Importance of M-Protein C Terminus as Substrate Antigen for Serodetection of Equine Arteritis Virus Infection

Abstract

Equine arteritis virus (EAV),

The bovine viral diarrhea virus (BVDV) NS3 protein, when expressed alone in mammalian cells, induces apoptosis which correlates with caspase-8 and caspase-9 activation

“…The BVDV NS3 antiserum used in this study was raised in rabbits against recombinant NS3∆50 (rNS3∆50) fusion proteins expressed in Escherichia coli [16,20] using the pEt-21b expression vector (Novagen, Madison, USA). The cells were washed in phosphate-buffered saline (PBS) solution, pH 7.3, and lysed in standard SDS-PAGE sample buffer.…”

“…Total cell extract proteins were fractionated by 15% SDS-PAGE under reducing conditions and electrotransferred onto nitrocellulose membranes. Western immunoblotting on cell extracts was performed using, as the blocking reagent solution, 5% nonfat dried milk solids and 0.05% Tween 20 in PBS [16]. The blot was incubated with rabbit preimmune serum and BVDV NS3∆50-specific antiserum for 2 h at room temperature.…”

Binding of Cellular Proteins to the Leader RNA of Equine Arteritis Virus

“…In order to delineate the region in the (+) strand of EAV leader RNA that was involved in interactions with cell proteins, overlapping deletion mutants of the leader RNA (+), strand were generated by inversed PCR [10] from plasmid pEAVL(+) using appropriate primers. These mutants spanned nucleic acids 1-70, 51-115, 96-160, and 140-206 of the EAV leader RNA (+) strand, respectively.…”

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

“…These studies indicated that the major N protein epitope that reacts with anti-EAV equine sera is located within the amino acid residue segment from 1 to 69 in the terminal region of the protein. Similarly, it has been demonstrated previously that only the carboxyl-terminal sequence (aa 88 to 162) of the M protein is necessary to identify equine serum antibodies specific to the EAV M protein; it was further suggested that this region should be useful for the serodetection of EAV-infected horses (37). On the basis of the foregoing data, it was decided to express the full-length GP5 , M , and N 1-110 proteins, various antigenic regions of GP5 (aa 1 to 116, 75 to 112, and 55 to 98), and the M 88-162 and N 1-69 proteins as bacterial fusion proteins for use in MIAs to detect antibodies to EAV.…”

“…The nucleotide sequence 5Ј-CACC-3Ј was added at the 5Ј end of each primer for directional cloning into the pET TOPO vector (Invitrogen, Carlsbad, CA) ( Table 1). The full-length ORFs 5, 6, and 7 (which encode full-length GP5, M, and N proteins, respectively), as well as the coding regions for the amino-terminal ectodomain (amino acids [aa] 1 to 116) and two antigenic regions (aa 55 to 98 and aa 75 to 112) of the GP5 protein (14,15,49), the antigenic carboxyl terminus (aa 88 to 162) of the M protein (37), and the antigenic amino terminus (aa 1 to 69) of the N protein (16), were PCR amplified from the plasmid containing the complete genomic sequence of the EAV virulent Bucyrus strain (pEAVrVBS; GenBank accession number DQ846751) (9) by using Pfu DNA polymerase enzyme (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The individual PCR products were concentrated using a Centricon centrifugal filter unit (Ultracel YM-30; Millipore, Billerica, MA) and purified using a commercial kit (QIAGEN, Valencia, CA).…”