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Short Communication |
Victorian Infectious Diseases Reference Laboratory, 10 Wreckyn Street, North Melbourne, Victoria 3051, Australia
Correspondence
Doris Chibo
Doris.chibo{at}mh.org.au
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are DQ243940 [GenBank] –DQ243962 [GenBank] and DQ243964 [GenBank] –DQ243987 [GenBank] .
A figure showing the phylogenetic analysis using PHYLIP on HCoV-229E isolates and strains identified between 1972 and 2004, and tables showing primers used and their nucleotide position relative to the HCoV-229E complete genome and the summary of amino acid changes in the S and N proteins of HCoV-229E variants studied are available as supplementary material in JGV Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). In animals they are significant veterinary pathogens, often causing severe disease (Holmes, 2001). Until recently, only two human coronaviruses were recognized, Human coronavirus 229E (HCoV-229E) and Human coronavirus OC43 (HCoV-OC43). These viruses have generally been associated with symptoms of the common cold (McIntosh, 1996) but there is growing evidence that they occasionally cause more severe infections (Pene et al., 2003; Vabret et al., 2003; Birch et al., 2005). In late 2002, a third human coronavirus (SARS-CoV) was implicated as the aetiological agent of severe acute respiratory syndrome (SARS) (Drosten et al., 2003). Since then, two more human coronaviruses have been identified, HCoV-NL63 associated with upper and lower respiratory tract infections (van der Hoek et al., 2004) and HCoV-HKU1 in patients with pneumonia (Woo et al., 2005). Based on their serological and genetic properties, the human coronaviruses are assigned to two of three existing antigenic serogroups: HCoV-229E and HCoV-NL63 in serogroup 1 (Siddell, 1995; van der Hoek et al., 2004); HCoV-OC43, HCoV-HKU1 and, most likely, SARS-CoV in serogroup 2 (Siddell, 1995; Snijder et al., 2003; Woo et al., 2005).
Coronaviruses contain four structural proteins, spike (S), membrane (M), small envelope (E) and nucleocapsid (N) (Brian & Baric, 2005). A fifth, the haemagglutinin-esterase (HE) protein, exists in some serogroup 2 coronaviruses (Holmes & Lai, 1996). Limited functional and structural information exists for the HCoV-229E S protein, although receptor-binding activity is associated with the S1 subunit (Bonavia et al., 2003). In the related non-human Infectious bronchitis virus, S1 is involved in the induction of neutralizing, serotype-specific and haemagglutination inhibiting antibodies (Cavanagh et al., 1988).
Both recombination and point mutations have the capacity to drive coronavirus evolution (Navas-Martin & Weiss, 2003). Although recombination has not been detected for HCoV-229E or HCoV-OC43, a recombination breakpoint in the SARS-CoV polymerase has been described (Rest & Mindell, 2003). Few studies on variation in the human coronavirus S protein have been reported. One study of three geographically and chronologically distinct HCoV-229E isolates found limited variation within S gene nucleotide sequences (Hays & Myint, 1998). In contrast, two distinct variants of HCoV-OC43 circulated in Belgium between 2003 and 2004 (Vijgen et al., 2005).
Prior to 2001, in our laboratory coronaviruses were identified using a method involving isolation from human fibroblasts, followed by confirmatory immune electron microscopy where these agents were suspected. Virus isolation attempts on respiratory specimens were discontinued in 2001 and replaced by a RT-PCR capable of differentiating between HCoV-229E and HCoV-OC43 (Birch et al., 2005). RNA was extracted from virus isolates and clinical material by using a High Pure Viral Nucleic Acid kit (Roche Diagnostics) and reverse transcribed using avian myeloblastosis virus reverse transcriptase (Promega) at 42 °C for 1 h using random hexamers. The full S and N gene sequences of HCoV-229E were amplified in a nested PCR (Supplementary Table S1 available in JGV Online) using a Qiagen Taq DNA polymerase kit (Qiagen). The cycling programmes consisted of an initial denaturation of 4 min at 94 °C, followed by 40 cycles (first round) or 25 cycles (second round) of 30 s at 94 °C, 30 s at 60 °C and 130 s for the S gene or 90 s for the N gene at 72 °C, with a final extension of 7 min at 72 °C. RT-PCR products were purified, sequenced in both directions using a cycle sequencing reaction (ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit; Perkin-Elmer) and analysed by using an ABI 3730S capillary sequencer.
Nucleotide sequences were analysed and amino acid sequences determined using BIOEDIT sequence alignment editor version 7.0.1 (Hall, 1999). Alignments of nucleotide sequences were made with Multalin (Corpet, 1988) and manual editing of alignments was performed using Genedoc version 2.5 (Nicholas & Nicholas, 1997). Expected transition/transversion ratios and gamma distribution parameter alpha were estimated using TREEPUZZLE version 5.2 (Schmidt et al., 2002). Phylogenetic trees based on the optimum alignment were constructed using DNAdist and neighbour-joining method with PHYLIP version 3.63 (Felsenstein, 1993) and parameters estimated from TREEPUZZLE. Unrooted phylograms were drawn with TREEVIEW version 1.5 (Page, 1996). Tests of selection were conducted using MEGA version 3.1 (Kumar et al., 2004). The codon-based model of Nei–Gojobori was used to compare synonymous (dS) and non-synonymous (dN) distances. Assumptions tested were purifying (dN<dS), neutral (dN=dS) and positive (dN>dS) selection. The probability computed was <0·05 for hypothesis rejection at the 5 % level. Potential N-glycosylation sites in the HCoV-229E S protein were predicted using the NetNGlyc 1.0 Server at the Centre for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/NetNGlyc/).
Between 1979 and 2004, HCoV-229E was isolated or detected by RT-PCR in 25 patients aged from 0·3 to 58 years of age (Table 1). Patients suffered from symptoms varying from mild upper respiratory tract infection to pneumonia, and three (patients 12, 18 and 19) were hospitalized. Several presented with fever, fatigue and cough, consistent with a case definition of influenza-like illness (Clothier et al., 2005). A total of 25 HCoV-229E sequences were analysed. Twenty-one were full-length S gene sequences, 13 from virus isolates and eight directly from clinical material. Partial S gene sequences were obtained from two samples of clinical material (patients 19 and 25 in Table 1). S gene sequences could not be obtained from the viruses detected in patients 18 and 24. All S gene sequences available were compared to an ATCC prototype strain (HCoV-229E 1973; GenBank accession no. DQ243963
[GenBank]
, containing 3522 nt, 1173 aa).
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). The 21 complete S gene sequences each contained 3513 nt (1170 aa). Deletion of 9 nt occurred in two discrete positions in each of these sequences, at 681–683 and 1057–1062. Without exception this resulted in 3 aa deletions compared to the prototype strain, aspartic acid at codon 228 and valine and tyrosine at codons 353 and 354, respectively. The partial sequences comprised nt 1–957 and 1806–3513 (patient 19) and 1–1788 (patient 25). The deletion pattern described above for full-length sequences also applied to these variants within the constraints of the sequence information available. Comparison of the full or partial S amino acid sequences with the prototype sequence identified 67 individual codon differences and the three deletions described above. The deletions and most dN changes occurred in the S1 domain, between aa 16 and 560. Thirty of 54 aa substitutions detected in this region were non-conservative compared with 7 of 13 substitutions identified outside the S1 domain (Table 2 and Supplementary Table S2 available in JGV Online). The putative N-glycosylation pattern (Asp–X–Ser/Thr) of the S protein of all variants differed slightly compared with the prototype. The 24 N-glycosylation sites in the prototype strain were also present in the variants, but two additional sites were generated in variants as a result of mutations to asparagine at codons 111 and 488. Binding of HCoV-229E to its cellular receptor human aminopeptidase N occurs through interaction with aa 417–517 of the S1 domain (Bonavia et al., 2003). Seven amino acid substitutions occurred in this region (Table 2). Three of them (Q430K, D444N and K488N) were non-conservative and included the generation of an N-glycosylation site at aa 488.
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The entire sequence of the N gene was obtained for 23 of 25 HCoV-229E variants; compared with the S gene, there was less heterogeneity in this gene and the most divergent variant (from patient 22 in Table 1
) differed by only 25 nt (9 aa) from the 1973 prototype strain (HCoV-229E 1973; GenBank accession no. DQ243939
[GenBank]
). Seven amino acid changes, four of which were non-conservative, were shared by each variant. Four changes clustered in a hot spot between aa 224 and 229 (Supplementary Table S3 available in JGV Online). Deletions were detected in only two N gene sequences. The first was a three base (GGT) in-frame deletion at nt position 480 in the virus from patient 3 resulting in the deletion of glycine at aa 160. The second was an A deletion from a string of five As starting at nt 611 in the variant from patient 14, producing a frameshift mutation and generation of a stop codon 52 aa downstream. This sequence was generated by direct RT-PCR from a clinical specimen and may be the result of amplification of non-infectious viral RNA.
Phylogenetic analysis of the S gene assigned the 23 variants to four distinct groups each containing temporally associated viruses (Fig. 1). The groups comprised viruses circulating during the periods 1979–1982, 1982–1984, 1990–1992 and 2001–2004. The expected transition/transversion ratio and gamma distribution parameter alpha were estimated as 1·62 and 0·16, respectively, from the dataset using TREEPUZZLE (Schmidt et al., 2002). These values were used in DNAdist (Kimura) to produce a more accurate tree topology. A strong correlation between bootstrap values and these groupings was found. The expected transition/transversion ratio and gamma distribution parameter alpha values estimated using the N gene dataset were 1·81 and 0·19, respectively. Phylogenetic analysis of the N gene showed clustering of variants similar to that obtained with the S gene. However, less sequence variation in the N gene made it difficult to resolve the viruses into four groups. Rather, two major clusters comprising groups 1 and 2, and groups 3 and 4, respectively, were identified (Supplementary Fig. S1 available in JGV Online).
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and Supplementary Table S2 available in JGV Online).
Alignment of S protein sequences and phylogenetic analysis of the S gene revealed several discrete time points where amino acid substitutions occurred and were then retained during subsequent years. These mutations may represent the outcome of natural evolution of the virus in the context of antibody pressure. Comparing rates of dS and dN nucleotide substitutions can test three forms of evolutionary selection on a gene. Equal dS and dN substitution rates demonstrate an absence of selection, whereas an excess of dS substitutions indicates purifying selection, operating to preserve protein structure and function. Excess dN substitution rates indicate positive selection and occur mainly in genes with adaptive functions (Bush, 2001). Most positively selected genes previously identified have been pathogen surface proteins (Yang & Bielawski, 2000). To avoid recognition by antibodies generated in response to prior antigen exposure, surface proteins of pathogens must change their structure. Hence, it is likely that evasion of the host immune system drives repeated amino acid replacements in surface proteins (Bush, 2001). Using the Nei–Gojobori method for estimating statistical differences between dS and dN substitution rates (Kumar et al., 2004), we confirmed that the HCoV-229E S gene sequences had undergone positive selection over time. Probabilities of 0·00 were obtained for purifying and neutral selection and both hypotheses were rejected. A probability of 1·0 was obtained when testing for positive selection and this hypothesis was therefore accepted.
We could not show evidence for the emergence of distinct subtypes of HCoV-229E as recently reported for HCoV-OC43, where strains circulating in Belgium between 2003 and 2004 showed sufficient unrelatedness at the amino acid level (3·1 %) to suggest the existence of two genetically distinct strains (Vijgen et al., 2005). For the HCoV-229E variants we studied, the overall amino acid difference between the 1979 strain and the 2004 strain was 3·3 %. When apparent antigenic drift occurred, the extent of amino acid difference between the old variant and its replacement was less than 1 %.
When the HCoV-229E S gene sequences from this study were compared to 26 sequences available through GenBank, only two variants were similar. The first, strain A162 (accession no. Y10051
[GenBank]
), was isolated in 1995 from an adult in Ghana (Hays & Myint, 1998). While the submitted sequence was 483 nt short of full-length, it grouped phylogenetically with coronaviruses circulating in Australia between 1990 and 1992 (Fig. 1). Also similar was strain 13–18, first isolated in 1999 in the USA. Although the sequence available was less than half that of the complete S gene, this virus grouped with variants circulating between 2001 and 2004 in Australia (Fig. 1). Hence, the time of circulation of distinct HCoV-229E variants in south-eastern Australia generally coincided with that of variants circulating elsewhere.
Overall, sequencing and analysis of the S and N gene products of HCoV-229E strains circulating in Victoria, Australia, between 1979 and 2004 has provided the first evidence for genetic drift and positive selection as part of the evolution of this virus. The similarity between those strains circulating in Victoria and a small number of strains identified in other geographical locations at similar times indicates that HCoV-229E, despite having the potential, has not undergone major recombination events since it was first isolated in 1967.
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Received 7 November 2005;
accepted 16 January 2006.
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