Evaluation of DNA Vaccine Candidates against Foot-and-Mouth Disease Virus in Cattle.

We evaluated four DNA vaccine candidates for their ability to produce virus-like particles (VLPs) and elicit a protective immune response against Foot-and-mouth disease virus (FMDV) in cattle. Two traditional DNA plasmids and two DNA minicircle constructs were evaluated. Both the pTarget O1P1-3C plasmid and O1P1-3C minicircle encoded a wild-type FMDV 3C protease to process the P1-2A polypeptide, whereas the O1P1-HIV-3CT minicircle used an HIV-1 ribosomal frameshift to down-regulate expression of a mutant 3C protease. A modified pTarget plasmid with a reduced backbone size, mpTarget O1P1-3CLT, used a 3C protease containing two mutations reported to enhance expression. All constructs produced mature FMDV P1 cleavage products in transfected cells, as seen by western blot analysis. Three constructs, O1P1-3C minicircles, pTarget O1P1-3C, and mpTarget O1P1-3CLT plasmids, produced intracellular VLP crystalline arrays detected by electron microscopy. Despite VLP formation in vitro, none of the DNA vaccine candidates elicited protection from clinical disease when administered independently. Administration of pTarget O1P1-3C plasmid enhanced neutralizing antibody titers when used as a priming dose prior to administration of a conditionally licensed adenovirus-vectored FMD vaccine. Further work is needed to develop these DNA plasmid-based constructs into standalone FMD vaccines in cattle.

Effect of Foot-and-Mouth Disease Virus 2B Viroporin on Expression and Extraction of Mammalian Cell Culture Produced Foot-and-Mouth Disease Virus-like Particles.

To improve the production of foot-and-mouth disease (FMD) molecular vaccines, we sought to understand the effects of the FMD virus (FMDV) 2B viroporin in an experimental, plasmid-based, virus-like particle (VLP) vaccine. Inclusion of the FMDV viroporin 2B into the human Adenovirus 5 vectored FMD vaccine enhanced transgene expression despite independent 2B expression negatively affecting cell viability. Evaluating both wildtype 2B and mutants with disrupted viroporin activity, we confirmed that viroporin activity is detrimental to overall transgene expression when expressed independently. However, the incorporation of 2B into an FMD molecular vaccine construct containing a wildtype FMDV 3C protease, a viral encoded protease responsible for processing structural proteins, resulted in enhancement of transgene expression, validating previous observations. This benefit to transgene expression was negated when using the FMDV 3CL127P mutant, which has reduced processing of host cellular proteins, a reversion resulting from 2B viroporin activity. Inclusion of 2B into VLP production constructs also adversely impacted antigen extraction, a possible side effect of 2B-dependent rearrangement of cellular membranes. These results demonstrate that inclusion of 2B enhanced transgene expression when a wildtype 3C protease is present but was detrimental to transgene expression with the 3CL127P mutant. This has implications for future molecular FMD vaccine constructs, which may utilize mutant FMDV 3C proteases.

Comparative Evaluation of the Foot-and-Mouth Disease Virus Permissive LF-BK αVß6 Cell Line for Senecavirus A Research.

Senecavirus A (SVA) is a member of the family Picornaviridae and enzootic in domestic swine. SVA can induce vesicular lesions that are clinically indistinguishable from Foot-and-mouth disease, a major cause of global trade barriers and agricultural productivity losses worldwide. The LF-BK αVß6 cell line is a porcine-derived cell line transformed to stably express an αVß6 bovine integrin and primarily used for enhanced propagation of Foot-and-mouth disease virus (FMDV). Due to the high biosecurity requirements for working with FMDV, SVA has been considered as a surrogate virus to test and evaluate new technologies and countermeasures. Herein we conducted a series of comparative evaluation in vitro studies between SVA and FMDV using the LF-BK αVß6 cell line. These include utilization of LF-BK αVß6 cells for field virus isolation, production of high virus titers, and evaluating serological reactivity and virus susceptibility to porcine type I interferons. These four methodologies utilizing LF-BK αVß6 cells were applicable to research with SVA and results support the current use of SVA as a surrogate for FMDV.

Transiently Transfected Mammalian Cell Cultures: An Adaptable and Effective Platform for Virus-like Particle-Based Vaccines against Foot-and-Mouth Disease Virus.

RNA viruses, such as foot-and-mouth disease virus (FMDV), have error-prone replication resulting in the continuous emergence of new viral strains capable of evading current vaccine coverage. Vaccine formulations must be regularly updated, which is both costly and technically challenging for many vaccine platforms. In this report, we describe a plasmid-based virus-like particle (VLP) production platform utilizing transiently transfected mammalian cell cultures that combines both the rapid response adaptability of nucleic-acid-based vaccines with the ability to produce intact capsid epitopes required for immunity. Formulated vaccines which employed this platform conferred complete protection from clinical foot-and-mouth disease in both swine and cattle. This novel platform can be quickly adapted to new viral strains and serotypes through targeted exchanges of only the FMDV capsid polypeptide nucleic acid sequences, from which processed structural capsid proteins are derived. This platform obviates the need for high biocontainment manufacturing facilities to produce inactivated whole-virus vaccines from infected mammalian cell cultures, which requires upstream expansion and downstream concentration of large quantities of live virulent viruses.

Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means.

BACKGROUND: Type I interferons are widely used in research applications and as biotherapeutics. Current assays used to measure interferon concentrations, such as plaque reduction assays and ELISA, are expensive, technically challenging, and may take days to provide results. We sought to develop a robust and rapid assay to determine interferon concentrations produced from transiently transfected cell cultures. METHOD: Indirect quantification of recombinant interferon was evaluated using a novel bi-cistronic construct encoding the Foot-and-mouth disease virus 2A translational interrupter sequence to yield equimolar expression of Gaussia princeps luciferase and porcine interferon α. Direct quantification was evaluated by expression of a novel fusion protein comprised of Gaussia princeps luciferase and porcine type I interferon. Plasmids encoding constructs are transiently transfected into cell cultures and supernatant harvested for testing of luminescence, ELISA determined concentration, and anti-viral activity against vesicular stomatitis virus. RESULTS: Bi-cistronic constructs, utilized for indirect quantification, demonstrate both luciferase activity and anti-viral activity. Fusion proteins, utilized for direct quantification, retained secretion and luminescence however only the interferon α fusion protein had antiviral activity comparable to wildtype porcine interferon α. A strong linear correlation was observed between dilution and luminescence for all compounds over a dynamic range of concentrations. CONCLUSION: The correlation of antiviral and luciferase activities demonstrated the utility of this approach, both direct and indirect, to rapidly determine recombinant interferon concentrations. Concentration can be determined over a more dynamic concentration range than available ELISA based assays using this methodology.

Detection of African swine fever virus utilizing the portable MatMaCorp ASF detection system.

African swine fever (ASF) has emerged as a major threat to domestic and wild suid populations, and its continued spread threatens commercial swine production worldwide. The causative agent of ASF, African swine fever virus (ASFV), possesses a linear, double stranded DNA genome. Traditional detection of ASFV relies on laboratory-based virus isolation or real-time PCR of samples, typically blood or spleen, obtained from suspect cases. While effective, these methodologies are not easily field deployable, a major limitation during disease outbreak and response management scenarios. In this report, we evaluated the MatMaCorp Solas 8® ASFV detection system, a field deployable DNA extraction and fluorescent detection device, for its ability to extract and detect ASFV from multiple sample types obtained from domestic swine experimentally infected with ASFV strain Georgia. We found that the MatMaCorp Solas 8® ASFV detection device, and affiliated MagicTip™ DNA extraction and C-SAND™ assay kits, readily detected ASFV in blood and spleen, as well as other sample types, including pinna, liver, skin, muscle and bone marrow.

Generation and characterization of genetically stable heterohybridomas producing foot-and-mouth disease virus-specific porcine monoclonal antibodies.

This report covers the methodology for generation of stable heterohybridoma clones producing Foot-and-mouth disease virus (FMDV) reactive porcine monoclonal antibodies (mAbs). Swine received five inoculations of an inactivated O1 Manisa FMDV vaccine prior to the harvest of splenocytes. Due to the lack of a species-specific hybridoma fusion partner, the Sp2/0 murine myeloma cell line was utilized for the formation of porcine-murine heterohybridoma clones. Twenty-nine FMDV-reactive parental clones were generated. Following sub-cloning and monitoring of reactivity over 20 serial passages, eleven subclones derived from unique parental origins were characterized and are reported herein. This methodology demonstrated the production of porcine mAbs by fusion of porcine splenocytes from immunized pigs with murine myeloma cells to generate heterohybridomas. The porcine immune response may differ from the murine immune response in relation to recognized epitopes. Therefore, application of this methodology may provide valuable resources for swine immunology and enhance the understanding of the mechanisms for antibody based protection from diseases in swine.

Evaluation of modified Vaccinia Ankara-based vaccines against foot-and-mouth disease serotype A24 in cattle.

To prepare foot-and-mouth disease (FMD) recombinant vaccines in response to newly emerging FMD virus (FMDV) field strains, we evaluated Modified Vaccinia virus Ankara-Bavarian Nordic (MVA-BN®) as an FMD vaccine vector platform. The MVA-BN vector has the capacity to carry and express numerous foreign genes and thereby has the potential to encode antigens from multiple FMDV strains. Moreover, this vector has an extensive safety record in humans. All MVA-BN-FMD constructs expressed the FMDV A24 Cruzeiro P1 capsid polyprotein as antigen and the FMDV 3C protease required for processing of the polyprotein. Because the FMDV wild-type 3C protease is detrimental to mammalian cells, one of four FMDV 3C protease variants were utilized: wild-type, or one of three previously reported mutants intended to dampen protease activity (C142T, C142L) or to increase specificity and thereby reduce adverse effects (L127P). These 3C coding sequences were expressed under the control of different promoters selected to reduce 3C protease expression. Four MVA-BN-FMD constructs were evaluated in vitro for acceptable vector stability, FMDV P1 polyprotein expression, processing, and the potential for vaccine scale-up production. Two MVA-BN FMD constructs met the in vitro selection criteria to qualify for clinical studies: MVA-mBN360B (carrying a C142T mutant 3C protease and an HIV frameshift for reduced expression) and MVA-mBN386B (carrying a L127P mutant 3C protease). Both vaccines were safe in cattle and elicited low to moderate serum neutralization titers to FMDV following multiple dose administrations. Following FMDV homologous challenge, both vaccines conferred 100% protection against clinical FMD and viremia using single dose or prime-boost immunization regimens. The MVA-BN FMD vaccine platform was capable of differentiating infected from vaccinated animals (DIVA). The demonstration of the successful application of MVA-BN as an FMD vaccine vector provides a platform for further FMD vaccine development against more epidemiologically relevant FMDV strains.

Effect of foot-and-mouth disease virus 3C protease B2 ß-strand proline mutagenesis on expression and processing of the P1 polypeptide using a plasmid expression vector.

The production of experimental molecular vaccines against foot-and-mouth disease virus utilizes the viral encoded 3C protease for processing of the P1 polyprotein. Expression of wild type 3C protease is detrimental to host cells. The molecular vaccine constructs containing the 3C protease L127P mutant significantly reduce adverse effects associated with protease expression while retaining the ability to process and assemble virus-like particles. In published 3C protease crystal structures, the L127 residue is contained within the B2 ß-strand as part of the A2-B2 ß-sheet. To provide insight into the mechanism by which the L127P mutant alters the properties of the 3C protease, we performed scanning proline mutagenesis of residues 123-128 of the B2 ß-strand and monitored expression and P1 processing. Simultaneously, we utilized random mutagenesis of the full 3C sequence to identify additional mutations presenting a phenotype similar to the L127P mutation. Six of the tested mutants enhanced expression over wild type, and the I22P, T100P and V124P mutations surpassed the L127P mutation in certain cell lines. These data areinterpreted in conjunction with published 3C protease crystal structures to provide insight into the mechanism by which these mutations enhance expression.

Production of foot-and-mouth disease virus capsid proteins by the TEV protease.

Protective immunity to viral pathogens often includes production of neutralizing antibodies to virus capsid proteins. Many viruses produce capsid proteins by expressing a precursor polyprotein and related protease from a single open reading frame. The foot-and-mouth disease virus (FMDV) expresses a 3C protease (3Cpro) that cleaves a P1 polyprotein intermediate into individual capsid proteins, but the FMDV 3Cpro also degrades many host cell proteins and reduces the viability of host cells, including subunit vaccine production cells. To overcome the limitations of using the a wild-type 3Cpro in FMDV subunit vaccine expression systems, we altered the protease restriction sequences within a FMDV P1 polyprotein to enable production of FMDV capsid proteins by the Tobacco Etch Virus NIa protease (TEVpro). Separate TEVpro and modified FMDV P1 proteins were produced from a single open reading frame by an intervening FMDV 2A sequence. The modified FMDV P1 polyprotein was successfully processed by the TEVpro in both mammalian and bacterial cells. More broadly, this method of polyprotein production and processing may be adapted to other recombinant expression systems, especially plant-based expression.

Foot-and-Mouth Disease (FMD) Virus 3C Protease Mutant L127P: Implications for FMD Vaccine Development.

The foot-and-mouth disease virus (FMDV) afflicts livestock in more than 80 countries, limiting food production and global trade. Production of foot-and-mouth disease (FMD) vaccines requires cytosolic expression of the FMDV 3C protease to cleave the P1 polyprotein into mature capsid proteins, but the FMDV 3C protease is toxic to host cells. To identify less-toxic isoforms of the FMDV 3C protease, we screened 3C mutants for increased transgene output in comparison to wild-type 3C using a Gaussia luciferase reporter system. The novel point mutation 3C(L127P) increased yields of recombinant FMDV subunit proteins in mammalian and bacterial cells expressing P1-3C transgenes and retained the ability to process P1 polyproteins from multiple FMDV serotypes. The 3C(L127P) mutant produced crystalline arrays of FMDV-like particles in mammalian and bacterial cells, potentially providing a practical method of rapid, inexpensive FMD vaccine production in bacteria.IMPORTANCE The mutant FMDV 3C protease L127P significantly increased yields of recombinant FMDV subunit antigens and produced virus-like particles in mammalian and bacterial cells. The L127P mutation represents a novel advancement for economical FMD vaccine production.

Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression.

BACKGROUND: The Gaussia princeps luciferase is used as a stand-alone reporter of transgene expression for in vitro and in vivo expression systems due to the rapid and easy monitoring of luciferase activity. We sought to simultaneously quantitate production of other recombinant proteins by transcriptionally linking the Gaussia princeps luciferase gene to other genes of interest through the foot-and-mouth disease virus 2A translational interrupter sequence. RESULTS: We produced six plasmids, each encoding a single open reading frame, with the foot-and-mouth disease virus 2A sequence placed either N-terminal or C-terminal to the Gaussia princeps luciferase gene. Two plasmids included novel Gaussia princeps luciferase variants with the position 1 methionine deleted. Placing a foot-and-mouth disease virus 2A translational interrupter sequence on either the N- or C-terminus of the Gaussia princeps luciferase gene did not prevent the secretion or luminescence of resulting chimeric luciferase proteins. We also measured the ability of another polycistronic plasmid vector with a 2A-luciferase sequence placed downstream of the foot-and-mouth disease virus P1 and 3C protease genes to produce of foot-and-mouth disease virus-like particles and luciferase activity from transfected cells. Incorporation of the 2A-luciferase sequence into a transgene encoding foot-and-mouth disease virus structural proteins retained luciferase activity and the ability to form virus-like particles. CONCLUSIONS: We demonstrated a mechanism for the near real-time, sequential, non-destructive quantitative monitoring of transcriptionally-linked recombinant proteins and a valuable method for monitoring transgene expression in recombinant vaccine constructs.

A colorimetric bioassay for high-throughput and cost-effectively assessing anti-foot-and-mouth disease virus activity.

Foot-and-mouth disease virus (FMDV) is one of the most contagious animal viruses. This virus is very sensitive to inhibition by type I interferons. Currently, a bioassay based on plaque reduction is used to measure anti-FMDV activity of porcine IFNs. The plaque reduction assay is tedious and difficult to utilize for high-throughput analysis. Using available FMDV susceptible bovine and porcine cells, we developed and tested a colorimetric assay based on cytopathic effect reduction for its ability to quantify FMDV-specific antiviral activity of bovine and porcine type I interferons. Our results show that this new method has significant advantages over other assays in terms of labor intensity, cost, high-throughput capability and/or anti-FMDV specific activity because of simpler procedures and direct measurement of antiviral activity. Several assay conditions were tested to optimize the procedures. The test results show that the assay can be standardized with fixed conditions and a standard or a reference for measuring antiviral activity as units. This is an excellent assay in terms of sensitivity and accuracy based on a statistical evaluation. The results obtained with this assay were highly correlated with a conventional virus titration method.

Mechanisms of foot-and-mouth disease virus tropism inferred from differential tissue gene expression.

Foot-and-mouth disease virus (FMDV) targets specific tissues for primary infection, secondary high-titer replication (e.g. foot and mouth where it causes typical vesicular lesions) and long-term persistence at some primary replication sites. Although integrin αVß6 receptor has been identified as primary FMDV receptors in animals, their tissue distribution alone fails to explain these highly selective tropism-driven events. Thus, other molecular mechanisms must play roles in determining this tissue specificity. We hypothesized that differences in certain biological activities due to differential gene expression determine FMDV tropism and applied whole genome gene expression profiling to identify genes differentially expressed between FMDV-targeted and non-targeted tissues in terms of supporting primary infection, secondary replication including vesicular lesions, and persistence. Using statistical and bioinformatic tools to analyze the differential gene expression, we identified mechanisms that could explain FMDV tissue tropism based on its association with differential expression of integrin αVß6 heterodimeric receptor (FMDV receptor), fibronectin (ligand of the receptor), IL-1 cytokines, death receptors and the ligands, and multiple genes in the biological pathways involved in extracellular matrix turnover and interferon signaling found in this study. Our results together with reported findings indicate that differences in (1) FMDV receptor availability and accessibility, (2) type I interferon-inducible immune response, and (3) ability to clear virus infected cells via death receptor signaling play roles in determining FMDV tissue tropism and the additional increase of high extracellular matrix turnover induced by FMDV infection, likely via triggering the signaling of highly expressed IL-1 cytokines, play a key role in the pathogenesis of vesicular lesions.

Differential mRNA translation in Medicago truncatula accessions with contrasting responses to ozone-induced oxidative stress.

Acute ozone is a model abiotic elicitor of oxidative stress and a useful tool for understanding biochemical and molecular events during oxidative signaling. Two Medicago truncatula accessions with contrasting responses to ozone were used to examine translational regulation during ozone stress. In ozone-resistant JE154, significant reduction in ribosome loading was observed within one hour of ozone treatment, suggesting energy homeostasis as a vital factor for oxidative stress management. Polysomal RNA-based expression profiling with Affymetrix arrays revealed extensive changes in the translatomes of both accessions. Messenger RNAs with low GC content in their 5' and 3'-UTRs were preferentially associated with polysomes during oxidative stress. Genebins analysis revealed extensive changes in various gene ontologies in both accessions. Extensive changes in nicotinate and nicotinamide metabolism genes were corroborated with increased levels of NAD(+) and NADH in JE154. The significantly lower NAD(+):NADH redox status in JE154, in conjunction with higher ATP amounts, provided a cellular milieu conducive for overcoming oxidative stress. Low levels of ATP, NADH, and suppression of antioxidant defense responses, abet build-up of ozone-derived ROS and ultimately lead to oxidative cell death in Jemalong.

Identification of stress-responsive genes in plants using suppression subtraction hybridization: ozone stress as an example.

Among the open-ended techniques for identifying differentially expressed genes in response to stress, the PCR-based suppression subtraction hybridization (SSH) is widely used. The popularity of this technique stems from the ease of conducting this procedure in any laboratory set up for basic molecular biology research. Further, the availability of a comprehensive kit for conducting suppression subtractions from BD Biosciences has made this technique easy to adapt and adopt to any biological system. In this chapter we describe in detail the SSH procedure and explain the subtle changes that have been incorporated to make this technique adaptable for identifying stress-responsive genes in plants.

Ozone responsive genes in Medicago truncatula: analysis by suppression subtraction hybridization.

Acute ozone is a model abiotic elicitor of oxidative stress in plants. In order to identify genes that are important for conferring ozone resistance or sensitivity we used two accessions of Medicago truncatula with contrasting responses to this oxidant. We used suppression subtraction hybridization (SSH) to identify genes differentially expressed in ozone-sensitive Jemalong and ozone-resistant JE154 following exposure to 300 nLL(-1) of ozone for 6h. Following differential screening of more than 2500 clones from four subtraction libraries, more than 800 clones were selected for sequencing. Sequence analysis of these clones identified 239 unique contigs. Fifteen novel genes of unknown functions were identified. A majority of the ozone responsive genes identified in this study were present in the Medicago truncatula EST collections. Genes induced in JE154 were associated with adaptive responses to stress, while in Jemalong, the gene ontologies for oxidative stress, cell growth, and translation were enriched. A meta-analysis of ozone responsive genes using the Genvestigator program indicated enrichment of ABA and auxin responsive genes in JE154, while cytokinin response genes were induced in Jemalong. In resistant JE154, down regulation of photosynthesis-related genes and up regulation of genes responding to low nitrate leads us to speculate that lowering carbon-nitrogen balance may be an important resource allocation strategy for overcoming oxidative stress. Temporal profiles of select genes using real-time PCR analysis showed that most of the genes in Jemalong were induced at the later time points and is consistent with our earlier microarray studies. Inability to mount an early active transcriptional reprogramming in Jemalong may be the cause for an inefficient defense response that in turn leads to severe oxidative stress and culminates in cell death.

Transcriptomic changes induced by acute ozone in resistant and sensitive Medicago truncatula accessions.

BACKGROUND: Tropospheric ozone, the most abundant air pollutant is detrimental to plant and animal health including humans. In sensitive plant species even a few hours of exposure to this potent oxidant (200-300 nL. L-1) leads to severe oxidative stress that manifests as visible cell death. In resistant plants usually no visible symptoms are observed on exposure to similar ozone concentrations. Naturally occurring variability to acute ozone in plants provides a valuable resource for examining molecular basis of the differences in responses to ozone. From our earlier study in Medicago truncatula, we have identified cultivar Jemalong is ozone sensitive and PI 464815 (JE154) is an ozone-resistant accession. Analyses of transcriptome changes in ozone-sensitive and resistant accession will provide important clues for understanding the molecular changes governing the plant responses to ozone. RESULTS: Acute ozone treatment (300 nL L-1 for six hours) led to a reactive oxygen species (ROS) burst in sensitive Jemalong six hours post-fumigation. In resistant JE154 increase in ROS levels was much reduced compared to Jemalong. Based on the results of ROS profiling, time points for microarray analysis were one hour into the ozone treatment, end of treatment and onset of an ozone-induced ROS burst at 12 hours. Replicated temporal transcriptome analysis in these two accessions using 17 K oligonucleotide arrays revealed more than 2000 genes were differentially expressed. Significantly enriched gene ontologies (GOs) were identified using the Cluster Enrichment analysis program. A striking finding was the alacrity of JE154 in altering its gene expression patterns in response to ozone, in stark contrast to delayed transcriptional response of Jemalong. GOs involved in signaling, hormonal pathways, antioxidants and secondary metabolism were altered in both accessions. However, the repertoire of genes responding in each of these categories was different between the two accessions. Real-time PCR analysis confirmed the differential expression patterns of a subset of these genes. CONCLUSION: This study provided a cogent view of the unique and shared transcriptional responses in an ozone-resistant and sensitive accession that exemplifies the complexity of oxidative signaling in plants. Based on this study, and supporting literature in Arabidopsis we speculate that plants sensitive to acute ozone are impaired in perception of the initial signals generated by the action of this oxidant. This in turn leads to a delayed transcriptional response in the ozone sensitive plants. In resistant plants rapid and sustained activation of several signaling pathways enables the deployment of multiple mechanisms for minimizing the toxicity effect of this reactive molecule.

Physiological and biochemical responses to acute ozone-induced oxidative stress in Medicago truncatula.

Oxidative signaling mediated by reactive oxygen species (ROS) is a central component of biotic and abiotic stresses in plants. Acute ozone (O(3)) fumigation is a useful non-invasive treatment for eliciting endogenous ROS in planta. In this study, 38 different accessions of the model legume, Medicago truncatula, from various geographical regions were fumigated with 300 nmol mol(-1) of O(3) for a period of six hours. Phenotypic symptoms were evaluated 24 and 48 h after the end of treatment. A majority of the accessions showed distinct visible damage. Eight accessions showing varying sensitivities to ozone were subjected to biochemical analysis to evaluate correlations between ozone damage and levels of ROS, antioxidants, and lipid peroxidation. Two-way analysis of variance indicated highly significant interactions between O(3) damage and levels of ROS, ascorbate, glutathione and lipid peroxidation. There were significant differences among the accessions for these traits before and after the end of O(3) fumigation, as indicated by equal variance Student's t-test. This study suggests that multiple physiological and biochemical mechanisms may govern O(3) tolerance or sensitivity. Surveying a large collection of germplasm led to identification of multiple resistant and sensitive lines for investigating molecular basis of O(3) phytotoxicity. The most resistant JE154 accession also showed enhanced tolerance to chronic O(3) and dehydration stress, suggesting germplasm with increased tolerance to acute O(3) can be a useful resource for improving resistance to multiple abiotic stressors.