Development of reverse-transcriptase, real-time PCR assays to distinguish the Southern African Territories (SAT) serotypes 1 and 3 and topotype VII of SAT2 of Foot-and-Mouth Disease Virus.

Foot-and-Mouth Disease Virus (FMDV), the causative agent of Foot-and-Mouth Disease, is a highly feared, economically devastating transboundary pathogen. This is due to the virus' extremely contagious nature and its ability to utilize multiple transmission routes. As such, rapid and accurate diagnostic testing is imperative to the control of FMD. Identification of the FMDV serotype is necessary as it provides the foundation for appropriate vaccine selection and aids in outbreak source tracing. With the vast genetic diversity, there is a desperate need to be able to characterize FMDV without relying on prior knowledge of viral serotypes. In this study, the Neptune bioinformatics tool was used to identify genetic signatures specific to each Southern African Territories (SAT) 1, 2 and 3 genomes but exclusionary to the other circulating FMDV serotypes (A, O, Asia1, and the heterologous SAT1, SAT2 and/or SAT3). Identification of these unique genomic regions allowed the design of TaqMan-based real-time reverse transcriptase PCR (rRT-PCR) primer/probe sets for SAT1, SAT2 and SAT3 viruses. These assays were optimized using prototypic FMDV cell culture isolates using the same reagents and thermocycling conditions as the FMDV pan-serotype 3D rRT-PCR assay. Cross-reactivity was evaluated in tandem with the FMDV pan-serotype 3D rRT-PCR utilizing representative strains from FMDV serotypes A, O, Asia1, SAT1, SAT2 and SAT3. The SAT1, SAT2, and SAT3 primer/probe sets were specific for the homologous serotype and exclusionary to all others. SAT1 and SAT3 primer/probe sets were able to detect several topotypes, whereas the SAT2 assay was revealed to be specific for topotype VII. The SAT2 topotype VII specificity was possibly due to the use of sequence data deposited post-2011to design the rRT-PCR primers and probes. Each assay was tested against a panel of 99 bovine tissue samples from Nigeria, where SAT2 topotype VII viruses were correctly identified and no cross-reactivity was exhibited by the SAT1 and 3 assays. These novel SAT1, SAT3 and SAT2 topotype VII rRT-PCR assays have the potential to detect and differentiate circulating FMD SAT viruses.

A Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay With Enhanced Capacity to Detect Vesicular Stomatitis Viral Lineages of Central American Origin.

Vesicular stomatitis virus (VSV) causes a disease in susceptible livestock that is clinically indistinguishable from foot-and-mouth disease. Rapid testing is therefore critical to identify VSV and rule out FMD. We previously developed and validated a multiplex real-time reverse transcription polymerase chain reaction assay (mRRT-PCR) for detection of both VS New Jersey virus (VSNJV) and VS Indiana virus (VSIV). However, it was subsequently apparent that this assay failed to detect some VSNJV isolates in Mexico, especially in genetic group II, lineage 2.1. In order to enhance the sensitivity of the mRRT-PCR for VSNJV, parts of the assay were redesigned and revalidated using new and improved PCR chemistries. The redesign markedly improved the assay by increasing the VSNJV detection sensitivity of lineage 2.1 and thereby allowing detection of all VSNJV clades. The new assay showed an increased capability to detect VSNJV. Specifically, the new mRRT-PCR detected VSNJV in 100% (87/87) of samples from Mexico in 2006-2007 compared to 74% for the previous mRRT-PCR. Furthermore, the analytical sensitivity of the new mRRT-PCR was enhanced for VSNJV. Importantly, the modified assay had the same sensitivity and specificity for VSIV as the previously published assay. Our results highlight the challenges the large genetic variability of VSV pose for virus detection by mRRT-PCR and show the importance of frequent re-evaluation and validation of diagnostic assays for VSV to ensure high sensitivity and specificity.

Molecular Characterization of Southern African Territories 2 (SAT2) Serotype of Foot-and-Mouth Disease Virus from Nigeria in 2017 to 2018.

This report describes the nucleotide sequences of eight Southern African Territories 2 (SAT2) serotype foot-and-mouth disease virus strains from 2017 to 2018 outbreaks in cattle in Nigeria. These viruses belong to topotype VII of SAT2 and were closely related to previous isolates from Nigeria and other West African countries.

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.

Foot-and-mouth disease virus detection on a handheld real-time polymerase chain reaction platform.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that requires rapid control. Early detection is critical but transportation of samples to laboratory delays testing. Sensitive and specific field-deployable assays are therefore desirable. Real-time reverse transcription polymerase chain reaction (RRT-PCR) and RRT-loop-mediated isothermal amplification assays for FMDV on portable platforms have been described but none of these are handheld. In this report, we have evaluated a handheld Biomeme two3™ Real-Time PCR Thermocycler (two3) as a field-deployable platform for FMDV RRT-PCR targeting the 3D gene segment. Two3's performance was compared with the laboratory-based reference assay on the ABI7500 platform. RNA extraction using a rapid Biomeme proprietary sample prep technology (M1) was compared with MagMax RNA extraction. Two3 successfully detected FMDV isolates for six serotypes (O, A, Asia 1, SAT 1, 2 and 3). Serotype C was excluded since it has not been detected in the field since 2004. The limits of detection for serial 10-fold dilutions of cell culture isolates were equal or one log different between two3 and ABI7500. Furthermore, two3 detected FMDV RNA in multiple sample types including serum, vesicular fluid, tissue suspensions, oral fluid, oral and nasal swabs. Two3 also detected FMDV RNA directly in vesicular fluid and other samples without prior RNA extraction. Comparison of the time to first detection of a positive result in serial samples in MagMax RNA extraction/ABI7500 (MgMx/ABI) system vs. M1 RNA extraction/Two3 system revealed similar or slightly better analytical sensitivity for the MgMx/ABI system. Overall, RNA extraction by M1 yielded good results and FMDV RNA detection on two3 was not significantly different from the ABI7500. Therefore, two3 could potentially enable sensitive penside detection of FMDV within an hour using M1-extracted RNA or direct testing of vesicular fluid and swabs without RNA extraction thereby ensuring prompt implementation of appropriate control measures.

Vesicular disease in pigs inoculated with a recent Canadian isolate of Senecavirus A.

The objective of this study was to investigate whether a virulent Canadian isolate of Senecavirus A (SVA) causes idiopathic vesicular disease (IVD) in pigs. Senecavirus A, which was first isolated in the United States in 2002 as Seneca Valley Virus, was linked to cases of porcine idiopathic vesicular disease in Canada in 2007 and in the United States in 2010. Since 2014, SVA outbreaks in Brazil, the US, Canada, China, Thailand, and Colombia point to an expanding global distribution and the need to study the pathogenicity of the virus. Unlike the prototype virus, recent US isolates of SVA have been shown to cause vesicular disease in pigs. We report vesicular disease in pigs following experimental inoculation with a 2016 Canadian isolate of SVA. All inoculated pigs developed vesicular lesions regardless of route of inoculation. Virus was detected in blood and oral fluids as well as on oral and fecal swabs. In addition, all pigs seroconverted to SVA by 6 days post-inoculation (DPI). This study confirms that recent Canadian isolates of SVA cause vesicular disease in pigs and highlights the importance of monitoring SVA for increased virulence.

L'objectif de la présente étude était d'examiner si un isolat canadien virulent de Senecavirus A (SVA) causait une maladie vésiculaire idiopathique (IVD) chez les porcs. Le SVA, qui fut isolé pour la première fois aux États-Unis en 2002 comme le virus de la vallée de Seneca, a été associé à des cas d'IVD porcine au Canada en 2007 et aux États-Unis en 2010. Depuis 2014, des épidémies de SVA au Brésil, aux États-Unis, au Canada, en Chine, en Thaïlande, et en Colombie indiquent une distribution globale en expansion et un besoin d'étudier la pathogénicité du virus. Contrairement au prototype du virus, des isolats récents de SVA aux États-Unis ont été démontrés comme causant une maladie vésiculaire chez les porcs. Nous rapportons ici une maladie vésiculaire chez des porcs à la suite de l'inoculation expérimentale d'un isolat canadien de SVA obtenu en 2016.Tous les porcs inoculés ont développé des lésions vésiculaires indépendamment de la voie d'inoculation. Le virus fut détecté dans le sang et les fluides oraux ainsi qu'à partir d'écouvillons oral et fécal. De plus, tous les porcs ont séro-convertis au SVA au 6e jour post-inoculation. Cette étude confirme que des isolats canadiens récents de SVA causent une maladie vésiculaire chez les porcs et souligne l'importance de surveiller l'augmentation de virulence du SVA.(Traduit par Docteur Serge Messier).

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.

Competitive Luminex immunoassays for detection of antibodies to foot-and-mouth disease and vesicular stomatitis viruses in multiple susceptible hosts.

Foot-and-mouth disease (FMD) and vesicular stomatitis (VS) cause such similar clinical signs and lesions that laboratory tests are required to distinguish between infections caused by each virus. Using mouse anti-foot-and-mouth disease virus (FMDV) 3B monoclonal or polyclonal anti-vesicular stomatitis virus-New Jersey (VSV-NJ) antibodies and recombinant FMDV 3ABC or VSV-NJ glycoprotein (G) antigens coated to MagPlex beads, competitive Luminex immunoassays (cLIAs) were developed for FMDV and VSV-NJ, respectively. The cLIAs successfully detected antibodies to FMDV 3ABC and VSV-NJ G in sera from infected animals. The diagnostic sensitivity and specificity were 93% and 98%, respectively for FMDV and 93% and 95.4%, respectively for VSV-NJ. These cLIAs are potential alternatives for competitive enzyme-linked immunosorbent assays (cELISAs) and provide the opportunity for multiplexing to reduce time and the amount of serum required for testing.

La fièvre aphteuse (FA) et la stomatite vésiculaire (SV) causent des signes cliniques et des lésions tellement similaires que des tests de laboratoire sont requis afin de distinguer entre les infections causées par chaque virus. En utilisant un anticorps monoclonal 3B de souris anti-virus de la fièvre aphteuse (VFA) ou un anticorps polyclonal anti-virus de stomatite vésiculaire-New Jersey (VSV-NJ) et des billes MagPlex enduites d'antigènes de VFA recombinant 3ABC ou de glycoprotéine G de VSV-NJ, des immuno-essais Luminex compétitifs (cLIAs) furent développés pour le VFA et le VSV-NJ, respectivement. Les cLIAs ont détecté avec succès des anticorps contre VFA 3ABC et VSV-NJ G dans le sérum d'animaux infectés. La sensibilité et spécificité diagnostiques étaient de 93 % et 98 %, respectivement pour le VFA et de 93 % et 95,4 %, respectivement pour le VSV-NJ. Ces cLIAs sont des alternatives potentielles pour les épreuves ELISA compétitives et fournissent l'opportunité de multiplexer afin de réduire le temps et la quantité de sérum requis pour les tests.(Traduit par Docteur Serge Messier).

Phylodynamics of parapoxvirus genus in Mexico (2007-2011).

In this study we report for the first time the phylodynamics of the parapoxvirus (PPV) genus in Mexico. Based on the analysis by PCR of 124 epithelial samples collected between 2007 and 2011 from naturally infected goats, sheep and cows in Mexico, we found that different PPV were present in 21 out of the 24 states sampled during this study. Our phylogenetic analysis confirmed the presence of different PPV species in Mexico, and their phylogenetic relationship with other PPV circulating in the US and Canada. Furthermore, we describe the existence of two different ORFV phylogenetic groups that are clearly host associated (sheep or goat). Evidence of directional selection at five specific amino acid residues in the enveloped glycoprotein B2L might help to support this host predilection. Collectively, the results generated in this study highlight the importance of PPV genus in Mexico and open the possibility for future studies describing with more detail the importance of this genus in North America.

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.

Genome wide analysis of the evolution of Senecavirus A from swine clinical material and assembly yard environmental samples.

Senecavirus A (SVA), previously known as Seneca Valley virus, was first isolated in the United States in 2002. SVA was associated with porcine idiopathic vesicular disease in Canada and the USA in 2007 and 2012, respectively. Recent increase in SVA outbreaks resulting in neonatal mortality of piglets and/or vesicular lesions in sows in Brazil, the USA and Canada point to the necessity to study the pathogenicity and molecular epidemiology of the virus. Here, we report the analysis of the complete coding sequences of SVA from 2 clinical cases and 9 assembly yard environmental samples collected in 2015 in Canada, along with 22 previously released complete genomes in the GenBank. With this combined data set, the evolution of the SVA over a 12-month period in 2015/2016 was evaluated. These SVA isolates were characterized by a rapid accumulation of genetic variations driven mainly by a high nucleotide substitution rate and purifying selection. The SVA sequences clustered in clearly defined geographical areas with reported cases of SVA infection. No transmission links were identified between assembly yards, suggesting that point source introductions may have occurred. In addition, 25 fixed non-synonymous mutations were identified across all analyzed strains when compared to the prototype SVA strain (SVV-001). This study highlights the importance of monitoring SVA mutations for their role in increased virulence and impact on SVA diagnostics.

Detection and serotype-specific differentiation of vesicular stomatitis virus using a multiplex, real-time, reverse transcription-polymerase chain reaction assay.

A multiplex, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed that allowed simultaneous detection and rapid differentiation of vesicular stomatitis virus strains--New Jersey (VSV-NJ) and Indiana 1, 2, and 3 (VSV-IN1-3). This assay involves use of a set of VSV universal primers located in the L gene that amplify VSV-IN1-3 and VSV-NJ using probes that allow differentiation of the major serotypes Indiana and New Jersey. The assay was evaluated using reference VSV, foot-and-mouth disease virus, swine vesicular disease virus, and vesicular exanthema of swine virus. To estimate diagnostic sensitivity, 159 epithelial samples collected between 1996 and 2002 from naturally infected cattle in Colombia were used. The assay cut off was calculated by testing RNA extracted from 150 virus-negative bovine tissues consisting of tongue, soft palate, muzzle, coronary band, and lymph node. All infected cattle were test positive for VS by results of real-time RT-PCR analysis; results for 156 of 159 (98.1%) agreed with the serotype determination from the complement-fixation test. Amplification did not occur in any of the negative bovine epithelial samples, allowing the cut-off values for the assay to be set. The real-time RT-PCR assay was documented to be sensitive and specific for the detection of VSV-NJ and VSV-IN (1-3) strains from field samples in a single reaction, thereby supporting use of this assay in the differential diagnosis of vesicular virus diseases in cattle.

Early protection in sheep against intratypic heterologous challenge with serotype O foot-and-mouth disease virus using high-potency, emergency vaccine.

In 2009-2011, spread of a serotype O foot-and-mouth disease virus (FMDV) belonging to the South East Asia topotype led to the culling of over 3.5 million cattle and pigs in Japan and Korea. The O1 Manisa vaccine (belonging to the Middle East-South Asian topotype) was used at high potency in Korea to limit the expansion of the outbreak. However, no data are available on the spread of this virus or the efficacy of the O1 Manisa vaccine against this virus in sheep. In this study, the early protection afforded with a high potency (>6 PD50) FMD O1 Manisa vaccine against challenge with the O/SKR/2010 virus was tested in sheep. Sheep (n=8) were vaccinated 4 days prior to continuous direct-contact challenge with donor sheep. Donor sheep were infected with FMDV O/SKR/2010 by coronary band inoculation 24h prior to contact with the vaccinated animals, or unvaccinated controls (n=4). Three of the four control sheep became infected, two clinically. All eight O1 Manisa vaccinated sheep were protected from clinical disease. None had detectable antibodies to FMDV non-structural proteins (3ABC), no virus was isolated from nasal swabs, saliva or oro-pharyngeal fluid and none became carriers. Using this model of challenge, sheep were protected against infection as early as 4 days post vaccination.

Development of a competitive enzyme-linked immunosorbent assay for detection of antibodies against the 3B protein of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is one of the most highly contagious and economically devastating diseases, and it severely constrains the international trade of animals. Vaccination against FMD is a key element in the control of FMD. However, vaccination of susceptible animals raises critical issues, such as the differentiation of infected animals from vaccinated animals. The current study developed a reliable and rapid test to detect antibodies against the conserved, nonstructural proteins (NSPs) of the FMD virus (FMDV) to distinguish infected animals from vaccinated animals. A monoclonal antibody (MAb) against the FMDV NSP 3B was produced. A competitive enzyme-linked immunosorbent assay (cELISA) for FMDV/NSP antibody detection was developed using a recombinant 3ABC protein as the antigen and the 3B-specific MAb. Sera collected from naive, FMDV experimentally infected, vaccinated carrier, and noncarrier animals were tested using the 3B cELISA. The diagnostic specificity was 99.4% for naive animals (cattle, pigs, and sheep) and 99.7% for vaccinated noncarrier animals. The diagnostic sensitivity was 100% for experimentally inoculated animals and 64% for vaccinated carrier animals. The performance of this 3B cELISA was compared to that of four commercial ELISA kits using a panel of serum samples established by the World Reference Laboratory for FMD at The Pirbright Institute, Pirbright, United Kingdom. The diagnostic sensitivity of the 3B cELISA for the panel of FMDV/NSP-positive bovine serum samples was 94%, which was comparable to or better than that of the commercially available NSP antibody detection kits. This 3B cELISA is a simple, reliable test to detect antibodies against FMDV nonstructural proteins.

Development and use of a biotinylated 3ABC recombinant protein in a solid-phase competitive ELISA for the detection of antibodies against foot-and-mouth disease virus.

A biotinylated 3ABC recombinant protein was developed and used in a competitive ELISA (cELISA) to detect foot-and-mouth disease virus (FMDV) antibodies in cattle, sheep and pigs. In this report, we describe the cloning and expression of 3ABC protein in Escherichia coli cells as fusion protein with 6xHis and biotin. This cELISA uses streptavidin to capture bacterially expressed and in vivo biotinylated 3ABC antigen. The antigen capture strategy provides a simple and reliable method, which does not require purification of recombinant antigen before the serological assay. An hyperimmune guinea pig antiserum produced against purified 6xHis-3ABC was used as competitor in the test. The potential use of this cELISA for the identification of antibodies induced by FMD virus infection from those induced by vaccination is discussed.

Development of neutralizing monoclonal antibodies against the pandemic H1N1 virus (2009) using plasmid DNA immunogen.

Reported here is the development and characterization of eighteen mouse monoclonal antibodies (MAbs) against the 2009 pandemic H1N1 influenza virus (A/Mexico/InDRE4487/2009). To our knowledge, this is the first report on pandemic (pH1) H1N1 MAbs developed using plasmid DNA encoding the viral surface glycoprotein, hemagglutinin (HA). All eighteen MAbs were specific for A/Mexico/InDRE4487/2009 HA. Ten MAbs were found to cross-react with A/Swine/Indiana/81 using a dot blot assay. However, there was no cross-reactivity detected against any other strains of influenza A viruses despite screening against all 16 hemagglutinin subtypes. Examination of these MAbs identified individual antibodies suitable for use in several practical applications including ELISA, immunoblot and immunofluorescence assays. Analysis of the kinetics of each MAb revealed significant binding affinities (K(D)<10(-8) M) confirming the antibodies are highly specific for A/Mexico/InDRE4487/2009 HA. Functional analysis demonstrated the panel of MAbs included antibodies with HA inhibition and virus neutralization activities. Not all MAbs inhibited hemagglutination or neutralized the virus. Furthermore, the panel of MAbs was not found to be cross-reactive against additional strains tested in hemagglutination inhibition assays. Finally, the MAbs were tested in competitive ELISA (cELISA) using reference serum antibodies developed against different clusters of H1 (pH1, α, ß, γ, and δ). The developed MAbs outcompeted serum antibodies of pH1 in 16/18, 15/18 (γ), 3/18 (α), 2/18 (δ1) and 1/18 (ß) samples. Overall, this panel of MAbs proved specific and highly sensitive for A/Mexico/InDRE4487/2009 HA and could potentially serve as immunodiagnostic tools for the rapid detection of this specific strain of influenza virus.

Development and characterization of neutralizing monoclonal antibodies against the pandemic H1N1 virus (2009).

The 2009 H1N1 influenza pandemic was a major international public health crisis which caused considerable morbidity and mortality worldwide. The goal of this study was to produce anti-H1 monoclonal antibodies (MAbs) for improving diagnostic immunological assays and to develop potential immunotherapeutics. Nine MAbs were produced after immunizing mice with recombinant hemagglutinin (HA) protein from A/California/06/09. Two spleenocyte myeloma fusions yielded 1588 hybridoma cultures. After screening the hybridoma culture supernatants for antibody reactivity to rHA, nine clones were selected for further characterization. Cross-reactivity studies of the anti-rHA antibodies against a panel of influenza viruses (H1-H16) revealed eight out of nine MAbs were specific to the pandemic H1 subtype, except for MAb F256G2sc1 which also cross-reacted with H5 subtype virus. All MAbs were of the IgG1κ isotype, except F256G2sc1 which was IgG2aκ. The anti-rHA MAbs had binding affinities to rHA that ranged from a K(D) (disassociation constant) of 1.34×10(-9)M (F255G7sc1) to the weakest affinity of 4.60×10(-8)M (F255G4sc1). Interestingly, in a plaque reduction neutralization assay, all MAbs except F255G3sc1 demonstrated neutralizing ability. Furthermore, all MAbs except F255G3sc1 and F255G9sc1 exhibited anti-hemagglutinin activity against pandemic H1N1 viruses, but not against classical North American swine influenza viruses of the same subtype. Immunofluorescence assay (IFA) demonstrated that all MAbs except F255G1sc1 and F255G3sc1 were able to detect 2009 pandemic H1N1 (2009) virus- infected MDCK cells. The MAbs were also evaluated for potential use in competitive ELISA (cELISA), and with the exception of F255G3sc1, all MAbs showed competitive activity with serum collected from pigs infected with pandemic H1N1 virus (2009). The developed MAbs have demonstrated utility as immunodiagnostic and research reagents, and their neutralizing capabilities also hold potential for designing antiviral drugs against pandemic influenza.

Production and diagnostic application of monoclonal antibodies against influenza virus H5.

Nine monoclonal antibodies (mAbs) against avian influenza virus (AI) H5 subtype from mice immunized with inactivated virus H5N1 (A/Turkey/ON/6213/66) were produced. Upon testing, the results indicated that the binding epitopes of eight out of the nine mAbs were conformational, while one mAb (#7) reacted with denatured H5N1 only. Two mAbs #10 and #11 reacted with all of the thirteen H5 strains tested indicating that the binding epitopes of these mAbs were conserved among these H5 subtypes. Possible applications of these mAbs in rapid tests for H5 antigen were explored. Double antibody sandwich (DAS) ELISAs were developed using two selected mAbs #10 and #11. This DAS ELISA detects specific H5 viruses and is able to identify all thirteen H5 strains tested. Three mAbs showed reactivity with AI H5 antigen for both immunofluorescence (IF) and immunohistochemistry. A cELISA used to screen chickens that had been infected with an H5 virus was developed with mAb #9 and recombinant H5 antigen. The sera from chickens that have been infected with an H5N1 virus were examined using the cELISA. 80% of the sera from H5 infected chickens showed a positive H5 specific antibody response at 7 days post-infection (dpi) and remained positive until the end of the experiment on day 30 (>40% inhibition). This panel of the AI H5 specific mAbs is valuable for the development of various immunoassays.

Simultaneous detection of antibodies to foot-and-mouth disease non-structural proteins 3ABC, 3D, 3A and 3B by a multiplexed Luminex assay to differentiate infected from vaccinated cattle.

For the first time, a multiplex bead immunoassay was used to test simultaneously, with a single sample, the immune response to foot-and-mouth disease non-structural proteins 3ABC, 3A, 3B and 3D from experimentally infected and vaccinated cattle. We cloned and expressed these non-structural proteins (NSPs) as recombinant antigens. The purified proteins were coupled to microspheres labeled with anti-His monoclonal antibody with different proportions of red and orange fluorescent dyes and reacted against serum specimens. Antibody reacting against different NSPs, and thus, the different colored beads, was detected by use of the Luminex system. This multiplex bead immunoassay can detect the immune response to NSPs in cattle as early as 7 days post-infection. In general antibodies to the protein 3D appeared early after infection and anti-3ABC antibodies were detected at higher levels than the other NSPs. A clear differentiation was established between infected and vaccinated or uninfected cattle. The multiplex bead immunoassay was compared to individual indirect enzyme-linked immunosorbent assays (iELISAs) for the same NSP's responses. Results indicated that this new assay had a high positive correlation with those generated by iELISA. The Luminex-based technology promises to be a sensitive and efficient method that permits multiplexed NSP antibody detection from a single sample and would therefore provide both a time and cost saving to the laboratory.