FMDV Strains Research Articles

Multiple incursions of foot-and-mouth disease virus serotype O into the Republic of Korea between 2010 and 2019

This study characterised type O foot-and-mouth disease (FMD) viruses recovered from outbreaks that were reported between 2010 and 2019 in the Republic of Korea. We used 96 newly generated whole-genome sequences (WGS) along with 131 already published WGSs from samples collected from countries in East and Southeast Asia. We identified at least eight independent introductions of O/SEA/Mya-98 and O/ME-SA/Ind-2001e FMDV strains into the Republic of Korea during the study period, which were closely related to the sequences of viruses circulating in the East and Southeast Asia neighbourhood with over 97 % nucleotide identity. Spatial-temporal transitions of O/SEA/Mya-98 lineage viruses recovered from the largest outbreak (2014–16) showed that after initial cases were detected within a 15-day period in July 2014, a single introduction of the same virus during December 2014 generated extensive forward virus transmission between farms that lasted until March 2016. We estimated that secondary transmissions were responsible for infection on 44 % FMD affected farms, over a total of 14 generations of infection. We eastimated a median evolutionry rate of 2.51 × 10−5 nt/site/day, which is similar for other FMD epidemic scenarios. These findings suggest that regular incursions of different FMDV lineages into the Republic of Korea have posed a continuous threat from endemic countries of East and Southeast Asia. These data highlight the importance of active cooperation and information exchange on FMD situation within Asian countries and assessment about the likely risk routes of virus movement is highly necessary to prevent further incursion and virus spread of FMDV in the Republic of Korea.

Calcium Chloride as a Novel Stabilizer for Foot-and-Mouth Disease Virus and Its Application in the Vaccine Formulation.

The thermal stability of the in-house-developed foot-and-mouth disease (FMD) type O and A viruses was evaluated, and the O Jincheon virus was found to exhibit the lowest thermal stability. To overcome this instability, we proposed a novel stabilizer, calcium chloride. The thermal stability of FMDVs increased up to a CaCl2 concentration of 10 mM, and it had a decreasing trend at >30 mM. The O Jincheon virus showed a significant decrease in the amount of antigen over time at 4 °C. In contrast, the samples treated with CaCl2 showed stable preservation of the virus without significant antigen loss. After the CaCl2-formulated vaccine was administered twice to pigs, the virus neutralization titer reached approximately 1:1000, suggesting that the vaccine could protect pigs against the FMDV challenge. In summary, the O Jincheon virus is difficult to utilize as a vaccine given its low stability during storage after antigen production. However, following its treatment with CaCl2, it can be easily utilized as a vaccine. This study evaluated CaCl2 as a novel stabilizer in FMD vaccines and may contribute to the development of stable vaccine formulations, especially for inherently unstable FMDV strains.

Phylogenetic and genotypic characteristics of the foot-and-mouth disease virus from outbreaks in southern Vietnam, 2019

In 2019, multiple FMD outbreaks occurred in swine farms vaccinated against FMDV in southern Vietnam. This study investigated the genotypic characteristics of FMDV strains from these outbreaks. Seven samples were collected from pigs exhibiting FMD clinical signs. All FMDV-positive samples were amplified and sequenced for the gene encoding the VP1. Results were analyzed and compared with sequences of reference strains and vaccine strains on GenBank. Phylogenetic analysis showed that all seven field isolates belonged to serotype O, topotype SEA/Mya-98. These strains shared high homology with strains from Vietnam (2018), Korea, and China, but low homology with vaccine strains. Moreover, 21 amino acid substitutions were found in the VP1 protein of the FMDV field strains, many of which were crucial antigenic determinants involved in the neutralization of FMDV. These findings suggest that the current vaccine may not be effective against the emerging FMDV strains in southern Vietnam.

Evaluation of Potential In Vitro Recombination Events in Codon Deoptimized FMDV Strains

Codon deoptimization (CD) has been recently used as a possible strategy to derive foot-and-mouth disease (FMD) live-attenuated vaccine (LAV) candidates containing DIVA markers. However, reversion to virulence, or loss of DIVA, from possible recombination with wild-type (WT) strains has yet to be analyzed. An in vitro assay was developed to quantitate the levels of recombination between WT and a prospective A24-P2P3 partially deoptimized LAV candidate. By using two genetically engineered non-infectious RNA templates, we demonstrate that recombination can occur within non-deoptimized viral genomic regions (i.e., 3'end of P3 region). The sequencing of single plaque recombinants revealed a variety of genome compositions, including full-length WT sequences at the consensus level and deoptimized sequences at the sub-consensus/consensus level within the 3'end of the P3 region. Notably, after further passage, two recombinants that contained deoptimized sequences evolved to WT. Overall, recombinants featuring large stretches of CD or DIVA markers were less fit than WT viruses. Our results indicate that the developed assay is a powerful tool to evaluate the recombination of FMDV genomes in vitro and should contribute to the improved design of FMDV codon deoptimized LAV candidates.

Design of multiplexing lateral flow immunoassay for detection and typing of foot-and-mouth disease virus using pan-reactive and serotype-specific monoclonal antibodies: Evidence of a new hook effect.

The foot-and-mouth disease (FMD) is the most important transboundary viral disease of livestock in the international context, because of its extreme contagiousness, widespread diffusion, and severe impact on animal trade and animal productions. The rapid and on-field detection of the virus responsible for the FMD represents an urgent demand to efficiently control the diffusion of the infection, especially in low resource setting where the FMD is endemic. Colorimetric lateral flow immunoassay (LFIA) is largely used for the development of rapid tests, due to the extreme simplicity, cost-effectiveness, and on-field operation. In this work, two multiplex LFIA devices were designed for the diagnosis of FMD and the simultaneous identification of major circulating serotypes of the FMD virus. The LFIAs relied on the sandwich-type immunoassay and combined a set of well-characterised monoclonal antibodies (mAb) pairs. One LFIA aimed at detecting and identifying O, A and Asia-1 serotypes, the second device enabled the detection and differentiation of the SAT 1 and SAT 2 serotypes. Both devices also incorporated a broad-specific test line reporting on infection from FMDV, regardless the strain and the serotype involved. Accordingly, five and four reactive zones were arranged in the two devices to achieve a total of six simultaneous analyses. The development of the two multiplex systems highlighted for the first time the relevance of the mAb positioning along the LFIA strip in connection with the use of the same or different mAb as capture and detector ligands. In fact, the excess of detector mAb typically employed for increasing the sensitivity of sandwich immunoassay induced a new type of hook effect when combined with the same ligand used as the capture. This effect strongly impacted assay sensitivity, which could be improved by an intelligent alignment of the mAb pairs along the LFIA strip. The analytical and diagnostic performances of the two LFIAs were studied by testing reference FMDV strains grown in cell cultures and some representative field samples (epithelium homogenates). Almost equivalent sensitivity and specificity to those of a reference Ag-ELISA kit were shown, except for the serotype SAT 2. These simple devices are suitable in endemic regions for in-field diagnosis of FMD accompanied by virus serotyping and, moreover, could be deployed and used for rapid confirmation of secondary outbreaks after FMD incursions in free-areas, thus contributing to promptly implement control measures.

Şap virüsü şuşlarına karşı poliklonal antikor üretimi

Antibodies(Abs) have been always a major place in diagnostic laboratories. Many diagnostic techniques like Enzyme-Linked Immuno Sorbent Assays (ELISA), immunofluorescence, Ab-microarray platforms, immunoblots, X-ray crystallography require the Abs. ELISA is the main test that used Abs for Foot and Mouth Disease (FMDV) serology. It needs polyclonal or monoclonal Abs to detect FMDV antigen or Abs. For this purpose, solid-phase competitive ELISA (SPCE) or liquid phase blocking ELISA (LPBE) and non-structural protein (NSP) ELISA are used. SPCE and LPBE have mainly used FMDV structural protein (SP)-Ab detection.
 In this study, it was aimed to produce a polyclonal Ab against FMDV ANep84 (Genotype VII) and OTUR07 (OPanAsia II), ATUR11 (A Iran05) strains for LPBE, FMDV SP-antibody detection. For this purpose, 4 guinea pigs and 6 rabbits were used for each serotype of FMDV. After producing Abs, checkerboard ELISA titration was performed to determine the optimal test dilution of Abs. Backgrounds, cross-reactions against three strains of FMDV were also checked. In conclusion, polyclonal Abs were produced against FMDV ANep84 (Genotype VII) and O Tur07 (O Pan Asia II), ATUR11 (A Iran05) strains, and standardized for LPBE test.

Development of a Potent Stabilizer for Long-Term Storage of Foot-and-Mouth Disease Vaccine Antigens.

A local virus isolate, O/SKR/JC/2014 (O JC), has been considered as a candidate vaccine strain in the development of a domestic foot-and-mouth disease (FMD) vaccine in Korea. However, producing and preserving a sufficient quantity of intact vaccine antigens from the O JC strain was difficult owing to its distinctive structural instability compared to other candidate vaccine strains. Based on this feature, the O JC strain was adopted as a model virus for the stabilization study to determine the optimal stabilizer composition, which enables long-term storage of the FMD vaccine antigen in both aqueous and frozen phases. In contrast to O JC vaccine antigens stored in routinely used Tris-buffered or phosphate-buffered saline, those stored in Tris-KCl buffer showed extended shelf-life at both 4 °C and −70 °C. Additionally, the combined application of 10% sucrose and 5% lactalbumin hydrolysate could protect O JC 146S particles from massive structural breakdown in an aqueous state for up to one year. The stabilizer composition was also effective for other FMDV strains, including serotypes A and Asia 1. With this stabilizer composition, FMD vaccine antigens could be flexibly preserved during the general production process, pending status under refrigeration and banking under ultrafreezing.

Studies on development of early immunity against type O FMD in naturally susceptible animals

FMD risk in the Russian Federation dictates the need for enhanced measures aiming to prevent the introduction of FMD virus and comprising systematic monitoring research and mass vaccination of susceptible animals in the buffer zone. Research into the development of vaccines for early protection confirm that their use induces the formation of virus-neutralizing antibodies in naturally susceptible animals in the outbreak area, which protects from FMD infection, limits its spread and contains it within the primary outbreak. Taking into account the high speed of the infection spread, such a control measure as using FMD vaccines which induce early protection should be adopted immediately after the occurrence of the outbreak. The article presents the results of the research into the formation of humoral immunity in naturally susceptible animals triggered by administration of inactivated emulsion FMD vaccines capable of ensuring early protection against type O FMD. Culture FMD virus of О/Primorsky/2012, О/Saudi Arabia/08 and О/Mongolia/2017 strains was used for vaccine production. Immunogenic activity of vaccines was tested in cattle, pigs, and sheep. It was found that monovalent emulsion FMD vaccine based on О/Mongolia/2017 strain induced the formation of virus-neutralizing antibodies in the quantity necessary to protect against the homologous strain in seven days after a single administration in the dose of 2 cm3 . Vaccines based on О/Saudi Arabia/08 and О/Primorsky/2012 FMDV strains can protect animals from infection with heterologous О/Mongolia/2017 strain at early stages if a double dose is administered. Vaccines based on the above-mentioned strains induce early immunity formation (seven days after vaccination) against type O FMD. We suggest using the given products in the zones of a higher risk of the virus introduction.

Hydrogen peroxide vapour is an effective replacement for Formaldehyde in a BSL4 Foot and mouth disease vaccine manufacturing facility.

An evaluation of the efficacy of 35% hydrogen peroxide vapour (HPV) against two strains of FMDV was conducted over a period of 6months. FMDV biological indicators were produced on-site using strains obtained from a commercial FMDV vaccine manufacturing process. FMDV biological indicators were distributed within a BSL4 laboratory and exposed to short duration hydrogen peroxide cycles. Variations in titre, support matrix (soiling), temperature and humidity were evaluated in a series of 16 exposures using over 200 individual FMDV indicators. Additional verification testing was performed in an operational material transfer lock to replicate real-world use. HPV was found to be efficacious in inactivating FMDV strains; the inoculum titre influenced the level of reduction achieved with the specified cycle. SIGNIFICANCE AND IMPACT OF THE STUDY: The classification of formaldehyde as a presumed human carcinogen has presented regulatory challenges for its continued use as a biocidal product. Institutions are actively seeking fumigants to replace formaldehyde and undertaking studies to validate biocidal efficacy, particularly in high-level biosafety facilities where the consequences of pathogen release can be extremely severe. This study builds on the already substantial scientific efficacy base of 35% hydrogen peroxide vapour and provides a comprehensive evaluation of the applicability of hydrogen peroxide vapour as a replacement for formaldehyde within a Foot & Mouth Disease (FMDV) vaccine manufacturing facility.

Validation of a high performance liquid chromatography method for quantitation of foot-and-mouth disease virus antigen in vaccines and vaccine manufacturing

Foot-and-mouth disease (FMD) is an infectious viral disease that affects the main meat and dairy production animals, including cattle, sheep, goats and swine. It is readily transmissible and countries where the disease is present suffer harsh international trade restrictions on livestock products and serious economic losses. Vaccines are important tools to contain outbreaks and maintain the status of free with or without vaccination, as defined by the World Organization for Animal Health (OIE). The efficacy of vaccines is reliant on the content and integrity of inactivated virus particles. The long-established method to quantify the viral content of vaccines along the manufacturing process and in the final product is the 140S sucrose density gradient analysis. This method has been a valuable tool for many decades. However, it requires gradient preparation for each sample, a lengthy ultracentrifugation and a manual UV reading of the gradient, rendering it highly operator dependent and almost impossible to automate. We present a method to quantify FMDV particles in vaccines and intermediate process samples that is based on separation of components by size exclusion high performance liquid chromatography (SE-HPLC) and measurement of virus by absorption at 254 nm. The method has been extensively validated; it is accurate, precise and linear. It is applicable to all FMDV strains and sample materials and has a good concordance with the 140S test. The proposed method uses off the shelf HPLC equipment and columns. It is easily automated for high throughput operation, affording a useful process analytical technology and a novel tool for control of final product by manufacturers and regulatory agencies.

Identification of a conserved linear epitope using monoclonal antibody against non-structural protein 3A of foot-and-mouth disease virus with potential for differentiation between infected and vaccinated animals

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral disease of cloven-hoofed animals. Vaccination is a key element in the control of FMD among countries where the disease is enzootic. Differentiating infected from vaccinated animals in herds after immunization is an important component of effective eradication strategies. Non-structural protein (NSP) 3A of FMDV is as part of a larger detected antigen that is used for this differential diagnosis. Here, we generated a specific monoclonal antibody (MAb) against FMDV non-structural protein called 3A10, and further defined the linear epitopes recognized by the MAb 3A10 using a series of peptides that expressed GST-fused protein. Using Western blot, it was showed that the 5-aa peptide 126ERTLP130 of 3A was the minimal epitope reactive to MAb 3A10. Alanine-scanning mutagenesis analysis revealed that Arg127 and Leu129 were crucial for MAb 3A10 binding to 126ERTLP130. Furthermore, sequence alignment analysis, indicated that the epitope 126ERTLP130 recognized by 3A10 was shown to be conserved among seven serotypes of FMDV strains. The synthetic peptide Elisa demonstrated that this epitope peptide could be recognized by sera from FMDV-infected pigs and cattle, but negative reactivity to unvaccinated and vaccinated healthy animal sera. Thus, the MAb reagents and the linear epitopes defined herein provide theoretical and technical support for the development of diagnostic tools for infection differentiating FMDV infected from vaccinated animals.

Serological Detection of FMD Serotypes by New Prepared Innovative Recombinant Hepta-Epitopic Peptide

Background: Foot-and-mouth disease (FMD) is a highly contagious and economically important disease that affects cloven-hoofed animals worldwide. In recent years, a series of outbreaks of FMD have occurred in many countries. Recombinant protein synthesis incorporating protective B- and T-cell epitopes are candidates for new safer and more effective (FMD) vaccines that have potential to provide protective immunity against diverse FMDV strains and to protect against future epidemics. Objectives: The aim of this study was to produce Recombinant Hepta-epitope peptide for detecting FMD O, A , Asia1 serotypes ,which can be used as a tool for diagnostic kits. Methods: In the present study, we designed and produced a Recombinant Hepta-epitopic peptide from FMDV epitopes of the viral proteins VP1, VP2, VP3, 3C (seven epitopic regions, amino acid residues VP1 140-160, Vp1 200-213, VP2 68-75 ,VP2 179-198 ,VP3129-148 ,VP3 190-199 , 3C121-135). Results: It was shown that the mentioned recombinant peptide could recognize the serum collected from cattle infected with FMD serotype A and O. Conclusion: Therefore we believe that this recombinant Hepta-epitopic peptide can be used for diagnostic serological assays. Furthermore this Recombinant peptide may be a potential candidate as an alternative vaccine against FMDV epidemic variants.

Foot-and-mouth disease virus type O specific mutations determine RNA-dependent RNA polymerase fidelity and virus attenuation

Previous studies have shown that the FMDV Asia1/YS/CHA/05 high-fidelity mutagen-resistant variants are attenuated (Zeng et al., 2014). Here, we introduced the same single or multiple-amino-acid substitutions responsible for increased 3Dpol fidelity of type Asia1 FMDV into the type O FMDV O/YS/CHA/05 infectious clone. The rescued viruses O-DA and O-DAMM are lower replication fidelity mutants and showed an attenuated phenotype. These results demonstrated that the same amino acid substitution of 3Dpol in different serotypes of FMDV strains had different effects on viral fidelity. In addition, nucleoside analogues were used to select high-fidelity mutagen-resistant type O FMDV variants. The rescued mutagen-resistant type O FMDV high-fidelity variants exhibited significantly attenuated fitness and a reduced virulence phenotype. These results have important implications for understanding the molecular mechanism of FMDV evolution and pathogenicity, especially in developing a safer modified live-attenuated vaccine against FMDV.

Evaluation of Infectivity, Virulence and Transmission of FDMV Field Strains of Serotypes O and A Isolated In 2010 from Outbreaks in the Republic of Korea.

Since the early 2000s outbreaks of foot-and-mouth disease (FMD) have been described in several previously FMD-free Asian nations, including the Republic of Korea (South Korea). One outbreak with FMD virus (FDMV) serotype A and two with serotype O occurred in South Korea in 2010/2011. The causative viruses belonged to lineages that had been spreading in South East Asia, far East and East Asia since 2009 and presented a great threat to the countries in that region. Most FMDV strains infect ruminants and pigs, as it happened during the outbreaks of FMDV serotype O in South Korea. Contrastingly, the strain of serotype A affected only ruminants. Based upon these findings, the intention of the work described in the current report was to characterize and compare the infectivity, virulence and transmission of both strains under laboratory conditions in cattle and pigs, by direct inoculation and contact exposure. As expected, FMDV serotype O was highly virulent in both cattle and swine by contact exposure and direct inoculation. Surprisingly, FMDV serotype A was highly virulent in swine, but was less infectious in cattle by contact exposure to infected swine or cattle. Interestingly, similar quantities of aerosolized FMDV RNA were detected during experiments with viruses of serotypes O and A. Specific virus-host interaction of A/SKR/2010 could affect the transmission of this strain to cattle, and this may explain in part the limited spread of the serotype A epizootic.

Phylogenic Analysis and Molecular Characterization of Slemani/Kurdistan/2013 Foot and Mouth Disease Virus Shows Circulation of New Genotype in Iraq

Foot-and-mouth disease (FMD) is endemic in Iraq. There were no much informations
 about FMD genotypes in Iraq. In this study, FMD type A virus was identified by RT-PCR from
 a cow in Kurdistan region. Viral protein1 (VP1) was amplified, sequenced and published in
 National Center for Biotechnology Information (NCBI) GenBank as Slemani/Kurdistan/2013.
 VP1 nucleic acid and amino acid sequence of Slemani/Kurdistan/2013 was comparatively
 analysed with all previously published Iraqi FMDV type A sequences available in NCBI Gen
 Bank from 1964 to 2013. The result indicated that Slemani/Kurdistan/2013 had 10 amino acid
 residues in immunogenic VP1 region, which were not present in other Iraqi FMD isolates.
 Furthermore, sequence alignment of Slemani/Kurdistan/2013 showed the amino acid identity
 ranged from 83 % to 93%, and the nucleotide identity ranged from 69% to 93% with other
 Iraqi FMDV type A isolates. Phylogenetic tree analysis showed that the identified field isolate
 differed from the other Iraqi subtype and could be placed in distinct sub lineages. It can be
 concluded that new genotype was circulated in Iraq. Therefore, monitoring the emergence of
 new FMDV strains in Iraq is important to enable appropriate vaccines selection.

Isolation and Molecular Characterization of Foot and Mouth Disease SAT2 Virus during Outbreak 2012 in Egypt

During April and May 2012, six outbreaks of FMD type SAT 2 were reported in Egyptian governorates: EL- Gharbiya (Kafr Qeretna, Al – Mahala Alkobra, Ebshaway Almalak, Qutoor), local cattle, buffaloes and dairy farms in Kafr El-Shaykh (Alsalmya, Fouah), AL - Minya (Village 3, Samalout, Mansafees, Abo Querkas), Dumyat (Ezbet 20, Kafr saad), ALexandria (Iraq Village, Al Aamerya), EL minufya (Monshaat Gerges, Ashmoun, Shatanoof, Ezbet Algalayla, Berket Alsabaa, Mahalet Sabak),Luxor (Al Gorna) and Al- Sharqiya (Al -Telleen, Menya Al kamh) Governorates. Suspected FMD virus samples (22 Tongue epithelium (TE), 12 vesicular fluid samples (VF)), in addition to, 10 cell culture grown virus of tongue epithelium origin, 6 cell culture grown virus of vesicular fluid origin. The assays used in this study involved CFT and Indirect ELISA typing kits for typing of FMDV virus samples; 3ABC-FMD for differentiation between vaccinated and infected animals in any revealed protective immune response in the examined sera from Egypt 2012 outbreak. PCR as an advanced confirmatory technique was done before submitting FMDV suspected clinical samples for Reference Laboratory of Foot and Mouth Disease in Pirbright, United Kingdom. Samples were isolated on primary bovine kidney and Baby Hamster kidney cell cultures, where all samples gave cytopathic effect. Also, all virus samples inoculated in swiss albino suckling baby mice showed pathognomonic paralysis effect of the virus in hind limbs of mice. Serum samples collected from suspected infected cattle were examined using FMD-3ABC. All positive samples to 3ABC antibodies indicated that the animals were infected by FMDV. FMD antibody detection revealed infection of 62%, 100%, 61%, 37.5%, 45.5% , 72.7% and 66.6% of sera collected from Al-Gharbiya, Kafr El-Shaykh, Dumyat, Alexandria, Al-Monufya, Luxor and Al-Sharqiya respectively. The obtained results of 3ABC antibodies were subsequently confirmed by Indirect ELISA Kit, which revealed that the causative agent of the outbreak was FMDV SAT2. The prefinal confirmatory technique was the Reverse transcription polymerase chain reaction (RT-PCR), which was done to detect clinically suspicious samples using target primers. Also, DNA sequencing of the current FMDV strain was performed and revealed that there was no significant divergence among the isolated strains as the divergence was 0.3 to 2.2% among them, and also there was no significant divergence between the isolated strains and FMDV SAT2/EGY/5/2012 and SAT2/EGY/15/2012 The closest percentage of identity of the isolated strains in comparison with reference strains was 89.4% with FMDV SAT2/LIB/2003. For official confirmation, the isolated viruses were sent to the Reference Laboratory for Foot and Mouth Disease in Pirbright, United Kingdom. Isolation of the causative agent is the first step for diagnosis of the disease. Finally, the PCR product was sequenced and analyzed using the gene bank data to confirm that the new out

Effects of amino acid substitutions in the VP2 B-C loop on antigenicity and pathogenicity of serotype Asia1 foot-and-mouth disease virus

BackgroundFoot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic variability. Studies of the antigenic diversity and determination of amino acid changes involved in this diversity are important to the design of broadly protective new vaccines. Although extensive studies have been carried out to explore the molecular basis of the antigenic variation of serotype O and serotype A FMDV, there are few reports on Asia1 serotype FMDV.MethodsTwo serotype Asia1 viruses, Asia1/YS/CHA/05 and Asia1/1/YZ/CHA/06, which show differential reactivity to the neutralizing monoclonal antibody (nMAb) 1B4, were subjected to sequence comparison. Then a reverse genetics system was used to generate mutant versions of Asia1/YS/CHA/05 followed by comparative analysis of the antigenicity, growth property and pathogenicity in the suckling mice.ResultsThree amino acid differences were observed when the structural protein coding sequences of Asia1/1/YZ/CHA/06 were compared to that of Asia1/YS/CHA/05. Site-directed mutagenesis and Immunofluorescence analysis showed that the amino acid substitution in the B-C loop of the VP2 protein at position 72 is responsible for the antigenic difference between the two Asia1 FMDV strains. Furthermore, alignment of the amino acid sequences of VP2 proteins from serotype Asia1 FMDV strains deposited in GenBank revealed that most of the serotype Asia1 FMDV strains contain an Asn residue at position 72 of VP2. Therefore, we constructed a mutant virus carrying an Asp-to-Asn substitution at position 72 and named it rD72N. Our analysis shows that the Asp-to-Asn substitution inhibited the ability of the rD72N virus to react with the MAb 1B4 in immunofluorescence and neutralization assays. In addition, this substitution decreased the growth rate of the virus in BHK-21 cells and decreased the virulence of the virus in suckling mice compared with the Asia1/YS/CHA/05 parental strain.ConclusionsThese results suggest that variations in domains other than the hyper variable VP1 G-H loop (amino acid 140 to 160) are relevant to the antigenic diversity of FMDV. In addition, amino acid substitutions in the VP2 influenced replicative ability and virulence of the virus. Thus, special consideration should be given to the VP2 protein in research on structure-function relationships and in the development of an FMDV vaccine.

A rapid molecular strategy for early detection and characterization of Vietnamese foot-and-mouth disease virus serotypes O, A, and Asia 1

A one-step RT-PCR method using newly designed primers VN-VP1F/VN-VP1R targeting the full VP1 capsid protein-coding gene, combined with direct sequencing of its PCR product, has been developed successfully for universal detection and characterization of Vietnamese FMDV serotypes O, A, and Asia 1 directly from clinical samples. The one-step RT-PCR amplified 821-bp dsDNA products covering the entire VP1 gene of FMDV serotypes O, A, and Asia 1. The obtained dsDNA products were suitable for direct sequencing, cloning, and other molecular epidemiology studies of Vietnamese FMDV strains, which eliminated the need for cell culture and virus purification. This one-step RT-PCR system was applied to detect and characterize 55 field FMDV strains, including 34 serotype O, 17 serotype A, and 4 serotype Asia 1 isolates collected from endemic outbreaks in Vietnam from 2005 to 2010. Interestingly, the PCR products obtained from the present PCR method could be used as DNA templates for the second PCR typing method using serotypes O, A, and Asia 1-specific primers (Le et al., 2011). The use of the second PCR amplification increased markedly the sensitivity of the test for FMDV detection. The present RT-PCR method promises to be an effective tool for molecular epidemiological studies of FMD in Vietnam.

Inactivation of foot-and-mouth disease virus in various bovine tissues used for the production of natural sausage casings

Bovine intestines, bladders and oesophagus are used for the production of natural casings ("beef casings") as edible sausage containers. Derived from cattle experimentally infected with FMDV (initial dosage 10(4) TCID(50)/mL, strain A Iran 97), these beef casings were treated with sodium chloride (NaCl) or phosphate supplemented salt (P-salt). In addition, different in-vitro experiments using beef casings were done on a small scale with other FMDV strains (A Turkey 06, C-Oberbayern and O(1) Manisa) as "proof of principle". Based on the combined results of the in-vivo and in-vitro experiments, it can be concluded that the storage period of 30 days at 20 °C in NaCl is sufficiently effective to inactivate a possible contamination with FMDV in beef casings and that the usage of P-salt does not clearly enhance the inactivation of FMDV infectivity. Storage of salted beef casings at about 20 °C for 30 days is already part of the Standard Operating Procedures (included in HACCP) of the international casing industry and can therefore be considered as a protective measure for the international trade in natural casings.

Molecular characterization of serotype Asia-1 foot-and-mouth disease viruses in Pakistan and Afghanistan; emergence of a new genetic Group and evidence for a novel recombinant virus

Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan. The FMD virus serotypes O, A and Asia-1 are responsible for the outbreaks in these countries. Diverse strains of FMDV, even within the same serotype, co-circulate. Characterization of the viruses in circulation can facilitate appropriate vaccine selection and tracing of outbreaks.The present study characterized foot-and-mouth disease serotype Asia-1 viruses circulating in Pakistan and Afghanistan during the period 1998–2009. Phylogenetic analysis of FMDV type Asia-1 revealed that three different genetic Groups of serotype Asia-1 have circulated in Pakistan during this time. These are Group-II, -VI and, recently, a novel Group (designated here as Group-VII). This new Group has not been detected in neighbouring Afghanistan during the study period but viruses from Groups I and -II are in circulation there. Using near complete genome sequences, from FMD viruses of serotypes Asia-1 and A that are currently circulating in Pakistan, we have identified an interserotypic recombinant virus, which has the VP2-VP3-VP1-2A coding sequences derived from a Group-VII Asia-1 virus and the remainder of the genome from a serotype A virus of the A-Iran05AFG-07 sub-lineage. The Asia-1 FMDVs currently circulating in Pakistan and Afghanistan are not efficiently neutralized by antisera raised against the Asia-1/Shamir vaccine strain. Thus, new Asia-1 vaccine strains may be required to block the spread of the current Asia-1 viruses.