Animal contacts among Middle East respiratory syndrome coronavirus (MERS-CoV) cases: UPDATE

Click on image to enlarge.

An update of the total number of cases where animal contact or exposure has been noted. 

Such mentions seem to have been on the rise since October 2013, but the Kingdom of Saudi Arabia (KSA) still has the lowest proportion of mentions among the list, despite the greatest number of MERS-CoV cases overall. 

Perhaps this disparity reflects a greater number of healthcare-associated (infected workers and hospital-acquired infections) transmission chains compared to other countries.

My last post on this back in 21-Mar-2014, was focussed on camels alone but this one differs in that it includes all animal mentions. Camels are most often identified though; 71% (15 of 21) of al cases noting animal exposure of any sort. 

Total cases with "animal" mentioned, according to my database, includes 21 distinct cases.

Monkey magic: Vero cells make more MERS-CoV RNA than any other animal's...

Apart from camel, goat, bat and human cells which draw the eye in studies on the source of MERS-CoV, did anyone notice the Vero cells? 

These cells are derived in the dim dark ages from African green monkey kidney (Cercopithecus aethiops). After infection, these monkey cells were shown to make more MERS-CoV RNA than any other cell line tested and the second greatest quantity of viral particles (after goat cells).

Check out Eckerle et al. over at the CDC's Emerging Infectious Diseases journal for a very nice graphic.

This may be an artefact of the adapted cell-line....but let's not forget to test those baboons hmm?

Most MERS may not have met a camel, but index cases may have

Donald G McNeil Jr., writing in the New York Times  a few days ago, posited the idea that camel contact, while not at all widespread among the majority of Middle East respiratory syndrome (MERS) cases, may play a role in the first cases that sparked clusters of infection; the so-called index cases.

These sorts of patterns, whether for camels, bats, baboons or cats, are key to understanding infection acquisition. 

We all look forward to reading more detailed local analyses of the sleuthing that seeks to define first contact with our MERS-CoV adversary...sometime in the future. 

Infection Scene Investigation (ISI): Kingdom of Saudi Arabia?

First noticed on Twitter from @crof.

Why only 181 nucleotides of T.perforatus MERS-CoV sequence?

In some of the many articles written about the new discovery this week, there were comments along the lines of  its amazing any sequence could be obtained from the samples cause they had sat for 48-hours at US customs and thawed. A more precise quote could be found here for example.

I have some thoughts on that - and these come from me, someone who has worked with a lot of clinical human specimens from which I've been able to amplify viral bits and pieces on a regular basis. Many small (200-600 basepairs[bp] fragments) but also longer pieces of >1,000bp, assembling small viral genomes from them. These samples may >10-years old, having been freeze-thawed numerous times after spending various amounts of time in courier vans, planes or sitting at room temperature before having nucleic acids extracted, tested and eventually (extracts may also sit around during testing and preparation and be freeze-thawed etc) frozen at -20°C or -80°C.

Keeping in mind that this issue of thawing might simply be a case of "hold your horses people". The EID paper was an early and quick report announcing the discovery of this MERS-CoV strain. So, my thoughts:

1. Because the materials that yielded the sequence (collected in October 2012) were described as "thawed" we can presume that the dry ice they were shipped with ran out during the transport to, or waiting time at, US customs. Once the refrigerant is all gone, the samples would come to room temperature as fast as the cardboard box and plastic receptacle it held, allowed. The publication described them as having been thawed for 48-hours.
2. How warm are we talking? The average temperature of Bisha (where the Taphozus perforatus bat was found, in an old date orchard outdoors) in October ranges from 15-20°C to 30-35°C. I don't know where the US customs site was so don't know that temp range - but expect it's less. So let's make some wholly unfounded assumptions:
• That this MERS-CoV strain can spread via the virus found in faecal pellets or other bat excreta. Perhaps as wind-blown dust or to other animals via a faecal-oral route. Even if the bats are hanging from a cave ceiling, but certainly when they are hanging outdoors, the virus must be capable of surviving in faecal pellets at a very high "room temperature" to complete a transmission event. If they can survive, that means intact virus - RNA genome + proteins + capsid + lipid envelope - the whole lot. For RT-PCR - you only need the RNA bit, not infectious virus. So, you're already lowering your expectations for what's required of a "successful" shipment.
• To confirm bat species, a genetic test was used which required the amplification of another piece of DNA - a region of the cytochrome B gene was amplified and sequenced. How large this fragment was, I'm not sure. However, a relatively large fragment of this gene can be used to differentiates bats, useful when you can't tell them apart by looking at physical features. Other work on opossums by the collaborator who helped sequence this region (Dr George Amato) in bats, employed >800bp of sequence. Why did this fragment amplify so well if the viral RNA did not? Perhaps because DNA is more hardy (various reasons) or because the bat blood or skin that it was amplified from, better protected the DNA from the thawing than bat faeces did for the viral RNA? Or...
3. Perhaps the primers used for other regions of the T.perforatus MES-CoV strain failed because the virus was too genetically distinct. I've had a look at the alignments and the primer binding sites can be found so it's probably not that. However, some of these primers that produce larger products are very degenerate (primers specially designed to account for nucleotide variation in a range of subtly different viruses or viral strains). 
• Degenerate PCR primers generally have much decreased sensitivity compared to 100% target-specific primers. This drop in ability to detect low amounts of RNA is the case even when using nested PCR - sorry if this has become to PCR technical! 
• The primers that did work for the T.perforatus bat MERS-CoV, Nested CII-MERS-RdRp, were much more target specific with only 1 degenerate base in 4 primers. That, combined with a drop in viral RNA amount, may well be why this 1 assay worked, worked where the others did not.
4. There was no mention in the EID paper of the use of an internal control RT-PCR target - a region of a gene in bat faeces (or blood or tissue depending on what was tested) that might allow some quality monitoring to see if there was truly decreased amounts of intact RNA in the October 2012 batch compared to that in the April 2013 batch of samples. That would be helpful to know which course to follow next.

So what does all this mean? Just me thinking in print I guess.

It's always important to maintain the cold chain from sample collection through to nucleic acid extraction and template addition to an RT-PCR/PCR tube. But I think we should look elsewhere for reasons why the T.perforatus MERS-CoV-positive sample has not yielded more than 1 fragment from the few assays used. 

I wouldn't be surprised if there was more sequence coming soon from this sample.

MERS-CoV genetic sequences found in Taphozous perforatus bat

Profs Ziad Memish and Ian Lipkin, and a team of collaborators including researchers from the EcoHelath Alliance, have published, in Emerging Infectious Diseases, their discovery of viral sequences in the faecal pellet of an Egyptian tomb bat.

Photo courtesy of Dr Jonathan H. Epstein.

Copyright EcoHealth Alliance 2013

MERS-CoV was only found in 1 of 29 Taphozous perforatus (Egyptian tomb bat, see some more detail on these in my next post) animals. These and 67 other bats captured in mist nets for this study, were observed nesting in abandoned ruins.

Samples from Bisha, Unaizah and Riyadh (Kingdom of Saudi Arabia) were snap-frozen on site, collected during October 2012 and April 2013. The October shipment was opened and thawed by US customs. Samples included wing biopsy, blood, throat swab, rectal swab and faecal pellets were collected for testing. Apart from RNA virus testing  bats were speciated by DNA analysis (cytochrome B gene). The T. perforatus bat identity could not be confirmed genetically because there was no reference sequence on GenBank - but it was similar to another member of the genus.

Helicase, RNA-dependent RNA polymerase (RdRp) and nucleocapsid or envelope regions were targeted for amplification and sequencing. 227/1003  samples (22.6%) were positive for an alpha or beta-CoV. 

The find, represented by a phylogenetic tree using on a 181nt RNA sequence fragment from the RNA-dependent RNA polymerase gene (100% identical to a sequence from the index case in Bisha, betaCoV 2c EMC/2012 over this region), secures bats as the/a primary animal source. So long as there was no contamination at customs or that the sequence actually came from a food source. Not too likely for either of those. 

Obviously more work will need to be done to find more instances, complete the genome (or at least sequence larger genetic fragments to make everyone happy) and isolate infectious virus - but this finding is a significant step in confirming a starting point for understanding how humans get infected by the MERS-CoV.

It's a shame this new fragment of the RdRp does not overlap with that sequence from the recent South African "nearest match" to MERS-CoV. In adjacent regions of the RdRp though, the South African virus does seems more genetically distant than this T. perforatus find.
  Perhaps we can re-visit the transmission chain issue with a view to how bats might infect a (probable) secondary host - say the camel for now - I'd suggest that palm trees might have a role in this as well as a possible role in direct human infections if sap/dates/drinks were consumed by the most at risk groups; elderly men with underlying conditions. Perhaps this consumption even has a role in them developing a chronic kidney-related disease? I previously wrote a little about this 19th June and on risk in a post 28th July.

H7N9 and H5N1 may have emerged from birds around Taihu Lake

Wang and colleagues from the Institute of Disease Control and Prevention of People’s Liberation Army, Beijing, China and from The University of Queensland, Australia writing in Emerging Infectious Diseases, note that most (71%) people in China infected with influenza A(H5N1) virus (spanning from 14/10/2004-17/05/2013) had direct contact with poultry or their excrement. That contact includes work-related handling of live and dead birds and their urine and faeces.
However, most of those infected by influenza A(H7N9) virus had indirect exposure to live birds, mainly through visiting live bird markets (LBMs).

Also of interest, most H7N9 cases were more closely clustered  compared to the more widely spread H5N1 cases.

The authors were looking for an overlap between the earliest known cases of infection of both viruses - perhaps to find a common source of the emergence of these zoonoses - and their studies suggested Taihu (Tai Lake) Lake which is near Anhui province and Shanghai municipality and borders Jiangsu and Zhejiang provinces. All place names that were foci of a lot of H7N9 activity earlier in the year.

The authors conclude that this region may be a key hotspot for spillover of avian influenza to humans. This would be a useful place to add to the emerging influenzavirus watch list and bird sampling/genotyping list. They also reaffirmed the role of LBM visits were another key risk factor for acquiring H7N9.

Camels being tested for MERS-CoV by Saudi Arabia?

An article on arabnews.com has Prof Ziad Memish saying that the Ministry of Health has "subjected samples from camels and sheep to lab tests" (not the long-delayed batch of samples Ian Lipkin recently received?).

Hopefully that means PCR-based testing with any of the various assays publicly available. 

The article notes the results will be announced after tests are completed.

This sounds a lot like reaction to the recent Lancet paper that found antibodies in dromedary or "Arabian" camels...the article does not note whether this is an ongoing study. Notably, camels were implicated early on the in the MERS-CoV outbreak.These new data will be very important in placing the Lancet article into context.

As Professor Marion Koopmans noted to me on Saturday,

"..we tried to rule out cross reactivity, and would not have published if we were not fairly sure that this is the case, but we will know for sure if someone finds the virus that triggers these antibodies."

Perhaps these will be the findings.

Camels carry signs of coronavirus contagion

Reusken and a European collaborative team have this morning described the first study looking for evidence of prior infection with the MERS-CoV, in animals. This evidence take the form of antibodies (immunoglobulin G or IgG ) made after the animal's immune system recognizes and then defends against future infection by that invader. 

The study used a very specific piece of the MERS-CoV Spike (S) protein. S is the bit of a CoV that sticks out and gives it the characteristic crown-like appearance under electron microscopy. The small piece of S acts as bait to detect the antibodies in serum samples and this interaction is identified by a fluorescent signal in the protein microarray system they used. 

These antibodies were found in the sera of 50 of 50 retired racing dromedary camels from Oman and from 1 in 7 Spanish (14 of 105) dromedary camels from the Canary islands. 

No antibodies were found in 80 cattle, 40 sheep, 40 goats or 34 other camelids.

The antibodies retained an ability to stop MERS-CoV infection in test that diluted the sera between 1:320-1:2560 for the Omani camels, and 1:20-1:320 for the Spanish camels 

Camels also had some signs of antibody reactivity to bovine coronavirus (BCoV), but the authors, after additional testing, concluded that the MERS-CoV reactivity was specific to that virus and not to BCoV. Sera from 2 human cases of infection by with the BCoV relative (both betacoronaviruses), HC0V-OC43, did not stop MERS-CoV from infecting cells - infection was not neutralized by the patient's HCoV-OC43 antibodies.

Camels were implicated earlier during the outbreak, in the death from MERS-CoV (then the "novel coronavirus" or nCoV) of a 73-year old male from Abu Dhabi, capital of the United Arab Emirates. 

"The patient owned racing camels. One of them got ill and was very weak; the patient was in close contact with that camel, and on the evening the camel got very sick, the patient developed flu-like symptoms. Three days later, he was in a medical unit in Abu Dhabi. There is another family member who also had close contact with the camel; he also got ill, but we could not follow up with that gentleman."

So this article points a finger at camels as some sort of host, possibly as an intermediate host between Pipistrellus spp and Rousettus aegypticus bats and humans. Perhaps the MERS-CoV story is akin to the Hendra virus story - bats contaminate horses and from there, close contact with horses can, on occasion  result in disease in humans. 

At the very least - there may be other animals involved yet and we still don't have viral RNA or a viral isolate from within a camel  - we now have a specific animal contact to track and trace for each human case. Perhaps specific risk avoidance measures can also be implemented, and the hotzones can communicate to their populations that close, perhaps any, contact with camels carries with it some risk of MERS-CoV infection  especially to be if you are in a category that places you at higher risk of severe outcomes from a MERS-CoV infection-older male, underlying conditions. 

Housing camels away from bats and areas known to be bat flyovers or frequented by feeding or birthing bats, or keeping camels under cover may all be helpful reduce transmission of the virus between these animals.

Jennifer Yang and Helen Branswell have breakdowns of this story as well.

Ian Lipkin has samples from animals for testing...and the hunt is away, like camels racing in the desert!

In a couple of Tweets from Jennifer Yang (@jyangstar; who writes for the Toronto Star) this morning, and from her article on MERS-CoV antibodies in camels, we heard some very encouraging news on the hunt for MERS-CoV origins and sources. 

One of the world's best virus hunters, Prof Ian Lipkin, has received 130 samples from animals and the molecular investigations to discover and characterise an animal host for the MERS-CoV (and probably a few other new viruses given the non-specific nature of the technologies available to him, and his traclk record) is now joined in earnest.

Further to last tweet: Ian Lipkin has just received 130 #MERS samples from KSA, including serum/swabs from 33 camels http://t.co/t6rymuqqA2
— Jennifer Yang (@jyangstar) August 8, 2013

and in a follow up.

@MackayIM Yep. Already running PCR; starting serology tomorrow.
— Jennifer Yang (@jyangstar) August 8, 2013

I'm certainly eager to see how Prof Lipkin's findings fit into with today's confirmation of a key role for camels in the transmission chain of MERS-CoV. More on that story shortly after the PDF becomes available from Lancet Diseases.

MERS-CoV numbers-where are we at?

As the dust settles from several days of new cases, and deaths and retrospective case identifications, I sit waiting for some caped crusader (no capes!) to step from the shadows and announce "I have the numbers you seek!" Okay, I'm a sucker for a caped crusader.
Alas, there are no such wonderful heroes to help fill the data gaps we lack among the MERS-CoV case data. There are plenty trying though. And so we watch the numbers climb, the cases spread, then contract (depending on which reliable source of information is speaking) and we wait for the likely spike in new cases due to the upcoming Hajj which, even with calls to reduce numbers, will likely go ahead as a mass gathering that puts MERS-CoV transmissibility to the test.

Sometimes we seem to hear a proposed new case or a death, and then we hear no more. 

Where is this virus coming from - animals, are older males with underlying conditions (and what precisely are all these conditions?) getting it from Pipistrellus sp. or perhaps Rousettus aegyptiacus bats via contamination of dates, date products of palm sap-derived drinks/alcohol? How can the world prepare, or understand whether it needs to prepare, for a novel virus when the region of its apparent origin (we don't know that either) has trouble sorting out whether members of its own populace are positive or not? A rough - what else can there be - count shows at least 23 dates of onset missing, 9 dates of death, 10 ages, 67 dates of hospitalisation and 11 sexes undefined for around 72 cases.

That (don't call me novel) coronavirus is back!

Media reports, FluTrackers and Avian Flu Diary describe five recent deaths and two other critically ill cases under close watch in the Al-Ahasa region of the Kingdom of Saudi Arabia, linked to infection with the newly identified human coronavirus HCoV-EMC. This virus was first isolated in September 2012 from a 60M (60-year-old male) with pneumonia and renal failure in Jeddah, KSA. 

Further evidence for bats as a major source of CoVs came in a recent study in Emerging Infectious Diseases. Yang and colleagues identified a novel CoV from each of 2 bat species. 

The newly identified betacoronaviruses (betaCoVs), Bat Rp-coronavirus/Shaanxi2011 and Bat Cp-coronavirus/Yunnan2011 (rolls of the tongue doesn't it?) were not that closely related to human betaCoVs but resided in the bat verison of the SARS-like COVs.