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. (1/63)

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)

African horse sickness in Portugal: a successful eradication programme. (2/63)

African horse sickness (AHS) was diagnosed for the first time in southern Portugal in autumn 1989, following outbreaks in Spain. AHS virus presence was confirmed by virus isolation and serotyping. An eradication campaign with four sanitary zones was set up by Central Veterinary Services in close collaboration with private organizations. Vaccination began on 6 October. In February 1990, vaccination was extended to all Portuguese equines (170000 animals). There were 137 outbreaks on 104 farms: 206 of the equidae present died (16%) or were slaughtered (14%); 81.5% were horses, 10.7% were donkeys and 7.8% were mules. Clinical AHS occurred more frequently in horses than donkeys and mules. In the vaccinated population, 82 animals (62.2% horses and 37.8% mules and donkeys), died or were slaughtered due to suspected or confirmed AHS. One year after ending vaccination, December 1991, Portugal was declared free of AHS. Cost of eradication was US$1955513 (US$11.5/Portuguese equine).  (+info)

Identification and differentiation of the nine African horse sickness virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2. (3/63)

This paper describes the first RT-PCR for discrimination of the nine African horse sickness virus (AHSV) serotypes. Nine pairs of primers were designed, each being specific for one AHSV serotype. The RT-PCR was sensitive and specific, providing serotyping within 24 h. Perfect agreement was recorded between the RT-PCR and virus neutralization for a coded panel of 56 AHSV reference strains and field isolates. Serotyping was achieved successfully with live and formalin-inactivated AHSVs, with isolates of virus after low and high passage through either tissue culture or suckling mouse brain, with viruses isolated from widely separated geographical areas and with viruses isolated up to 37 years apart. Overall, this RT-PCR provides a rapid and reliable method for the identification and differentiation of the nine AHSV serotypes, which is vital at the start of an outbreak to enable the early selection of a vaccine to control the spread of disease.  (+info)

Effects of chlorine, iodine, and quaternary ammonium compound disinfectants on several exotic disease viruses. (4/63)

The effects of three representative disinfectants, chlorine (sodium hypochlorite), iodine (potassium tetraglicine triiodide), and quaternary ammonium compound (didecyldimethylammonium chloride), on several exotic disease viruses were examined. The viruses used were four enveloped viruses (vesicular stomatitis virus, African swine fever virus, equine viral arteritis virus, and porcine reproductive and respiratory syndrome virus) and two non-enveloped viruses (swine vesicular disease virus (SVDV) and African horse sickness virus (AHSV)). Chlorine was effective against all viruses except SVDV at concentrations of 0.03% to 0.0075%, and a dose response was observed. Iodine was very effective against all viruses at concentrations of 0.015% to 0.0075%, but a dose response was not observed. Quaternary ammonium compound was very effective in low concentration of 0.003% against four enveloped viruses and AHSV, but it was only effective against SVDV with 0.05% NaOH. Electron microscopic observation revealed the probable mechanism of each disinfectant. Chlorine caused complete degeneration of the viral particles and also destroyed the nucleic acid of the viruses. Iodine destroyed mainly the inner components including nucleic acid of the viruses. Quaternary ammonium compound induced detachment of the envelope of the enveloped viruses and formation of micelle in non-enveloped viruses. According to these results, chlorine and iodine disinfectants were quite effective against most of the viruses used at adequately high concentration. The effective concentration of quaternary ammonium compound was the lowest among the disinfectants examined.  (+info)

Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries. (5/63)

VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.  (+info)

Variation of African horsesickness virus nonstructural protein NS3 in southern Africa. (6/63)

NS3 protein sequences of recent African horsesickness virus (AHSV) field isolates, reference strains and current vaccine strains in southern Africa were determined and compared. The variation of AHSV NS3 was found to be as much as 36.3% across serotypes and 27.6% within serotypes. NS3 proteins of vaccine and field isolates of a specific serotype were found to differ between 2.3% and 9.7%. NS3 of field isolates within a serotype differed up to 11.1%. Our data indicate that AHSV NS3 is the second most variable AHSV protein, the most variable being the major outer capsid protein, VP2. The inferred phylogeny of AHSV NS3 corresponded well with the described NS3 phylogenetic clusters. The only exception was AHSV-8 NS3, which clustered into different groups than previously described. No obvious sequence markers could be correlated with virulence. Our results suggest that NS3 sequence variation data could be used to distinguish between field isolates and live attenuated vaccine strains of the same serotype.  (+info)

Membrane association of African horsesickness virus nonstructural protein NS3 determines its cytotoxicity. (7/63)

The smallest RNA genome segment of African horsesickness virus (AHSV) encodes the nonstructural protein NS3 (24K). NS3 localizes in areas of plasma membrane disruption and is associated with events of viral release. Conserved features in all AHSV NS3 proteins include the synthesis of a truncated NS3A protein from the same open reading frame as that of NS3, a proline-rich region, a region of strict sequence conservation and two hydrophobic domains. To investigate whether these features are associated with the cytotoxicity of NS3 or altered membrane permeability, a series of mutants were constructed and expressed in the BAC-TO-BAC baculovirus-expression system. Our results indicate that mutations in either of the two hydrophobic domains do not prevent membrane targeting of the mutant proteins but abolish their membrane anchoring. This prevents their localization to the cell surface and obviates their cytotoxic effect. The cytotoxicity of NS3 is therefore dependent on its membrane topography and thus involves both hydrophobic domains. NS3 has many of the characteristics of lytic viral proteins that play a central role in viral pathogenesis through modifying membrane permeability.  (+info)

Definition of neutralizing sites on African horse sickness virus serotype 4 VP2 at the level of peptides. (8/63)

The antigenic structure of African horse sickness virus (AHSV) serotype 4 capsid protein VP2 has been determined at the peptide level by PEPSCAN analysis in combination with a large collection of polyclonal antisera and monoclonal antibodies. VP2, the determinant for the virus serotype and an important target in virus neutralization, was found to contain 15 antigenic sites. A major antigenic region containing 12 of the 15 sites was identified in the region between residues 223 and 400. A second domain between residues 568 and 681 contained the three remaining sites. These sites were used for the synthesis of peptides, which were later tested in rabbits. Of the 15 synthetic peptides, three were able to induce neutralizing antibodies for AHSV-4, defining two neutralizing epitopes, 'a' and 'b', between residues 321 and 339, and 377 and 400, respectively. A combination of peptides representing both sites induced a more effective neutralizing response. Still, the relatively low neutralization titres make the possibility of producing a synthetic vaccine for AHSV unlikely. The complex protein-protein interaction of the outer shell of the viral capsid would probably require the presence of either synthetic peptides in the correct conformation or peptide segments from the different proteins VP2, VP5 and VP7.  (+info)