TITLE: Protocol for the field detection of the European filovirus (Lloviu cuevavirus)
AUTHORS: Gábor Tóth, Sándor Boldogh, Ágnes Balázs-Nagy, Tamás Görföl, Gabor Kemenesi

[DESCRIPTION]
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.



  




 


[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.

 


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
  
Link: https://www.researchgate.net/publication/228985859_Illustrated_identification_key_to_the_bats_of_Europe

Dietz, C. & Kiefer A. 2018. Bats of Britain and Europe. Bloomsbury Publishing, 400 pp. 
Link: https://www.nhbs.com/en/title?slug=bats-of-britain-and-europe-book





[STEPS]
SECTION: Bat sampling
1. 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.

  

  

SECTION: Bat sampling
3. 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.

  

 

SECTION: RNA extraction
5. 
All the RNA extraction kits were used according to the manufacturers. 

User manual for Direct-zol RNA Miniprep Kits  (Zymo Research)
  
User manual for QIAamp MinElute Virus Spin Kit (Qiagen)
  
User manual for QIAamp Viral RNA Mini Kit (Qiagen)
  
User manual for RNAdvance Viral XP (Beckman Coulter)
  EZT KI KELL VENNI!



 

SECTION: LLOV Real-time RT-PCR
6. 
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 20 min 
95 °C 5 min 
25 °C 30 s 

User manual for Agilent AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies)
  

 

SECTION: Bat sampling
2. 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. 
 
  

SECTION: Bat sampling
4. The collected blood samples should be placed directly into 200 μl viral transport medium.


 

SECTION: LLOV Real-time RT-PCR
7. 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 3 min         1
Denaturation                         95 °C 15 s        45
Annealing/elongation          60 °C 45 s        45
Cooling                                   25 °C 30 s         1

User manual for Brilliant III Ultra-Fast QPCR Master Mix  (Agilent Technologies)
  


 




8.