Generation of a Candidate Live Marker Vaccine for Equine Arteritis Virus by Deletion of the Major Virus Neutralization Domain

Abstract

Equine arteritis virus (EAV) is an enveloped plus-strand RNA virus of the family. By using an infectious cDNA we have now generated, in the controlled background of a nonvirulent virus, a mutant EAV from which this immunodominant domain was deleted. This virus, EAV-G L ⌬, replicated to normal titers in culture cells, although at a slower rate than wild-type EAV, and caused an asymptomatic infection in ponies. The antibodies induced neutralized the mutant virus efficiently in vitro but reacted poorly to wild-type EAV strains. Nevertheless, when inoculated subsequently with virulent EAV, the immunized animals, in contrast to nonvaccinated controls, were fully protected against disease; replication of the challenge virus occurred briefly at low though detectable levels. The levels of protection achieved suggest that an immune effector mechanism other than VNAb plays an important role in protection against infection. As expected, infection with EAV-G L ⌬ did not induce a measurable response in our G Lpeptide ELISA while the challenge infection of the animals clearly did. EAV-G L ⌬ or similar mutants are therefore attractive marker vaccine candidates, enabling serological discrimination between vaccinated and wild-type virus-infected animals.Equine arteritis virus (EAV), a plus-strand RNA virus of the family Arteriviridae (order Nidovirales), is a worldwide pathogen for horses and donkeys. The virus was first isolated from lung tissue of fetuses aborted during an outbreak in Ohio in 1953 (25, 27) and became the prototype arterivirus; it was joined later by the lactate dehydrogenase-elevating virus of mice, simian hemorrhagic fever virus, and porcine reproductive and respiratory syndrome virus. Though the virus infects cells from quite a variety of origins in vitro, its host range in nature is narrowly restricted to equids (17,19,36,52,54).In the horse clinical signs of infection vary widely and appear to depend on the particular strain and dose of the virus, the age and physical condition of the animal, and possibly also the route of infection (58, 60). While epidemiological studies indicate that natural infections often occur asymptomatically (16,46,61), affected animals may develop moderate-to-severe symptoms as well. Lethal infections of horses with EAV have been reported only under experimental conditions (12, 29).While infected horses usually recover after cessation of viremia, foal death as a result of rapidly progressive bronchointerstitial pneumonia and intestinal necrosis can occur in young and adolescent pregnant mares. In mares EAV infection does not seem to affect fertility. Stallions may, however, temporarily exhibit fertility problems due to a reduced production of morphologically normal sperm cells during the first months after infection (48).Transmission of EAV occurs via the respiratory route through direct contact with infectious aerosolized nasopharyngeal secretions from acutely infected horses or other secretions such as urine; feces and vaginal fluids; and fetal, placental, and amniotic materia...

Structural Protein Requirements in Equine Arteritis Virus Assembly

“…However, to improve the detection of the GP 3 and GP 4 proteins, this time the labeling procedure was performed with [ 35 S]cysteine instead of [ 35 S]methionine. After labeling, the culture supernatants were mixed with concentrated lysis buffer, and immunoprecipitations were performed with antisera specific for E, GP 2b , GP 3 , and GP 4 immunoprecipitates were analyzed by SDS-PAGE under reducing conditions (Fig. 2).…”

“…On the other hand, arteriviruses appear surprisingly tolerant of deletions in and replacements of their GP 5 ectodomain. EAV deletion mutants lacking amino acids 66 through 112, 62 through 101, or 52 to 79 of GP 5 replicate in vitro (1,3,26). Moreover, replacement of the entire GP 5 ectodomain of EAV by that of PRRSV or LDV yielded replication-competent viruses (9).…”

“…The GP 2b (previously named G S ), GP 3 , and GP 4 proteins are all minor envelope glycoproteins (6,43). Recently, we demonstrated that these proteins occur in virions as a covalently associated heterotrimeric complex (42).…”

“…The small hydrophobic envelope protein (E) of 6.5 to 10.5 kDa does not contain N-linked glycans and has been detected in virus parti-cles in higher amounts than the minor envelope glycoproteins but in lower quantities than the GP 5 and M proteins (27). The E, GP 2b , GP 3 , GP 4 , GP 5 , M, and N proteins are encoded by open reading frames (ORFs) 2a, 2b, 3, 4, 5, 6, and 7, respectively. By separately knocking out the expression of each of these ORFs in the context of a (full-length) EAV cDNA clone, it has been shown that all seven structural proteins are required for the production of infectious progeny virus (21,27,32).…”

Equine viral vaccines: the past, present and future

“…Castillo-Olivares et al [17] developed a candidate live marker vaccine (EAV-G L ∆) for EAV by deletion of the major virus neutralising domain of the G L protein. A peptide, contained within the deleted sequence forms the basis of a discriminating ELISA assay [71].…”