Standard Operating Procedure for Lyssavirus Surveillance of the Bat Population in Taiwan

1. Safety precautions when handling lyssaviruses

1. Ensure that all laboratory workers handling bat specimens receive pre-exposure rabies prophylaxis¹⁷. Monitor the rabies antibody levels of the workers beforehand and re-examine them every 6 months¹⁷. Follow-up rabies vaccination is required for those whose antibody levels are lower than 0.5 IU/mL¹⁷.
2. Depending on the biosafety regulations of the country where the laboratory is located, ensure that the following procedures are performed in suitable biosafety level laboratories (e.g., BSL-2 laboratories in Taiwan and BSL-3 laboratories in Australia), and that workers are currently qualified and wear proper personal protective equipment¹⁸.
   NOTE: Rabies vaccination provides little to no protection from lyssaviruses belonging to phylogroups II and III¹⁸. Workers must be informed that several zoonotic pathogens have been identified in bats¹⁹ and should handle samples under suitable biosafety level laboratory conditions with proper personal protective equipment.

2. Sample collection

1. Use found weak or sick bats or carcasses.
   NOTE: Weak or sick bats are delivered to the Bat Conservation Society of Taipei for veterinary care or research, while carcasses are submitted directly to the Animal Health Research Institute. No healthy bats have been euthanized in this surveillance program.
2. Have a bat ecologist identify bat species through morphological characteristics²⁰.
3. Perform DNA barcoding of the bat species when lyssavirus positive is diagnosed, using previously published procedures²¹.
4. Submit an information sheet of each bat carcass (collection site, species, clinical signs, etc.).

3. Necropsy of bat specimens

1. Prepare materials.
   1. Prepare a clean dissection board and place a sterile absorbent pad for necropsy.
   2. Prepare collection tubes for collecting bat organs.
   3. Prepare disposable tweezers and scalpels for necropsy. Change tools between each bat’s necropsy. Prepare cotton balls moistened with 70% ethanol for cleaning tweezers and scalpels during sampling.
   4. Prepare two microscope slides for the FAT. Collect fresh specimens and fix them in formalin solution for histopathological examination.
2. Examine all external orifices before necropsy. Photograph the bat’s external features, especially the head, ears, and wings, for species differentiation.
3. Collect an oral swab sample. Place the bat in ventral recumbency on the board and fix the bat’s head with tweezers. Cut the skin along the midline of the calvaria with a scalpel and pull the skin to the lateral sides. Cut the skull along the midline of the calvaria with a scalpel and open it with tweezers to expose the brain tissue.
4. Remove the brain tissue from the skull and place it on a sterile tongue depressor, and make impression smears from the brain tissue (see step 4.2). Collect a small piece of fresh brain tissue and fix it in formalin for histopathological examination. Contain the remaining brain tissue in a tube for nucleic acid extraction.
5. Clean tweezers and scalpel with cotton balls moistened with 70% ethanol to remove retained tissues between specimen collection.
6. Place the bat on the board in dorsal recumbency and fix it on the board with needles at both sides of the axilla and tail heel.
7. Incise the skin along the midline of the body from mandible to anus. Lift and separate the skin and underlying muscle tissues with tweezers. Collect the salivary glands, which are near the mandibular bone.
8. Lift the sternum slightly with tweezers, and cut the sternum and abdominal wall along the midline with a scalpel. Cut the clavicles with a scalpel. Fix the left and right rib cages to the board with needles to open the thoracic cavity.
9. Record the gross lesions and the degree of post-mortem change.
10. Remove the visceral tissues (i.e., heart, lungs, liver, kidneys, intestines) from the carcass using tweezers and a scalpel. Collect the visceral specimens as needed for future research.
    NOTE: Collection of duplicate samples is recommended. One should be collected for molecular diagnosis, and the other should be frozen at -80 °C with or without viral transport medium for viral culture²².

4. Direct fluorescent antibody test (FAT)

1. Make impression smears of the brain tissue for the FAT. Perform the FAT as previously described^18,23 with the following modifications.
2. Gently separate the brain tissue from the connected nerve tissue with tweezers and transfer the brain tissue to a sterile tongue depressor. Cut the cross-section of the brain, including the brain stem and cerebellum^18,23. Make impression smears of the brain tissue by lightly touching the cut surface of the brain tissue, and press the slide on lens tissue to remove excess tissue.
3. Fix the slides with acetone at -20 °C for 30 min. Dry the tested slides and the positive and negative controls before staining with conjugate.
4. Using each of the two commercially available FITC-conjugated anti-rabies antibodies for staining lyssavirus antigen is highly recommended²³. Determine the working concentration of the commercial conjugate before the first staining. Drop the diluted conjugates through a 0.45 μM syringe filter onto the slides and incubate the slides at 37 °C for 30 min within a wet chamber.
5. Drain the excess conjugate from the slides and wash the slides with phosphate-buffered saline (PBS) after incubation.
6. Drop a small amount of 10% glycerol on the slides and cover with cover slides.
7. Examine the slides with a fluorescent microscope.

5. Nucleic acid extraction

1. Add the proper volume of MEM-10 (minimum essential medium supplemented with 10% fetal bovine serum) to the brain tissue (10% w/v).
2. Homogenate the brain tissue with a 5 mm steel bead in a homogenizer instrument and centrifuge at 825 x g for 10 min.
3. Extract the nucleic acid, the final volume of which is 50 μL, within 200 μL of the supernatant using the commercially available total nucleic acid extraction kit with instrument.

6. RT-PCR and phylogenetic analysis

NOTE: Several primer sets have been published to detect all known lyssaviruses or specific lyssaviruses. The protocol described here is an example that our laboratory uses and may not fit all experimental needs. Select suitable primers according to laboratory needs.

1. Prepare the one-step RT-PCR reagent as follows: add 5 μL of extracted nucleic acid to the reaction mixture containing 2.5 μL of 10x reaction buffer, 0.5 μL of forward and reverse primers (10 μM of each), 4 μL of 1.25 mM dNTP, 0.3 μL of RNase inhibitor (40 U/µL), 0.3 μL of reverse transcriptase (10 U/µL), 0.4 μL of DNA polymerase (5 U/µL), and 11.5 μL of DEPC-treated water.
   NOTE: The primer set used in this protocol is JW12 (5'-ATGTAACACCYCTACAATG-3') and N165-146 (5'-GCAGGGTAYTTRTACTCATA-3')²⁴. Modify the preparation of reagents and cycling conditions according to the primer sets used.
2. Perform the cycling under the following conditions: incubation at 42 °C for 40 min; initial denaturation at 94 °C for 10 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; and finally, further extension at 72 °C for 10 min.
3. Use another or more primer sets to increase the diagnostic sensitivity.
   1. Use N113F (5'-GTAGGATGCTATATGGG-3') and N304R (5'-TTGACGAAGATCTTGCTCAT-3')^25,26 to prepare the one-step RT-PCR reagent as follows: add 5 μL of extracted nucleic acid to a reaction mixture containing 5 μL of 10x buffer, 5 μL of forward and reverse primers (4 μM of each), 5 μL of 1.25 mM dNTP, 0.5 μL of RNase inhibitor (40 U/µL), 0.2 μL of reverse transcriptase (10 U/µL), 1 μL of DNA polymerase (5 U/µL), and 23.3 μL of DEPC-treated water.
   2. Perform the cycling under the following conditions: incubation at 42 °C for 40 min; initial denaturation at 95 °C for 5 min; 35 cycles of 95 °C for 1 min, 55 °C for 1 min and 20 s, and 72 °C for 1 min; and finally, further extension at 72 °C for 10 min.
      NOTE: Depending on laboratory needs, choose suitable primers for diagnosis. N113F was originally designed for rabies virus amplification, but it may not work well for other lyssaviruses. The set of N113F and N304R works well for rabies virus (Taiwan ferret badger variant) and Taiwan bat lyssavirus. It will be easier to obtain whole nucleoprotein sequences by using the set of JW12 and N304R primers if the lyssavirus is amplified by both of the above two primer sets.
4. Analyze the PCR product on 2% agarose gel electrophoresis and visualize by UV light illumination.
5. Sequence the PCR product by commercial sequencing service.
6. Enter or upload the sequence to the webpage of the Nucleotide Basic Local Alignment Search Tool (BLAST). Select the "others (nr etc.)" database and enter the organism as Lyssavirus. Select the MegaBlast algorithm and run the BLAST.

7. Virus isolation

NOTE: Perform virus isolation when either 1) the FAT or 2) the RT-PCR indicates positivity.

1. Homogenize the brain specimen in a 10% (w/v) suspension in MEM-10. Centrifuge at 825 x g for 10 min.
2. Inoculate 200 µL of supernatant with a suspension of 3 x 10⁶ MNA (mouse neuroblastoma) cells in 1 mL of MEM-10 for 1 h at 37 °C with 1% CO₂. Transfer the brain homogenate-cell suspension to a 25 cm² flask and add 6 mL of MEM-10.
3. Cultivate 1 mL of the brain homogenate-cell suspension in a 4 well Teflon-coated glass slide with 6 mm diameter at the same time.
4. After 3–4 days of incubation at 37 °C with 1% CO₂, fix the cells on the 4 well slide with 100% acetone (v/v).
5. Stain the slides with two FITC-conjugated anti-rabies antibodies following steps 4.4–4.7. The cells are infected when the intracytoplasmic inclusions are investigated. Record the percentage of infected cells.
6. Perform trypsinization and subculture of the inoculated cell culture when the slides are stained as negative:
   1. Remove medium and rinse the flask with 5 mL of PBS.
   2. Add 1 mL of trypsin to the flask and firmly strike the bottom of the flask.
   3. Add 6 mL of MEM-10 and resuspend the cells.
   4. Put the cell suspension into a new tissue flask (6 mL) and onto a 4 well slide (1 mL).
7. Repeat steps 7.4–7.6 until 100% infectivity is reached.
8. Collect the supernatant after 24 h of incubation.