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(A) Representation of the 3 part of the EAV genome. Black box in ORF5, region deleted in the EAV-G L mutant. (B) Hydropathy profile of the EAV G L protein as determined by the method of Kyte and Doolittle (39) with a seven-residue moving window. Peaks extending upward indicate hydrophobic domains, and those pointing downward correspond to hydrophilic regions. Black bar, region deleted in the EAV-G L mutant.
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Equine arteritis virus (EAV) is an enveloped plus-strand RNA virus of the family Arteriviridae (order Nidovirales) that causes respiratory and reproductive disease in equids. Protective, virus-neutralizing antibodies (VNAb) elicited by
infection are directed predominantly against an immunodominant region in the membrane-proximal domain of the viral...
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... delete the immunodominant domain of G L and thereby obtain a potential marker vaccine virus, an EAV infectious cDNA mutant clone with a deletion of the gene segment specifying residues 66 to 112 of the G L protein was constructed (Fig. 1). For this purpose we used the primers 984 (positive polarity) and 983 (negative polarity), and infectious cDNA clone pEAN515 served as the template. Primer 984 hybridizes up- stream of a unique BglII site. Primer 983 hybridizes just up- stream of the proposed deletion and contains an extension corresponding to the oligonucleotide ...
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The replicase of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) is expressed in the form of two polyproteins (the open reading frame 1a [ORF1a] and ORF1ab proteins). Three viral proteases cleave these precursors into 12 nonstructural proteins, which direct both genome replication and subgenomic mRNA transcription. Immunofluor...
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... Mature MoDC infected with the low pathogenic strains too were largely unaffected and will contribute to the development of an immune response. It is well documented that CD8 + cytotoxic T lymphocytes (CTL) play a key role in the clearance of viral infections and a previous study specifically implicated these to target EAVinfected cells [55]. Indeed, iMoDC and mMoDC infected with less pathogenic strains may still have the ability to stimulate CD8 + T cells leading to clearance of these infected cells. ...
Equine viral arteritis is an infectious disease of equids caused by equine arteritis virus (EAV), an RNA virus of the family Arteriviridae. Dendritic cells (DC) are important modulators of the immune response with the ability to present antigen to naïve T cells and can be generated in vitro from monocytes (MoDC). DC are important targets for many viruses and this interaction is crucial for the establishment—or rather not—of an anti-viral immunity. Little is known of the effect EAV has on host immune cells, particularly DC. To study the interaction of eqDC with EAV in vitro, an optimized eqMoDC system was used, which was established in a previous study. MoDC were infected with strains of different genotypes and pathogenicity. Virus replication was determined through titration and qPCR. The effect of the virus on morphology, phenotype and function of cells was assessed using light microscopy, flow cytometry and in vitro assays. This study confirms that EAV replicates in monocytes and MoDC. The replication was most efficient in mature MoDC, but variable between strains. Only the virulent strain caused a significant down-regulation of certain proteins such as CD14 and CD163 on monocytes and of CD83 on mature MoDC. Functional studies conducted after infection showed that EAV inhibited the endocytic and phagocytic capacity of Mo and mature MoDC with minimal effect on immature MoDC. Infected MoDC showed a reduced ability to stimulate T cells. Ultimately, EAV replication resulted in an apoptosis-mediated cell death. Thus, EAV evades the host anti-viral immunity both by inhibition of antigen presentation early after infection and through killing infected DC during replication.
... Pseudorabies virus and bovine herpes virus marked vaccines were among the first to be developed and deployed in the field [26,27]. These vaccines were followed by the development of negative marked vaccines for RNA viruses, such as classical swine fever virus [28], Newcastle disease virus [29], and Equine Arteritis virus [30] through epitopes deletion strategy. These studies on RNA viruses provided insight into the development of negative marker vaccine against FMDV. ...
... Pseudorabies virus and bovine herpes virus marked vaccines were among the first to be developed and deployed in the field [26,27]. These vaccines were followed by the development of negative marked vaccines for RNA viruses, such as classical swine fever virus [28], Newcastle disease virus [29], and Equine Arteritis virus [30] through epitopes deletion strategy. These studies on RNA viruses provided insight into the development of negative marker vaccine against FMDV. ...
Foot-and-mouth disease (FMD) is a highly contagious, economically important disease of transboundary importance. Regular vaccination with chemically inactivated FMD vaccine is the major means of controlling the disease in endemic countries like India. However, the traditional inactivated vaccines may sometimes contain traces of FMD viral (FMDV) non-structural protein (NSP), therefore, interfering with the NSP-based serological discrimination between infected and vaccinated animals. The availability of marker vaccine for differentiating FMD infected from vaccinated animals (DIVA) would be crucial for the control and subsequent eradication of FMD in India. In this study, we constructed a negative marker FMDV serotype O virus (vaccine strain O IND R2/1975), containing dual deletions of amino acid residues 93-143 and 10-37 in the non-structural proteins 3A and 3B, respectively through reverse genetics approach. The negative marker virus exhibited similar growth kinetics and plaque morphology in cell culture as compared to the wild type virus. In addition, we also developed and evaluated an indirect ELISA (I-ELISA) targeted to the deleted 3AB NSP region (truncated 3AB) which could be used as a companion differential diagnostic assay. The diagnostic sensitivity and specificity of the truncated 3AB I-ELISA were found to be 95.5% and 96%, respectively. The results from this study suggest that the availability negative marker virus and companion diagnostic assay could open a promising new avenue for the application of DIVA compatible marker vaccine for the control of FMD in India. © 2015 The International Alliance for Biological Standardization.
... Experimental EAV vaccines have also been recently developed using recombinant DNA technology but none of these vaccines have reached the market. 17,84 ...
Equine arteritis virus (EAV), the causative agent of equine viral arteritis (EVA), is a respiratory and reproductive disease that occurs throughout the world. EAV infection is highly species-specific and exclusively limited to members of the family Equidae, which includes horses, donkeys, mules, and zebras. EVA is an economically important disease and outbreaks could cause significant losses to the equine industry. The primary objective of this article is to summarize current understanding of EVA, specifically the disease, pathogenesis, epidemiology, host immune response, vaccination and treatment strategies, prevention and control measures, and future directions.
Copyright © 2014 Elsevier Inc. All rights reserved.
... d/or deletions into the virus genome to produce attenuation and to minimize the likelihood of reversion to virulence (de Vries et al., 2000de Vries et al., , 2001). Obviously, the substitutions or deletions that are introduced into the cloned virus genome must not hinder the recombinant virus from inducing protective immunity in vaccinated animals. Castillo-Olivares et al. (2003) described the generation of a candidate live marker vaccine for EAV by deletion of the major neutralization domain (aa 66–112) in the GP5 protein. This recombinant (deletion mutant) virus replicated to normal titer in cell culture, but at a lower rate than parental virus. Furthermore, two ponies immunized with this deletion mutant virus ...
The advent of recombinant DNA technology, development of infectious cDNA clones of RNA viruses, and reverse genetic technologies have revolutionized how viruses are studied. Genetic manipulation of full-length cDNA clones has become an especially important and widely used tool to study the biology, pathogenesis, and virulence determinants of both positive and negative stranded RNA viruses. The first full-length infectious cDNA clone of equine arteritis virus (EAV) was developed in 1996 and was also the first full-length infectious cDNA clone constructed from a member of the order Nidovirales. This clone was extensively used to characterize the molecular biology of EAV and other Nidoviruses. The objective of this review is to summarize the characterization of the virulence (or attenuation) phenotype of the recombinant viruses derived from several infectious cDNA clones of EAV in horses, as well as their application for characterization of the molecular basis of viral neutralization, persistence, and cellular tropism.
... Herpesvirus negative marker vaccines were first developed in 1980, and their use has contributed to disease control and eradication in some countries [11,37,38]. Following the successes of these vaccines, negative marker vaccines of some animal diseases have been developed by identifying and selecting nonessential genes [35,[39][40][41][42], and some of them have successfully been used to control disease outbreaks [9,10]. ...
Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals in the world. The disease can be effectively controlled by vaccination of susceptible animals with the conventional inactivated vaccine. However, one major concern of the inactivated FMD virus (FMDV) vaccine is that it does not allow serological discrimination between infected and vaccinated animals, and therefore interferes with serologic surveillance and the epidemiology of disease. A marker vaccine has proven to be of great value in disease eradication and control programs. In this study, we constructed a marker FMDV containing a deletion of residues 93 to 143 in the nonstructural protein 3A using a recently developed FMDV infectious cDNA clone. The marker virus, r-HN/3A93-143, had similar growth kinetics as the wild type virus in culture cell and caused a symptomatic infection in pigs. Pigs immunized with chemically inactivated marker vaccine were fully protected from the wild type virus challenge, and the potency of this marker vaccine was 10 PD50 (50% pig protective dose) per dose, indicating it could be an efficacious vaccine against FMDV. In addition, we developed a blocking ELISA targeted to the deleted epitope that could clearly differentiate animals infected with the marker virus from those infected with the wild type virus. These results indicate that a marker FMDV vaccine can be potentially developed by deleting an immunodominant epitope in NSP 3A.
... The cell-mediated immune (CMI) response to arterivirus infection has not been characterized in great detail. Cellmediated cytotoxic responses against EAV were studied in experimentally infected ponies, in which CD8 + T-cellmediated cytotoxicity was virus strain-specific and genetically restricted (Castillo-Olivares et al., 2003). The EAVspecific CTL precursors persisted for at least 1 year after infection. ...
... Horses vaccinated with this candidate vaccine produced neutralizing antibodies, shed little or no virus and developed only mild symptoms after a challenge (Balasuriya et al., 2002). Using reverse genetics, a candidate EAV live marker vaccine was constructed by deletion of the major neutralization domain of GP5 (Castillo-Olivares et al., 2003). This recombinant virus produced an asymptomatic infection in ponies, which were subsequently protected against a challenge with virulent EAV. ...
Arteriviruses are positive-stranded RNA viruses that infect mammals. They can cause persistent or asymptomatic infections, but also acute disease associated with respiratory syndrome, abortion or lethal haemorrhagic fever. During the past two decades, porcine reproductive and respiratory syndrome virus (PRRSV) and - to a lesser extent - equine arteritis virus (EAV) have attracted attention as veterinary pathogens with significant economic impact. Particularly noteworthy were the 'porcine high fever disease' outbreaks in South-East Asia and the emergence of new virulent PRRSV strains in the U.S. Recently, the family was expanded with several previously unknown arteriviruses isolated from different African monkey species. At the molecular level, arteriviruses share an intriguing but distant evolutionary relationship with coronaviruses and other members of the order Nidovirales. On the other hand, several of their characteristics are unique, including virion composition and structure, and the conservation of only a subset of the replicase domains encountered in nidoviruses with larger genomes. During the past 15 years, the advent of reverse genetics systems for EAV and PRRSV has changed and accelerated the structure-function analysis of arterivirus RNA and protein sequences. These systems now also facilitate studies into host immune responses and arterivirus immune evasion and pathogenesis. In this review, we will summarize recent advances in the areas of arterivirus genome expression, RNA and protein functions, virion architecture, virus-host interactions, immunity and pathogenesis. We will also briefly review the impact of these advances on disease management, the engineering of novel candidate live vaccines and the diagnosis of arterivirus infection.
... While technically straightforward in the case of some double-stranded DNA viruses like Pseudorabies Virus (Suid Herpesvirus 1), it is very difficult to delete an entire gene of a smaller RNA genome virus like PRRSV, whose genes are all essential for the productive viral infection [20][21][22][23]. An alternative approach to develop live-attenuated DIVA vaccines for RNA viruses is to selectively eliminate a small protein fragment or an epitope, instead of deleting the whole protein [24][25][26]. Previously, we identified several immunodominant B-cell epitopes located in non-structural protein 2 (nsp2) and in different structural proteins of a type-II PRRSV strain FL12 [27]. ...
... The cell-mediated immune response (CMI) to EAV is poorly characterized. Castillo-Olivares et al. (2003) described EAV-specific cytotoxic T lymphocyte (CTL) responses using peripheral blood mononuclear cells (PBMCs) from convalescent EAV-infected (experimental) ponies. The data showed that cytotoxicity induced by EAVstimulated PBMCs was virus-specific, genetically restricted, and mediated by CD8 + T cells, and that EAV-specific CTL precursors persist for at least 1 year after infection. ...
... Several laboratories have developed and evaluated enzyme-linked immunosorbent assays (ELISAs) to detect antibodies to EAV using whole virus, synthetic peptides, or recombinant viral proteins (e.g. GP5, M, and N) as antigens (Castillo-Olivares et al., 2003;Cook et al., 1989;Duthie et al., 2008;Hedges et al., 1998;Kondo et al., 1998;Nugent et al., 2000;Starik et al., 2001;Wagner et al., 2003). Various studies have shown that the source of antigen as well as the sera evaluated can markedly influence the results obtained with EAV protein specific ELISAs and competitive ELISA. ...
... All vaccinated horses developed high VN antibody titers regardless of age and VN antibodies were still detectable over a year later (Giese et al., 2002). Castillo-Olivares et al. (2003) described creation of a candidate live marker vaccine for EAV by deletion of the major neutralization domain (aa 66-112) in the GP5 protein in an infectious cDNA clone. Similarly, the EAV030 cDNA clone has been used to develop disabled infectious single-cycle (DISC) mutants using complementing cell lines expressing minor structural proteins (GP2 [formally G S ], GP3, and GP4) (Zevenhoven- Dobbe et al., 2004). ...
... Modified-live vaccines containing such genetic markers have been developed for various animal RNA and DNA viruses, either by insertion of foreign antigens to create a positive marker or by deletion of an immunogenic domain (epitope) to create a negative marker. The antibody responses to the foreign insert or deleted epitope were used to differentiate vaccinated animals from naturally infected animals (Moormann et al., 1990;Kaashoek et al., 1994;van Oirschot et al., 1996;Walsh et al., 2000aWalsh et al., , 2000bWidjojoatmodjo et al., 2000;Mebatsion et al., 2002;Castillo-Olivares et al., 2003). ...
Our knowledge about the structure and function of the nonstructural proteins (nsps) encoded by the arterivirus replicase gene has advanced in recent years. The continued characterization of the nsps of the arterivirus prototype equine arteritis virus has not only corroborated several important functional predictions, but also revealed various novel features of arteriviral replication. For porcine reproductive and respiratory syndrome virus (PRRSV), based on bioinformatics predictions and experimental studies, a processing map for the pp1a and pp1ab replicase polyproteins has been developed. Crystal structures have been resolved for two of the PRRSV nonstructural proteins that possess proteinase activity (nsp1α and nsp4). The functional characterization of the key enzymes for arterivirus RNA synthesis, the nsp9 RNA polymerase and nsp10 helicase, has been initiated. In addition, progress has been made on nsp functions relating to the regulation of subgenomic mRNAs synthesis (nsp1), the induction of replication-associated membrane rearrangements (nsp2 and nsp3), and an intriguing replicative endoribonuclease (nsp11) for which the natural substrate remains to be identified. The role of nsps in viral pathogenesis and host immunity is also being explored, and specific nsps (including nsp1α/β, nsp2, nsp4, nsp7, and nsp11) have been implicated in the modulation of host immune responses to PRRSV infection. The nsp3-8 region was identified as containing major virulence factors, although mechanistic information is scarce. The biological significance of PRRSV nsps in virus-host interactions and the technical advancements in engineering the PRRSV genome by reverse genetics are also reflected in recent developments in the area of vaccines and diagnostic assays.
