Cytokine mRNA Expression Profile in Target Organs of IFNAR (-/-) Mice Infected with African Horse Sickness Virus.

African horse sickness (AHS) is a highly severe disease caused by a viral etiological agent, African horse sickness virus (AHSV). It is endemic in sub-Saharan Africa, while sporadic outbreaks have occurred in North Africa, Asia, and Europe, with the most recent cases in Thailand. AHSV transmission between equines occurs primarily by biting midges of the genus Culicoides, especially C. imicola, with a wide distribution globally. As research in horses is highly restricted due to a variety of factors, small laboratory animal models that reproduce clinical signs and pathology observed in natural infection of AHSV are highly needed. Here, we investigated the expression profile of several pro-inflammatory cytokines in target organs and serum of IFNAR (-/-) mice, to continue characterizing this established animal model and to go deep into the innate immune responses that are still needed.

African horse sickness virus NS4 is a nucleocytoplasmic protein that localizes to PML nuclear bodies.

African horse sickness virus (AHSV) is the causative agent of the often fatal disease African horse sickness in equids. The non-structural protein NS4 is the only AHSV protein that localizes to the nucleus. Here we report that all AHSV reference and representative field strains express one of the two forms of NS4, i.e. NS4-I or NS4-II. Both forms of NS4 are nucleocytoplasmic proteins, but NS4-I has a stronger nuclear presence whilst NS4-II has a proportionally higher cytoplasmic distribution. A subtype of NS4-II containing a nuclear localization signal (NLS), named NLS-NS4-II, displays distinct punctate foci in the nucleus. We showed that NS4 likely enters the nucleus via passive diffusion as a result of its small size. Colocalization analysis with nuclear compartments revealed that NS4 colocalizes with promyelocytic leukaemia nuclear bodies (PML-NBs), suggesting a role in the antiviral response or interferon signalling. Interestingly, we showed that two other AHSV proteins also interact with nuclear components. A small fraction of the NS1 tubules were present in the nucleus and associated with PML-NBs; this was more pronounced for a virus strain lacking NS4. A component of nuclear speckles, serine and arginine rich splicing factor 2 (SRSF2) was recruited to viral inclusion bodies (VIBs) in the cytoplasm of AHSV-infected cells and colocalized with NS2. Nuclear speckles are important sites for cellular mRNA transcript processing and maturation. Collectively, these results provide data on three AHSV non-structural proteins interacting with host cell nuclear components that could contribute to overcoming antiviral responses and creating conditions that will favour viral replication.

Targeted mutational analysis to unravel the complexity of African horse sickness virus NS3 function in mammalian cells.

The African horse sickness virus non-structural protein 3 (NS3) is involved in the final stages of infection. To gain insight into the function of different NS3 domains, we generated reverse genetics-derived mutants, each expressing a modified version of the protein. A functional comparison of these mutants to the wild-type virus in mammalian cells indicated the variable contribution of the different domains to the cytopathic effect and in ensuring effective virus trafficking and release. The transmembrane domains were determined as essential mediators of NS3 localisation, as the abnormal processing of these mutant proteins resulted in their nuclear localisation and interaction with NS1. NS3 cytoplasmic domain disruptions resulted in increased cytosolic virus particle accumulation and abnormal virion tethering to plasma membranes. Other aspects of infection were also affected, such as VIB formation and distribution of the outer capsid proteins. Overall, these results illustrate the intricate role of NS3 in the infection cycle.

Analysis of the three-dimensional structure of the African horse sickness virus VP7 trimer by homology modelling.

VP7 is the major core protein of orbiviruses and is essential for virion assembly. African horse sickness virus (AHSV) VP7 self-assembles into highly insoluble crystalline particles - an attribute that may be related to the role of AHSV VP7 in virus assembly but also prevents crystallization. Given that this inherent insolubility is unique to AHSV VP7, we use amino acid sequence conservation analysis between AHSV VP7 and other orbiviruses to identify putative key residues that drive AHSV VP7 self-assembly. A homology model of the AHSV VP7 trimer was generated to analyze surface properties of the trimer and to identify surface residues as candidates for the AHSV VP7 trimer-trimer interactions that drive AHSV VP7 self-assembly. Nine regions were identified as candidate residues for future site-directed mutagenesis experiments that will likely result in a soluble AHSV VP7 protein. Additionally, we identified putative residues that function in the intermolecular interactions within the AHSV VP7 trimer as well as several epitopes. Given the many previous efforts of solubilizing AHSV VP7, we propose a useful strategy that will yield a soluble AHSV VP7 that can be used to study AHSV assembly and increase yield of recombinant vaccine preparations.

A Dual Laser Scanning Confocal and Transmission Electron Microscopy Analysis of the Intracellular Localization, Aggregation and Particle Formation of African Horse Sickness Virus Major Core Protein VP7.

The bulk of the major core protein VP7 in African horse sickness virus (AHSV) self-assembles into flat, hexagonal crystalline particles in a process appearing unrelated to viral replication. Why this unique characteristic of AHSV VP7 is genetically conserved, and whether VP7 aggregation and particle formation have an effect on cellular biology or the viral life cycle, is unknown. Here we investigated how different small peptide and enhanced green fluorescent protein (eGFP) insertions into the VP7 top domain affected VP7 localization, aggregation, and particle formation. This was done using a dual laser scanning confocal and transmission electron microscopy approach in conjunction with analyses of the solubility, aggregation, and fluorescence profiles of the proteins. VP7 top domain modifications did not prevent trimerization, or intracellular trafficking, to one or two discrete sites in the cell. However, modifications that resulted in a misfolded and insoluble VP7-eGFP component blocked trafficking, and precluded protein accumulation at a single cellular site, perhaps by interfering with normal trimer-trimer interactions. Furthermore, the modifications disrupted the stable layering of the trimers into characteristic AHSV VP7 crystalline particles. It was concluded that VP7 trafficking is driven by a balance between VP7 solubility, trimer forming ability, and trimer-trimer interactions.

Requirements and comparative analysis of reverse genetics for bluetongue virus (BTV) and African horse sickness virus (AHSV).

BACKGROUND: Bluetongue virus (BTV) and African horse sickness virus (AHSV) are distinct arthropod borne virus species in the genus Orbivirus (Reoviridae family), causing the notifiable diseases Bluetongue and African horse sickness of ruminants and equids, respectively. Reverse genetics systems for these orbiviruses with their ten-segmented genome of double stranded RNA have been developed. Initially, two subsequent transfections of in vitro synthesized capped run-off RNA transcripts resulted in the recovery of BTV. Reverse genetics has been improved by transfection of expression plasmids followed by transfection of ten RNA transcripts. Recovery of AHSV was further improved by use of expression plasmids containing optimized open reading frames. RESULTS: Plasmids containing full length cDNA of the 10 genome segments for T7 promoter-driven production of full length run-off RNA transcripts and expression plasmids with optimized open reading frames (ORFs) were used. BTV and AHSV were rescued using reverse genetics. The requirement of each expression plasmid and capping of RNA transcripts for reverse genetics were studied and compared for BTV and AHSV. BTV was recovered by transfection of VP1 and NS2 expression plasmids followed by transfection of a set of ten capped RNAs. VP3 expression plasmid was also required if uncapped RNAs were transfected. Recovery of AHSV required transfection of VP1, VP3 and NS2 expression plasmids followed by transfection of capped RNA transcripts. Plasmid-driven expression of VP4, 6 and 7 was also needed when uncapped RNA transcripts were used. Irrespective of capping of RNA transcripts, NS1 expression plasmid was not needed for recovery, although NS1 protein is essential for virus propagation. Improvement of reverse genetics for AHSV was clearly demonstrated by rescue of several mutants and reassortants that were not rescued with previous methods. CONCLUSIONS: A limited number of expression plasmids is required for rescue of BTV or AHSV using reverse genetics, making the system much more versatile and generally applicable. Optimization of reverse genetics enlarge the possibilities to rescue virus mutants and reassortants, and will greatly benefit the control of these important diseases of livestock and companion animals.

VP2 Exchange and NS3/NS3a Deletion in African Horse Sickness Virus (AHSV) in Development of Disabled Infectious Single Animal Vaccine Candidates for AHSV.

UNLABELLED: African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African horse sickness (AHS) in equids, with a mortality rate of up to 95% in naive horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate "synthetic" reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2, which is the serotype-determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replication in vitro. NS3/NS3a of AHSV is, however, involved in the cytopathogenic effect in mammalian cells and is very important for virus release from cultured insect cells in particular. Similar to the concept of the bluetongue disabled infectious single animal (BT DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using exchange of genome segment 2 encoding VP2 protein (Seg-2[VP2]), we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes. IMPORTANCE: African horse sickness virus is transmitted by species of Culicoides biting midges and causes African horse sickness in equids, with a mortality rate of up to 95% in naive horses. African horse sickness has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are supposed to be competent vectors. By using reverse genetics, viruses of all nine serotypes were constructed by the exchange of Seg-2 expressing the serotype-determining VP2 protein. Furthermore, we demonstrated that the nonstructural protein NS3/NS3a is not essential for virus replication in vitro. However, the potential spread of the virus by biting midges is supposed to be blocked, since the in vitro release of the virus was strongly reduced due to this deletion. VP2 exchange and NS3/NS3a deletion in African horse sickness virus were combined in the concept of a disabled infectious single animal vaccine for all nine serotypes.

Characterising Non-Structural Protein NS4 of African Horse Sickness Virus.

African horse sickness is a serious equid disease caused by the orbivirus African horse sickness virus (AHSV). The virus has ten double-stranded RNA genome segments encoding seven structural and three non-structural proteins. Recently, an additional protein was predicted to be encoded by genome segment 9 (Seg-9), which also encodes VP6, of most orbiviruses. This has since been confirmed in bluetongue virus and Great Island virus, and the non-structural protein was named NS4. In this study, in silico analysis of AHSV Seg-9 sequences revealed the existence of two main types of AHSV NS4, designated NS4-I and NS4-II, with different lengths and amino acid sequences. The AHSV NS4 coding sequences were in the +1 reading frame relative to that of VP6. Both types of AHSV NS4 were expressed in cultured mammalian cells, with sizes close to the predicted 17-20 kDa. Fluorescence microscopy of these cells revealed a dual cytoplasmic and nuclear, but not nucleolar, distribution that was very similar for NS4-I and NS4-II. Immunohistochemistry on heart, spleen, and lung tissues from AHSV-infected horses showed that NS4 occurs in microvascular endothelial cells and mononuclear phagocytes in all of these tissues, localising to the both the cytoplasm and the nucleus. Interestingly, NS4 was also detected in stellate-shaped dendritic macrophage-like cells with long cytoplasmic processes in the red pulp of the spleen. Finally, nucleic acid protection assays using bacterially expressed recombinant AHSV NS4 showed that both types of AHSV NS4 bind dsDNA, but not dsRNA. Further studies will be required to determine the exact function of AHSV NS4 during viral replication.

Recovery of African horse sickness virus from synthetic RNA.

African horse sickness virus (AHSV) is an insect-vectored emerging pathogen of equine species. AHSV (nine serotypes) is a member of the genus Orbivirus, with a morphology and coding strategy similar to that of the type member, bluetongue virus. However, these viruses are distinct at the genetic level, in the proteins they encode and in their pathobiology. AHSV infection of horses is highly virulent with a mortality rate of up to 90 %. AHSV is transmitted by Culicoides, a common European insect, and has the potential to emerge in Europe from endemic countries of Africa. As a result, a safe and effective vaccine is sought urgently. As part of a programme to generate a designed highly attenuated vaccine, we report here the recovery of AHSV from a complete set of RNA transcripts synthesized in vitro from cDNA clones. We have demonstrated the generation of mutant and reassortant AHSV genomes, their recovery, stable passage, and characterization. Our findings provide a new approach to investigate AHSV replication, to design AHSV vaccines and to aid diagnosis.

Structural insight into African horsesickness virus infection.

African horsesickness (AHS) is a devastating disease of horses. The disease is caused by the double-stranded RNA-containing African horsesickness virus (AHSV). Using electron cryomicroscopy and three-dimensional image reconstruction, we determined the architecture of an AHSV serotype 4 (AHSV-4) reference strain. The structure revealed triple-layered AHS virions enclosing the segmented genome and transcriptase complex. The innermost protein layer contains 120 copies of VP3, with the viral polymerase, capping enzyme, and helicase attached to the inner surface of the VP3 layer on the 5-fold axis, surrounded by double-stranded RNA. VP7 trimers form a second, T=13 layer on top of VP3. Comparative analyses of the structures of bluetongue virus and AHSV-4 confirmed that VP5 trimers form globular domains and VP2 trimers form triskelions, on the virion surface. We also identified an AHSV-7 strain with a truncated VP2 protein (AHSV-7 tVP2) which outgrows AHSV-4 in culture. Comparison of AHSV-7 tVP2 to bluetongue virus and AHSV-4 allowed mapping of two domains in AHSV-4 VP2, and one in bluetongue virus VP2, that are important in infection. We also revealed a protein plugging the 5-fold vertices in AHSV-4. These results shed light on virus-host interactions in an economically important orbivirus to help the informed design of new vaccines.

Characterization of the nucleic acid binding activity of inner core protein VP6 of African horse sickness virus.

Minor structural protein VP6 is the putative helicase of African horse sickness virus (AHSV), of the genus Orbivirus in the Reoviridae family. We investigated how the protein interacts with double-stranded (ds) RNA and other nucleic acids. Binding was assayed using an electrophoretic migration retardation assay and a nucleic acid overlay protein blot assay. VP6 bound double and single stranded RNA and DNA in a NaCl concentration sensitive reaction. Of six truncated VP6 peptides investigated, two partially overlapping peptides were found to bind dsRNA at pH 7.0, while other peptides with the same overlap did not. The distinction between the peptides appeared to be the pI which ranged from more than 8.0 to just above 6.0. Changing the pH of the binding buffer modified the binding activity. Regardless of assay conditions, only peptides with a specific region of amino acids in common, showed evidence of binding activity. No sequence homology was identified with other binding domains, however, the presence of charged amino acids are assumed to be important for binding activity. The results suggested dsRNA binding in the blot assay was strongly affected by the net charge on the peptide.

A first full outer capsid protein sequence data-set in the Orbivirus genus (family Reoviridae): cloning, sequencing, expression and analysis of a complete set of full-length outer capsid VP2 genes of the nine African horsesickness virus serotypes.

The outer capsid protein VP2 of African horsesickness virus (AHSV) is a major protective antigen. We have cloned full-length VP2 genes from the reference strains of each of the nine AHSV serotypes. Baculovirus recombinants expressing the cloned VP2 genes of serotypes 1, 2, 4, 6, 7 and 8 were constructed, confirming that they all have full open reading frames. This work completes the cloning and expression of the first full set of AHSV VP2 genes. The clones of VP2 genes of serotypes 1, 2, 5, 7 and 8 were sequenced and their amino acid sequences were deduced. Our sequencing data, together with that of the published VP2 genes of serotypes 3, 4, 6 and 9, were used to generate the first complete sequence analysis of all the (sero)types for a species of the Orbivirus genus. Multiple alignment of the VP2 protein sequences showed that homology between all nine AHSV serotypes varied between 47.6 % and 71.4 %, indicating that VP2 is the most variable AHSV protein. Phylogenetic analysis grouped together the AHSV VP2s of serotypes that cross-react serologically. Low identity between serotypes was demonstrated for specific regions within the VP2 amino acid sequences that have been shown to be antigenic and play a role in virus neutralization. The data presented here impact on the development of new vaccines, the identification and characterization of antigenic regions, the development of more rapid molecular methods for serotype identification and the generation of comprehensive databases to support the diagnosis, epidemiology and surveillance of AHS.

Expression of the major core structural proteins VP3 and VP7 of African horse sickness virus, and production of core-like particles.

The genome segments encoding the seven structural proteins of African horse sickness virus (AHSV), including the largest coding for VP1, were cloned and sequenced. Analysis of the VP1 sequence supports the putative identity of this protein as an RNA polymerase. The genes encoding the two major core proteins, VP3 and VP7, were also cloned and expressed by both in vitro translation and by means of recombinant baculoviruses. Co-infection of insect cells with VP3 and VP7 recombinant baculoviruses resulted in the intracellular formation of multimeric particles with a diameter of 72 nm, which structurally resembled authentic AHSV cores (core like particles: CLP). The complete genome of AHSV has now been cloned and sequenced.

Characterization of tubular structures composed of nonstructural protein NS1 of African horsesickness virus expressed in insect cells.

The characteristic tubules that are produced during the orbivirus infection cycle are composed of a major viral nonstructural protein, NS1. To characterize the NS1 gene and gene product of African horsesickness virus (AHSV), a full-length cDNA copy of the NS1 gene of AHSV-6 was cloned and the nucleotide sequence determined. NS1 was highly conserved within the AHSV serogroup with between 95-98% conservation of amino acids among NS1 of AHSV-6, AHSV-4 and AHSV-9. The structure of AHSV NS1 tubules was investigated by in vitro translation of the AHSV-6 NS1 gene followed by expression of the gene in insect cells. The NS1 protein assembled in tubular structures with a diameter of approximately 23 nm and lengths of up to 4 microns. The absence of a ladder-like structure and lower sedimentation value of AHSV NS1 tubules clearly distinguished them from those of bluetongue virus.

The multimeric nonstructural NS2 proteins of bluetongue virus, African horsesickness virus, and epizootic hemorrhagic disease virus differ in their single-stranded RNA-binding ability.

The structure and single-stranded (ss) RNA-binding by the nonstructural protein NS2 of three different orbiviruses were studied and compared. African horsesickness virus (AHSV), bluetongue virus (BTV), and epizootic hemorrhagic disease virus (EHDV) were analyzed in recombinant baculovirus-infected cells and in cells infected with BTV and AHSV. Sedimentation analysis and nonreducing SDS-PAGE revealed that NS2 of all three orbiviruses is a 7S multimer with both inter- and intramolecular disulfide bonds, probably consisting of six or more NS2 molecules. The 7S NS2 multimer of all three viruses binds ssRNA but there is a marked disparity in the ssRNA-binding ability between the three proteins. At physiological salt concentration, BTV NS2 binds ssRNA very efficiently, whereas AHSV NS2 shows only a low efficiency for binding ssRNA. EHDV NS2 binds with intermediate efficiency. The result was the same irrespective of whether poly(U)-Sepharose or viral mRNA was used, indicating that ssRNA-binding by NS2 is nonspecific. The difference in RNA-binding ability may be related to the alpha-helix content of the respective proteins. NS2 of BTV has the highest predicted alpha-helix content followed by EHDV and AHSV. The ability of the NS2 proteins to form virus inclusion body-like structures in baculovirus-infected cells is not affected by the ssRNA-binding disparity.

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.

The 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 (24 K) 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.

The complete sequences of African horsesickness virus serotype 4 (vaccine strain) RNA segment 2 and 6 which encode outer capsid protein.

The complete sequences of RNA segment 2 and segment 6 of African horsesickness virus serotype 4 (AHSV-4) vaccine strain were determined from cDNA clones inserted into pBR 322. The RNAs of segment 2 and 6 are 3229, 1566 bp long respectively and both contain an open reading frame encoding proteins VP2 and VP5 of 1060, 505 amino acid residues. The estimated molecular weight of VP2 was 124,178 dalton and that of VP5 was 56,793 dalton. Their noncoding end sequences were 5'GTTTAA . . . and . . . ACATAC3' (segment 2), 5'GTTTAT . . . and . . . ACTTAC3' (segment 6). They were different from orbivirus characteristic terminal sequences, which were 5'GTTAAA . . . and . . . ACTTAC3'. The comparison of both sequences of AHSV-4 segment 2 and 6 with those of segment 2 and 5 of bluetongue virus (BTV) serotype 10 revealed 53% nucleotide similarity and 23% amino acid similarity (segment 2), and 58% nucleotide similarity and 46% amino acid similarity (segment 6). In the same way, the comparison of both sequences of the vaccine strain with those of the virulent strain segment 2 and segment 6 of AHSV-4 revealed 91% nucleotide and 96% amino acid similarity (segment 2), and 98% nucleotide and 98% amino acid similarity (segment 6).