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Volume 17, Number 5–May 2011
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Genetic Characterization of West Nile Virus Lineage 2, Greece, 2010
Anna Papa, Tamás Bakonyi, Kyriaki Xanthopoulou, Ana Vázquez, Antonio Tenorio, and Norbert Nowotny

Author affiliations: Aristotle University, Thessaloniki, Greece (A. Papa, K. Xanthopoulou); Faculty of Veterinary Science, Budapest, Hungary (T. Bakonyi); University of Veterinary Medicine, Vienna, Austria (T. Bakonyi, N. Nowotny); and Instituto de Salud Carlos III, Majadahonda, Spain (A. Vázquez, A. Tenorio)

Suggested citation for this article

Abstract
We conducted a complete genome analysis of a West Nile virus detected in Culex pipiens mosquitoes during a severe outbreak of human West Nile disease in Greece 2010. The virus showed closest genetic relationship to the lineage 2 strain that emerged in Hungary in 2004; increased virulence may be associated with amino acid substitution H249P.

West Nile virus (WNV) is a flavivirus maintained in an enzootic cycle between bird amplifying hosts and ornithophilic mosquito vectors, mainly Culex species; humans, horses, and other mammals are incidental hosts. Although most human WNV infections remain subclinical, febrile illness develops in ≈20% of infected persons and neuroinvasive disease in <1%. Severe disease is more frequent among the elderly and immunocompromised (1). WNV strains are classified into at least 7 putative genetic lineages (2). Lineage 1 strains are the most widespread, found in Africa, Europe, Asia, Australasia ("Kunjin virus"), and America, while lineage 2 strains are mainly distributed in sub-Saharan Africa and Madagascar. WNV of proposed lineage 3 ("Rabensburg virus") is circulating in certain Culex and Aedes species mosquitoes in southern Moravia, Czech Republic, close to the Austrian border, without recognized pathogenicity for mammals (3). Strain LEIV-Krns88-190, isolated from Dermacentor marginatus ticks from the Caucasus represents proposed lineage 4 of WNV. A new lineage, lineage 5, has been proposed for Indian isolates previously comprising lineage 1c, and a reclassification as lineage 6 has been proposed for the Sarawak Kunjin virus strain, which is markedly different from the other Kunjin viruses. Furthermore, a seventh lineage has been suggested for the African Koutango virus, which is closely related to WNV (2), and an eighth lineage has been proposed on the basis of WNV sequences detected in Cx. pipiens mosquitoes captured in southern Spain in summer 2006 (4). WNV strains differ considerably in virulence and neuroinvasiveness. Since neuroinvasive isolates mainly belonged to lineage 1, lineage 2 strains were previously considered to be less virulent. Recent studies, however, indicate that several highly virulent and neuroinvasive strains of lineage 2 WNV were detected in southern Africa (5). Until 2004, only lineage 1 and 3 WNV strains had been found in Europe. A lineage 2 strain emerged in 2004 in Hungary in birds of prey (6), which established itself in the region and exhibited explosive geographic spread in 2008 throughout Hungary and into eastern Austria. Besides deaths in birds and domesticated mammals, human neurologic WNV cases have been diagnosed in the affected regions during the epidemic seasons since 2004. However, the human cases of WNV neuroinvasive disease have been comparatively rare and rather mild with no deaths. In Greece, where WNV cases had not previously been reported, a 2007 study, conducted in an area near a delta where 4 rivers enter the Aegean Sea, 4 (1%) of 392 persons exhibited neutralizing WNV antibodies, 2 with high titers. These findings suggested that WNV, or an antigenically closely related flavivirus, circulates, at least locally, in rural areas in Greece (7). This area was the focus of a large WNV outbreak in summer–autumn 2010 (8). Up to October 4, 2010, a total of 191 neuroinvasive human cases have been laboratory diagnosed, with 32 deaths, all in elderly patients. Soon after the first human cases were recognized, mosquitoes were trapped at the sites where the cases occurred and tested for WNV. One pool, consisting of 50 Cx. pipiens mosquitoes, trapped during the night of August 10, 2010, in Nea Santa (40.84194°N, 22.91499°E), a village 30 km northeast of Thessaloniki, was found to be positive for WNV RNA by reverse transcription–PCR. Sequencing of a 146-nt fragment of the nonstructural protein 5 (NS5) gene gave the first evidence that the virus belongs to lineage 2, and that it is highly similar to the strain that emerged in Hungary in 2004 and to isolates from fatal human and animal cases in South Africa (9). The purpose of this study was to establish the complete genomic sequence of the WNV identified in a pool of mosquitoes trapped at the site of an ongoing West Nile disease epidemic in humans, and investigate it for potential (neuro)virulence/pathogenicity markers. full-text: West Nile Virus, Greece | CDC EID


Suggested Citation for this Article
Papa A, Bakonyi T, Xanthopoulou K, Vázquez A, Tenorio A, Nowotny N. Genetic characterization of West Nile virus lineage 2, Greece, 2010. Emerg Infect Dis [serial on the Internet]. 2011 May [date cited].
http://www.cdc.gov/EID/content/17/5/920.htm


DOI: 10.3201/eid1705.101759


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Norbert Nowotny, Zoonoses and Emerging Infections Group, Clinical Virology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Veterinärplatz 1, A-1210 Vienna, Austria; email: norbert.nowotny@vetmeduni.ac.at