Feb 23, 2023
Protocol for the field detection of the European filovirus (Lloviu cuevavirus)
This protocol is a draft, published without a DOI.
- Gábor Tóth1,
- Sándor Boldogh2,
- Ágnes Balázs-Nagy3,
- Tamás Görföl1,
- Gabor Kemenesi4
- 1National Laboratory of Virology (Hungary);
- 2Aggtelek National Park Directorate;
- 3Hungarian Army Medical Centre;
- 4University of Pecs
Protocol Citation: Gábor Tóth, Sándor Boldogh, Ágnes Balázs-Nagy, Tamás Görföl, Gabor Kemenesi 2023. Protocol for the field detection of the European filovirus (Lloviu cuevavirus). protocols.io https://protocols.io/view/protocol-for-the-field-detection-of-the-european-f-bqwxmxfn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: December 20, 2020
Last Modified: February 23, 2023
Protocol Integer ID: 45751
Keywords: Lloviu cuevavirus, bat sampling, European filovirus, field detection
Abstract
Lloviu cuevavirus (LLOV) is the only filovirus endemic in Europe and the only known host for this virus is the Miniopterus schreirbersii bat which is widespread throughout the southern part of the continent. LLOV was originally emerged in 2002 in Spain and re-emerged in 2016 in Hungary. Hitherto just these two studies provided information about the detection of LLOV in the nature. There is a huge lack of understanding in the circulation of this virus and the potential of zoonotic transmission, hence there is an urgent need to develop standardised protocols for the detection LLOV to deepen our knowledge about the spatio-temporal distribution of this virus, which is still circulating in Hungary.
The distribution area of Miniopterus schreirbersii in the IUCN database.
Note
Basic literature of the virus:
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Guidelines
Bat handling
All bat species in Europe are strictly protected under the Flora, Fauna, Habitat Guidelines of the European Union (92/43/EEC) and the Agreement on the Conservation of Populations of European Bats (www.eurobats.org). It is important that the bat handling procedures should be carried out by a trained chiropterologist with the appropriate license. The guidelines for the bat examinations and sampling are available at the publication of Sikes et al.
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All bats should be identified for species by an experienced chiropterologist. For the European bats there is an excellent work by Christian Dietz and Otto Von Helversen which presents all the identification keys in details.
Title: Illustrated identification key to the bats of Europe
Date: January 2004
DietzvonHelversen2004Identificationkeybatscomplete.pdf Dietz, C. & Kiefer A. 2018. Bats of Britain and Europe. Bloomsbury Publishing, 400 pp.
Materials
Bat sampling
-PPE: leather gloves, latex gloves, masks(FFP3). protective gowns
-Disposable or autoclavable bags for animal handling
-1,5 ml Eppendorf tubes
-POCT Minivette 100 μl neutral (Sarstedt)
-27-29G needle
-EMOFIX haemostatic barrier ointment (PharmaQ)
-Minivette POCT 100µl (Sarstedt)
-Viral transport medium
Viral RNA extraction
-Direct-zol RNA Miniprep Kits (Zymo Research)
-QIAamp MinElute Virus Spin Kit (Qiagen)
-QIAamp Viral RNA Mini Kit (Qiagen)
-Centrifuge for 1.5 ml tubes
-RNAdvance Viral XP (Beckman Coulter)
-Magnetic rack for 1.5 ml tubes
-Pipettes from volume 0,5 μl to 1000 μl
LLOV Real-time RT-PCR
-Agilent AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies)
-Brilliant III Ultra-Fast QPCR Master Mix (Agilent Technologies)
-MyGo Mini or MyGo Pro PCR Instrument (IT-IS Life Science Ltd.)
Electrical supply
-Power generator (~2000 watt)
Safety warnings
Personal protective equipment - PPE
Bats harbour enormous number of pathogens with zoonotic potential and every person who works with bats must avoid the spillover. Wearing Personal Protective Equipment (PPE), like gloves, leather gloves and masks (FFP3) should be the gold standard to avoid the zoonotic and reverse zoonotic transmission of different pathogens.
Bat sampling
Bat sampling
15m
15m
1, The bat handler should hold the animal by the abdominal side faced upwards. Pressing the chest too hard must be avoided as this can cause injuries for the bat. The handler should place the index finger under the interfemoral vein (hand protection is necessary to avoid the puncture of the human finger). When the uropatagium is stretched, you should make a gentle puncture on the vein with a 27-29 G needle.
1. Puncture of the uropatagial vein. The photo was taken for educational purposes and this is the reason why one of the bat handler not weared gloves in this picture to make easier to explain the main rules and tricks of bat handling.
2. It is recommended to wait for a bigger, unified, intact blood droplet, because it is much easier to collect it with the capillary.
2, After the appearance of the first blood droplet, the collection of blood can be started using Minivette POCT 100µl. It is recommended to hold the capillary in a lower angle and just touch the upper surface of the blood droplet.
3. Collection of blood droplet with the capillary.
3, According to the recommendation of Hooper et al. (2014), themaximumamount of blood should not exceed the 1% of the body mass of the bat.IncaseofMiniopterusschreibersii,whichbodymassextendbetween8-13gram,the limit is80-130 µl. After the collection of the appropriate amount of blood, further bleeding must be stopped with the application of EMOFIX haemostatic barrier ointment. After the treatment, animals should be put back to their own bags for 10 minutes. Before releasing the bats, their condition (e.g. the stop of bleeding, overall status) should be carefully verified.
4. The location of the puncture is covered with EMOFIX haemostatic barrier ointment.
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The collected blood samples should be placed directly into 200 μl viral transport medium.
Note
If you would like to use the samples for other analysis as well (e.g. for serology) you can centrifuge the blood samples on 2500 x g for 5 minutes to separate the serum and blood residues before the nucleic acid extraction step.
RNA extraction
RNA extraction
All the RNA extraction kits were used according to the manufacturers.
User manual for Direct-zol RNA Miniprep Kits (Zymo Research)
_r2050_r2051_r2052_r2053_direct-zol_rna_miniprep.pdf User manual for QIAamp MinElute Virus Spin Kit (Qiagen)
HB-0323-005_HB_QA_ME_Virus_Spin_Kit_0220_WW.pdf User manual for QIAamp Viral RNA Mini Kit (Qiagen)
HB-0354-007_HB_QA_Viral_RNA_Mini_0720_WW (1).pdf User manual for RNAdvance Viral XP (Beckman Coulter)
C58637AB.pdf EZT KI KELL VENNI!Note
RNAdvance ViralXP (BeckmanCoulter) has the advantage that this RNA extraction method does not need any centrifugation, which is really promising in the field application of the protocol, because you do not have to use electrical power till the cDNA preparation.
LLOV Real-time RT-PCR
LLOV Real-time RT-PCR
1h 30m
1h 30m
cDNA preparation
Kit: Agilent AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies)
Component Volume
2x Master mix 10 µL
Random Hexamer 1 µL
Affinity Script RT/ RNase Block Enzyme Mix 1 µL
Nuclease-free water 3 µL
RNA template 5 µL
Incubate the reaction as follows:
42 °C 00:20:00
95 °C 00:05:00
25 °C 00:00:30
User manual for Agilent AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies)
600559.pdf Note
The advantageof the separation of the qPCR and the cDNA synthesis is that you can screen your samples to various pathogens but,if you are focused to a specific RNA virus, you have the opportunity to use another kit to save time.
User manual for qRT-PCR Brilliant III Probe Master Mix (Agilent Technologies)
600884.pdf 30m
Real-time PCR
Kit: Brilliant III Ultra-Fast QPCR Master Mix (Agilent Technologies)
Component Volume
Master mix 10 µL
FiloAneo 50 μM (froward primer) 0.4 µL
FiloBNeo 50 μM (reverse primer) 0.4 µL
Lloviu-S 50 μM (probe,FAM) 0.2 µL
Nuclease-free water: 4 µL
cDNA 5 µL
Set-up the following program on the thermal cycler:
Step Temperature Time Cycles
Initial Denaturation 95 °C 00:03:00 1
Denaturation 95 °C 00:00:15 45
Annealing/elongation 60 °C 00:00:45 45
Cooling 25 °C 00:00:30 1
User manual for Brilliant III Ultra-Fast QPCR Master Mix (Agilent Technologies)
600880.pdf Note
Primers:
LLOV-Fw-scr1: 5’-AAGCATTTCCGAGTAATATGATGGTTG-3’
LLOV-Rev-scr1: 5’-TACATGGTCTCCTAGATTGCCCTG-3’
LLOV-Prob-scr1: 5’-FAM-CCTGATGAAGGAGAGTTTCTTTCTG-ZEN-3
The source of this primer set:
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This figure shows the binding sites of the screening primers (green triangles) on the schematic illustration of LLOV genome.
55m
Citations
Sikes RS, Animal Care and Use Committee of the American Society of Mammalogists.. 2016 Guidelines of the American Society of Mammalogists for the use of wild mammals in research and education.
https://doi.org/10.1093/jmammal/gyw078Step 3
Hooper SE, Amelon SK. Handling and blood collection in the little brown bat (Myotis lucifugus).
https://doi.org/10.1038/laban.543