Generation and Characterization of Recombinant ECTV

The production of ECTV stocks for infection of mice and the determination of titers in stocks and organs were done as described previously [8 (link)]. To generate ECTV 189898-p7.5-EGFP, we adapted the method described by Johnston and McFadden [60 (link)]. Briefly, a construct containing the ECTV Moscow fragment 189543–189897, the VACV early/late promoter p7.5, the sequence of EGFP, and the ECTV Moscow fragment 189950–190297, in that order, was made by recombinant PCR and cloned into plasmid Bluescript II SK+ to generate the targeting vector pBS-EVM189898-p7.5-EGFP. This targeting vector was used to transfect mouse A9 cells using Lipofectamine 2000 as per manufacturer's instructions (Invitrogen). The transfected cells were infected with wild-type ECTV (Moscow strain, 0.3 pfu/cell) in 6-well plates. 2 d later, transfected/infected A9 cells were harvested using a rubber policeman, frozen and thawed, and different dilutions of cell lysates were used to infect BSC-1 cells in 6-well plates. 2 h after infection, the cells were overlaid with media containing 0.5% agarose. 4 d later, green-fluorescent plaques were picked with a pipette tip and used to infect a new set of cells. The purification procedure was iterated five times until all plaques were fluorescent. The resulting virus, ECTV 189898-p7.5-EGFP, carries EGFP in a non-coding region and is as pathogenic as wild-type ECTV Moscow (not shown). For preparation of ECTV stock for infection of different cell lines, A9 cells were infected with 0.2 pfu ECTV/cell, and incubated at 37 °C, 5% CO₂. After 5 d, the cells were collected, frozen and thawed three times, and then sonicated in a water-bath sonicator. The solid material was pelleted by centrifugation, and the supernatant was stored in aliquots at −80 °C. The DAP10-deficient mice [61 (link),62 (link)] (generously provided by Dr. Joe Phillips) and DAP12-deficient mice [63 (link)] on the C57BL/6 background were bred at UCSF. All the other mice were bred at the Fox Chase Cancer Center Laboratory Animal Facility in specific pathogen-free rooms or were purchased from Jackson Laboratories. IFN-γ-deficient C57BL/6 mice were generously provided by Dr. Glenn Rall. For infections, sex-matched animals 8–12 wk old were transferred to a biosafety level 3 room. For ECTV infection, anesthetized mice were infected in the left footpad with 25 μl PBS containing 3 × 10³ pfu ECTV. Following infections, mice were observed daily for signs of disease (lethargy, ruffled hair, weight loss, skin rash, and eye secretions) and imminent death (unresponsiveness to touch and lack of voluntary movements). Moribund mice were euthanized by halothane inhalation. All of the experimental protocols involving animals were approved by the Fox Chase Cancer Center Institutional Animal Care and Use Committee.
For ECTV infection of cells, 3–5 × 10⁵ cells were plated in 6-well plates and cultured overnight to allow cells to adhere. The cells were then infected with 0.5 pfu ECTV/cell for 18 h, collected, washed, stained, and analyzed for surface expression of various markers. For ECTV infection of peritoneal cells, the mice were euthanized by halothane inhalation and injected i.p. with PBS, the abdomen massaged gently, and the peritoneal cells were collected by aspiration and washed.