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).     

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.     

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.     

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.     

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.     

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.     

Complete genomic sequences of the GP5 protein gene of bluetongue virus serotype 11 and 17.

The complete nucleotide sequences of full-length copies of genomic segment 6 or M3 of US bluetongue virus serotype 11 and 17 consisted of 1638 nucleotides. The plus-strand contained an open reading frame for a protein of 526 amino acids which was equivalent to about 59,000 Da, similar to the molecular weight of GP5 as determined by SDS-PAGE analysis. This long open reading frame was flanked by a 5' non-coding region of 29 nucleotides and a 3' non-coding region of 28 bases. When the predicted amino acid sequences of GP5 of BTV-11 and -17 were aligned and compared with those of BTV-2, -10, -13, -1AU and -1SA, four major highly conserved domains interrupted by several variable regions were detected. The potential significance of these discrete domains is discussed. Evolutionary and phylogenetic characteristics of these US BTV serotypes were consistent with our finding concerning BTV-1AU and -1SA.     

Complete genome sequence of a raccoon rabies virus isolate

The entire genome of a mid-Atlantic raccoon strain rabies virus (RRV) isolated in Canada was sequenced; this is the second North American wildlife rabies virus isolate to be fully characterized. The overall organization and length of the genome was similar to that of other lyssaviruses. The nucleotide sequence identity of the raccoon strain ranged between 32.7% and 85.0% when compared to other lyssaviruses, while the deduced amino acid sequence identity ranged between 22.9% and 94.2% with the nucleoprotein and polymerase being the most conserved. Notable features of RRV include the phosphoprotein's four amino acid extension compared to most other rabies viruses, and a nucleotide substitution immediately prior to the normal start codon that results in an additional methionine at the beginning of the L protein. This is the first report of the RRV L gene sequence and its 2128 amino acid product. Rates of non-synonymous and synonymous nucleotide changes within the lyssavirus L gene identified the conserved blocks II, III and IV as being most constrained. Analysis of L gene codon substitution patterns favoured models that supported positive selection, but only one site, corresponding to Leu62 of the RRV L protein, was identified as being under weak positive selection.     

蓝舌病毒两外壳蛋白VP2和VP5在昆虫细胞中的表达 …

目的 研究蓝舌病毒（ＢＴＶ）ＶＰ２与ＶＰ５的免疫学特性,为ＢＴＶ基因工程疫苗研究和病毒样颗粒装配打下基础。方法 将ＢＴＶ１０ ＶＰ２和ＶＰ５基因分别插入杆状病毒表达载体ｐＦａｓｔＢａｃ１,转染昆虫细胞获得重组杆状病毒。用ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ检测重组杆状病毒对ＶＰ２和ＶＰ５的表达,运用组织培养中和试验和直接血凝试验检测表达产物的生物学活性。     

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.     

Complete genome sequence of a novel porcine parainfluenza virus 5 isolate in Korea

A novel cytopathogenic paramyxovirus was isolated from a lung sample from a piglet, using continuous porcine alveolar macrophage cells. Morphologic and genetic studies indicated that this porcine virus (pPIV5) belongs to the species Parainfluenza 5 in the family Paramyxoviridae. We attempted to determine the complete nucleotide sequence of the first Korean pPIV5 isolate, designated KNU-11. The full-length genome of KNU-11 was found to be 15,246 nucleotides in length and consist of seven nonoverlapping genes (3′-N-V/P-M-F-SH-HN-L-5′) predicted to encode eight proteins. The overall degree of nucleotide sequence identity was 98.7 % between KNU-11 and PIV5 (formerly simian virus 5, SV5), a prototype paramyxovirus, and the putative proteins had 74.4 to 99.2 % amino acid identity to those of PIV5. Phylogenetic analysis further demonstrated that the novel pPIV5 isolate is a member of the genus Rubulavirus of the subfamily Paramyxovirinae. The present study describes the identification and genomic characterization of a pPIV5 isolate in South Korea.     

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.     

Complete nucleotide sequence and molecular probing of potato virus S genome

Complete genomes of three isolates of Potato virus S (PVS) were cloned and sequenced. The PVS ORF-1 was characterized for the first time. It encodes a putative replication protein (RPT) that shares the highest homology (about 52%) with that of Blueberry scorch virus (BlScV). ORF-1 motifs, characteristic for carlaviruses were found for methyltransferase (MTR), helicase (HEL) and RNA-dependent RNA polymerase (RdRp). The complete sequence of PVS genome enabled to develop an immunocapture RT-PCR probing of the PVS genome. Using this system, the sequence variability of 11 genome zones was examined for 34 PVS isolates including 15 PVS-CS variants that caused a systemic infection in Chenopodium quinoa. A broad variability between PVS isolates and diverse sequence variants was found. cDNA fragments covering the coat protein (CP) leader and CP-coding region (approx. 420 bp) were pooled for PVS-O and Chenopodium-systemic PVS isolates (PVS-CS) and corresponding cDNA libraries were screened for sequence variants. Both cDNA pools differred mainly in the 5'-end of the CP gene. Methionine at the position 17 in combination with serine at the position 34 were frequently associated with the CS character of PVS. In general, hydrophobic and polar amino acids were characteristic for the positions 17 and 34, respectively in PVS-CS isolates. Genome probing and evolutionary distances suggested that the PVS-CS isolates analyzed were close to the ordinary European isolates of ordinary strain of PVS (PVS-O) but distant to the original Andean strain of PVS (PVS-A).     

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.     

The nucleotide sequence of the gene encoding the F protein of canine distemper virus: a comparison of the deduced amino acid sequence with other paramyxoviruses

The nucleotide sequence of the gene encoding the fusion protein of canine distemper virus was determined from cDNA clones derived from virus genome RNA and poly(A)+ RNA extracted from infected cells. The mRNA encoding the F protein is about 2300 nucleotides in length including the 3' poly(A) tail. There is a large open reading frame from nucleotides 86 to 2071 which begins at the first AUG codon in the F mRNA. This reading frame encodes a protein of 662 amino acid residues with a calculated mol. wt. of 73001. The first major hydrophobic domain in the amino acid sequence of the deduced protein (residues 104 to 130) may represent all or part of a signal sequence for cleavage of the N terminal part of the F2 protein. There are four potential N glycosylation sites in the F protein located within the F2 part of the molecule or the putative signal sequence, and one in the F1 portion. A second hydrophobic region corresponds to the proteolytic cleavage site which generates the F2 and F1 subunits. This stretches from residue 225 to 262 and the N terminal part of the F1 protein shows sequence conservation with the other paramyxoviruses. A third major hydrophobic domain near the C terminus of the F protein probably represents the membrane anchor for the F protein (residues 602 to 630). The F1 proteins of six paramyxoviruses are compared and shown to have substantial conservation of those residues important in the maintenance of tertiary structure of this protein.     

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.     

RNA segment 5 of broadhaven virus, a tick-borne orbivirus, shows sequence homology with segment 5 of bluetongue virus

The sequence of Broadhaven (BRD) virus segment 5, the major genetic determinant of serotype, is 1658 nucleotides in length and contains a single open reading frame (ORF) having the coding capacity for a protein of Mr 52.5K. Comparison of the ORF of segment 5 of BRD virus with published sequences of bluetongue virus (BTV) revealed 30% nucleotide homology and 31% amino acid homology with the protein encoded by segment 5 of BTV serotype 10. Significant homology was not shown with segment 2 of BTV, the major genetic determinant of the BTV serotype. The sequences at the 3' and 5' ends determined for BRD segment 5 were similar to the respective 3' and 5' regions of BTV. The sequence data provide evidence of an evolutionary relationship between two ecologically distinct groups of orbiviruses and demonstrate changes that have occurred in the functions of genetically related genomic segments.