Task for perform 2. Continue field expeditions, SOPs trouble shootings, first analysis on positive rates and typing data. (Year 1, Year 2, Year 3)

Subtask 2. 1. Continue collection of bats and biological specimens (NCDC, IECVM, US-CDC and USGS);

Subtask 2. 2. Follow up on the data quality stored and data base management and curation (Virginia Tech and US-CDC).

Subtask 2. 3. Follow up on correct execution of SOPs (QA/QC activities), review data of screening tests conducted on the first year, trouble shooting of laboratory procedures in case of necessary. Preliminary analysis of typing data (Activities coordinated by US-CDC, NCDC).

Field activities will include bat capture, sampling, field work and biomaterial transportation to the laboratory in compliance to current biosafety/biosecurity and bio-ethic protocols. Bats will be trapped from different urban roosts and in wild ecosystems according to CDC/USGS best practices. The expected number of examined animals for three years of the project is about 850 in Ukraine and 650 in Georgia. Bats (in Ukraine and Georgia) will be mist-netted, captured and sampled in breeding season (May-July), during migration (August-September) and at the beginning of hibernation (November-December) in different geographic areas of Ukraine and Georgia. The field sites in Ukraine will located in three different regions: Carpathian mountain region (West Ukraine), forest regions (the North Ukraine), steppe and forest-steppe regions (the Eastern and Central Ukraine). There will three regions covered across Georgia: West Georgia Imereti and Samegrelo region, East Georgia region (Kakheti) and East Georgia region (Gardabani Managed Reserve) (Fig. 1).

Sampling. Two types of sampling (lethal and non-lethal) will be conducted during this investigation, no more than 120 bats in Georgia and no more than 150 bats in Ukraine during three years will be euthanatized. Nonlethal samplings will consist of collection of feces or rectal swabs, urine, oropharyngeal swabs will be obtained from live animals. Alternatively, collecting fresh feces from caves or roost floors may be used for overall screening of roosts. In the case of mono species colonies fresh feces will be used only. The species of bats in the colony will be clearly identified. In the case of several bat’s species in the colony, samples will not be taken. Individually located faces will be sampled, which will be considered as the samples from one animal. When using fresh feces collected from the colony, the percentage of positive single feces samples will be evaluated. The last one will indicate about the total colony rate of infectivity. The size of the colony and the total number of animals will also be taken into account. Tissue will be collected as biological samples from dead or euthanized animals. Also we will collect GIS data about study site (position data, place of sampling, urban or wild place), environment characteristics (landscape, weather conditions, season of year) and bat (number of animals, number of species in the place, physiological and health status, age, gender, etc. ).

Sample transportation and storage. Common SOPs regarding transportation, storage, disinfection and utilization of samples according to CBEP and US CDC standards will be developed before the beginning of the project. Appropriate records for incoming biological specimens will be kept in PACS system in laboratories. Collected samples will be transported in dry shipping liquid nitrogen tanks. Frozen samples will be stored in -80⁰C Ultra freezers in BSL 2 facility. All samples will be autoclaved after completion of all studies (at least 12 months after the project end date). Access (only for authorized persons) to all samples collected and data generated during the project will be up to and including at least 12 months after the project end date.

Landscape Characterization. To explore patterns of landscape characteristics in the study area, we will 16-day composite enhanced vegetation index (EVI) images at 250 m spatial resolution from the MODIS sensor (MOD13A1) on the Terra satellite (https: //lpdaac. usgs. gov/data_access/data_pool).

Urbanization Estimation. We will use nighttime-light satellite-derived data as a proxy of levels of urbanization. Urbanization levels will be characterized in the form of a nighttime-light satellite image at ~0. 75 km resolution matching our first fieldwork period. Data will be collected by the VIIRS sensor, at the Suomi NPP satellite (http: //1. usa. gov/1FQvs5r).

Bat Diversity Estimation. Mammals will be captured across Ukraine and Georgia based on vegetation phenology and urbanization level, ranging from urban to dense forest, to achieve different species configurations and different levels of biodiversity.

Task for perform 3. Continue laboratory studies on biological material. (Year 1, Year 2, Year 3).

Subtask 3. 1. Processing of specimens (feces, urine and swab samples to detect nucleic acid of all agents of interest via PCR.

Subtask 3. 2. Typing of positive samples through sequencing and phylogenetic inference.

Subtask 3. 3. Full genome characterization through NGS data, standardization of an analytical pipeline (US-CDC trainings), phylogenetic analysis and application of comparative genomics approaches to determine relatedness to previously identified pathogens (Training activities lead by US-CDC).

Subtask 3. 4. Implementation of data integration applications for special visualization of all generated data (sequence data, environmental variables), comparative genomic training. This activity will be lead by US CDC.

Subtask 3. 5. Data integration continuation, development of ecological niche models (activity lead by Virginia Tech and US-CDC).

Laboratory studies. The project will focus on detection (PCR only) of Filoviruses, Paramyxoviruses, Lyssaviruses, Orthomyxoviruses, Brucella spp, Leptospira spp, Yersinia spp and in both countries and additional Coronaviruses (only in Ukraine). Study of coronaviruses in Georgia is included in other current project. During laboratory studies we will determine the presence of pathogens in the collected field samples. The presence of pathogens will be determined by genome detection in biological material. For detection all pathogens (except orthomyxoviruses) we will use SOP and methodology that will be provided CDC team. SOP and methodology for detection orthomyxoviruses will be provided USGS team.

Regarding viral agents, automated total RNA extraction for solid tissue pools (stool pellets, rectal swabs, lung, kidney, spleen, liver and intestines) per individual will be carried out. Total RNA pools made by mixing individual total RNA of 3-5 individuals of the same species and the same collection point will be prepared and subsequently screened by two to four independent real time RT-PCR or nested pan viral group end point PCR assays per each viral family or genus of interest. Two assays would be used for coronaviruses, two for filoviruses, four for paramyxoviruses, two for lyssaviruses, and one for orthomyxoviruses.

All assay’s designs use broadly reactive primers and probes to increase the detection sensitivity for new viral species. Samples in all positive pools will be re-screened by real time RT-PCR to determine the exact number of positive individuals per pool. Subsequent, individual end point RT-PCR in all positive individuals will target the amplification of highly informative genes or loci (2 to 3 kb), which then will be Sanger sequenced to determine the diversity spectrum for each viral family. The presence of pathogens will be calculated according to the results of laboratory studies. Statistical analyses will performed using general statistics methods (Pearson chi-square (χ ²) or Fisher’s exact tests). Statistical analysis will be carried out using software program R (R Core Team 2019) or other.

Appropriate protocols for geocoding and importing data on the EDP’s will be developed to ensure that data are captured in an appropriately formatted spreadsheet or comma-delimited text file. Once an array of disease data has been converted into digital GIS formats, will focus on concepts regarding spatial analysis, cartography, and methods for producing different types of maps for visualizing disease distributions. Topics covered will include: map projections, data selection, vector data analysis, raster analysis, data integration, data symbolization, and map design.

Place of project implementation and its technical equipment

In Ukraine the project will be implemented at the National Scientific Center " Institute of Experimental and Clinical Veterinary Medicine" of the NAAS (83 Pushkinskaya Street, Kharkiv). Units of NSC " IECVM" that will be involved in the project are equipped with basic machinery for conventional PCR, qPCR, low-temperature refrigerators, class 2 biosafety rooms, centrifuges with cooling, water baths, autoclaves, drying chambers and computer for data analysis. In addition, all rooms involved in the project have ventilation with HEPA filters providing a pressure difference in accordance with the BSL2 + classification.

In Georgia the project will be implemented at National Center for Disease Control and Public Health; Richard G. Lugar Center for Public Health Research (NCDC, Georgia) provides national leadership in preventing and controlling communicable and non-communicable diseases, disease surveillance, immunization, laboratory work, research, and responding to public health emergencies. The Lugar center is top-tiered institution in NCDC’s laboratory network and serves as a reference laboratory of the Georgia’s public health system. The Lugar Center’s possesses a BSL-3 facility and BSL-2 space with following laboratories: Bacteriology, Virology, Molecular Biology/Genomics, Cell culture, Parasitology, Entomology, Vivarium, and the National Repository of human and animal EDPs. Since the Lugar Center is Georgia’s only facility where the work with EDPs is conducted, safety and security are our primary concerns. We have robust engineering control in place, with double redundancy for all major facility systems. We have general and EDP-specific emergency response plans, planned emergency drills and identified first responders.

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