Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus.
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African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned in Escherichia coli using the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151-200) and II (residues 83-120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule. Neutralizing epitopes were defined at positions 85-92 (PDPLSPGE) for MAb 10AE12 and at 179-185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques. These data will be especially useful for vaccine development and diagnostic purposes. (+info)
Identification and PCR-restriction fragment length polymorphism analysis of a variant of the Ibaraki virus from naturally infected cattle and aborted fetuses in Japan.
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One hundred fourteen field isolates of the Ibaraki virus (IBAV), a member of the epizootic hemorrhagic disease virus serotype 2 (EHDV-2), were isolated from blood samples of affected and apparently healthy cattle and Culicoides biting midges and from blood samples of dams and internal organs of aborted fetuses during an outbreak of Ibaraki disease in the southern part of Japan in 1997. In this outbreak, 242 cattle showed typical symptoms of the disease, and several hundred dams had miscarriages or stillbirths. The viruses that induced typical Ibaraki disease and reproductive problems among cattle were identical and were antigenically closely related to but distinct from previous isolates of IBAV and EHDV-2. The virus was considered to be a putative agent of this outbreak. Reverse transcription-PCR based on segment 3 of the RNA genome of EHDV-2 and restriction fragment length polymorphism analysis of the PCR products were conducted to compare the genomes of the viruses. The results suggested that the virus isolated in 1997 was a variant of IBAV and might be exotic. (+info)
Development of an enzyme-linked immunosorbent assay for the detection of antibody to epizootic hemorrhagic disease of deer virus.
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An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1- and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo. (+info)
Cloning and expression of the M5 RNA segment encoding outer capsid VP5 of epizootic hemorrhagic disease virus Japan serotype 2, Ibaraki virus.
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The complete nucleotide sequence of a cDNA clone representing the M5 RNA segment of epizootic hemorrhagic disease virus Japan serotype 2 (EHDV-2), Ibaraki virus, was determined. The M5 segment is 1641 base pairs long with the single open reading frame which predicts a polypeptide of 527 amino acids. The comparison of the amino acid sequence of the VP5 with those of EHDV-1, bluetongue virus serotype 10, and African horse sickness virus serotype 4 revealed that the protein shared 67%, 57% and 42% homologies, respectively. In addition, the VP5 protein was expressed in insect cells by recombinant baculovirus, which could be recognized by the mouse anti-EHDV-2 sera at a position of the expected 59 kDa on immunoblot analysis. (+info)
The complete nucleotide sequence of segment L2 of Ibaraki virus encoding for the antigen recognized by neutralizing antibodies.
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The complete nucleotide sequence of cDNA clones representing the L2 dsRNA from Japan isolate of epizootic hemorrhagic disease serotype 2 (EHDV-2JPN) was determined. The EHDV-2JPN L2 gene is 3002 base pairs long with a single open reading frame of 2949 bp which predicts a polypeptide of 982 amino acid residues. Comparison of VP2 sequence between Japan and North American Isolates of EHDV-2 showed a 72% homology in spite of the same serotype, although those among the North American isolates showed a high genetic identity (>97%). (+info)
The complete nucleotide sequences of L3 and S7 segments of Ibaraki virus encoding for the major inner capsid proteins, VP3 and VP7.
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The complete nucleotide sequences of the genes encoding two of the major inner capsid proteins of Ibaraki virus (IBAV), belonging to epizootic hemorrhagic disease virus serotype 2 (EHDV-2) were determined. The L3 RNA segment is 2768 nucleotides in length which encodes VP3 polypeptides of 899 amino acid residues (M.W. 103 kDa). The S7 RNA segment, which encodes the VP7 core protein, is 1162 nucleotides in length and encodes 349 amino acids (M.W. 38 kDa). These RNA segments had the characteristic consensus motifs of Orbivirus RNA segments in termini, namely 5'-GUUAAA... and ...ACUUAC-3'. The comparison of the IBAV L3 and S7 sequences with those of other two EHDV-2 isolates revealed the higher homologies of 93% and 92% against EHDV-2 Australia isolate (EHDV-2AUS) and lower homologies of 80% and 81% against EHDV-2 North America isolate, respectively. The phylogenetic analysis based on L3 and S7 genes also indicated close relationships between IBAV and EHDV-2AUS. (+info)
Incursion of epizootic hemorrhagic disease into the Okanagan Valley, British Columbia in 1999.
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In September 1999, unusually high mortality rates in white-tailed deer and California bighorn sheep occurred in the southern Okanagan Valley. Necropsy and histopathologic findings were compatible with epizootic hemorrhagic disease (EHD); the presence of virus was not demonstrated. Subsequent serologic and polymerase chain reaction assays on sentinel cattle suggested an EHD virus incursion. (+info)
Analysis of intratypic variation evident in an Ibaraki virus strain and its epizootic hemorrhagic disease virus serogroup.
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A new strain of Ibaraki virus (IBAV) was isolated from cattle showing atypical symptoms of Ibaraki disease. The isolate was genetically characterized, and the genetic diversity and evolution of the capsid proteins of viruses in the epizootic hemorrhagic disease virus (EHDV) serogroup were investigated. The nucleotide sequences of the isolate's viral RNA segments 2, 3, 6, and 7, which encode the viral structural proteins VP2, VP3, VP5, and VP7, respectively, were determined and were then compared against those of the existing strains of IBAV and EHDV, to which IBAV belongs serologically. The nucleotide sequences of segments 3 and 7 were conserved within the EHDV serogroup, particularly well among the strains of IBAV and Australian EHDV. The similarity of the sequence of segment 6 of the isolate to sequences of corresponding segments of the other strains of IBAV and EHDV was found to be about 93%. The similarity of segment 2 of the isolate to segments 2 of the other strains of IBAV and EHDV was less than 70%. Phylogenetic analysis based on the deduced amino acid sequences of segments 3 and 7 revealed that the viruses differed according to their geographical distributions. However, the new isolate of IBAV was categorized as having a distinct lineage in the phylogenetic tree of VP2. These results suggest that the isolate was modified by a reassortment of segment 2 and that it exhibits unique genetic and antigenic characteristics. (+info)