The complete nucleotide sequence of the outer coat protein, VP5, of the Australian bluetongue virus (BTV) serotype 1 reveals conserved and non-conserved sequences

The complete sequence of the outer coat protein, VP5, of the Australian BTV serotype 1 was determined and found to be 1634 nucleotides in length. One single open reading frame of 526 amino acids was observed defining a protein of Mr 59,252 and having a charge of +0.5 at neutral pH. When compared to VP5 of BTV serotype 10 from the United States of America (US) (Purdy et al., 1986, J. Gen. Virol. 67, 957) a homology of 68% at the nucleotide level and 76% at the amino acid level, was observed. However, this conservation at the protein level was more apparent in certain regions of the gene. In four main regions the conservation varied from 83-91% while in the remaining regions the homology dropped to between 56-62%. Many of the amino acid substitutions were conservative in nature, raising the apparent overall homology to 87%. Comparisons of the hydropathy profiles of the two proteins again revealed a remarkable degree of conservation. The importance of these observations is discussed.     

The complete sequence of bluetongue virus serotype 10 segment 3 and its predicted VP3 polypeptide compared with those of BTV serotype 17

The complete sequence of the large RNA segment 3 (L3) of bluetongue virus serotype 10 (BTV-10) has been determined from DNA copies of the viral RNA cloned in the E. coli plasmid pBR322. The L3 viral RNA is 2772 nucleotides long with a single open reading frame of 2706 nucleotides. The L3 predicted primary gene product (VP3) is 103 342 daltons and has a net charge at neutral pH of -5. The sequence of the L3 RNA species differs by 126 point mutations from that of BTV-17 (i.e., 95.5% homology; see M. Purdy, J. Petre and P. Roy, J. Virol. 51, 754-759, 1984). The predicted L3 primary gene products of the two viruses differ by 9 amino acids. These differences correspond to 9 point mutations and represent 0.15% of the sites where nucleotide substitution could cause an amino acid change. By contrast, another 114 point mutations in the genome correspond to 6.5% of the available sites where nucleotide substitutions could be silent (i.e., where a nucleotide substitution may not cause an amino acid change). Three point mutations are in the 3' non-coding region of the RNA species. The quantitative differences between the coding and silent mutations are interpreted as representing the result of gene product conservation.     

Identification of a bluetongue virus serotype 1-specific ovine helper T-cell determinant in outer capsid protein VP2.

Ovine T-cell lines (including one clone [101A]), which are specific for Bluetongue virus serotype 1 (BTV1), have been established and characterized. Although these T-cell lines react with different isolates of BTV1 (including those from South Africa, Australia, Nigeria, and Cameroon), they do not react with heterologous BTV serotypes. Antigen specificity of these T-cells was studied using purified virus particles, infectious subviral particles (ISVP) and cores, or using individual BTV structural proteins that were either isolated by SDS-PAGE or expressed by recombinant strains of vaccinia virus. The results showed that each of the T-cell lines reacted with outer capsid protein VP2 (the BTV protein exhibiting most serotype-specific variation and the major neutralization antigen). However, all of the uncloned T-cell lines also reacted with either the core structural proteins or the outer capsid protein VP5. In contrast, the T-cell clone 101A only reacted with outer capsid protein VP2. Cell surface marker analysis showed that 101A has a helper T-cell phenotype (CD5+, CD4+, CD8-, T-19-). The T-cell lines and clone 101A all produced large amounts of interleukin 2 (IL-2) when stimulated with purified BTV1 virus particles, or with VP2 (up to 120 IU/ml from 2 x 10(5) T-cells). BTV serotype-specific antigenic sites, for B cells and at least one site for ovine helper T-cells, are therefore located within VP2.     

Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype

Analyses of reassortant and parental strains of BTV serotypes 3 and 10, in serum neutralization tests, confirmed the major role of outer capsid protein VP2 in determination of virus serotype and its involvement in serum neutralization. However, a reassortant BTV strain (R70), containing protein VP5 derived from BTV 3 and VP2 derived from BTV 10, cross-neutralized with both parental virus strains (BTV 3 and BTV 10). It is concluded that VP5 also plays some part in serotype determination of these virus isolates, as analyzed by serum-neutralization, but its role may be less significant than that of VP2.     

The amino acid sequence of the outer coat protein VP2 of neutralizing monoclonal antibody-resistant, virulent and attenuated bluetongue viruses.

Monoclonal antibodies which reacted with four different epitopes were used to select neutralization-resistant variants of Australian bluetongue virus serotype 1 (BTV1AUS; isolate CS156). Nucleotide sequencing of the VP2 outer coat protein gene of these variants showed that two of them contained alterations within the previously defined neutralization site at amino acids 328 to 335 (Gould et al., 1988). Comparison of VP2 sequences of several BTV serotypes, in addition to nucleotide sequence changes in a number of variants, suggested that this neutralization site was larger and contained 19 amino acids, the conformation of which could be affected by other regions of the VP2 protein. Nucleotide sequencing of neutralization-resistant variants revealed a total of four other regions of VP2 affecting the ability of monoclonal antibodies to neutralize the virus and these results support the notion that the neutralization site in VP2 was conformation dependent. The complete nucleotide sequence of the VP2 gene of virulent BTV1AUS (C5156) was determined directly from viral nucleic acid isolated from the blood of a sheep suffering clinical bluetongue disease. Comparison of the VP2 sequence of this virulent virus with that previously published for an avirulent, laboratory strain (Gould, 1988), indicated that the passage of virulent virus approximately 20 times in tissue culture over the last decade, not only led to attenuation but resulted in the appearance of ten nucleotide changes in the VP2 gene. Six of these nucleotide changes were silent, two resulted in conservative amino acid substitutions and two generated radical amino acid changes. However, in a separate experiment, a single passage of the virulent virus in tissue culture while leading to attenuation did not result in a nucleotide change in the VP2 outer coat protein gene.     

Morphogenesis of a bluetongue virus variant with an amino acid alteration at a neutralization site in the outer coat protein, VP2

Neutralization-resistant variants of bluetongue virus, selected with a monoclonal antibody to the outer coat protein VP2, have been used to delineate a neutralization epitope on the VP2 protein. Comparison of the RNA 2 sequence of four variants with that of the wild-type virus indicated that each variant contained a single nucleotide substitution which in turn resulted in a single amino acid alteration in VP2. The changes were clustered within a span of eight amino acids at positions 328 to 335 in the VP2 protein. In addition, analyses of cells infected with wild-type and a variant virus V35B2 have provided information on the site of VP2 addition to virus particles during morphogenesis. Electron microscopic examination revealed few virus-like particles around virus inclusion bodies (VIB) in wild-type virus-infected cells and cytoskeletons. In contrast, VIB in cells infected with the neutralization-resistant variant V35B2 were surrounded by particles identified as virus cores on the basis of their size and morphology. Probing of cytoskeletons with gold-labeled anti-VP2 monoclonal antibody revealed that in wild-type virus-infected cells the antibodies reacted weakly with VIB and only at locations where virus particles appeared to be leaving. The core-like particles surrounding VIB in V35B2-infected cells labeled very weakly with the anti-VP2 antibody. In contrast, wild-type and V35B2 virus particles which bound to the cytoskeleton at locations distal to VIB and those outside the infected cell bound significant amounts of antibody. These results suggest that although some VP2 may be added to developing virus particles at the periphery of VIB, the remainder of the VP2 protein is added outside the VIB either in the cytosol or following attachment of the particles to the cytoskeleton.     

Nucleotide and deduced amino acid sequence of the nonstructural phosphoprotein, NS2, of bluetongue virus serotype 17: comparison to two isolates of serotype 10

The nucleotide sequence of bluetongue virus (BTV) serotype 17 segment 8 from North America (NA) coding for the nonstructural phosphoprotein, NS2, was determined. This segment contains 1125 base pairs and codes for a protein of 40,581 daltons containing 354 amino acids with a net charge of -8.5 at pH 7.0. The carboxyl terminal portion of the protein is very hydrophilic and has a high degree of potential alpha-helix. Serine is the major, if not the exclusive, phosphorylated amino acid residue and ten of the twenty serine residues present in NS2 are found in consensus phosphorylation sites. Comparison of the nucleotide sequence of BTV-17NA segment 8 with the sequence of BTV-10NA and BTV-10 South Africa (SA) revealed a greater degree of homology between different serotypes within the same geographical area, i.e., 17NA and 10NA, than between isolates of the same serotype located in different areas, i.e., 10NA and 10SA. The same homology relationship as above was found at the amino acid level.     

Relationships amongst bluetongue viruses revealed by comparisons of capsid and outer coat protein nucleotide sequences

Sequence data from the gene segments coding for the capsid protein. VP3, of all eight Australian bluetongue virus serotypes were compared. The high degree of nucleotide sequence homology for VP3 genes amongst BTV isolates from the same geographic region supported previous studies (Gould, 1987; 1988b, c; Gould et al., 1988b) and was proposed as a basis for "topotyping" a bluetongue virus isolate (Gould et al., 1989). The complete nucleotide sequences which coded for the VP2 outer coat proteins of South African BTV serotypes 1 and 3 (vaccine strains) were determined and compared to cognate gene sequences from North American and Australian BTVs. These VP2 comparisons demonstrated that BTVs of the same serotype, but from different geographical regions, were closely related at the nucleotide and amino acid levels. However, close inter-relationships were also demonstrated amongst other BTVs irrespective of serotype or geographic origin. These data enabled phylogenic relationships of the BTV serotypes to be analysed using VP2 nucleotide sequences as a determinant.     

Complete nucleotide sequence of segment 5 of epizootic haemorrhagic disease virus; the outer capsid protein VP5 is homologous to the VP5 protein of bluetongue virus

The complete nucleotide sequence of a cDNA clone representing the segment 5 RNA of epizootic haemorrhagic disease virus (EHDV) United States serotype 1 was determined. The 5' and 3' termini of the RNA are complementary and are capable of forming secondary structures. The comparison of the predicted amino acid sequence of the encoded outer capsid protein (VP5) with the sequences of VP5 from four serotypes of bluetongue virus, the prototype orbivirus, revealed that the protein shares 59% to 62% homologies with various BTV serotypes, including a single conserved glycine residue at the amino terminus. The sequence has been submitted to the Genebank databox (X55782).     

Complete sequence of neutralization protein VP2 of the recent US isolate bluetongue virus serotype 2: its relationship with VP2 species of other US serotypes

In order to determine the relationship between the most recently isolated bluetongue virus serotype (BTV-2), and other US serotypes, the complete nucleotide sequence was determined from cDNA clones representing the L2 dsRNA of BTV-2, the gene that codes for the outer capsid neutralization antigen (VP2). The predicted amino acid sequence of the protein was compared with the VP2 sequences of the US serotypes BTV-10, BTV-11, BTV-13 and BTV-17. The VP2 protein of BTV-2 was found to exhibit 47% homology with the VP2 species of BTV-13, but only 40-41% homology with the VP2 species of earlier US isolates, BTV-10-, and -17. However, Diagon comparisons and hydropathic plots of all five VP2 species indicated that all of them are structurally very similar.     

Production and characterization of the neutralization antigen VP2 of bluetongue virus serotype 10 using a baculovirus expression vector

DNA representing RNA segment 2 of bluetongue virus (BTV) serotype 10, corresponding to the gene that codes for the BTV neutralization antigen VP2, has been inserted into a baculovirus transfer vector in lieu of the 5' coding region of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV). After cotransfection of Spodoptera frugiperda cells with wild-type AcNPV DNA in the presence of the derived recombinant transfer vector DNA, polyhedrin-negative recombinant baculoviruses were recovered. When S. frugiperda cells were infected with one of these recombinant viruses, a protein that was similar in size and antigenic properties to the BTV VP2 protein was synthesized. Antibodies raised in mice or rabbits to the baculovirus expressed VP2 protein neutralized the infectivity of BTV-10 virus and to lesser extents BTV serotype 11 and 17 viruses but not BTV-13 virus.     

Nucleotide and deduced amino acid sequences of the non-structural protein, NS1, of Australian and South African bluetongue virus serotype 1

The sequence of the sense strand of RNA segment 5 of both Australian and South African bluetongue virus (BTV) serotype 1 has been determined and found to be 1771 and 1773 nucleotides in length, respectively. Both coding sequences of 1656 nucleotides were flanked by a 5' non-coding sequence of 34 nucleotides and 3' non-coding regions of 78 and 80 nucleotides, respectively. The methionine codons at residues 35-37 were assumed to initiate the synthesis of 64.6 or 64.415 kDa proteins which had calculated net charges of +5 or +4 at neutral pH, respectively. The encoded NS1 proteins had a very high molar ratio of cysteine residues. A variable region of approximately 45 nucleotides at the 3'-terminus of RNA segment 5 of South African and Australian BTV-1 and the RNA segment 6 of the North American BTV-10 was shown to be unusually rich in A + T residues (approximately 80-82%) compared with other BTV gene segments so far sequenced which have between 52 and 56% A + T. These regions were thought to be responsible for the variable migration of RNA 5 segments on electrophoresis in polyacrylamide gels in the presence of urea. This variability in the apparent molecular weight of RNA 5 segments was not restricted to BTV amongst Australian orbiviruses tested, nor was the apparent molecular weight for RNA 5 identical for different isolates of the same BTV serotype, indicating that this A + T rich region was highly variable. Comparison of the nucleotide and amino acid sequence divergence of the Australian and South African BTV RNA segments 5 to that for the North American BTV-10 RNA segment 6 (which codes for NS1) revealed the same relationships as those found for the core protein VP3 gene sequences, in that although all NS1 proteins were very similar in their amino acid sequences, their genes were more variable. The Australian NS1 sequence differed from both the South African and North American genes by 20% at the nucleotide level, whereas the North American and South African sequences diverged by only 11%. Hybridization analyses showed that RNA segment 5 DNA probes were capable of delineating the geographical origin of a BTV isolate, as had been observed for VP3 probes; however, other probes were also generated which were capable of unambiguously differentiating BTV isolates from other orbiviruses tested.     

Complete nucleotide and deduced amino acid sequence of genome segment 5 encoding the outer capsid protein, VP5, of a U.S. isolate of bluetongue virus serotype 11.

The complete nucleotide sequence of the RNA genome segment coding for the outer capsid protein, VP5, of the United States prototypic strain of bluetongue virus (BTV) serotype 11 was determined from two overlapping cDNA clones. The genome segment was found to be 1638 nucleotides in length with a single open reading frame coding for a 526 amino acid protein of MW 59,278 and having a net charge of -4.0 at neutral pH. Comparisons of the predicted amino acid sequence of VP5 of BTV 11 with those of the United States serotypes 2, 10, and 13 and two isolates of BTV 1 from Australia and South Africa confirmed earlier reports that VP5 is a conserved protein with no clear regions of variability. A computer generated consensus sequence suggested VP5 of BTV 2 to be representative of the average VP5 sequences reported thus far.     

Nucleotide sequence of the genome segment encoding nonstructural protein NS1 of bluetongue virus serotype 20 from Australia

The nucleotide sequence of the genome segment (S6) encoding the nonstructural protein NS1 of an Australian isolate of bluetongue virus serotype 20 (BTV 20) has been determined from a series of overlapping cDNA clones synthesized using two terminal 15-mer oligonucleotides as primers. The gene consists of 1769 nucleotides with an open reading frame between nucleotides 35 and 1690 encoding a protein of 552 amino acids (molecular weight 64,506 Da; net charge –2 at pH 7). Comparison of the nucleotide and deduced amino acid sequence of this genome segment with cognate segments of isolates of BTV 1 from Australia and South Africa, and BTV 10 and BTV 17 from the United States, revealed homologies of 98%, 80%, 79%, and 79%, respectively, at the nucleotide level and 98%, 90%, 89%, and 90% identity, respectively, at the amino acid level. The data indicate that the evolutionary divergence between NS1 genes of two different Australian BTV serotypes (BTV 20 and BTV 1) is less than that between isolates of the same (BTV 1) or different serotypes from different geographical locations.The EMBL Data Library sequence accession Code is X56735 BLUETONGUE VIRUS RNA SEGMENT 6.     

Changes in the outer capsid proteins of bluetongue virus serotype ten that abrogate neutralization by monoclonal antibodies

Six neutralizing monoclonal antibodies (Mabs) and nine neutralization resistant viral variants (escape-mutant viruses (EMVs)) were used to further characterize the neutralization determinants of bluetongue virus serotype 10 (BTV10). The EMVs were produced by sequential passage of a highly cell culture adapted United States prototype strain of BTV10 in the presence of individual neutralizing Mabs. Mabs were characterized by neutralization and immune precipitation assays, and phenotypic properties of EMVs were characterized by neutralization assay. Sequencing of the gene segments encoding outer capsid proteins VP2 and VP5 identified mutations responsible for the altered phenotypic properties exhibited by individual EMVs. Amino acid substitutions in VP2 were responsible for neutralization resistance in most EMVs, whereas an amino acid substitution in VP5, without any change in VP2, was responsible for the neutralization resistance of one EMV. The data confirm that VP2 contains the major neutralization determinants of BTV, and that VP5 also can influence neutralization of the virus. The considerable plasticity of the neutralization determinants of BTV has significant implications for future development of non-replicating vaccines.     

A comparison of the VP1, VP2, and VP4 regions for molecular typing of human enteroviruses

The VP4, VP2, and VP1 gene regions were evaluated for their usefulness in typing human enteroviruses. Three published RT‐PCR primers sets targeting separately these three gene regions were used. Initially, from a total of 86 field isolates (36 HEV‐A, 40 HEV‐B, and 10 HEV‐C) tested, 100% concordance in HEV‐A was identified from all three gene regions (VP4, VP2, and VP1). However, for HEV‐B and HEV‐C viruses, only the VP2 and VP1 regions, and not VP4, showed 100% concordance in typing these viruses. To evaluate further the usefulness of VP4 in typing HEV‐A enteroviruses, 55 Japanese and 203 published paired VP4 and VP1 nucleotide sequences were also examined. In each case, typing by VP4 was 100% in concordance with typing using VP1. Given these results, it is proposed that for HEV‐A enteroviruses, all three gene regions (VP4, VP2, and VP1), would be useful for typing these viruses. These options would enhance the capability of laboratories in identifying these viruses and would greatly help in outbreaks of hand, foot, and mouth disease. J. Med. Virol. 82:649–657, 2010. © 2010 Wiley‐Liss, Inc.     

Identification of a novel bluetongue virus 1-specific B-cell epitope using a monoclonal antibody against the VP2 protein

Bluetongue virus (BTV) VP2 is an important antigenic protein that can be used for the differential diagnosis of different BTV serotypes. Here, we generated a serotype-specific monoclonal antibody (mab) against BTV1. A series of peptides synthesized based on the amino acid sequence of BTV1 VP2 were screened to define ¹¹⁵AQPLKVGL¹²² as the minimal linear peptide epitope recognized by mab 4B6. Using an immunofluorescence assay (IFA), we found that mab 4B6 reacted strongly with BTV1, but did not react with other BTV serotypes (BTV2-24). The 4B6 will serve as a novel reagent in the development of diagnostic tests for BTV1 infection.