H. Huismans

In vitro Transcription and Translation of Bluetongue Virus mRNA

Fractionation of in vitro transcribed bluetongue virus (BTV) mRNA by agarose gel electrophoresis resulted in the separation of eight of the 10 species. The relative molar ratio of the mRNAs confirmed that mRNA 5 was transcribed more frequently than would be predicted from the size of the S5 genome segment, while mRNA 10 was transcribed less frequently. In vitro translation of unfractionated BTV mRNAs resulted in the synthesis of the seven known structural proteins (P1 to P7) and two known non-structural proteins (NS1 and NS2). Two additional non-structural proteins (NS3 and NS3A) with Mr of 28K and 25K respectively were identified. The protein coding assignments for the medium- and small-sized double-stranded RNA genome segments of BTV serotype 10 were found to correspond to those reported for BTV-1 and BTV-17. The peptide maps of NS1, NS2, NS3 and NS3A synthesized in vitro corresponded to those of their counterparts synthesized in infected cells. Protein NS3A appeared to be a truncated form of NS3, since its peptide map completely overlapped that of NS3. Proteins NS3 and NS3A were present in very small amounts in the soluble fraction of the cytoplasm of infected cells, and were synthesized in variable amounts in vitro, whereas the other nine viral proteins were synthesized in constant molar ratios. A difference in the relative molar ratios in which some of the BTV proteins were synthesized in vitro and in vivo was observed. In vivo, protein NS1 was translated in the largest amount but in vitro, NS2 was the most efficiently translated protein. Conversely, protein P6 was translated much more efficiently in vitro than in vivo.

Characterization and cloning of the African horsesickness virus genome

The dsRNA profiles of all nine African horsesickness virus (AHSV) serotypes were compared by agarose gel electrophoresis and PAGE. The agarose profiles were identical, but a unique profile was obtained for each of the nine serotypes by PAGE. Nine of the 10 dsRNA genome segments of AHSV-3 were cloned and the clones were used in dot-spot and Northern blot hybridization experiments to determine intra- and inter-serogroup nucleic acid similarities. Segments 1, 3, 4, 5, 7 and 8 were highly conserved in the AHSV serogroup and no genetic relationship with any of the other orbiviruses was observed. Of these segments 3, 5 and 8 showed the largest degree of cross-hybridization to the cognate genes of all the serotypes. These clones did not cross-hybridize to other orbiviruses such as epizootic haemorrhagic disease virus, bluetongue virus or equine encephalosis virus and are therefore recommended for use as group-specific probes for the identification of the AHSV serogroup. Genome segments 6 and 10 showed an intermediate degree of conservation, whereas segment 2 is serotype-specific and therefore probably codes for the outer capsid protein VP2.

A comparison of the genes which encode non-structural protein NS3 of different orbiviruses

The segment 10 (S10) genes of African horsesickness virus (AHSV), Palyam virus and epizootic haemorrhagic disease virus were translated in vitro in a rabbit reticulocyte lysate system. Each of the S10 genes encoded two proteins NS3 and NS3A, which were shown to be related by peptide mapping. Cloned copies of the S10 genes of two AHSV serotypes (AHSV-3 and AHSV-9) and Palyam virus were sequenced and compared to each other and to the nucleotide sequence of bluetongue virus (BTV) gene S10. Two in-phase ATG translation initiation codons reported for the S10 genes of BTV-10 and BTV-1 were conserved in the S10 genes of AHSV-3, AHSV-9 and Palyam virus, and would be able to initiate synthesis of NS3 and NS3A respectively. Comparison of the amino acid sequences of NS3 of AHSV-3 and AHSV-9 identified two areas of approximately 45 amino acids which displayed high (98%) similarity. One of these areas corresponded to the only region which displayed more than 50% amino acid similarity between NS3 of BTV, AHSV and Palyam virus. This region could represent an important structural or catalytic site of the protein. The overall amino acid similarity outside this conserved region was between 13% and 29%.

A comparison of the nucleotide sequences of cognate NS2 genes of three different orbiviruses

The genes encoding nonstructural protein NS2 of African horsesickness virus (AHSV) and epizootic hemorrhagic disease virus (EHDV) were cloned, sequenced, and compared to the NS2 gene of bluetongue virus (BTV). Nucleotide similarity ranged from 53 to 60%. The length of the proteins varied from 376 amino acids (EHDV) to 365 amino acids (AHSV). The N-terminal half of NS2 is more conserved (+/- 58% similarity) among the three orbiviruses, while the C-terminal half contains a 120 amino acid region of low similarity (18%). The variable region has a high content of alpha-helix conformation and a hydrophilic character. A short region of 9 amino acids contains 5 amino acids that are either similar or identical in single-stranded RNA binding proteins of BTV, EHDV, AHSV, reovirus and rotavirus.

Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries

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.

Expression of nonstructural protein NS3 of African horsesickness virus (AHSV): evidence for a cytotoxic effect of NS3 in insect cells, and characterization of the gene products in AHSV infected Vero cells.

SummaryThe smallest genome segment of African horsesickness virus (AHSV), segment 10 (S10), encodes two minor nonstructural proteins, NS3 and NS3A. While the cognate bluetongue virus (BTV) proteins have been suggested to play a role in the release of virus particles from infected cells, no function has yet been ascribed to AHSV NS3/NS3A. When the AHSV-3 S10 gene was expressed in a baculovirus system only a single NS3 protein (24K) was synthesized, at lower levels than expected. It was shown that this could be due to a membrane association of NS3, leading to an alteration in host cell membrane permeability and eventual cell death. Based on computer predictions a general model for the membrane-associated topology of NS3 of five different orbiviruses was proposed. Studies on AHSV-3 infected Vero cells showed that equimolar amounts of NS3 and NS3A were synthesized. No evidence was found for the glycosylation of NS3. The S10 genes and NS3/3A proteins of AHSV-3 and AHSV-7 were shown to be closely related, and clearly distinct from the cognate proteins of the other 7 AHSV serotypes. This distinguishes the AHSV S10 gene product from that of BTV NS3, which appears to be much more conserved.

Intracellular production of African horsesickness virus core-like particles by expression of the two major core proteins, VP3 and VP7, in insect cells.

To gain more insight into the structure of the African horsesickness virus (AHSV) core particle, we have cloned, partially characterized and expressed the two major core proteins, VP3 and VP7, of AHSV-9. VP7 was found to be highly conserved amongst different serotypes. The VP3 and VP7 genes were subsequently expressed in insect cells by means of recombinant baculoviruses. VP7 was synthesized to very high levels and aggregated into distinctive crystals. Co-expression of VP3 and VP7 resulted in the intracellular formation of core-like particles which structurally resembled empty AHSV cores.

The Characterization of Equine Encephalosis Virus and the Development of Genomic Probes

Equine encephalosis virus (EEV) is an orbivirus associated with a peracute illness of horses in southern Africa. The virus has now been partially purified for the first time and characterized on a molecular level. The virion is composed of 10 dsRNA segments and a protein capsid consisting of at least seven structural proteins that vary in Mr from 36,000 to 120,000. Partial clones of six of the dsRNA segments of EEV serotype Cascara were obtained and analysed for possible use as serotype-specific or group-specific probes in the detection of EEV dsRNA. Cloned fragments of genome segments 3, 8 and 10 were found to show high conservation of these segments, hybridizing to dsRNA from the six EEV serotypes under conditions that indicated more than 90% sequence homology. The genome segment 2-specific probe did not hybridize with dsRNA from any of the other EEV serotypes, suggesting that this segment encodes the serotype-specific antigen of EEV. Cross-hybridization of probes from genome segments 3 and 5 with dsRNA from bluetongue virus (BTV), epizootic haemorrhagic disease virus (EHDV) and African horse sickness virus (AHSV) indicated that EEV is more closely related to BTV and EHDV than to AHSV. Both probes can be used to distinguish between EEV and AHSV dsRNA.

Synthesis of the virus-specified tubules of epizootic haemorrhagic disease virus using a baculovirus expression system.

The formation of virus-specific tubules is one of the most characteristic features in the orbivirus infection cycle, yet little is known about their role in virus replication. The tubuli are composed of a major nonstructural protein, NS1. We have investigated the expression of the NS1-encoding gene of epizootic haemorrhagic disease virus serotype 2 (Alberta-strain) by producing a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV). Prior to cloning in the baculovirus transfer vector, pAcYM1 and cotransfection with AcNPV DNA, the NS1 gene was tailored by means of a polymerase chain reaction method to remove G/C tails. The baculovirus recombinant, AcNPV-EHDV2 NS1, expressed large amounts of a 55 K protein which could be purified by sucrose gradient sedimentation as a tubular complex. It appeared that the tubules could break up into 50 nm diameter circular units, which in turn were composed of approximately 16 subunits. The circular units appeared to be hollow and stacked on top of one another (100 units/micron tubule length), giving the tubules a segmented, ladderlike appearance. A large excess of EHDV2-specific tubuli could also be demonstrated in AcNPV-EHDV2 NS1-infected Spodoptera frugiperda cells by electron microscopic examination of thin sections. With pulse-labelling experiments it was shown that, regardless of the level of NS1 expression, the majority of NS1 synthesized in a 30 min period could only be recovered in a particulate form.

Variation of African horsesickness virus nonstructural protein NS3 in southern Africa

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.