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 bee....

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 laborat....

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