Foot-and-mouth Disease Virus Serotype Research Articles

Effects of two amino acid substitutions in the capsid proteins on the interaction of two cell-adapted PanAsia-1 strains of foot-and-mouth disease virus serotype O with heparan sulfate receptor.

BackgroundSome cell-adapted strains of foot-and-mouth disease virus (FMDV) can utilize heparan sulfate (HS) as a receptor to facilitate viral infection in cultured cells. A number of independent sites on the capsid that might be involved in FMDV-HS interaction have been studied. However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O.To identify the molecular determinant(s) for the interaction of O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc with HS receptor, several chimeric viruses and site-directed mutants were generated by using an infectious cDNA of a non-HS-utilizing rescued virus (Cathay topotype) as the genomic backbone. Phenotypic properties of these viruses were determined by plaque assays and virus adsorption and penetration assays in cultured cells.ResultsOnly two of the rescued viruses encoding VP0 of O/Tibet/CHA/6/99tc or VP1 of O/Fujian/CHA/9/99tc formed plaques on wild-type Chinese hamster ovary (WT-CHO; HS+) cells, but not on HS-negative pgsD-677 cells. The formation of plaques by these two chimeric viruses on WT-CHO cells could be abolished by the introduction of single amino acid mutations Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc and Lys-1083 → Glu in VP1 of O/Fujian/CHA/9/99tc, respectively. Nonetheless, the introduced mutation Leu-2080 → Gln in VP2 of O/Fujian/CHA/9/99tc for the construction of expectant recombinant plasmid led to non-infectious progeny virus in baby hamster kidney 21 (BHK-21) cells, and the site-directed mutant encoding Glu-1083 → Lys in VP1 of O/Tibet/CHA/6/99tc did not acquire the ability to produce plaques on WT-CHO cells.Significant differences in the inhibition of the infectivity of four HS-utilizing viruses by heparin and RGD-containing peptide were observed in BHK-21 cells.Interestingly, the chimeric virus encoding VP0 of O/Fujian/CHA/9/99tc, and the site-directed mutant encoding Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc could bind to HS, but there was no expression of the 3A protein of these two viruses in WT-CHO cells.ConclusionThe results suggest that the cooperation of certain specific amino acid residues in the capsid proteins of these two cell-adapted PanAsia-1 strains is essential for viral infectivity, the heparin affinity and the capability on FMDV-HS interaction.

Foot-and-mouth Disease Transmission in Africa: Implications for Control, a Review.

In Africa, for the control of foot-and-mouth disease (FMD), more information is needed on the spread of the disease at local, regional and inter-regional level. The aim of this review is to identify the role that animal husbandry, trade and wildlife have on the transmission of FMD and to provide a scientific basis for different FMD control measures in Africa. Review of literature, published reports and databases shows that there is more long distance spread of FMD virus serotypes within North, West, Central and East Africa than in southern Africa. In North, West, Central and East Africa migratory animal husbandry systems often related with search for grazing and water as well as trade are practiced to a greater extent than in southern Africa. In southern Africa, the role of African buffalo (Syncerus caffer) is more extensively studied than in the other parts of Africa, but based on the densities of African buffalo in Central and East Africa, one would assume that buffalo should also play a role in the epidemiology of FMD in this part of Africa. More sampling of buffalo is necessary in West, Central and East Africa. The genetic analysis of virus strains has proven to be valuable to increase our understanding in the spread of FMD in Africa. This review shows that there is a difference in FMD occurrence between southern Africa and the rest of the continent; this distinction is most likely based on differences in animal husbandry and trade systems. Insufficient data on FMD in wildlife outside southern Africa is limiting our understanding on the role wildlife plays in the transmission of FMD in the other buffalo inhabited areas of Africa.

Expression of porcine fusion protein IRF7/3(5D) efficiently controls foot-and-mouth disease virus replication.

Several studies have demonstrated that the delivery of type I, II, or III interferons (IFNs) by inoculation of a replication-defective human adenovirus 5 (Ad5) vector expressing IFNs can effectively control foot-and-mouth disease (FMD) in cattle and swine during experimental infections. However, relatively high doses are required to achieve protection. In this study, we identified the functional properties of a porcine fusion protein, poIRF7/3(5D), as a biotherapeutic and enhancer of IFN activity against FMD virus (FMDV). We showed that poIRF7/3(5D) is a potent inducer of type I IFNs, including alpha IFN (IFN-α), IFN-β, and IFN-ω but not type III IFN (interleukin-28B), without inducing cytotoxicity. Expression of poIRF7/3(5D) significantly and steadily reduced FMDV titers by up to 6 log10 units in swine and bovine cell lines. Treatment with an IFN receptor inhibitor (B18R) combined with an anti-IFN-α antibody neutralized the antiviral activity in the supernatants of cells transduced with an Ad5 vector expressing poIRF7/3(5D) [Ad5-poIRF7/3(5D)]. However, several transcripts with known antiviral function, including type I IFNs, were still highly upregulated (range of increase, 8-fold to over 500-fold) by poIRF7/3(5D) in the presence of B18R. Furthermore, the sera of mice treated with Ad5-poIRF7/3(5D) showed antiviral activity that was associated with the induction of high levels of IFN-α and resulted in complete protection against FMDV challenge at 6, 24, or 48 h posttreatment. This study highlights for the first time the antiviral potential of Ad5-poIRF7/3(5D) in vitro and in vivo against FMDV. FMD remains one of the most devastating diseases that affect livestock worldwide. Effective vaccine formulations are available but are serotype specific and require approximately 7 days before they are able to elicit protective immunity. We have shown that vector-delivered IFN is an option to protect animals against many FMDV serotypes as soon as 24 h and for about 4 days postadministration. Here we demonstrate that delivery of a constitutively active transcription factor that induces the production of endogenous IFNs and potentially other antiviral genes is a viable strategy to protect against FMD.

Investigation of foot-and-mouth disease outbreaks in the Mbala and Kazungula districts of Zambia.

Foot-and-mouth disease (FMD) is an acute, highly contagious viral infection of domestic and wild cloven-hoofed animals. It is known to be endemic in Zambia, with periodic outbreaks occurring in different geographical areas of the country. This study was conducted to investigate the presence of FMD virus (FMDV) in reported FMD-suspected cases in cattle from the Kazungula and Mbala districts of Zambia. Sixty epithelial tissues or oesophageal-pharyngeal (OP) scrapings (probang samples) were collected from Mbala (n = 51) and Kazungula (n = 9) and examined for FMDV. The FMDV viral RNA and serotypes were examined by realtime reverse transcription polymerase chain reaction (qRT-PCR) and antigen Enzyme- linked immunosorbent assay (ELISA), respectively. Twenty-two samples (36.7%) were positive for the FMDV genome by qRT-PCR with Cycle threshold (Ct) values ranging from 13 to 31. The FMDV-positive samples from epithelial tissues showed relatively higher Ct values compared to those obtained from OP scrapings, irrespective of geographical location. Forty percent (40%; n = 4) of epithelial tissues from Mbala were serotyped into SAT 2 serotype by antigen ELISA. Kazungula samples were serotyped into SAT 1. These findings indicated that Mbala and Kazungula districts had FMD outbreaks in 2012 that were ascribed to at least FMDV serotype SAT 2 and SAT 1 field strains. Furthermore, regular interaction between buffalos from the Mosi-o Tunya Park and domestic animals from surrounding areas could contribute to the occurrence of regular FMD outbreaks in Kazungula, whilst the uncontrolled animal movements across borders between Mbala and Nsumbawanga could be responsible for disease outbreaks in Mbala. In-depth molecular biological studies, including sequencing and phylogeny of the viruses, should be conducted to elucidate the complex epidemiology of FMD in Zambia, thereby providing valuable information needed for the rational control strategy of FMD in Zambia and neighbouring countries.

Rapid, sensitive and effective diagnostic tools for foot-and-mouth disease virus in Africa.

Speed is paramount in the diagnosis of highly infectious diseases, such as foot-and-mouth disease (FMD), as well as for emerging diseases; however, simplicity is required if a test is to be deployed in the field. Recent developments in molecular biology have enabled the specific detection of FMD virus (FMDV) by reverse-transcription loop-mediated isothermal amplification (RT-LAMP), real-time reverse-transcription polymerase chain reaction (RT-qPCR) and sequencing. RT-LAMP enables amplification of the FMDV RNA-dependent RNA polymerase 3D(pol) gene at 63 °C (in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase) for 1 h, whilst RT-qPCR amplifies the same gene in approximately 2 h 30 min. In this study, we compared the sensitivity and effectiveness of RT-LAMP against RT-qPCR for the detection of the FMDV 3D(pol) gene in 179 oesophageal-pharyngeal scraping samples (collected by probang) obtained from clinically healthy cattle and buffalo in Malawi, Mozambique and Tanzania in 2010. The FMDV detection rate was higher with RT-LAMP (30.2%; n = 54) than with RT-qPCR (17.3%; n = 31). All samples positive by RT-qPCR (Cq ≤ 32.0) were also positive for the RT-LAMP assay; and both assays proved to be highly specific for the FMDV target sequence. In addition, the VP1 sequences of 10 viruses isolated from positive samples corresponded to the respective FMDV serotypes and genotypes. Our findings indicate that the performance of RT-LAMP is superior to RT-qPCR. Accordingly, we consider this test to have great potential with regard to the specific detection and surveillance of infectious diseases of humans and animals in resource-compromised developing countries.

Serotype Diversity of Foot-and-Mouth-Disease Virus in Livestock without History of Vaccination in the Far North Region of Cameroon.

Little information is available about the natural cycle of foot-and-mouth disease (FMD) in the absence of control measures such as vaccination. Cameroon presents a unique opportunity for epidemiological studies because FMD vaccination is not practiced. We carried out a prospective study including serological, antigenic and genetic aspects of FMD virus (FMDV) infections among different livestock production systems in the Far North of Cameroon to gain insight into the natural ecology of the virus. We found serological evidence of FMDV infection in over 75% of the animals sampled with no significant differences of prevalence observed among the sampled groups (i.e. market, sedentary, transboundary trade and mobile). We also found antibodies reactive to five of the seven FMDV serotypes (A, O, SAT1, SAT2 and SAT3) among the animals sampled. Finally, we were able to genetically characterize viruses obtained from clinical and subclinical FMD infections in Cameroon. Serotype O viruses grouped into two topotypes (West and East Africa). SAT2 viruses grouped with viruses from Central and Northern Africa, notably within the sublineage causing the large epidemic in Northern Africa in 2012, suggesting a common origin for these viruses. This research will guide future interventions for the control of FMD such as improved diagnostics, guidance for vaccine formulation and epidemiological understanding in support of the progressive control of FMD in Cameroon.

Investigation of foot-and-mouth disease outbreaks in Chikkaballapur district of Karnataka

The present study was undertaken to investigate foot-and-mouth disease (FMD) outbreaks in various villages of Chikkaballapur district in Karnataka. The disease was recorded in both small and large ruminant population. Serum samples (357) were collected from clinically sick, recovered and in-contact animals, and clinical samples (31) were collected from sick or recovering animals. In 3AB non-structural protein (NSP) ELISA, 42.85% cattle, 59.93% sheep and 73.91% goats were tested positive indicating an extensive FMD virus (FMDV) activity. The serum samples were also tested in liquid phase blocking (LPB) ELISA to assess the protective antibody titre, which were 42.85%, 6.72% and 8.69% for cattle, sheep and goats, respectively. Hence, it is presumed that poor herd immunity might have been one of the contributing factors for the spread of the disease. The outbreaks were caused by FMDV serotype O and Asia 1 as confirmed in serotype differentiating antigen detection ELISA and multiplex PCR. The VP1 region based phylogenetic analysis indicated the involvement of both Ind2001 and recently identified Ind2011 lineage of serotype O and lineage C of serotype Asia 1 in the outbreaks. The study revealed the epidemiological complexity of FMD in the field with respect to the spectrum of species of animals affected and the contemporaneous involvement of multiple serotypes and lineages.

Complete Genome Sequence of Foot-and-Mouth Disease Virus Serotype O Isolated from Bangladesh

Foot-and-mouth disease (FMD) is a highly infectious enzootic disease caused by FMD virus. The complete genome sequence of a circulatory FMD virus (FMDV) serotype O isolated from Natore, Bangladesh, is reported here. Genomic analysis revealed antigenic heterogeneity within the VP1 region, a fragment deletion, and insertions at the 5′ untranslated region (UTR) and 3A region compared to the genome of the available vaccine strain.

Antigenic variation of foot-and-mouth disease virus serotype A

The current measures to control foot-and-mouth disease (FMD) include vaccination, movement control and slaughter of infected or susceptible animals. One of the difficulties in controlling FMD by vaccination arises due to the substantial diversity found among the seven serotypes of FMD virus (FMDV) and the strains within these serotypes. Therefore, vaccination using a single vaccine strain may not fully cross-protect against all strains within that serotype, and therefore selection of appropriate vaccines requires serological comparison of the field virus and potential vaccine viruses using relationship coefficients (r1 values). Limitations of this approach are that antigenic relationships among field viruses are not addressed, as comparisons are only with potential vaccine virus. Furthermore, inherent variation among vaccine sera may impair reproducibility of one-way relationship scores. Here, we used antigenic cartography to quantify and visualize the antigenic relationships among FMD serotype A viruses, aiming to improve the understanding of FMDV antigenic evolution and the scope and reliability of vaccine matching. Our results suggest that predicting antigenic difference using genetic sequence alone or by geographical location is not currently reliable. We found co-circulating lineages in one region that were genetically similar but antigenically distinct. Nevertheless, by comparing antigenic distances measured from the antigenic maps with the full capsid (P1) sequence, we identified a specific amino acid substitution associated with an antigenic mismatch among field viruses and a commonly used prototype vaccine strain, A22/IRQ/24/64.

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

BackgroundFoot-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.ResultsFour 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.ConclusionThese data demonstrate that IRES-specific miRNAs can significantly inhibit FMDV infection in vitro and in vivo.

Geo-spatial distribution of serologically detected bovine Foot and Mouth Disease (FMD) serotype outbreaks in Ilesha Baruba, Kwara State-Nigeria

In this study, geo-spatial distribution of bovine Foot and Mouth Disease (FMD) outbreaks were investigated to establish the prevalence and distribution of FMD serotypes in Ilesha Baruba, Kwara state-Nigeria. This purposive study involved 15 FMD outbreaks occurred during December 2010 to March 2011. For geo-spatial analysis, the outbreak locations were indexed using Global Positioning Service (GPS) device (EtrexR). Randomly sampled bovine sera (n=64) from herd representatives were subjected to FMD 3ABC enzyme-linked immunosorbent assay (FMD 3ABC ELISA) and solid-phase competitive ELISA (SP-cELISA), for the screening and serotyping of FMD virus, respectively. The serotypes were indexed with the specific outbreak locations. Through ELISA, the FMD serotypes detected in this study were- serotype O (83%; n=53/64), serotype A (7.8%; n=5/64), serotype vaccine O (1.6%; n=1/64)), and serotype vaccine SAT2 (1.6%; n=1/64). Multiple serotypes were observed in two different combinations; these were O and A (4.7%; n=3/64), and O and SAT2 (1.6%; n=1/64). FMD multiple serotype infections were associated with absence of cross-immunity between serotypes and cross reactivity enhanced by clustered herds, highland study area topography, road and river interconnectivity, possible human settlements, activities and traffic. This study provides baseline information on geo-spatial distribution, and identification of prevalent FMD serotypes in Ilesha Baruba, Kwara state-Nigeria.

Evolutionary Analysis and Prediction of Peptide Vaccine Candidates for Foot-and-Mouth-Disease Virus Types A and O in Bangladesh.

Foot-and-mouth disease (FMD), an endemic disease of cloven-hoofed animals, causes an annual economic loss of US$60–150 million in Bangladesh. There is no cross-protection among the foot-and-mouth disease virus (FMDV) serotypes and vaccination escape mutation may happen. Peptide vaccine is a safer alternative. The aim of this study is to predict and map the B and T cell epitopes of VP1 proteins of FMDV serotypes O and A that were circulating in Bangladesh from 2011 to 2013. Using evolutionary and computational approach (BCPred, BepiPred, DiscoTope, ElliPro, and ProPred-I, IEDB analysis for MHC-I prediction), a total of 11 B and T cell epitopes were predicted. Also, the three-dimensional (3D) structure of VP1 protein showed that the predicted five epitopes residing on N- and C-termini can be considered as good vaccine candidates, and epitopes on the G–H loop can serve as receptor recognition sites for vaccine design. The scores of predicted epitopes of one method were cross-checked with other one for potential epitope mining. Within the VP1 antigenic sites, significant evidence of positive selection was present indicating evolution of VP1 under high immune surveillance.

Collection of Oral Fluids Using Cotton Ropes as a Sampling Method to Detect Foot-and-Mouth Disease Virus Infection in Pigs

In high-density farming practices, it is important to constantly monitor for infectious diseases, especially diseases that have the potential to spread rapidly between holdings. Pigs are known to amplify foot-and-mouth disease (FMD) by excreting large amounts of virus, and it is therefore important to detect the virus quickly and accurately to minimize the spread of disease. Ropes were used to collect oral fluid samples from pigs, and each sample was compared to saliva samples collected from individual animals by detecting FMD virus RNA using real-time PCR. Two different experiments are described where groups of pigs were infected with different serotypes of FMD virus, either with or without vaccination, and unvaccinated pigs were kept in aerosol contact. The sensitivity of the rope sampling varied between 0.67 and 0.92, and the statistical agreement between this method and individual sampling ranged from substantial to moderate for the two different serotypes. The ease of collecting oral fluids using ropes together with the high sensitivity of subsequent FMD detection through PCR indicates that this could be a useful method to monitor pig populations for FMD virus infection. With further validation of the sensitivity of detection of FMD virus RNA, this can be a cost-effective, non-invasive diagnostic tool.

Reconstructing the origin and transmission dynamics of the 1967–68 foot-and-mouth disease epidemic in the United Kingdom

A large epidemic of foot-and-mouth disease (FMD) occurred in the United Kingdom (UK) over a seven month period in Northwest England from late 1967 to the summer of 1968. This was preceded by a number of smaller FMD outbreaks in the country, two in 1967, in Hampshire and Warwickshire and one in Northumberland during 1966. The causative agent of all four events was identified as FMD virus (FMDV) serotype O and the source of the large epidemic was attributed to infected bone marrow in lamb products imported from Argentina. However, the diagnostic tools available at the time were unable to entirely rule out connections with the earlier UK FMD outbreaks, as well as other potential sources from Europe. The aim of this study was to apply molecular sequencing to investigate the likely source of this epidemic using VP1 region and full genome (FG) sequences determined directly from clinical epithelium samples (n=13) or cell culture isolates (n=6), from this and contemporary outbreaks in the UK, Europe and South America. Analysis of the VP1 sequences provided evidence for at least three separate incursions of FMDV into the UK including one independent introduction that was responsible for the main 1967/68 epidemic. Analysis of FG sequences from the main 1967/68 outbreak (n=10) revealed nucleotide substitutions at 94 genomic sites providing evidence for the linear accumulation of nucleotide substitutions (rate=2.42×10−5nt substitutions/site/day). However, there were five samples where this linear relationship was absent, indicating evolutional dormancy of the virus, presumably outside a host. These results help define the evolutionary dynamics of FMDV during an epidemic and contribute to the knowledge and understanding from which to base future outbreak control strategies.

A partial deletion in non-structural protein 3A can attenuate foot-and-mouth disease virus in cattle

The role of non-structural protein 3A of foot-and-mouth disease virus (FMDV) on the virulence in cattle has received significant attention. Particularly, a characteristic 10–20 amino acid deletion has been implicated as responsible for virus attenuation in cattle: a 10 amino acid deletion in the naturally occurring, porcinophilic FMDV O1 Taiwanese strain, and an approximately 20 amino acid deletion found in egg-adapted derivatives of FMDV serotypes O1 and C3. Previous reports using chimeric viruses linked the presence of these deletions to an attenuated phenotype in cattle although results were not conclusive. We report here the construction of a FMDV O1Campos variant differing exclusively from the highly virulent parental virus in a 20 amino acid deletion between 3A residues 87–106, and its characterization in vitro and in vivo. We describe a direct link between a deletion in the FMDV 3A protein and disease attenuation in cattle.

A Portable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Foot-and-Mouth Disease Virus

Foot-and-mouth disease (FMD) is a trans-boundary viral disease of livestock, which causes huge economic losses and constitutes a serious infectious threat for livestock farming worldwide. Early diagnosis of FMD helps to diminish its impact by adequate outbreak management. In this study, we describe the development of a real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of FMD virus (FMDV). The FMDV RT-RPA design targeted the 3D gene of FMDV and a 260 nt molecular RNA standard was used for assay validation. The RT-RPA assay was fast (4–10 minutes) and the analytical sensitivity was determined at 1436 RNA molecules detected by probit regression analysis. The FMDV RT-RPA assay detected RNA prepared from all seven FMDV serotypes but did not detect classical swine fever virus or swine vesicular disease virus. The FMDV RT-RPA assay was used in the field during the recent FMD outbreak in Egypt. In clinical samples, reverse transcription polymerase chain reaction (RT-PCR) and RT-RPA showed a diagnostic sensitivity of 100% and 98%, respectively. In conclusion, FMDV RT-RPA was quicker and much easier to handle in the field than real-time RT-PCR. Thus RT-RPA could be easily implemented to perform diagnostics at quarantine stations or farms for rapid spot-of-infection detection.

Antigenic site variation in foot-and-mouth disease virus serotype O grown under vaccinal serum antibodies in vitro

Foot-and-mouth disease virus (FMDV) is constantly evolving under neutralizing antibody pressure in either naturally infected or vaccinated animals. This study was carried out to understand the dynamics of evolution of antigenic sites. Neutralizing antibody-resistant populations of three strains of FMDV serotype O (INDR2/1975, IND120/2002 and IND271/2001) were isolated by serial propagation in BHK-21 cells in the presence of sub-neutralizing level of bovine vaccinal sera (BVS). In the partial neutralization escape variants, fixation of aa substitutions were observed at critical residues of all established antigenic sites of serotype O {144 of VP1 (site 1), 45 and 48 of VP1 (site 3), 72 and 134 of VP2 (Site 2)} except site 4 and 5. In majority of the variant populations, site 3 was found to be substituted and therefore immunodominance may not be associated with a particular site, rather it appears to be a virus strain and infected host specific affair. Substitutions were also observed in proximity to the identified residues {41 and 51 (βB-βC loop), 133, 140 and 143 (βG-βH loop), 201, 204 and 209 (C terminus) of VP1, 71 and 75 (βB-βC loop), 131 (βE-αB region), 174 and 179 (βG-βH loop) and 219 (C terminus) of VP3} within antigenic sites of serotype O or other serotypes which could be significant in terms of neutralizing antibody binding and immune escape. Presence of similar residues in the Indian field viruses as selected in the variants supports the importance of these sites in antigenic diversification of serotype O FMD virus.

Diagnosis of Foot-and-Mouth Disease Virus by Real Time Reverse Transcription Polymerase Chain Reaction Assay in Iran

Background and Aims: Accurate and rapid diagnosis is necessary for effective control and prevention of foot-and-mouth disease (FMD). In present study, was evaluated real time reverse transcription-polymerase chain reaction (rRT-PCR) assay along with diagnostic routine methods for the detection of all seven serotypes of FMD virus (FMDV), namely O, C, A, SAT1, 2, 3 and Asia 1 in biological samples at the reference laboratory for FMD, Iran. Materials and Methods: Two different RT-PCR assays targeting two different regions 5´ untranslated region (5´-UTR) and RNA polymerase (3D) of the FMDV genome were used to confirm the presence of FMDV in epithelial suspensions. Results: In the two methods the viral RNA in all tested archival serotypes of FMDV were detected. Specificity of this reaction was confirmed by the use of swine vesicular disease virus and blue-tongue. The amount of cycle threshold (CT) value of all seven serotypes was different and the lowest and highest of CT value achieved for SAT3, A, O types and SAT2, C types, respectively. Conclusion: The results showed that RT-PCR was more sensitive and effective than routine diagnostic methods. Furthermore, RT-PCR as a strong and valuable tool concomitant with diagnostic routine methods facilitate monitoring the fields FMDV strains and suggested that the use of the multiple diagnostic targets could enhance the sensitivity of the molecular methods for the detection of FMDV.

The effects of the codon usage and translation speed on protein folding of 3Dpol of foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) can cause a highly infectious disease of cloven-hoofed livestock including sheep, domestic pig and cattle. As for the sever economic and social effects of a foot-and-mouth disease (FMD) outbreak, FMD has been recognized as the most important constraint to international trade in animals and animal products by the World Organization for Animal Heath (OIE) (Leforban 1999). FMDV is one of the members in the Aphthovirus genus which is classified into the Picornaviridae family. This virus exists in the form of seven different serotypes: Asia 1, O, C, A and South African Territories 1 (SAT 1), SAT 2 and SAT 3, but a many subtypes have evolved within each serotype. The genome of FMDV consists of a single open reading frame (ORF) flanked by a 5′ untranslated region and a 3′untranslated region. The ORF encodes 12 viral proteins, from 5′ termination to 3′ termination, including the leader protease (L), four capsid proteins (VP4, VP2, VP3 and VP1) and the nonstructural proteins 2A, 2B, 2C, 3A, 3B, 3C and 3D (Cooke and Westover 2008; Klein 2009). In Picornaviridae family, the 3D, namely RNAdependent RNA polymerase, is responsible for synthesis of RNA virus genomes. Under physiological conditions, 3D catalyze the formation of phosphodiester bonds between ribonucleotides in an RNA template-dependent fashion. However, because of the limited template copying fidelity of 3D, FMDV genome replication reflects the error-prone nature(Pariente et al. 2003). Given the important function of 3D for virus multiplication, the FMDV 3D has been the subject of intensive study for a long time. The 3D was first described in polymerase complex in the infected cells by FMDV. This complex was originally called the virus infection-associated antigen (VIAA), since it could be recognized by positive serum against FMDV (Cowan and Graves 1966). Subsequently, this VIAA was identified to contain the 3D (Lowe and Brown 1981; Newman et al. 1979; Robertson et al. 1983). Among the different FMDV serotypes or subtypes, the amino acid sequence for 3D of FMDV are conserved to some degree (Martinez-Salas et al. 1985; Villaverde et al. 1988), furthermore, some variable residues, invariable residues and peptide motifs were thought to play important roles in sustaining the functional integrity of the 3D (Carrillo et al. 2005; Doherty et al. 1999; George et al. 2001; Kaku et al. 1999; Xiang et al. 1998). However, these observations fail to indicate the overall structure of FMDV 3D. Fortunately, at this time, crystallization structure of FMDV 3D has been identified and the structure-function relationships were analyzed. The two studies help researchers to understand FMDV genome synthesis at the molecular level, account for the error-prone properties of the replication machinery (Ferrer-Orta et al. 2004, 2009). The primary sequence was regarded as the determinant of the protein secondary structure, hence, some studies of various protein structures were carried out based on this theory (Chou and Fasman 1974, 1978; Lim 1974; Qian and Sejnowski 1988; Xiao-xia Ma and Yu-ping Feng contributed equally to this work.

Development of a quick and simple detection methodology for foot-and-mouth disease virus serotypes O, A and Asia 1 using a generic RapidAssay Device

BackgroundOutbreaks of Foot-and-mouth disease (FMD) have resulted in tremendous economic losses. Thus, the development of a rapid and easily performed test for FMD detection is important for controlling a FMD outbreak and containing its spread. The purpose of this project is to develop a lateral flow immunochromatographic (LFI) strip test for rapid detection of FMD virus serotypes O, A and Asia 1.MethodsSpecific monoclonal antibodies (mAbs) against each serotype were produced and used as the capture mAbs. A serotype independent mAb was selected and used as the detection mAb with the aim of subsequently developing a multi-serotype strip test. A new generation of the generic RapidAssay Device (gRAD) was used for the test.ResultEach strip test can specifically detect the FMDV O, A or Asia 1 viruses, but not other vesicular disease viruses. The LFI strip tests for serotypes A and Asia 1 were able to identify all tested serotype A (n= 39) and Asia 1 field isolates (n=17). Whereas the test for serotype O detected 45 out of 46 field isolates. The sensitivity of this strip test was comparable with the double antibody sandwich ELISA for viral antigen detection. All vesicular fluid and epithelium samples collected from experimentally infected animals with serotype O, A and Asia 1 were identified as positive by the LFI strip test. Swab samples (n=11) collected over the lesion area from experimentally inoculated animals (serotype A) were examined. All of them demonstrated positive results using the LFI serotype A strip test and double antibody sandwich (DAS) ELISA.ConclusionsThe ability of strip tests to produce rapid results and high specificity makes it a valuable tool for early detection of FMDV O, A and Asia 1 in the field.