canping-huang-phd-novel-virus-discovery-in-bat-isn-translation

3.1 Materials and Methods
3.1.1 Reagents and materials
The reagents, materials and equipment are the same as in Chapter 1.
3.1.2 Sample Collection
With the assistance of the staff from Yunnan Centers for Disease Control and Prevention
and the local Center for Disease Control, we caught 87 bats in an abandoned mining cave at
Tongguan Town, Mojiang Hani Autonomous County (N 23°10'36 E 101°21'28"). Of all the
bats, 84 were Rhinolophus sinicus and 3 were Miniopterus fuliginosus. At night, we set up a
rat trap in the cave. The following day we caught a wild rat. After we euthanized all the bats,
we collected tissue samples from the intestine, lung, liver, and spleen. Because of the size of
the bats, we weren’t able to take anal swabs. We followed the same procedures with the
wild rat. All tissue samples were kept separately in 2 ml Cryogenic Tubes. These were kept
in individual gauze bags and stored in liquid nitrogen immediately. They were sent to the
laboratory of the local Center for Disease Control to be stored until used for testing. This
study passed the review of the Ethics and Animal Welfare and Use Committee of the
Institute for Viral Disease Control and Prevention of the Chinese Center for Disease Control
and Prevention.
3.1.3 Sample Handling
The samples were taken out from the liquid Nitrogen in batches and were lined up in
numerical order. After defrosting, we removed an appropriate amount of tissue and ground
it in a sterilized glass hand mill. Grinding was done on ice. A small amount of antibiotic
(50,000 µg/mL vancomycin, 50,000 µg/mL amikacin, 10,000 units/ mL nystatin) was
added to the culture medium. After grinding, culture medium was added up to 1 ml and the
sample was transferred to a centrifuge tube. After centrifugation at 4° C at high speed,
leaving behind the pellet, the supernatant was carefully transferred to a new centrifuge
tube. 100 ul of supernatant was taken for nucleic acid extraction. The remainder was put
back into the -80° C freezer for storage.
3.1.4 Nucleic Acid Extraction
Nucleic acid extraction was done using the Qiagen Nucleic Acid test kit. For the full
procedure, see chapter two. The extracted nucleic acid was immediately used for reverse
transcription testing or kept in a -80° C freezer for storage.
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A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of
Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,
2016. Translation completed for Independent Science News in March 2021.