Sunday, 22 November 2015

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]


Friday, 20 November 2015

Liberia reports another case of Ebola virus infection...

Reset the contacts clocks. Liberia has had a second setback in the fight to rid West Africa of human Ebola virus (EBOV) infections. 

While we were academically aware this could happen again, I'd venture to say we probably thought the next case of Ebola virus disease (EVD) would return to Sierra Leon or Guinea. Liberia which was declared free of known transmission back in 03-SEPT-2015...78-days or 2-months and 17-days ago, and was without a case for 42 days before that.

This was where I learned about the new case from...
It has now been confirmed that a 15-year old male, the beginning of Liberia's "fourth wave" of EVD, acquired EBOV from....somewhere...somehow.[9] 

15M became ill from 13-Nov and was confirmed as EBOV infected 19-Nov.[7,8,9] He died 24-NOV.[12]

UPDATE #1: tally now stands at 3 cases - the 2 new cases among family members of 15M include his 8-year old brother and his father.[8,9] It has been said that 15M had no known contact with a survivor or relevant travel history.[9] 15M was previously described as 10M, but age amended in [9])

UPDATE #2: 153 contacts being monitored.[10]
UPDATE #3: WHO lists boy as 10-year old in 23NOV update. Aiyahh!  
UPDATE #4: Announcement of 10M death.[12]
UPDATE #5: Guess what? WHO says 15M.[13] Father, 40M.[13] 149 contacts including 10 healthcare workers.

Information on how the initial infection was acquired remains scant.

Time will may tell us more.


Monday, 16 November 2015

National Health and Medical Research Council: project grant funding successes for supply in 2016...

Just a short one.

Putting up a graph I posted on Twitter 09-NOV-2015 in case anyone wanted the chart.

This is a better quality image.

Click on image to enlarge

You can move money around to young or old or male or female, and dress it up as people support or project support - but until the pot of cash grows, or researchers stop applying, success rates will remain dismal or get worse.

Perhaps the Medical Research Future Fund will do this. At some point. If the will remains.....sigh


...the camel data review will return soon.

Saturday, 7 November 2015

Congratulations to Sierra Leone for defeating its Ebola virus disease epidemic!

Edited by Katherine Arden, PhD.

"On Behalf of the Sierra Leone National Ebola Response Centre (NERC) and the World Health Organisation (WHO)

Note to Correspondents

Subject: Ebola transmission in Sierra Leone over. 
Nation enters 90-day enhanced surveillance period
On 7 November 2015, if no new case of Ebola virus disease is recorded, Sierra Leone will have met the criteria set by the World Health Organization (WHO) for declaring the end of Ebola transmission. If Sierra Leone meets that milestone, on that day, the WHO will declare the end of Ebola transmission in Sierra Leone, at an event organised by the Government of Sierra Leone through the National Ebola Response Centre (NERC)."

This is the message that greets you on the NERC website today. Ebola virus transmission has finally been kicked out of Sierra Leone after a 42 period with no cases confirmed. Getting to zero is now, Got to zero!


It has been a hard fought battle with many, many losses. Battling the Ebola virus has also provided many teaching moments for the it has been for Liberia and still is for Guinea...and the world.

Within the next 90 day enhanced surveillance period and in the months and months to come, we may see a case or cases pop up and clusters may result. But Sierra Leone knows what Ebola virus disease is and how to deal with it. It won't be caught out the same way again. 

Many more teaching moments undoubtedly remain. But each will surely be faced with the same strength and passion that drove the nation to defeat an epidemic the likes of which the world had never before seen. 

The people of Sierra Leone made many new friends during this tragedy and hopefully they will always be but a call or a text or an email away. Far too many of those incredibly brave local and international health workers, burial teams, laboratory specialists and ambulance drivers paid for their efforts with their lives. So to them, to those who survived, to all the contact tracers, the social anthropologists, the psychosocial experts, the survivor clinics, the organizers, the facilitators, the doers and the thinkers from within and outside Sierra Leone, we give our heartfelt thanks for your work and your many sacrifices. You together with the people of Sierra Leone all contained and defeated Ebola virus disease and you did it in the face of often overwhelming odds. 

Enjoy the parties. Remember the lessons. Be vigilant.


Thursday, 5 November 2015

Updating the very model of a modern mammal-camel....

The new findings from the case-control study out of the Kingdom of Saudi Arabia (and US CDC) deserve an update of my old model of how one might become infected with MERS-CoV after exposure to an infected camel.[1,2]

Some of the possible ways in which MERS-CoV may be spread from an infected
camel to a human in direct or close contact with the camel or with surfaces
onto which MERS-CoV-laden camel excretions or secretions have been deposited.
The major change is the removal of the ingestion options. As readers of this blog will know, I've never been a "believer" in that route of infection, and the new study would seem to support that gut feeling with some facts.

As ever, the distinction between direct contact and being close enough to be exposed to droplets that are inhaled, has not been possible and wasn't attempted. The word "droplet" does not appear anywhere in the paper. In fact, animal contact and droplet-producing processes are all rolled together in the new study under the direct contact banner - so I have retained droplets among the possible risks shown in the figure.



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.


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, 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.

  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.

Friday, 23 October 2015

Markets that deal in camels may help spread MERS-CoV variants..

This camel/MERS-CoV study from Farag and colleagues, serves as follow-up of sorts to my last post. The paper, which was published in July 2015's Infection, Ecology and Epidemiology, is entitled High proportion of MERS-CoV shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases, Qatar 2014.[1]

The authors remind us in the background that the routes of direct or indirect zoonotic transmission are still unknown but that a "large proportion of MERS cases" are suspected to have resulted from zoonotic transmission.

105 dromedary camels (DCs) either from a market sale or directly from Qatar or the Kingdom of Saudi Arabia (KSA) were sampled in February (n=53) and March (n=52), 2014. Samples included nasal, oral, rectal and bronchial swabs and lymph nodes from animals grouped into age 3 groups: 0 to 6 months (n=41), 7 to 12 months (n=35) or greater than 12 months (n=29) of age. Testing for virus was by Corman et al's UpE and N gene real-time RT-PCRs.[2] Testing for antibodies was via the detection of a reaction to the MERS-CoV, severe acute respiratory syndrome (SARS)-CoV and human CoV (HCoV)-OC43 spike domain S1 antigen using the protein-microarray method described previously by this group.[4]

  • 59% of DCs had at least one MERS-CoV RNA positive sample but no significant difference in viral load was apparent between sample types or ages
    • 61/101 (60.3%) of DC's nasal samples had RNA detected
    • 23/102 (22.5%) of DC's saliva samples had RNA detected
    • 15/103 (14.6%) of DC's rectal samples had RNA detected
    • 7/101 (6.9%) of DC's bronchial samples had RNA detected 
    • 5/53 (9.4%) of DC's lymph nodes had RNA detected
  • 5 different MERS-CoV variants (subtly different versions of MERS-CoV) were circulating in Qatar among the sampled animals at this time according to RT-PCR/sequencing method that targets a fragment of the S2 domain of the MERS-CoV Spike gene.[3]
  • 100/103 (97%) animals were reactive for IgG, and most of 53 animals tested, had antibodies capable of specifically neutralizing cellular infection by MERS-CoV as determined by a 90% plaque reduction neutralization test (PRNT90; [5])
  • Antibody levels and viral load did not correlate suggesting - based on this subset of the immune response - that reinfection may be possible since protection may be limited, as it is among humans with the 4 known HCoVs. The authors note that this may prove a challenge for any future DC vaccine which would need to produce a protective effect to meets its need
  • No age-specific differences were found in MERS-CoV RNA shedding - usually younger DCs are distinctly more likely to be shedding viral RNA than older DCs

The authors noted here that discrepancies do exist between their study and those of some others - specifically, that others have not found viral RNA in faeces - but those studies also tested fewer animals. It is important, when percentages are not high, to test enough animals to see the full extent of MERS-CoV shedding and potential transmission routes.

DCs from different regions within Qatar and outside Qatar, may be shedding MERS-CoV while in DC markets and holding pens, sometimes for weeks, awaiting slaughter. 

Camel markets are thus a likely high risk area for acquiring a MERS-CoV infection - and multiple variants can be circulating here. 

In previous Qatari investigations, human cases have been linked with visits to the areas studied here and have also included DC slaughterer cases, supporting the notion that humans with DC exposures (presumably when they are infected with MERS-CoV) are at risk of becoming infected themselves. 

Yet this study did not manage to capture the process of transmission in action. It is that process that holds such importance for this chapter on MERS-CoV and especially for those who disbelieve the role of DCs in human MERS cases. 

In the next post, we will re-visit a study that did seem to capture DC>human infection.

  1. High proportion of MERS-CoV shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases, Qatar 2014.

Tuesday, 20 October 2015

If you are often in contact with camels are you more likely to acquire MERS-CoV? [spoiler: yep]

This dromedary camel (DC)/Middle East respiratory syndrome (MERS) themed post is a quick review of a paper from 2015 by Reusken and a team of absolute champions in this space. 

It, as many have been, was published in the Emerging Infectious Disease journal, listed in its August issue (but online earlier) and entitled, Occupational Exposure to Dromedaries and Risk for MERS-CoV Infection, Qatar, 2013–2014.[1]

The study examined 498 sera from humans in Qatar split into different exposures types. Included were European (the Netherlands and Germany) human sera for use as controls - collected from a part of the world where there was not expected to have been any MERS-coronavirus (CoV) exposure and so no antibodies were expect to be present; a test for the tests.

As an aside, we've seen some great informative MERS-CoV/camel studies come out of Qatar. I love watching good collaborations pay dividends.

The 498 sera breakdown as follows:
  • 294 from those with daily DC exposure
    • Cohort A: 109 camel (A1; n=5) and sheep (A2; n=104) slaughterers
    • Cohort B: 8 central animal market (CAM) workers
    • Cohort C: 22 healthy males living & working at Al Shahaniya barn complex adjacent to DC race track
    • Cohort D: 155 healthy males living & working at DC farm
  • 204 from those without camel contact
    • Cohort E: 56 samples from construction workers
    • Cohort F: 10 people living in a complex with 200 sheep barns
    • Cohort G: 138 specificity testing samples (66 from the Netherlands and Germany who had recent CoV infection (G1) and 72 from the Netherlands obtained for Bordetella pertussis infection testing (G2)
The antibody testing regimen relied on a multi-tier approach (the best ones do, until we're sure that any single assay can cope with all the variables):
  • Tier 1: IgG antibodies were sought using the MERS-CoV, severe acute respiratory syndrome (SARS)-CoV, human CoV (HCoV)-OC43 spike domain S1 antigen protein-microarray method used previously by this group [2]
    • 20/294 samples (6.8%) reacted (had IgG antibody in them) - none were from controls sera or from those without DC contact
    • 4/22 Cohort C, 8/155 Cohort D, 3/104 Cohort A2 and 4/5 Cohort A1 samples were reactive
    • All samples from A1, A2, B, C, D, E, F and G1 showed responses to HCoV-OC43 S1
    • None of 498 sera reacted to SARS-CoV S1
  • Tier 2: A 90% plaque reduction neutralization test (PRNT90 [4]) was used to show whether antibodies in samples could specifically stop MERS-CoV from infecting cells after sera and virus were co-incubated ahead of infection of a cell line
    • the 20 IgG reactive samples from Cohort A to D were tested and 10 were able to neutralize infection
    • 34/35 samples from those with camel contact (Cohorts A1, B and C) that were IgG non-reactive, also had no neutralizing antibody
  • "Tier 3": Use of a whole MERS-CoV immunofluorescence assay (IFA). However, the results from testing 8 reactive samples (5 of which were positive by IFA) were not included
This paper has a nice central finding which goes something like: if you don't have contact with camels, you don't get infected by MERS-CoV. If you do regularly have contact with camels - you are much more likely to get infected as determined by you having developed antibodies to that virus; you were infected but you fought off the infection. A similar finding came out of the larger serosurvey from the Kingdom of Saudi Arabia.[3] 

I do wonder about the reactive sheep slaughterers though (Cohort A2) - where did those infections come from?  

The authors also addressed why other serologic studies of humans with occupational exposures have not found reactive sera-those studies hardly ever documented infected camels at the workplaces and there may not have been any (for some significant period of time presumably). More infected camels may be associated with more human infections. No surprise. The authors had found, outside this publication, that 60% of camels at the CAM and slaughterhouse were shedding MERS-CoV. This discrepancy has been a question of mine for a long while - and I like this answer.

Interestingly, the participants with antibodies don't recall being seriously sick. So you may get infected and just think you have the flu, or a cold, or nothing at all. This result may further confuse camel-deniers who do not have any background in the wide spectrum of outcomes one can expect after infection by any virus. Nonetheless, such apparently unnoticeable infections add more weight to the story that the current proportion of fatal cases is an exaggeration. So we learned yesterday that MERS (the disease) is rare, that camel contact makes up only a proportion of the likely sources of infection and now we see that you may not even get sick if you do get infected. A few things to digest there.

Also very interesting to me is that the neutralizing antibody titres were lower than had been found elsewhere. The authors suggest this may be due to these infections producing only mild disease. Without a prospective study though, it's very hard to be sure about the true disease severity - recall bias can be a pest. This is an area that needs a more focussed study; do our antibody tools detect mild and asymptomatic cases as reliably as severe MERS cases, for how long and in all cases of infection?

Its feels like its getting pretty hard to mount any realistic case for why we should ignore the role of camels in infecting us with MERS-CoV - even if they do so rarely, and perhaps often without serious complications.