Experimental design, monitoring, and specimen collection

The 24 ERBs were divided into 2 study arms: 9 bats were assigned to the serial sampling arm and 15 bats were assigned to the serial euthanasia arm. Bats in the serial sampling arm were acclimated to the BSL-4 laboratory for 5 days before beginning the study. To mimic a tick bite, all bats were inoculated by the intradermal route in the subcaudal abdominal region under isoflurane anesthesia at 0 DPI. 7 bats (4 males and 3 females) received 4.0 log₁₀ tissue culture infectious dose 50 (TCID₅₀) of the UGA-Tick-20170128 strain of KASV (Vero E6 + 2 passages; mycoplasma-free; sequence identical to GenBank Accession Numbers {"type":"entrez-nucleotide","attrs":{"text":"MT309090","term_id":"1904824910","term_text":"MT309090"}}MT309090, {"type":"entrez-nucleotide","attrs":{"text":"MT309094","term_id":"1904824918","term_text":"MT309094"}}MT309094, and {"type":"entrez-nucleotide","attrs":{"text":"MT309097","term_id":"1904824924","term_text":"MT309097"}}MT309097 [Vero E6 + 1]) prepared in 0.1 mL of sterile phosphate buffered saline (PBS), and 2 negative control (NEG CO) bats (2 males) received 0.1 mL of sterile PBS. The KASV inoculum dose was selected based on the estimated volume of saliva secreted by feeding Ornithodoros spp. ticks (10 µL)³⁴ and the KASV titer of a pool of 5 unengorged O. (R.) faini ticks (4.1 log₁₀TCID₅₀ equivalents (eq)/10 µL). The KASV-inoculated bats were housed in a single flight cage maintained within a bio-flow isolator with HEPA-filtered inlet and exhaust air supplies (Duo-Flow Mobile Units, Lab Products Inc., Seaford, DE), while the NEG CO bats were housed in a non-human primate-sized cage maintained within a second bio-flow isolator unit. Temperatures, blood, duplicate oral swabs, rectal swabs, and urine (opportunistically) were collected from all bats daily (beginning at 0 DPI for temperatures and blood, and 1 DPI for oral swabs, rectal swabs, and urine) to the end of the study, while weights were taken on a weekly basis. At 18 and 20 DPI, the bats were euthanized by an overdose of isoflurane followed by cardiac exsanguination.

Blood was taken from the cephalic vein using a sterile lancet (C&A Scientific, Manassas, VA), polyester-tipped applicators (Fisher Scientific, Grand Island, NY) were used to swab the oral mucosa, and rectal swabs were obtained opportunistically at the time rectal temperatures were taken by repurposing the plastic thermometer probe cover (MABIS Healthcare, Waukegan, IL). Opportunistic urine collection was attempted by allowing a single bat to hang in a sterile mouse cage fitted with an inverted wire top (Thoren Caging, Hazleton, PA) and later collecting the accumulated urine. Aliquots of whole blood were used to monitor for viremia (KASV RNA by quantitative reverse transcriptase-polymerase chain reaction [qRT-PCR]) and antibody responses (anti-KASV IgG by indirect enzyme-linked immunosorbent assay [ELISA]). Oral swabs, rectal swabs and urine were used to detect virus shedding (KASV RNA by qRT-PCR).

Bats in the serial euthanasia arm were acclimated to the BSL-4 laboratory for 10 days before beginning the study. All bats were inoculated by the intradermal route in the subcaudal abdominal region under isoflurane anesthesia at 0 DPI. 12 bats (8 males and 4 females) received 4 log₁₀TCID₅₀ of the UGA-Tick-20170128 strain of KASV prepared in 0.1 mL of sterile PBS and 3 NEG CO bats (1 males and 2 females) received 0.1 mL of sterile PBS. The KASV-inoculated bats were housed in primate-sized cages according to the DPI of euthanasia (3, 6, 9, and 12; each euthanasia group included 2 males and 1 female) that were maintained within a bio-flow isolator unit, while the NEG CO bats were housed in a non-human primate-sized cage maintained within a second bio-flow isolator unit. All bats were euthanized by an overdose of isoflurane followed by cardiac exsanguination. Temperatures, weights, and blood were taken from all bats at 0 DPI and at euthanasia. Aliquots of whole blood collected at 0 DPI and at euthanasia were used to assess viremia and antibody responses. Serum collected at euthanasia was used to perform clinical chemistries and attempt virus isolation (if corresponding whole blood specimen was positive for KASV RNA). Tissues (skin at the inoculation site, skin distal to the inoculation site, axillary lymph node, salivary gland, gonads, inguinal lymph node, small intestine, liver, spleen, heart, lung, kidney, colon/rectum, and brain) collected at necropsy from all bats in the serial euthanasia arm and randomly-selected bats euthanized at 18 (n = 3 bats) and 20 DPI (n = 3 bats) from the serial sampling arm were tested for KASV RNA by qRT-PCR to determine virus-tissue tropism. Following necropsy, bat carcasses and remaining tissues were immersed in 10% neutral buffered formalin for a minimum of 7 days in BSL-4 containment, with a complete formalin replacement occurring at least 3 days prior to processing for histopathology.