South African WNV isolates SPU116/89, SA93/01, SA381/00, and H442 were obtained from the Special Pathogens Unit, National Institute for Communicable Diseases, South Africa, as freeze-dried mouse brain passages 2–4. They were replicated by 1 passage in Vero cells for this study.

Viral RNA was extracted from cell culture supernatant with the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. For cDNA synthesis, 10 μL of RNA and 0.4 μg of random hexanucleotides (Roche Diagnostics, Mannheim, Germany) were incubated at 65°C for 10 min before cooling on ice. Then 1× Expand Reverse Transcriptase buffer, 100 mmol/L dithiothreitol, 200 μmol of each deoxynucleotide triphosphate, 20 U RNase Inhibitor and 50 U Expand Reverse Transcriptase (Roche Diagnostics) were added and incubated at 30°C for 10 min, followed by 1 h at 43°C. For PCR amplification, 10 μL of the cDNA reaction was added to the PCR master mix consisting of 3.75 U of Expand High Fidelity Polymerase and 30 pmol of each specific primer (primer sequences available on request) and cycled as follows: 94°C for 2 min (94°C for 15 s, followed by primer-specific annealing temperature for 30 s, 72°C for 2 min) × 35 and 72°C for 7 min. Expand Long Template PCR Polymerase (Roche Diagnostics) was used for products >2 kb with 300 μmol of each dNTP, 1× buffer, and 30 pmol of each specific primer and cycled at 94°C for 2 min (94°C for 10 s, 50°C for 30 s, 68°C for 3 min) × 10; followed by 30 cycles of 94°C for 15 s, 50°C for 30 s, 68°C for 5 min plus 5 s per cycle, and 72°C for 7 min.

PCR products were purified with Wizard SV gel and PCR clean-up system (Promega, Southampton, UK). DNA cycle sequencing was performed with the BigDye Terminator V3.1 kit and analyzed on an ABI PRISM 3100/3130 genetic analyzer (both from Applied Biosystems, Foster City, CA, USA).

Genome editing and assembling were performed by using Vector NTI 9.1.0 (Invitrogen, Carlsbad, CA, USA); multiple sequence alignments, with ClustalW (11); and amino acid analysis, with GeneDoc for Windows (12). Amino acid changes considered to have a potential effect on the secondary structure of the proteins included substitution of hydrophilic for hydrophobic amino acids or vice versa and substitutions of cysteine, glycine, and proline residues (12).

Comparisons are relative to the top sequence (SA381/00); numbering refers to the sequence position of isolate SA381/00. Neighbor-joining trees were drawn with MEGA version 3.1 (13) by using the Kimura-2 distance-parameter and a bootstrap confidence level of a 1,000 replicates. Nucleotide and amino acid p-distances (the number of pairwise nucleotide or amino acid differences divided by the total number of nucleotides or amino acids in the sequenced region) were calculated by using MEGA version 3.1. Signalase cleavage predicted scores were calculated with AnalyzeSignalase 2.03 (14).

Four lineage 2 WNV strains isolated from patients in South Africa who had mild or severe WNV infections were selected for genome sequencing. Phenotypic pathogenicity data for these strains (H442, SPU116/89, SA93/01, SA381/00) in humans and mice are summarized in Table 1. Detailed clinical data for all 4 strains have been described by Burt et al. (3), and mouse neuroinvasive experiments and gene expression data for H442, SPU 116/89, and SA381/00 have been described by Venter et al. (4). Strain SA93/01 has been shown to be highly neuroinvasive in a mouse model (M. Venter, unpub. data), similar to SPU116/89 and H442 strains, whereas SA381/00 has been classified as being of low neuroinvasive phenotype in mice. H442 and SA381/00 caused fever, rash, myalgia, and arthralgia in human patients; SA93/01 caused nonfatal encephalitis in 2, and SA116/89 caused fatal hepatitis (3).

The 4 South African strains were compared with strains that were known to be highly or less neuroinvasive in mice or that had been reported to be highly pathogenic or attenuated. Lineage 2 strains for which both full genome sequences and neuronvirulence data in mice were available included isolate B956D117B3 (21) and Madagascar strain AnMg798. B956D117B3 is a passaged clone of reduced virulence (7) of the prototype strain (B956), which was originally associated with fever in a patient and was neurotropic in mice (22); AnMg798 is not neuroinvasive (2). Lineage 1 strains included the highly pathogenic and neuroinvasive NY385–99 strain (2), the attenuated non-neuroinvasive strain TM171–03 isolated in Mexico in 2003 (19), hamster-passaged attenuated clones of NY-385–99 (clone TYP-9376 and clone 9317B) (18), and a non-neuroinvasive Kunjin virus strain MRM61C (Table 1).

Phylogenetic analysis confirmed that the South African strains described here belong within lineage 2 (Figure 1). SA93/01 and SPU116/89 clustered together; H442 and SA381/00 were on separate branches within lineage 2 with respect to the full genome sequences or with respect to individual E, NS3, and NS5 genes (data not shown). Although the Indian strain clustered with lineage 1, p-distance analysis suggested that it was as distant to the lineage 1 strains (20% differences) as to the lineage 2 strains (21%–22%) relative to <5% differences within lineage 1C and 12% differences between 1A and 1B. It was therefore termed lineage 5, as suggested by Bondre et al. (23).

The complete genome sequences of strains H442, SPU116/89, SA381/00, and SA93/01 were deposited in GenBank (accession nos. EF429197–200). The termini were amplified with primers designed from other lineage 2 full genome sequences. If one assumes that the 5′ and 3′ termini are identical in length to other published strains, these genomes were 11052 nt (SPU116/89, SA381/00, and SA93/01) and 11051 nt (H442) long. South African strains had overall nucleotide p-distances of 0.0278 (97.2% similarity) to each other (Table 2), with <1% amino acid differences over the complete genome despite having been isolated as many as 50 years apart. The highest percentage of amino acid differences in the individual proteins of the lineage 2 strains were in the NS proteins, especially the NS5 protein. Table 3 shows the differences between individual proteins of the South African lineage 2 strains.

The 2 strains from North America, New York (NY-385–99) and Mexico (TM171–03), were similarly conserved. In contrast, the Madagascar strain, AnMg798, differed by >3% at the amino acid level from all lineage 2 strains from South Africa or Uganda B956D117B3, and the lineage 1 and 2 strains differed by >6% amino acids from each other.

Few amino acid differences were observed between the structural proteins of the South African strains (Figure 2). SA381/00 had only 1 difference in the premembrane (prM) protein at position 105 relative to the highly neuroinvasive strains (Ala105Val) (Figure 2). Two differences, (Ala54Gly and Thr70Pro) could result in structural changes in the E protein of H442, which was isolated 50 years earlier than strains SPU116/89, SA93/01, and SA381/00. The attenuated lineage 2 strain B956D117B3 and the non-neuroinvasive Madagascar strain AnMg798 contained differences in the glycosylation site of the E protein relative to the South African strains (residues 154–157 deleted in B956D117B3, and Ser156Pro in AnMg798). Either of these changes would prevent glycosylation. Further substitutions of hydrophilic amino acids for proline and glycine residues with potentially structural implications were found in AnMg798 at positions 156, 199, and 230.

The NS3, NS4A/B, and NS5 proteins were the most variable viral proteins. In strain SA381/00, the least virulent of the 4 strains, a hydrophobic amino acid in contrast to a hydrophilic amino acid (Ser160Ala) and Arg298Gly could alter the structure of the SA381/00 NS3 protein. In the highly pathogenic strain SPU116/89, a hydrophobic-to-hydrophilic mutation (Ala79Thr) in NS4B is found relative to the other strains. Other amino acid changes with potential structural implications were for strain B956D117B3 at positions 18 and 145 of the NS4A gene and 14 of the NS4B gene and for strain AnMg798 at positions 14 and 27 in the NS4B gene of (Figure 2).

The NS5 protein was the most variable. Several positions were identified where the South African strains associated with mild infections (SA381/00 and H442) and the 2 other lineage 2 strains (AnMg798,B956D117B3) associated with reduced virulence in mice had the same amino acid changes relative to strains that caused severe disease (SPU116/89 and SPU93/01). These included hydrophilic versus hydrophobic amino acids in position 614 and hydrophobic (mild) versus hydrophilic (pathogenic) in positions 625 and 626 of the NS5 protein. SPU116/89, isolated from a patient with necrotic hepatitis, was found to have amino acid changes that affect the hydrophobicity of the NS5 protein relative to all other strains in positions 197, 623, 635, 641, and 643.

Approximately 98.6% identity existed between the 5′ noncoding regions of SA381/00 and the 3 remaining South African strains; the other strains were 100% conserved. For the 3′ noncoding regions, the overall identity was 98.5% (99% between SPU381/00 and H442 and 98% between SPU116/89 and SA381/00). Noteworthy nucleotide differences in the 3′ noncoding regions were a 2-nt deletion at nt 10439 and nt 10440 in strain H442 and a 76-bp deletion in the 3′ noncoding region from nt 10404 through nt 10479 in the attenuated strain B956D117B3, which was not present in the prototype strain (B956) or in any of the South African strains. Strain AnMg798 had deletions overlapping those of strain B956D117B3 at position 10411 to 10487 and from 10501 to 10512 and 10951 (Figure 2, panel B). The sequence of the AnMg798 strain is incomplete in GenBank and ended at position 10866 (16).

The E protein glycosylation motif previously identified in lineage 1 at positions 154–156 (NYS) (6) was present in all 4 South African strains. However, as a result of a proline substitution at position 156, the site was not predicted to be glycosylated in strain AnMg798. The glycosylation motif is deleted completely in strains B956 and B956D117B3 (Table 1).

Signalase prediction algorithms were used to analyze the signal peptidase cleavage sites (14); no differences were found in cleavage efficiency between the highly and less pathogenic strains (Table 4). The only meaningful difference was observed in the capsid (C)–PrM cleavage region, as indicated by the Student t test probability calculated in Table 4, where the lineage 2 strains were predicted to be cleaved more efficiently than lineage 1 stains. Only slight differences were apparent in the PrM-E site; no differences were apparent in any other cleavage regions between lineage 1 and 2 strains.