Field Information Report — pp. 5–8

PEDv Virus Expansion ae e PEDvcan be grown on Vero-76 or Vero-81 cell lines. The virus grows at the same efficiency in ab conditions. e Vero-81 cells seem to be more resilient to Trypsin and may be the preferred cell line for assays requiring long incubations in the PEDv Maintenance Media. Materials: o PEDv Maintenance Media o 5ml Sterile Individually wrapped pipettes for Pipet Aid o 10ml Sterile Individually wrapped pipettes for Pipet Aid o Sterile 1x ▮ (no ▮ [redacted] ▮ o 70% EtOH o Water bath set at 37°C o 500ml! Plastic waste container o Gloves o Laminar flow hood o Paper Towels o Confluent flasks of Vero cells o 1imL of PEDv o Conical holder/Rack © Sterile 50ml conicals o Microcentrifuge tubes Safety: ▮ Wear gloves and lab coat while performing this procedure. ▮ This procedure is performed in a laminar flow hood ▮ Complete all training required by the University of Minnesota and the Department of Environmental and Health & Safety guidelines Method 1) Heat ▮ in water bath for 15 minutes 2) Bring PEDv aliquot to room temperature 3) Sterilize workspace in laminar flow hood by spraying area with 70% EtOH and wiping with paper towels 4) Sterilize Pipet Aid and plastic waste container by spraying with 70% EtOH and wiping with paper towels prior to placing in hood. 5) Spray gloved hands with 70% EtOH and rub hands together until EtOH has evaporated before opening incubator and handling cell culture flasks 6) Bring confluent T75 flask of Vero cells into the hood 7) Sterilize the exterior of ▮ bottle by spraying with 70% EtOH and wiping with paper towels. Place in hood 8) Draw up media from T75 flask of cells with Pipet Aid and a 10m pipette. Draw from lower left corner of flask without disrupting the monolayer 9) Discard media in plastic waste container and pipette tip in the tip waste container 10)Unscrew cap of ▮ bottle and let the cap sit loosely on top of the bottle ▮ Use a 5m! pipette and Pipet Aid to draw up 3ml of ▮ Use dominant hand to control Pipet Aid and the other to lift loosened ▮ cap. 12) Place cap back on ▮ bottle after drawing up desired volume. 13) Gently dispense 3ml of 1x ▮ onto Vero cells by uncapping the flask and holding the flask at a 45° angle. Gently dispense the ▮ by the top opening of the flask and let the ▮ slowly run down the length of flask. 14) Cap the flask and rotate flask so ▮ has covered monolayer of cells 15) Uncap flask and draw up the ▮ with 5ml pipette. Draw from lower left corner of flask without disrupting the monolayer 16) Discard ▮ in the plastic waste container. 17) Inoculate cells by adding 1m] of PEDv to cells. 18) Recap flask and place in incubator (37°C, 5% COz) for 1 hour 19)During incubation, place PEDv Maintenance Media in water bath 20) After 1 hour incubation, follow the same procedure in taking the flask out of the incubator and placing it in the laminar flow hood 21) Sterilize the exterior of PEDv Maintenance Media by spraying with 70% EtOH and wiping with paper towels. Place in hood. ▮ Add 8ml of warm PEDv Maintenance Media to flask 23) Collect 200ul aliquot for ▮ Store at -20°C 24) Incubate at 37°C, 5% CO2 for 3 days. Virus induced cyotopathic effect should be visible under the microscope in Vero-76 by day 3 post infection 3 days later... 25) On day 3 post infection, freeze and thaw the flask 3 times at -20°C and room temperature. Follow instructions on Step 5 to take flask out of incubator 26) Sterilize laminar flow hood as instructed in Step 3 27) Sterilize Pipette Aid and conical holder/rack as instructed on Step 4 28) With a 10m] pipette and Pipette Aid, draw up everything in flask and transfer to a sterile 50ml conical 29) Centrifuge conical at 1500rpm for 10 minutes at room temperature 30) Collect supernatant and transfer to a new 50m! conical. Discard old conical with pellet 31) Collect 200ul aliquot for ▮ If continuing to expand: a) Repeat Steps 6-16 b) Use 4ml of newly collected virus to inoculate new T75s confluent with Vero cells. c) Repeat steps 18-22 d) Incubate at 37°C, 5% CO2 e) Check daily for ▮ Do not incubate for more than 3 days If harvesting: a) Filter newly collected virus in .22um filter b) Aliquot into desired volume c) Place and store at -80°C DEPARTMENT OF HOMELAND SECURITY CUSTOMS AND BORDER PROTECTION FIELD INFORMATION REPORT Product Title: University of Minnesota Professor and Virologist Fang Li and Associates Product ID: (U) FOR OFFICIAL USE ONLY (FOUO) WARNING: This document is UNCLASSIFIED//FOR OFFICIAL USE ONLY(U//FOUO). It contains information that may be exempt from public release under the Freedom of Information Act (5U.S.C. 552). It is to be controlled, stored, handled, transmitted, distributed, and disposed of in accordance with DHS policy relating to FOUO information and is not to be released to the public, the media, or other personnel who do not have a valid need-to-know without prior approval of an authorized DHS official. (U) FOR OFFICIAL USE ONLY/LAW ENFORCEMENT SENSITIVE (FOUO/LES) WARNING: This document is FOR OFFICIAL USE ONLY/LAW ENFORCEMENT SENSITIVE (FOUO/LES). This document may be shared with DHS personnel who have a need to know but any sharing outside of DHS requires the written approval of the author. If approved for dissemination outside of DHS, the report may only be used for intelligence and investigative lead purposes. Any other use is prohibited unless prior written authorization is received from the author. Prior written authorization from CBP Office of Chief Counsel is required before providing this material to the Department of Justice or other authority for purposes of litigation, including disclosure in a civil or criminal case. (U) US PERSON INFORMATION (USPER) WARNING: This product contains U.S. person information that has been deemed necessary for the intended recipient to understand, assess, or act on the information provided. It has been highlighted in this document with the label USPER and should be handled in accordance with the recipient's intelligence oversight/information handling procedures. U.S. person information must be protected in accordance with all relevant laws and policies of the receiving entity.