Margarita Sáiz

Foot-and-mouth disease virus: biology and prospects for disease control.

Foot-and-mouth disease virus (FMDV) is the causative agent of a disease that constitutes one of the main animal health concerns, as evidenced by the devastating outbreaks that occurred in different areas of the world over the last few years. In this review, we summarise important features of FMDV, aspects of its interactions with cells and hosts as well as current and new strategies for FMD control by vaccination.

Recovery of Infectious Foot-and-Mouth Disease Virus from Suckling Mice after Direct Inoculation with In Vitro-Transcribed RNA

ABSTRACT We assayed the infectivity of naked foot-and-mouth disease virus (FMDV) RNA by direct inoculation of suckling mice. Our results demonstrate that transcripts generated from full-length cDNA clones were infectious, as was virion-extracted RNA. Interestingly, infectious virus could be recovered from a mutant transcript encoding amino acid substitution L-147→P in capsid protein VP1, known to be noninfectious for BHK-21 cells. The model described here provides a useful tool for virulence studies in vivo, bypassing possible selection of variants during viral replication in cell culture.

Attenuated Foot-and-Mouth Disease Virus RNA Carrying a Deletion in the 3′ Noncoding Region Can Elicit Immunity in Swine

ABSTRACT We constructed foot-and-mouth disease virus (FMDV) mutants bearing independent deletions of the two stem-loop structures predicted in the 3′ noncoding region of viral RNA, SL1 and SL2, respectively. Deletion of SL2 was lethal for viral infectivity in cultured cells, while deletion of SL1 resulted in viruses with slower growth kinetics and downregulated replication associated with impaired negative-strand RNA synthesis. With the aim of exploring the potential of an RNA-based vaccine against foot-and-mouth disease using attenuated viral genomes, full-length chimeric O1K/C-S8 RNAs were first inoculated into pigs. Our results show that FMDV viral transcripts could generate infectious virus and induce disease in swine. In contrast, RNAs carrying the ΔSL1 mutation on an FMDV O1K genome were innocuous for pigs but elicited a specific immune response including both humoral and cellular responses. A single inoculation with 500 μg of RNA was able to induce a neutralizing antibody response. This response could be further boosted by a second RNA injection. The presence of the ΔSL1 mutation was confirmed in viruses isolated from serum samples of RNA-inoculated pigs or after transfection and five passages in cell culture. These findings suggest that deletion of SL1 might contribute to FMDV attenuation in swine and support the potential of RNA technology for the design of new FMDV vaccines.

Response to retreatment with Interferon-α plus ribavirin in chronic hepatitis C patients is independent of the NS5A gene nucleotide sequence

OBJECTIVE:Interferon-α plus ribavirin is an effective treatment for chronic hepatitis C patients. We evaluated whether the response to this combined therapy correlated with the presence of mutations in a region of 372 nucleotides within the NS5A gene.METHODS:Sixty-two patients, 42 nonresponders and 20 relapsers to a previous course of interferon-α, received 3 million units thrice weekly of interferon-α-2b and 1–1.2 g daily of ribavirin for 12 months. Basal biochemical and virological (HCV RNA and genotype) parameters were determined. Clinical examinations were carried out at 1, 2, 3, 6, and 12 months. In addition, nucleotide sequencing of the NS5A gene was determined for viral samples obtained from 38 of these patients at the baseline of the combined therapy, as well as in 15 of them before initiating the previous course of interferon as monotherapy.RESULTS:On finishing the 12 months, 36 patients (58.1%) had normal aminotransferases and 25 (40.3%) cleared viremia. Nucleotide sequencing indicated the same level of genetic variability within the group of responder and nonresponder patients all along the 124 amino acid residues of the NS5A gene studied. Neither the type of amino acid substitution nor the number of them was significantly different in one group relative to the other.CONCLUSIONS:Therapy with interferon-α-2b plus ribavirin was well tolerated, achieving an end-of-treatment response in 25 (40.3%) patients. Response did not correlate with the presence of mutations in the NS5A gene analyzed, including the interferon sensitivity determining region (ISDR) and its flanking sequences.

RNA Structural Domains in Noncoding Regions of the Foot-and-Mouth Disease Virus Genome Trigger Innate Immunity in Porcine Cells and Mice

ABSTRACT The induction of type I interferons (alpha/beta interferon [IFN-α/β]) in response to viral infection is a crucial step leading to the antiviral state in the host. Viruses produce double-stranded RNA (dsDNA) during their replication cycle that is sensed as nonself by host cells through different receptors. A signaling cascade then is activated to block viral replication and spread. Foot-and-mouth disease virus (FMDV) is a picornavirus that is highly sensitive to IFN, and it causes one of the worlds most important animal diseases. In this study, we showed the ability of structural domains predicted to enclose stable dsRNA regions in the 5′- and 3′-noncoding regions (NCRs) of the FMDV genome to trigger an IFN-α/β response in porcine kidney cultured cells and newborn mice. These RNAs, generated by in vitro transcription, were able to stimulate IFN-β transcription and induce an antiviral state in SK-6 cells. The induction levels elicited by the different NCR RNAs were compared. Among them, the 3′NCR was identified as a potent IFN activator, and the features in this region involved in signaling have been analyzed. To address whether the FMDV NCR transcripts were able to trigger the innate immune response in vivo, Swiss suckling mice were inoculated intraperitoneally with the RNAs. All transcripts induced the innate response in transfected animals, measured as IFN-α/β protein levels, antiviral activity in sera, and reduced susceptibility to FMDV infection. Our work provides new insight into innate responses against FMDV and identifies these small noninfectious RNA molecules as potential adjuvants for vaccine improvement and antiviral strategies against picornaviruses.

Incidence and characterization of bean common mosaic virus isolates in Spanish bean fields

A 5-yr survey for the presence of bean common mosaic potyvirus (BCMV) in the major bean (Phaseolus vullgaris) growing areas of Spain was conducted, and the viral isolates were characterized biologically and serologically. Both serotype A and B were found. Necrosis-inducing serotype A isolates, found for the first time in Spain, resembled NL-3. Serotype B isolates resembled NL-4 and US-5. The complex antigenic relationships of Spanish BCMV isolates, as determined with specific monoclonal and polyclonal antibodies, and the incidence and distribution of serotypes are discussed

Tolerance to mutations in the foot-and-mouth disease virus integrin-binding RGD region is different in cultured cells and in vivo and depends on the capsid sequence context.

Engineered RNAs carrying substitutions in the integrin receptor-binding Arg-Gly-Asp (RGD) region of foot-and-mouth disease virus (FMDV) were constructed (aa 141-147 of VP1 capsid protein) and their infectivity was assayed in cultured cells and suckling mice. The effect of these changes was studied in the capsid proteins of two FMDVs, C-S8c1, which enters cells through integrins, and 213hs(-), a derivative highly adapted to cell culture whose ability to infect cells using the glycosaminoglycan heparan sulfate (HS) as receptor, acquired by multiple passage on BHK-21 cells, has been abolished. The capsid sequence context determined infectivity in cultured cells and directed the selection of additional replacements in structural proteins. Interestingly, a viral population derived from a C-S8c1/L144A mutant, carrying only three substitutions in the capsid, was able to expand tropism to wild-type (wt) and mutant (mt) glycosaminoglycan-deficient CHO cells. In contrast, the 213hs(-) capsid tolerated all substitutions analysed with no additional mutations, and the viruses recovered maintained the ability of the 213hs(-) parental virus to infect wt and mt CHO cells. Viruses derived from C-S8c1 with atypical RGD regions were virulent and transmissible for mice with no other changes in the capsid. Substitution of Asp143 for Ala in the C-S8c1 capsid eliminated infectivity in cultured cells and mice. Co-inoculation with a neutralizing monoclonal antibody directed against the type C FMDV RGD region abolished infectivity of C-S8c1 virus on suckling mice, suggesting that FMDV can infect mice using integrins. Sequence requirements imposed for viral entry in vitro and in vivo are discussed.

Synthetic RNAs Mimicking Structural Domains in the Foot-and-Mouth Disease Virus Genome Elicit a Broad Innate Immune Response in Porcine Cells Triggered by RIG I and TLR Activation

The innate immune system is the first line of defense against viral infections. Exploiting innate responses for antiviral, therapeutic and vaccine adjuvation strategies is being extensively explored. We have previously described, the ability of small in vitro RNA transcripts, mimicking the sequence and structure of different domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs), to trigger a potent and rapid innate immune response. These synthetic non-infectious molecules have proved to have a broad-range antiviral activity and to enhance the immunogenicity of an FMD inactivated vaccine in mice. Here, we have studied the involvement of pattern-recognition receptors (PRRs) in the ncRNA-induced innate response and analyzed the antiviral and cytokine profiles elicited in swine cultured cells, as well as peripheral blood mononuclear cells (PBMCs).

Delivery of synthetic RNA can enhance the immunogenicity of vaccines against foot-and-mouth disease virus (FMDV) in mice.

We have recently described the antiviral effect in mice of in vitro-transcribed RNAs mimicking structural domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome RNA. These small, synthetic and non-infectious RNA molecules (ncRNAs) are potent type-I interferon (IFN) inducers in vivo. In this work, the immunomodulatory effect of the ncRNA corresponding to the internal ribosome entry site (IRES) on immunization with two different FMD vaccine formulations, both based on inactivated virus, including or not a commercial adjuvant, was analyzed in the mice model. The effect of the time interval between RNA inoculation and immunization was also studied. RNA delivery consistently increased the titers of specific anti-FMDV antibodies, including neutralizing antibodies, elicited after vaccination. Moreover, at day 2 after immunization, significant differences in mean antibody titers could be detected between the groups of mice receiving either vaccine co-administered with the RNA and the control group, unlike those immunized with the vaccine alone. When vaccinated mice were challenged with FMDV, the mean values of viral load were lower in the groups receiving the RNA together with the vaccine. Our results show the enhancing effect of the IRES RNA on the immune response elicited after vaccination and suggest the potential of this molecule as an adjuvant for new FMD vaccine design.

Protection against West Nile Virus Infection in Mice after Inoculation with Type I Interferon-Inducing RNA Transcripts

West Nile virus (WNV) is a neurovirulent single stranded RNA mosquito-borne flavivirus, whose main natural hosts are birds, but it also infects humans and horses. Nowadays, no human vaccine is commercially available and clinical treatment is only supportive. Recently, it has been shown that RNA transcripts, mimicking structural domains in the non-coding regions (NCRs) of the foot-and mouth disease virus (FMDV) induce a potent IFN response and antiviral activity in transfected cultured cells, and also reduced mice susceptibility to FMDV. By using different transcripts combinations, administration schedules, and infecting routes and doses, we have demonstrated that these FMDV RNA transcripts protect suckling and adult mice against lethal challenge with WNV. The protective activity induced by the transcripts was systemic and dependent on the infection route and dose. These results confirm the antiviral potential of these synthetic RNAs for fighting viruses of different families relevant for human and animal health.