Institute of Biological Sciences, Faculty of Science1, Department of Medical Microbiology, Faculty of Medicine2 and Institute of Postgraduate Studies and Research3, University of Malaya, 50603 Kuala Lumpur, Malaysia
Author for correspondence: K. B. Chua. Fax +60 3 7958 4844. e-mail chuakb{at}ummc.edu.my
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During the peak outbreak of NiV encephalitis, 94 patients with NiV infection were treated in the University Malaya Medical Centre (UMMC) (Goh et al., 2000 ). NiV could be isolated from the upper respiratory secretions, urine and CSF of patients with acute illness (Chua et al., 2001
). The presence of NiV in the CSF of patients is associated with high mortality (Chua et al., 2000b
). In the same study, NiV was isolated from CSF of 17 out of 27 fatal cases and only 1 of 57 nonfatal cases (Chua et al., 2000b
). The complete genomes of two NiV isolates were completely sequenced by PCR using pairs of virus-specific primers, primer walking strategy, a modified method of rapid amplification of cDNA ends according to Tillett et al. (2000)
and viral RNA end-to-end ligation followed by inverted PCR. The NiV isolated from the CSF of the sole surviving encephalitic patient was designated as UMMC1, while the isolate from the throat secretion of the same patient was designated as UMMC2. The complete nucleoptide (nt) sequences of both isolates were deposited in GenBank (accession nos AY029767 and AY029768). The complete nt sequence of UMMC1 was used as the reference for further analysis and comparison with genomic sequences of other paramyxoviruses.
The complete genomic sequence of UMMC1 comprises 18246 nt, with a 55 nt leader at its 3' end and a 33 nt trailer at its 5' end. It has nucleocapsid (N), phosphoprotein (P), matrix (M), fusion protein (F), attachment protein (G) and large polymerase (L) genes. The complete nt sequence of UMMC2 also comprises 18246 nt. Pair-wise alignment of the two genomes shows that UMMC2 differs from UMMC1 by only 4 nt, at positions 2381 (TC) at the 3' non-coding region of the P/V/C gene, 11315 (G
A) at the 3' non-coding region of the L gene, 16160 (C
T) at the coding region of the L gene [no resultant amino acid (aa) change] and 16345 (T
C) at the coding region of the L gene (phenylalanine
serine). In comparison with the published partial nt sequence of NiV (11200 nt; GenBank accession nos AF212302, AF238466 and AF238467) (Harcourt et al., 2000
) extending from the beginning of the N gene (nt 56) to the end of the G gene (nt 11255), UMMC2 has an identical sequence, whereas UMMC1 differs by 1 nt (at nt position 2381, T
C) in the region studied.
The genetic relationship of NiV with other paramyxoviruses, including Hendra virus (HeV), based on the N, P/V/C, M, F and G genes has been presented by Harcourt et al. (2000) . Table 1
gives a comparison of the open reading frames (ORF) and the 5' and 3' non-coding nt sequences of all the genes in UMMC1 and HeV (genome size of 18234 nt, GenBank accession no. AF017149). On the whole, the coding sequences of both viruses have a higher level of identity than their non-coding sequences, with the highest identity (78%) seen in the coding sequence of the N gene.
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The NiV 3' leader sequence is 55 nt long, identical in length to the 3' leader sequences of other members of the subfamily Paramyxovirinae. Overall, the 3' leader sequence of NiV is very closely related to that of HeV and both are more closely related to the 3' leader sequences of the morbilliviruses and respiroviruses than to those of the rubulaviruses. Unlike HeV, NiV has an A residue at position 4 in lieu of a G and is similar to all other paramyxoviruses. Unlike the 3' leader sequences, the 5' trailer sequences differ quite substantially in length within the subfamily. The trailer sequence of NiV is 33 nt long, similar in length to that of HeV; however, the two sequences differ by 3 nt (at positions 19, 20 and 21, counting from the 5' end). Despite the variation in length of the 5' trailer sequences among the paramyxoviruses, the last four nt (TGGT) are conserved except for NiV and HeV, where the internal T of TGGT is replaced by C, and the 3' nt is C in all except for members of the Rubulavirus genus. Sequence comparison analyses revealed that NiV, like HeV, is similar to members of the Morbillivirus and Respirovirus genera as it has a conserved intergenic trinucleotide, 3'-GAA-5'. The transcriptional initiation and stop signals of all NiV genes are very closely related to HeV, to the extent that the transcriptional stop signals of the F genes of both viruses begin with the nt T instead of an A as found in the stop signals of other genes.
The complete nt sequences of the L genes of UMMC1 and UMMC2 were determined by primer walking strategy. The highly conserved transcriptional initiation and stop signals were identified. The length of the L gene mRNA was calculated to be 6955 nt (nt 11258 to 18213). The first and second AUG codons in the NiV mRNA are located at nt 20 and 113 of the first reading frame. Neither are in the favourable translation initiation context (Kozak, 1987 ) and both are rapidly terminated by two in-frame stop codons. The third AUG codon is located in the third reading frame, with favourable translation initiation context, and the ORF extends from nt 11412 to 18146. There are three more AUG codons in the first reading frame, 400 nt downstream from the third AUG codon; none are in the favourable Kozaks context of translation initiation and all are rapidly terminated by stop codons. The use of the third AUG codon as the initiation site of translation of the NiV L gene was supported by the conserved N-terminal aa sequence of the deduced L protein in comparison with the L protein sequences of HeV, morbilliviruses and respiroviruses. The 5' untranslated region (UTR) (153 nt), ORF (6735 nt) and 3' UTR (67 nt) of the NiV L gene are of the same lengths as the respective regions of the HeV L gene (Table 1
).
Sequence analysis of the deduced NiV L protein revealed a large protein of 2244 aa with a molecular mass of 257288 Da, very close to that of the HeV L protein (257280 Da). It is a basic protein with a pI of 7·33. The NiV L protein is also rich in leucine (235 molecules) and isoleucine (196 molecules), both of which account for 19·2% of the total aa. The linear domains and motifs of L proteins suggested by Poch et al. (1990) could be easily identified in the L proteins of both NiV and HeV. Both share a high degree of similarity at the aa level (85%) except at two regions, one (aa 611707) between domains II and III and the other (aa 12981325) within domain V (Poch et al., 1990
) (data not shown). The similarity between these two L proteins increases to 88% if the two hypervariable regions are excluded from the analysis. A sequence analysis of the most conserved domain (domain III) and the four conserved motifs (motifs A, B, C and D) therein showed that the C motif of NiV and HeV is GDNE instead of GDNQ, an unusual finding because GDNQ is conserved among the L proteins of all non-segmented negative-strand RNA viruses, except for the L protein of Tupaia paramyxovirus (Tidona et al., 1999
).
Table 2 shows the degrees of similarity of the various proteins of NiV with those of representative viruses within the family Paramyxoviridae by pair-wise alignment using the Clone Manager 5 and Align Plus-4 programme package (S&E Software). Overall, the various proteins of NiV have the highest similarities with the respective proteins of HeV, followed by those of the members of the Morbillivirus and Respirovirus genera. Within the family Paramyxoviridae, the degree of similarity drops drastically to approximately 35% with members of the Morbillivirus genus and further decreases to approximately 30% with members of the Respirovirus genus. The degrees of similarity never reached 20% with similar proteins of members of the subfamily Pneumovirinae. Among the six proteins of NiV, the P protein always has the lowest similarity with the P proteins of other paramyxoviruses. The topology of the phylogenetic tree based on the complete L protein sequences (Fig. 1
) demonstrates that NiV and HeV are closely related, forming a unique group that is clearly distinct from any of the established genera within the Paramyxovirinae subfamily.
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References |
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Received 30 April 2001;
accepted 6 June 2001.