An Assessment of West Nile and Usutu Viruses' Seroprevalence in Hospitalized Patients: A Preliminary Study on Flavivirus Exposure in Eastern Romania.

WNV and USUV are closely related epornitic flaviviruses transmitted by Culex mosquitoes which can cause febrile and neurodegenerative disease in humans. The impact of both viruses on public health has increased in the recent decades. AIM: The aim of the study was to evaluate the seroprevalence of WNV and USUV in hospitalized patients from eastern Romania who did not show symptoms corresponding to the case definition. METHODS: Human blood samples from the hospitalized patients were collected in 2015 and from April to September 2019 in Iasi County, Romania. The samples were screened by ELISA for anti-WNV IgG, IgM, and anti-USUV IgG antibodies. RESULTS: A cumulative seroprevalence of 3.4% was recorded for anti-WNV IgG antibodies and 9.1% for anti-WNV IgM. No sample was positive for anti-USUV antibodies. CONCLUSION: The cumulative seroprevalence observed provides support for the consideration of WNV as being endemic in the east of Romania. The absence of anti-USUV antibodies may be related to cross-reactivity and cohort size, thus, USUV should be considered in clinical practice and become an objective for active surveillance in Romania.

First Detection of Hepatitis E Virus (Rocahepevirus ratti Genotype C1) in Synanthropic Norway Rats (Rattus norvegicus) in Romania.

Hepatitis E virus (HEV) is an emerging zoonotic pathogen with different viral genera and species reported in a wide range of animals. Rodents, particularly rats, carry the specific genus rat HEV (Rocahepevirus genus, genotype C1) and are exposed occasionally to HEV-3 (Paslahepevirus genus, genotype 3), a zoonotic genotype identified in humans and widely distributed in domestic and feral pigs. In this study, the presence of HEV was investigated in synanthropic Norway rats from Eastern Romania, in areas where the presence of HEV-3 was previously reported in pigs, wild boars and humans. Using methods capable of detecting different HEV species, the presence of HEV RNA was investigated in 69 liver samples collected from 52 rats and other animal species. Nine rat liver samples were identified as being positive for rat HEV RNA (17.3%). High sequence identity (85-89% nt) was found with other European Rocahepevirus. All samples tested from other animal species, within the same environment, were negative for HEV. This is the first study to demonstrate the presence of HEV in rats from Romania. Since rat HEV has been reported to cause zoonotic infections in humans, this finding supports the need to extend the diagnosis of Rocahepevirus in humans with suspicion of hepatitis.

Detection of West Nile Virus Lineage 2 in Eastern Romania and First Identification of Sindbis Virus RNA in Mosquitoes Analyzed using High-Throughput Microfluidic Real-Time PCR.

The impact of mosquito-borne diseases on human and veterinary health is being exacerbated by rapid environmental changes caused mainly by changing climatic patterns and globalization. To gain insight into mosquito-borne virus circulation from two counties in eastern and southeastern Romania, we have used a combination of sampling methods in natural, urban and peri-urban sites. The presence of 37 mosquito-borne viruses in 16,827 pooled mosquitoes was analyzed using a high-throughput microfluidic real-time PCR assay. West Nile virus (WNV) was detected in 10/365 pools of Culex pipiens (n = 8), Culex modestus (n = 1) and Aedes vexans (n = 1) from both studied counties. We also report the first molecular detection of Sindbis virus (SINV) RNA in the country in one pool of Culex modestus. WNV infection was confirmed by real-time RT-PCR (10/10) and virus isolation on Vero or C6/36 cells (four samples). For the SINV-positive pool, no cytopathic effectwas observed after infection of Vero or C6/36 cells, but no amplification was obtained in conventional SINV RT-PCR. Phylogenetic analysis of WNV partial NS5 sequences revealed that WNV lineage 2 of theCentral-Southeast European clade, has a wider circulation in Romania than previously known.

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.

Seasonal dynamics of Phlebotomus neglectus (Diptera: Psychodidae) in cave microhabitats in Romania and the rediscovery of Sergentomyia minuta (Rondani, 1843) after 50 years.

BACKGROUND: In a countrywide study aiming to update the knowledge on diversity of sand fly species in Romania, a sand fly population was observed in an isolated system of cave microhabitats. The caves are located in the protected area of Canaraua Fetii, Dobrogea region, southeastern Romania. The highest sand fly diversity was recorded in this area between 1968 and 1970. This work presents a study conducted to estimate the seasonal variation of the sand fly species in correlation with the particular environmental factors of the isolated system of cave microhabitats. METHODS: Sand flies were collected between May and October 2020 from one trapping site of interest in Canaraua Fetii. The trapping site consisted of a cave entrance. CDC miniature light traps and sticky traps were used to collect insects from the exterior walls of the cave entrance. Species identification of collected sand flies was done using morphological keys. Statistical analysis of the trapping and climatic data was performed. RESULTS: From all collected sand flies, 99.7% (818/822) were Phlebotomus neglectus, 0.1% (1/822) Ph. balcanicus and 0.2% (2/822) Sergentomyia minuta. Sand fly activity was first observed on 2 July and last on 24 September. A monomodal abundance trend was present, with the peak activity between 16 and 17 July. The analysis of the climatic data showed correlations between the total number of captured sand flies and both average temperature and average relative humidity. The total number of collected specimens was statistically higher when CDC miniature light traps were used compared to sticky traps. The number of females on the sticky traps was significantly higher than the number of males on the same trap type. Compared with the sticky traps, significantly more males were collected by CDC miniature light traps. This is the first record of Se. minuta in Romania after 50 years of no records (despite the trapping effort of the last 5 years in the country). Also, Ph. sergenti, previously present in this location, was not found. CONCLUSIONS: In the investigated natural habitat, the diversity of the sand fly species appears to have changed, with the predominance of Ph. neglectus instead of Ph. balcanicus and Se. minuta (recorded as the two predominant species in 1968-1970). A monomodal abundance trend was observed as in other regions of the country. The sand fly activity in this particular cave microhabitat appears to be longer than in other regions in Romania. Longer sand fly activity increases the zoonotic risk of various pathogenic species' transmission, with an impact on public health, as sand flies are important insect vectors.

Common European Origin of Hepatitis E Virus in Human Population From Eastern Romania.

The purpose of this research was to improve the epidemiological data on HEV infection in the human population in Romania. The analysis targeted hospitalized subjects with acute hepatitis (n = 94) of unknown etiology from the Infectious Diseases Regional Hospital in Iasi. Moreover, patients without liver disease (n = 40) from a different county hospital located in Eastern Romania were included. The presence of HEV infection and first characterization of human HEV strains was determined using serological and molecular assays. The apparent HEV seroprevalence varied between 29.16% (95% CI, 16.31-42.03) and 32.5% (95% CI, 17.98-47.02) according to patient grouping. Molecular analysis enhanced the detection of two HEV isolates, that clustered in subtype HEV-3c, the most commonly identified subtype in Europe. Identification of acute hepatitis E cases, together with the first detection and molecular characterization of human HEV in Romania represent the originality attributes of the present study.

Molecular Evidence of Rickettsia spp., Anaplasma phagocytophilum, and "Candidatus Neoehrlichia mikurensis" in Ticks from Natural and Urban Habitats in Eastern Romania.

Ixodid ticks are competent vectors for multiple pathogens, several of which cause infections in human. The medical importance of tick-borne pathogens is well known, yet unanswered questions remain regarding the occurrence of pathogens such as Rickettsia spp., Anaplasma phagocytophilum, and "Candidatus Neoehrlichia mikurensis" in questing ticks in Romania. Our objectives were to identify three emerging tick-borne zoonotic pathogens in eastern Romania, to assess their prevalence, establish co-infection rates, and to compare infection levels of selected pathogens in questing ticks collected from one suburban area in the city of Iasi and one forested area located in the Danube Delta Biosphere Reserve. We collected 490 questing nymphs or adult ticks (467 Ixodes ricinus, 4 Dermacentor reticulatus, and 19 Haemaphysalis punctata). We individually analyzed ticks for the presence of Rickettsia spp., A. phagocytophilum, and "C. N. mikurensis." Rickettsia spp. was detected in 9.4% of ticks from both sampling areas. Rickettsia spp. included R. helvetica (n = 17 I. ricinus ticks), R. monacensis (n = 28 I. ricinus ticks), and R. raoultii (n = 1 D. reticulatus). "C. N. mikurensis" had an infection rate of 4.9% while A. phagocytophilum was detected only in the forested area with a global prevalence of 1.2%. The overall prevalence of ticks infected with at least one pathogen was 15.5%, and 5.3% of infected ticks were tested positives for dual pathogen association. Our study documents the presence of pathogens in questing ticks in the urban recreational areas of Iasi and forested areas located in the Danube Delta Biosphere Reserve. Worth mentioning, is the presence of "C. N. mikurensis" in ticks from eastern Romania, an agent just recently described in Romania, and the existence of co-infections in ticks at a similar prevalence to other European countries.

Borrelia Diversity and Co-infection with Other Tick Borne Pathogens in Ticks.

Identifying Borrelia burgdorferi as the causative agent of Lyme disease in 1981 was a watershed moment in understanding the major impact that tick-borne zoonoses can have on public health worldwide, particularly in Europe and the USA. The medical importance of tick-borne diseases has long since been acknowledged, yet little is known regarding the occurrence of emerging tick-borne pathogens such as Borrelia spp., Anaplasma phagocytophilum, Rickettsia spp., Bartonella spp., "Candidatus Neoehrlichia mikurensis", and tick-borne encephalitis virus in questing ticks in Romania, a gateway into Europe. The objective of our study was to identify the infection and co-infection rates of different Borrelia genospecies along with other tick-borne pathogens in questing ticks collected from three geographically distinct areas in eastern Romania. We collected 557 questing adult and nymph ticks of three different species (534 Ixodes ricinus, 19 Haemaphysalis punctata, and 4 Dermacentor reticulatus) from three areas in Romania. We analyzed ticks individually for the presence of eight different Borrelia genospecies with high-throughput real-time PCR. Ticks with Borrelia were then tested for possible co-infections with A. phagocytophilum, Rickettsia spp., Bartonella spp., "Candidatus Neoehrlichia mikurensis", and tick-borne encephalitis virus. Borrelia spp. was detected in I. ricinus ticks from all sampling areas, with global prevalence rates of 25.8%. All eight Borrelia genospecies were detected in I. ricinus ticks: Borrelia garinii (14.8%), B. afzelii (8.8%), B. valaisiana (5.1%), B. lusitaniae (4.9%), B. miyamotoi (0.9%), B. burgdorferi s.s (0.4%), and B. bissettii (0.2%). Regarding pathogen co-infection 64.5% of infected I. ricinus were positive for more than one pathogen. Associations between different Borrelia genospecies were detected in 9.7% of ticks, and 6.9% of I. ricinus ticks tested positive for co-infection of Borrelia spp. with other tick-borne pathogens. The most common association was between B. garinii and B. afzelii (4.3%), followed by B. garinii and B. lusitaniae (3.0%). The most frequent dual co-infections were between Borrelia spp. and Rickettsia spp., (1.3%), and between Borrelia spp. and "Candidatus Neoehrlichia mikurensis" (1.3%). The diversity of tick-borne pathogens detected in this study and the frequency of co-infections should influence all infection risk evaluations following a tick bite.