Detection and Early Warning of West Nile Virus Circulation in Central Macedonia, Greece, Using Sentinel Chickens and Mosquitoes

Abstract

Following the West Nile Virus (WNV) epidemic in 2010 in Central Macedonia, Greece, which resulted in 197 human neuroinvasive disease cases, we determined the seasonal appearance and prevalence of the virus in 2011 by testing weekly for WNV genomic RNA in mosquitoes collected in carbon dioxide-baited traps, and for anti-WNV antibodies in sentinel chickens. Preliminary findings of the surveillance program regarding the circulation of "Nea Santa-Greece-2010" in sentinel chickens were rapidly communicated to public health authorities. In the present article, the full 2011 data produced by this surveillance program are presented. We detected enzootic circulation of WNV in chickens 1 month prior to the onset of the first human cases in 2011. Culex pipiens and Cx. modestus were abundant throughout the sampling period and at all sites of increased transmission. Molecular identification and phylogenetic analysis of WNV isolates from two chickens and one Cx. pipiens mosquito pool suggested that: (1) the virulent "Nea Santa-Greece-2010" WNV lineage 2 strain responsible for the 2010 epidemic was actively circulating in 2011, and (2) all Greek isolates belong to a distinct recent evolutionary clade. In Europe, where numerous strains of different virulence coexist, sequencing information for WNV is important for phylogeography and identification of virulent strains for human health risk assessment.

Evaluation of a West Nile virus surveillance and early warning system in Greece, based on domestic pigeons

“…This enabled the timely dissemination of information to public health authorities, in order to increase preparedness and implementation of adapted vector control measures and minimize the impact of the epidemic for humans and horses. The sensitivity of this WNV surveillance system based on pigeons is comparable to other surveillance systems applied for WNV surveillance in other countries [30][31][32][33][34][35], as well as in Greece [14,17,18]. In our case, and in order to increase the possibilities to detect seroconverted pigeons early in the epidemic seasons, we monitored mosquito populations and environmental temperatures, before the initiation of the pigeon samplings.…”

“…Since it would be difficult to get permission from pigeon owners for repetitive blood sampling, surveillance systems like the one we describe are not ideal for fast virus isolation and molecular characterization. Consequently, weekly sampling from captive chickens and retrospective RT-PCR analysis in samples collected from seroconverted birds proved to be a more efficient surveillance approach [14,17,18]. However, it should be emphasized that, the success of early warning surveillance systems with captive birds depends heavily on the identification of WNV initial enzootic transmission foci.…”

“…For the molecular detection of WNV, RNA extracts were examined using a one tube real-time RT-PCR protocol, as described by Chaskopoulou et al [14].…”

“…Two other epidemics occurred the following years, with 76 human WNND cases, as well as 8 fatalities in 2011 and 109 reported WNND cases in humans and 16 fatalities in 2012 [10,11]. A lineage 2 WNV strain named "Nea Santa-Greece-2010" was detected in pools of Culex pipiens mosquitoes [12][13][14], a donated human blood sample [15], a Belgian traveler returning from Greece [16], captive sentinel chickens [14,17,18] and an Eurasian magpie (Pica pica) [19], between 2010 and 2012.…”

Evaluation of Cross-Protection of a Lineage 1 West Nile Virus Inactivated Vaccine against Natural Infections from a Virulent Lineage 2 Strain in Horses, under Field Conditions

“…Extracts were examined using a WNV-specific, one-tube real-time reverse transcription (RT)-PCR, using the primer pair WNPolUp (5=-TTTTGGGAGATGGTG GATGARGA-3=) and WNPolDo2 (5=-CCACATGAACCAWATGGCTC TGC-3=) at a final concentration of 0.6 M each, and the TaqMan probe WNPolProb2 (5=-FAM-TCTCTCTCTTTCCCATCATGTTGT-ZNA5-BHQ1-3= at a final concentration of 0.2 M), targeting a 144-bp part of the nonstructural protein 5 (NS5) genomic region of WNV. The limit of detection was previously determined to be 1 TCID 50 /ml (40). Amplification reactions were run in a total volume of 25 l using 5 l of RNA extract and 20 l of reaction buffer of a commercial RT-PCR kit (OneStep RT-PCR kit; Qiagen, Hilden, Germany).…”