDV evolution and vaccine design are discussed.

International audience; The antigenic properties and genetic stability of a multiply passaged foot-and-mouth disease virus (FMDV) clone C-S8c1 with an Arg-Gly-Gly triplet (RGG) instead of the Arg-Gly-Asp (RGD) integrin-recognition motif at positions 141 to 143 of capsid protein VP1 are described. Clear antigenic differences between FMDV RGG and clone C-S8c1 have been documented in ELISA, enzyme-linked immunoelectrotransfer (Western) blot and neutralization assays using site A-specific monoclonal antibodies and anti-FMDV polyclonal antibodies from swine and guinea pigs. The results validate with a live virus the role of the RGD (in particular Asp-143) in recognition of (and neutralization by) antibodies, a role previously suggested by immunochemical and structural studies with synthetic peptides. The FMDV RGG was genetically stable in a large proportion of serial infections of BHK-21 cells. However, a revertant virus with RGD was generated in one out of six passage series. Interestingly, this revertant FMDV did not reach dominance but established an equilibrium with its parental FMDV RGG, accompanied by an increase of quasispecies complexity at the sequences around the RGG triplet. FMDV RGG exhibited a selective disadvantage relative to other RGD-containing clones isolated from the same parental FMDV population. The results suggest that large antigenic variations can be prompted by replacements at critical capsid sites, including those involved in receptor recognition. These critical replacements may yield viruses whose stability allows them to replicate efficiently and to expand the sequence repertoire of an antigenic site.

The three-dimensional structures of the Fab fragment of a neutralizing antibody raised against a foot-and-mouth disease virus (FMDV) of serotype C1, alone and complexed to an antigenic peptide representing the major antigenic site A (G-H loop of VP1), have been determined. As previously seen in a complex of the same antigen with another antibody which recognizes a different epitope within antigenic site A, the receptor recognition motif Arg-Gly-Asp and some residues from an adjacent helix participate directly in the interaction with the complementarity-determining regions of the antibody. Remarkably, the structures of the two antibodies become more similar upon binding the peptide, and both undergo considerable induced fit to accommodate the peptide with a similar array of interactions. Furthermore, the pattern of reactivities of five additional antibodies with versions of the antigenic peptide bearing amino acid replacements suggests a similar pattern of interaction of antibodies raised against widely different antigens of serotype C. The results reinforce the occurrence of a defined antigenic structure at this mobile, exposed antigenic site and imply that intratypic antigenic variation of FMDV of serotype C is due to subtle structural differences that affect antibody recognition while preserving a functional structure for the receptor binding site., Work at Centre de Investigació i Desenvolupament in Barcelona was supported by grants PB92-0707 and PB 95-0218 from DGICYT, that in Madrid was supported by grant PB 94-0034-C02-01 from DGICYT and Fundación Ramón Areces, and that at Universitat de Barcelona was supported by grants PB94-0845 and PB95-1131 from DGICYT. The Barcelona groups are part of CERBA from Generalitat de Catalunya

Foot-and-mouth disease virus (FMDV) shows a dual potential to be cytolytic or to establish persistent infections in cell culture. FMDV R100, a virus rescued after 100 passages of carrier BHK-21 cells persistently infected with FMDV clone C-S8c1, showed multiple genetic and phenotypic alterations relative to the parental clone C-S8c1. Several FMDV R100 populations have been subjected to 100 serial cytolytic infections in BHK-21 cells, and the reversion of phenotypic and genetic alterations has been analyzed. An extreme temperature sensitivity of R100 reverted totally or partially in some passage series but not in others. The small-plaque morphology reverted to normal size in all cases. The hypervirulence for BHK-21 cells did not revert, and even showed an increase, upon cytolytic passage. Most of the mutations that had been fixed in the R100 genome during persistence did not revert in the course of cytolytic passages, but the extended polyribocytidylate tract of R100 (about 460 residues, versus 290 in C-S8c1) decreased dramatically in length, to the range of 220 to 260 residues in all passage series examined. In passages involving very large viral populations, a variant with two amino acid substitutions (L-144-->V and A-145-->P) next to the highly conserved Arg-Gly-Asp (RGD motif; positions 141 to 143) within the G-H loop of capsid protein VP1 became dominant. A clonal analysis allowed isolation of a mutant with the single replacement A-145-->P. Viral production and growth competition experiments showed the two variants to have a fitness very close to that of the parental virus. The results provide evidence that the repertoire of variants that could potentially become dominant in viral quasispecies may be influenced by the population size of the evolving virus. The net results of a series of persistent-infection passages followed by a series of cytolytic passages was progressive genomic diversification despite reversion or stasis of phenotypic traits. Implications for the evolution of RNA viruses are discussed.

A hallmark of RNA genomes is the error-prone nature of their replication and retro-transcription. The major biochemical basis of the limited replication fidelity is the absence of proofreading/repair and postreplicative error correction mechanisms that normally operate during replication of cellular DNA. In spite of this unique feature of RNA replicons, the dynamics of viral populations seems to follow the same basic principles that classical population genetics has established for higher organisms. Here we review recent evidence of the profound effects that genetic bottlenecks have in enhancing the deleterious effects of Muller's ratchet during RNA virus evolution. The validity of the Red Queen hypothesis and of the competitive exclusion principle for RNA viruses are viewed as the expected result of the highly variable and adaptable nature of viral quasispecies. Viral fitness, or ability to replicate infectious progeny, can vary a million-fold within short time intervals. Paradoxically, functional and structural studies suggest extreme limitations to virus variation. Adaptability of RNA viruses appears to be based on the occupation of very narrow portions of sequence space at any given time., Work in Madrid supported by grants DCICYT PB94-0034-C02-01, FIS 95/0034-1, and Fundación Ramón Areces. Work in Valencia supported by grant DCICYT PB94-0034-C02-02. and in La Jolla by grant AI-14627 from the National Institute of Allergy and Infectious Diseases. N.S. was supported by a felloship from Comunidad Autónoma de Madrid, and S.F.E. and J.Q. by fellowships from Ministerio de Educación y Ciencia (Spain).

Hypervirulent variants of foot-and-mouth disease virus (FMDV) of serotype C arise upon serial cytolytic or persistent infections in cell culture. A specific mutation in the internal ribosome entry site of persistent FMDV was previously associated with enhanced translation initiation activity that could contribute to the hypervirulent phenotype for BHK-21 cells. Here we report that several hypervirulent FMDV variants arising upon serial cytolytic passage show an invariant internal ribosome entry site but have a number of mutations affecting structural and nonstructural viral proteins. The construction of chimeric type O-type C infectious transcripts has allowed the mapping of a major determinant of hypervirulence to the viral capsid. Tissue culture-adapted FMDV displayed enhanced affinity for heparin, but binding to cell surface heparan sulfate moieties was not required for expression of the hypervirulent phenotype in Chinese hamster ovary (CHO) cells. Virulence was identical or even higher for glycosaminoglycan-deficient CHO cells than for wild-type CHO cells. FMDV variants with decreased affinity for heparin were selected from a high-binding parental population and analyzed. Substitutions associated with decreased heparin binding were located at positions 173 of capsid protein VP3 and 144 of capsid protein VP1. These substitutions had a moderate effect on virulence for BHK-21 cells but completely abrogated infection of CHO cells. The comparative results with several FMDV isolates show that (i) increased affinity for heparin and alterations in cell tropism may be mediated by a number of independent sites on the viral capsid and (ii) the same capsid modifications may have different effects on different cell types.

Dendritic cells (DCs) comprise the major antigen-presenting cells (APCs) of the host, uniquely programmed to stimulate immunologically naïve T lymphocytes. Viruses that can target and disorder the function of these cells enjoy a selective advantage. The cellular receptor for lymphocytic choriomeningitis virus (LCMV), Lassa fever virus (LFV), and several other arenaviruses is alpha-dystroglycan (alpha-DG). Among cells of the immune system, CD11c+ and DEC-205+ DCs primarily and preferentially express alpha-DG. By selection, strains and variants of LCMV generated as quasi-species that bind alpha-DG with high affinity replicate in the majority of CD11c+ and DEC-205+ (75%) DCs, causing a generalized immunosuppression, and establish a persistent infection. In contrast, viral strains and variants that bind with low affinity to alpha-DG display minimal replication in CD11c+ and DEC-205+ DCs (10%), rarely replicate in the white pulp, and generate a robust anti-LCMV CTL response that clears the virus infection. Hence, receptor-virus interaction on DCs in vivo is an essential step in the initiation of virus-induced immunosuppression and viral persistence. Investigation into the mechanism of how virus-infected DCs cause immunosuppression reveals loss of MHC class II surface expression and costimulatory molecules on surface of such DCs. As a consequence DCs are unable to act as APCs, initiate immune responses, and have a defect in migration into the T cell area. These data indicate that LCMV infection influences DC maturation and migration, leading to decreased T cell stimulatory capacity of DCs, events essential for the initiation of immune responses. Because several other viruses known to cause immunosuppression (HIV, measles) interact with DCs, the observations noted here are likely a common selective mechanism by which viruses also are able to evade the host's immune system.

Evidence for a mechanism of initiation of viral persistence in which the cell, and not the virus, plays a critical role has been obtained using the important animal pathogen foot-and-mouth disease virus (FMDV). We have developed a virulence assay consisting of quantification of the ability of virus to kill cells and of cells to divide in the presence of virus and to initiate a carrier state. Cells were cured of FMDV at early times following a cytolytic infection of BHK-21 monolayers with FMDV. When cured cells were subjected to the virulence assay they showed an increased ability to survive a second infection by FMDV but not by other RNA viruses. This altered phenotype was maintained as a stable genetic trait. When the virus present in such early surviving cells was used to infect BHK-21 cells, it proved to be as virulent as the initial cytolytic FMDV and, furthermore, its ability to kill BHK-21 cells increased upon replication in the surviving cells. Both the level of genetic heterogeneity and the rate of evolution of FMDV were similar to those previously documented during acute and persistent FMDV infections. The results suggest that, in contrast to most other viral systems, the critical element in the establishment of a persistent infection of BHK-21 cells with FMDV is the ability of the host cells to vary genetically and phenotypically, which promotes selection of cells with increased resistance to virus. The possible relevance of this mechanism to viral persistence in vivo is discussed.

8 páginas, 4 figuras, 1 tabla., Foot-and-mouth disease virus (FMVD), one of the most contagious viruses of cloven-hoofed animals, may cause a prolonged, asymptomatic but persistent infection in ruminants, named the "carrier state". However, it remains an open question whether this carrier state occurs in pigs. Here we present quantitative analyses of the duration of FMDV RNA and infectivity in lymphoid and epithelial tissues in experimentally infected pigs with FMDV C-S8c1. The data indicated that although FMDV RNA remained in blood until day 14 post-infection (pi), viremia was cleared by day 7 pi. However, all tissues tested were positive for FMDV until day 14-17 pi. Interestingly, the specific infectivity of FMDV in these tissues was in some cases even higher than the FMDV C-S8c1. We therefore propose that a "pseudopersistent state" may occur in pigs in which virus replicates in lymphoid tissues for a prolonged period of time, thereby representing a potential source of virus., This research was supported by grant AGL2004-0049 and AGL2007-61374 from Ministerio de Educación y Ciencia, Spain and EU, Netwok of Excellence, EPIZONE (Contract # FOOD-CT-2006-016236). F Diaz-San Segundo was supported by a fellowship of INIA, T Rodriguez-Calvo was supported by a contract from Comunidad Autónoma de Madrid and M Sanz-Ramos was supported by an FPU fellowship from Ministerio de Educación y Ciencia.