Pathology, virulence-associated gene profiling, antimicrobial susceptibility, and pathogenicity of untypeable capsular serotypes of Pasteurella multocida isolated from slaughtered pigs of India.

Pasteurella multocida is widely distributed in all pig-rearing countries, affecting the economic viability and profitability of pig production. The present research highlights the molecular characterization and pathology of untypeable capsular serotypes of P. multocida in slaughtered pigs from prominent pig-rearing states of India. The prevalence of Pasteurellosis was 27.17% by Pasteurella multocida specific Pasteurella multocida specific PCR (PM-PCR). assay, while isolation rate was 7.62%. The microscopic lesions of bronchopneumonia, tonsillitis, and the presence of bacterial antigens in immunohistochemistry confirmed P. multocida with pathologies. In capsular typing, the majority of the isolates were untypeable with prevalence of 52.15% and 43.58% in molecular and microbiological methods, respectively. All the isolates showed the uniform distribution of virulence genes such as exbB, nanB, sodC, plpB, and oma87 (100%), while the variations were observed in ptfA, hasR, ptfA, pfhA, hsf-1, and plpE genes. The untypeable isolates showed higher prevalence of hsf-1 gene as compared to others. The untypeable serotypes showed a higher degree of resistance to ampicillin, amoxicillin, and penicillin antibiotics. The mouse pathogenicity testing of untypeable capsular isolates confirmed its pathogenic potential. The higher frequency of pathogenic untypeable isolates with antibiotic resistance profile might pose a serious threat to the pigs, and therefore, preventive measures should be adopted for effective control.

Development of TaqMan Probe-Based One-Step RT-qPCR Assay Targeting 2B-NSP Coding Region for Diagnosis of Foot-and-Mouth Disease in India.

A one-step TaqMan probe-based RT-qPCR assay in the duplex format simultaneously targeting FMD Virus (FMDV) 2B NSP-coding region and 18S rRNA housekeeping gene was developed and evaluated. The duplex RT-qPCR assay specifically detected FMDV genome in both infected cell culture suspensions and a variety of clinical samples such as FMD-affected tongue/feet epithelium, oral/nasal swabs, milk and oro-pharyngeal fluids. The RT-qPCR assay was found to be highly sensitive, since the assay was 105-fold more sensitive than the traditional FMDV detecting antigen-ELISA (Ag-ELISA) and 102-fold better sensitive than both virus isolation and agarose gel-based RT-multiplex PCR. In addition, the assay could detect up to 100 copies of FMDV genome per reaction. In the epithelial samples (n = 582) collected from the FMD-affected animals, the diagnostic sensitivity was 100% (95% CI 99-100%). Similarly, all the FMDV-negative samples (n = 65) tested were confirmed negative by the new RT-qPCR assay, corresponding to 100% diagnostic specificity (95% CI = 94-100%). Further, the duplex RT-qPCR assay proved to be robust, showing an inter-assay co-efficient of variations ranging from 1.4 to 3.56% for FMDV-2B gene target, and from 2 to 4.12% for 18S rRNA gene target. While analyzing FMDV-infected cell culture suspension, a fairly strong positive correlation (correlation coefficient = 0.85) was observed between 2B-based RT-qPCR and WOAH-approved 5'UTR RT-qPCR assays. Therefore, the one-step RT-qPCR assay developed here with an internal control could be used for rapid, effective, and reliable detection of FMDV in pan-serotypic manner, and has the potential for routine diagnosis of FMDV in high throughput manner.

A single amino acid substitution in the VP2 protein of Indian foot-and-mouth disease virus serotype O vaccine strain confers thermostability and protective immunity in cattle.

Foot-and-mouth disease (FMD) is a significant threat to animal health globally. Prophylactic vaccination using inactivated FMD virus (FMDV) antigen is being practised for the control in endemic countries. A major limitation of the current vaccine is its susceptibility to high environmental temperature causing loss of immunogenicity, thus necessitating the cold chain for maintenance of its efficacy. Hence, the FMD vaccine with thermostable virus particles will be highly useful in sustaining the integrity of whole virus particle (146S) during storage at 4°C. In this study, 12 recombinant mutants of Indian vaccine strain of FMDV serotype O (O/IND/R2/1975) were generated through reverse genetics approach and evaluated for thermostability. One of the mutant viruses, VP2_Y98F was more thermostable than other mutants and the parent FMDV. The oil-adjuvanted vaccine formulated with the inactivated VP2_Y98F mutant FMDV was stable up to 8 months when stored at 4°C and induced protective antibody response till dpv 180 after primary vaccination. It is concluded that the VP2_Y98F mutant FMDV was thermostable and has the potential to replace the parent vaccine strain.

One-step SYBR green-based real-time RT-PCR assay for detection of foot-and-mouth disease virus circulating in India.

Rapid, sensitive, and reliable laboratory detection of foot-and-mouth disease virus (FMDV) infection is essential for containing and controlling virus infection in any geographical area. In this report a SYBR green-based 3Dpol-specific one-step real-time RT-PCR (rRT-PCR) assay was developed for the pan-serotype detection of FMDV in India. The detection limit of the SYBR green-based rRT-PCR was 10-2 TCID50/50 µl, which is 10 times more sensitive than the traditional agarose gel electrophoresis-based RT-multiplex PCR (RT-mPCR). The standard curve exhibited a linear range across 8-log10 units of viral RNA dilution. The reproducibility and specificity of this assay were reasonably high suggesting that the 3Dpol-specific SYBR green rRT-PCR could detect FMDV genome specifically and with little run-to-run variation. The new 3Dpol-specific SYBR green rRT-PCR assay was evaluated alongside the established RT-mPCR using the archived FMDV isolates and clinical field samples from suspected FMD outbreaks. A perfect concordance was observed between the new rRT-PCR and the traditional RT-mPCR on viral RNA in the archived FMDV cell culture isolates tested. Furthermore, 73% of FMDV-suspected clinical samples were detected positive through the 3Dpol-specific SYBR green rRT-PCR, while the detection rate through the traditional RT-mPCR was 57%. Therefore, the SYBR green-based 3Dpol-specific one-step rRT-PCR could be considered as a valuable assay with higher diagnostic sensitivity to complement the routine assays that are being used for FMD virus diagnosis in India.

Infection and protection responses of deletion mutants of non-structural proteins of foot-and-mouth disease virus serotype Asia1 in guinea pigs.

The development of a negative marker vaccine against the foot-and-mouth disease virus (FMDV) will enhance the capabilities to differentiate vaccinated from infected animals and move forward in the progressive control pathway for the control of FMD. Here, we report the development of mutant FMDV of Asia1 with partial deletion of non-structural proteins 3A and 3B and characterization of their infectivity and protection response in the guinea pig model. The deleted FMDV Asia1/IND/63/1972 mutants, pAsiaΔ3A and pAsiaΔ3A3B1 were constructed from the full-length infectious clone pAsiaWT, the viable virus was rescued, and the genetic stability of the mutants was confirmed by 20 monolayer passages in BHK21 cells. The mutant Asia1 viruses showed comparable growth pattern and infectivity with that of AsiaWT in the cell culture. However, the AsiaΔ3A3B1 virus showed smaller plaque and lower virus titer with reduced infectivity in the suckling mice. In guinea pigs, the AsiaΔ3A3B1 virus failed to induce the disease, whereas the AsiaΔ3A virus induced typical secondary lesions of FMD. Vaccination with inactivated Asia1 mutant viruses induced neutralizing antibody response that was significantly lower than that of the parent virus on day 28 post-vaccination (dpv) in guinea pigs (P < 0.05). Furthermore, challenging the vaccinated guinea pigs with the homologous vaccine strain of FMDV Asia1 conferred complete protection. It is concluded that the mutant AsiaΔ3A3B1 virus has the potential to replace the wild-type virus for use as a negative marker vaccine after assessing the vaccine worth attributes in suspension cell and protective efficacy study in cattle.Key points• Deletion mutant viruses of FMDV Asia1, developed by PCR-mediated mutagenesis of NSP 3A and 3B1, were genetically stable.• The growth kinetics and antigenic relatedness of the mutant viruses were comparable with that of the wild-type virus.• Vaccination of guinea pigs with the deletion mutant viruses conferred complete protection upon challenge with the homologous virus.

Regenerated silica-based RNA purification columns to address the short supply of RNA purification kits for COVID-19 diagnosis.

BACKGROUND: RT-qPCR technique is the current world-wide method used for the early detection of SARS-CoV2 RNA in the suspected clinical samples. Viral RNA extraction is the key pre-analytical step for SARS-CoV2 detection which often achieved using commercial RNA-extraction kits. However, due to the COVID-19 pandemic, bulk production and the supply chains for the commercial RNA-extraction kit have been seriously compromised. The shortage of commercial RNA-extraction kit is even more acute in developing country. Furthermore, use of one-off design RNA-columns can generate plastic wastes that have an environmental pollution effect. METHODS AND RESULTS: To address these issues, in this study, we used warm alkaline solution containing Triton X-100 for the complete removal of the residual SARS-CoV2 RNA from the used RNA-binding silica column. Columns regenerated using the alkaline solution have the viral RNA purification capability that is comparable to the fresh silica columns. We also demonstrated that RNA-binding silica columns can be regenerated and reused for a minimum of five-times. CONCLUSIONS: Therefore, the use of the RNA-column regeneration method may benefits several SARS-CoV2 diagnostic laboratories throughout the world by cutting down the requirement of commercial RNA-purification column.

Development and Validation of a Mucosal Antibody (IgA) Test to Identify Persistent Infection with Foot-and-Mouth Disease Virus.

It is well known that approximately 50% of cattle infected with foot-and-mouth disease (FMD) virus (FMDV) may become asymptomatic carrier (persistently infected) animals. Although transmission of FMDV from carrier cattle to naïve cattle has not been demonstrated experimentally, circumstantial evidence from field studies has linked FMDV-carrier cattle to cause subsequent outbreaks. Therefore, the asymptomatic carrier state complicates the control and eradication of FMD. Current serological diagnosis using tests for antibodies to the viral non-structural proteins (NSP-ELISA) are not sensitive enough to detect all carrier animals, if persistently infected after vaccination and do not distinguish between carriers and non-carriers. The specificity of the NSP ELISA may also be reduced after vaccination, in particular after multiple vaccination. FMDV-specific mucosal antibodies (IgA) are not produced in vaccinated cattle but are elevated transiently during the acute phase of infection and can be detected at a high level in cattle persistently infected with FMDV, irrespective of their vaccination status. Therefore, detection of IgA by ELISA may be considered a diagnostic alternative to RT-PCR for assessing FMDV persistent infection in ruminants in both vaccinated and unvaccinated infected populations. This study reports on the development and validation of a new mucosal IgA ELISA for the detection of carrier animals using nasal, saliva, and oro-pharyngeal fluid (OPF) samples. The diagnostic performance of the IgA ELISA using nasal samples from experimentally vaccinated and infected cattle demonstrated a high level of specificity (99%) and an improved level of sensitivity (76.5%). Furthermore, the detection of carrier animals reached 96.9% when parallel testing of samples was carried out using both the IgA-ELISA and NSP-ELISA.

Emergence of foot-and-mouth disease virus serotype Asia1 group IX in India.

Foot-and-mouth disease virus (FMDV) serotype Asia1 is prevalent in India and is responsible for a minor proportion of FMD outbreaks. Globally, serotype Asia1 is grouped into nine different groups (GI-IX) based on genetic analysis. In India, only Asia1/G-III and Asia1/G-VIII have been documented so far. Phylogenetic analysis of recent serotype Asia1 isolates from India revealed the emergence of Asia1/G-IX. The Asia1/G-IX lineage shares recent common ancestry with Asia1/G-VIII dating to 2016. The root state posterior probabilities of Asia1/G-VIII are inclusive and there may have been either an incursion of the virus from Bangladesh, where it was first identified, or in situ evolution of the virus within India, which is an intriguing possibility.

Genome Sequences of Seven Foot-and-Mouth Disease Virus Isolates Reveal Diversity in the O/ME-SA/Ind2001 Lineage in India between 1997 and 2009.

We report the genome sequences of seven foot-and-mouth disease (FMD) virus (FMDV) isolates collected in India between 1997 and 2009. The strains represented four sublineages within the O/ME-SA/Ind2001 lineage. These viruses provide insights into FMDV diversity and evolution in India and may influence future control measures, including vaccine selections.

Immunogenicity and protective efficacy of 3A truncated negative marker foot-and-mouth disease virus serotype A vaccine.

Foot-and-mouth disease (FMD) is a highly contagious, economically significant disease of cloven-hoofed animals caused by FMD virus (FMDV) of the Picornaviridae family. Vaccination of susceptible animals with inactivated virus vaccine is the standard practice for disease control. The prophylactic use of the inactivated vaccines has reduced the disease burden in many countries endemic to FMD. In the process of implementation of the mass vaccination program and disease eradication, it is essential to differentiate infected from vaccinated animals (DIVA) where a large proportion of the animal population is vaccinated, and disease-free zones are being established, to help in sero-surveillance of the disease. In such a scenario, the use of a negative marker vaccine is beneficial to rule out false-positive results in a disease-free zone. Here we report the construction and rescue of an infectious cDNA clone for FMDV serotype A Indian vaccine strain lacking 58 amino acid residues (87-144 amino acid position) in the carboxy-terminal region of the viral 3A protein. The recombinant deletion mutant virus showed similarity in the antigenic relationship with the parental strain. Immunization of guinea pigs with the inactivated vaccine formulated using the deletion mutant virus induced potent immune response with 100% protective efficacy upon challenge with homologous virus. Further, we show that sera from the guinea pigs infected with the deletion mutant virus did not show reactivity in an indirect ELISA test targeting the deleted portion of 3A protein. We conclude that the recombinant deletion mutant virus vaccine along with the newly developed companion indirect ELISA targeting portion of FMDV 3A protein could be useful in the implementation of a precise DIVA policy in our country when we reach FMD free status with vaccination.

Differential antibody responses to the major antigenic sites of FMD virus serotype O after primo-vaccination, multiply-vaccination and after natural exposure.

Foot and mouth disease (FMD) virus serotype O is the predominant cause of FMD outbreaks in several regions of the world including India. Five independent neutralizing antigenic sites have been identified on the capsid surface of FMD virus serotype O. The relative importance of these neutralizing sites in eliciting antibody responses in the polyclonal sera collected from un-infected vaccinated (both primo and multiply-vaccinated) and naturally infected cattle populations were determined through a combination of reverse genetics and serology. The known critical amino acid residues present on the five antigenic sites of FMD virus serotype O Indian vaccine strain O IND R2/1975 were mutated through site-directed mutagenesis. The mutant viruses were rescued in cell-culture and analyzed through virus-neutralization assays along with parent virus using the polyclonal sera collected from three groups of cattle. In the polyclonal sera from primo-vaccinated cattle, significantly higher level of antibodies were directed towards antigenic site 2. In contrast, in polyclonal sera from multiply vaccinated animals, both antigenic sites 1 and 2 were equally important. In case of naturally infected animals, antibody responses were elicited against all the five antigenic sites. Although a drop in neutralization titres was observed for all the mutants, in one instance, increase in titre was noticed for a site 3 mutant. The findings from this study extend our knowledge on the antibody immunodominace following FMDV vaccination and infection, and may improve our strategies for vaccine strain selection and rational vaccine design.

Genome Sequences of 18 Foot-and-Mouth Disease Virus Outbreak Strains of Serotype O Sublineage Ind2001d from India, 2013 to 2014.

We report the full polyprotein-coding sequences and partial untranslated regions (UTRs) of 18 foot-and-mouth disease (FMD) viruses from 4 outbreaks in India in 2013 and 2014. All strains grouped within the O/ME-SA/Ind2001d sublineage. These genomes update knowledge of FMD virus (FMDV) diversity in South Asia and may contribute to molecular epidemiology studies and vaccine selections.

Genetic and antigenic variation of foot-and-mouth disease virus during persistent infection in naturally infected cattle and Asian buffalo in India.

The role of foot-and-mouth disease virus (FMDV) persistently infected ruminants in initiating new outbreaks remains controversial, and the perceived threat posed by such animals hinders international trade in FMD-endemic countries. In this study we report longitudinal analyses of genetic and antigenic variations of FMDV serotype O/ME-SA/Ind2001d sublineage during naturally occurring, persistent infection in cattle and buffalo at an organised dairy farm in India. The proportion of animals from which FMDV RNA was recovered was not significantly different between convalescent (post-clinical) and sub-clinically infected animals or between cattle and buffalo across the sampling period. However, infectious virus was isolated from a higher proportion of buffalo samples and for a longer duration compared to cattle. Analysis of the P1 sequences from recovered viruses indicated fixation of mutations at the rate of 1.816 x 10-2substitution/site/year (s/s/y) (95% CI 1.362-2.31 x 10-2 s/s/y). However, the majority of point mutations were transitional substitutions. Within individual animals, the mean dN/dS (ω) value for the P1 region varied from 0.076 to 0.357, suggesting the selection pressure acting on viral genomes differed substantially across individual animals. Statistical parsimony analysis indicated that all of the virus isolates from carrier animals originated from the outbreak virus. The antigenic relationship value as determined by 2D-VNT assay revealed fluctuation of antigenic variants within and between carrier animals during the carrier state which suggested that some carrier viruses had diverged substantially from the protection provided by the vaccine strain. This study contributes to understanding the extent of within-host and within-herd evolution that occurs during the carrier state of FMDV.

Mutation in the VP2 gene of P1-2A capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype O.

Foot-and-mouth disease (FMD) is an economically important, global disease of cloven-hoofed animals. The conventional vaccine could bring down the incidence of disease in many parts of the world but has many limitations and in India, the disease is enzootic. More promisingly, the alternate vaccine candidates, virus-like particles (VLPs) are as immunogenic as a native virus but are more labile to heat than the live virus capsids. To produce stable VLPs, a single amino acid residue was mutated at 93 and 98 positions at VP2 inter-pentamer region of the P1-2A gene of FMD virus serotype O (IND/R2/75). The mutated capsid protein was expressed in insect cells and characterized for temperature and varying pH stability. Out of S93Y, S93F, S93C, S93H, and Y98F mutant, VLPs, S93Y, S93F, and Y98F showed improved stability at 37 °C for 75 days compared to wild capsid, which was evaluated by sandwich ELISA. Further, the stability analysis of purified VLPs either by differential scanning fluorescence (DSF) stability assay at different temperatures and pH conditions or by dissociation kinetics showed that the Y98F mutant VLPs were more stable than S93Y, S93F, S93C, and S93H mutant and wild-type VLPs. Immunization of guinea pigs with Y98F VLPs induced neutralizing antibodies and 60% of the animals were protected from the FMDV "O" 100 GPID50 challenge virus.

Evidence of subclinical foot-and-mouth disease virus infection in young calves born from clinically recovered cow under natural condition.

Foot-and-mouth disease (FMD) is a highly contagious and economically important, transboundary viral disease of cloven-hoofed animals. It is known that an asymptomatic, persistent FMD virus (FMDV) infection may occur subsequent to acute or subclinical FMDV infection in adult ruminants. However, virus persistence in young calves has not been studied. In the current investigation, FMDV infection parameters were examined for calves born to FMD-clinically recovered cows (CRC), asymptomatic cows from infected herds (ASC) and cows from with no history of FMD (NHF). The study was conducted in natural condition after FMD outbreaks in two dairy herds in India. No calves described herein had any clinical signs of FMD. Six out of 12 calves born to CRC had detectable FMDV RNA in oesophageal-pharyngeal fluid consistent with asymptomatic FMDV infection. Three of the 12 calves of CRC group had seroreactivity against FMDV non-structural proteins. One calf had detectable FMDV RNA at two consecutive samplings at 2 months apart. However, infectious FMDV was not isolated from any calf in the study. None of the calves in the ASC or NHF groups had any evidence of FMDV infection. Overall, these data are consistent with earlier report on calves having been infected in utero. Further investigation of FMDV persistence in calves under controlled conditions may lead to greater understanding of the viral pathogenesis.

Uncleaved 2A-peptide of foot-and-mouth disease virus can display foreign epitope-tag at the virion surface.

Foot-and-mouth disease virus (FMDV) capsid precursor protein P1-2A is cleaved by viral-encoded 3C protease (3Cpro) to generate VP0, VP3, VP1 and 2A proteins. It was reported earlier that substitution of a single amino acid residue within the 2A peptide sequence (L2P) blocked the 3Cpro mediated VP1/2A cleavage and produced 'self-tagged' FMDV particles containing uncleaved 2A-peptide. To determine whether the uncleaved 2A-peptide can function as a target structure to harbour and display exogenous epitope on FMDV particles, a full-length FMDV cDNA clone containing a HA-tag within the uncleaved 2A-peptide sequence was constructed. Subsequently, chimeric marker FMDV, displaying a HA-tag on the viral surface was rescued through reverse genetics approach. The 2A-HA epitope tag-inserted recombinant chimeric FMDV serotype O was genetically stable through up to ten serial passages in cell culture and exhibited growth properties similar to the parental virus. Furthermore the surface displayed HA-epitope tag was able to react with anti-HA antibodies as determined by various immuno-assays. The results from our study suggest that the uncleaved 2A-peptide of FMDV is suitable to present foreign antigenic epitopes on the surface of FMD virion.

Antigenic variability of foot-and-mouth disease virus serotype O during serial cytolytic passage.

The emergence and disappearance of antigenic variants of foot-and-mouth disease virus (FMDV) during a field outbreak occurs periodically due to the volatile nature of its genome. In the present analysis, change in antigenic behavior of serotype O FMDV during the serial cytolytic passage in the absence of immune pressure was observed. Initially, the isolate showed a poor antigenic match (relationship value <0.3) with the serotype O vaccine strain and upon serial passage increase in relationship value was observed. Comparison of capsid sequence revealed substitution at four positions (VP3:K58 â†’ E and P158 â†’ S, VP1:E83 â†’ K and R172 â†’ Q) acquired during the serial passage. Examination of passage level and amino acid substitution revealed the critical role of position VP3-58 that was identified earlier as crucial for antigenic site IV, in the observed antigenic variability. The role of position VP3-58 was further confirmed using reverse genetics approach.

Foot-and-Mouth Disease Virus-Associated Abortion and Vertical Transmission following Acute Infection in Cattle under Natural Conditions.

Foot-and-mouth disease (FMD) is a highly contagious and economically important viral disease of cloven-hoofed animals, including domestic and wild host species. During recent FMD outbreaks in India, spontaneous abortions were reported amongst FMD-affected and asymptomatic cows. The current study was an opportunistic investigation of these naturally occurring bovine abortions to assess causality of abortion and vertical transmission of FMDV from infected cows to fetuses. For this purpose, fetal tissue samples of eight abortuses (heart, liver, kidney, spleen, palatine tonsil, umbilical cord, soft palate, tongue, lungs, and submandibular lymph node) were collected and screened by various detection methods, including viral genome detection, virus isolation, and immunomicroscopy. Amongst these cases, gross pathological changes were observed in 3 abortuses. Gross pathological findings included blood-tinged peritoneal and pleural effusions and myocarditis. Hearts of infected calves had mild to moderate degeneration and necrosis of the myocardium with moderate infiltration by mixed inflammatory cells. Localization of FMDV antigen was demonstrated in lungs and soft palate by immunomicroscopy. FMDV serotype O viral genome was recovered from 7 of 8 cases. Infectious FMDV serotype O was rescued by chemical transfection of the total RNA extracted from three soft palate samples and was sequenced to confirm 100% identity of the VP1 (capsid) coding region with isolates collected from infected cattle during the acute phase of infection. Based upon these findings, it may be concluded that FMDV-associated abortion occurred among the infected pregnant cows included within this study and FMDV was subsequently transmitted vertically to fetuses. This is the first documentation of FMDV-associated abortions in cattle.

Evaluation of FTA(®) card for the rescue of infectious foot-and-mouth disease virus by chemical transfection of extracted RNA in cultured cells.

Foot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. Inadequate storage and shipment of suspected clinical samples can compromise the ability to detect and characterise FMD virus (FMDV) in endemic countries, thereby, leading to the loss of valuable virological and epidemiological data. This study, investigates the potential of using FTA(®) cards for dry transportation of clinical samples and subsequent recovery of infectious FMDV by chemical transfection of FTA(®) card fixed RNA as an alternative to the conventional cell culture based virus isolation method. A higher proportion of infectious FMDV was rescued from clinical samples (cell culture isolates, tongue epithelial suspension and impression smears) by the FTA(®) card fixed RNA transfection method (76%) compared to the conventional cell culture based virus isolation (56%), suggesting a better performance of the current RNA transfection procedure. Furthermore, it was possible to rescue live virus by the transfection of RNA extracted from FTA(®) card impregnated with clinical samples that had been stored at varying temperature (4-37 °C) up to a period of six weeks. The VP1 sequence data and antigenic relationships with the vaccine strains, between viruses rescued by FTA(®) card fixed RNA transfection and conventional cell culture, were comparable. Therefore, these results support the use of the FTA(®) card for the economic, dry, non-hazardous transport of FMD suspected clinical samples from the site of collection to national/international reference laboratories.

Chimeric foot-and-mouth disease virus serotype O displaying a serotype Asia1 antigenic epitope at the surface.

OBJECTIVE: To determine whether the G-H loop of foot-and-mouth disease virus (FMDV) serotype O can function as a target structure to harbour and display serotype Asia1 antigenic epitope at the surface. RESULTS: Using reverse genetics, FMDV serotype O IND R2/1975 displaying a FMDV serotype Asia1 B cell epitope at the capsid surface was constructed. The epitope-inserted recombinant chimeric virus was genetically stable up to ten serial passages in cell culture and exhibited growth properties similar to the parental serotype O virus. Furthermore, the surface-displayed Asia1 epitope able to react with serotype Asia1 specific antibodies in a competitive ELISA. Importantly, the recombinant chimeric virus showed neutralizing activity to both serotype O and Asia1 polyclonal antibodies. CONCLUSION: The capsid protein of FMDV serotype O can effectively display potent epitope of other serotypes, making this an attractive approach for the design of new generation bi-valent FMD vaccines.