Bat sampling techniques

1. About bats

1.a Main points

The important elements to remember about bats are that:

  • They come in many different species, some very small (too small for anal swabs).
  • They can be insectivore or fructivore.
  • They use a lot of energy to fly.
  • They fly out at night to get food.
  • They go into torpor in the cold months, since they would likely use more energy than they could collect when flying at night in cold weather, which is made worse by the fact that the source of this energy (insects or fruits) itself is largely seasonal.
  • Their roosting places are typically trees (if the weather is not too cold) or caves (old mines will do too).
  • They sometimes migrate from winter roosting places to summer locations.
  • They keep warm in their roosting place by staying close together.
  • They often mix with other species in the same cave.
  • They harbour many different viruses which they share easily in their roost or when migrating.
  • They typically are asymptomatic to such viruses, possibly because of a high body temperature and natural resistance.

1.b When do bats go into torpor in Yunnan?

Simple answer: they hibernate when the temperature at night outside and the limited availability of food (fruits or insects) does not make it worth flying out.

Average monthly temperature in Kunming (Yunnan), China (celsius)

2. Bat Sampling

Bat sampling can take different forms:

  • blood sampling
  • fecal sampling via anal swabs
  • feces sampling via splat sheets
  • tissue (for cell cultures of for sequencing the bat itself for identification)

2.a Direct sampling (i.e: caught bats):

One important finding of SZL was the traces of BatCoV in bats feces are typically ephemeral, while blood sampling of the same bats will often show BatCoV antigens.

  • Tissue sampling is obviously destructive and often limited to a few bats, maybe the ones injured during capture — wand which would typically be euthanised anyway.
  • Anal swabs is not practical for some very small bat species
  • Some bat species are notoriously difficult to catch.
Positioning a net to capture bats on their way back to a cave. Note the absence of PPE or gloves. From Shi Zheng Li’s presentation
The catch of the day. Bats hanging in cloth bags.
Olival from EcoHealth Alliance releasing bats from one of these typical cloth bags hanging from a rope behind him.. Field lab in Thailand. Source.
The USAID PREDICT team in Thailand takes blood from a fruit bat to screen for viruses. / Montakan Tanchaisawat, USAID. Source.
A fruit bat gets a sip of nourishment after veterinarians take a blood sample in Chonburi, Thailand. / Richard Nyberg and Montakan Tanchaisawat, USAID. Source. Note the typical white bag the bat is in.

2.b Indirect sampling: Splat Sheets

Means non-invasive, the bats are not handled. Can be used for urine too.

A veterinarian scoops up bat droppings to test for viruses in Chonburi, Thailand. / Montakan Tanchaisawat, USAID. Source

2.c Splitting the samples

Often when PREDICT/USAID is involved, samples get split, especially if they are blood samples. One sample goes to the US, the other one to a local country lab (or to China as was requested by EHA for years 4 and 5 of PREDICT-2), as described by David Quanmen in Spillover (click ‘search inside’).

Source: Spillover (use ‘search inside’)

2.d Safety Recommendations

Practically the main concern of sampling teams seems to be rabies. All members of the sampling teams have to be vaccinated against rabies as bites when handling bats are rather common.

SEABCRU recommendations extract
SEABCRU recommendations extract

2.e Safety practices

While rabies vaccination of field samplers seems widespread, as far as BatCoVs are concerned, a rather relaxed attitude is often prevalent.

Source: NPR
Shi Zheng Li handling bats with students. Note the absence of mask for one of them. Source
Safe (left) and not so safe (right and top). From Shi Zheng Li’s presentation
Source: WHO report Annex D7 (Feb 2021)

2.f Accidents

As it is, accidents do happen. Michel Callahan (who worked for USAID, DARPA and the CTR programs) mentioned some:

Source
SEABCRU recommendations extract
Source

3. Virus Identification

3.a Background:

  • A sample done to collect viruses could contain any type of virus, but will be typically checked using a not-so-broad PCR test or less specific metagenomic analysis.
  • If the test comes back positive, that sample is said to be “positive” which is still rather vague, as in positive for a bat-cov or a SARS-like BatCov.

3.b PCR Testing:

In DNA metabarcoding, PCR is conducted using genetic markers (i.e., DNA barcodes) that target specific taxonomic groups, the PCR products are sequenced using NGS, and the data are compared to DNA sequence databases to categorize and annotate the diversity of DNA sequences in a sample (Huson et al., 2016; Pompanon et al., 2012; Taberlet et al., 2012). See https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050187/

3.c NGS Metagenomic analysis:

RNA virus metagenomics has developed rapidly with the availability of next generation sequencing platforms (NGS). By contrast to PCR testing, metagenomic analysis does not need a targeted primer and will detect any virus in the sample, including new ones. When applied to feces of insectivorous bats, the technique will detect bat viruses, but also insect viruses (the bats eat the insects) and plant viruses (the bats eats insects that eats the plants…).

4. Sequencing:

  • Some viruses are fully sequenced [typically a consensus sequence], some only partially [the RdRp]
  • The RdRp is typically used to ‘index’ coronaviruses. When sequencing was still expensive, only the RdRp may have been sequenced.
  • It is now much easier to sequence the whole lot with NGS techniques (New Generation Sequencing, see https://pubmed.ncbi.nlm.nih.gov/31996413/).
  • A full sequence is subject to the quality of the sample and the quality of the reads. Quite often a ‘consensus’ sequence is built from the various reads.

5. Isolation & Reverse Genetics:

To do:

  • Difficulty of isolation
  • Role of Reverse Genetics

6. WIV Collections:

Vocabulary:

  • Sample: biological sample (blood, feces, etc) — does not necessarily have any virus (may depend on the season, location, etc)
  • Positive sample: a sample that was came back positive for a certain range of viruses (via PCR or metagenomic analysis)
  • Isolate: a cultured (live) virus, ‘isolated’ from any other virus. Many times used wrongly as a synonym of strain.
  • Virus: usually used as meaning a specific virus, main be represented by different strains
  • Strain: ambiguous. Sometimes used as a semantical distinction for a specific physical instance of a virus, which could be as simple as different cultures (different Petri dishes), or different samples containing the same virus. Sometimes used as a group of very similar viruses.
  • Recombinant or chimeric virus: an artificially created virus. There are many techniques: In the WIV it was very commonly the BAC infectious clone. Usually starts with a live isolate and then a gene or segment is swapped by another.
  • TO DO: explain reverse engineering techniques.

The WIV had:

  • the largest virus collection in Asia and one of the largest in the world, if not the largest [source: https://t.co/i3ZMCGfHBV?amp=1]
  • collected around 15,000 bat samples (source: https://t.co/i3ZMCGfHBV?amp=1 gives 15,000 (China + Africa) and based on series at ID 9508 in Mar 2016 as per Francisco + ScienceMag) [other source: 19,000]
  • collected 50,000 tick samples [source: https://t.co/i3ZMCGfHBV?amp=1]
  • a library of 1,400 types of viruses across all species (animal, insects, etc)[source: https://t.co/i3ZMCGfHBV?amp=1, Francisco 1,353] including
  • At least 957 CoVs (source: Francisco)
  • At least 118 MERS related viruses (source: Francisco, + paper)
  • For a total of 60,000 strains across all species (animal, insects, etc) [source: https://t.co/i3ZMCGfHBV?amp=1]
  • identified 500 of coronaviruses (source: Daszak interview + ScienceMag), including at least 50 close to SARS
  • isolated 19 coronaviruses by June2016 — no data since then [source: Francisco]

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Gilles Demaneuf

Gilles Demaneuf

Opinions, analyses and views expressed are purely mine and should not in any way be characterised as representing any institution.