Detection of West Nile Virus Lineage 2 in Culex Mosquitoes,Greece,2012

Introduction

A n outbreak of human West Nile virus (WNV) infections is ongoing in Greece for a third consecutive year. From the beginning of 2012 until August 30, 2012, 110 laboratory-diagnosed cases have been reported to KEELPNO (Hellenic Centre for Disease Control & Prevention), six of them with a fatal outcome. Among cases, 76 presented with West Nile neuroinvasive disease (WNND) and 34 with mild symptoms (Hellenic Centre for Disease Control and Prevention 2012). In 2010, apart from the West Nile fever (WNF) cases (with no neurological symptoms), a total of 197 patients with WNND (encephalitis and/or meningitis and/or acute flaccid paralysis) were reported, of whom 33 (17%) died (Danis et al. 2011b). The respective number for 2011 was 76, with 9 (11.8%) fatalities (Danis et al. 2011a, Hellenic Centre for Disease Control and Prevention 2011). A WNV lineage 2 strain (Nea Santa-Greece-2010) was detected in Culex mosquitoes collected at the places where encephalitis cases had been observed (Papa et al. 2011b). Similar sequences were obtained later from an Eurasian magpie (Valiakos et al. 2011), sentinel chickens (Chaskopoulou et al. 2011), and additional pools of mosquitoes collected in 2011 (Danis et al. 2011a), whereas the virus was isolated from an asymptomatic blood donor who presented mild symptoms a few days after the donation (Papa et al. 2012). The genetic characterization of the Nea Santa strain revealed that it was genetically closer to a Hungarian WNV lineage 2 strain; however, it has the H₂₄₉P substitution in the NS3 protein (Papa et al. 2011a). This specific mutation has been previously associated with increased pathogenicity and thermotolerance in WNV lineage 1 strains (Brault et al. 2007). The majority of cases during the outbreak of 2010 were observed in central Macedonia, northern Greece, whereas in 2011 the virus also spread in the central part of the country. The geographic distribution during 2012 is wider, and new areas, where cases have not previously reported, are highly affected, like the Xanthi Prefecture (eastern Macedonia and the Thrace region), where 20 WNND cases have been diagnosed in the laboratory up to now (Hellenic Centre for Disease Control and Prevention 2012, our laboratory data). Nine additional WNND cases have been diagnosed in the neighboring Kavala Prefecture (in the same region), where one WNND case was observed in 2010. The Nestos River is the natural border of the two prefectures, ending up in a 125,000-acre delta, which, together with the adjoining lagoons and lakes, constitutes a major wetland where numerous local and migrating birds live. The first WNF case in the Xanthi Prefecture was diagnosed on August 3, 2012. Soon after, the number of cases (either WNF or WNND) started to increase rapidly.

During August 6–22, 2012, CO₂ traps were set in 12 sites of the Xanthi Prefecture, and especially in the two municipalities where the highest incidence of WNND cases was observed—Topeiros and Avdira (78.4 and 53.11 per 100,000 population, respectively) (Fig. 1). A total number of 595 Culex spp. mosquitoes were collected, 292 from the Topeiros municipality, and 303 from the Avdira municipality, grouped in eight and six pools, respectively. Using an RT-nested PCR with generic primers (Sanchez-Seco et al. 2005), a PCR product of the expected size was obtained from two pools of Culex spp. mosquitoes, both collected in Melissa Village, in the Topeiros municipality (Fig. 1). Sequencing of the PCR products gave the first evidence that the strain belonged to WNV lineage 2, whereas further application of a second PCR (Papa et al. 2012) showed that the sequences were identical with the respective ones of Nea Santa, Greece in 2010, thus containing the H₂₄₉P substitution in the NS3 protein. The minimum infection rate (MIR) for the Topeiros municipality was estimated to be 6.84.

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FIG. 1.

Locations (triangle) in Xanthi prefecture where the collection of the Culex spp. mosquitoes was performed. The location where the two WNV-positive mosquito pools were collected is indicated with a circle. The map of Greece is shown in the inset: the Eastern Macedonia and Thrace region is seen in light grey, with the Xanthi prefecture in dark gray.

As in the previous 2 years, the WNV strain detected in Culex mosquitoes in 2012 in Greece belongs to the lineage 2, and carries the H₂₄₉P substitution, suggesting that this strain is established in Greece and has caused outbreaks every year since 2010. Because it seems that WNV is likely to remain of public health concern in the next years, prevention and control measures have to be taken and enhanced surveillance is needed.