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1 Department of Pathology, University of Texas Medical Branch at Galveston, 301 University Blvd, Galveston, TX 77555-0609, USA
2 University of California at Berkeley, Berkeley, CA 94720, USA
3 School of Natural Sciences, University of Texas, Austin, TX 78712, USA
4 Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, 301 University Blvd, Galveston, TX 77555-0609, USA
5 Sealy Center for Vaccine Development and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch at Galveston, 301 University Blvd, Galveston, TX 77555-0609, USA
Correspondence
Alan D. T. Barrett
abarrett{at}utmb.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary tables are available in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Monath, 1999). Infection of humans with YFV may result in a haemorrhagic fever with high mortality (Tomori, 1999). Transmission cycles involve both a jungle/sylvatic cycle and an urban cycle, with an additional intermediate ‘savannah’ cycle in Africa.
YFV is the prototype member of the family Flaviviridae, the genus Flavivirus, consisting of a single-stranded, positive-sense RNA genome, almost 11 kb in length. A type I 5'-terminal cap (m7GpppAmp) precedes the genome. The genome is flanked by 5' and 3' non-coding regions (NCR) and translated as a single open reading frame (ORF) that is co- and post-translationally cleaved into 10 proteins, including three structural proteins (C, prM/M and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) (Barrett & Monath, 2003).
Understanding genetic relationships of this medically important viral pathogen is valuable for monitoring of future YFV outbreaks. Based on the examination of prM/M, E and 3'NCR of the YFV genome, previous studies have identified seven genotypes of YFV (Mutebi et al., 2001; Wang et al., 1996). Five African genotypes have been proposed, which include west African genotype I (strains from Nigeria and Ivory Coast), west African genotype II (strains from Senegal, plus additional Nigerian and Ivory Coast strains), east African genotype (strains from Kenya and Uganda), east/central African genotype [strains from Zaire (Democratic Republic of Congo), Uganda, Ethiopia and central African Republic] and Angolan genotype (Mutebi et al., 2001). Two South American genotypes have also been proposed: South American genotype I (Brazil, Columbia, Panama and Trinidad) and South American genotype II (predominantly Peru, plus a few isolates from Brazil and Trinidad) (Wang et al., 1996; Bryant & Barrett, 2003; Barrett & Monath, 2003).
In the present study, we sought to expand upon these genetic relationships using full genome analysis of east African (Uganda48a), east/central African (Ethiopia61b) and Angolan (Angola71) genotypes. Strain Angola71 was isolated from a human in early 1971, during a YF outbreak that occurred in this region during this time period (Pinto & Filipe, 1973). Prior to this outbreak, there had been no reported outbreaks in this country since 1872. Factors that led to the emergence of a YF outbreak in a region that had previously been free of the disease for nearly a century are unknown; however, genomic analysis of this particular virus strain may reveal potential contributing molecular characteristics. In addition, YFV strain Ethiopia61b was isolated during a human epidemic that occurred in this region in 1961 and 1962 (Andral et al., 1968; Serie et al., 1968), while Uganda48a was isolated in Uganda in 1948 from an unknown source, likely from a sylvan outbreak in this region at the time (Smithburn et al., 1949). Using these strains, we sought to expand upon the very limited full genome database for YFV, of which this report is the first to present genomic sequences of strains from east and central Africa.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Three virus genomes were sequenced in this study: the east African strain Uganda48a, the east/central African strain Ethiopia61b and the Angolan strain Angola71. These strains were obtained as lyophilized stocks from the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch at Galveston. Viruses were reconstituted and passaged once in Vero cells to generate seed stocks, and were then given one additional passage in Vero cells to produce working stocks.
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). Briefly, viral RNA was extracted using the Qiagen viral RNA extraction kit according to the manufacturer's instructions, followed by Superscriptase II or Titan RT-PCR protocols. Amplified fragments were visualized by ethidium bromide agarose gel staining, extracted from the gels and either sequenced directly if sufficient quantities of the DNA were recovered or cloned into the pGEM T-easy vector (Promega). For cloned PCR products, three separate clones were sequenced to ensure that the sequence was representative of the consensus sequence. YFV primers used in this study are listed in Supplementary Table S1 (available in JGV Online). Oligonucleotide primers were obtained from Sigma-Genosys.
Phylogenetic and sequence analyses.
Sequence analysis was performed using Contig Express. Translations and alignments were performed using Vector NTI. Percentage similarities and differences were calculated using MegAlign (DNASTAR). NetNGlyc (Gupta et al., 2004; http://www.cbs.dtu.dk/services/NetNGlyc/) was utilized for glycosylation site prediction. PAUP (Swofford, 2003) was utilized for the construction of phylogenetic trees by the neighbour-joining method (HKY85 parameter). In addition, GCUA version 1.1 (McInerney, 1998) was used for codon usage analysis and percentage GC calculations. For genomic codon usage analysis, the first 10 000 nt of the ORF was utilized (GCUA 1.1 is limited to 10 000 nt).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Mutebi et al., 2001; Bryant et al., 2003), including five in Africa: west Africa I, west Africa II, Angola, east Africa and east/central Africa genotypes. Genomic sequences are available for the west Africa I genotype (Ivory Coast strain 85-82H; Pisano et al., 1997) and the west Africa II genotype: Asibi (Hahn et al., 1987), Gambia01 (Colebunders et al., 2002) and French viscerotropic virus (FVV) (Wang et al., 1995), while there are no genomic sequences available for the other three African genotypes. We determined the nucleotide sequence of the genomes of Uganda48a (east Africa genotype), Ethiopia61b (east/central Africa genotype) and Angola71 (the only known member of the Angolan genotype). All three viruses had genomes of 10 823 nt in length, with two copies of the repeat nucleotide sequence elements (RYF) in the 3'NCR that has been shown previously to characterize strains of this lineage (Mutebi et al., 2004).
Nucleotide sequence variation among diverse YFV strains
Uganda48a, Ethiopia61b and Angola71 were compared to previously published African YFV genomic sequences. In the neighbour-joining phylogenetic tree shown in Fig. 1, Uganda48a and Ethiopia61b showed a high level of identity with each other but were both distinct from Angola71. Together, these newly sequenced strains formed a separate clade from the west African strains. In addition, phylogenetic relationships similar to those shown in Fig. 1 were observed when phylogenetic trees were constructed for each structural and non-structural protein gene independently, as well as the 3'NCR (data not shown).
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). All three newly sequenced strains were approximately 25 % divergent in nucleotide sequence and 7 % divergent in amino acid sequence from the prototype Asibi strain (Table 2). Consistent with their classification as separate genotypes, Uganda48a and Angola71 were 17·1 % divergent in nucleotide sequence and 3·3 % divergent in amino acid sequence from each another. Interestingly, sequence divergence between Uganda48a and Ethiopia61b was much less pronounced, with these strains differing by 7·3 % at the nucleotide level and 1·5 % in amino acid sequence. The west Africa I genotype was 9 % divergent in nucleotide composition from the west Africa II genotype, 24·6 % divergent from the Angolan and east/central African genotypes and 24·2 % divergent from the east African genotype.
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, were supported by this study. In addition, various nucleotides differentiated the genotypes identified by Mutebi et al. (2001). Lineage and genotype-specific nucleotides for the NS2A gene, a highly variable area of the genome, are shown in Supplementary Table S2 (available in JGV Online). Compared to the Asibi prototype, Angola71, Uganda48a and Ethiopia61b strains possessed 59 common nucleotide substitutions in NS2A. In addition, Uganda48a possessed 15 genotype-specific nucleotide substitutions in this region, while Ethiopia61b had 19 genotype-specific nucleotide substitutions and Angola71 had 35 genotype-specific nucleotide substitutions. West Africa I genotype Ivory Coast possessed 24 genotype-specific nucleotides in NS2A that differentiated it from west Africa II genotype strains.
Amino acid sequence variation between various YFV genotypes
Differences in amino acid composition ranged from 0·2 to 7·6 % for the various genotype-representative YFV strains compared with the prototype Asibi strain (Table 2
). Alignment of the ORF of the various strains is shown in Fig. 2. Several amino acid substitutions characterized strains from west Africa, east Africa and Angola. Angola71, Uganda48a and Ethiopia61b shared many common amino acid substitutions and together comprised the east and central African lineage. The Angolan genotype possessed 52 additional genotype-specific amino acid substitutions (six in the C protein, one in prM/M, six in NS1, seven in NS2A, two in NS2B, seven in NS3, three in NS4A, two in NS4B and 18 in NS5), while the east African genotype-representative strain Uganda48a had 21 genotype-specific substitutions (four in the C protein, one in prM/M, one in E, four in NS1, two in NS2A, two in NS4B and seven in NS5) and Ethiopia61b had 23 (two in the C protein, three in prM/M, one in E, two in NS1, four in NS2A, four in NS3, one in NS4A and six in NS5). The west Africa I-representative strain, Ivory Coast, had 37 aa substitutions that differentiated it from west Africa II genotype strains (three in C, six in E, two in NS1, two in NS2A, one in NS2B, four in NS3, one in NS4A, four in NS4B and 14 in NS5; Supplementary Table S3 available in JGV Online).
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) (Fig. 3a). Since this region of NS4B is highly variable between YFV strains, we also compared these strains to other available flavivirus NS4B sequences in the alignment shown in Fig. 3(b). As Yokose virus (a flavivirus isolated from a bat in Japan) is a close relative to YFV (Fig. 4), it is notable that this virus also possesses a cluster of amino acids in NS4B similar to the east and central African strains of YFV, but not west African strains (Fig. 3b). In addition to this variable region in NS4B, there was also a highly variable cluster of amino acids in the C protein of Uganda48a, Ethiopia61b and Angola71 (residues 103–110) and several regions of variability in NS5 of these strains (residues 287–295, 524–527, 556–562 and 632–660) compared with west African strains.
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. NS2A possessed high levels of intergenotype variation in codon usage, with 19 aa exhibiting differences in codon usage. In addition to differences observed when individual viral genes were assessed, examination of genomic ORF (nt 1–10 000) revealed more similar RSCU values between the various genotypes, as well as RSCU values closer to 1 for several of the amino acids when compared to values for single genes (e.g. Asp, Asn, Gln, Phe, Lys and Cys) (Table 3).
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), the percentage GC at third base codon positions (GC3s) for each gene and ORF were plotted against Nc values for the representative west African strain Asibi and east/central African strain Ethiopia61b. Nc is a measure of codon usage bias that is independent of amino acid composition and gene length, and ranges from 20 to 61 when there is extreme bias and no bias, respectively (Wright, 1990). Consistent with previous studies that investigated codon usage bias in flaviviruses (Jenkins et al., 2001; Jenkins & Holmes, 2003), in the plot of Nc against GC3s for YFV genomic sequences (Fig. 4; Table 4), some points nearly overlap the curve, while others lie farther away, suggesting that base composition as well as other factors may contribute to the observed codon usage bias.
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; Ballinger-Crabtree & Miller, 1990; Muylaert et al., 1996). Here, we used YFV genomic sequences to examine potential N-linked glycosylation sites by sequence motif prediction (Asn–X–Ser/Thr). As expected, all virus strains examined possessed two N-linked glycosylation motifs in prM at amino acid positions 13 and 29, plus an additional Asn–X–Ser/Thr sequence at position 51 that may not be glycosylated in vivo, due to the presence of a proline immediately following the motif. Consistent with previous results, a glycosylation motif was also present at position 55 of M; however, this site is also unlikely to be glycosylated due to the hydrophobicity of this region (Ballinger-Crabtree & Miller, 1990).
In the E protein, all YFV strains examined had one site at position 309 and one site at position 469 which, like position 55 of M, may not be glycosylated due to its location in a hydrophobic region of the protein. N-linked glycosylation sites in NS1 were at Asn 130 and Asn 208, consistent with previous findings (Muylaert et al., 1996). All strains also possessed two possible sites in NS4B (positions 63/64 and 89/90) and two possible sites in NS5 (positions 226/227 and 232/233).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and extends upon previous findings that suggested high levels of genetic variation among YFV isolates (Lepiniec et al., 1994; Chang et al., 1995; Wang et al., 1996; Mutebi et al., 2001; Bryant et al., 2003). Nucleotide and amino acid variation between genomes (Table 2) supports previous observations that proposed at least five genotypes of YFV in Africa. Genotype-specific amino acid substitutions (Supplementary Table S3 available in JGV Online) may aid in identification of amino acids important for the virulence of a particular strain, once phenotypic traits of these viruses have been compared.
In particular, a deletion of Thr (or His when compared to CAR77-900) identified in the N terminus of NS4B of strains from west Africa may be of potential significance (Fig. 3). While little is known about the structure and function of YFV NS4B, for other members of the family Flaviviridae there is evidence that the protein may play a role in vector competence (Hanley et al., 2003), may serve as an interferon antagonist (Munoz-Jordan et al., 2003; Jones et al., 2005) and may facilitate virus replication through association with intracellular membranes (Elazar et al., 2004; Gao et al., 2004). Furthermore, the deletion is located in an area of the protein that is highly variable for east and central African strains compared with the published west African YFV strains (Figs 2 and 3) and is predicted to be outside the membrane in hydrophobicity plots (data not shown). Comparison of this region of YFV NS4B with the same region of other flaviviruses (Fig. 3b) revealed that Yokose virus, a close relative of YFV, also possesses an amino acid at the position of the additional amino acid in the east and central African strains, and has an amino acid signature that is more similar to these strains than west African strains of YFV. In addition, regions of the ORF that were highly variable between eastern and central African strains, and strains from western Africa, were also observed in C and NS5 proteins. The significance of this variability is unknown; however, C protein substitutions were located in the C-terminal hydrophobic sequence that is cleaved from the protein in the mature virion (Chambers et al., 1990), and NS5 substitutions were located outside the polymerase motifs (Lai et al., 1999), which were conserved for all strains examined.
Results of codon usage analysis revealed more similar codon usage when genomic sequences were examined than when individual genes were studied (Table 3). The cause of codon usage bias in YFV may be a combination of factors, including mutational bias (due to genomic compositional constraints) and/or translational selection on areas of the YFV genome. GC3s were therefore plotted against Nc values and compared to the expected Nc if base composition is the only factor involved (solid curve) (Fig. 4). Results suggested that while base compositional constraints may account for most of the codon usage bias in some genes (e.g. NS2B of Ethiopia61b that overlaps the curve), it is unlikely to be the single determining factor, as some points are located farther from the expected Nc curve. This is consistent with previous studies suggesting that while base composition may account for the majority of codon usage bias between different flavivirus groups, other factors may also be involved (Jenkins et al., 2001; Jenkins & Holmes, 2003). It is therefore possible that translational selection, involving increased translational efficiency due to preferential usage of a codon for which there is an abundant cognate tRNA, may also contribute to the observed codon usage bias. For example, vector and vertebrate host species vary according to geographical location for YFV. Since these different cell types may exhibit very different codon/translational preferences, this could contribute to the differences in codon usage bias for various genotypes. This possibility may be unlikely, however, since previous studies did not find a correlation between codon usage bias and arthropod association (tick vs mosquito vs no vector) (Jenkins et al., 2001). In order to fully explore the potential importance of translational selection in the codon usage bias of YFV, experiments must be undertaken to determine the cellular levels of tRNA availability in response to viral gene expression.
Since glycosylation of YFV M, E and NS1 proteins may be important for the function of these proteins (Ruiz-Linares et al., 1989; Ballinger-Crabtree & Miller, 1990; Muylaert et al., 1996), we also analysed sequences of the various YFV genomes for potential N-linked glycosylation sites. Positions of N-linked glycosylation motifs in E, prM/M and NS1 were conserved for all virus strains. In addition, conserved N-linked glycosylation motifs were also detected in the NS4B and NS5 proteins. Whether or not these additional sites are glycosylated in vivo remains to be determined. While topology predictions of NS4B indicate three major hydrophobic segments (which are therefore unlikely to be glycosylated), predicted N-linked glycosylation sites are located outside these regions. YFV NS5 serves as the viral RNA polymerase and is therefore unlikely to be glycosylated.
In conclusion, this study supports the existence of previously identified genotypes of YFV, on the basis of full-genome phylogenetic analysis. High degrees of genetic diversity are found within the YFV species, while potential glycosylation sites are generally conserved. Phenotypic evaluation of differences among these viruses remains to be assessed, in particular, the potential significance of highly variable regions in C and NS5, as well as the Thr/His deletion in NS4B of west African viruses. Identification of genetic relationships and potential corresponding phenotypic diversity of YFV is important for monitoring of future outbreaks.
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ACKNOWLEDGEMENTS |
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Received 3 June 2005;
accepted 1 December 2005.
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