Nipah virus: a recently emergent deadly paramyxovirus.

A paramyxovirus virus termed Nipah virus has been identified as the etiologic agent of an outbreak of severe encephalitis in people with close contact exposure to pigs in Malaysia and Singapore. The outbreak was first noted in late September 1998 and by mid-June 1999, more than 265 encephalitis cases, including 105 deaths, had been reported in Malaysia, and 11 cases of encephalitis or respiratory illness with one death had been reported in Singapore. Electron microscopic, serologic, and genetic studies indicate that this virus belongs to the family Paramyxoviridae and is most closely related to the recently discovered Hendra virus. We suggest that these two viruses are representative of a new genus within the family Paramyxoviridae. Like Hendra virus, Nipah virus is unusual among the paramyxoviruses in its ability to infect and cause potentially fatal disease in a number of host species, including humans.

Deduced amino acid sequences surrounding the fusion glycoprotein cleavage site and of the carboxyl-terminus of haemagglutinin-neuraminidase protein of the avirulent thermostable vaccine strain I-2 of Newcastle disease virus.

A single-tube RT-PCR technique generated a 387 bp or 300 bp cDNA amplicon covering the F(0) cleavage site or the carboxyl (C)-terminus of the HN gene, respectively, of Newcastle disease virus (NDV) strain I-2. Sequence analysis was used to deduce the amino acid sequences of the cleavage site of F protein and the C-terminus of HN protein, which were then compared with sequences for other NDV strains. The cleavage site of NDV strain I-2 had a sequence motif of (112) RKQGRLIG(119), consistent with an avirulent phenotype. Nucleotide sequencing and deduction of amino acids at the C-terminus of HN revealed that strain I-2 had a 7-amino-acid extension (VEILKDGVREARSSR. This differs from the virulent viruses that caused outbreaks of Newcastle disease in Australia in the 1930s and 1990s, which have HN extensions of 0 and 9 amino acids, respectively. Amino acid sequence analyses of the F and HN genes of strain I-2 confirmed its avirulent nature and its Australian origin.

Genome sequence of the thermostable Newcastle disease virus (strain I-2) reveals a possible phenotypic locus.

The complete genome sequence of the Australian I-2 heat-tolerant Newcastle disease virus (NDV) vaccine (master seed stocks) was determined and compared to the sequence of the parent virus from which it had been derived after exposure of the parent stock at 56 degrees C for 30 min. Nucleotide changes were observed at a number of positions with synonymous mutations being greater than those observed for non-synonymous mutations. Sequence data for the HN gene of a parental culture of V4 and two heat-tolerant variants of V4 were obtained. These were compared with the data for the I-2 viruses and with published sequences for parental V4 and for a number of ND vaccine strains. Sequence analyses did not reveal the ARG(303) deletion in the HN protein, previously claimed to be responsible for the thermostable phenotype. No consistent changes were detected that would indicate involvement of the HN protein in heat resistance. The majority of alterations were observed in the L protein of the virus and it is proposed that these alterations were responsible for the heat-tolerant phenotype of the I-2 NDV vaccine.

The crystal structure of plasminogen activator inhibitor 2 at 2.0 A resolution: implications for serpin function.

BACKGROUND: Plasminogen activator inhibitor 2 (PAI-2) is a member of the serpin family of protease inhibitors that function via a dramatic structural change from a native, stressed state to a relaxed form. This transition is mediated by a segment of the serpin termed the reactive centre loop (RCL); the RCL is cleaved on interaction with the protease and becomes inserted into betasheet A of the serpin. Major questions remain as to what factors facilitate this transition and how they relate to protease inhibition. RESULTS: The crystal structure of a mutant form of human PAI-2 in the stressed state has been determined at 2.0 A resolution. The RCL is completely disordered in the structure. An examination of polar residues that are highly conserved across all serpins identifies functionally important regions. A buried polar cluster beneath betasheet A (the so-called 'shutter' region) is found to stabilise both the stressed and relaxed forms via a rearrangement of hydrogen bonds. CONCLUSIONS: A statistical analysis of interstrand interactions indicated that the shutter region can be used to discriminate between inhibitory and non-inhibitory serpins. This analysis implied that insertion of the RCL into betasheet A up to residue P8 is important for protease inhibition and hence the structure of the complex formed between the serpin and the target protease.

Limited proteolysis study of structure-function relationships in Sh I, a polypeptide neurotoxin from a sea anemone.

The structure-function relationships of the neurotoxic polypeptide Sh I, from the sea anemone Stichodactyla helianthus, have been studied using limited proteolysis with trypsin and endoproteinase Lys-C. Major products from each of the proteolytic digests were characterised using N-terminal peptide sequencing and amino-acid analysis or mass spectrometry. Of the six possible tryptic cleavage sites in Sh I, the bonds adjacent to Arg-13 and Lys-47 were found to be the most susceptible, complete cleavage occurring within minutes. Cleavages adjacent to Lys-32 and Lys-46 proceeded more slowly and cleavage adjacent to Arg-45 was the slowest. The sixth potential site, adjacent to Lys-4, was not cleaved at all. All derivatives were inactive as crustacean neurotoxins. Cleavage with endoproteinase Lys-C generated two major products. Derivatives cleaved adjacent to Lys-32 and either Lys-46 or Lys-47 were isolated. Both were inactive, indicating that either cleavage adjacent to Lys-32 or the removal of the C-terminal lysine residue(s) was sufficient to abolish activity. Lys-4 again was refractory to cleavage. The sequence of cleavage events correlated well with the static accessibility of the lysyl and arginyl side chains and to a lesser extent with the accessibility of the carbonyl oxygen of susceptible peptide bonds, as measured from the solution structure of Sh I determined by 1H-NMR. In the case of Lys-4, the lack of cleavage by trypsin and endoproteinase Lys-C may reflect a lack of flexibility in this region. The effects of the various cleavages on biological activity emphasise that the surface of the protein near the reverse turn encompassing Asp-6, Asp-7 and Glu-8 is essential for activity.

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.

Conserved and non-conserved regions of the outer coat protein, VP2, of the Australian bluetongue serotype 1 virus, revealed by sequence comparison to the VP2 of North American BTV serotype 10.

The complete nucleotide sequence of the outer coat protein, VP2, of the Australian BTV serotype 1 was determined and found to be 2940 nucleotides in length. An open reading frame of 961 amino acids was found, defining a protein of 112,115 Daltons having a charge of +15 at neutral pH. This coding region was flanked by 5' and 3' non-coding regions of 17 and 37 nucleotides, respectively. When compared to VP2 of the North American BTV serotype 10 (Purdy et al., 1985, J. Virol. 55, 826-830) a homology of 52% at the nucleotide level, and 40% at the amino acid level, was observed. Significant conservation of amino acid sequence was found in eleven distinct regions; the overall hydropathy of the protein, as well as 9 of its 12 cysteine residues, was conserved. The importance of this conservation, in relation to similar observations for the other outer coat protein, VP5, is discussed.

Comparison of the deduced matrix and fusion protein sequences of equine morbillivirus with cognate genes of the Paramyxoviridae.

The nucleotide sequence of the matrix protein of equine morbillivirus (EMV) was determined to be 1062 nucleotides and coded for a deduced protein of M(r) 40148 having a net charge of + 19 at neutral pH. The matrix protein gene was separated from the P and F genes by intercistronic regions of 546 and 469 nucleotides, respectively. The nucleotide sequence which coded for the F protein was 1641 nucleotides and coded for a deduced protein of 546 amino acids having an M(r) of 60,447 and a charge + 4 at neutral pH. Partial sequence information was also determined for the P/V proteins. M, P and F protein sequence comparisons revealed that a greater homology existed between EMV and known members of the morbillivirus genus than with other members of the Paramyxoviridae and that this homology resided within the central half of the protein for the fusion protein, the C-terminal half of the matrix protein and at certain sites with the P protein. Far greater homology was seen between the morbilliviruses and EMV than for the other paramyxoviridae. It was inferred from phylogenetic analyses that EMV was a distantly related member of the morbillivirus genus. A conserved sequence of 18 nucleotides (assumed to be the transcriptional editing site) was present in the P gene of EMV. Insertion of a single nucleotide residue within this site generated the C-terminus of a V-like, cysteine rich protein. Likewise, a conserved 'CTT' intergenic region presumed to be the transcription termination and polyadenylation signal was present in EMV between the P-M-F genes. The close sequence homology of these sites with that of morbilliviruses also inferred that EMV was a member of the morbillivirus genus.

Isolation and characterization of orbivirus genotypic variants.

Orbivirus variants containing either RNA deletions or concatemeric RNAs have been isolated. A variant of Ibaraki virus (a member of the epizootic hemorrhagic disease of deer serogroup) contained an RNA 9 segment which had terminal sequences identical to RNA 9 of wild type virus but was approximately 140 base pairs (bp) shorter. In vitro translation showed that whereas RNA 9 of wild type virus generated the minor structural protein VP6 (molecular weight 38 K), the variant RNA 9 coded for a 32 K protein. Analysis of hybrid molecules formed after melting and reannealing mixtures of [32P]-pCp-labeled wild type and variant RNA 9 molecules indicated that generation of variant RNA 9 may have involved the loss of approximately 150 bp at a location 148 bp from one end of the wild type RNA molecule. Analysis of minor proteins generated by premature termination during in vitro translation of wild type and variant RNA 9 suggested that the deletion occurred towards the 3' end of the positive strand of wild type RNA 9. RNA genome segments 10 and 9 of bluetongue virus type 21 and Bunyip Creek (a Palyam serogroup member) respectively, were observed to form concatemers. Molecular weight estimates and T1 RNase mapping suggested that the concatemers were dimers in a 5'-3' to 5'-3' orientation. In vitro translation of dimeric RNAs yielded products apparently identical to those generated by monomeric RNAs. The possible ramifications of these results with respect to orbivirus evolution are discussed.

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.

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.

Phylogenetic analyses of the complete nucleotide sequence of the capsid protein (VP3) of Australian epizootic haemorrhagic disease of deer virus (serotype 2) and cognate genes from other orbiviruses.

The complete nucleotide sequence of the minor capsid protein (VP3) of epizootic haemorrhagic disease of deer virus (EHDV; Australian serotype 2) was determined using a combination of cloning and sequencing methods. Gene segment 3 that coded for the EHDV VP3 capsid protein was 2768 nucleotides in length with a coding region of 2697 nucleotides flanked by 5' and 3' non-coding regions of 17 and 53 nucleotides, respectively. A protein of 899 amino acids (Mr 103,160) having no overall charge at neutral pH was deduced from the nucleotide sequence. Comparisons with equivalent regions from the other Australian EHDV serotypes showed the VP3 genes and the segments that coded for them were similar, varying by a maximum of 5%. Comparisons with known cognate genes from bluetongue viruses showed that their VP3 genes and the proteins translated from them were remarkably similar to those of EHDV, having approximately 70% to 80% homology at either level, respectively. In an attempt to delineate the evolution of orbiviruses, we have obtained sequence data from the VP3 genes from representative members of all Australian orbiviruses now known. Computer analyses of this data enabled a phylogenetic tree for the orbiviruses to be proposed that incorporated the concept of topotypes.

Detection and characterisation of bluetongue virus using the polymerase chain reaction.

Pairs of oligodeoxynucleotide primers whose sequences were based on those of RNA segment 3 that encodes the bluetongue virus serogroup-reactive protein VP3, were synthesized for three BTVs from different geographic regions of the world and for seven Australian orbiviruses. Each pair of primers was then tested for the synthesis of cDNA and in subsequent polymerase chain reactions (PCR) with all ten virus groups. All primers were serogroup-specific at low or high stringency. One pair of primers was specifically designed for its ability to serogroup a BTV isolate irrespective of its geographic origin. At either high or low stringency, this primer-pair resulted in a common and specific PCR product for each of the BTVs tested but not for the other orbiviruses. Eight pairs of primers based on RNA2 sequences (the gene segment encoding the serotype-specific protein VP2) were also synthesized for the eight Australian serotypes of BTV. Each primer-pair was serotype-specific at low or high stringency except for the BTV16A pair, which cross-reacted with BTV3A and also gave a non-specific product that differed in Mr from the authentic PCR product. Using the PCR and BTV1A RNA3-based primers, BTV1A was detected in blood samples from two sheep at 9 days post inoculation. Virus was found in the platelet, buffy-coat and packed red blood cell fractions, but not in whole blood.

Characterisation of Wongorr virus, an Australian orbivirus.

Sequence analyses of VP3 gene segments of Wongorr virus isolates from the Northern Territory of Australia were compared with the cognate gene segments from Picola and Paroo River viruses. Previous serological investigations had demonstrated some relationships between these viruses, however VP3 gene sequence and phylogenetic analyses placed these viruses within the same serogroup which was distinct from other described orbivirus serogroups. A polymerase chain reaction (PCR) was developed for the detection of this serogroup and used to identify and determine partial sequence data for other isolates of the virus. Wongorr virus and the other tick and mosquito-borne orbiviruses (Kemerovo and Corriparta), were more closely related than the Culicoides transmitted orbiviruses, such as bluetongue (BTV) and African horse sickness virus (AHSV) which were shown to be on a separate branch of the orbivirus phylogenetic tree.

Phylogenetic comparison of the serotype-specific VP2 protein of bluetongue and related orbiviruses.

Regions of the VP2 gene from various bluetongue virus serotypes were sequenced and phylogenetic comparisons were performed. The sequences were characteristic for each BTV serotype and isolates of the same serotype could be grouped geographically, mimicking the topotyping characteristics of BTV VP3 gene sequences. PCR amplification and sequence analysis were used to show the close relationship between Caribbean BTV isolates and South African BTV isolates of the same serotype. Similarly, Australian BTV isolates showed a close genetic relationship with Asian BTV isolates of the same serotype. A multiple amino acid sequence alignment of fifteen BTV serotypes and other orbiviruses over a proposed major neutralization site showed this region (317 335 aa.) was highly variable and nucleotide sequences showed that BTV serotypes could be grouped into nucleotypes, or related serotypes, in broad agreement with the inter-relationships postulated by Erasmus (1990), using plaque-reduction tests.

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.

Expression of the outer capsid proteins VP2 and VP5 of bluetongue virus in Saccharomyces cerevisiae.

cDNAs transcribed from bluetongue virus serotype 1 (Australia) ds RNA 2 and ds RNA 6 coding for the major neutralising antigen VP2 and the outer capsid protein VP5, respectively, were amplified in polymerase chain reactions and ligated downstream of the copper-inducible metallothionein promoter in the yeast expression plasmid pYELC5. Saccharomyces cerevisiae transformed with the recombinant plasmid pYELC5-VP2 expressed full-length VP2 only following induction with 1 mM CuSO4 and reached the maximum level after 6 h. In contrast, S. cerevisiae transformants harboring the recombinant plasmid pYELC5-VP5 expressed VP5 constitutively, although induction increased the level to a maximum after 4 h. A sheep trial was done testing the recombinant proteins, however it was shown that none of these were effective immunogens for eliciting a protective response against a subsequent challenge with bluetongue virus. An analysis of the yeast expression products for the VP2 outer coat protein using a panel of monoclonal antibodies showed that the yeast expressed VP2 was in a conformation different from native VP2 and hence probably unable to elicite an appropriate protective immune response.

Cloning and nucleotide sequence determination of the major envelope glycoprotein (gp55) gene of hog cholera virus (Weybridge).

A 1.7 kb cDNA fragment corresponding to the coding region of the major envelope glycoprotein (gp55) of pestivirus hog cholera (Weybridge) was obtained using the polymerase chain reaction (PCR), and then cloned into pUC 8. The deduced amino acid sequence of gp55 showed a strong homology to that of HCV strains Brescia (94%) and Alfort (90%), and to a lesser extent to the closely related gp53 of bovine viral diarrhoea virus strain, NADL (65%). Eighteen cysteine residues were identified in the sequenced region, all of which were conserved between the gp55/gp53 sequences. This suggests that although the homology at the protein level may vary, there are strong conformational motifs which are conserved among the pestivirus envelope proteins.

Expression of the non-structural protein NS1 of bluetongue virus in bacteria and yeast: identification of two antigenic sites at the amino terminus.

cDNA transcribed from bluetongue virus serotype 1 (Australia) dsRNA 5 coding for non-structural protein NS1 was amplified in a polymerase chain reaction and ligated downstream of the T7 RNA polymerase promoter in the bacterial expression plasmid pET-5b, as a fusion protein with glutathione S-transferase using the pGEX bacterial expression system or the metallothionein promoter in the yeast expression plasmid pYELC5. The linear epitopes bound by six monoclonal antibodies to NS1 were localised to two antigenic regions at the amino terminus by Western blots using a series of carboxy-terminal truncations of the NS1 protein overexpressed in Escherichia coli. Expression of truncated NS1 genes using the pGEX expression system in E. coli enabled a more detailed map of the two epitopes to be constructed. The first epitope is thought to lie between amino acid residues 40-59, while the second is defined by the peptide sequences flanking amino acid 96.

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.