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.

BACKGROUND: Outbreaks 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. METHODS: Specific 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. RESULT: Each 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. CONCLUSIONS: The 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.

Research Alliance for Veterinary Science and Biodefense BSL-3 Network (RAV3N): Report on network origin and phase I activities.

Recent disease events have heightened awareness for the need for collaboration between the nation's public health and veterinary infectious disease communities to improve preparedness for current and future biological threats. To address this need, the U.S. Department of Agriculture's National Bio- and Agro-Defense Facility (USDA NBAF) has partnered with Texas A&M University through its Global Health Research Complex (TAMGHRC) to establish the Research Alliance for Veterinary Science and Biodefense BSL-3 Network (RAV3N). As a collaborative network of U.S. university and federal BSL-3Ag/BSL-3/BSL-4 laboratory research facilities, the objective of RAV3N is to establish strategic and coordinated approaches for harnessing collective large-animal biocontainment infrastructure and research capacity to improve bio-surveillance, diagnostics, and countermeasure development against high-consequence pathogens of veterinary and zoonotic importance. Here, we describe the origin and development of RAV3N, detail phase I activities, and summarize the proceedings of its first membership meeting held in August 2022.

Molecular and Pathological Characterization of Classical Swine Fever Virus Genotype 2 Strains Responsible for the 2013-2018 Outbreak in Colombia.

Classical swine fever (CSF) is a highly contagious transboundary viral disease of domestic and wild pigs. Despite mass vaccination and continuous eradication programs, CSF remains endemic in Asia, some countries in Europe, the Caribbean and South America. Since June 2013, Northern Colombia has reported 137 CSF outbreaks, mostly in backyard production systems with low vaccination coverage. The purpose of this study was to characterize the virus responsible for the outbreak. Phylogenetic analysis based on the full-length E2 sequence shows that the virus is closely related to CSF virus (CSFV) genotype 2.6 strains circulating in Southeast Asia. The pathotyping experiment suggests that the virus responsible is a moderately virulent strain. The 190 nucleotide stretch of the E2 hypervariable region of these isolates also shows high similarity to the CSFV isolates from Colombia in 2005 and 2006, suggesting a common origin for the CSF outbreaks caused by genotype 2.6 strains. The emergence of genotype 2.6 in Colombia suggests a potential transboundary spread of CSFV from Asia to the Americas, complicating the ongoing CSF eradication efforts in the Americas, and emphasizes the need for continuous surveillance in the region.

Meat Exudate for Detection of African Swine Fever Virus Genomic Material and Anti-ASFV Antibodies.

African swine fever (ASF) is one of the most important viral diseases of pigs caused by the ASF virus (ASFV). The virus is highly stable over a wide range of temperatures and pH and can survive in meat and meat products for several months, leading to long-distance transmission of ASF. Whole blood, serum, and organs from infected pigs are used routinely as approved sample types in the laboratory diagnosis of ASF. However, these sample types may not always be available. Here, we investigated meat exudate as an alternative sample type for the detection of ASFV-specific nucleic acids and antibodies. Pigs were infected with various ASFV strains: the highly virulent ASFV Malawi LIL 18/2 strain, the moderately-virulent ASFV Estonia 2014 strain, or the low-virulent ASFV OURT/88/3 strain. The animals were euthanized on different days post-infection (dpi), and meat exudates were collected and tested for the presence of ASFV-specific nucleic acids and antibodies. Animals infected with the ASFV Malawi LIL 18/2 developed severe clinical signs and succumbed to the infection within seven dpi, while pigs infected with ASFV Estonia 2014 also developed clinical signs but survived longer, with a few animals seroconverting before succumbing to the ASFV infection or being euthanized as they reached humane endpoints. Pigs infected with ASFV OURT/88/3 developed transient fever and seroconverted without mortality. ASFV genomic material was detected in meat exudate from pigs infected with ASFV Malawi LIL 18/2 and ASFV Estonia 2014 at the onset of viremia but at a lower amount when compared to the corresponding whole blood samples. Low levels of ASFV genomic material were detected in the whole blood of ASFV OURT/88/3-infected pigs, and no ASFV genomic material was detected in the meat exudate of these animals. Anti-ASFV antibodies were detected in the serum and meat exudate derived from ASFV OURT/88/3-infected pigs and in some of the samples derived from the ASFV Estonia 2014-infected pigs. These results indicate that ASFV genomic material and anti-ASFV antibodies can be detected in meat exudate, indicating that this sample can be used as an alternative sample type for ASF surveillance when routine sample types are unavailable or are not easily accessible.

Rapid and highly sensitive portable detection of African swine fever virus.

African swine fever (ASF) continues to spread across Asia, devastating pig populations. The disease is nearly 100% fatal in pigs, and currently, there is no effective vaccine available. Therefore, early detection of ASF is critical for effective disease control. The testing process usually requires samples to be shipped to a central laboratory, which may take many hours of travel or shipping time, delaying the results needed for a rapid response. The ability to confirm ASFV-infected animals on-site or in a regional laboratory that has limited technical capacity and/or infrastructure should eliminate these issues. This study describes the successful transfer of a highly sensitive and specific laboratory-validated real-time PCR assay to a portable pen-side thermocycler, which can be operated in the field for rapid detection of ASFV following a quick manual nucleic acid extraction from a wide array of clinical samples including aggregate samples such as oral fluids. The performance of the portable assay was comparable to the laboratory-based assay. The true portability of the assay was evaluated in seven ASF-suspected farms in Vietnam by testing eighty-nine freshly collected whole blood samples on-site. The results obtained on-site were in agreement with the laboratory data obtained the following day. Availability of this field-deployable molecular assay would eliminate the need to ship samples to a central laboratory, when rapid laboratory results are required, ultimately improving the response time.

Evaluation of oral fluid as an aggregate sample for early detection of African swine fever virus using four independent pen-based experimental studies.

The sustained spread of African swine fever (ASF) virus throughout much of the world has made ASF a global animal health priority, with an increased emphasis on enhancing preparedness to prevent, detect and respond to a potential outbreak of ASF virus (ASFV). In the event of ASFV entry to the North American swine population, enhanced surveillance and diagnostic testing strategies will be critical to facilitate progressive response and eradication of the disease. Compared to individual animal sampling, pen-based oral fluid collection for active surveillance is a non-invasive alternative that is less resource and time-intensive. To evaluate the feasibility of using rope-based oral fluid for early detection of ASFV, four independent animal experiments were conducted in weaned pigs housed in numbers that mimic the industry settings, utilising either highly virulent ASFV Georgia 2007/1 strain or moderately virulent ASFV Malta'78 strain. Pen-based oral fluid and individual oropharyngeal swabs were collected daily and blood samples from each animal were collected every other day. All samples were subsequently tested for ASFV by real-time PCR. ASFV genome was detected in individual blood samples as early as one day post-infection and detected in oral fluids at low-to-moderate levels as early as 3-5 days post-infection in all four independent experiments. These results suggest that pen-based oral fluid samples may be used to supplement the use of traditional samples for rapid detection of ASFV during ASF surveillance.

Diagnosis of caprine mucopolysaccharidosis type IIID by real-time polymerase chain reaction-based genotyping.

Mucopolysaccharidosis type IIID is caused by a deficiency of N-acetylglucosamine-6-sulfatase, which is one of the enzymes involved in the catabolism of heparin sulfate. Simple molecular marker assays underpin modern routine animal breeding and research activities worldwide. With the rapid growth of single nucleotide polymorphism (SNP) resources for many important animal genetic disorders, the availability of routine assays for genotyping SNPs is of increased importance. In the current study, real-time polymerase chain reaction (PCR) is demonstrated to provide a valuable approach as a rapid and accurate alternative to a previously developed gel-based PCR as a straightforward and efficient assay for the diagnosis of caprine mucopolysaccharidosis IIID.

Improvement and optimization of a multiplex real-time reverse transcription polymerase chain reaction assay for the detection and typing of Vesicular stomatitis virus.

An improvement to a previously reported real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay for the detection of Vesicular stomatitis virus (VSV) is described. Results indicate that the new assay is capable of detecting a panel of genetically representative strains of VSV present in North, Central, and South America. The assay is specific for VSV and allows for simultaneous differentiation between Vesicular stomatitis Indiana virus and Vesicular stomatitis New Jersey virus. This real-time RT-PCR is able to detect current circulating strains of VSV and can be used for rapid diagnosis of VSV and differentiation of VSV from other vesicular diseases, such as foot-and-mouth disease.

An improved real-time polymerase chain reaction for the simultaneous detection of all serotypes of Epizootic hemorrhagic disease virus.

Epizootic hemorrhagic disease virus (EHDV) is a significant pathogen of wild and sometimes domestic ungulates worldwide. Rapid and reliable methods for virus detection and identification play an essential part in the control of epizootic hemorrhagic disease (EHD). In the present study, a 1-step real-time polymerase chain reaction (PCR) group-specific assay was developed. The assay detects genome segment 5 (NS1) from all of the 8 serotypes of EHDV. Assay sensitivity was evaluated relative to a conventional gel-based nested PCR using cell culture-derived virus and diagnostic samples from clinically affected white-tailed deer (Odocoileus virginianus). The assay reliably amplified the NS1 gene from any of the EHDV strains tested, including isolates from each of the 8 EHDV serotypes. No cross-reactions were detected when all 24 serotypes of Bluetongue virus, a closely related member of the genus Orbivirus, were tested. A panel of 76 known EHDV-positive clinical samples was used to compare the performance of the assay relative to a previously reported real-time PCR assay. Results indicated that there was no statistically significant difference between the threshold cycle values obtained with both assays. A collection of 178 diagnostic samples submitted for EHD diagnosis was also used for test evaluation. The assay could be applied for rapid detection of EHDV in clinical samples from susceptible ruminants during an outbreak of the disease. In addition, this PCR assay has the benefits of being reliable and simple and could provide a valuable tool for studying the epidemiology of EHDV infection in susceptible ruminants by facilitating the detection of EHDV, regardless of the serotype.

Development and evaluation of swine vesicular disease isotype-specific antibody ELISAs based on recombinant virus-like particles.

Swine vesicular disease (SVD) is a contagious viral disease of pigs. The clinical signs of SVD are indistinguishable from other vesicular diseases, such as senecavirus A infection (SVA) and foot-and-mouth disease (FMD). Rapid and accurate diagnostic tests of SVD are considered essential in countries free of vesicular diseases. Competitive ELISA (cELISA) is the serological test used routinely. However, although cELISA is the standard test for SVD antibody testing, this test produces a small number of false-positive results, which caused problems in international trade. The current project developed a SVD isotype antibody ELISA using recombinant SVD virus-like particles (VLP) and an SVD-specific monoclonal antibody (mAb) to reduce the percentage of false positives. The diagnostic specificities of SVD-VLP isotype ELISAs were 98.7% and 99.6% for IgM and IgG. The SVD isotype ELISAs were SVD-specific, without cross-reactivity to other vesicular diseases. A panel of 16 SVD-positive reference sera was evaluated using the SVD-VLP isotype ELISAs. All sera were correctly identified as positive by the two combined SVD-VLP isotype ELISAs. Comparison of the test results showed a high level of correlation between the SVDV antigen isotype ELISAs and SVD-VLP isotype ELISAs. 303 sera from animals lacking clinical signs and history of SVDV exposure were identified positive using SVD cELISA. These samples were examined using SVD-VLP isotype ELISAs. Of the 303 serum samples, five were positive for IgM, and five of 303 were positive for IgG. Comparable to virus neutralization test results, SVD isotype ELISAs significantly reduced the false-positive samples. Based on above test results, the combined use of cELISA and isotype ELISAs can reduce the number of false-positive samples and the use of time-consuming virus neutralization tests, with benefit for international trade in swine and related products.

Optimization of immunohistochemical and fluorescent antibody techniques for localization of Foot-and-mouth disease virus in animal tissues.

Immunohistochemical (IHC) and fluorescent antibody (FA) techniques were optimized for the detection of Foot-and-mouth disease virus (FMDV) structural and nonstructural proteins in frozen and paraformaldehyde-fixed, paraffin-embedded (PFPE) tissues of bovine and porcine origin. Immunohistochemical localization of FMDV was compared with 7 detection systems, 8 primary antibodies, and 11 epitope retrieval techniques. All serotypes tested (O, A, Asia, C [cryosection]; O, A, Asia [PFPE]) were localized in association with mature vesicles. Multi-label FA was used in conjunction with IHC and conventional histopathology to characterize vesicle maturation in 4 steers and 2 pigs experimentally infected with FMDV. At the edge of advancing vesicles, a consistent finding was acantholytic degeneration of basal keratinocytes surrounding dermal papillae with suprabasilar clefts and microvesiculation. Progression of microvesiculation led to coalescence with the expanding vesicle. Cells at the leading edge of vesicles were positive for FMDV antigens by IHC and FA. Cell marker profile of these cells by FA was consistent with keratinocytes (i.e., cytokeratin [CK]-positive, S100-negative, MHC-II-negative). In rare instances, CK-negative, MHC-II- positive, and FMDV-positive cells (presumptive dendritic cells or macrophages) were identified within dermis subjacent to vesicles.

Development and application of monoclonal antibodies against avian influenza virus nucleoprotein.

Rapid and accurate diagnosis of avian influenza (AI) infection is important for an understanding epidemiology. In order to develop rapid tests for AI antigen and antibody detection, two monoclonal antibodies (mAbs) against influenza nucleoprotein (NP) were produced. These mAbs are designated as F26-9 and F28-73 and able to recognize whole AI virus particles as well as the recombinant NP. Both of the mAbs were tested in a slot blot for their reactivity against 15 subtypes of influenza virus; F28-73 reacted with all tested 15 subtypes, while F26-9 failed to react with H13N6 and H15N8. The mAb binding epitopes were identified using truncated NP recombinant proteins and peptide array techniques. The mAb F26-9 reacted with NP-full, NP-1 (638bp), NP-2 (315bp), NP-4 (488bp), and NP-5 (400bp) in the Western blot. The peptide array results demonstrated that the mAb F26-9 reacted with NP peptides 15-17 corresponding to amino acids 71-96. The mAb F28-73 recognized the NP-full, -1 and -4 fragments, but failed bind to NP-2, -3, -5, and any peptides. This antibody-binding site is expected to be contained within 1-162 amino acids of AI NP, although the exact binding epitope could not be determined. The two mAbs showed reactivity with AI antigen in immunofluorescence, immunohistochemistry and immune plaque assays. Immune response of AI infected animals was determined using the mAb F28-73 in a cELISA. All tested chickens were positive at 11 days post-infection and remained positive until the end of the experiment on day 28 (>50% inhibition). The two mAbs with different specificities are appropriate for developing various tests for diagnosis of AI infection.

Serotype-independent detection of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) causes a highly contagious vesicular disease affecting cloven hoofed animals and is considered the most economically important disease worldwide. Recent FMD outbreaks in Europe and Taiwan and the associated need for rapid diagnostic turnaround have identified limitations that exist in current diagnostic capabilities. To aid improved diagnosis, a serotype-independent FMDV antigen capture assay was developed using antibodies directed against a highly conserved cross-reactive protein fragment (1AB') located within the structural protein 1AB. Cattle sera raised against all 7 serotypes of FMDV bound purified 1AB' demonstrating its immunogenicity in infected animals. Polyclonal anti-1AB' antiserum was produced in chickens and applied as a universal detector of FMDV antigen. Western blot analysis and ELISA both demonstrated that anti-1AB' serum could recognize FMDV antigens independent of serotype. Two recently characterized anti-FMDV monoclonal antibodies were also evaluated for their ability to capture FMDV antigen independently of serotype. When used in combination with chicken anti-1AB' antibodies in an antigen capture ELISA format, all serotypes of FMDV were detected. These data represent the first demonstration of the use of serotype-independent FMDV antigen capture reagents which may enable the development of rapid laboratory based assays or perhaps more significantly, rapid field-based pen-side or point of entry border control diagnostic tests.

Preliminary evaluation of diagnostic accuracy and precision of a competitive ELISA for detection of antibodies to Rift Valley fever virus in cattle and sheep sera.

Rift Valley fever virus (RVFV), a mosquito-borne, zoonotic pathogen, is endemic to sub-Saharan Africa and has spread beyond the continent to the Arabian Peninsula. The high likelihood of RVFV's spread to other non-endemic countries spurs the need for development and implementation of rapid diagnostic tests and surveillance programs. In this preliminary evaluation, we assessed the diagnostic accuracy and precision of a recombinant RVFV nucleoprotein based competitive ELISA (cELISA) assay to detect RVFV antibodies compared to a plaque reduction neutralization test (PRNT80), using sera of cattle and sheep that were experimentally infected with either the MP-12 RVFV vaccine or a wild-type RVFV strain, as well as using known RVFV negative sera. With the manufacturer recommended 60% inhibition for the cELISA and a 1:40 titer for the PRNT80, the sensitivity and specificity of the cELISA assay was determined to be 95.1% (95% CI = 83.5-99.4%) and 91.8% (95% CI = 85.0-96.2%), respectively. Antibodies to RVFV were first detected at 5 days post inoculation (dpi) in both species' sera. The prototype cELISA is an easy to implement, sensitive, specific, and safe test for the detection of antibodies to RVFV that can be used in surveillance programs.

An improved, rapid competitive ELISA using a novel conserved 3B epitope for the detection of serum antibodies to foot-and-mouth disease virus.

The highly contagious foot-and-mouth disease virus (FMDV) afflicts cloven-hoofed animals, resulting in significant costs because of loss of trade and recovery from disease. We developed a sensitive, specific, and rapid competitive ELISA (cELISA) to detect serum antibodies to FMDV. The cELISA utilized a monoclonal blocking antibody specific for a highly conserved FMDV nonstructural 3B epitope, a recombinant mutant FMDV 3ABC coating protein, and optimized format variables including serum incubation for 90 min at 20-25°C. Samples from 16 animals experimentally infected with one FMDV serotype (A, O, Asia, or SAT-1) demonstrated early detection capacity beginning 7 d post-inoculation. All samples from 55 vesicular stomatitis virus antibody-positive cattle and 44 samples from cloven-hoofed animals affected by non-FMD vesicular diseases were negative in the cELISA, demonstrating 100% analytical specificity. The diagnostic sensitivity was 100% against sera from 128 cattle infected with isolates of all FMDV serotypes, emphasizing serotype-agnostic results. Diagnostic specificities of U.S. cattle ( n = 1135) and swine ( n = 207) sera were 99.4% and 100%, respectively. High repeatability and reproducibility were demonstrated with 3.1% coefficient of variation in percent inhibition data and 100% agreement using 2 kit lots and 400 negative control serum samples, with no difference between bench and biosafety cabinet operation. Negative results from vaccinated, uninfected cattle, pig, and sheep sera confirmed the DIVA (differentiate infected from vaccinated animals) capability. This rapid (<3 h), select agent-free assay with high sensitivity and specificity, DIVA capability, and room temperature processing capability will serve as a useful tool in FMDV surveillance, emergency preparedness, response, and outbreak recovery programs.

Identification of a major antibody binding epitope in the non-structural protein 3D of foot-and-mouth disease virus in cattle and the development of a monoclonal antibody with diagnostic applications.

Detection of FMDV non-structural protein 3D antibodies has been used as a complementary method for sero-epidemiological studies as an indirect indicator of FMDV infection. In order to develop a sensitive cELISA to detect FMDV antibodies, immune dominant epitopes in FMDV-3D protein were identified by peptide array analysis. Monoclonal antibodies were then raised to a selected epitope and used in cELISA. Ninety two peptides corresponding to the complete amino acid sequence of FMDV-3D were synthesized. The sera from 15 FMDV infected cows were tested for binding to the peptides in an indirect ELISA. One major peptide (3D-4) was recognized by antisera in 12 of the 15 infected cows (80%). The sequence was formed by amino acid residues 16-30 of FMDV-3D. The mAbs produced from the mice immunized with native 3D showed neither reactivity to this epitope nor competition with sera from FMDV infected cattle. However, the mAbs produced from the mice immunized with native 3D and boosted with the peptide 3D-4 showed reactivity with native 3D, recombinant 3D as well as competition with sera of FMDV infected cattle and sheep in ELISA assays. Immune response to FMDV-3D was determined using a cELISA. All cattle and sheep tested were positive at 9 dpi and remained positive until the end of the experiment on days 28-31 (>50% inhibition). This demonstrated that mAbs directed to the peptide 3D-4 were effective competitors to the polyclonal antibodies against 3D in infected sera. The approach described here provides a useful tool for specific mAb production in the development of new diagnostic tests.

Production and characterization of two serotype independent monoclonal antibodies against foot-and-mouth disease virus.

Two foot-and-mouth disease virus (FMDV) monoclonal antibodies (mAbs) were produced from mice immunized with either FMDV serotype A, subunit (12S) or FMDV serotype O, whole virus (140S). Both mAbs (F1412SA and F21140SO) recognized all seven serotypes of FMDV in a double antibody sandwich (DAS) ELISA, suggesting that the binding epitopes of the two mAbs are conserved between serotypes. These mAbs are IgG1 isotype and contain kappa light chains. In order to define the mAb binding epitopes, the reactivity of these mAbs against trypsin-treated and denatured FMDV were examined using an indirect ELISA. The binding site of the mAb, F1412SA is trypsin sensitive and the epitope is linear. Both ELISA and Western blot results suggested that the polypeptide VP2 contributed to the immunodominant site. This mAb showed reactivity to VP2 peptide (DKKTEETTILEDRIL). The mAb, F21140SO, recognized an epitope which is trypsin resistant and discontinuous. This mAb binding to FMDV is dependent on conformational structures of intact viral (140S) or subunit (12S) particle, since it failed to recognize any viral protein in Western blot. This conformational and highly conserved epitope is the first identified epitope among all seven FMDV serotypes. Because the use of mAbs increases the specificity, accuracy and efficiency of diagnostic tests compared to polyclonal antisera, these two mAbs with different specificities are suitable for type-independent diagnosis of FMDV, such as DAS ELISA, or could be adapted to immuno-chromatographic or flow-through rapid test.

Performance of a foot-and-mouth disease virus reverse transcription-polymerase chain reaction with amplification controls between three real-time instruments.

The foot-and-mouth disease virus (FMDV) is a member of the picornavirus family, possessing an 8-kb single-stranded RNA genome of positive polarity. It is highly contagious among several livestock species and can lead to severe economic consequences, as evidenced by the UK outbreak in 2001. The usage of real-time polymerase chain reaction has facilitated rapid detection of FMDV. Several real-time PCR instruments are available with various capabilities, such as portability and high sample volume analysis. Primers and a dual-labeled TaqMan probe were optimized to detect a single, highly conserved 88-bp segment of the FMDV 3D (RNA polymerase) gene. To increase the confidence of the RT-PCR result, a positive amplification control was synthesized to detect potential false-positive results due to contamination if a wild-type virus is used as positive control. In addition, a preventative measure against false-negative results was developed in which endogenous beta actin mRNA is coamplified by RT-PCR. Assay performance was compared on the LightCycler1.2 (Roche), the SmartCyclerII (Cepheid), and the SDS 7900HT (ABI). These assays successfully identified the FMDV genome and beta actin mRNA from several sources of infected nasal and oral swabs, as well as probang samples.

Toward a multiplexed serotyping immunoassay for foot-and-mouth disease virus.

Initial results demonstrating the feasibility of a multiplexed liquid array immunoassay for foot-and-mouth disease viral antigen detection and simultaneous serotype differentiation are presented. Serotype-specific antibodies from rabbit and guinea pig hyperimmunesera were isolated and prepared for use in a multiplexed, bead-based assay. The performance of all of the available antibodies as both capture and detector reagents was evaluated in the multiplexed system to establish a combination exhibiting the highest homotypic responses and lowest heterotypic reactions. The multiplexed assay was evaluated against inactivated cell culture supernatant samples of the same subtype as the virus used to raise the capture and detector antibodies. Distinct serotype differentiation was observed, except in the case of serotype SAT1. Subsequently, cell culture supernatant samples from a larger pool of viral subtypes were analyzed. Distinct serotype differentiation was obtained when analyzing cell culture supernatant samples from viral serotypes C, Asia, and SAT3, irrespective of the subtype. However, limitations of the current antibody pairs were realized in some inconclusive results obtained when analyzing samples from a broader range of O, A, and SAT2 subtypes. The results obtained in this initial study will be used to further optimize the assay using polyvalent or monoclonal antibodies and move toward the analysis of clinical samples.