Complete nucleotide sequences of Nipah virus isolates from Malaysia

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1 Journal of General Virology (2001), 2, Printed in Great Britain... SHORT COMMUNICATION Complete nucleotide sequences of virus isolates from Malaysia Y. P. Chan, 1 K. B. Chua, 2 C. L. Koh, 1 M. E. Lim 3 and S. K. Lam 2 Institute of Biological Sciences, Faculty of Science 1, Department of Medical Microbiology, Faculty of Medicine 2 and Institute of Postgraduate Studies and Research 3, University of Malaya, 003 Kuala Lumpur, Malaysia We have completely sequenced the genomes of two virus (NiV) isolates, one from the throat secretion and the other from the cerebrospinal fluid (CSF) of the sole surviving encephalitic patient with positive CSF virus isolation in Malaysia. The two genomes have 1 24 nucleotides each and differ by only 4 nucleotides. The NiV genome is 12 nucleotides longer than the virus (HeV) genome and both genomes have identical leader and trailer sequence lengths and hexamer-phasing positions for all their genes. Both NiV and HeV are also very closely related with respect to their genomic end sequences, gene start and stop signals, P gene-editing signals and deduced amino acid sequences of nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, glycoprotein and RNA polymerase. The existing evidence demonstrates a clear need for the creation of a new genus within the subfamily Paramyxovirinae to accommodate the close similarities between NiV and HeV and their significant differences from other members of the subfamily. An outbreak of severe febrile encephalitis associated with fatal human cases was reported in the northern part of peninsular Malaysia in late September 199 (CDC, 1999 a). The outbreak was associated with, and preceded by, respiratory illness and encephalitis in pigs in the same district (Mohd Nor et al., 2000). By February 1999, similar diseases in pigs and humans were recognized in the central and southern parts of peninsular Malaysia, as a consequence of movement of a large number of pigs from the initial outbreak area southward. In March 1999, a cluster of 11 cases of respiratory and encephalitis illness with one death was noted in Singapore among abattoir workers who handled pigs from outbreak regions in Malaysia (Paton et al., 1999). A novel paramyxovirus, named virus (NiV), was first isolated from the cerebrospinal fluid (CSF) Author for correspondence: K. B. Chua. Fax chuakb ummc.edu.my of an encephalitic patient from Sungai village and subsequently identified as the aetiological agent responsible for the outbreak (Chua et al., 1999, 2000a). The outbreak in Singapore ended when the importation of pigs from Malaysia was prohibited and the outbreak in Malaysia ceased when over a million pigs were culled within the outbreak areas and their immediate surrounding areas. A total of 2 cases of encephalitis including 10 deaths associated with the outbreak were recorded by May 1999 in Malaysia (CDC, 1999b; Chua et al., 2000a). 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 1 out of 2 fatal cases and only 1 of 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 AY029 and AY029). 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 124 nt, with a nt leader at its 3 end and a 33 nt trailer at its 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 1 24 nt. Pair-wise alignment of the two genomes shows that UMMC2 differs from UMMC1 by only 4 nt, at positions 231 (T C) at the 3 non-coding region of the P V C gene, 1131 (G A) at the 3 non-coding region of the L gene, 110 (C T) at the coding region of the L SGM CBFB IP: On: Wed, 11 Apr :3:4

2 Y. P. Chan and others Table 1. Comparison of the nt sequences of the N, P, M, F, G and L genes of NiV and HeV ORF UTR 3 UTR Gene Virus Length (%) Length (%) Length (%) N P M F G L gene [no resultant amino acid (aa) change] and 134 (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, AF234 and AF234) (Harcourt et al., 2000) extending from the beginning of the N gene (nt ) to the end of the G gene (nt 112), UMMC2 has an identical sequence, whereas UMMC1 differs by 1 nt (at nt position 231, T C) in the region studied. The genetic relationship of NiV with other paramyxoviruses, including 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 and 3 non-coding nt sequences of all the genes in UMMC1 and HeV (genome size of nt, GenBank accession no. AF01149). On the whole, the coding sequences of both viruses have a higher level of identity than their noncoding sequences, with the highest identity (%) seen in the coding sequence of the N gene. It has been suggested that Paramyxovirinae genomes are replicated efficiently only when they are in multiples of nt and this has been dubbed the rule-of-six (Calain & Roux, 1993; Kolakofsky et al., 199). This is because the template for paramyxovirus RNA synthesis is not naked RNA but the helical ribonucleoprotein complex core of the virus, a structure in which nt hexamers are believed to be associated with individual nucleocapsid protein molecules (Kolakofsky et al., 199). As a consequence, many members of the family Paramyxoviridae have a genome length in a multiple of nt. The genome length of NiV (124) conforms to this rule. Furthermore, it has been found that, within a genus, the transcriptional start site for each gene tends to be conserved in relation to the hexamer-phasing position (Kolakofsky et al., 199; de Leeuw & Peeters, 1999). The hexamer-phasing position of the NiV N gene is the same as that of the cognate genes of members within the subfamily as a result of the uniform length of their 3 -leader sequences. However, like HeV, NiV has a hexamer-phasing pattern of 2, 3, 4, 4, 4, 3, which is significantly different from the hexamer-phasing patterns of other viruses in the subfamily. Both are also unique in that they use hexamer-phasing position for the P-editing site. This pattern of hexamer-phasing positioning is not used by any other viruses within the subfamily for either transcriptional start or P-editing site (Wang et al., 2000). The NiV 3 leader sequence is 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 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 end). Despite the variation in length of the 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 CBFC IP: On: Wed, 11 Apr :3:4

3 Nucleotide sequences of viruses Table 2. Pair-wise alignments of various proteins of NiV and other paramyxoviruses to demonstrate the degrees of similarity with respect to the NiV proteins Abbreviations: NiV, virus; HeV, virus; TPMV, Tupaia paramyxovirus; MeV, measles virus; CDV, canine distemper virus; SaV, Salem virus; HPIV3, human parainfluenza virus 3; SeV, Sendai virus; HPIV1, human parainfluenza virus 1; MuV, mumps virus; SV, simian virus ; SV41, simian virus 41; HPIV2, human parainfluenza virus 2; HPIV4a, human parainfluenza virus 4a; HPIV4b, human parainfluenza virus 4b; PoRV, porcine rubulavirus; MaV, Mapuera virus; NDV, Newcastle disease virus; RSV, respiratory syncytial virus; and BRSV, bovine respiratory syncytial virus. Virus Accession no. Sequence similarity (%)* NiV AY029 N P M F A L HeV AF TPMV AF MeV AF CDV AF SaV AAF HPIV3 Z SeV M HPIV1 X MuV AB SV AF SV41 X HPIV2 X HPIV4a P HPIV4b P PoRV P MaV X12 2 NDV AF RSV AF BRSV AF * Percentage similarity of nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), attachment (i.e. HN, H or G) protein (A) and large protein or RNA polymerase (L) of other paramyxoviruses with respect to the corresponding protein of NiV. the Morbillivirus and Respirovirus genera as it has a conserved intergenic trinucleotide, 3 -GAA-. 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 9 nt (nt 112 to 1213). 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, 19) 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 to 114. There are three more AUG codons in the first reading frame, 400 nt downstream from the third AUG codon; none are in the favourable Kozak s 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 untranslated region (UTR) (13 nt), ORF (3 nt) and 3 UTR ( 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 2 2 Da, very close to that of the HeV L protein (220 Da). It is a basic protein with a pi of 33. The NiV L protein is also rich in leucine (23 molecules) and isoleucine (19 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 (%) except at two regions, one (aa 11 0) between domains II IP: On: Wed, 11 Apr :3:4 CBFD

4 Y. P. Chan and others Fig. 1. Phylogenetic tree based on complete L protein sequences of viruses within the subfamily Paramyxovirinae. The unrooted bootstrapped neighbour-joining tree was generated by using the ClustalX and PHYLIP programs and the branch lengths represent relative genetic distances. Members within the genus are enclosed in dotted circles. The topology of the tree supports the view that NiV and HeV should be put into a new genus. Abbreviations are given in the legend to Table 2, except for RPV, rinderpest virus and BPIV3, bovine parainfluenza virus 3. GenBank accession numbers (in parentheses) are as follows: MuV (P30929), SV (Q434), SV41 (JQ10), HPIV2 (P2), PoRV (CAA0), NDV (P1120), MeV (P12), CDV (Y0929), RPV (Z309), SeV (P029), HPIV3 (P12), BPIV3 (AF1), TPMV (AAF3393), HeV (AF01149), NiV (AY029). and III and the other (aa ) within domain V (Poch et al., 1990) (data not shown). The similarity between these two L proteins increases to % 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- CBFE IP: On: Wed, 11 Apr :3:4

5 Nucleotide sequences of viruses 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 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 3% 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. The data reported here on the NiV genome suggest that this newly identified NiV is a member of the subfamily Paramyxovirinae. The data also show that NiV is closely related to HeV. The existing evidence demonstrates a clear need for the creation of a separate genus within the subfamily to accommodate the close similarities between NiV and HeV and their significant differences from other existing members of the subfamily Paramyxovirinae. The name Henipavirus ( ) has been proposed for a new genus that would include HeV as the type species and NiV as the second member of the genus (Wang et al., 2000). This project is funded by research grant IRPA no from the Ministry of Science, Technology and the Environment, Malaysia. We thank Dr Linfa Wang, Australia Animal Health Laboratory, Geelong, Australia for useful advice in preparation of the manuscript. References Calain, P. & Roux, L. (1993). The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. Journal of Virology, CDC (1999a). Outbreak of -like virus Malaysia and Singapore Morbidity and Mortality Weekly Report 4, CDC (1999b). Update: outbreak of virus Malaysia and Singapore. Morbidity and Mortality Weekly Report 4, Chua, K. B., Goh, K. J., Wong, K. T., Kamarulzaman, A., Tan, P. S. K., Ksiazek, T. G., Zaki, S. R., Paul, G., Lam, S. K. & Tan, C. T. (1999). Fatal encephalitis due to virus among pig farmers in Malaysia. Lancet 34, Chua, K. B., Bellini, W. J., Rota, P. A., Harcourt, B. H., Tamin, A., Lam, S. K., Ksiazek, T. G., Rollin, P. E., Zaki, S. R., Shieh, W.-J., Goldsmith, C. 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Journal of Virology 4, Received 30 April 2001; Accepted June 2001 IP: On: Wed, 11 Apr :3:4 CBFF