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.

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.

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.

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.

Correction: Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses.

[This corrects the article DOI: 10.1371/journal.pntd.0002157.].

West Nile virus lineage 2 in Romania, 2015-2016: co-circulation and strain replacement.

BACKGROUND: West Nile virus (WNV) is endemic in southeastern Romania and, after the unprecedented urban epidemic in Bucharest in 1996 caused by lineage 1 WNV, cases of West Nile fever have been recorded every year. Furthermore, a new outbreak occurred in 2010, this time produced by a lineage 2 WNV belonging to the Eastern European clade (Volgograd 2007-like strain), which was detected in humans and mosquitoes in the following years. RESULTS: We report here, for the first time, the emergence, in 2015, of lineage 2 WNV belonging to the monophyletic Central/Southern European group of strains which replaced in 2016, the previously endemized lineage 2 WNV Volgograd 2007-like strain in mosquito populations. The emerged WNV strain harbors H249P (NS3 protein) and I159T (E glycoprotein) substitutions, which have been previously associated in other studies with neurovirulence and efficient vector transmission. CONCLUSIONS: In 2016, both early amplification of the emerged WNV and complete replacement in mosquito populations of the previously endemized WNV occurred in southeastern Romania. These events were associated with a significant outbreak of severe West Nile neuroinvasive disease in humans.

Re-emergence of severe West Nile virus neuroinvasive disease in humans in Romania, 2012 to 2017-implications for travel medicine.

BACKGROUND: In Romania, after a major outbreak in 1996, West Nile neuroinvasive disease (WNND) was reported only in a limited number of cases annually. During 2016-2017, a significant increase in the number of WNND cases was reported at the national level, associated with high mortality rates. METHODS: A retrospective analysis of all cases confirmed with WNND, hospitalized during 2012-2017 in a single tertiary facility from Bucharest was performed in order to determine the annual prevalence and mortality rate and the risk factors associated with a severe outcome. RESULTS: 47 cases were confirmed as WNND. The mortality rate was 25.5%, all death occurred during 2016-2017. Coma, confusion, obtundation, sleepiness and depressed deep tendon reflexes were symptoms predicting a severe outcome. In a univariate analysis age (p < 0.001), associated cancers (p = 0.012) and low levels of chloride in the CSF (p = 0.008) were risk factors for mortality. In a multinomial logistic analysis, age older than 75 years remained the only independent predictor of death in WNND. CONCLUSIONS: The increase in both the number and the mortality rate of WNND cases suggest a changing pattern of WNV infection in Romania. Public health authorities and clinicians should be aware of the risk of severe WNV infection in travelers returning from Romania.

First Two Imported Cases of Zika Virus Infections in Romania.

We report the first two cases of imported Zika virus (ZIKV) infection in Romanian patients returning from areas with ongoing outbreaks and challenges for laboratory diagnostic; first one with a classical pattern of acute flaviviral infection and the second one with an interesting pattern of a secondary flaviviral (ZIKV) infection in a yellow fever-vaccinated child living abroad in an endemic area.

Transmission Dynamics of the West Nile Virus in Mosquito Vector Populations under the Influence of Weather Factors in the Danube Delta, Romania.

Mosquitoes were collected in the Danube Delta during the active seasons of 2011-2013. For Culex spp. mosquitoes, the abundance was calculated. Culex pipiens (sensu lato), (s.l.) and Culex modestus pools were tested for the presence of West Nile virus (WNV) genome, and the maximum likelihood of the infection rate was established. Mean daily temperatures and precipitation were obtained for the closest meteorological station. A negative binominal model was used to evaluate linkages between the temperature/precipitation and mosquito population size. A zero-inflated negative binomial model was used to test the relationship between the temperature and the infection rate. A single complex model for infection rate prediction was also used. The linkages were calculated for lag 0 and for 10 days earlier (lag 1), 20 days earlier (lag 2), and 30 days earlier (lag 3). Significant positive linkages (P < 0.001) were detected between temperature and mosquito population size for lag 1, lag 2, and lag 3. The linkages between temperature and infection rates were positive and significant for lag 2 and lag 3. Negative significant (P < 0.001) results were detected between precipitation and infection rates for lags 1, 2, and 3. The complex model showed that the best predictors for infection rate are the temperature, 20 days earlier (positive linkage) and the precipitation, 30 days earlier (negative linkage). Positive temperature anomalies in spring and summer and rainfall decrease contributed to the increase in the Culex spp. abundance and accelerated the WNV amplification in mosquito vector populations in the following weeks.

Variation in interferon sensitivity and induction between Usutu and West Nile (lineages 1 and 2) viruses.

Given the pivotal role of monocyte-derived dendritic cells (DCs) in determining the magnitude of the antiviral innate immune response, we sought to determine whether Usutu virus (USUV) and West Nile virus (WNV) lineages (L)1 and L2 can infect DCs and affect the rate of type I interferon (IFN) activation. The sensitivity of these viruses to types I and III IFNs was also compared. We found that USUV can infect DCs, induce higher antiviral activities, IFN alpha subtypes and the IFN stimulated gene (ISG)15 pathway, and is more sensitive to types I and III IFNs than WNVs. In contrast, we confirmed that IFN alpha/beta subtypes were more effective against WNV L2 than WNV L1. However, the replication kinetics, induction of IFN alpha subtypes and ISGs in DCs and the sensitivity to IFN lambda 1-3 did not differ between WNV L1 and L2.

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.

Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013.

BACKGROUND: Dengue fever is the commonest arthropod-borne infection worldwide. In recent years, rapid growth in global air travel has resulted in a considerable increase in the incidence of imported cases. In Romania it is now the second most frequent cause for hospitalization (after malaria) in patients arriving from tropical regions. METHODS: Serological and molecular diagnostics were applied to samples obtained between 2008 and 2013 from travelers with suspected dengue. Molecular typing was performed by RT-PCR followed by sequencing of the E-NS1 junction. RESULTS: Twelve of 37 suspected cases were confirmed and three remained probable. The infections were acquired in endemic regions in Asia, Africa and in Europe (Madeira Island). Dengue virus nucleic acid was detected and sequenced in nine cases. Phylogenetic analysis indicated that the viruses were of genotypes I and V of serotype 1, cosmopolitan genotype of serotype 2 and genotypes I and III of serotype 3. CONCLUSIONS: Romanian tourists traveling to dengue-endemic countries are at risk of acquiring dengue infection. Appropriate prevention measures prior to travel and upon return should be taken, particularly as the dengue secondary vector Aedes albopictus is now established in Bucharest.

Dobrava virus carried by the yellow-necked field mouse Apodemus flavicollis, causing hemorrhagic fever with renal syndrome in Romania.

Hemorrhagic fever with renal syndrome (HFRS) has been confirmed by serological methods during recent years in Romania. In the present study, focus-reduction neutralization tests (FRNT) confirmed Dobrava hantavirus (DOBV) as the causative agent in some HFRS cases, but could not distinguish between DOBV and Saaremaa virus (SAAV) infections in other cases. DOBV was detected by a DOBV-specific TaqMan assay in sera of nine patients out of 22 tested. Partial sequences of the M genomic segment of DOBV were obtained from sera of three patients and revealed the circulation of two DOBV lineages in Romania. Investigation of rodents trapped in Romania found three DOBV-positive Apodemus flavicollis out of 83 rodents tested. Two different DOBV lineages were also detected in A. flavicollis as determined from partial sequences of the M and S genomic segments. Sequences of DOBV in A. flavicollis were either identical or closely related to the sequences obtained from the HFRS patients. The DOBV strains circulating in Romania clustered in two monophyletic groups, together with strains from Slovenia and the north of Greece. This is the first evidence for the circulation of DOBV in wild rodents and for a DOBV etiology of HFRS in Romania.

Indirect immunofluorescence assay for the simultaneous detection of antibodies against clinically important old and new world hantaviruses.

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n=97); SEOV (China, n=5); DOBV (Romania, n=7); SNV (Canada, n=23); ANDV (Argentina and Chile, n=52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n=177; 96%) and/or IgM (n=131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).

Permissive summer temperatures of the 2010 European West Nile fever upsurge.

BACKGROUND: In the summer of 2010, Europe experienced outbreaks of West Nile Fever (WNF) in humans, which was preceded by hot spells. The objective of this study was to identify potential drivers of these outbreaks, such as spring and summer temperatures, relative humidity (RH), and precipitation. METHODS: Pearson and lag correlations, binary and multinomial logistic regressions were used to assess the relationship between the climatic parameters and these outbreaks. RESULTS: For human morbidity, significant (<0.05) positive correlations were observed between a number of WNF cases and temperature, with a geographic latitude gradient: northern ("colder") countries displayed strong correlations with a lag of up to four weeks, in contrast to southern ("warmer") countries, where the response was immediate. The correlations with RH were weaker, while the association with precipitation was not consistent. Horse morbidity started three weeks later than in humans where integrated surveillance was conducted, and no significant associations with temperature or RH were found for lags of 0 to 4 weeks. CONCLUSIONS: Significant temperature deviations during summer months might be considered environmental precursors of WNF outbreaks in humans, particularly at more northern latitudes. These insights can guide vector abatement strategies by health practitioners in areas at risk for persistent transmission cycles.

First serologic evidence for the circulation of Crimean-Congo hemorrhagic fever virus in Romania.

Serum samples from sheep in localities situated in the county of Tulcea, Northern Dobrogea, were tested with an IgG sandwich ELISA using a recombinant Crimean-Congo hemorrhagic fever virus (CCHFV) antigen. In all, 131 sera out of 471 tested (27.8%) had IgG antibodies specific to CCHFV. This is the first evidence for the circulation of CCHFV virus in Romania.

Hantavirus infection--hemorrhagic fever with renal syndrome: the first case series reported in Romania and review of the literature.

Hemorrhagic fever with renal syndrome (HFRS) is a zoonotic infectious disease caused by Hantaviruses, a group of RNA viruses belonging to the Bunyaviridae family. Humans may get the disease by contamination with excreta of carrier rodents. The disease typically manifests with the triad fever-thrombocytopenia-acute kidney injury (AKI). Although its global prevalence seems to be increasing, Hantavirus infection is still commonly overlooked, because of its clinical polymorphism and non-specific symptoms, particularly in mild cases. Until recently, the disease was virtually unknown in Romania, due to lack of physicians' awareness and of adequate laboratory diagnostic techniques. In this article, we present the first six cases of HFRS diagnosed in our country, based on serology testing. We review the existing literature on HFRS and discuss our findings in comparison with other reports. All our patients presented with fever, flu syndrome, bleeding, gastrointestinal symptoms, and oliguria. Among laboratory abnormalities, elevated serum creatinine and liver enzymes, high C-reactive protein, leukocytosis, low platelet count, and hematuria were constantly seen. Five patients required hemodialysis. All patients survived and five of them completely recovered their renal function, while only one patient retained a mild impairment of the glomerular filtration rate. From a clinical viewpoint, we believe that Hantavirus infection should be considered in all patients presenting with fever, thrombocytopenia, and AKI, when specific serology testing would be indicated. From a public health perspective, we suggest that future efforts in our country should be directed toward (1) increasing the understanding and the awareness of this disease among health care professionals, (2) educating the population at risk on the application of prophylactic measures, (3) expanding the availability of diagnostic laboratory tools, and (4) developing research on national zoonotic virus reservoirs.