Computational insights into RNAi-based therapeutics for foot and mouth disease of Bos taurus.

Foot-and-mouth disease (FMD) endangers a large number of livestock populations across the globe being a highly contagious viral infection in wild and domestic cloven-hoofed animals. It adversely affects the socioeconomic status of millions of households. Vaccination has been used to protect animals against FMD virus (FMDV) to some extent but the effectiveness of available vaccines has been decreased due to high genetic variability in the FMDV genome. Another key aspect that the current vaccines are not favored is they do not provide the ability to differentiate between infected and vaccinated animals. Thus, RNA interference (RNAi) being a potential strategy to control virus replication, has opened up a new avenue for controlling the viral transmission. Hence, an attempt has been made here to establish the role of RNAi in therapeutic developments for FMD by computationally identifying (i) microRNA (miRNA) targets in FMDV using target prediction algorithms, (ii) targetable genomic regions in FMDV based on their dissimilarity with the host genome and, (iii) plausible anti-FMDV miRNA-like simulated nucleotide sequences (SNSs). The results revealed 12 mature host miRNAs that have 284 targets in 98 distinct FMDV genomic sequences. Wet-lab validation for anti-FMDV properties of 8 host miRNAs was carried out and all were observed to confer variable magnitude of antiviral effect. In addition, 14 miRBase miRNAs were found with better target accessibility in FMDV than that of Bos taurus. Further, 8 putative targetable regions having sense strand properties of siRNAs were identified on FMDV genes that are highly dissimilar with the host genome. A total of 16 SNSs having > 90% identity with mature miRNAs were also identified that have targets in FMDV genes. The information generated from this study is populated at http://bioinformatics.iasri.res.in/fmdisc/ to cater the needs of biologists, veterinarians and animal scientists working on FMD.

The role of viral particle integrity in the serological assessment of foot-and-mouth disease virus vaccine-induced immunity in swine.

The efficacy of foot-and-mouth disease virus (FMDV) inactivated vaccines is mainly dependent on the integrity of the whole (146S) viral particles. If the intact capsids disassemble to 12S subunits, antibodies against internal-not protective epitopes, may be induced. Serological correlates with protection may be hampered if antibodies against internal epitopes are measured. Here we compared the performance of different ELISAs with the virus-neutralization test (VNT) that measures antibodies against exposed epitopes. Sera from pigs immunized with one dose of an expired commercial FMDV vaccine were used. This vaccine contained about 50% of O1/Campos and over 90% of A24/Cruzeiro strains total antigen as whole 146S particles. Specific-total antibodies were measured with the standard liquid-phase blocking ELISA (LPBE). We also developed an indirect ELISA (IE) using sucrose gradient purified 146S particles as capture antigen to titrate total antibodies, IgM, IgG1 and IgG2. A good correlation was found between VNT titers and IgG-ELISAs for A24/Cruzeiro, with the lowest correlation coefficient estimated for IgG2 titers. For O1/Campos, however, the presence of antibodies against epitopes different from those of the whole capsid, elicited by the presence of 12S particles in the vaccine, hampered the correlation between LPBE and VNT, which was improved by using purified O1/Campos 146S-particles for the liquid-phase of the LPBE. Interestingly, 146S particles but not 12S were efficiently bound to the ELISA plates, confirming the efficiency of the IE to detect antibodies against exposed epitopes. Our results indicate that any serological test assessing total antibodies or IgG1 against epitopes exposed in intact 146S-particles correlate with the levels of serum neutralizing antibodies in vaccinated pigs, and might potentially replace the VNT, upon validation. We recommend that antigen used for serological assays aimed to measure protective antibodies against FMDV should be controlled to ensure the preservation of 146S viral particles.

Knowledge, attitudes, and practices (KAP) related to brucellosis and factors affecting knowledge sharing on animal diseases: a cross-sectional survey in the dry zone of Sri Lanka.

Farmers' lack of knowledge is assumed to have affected the presence of brucellosis in Sri Lanka for decades. This study, carried out in the Ampara district in the dry zone of Sri Lanka, revealed that there is a significant knowledge gap for brucellosis compared to foot and mouth disease (FMD) (p < 0.001). Only 8.3% of farmers knew that brucellosis causes cattle abortions. Only 2.6% knew that it is zoonotic. The difference in knowledge of the symptoms and transmission of brucellosis and FMD was significant (p < 0.001). Farmers' attitudes and practices related to the spread of the disease were poor. Farmers' education and spoken language had a negative influence on knowledge. Young people and those with strong social relationships were efficient in knowledge sharing. It can be concluded that brucellosis knowledge, attitudes, and practices are poor; thus, there is a need for more attention in disease control policymaking. Backward farmer groups should be the focus in animal health extension programs.

Global Foot-and-Mouth Disease Research Update and Gap Analysis: 1 - Overview of Global Status and Research Needs.

The Global Foot-and-mouth disease (FMD) Research Alliance periodically reviews the state of FMD research to assess progress and to identify new priorities. In this supplement we provide an update of global FMD research, comprising (i) this overview paper, which includes background information with key findings, and papers covering (ii) epidemiology, wildlife and economics, (iii) vaccines, (iv) diagnostics, (v) biotherapeutics and disinfectants, (vi) immunology and (vii) pathogenesis and molecular biology. FMD research publications were reviewed (2011-2015) and activity updates were obtained from 33 FMD research institutes from around the world. Although a continual threat, FMD has been effectively controlled in much of the world using existing tools. However, control remains a challenge in most developing countries, where little has been done to understand the ongoing burden of FMD. More research is needed to support control in endemically infected countries, particularly robust field studies. Traditional FMD vaccines have several limitations including short duration and spectrum of protection, cold chain requirements, and the costs and biosecurity risks associated with vaccine production. Significant progress has been made in the development of novel vaccine candidates, particularly in the use of recombinant vaccines and virus-like particles as an alternative to traditional inactivated whole virus vaccines. Continued investment is needed to turn these developments into improved vaccines produced at scale. Increased knowledge of cellular and mucosal immunity would benefit vaccine development, as would further advances in our ability to enhance vaccine capsid stability. Developments in molecular biology and phylogenetics underlie many of the recent advances in FMD research, including improved vaccines and diagnostics, and improved understanding of FMD epidemiology. Tools for genetic analyses continue to become both more powerful and more affordable enabling them to be used to address an ever-expanding range of questions. This rapidly advancing field potentiates many areas of FMD research and should be prioritized.

Injuries to staff engaged in foot-and-mouth disease eradication in Japan.

BACKGROUND: In April 2010, a large-scale outbreak of foot-and-mouth disease, a highly infectious disease affecting cloven-hoofed animals, was reported in Miyazaki prefecture in Japan. Many staff were dispatched to the epidemic area to undertake containment measures. Various injuries were reported among the dispatched staff. AIMS: To study the characteristics of injuries that occurred during containment measures and to identify the characteristics of those injured. METHODS: We analysed records of injuries that occurred among staff dispatched from national organizations and prefectures other than Miyazaki prefecture. Based on these records, a qualitative analysis was conducted for veterinarians and non-veterinarian support staff as each group played a different role during the outbreak. RESULTS: Forty-seven veterinarians and 183 support staff were injured. The injury risk was significantly higher for support staff (13%) than for veterinarians (5%; P < 0.001). The most frequently reported injury resulted from exposure to disinfectant and the next was abrasions caused by friction from protective clothing and rubber boots. Among veterinarians, needlestick accidents were also frequently reported. CONCLUSIONS: Applying a less hazardous disinfectant and improving protective equipment to prevent skin abrasion may be effective in reducing the risk of these injuries among staff conducting containment measures. Reducing injuries among staff in such circumstances will assist effective control measures.

HSV-1 amplicon vectors as genetic vaccines.

HSV-1 amplicon vectors have been used as platforms for the generation of genetic vaccines against both DNA and RNA viruses. Mice vaccinated with such vectors encoding structural proteins from both foot-and-mouth disease virus and rotavirus were partially protected from challenge with wild-type virus (D'Antuono et al. Vaccine 28: 7363-7372, 2010; Laimbacher et al. Mol Ther 20: 1810-1820, 2012), indicating that HSV-1 amplicon vectors are attractive tools for the development of complex and safe genetic vaccines. This chapter describes the use of HSV-1 amplicon vectors that encode individual or multiple viral structural proteins from a polycistronic transgene cassette in mammalian cells. More precisely, amplicon vectors that encode multiple structural viral proteins support the in situ production of viruslike particles (VLPs) in vector-infected cells. The expression of the viral genes is confirmed by Western blot and immune fluorescence analysis, and the generation of VLPs in vector-infected cells is demonstrated by electron microscopy.

Multiple microRNAs targeted to internal ribosome entry site against foot-and-mouth disease virus infection in vitro and in vivo.

BACKGROUND: Foot-and-mouth disease virus (FMDV) causes a severe vesicular disease in domestic and wild cloven-hoofed animals. Because of the limited early protection induced by current vaccines, emergency antiviral strategies to control the rapid spread of FMD outbreaks are needed.Here we constructed multiple microRNAs (miRNAs) targeting the internal ribosome entry site (IRES) element of FMDV and investigated the effect of IRES-specific miRNAs on FMDV replication in baby hamster kidney (BHK-21) cells and suckling mice. RESULTS: Four IRES-specific miRNAs significantly reduced enhanced green fluorescent protein (EGFP) expression from IRES-EGFP reporter plasmids, which were used with each miRNA expression plasmid in co-transfection of BHK-21 cells. Furthermore, treatment of BHK-21 cells with Bi-miRNA (a mixture of two miRNA expression plasmids) and Dual-miRNA (a co-cistronic expression plasmid containing two miRNA hairpin structures) induced more efficient and greater inhibition of EGFP expression than did plasmids carrying single miRNA sequences.Stably transformed BHK-21 cells and goat fibroblasts with an integrating IRES-specific Dual-miRNA were generated, and real-time quantitative RT-PCR showed that the Dual-miRNA was able to effectively inhibit the replication of FMDV (except for the Mya98 strain) in the stably transformed BHK-21 cells.The Dual-miRNA plasmid significantly delayed the deaths of suckling mice challenged with 50× and 100× the 50% lethal dose (LD50) of FMDV vaccine strains of three serotypes (O, A and Asia 1), and induced partial/complete protection against the prevalent PanAsia-1 and Mya98 strains of FMDV serotype O. CONCLUSION: These data demonstrate that IRES-specific miRNAs can significantly inhibit FMDV infection in vitro and in vivo.

Novel antiviral therapeutics to control foot-and-mouth disease.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. Vaccines require ∼7 days to induce protection; thus, before this time, vaccinated animals are still susceptible to the disease. Our group has previously shown that swine inoculated with 1×10(11) focus forming units (FFU) of a replication-defective human adenovirus containing the gene for porcine interferon alpha (Adt-pIFN-α) are sterilely protected from FMDV serotypes A24, O1 Manisa, or Asia 1 when the animals are challenged 1 day postadministration, and protection can last for 3-5 days. Polyriboinosinic-polyribocytidylic acid stabilized with poly-l-lysine and carboxymethyl cellulose (poly ICLC) is a synthetic double-stranded RNA that is a viral mimic and activates multiple innate immune pathways through interaction with toll-like receptor 3 and MDA-5. It is a potent inducer of IFNs. In this study, we initially examined the effect of poly IC and IFN-α on FMDV replication and gene induction in cell culture. Poly ICLC alone or combined with Adt-pIFN-α was then evaluated for its therapeutic efficacy in swine against intradermal challenge with FMDV A24, 1 day post-treatment. Groups of swine were subcutaneously inoculated either with poly ICLC alone (4 or 8 mg) or in combination with different doses of Adt-pIFN-α (2.5×10(9), 1×10(9), or 2.5×10(8) FFU). While different degrees of protection were achieved in all the treated animals, a dose of 8 mg of poly ICLC alone or combined with 1×10(9) FFU of Adt-pIFN-α was sufficient to sterilely protect swine when challenged 24 h later with FMDV A24. IFN-stimulated gene (ISG) expression in peripheral blood mononuclear cells at 1 day post-treatment was broader and higher in protected animals than in nonprotected animals. These data indicate that poly ICLC is a potent stimulator of IFN and ISGs in swine and at an adequate dose is sufficient to induce complete protection against FMD.

Cationic microparticle [poly(D,L-lactide-co-glycolide)]-coated DNA vaccination induces a long-term immune response against foot and mouth disease in guinea pigs.

BACKGROUND: Foot and mouth disease (FMD) can be controlled by regular vaccination and restriction of the movement of infected animals in the endemic countries. Although presently used, tissue culture inactivated vaccine gives protection, it has several limitations, including a short duration of immunity. DNA vaccine delivered through microparticles could comprise an alternative approach to conventional vaccine when aiming to circumvent these limitations. METHODS: We constructed the expression plasmid (pVAC-1D) containing 1D gene FMD virus serotype Asia 1. Poly(D,L-lactide-co-glycolide) (PLG) microparticles were prepared and coated with the pVAC-1D plasmid. Guinea pigs were vaccinated with PLG-coated and naked DNA vaccine constructs intramuscularly. The humoral response was measured by an enzyme-linked immunosorbent assay (ELISA) and the serum neutralization test (SNT). Analysis of the persistence and the expression of pVAC-1D plasmid construct was carried out by quantitative polymerase chain reaction (qPCR). RESULTS: The humoral response lasted for 1 year, as measured by ELISA and SNT. Analysis of the persistence and the expression of pVAC-1D plasmid construct by qPCR has shown that pVAC-1D expression was seen for a longer duration compared to the naked DNA vaccine. Microparticles coated plasmid DNA-injected guinea pigs were protected when challenged with FMD virus. CONCLUSIONS: The present study has shown that the delivery of plasmid coated on cationic PLG microparticles enhance the duration of immunity of the DNA vaccine constructs.

Bovine type III interferon significantly delays and reduces the severity of foot-and-mouth disease in cattle.

Interferons (IFNs) are the first line of defense against viral infections. Although type I and II IFNs have proven effective to inhibit foot-and-mouth disease virus (FMDV) replication in swine, a similar approach had only limited efficacy in cattle. Recently, a new family of IFNs, type III IFN or IFN-λ, has been identified in human, mouse, chicken, and swine. We have identified bovine IFN-λ3 (boIFN-λ3), also known as interleukin 28B (IL-28B), and demonstrated that expression of this molecule using a recombinant replication-defective human adenovirus type 5 (Ad5) vector, Ad5-boIFN-λ3, exhibited antiviral activity against FMDV in bovine cell culture. Furthermore, inoculation of cattle with Ad5-boIFN-λ3 induced systemic antiviral activity and upregulation of IFN-stimulated gene expression in the upper respiratory airways and skin. In the present study, we demonstrated that disease could be delayed for at least 6 days when cattle were inoculated with Ad5-boIFN-λ3 and challenged 24 h later by intradermolingual inoculation with FMDV. Furthermore, the delay in the appearance of disease was significantly prolonged when treated cattle were challenged by aerosolization of FMDV, using a method that resembles the natural route of infection. No clinical signs of FMD, viremia, or viral shedding in nasal swabs was found in the Ad5-boIFN-λ3-treated animals for at least 9 days postchallenge. Our results indicate that boIFN-λ3 plays a critical role in the innate immune response of cattle against FMDV. To this end, this work represents the most successful biotherapeutic strategy so far tested to control FMDV in cattle.

Foot and mouth disease and similar virus infections in camelids: a review.

Foot and mouth disease (FMD) remains the most important animal disease. The FMD virus is highly contagious and occurs almost exclusively among cloven-hoofed animals such as cattle, sheep, goats, Bactrian camels and swine. Old World camels (OWCs) and New World camels (NWCs) inhabit FMD-endemic countries in South America, North and East Africa, and the Middle and Far East. Results of experimental infection of OWCs with the virus, and several clinical observations from the field over a century, confirm that the two closely related camel species of Bactrian and dromedary camels possess noticeably different susceptibilities to FMD. It is now certain that Bactrian camels can contract the disease. In contrast, dromedaries are not susceptible to FMD and do not transmit infection, even when in close contact with susceptible animals. The susceptibility of NWCs to the FMD virus has been demonstrated in the field and in experimental infection trials. However, these animals are not very susceptible and do not represent a serious risk in transmitting FMD to susceptible animal species.

Multiple shRNAs driven by U6 and CMV promoter enhances efficiency of antiviral effects against foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is an economically significant animal disease because of the speed of its transmission. The current vaccine for FMD provides no protection until 7 days post-vaccination, thus reducing its effectiveness in the case of an outbreak. Small interfering RNA (siRNA) is a promising antiviral approach because it can induce a protective response much more rapidly. This study is the first report to apply multiple short hairpin RNA (shRNA) expression systems to inhibit foot-and-mouth disease virus (FMDV) replication. Three different shRNAs, one targeting 2B region and two targeting 3C region, were driven by three RNA Polymerase III (Pol III) promoters, U6 or a combination of two U6 promoters and one RNA Polymerase II (Pol II) promoter, CMV. The adenoviruses simultaneously expressing three different shRNAs in a single construct had significantly enhanced antiviral effects compared with those expressing only a single shRNA, those expressing double shRNAs or a mixture of adenoviruses expressing a single shRNA and the adenovirus expressing double shRNAs, both in vitro and in vivo. The adenoviruses had broad antiviral effects against seven serotypes of FMDV, including O, A, Asia1, C, SAT1, SAT2, and SAT3 in vitro, but differed in their efficacy. The adenovirus expressing multiple shRNAs driven by three U6 promoters had strong antiviral effects in suckling mice challenged with O, A, and Asia1 serotype of FMDV.

New vaccine design based on defective genomes that combines features of attenuated and inactivated vaccines.

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.

Therapeutic application of RNA interference against foot-and-mouth disease virus in vitro and in vivo.

Foot-and-mouth disease (FMD) is an economically important animal disease because of the speed of its transmission. Routine vaccination may not be effective for early protection in an outbreak situation. Small interfering RNA (siRNA) can be used in a rapid and effective antiviral approach. However, siRNA has limitations when used in disease prevention, such as a short duration of action. In this study, we have demonstrated that treatment with siRNA after FMD virus (FMDV) infection has an antiviral effect and could be effective in control of FMDV. We applied adenoviruses expressing siRNA both before and after FMDV infection in vitro and in vivo. Treatment after FMDV infection gave effective viral inhibition, but a combination of treatment before and after FMDV infection gave the best results in IBRS-2 cells. We obtained high survival rates in suckling mice by the use of therapeutic injections following challenge. The results of this study suggest that treatment with siRNA could enhance antiviral effects and may be helpful in the control of FMDV in an outbreak.

Inhibition of foot-and-mouth disease virus replication in vitro and in vivo by small interfering RNA.

By using bioinformatics computer programs, all foot-and-mouth disease virus (FMDV) genome sequences in public-domain databases were analyzed. Based on the results of homology analysis, 2 specific small interfering RNA (siRNA) targeting homogenous 3D and 2B1 regions of 7 serotypes of FMDV were prepared and 2 siRNA-expression vectors, pSi-FMD2 and pSi-FMD3, were constructed. The siRNA-expressing vectors were used to test the ability of siRNAs to inhibit virus replication in baby hamster kidney (BHK-21) cells and suckling mice, a commonly used small animal model. The results demonstrated that transfection of BHK-21 cells with siRNA-expressing plasmids significantly weakened the cytopathic effect (CPE). Moreover, BHK-21 cells transiently transfected with short hairpin RNA (shRNA)-expressing plasmids were specifically resistant to the infection of the FMDV serotypes A, O, and Asia I and this the antiviral effects persisted for almost 48 hours. We measured the viral titers, the 50% tissue culture infective dose (TCID50) in cells transfected with anti-FMDV siRNAs was found to be lower than that of the control cells. Furthermore, subcutaneous injection of siRNA-expressing plasmids in the neck of the suckling mice made them less susceptible to infection with O, and Asia I serotypes of FMDV.

Fiebre Aftosa: Diagnóstico diferencial con otras enfermedades vesiculares en el hombre / The Aphthous Fever: Differential diagnosis with another vesicular diseases in the man

La Fiebre Aftosa es una enfermedad viral ocasionada por un picornavirus del género aphtovirus. Es una zoonosis de muy baja incidencia en humanos pero muy contagiosa entre animales susceptibles, por lo que es considerada la enfermedad animal más importante del mundo debido a sus implicaciones económicas y políticas. El hombre es un huésped incidental que rara vez se infecta y enferma. Los síntomas iniciales son inespecíficos, pudiendo ocasionar lesiones vesículo-ulcerativas en la mucosa bucal y en las palmas de las manos y plantas de los pies. El curso de la enfermedad es corto y benigno. Esta entidad no debe ser confundida con otras enfermedades virales, fundamentalmente las ocasionadas por enterovirus que presentan una nomenclatura o una clínica similar (AU)

Epidemiology, surveillance and control of the principal infectious animal diseases in Africa.

The author presents detailed information on traditional methods, the majority of which remain in use, for the recognition, prevention and treatment of the principal infectious diseases prevalent on the African continent. The information provided relates to the observations and practices of peoples in three main regions, namely West, East and Southern Africa. Data are presented for ten diseases of major importance, with the widest range of practices being recorded for the control of foot and mouth disease, rinderpest and anthrax.