Camel cough, coronavirus caught? [UPDATED]

Are camels the main source of human infection
by the Middles East respiratory syndrome
coronavirus (MERS-CoV)?

I awoke to find the world has learned of a camel that tested positive using a Middle East respiratory syndrome coronavirus PCR. According to the Kingdom of Saudi Arabia's Ministry of Health announcement (on the Arabic language and not English language page), the camel was owned by by a recent case (43M from Jeddah, reported on 7th of Nov- FluTracker's #156) and was showing signs of disease. The fact that it was ill may suggest it was the source, but we don't yet know which illness came first, the camel or the man. We do know (from CIDRAP/WHO) that 43M became ill on Oct-27 and has been in hopsital since Nov-3.

This is in line with recent studies finding antibodies to a MERS-CoV-like virus. I've previously reported on that here and here.

The PCR positive has not yet been genotyped (had its DNA sequence determined, and by inference, any of its RNA genome - a way of measuring the similarity, or not, to known "human" MERS-CoV). That work is ongoing. Hopefully we won't have to wait until the entire genome is achieved as that can be a lengthy process.

For the record, if you are relying on Google translate, the Arabic for beauty (jamaal, الجمال)  is derived from the Arabic word for camel - so read "beauty" as meaning camel (h/t Mike Coston).

I found it interesting that 43M was a healthy adult and yet he was ill enough to require intensive care. He wasn't old and had no underlying comorbidities. Does this hint towards his acquisition of MERS-CoV being from an animal source rather than a secondary human exposure? The latter often seems to result in milder disease or asymptomatic detection (mostly based on contacts of known cases). It's as if passage to another healthy human is via a smaller dose (reduced viral load after growth in 1st human?) or is the virus changed by growth in a human rendering it less capable of severe disease (perhaps seen as less "foreign" by our immune system?)?

A model of possible MERS-CoV acquisition. Is the camel the
central player or a secondary host?

The KSA Ministry of Agriculture is helping to try and grow the virus. MERS-CoV is certainly culturable - and with a positive camel at hand, getting fresh samples should yield results quickly.

Its great to hear about this in real time. No waiting on the publication process. 43M was only reported 5-days ago (my time; not sure when he became symptomatic of course). Well done KSA MOH!

We don't need to find every camel to be MERS-CoV-positive for them to be a likely source of infections. We know that cases have been sporadic and widespread and that genetically, a number of different introductions of MERS-CoV have occurred into the Arabian peninsula's human population.

Could this finding be used in a quick retrospective analysis? Among otherwise healthy MERS-CoV-positive humans, does evidence of contact with camels more often link to severe disease outcomes than if there has definitely been no contact at all? The corollary then is that milder disease results when acquisition is via a human-to-human route among the otherwise healthy. Keeping in mind a quote from Dr. House, M.D. "I don't ask why patients lie, I just assume they all do." Patients may not always want to own up to something, for whatever reason.

Some questions that remain from this finding:

• Which diagnostic PCR was used? Presumably the well-validated version suggested by the WHO. We don't yet know if this assay cross reacts with any as-yet-unknown-but-very-closely-related CoVs such as one that may reside in camels. My bet is that it doesn't.
• Is it MERS-CoV or a camel cousin to that virus?
• Does this virus, MERS-CoV or very close relative, actually cause disease in camels? It's possible that 43M's ill camel was symptomatic due to another viral or bacterial infection.
• Did the camel transmit virus to the human or did the human transmit the virus to the camel?
• If acquired via camel-to-human (seems most likely), how did 43M acquire the infection from the camel? Airborne drops or aerosols, faecal-oral, scratch/broken skin, direct contact with secretions...?
• Where did the camel get its infection from? Another camel (enzootic within the camel population?) or from a primary host or other secondary vector (bat, baboon...whatever?)

References for further reading

1. PCR assay for MERS-CoV.
   http://virologydownunder.blogspot.com.au/2013/09/mers-cov-who-testing-guidelines.html
2. KSA MOH report on camel MERS-CoV-assay. positive
   http://www.moh.gov.sa/CoronaNew/PressReleases/Pages/mediastatemenet-2013-11-11-001.aspx
3. 43M report by KSA MOH.
   http://www.moh.gov.sa/en/CoronaNew/PressReleases/Pages/mediastatemenet-2013-11-07-001.aspx
4. Helen Branswell's article.
   http://www.theprovince.com/health/Saudi+officials+find+camel+infected+with+MERS+owned+disease/9152077/story.html
5. BBC health news article.
   http://www.bbc.co.uk/news/health-24901531
6. MERS-CoV can grow in cell culture.
   http://virologydownunder.blogspot.com.au/2013/10/the-mers-receptor-story-to-date.html
7. Mike Coston's Avian FluDiary Post.http://afludiary.blogspot.com.au/2013/11/ksa-mers-investigationtesting-beast-not.html
8. MERS-CoV-like antibodies in Omani and Spanish camels.
   http://virologydownunder.blogspot.com.au/2013/08/camels-carry-signs-of-coronavirus.html
9. Most MERS cases may not have met a camel.
   http://virologydownunder.blogspot.com.au/2013/09/most-mers-may-not-have-met-camel-but.html
10. CIDRAP on camel case and WHO update.
    http://www.cidrap.umn.edu/news-perspective/2013/11/reports-mers-cov-found-saudi-patients-camel

Break out the bug zapper: DENV-5 is the new dengue virus in town!

A report from the Third International Conference on Dengue and Dengue Haemorrhagic Fever describes the discovery, by researchers from the University of Texas Medical Branch, of a new type of dengue virus (DENV). he virus was found during screening of samples from 2007, collected from Malaysia's northern Sarawak state.

Click to enlarge. An alignment of the previously known 4 dengue virus complete genome sequence. 
The GenBank accession number is shown next to the serotype's name.
They share 68% olignucleotide identity. Aligned using Geneious 6.1.6.

Dengue viruses have an ~11 kilobase, positive-sense, RNA genome enveloped in a lipid bilayer membrane (taken from the host cell upon virion exit) resulting in a 50 nanometer particle. 

Dengue viruses belong to the Family Flaviviridae, Genus Flavivirus and belong to the Species Dengue virus. The viral genome produces a single polyprotein that is cut into 10 proteins (called C, M, E, NS1, NS2A, NS2B, NS3, NS4a, NS4b, NS5). M and E are embedded in the viral membrane.

New virions are assembled on the surface of the endoplasmic reticulum. Dengue virus is transmitted to non-human primates and humans via a mosquito vector (primarily of the genus Aedes) and infection can result in dengue haemorrhagic fever.

This virus, DENV-5 (preusmably), was discovered by Dr Nikolaos Vasilakis and colleagues. It is the 5th member of the species and the first addition in 50-years. DENV-1 to DENV-4, called serotypes (because they interact differently with our immune response to them) are approximately 65% identical in sequence.

How this latest discovery will impact on existing efforts to interrupt, treat or prevent infection and disease remain to be seen. As does a full research publication.

Thanks to FluTrackers for their earlier post on this.

Further Reading:

1. http://www.nature.com/scitable/topicpage/dengue-viruses-22400925

Got a spare $6.3-billion? Experts could use it to discover the missing 320,000+ mammalian viruses we don't yet know about

...or just $1.4-billion for 85% of those. And that's not including the non-mammalian ones. This is according to a new paper in mBio today by Andrew and colleagues from a collaborative team including Prof Ian Lipkin, from Columbia University's Mailman School of Public Health. 

The study advocates for a much more structured, systematic approach to discovery and notes that existing studies, such as the U.S. Agency for International Development's (USAID) Emerging Pandemic Threats (EPT) program including the PREDICT project (more detail in Lancet article here), have made headway into the list already. Each adding valuable assets to our virology intelligence archive.

Finding these viruses, and the animals they reside in, is key to limiting zoonoses. Sure, discovery does not equal simultaneous cure, but ignorance does equal surprise outbreak and death. Most emerging human infectious are caused by animal viruses infecting us. This is well defined by the One Health concept which promotes investigation of all aspects of the network of links between humans and their hairier, more leggy or winged co-habitants.

If we ever want to get ahead of the curve, investing in this sort of research is essential to allow us to know our enemy. It let's us be ready to meet them at the door instead of scrambling to action when they kick our door in! And it is a scramble; just look through the literature and media surrounding any virus that has spilt over from animals to humans in recent decades....a degree of controlled panic over the many things we don't yet know in the early stages of an unexpected emergence. For example: 

• We'd have no laboratory testing methods (culture, PCR or serology) nor the procedures to confirm weird results.
• Which country would "own" the virus, what would we call it (mock you may, but a lot of electrons and ink have been wasted on that story for the MERS-CoV for instance) and how long would it take before commercial detection kits were available (for MERS-CoV - its been nearly 15-months since the first cases in Jordan and still nothing well validated and widely available for use by non-reference laboratories)?
• When would we have enough of the virus to make positive controls for those tests or to kick off research into how the virus does what it does?
• We don't yet know what it does! What is the clinical spectrum of disease, how big is the iceberg let alone it's tip; what are the signs and symptoms; what does it do in different patient groups - those with and without comorbidities, different ages and sex?
• What is the proportion of fatal cases?
• Where did the invader came from?
• How best to handle the pathogen in hospital settings
• How fast and to how many does each case transmit (it will be a while until we can calculate the R₀)?
• What drugs do we already have that can moderate disease?
• How long will it take for an antiviral or vaccine, if they can be prepared, to be available and how long thereafter will antiviral resistance become an issue?
• How many that the virus infects will die?
• Does the virus interact with other viruses, bacteria, fungi or parasites?
• Does it have a peak season and is that affected by the environment?

Pretty much ALL of these things can be addressed if we invest in finding the culprits, their host and begin to unravel how they tick sooner rather than (too) later. Sure, they may never spillover, but when just 1 does, the impact is felt around the world, be it from loss of life, financial instability, healthcare burden, travel and tourism decline, animal culling or just a global feeling of insecurity. Any 1 virus outbreak can wield a lot of power in today's highly interconnected world. 

Count VDU in the cheer squad for this sort of proactive research. Money well invested.

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.