West Nile virus circulation in south-eastern Romania , 2011 to 2013

S Dinu1,2, A I Cotar3,4, I R Pănculescu-Gătej3, E Fălcuţă3, F L Prioteasa3, A Sîrbu5, G Oprişan1, D Bădescu3, P Reiter6, C S Ceianu (ceianu@cantacuzino.ro)3 1. Molecular Epidemiology Laboratory, “Cantacuzino” National Institute of Research-Development for Microbiology and Immunology, Bucharest, Romania 2. Genetics Department, Faculty of Biology, University of Bucharest, Bucharest, Romania 3. Vector-Borne Infections and Medical Entomology Laboratory, “Cantacuzino” National Institute of Research-Development for Microbiology and Immunology, Bucharest, Romania 4. The European Programme for Public Health Microbiology Training (EUPHEM), European Centre for Disease Prevention and Control (ECDC), Stockholm, Sweden 5. National Institute of Public Health–National Centre for Surveillance and Control of Communicable Diseases, Bucharest, Romania 6. Unité Insectes et Maladies Infectieuses, Institut Pasteur, Paris, France


Introduction
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].
Following the unprecedented epidemic of West Nile fever in south-eastern Romania in 1996 [22], caused by a lineage 1 strain [23], scattered human cases were recorded every year until a second significant outbreak occurred in 2010 [14].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 [14].
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 [14], 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 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].

Human cases
Sera collected in 2012 and 2013 from patients with confirmed WNV neuroinvasive infection living in southeastern 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).

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 [26] 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 [27], and pooled by species, sex and physiological age in pools never exceeding 50 individuals.

Molecular analysis
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 [5].All amplicons were sequenced (3130 Genetic Analyzer, Applied Biosystems) and the resulting sequences were aligned with ClustalW, BioEdit version 7.0.5.3 [30].Maximum-likelihood phylogenetic analysis was conducted with Mega 6 software [31], which was also used for choosing the fittest nucleotide substitution model.The reliability of the phylogenetic trees was tested with 1,000 bootstrap replicates.

Human samples
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.

Mosquito samples
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 ( major groups that differed by a small number of synonymous and non-synonymous nucleotide substitutions. 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).
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 [32].

Discussion
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  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 [32].
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 [34] and in North America [35,36].Vertical transmission has also been documented in overwintering Culex spp.females [37] and is thought to represent a mechanism for WNV maintenance [38].The persistence of WNV in a temperate climate may be achieved by overwintering of infected arthropod vectors and by long-term infection in birds [39], 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 [40], 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 [7] and in Culex sp.mosquitoes and collared doves (Streptopelia decaocto) in Italy [42].During the period from 2010 to 2013, lineage 2 WNV isolates similar to those in central Europe caused major outbreaks in Greece [10][11][12][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 [43].
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 [44].
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 [45].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 [46].Lineage 4 (isolate LEIV-Krnd88-190) was first identified in 1988 in Dermacentor marginatus ticks collected in the north-west Caucasus [32].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 [46].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 [48].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 [47].

Conclusion
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 [14] 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.Analysis was conducted on a 365 nt sequence (positions 9,479-9,843, in isolate LEIV-Krnd88-190, GenBank acc.no.AY277251) using maximum-likelihood method, Kimura 2-parameter model, 1,000 bootstrap replicates.A sequence obtained from a Japanese encephalitis virus isolate (GenBank acc.no.L48961) was used as an outgroup.
Lineages were defined as previously proposed [48].

Figure 1
Figure 1Distribution of confirmed human cases of West Nile fever selected for molecular investigation and mosquito collection sites by county,Romania, 2011Romania,  -2013

Figure 4
Figure 4Phylogenetic tree of lineage 4 West Nile viruses based on NS5 partial sequences,Romania, 2012Romania,  -2013 . 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

Table 2
Mosquito species found to be positive for West Nile virus by sequencing and real-time RT-PCR,Romania, 2011Romania,  -2013 NA: not applicable.a West Nile virus lineage 2. b 46 pools of females and one pool of males.c West Nile virus lineage 4

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 (Table2).Two partial E sequences were also obtained from Cx. pipiens s.l.mosquitoes collected in the Danube Delta in 2011 and 2012.tered 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