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

Summary

This protocol introduces a standard laboratory operating procedure for diagnostic testing of lyssavirus antigens in bats in Taiwan.

Abstract

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are natural reservoirs of most lyssaviruses, a lyssavirus surveillance program of bats has been conducted in Taiwan since 2008 to understand the ecology of these viruses in bats. In this program, non-governmental bat conservation organizations and local animal disease control centers cooperated to collect dead bats or bats dying of weakness or illness. Brain tissues of bats were obtained through necropsy and subjected to direct fluorescent antibody test (FAT) and reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus antigens and nucleic acids. For the FAT, at least two different rabies diagnosis conjugates are recommended. For the RT-PCR, two sets of primers (JW12/N165-146, N113F/N304R) are used to amplify a partial sequence of the lyssavirus nucleoprotein gene. This surveillance program monitors lyssaviruses and other zoonotic agents in bats. Taiwan bat lyssavirus is found in two cases of the Japanese pipistrelle (Pipistrellus abramus) in 2016–2017. These findings should inform the public, health professionals, and scientists of the potential risks of contacting bats and other wildlife.

Introduction

Viruses within the genus Lyssavirus are zoonotic pathogens. There are at least seven lyssavirus species associated with human cases¹. In addition to the 16 species in this genus^1,2,3, Taiwan bat lyssavirus (TWBLV)⁴ and Kotalahti bat lyssavirus⁵ have been recently identified in bats, but their taxonomic statuses have yet to be determined.

Bats are the natural hosts of most lyssaviruses, with the exception of Mokola lyssavirus and Ikoma lyssavirus, which have yet not been identified in any bats^1,2,3,6. The information on lyssaviruses in Asian bats is still limited. Two uncharacterized lyssaviruses in Asian bats (one in India and the other in Thailand)^7,8 have been reported. One human rabies case associated with a bat bite in China was reported in 2002, but the diagnosis was made only by clinical observation⁹. In Central Asia, Aravan lyssavirus was identified in the lesser mouse-eared bat (Myotis blythi) in Kyrgyzstan in 1991, and Khujand lyssavirus was identified in the whiskered bat (Myotis mystacinus) in Tajikistan in 2001¹⁰. In South Asia, Gannoruwa bat lyssavirus was identified in the Indian flying fox (Pteropus medius) in Sri Lanka in 2015³. In Southeast Asia, several serological studies on bats in the Philippines, Thailand, Bangladesh, Cambodia, and Vietnam showed variable seroprevalence^{11,12,13,14,15}. Although Irkut lyssavirus was identified in the greater tube-nosed bat (Murina leucogaster) in Jilin Province, China in 2012¹⁶, the exact species and locations of lyssaviruses in East Asian bat populations remain unknown.

To assess the presence of lyssavirus in Taiwanese bat populations, a surveillance program employing both direct FAT and RT-PCR was initiated. Taiwan bat lyssavirus was identified in two cases of the Japanese pipistrelle (Pipistrellus abramus)⁴ in 2016–2017. In the present article, a laboratory standard operating procedure is introduced for lyssavirus surveillance of the bat population in Taiwan. The flow chart of bat lyssavirus diagnosis in our laboratory is presented in Figure 1.

Protocol

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.

Results

From 2014 to May 2017, 332 bat carcasses from 13 species were collected for surveillance. Two tested positive. In the first bat case, the brain impression tested negative using the FAT with one of the commercial FITC-conjugated anti-rabies antibodies (Figure 2), while the RT-PCRs employing each of the two primer sets (JW12/N165-146, N113F/N304R) yielded positive results (Figure 3). A 428 bp sequence of amplicon (amplified with N113F/N304R and containing the part...

Discussion

This laboratory standard operating procedure (SOP) provides a serial process for testing bat samples for the presence of lyssavirus antigens in Taiwan. The key steps include the employment of FAT and RT-PCR. The selection of suitable samples and successful isolation of the virus are also important. Additionally, some troubleshooting was conducted during the monitoring of bat lyssaviruses. The major difference was the target animals. Initially (2008–2009), the target animals of bat lyssavirus surveillance were live ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2.5% Trypsin (10x)	Gibco	15090046	Trypsin
25 cm2 flask	Greiner bio-one	690160
Acetone	Honeywell	32201-1L
Agarose I	VWR Life Science	97062-250
Alcohol	NIHON SHIYAKU REAGENT	NS-32294
AMV Reverse Transcriptase	Promega	M5101
Antibiotic-Antimycotic(100X)	Gibco	15240-062	MEM-10
Blade	Braun	BA215
Centrifuge	eppendorf	5424R
Chemilumineance system	TOP BIO CO.	MGIS-21-C2-1M
Collection tube	Qiagen	990381
Collection tube	SSI	2341-SO
Cover slide	Muto Pure chemical Co., LTD.	24505
DNA analyzer	Applied Biosystems	3700XL
Fetal bovine serum	Gibco	10437028	MEM-10
FITC Anti-Rabies Monoclonal Globulin	Fujirebio Diagnostic Inc.	800-092	FITC-conjugated anti-rabies antibodies: reagent B
Four-well Teflon-coating glass slide	Thermo Fisher Scientific	30-86H-WHITE
Gel Electrophoresis System	Major Science	MJ-105-R
HBSS (1x)	Gibco	14175095	Trypsin
Incubator	ASTEC	SCA-165DS
Inverted Microscope	Olympus	IX71
L-Glutamine 200 mM (100x)	Gibco	A2916801	MEM-10
LIGHT DIAGNOSTICS Rabies FAT reagent	EMD Millipore Corporation	5100	FITC-conjugated anti-rabies antibodies: reagent A
MagNA Pure Compact Instrument	Roche	03731146001
MagNA Pure Compact NA Isolation Kit 1	Roche	03730964001
MEM (10x)	Gibco	11430030	MEM-10
MEM NEAA (100x)	Gibco	11140050	MEM-10
MEM vitamin solution	Gibco	11120052	MEM-10
NaHCO3	Merck	1.06329.0500	MEM-10
Needle	Terumo	NN*2332R9
PBS	Medicago	09-8912-100
Primer synthesis	Mission Biotech
RNasin ribonuclease inhibitor	Promega	N2111
Sequencing service	Mission Biotech
Slide	Thermo Scientific	AA00008032E00MNT10
Sodium Pyruvate (100 mM)	Gibco	11360070	MEM-10
Stainless Steel Beads	QIAGEN	69989
Sterile absorbent pad	3M	1604T-2
Syringe filter	Nalgene	171-0045
Taq polymerase	JMR Holdings	JMR-801
Thermal cycler	Applied Biosystems	2720
TissueLyser II	QIAGEN	85300
Tongue depressor	HONJER CO., LTD.	122246
Tweezer	Tennyson medical Instrument developing CO., LTD.	A0601
Tylosin Tartrate	Sigma	T6271-10G	MEM-10