Gordon Ward

Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine.

We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.

A pandemic strain of calicivirus threatens rabbit industries in the Americas

Rabbit Hemorrhagic Disease (RHD) is a severe acute viral disease specifically affecting the European rabbit Oryctolagus cuniculus. As the European rabbit is the predominant species of domestic rabbit throughout the world, RHD contributes towards significant losses to rabbit farming industries and endangers wild populations of rabbits in Europe and other predatory animals in Europe that depend upon rabbits as a food source. Rabbit Hemorrhagic Disease virus (RHDV) – a Lagovirus belonging to the family Caliciviridae is the etiological agent of RHD. Typically, RHD presents with sudden death in 70% to 95% of infected animals. There have been four separate incursions of RHDV in the USA, the most recent of which occurred in the state of Indiana in June of 2005. Animal inoculation studies confirmed the pathogenicity of the Indiana 2005 isolate, which caused acute death and pathological changes characterized by acute diffuse severe liver necrosis and pulmonary hemorrhages. Complete viral genome sequences of all USA outbreak isolates were determined and comparative genomics revealed that each outbreak was the result of a separate introduction of virus rather than from a single virus lineage. All of the USA isolates clustered with RHDV genomes from China, and phylogenetic analysis of the major capsid protein (VP60) revealed that they were related to a pandemic antigenic variant strain known as RHDVa. Rapid spread of the RHDVa pandemic suggests a selective advantage for this new subtype. Given its rapid spread, pathogenic nature, and potential to further evolve, possibly broadening its host range to include other genera native to the Americas, RHDVa should be regarded as a threat.

Construction and evaluation of an attenuated vaccine for foot-and-mouth disease: difficulty adapting the leader proteinase-deleted strategy to the serotype O1 virus

Over the last few years we have utilized a system to genetically engineer foot-and-mouth disease virus (FMDV) to produce live-attenuated vaccine candidates. These candidates have been generated in the genetic background of a tissue culture-adapted strain of serotype A12 virus. Based on this A12 system, we created a virus lacking the sequence encoding the leader (L) proteinase (Piccone et al., 1995), and demonstrated that this leaderless virus, A12-LLV2 was avirulent in bovine and swine, and could be used as an attenuated vaccine (Mason et al., 1997; Chinsangaram et al., 1998). The current study shows that a similar leader-deleted chimeric virus containing the genome of the type A12 virus with a substituted type O1 capsid coding region from a bovine-virulent virus can be constructed, and that the virus has low, but detectable virulence in swine. A second chimera specifying a tissue culture-adapted type O1 capsid lacking the RGD cell binding site, was avirulent in swine, but was not sufficiently immunogenic to provide protection from challenge. These results are described with respect to mechanisms of attenuation and antigen formation in live-attenuated virus-inoculated animals.

FOOT-AND-MOUTH DISEASE IN NORTH AMERICAN BISON (BISON BISON) AND ELK (CERVUS ELAPHUS NELSONI): SUSCEPTIBILITY, INTRA- AND INTERSPECIES TRANSMISSION, CLINICAL SIGNS, AND LESIONS

There is limited information about the pathogenesis and epidemiology of foot-and-mouth disease (FMD) in North American bison (Bison bison) or elk (Cervus elaphus nelsoni). In these two experimental infection studies, we compared the susceptibilities of bison and elk to FMD virus (FMDV), respectively, with that of cattle; determined whether intra- and interspecies transmission could occur in bison and cattle, and elk and cattle; determined suitability of conventional available laboratory tests to detect FMDV infection in bison and elk; and investigated whether bison or elk are efficient long-term carriers of FMDV. In both studies, after a period of acclimation to the containment at Plum Island Animal Disease Center, animals were infected by intraepithelial tongue inoculation with 10,000 bovine tongue infective doses of FMDV, strain O1 Manisa. Inoculated animals were kept with contact animals; subsequently, inoculated and/or exposed contact animals were placed in rooms with unexposed animals. All bison developed oral mucosal and foot lesions similar to those of cattle. Bison developed fever, lameness, inappetence, and ptyalism. Physical examinations on bison revealed numerous small vesicles and erosions affecting tongue, gingiva, muzzle, hard and soft palates, coronary bands, and interdigital skin. Inoculated elk developed transient fever and mild focal tongue and foot lesions. Contact elk developed neither clinical signs nor gross pathologic lesions of FMD. At necropsy, lesions in bison included numerous extensive vesicles, erosions, and/or ulcers in the oral cavities, feet, and rumen pillars depending on the stage of disease. Less extensive oral, foot, and rumen lesions were present in the inoculated elk. All bison and inoculated elk developed antibodies to FMDV and were positive for FMDV by reverse transcription-polymerase chain reaction (RT-PCR). Transmission occurred between cattle and bison, and bison and bison. It did not occur between elk and cattle. Elk-to-elk transmission studies resulted in only one contact elk developing serologic evidence of a subclinical infection. Other exposed elk developed neither clinical, pathologic, virologic, nor serologic evidence of disease. FMDV was not isolated from animals past 28 days postinfection.

Analysis of clinical samples for early detection of classical swine fever during infection with low, moderate, and highly virulent strains in relation to the onset of clinical signs.

Classical swine fever (CSF) is a transboundary viral disease affecting swine. The clinical course of disease and the best diagnostic samples for early detection were examined using low, moderate, and highly virulent strains of CSFV inoculated into 8-12 week old domestic pigs. Clinical signs were monitored and recorded. Nasal swabs, tonsil scrapings, blood and tonsils were tested using virus isolation, immunohistochemistry, and real-time reverse transcriptase PCR (rRT-PCR).Severe clinical signs appear 3 days post infection (dpi) with the highly virulent strain, correlating with positive tonsil scrapings, tonsil and blood by virus isolation and rRT-PCR (83-100%), whereas nasal swabs become comparable by 5dpi (89-100%). The moderate strain caused less severe clinical signs between 5 and 7dpi, with tonsil scrapings, tonsil and blood positive by 7dpi (83-100%), and nasal swabs were comparable at 10dpi (67-90%). The low virulent strain showed mild clinical signs at 7dpi, with blood, tonsil and tonsil scrapings positive by virus isolation and rRT-PCR. Except for one sample at 10dpi, nasal swabs remained negative throughout the course of infection. This study indicates that irrespective of virulence, whole blood and tonsil scrapings are the sample of choice for early detection of CSFV in live pigs.

FOOT-AND-MOUTH DISEASE IN A SMALL SAMPLE OF EXPERIMENTALLY INFECTED PRONGHORN (ANTILOCAPRA AMERICANA).

Abstract There is limited information on the pathogenesis and epidemiology of foot-and-mouth disease (FMD) in North American wildlife and none concerning pronghorn (Antilocapra americana). In an experimental study of 13 pronghorn and six steers (Bos taurus), we compared the susceptibility of pronghorn to FMD virus (FMDV) strain O, with that of cattle (Bos taurus). We also determined the potential for intra- and interspecies transmission of FMDV strain O in pronghorn and cattle, assessed the application of conventional laboratory tests in their suitability to detect FMDV infection in pronghorn, and evaluated the potential role of pronghorn as efficient long-term carriers of FMDV. After acclimation to containment at Plum Island Animal Disease Center, two pronghorn and one steer were each infected by intraepithelial tongue inoculation with 10,000 bovine tongue infective doses of FMDV, strain O1 Manisa. Inoculated animals were housed with contact animals. When contact-exposed animals developed fever they were placed in rooms with previously unexposed animals. All inoculated and exposed cattle and pronghorn developed clinical disease typical of FMD. Pronghorn developed severe foot lesions and mild to moderate oral lesions, primarily on the tongue. Duration of clinical signs in both species was 2–3 wk with foot abnormalities evident to the end of the study (51 d postexposure). Other lesions included pancreatitis, myositis of the tongue, and secondary lesions including pleuritis, pneumonia, decubital ulcers, and tenosynovitis. Virus transmission occurred between pronghorn, from cattle to pronghorn, and from pronghorn to cattle. Conventional laboratory tests detected virus and antibodies against nonstructural and structural FMDV proteins in pronghorn and cattle. Virus was present in some animals for 1 wk but was not detectable by virus isolation or PCR at 3 wk postinfection or afterward.

Development and validation of a highly sensitive real-time PCR assay for rapid detection of parapoxviruses

Parapoxviruses (PaPVs) cause widespread infections in ruminants worldwide. All PaPVs are zoonotic and may infect humans after direct or indirect contact with infected animals. Herein we report the development and validation of a highly sensitive real-time PCR assay for rapid detection of PaPVs. The new assay (referred to as the RVSS assay) was specific for PaPVs only and had no cross-reactivity against other pox viruses. Using a recombinant plasmid as positive control, the analytical sensitivity of the assay was determined to be 16 genome copies of PaPV per assay. The amplification efficiency estimate (91–99%), the intra- and interassay variability estimate (standard deviation [SD]: 0.28–1.06 and 0.01–0.14, respectively), and the operator variability estimate (SD: 0.78 between laboratories and 0.28 between operators within a laboratory) were within the acceptable range. The diagnostic specificity was assessed on 100 specimens from healthy normal animals and all but 1 tested negative (99%). The diagnostic sensitivity (DSe) was assessed on 77 clinical specimens (skin/scab) from infected sheep, goats, and cattle, and all tested positive (100%). The assay was multiplexed with beta-actin as an internal positive control, and the multiplex assay exhibited the same DSe as the singleplex assay. Further characterization of the PaPV specimens by species-specific real-time PCR and nucleotide sequencing of the PCR products following conventional PCR showed the presence of Orf virus not only in sheep and goats but also in 1 bovid. The validated RVSS assay demonstrated high specificity, sensitivity, reproducibility, and ruggedness, which are critical for laboratory detection of PaPVs.