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

Lyssavirus is a zoo-no-tic pathogens. To assess the presence of Lyssavirus in Taiwanese bat populations, we initiated a surveillance program. And it successfully identified four Lyssaviruses from 2016 to 2018.

This protocol is a step-by-step introduction to vi-al Lyssavirus surveillance in Taiwan. We hope it will be useful to researches who are interested in vi-al Lyssavirus surveillance. It is important to connect the correct tac-keg symbol.

A dead or dying bat is more suitable than a live bat for Lyssavirus surveillance. Depending on the bio-safety regulations of the country where the laboratory is located;perform the following procedures in a suitable bio-safety level laboratory. And ensure that the researchers are currently qualified and wear proper personal protective equipment.

Upon the collection of weak or sick bats or carcasses, have a bat ecologist identify the bat species by the specimen's morphological characteristics. Submit an information sheet to the laboratory, including the collection site, species, clinical signs and other relevant data with each bat specimen. Before beginning the necroscopy, examine all the external orifices and photograph the bats external features, especially the head, ears and wings for species differentiation.

To collect an oral swab sample place the bat in ventral recumbency on a sterile dissection board and fix the head with tweezers. Use a scalpel to cut the skin along the midline of the calf area;and pull the skin to the lateral sides, to make an incision in the skull along the midline of the calf area. Open the skull with tweezers to expose the brain tissue and use the tweezers to gently separate the brain tissue from the connected nerve tissue.

Cut the cross-section of the brain, including the brain stem and cerebellum, and lightly touch the cut surface of the brain tissue with glass slides to make smear impressions. Press the slide on lens tissue to remove the excess tissue and fix the smear impression slides in minus 20 degree celsius acetone for 30 minutes. Then fix a small piece of fresh brain tissue, in formalin, for histopathological examination.

And place the rest of the tissue in emi-em 10 for nucleic acid extraction. Next, incise the skin from the manifold to the anus along the midline of the body, and use the tweezers to lift and separate the skin and underline muscle tissues;to allow collection of the salivary glands, located near the mandibular bone. Slightly lifting the sternum with the tweezers, use the scalpel to cut the sternum and abdominal wall along the midline and cut the clavicles.

Use needles to fix the left and right rib cages to the dissection board, to open the thoracic cavity. And record the gross legions and the degree of post mortem change. Then use the tweezers and scalpel to remove the visceral tissues as appropriate for the planned downstream analysis.

To perform a direct fluorescent antibody test at the end of the fixation, dry the smear impression slides and select at least two fluorescence conjugated anti-rabies antibodies for the analysis. Filter one antibody onto each slide through a 0.45 micrometer strainer. And incubate the slides for 30 minutes at 37 degrees celsius in a humid chamber.

At the end of the incubation, drain off the excess conjugate and wash the slides with PBS. Then use a small volume of 10%glycerol to mount cover-slips onto the slides and image the slides by fluorescence microscopy. When either the fluorescent antibody test or the RT-PCR analysis indicates positivity, homogenize the brain specimen in the 10%suspension in emi-en 10 and sediment the tissues fleu-rry by centrifugation.

Inoculate 200 microliters of the super-nat-en with three times 10 to the 6th mouse neuroblastoma cells and one milliliter of emi-en 10 for one hour at 37 degrees celsius and 1%carbon dioxide. And transfer the brain homogeneous-cell suspension to a 25 centimeter squared flask, containing six milliliters of fresh emi-en 10. At one milliliter of brain homogeneous-cell suspension into a four-well Teflon-coated glass slide, with a six millimeter diameter and place the slide at 37 degrees celsius and 1%carbon dioxide for three to four days.

After fixation with 100%acetone, stain the slides with the same two fluorescence conjugated anti-rabies antibodies as used for the fluorescence antibody test. And visualize the slides by fluorescent microscopy to determine the number of intracida-plas-mic inclusions. After trypsinizing and sub-culturing the inoculated cell culture according to standard protocols, return the cold-culture to the cell culture incubator for another three to four days with repeat counting of the number of infected cells as just demonstrated until a 100%infectivity is reached.

From January 2014 to May 2017, 332 bat carcasses from 13 species were collected for surveillance. In this representative analysis of two positive bat cases, the brain impression tested negative using the fluorescent antibody test with one of the commercial, fit-sy conjugated anti-rabies antibodies, while the RT-PCR employing each of the two different primer sets, yielded positive results. Subjection of the obtained ampli-con sequence to blast querying in the Gen bank database revealed that sequence was similar to Lyssaviruses with identities of less than 79%supporting the identities of the detected Lyssaviruses.

Two Lyssaviruses were subsequently successfully isolated from the two brains as confirmed by fluorescence antibody test and sequencing. The key step for determining Lyssavirus positivity are the fluorescence antibody test and the RT-CPR analysis. The use of two or more anti-rabies antibodies in the primer sets is highly recommended.

In addition to Lyssavirus other zoo-not-ical bat pathogens can be monitored using closed diagnostical methods. The data can then be used to inform the public about avoiding contact with bats. The more we are able to study bat Lyssavirus, the more we will be able to understand its evolution and impact on public health.