Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 genus1,2,3, 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 loca.......

Representative 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 .......

Acknowledgements

We thank Tien-Cheng Li, Yi-Tang Lin, Chia-Jung Tsai, and Ya-Lan Li for their assistance during this study. This study was supported by grant no. 107AS-8.7.1-BQ-B2 (1) from the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, Taiwan.

....

Materials

NameCompanyCatalog NumberComments
2.5% Trypsin (10x)Gibco15090046Trppsin
25 cm2 flaskGreiner bio-one690160
AcetoneHoneywell32201-1L
Agarose IVWR Life Science97062-250
AlcoholNIHON SHIYAKU REAGENTNS-32294
AMV Reverse TranscriptasePromegaM5101
Antibiotic-Antimycotic(100X) Gibco15240-062MEM-10
BladeBraunBA215
Centrifugeeppendorf5424R
Chemilumineance systemTOP BIO CO.MGIS-21-C2-1M
Collection tubeQiagen990381
Collection tubeSSI2341-SO
Cover slideMuto Pure chemical Co., LTD.24505
DNA analyzerApplied Biosystems3700XL
Fetal bovine serumGibco10437028MEM-10
FITC Anti-Rabies Monoclonal GlobulinFujirebio Diagnostic Inc.800-092FITC-conjugated anti-rabies antibodies: reagent B
Four-well Teflon-coating glass slideThermo Fisher Scientific30-86H-WHITE
Gel Electrophoresis SystemMajor ScienceMJ-105-R
HBSS (1x)Gibco14175095Trppsin
IncubatorASTECSCA-165DS
Inverted MicroscopeOlympusIX71
L-Glutamine 200 mM (100x)GibcoA2916801MEM-10
LIGHT DIAGNOSTICS Rabies FAT reagentEMD Millipore Corporation5100FITC-conjugated anti-rabies antibodies: reagent A
MagNA Pure Compact InstrumentRoche03731146001
MagNA Pure Compact NA Isolation Kit 1Roche03730964001
MEM (10x)Gibco11430030MEM-10
MEM NEAA (100x)Gibco11140050MEM-10
MEM vitamin solutionGibco11120052MEM-10
NaHCO3Merck1.06329.0500MEM-10
NeedleTerumoNN*2332R9
PBSMedicago09-8912-100
Primer synthesisMission Biotech
RNasin ribonuclease inhibitorPromegaN2111
Sequencing serviceMission Biotech
SlideThermo ScientificAA00008032E00MNT10
Sodium Pyruvate (100 mM)Gibco11360070MEM-10
Stainless Steel BeadsQIAGEN69989
Sterile absorbent pad3M1604T-2
Syringe filterNalgene171-0045
Taq polymeraseJMR HoldingsJMR-801
Thermal cyclerApplied Biosystems2720
TissueLyser IIQIAGEN85300
Tongue depressorHONJER CO., LTD.122246
TweezerTennyson medical Instrument developing CO., LTD.A0601
Tylosin TartrateSigmaT6271-10GMEM-10

References

  1. Kuzmin, I. V., Rupprecht, C. E., Nagarajan, T. Basic facts about lyssavirus. Current laboratory techniques in rabies diagnosis, research, and prevention, volume 1. , 3-21 (2014).
  2. Aréchiga Ceballos, N., et al. Novel lyssavirus in bat, Spain. Emerging Infectious....

Explore More Articles

LyssavirusBat PopulationSurveillanceVirologyNecropsyBrain TissueDiagnostic TestingSample CollectionBiosafetyTaiwan

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved