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.

Adenovirus-Mediated FasL Minigene Transfer Endows Transduced Cells with Killer Potential.

Fas ligand (First apoptosis signal ligand, FasL, also known as CD95L) is the common executioner of apoptosis within the tumor necrosis factor (TNF) superfamily. We aimed to induce functional FasL expression in transduced cells using an adenovirus vector, which has the advantage of strong and transient induction of the gene included in the adenoviral genome. Here, we report that the adenovirus carrying a truncated FasL gene, named FasL minigene, encoding the full-length FasL protein (Ad-gFasL) is more efficient than the adenovirus carrying FasL cDNA (Ad-cFasL) in the induction of FasL expression in transduced cells. FasL minigene (2887 bp) lacking the second intron and a part of the 3'-UTR was created to reduce the gene length due to the size limitation of the adenoviral genome. The results show that, in transduced hepatocytes, strong expression of mRNA FasL appeared after 10 h for Ad-gFasL, while for Ad-cFasL, a faint expression appeared after 16 h. For Ad-gFasL, the protein expression was noticed starting with 0.5 transfection units (TU)/cell, while for Ad-cFasL, it could not be revealed. FasL-expressing endothelial cells induced apoptosis of A20 cells in co-culture experiments. FasL-expressing cells may be exploitable in various autoimmune diseases such as graft-versus-host disease, chronic colitis, and type I diabetes.

Impact of genetic testing and family health history of cystic fibrosis in the early prenatal diagnosis and prevention of a new case of genetic disorder.

Cystic fibrosis (CF) is a multi-system autosomal recessive disorder, results of mutations in the CF transmembrane conductance regulator (CFTR) gene, located on the long arm of chromosome 7. We present a special family couple with particular medical history of CF, who comes to our Clinic for genetic tests and a prenatal genetic counseling, to prevent the birth of a new affected CF child. Genetic analysis showed that the first affected child, a daughter, is compound heterozygous for two clinically significant recessive mutations: c.1521_1523delCTT; p.Phe508del, inherited from her mother, who carries the same CFTR mutation, and c.1853_1863delTTTTGCATGAA; p.IIe618Argfs 2, inherited from her father, who is heterozygous, healthy carrier, for the same CFTR mutation. In our case report, early prenatal genetic testing, pre- and post-test genetic counseling was crucial in the management of the present pregnancy, to prevent the birth of a new affected CF child.

The crucial role of SRY gene in the determination of human genetic sex: 46,XX disorder of sex development.

Prenatal diagnosis of disorder of sex development (DSD) is very rare and is estimated to occur in 1∕2500 pregnancies. A group of DSDs are the 46,XX testicular DSD. Today, the incidence of 46,XX testicular DSD is estimated at 1∕20 000 newborn males. A majority of males with DSD have an unbalanced X;Y exchange involving the pseudoautosomal region, with translocation of the sex-determining region of the Y (SRY) gene onto Xp23.3. We present a rare case of very early prenatal diagnosis and management of a fetus with SRY-positive 46,XX testicular DSD.