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.

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.

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.

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.

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.

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.

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.

Application of the susceptible-infected-recovered deterministic model in a GII.P17 emergent norovirus strain outbreak in Romania in 2015.

PURPOSE: This study shows the epidemiological profile of the first gastroenteritis outbreak of GII.P17 in the Romanian territory. An outbreak with such large amplitude in a European territory was previously undocumented. PATIENTS AND METHODS: Using a cross-sectional design, with the susceptible-infected-recovered (SIR) deterministic compartmental model for a fixed population, and the cluster method for establishing the high-incidence zones, we carried out our investigation by means of questionnaires containing personal data, affected collectivities, disease onset and duration, symptoms displayed, medical assistance provided, previous antibiotic intake where applicable, food consumption and water sources, and sanitation conditions. The confirmation of cases was done based on the typical norovirus gastroenteritis symptomatology and using three laboratory confirmations (by molecular diagnosis) for GII.P17-GII.17 genotype noroviruses from three patients. RESULTS: A gastroenteritis outbreak occurred in October-November 2015, affecting 328 people in Arad, a county in Western Romania, covering 44 neighbouring localities with a total population of 35,440 people. The study detected an inter-human transmission of the infection, with an intrafamilial risk of disease of 2.26 (95% CI 1.76 to 2.90) compared with the community transmission (in school collectivity). The basic reproduction number Ro dropped from 1.26 to 0.18 during weeks 43:44, after controlling the transmission by decontamination and isolation. CONCLUSION: SIR made it possible to highlight the expansion of the emerging norovirus strain infection from community to family collectivities. This study provides practical solutions to limit disease cases, even in the absence of etiology, and shows the importance of sometimes underestimated traditional control methods.

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.

Molecular Typing of Escherichia coli O157 Isolates from Romanian Human Cases.

Verocytotoxin-producing Escherichia coli (VTEC) of serogroup O157 are among the most important causes of severe cases of foodborne disease and outbreaks worldwide. As little is known about the characteristic of these strains in Romania, we aimed to provide reference information on the virulence gene content, phylogenetic background, and genetic diversity of 7 autochthonous O157 strains collected during 2016 and 2017 from epidemiologically non-related cases. These strains were typed by a combination of phenotypic and molecular methods routinely used by the national reference laboratory. Additionally, 4 of them were subjected to whole-genome sequencing (WGS), and public web-based tools were used to extract information on virulence gene profiles, multilocus sequence types (MLST), and single nucleotide polymorphism (SNP)-based phylogenetic relatedness. Molecular typing provided evidence of the circulation of a polyclonal population while distinguishing a cluster of non-sorbitol-fermenting, glucuronidase-negative, phylogenetic group E, MLST 1804 strains, representing lineage II and clade 7, which harbored vtx2c, eae-gamma, and ehxA genes. A good correlation between the routine typing methods and WGS data was observed. However, SNP-based genotyping provided a higher resolution in depicting the relationships between the O157:H7 strains than that provided by Pulse-field gel electrophoresis. This study should be a catalyst for improved laboratory-based surveillance of autochthonous VTEC.

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.

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.

Impact of hepatitis C virus core mutations on the response to interferon-based treatment in chronic hepatitis C.

AIM: To determine whether hepatitis C virus (HCV) core substitutions play a role in the response to interferon-based treatment in Caucasian patients. METHODS: One hundred eight HCV chronically infected patients initiating treatment with pegylated IFN plus ribavirin for 48 wk were tested for baseline substitutions at codons 70 and 91 of the viral core protein (BigDye Terminator vers.3.1, Applied Biosystems,) and for genetic polymorphisms in host IL28B gene rs12979860 (Custom TaqMan 5' allelic discrimination assay; Applied Biosystems). RESULTS: Of the patients, all were infected with HCV genotype 1b, 44.4% had low baseline HCV viral load, and 37.9% had mild/moderate fibrosis. Only 38.9% achieved therapeutic success, defined as sustained virological response (SVR). Eighty-eight percent of the patients presented at least one substitution at core position 70 (R70Q/H) or/and position 91 (L91M). The favorable IL28B CC polymorphism was detected in only 17.6% of the patients. In the univariate analysis, young age (P < 0.001), urban residence (P = 0.004), IL28B CC genotype (P < 0.001), absence of core mutations (P = 0.005), achievement of rapid virologic response (P < 0.001) and early virological response (P < 0.001) were significantly correlated with SVR. A multivariate analysis revealed three independent predictors of therapeutic success: young age (P < 0.001), absence of core substitutions (P = 0.04) and IL28B CC genotype (P < 0.001); the model correctly classified 75.9% of SVR cases with a positive predictive value of 80.7%. CONCLUSION: HCV core mutations can help distinguish between patients who can still benefit from the affordable IFN-based therapy from those who must be treated with DAAs to prevent the evolution towards end-stage liver disease.

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.

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.