Back to the bats for MERS-CoV...

It's a smoking bat. Get it? Hmm.

In late February Munster and a team of United States' researchers infected bats with MERS-CoV. 

The aim here was to seek out information about a possible origin for MERS-CoV in bats and perhaps find an animal source from which camels may have first been infected. This path is suspected because of the genetic similarities between MERS-CoV and other bat coronaviruses which make bats an attractive reservoir for MERS-CoV...or an earlier form of MERS-CoV.

It's worth reinforcing that there is as yet no "smoking bat" - no bat species has been found to harbour infectious MERS-CoV in the wild or more than a diagnostic PCR fragment's worth of genetic material. To date.

The team used a bat species called Artibeus jamaicensis - the Jamaican fruit bat. 

They first tested whether the bat's version of the molecular known to be the human MERS-CoV receptor,  dipeptidyl peptidase 4 (DPP4), could act in that role. To do this they cloned the relevant bat sequence and introduced it into some some cells that usually didn't permit MERS-CoV to infect and replicate wihtin them. When the introduced DPP4 material was expressed, the cells permitted MERS-CoV infection and replication; Jamaican fruit bat DPP4 could act as the MERS-CoV receptor, just like human DPP4 does.

From the Histology Lecture Image Gallery at Yale
Medical Cell Biology.[3]

Next the team infected 10 A.jamaicensis bats via the nose and body cavity; none showed signs of disease, lost weight or were found to have a rise in their temperature but 8 shed virus - moreso from their respiratory than their gastrointestinal tracts.

When the bats were killed at different times and examined, the lower respiratory tract was found to contain the highest level of virus, but MERS-CoV was also found throughout the bodies of the bats, possibly spread via a blood-borne path since viral RNA was detected in the blood 2 and 4 days after inoculation. 

Signs of mild tissue damage could only be found in 2 bats - both in the respiratory tract with signs that virus infected Type I pneumocytes. Type I pneumocytes form part of the alveolar gas exchange barrier. In humans, Type II pneumocytes were identified as MERS-CoV targets in the only human autopsy conducted to date.[2] Type II pneumocytes are responsible for secreting surfactant which helps stop the lung's air sacs from sticking to themselves, they metabolize drugs, move water across epithelium (tissues that line the body's hollow cavities in this case) and repair injured alveoli.[4]

Only 1 bat developed a MERS-CV a specific antibody response.

The discussion had some interesting points including....

• A.jamaicensis may be a good model system for studies of how bats and their coronaviruses co-exist
• MERS-CoV "maintains the ability to efficiently replicate" in bats which the authors take to support the hypothesis that bats are the ancestral reservoir for MERS-CoV. Or maybe its ancestor?
• Because bat coronaviruses are usually bat gut pathogens, but MERS-CoV was a bat respiratory pathogen, this might suggest MERS-CoV evolved to be so in camels. Orrrr...it might not.
• In the absence of disease, a detectable antibody response may not occur in humans as was found in bats, or it may be delayed in humans as has been previously reported. 
• In most bats and bat tissues, MERS-CoV RNA could not be detected beyond a 2 week period after inoculation - but in 1 bat, the small intestine was still positive at day 28

I'll get back onto the camel literature reviews next.

References...

1. Replication and shedding of MERS-CoV in Jamaican fruit bats (Artibeus jamaicensis)
   http://www.nature.com/articles/srep21878
2. Clinicopathologic, Immunohistochemical, and Ultrastructural Findings of a Fatal Case of Middle East Respiratory Syndrome Coronavirus Infection in the United Arab Emirates, April 2014
   http://ajp.amjpathol.org/article/S0002-9440(15)00647-1/abstract
3. http://medcell.med.yale.edu/medcell.php#
4. http://www.ncbi.nlm.nih.gov/pubmed/3285521

Updating the animal "to-do" list...

I have a couple of talks coming up, so I'm making graphics again. And I like to share those. 

This one is an update on some of the creatures that could also be considered suspects in the hunt for sources of MERS-CoV infection of humans.

Of course, camels are the ones we know to be a true risk for infection and there was that 1 bat that was positive for a very small diagnostic PCR product. Cattle contact was also recently listed, with little detail, as a significant risk factor among those acquiring MERS-CoV infection and we also know that cells from camels, horses, alpacas, cattle and goats can be infected and host genome or virus replication of MERS-CoV in the lab, or have the MERS-CoV cellular receptor, DPP4, on their surface.[1,2]

I heard that there will be more bat testing in the future, but we haven't read of any MERS-CoV targeted bat studies since 2013.

So here is the long laundry list of animal testing that needs more work - many of which have been tested in small numbers over limited time periods already - in a graphical form.

Click on image to enlarge.
You can also access this from Figshare.[3]

References...

1. http://www.ncbi.nlm.nih.gov/pubmed/25656066
2. http://wwwnc.cdc.gov/eid/article/22/1/15-1340_article
3. http://figshare.com/articles/Creatures_of_interest_to_how_humans_acquire_a_MERS_CoV_infection/1609604

It was the camel, in the library, with the MERS-CoV...

In a paper out overnight, which is assigned to the January 1st 2016 edition of Emerging Infectious Diseases (why do you do this to us EID?!), Alraddadi and colleagues (overwhelmingly from the Kingdom of Saudi Arabia with help from the Centers for Disease Control and Prevention in the United States) have published Risk Factors for Primary Middle East Respiratory Syndrome Coronavirus Illness in Humans, Saudi Arabia, 2014. 

This is a long awaited case control study. Long awaited.

From [2]

It tells us that direct contact with dromedary camels (including the act of milking them) in Saudi Arabia, in the 2 weeks prior to symptoms ascribed to a confirmed MERS-CoV infection, is a significant risk factor for developing Middle East respiratory syndrome (MERS) disease. Cattle contact also fell out as a significant risk. 

However, cases were no more likely than controls to report exposure to bats, goats, horses, sheep or consumption of fruits, vegetables, or animal products, including uncooked meat, unpasteurized animal milk, or dromedary urine. 

The study also reminds us that the host factors of diabetes, heart diseases and smoking are associated with MERS (the disease, not how likely you are to get infected). If you do not have these then you may be more likely to have mild or asymptomatic outcomes if you were to be exposed and infected by MERS-CoV.

These are astounding findings that will take many by surprise and revolutionize out understanding of MERS (the disease) and MERS-CoV (the virus) throughout the Arabian Peninsula. 

Said no-one. Ever.

Ridiculous sarcasm aside though, much kudos to the Saudi research community! This case-control study, a long-awaited piece of work, was a camel that had to be broken by them for them, and now it has been. A win for science and for the region's science.

I hope the study helps to confirm the sizable pool of research that has come before.

But let's not lose sight of the camel in the room; most human cases of MERS come from other human infections closely associated with healthcare settings.

Defeating MERS and MERS-CoV requires battles on many fronts. As usual for any emerging viral disease. 

But then, it's a OneHealth kinda world.

References...

1. http://wwwnc.cdc.gov/eid/article/22/1/15-1340_article
2. http://virologydownunder.blogspot.com.au/2014/05/camels-at-centre-aerosol-all-around.html

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

Evidence that Reston ebolavirus resides in live bats in the Philippines...

Update #1 18JUN2015

Jayme and colleagues find some
"smoking bats"-possible bat reservoir
species for Reston ebolavirus
in the Philippines.

In what I think is only the second example of this, a new collaborative study from Jayme and a team of eminent researchers in the Philippines, Australia, Vietnam and the United States, have reported the finding of Reston ebolavirus (RESTV) viral RNA and antibodies to viral infection in a range of different bat species....some more "smoking bats" - bats with more than just past evidence, sometimes considered vague and unreliable, of an ebolavirus being hosted by the animal.

The finding of RNA is not the same as actual infectious virus, but RNA is a very specific marker for the virus nonetheless. And the authors note that they didn't want to kill the bats so only a small volume of sample was available-not enough for culture.

Leroy and colleagues had previously reported finding Zaire ebolavirus RNA and antibodies against this species of virus in Hypsignathus monstrosus, Epomops franqueti and Myonycteris torquatebats, all fruit-eating megabats of the family Pteropodidae. These are considered to be important reservoir hosts, yet they do not show signs of disease.[2] 

According to one of the authors on the latest study, bats in the Philippines also seemed clinically well...

@MackayIM all bats we sampled appeared clinically healthy.

— Jonathan Epstein (@EpsteinJon) July 17, 2015

Locating the Philippine RESTV sequences
on the ebolavirus phylogenetic tree.
Jayme et al. Virology J. (2105) 12:107.[1]

Jayme's findings are important to the story of RESTV importations to animal facilities in the United States from the Philippines which occurred multiple times between 1989 to 1996. These fed into the dramatized retelling we know of as The Hot Zone. There were also signs of antibodies to the virus in humans working with infected non human primates in the Philippines in 1994, 1996 and 2008.

The amount of viral RNA in most of the bats was quite low - but was usually repeatably detectable. I'm a firm believer in PCR giving a specific signal when there is something specific present to detect (assuming it was done in a professional laboratory setting that reduces the risk of false positives-which it was in this instance). So low viral loads are not no viral loads.

RESTV RNA was repeatably found in oropharyngeal swabs taken from bats assigned to the following species:

• Cynopterus brachyotis (Lesser dog-faced fruit bat; range; fruit bat)
• Miniopterus australis (Little long-fingered or little bent-wing bat; range; insectivorous bat)
• Miniopterus schreibersii (Schreiber's Bent-winged Bat; range; insectivorous bat)

...and in one sample from:

• Chaerephon plicata (Wrinkle-lipped Free-tailed Bat; range; insectivorous bats)

What's particularly interesting to me is that some of these bat species are found in Australia. However, keep in mind that the range of some (?many) bats may be underestimated. The example here is using the IUCN Red List's described range for M. schreibersii-apparently it's a bat that inhabits an area around the Mediterranean.[4] Last I looked, the Philippines is a bit south of there. In the past, as Wikipedia lists, a much bigger range was ascribed to this bat, also including Australia,[5] Guinea, Liberia and Sierra Leone - among many others. Looks like there may be lots of work to do in the area of bat census.

Jayme and colleagues also sampled the blood of 61 flying foxes (of the fruit-eating bat family Pteropodidae) and antibodies were found by ELISA and Western blot in 3 Acerodon jubatus (giant golden crowned flying fox; range) bats and by ELISA alone in a Pteropus vampyrus (Large flying fox; range). If you trust the test, then this indicates past exposure.

Superman and the Joker know very well - Bats can be very tricky. But at least this finding helps to further address the Riddle(r) of the reservoir. Now, if only we could only nail down the specific culprit(s) in West Africa.

References...

1. Molecular evidence of Ebola Reston virus infection in Philippine bats
   http://www.virologyj.com/content/12/1/107?utm_content=bufferf022a&utm_medium=social&utm_source=twitter.com&utm_campaign=buffer
2. Fruit bats as reservoirs of Ebola virus
   http://www.nature.com/nature/journal/v438/n7068/abs/438575a.html
3. Many details about bats to be found at the excellent IUCN Red List
   http://www.iucnredlist.org/
4. Population Structure of a Cave-Dwelling Bat, Miniopterus schreibersii: Does It Reflect History and Social Organization?
   http://jhered.oxfordjournals.org/content/100/5/533.full
5. Seasonal movements of the Schreibers’ bat, Miniopterus schreibersii, in the northern Iberian Peninsulahttp://www.tandfonline.com/doi/abs/10.1080/11250000801927850#.Vamrtvnzp1M

Updates...

1. Added bat specie range data (and discussion) from IUCN Red List and Wikipedia.

Liberia gave Ebola the boot...and a virus may soon be removed from the wild

The people of Liberia have earned our respect, some time for national celebrations and frankly any other rewards that may flow from denying the Makona variant of Ebola virus any hosts among their community. 

The world considered this viral species to be one of the list-toppers when it came to ranking the causes of the most scary acute infectious diseases. Ebola virus has been the basis for all sorts of 'end-of 'the-world' mutating virus horror movies, books, and TV shows. It's not at all surprising that the public view of an Ebola virus infection had long been one of blood, fear and terror.

Figure 1. The decline of the Makona variant of
Ebola virus in Guinea, Sierra Leone and Liberia
(now free of EVD transmission).
Click on image to enlarge.

Behavioural change was a major factor in reducing virus transmission in Liberia. Alongside that was a broad range of aid given from within and beyond Africa's nations. By working together, a widespread outbreak that was not initially thought likely to happen at all, was routed. 

For now. 

Liberia is not immune to new cases of Ebola virus disease (EVD) crossing its borders or popping up due to a new animal-to-human jump (a zoonosis). That could happen any day - it might be happening now. But those who are still on watch will be searching out new cases while the remaining sites of transmission - Guinea and Liberia - do their best to deny Ebola virus a chance to replicate and spread. The people of Liberia will keep watch help because they have learned very tough lessons about viruses, epidemiology and communication. At least 10,604 suspect, probable and confirmed EVD cases, 4,769 deaths and way too many stories of sadness and families destroyed are a very strict teacher. 

Figure 2. The number of confirmed EVD
cases (yellow) grinds to a standstill. Only
9 cases in the week to 10th May 2015.
Click on image to enlarge.

The crude prediction in Figure 1 suggests that zero cases across all three countries could happen at the end of May, but many stars must align for that to be a real event. 

Human factors - the causal and sustaining variables of any outbreak of infectious disease in humans and sometimes animals - remain very much in play. But once that tri-country zero case value is attained, we have 42 days of watching and waiting - from the time the final case tests negative. 

New cases may arise from sources as-yet-unknown. But even if they do keep popping up, it seems very unlikely that widespread transmission will amplify to earlier levels (see the steep slopes in Figure 2) unless a major lapse in attention occurs. Hence,the need for continued vigilance - and Liberia remains on alert for a further 90 days. That more recent figure comes about because we know that infectious Ebola virus can persist in some body sites for many weeks after signs of disease have passed. Whether that virus reservoir is present in every person and whether it actually does cause new Ebola virus infections remain unproven. When you consider what can happen when one person gets infected by an Ebola virus in a tiny remote village in a country that is ill prepared to cope with it and has traditions that lend themselves to its spread...even minor risks rightly come under more intense scrutiny.

What next for this particular virus though? The only place where the Makona variant of this member of the Zaire ebolavirus species will soon exist, is in the freezer of (hopefully) very biosecure laboratories in the US, UK, Africa, Russia, China and probably other laboratories in countries that hosted, evacuated or repatriated cases of EVD. 

There is no sign at all - and this is because of the continued efforts and focus of many currently working throughout west Africa - of the fabled "endemic Ebola" becoming a reality. Unless you mean enzootic 'Ebola'- in which case , it already is, I suspect. It seems very, very likely that the forests of west Africa continue to shelter animal hosts with less mutated versions of this and other ebolaviruses (and filoviruses and who-knows-what else). The host species and route(s) of transmission to humans are yet to be confirmed but for now, we are not too far off eradicating one unwanted viral scourge from the wild. Impressive what we can do when we pull together.

Bats in a tree...

Meliandou and the burnt tree that
once housed a bat colony (from Fig 3, [1]).

While not snakes on a plane, I'm fairly sure the level of swearing has at times been at least as bad among those suffering from and dealing with the possible fall-out from these bats - if in fact they were the source for the biggest Ebola virus disease (EVD) epidemic on record.

A recent animal counting, trapping and testing study in Guinea included sampling in and around the village of Meliandou.[1] This village is, to the best of our knowledge, the site of the first animal-to-human, or zoonotic, transmission of the Ebola virus variant called Makona.[2]

The study team, made up of researchers affiliated with Germany, Sweden, Core d'Ivoire and Canada, did not find any decline in numbers of usually susceptible larger mammals around the index village; a sign during other outbreaks, of active local ebolavirus "activity". The team also found that primate hunting was not a big thing in this region, which is rather devoid of these and other Ebola virus mammalian host animals (including few of the Duiker, or forest antelope). Fruit bat hunting was common though.

The team captured 169 bats representing at least 13 different species and 6 families. But in the house of the 2-year old boy considered the epidemic's index case, fruits bats were not eaten and no bat hunters resided there. No Ebola virus RNA was detected in any bats and antibody screening results from bat blood were inconclusive. 

These findings led the authors to study Meliandou, resulting in an hypothesis that a nearby hollow tree that once housed a large colony of free-tailed bats [locally described as lolibelo - small and smelly bats - otherwise known to belong to the species of insectivorous bat, Mops condylurus of the family Molossidea; [3], may have been the source of  infection. Why only one child was infected this way when the tree was a site of frequent play by many children is not known. The tree was burned out in March 2014 which caused many bat deaths, some of which were collected for consumption. Sequencing of a PCR-amplified mitochondrial DNA segment found that in 5 of 11 ash and soil samples from around the tree, contained traces of Mops condylurus genetic material. So that species was at least there.

So, this is all quite far from a conclusive link between the 2-year old boy and these bats. But it does read as though every avenue has been tested in this village, perhaps apart from better animal antibody testing (serology), and some serology on the blood of those villagers who remain alive in Meliandou. 

Serology testing is going to be very important for answering many questions around EVD and this outbreak and epidemic. 

Of course this will raise the usual question of whether we cull all bats to prevent this from ever happening again. Don't be ignorant! Bats have very important roles in pollinating and thus in keeping our ecosystem going. Should we kill all bees because they sting us? I'm pretty sure I've been stung by a bee more times than I've had Ebola/Hendra/SARS/Nipah/MERS/Lyssavirus or any other bat-hosted virus infection. Killing off everything to prevent a very rare zoonotic event when better knowledge can resolve the problem is just a typically short-sighted and knee-jerk human reaction (not a fan-can you guess?).

One question that does still remain, and one that is of extreme interest to me, is how often mild disease results from an Ebola virus infection? Good, robust serology methods to the rescue.


References...

1. Investigating the zoonotic origin of the West African Ebola epidemic. EMBO Molecular Medicine(2014). http://embomolmed.embopress.org/content/embomm/early/2014/12/29/emmm.201404792.full.pdf
2. Nomenclature- and Database-Compatible Names for the Two Ebola Virus Variants that Emerged in Guinea and the Democratic Republic of the Congo in 2014. Viruses 2014, 6(11), 4760-4799.
   http://www.mdpi.com/1999-4915/6/11/4760
3. Mops condylurus via the IUCN Red List of threatened species (listed as of least concern)
   http://www.iucnredlist.org/details/full/13838/0

Stuff from the literature: very SARS-like coronavirus in Chinese horsehoe bats...

The smoking bat for SARS-CoV?

Xing-Yi Ge and colleagues from China, USA, Australia and Singapore described some new severe acute respiratory syndrome (SARS)-like coronaviruses (SL-CoVs) in bats, publishing in Nature last month.

These discoveries were especially notable (not that any new virus discovery isn't) because they displayed more "SARS-like" properties than many earlier so-called SARS-like CoVs. One could grow in the same line of lab cells and also in human cells, it could be visualized by electron microscopy and it could use the same receptor as the SARS-CoV (angiotensin converting enzyme II; ACE2) . Plus, they were genetically very similar.

The bat species was confirmed by gene sequencing to be Rhinolophus sinicus, family Rhinolophidae; the Chinese rufous horseshoe bat.

Throat and faecal samples (anal swabs and faeces) were screened using RT-PCR with primers towards the conserved RNA-dependent RNA polymerase region (RdRp) and new primers were designed to detect other regions of any discoveries. 27 of 117 samples were CoV POS and had sequences determined.

Two novel (and 5 previously identified) SL-CoVs, each with a 29,787+ base pair RNA genome and sharing 95% nucleotide identity with the Tor2 strain of the SARS-CoV which is higher than previous SL-CoVs from China. The receptor binding domain (RBD) of the new CoVs shared 85-96% amino acid identify with the SARS-CoV. These were called:

1. RsSHC014
2. Rs3367

Vero cells were used to attempt growth of SL-CoV virions that were first concentrated from samples. This succeeded for one sample, a variant of Rs2267 (99.9% nucleotide identity with Rs3367) and they named this isolate SL-CoV-WIV1. This success is something that hasn't been achieved with the majority of recently identified bat CoVs.

WIV1 also grew, although less efficiently, in:

• human alveolar basal epithelial (A549) cells
• pig kidney (PK-15) cells
• R.sinicus kidney (RSKT) cells

...but not in...

• Human cervix (HeLa) cells
• Syrian golden hamster kidney (BHK21) cells
• Myotis davidii kidney (BK) cells
• Myotis chinensis kidney (MCKT) cells
• Rousettus leschenaulti kidney (RLK) cells
• Pteropus alecto kidney (PaKi) cells

So we have much more convincing evidence that the SARS-CoV is likely to have originated from a bat.

h/t to @MERS_inSAUDI

Is there a better smoking bat or camel?

That teensy fragment of Middle East respiratory syndrome coronavirus (MERS-CoV) sequence (yes, I called it a fragment of that virus) from a Taphozous perforatus bat caused a lot of hassle last week,certainly a disproportionate amount to it's representation of only 0.5% of a MERS-CoV genome. Similarly, the report of MERS-CoV protein-reactive antibodies in camels some weeks back.

In a New York Times (NYT) article discussing the discovery of the 180-203 nucleotide (nt) gene fragment in a Saudi Arabian tomb bat, Donald McNeil opened with..

Health officials confirmed Wednesday that bats in Saudi Arabia were the source of the mysterious virus that has sickened 96 people in the Middle East, killing 47 of them.

The size range represents numbers used in various articles and of the fragment from the public sequence database GenBank (203nt) -"~190 nt" noted in the actual scientific publication). By the way, has so much ever before been written about so tiny a sequence?

Because he did not include in this line, or his article, a list of all the various possible scientific shortcomings, he didn't write in more detail about the difficulties with linking a virus in any sample to the cause of a disease  in humans, he forgot to specify that this was not the actual virion that caused MERS in the human index case in Bisha, he left out the PCR-101 section on why detecting a genetic sequence is not the same as isolating an infectious virus or how to interpret a PCR fragment's sequence....he was, in some circles, criticised. 

Okay, so the bat study did not isolate infectious virus, could not obtain any other sequence, found sequence in a bat from the family Emballonuridae rather than the "expected" Vespertilionidae bats and the positive sample was from bat faeces rather than blood or some other sample more convincing. Perhaps insects, food for T.perforatus, carry MERS-like CoVs? No-one has ever found that though. Is there evidence for 2 CoVs to be completely different except for a stretch of ~100% identity? Don't know, but don't think so. As Prof Andrew Rambaut noted in his very detailed analysis of this fragment, it does differ by 1 nucleotide from many MERS-CoV sequences (so its 99.5%-100% identical). Is there a more likely animal carrying a more similar MERS-CoV strain of virus that is spilling over to humans causing MERS cases? None that has been publicised to date. And therein likes my beef with some of the criticisms I have read this week. 

So far, this finding is the best lead we have in finding an animal source. There is no evidence to dispute the link, any more than there is evidence to prove it. Yes, there may be a another smoking bat or camel or something else out there. But it hasn't been found yet. And the public might like to hear how researchers are progressing rather than wait the very long time it will take to dot Is and cross Ts on the final MERS-CoV life-cycle, once they determine it.

And by the way, there is no other CoV sequence on GenBank that has >90% nucleotide identity with the T.perforatus sequence, except for the human MERS-CoV sequences. That doesn't exclude there being some other recombinant or novel CoV out there, but that is pure speculation; more so than saying that this new sequence represents a strain of the MERS-CoV found in humans.

In succinctly summarising some of the criticism, an article in CIDRAP presents a great overview and hints at what this criticism implies; that stories in the media must get every detail spot on or the writer may be portrayed as a poor scientist.

What? Wait. Seriously??

This was a newspaper article in the New York Times people. It was about 870 words long. It won't be setting global health policy nor will it be creating a WHO disease notification stating Taphozous perforatus bats, in particular, are the primary source of all MERS cases. Or of any cases. I don't doubt this is a prestigious newspaper but this story will likely be nest week's fish 'n chip wrappings (does anyone still use newspaper to wrap fish 'n chips? Is there a digital equivalent of - "yesterday's homepage, tomorrow's archive"?). In my opinion, and I don't mean to speak for all, scientists and health policy makers know that a newspaper is not a peer-reviewed scientific journal and that it's intent is to inform it's readers so they'll come back.

Were the many readers of the NYT misinformed? Perhaps the "health officials" could have been better defined by the NYT article. Presumably it's the authors-researchers may have been a better descriptive (as was used in a follow-up piece), probably more in tune with the public's perception of us. Beyond that the article did a good job of presenting the results of a research paper's relevant findings to the wider audience. They also both caution against over-interpreting the data. The NYT article has that well covered; a transmission route is not clear, more testing needed, more work being done, sample degradation due to a break in the cold chain, it was only 1 bat.

I very much agree with comments in Robert Roos CIDRAP article; the critics are getting carried away. There are many different levels of science communication that reach the general community - the popular press are not Lancet, and vice versa.

If you really want to pick holes in a part of the coverage, you might well ask why so many are looking at a 180-190nt fragment when the ends of that PCR-amplified fragment actually reflect the commercially made oligonucleotide "primers", not the (likely) viral template at all.  The actual fragment that should be analysed from the T.perforatus bat droppings is, at best, 156nt (but only 137nt if the internal nested PCR product was sequenced but the product on GenBank, 203nt long, includes both internal and external primer sequences in it-PCR speak here, sorry). Probably won't change the outcome of any analyses to date (156 still encompasses the nucleotide variation and is still differs by 11% from any non-MERS-CoV sequence), but it is a different number. 

I'm sure a newspaper headline "156 nucleotides of a 30,130 nucleotide genome possibly related to the mystery virus that may have directly or indirectly killed 47 people in Saudi Arabia" would be a real page turner.

So, let's keep up the good work of presenting and trying to deconvolute our own studies, let's keep the public interested and informed without overcooking the message, let's allow for imperfection (as readers of this blog will be all too familiar with), but perhaps let's keep the peer reviews to the scientific literature where they are in demand and required...and keep perspective on these new findings when they come to our attention through the popular press.

A model of MERS-CoV acquisition (ver1)

With thanks to David Spalten (@dspalten) for discussion and considerations and AtRG for advice.

First we heard about Middle East respiratory syndrome coronavirus (MERS-CoV)-related viruses in bats in South Africa, then we read of antibodies in camels that reacted to MERS-CoV more than the most likely (known) other CoV to infect cattle, and most recently we were absorbed by the discovery of a probable parental strain of the MERS-CoV in the faeces of a Taphozous perforatus insectivorous bats.

We've also heard that most patients have not had direct or obvious contact with bats and we also know that pasteurised camel milk products should be safe. But that still leaves many stones unturned.

So if we can assume that the most likely route of acquisition of MERS-CoV is through the upper respiratory tract and that the spillover events come from animals (I'm including human-to-human exposures in this figure) then we need to consider how that might happen. I've included the animals above as well as baboons as they seem highly mobile, interact well with humans, visit mountains and caves (where bats are likely to hang out") and are found in the KSA. I've added ingestion but I don't really imagine how this could result in a respiratory infection, and MERS-CoV gastrointestinal involvement seems infrequent.

I don't live in the Kingdom of Saudi Arabia or in the Middle East and I do not profess to know much of the environment so what follows is "remote guestimation" at best. But I've thrown together some of the possible routes and animal players into a figure which may have some degree of reality buried in there somewhere. It may also spark an idea or two among those who do know what they're talking about.

So, here is my model of how humans may indirectly get a zoonotic infection from a primary or secondary animal host...

A model of MERS-CoV acquisition. Click to enlarge.

I'd be most happy to take suggestions for improvement of the figure. I know some of you like to use the graphics from the blog and Virology Down Under (which I strongly support, asking only for a specific reference to their source) so if they can be made more robust, I am very happy to do so. Get to me via the comment section below or on Twitter (see top right).

Prof Lipkin: There is no more sequence coming from that bat sample

Many thanks to Prof Ian Lipkin's indulgence of my eMail questions.
Also, check out the TWiV webcast by Prof Lipkin.

So, I guess to carry on from last night's post....I stand surprised. 

Not even next-generation sequencing could pull any more sequence from the MERS-CoV-positive T.perforatus bat samples that thawed after the dry shipper (not a box+dry ice as I previously guessed, but a vacuum sealed vessel previously brought to -150°C then all free liquid nitrogen removed for transport; shipped by FedEx) was opened and the cold chain interrupted after arriving from the Kingdom of Saudi Arabia (KSA). 

According to Prof Lipkin, in an email exchange we had last night, the group also tried a couple of runs of next-gen sequencing.

..we tried two separate ion torrent runs with no joy.

So why was only 1 October 2012 sample positive for the MERS-CoV strain? Prof Lipkin concludes that..

..the concentration of template was already extremely low in the sample at the time of field collection and lower still at the time of arrival in our Center. I would not be surprised if two aliquots of the same sample yielded different results in different labs. However, we will never have an opportunity to know because there is no more sample to test.

At the time of receiving the October samples (no MERS-CoV was found in the April samples), no viral gene/gene fragment/genome cloning had been done in Prof Lipkin's lab. A common potential source of PCR contamination ruled out. 

How does your group know that this 182 basepair nucleotide sequence was not a contaminant from somewhere else? 

The one sample came up positive repeatedly with the same assay. No other sample did so. We have recovered no other fragments that correlate with a MERS-like CoV in samples collected in the October 2012 or in the subsequent April 2013 field collections.

Antibodies were not sought in the massive 10μl of bat blood obtained per bat (the bats were released after sampling). But do these findings exclude the possibility that other bats, like those from genus Pipistrellus and Neoromicia (both from the family Vespertilionidae), or genus Nycteris, family Nycteridae, may be a host for MERS-CoV? At a World Health Organisation meeting in Cairo, Prof Lipkin told the the audience that..

...our findings don't exclude the presence of virus in a Vesper bat and that we were doing everything anyone suggested to test alternative explanations, including reagent contamination. We went back to the original materials using every specific and consensus primer set we and others had designed until all of the original material was exhausted. The results were the same. I sat on these data for months hoping to find another positive bat in subsequent field expeditions where we could report more sequence.

The decision to report it now was multifactorial. First and foremost, we tested every possible alternative explanation for the sample coming up positive other than that this fragment is bona fide-we can't find an alternative explanation. Second, there are no other reports from animals in KSA-I discussed phylogenetic analyses with several people in light of what was found elsewhere in Africa in Vesper bats. This fragment, although short and located in the RdRp is informative....Third, the scientists who did the work in the US and the Ministry of Health of KSA wanted to see it reported. There is a point where one has to get the work out in the public domain.

What's next in the search for animals hosting this virus and in trying to confirm what the group has just reported? There will also be a new European collaborative report (UK and KSA) coming out very soon that has new human MERS-CoV sequences suggesting multiple human introductions (animal to human?) with much more sequence variation in the MERS-CoV genome than we have seen thus far. This will further support the conclusion that the T.perforatus CoV is one and the same virus as that which infects humans.

...field expeditions should begin in the next few months and we will look again. The amount of time and resource invested already is far more than intended. I've never put in so much to recover so little.

Thank you to Prof Lipkin. This gives a some valuable insight into his careful efforts to deduce what animals may host a MERS-CoV strain,m as the first step in tracking how humans in the KSA are getting infected. It also highlights that finding even a basic piece of information requires many steps, lots of people, much effort and some luck. But if virus hunting was easy, everyone would do it right?

Some slight editing for brevity, and to account for mobile phone thumbs, was undertaken by VDU.