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.     

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.     

Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparison to other European strains

During 2006 the first outbreak of bluetongue ever recorded in northern Europe started in Belgium and the Netherlands, spreading to Luxemburg, Germany and north-east France. The virus overwintered (2006-2007) reappearing during May-June 2007 with greatly increased severity in affected areas, spreading further into Germany and France, reaching Denmark, Switzerland, the Czech Republic and the UK. Infected animals were also imported into Poland, Italy, Spain and the UK. An initial isolate from the Netherlands (NET2006/04) was identified as BTV-8 by RT-PCR assays targeting genome segment 2. The full genome of NET2006/04 was sequenced and compared to selected European isolates, South African vaccine strains and other BTV-8 strains, indicating that it originated in sub-Saharan Africa. Although NET2006/04 showed high levels of nucleotide identity with other 'western' BTV strains, it represents a new introduction and was not derived from the BTV-8 vaccine, although its route of entry into Europe has not been established.     

Development and initial evaluation of a real-time RT-PCR assay to detect bluetongue virus genome segment 1

Since 1998, multiple strains of bluetongue virus (BTV), belonging to six different serotypes (types 1, 2, 4, 8, 9 and 16) have caused outbreaks of disease in Europe, causing one of the largest epizootics of bluetongue ever recorded, with the deaths of >1.8 million animals (mainly sheep). The persistence and continuing spread of BTV in Europe and elsewhere highlights the importance of sensitive and reliable diagnostic assay systems that can be used to rapidly identify infected animals, helping to combat spread of the virus and disease. BTV has a genome composed of 10 linear segments of dsRNA. We describe a real-time RT-PCR assay that targets the highly conserved genome segment 1 (encoding the viral polymerase--VP1) that can be used to detect all of the 24 serotypes, as well as geographic variants (different topotypes) within individual serotypes of BTV. After an initial evaluation using 132 BTV samples including representatives of all 24 BTV serotypes, this assay was used by the European Community Reference Laboratory (CRL) at IAH Pirbright to confirm the negative status of 2,255 animals imported to the UK from regions that were considered to be at risk during the 2006 outbreak of BTV-8 in Northern Europe. All of these animals were also negative by competition ELISA to detect BTV specific antibodies and none of them developed clinical signs of infection. These studies have demonstrated the value of the assay for the rapid screening of field samples.     

Sequence analysis and expression of a cDNA clone encoding a 98-kDa allergen in Dermatophagoides farinae

BACKGROUND: The important dust mite allergens identified to date are of molecular weights ranging from 14 to 60 kDa. Our previous protein study indicated that the 98-kDa native paramyosin in Dermatophagoides farinae mite showed IgE reactivity with 82% of the mite-sensitive asthmatic patients suggesting that it is a novel major mite allergen. This study described the isolation and characterization of the cDNA clone encoding the 98-kDa mite allergen. METHODS: A Dermatophagoides farinae cDNA library was constructed in lambda ZAPII vector and the library was immunoscreened with a monoclonal antibody 642. The cDNA insert was sub-cloned into M13 sequencing vector for single-stranded sequencing. The whole cDNA insert was expressed in pGEX-2T Escherichia coli expression system as a fusion protein with GST. The allergenicity of the recombinant peptides was tested by skin tests and IgE immunoassay. The IgE and IgG immunoassays were performed with sera from 20 mite-allergic patients. RESULTS: The cDNA clone Df642 was 2134 bp long, coding for a polypeptide of 711 amino acid residues. Protein sequence analysis and alignment confirmed that the deduced polypeptide is a mite paramyosin which is truncated slightly at the N- and C-terminuses. In vivo skin tests and in vitro IgE-binding study showed that 62% (13/21) and 50% (10/20) of the mite-sensitive asthmatic patients reacted positively with the recombinant Dermatophagoides farinae paramyosin, respectively. CONCLUSION: The study indicated that 98-kDa mite paramyosin is an important allergen.     

Expression of bluetongue virus serotype 17 NS1 protein from a cloned gene

A full-length copy of the coding region of segment 6 from bluetongue virus (BTV) serotype 17 was constructed from five overlapping cDNA clones. The gene coding for the NS1 protein was cloned into an expression plasmid under the control of a bacteriophage T7 promoter and expressed both in vitro and in Escherichia coli BL21(DE3) cells which contain a T7 RNA polymerase gene in their chromosome. Expression in both systems resulted in the synthesis of a protein comigrating with NS1 and a minor polypeptide comigrating with another viral-induced protein, NS1a, sometimes seen in BTV-infected cells. The proteins induced in E. coli were synthesized to high levels as insoluble products.     

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 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.     

Vaccinia virus expression of the VP7 protein of South African bluetongue virus serotype 4 and its use as an antigen in a capture ELISA

Summary Recombinant vaccinia viruses expressing the VP7 core protein of South African bluetongue virus serotype 4 (SA-BTV4) were identified by polymerase chain reaction amplification. Expression of VP7 was verified by radio-immunoprecipitation and a F(ab)₂-based ELISA. Antibodies to VP7 were detected in sera from sheep that had been infected with 20 different virulent BTV serotypes by using the vaccinia virus (VV) expressed VP7 as antigen in a capture ELISA. F(ab)₂-immobilised VV-expressed SA-BTV4 VP7 cross-reacted with sera directed against all 9 African horsesickness virus serotypes and epizootic haemorrhagic disease virus serotype 2.     

The complete nucleotide sequence of bluetongue virus serotype 1 RNA3 and a comparison with other geographic serotypes from Australia, South Africa and the United States of America, and with other orbivirus isolates

The sequence of the RNA segment 3 of bluetongue virus (BTV) serotype 1 from Australia is presented along with its deduced amino acid sequence. DNA copies of this genome segment were inserted either into the E. coli plasmid pBR322 by homopolymeric tailing or by direct insertion of double-stranded DNA fragments generated by restriction endonuclease cleavage into the appropriate M13 bacteriophage vectors (Vieira, J. and Messing, J., 1982, Gene 19, 259-268). Direct comparisons were made to the nucleotide sequence data of Purdy, M. et al., 1984 (J. Virol. 51, 754-759) and Ghiasi, H. et al., 1985 (Virus Res. 3, 181-190) for the United States of America (US) isolates of BTV, serotypes 10 and 17, respectively. A method for the rapid cloning, sequencing and alignment of orbivirus RNA 3 segments was utilised to compare other geographical isolates of BTV, as well as those of other orbivirus serotypes, in particular, epizootic haemorrhagic disease of deer virus (EHDV) and Warrego. The comparison of this sequence data reveals that BTV isolates can be separated into distinct geographical types which in turn are distinct from the other orbivirus isolates studied. The sequence conservation at the amino acid level for the gene product of RNA3 (VP3) does not enable distinctions to be made amongst the BTV isolates at a geographical level, but does afford easy distinction into the different orbivirus groups. A possible evolutionary schematic is presented for the orbiviruses studied.     

Isolation and identification of a variant of bluetongue virus serotype 11 from a ram in a bluetongue outbreak in western Texas.

A field strain of bluetongue virus was isolated from a blood sample of a ram during an outbreak of bluetongue in November 1985 in western Texas. In this bluetongue outbreak at least 25 of the 2,000 sheep were infected. Isolation was made by intravenous inoculation of 11-day-old embryonated chicken eggs. The serotype was identified as serotype 11 by serum neutralization tests. The genomic pattern on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of the new isolate is similar to that of bluetongue virus prototype 11. Comparisons were also made with proteins labeled in vivo with [3H]leucine and separated by SDS-PAGE. We conclude that this virus belongs to serotype 11, with slight differences in both genome and protein electrophoretic patterns.     

Sequence and phylogenetic analysis of the S1 genome segment of turkey-origin reoviruses

Based on previous reports characterizing the turkey-origin avian reovirus (TRV) σB (σ2) major outer capsid protein gene, the TRVs may represent a new group within the fusogenic orthoreoviruses. However, no sequence data from other TRV genes or genome segments has been reported. The σC protein encoded by the avian reovirus S1 genome segment is the cell attachment protein and a major antigenic determinant for avian reovirus. The chicken reovirus S1 genome segment is well characterized and is well conserved in viruses from that species. This report details the amplification, cloning and sequencing of the entire S1 genome segment from two and the entire coding sequences of the σC, p10 and p17 genes from an additional five TRVs. Sequence analysis reveals that of the three proteins encoded by the TRV S1 genome segment, σC shares at most 57% amino acid identity with σC from the chicken reovirus reference strain S1133, while the most similar p10 and p17 proteins share 72% and 61% identity, respectively, with the corresponding S1133 proteins. The most closely related mammalian reovirus, the fusogenic Nelson Bay reovirus, encodes a σC protein that shares from 25% to 28% amino acid identity with the TRV σC proteins. This report supports the earlier suggestion that the TRVs are a separate virus species within the Orthoreovirus genus, and may provide some insight into TRV host specificity and pathogenesis. NC/SEP–R44/03 S1 genome segment DQ525419 NC/98 S1 genome segment DQ995806 TRV sigmaC sequences DQ996601–DQ996605 TRV p10 sequences DQ996606–DQ996610 TRV p17 sequences DQ996611–DQ996615     

A European field strain of bluetongue virus derived from two parental vaccine strains by genome segment reassortment

Since 1998, nine bluetongue virus (BTV) strains from serotypes 1, 2, 4, 8, 9 and 16 have invaded Europe, killing >2 million animals (mainly sheep). Live vaccine strains of BTV-2, 4, 9 and 16 have also been used in the region. The BTV genome is composed of ten linear-segments of dsRNA, and events in Europe have provided opportunities for different strains to exchange/reassort genome segments, generating novel progeny-viruses. Genome segment 2 (Seg-2) encodes outer capsid protein VP2, the primary determinant of virus serotype, while Seg-5 encodes NS1, which forms 'tubules' within the cell-cytoplasm. Seg-2 and Seg-5 from 15 European isolates, and vaccine/reference strains, of BTV-2 and BTV-16, were sequenced. Isolates from the same serotype showed >92% nt identity in Seg-5, but <84% identity between types. However, published data for Seg-5 of BTV-16 from Italy 2002 showed <83% nt identity with other BTV-16 strains, but was identical to the BTV-2 vaccine strain (used in Italy during 2002, and annually in a multivalent vaccine in Israel since 1995) indicating that ITL2002 is a reassortant between the BTV-2 and BTV-16 vaccine strains. This represents the first detection of a reassortant BTV strain within Europe, highlighting concerns about the use of live BTV vaccines in the region.