Endemska populaciona dinamika virusa Zapadnog Nila u komarcima sa teritorije Srbije

Abstract

Prvi slučajevi infekcije virusom Zapadnog Nila (VZN) u Srbiji zabeleženi su 2012. godine, dok je 2018., prema izveštaju Evropskog centra za kontrolu i prevenciju bolesti (ECDC), Srbija prijavila najveći broj obolelih (385) od svih zemalja jugoistočne Evrope. Cilj ove studije je bio da se genetički okarakterišu sojevi VZN-a, koji cirkulišu u Srbiji. U studiji je ispitano 200 pulova komaraca, skupljenih u Beogradu i široj okolini, u okviru redovnog nadzora koji sprovodi Zavod za biocide i medicinsku ekologiju u Beogradu u periodu od 2018-2020. Prisustvo VZN-a utvrđeno je reakcijom lančanog umnožavanja (engl. polymerase chain reaction – PCR), za detekciju NS5 gena, sa primenom Sangerovog DNK sekvenciranja dobijenih PCR produkata i njihovom preliminarnom identifikacijom primenom BLAST alata u NCBI bazi podataka. Konstrukcija filogenetskog stabla rađena je primenom metode Bajesove statistike implementirane u MrBayes softverski paket, uz odgovarajući evolutivni model koji je izabran u jModelTest softveru. Iz 200 analiziranih pulova komaraca, koji su skupljeni u periodu od 2018-2020, dobili smo 45 sekvenci NS5 gena VZN-a. Rezultati studije su pokazali da je 22.5% (45/200) pulova komarca bilo pozitivno na prisustvo VZN-a. Najveći procenat pozitivnih pulova je iz 2018. godine i iznosi 57.8% (26/45), zatim iz 2019 26.7% (12/45) i iz 2020 15.5% (7/45). Identifikacija dobijenih sekvenci pokazala je da je u Srbiji trenutno u cirkulaciji samo linija 2 VZN-a. Dalja analiza je potvrdila da su sojevi VZN-a iz Srbije najsličniji izolatima iz Grčke. Dodatno, u jednom pulu komaraca, uzorkovanom u okolini Šapca 2019. godine, identifikovano je prisustvo Usutu virusa. Dobijeni rezultati su potvrdili da u Srbiji cirkuliše linija 2 VZN-a kao i da postoji potreba za kontinuiranim molekularno-bioinformatički nadzorom nad ovim i sličnim zoonotskim virusima.

The first cases of West Nile virus (WNV) infection in Serbia recorded in 2012, while in 2018, according to the report of the European Centre for Disease Control and Prevention (ECDC), Serbia reported the highest number of cases (385) in the Southeast Europe. The aim of this study was to genetically characterize strains of WNV circulating in Serbia. The present study included 200 pools of mosquitoes, collected in Belgrade and the surrounding area by the Institute for Biocides and Medical Ecology in Belgrade in the period from 2018-2020, as part of regular surveillance. The presence of WNV was detected by Nested-PCR (Polymerase chain reaction) method together with specific primers for detection of partial NS5 gene. All obtained specific PCR products were directly sequenced in both directions. Preliminary sequence identification was done by BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Bayesian method was employed to infer the evolutionary relationship of studied sequence dataset, using MrBayes software with the best-fitting nucleotide substitution model for the final sequence dataset, as selected by jModelTest 2. In total, 45 partial NS5 gene WNV sequences were obtained from mosquito pools collected in Serbia between 2018 and 2020. The results obtained in this study showed that 22.5% (45/200) of mosquito pools were positive for the presence of WNV. The highest percentage of positive pools was detected in 2018 (57.8% (26/45)), followed by 2019 (26.7% (12/45)) and 2020 (15.5% (7/45)). Phylogenetic analysis conformed that only linage 2 of WNV is circulating in Serbia. Majority of Serbian isolates clustered with Greek strains, thus forming the Balkan cluster. Of note, in one mosquito pool sampled in the Western Serbia in 2019, we detected the presence of Usutu virus. Results obtained in the present study confirmed the presence of WNV linage 2 in Serbia and the necessity for continuous molecular-bioinformatic monitoring of zoonotic viruses.