Foot-and-mouth disease virus strains isolated in Vietnam during 2010-2019: genetic characterization and antigenic relatedness to the Euro SA vaccine.

Foot-and-mouth disease is a highly contagious disease that affects cloven-hoofed animals. It has an important socio-economic impact on the livestock industry because it produces a drastic decrease of productivity. The disease has been successfully eradicated from some regions, including North America and Western Europe, but it is still endemic in developing countries. Agriculture plays an important role in the national economy of Vietnam, to which animal production contributes a great proportion. The concurrent circulation of foot-and-mouth disease virus (FMDV) serotypes O, A, and Asia 1 has been detected in recent years, but serotype O remains the most prevalent and is responsible for the highest numbers of outbreaks. Appropriate vaccine strain selection is an important element in the control of FMD and is necessary for the application of vaccination programs in FMD-affected regions. Here, we present updated information about the genetic and antigenic characteristics of circulating strains, collected from endemic outbreaks involving types O and A, between 2010 and 2019. Neutralizing assays showed a good in vitro match between type O strains and the monovalent O1 Campos vaccine strain. High r1 values were obtained (above 0.7) when testing a swine serum pool collected 21 days after vaccination, but the O/VTN/2/2019 strain was an exception. An EPP estimation resulted in a median neutralizing titre of about 1.65 log10, indicating that good protection could be achieved. For type A Asia SEA 97 lineage strains, acceptable individual neutralizing titres were obtained with estimated EPP values over 80% for different combinations of vaccine strains. Taking into account that the r1 value is one tool of a battery of tests that should be considered for estimating the cross-protection of a field strain against a vaccine strain, an in vivo challenge experiment was also performed, yielding a PD50 value of 8.0. The results indicate that South American strains could be potentially used for controlling outbreaks involving these lineages. This study demonstrates the importance of considering strain characteristics when choosing vaccine strains and controls.

Broad immunogenic spectrum of monovalent and trivalent foot-and-mouth disease virus vaccines containing O1 campos, A24 cruzeiro and A Argentina 2001 strains against circulating viral lineages in cattle and pigs.

FMD remains endemic in many Asian and African countries where multiple variants of serotypes O and A, among others, currently circulate. Due to lack of cross-protection between serotypes and incomplete protection between some strains even within a serotype, an important challenge for the application of effective vaccination programs is to select highly immunogenic and widely cross-reactive vaccine strains. Adaptation of a candidate field virus for use as a vaccine can be quite complex, so that whenever possible, the use of well-established vaccine viruses could have enormous advantages. FMD vaccine strains harmonized for use in South America have shown excellent results in FMD control, not only in the region, where it is still used systematically as a preventive measure, but also more recently in some Asian countries. To gain further insight into the immunogenic spectrum of these strains, VN tests (VNT) were performed with sera from cattle and/or pigs vaccinated with monovalent (type O) or trivalent (types O and A) formulations against 122 type O and 32 type A field viruses isolated from 35 countries in Asia and Africa, belonging to different lineages. Almost all VNT titers obtained were within the expected protective level, indicating the wide immunogenic spectrum of high potency FMD vaccines formulated with O1 Campos, A24 Cruzeiro and A Argentina 2001 South American vaccine strains belonging to EURO-SA topotypes against currently active viruses from other topotypes. These in vitro results are in line with previously reported in vivo challenge tests in pigs against three A/ASIA/Sea-97 isolates and two isolates belonging to type O lineages O/SEA/Mya-98 and O/ME-SA/Ind-2001e.

Bovine Dendritic Cell Activation, T Cell Proliferation and Antibody Responses to Foot-And-Mouth Disease, Is Similar With Inactivated Virus and Virus Like Particles.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that causes severe economic losses in the livestock industry. Currently available vaccines are based on the inactivated FMD virus (FMDV). Although inactivated vaccines have been effective in controlling the disease, they have some disadvantages. Because of these disadvantages, investigations are being made to produce vaccines in low containment facilities. The use of recombinant empty capsids (also referred as Virus Like Particles, VLPs) has been reported to be a promising candidate as a subunit vaccine because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. Mignaqui and collaborators have produced recombinant FMDV empty capsids from serotype A/ARG/2001 using a scalable technology in mammalian cells that elicited a protective immunity against viral challenge in a mouse model. However, further evaluation of the immune response elicited by these VLPs in cattle is required. In the present work we compare the effect that VLPs or inactivated FMDV has on bovine dendritic cells and the humoral response elicited in cattle after a single vaccination.

Foot-and-Mouth Disease: Optimization, Reproducibility, and Scalability of High-Yield Production of Virus-Like Particles for a Next-Generation Vaccine.

Inactivated Foot-and-Mouth Disease (FMD) vaccine has proven to be effective in the control of the disease. However, its production has some disadvantages, including the costly biosafety facilities required for the production of huge amounts of growing live virus, the need of an exhaustive purification process to eliminate non-structural proteins of the virus in the final formulations in order to differentiate infected from vaccinated animals and variable local regulatory restrictions to produce and commercialize the vaccine. Thus, a novel vaccine against FMD that overcome these restrictions is desirable. Although many developments have been made in this regard, most of them failed in terms of efficacy or when considering their transferability to the industry. We have previously reported the use of transient gene expression in mammalian cells to produce FMD virus-like particles (VLPs) as a novel vaccine for FMD and demonstrated the immunogenicity of the recombinant structures in animal models. Here, we report the optimization of the production system by assaying different DNA:polyethylenimine concentrations, cell densities, and direct and indirect protocols of transfection. Also, we evaluated the reproducibility and scalability of the technology to produce high yields of recombinant VLPs in a cost-effective and scalable system compatible with industrial tech-transfer of an effective and safe vaccine.

Combined administration of synthetic RNA and a conventional vaccine improves immune responses and protection against foot-and-mouth disease virus in swine.

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease and a major concern in animal health worldwide. We have previously reported the use of RNA transcripts mimicking structural domains in the non-coding regions of the FMDV RNA as potent type-I interferon (IFN) inducers showing antiviral effect in vivo, as well as their immunomodulatory properties in combination with an FMD vaccine in mice. Here, we describe the enhancing effect of RNA delivery on the immunogenicity and protection induced by a suboptimal dose of a conventional FMD vaccine in pigs. Animals receiving the RNA developed earlier and higher levels of neutralizing antibodies against homologous and heterologous isolates, compared to those immunized with the vaccine alone, and had higher anti-FMDV titers at late times post-vaccination. RNA delivery also induced higher specific T-cell response and protection levels against FMDV challenge. Peripheral blood mononuclear cells from pigs inoculated with RNA and the vaccine had a higher IFN-γ specific response than those from pigs receiving the vaccine alone. When challenged with FMDV, all three animals immunized with the conventional vaccine developed antibodies to the non-structural viral proteins 3ABC and two of them developed severe signs of disease. In the group receiving the vaccine together with the RNA, two pigs were fully protected while one showed delayed and mild signs of disease. Our results support the immunomodulatory effect of these RNA molecules in natural hosts and suggest their potential use for improvement of FMD vaccines strategies.

GABA induction of the Saccharomyces cerevisiae UGA4 gene depends on the quality of the carbon source: role of the key transcription factors acting in this process.

Yeast cells are able to adapt their metabolism according to the quality of both carbon and nitrogen sources available in the environment. Saccharomyces cerevisiae UGA4 gene encodes a permease capable of transporting γ-aminobutyric acid (GABA) into the cells. Yeast uses this amino acid as a nitrogen source or as a carbon skeleton that enters the tricarboxylic acid cycle. The quality of the carbon source modulates UGA4 expression through two parallel pathways, each one acting on different regulatory elements, the UAS(GATA) and the UAS(GABA). In the presence of a fermentable carbon source, UGA4 expression is induced by GABA while in the presence of a non-fermentable carbon source this expression is GABA-independent. The aim of this work was to study the mechanisms responsible for the differences in the profiles of UGA4 expression in both growth conditions. We found that although the subcellular localization of Gln3 depends on the carbon source and UGA4 expression depends on Tor1 and Snf1, Gln3 localization does not depend on these kinases. We also found that the phosphorylation of Gln3 is mediated by two systems activated by a non-fermentable carbon source, involving the Snf1 kinase and an unidentified TORC1-regulated kinase. We also found that the activity of the main transcription factors responsible for UGA4 induction by GABA varies depending on the quality of the carbon source. In a fermentable carbon source such as glucose, the negative GATA factor Dal80 binds to UGA4 promoter; only after the addition of the inducer, the positive factors Uga3, Dal81 and Gln3 interact with the promoter removing Dal80 and leading to gene induction. In contrast, in the non-fermentable carbon source acetate the negative GATA factor remains bound to UGA4 promoter in the presence or absence of GABA, the positive factors are not detected bound in any of these conditions and in consequence, UGA4 is not induced.

Interplay between the transcription factors acting on the GATA- and GABA-responsive elements of Saccharomyces cerevisiae UGA promoters.

γ-Aminobutyric acid (GABA) transport and catabolism in Saccharomyces cerevisiae are subject to a complex transcriptional control that depends on the nutritional status of the cells. The expression of the genes that form the UGA regulon is inducible by GABA and sensitive to nitrogen catabolite repression (NCR). GABA induction of these genes is mediated by Uga3 and Dal81 transcription factors, whereas GATA factors are responsible for NCR. Here, we show how members of the UGA regulon share the activation mechanism. Our results show that both Uga3 and Dal81 interact with UGA genes in a GABA-dependent manner, and that they depend on each other for the interaction with their target promoters and the transcriptional activation. The typical DNA-binding domain Zn(II)(2)-Cys(6) of Dal81 is unnecessary for its activity and Uga3 acts as a bridge between Dal81 and DNA. Both the trans-activation activity of the GATA factor Gln3 and the repressive activity of the GATA factor Dal80 are exerted by their interaction with UGA promoters in response to GABA, indicating that Uga3, Dal81, Gln3 and Dal80 all act in concert to induce the expression of UGA genes. So, an interplay between the factors responsible for GABA induction and those responsible for NCR in the regulation of the UGA genes is proposed here.

Common features and differences in the expression of the three genes forming the UGA regulon in Saccharomyces cerevisiae.

The three genes that form the UGA regulon in Saccharomyces cerevisiae are responsible for the transport and degradation of γ-aminobutyric acid (GABA) in this organism. Despite the differences in the sequence of their promoters, these genes similarly respond to GABA stimuli. The expression of UGA1, UGA2 and UGA4 depends on GABA induction and nitrogen catabolite repression (NCR). The induction of these genes requires the action of at least two positive proteins, the specific Uga3 and the pleiotropic Uga35/Dal81 transcription factors. Here we show that all the members of the UGA regulon, as was already demonstrated for UGA4, are negatively regulated by extracellular amino acids through the SPS amino acid sensor. We also show that this negative effect is caused by a low availability of Uga35/Dal81 transcription factor and that Leu3 transcription factor negatively regulates UGA4 and UGA1 expression but it does not affect UGA2 expression.

Uga3 and Uga35/Dal81 transcription factors regulate UGA4 transcription in response to gamma-aminobutyric acid and leucine.

The Saccharomyces cerevisiae UGA4 gene encodes a permease capable of importing gamma-aminobutyric acid (GABA) and delta-aminolevulinic acid (ALA) into the cell. GABA-dependent induction of this permease requires at least two positive-acting proteins, the specific factor Uga3 and the pleiotropic factor Uga35/Dal81. UGA4 is subjected to a very complex regulation, and its induction is affected by the presence of extracellular amino acids; this effect is mediated by the plasma membrane amino acid sensor SPS. Our results show that leucine affects UGA4 induction and that the SPS sensor and the downstream effectors Stp1 and Stp2 participate in this regulation. Moreover, we found that the Uga3 and Uga35/Dal81 transcription factors bind to the UGA4 promoter in a GABA-dependent manner and that this binding is impaired by the presence of leucine. We also found that the Leu3 transcription factor negatively regulates UGA4 transcription, although this seems to be through an indirect mechanism.

New insights into the regulation of the Saccharomyces cerevisiae UGA4 gene: two parallel pathways participate in carbon-regulated transcription.

The Saccharomyces cerevisiae UGA4 gene, which encodes the gamma-aminobutyric acid (GABA) and delta-aminolaevulinic acid (ALA) permease, is well known to be regulated by the nitrogen source. Its expression levels are low in the presence of a rich nitrogen source but are higher when a poor nitrogen source is used. In addition, GABA can induce UGA4 expression when cells are grown with proline but not when they are grown with ammonium. Although vast amounts of evidence have been gathered about UGA4 regulation by nitrogen, little is known about its regulation by the carbon source. Using glucose and acetate as rich and poor carbon source respectively, this work aimed to shed light on hitherto unclear aspects of the regulation of this gene. In poor nitrogen conditions, cells grown with acetate were found to have higher UGA4 basal expression levels than those grown with glucose, and did not show UGA4 induction in response to GABA. Analysis of the expression and subcellular localization of the transcription factors that regulate UGA4 as well as partial deletions and site-directed mutations of the UGA4 promoter region suggested that there are two parallel pathways that act in regulating this gene by the carbon source. Furthermore, the results demonstrate the existence of a new factor operating in UGA4 regulation.