Close Relationship between West Nile Virus from Turkey and Lineage 1 Strain from Central African Republic

We sequenced West Nile viruses (WNVs) from Turkey and found close relationships to WNV lineage 1 strain ArB310/67 from the Central African Republic, distinct from other WNVs circulating in the Mediterranean Basin, eastern Europe, and the Middle East. These findings suggest independent introductions of WNV strains from Africa to the Middle East.


W est Nile virus (WNV; family Flaviviridae, genus
Flavivirus) (1) is a mosquito-borne avian virus that can cause febrile illness and potentially life-threatening neuroinvasive disease in mammalian hosts, predominantly in humans and horses (2). WNV is widely distributed in Africa; the Middle East; central, southern and eastern Europe; southwestern Russia; southwestern Asia and Australia (3); and North, Central, and South America (4,5).
Initial evidence for WNV circulation in Turkey was provided by serosurveillance reports (6). Since 2009, sporadic human and equine cases and limited disease outbreaks have demonstrated the occurrence of symptomatic infections comprising self-limiting febrile diseases as well as central nervous system infections resulting in occasional deaths (6)(7)(8). Current data reveal widespread WNV circulation in Turkey, including virus detection in competent mosquito species (8)(9)(10). To determine the lineage and relationships of WNVs circulating in Turkey, we analyzed the complete genome sequence of the initially reported WNV isolate from Turkey and several partial sequences from isolates detected from various sources and locations in this country during 2011-2013.
To evaluate whether T2 is the main WNV strain circulating in Turkey or whether divergent strains are co-circulating, we investigated another 37 WNV isolates, detected during 2011-2013 from different geographic regions of Turkey and from human, equine, and mosquito samples. From these samples, only partial envelope (E) gene sequences were available; these sequences consisted of 212-256 nt and corresponded to amino acid positions 174-255 in the T2/NY99 WNV polyprotein and have been deposited in GenBank (accession nos. JN828805, JX310862, JX310863, KC290932-KC290942, KC466019-KC466021, KF437832, and KJ433822-KJ433840). These 37 partial E gene sequences exhibited 98.8%-100% nucleotide identities to each other, regardless of collection date, geographic area, and source of isolation (data not shown). Neighborjoining phylogenetic analysis of a common 183-nt stretch of these isolates revealed that all but 1 clustered together with the T2 and ArB310/67 strains (Figure 2). Maximumlikelihood and UPGMA analyses yielded similar results (data not shown). One isolate (GenBank accession no. JN828805), identified from a horse in February 2011 in Central Anatolia, clustered in a sister subclade with 2 other older WNV strains from Africa (strain IBAN7019, isolated in 1965 in Nigeria, and strain ArD27875, isolated in 1979 in Senegal).
WNV lineage 1 strains have a widespread geographic distribution throughout Africa, Europe, the Middle East, and North America, and have been divided into 3 clades, of which clade 1a contains the largest number of strains; these strains can be further grouped into 6 distinct clusters (12). The phylogenetic analyses in our study confirm that the various clusters are well separated and that the T2 strain and 36 of 37 other WNV strains from Turkey are located in cluster 6, together with strain ArB310/67 from the Central African Republic (Figures 1, 2). However, subclustering of the sequences in cluster 6 ( Figure 2) is statistically poorly supported because of the relatively short sequences analyzed and the few common nucleotide substitutions.
Amino acid substitutions that delineate WNV strains belonging to cluster 1 (NS4B-S11N), cluster 3 (NS2A-A224T), cluster 4 (E-T126I, NS4A-V85A), and the eastern European subtype of cluster 2 (NS1-L206F, NS2B-A103V, NS3-T249P and NS5-T898I) were not found in the T2 WNV strain from Turkey (12). Likewise, substitutions that have become established in the North America WNV populations (E-K291R, NS4A-A85T, and NS5-K318R) Figure 1. Neighbor-joining phylogenetic tree constructed on the basis of the complete genomic nucleotide sequences of selected West Nile virus (WNV) strains, including isolate T2 from Turkey (black dot) and the closely related strain ArB310/67 from the Central African Republic (black diamond). Major WNV lineages, clades, and clusters are indicated, except for the local cluster 3 (12). Bootstrap values of major branches are given for 1,000 replicates. Virus strains are described by GenBank accession number, name, and country of isolation. Japanese encephalitis virus strain GP78 served as outgroup. Scale bar indicates substitutions per site.
were absent from this strain. However, the NS1-A70S substitution noted in cluster 2 viruses was observed as A70V in strain T2. Furthermore, NS3-P249T and NS5-V258A substitutions, reported in several strains of the Mediterranean subtype (12), were detected in the T2 strain. These findings suggest that the T2 strain evolved independently from other Mediterranean strains from a common ancestor of African origin.
The T2 strain retained the original WNV envelope protein glycosylation motif (E154-156, NYS) and lacked substitutions associated with neurovirulence attenuation (NS4b-C102S, NS2A-A30P) and increased virulence for crows and humans (NS3-T249P) (12,13). However, the E-V159A change in the WN02 genotype that was associated with a shorter extrinsic incubation period in Culex spp. mosquitoes (14) was noted as V159F in this isolate. A few other amino acid variations with unknown implications were also observed (data not shown).

Conclusions
The evaluation of the complete nucleotide and amino acid sequences of the T2 WNV isolate from Turkey revealed a close genetic relationship with strain ArB310/67, isolated in 1967 in the Central African Republic. These 2 virus strains form a distinct genetic cluster within WNV lineage 1a strains. This observation is supported by analysis of partial E gene sequences detected in temporally and spatially separated sources in Turkey. The investigated circulating WNV strains from Turkey proved to be genetically distinct from viruses circulating in eastern Europe, the Mediterranean region, and the Middle East. However, several amino acid substitutions detected in viruses from throughout the Mediterranean region were identified in strain T2, including those that might affect virus replication in vector mosquitoes. A recent report on a lineage 2 WNV obtained from a human patient in Iran in 2009 also revealed 99% nucleotide identity to a WNV strain collected in the Central African Republic (15). These findings suggest independent introductions of WNV strains of African origin to the Middle East, potentially by migratory birds, and the accumulation of adaptive changes during circulation.