Department of Genetics, Faculty of Biological and Agricultural Sciences, University of Pretoria, Lunnon Road, Hillcrest, Pretoria 0002, South Africa1
Biochemistry Division, Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa2
Author for correspondence: H. Huismans. Fax +27 12 3625327. e-mail hhuisman{at}postino.up.ac.za
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Abstract |
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Introduction |
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AHSV is a member of the family Reoviridae, genus Orbivirus, of which bluetongue virus (BTV) is the prototype member. The virion consists of a double-layered protein coat. The outer capsid (VP2 and VP5) probably mediates infection of mammalian cells, while the inner core (VP3 and VP7) remains intact inside infected cells as a replication complex. The viral genome has ten double-stranded (ds)RNA genome segments that encode at least seven structural (designated VP1 to VP7) and four nonstructural (designated NS1, NS2, NS3 and NS3A) proteins (reviewed by Roy et al., 1994 ). The functions of the nonstructural proteins are still under investigation. The smallest genome segment, S10, encodes the membrane-associated nonstructural proteins NS3 and NS3A (Van Staden & Huismans, 1991
; Stoltz et al., 1996
). Baculovirus-expressed NS3 is cytotoxic in insect cells (Sf9 cells), possibly as a result of membrane damage (Van Staden et al., 1998
), and is associated with events of AHSV release from infected Vero cells (Stoltz et al., 1996
). AHSV NS3 is likely to have an analogous function to NS3 of BTV, which is proposed to play a role in the final stages of BTV morphogenesis and release of virions (Hyatt et al., 1993
). In addition, AHSV NS3 may be involved in determining the virulence potential of a particular strain in a mouse model system (OHara et al., 1998
) and influencing the timing of virus release from infected cells (Martin & Meyer, 1998
). A number of conserved domains of AHSV NS3 have been identified (Van Staden et al., 1995
). A possible membrane insertion peptide proposed by Jensen & Wilson (1995)
for oribivirus NS3 proteins includes four highly positive charged residues preceding the first hydrophobic domain of 16 residues and a strong polar stretch of 7 residues distal to the hydrophobic domain. However, the membrane targeting signal of the orbivirus NS3 protein has not been identified.
Published phylogenetic studies on AHSV NS3 gene and protein sequences describe three distinct NS3 genetic lineages designated groups ,
and
, respectively, with a maximum variation of 35% between different serotypes (Sailleau et al., 1997
; Martin & Meyer, 1998
). BTV NS3 groups into three monophyletic clusters (Bonneau et al., 1999
) independent of BTV serotype, year of isolation, geographical origin and host species of isolation (Pierce et al., 1998
), and variation within the NS3 protein of BTV isolates is reported to be only 7% (Hwang et al., 1992
). Limited NS3 sequence data for other orbiviruses, however, restrict a comprehensive comparison of the typical variation for this nonstructural protein of the orbivirus serogroup. Genetic characterization of the corresponding protein to NS3 in rotavirus (family Reoviridae) NSP4 suggests the presence of four NSP4 genotypes of which the most recently recognized, genotype D, is the most divergent (Kirkwood & Palombo, 1997
; Ciarlet et al., 2000
). The variation between the NSP4 protein of genotype A and B rotavirus isolates is greater than that of BTV NS3 and approaches 20%.
Since NS3 is a potential contributing factor to AHSV virulence, it was of interest to investigate the genetic variation between recent South African AHSV field isolates in comparison to vaccine and laboratory reference strains of different serotypes. It may be possible to use NS3 sequence data together with serotyping to distinguish between different outbreaks of the same serotype as well as between field isolates and live attenuated vaccine strains. We found that the variation of AHSV NS3 is as much as 36·3% across AHSV serotypes and 27·6% within a serotype. NS3 proteins of vaccine and field isolates of the same serotype were found to differ between 2·3% and 9·7%. The inferred phylogeny of AHSV NS3 indicated an alternative grouping of AHSV-8 NS3 proteins. We also identified a highly conserved possible myristylated region that is followed by basic amino acids within the amino-terminal conserved domain of orbivirus NS3 proteins.
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Methods |
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DNA sequencing and analysis.
S10 PCR amplicons were purified using a commercial purification kit (Roche Diagnostics) and sequenced according to the manufacturers recommendations using an ABI 377 automated sequencer (Perkin Elmer). In addition to the above-mentioned primers, an additional internal primer, NS3C2.rev (5' gccccactcgcaccag) was designed to sequence the 5' region of the gene. The AHSV S10 gene sequences were translated into subsequent NS3 amino acid sequences and aligned using ClustalX (Thompson et al., 1997 ). The aligned NS3 sequences were used to generate a table of pairwise distances (PAUP version 4.0b3) to evaluate the variation within AHSV NS3. Accession numbers for the AHSV protein sequences retrieved from GenBank used in variation comparisons are listed in Table 2
. The phylogeny of AHSV NS3 was investigated with the construction of phylogenetic trees using the neighbour-joining and parsimony methods (PAUP version 4.0b3). A gamma test was done on the aligned nucleotide sequences to evaluate whether the genes displayed unequal rates of mutation. Bootstrap analysis was done (1000 replicates) and confidence scores are shown on the branches of the neighbour-joining tree (PAUP version 4.0b3).
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Results |
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The NS3 sequences of the virulent field isolates were compared to the avirulent vaccine strain NS3 sequences of the corresponding serotype. The amount of variation was found to be 2·3% for AHSV-2, 7·48·8% for AHSV-3, 2·36·0% for AHSV-6, 6·06·5% for AHSV-7 and 7·89·7% for AHSV-8 (Fig. 4 a). With the exception of AHSV-8 vaccine and reference strains (26·2% variation), comparisons of NS3 sequence variation of vaccine and reference strains within a particular serotype ranged between 1·83·2%. The AHSV-2 vaccine strain was produced from the reference strain 82/61. It is not known if the AHSV-3 and -6 vaccine strains are attenuated forms of the respective reference strains used in this study. The high level of NS3 sequence variation between the AHSV-8 vaccine and reference strains suggests that the vaccine strain of AHSV-8 was not derived directly from the AHSV-8 reference strain analysed in this study.
In comparison to the other AHSV proteins (Fig. 4b), NS3 is the second most variable virus protein with approximately 20% less variation than the observed 56% variation between the outer capsid proteins (VP2) of different serotypes. Other AHSV proteins (VP3, VP5, VP6, VP7 and NS1) have an internal variation ranging between 19% (VP5) and 0·2% (VP7). As expected, the sequence conservation of the inner core proteins (VP3 and VP7) is significantly higher than that of the outer capsid proteins (VP5 and VP2).
To investigate the phylogeny between virulent field isolates, vaccine strains and laboratory reference strains, the aligned NS3 amino acid sequences were used to construct phylogenetic trees (PAUP version 4.0b3) (Fig. 5). The estimated value of the gamma shape parameter was 0·58 and the small distances obtained using this value in a gamma test indicated that the NS3 gene was subject to unequal rates of mutation. Certain algorithms used to construct phylogenetic trees, such as that used by the unweighted pairgroup method with arithmetic mean (UPGMA), assume equal rates of mutation and were therefore not used in this analysis. Results of the aligned S10 gene nucleotide sequences and inferred phylogenetic trees generated the same results as found for the NS3 protein sequences and are thus not shown. The NS3 proteins of related orbiviruses retrieved from GenBank (Table 2
), namely BTV, EHDV, CHV and BRDV (selected as outgroup), were included for comparative purposes.
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The inferred phylogenetic trees of the isolates that we included indicated the presence of the same three distinct NS3 phylogenetic clusters previously described, viz. ,
and
(Martin & Meyer, 1998
; Sailleau et al., 1997
) with AHSV-4,-5, -6, -8 and -9 clustering in
, AHSV-3, -7 and -8 in
and AHSV-2 in
. The
and
clusters were found to share the most recent common ancestor and are therefore closer related to one another than to the
cluster of AHSV-2 viruses. NS3 of the AHSV-8 isolates that we studied clustered into two different phylogenetic groups and did not cluster with AHSV-2 as reported previously. The AHSV-8 field and vaccine strains grouped with AHSV-4, -5, -6 and -9 (
), while the AHSV-8 reference strain grouped with AHSV-3 and AHSV-7 (
). The
cluster showed that the AHSV-2 reference and vaccine strain NS3 proteins are more closely related to one another than to NS3 of the AHSV-2 field strain. The grouping pattern of the
cluster indicated no sub-clustering of NS3 based on AHSV serotype. NS3 of AHSV-3 vaccine and reference strains were sisters while the NS3 of AHSV-7 vaccine and AHSV-8 reference strains were closely related. The AHSV-7 field isolates were closely related while NS3 of AHSV-3 field isolates did not share the same common internal node in the tree and are therefore more distantly related. The largest cluster in this study,
, grouped NS3 of all the AHSV-4, -5, -6 and -9 isolates and three of the AHSV-8 isolates together. The AHSV-6 NS3 proteins were found to form a distinct lineage within this cluster. The AHSV-6 field isolates were closely related to one another and the AHSV-6 vaccine and reference strains were sisters in this sub-cluster. The AHSV-8 NS3 proteins were more dispersed through this cluster. The AHSV-8 field isolates grouped in a separate lineage to that of the AHSV-8 vaccine strain, which was in turn closely related to the AHSV-5 and -9 reference strains.
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Discussion |
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This AHSV NS3 phylogenetic study confirms the grouping of the majority of AHSV NS3 proteins into three distinct phylogenetic lineages (Martin & Meyer, 1998 ). In exception, NS3 of four different AHSV-8 isolates in this study grouped in two separate clusters (
and
) and not together with NS3 of serotypes 1 and 2 in the
cluster as predicted from the literature. Therefore, it may be assumed that although three distinct NS3 phylogenetic clusters are evident, the placement of a specific AHSV NS3 is not exclusively defined by the serotype. Reassortment of AHSV genome segments may be able to explain some of this large variation. Reassortment is a natural occurrence in the case of viruses with segmented genomes such as orthomyxo- and reoviruses. Multiple AHSV serotypes simultaneously present in zebras (Barnard, 1993
) and mixed AHSV infections in horses could facilitate S10 reassortment between virus populations of different serotypes. The serotype groupings in the NS3 phenogroups (Fig. 5
) appear to indicate a certain tendency for serotypes (i.e. those within the same NS3 cluster) to exchange the NS3 gene more readily. These serotypes may further have a higher incidence of co-circulation. BTV NS3 also shows extensive shifting as NS3 clusters do not conform with BTV serotypes (Pierce et al., 1998
). AHSV field isolates of the same season (usually commencing from early January to late May) and same serotype are closely related, while those of different seasons are more distantly related. The close relatedness of the NS3 sequence of viruses of the same serotype that were isolated from nearby geographical locations agrees with generally accepted epidemiological principles. In no instance was NS3 of a vaccine strain identical to NS3 of field isolates of the same serotype.
The origin of the observed NS3 variation in AHSV is not clear and can include many different variables. For example, the intermediate vector (Culicoides imicola) may tolerate a large amount of random variation in NS3 without an adverse affect on virion viability. NS3 is membrane-associated and may be under some immunological pressure, unlike the inner capsid proteins and other nonstructural proteins (NS1 and NS2) which seem to remain within cells. The large variation in the NS3 region between the two hydrophobic membrane-spanning domains illustrates that this area is able to tolerate a large amount of variation. A particularly intriguing question that remains to be investigated is why BTV NS3 seems to be so much more conserved. It has been proposed that variation of BTV NS3 is limited by structural constraints important for its function (Pierce et al., 1998 ). Why these limitations do not apply to the same degree for AHSV NS3 remains uncertain.
Despite the extensive variation, higher-order structures of AHSV NS3 predicted to be of importance, for example membrane-anchoring domains, are highly conserved. A highly conserved myristylation motif was also identified within the conserved N-terminal region of the orbivirus NS3 proteins. Although this sequence is common to a vast number of proteins and, therefore, does not imply that a protein is myristylated, it is nevertheless of interest in view of the fact that a major function of protein acylation is membrane targeting and association. Myristylation alone does, however, not provide sufficient energy to attach the protein to the phospholipid bilayer (McLaughlin & Aderem, 1995 ). Membrane-associated proteins of other viruses, such as Gag of human immunodeficiency virus (HIV)-1 (Zhou et al., 1994
) and Src of Rous sarcoma virus (Silverman & Resh, 1992
), contain a region of basic amino acid residues that stabilize membrane interactions. We identified a similar bipartite motif in all orbivirus NS3 proteins investigated that may function as a membrane-targeting signal of cleaved orbivirus NS3 proteins.
The two hydrophobic regions of AHSV NS3 have been associated with the cytotoxic effect of this protein (Van Staden et al., 1998 ). Any variation in these hydrophobic regions would therefore have the potential to abolish or alter the cytotoxic effect of NS3. This investigation did not show any differences between the hydrophobic domains of vaccine and field isolates that could be used as a virulence marker. Avirulence in rotavirus infections can be associated with mutations in NSP4, in particular between amino acids 131 and 140, that mediate binding to another viral protein, VP4 (Zhang et al., 1998
). It was also observed that a single codon difference between the authentic sequence of rotavirus SA11 NSP4 and the commonly used NSP4 cDNA clone (amino acid 47) enhances the cytotoxicity of authentic NSP4 and calcium influx properties when expressed as a baculovirus recombinant (Tian et al., 2000
). The significance of single amino acid differences can therefore not be underestimated.
This study is the first investigation of the variation in NS3 between recent virulent field isolates of AHSV and their relatedness to the current vaccine strains. Sequencing of the comparatively small NS3 gene in outbreaks of the disease may provide significant epidemiological information as to the origin of a virus involved in an outbreak. This is supported by preliminary, unpublished results on a recent outbreak of AHSV-7 in a region of South Africa (Western Cape) that has been free from AHSV for a large number of years.
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Acknowledgments |
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Footnotes |
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References |
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Received 23 June 2000;
accepted 15 September 2000.