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.

1. Safety precautions when handling lyssaviruses
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Sample collection
<ol>
	<li>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.</li>
	<li>Have a bat ecologist identify bat species through morphological characteristics20.</li>
	<li>Perform DNA barcoding of the bat species when lyssavirus positive is diagnosed, using previously published procedures21.</li>
	<li>Submit an information sheet of each bat carcass (collection site, species, clinical signs, etc.).</li>
</ol>
3. Necropsy of bat specimens
<ol>
	<li>Prepare materials.
	<ol>
		<li>Prepare a clean dissection board and place a sterile absorbent pad for necropsy.</li>
		<li>Prepare collection tubes for collecting bat organs.</li>
		<li>Prepare disposable tweezers and scalpels for necropsy. Change tools between each bat&#8217;s necropsy. Prepare cotton balls moistened with 70% ethanol for cleaning tweezers and scalpels during sampling.</li>
		<li>Prepare two microscope slides for the FAT. Collect fresh specimens and fix them in formalin solution for histopathological examination.</li>
	</ol>
	</li>
	<li>Examine all external orifices before necropsy. Photograph the bat&#8217;s external features, especially the head, ears, and wings, for species differentiation.</li>
	<li>Collect an oral swab sample. Place the bat in ventral recumbency on the board and fix the bat&#8217;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.</li>
	<li>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.</li>
	<li>Clean tweezers and scalpel with cotton balls moistened with 70% ethanol to remove retained tissues between specimen collection.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Record the gross lesions and the degree of post-mortem change.</li>
	<li>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 &#176;C with or without viral transport medium for viral culture22.</li>
</ol>
4. Direct fluorescent antibody test (FAT)
<ol>
	<li>Make impression smears of the brain tissue for the FAT. Perform the FAT as previously described18,23 with the following modifications.</li>
	<li>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 cerebellum18,23. 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.</li>
	<li>Fix the slides with acetone at -20 &#176;C for 30 min. Dry the tested slides and the positive and negative controls before staining with conjugate.</li>
	<li>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 &#956;M syringe filter onto the slides and incubate the slides at 37 &#176;C for 30 min within a wet chamber.</li>
	<li>Drain the excess conjugate from the slides and wash the slides with phosphate-buffered saline (PBS) after incubation.</li>
	<li>Drop a small amount of 10% glycerol on the slides and cover with cover slides.</li>
	<li>Examine the slides with a fluorescent microscope.</li>
</ol>
5. Nucleic acid extraction
<ol>
	<li>Add the proper volume of MEM-10 (minimum essential medium supplemented with 10% fetal bovine serum) to the brain tissue (10% w/v).</li>
	<li>Homogenate the brain tissue with a 5 mm steel bead in a homogenizer instrument and centrifuge at 825 x g for 10 min.</li>
	<li>Extract the nucleic acid, the final volume of which is 50 &#956;L, within 200 &#956;L of the supernatant using the commercially available total nucleic acid extraction kit with instrument.</li>
</ol>
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.
<ol>
	<li>Prepare the one-step RT-PCR reagent as follows: add 5 &#956;L of extracted nucleic acid to the reaction mixture containing 2.5 &#956;L of 10x reaction buffer, 0.5 &#956;L of forward and reverse primers (10 &#956;M of each), 4 &#956;L of 1.25 mM dNTP, 0.3 &#956;L of RNase inhibitor (40 U/&#181;L), 0.3 &#956;L of reverse transcriptase (10 U/&#181;L), 0.4 &#956;L of DNA polymerase (5 U/&#181;L), and 11.5 &#956;L of DEPC-treated water. 
	NOTE: The primer set used in this protocol is JW12 (5&#39;-ATGTAACACCYCTACAATG-3&#39;) and N165-146 (5&#39;-GCAGGGTAYTTRTACTCATA-3&#39;)24. Modify the preparation of reagents and cycling conditions according to the primer sets used.</li>
	<li>Perform the cycling under the following conditions: incubation at 42 &#176;C for 40 min; initial denaturation at 94 &#176;C for 10 min; 35 cycles of 94 &#176;C for 30 s, 55 &#176;C for 30 s, and 72 &#176;C for 30 s; and finally, further extension at 72 &#176;C for 10 min.</li>
	<li>Use another or more primer sets to increase the diagnostic sensitivity.
	<ol>
		<li>Use N113F (5&#39;-GTAGGATGCTATATGGG-3&#39;) and N304R (5&#39;-TTGACGAAGATCTTGCTCAT-3&#39;)25,26 to prepare the one-step RT-PCR reagent as follows: add 5 &#956;L of extracted nucleic acid to a reaction mixture containing 5 &#956;L of 10x buffer, 5 &#956;L of forward and reverse primers (4 &#956;M of each), 5 &#956;L of 1.25 mM dNTP, 0.5 &#956;L of RNase inhibitor (40 U/&#181;L), 0.2 &#956;L of reverse transcriptase (10 U/&#181;L), 1 &#956;L of DNA polymerase (5 U/&#181;L), and 23.3 &#956;L of DEPC-treated water.</li>
		<li>Perform the cycling under the following conditions: incubation at 42 &#176;C for 40 min; initial denaturation at 95 &#176;C for 5 min; 35 cycles of 95 &#176;C for 1 min, 55 &#176;C for 1 min and 20 s, and 72 &#176;C for 1 min; and finally, further extension at 72 &#176;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.</li>
	</ol>
	</li>
	<li>Analyze the PCR product on 2% agarose gel electrophoresis and visualize by UV light illumination.</li>
	<li>Sequence the PCR product by commercial sequencing service.</li>
	<li>Enter or upload the sequence to the webpage of the Nucleotide Basic Local Alignment Search Tool (BLAST). Select the &#34;others (nr etc.)&#34;&#160;database and enter the organism as Lyssavirus. Select the MegaBlast algorithm and run the BLAST.</li>
</ol>
7. Virus isolation
NOTE: Perform virus isolation when either 1) the FAT or 2) the RT-PCR indicates positivity.
<ol>
	<li>Homogenize the brain specimen in a 10% (w/v) suspension in MEM-10. Centrifuge at 825 x g for 10 min.</li>
	<li>Inoculate 200 &#181;L of supernatant with a suspension of 3 x 106 MNA (mouse neuroblastoma) cells in 1 mL of MEM-10 for 1 h at 37 &#176;C with 1% CO2. Transfer the brain homogenate-cell suspension to a 25 cm2 flask and add 6 mL of MEM-10.</li>
	<li>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.</li>
	<li>After 3&#8211;4 days of incubation at 37 &#176;C with 1% CO2, fix the cells on the 4 well slide with 100% acetone (v/v).</li>
	<li>Stain the slides with two FITC-conjugated anti-rabies antibodies following steps 4.4&#8211;4.7. The cells are infected when the intracytoplasmic inclusions are investigated. Record the percentage of infected cells.</li>
	<li>Perform trypsinization and subculture of the inoculated cell culture when the slides are stained as negative:
	<ol>
		<li>Remove medium and rinse the flask with 5 mL of PBS.</li>
		<li>Add 1 mL of trypsin to the flask and firmly strike the bottom of the flask.</li>
		<li>Add 6 mL of MEM-10 and resuspend the cells.</li>
		<li>Put the cell suspension into a new tissue flask (6 mL) and onto a 4 well slide (1 mL).</li>
	</ol>
	</li>
	<li>Repeat steps 7.4&#8211;7.6 until 100% infectivity is reached.</li>
	<li>Collect the supernatant after 24 h of incubation.</li>
</ol>

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No conflicts of interest are declared.

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&#8211;2009), the target animals of bat lyssavirus surveillance were live ...

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 been identified in any bats1,2,3,6. 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 seroprevalence11,12,13,14,15. 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&#8211;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.

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

standard operating procedure for lyssavirus surveillance of the bat population in taiwan

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&#8211;2017. These findings should inform the public, health professionals, and scientists of the potential risks of contacting bats and other wildlife.

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...

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

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

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are ...

standard-operating-procedure-for-lyssavirus-surveillance-bat

Research

JoVE Journal

Environment

7.4K Views.  Council of Agriculture, Executive Yuan. Lyssavirus is een dierentuin-no-tic pathogenen. Om de aanwezigheid van Lyssavirus in Taiwanese vleermuispopulaties te beoordelen, hebben we een bewakingsprogramma gestart. En het identificeerde met succes vier Lyssavirussen van 2016 tot 2018.Dit protocol is een stapsgewijze inleiding tot vi-al Lyssavirus surveillance in Taiwan. We hopen dat het nuttig zal zijn om onderzoek te doen naar mensen die geïnteresseerd zijn in vi-al Lyssavirus surveillance. Het is belangrijk om het juiste tac...

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

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Genetics

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

Immunology and Infection

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to confirm rabies diagnosis but have only recently been accepted by the OIE (World Organisation for Animal Health) as a primary method to detect rabies infection. Real-time RT-PCR assays provide real-time data, and are closed-tube systems, minimizing the risk of contamination during setup. DNA intercalating fluorochrome real-time RT-PCR assays do not require expensive probes, minimizing the cost per sample, and when the primers are designed in conserved regions, assays that are specific across virus genera rather than specific to just one virus species are possible. Here we describe a pan-lyssavirus SYBR real-time RT-PCR assay that detects lyssaviruses across the Lyssavirus genus, including the most divergent viruses IKOV, WCBV and LLEBV. In conjunction with dissociation curve analysis, this assay is sensitive and specific, with the advantage of detecting all lyssavirus species. The assay has been adopted in many diagnostic laboratories with quality assured environments, enabling robust, rapid, sensitive diagnosis of animal and human rabies cases.

This real-time RT-PCR using dsDNA intercalating dye is suitable to diagnose lyssavirus infections. The method begins with RNA extracted from rabies suspected ante-mortem or post-mortem samples, detailing master mix preparation, RNA addition, setup of the real-time machine and correct interpretation of results.

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to ...

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer&#39;s protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping.

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Biology

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects' feeding behavior. A convenient method that will be useful for research on insects' saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective ...

Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes

With the broad application of sequencing technologies, many novel virus-like sequences have been discovered in arthropods, including mosquitoes. The two main categories of these new mosquito-associated viruses are &#34;mosquito-borne viruses (MBVs)&#34; and &#34;mosquito-specific viruses (MSVs)&#34;. These novel viruses might be pathogenic to both vertebrates and mosquitoes, or they could just be symbiotic with mosquitoes. Entity viruses are essential to confirm the biological characters of these viruses. Thus, a detailed protocol has been described here for virus isolation and amplification from field-collected mosquitoes. First, the mosquito samples were prepared as supernatants of mosquito homogenates. After centrifugation twice, the supernatants were then inoculated into either mosquito cell line C6/36 or vertebrate cell line BHK-21 for virus amplification. After 7 days, the supernatants were collected as the P1 supernatants and stored at -80 &#176;C. Next, P1 supernatants were passaged twice more in C6/36 or BHK-21 cells while the cell status was being checked daily. When cytopathogenic effect (CPE) on the cells was discovered, these supernatants were collected and used to identify viruses. This protocol serves as the foundation for future research on mosquito-associated viruses, including MBVs and MSVs.

Numerous novel virus-like sequences have been found in mosquitoes due to the extensive use of sequencing technologies. We provide an effective procedure for isolating and amplifying viruses using vertebrate and mosquito cell lines, which might serve as the basis for future studies on mosquito-associated viruses, including mosquito-borne and mosquito-specific viruses.

With the broad application of sequencing technologies, many novel virus-like sequences have been discovered in arthropods, including mosquitoes. The two ...

Postmortem Diagnosis of Rabies in Animals by the Updated, Multiplexed LN34 Real-Time Reverse Transcription-Polymerase Chain Reaction Assay

Rabies is a fatal zoonotic disease caused by Lyssavirus rabies (RABV) and related negative strand RNA viruses from the Lyssavirus genus (family Rhabdoviridae). The LN34 assay targets the highly conserved leader region and nucleoprotein gene of the lyssavirus genome and utilizes degenerate primers and a TaqMan probe containing locked nucleotides to detect RNA across the diverse Lyssavirus genus. A negative finding for rabies should be made only if a full cross-section of the brain stem and three lobes of the cerebellum are examined; however, identification of lyssavirus RNA in any tissue is diagnostic of rabies infection. Tissue is collected and homogenized in TRIzol reagent, which also inactivates the virus. RNA extraction is performed using a spin column-based commercial extraction kit. Master mixes are prepared in a clean space and aliquoted into a 96 well plate before adding sample RNA. In clinical settings, each sample is tested by real-time RT-PCR for the presence of lyssavirus RNA in triplicate and singly for host &#946; -actin mRNA. Positive and negative controls are included at extraction and real-time RT-PCR steps of the protocol. Data analysis involves manual adjustment of the thresholds to standardize Ct values across instrument runs. Positive results are determined by the presence of typical amplification in the pan-lyssavirus assay (Ct &#8804; 35). Negative results are determined by the absence of typical amplification in the pan-lyssavirus assay and detection of host &#946;-actin mRNA (Ct &#8804; 33). Observation of values outside of these ranges or failure of the assay controls can invalidate the run or result in inconclusive results for a specimen. The protocol should be closely followed to ensure high assay sensitivity and specificity. Procedural modifications can affect assay performance and lead to false positive, false negative, or uninterpretable results.

This protocol demonstrates the pan-lyssavirus LN34 real-time reverse transcription-polymerase chain reaction (RT-PCR) assay from tissue collection to result interpretation, including updates to primer sequences and formulations to improve assay performance for some non-rabies lyssaviruses and lagomorphs. We also demonstrate the assay setup for a single-well LN34 multiplexed (LN34M) format.

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

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