21 May 2015
West Nile virus circulation in south-eastern Romania, 2011 to 2013
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.
WNV is by far the most widely distributed arbovirus. It belongs to the Japanese encephalitis antigenic complex of the family Flaviviridae, transmitted in an avian cycle by ornithophilic mosquitoes, mainly of the genus Culex. Mammals can also be infected, but are considered dead-end hosts because viraemia is generally too low to infect mosquitoes. Eighty per cent of human infections are asymptomatic, and less than 1% of clinical cases lead to neuroinvasive disease [1,2].
Evidence of WNV circulation in Europe dates back to the early 1960s , lineage 1 isolates being responsible for the major outbreaks. A recent retrospective study indicates the circulation of lineage 2 WNV in birds (Sylvia nisoria) in Cyprus as early as 1968 . Starting in 2004, lineage 2 WNV was identified in Hungary in birds of prey and was subsequently found again in Hungary in 2005 and in Austria in 2008 and 2009 [5-7]. Erroneously considered to be non-pathogenic for humans and with a distribution restricted only to sub-Saharan Africa and Madagascar , lineage 2 isolates caused outbreaks of WNV infection in Russia (2007), Greece (2010–2013), Romania (2010), Italy (2011, 2012), Serbia (2012, 2013) and Croatia (2012, 2013) [9-21].
Following the unprecedented epidemic of West Nile fever in south-eastern Romania in 1996 , caused by a lineage 1 strain , scattered human cases were recorded every year until a second significant outbreak occurred in 2010 . In the latter, the affected area also included counties in the north-east of the country as well as in Transylvania, beyond the Carpathian mountains. The WNV detected in one human serum was lineage 2, closely similar to an isolate from a patient during the outbreak in Volgograd, Russia in 2007 .
According to the data provided by the National Institute of Public Health, laboratory-based surveillance of neuroinvasive West Nile infection was carried out in 2012 as previously described , and detected one probable and 14 confirmed cases among 128 suspected cases. The majority of West Nile fever cases were recorded in south-eastern Romania: seven in Bucharest city, two (one probable and one confirmed case) in the adjacent Ilfov county and in Ialomiţa (one case), Brăila (two cases), and Giurgiu (two cases) counties. A single case was also recorded in Iaşi, north-eastern Romania. Dates of onset were from 31 July to 11 September 2012. One death was recorded. In 2013, 22 confirmed and two probable cases were recorded among 142 suspected cases tested. Dates of onset were from 17 July to 19 September 2013. No deaths were reported (source: National Centre for Surveillance and Control of Communicable Diseases, National Institute of Public Health). The majority of West Nile fever cases in that year also occurred in south-eastern Romania: Ialomiţa (four confirmed cases), Brăila (one probable and four confirmed cases), Tulcea (one probable case and two confirmed cases), Constanţa (two confirmed cases), Bucharest city (one confirmed case) and the adjacent Ilfov county (one confirmed case). Another five cases were recorded in the north-eastern part of the country: Iaşi (two confirmed cases), Galaţi (two confirmed cases) and Bacău (one confirmed case). Bacău county is a new affected area. Three cases were recorded in the central part of the country (Mureş and Sibiu counties). Sera from these three cases were also tested for the presence of tick-borne encephalitis virus-specific antibodies because this flavivirus had previously been found to be circulating in this area.
Here we present the molecular characterisation of WNV circulating between 2011 and 2013 in humans and mosquitoes in south-eastern Romania, an area of endemic WNV circulation as shown by previous human and animal host surveillance studies [24,25].
Sera collected in 2012 and 2013 from patients with confirmed WNV neuroinvasive infection living in south-eastern Romania were included in this study (Figure 1). Only samples collected in the first seven days post onset were selected for molecular investigation. Viral RNA was extracted from sera using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany).
Figure 1. Distribution of confirmed human cases of West Nile fever selected for molecular investigation and mosquito collection sites by county, Romania, 2011–2013
Mosquito collection and processing
Adult mosquitoes were collected from two main areas: Tulcea county and Ilfov county including Bucharest. Four sites were investigated in Tulcea county: Mila 26 (in the core of the Danube Delta), Sălcioara and Grindul Lupilor (on the Razim lagoon shore) and Tulcea city (Figure 1). In Tulcea county, mosquitoes were collected by overnight capture in cylindrical traps baited with birds (chickens) and small rodents (guinea pigs) or collected from vegetation with a backpack aspirator. In the Danube Delta and the lagoon shore, collections were performed for periods of five to eight days in August and September 2011 and from May to October in 2012 and 2013. In Bucharest and the periurban area of the city, mosquito captures were performed between July and September (2011–13) using CDC Gravid Traps  and BG Sentinel Traps (Biogents AG, Germany). Mosquitoes were also collected from resting sites such as hallways of buildings using hand aspirator.
For RNA extraction (QIAamp Viral RNA Mini Kit, Qiagen, Hilden, Germany), the mosquitoes were processed as follows: they were identified using an entomological key , and pooled by species, sex and physiological age in pools never exceeding 50 individuals.
All samples (mosquito pools and human sera) were screened for the presence of WNV genome by one-step real-time RT-PCR using a commercial kit (West Nile Virus Real-TM, Sacace Biotechnologies). Positive samples were further tested by RT-PCR using primers VD8, FU2, cFD3 and WNV9368f, targeting the NS5 genomic region [5,28,29]. When possible, a fragment spanning the envelope glycoprotein region (E) was amplified using primers WNVII 870f and WNVII 1630r . All amplicons were sequenced (3130 Genetic Analyzer, Applied Biosystems) and the resulting sequences were aligned with ClustalW, BioEdit version 126.96.36.199 . Maximum-likelihood phylogenetic analysis was conducted with Mega 6 software , which was also used for choosing the fittest nucleotide substitution model. The reliability of the phylogenetic trees was tested with 1,000 bootstrap replicates.
In 2012, serum samples from eight patients with serologically confirmed WNV infection, all in the acute phase of the disease, were tested by real-time RT-PCR. All patients lived in south-eastern Romania: Bucharest city (four cases) and the counties of Brăila (two cases), Giurgiu (one case) and Ialomiţa (one case). WNV genome was detected by real-time RT-PCR (Ct values: 28.1–34.4) in four of these samples: Bucharest city (two cases), Brăila (one case) and Ialomiţa (one case) (Figure 1).
In 2013, nine serum samples fulfilled the inclusion criteria: Bucharest city (two cases) and Brăila (three cases), Constanţa (one case), Ialomiţa (two cases), Tulcea (one case) counties. WNV genome was found in the samples from one patient living in Bucharest city and two from Ialomiţa county (Ct values: 29.85–33.65) (Figure 1).
In summary, during the two years of investigation, we detected seven positive serum samples in the PCR screening assay. Only one sample from 2012 and two from 2013 yielded an NS5 amplicon suitable for obtaining a DNA sequence. None of the above samples yielded an E amplicon suitable for sequencing.
In 2011 to 2013, we collected and analysed 30,855 mosquitoes. About 75% of these insects were captured in the Danube Delta, and 189 of the 1,185 mosquito pools tested were real-time RT-PCR-positive for WNV genome (Table 1). As expected, the majority of these pools consisted of Culex pipiens s.l., followed by Cx. modestus. Other mosquito species were also found real-time RT-PCR-positive for WNV genome (Table 2). Of interest was the detection of WNV genome in a pool of Cx. pipiens s.l. males collected in 2012 in Bucharest. Unfortunately, no amplicon for sequencing could be obtained from this sample. During the three years of the study, we obtained 30 DNA sequences for WNV NS5 derived from mosquito pools containing Cx. pipiens s.l., Cx. modestus, Anopheles hyrcanus, Coquillettidia richiardii and Uranotaenia unguiculata species (Table 2). Two partial E sequences were also obtained from Cx. pipiens s.l. mosquitoes collected in the Danube Delta in 2011 and 2012.
Table 1. Mosquito pools tested for West Nile virus genome, Romania, 2011–2013
Table 2. Mosquito species found to be positive for West Nile virus by sequencing and real-time RT-PCR, Romania, 2011–2013
In 2012 and 2013, we identified the WNV lineage 2 genome in sera collected from three patients with meningoencephalitis living in south-eastern Romania. The first detection was in August 2012 in the acute phase serum of a resident of Bucharest. In September 2013, similar WNV isolates were found in a patient living in Bucharest and in a resident of Ialomiţa county.
Our phylogenetic analysis based on NS5 partial sequences (Figure 2) indicated that the viruses in circulation in Romania between 2011 and 2013 were very similar to a lineage 2 WNV isolated during the outbreak in Volgograd, Russia, in 2007; all our mosquito and human-derived sequences were more than 99% similar to that isolate. The same strain of lineage 2 WNV, 99% identical to Volgograd 2007, has been circulating in Bucharest since 2010 . Although the investigated genomic region is strongly conserved, a bootstrap value of 79% from the node relating the Romanian sequences to the Volgograd 2007 isolate indicated the robustness of the analysis. It is worth mentioning that the sequences obtained from human specimens clustered with two sequences derived from two pools of Cx. pipiens s.l. females collected in 2012 and 2013 in Bucharest area. All sequences derived from mosquitoes collected in the same ecosystem (Danube Delta, Mila 26 collecting site), regardless of the year of collection, clustered in a single subclade comprised of three major groups that differed by a small number of synonymous and non-synonymous nucleotide substitutions.
Figure 2. Phylogenetic tree of lineage 2 West Nile viruses based on NS5 partial sequences, Romania, 2011–2013
Furthermore, the phylogenetic analysis based on E partial sequences confirmed the topology of the NS5 tree, placing the Romanian sequences in the same clade with the isolate obtained in 2007 in Volgograd, Russia (Figure 3).
Figure 3. Phylogenetic tree of lineage 2 West Nile viruses based on envelope glycoprotein (E) sequences, Romania, 2011–2012
An interesting finding was the detection of lineage 4 WNV in four pools of Ur. unguiculata mosquito collected in 2012 and 2013 (Figure 4). Similar strains have already been reported by Russian authors from this mosquito species, known to feed on amphibians, and from frogs collected in Volga Delta, as well as from Dermacentor marginatus ticks collected in the Caucasus .
Figure 4. Phylogenetic tree of lineage 4 West Nile viruses based on NS5 partial sequences, Romania, 2012–2013
NS5 and E partial sequences described in this study are available in GenBank under the following accession numbers: HE984574, HE984575, HG328830, HG328831, HG514461-HG514468, HG918026-HG918037, LK022077-LK022085, LN808678 and LN808682.
As shown by sequencing, Volgograd 2007-like lineage 2 WNV isolates were found both in patients with neurological WNV infections (2012 and 2013) and in mosquito vectors (2011–13) in south-eastern Romania.
In 2011, Volgograd 2007-like isolates were detected in two pools of Cx. pipiens s.l. mosquitoes collected from the Danube Delta. In 2012, in this area, the same virus was found in nine mosquito pools consisting of specimens belonging to three species: Cx. pipiens s.l. (five pools) and Cx. modestus (three pools) mosquitoes, already known as WNV principal vectors in Europe [23,33], but also in An. hyrcanus (one pool). The results for 2013 in the Danube Delta indicated the presence of the same lineage 2 WNV in 15 mosquito pools belonging to two species: Cx. pipiens s.l. (13 pools) and Cq. richiardii (two pools). Also, in 2012 and 2013, we detected Volgograd 2007-like isolates in two pools of Cx. pipiens s.l. mosquitoes collected in Bucharest city. In Europe, WNV has been previously detected in Cq. richiardii and An. hyrcanus in the Volga Delta .
WNV genome was also detected in one pool of Cx. pipiens s.l. males collected in 2012 in Bucharest city. To our knowledge, this is the first direct field evidence of vertical transmission of WNV in Europe. WNV has previously been detected in Culex spp. males in Kenya  and in North America [35,36]. Vertical transmission has also been documented in overwintering Culex spp. females  and is thought to represent a mechanism for WNV maintenance . The persistence of WNV in a temperate climate may be achieved by overwintering of infected arthropod vectors and by long–term infection in birds , and may explain endemic WNV circulation. Indeed, maintenance of this WNV lineage 2 Volgograd-like strain in the same area for three years has been documented by us.
Initially considered to be non-pathogenic for humans , lineage 2 WNV was detected in Europe in 2004, in a goshawk (Accipiter gentilis) in south-eastern Hungary. It then became established and caused sporadic cases of infection in wild birds, sheep, horses and humans [5,41]. In the following years, similar isolates were detected in birds of prey in eastern Austria  and in Culex sp. mosquitoes and collared doves (Streptopelia decaocto) in Italy . During the period from 2010 to 2013, lineage 2 WNV isolates similar to those in central Europe caused major outbreaks in Greece [10-13]. The Greek isolates from 2010 and 2012 had unique amino acid substitutions (V119I in NS2B; H249P in NS3; S14G, T49A and V113M in NS4) compared with the isolates from Hungary and Austria, which might explain their high pathogenicity .
Although there is a high degree of similarity (96%) between our sequences and those obtained from other isolates circulating in central and southern Europe in recent years, our sequences clearly clustered with the Volgograd 2007 isolate. This distribution of a distinct strain of lineage 2 WNV in eastern Europe may be related to the Mediterranean/Black Sea flyway of northward migrating birds, as it is documented that birds play a crucial role in the spread of the virus . The Volga and Danube deltas are also connected by autumn migration, and there is evidence that WNV may be introduced as birds travel to their owerwintering sites in Africa . Our findings, together with other published data suggest that at least two independent introduction events of two different lineage 2 WNV strains occurred in Europe, followed by their subsequent endemisation.
The presence of WNV lineage 4 identified in two consecutive years (2012–13) in four pools of Ur. unguiculata mosquitoes captured in the Danube Delta is worth mentioning. Studies between 2002 and 2006 in the Volga Delta, an ecosystem similar to that of the Danube Delta, demonstrated the presence of WNV lineage 4 in Ur. unguiculata and in the lake frog Rana ridibunda . Lineage 4 (isolate LEIV-Krnd88–190) was first identified in 1988 in Dermacentor marginatus ticks collected in the north-west Caucasus . Attempts to propagate the virus in suckling mice or in mammalian or mosquito cell lines have failed. The strains of this lineage seem to be associated with arthropods and amphibians and their pathogenicity for vertebrates is not characterised . Recent studies conducted in Spain and Austria [47,48] reported two WNV strains of unknown pathogenicity which can be assigned to new lineages closely related to lineage 4. The Spanish WNV was found in Cx. pipiens mosquitoes, while the Austrian virus was detected in Ur. unguiculata mosquitoes. It has been proposed based on NS5 partial sequences that WNV isolates previously found in Ur. unguiculata mosquitoes and Dermacentor marginatus ticks from Russia should be grouped in a clade designated lineage 4 or 4a, while the sequence from Spain should be classified in lineage 4b or 6, and the WNV sequence identified in mosquitoes from Austria should be comprised in lineage 4c or 9 . As shown in Figure 4, WNV sequences derived from Ur. unguiculata mosquitoes collected in Romania clearly cluster in the proposed lineage 4/4a, along with the Russian isolates. It has been speculated that less or non-pathogenic WNV strains may infect birds, conferring them immunity, thus limiting the spread of pathogenic strains .
The neurovirulent strain Volgograd 2007-like of lineage 2 WNV has been circulating in Romania in mosquito populations and causing disease in humans since at least 2010, as shown by previous  and present findings. The distribution of this strain may be linked to the flyways connecting Africa to eastern Europe and the Danube and Volga deltas, followed by virus maintenance and endemic circulation in the region.
This study was funded by European Union (EU) grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext256 (www.edenext.eu). The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission.
Conflict of interest
SD, IRPG and GO set up and performed molecular diagnostic, sequencing and phylogenetic analysis. IRPG, DB and CSC performed serological diagnosis of human cases. AIC and IRPG performed molecular detection tests in mosquitoes pools. AS managed West Nile fever surveillance program. FLP and EF performed field collection of mosquitoes, their identification and processing for molecular analyses. SD and CSC wrote the paper. PR coordinated the entomological work and provided a critical review of the manuscript.
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