Genome characterisation of norovirus GII.P17-GII.17 detected during a large gastroenteritis outbreak in Romania in 2021.

Norovirus (NoV) is one of the leading causes of acute gastroenteritis worldwide. Genotype GII.P17-G.II.17 emerged in Asia between 2013 and 2015 and transiently replaced the GII.4 Sydney 2012 variant circulating at that time. We present the genome characterisation of a GII.P17-GII.17 strain causing a large outbreak in Romania in 2021. Our study shows that the 2021 strain belongs to a novel cluster of genotype GII.17, different from the two previously recognised P.17 clusters. Distinctive substitutions in predicted conformational epitopes of VP1 were identified for this new cluster. Also, our phylogenetic analysis showed the existence of another P.17 cluster grouping strains from France and Canada.

Emergence of Toscana Virus, Romania, 2017-2018.

We describe a series of severe neuroinvasive infections caused by Toscana virus, identified by real-time reverse transcription PCR testing, in 8 hospitalized patients in Bucharest, Romania, during the summer seasons of 2017 and 2018. Of 8 patients, 5 died. Sequencing showed that the circulating virus belonged to lineage A.

Molecular characterisation of human Shiga toxin-producing Escherichia coli O26 strains: results of an outbreak investigation, Romania, February to August 2016.

IntroductionAt the beginning of 2016, an increase in paediatric haemolytic uremic syndrome (HUS) cases was observed in Romania. The microbiological investigations allowed isolation of Shiga toxin-producing Escherichia coli (STEC) O26 as the causative agent from most cases. Methods: An enhanced national surveillance of HUS and severe diarrhoea was established across the country following the identification of the first cases and was carried out until August 2016. A total of 15 strains were isolated from 10 HUS and five diarrhoea cases. Strains were characterised by virulence markers (i.e. stx type/subtype, eae, ehxA genes), phylogroup, genetic relatedness and clonality using PCR-based assays, PFGE and multilocus sequence typing (MLST). The first six strains were further characterised by whole genome sequencing (WGS). Results: Five PCR-defined genotypes were distinguished. All strains from HUS cases harboured stx2a and eae, with or without stx1a, while strains from diarrhoea cases carried exclusively stx1a and eae genes. PFGE resolved strains into multiple pulsotypes, compatible with a certain geographic segregation of the cases, and strains were assigned to phylogroup B1 and sequence type (ST) 21. WGS confirmed the results of conventional molecular methods, brought evidence of O26:H11 serotype, and complemented the virulence profiles. Discussion/conclusion: This first description of STEC O26 strains from cases in Romania showed that the isolates belonged to a diverse population. The virulence content of most strains highlighted a high risk for severe outcome in infected patients. Improving the national surveillance strategy for STEC infections in Romania needs to be further considered.

Molecular identification of emergent GII.P17-GII.17 norovirus genotype, Romania, 2015.

The novel GII.P17-GII.17 norovirus genotype has been reported as cause of gastroenteritis outbreaks in China and Japan since the winter season 2014/15, replacing the pandemic strain GII.4 Sydney 2012. These emergent strains have also been sporadically reported on other continents than Asia. GII.P17-GII.17 isolates, similar to Kawasaki308 2015, were identified in three patients during a large outbreak of acute gastroenteritis affecting 328 people in Romania, in neighbouring localities, in 2015.

Retrospective study reveals the circulation of norovirus genotype GII.P21-GII.2 in Romania

Noroviruses are the leading cause of acute gastroenteritis, causing significant economic burden globally. Infection is self-limiting, occurring as sporadic cases or producing outbreaks associated with consumption of contaminated water or food. All age groups are affected and person to person transmission is frequent. Except a recent outbreak in Romania caused by the emergent genotype GII.P17-GII.17, few data regarding the circulation of noroviruses in our country are available. We retrospectively analyzed stool samples from acute gastroenteritis patients hospitalized in Romania between 2005 and 2008. Noroviruses were detected by RT-PCR and phylogenetic analysis was inferred from partial sequences spanning ORF1 and ORF2. Recombinant GII.P21-GII.2 isolates were found in two adult patients from a cluster of acute gastroenteritis in 2006. Molecular analysis based on partial genomic sequences indicated high degree of similarity between the two isolates and grouped them with cosmopolitan strains circulating in the same period of time. Along with the high rate of mutation, recombination is an important driving force in norovirus evolution. GII.P21 isolates, formerly known as GII.b recombinants, have been detected in Europe since 2000 and associated with sporadic cases and outbreaks of gastroenteritis worldwide. This is the first work describing norovirus GII.P21-GII.2 identified in Romania.

Established Population of the Invasive Mosquito Species Aedes albopictus in Romania, 2012-14.

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.

SCREENING OF PROTEASE INHIBITORS RESISTANCE MUTATIONS IN HEPATITIS C VIRUS ISOLATES INFECTING ROMANIAN PATIENTS UNEXPOSED TO TRIPLE THERAPY.

Although the European recommendations include the use of new antiviral drugs for the treatment of hepatitis C, in Romania the current treatment remains interferon plus ribavirin. First generation viral protease inhibitors (i.e. boceprevir, telaprevir), which have raised the chances of obtaining viral clearance in up to 70% of infection cases produced by genotype 1 isolates, have not been introduced yet as standard treatment in our country. The success of these new antivirals is limited by the occurrence and selection of resistance mutations during therapy. We set-up a molecular study aiming to detect any resistance mutations to boceprevir and telaprevir harbored by hepatitis C isolates infecting Romanian patients naïve to viral protease inhibitors. Since these new antivirals are efficient and approved for genotype 1 infection, viral samples were genotyped following a protocol previously developed by our research group. We analyzed by both population sequencing and molecular cloning and sequencing the NS3 protease region of hepatitis C virus isolates infecting patients which were not previously exposed to boceprevir and telaprevir. All the analyzed samples were subtype 1b and resembled the samples collected in recent years from Romanian patients. Molecular cloning followed by sequencing showed great intra-host diversity, which is known to represent the source of isolates with different resistance phenotypes. Both population sequencing and molecular cloning followed by clone sequencing revealed two boceprevir resistance mutations (T54S and V55A), respectively, a telaprevir resistance mutation (T54S) in the sequences obtained from a patient with chronic hepatitis C. To our knowledge, this is the first study indicating the existence of pre-treatment resistance mutations to boceprevir and telaprevir in hepatitis C virus isolates infecting Romanian patients.

GENOMIC ANALYSIS OF HEPATITIS B VIRUS STRAINS INFECTING ROMANIAN PATIENTS.

Chronic hepatitis B is widespread and represents an important cause of morbidity and mortality due to the evolution to cirrhosis and hepatocellular carcinoma. This study was designed to improve the national laboratory surveillance of hepatitis B virus (HBV) infection, focusing on genomic analysis of isolates from Romanian patients. Sera from ten patients with HBV were collected and analyzed. Phylogenetic analysis was conducted on a DNA fragment spanning almost the entire genome. The occurrence of mutations was assessed for each open reading frame in the viral genome. Phylogenetic analysis revealed five isolates belonging to genotype A (subgenotype A2) and other five clustering with genotype D strains (subgenotype D1). Two patients treated with lamivudine were found to carry isolates harboring rtM204V lamivudine resistance mutation. An HBV isolate displaying a lamivudine complex resistance pattern, rtM204I in conjunction with rtL180M and rtA200V, was found in a lamivudine naïve patient. All samples harbored sA105P substitution, usually found in HBIg therapy escape isolates. Three of the studied strains were simultaneously displaying T1753, T1762 and A1764 mutations which in vitro induce enhanced genome replication and reduction of HBeAg expression. The sequence obtained from a patient with decompensated liver cirrhosis presents a novel type of insertion consisting of nine nucleotides between positions 260 and 261 in the X gene. Despite the small number of samples, our findings suggest the need to determine the drug resistance pattern for each patient before taking a therapeutic decision and also highlight the necessity of knowing the real level of drug resistance among HBV strains circulating in Romania.

New diphtheria toxin repressor types depicted in a Romanian collection of Corynebacterium diphtheriae isolates.

Corynebacterium diphtheriae is the etiological agent of diphtheria, a potential fatal disease caused by a corynephage toxin. The expression of this diphtheria toxin is controlled via an iron-dependent repressor with various functions (DtxR). Some mutations in the dtxR gene are associated with diminished activity or even with total loss of DtxR function. We conducted a molecular study to characterize the dtxR alleles harbored by 34 isolates of C. diphtheriae recovered from Romanian patients between 1961 and 2007. Three of the seven alleles identified in this study have not previously been described. Two new DtxR types were identified, one of which has an unusual polypeptide length. All the new DtxR types were found in toxigenic isolates, suggesting that they effectively regulate the expression of diphtheria toxin. Furthermore, one of the new DtxR identified was also found in a non-toxigenic isolate, making it a potential source of toxigenic isolates after lysogenic conversion.

Shiga Toxin-Producing Escherichia coli Strains from Romania: A Whole Genome-Based Description.

The zoonotic Shiga toxin-producing Escherichia coli (STEC) group is unanimously regarded as exceptionally hazardous for humans. This study aimed to provide a genomic perspective on the STEC recovered sporadically from humans and have a foundation of internationally comparable data. Fifty clinical STEC isolates, representing the culture-confirmed infections reported by the STEC Reference Laboratory between 2016 and 2023, were subjected to whole-genome sequencing (WGS) analysis and sequences were interpreted using both commercial and public free bioinformatics tools. The WGS analysis revealed a genetically diverse population of STEC dominated by non-O157 serogroups commonly reported in human STEC infections in the European Union. The O26:H11 strains of ST21 lineage played a major role in the clinical disease resulting in hospitalisation and cases of paediatric HUS in Romania surpassing the O157:H7 strains. The latter were all clade 7 and mostly ST1804. Notably, among the Romanian isolates was a stx2a-harbouring cryptic clade I strain associated with a HUS case, stx2f- and stx2e-positive strains, and hybrid strains displaying a mixture of intestinal and extraintestinal virulence genes were found. As a clearer picture emerges of the STEC strains responsible for infections in Romania, further surveillance efforts are needed to uncover their prevalence, sources, and reservoirs.

COVID-19 Vaccine Effectiveness in Autumn and Winter 2022 to 2023 Among Older Europeans.

Importance: In the context of emerging SARS-CoV-2 variants or lineages and new vaccines, it is key to accurately monitor COVID-19 vaccine effectiveness (CVE) to inform vaccination campaigns. Objective: To estimate the effectiveness of COVID-19 vaccines administered in autumn and winter 2022 to 2023 against symptomatic SARS-CoV-2 infection (with all circulating viruses and XBB lineage in particular) among people aged 60 years or older in Europe, and to compare different CVE approaches across the exposed and reference groups used. Design, Setting, and Participants: This case-control study obtained data from VEBIS (Vaccine Effectiveness, Burden and Impact Studies), a multicenter study that collects COVID-19 and influenza data from 11 European sites: Croatia; France; Germany; Hungary; Ireland; Portugal; the Netherlands; Romania; Spain, national; Spain, Navarre region; and Sweden. Participants were primary care patients aged 60 years or older with acute respiratory infection symptoms who were recruited at the 11 sites after the start of the COVID-19 vaccination campaign from September 2022 to August 2023. Cases and controls were defined as patients with positive and negative, respectively, reverse transcription-polymerase chain reaction (RT-PCR) test results. Exposures: The exposure was COVID-19 vaccination. The exposure group consisted of patients who received a COVID-19 vaccine during the autumn and winter 2022 to 2023 vaccination campaign and 14 days or more before symptom onset. Reference group included patients who were not vaccinated during or in the 6 months before the 2022 to 2023 campaign (seasonal CVE), those who were never vaccinated (absolute CVE), and those who were vaccinated with at least the primary series 6 months or more before the campaign (relative CVE). For relative CVE of second boosters, patients receiving their second booster during the campaign were compared with those receiving 1 booster 6 months or more before the campaign. Main Outcomes and Measures: The outcome was RT-PCR-confirmed, medically attended, symptomatic SARS-CoV-2 infection. Four CVE estimates were generated: seasonal, absolute, relative, and relative of second boosters. CVE was estimated using logistic regression, adjusting for study site, symptom onset date, age, chronic condition, and sex. Results: A total of 9308 primary care patients were included, with 1687 cases (1035 females; median [IQR] age, 71 [65-79] years) and 7621 controls (4619 females [61%]; median [IQR] age, 71 [65-78] years). Within 14 to 89 days after vaccination, seasonal CVE was 29% (95% CI, 14%-42%), absolute CVE was 39% (95% CI, 6%-60%), relative CVE was 31% (95% CI, 15% to 44%), and relative CVE of second boosters was 34% (95% CI, 18%-47%) against all SARS-CoV-2 variants. In the same interval, seasonal CVE was 44% (95% CI, -10% to 75%), absolute CVE was 52% (95% CI, -23% to 82%), relative CVE was 47% (95% CI, -8% to 77%), and relative CVE of second boosters was 46% (95% CI, -13% to 77%) during a period of high XBB circulation. Estimates decreased with time since vaccination, with no protection from 180 days after vaccination. Conclusions and Relevance: In this case-control study among older Europeans, all CVE approaches suggested that COVID-19 vaccines administered in autumn and winter 2022 to 2023 offered at least 3 months of protection against symptomatic, medically attended, laboratory-confirmed SARS-CoV-2 infection. The effectiveness of new COVID-19 vaccines against emerging SARS-CoV-2 variants should be continually monitored using CVE seasonal approaches.

Comparison of truncated human angiotensin-converting enzyme 2 (hACE2) expression in pET28a(+) versus pET-SUMO vector and two Escherichia coli strains.

PURPOSE: Truncated human angiotensin-converting enzyme 2 (hACE2) expression rises a great scientific interest, considering its possible therapeutic and diagnostic applications. A promising research direction is the therapeutic use of smaller hACE2 versions with high binding affinity as decoy receptors for S1 glycoprotein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Another possible application is the use of these truncated versions for the functionalization of appropriate nanomaterials for constructing novel biosensors with a rapid and sensitive response for coronavirus disease 2019 (COVID-19) detection. The present study aimed to find a suitable system for high yield expression of different versions of truncated hACE2. MATERIALS AND METHODS: The encoding DNA for the hACE2 fragments (7-507 aa, 16-128 aa, and 30-357 aa) was obtained by PCR amplification using as template pcDNA3.1-hACE2 plasmid and further cloned into pET28a(+) and pET-SUMO vectors. The positive clones were selected and the correct DNA insertion was confirmed through gene sequencing. The truncated hACE2 proteins were further expressed in two E. coli strains, Rosetta(DE3) and BL21(DE3). RESULTS: For all three truncated hACE2 mini proteins, pET28a(+) does not lead to protein expression, regardless of the bacterial strain. The situation changes with the use of the pET-SUMO expression system when all hACE2 fragments are expressed, but with higher efficiency in E. coli BL21(DE3) than E. coli Rosetta. CONCLUSION: In the present study, we showed that different versions of recombinant hACE2 are successfully expressed in E. coli BL21(DE3) by using pET-SUMO expression system.

Sequencing and Partial Molecular Characterization of BAB-TMP, the Babeș Strain of the Fixed Rabies Virus Adapted for Multiplication in Cell Lines.

The rabies virus is a major zoonosis that causes severe nervous disease in humans, leading to paralysis and death. The world's second anti-rabies center was established in 1888 by Victor Babeș, in Bucharest, where an eponymous strain of rabies was isolated and used to develop a method for immunization. The Babeș strain of the rabies virus was used for over 100 years in Romania to produce a rabies vaccine for human use, based on animal nerve tissue, thus having a proven history of prophylactic use. The present study aimed to sequence the whole genome of the Babeș strain and to explore its genetic relationships with other vaccine strains as well as to characterize its relevant molecular traits. After being adapted for multiplication in cell lines and designated BAB-TMP, 99% of the viral genome was sequenced. The overall organization of the genome is similar to that of other rabies vaccine strains. Phylogenetic analysis indicated that the BAB-TMP strain is closely related to the Russian RV-97 vaccine strain, and both seem to have a common ancestor. The nucleoprotein gene of the investigated genome was the most conserved, and the glycoprotein showed several unique amino acid substitutions within the major antigenic sites and linear epitopes.

First Detection and Molecular Characterization of Usutu Virus in Culex pipiens Mosquitoes Collected in Romania.

Usutu virus (USUV) is an emergent arbovirus in Europe causing mortality in bird populations. Similar to West Nile virus (WNV), USUV is maintained in sylvatic cycles between mosquito vectors and bird reservoirs. Spillover events may result in human neurological infection cases. Apart from indirect evidence provided by a recent serological study in wild birds, the circulation of USUV in Romania was not assessed. We aimed to detect and molecular characterize USUV circulating in mosquito vectors collected in South-Eastern Romania-a well-known WNV endemic region-during four transmission seasons. Mosquitoes were collected from Bucharest metropolitan area and Danube Delta, pooled, and screened by real-time RT-PCR for USUV. Partial genomic sequences were obtained and used for phylogeny. USUV was detected in Culex pipiens s.l. female mosquitoes collected in Bucharest, in 2019. The virus belonged to Europe 2 lineage, sub-lineage EU2-A. Phylogenetic analysis revealed high similarity with isolates infecting mosquito vectors, birds, and humans in Europe starting with 2009, all sharing common origin in Northern Italy. To our knowledge, this is the first study characterizing a strain of USUV circulating in Romania.

Using Rapid Analyte Measurement Platform (RAMP) as a Tool for an Early Warning System Assessing West Nile Virus Epidemiological Risk in Bucharest, Romania.

West Nile virus (WNV) is the most widely spread arbovirus in the world. Early detection of this virus in mosquito populations is essential for implementing rapid vector control measures to prevent outbreaks. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is a powerful tool for the detection of WNV in mosquito pools, but it is a time- and resource-consuming assay. We used a Rapid Analyte Measurement Platform (RAMP) assay in a vector surveillance program for rapid detection of WNV in mosquitoes collected in Bucharest city, Romania, in 2021. The positive mosquito pools were tested for confirmation with real-time RT-PCR. Three out of the 24 RAMP assay positive pools were not confirmed by real-time RT-PCR. We consider that RAMP assay can be used as a fast and reliable method for the screening of WNV presence in mosquito pools, but we recommend that samples with values ranging from 30 to 100 RAMP units should fall in a grey zone and should be considered for real-time RT-PCR confirmation.

Distribution of Insecticide Resistance Genetic Markers in the West Nile Virus Vector Culex pipiens from South-Eastern Romania.

Culex pipiens pipiens and Culex pipiens molestus mosquitoes are the vectors of West Nile virus in south-eastern Romania, an area of intense circulation and human transmission of this virus. The level of insecticide resistance for the mosquito populations in the region has not been previously assessed. Culex pipiens mosquitoes collected between 2018 and 2019 in south-eastern Romania from different habitats were subjected to biotype identification by real-time PCR. Substitutions causing resistance to organophosphates and carbamates (F290V and G119S in acetylcholinesterase 1) and to pyrethroids (L1014F in voltage gated Na+ channel) were screened by PCR or sequencing. Substitutions F290V and G119S were detected at very low frequencies and only in heterozygous state in Culex pipiens molestus biotype specimens collected in urban areas. The molestus biotype population analysed was entirely homozygous for L1014F, and high frequencies of this substitution were also found for pipiens biotype and hybrid mosquitoes collected in urban and in intensive agriculture areas. Reducing the selective pressure by limiting the use of pyrethroid insecticides only for regions where it is absolutely necessary and monitoring L1014F mutation should be taken into consideration when implementing vector control strategies.

The frequency and biodiversity of poliovirus and non-polio enterovirus strains isolated from healthy children living in a limited area in Romania.

The risk of importation and transmission of poliovirus strains to small susceptible groups within populations will remain until polio is eradicated globally. We investigated the circulation and biodiversity of enteroviruses in a group of children under 6 years of age with low vaccine coverage against polio. Only vaccine Sabin strains and viruses of the human enterovirus species B were isolated from the group. Evidence of inter-human circulation of Sabin strains was found.

Q fever endocarditis in Romania: the first cases confirmed by direct sequencing.

Infective endocarditis (IE) is a serious, life-threatening disease with highly variable clinical signs, making its diagnostic a real challenge. A diagnosis is readily made if blood cultures are positive, but in 2.5 to 31% of all infective endocarditis cases, routine blood cultures are negative. In such situations, alternative diagnostic approaches are necessary. Coxiella burnetii and Bartonella spp. are the etiological agents of blood culture-negative endocarditis (BCNE) most frequently identified by serology. The purpose of this study is to investigate the usefulness of molecular assays, as complementary methods to the conventional serologic methods for the rapid confirmatory diagnostic of Q fever endocarditis in patients with BCNE. Currently, detection of C. burnetii by culture or an antiphase I IgG antibody titers >800 represents a major Duke criterion for defining IE, while a titers of >800 for IgG antibodies to either B. henselae or B. quintana is used for the diagnosis of endocarditis due to Bartonella spp. We used indirect immunofluorescence assays for the detection of IgG titers for C. burnetii, B. henselae and B. quintana in 57 serum samples from patients with clinical suspicion of IE. Thirty three samples originated from BCNE patients, whereas 24 were tested before obtaining the blood cultures results, which finally were positive. The results of serologic testing showed that nine out of 33 BCNE cases exhibited antiphase I C. burnetii IgG antibody titer >800, whereas none has IgG for B. henselae or B. quintana. Subsequently, we used nested-PCR assay for the amplification of C. burnetii DNA in the nine positive serum samples, and we obtained positive PCR results for all analyzed cases. Afterwards we used the DNA sequencing of amplicons for the repetitive element associated to htpAB gene to confirm the results of nested-PCR. The results of sequencing allowed us to confirm that C. burnetii is the causative microorganism responsible for BCNE. In conclusion, the nested PCR amplification followed by direct sequencing is a reliable and accurate method when applied to serum samples, and it may be used as an additional test to the serological methods for the confirmatory diagnosis of BCNE cases determined by C. burnetii.

A Snapshot of the Genetic Diversity of Salmonella Enteritidis Population Involved in Human Infections in Romania Taken in the European Epidemiological Context.

In the absence of consistent national molecular typing data to enhance the surveillance of Salmonella Enteritidis, it was considered useful to collect baseline information on the genetic diversity and antibiotic susceptibility of strains isolated in Romania between January 2016 and April 2020 and compare them to strains described in major international outbreaks of the same period. A collection of 245 clinical isolates were genotyped by a standardised multiple-locus variable-number of tandem repeats analysis (MLVA) 5-loci protocol and screened for antimicrobial resistance against 15 compounds. Twenty strains were further subjected to whole genome sequencing (WGS) and compared to epidemiologically relevant high-throughput sequencing data available in European databases. Twenty-seven MLVA genotypes were identified, of which three, commonly reported in Europe between 2016-2020, covered 72% of the collection. Antibiotic resistance was detected in 30% of the strains, with resistance to nalidixic acid and ciprofloxacin as the most common phenotype, and also associated with two prevalent MLVA clones. WGS-derived multilocus sequence typing (MLST) revealed a single sequence type (ST11) further resolved into 10 core-genome MLST complex types. The minimum spanning tree constructed from the cgMLST data clustered Romanian and international strains, which shared more than 95% of the core genes, revealing links with a contemporaneous multi-country outbreak. This study could be regarded as a forerunner to the advent of using this integrative approach in the public health practice at a national level and thus contribute to the concerted actions at a European level to stop outbreaks.

Characterization and Host-Feeding Patterns of Culex pipiens s.l. Taxa in a West Nile Virus-Endemic Area in Southeastern Romania.

Culex pipiens sensu lato has been documented as West Nile virus (WNV) vector in southeastern Romania. Bucharest, the densely populated capital city of Romania, and the surrounding Ilfov county are WNV hotspots. In this area, the morphologically indistinguishable biotypes of Cx. pipiens, namely pipiens and molestus, are usually differentiated by their behavioral and physiological traits. Their involvement in WNV transmission, as suggested by entomological investigations, was not previously documented for each biotype. We used a Real-Time PCR assay based on CQ11 microsatellite to identify the Cx. pipiens biotypes and their hybrids collected in various habitats in the Bucharest metropolitan area. A sympatric distribution of both biotypes was observed, with a preference of green areas for pipiens, and human settings and animal farmlands for molestus. In the latter habitats, pipiens and molestus were found in mixed aboveground populations. A low number of hybrids was found suggesting existence of reproductive isolation. In subway tunnels molestus was dominant with a higher number of hybrids recorded than aboveground. Blood-engorged mosquitoes were identified to biotype and the blood meal source identified by DNA barcoding. Overall, Cx. pipiens s.l. fed mainly on birds, commonly on house sparrows, collared doves, and blackbirds, which are potential WNV-amplifying hosts. The preference for avian hosts was expressed strongest by pipiens biotype, while molestus was substantially less specific, feeding on avian and mammal hosts with similar frequency, with humans representing 20% of the hosts. Hybrids had a host choice closer to that of molestus. These findings highlight the role of pipiens biotype as enzootic/epizootic vector, and specifically show molestus as the bridge vector for WNV. The pipiens and molestus biotypes show important differences in habitat preferences, including oviposition; these findings demonstrate that targeted mosquito control to limit WNV transmission may be possible.