Monday, 2 November 2015

MERS in bats..what have we actually found so far?

Only 1 MERS-CoV sequence. In 1 bat.

That's the short answer.

Researchers found a Middle East respiratory syndrome (MERS) coronavirus (CoV) sequence in a bat. They've found lots of other coronavirus sequences in bats before and after that. Heaps of them. But from different CoVs. I'm not even sure how many dromedary camels (DCs) have tested positive for viral RNA or MERS-CoV-specific (as far as we know) antibodies.

One bat.

I'm deviating from the camel literature reviews for this post to go back to the paper that describes that one sequence found in that one bat. I had asked for a little more info on the paper from the authors but they are busy and I have little patience so I'll update this post if that information comes my way. Worthy of note is that some of the specifics about which CoV came from what sample and whether that was from a live bat or old dried faecal pellets can be a bit hard to decipher.

Oh, and I have posted on this paper before by the way:
  1. MERS-CoV genetic sequences found in Taphozous perforatus bat.(22AUG2013; [6])
  2. Taphozous perforatus - The Egyptian Tomb Bat.(22AUG2013; [4])
  3. MERS-CoVs: South African bats vs Saudi Arabian bats.(23AUG2013; [3])
  4. T.perforatus MERS-CoV strain sequence, and others, online...(26AUG2013; [7])
  5. A model of MERS-CoV acquisition (ver1).(30AUG2013; [7])
  6. Is there a better smoking bat or camel?(01SEPT2013; [5])
On to this post. The paper in question comes from Professors Memish, Lipkin and crew. Good pedigree. Sadly, not an ongoing collaboration.[1] The paper, in Emerging Infectious Diseases' November 2013 edition was entitled Middle East Respiratory Syndrome Coronavirus in Bats, Saudi Arabia.

The samples were tested by eight different PCR methods:
  1. A nested pan-CoV reverse-transcription polymerase chain reaction (RT-PCR; "pan"meaning an assay that theoretically detects all known and perhaps as-yet-undiscovered CoVs; assay called 'PLQ') targeting the RNA dependent RNA polymerase (RdRp)
  2. A nested pan-CoV RT-PCR assay (called WT-CoV) targeting RdRp region
  3. A semi-nested MERS-CoV RT-PCR assay (called EMC-SeqRdRp) targeting RdRp region
  4. A semi-nested MERS-CoV RT-PCR assay (called EMC-SeqN) targeting the nucleocapsid (N) region
  5. A nested pan-CoV RT-PCR assay (called NM-CoV) targeting the helicase region
  6. A nested MERS RT-PCR assay (called NM-HCOV) targeting RdRp region
  7. A semi-nested MERS RT-PCR assay (called NM-NSeq) targeting the N region
  8. A real-time RT-PCR (RT-rtPCR) assay (called upE [7]) targeting upstream of the E region
  9. An RT-rtPCR assay (called ORF1b) targeting the ORF 1b region.[7]
Samples included those from a known number of bats (some with multiple samples taken) and also samples of opportunity - bat faecal pellets that could not be matched to a bat so bat numbers could not be estimated. Samples were collected in two rounds (whether a MERS-CoV sequence or any other fragment of CoV RNA genome was identified, is indicated within brackets):
  1. The first in October 2012, shortly after the first human MERS case was identified in Bisha (the MERS-CoV variant represented by Human betacoronavirus 2c EMC/2012, complete genome, on GenBank as JX869059 [8]; 96 bats) 
    • 314 samples from which 8 (2.5% of samples; from 8 distinct bats I think) were positive for a CoV, 1 of which was MERS-CoV
    • 96 bats were tested encompassing 7 species...
      • Rhinopoma hardwickii (CoVs detected)
      • Rhinopoma microphyllum
      • Taphozous perforatus (MERS-CoV & other CoVs detected)
      • Pipistrellus kuhlii (CoVs detected)
      • Eptesicus bottae
      • Eidolon helvum (CoVs detected)
      • Rosettus aegyptiacus
      • From 29 T.perforatus bats in Bisha ruins...
        • 29 yielded throat swabs
        • 25 yielded faecal pellets (2 CoV positives; 1 yielded  a MERS-CoV sequence)
        • 8 yielded urine samples
        • 22 yielded sera
        • 10 yielded roost faeces samples (1 CoV positive)
      • From 25 E.helvum bats in Bisha town centre
        • 25 yielded throat swabs
        • 25 yielded faecal pellets (5 CoV positives)
        • 13 yielded urine samples
        • 19 yielded sera
      • From 3 R.aegypticus bats in Bisha town centre
        • 3 yielded throat swabs
        • 3 yielded faecal pellets
        • 1 yielded urine sample
        • 2 yielded sera
      • From 36 R.hardwickii bats in Naqi and Old Naqi
        • 36 yielded throat swabs
        • 35 yielded faecal pellets
        • 4 yielded urine samples
        • 15 yielded roost faeces samples
      • From 1 R.microphyllum bat in Old Naqi
        • 1 yielded a throat swab
        • 1 yielded a faecal pellet
      • From 1 E.bottae bat in Bisha ruins
        • 1 yielded throat swab
        • 1 yielded faecal pellets
        • 1 yielded urine sample
        • 32 yielded roost faces samples
      • From 1 P.kuhlii bat in Bisha ruins
        • 1 yielded throat swab
        • 1 yielded faecal pellets
  2. The second in April  2013 (mostly faecal pellets and samples; 14  bats)
    • 689 samples, 219 (31.8% of samples) positive for a CoV
    • 14 bats and a lot of faeces not associated with bats, were tested..
      • From R.hardwickii bats in Greater Bisha area
        • 209 yielded roost faeces samples (93 CoV positives)
      • From T.perforatus bats in Bisha ruins
        • 203 yielded roost faeces samples
      • From 9 P.kuhlii bats in Greater Unaizah area
        • 9 yielded throat swabs
      • From 5 P.kuhlii bats in Greater Riyadh area
        • 5 yielded throat swabs
      • Also from P.kuhlii bats in Greater Unaizah area
        • 263 yielded roost faeces samples (126 CoV positives)
So in total, 1,003 samples were tested and 1 MERS-CoV hit was returned while 226 other coronaviruses were confirmed by sequencing. The authors attribute the big difference between finding 8 CoVs in the October 2012 bat sampling (2.5% of samples) and 219 in the April 2013 sampling (31.8% of samples) to a cold chain failure after the arrival of samples back to the United States for testing. There were also fewer roost faeces samples in the October 2012 vs. April 2013 batch (52 vs. 472). No April 2013 T.perforatus bats, from which the October 2012 MERS-CoV sequence was obtained, yielded any CoV sequences. 

And what of that 1 MERS-CoV sequence? We don't know precisely which of the 8 PCR assays amplified it though (probably #3 or #6 above). We do know it's very short and that it could not be confirmed by other PCR assays. 

We know that to date there is no other bat CoV, anywhere, that has a sequence that is 100% identical to a MERS-CoV variant's sequence, except for the T.perfortaus faecal pellet sequence; not Neoromicia/PML-PHE1/RSA/2011 (but close), not Bat HKU4, Bat HKU5, Bat HKU9, and not Bat HKU10...just human and camel MERS-CoV variants. 

But it is of interest that two of these camel variants are called NRCE-HKU205 and NRCE-HKU270 from camels in Egypt. The sequence of these MERS-CoV variants in other places across the genome is relatively different from the majority of MERS-CoV variants from humans and camels. This may provide support for the existence of other different MERS-CoV variants out there, that look like the MERS-CoV we know in small parts of their genomes, but are otherwise quite distinct. And perhaps they reside in other camels outside the Arabian peninsula, or in bats. 

The T.perforatus faecal pellet sequence is a diagnostic sequence as far as we know. It most likely came from a MERS-CoV virus or a variant or ancestor we have not yet met. Or...a contaminant from someone or something else with a MERS-CoV infection of course. 

So, to all the people who continue to insist that bats are a current player in human cases of MERS, I suggest you organize some funding and do some collaborative bat testing because so far there is very limited evidence of there being a bat host for MERS-CoV. 

Just 1 MERS-CoV sequence. 

From 1 bat.

References...
  1. http://virologydownunder.blogspot.com.au/2014/02/coming-back-to-merserable-data.html
  2. Middle East Respiratory Syndrome Coronavirus in Bats, Saudi Arabia
    Memish ZA, Mishra N, Olival KJ, Fagbo SF, Kapoor V, Epstein JH, Alhakeem R, Durosinloun A, Al Asmari M, Islam A, Kapoor A, Briese T, Daszak P, Al Rabeeah AA, Lipkin WI.
    http://dx.doi.org/10.3201/eid1911.131172
  3. http://virologydownunder.blogspot.com.au/2013/08/mers-covs-south-african-bats-vs-saudi.html
  4. http://virologydownunder.blogspot.com.au/2013/08/taphozous-perforatus-egyptian-tomb-bat.html
  5. http://virologydownunder.blogspot.com.au/2013/09/is-there-better-smoking-bat-or-camel.html
  6. http://virologydownunder.blogspot.com.au/2013/08/mers-cov-genetic-sequences-found-in.html
  7. http://virologydownunder.blogspot.com.au/2013/06/new-mers-cov-genomes-dont-impact-on.html
  8. http://www.ncbi.nlm.nih.gov/nuccore/JX869059