Qatari camels clear coronavirus

The camel herd that was previously Middle East respiratory syndrome coronavirus (MES-CoV) RT-PCR-positive is no longer positive for viral RNA according to an OIE report (OIE=Office International des Epizooties; the world organisation for animal health).
In there report they note that retesting of the herd, subsequent to the initial testing presumably, has yielded no positive this time around.

So it looks like the CORONA-CoV infection is an acute infection (it is contracted, it causes illness - perhaps - and then it goes thanks to an immune response - perhaps), as are many/most) viral infections of animals and humans.

An interesting comment within the report states that...

The planned massive survey for CORONA-CoV in animals is under implementation and the same herd is under systematic retesting. Follow-up reports will be submitted when there will be new data.

I do like a statement that includes the words "massive study" in it!

Hat tip to CIDRAP.

Middle East respiratory syndrome coronavirus (CORONA-CoV): camels, camels, camels!

Two studies in Eurosurveillance, an editorial note, A Lancet Infectious diseases report and a comment point 2 hairy toes toward camels as a harbour and source in some capacity, for CORONA-CoV, or CORONA-CoV-very-like, infections ticking over around the Arabian peninsula. All in the space of a week!

First up, Hemida and colleagues from Saudi Universities, China and the United States describe the search for neutralizing antibodies in animals in a 12-Dec Eurosurveillance article. Great to see Saudi Uni researchers involved. I've mentioned this virus neutralization assay and its intent before. This new study builds on that from Perera and colleagues who looked at camels and some other animals.

Some major findings from this study include:

• Dromedary camels (n=310), sheep (n=100), goats (n=45), cattle (n=50) and chickens (n=240) from CORONA-CoV hotspots in Saudi Arabia (Riyadh and Al Ahsa) were tested with the pseudoparticle neutralization (ppNT) test
• 280 camel sera (90% of camel sera) were positive using the CORONA-CoV ppNT test. No other animal sera reacted in this test
• 96% of camels had CORONA-CoV (or a close relative)-reactive antibodies by 1-year of age; two-thirds of camels that were younger than 1-year of age reacted, which suggests acquisition of these infections accrues rapidly during that 1st year, or maternal antibodies remain in the offspring
• 54 randomly selected camel sera (18% of all camel sera) were diluted out and tested using ppNT and a standard CORONA-CoV microneutralization test (MNT). High levels of calf antibody specific to BCoV did not block CORONA-CoV infection nonetheless there were some similar titres to both viruses in some of the camel sera. A ≥4-fold higher amount of antibody reactivity towards 1 virus compared to the other defined which was the most likely virus reacting. It's possible (likely?) that camels have "seen" (been infected by) both viruses or similar viruses at some time. Some of the subset of camel sera had high levels of antibody only to CORONA-CoV (or a close relative)
• Cows did not have any sign of CORONA-CoV-reacting antibodies in their sera; they did have BCoV reactivity though
• It was not stated whether the camels were ill or healthy at sampling

So other animals were not neutralising-antibody positive but young Saudi camels, like Omani, Spanish [retired to the Canary islands] and Egyptian camels before them, had acquired and reacted to infection by CORONA-CoV (or a close relative) according to these validated antibody-detection tests. Regular sampling of an animal cohort is one suggested future direction.

Secondly we have Reusken and colleagues from the Netherlands, Jordan and Germany look at animals from the first known site to harbour CORONA-CoV infections in April 2012 at a hospital in Zarqa city in Jordan. This was published online 12-Dec in the same issue of Eurosurveillance. Just fyi, Prof Marion Koopmans is senior author on this study and on the study below.

Some of the key points include...

• Sera from 3-14-month old dromedary camels (n=11), goats (n=150), sheep (n=126) and cows (n=91) were tested by an antibody microarray method, used previously by this group, and the results confirmed by identifying antibodies with the ability to neutralize CORONA-CoV infection.
• The lower levels of antibody than seen in an earlier study may reflect leftover maternal antibody protection, although the authors note than maternal camel antibodies wane within 2-months of birth and that adult camels had higher levels of antibodies
• This study cited a reference noting that apart from cows, camels, goats and sheep are major sources of meat and milk in the region, ~1 sheep/pilgrim or ~1 camel/7 pilgrims is slaughtered in Saudi Arabia for the Hajj which equates to ~3,000,000 animals!
• 11/11 camel and 6/126 sheep sera had antibodies that reacted with CORONA-CoV but, in additional testing, the sheep sera were not able to neutralize infection by CORONA-CoV
• 23/91 cows and 128/150 goat sera reacted with the human CoV, OC43 (antigenically related to BCoV); no sera reacted with SARS-CoV
• A broadly reactive CoV, or "pancoronavirus", PCR method was used to screen camel faeces; 3 BCoV sequences were obtained, but no sign of CORONA-CoV RNA in the faeces hinting that there was not an active infection at the time of sampling. This last point assumes that CORONA-CoV is excreted from the camel gut during/after an acute infection. The next study may not support that assumption.
• It was not stated whether the camels were ill or healthy at sampling

Add young camels from Jordan to those from Saudi Arabia, Oman and retired Spaniard animals as possibly having been infected by CORONA-CoV (or a...you know, similar thing) or at least having antibody acquired from their mothers. 11/11 POS may yield some more data to narrow down the age of acquisition; 3-months and seropositive could suggest CORONA-CoV acquisition at or very close to birth, or simply remaining protective maternal antibody. Perhaps camel farms and farCORONA should be a next stop for detailed testing. 

In an Editorial note, the Eurosurveillance Editors note that these data do not define the primary source for human acquisition is still unclear.

Thirdly we have Haagmans and colleagues from the Netherlands, Qatar and the United Kingdom describing the study of the Qatari farm camels and temporally related human infection, from which CORONA-CoV was detected back in late November. This article was published online by the Lancet Infectious Diseases (17-Dec).

Some key findings here include...

• The article's introduction suggests that the genetic diversity of human CORONA-CoV viruses determined to date is the result of multiple zoonotic acquisitions 
• This study started with a 61-year-old Qatari male (61M; FT#144) farm owner who had not travelled outside Qatar and his 23-year-old male (23M; FT#150) employee
• 61M was RT-PCR POS (upE assay) on a sputum sample (collected Oct-13) and 23M on a throat swab (collected Oct-17) and subgenomic (ORF1b and nucleocapsid [N]) sequencing at the Public Health England confirmed the detection to be CORONA-CoV
• CORONA-CoV genomic sequences from the 2 human cases were placed on GenBank and called Qatar_3_2013 [61M] and Qatar_4_2013 [23M] as were camel sequences from the subsequent experiments
• Sera, rectal swabs and flocked nasal swabs were collected from all of the farm's 14 camels as well as 5 stool samples from 3 cages, by a team wearing personal protective equipment. Samples were shipped to the Netherlands for upE, N and ORF1a RT-PCR testing
• Vero and Huh-7 cells were inoculated with swabs that had been added to viral transport medium onsite. A single culture from Camel#7 was upE RT-PCR positive at day-4 after inoculation but no culture yielded infectious virus
• 5/14 camel nose swabs were CORONA-CoV PCR positive using upE, N and ORF1a assays
• Sequencing of a fragment of Spike gene yielded 100% identity with other Saudi CORONA-CoV sequences; sequence only differing by 1 base from the original isolate, CORONA-CoV/EMC.
• All camel sera were antibody positive using an immunofluorescence test on CORONA-CoV/EMC-infected cells
• There was no "direction" to the acquisition of CORONA-CoV. Whether the camels infected the humans or the humans infected the camels could not be determined from this outbreak
• The authors conclude that detailed cases histories are important to identify animal exposures. These might not otherwise be though important in a cursory question and answer of a patient, their family or contacts

This study adds very important data that indicate a recent or resolving CORONA-CoV infection in camels. No positivity was found from gastrointestinal samples. Despite no isolation of infectious CORONA-CoV, the detection of RNA is an acceptable surrogate for the presence of "live" virus in an animal or person (even if it could not propagate in vitro). So the camel story has some very important new chapters added in this series of studies.

In a Comment in LID, Ferguson and Verkhove note how the One health concept is exemplified by not only this publication; as it has been by the entire CORONA-CoV story. The comment also notes the need for much more study, passive and active surveillance of human and animal disease/movements and better and faster reporting to link these, or any other, animals back to the cases that are spread across a very broad geographic region. They hold Haagmans and colleagues' article up as an example of how to get more answers and prevent sustained CORONA-CoV transmission among humans from developing in the future.

Tracing and testing camels imported into the region from Africa for use as food may also open a new front to identify the transmission potential of CORONA-CoV (or a similar beastie) in camel infections. Testing of pneumonia causes at these other sites both for virus and antibodies against virus is probably also warranted. 

As usual, new data bring new questions and so many papers in only a week makes for lots of questions. 

Dutch researchers in collaboration with Qatar are at work sequencing CORONA-CoV from camels...

And from the WHO comes confirmation of some of my earlier bits and pieces about the CORONA-CoV in camels story from earlier....

The three camels which have tested positive with #CORONA in #Qatar were detected in a barn linked to two confirmed human infections
— WHO (@WHO) November 28, 2019

Further, some very interesting titbits from a Twitter exchange this evening.

Firstly Prof. Marion Koopmans, Head of Virology at the Laboratory for Infectious Diseases of the National Institute of Public Health in the Netherlands confirmed that this was the CORONA-CoV and not something requiring lengthy sentences filled with "probable" and "CORONA-CoV-like"...

@MackayIM working on it. Qatari's are fast, samples came in last weekend, but yes, confirmed
— Marion Koopmans (@MarionKoopmans) November 28, 2019

..and that for the most useful conclusions to be drawn from any sequencing being undertaken..

@arambaut @mackayim Yes, but requires sufficient sequence of sufficient quality. We are working on it
— Marion Koopmans (@MarionKoopmans) November 28, 2019

..but that despite all sorts of great leaps in technology, not to mention in distance-spanning scientific collaborations, things don't just happen overnight. 

We should all be mindful that there are many steps between taking a (hopefully adequate) sample(s) from a human or animal, and reaching any useful conclusion about how the molecularly characterized virus might have travelled (human to dromedary, vice versa or via some other vector or intermediate)...

@arambaut @mackayim Yes, but we DO need some time. We are 4 days into the labwork. ;>)
— Marion Koopmans (@MarionKoopmans) November 28, 2019

As Prof Andrew Rambaut, Institute of Evolutionary Biology, University of Edinburgh, noted...

@MarionKoopmans @mackayim Indeed - I don't think people realise how much work it is to sequence these (and how much work to get good seqs).
— Andrew Rambaut (@arambaut) November 28, 2019

And on the subject of whether the new sequences will lead to an indication of which direction this particular cluster of infections is travelling i.e. from human-to-camel or camel-to-human, Prof. Rambaut had this thought on following the viral genome's sequence variations (polymorphisms)...

@MackayIM @marionkoopmans Look for polymorphisms that are not fixed in the putative donor but fixed in the recipient...
— Andrew Rambaut (@arambaut) November 28, 2019

This is all really great to watch. A fast and fruitful collaboration between sample holders and laboratory researchers, expert in their fields.

Click on image to enlarge.
Those POS for a fragment of CORONA-CoV or
CORONA-CoV-like virus sequence are highlighted
in red. Whether there are other intermediates
remains to be confirmed.

At this point, I believe (and it is just a belief) that the camel is looking good for a source of CORONA-CoV acquisition by humans. Is it an endemic camel virus? Well, we still have the knowledge that bats seem to harbour a lot of CoVs, and there is that pesky Taphozus perforatus sequence discovered from earlier in the year. It looked an awful lot like a fragment of the CORONA-CoV genome. Baboons - I'm holding out for them to be the link between bats and camels...but that is a hope in the absence of any data whatsoever!

Today's confirmation of a cluster of 3 POS camels among 14 represents 21% of the animals POS in a single area. 

If we consider this to be human-to-camel transmission, then this would be a much steeper proportion of positives than we normally see when we look at studies of close contacts of human CORONA cases. Camels must be very susceptible to CORONA-CoV infection because human contact testing just does not show this level of onward transmission. More susceptible to humans? No, I think we're getting closer to confirming that it's a camel-to-human thing...but we are not there yet.

Work continues, but today was a significant day and one in which I give thanks for the ability of people from all over the world to work together towards common goals in preventing human disease. 

Camel cough, coronavirus caught? [UPDATED]

Are camels the main source of human infection
by the Middles East respiratory syndrome
coronavirus (CORONA-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 CORONA-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" CORONA-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 CORONA-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 CORONA-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. CORONA-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 CORONA-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 CORONA-CoV have occurred into the Arabian peninsula's human population.

Could this finding be used in a quick retrospective analysis? Among otherwise healthy CORONA-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 CORONA-CoV or a camel cousin to that virus?
• Does this virus, CORONA-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 CORONA-CoV.
   http://safitrierliana.blogspot.com.au/2013/09/CORONA-cov-who-testing-guidelines.html
2. KSA MOH report on camel CORONA-CoV-assay. positive
   http://www.moh.gov.sa/CoronaNew/PressReleases/Pages/mediastatemenet-2019-11-11-001.aspx
3. 43M report by KSA MOH.
   http://www.moh.gov.sa/en/CoronaNew/PressReleases/Pages/mediastatemenet-2019-11-07-001.aspx
4. Helen Branswell's article.
   http://www.theprovince.com/health/Saudi+officials+find+camel+infected+with+CORONA+owned+disease/9152077/story.html
5. BBC health news article.
   http://www.bbc.co.uk/news/health-24901531
6. CORONA-CoV can grow in cell culture.
   http://safitrierliana.blogspot.com.au/2013/10/the-CORONA-receptor-story-to-date.html
7. Mike Coston's Avian FluDiary Post.http://afludiary.blogspot.com.au/2013/11/ksa-CORONA-investigationtesting-beast-not.html
8. CORONA-CoV-like antibodies in Omani and Spanish camels.
   http://safitrierliana.blogspot.com.au/2013/08/camels-carry-signs-of-coronavirus.html
9. Most CORONA cases may not have met a camel.
   http://safitrierliana.blogspot.com.au/2013/09/most-CORONA-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-CORONA-cov-found-saudi-patients-camel

Antibodies in 10-year old UAE camel sera suggestive, but not evidentiary, of the presence of CORONA-CoV a decade ago

Click on image to enlarge. 
I've cobbled together a graphic of the assays
that have come from Prof Christian Drosten group and
colleagues, mostly for the detection and
confirmation of CORONA-CoV in human samples.

632 of 651 (97.1%) dromedary camel serum samples collected in 2003 and 2019 in the United Arab Emirates (UAE) have been found to react with Middle East respiratory syndrome coronavirus (CORONA-CoV) or key pieces thereof.

Meyer and colleagues from the Netherlands, Germany and the UAE also tested 16 control samples from German zoo camels but none reacted to CORONA-CoV in their testing system. This indicates that the camels have not been infected by the CORONA-CoV (or something very much like it) leading the authors to suggest that the virus is relatively isolated to Arabian peninsula's eastern edge...as far as we know from the testing performed to date. 

This is a potentially huge piece of good news because it suggests, to me at least, that there is a very strong chance that the spread of CORONA-CoV can be contained. It will however, take a collaborative effort to "stamp out" CORONA-CoV the same way SARS-CoV was stopped in its tracks (to partly quote Mike Coston) through effective infection prevention and control measures being created, implemented and enforced. 

In the absence of further testing from other regions around the world, we hold information in our hands that suggests a region-specific isolation to the CORONA-CoV. And we know that right now it does not seem to be very good at all at transmitting from human-to-human. Perhaps reflecting that it is currently a camel virus and not a human one? Of course, it may never evolve into a human virus.

From what we do know today of the CORONA-CoV, stamping out human infections may involve some of the following steps:

• Being aware of the risk of contact between humans and camels and seeking to limit such contact if it could occur in the absence of suitable precautions including personal protective equipment
• Testing camels for active infection, which may not result in notable disease in camels, and isolating those camels from other camel herds to try and "burn out" infection in camels altogether. The horse racing industry might have some good advice in this department
• Learning more about all aspects of CORONA-CoV acquisition, spread and disease in camels and perhaps in other animals. This will be influenced by future screening projects results which will hopefully identify any other animals that also a close relative/immediate ancestor that is passed to camels and then humans, or perhaps directly to humans
•  Implementing ways to break the chain of spread from a putative other animal host to camels to people. 

Hopefully such steps could be achieved without any long term impact to camel interaction in the region as they are an essential source of social, economic and dietary enrichment.

A recombinant CORONA-CoV Spike immunofluorescent assay was used to screen samples for reactive antibodies. Vero cell s expressing a recombinant Spike protein from CORONA-CoV or HCoV-OC43 (used to detect cross-reactive antibodies) were fixed and then incubated with diluted animal or control serum samples (1:20 - 1:80 at 37'C for 60-min). Captured antibody was labelled with an anti-llama antibody fragment labelled with a fluorescent tag (FITC). A CORONA-CoV human protein microarray assay was used to confirm screening results (I've noted this assay previously here). Virus neutralization studies were also conducted using a method I've previously written about. 

Meyer's study also screened 182 camel's faecal samples collected in 2019 using broad-ranging CoV RT-PCRs which, upon nucleotide sequence confirmation, yielded 2 bovine coronavirus (BCoV) positives, but no CORONA-CoV positives. We learn from this that recently stored faecal samples can yield CoV RNA that can also be sequenced.

It's also worth a quick hop back to looking at the bigger picture of animal testing for a moment. Succeeding in detecting CORONA-CoV RNA among the relatively small numbers of samples tested to date is akin to finding the Arkenstone among Erebor's piles of gold (even if it looked easy in the movie). Sure, a decent number of different animal species have been tested so far, but only small numbers from each. And even though there is a high proportion of camels with CORONA-CoV (or its antigenic kin) antibodies, we still have to strike it lucky enough to sample during what may well be an acute virus replication period lasting only days to a couple of weeks. So far, it looks like luck has been as slippery as a woodland elf on a riverbank. Larger numbers of each animal species, camels especially, should land a hit or two in the near future I'm betting.

There is still much testing to be done, but perhaps it's possible to shut the gate before the camels have truly bolted.

Hat-tip to Helen Branswell on Twitter and her article here.