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. 2005 Mar;79(6):3807-21.
doi: 10.1128/JVI.79.6.3807-3821.2005.

Size heterogeneity in the 3' noncoding region of South American isolates of yellow fever virus

Juliet E Bryant  1 , Pedro F C Vasconcelos, Rene C A Rijnbrand, J P Mutebi, Stephen Higgs, Alan D T Barrett
Affiliations

Size heterogeneity in the 3' noncoding region of South American isolates of yellow fever virus

Juliet E Bryant et al. J Virol. 2005 Mar.

Abstract

The 3' noncoding region (3' NCR) of flaviviruses contains secondary and tertiary structures essential for virus replication. Previous studies of yellow fever virus (YFV) and dengue virus have found that modifications to the 3' NCR are sometimes associated with attenuation in vertebrate and/or mosquito hosts. The 3' NCRs of 117 isolates of South American YFV have been examined, and major deletions and/or duplications of conserved RNA structures have been identified in several wild-type isolates. Nineteen isolates (designated YF-XL isolates) from Brazil, Trinidad, and Venezuela, dating from 1973 to 2001, exhibited a 216-nucleotide (nt) duplication, yielding a tandem repeat of conserved hairpin, stem-loop, dumbbell, and pseudoknot structures. YF-XL isolates were found exclusively within one subclade of South American genotype I YFV. One Brazilian isolate exhibited, in addition to the 216-nt duplication, a deletion of a 40-nt repeated hairpin (RYF) motif (YF-XL-DeltaRYF). To investigate the biological significance of these 3' NCR rearrangements, YF-XL-DeltaRYF and YF-XL isolates, as well as other South American YFV isolates, were evaluated for three phenotypes: growth kinetics in cell culture, neuroinvasiveness in suckling mice, and ability to replicate and produce disseminated infections in Aedes aegypti mosquitoes. YF-XL-DeltaRYF and YF-XL isolates showed growth kinetics and neuroinvasive characteristics comparable to those of typical South American YFV isolates, and mosquito infectivity trials demonstrated that both types of 3' NCR variants were capable of replication and dissemination in a laboratory-adapted colony of A. aegypti.

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Figures

FIG. 1.
FIG. 1.
(A) Schematic diagram highlighting conserved features of the prototype YFV 3′ NCR (modified from reference 28). RYF, repeated dual F-G hairpins; CS2, ∼24 nt comprising part of dumbbell B; cyc, conserved cyclization domain; LSH, long stable hairpin. (B) Consensus alignment of YFV 3′ NCR sequences (domains I and II). The numbers of isolates used to build the consensus sequences are provided in parentheses (details in Table 1). Nucleotides identical to the reference Angola71 isolate are indicated by dots. Alignment was optimized by the insertion of gaps (∼). Ambiguity codes are as follows: M = A or C; R = A or G; W = A or T; S = C or G; Y = C or T; K = G or T; V = A, C,or G; H = A, C, or T; D = A, G, or T; B = C, G, or T. Regions involved in basepairing are boxed and labeled to correspond with stem structures as indicated in the schematic in panel A.
FIG. 1.
FIG. 1.
(A) Schematic diagram highlighting conserved features of the prototype YFV 3′ NCR (modified from reference 28). RYF, repeated dual F-G hairpins; CS2, ∼24 nt comprising part of dumbbell B; cyc, conserved cyclization domain; LSH, long stable hairpin. (B) Consensus alignment of YFV 3′ NCR sequences (domains I and II). The numbers of isolates used to build the consensus sequences are provided in parentheses (details in Table 1). Nucleotides identical to the reference Angola71 isolate are indicated by dots. Alignment was optimized by the insertion of gaps (∼). Ambiguity codes are as follows: M = A or C; R = A or G; W = A or T; S = C or G; Y = C or T; K = G or T; V = A, C,or G; H = A, C, or T; D = A, G, or T; B = C, G, or T. Regions involved in basepairing are boxed and labeled to correspond with stem structures as indicated in the schematic in panel A.
FIG. 1.
FIG. 1.
(A) Schematic diagram highlighting conserved features of the prototype YFV 3′ NCR (modified from reference 28). RYF, repeated dual F-G hairpins; CS2, ∼24 nt comprising part of dumbbell B; cyc, conserved cyclization domain; LSH, long stable hairpin. (B) Consensus alignment of YFV 3′ NCR sequences (domains I and II). The numbers of isolates used to build the consensus sequences are provided in parentheses (details in Table 1). Nucleotides identical to the reference Angola71 isolate are indicated by dots. Alignment was optimized by the insertion of gaps (∼). Ambiguity codes are as follows: M = A or C; R = A or G; W = A or T; S = C or G; Y = C or T; K = G or T; V = A, C,or G; H = A, C, or T; D = A, G, or T; B = C, G, or T. Regions involved in basepairing are boxed and labeled to correspond with stem structures as indicated in the schematic in panel A.
FIG. 2.
FIG. 2.
(A) Nucleotide substitutions within the 3′ NCR of South American YFVs compared to the prototype Asibi isolate. Substitutions shared among all South American isolates are indicated in red; sites that are partially conserved within genotypes I and II are indicated in black. (B) Schematic diagram showing the positions of informative sites within multiple sequence alignment of partial 3′ NCR sequences. Note that the alignment ends after the b4 loop.
FIG. 3.
FIG. 3.
RT-PCR amplification of YF-XL isolates using the emf-vd8 primer pair results in double bands (A), whereas amplification using the sNS5i-aNS5i primer pair results in a larger fragment size (B).
FIG. 4.
FIG. 4.
Alignment of the duplicated region of 19 YF-XL isolates, genomic positions 10533 to 10749 (3′ NCR 182 to 398; Asibi reference numbering). Sequences shown in red are the downstream duplicates of the sequences shown in black. Multiple isolates with 100% identity are represented by a single sequence. Regions involved in basepairing are boxed and labeled to correspond to stem structures as indicated in Fig. 1A and 2A. Hatched boxes indicate pseudoknot binding sites; red boxes indicate conserved substitutions among duplicated structures.
FIG. 5.
FIG. 5.
Comparison of growth characteristics of YF-XL, YF-XL-ΔRYF, and YF-std isolates in Vero and C6/36 cells. Cultures were infected with equal volumes of virus stocks at an approximate MOI of 0.03; 0.5-ml aliquots of supernatant were sampled with replacement for 12 consecutive days. The values represent the mean infectivity titers by endpoint assay; error bars indicate the standard deviations of the results for triplicate samples. (A) YF-std in C6/36 cells; (B) YF-XL in C6/36 cells; (C) YF-std in Vero cells; (D) YF-XL in Vero cells; (E) YF-XL-ΔRYF compared to mean of four YF-std isolates, each evaluated in duplicate.
FIG. 6.
FIG. 6.
Neighbor-joining phylogenetic tree based on partial NS5-3′ NCR sequences (576 nt) of 117 South American isolates. To obtain sequences of identical lengths, the alignment was trimmed at the 3′ side of pk1′; thus, the alignment did not incorporate the duplicate copy of domain II structures for the YF-XL isolates. Sequences with 100% identity are represented by single branches. Isolates with no color highlighting have undetermined 3′ NCR status, because sequence for the genome terminus was not obtained.
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