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Currently, five invasive Aedes mosquito species are of concern in Europe according to the European Centre for Disease Prevention and Control: Aedes aegypti, Ae. albopictus, Ae. atropalpus, Ae. japonicus, and Ae. koreicus. Among these, only Ae. albopictus was reported to occur in Romania, in Bucharest. The aim of this study was to update the knowledge on the distribution of this invasive mosquito species in Romania, by investigating new potential locations. Monitoring of Ae. albopictus was carried out between May 2017 and October 2018. Three types of traps (CDC-Gravid Traps, CDC miniature Light Traps, ovitraps) were placed in 53 localities in 13 counties at sites suitable for container-breeding mosquitoes. Collected adult mosquitoes were counted and identified according to morphological criteria. Larvae were found present in domestic containers and rain catch basins. Aedes albopictus adults and eggs were collected in 10 localities in eight counties across Romania. Our study confirms nine new localities and seven counties where Ae. albopictus became established in Romania, highlighting the need for surveillance to further assess the species' distribution and abundance, as well as the pathogen transmission risk related to that vector species.

During an entomological investigation carried out in Bucharest and surroundings in fall of 2012, 45 adult mosquitoes (38 females and 7 males) of Aedes albopictus were collected in a neighborhood from the southern area of the city. The morphological identification of the species was further confirmed by sequencing 2 mitochondrial DNA markers: the cytochrome c oxidase subunit I and NADH dehydrogenase subunit 5 genes. Aedes albopictus was collected again in 2013 in the same area from July until October. During late summer the species was found also in another location in the city, downtown Bucharest. Larvae were found in water barrels and other types of household containers, as well as in rain catch basins. In 2014, following a nuisance complaint of a Bucharest inhabitant, the entomological investigation found aggressive Ae. albopictus adults on his property that harbored many mosquito larvae in container-type breeding habitats. These findings are the 1st records of this invasive species and of its breeding population in Romania, and show maintenance of the species over 2 winter seasons. Surveillance of the species outside the area of the capital city was not performed, therefore it is not known whether Ae. albopictus has been introduced in other regions of the country. The presence of Ae. albopictus has been reported every year (2012-14) to competent public health authorities, stressing on the importance of surveillance and of implementation of control measures.

Lineage 2 West Nile virus (WNV), previously found only in sub-Saharan Africa and Madagascar, was identified in Hungary in 2004 and has rapidly expanded in Europe in the past decade. Following a significant outbreak of West Nile fever with neurological cases caused by lineage 1 WNV in Romania in 1996, scattered cases have been recorded in the south-east of the country in each transmission season. Another outbreak, affecting a larger area and caused by lineage 2 WNV, was recorded in 2010. We analysed human sera from neuroinvasive West Nile fever cases and mosquitoes, sampled in south-eastern Romania between 2011 and 2013, for the presence of WNV genome, and obtained partial NS5 and envelope glycoprotein sequences. Human- and mosquito-derived WNV sequences were highly similar (99%) to Volgograd 2007 lineage 2 WNV and differed from isolates previously detected in central and southern Europe. WNV was detected in one pool of Culex pipiens s.l. males, documenting vertical transmission. Lineage 4 WNV, of unknown pathogenicity to mammals, was found in the amphibian-feeding mosquito Uranotaenia unguiculata from the Danube Delta. Our results present molecular evidence for the maintenance of the same isolates of Volgograd 2007-like lineage 2 WNV in south-eastern Romania between 2011 and 2013.