Development of a Fluorescent-Microsphere Immunoassay for Detection of Antibodies Specific to Equine Arteritis Virus and Comparison with the Virus Neutralization Test

Abstract

The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5 ], M 1-162 , and N 1-110 ), as well as partial sequences of these structural proteins (GP5 1-116 , GP5 75-112 , GP5 55-98 , M 88-162 , and N 1-69 ) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5 55-98 protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5 55-98 MIA and VNT outcomes correlated significantly (r ‫؍‬ 0.84; P < 0.0001). Although the GP5 55-98 MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses (61). EAV is a small enveloped virus with a positivesense, single-stranded RNA genome of 12.7 kb and belongs to the family Arteriviridae (genus Arterivirus, order Nidovirales), which also includes porcine reproductive and respiratory syndrome virus, simian hemorrhagic fever virus, and lactate dehydrogenase-elevating virus of mice (13, 57). The EAV genome includes nine functional open reading frames (ORFs) (55, 57). ORFs 1a and 1b encode two replicase polyproteins (pp1a and pp1ab) (25,55,57), and the remaining seven ORFs (2a, 2b, and 3 to 7) encode the structural proteins of the virus. These include four membrane glycoproteins, GP2 (25 kDa), GP3 (36 to 42 kDa), GP4 (28 kDa), and GP5 (30-44 kDa), encoded by ORFs 2b, 3, 4, and 5, respectively; two unglycosylated membrane proteins, E (8 kDa) and M (17 kDa), encoded by ORFs 2a and 6; and the phosphorylated nucleocapsid protein N (14 kDa), encoded by ORF 7 (23, 58, 64). The major envelope glycoprotein GP5 expresses the known neutralization determinants of EAV. The two major envelope proteins GP5 and M form a disulfide-linked heterodimer in the virus particle, and this association is critical for their maturation and for the expression of some of the neutralization epitopes in authentic form (3,24,56).Serological and clinical studies indicate that EAV is widely distributed in equine populations around the world (35,46,48). Although there is considerable variation in the sequences of...

Comparison of Immunologic Responses Following Intranasal and Oral Administration of a USDA-Approved, Live-Attenuated Streptococcus equi Vaccine

“…Microspheres coupled with partial glycoprotein (GP) 555-98 had a sensitivity and specificity of 93.1% and 93.9% compared to VNT. The MIA results correlated significantly to VNT; but sera with low antibody titers (1:4 and 1:8) gave false negative results on MIA indicating that the MIA can only detect higher titers (>/= 1:16) to EAV reliably (Go et al 2008). …”

“…). An MIA detecting equine arteritis virus (EAV) antibodies was compared to VNT (Go et al 2008). Microspheres coupled with partial glycoprotein (GP) 555-98 had a sensitivity and specificity of 93.1% and 93.9% compared to VNT.…”

“…Microsphere immunoassays have been developed to detect serologic responses to a variety of equine diseases including West Nile virus (WNV) (Balasuriya et al 2006), flavivirusassociated neurologic diseases , equine viral arteritis (Go et al 2008), African horse sickness (Sanchez-Matamoros et al 2015), Lyme disease (Wagner et al 2011), andHendra virus (McNabb et al 2014). An MIA incorporating recombinant WNV envelope protein (rE) was found to have diagnostic sensitivity and specificity (99.3% and 97.4%, respectively) compared to plaque reduction neutralization test (PRNT) (Balasuriya et al 2006).…”

Expression of M Protein from LP02/C Equine Arteritis Virus Inhibits Growth of Escherichia Coli M15-pQE30 System

“…As all gene products that affect cells kinetics growth are considered as toxic, we argue that the lower yields in EAV-M recovery could be attributed to an associated toxicity of EAV-M protein expression in this system or also to instability of the construction, as other expression system of EAV-M protein did not evidence this growth inhibition [15,23,24].…”

Equine viral arteritis: Current status and prevention

“…Assays using RT-PCR have several potential advantages over the current virus isolation procedure. However, further standardization and validation is absolutely necessary before RT-PCR is adopted as a reliable screening assay to evaluate clinical specimens, such as those from cases of abortion and semen from horses that are suspect carriers of the virus [82,83].…”