VECTORSCAN

Investigating human TTV (Torque Teno Virus) as new emerging viruses from arthropod vectors -

Project VECTORSCAN - PN-III-P1-1.1-PD-2019-0496

Contract no. 158/2020 University of Bucharest

Department of Genetics

Budget 246.950,00 RON / 49.900,00 EURO

Duration: 24 months (september 2020 - august 2022)

Finance Agency: RO-UEFISCDI

Team:

 Abstract:

Torque Teno Virus (TTV) is an emergent virus infecting humans. Despite being found in worldwide population and in almost every type of cell of the human body, TTV still eludes the pathogen stigma. Many other aspects of TTV biology remain untackled or unanswered, including the possibility of vector-borne transmission of TTV. The presence of human TTV in mosquitoes and the potential of vector transmission route or cycle of TTV have never been tested before.

The project aims at a world premiere in estimating the potential of horizontal (from infected mosquitoes to human hosts) and vertical (from infected females and/or males to offspring) transmission of TTV in mosquitoes employing a multidisciplinary approach and modern techniques.

This project aims to identify and evaluate a new possible transmission route or cycle of transmission for human Torque Teno Virus (TTV) - vector-borne transmission – by detecting and characterizing viral isolates from mosquito vectors collected in Romania.

The specific objectives are : optimization of TTV DNA detection methods using mosquitoes as biological samples, detection and molecular characterization of TTV DNA, as well as metagenomic (virome) analysis of mosquitoes collected for the proposed project.

The estimated results of the project consists in the evaluation of a new transmission route or cycle of TTV, as well as the understanding of the biology and ecology of TTV by identifying new hosts - mosquitoes - and identifying new cellular tropisms. The project undertakes the promotion of broad dissemination of the results by publication of original research paper and scientific communications at international conferences.

VECTORSCAN - Summary update 2022

In the third phase of the project the mosquito collection was enriched with samples collected in 2021. A total number of 13368 mosquitoes grouped in 408 pools were analyzed throughout the entire project duration. TTV DNA presence was confirmed in 15 pools (~4%). All positive samples derived from pools of female mosquitoes, both Culex pipiens (n=9) and Aedes albopictus (n=6). TTV DNA was not found in any pool of male mosquitos, suggesting an unlikely vertical transmission of TTV in mosquitoes. Also, none of the samples collected in the Danube Delta were positive for TTV DNA; as the main source of blood meal for these mosquitoes are birds, the lack of TTV DNA in these samples is supporting the hypothesis of horizontal transmission of the virus from infected humans to mosquitoes.

The main activities were, however, focused on obtaining suitable material for the mosquito metagenome (virome) analysis. Since TTV is a virus with single-stranded DNA genome and most next-generation sequencing library preparation strategies eliminate single-stranded DNA, coupled with the fact that TTV is scarce in mosquitoes, its viral DNA had to me enriched. Thus, mosquito samples were pooled together by sex, species, and collection date and site in pools of about 250 individuals. DNA was extracted and amplified using a polymerase which amplifies preferentially circular single-stranded DNA. Samples prepared this way were subjected to paired-end next-generation sequencing on the NovaSeq platform. The obtained data is currently undergoing bioinformatic analysis.

Publications:

1. Phylogenetic analysis of torque teno virus in Romania: possible evidence of distinct geographical distribution, Sonia Spandole-Dinu, Dănuţ Cimponeriu, Ileana Stoica, Oana Apircioaie, Larisa Gogianu, Lavinia Mariana Berca, Silvia Nica, Mihai Toma, Remus Nica. Archives of Virology, published online 13 August 2022, doi 10.1007/s00705-022-05559-8.

2. Detection of torquetenovirus DNA in unfed mosquitoes collected in Romania, Sonia Spandole-Dinu, Liviu Prioteasa, Georgiana Victorița Tiron, Ioana Georgeta Stancu, Elena Fălcuță, Ani Ioana Cotar, Dănuț Cimponeriu, Ileana Stoica. 32nd European Congress of Clinical Microbiology and Infectious Diseases, 23 – 26 April 2022, Lisbon, Portugal.

VECTORSCAN - Summary update 2021

The main activities in the second phase of the projects were to obtain the genetic material from the biological specimens collected in the previous phase and to detect and characterize the TTV isolates infecting mosquitoes by using molecular tools.

Total DNA from 379 pools obtained from 12643 mosquitoes specimens collected between 2013 and 2020 from various types of urban and natural habitats in Romania was extracted and purified using methods optimized in the previous year. The specimens analyzed belonged to the species Culex pipiens (n = 11564), Aedes albopictus (n = 1733), Culex modestus (n = 191), and to the species Coquillettidia richardii (n = 103).

Bioinformatic analysis of TTV genome was performed and primers spanning 2 untranstaled regions (3’UTR and 5’UTR) and one coding region (hipervariable region of ORF1) were selected for identification of TTV DNA in mosquito samples. PCRs were optimized using template DNA obtained from mosquito homogenates.

TTV DNA screening of mosquito samples was assessed by end-point PCR and real-time PCR. Five out of 379 (1.32%) pools of mosquitoes were confirmed TTV DNA-positive by sequencing. The sequences were submitted to European Archive of Nucleotide (ENA) and are currently under curation. All positive pools were made out of female specimens collected from Bucharest; two of the pools contained Aedes albopictus mosquitos, while the other 3 were comprised of Culex pipiens.

The phylogenetic analysis showed sequences obtained from 3 pools clustering with sequences from human TTV isolates, as well as unassigned anellovirus species with worldwide distribution. The other 2 sequences, spanning a different region of TTV genome, exhibited high similarities with human TTV isolates from Romania and other European countries (e.g. Poland, Germany).

VECTORSCAN - Summary update 2020

The first phase of the project focused on the collection of biological material and optimization of DNA extraction and purification.

A total number of 7124 mosquito females were collected between June and September 2020 from 18 sites located in various types of urban habitats (aquatic areas, green areas and residential areas) in Bucharest. Mosquito collection and identification were carried out in collaboration with dr. Elena Fălcuță and dr. Liviu Florian Prioteasa, Medical Entomology Laboratory, I.N.C.D.M.M. "Cantacuzino". The specimens collected belong to the species Culex pipiens (n = 6893) known as the main vector of West Nile virus in Romania (Savage, 1999; Dinu, 2015), and to the species Aedes albopictus (n = 231), an invasive species reported in Bucharest since 2012 (Prioteasa, 2015) and known as a vector for several arboviruses, but also for the parasite Dirofilaria. Mosquitoes were pooled by species, sex, physiological age, place of collection in pools of 14-50 specimens, resulting in 194 pools of Culex pipiens and 6 pools of Aedes albopictus. The biological material was homogenized and stored at -70^oC. This activity was carried out with the support of dr. Ani Ioana Cotar, Laboratory of Infections transmitted by vectors, I.N.C.D.M.M. "Cantacuzino".

Twelve pools from the previous collections of Vector-Borne Infections and Medical Entomology laboratories within I.N.C.D.M.M. "Cantacuzino” were randomly selected to optimize DNA extraction and purification method. DNA quality testing was performed by PCR amplification of a fragment of the mitochondrial gene encoding Cytochrome c oxidase subunit I. Amplification products were obtained for all DNA extracts tested, which demonstrates that the extracted DNA is qualitatively suitable for the PCR technique.