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]

Findings...

• 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

Discussion...

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.

References...

1. High proportion of MERS-CoV shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases, Qatar 2014.
   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505336/
2. http://www.ncbi.nlm.nih.gov/pubmed/23041020 
3. http://www.ncbi.nlm.nih.gov/pubmed/25728084
4. http://virologydownunder.blogspot.com.au/2015/10/if-you-are-often-in-contact-with-camels.html
5. http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(13)70164-6/abstract

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.

References...

1. http://wwwnc.cdc.gov/eid/article/21/8/15-0481_article
2. http://virologydownunder.blogspot.com.au/2015/10/kenyan-camel-coronaviruses.html
3. http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(15)70090-3/abstract
4. http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(13)70164-6/abstract

Can MERS-CoV be found in the upper and lower respiratory tract of infected camels? You bet your single hump it can!

Today's review is of a paper listed as published in Emerging Infectious Diseases in July 2015, authored by Khalafalla and colleagues from King Faisal University in the Kingdom of Saudi Arabia and from the CDC in the United States of America.[1]

Al Omran City (also Al Umran) city is located 
just adjacent to Al Hofuf on this map.

The introduction tells us that bats "seem to be the reservoir host" but are not the likely ongoing source of human Middle East respiratory syndrome (MERS) cases in the KSA. An assumption based on the finding of 1 small yet diagnostic MERS-CoV sequence in 1 bat from 1,003 samples, once.[5,6] It also reminds us that up to this point in time, camels were mostly sampled from the nose and eye during MERS-CoV investigations. We still didn't know whether other parts of the camel respiratory tract could test positive for MERS coronavirus (MERS-CoV). This is important knowledge as it pertains to virus transmission from camel-to-camel and camel-to-human.

This study focused on the dromedary camel (DC; Camelus dromedarius) collected samples
from around the Al-Ahsa area. These comprised two sample populations collected during a year (April 2013-May 2014):

1. Tissue from at least lung lobe of camels slaughtered at the Al Omran Abattoir, Al Omran City. Animals were kept in groups of 10-15 for up to 4 days in stock markets prior to slaughter
   ..8 batches of samples were collected (a batch every 1-2 months) from 91 carcasses in total
   ..28 young animals (<4 years of age) and 63 adult animals (4 or more years old) were sampled
2. Over the same period, age-matched nasal swab samples were collected from Al Omran abattoir, Al Ahsa livestock market and the King Faisal University veterinary hospital
   ..96 animals were swabbed; 36 young animals and 60 adults
   ..only 2/94 animals were visibly unwell - the 2 had nasal and lacrimal discharge

Samples were tested by a pancoronavirus conventional RT-PCR assay [4] as well as two real-time RT-PCR assays [2,3] No culture of virus was attempted so we must extrapolate from the RT-PCR findings to assume a positive finding of MERS-CoV RNA represents replicating virus-at some point during the infection anyway (a safe assumption).

The findings...

• 84 of 187 DCs (44.9%) tested positive, most often during the cooler months (NOV2013-JAN2014) and more often from young camels than adults
• 59 of 91 (61.5%) DC carcasses had MERS-CoV RNA detected
• 28 of 86 (29.2%) nasal swabs were positive
• 4 samples yielded a spike gene sequence-these differed from each other but clustered with other human and camel MERS-CoV spike gene sequence

The authors are clearly not exuberant that their findings have shed much new light on the camel>human debate and call for longitudinal studies to better understand how MERS-CoV spreads among DCs. A good suggestion indeed. 

Nonetheless, this study adds pieces to the story; DCs appear to be infected throughout their respiratory tract, not just at the openings usually swabbed. And if the seasonality of MERS-CoV in DCs hinted at by this study at this locale, does not overlap perfectly with human cases at the same time and place, that is most likely because camel>human infections are very rare. Also this mismatch is likely because most human infections are not due to camel/human interactions, but are acquired from human-to-human infections, thanks to errors in the management of a sick index case. That case's uncontrolled infection is what usually results in many other patients, healthcare workers and visitors becoming infected. 

That's the camel in the room that seems to be overlooked so frequently. 

Another thing that seem lost in translation - MERS itself is a relatively rare human disease and when you consider that camel>human transmission is only a fraction of that.... 

Some seem to think that lots of infected camels must equate to lots of infected humans if this crazy theory about camels being the source of human cases is to be believed. Sorry. Not the case (unless reported contacts are much more frequent than we are being told). 

There are definitely human MERS-CoV infections who only had DC, not infected human, exposures. Unarguably we do need to do better to try and catch transmission 'in the act' and show how it happened in order to dot the 'i' and cross the 't'. The same also applies to a lot of zoonoses. Until those breakthroughs though - we have a lot of data which can be used to better protect people from getting infected by MERS-CoV. 

Seems pretty dumb to wait on more convenient data while people still get infected, become sick and often die.
On balance, separating camels from humans, being better protected when in contact with camels, and improving infection prevention and control in hospitals may even obviate the need for vaccination. Gasp.

References...

1. MERS-CoV in Upper Respiratory Tract and Lungs of Dromedary Camels, Saudi Arabia, 2013–2014Abdelmalik I. Khalafalla, Xiaoyan Lu, Abdullah I.A. Al-Mubarak, Abdul Hafeed S. Dalab, Khalid A.S. Al-Busadah, and Dean D. Erdman
   http://wwwnc.cdc.gov/eid/article/21/7/15-0070_article
2. http://www.ncbi.nlm.nih.gov/pubmed/23041020
3. http://www.ncbi.nlm.nih.gov/pubmed/24153118
4.  http://www.ncbi.nlm.nih.gov/pubmed/19057882
5. http://wwwnc.cdc.gov/eid/article/19/11/pdfs/13-1172.pdf
6. http://virologydownunder.blogspot.com.au/2013/08/mers-cov-genetic-sequences-found-in.html

A negative viral culture result is not the end of the story...just a negative result

Lately, for both Ebola virus and Middle East respiratory syndrome coronavirus, there have been instances where I've been reminded that one must not rely on the growth of an infectious virus from a sample to be sure that there is virus in that sample.

Past diagnostic methods have failed to isolate many newly identified viruses (NIVs), which is not surprising considering that those culture-based methods can be over 100-fold less sensitive than current molecular (PCR-based) tests [1,2,3,4] and that many new viruses do not grow in traditional culture at all.

So when an RT-PCR or PCR result cannot be confirmed by the culture of a virus from the same sample, that really doesn't mean more than...that. 

Virus may be present, but our relatively insensitive culture techniques, which haven't really advanced in a long time, may just fail to get it growing. 

Viral isolation by cell culture is really a dying art form. And it is a very lengthy, demanding and sometimes subjective art form at that requiring particularly skilled artistes. 

References...

1. Templeton,K.E. et al. Improved diagnosis of the etiology of community-acquired pneumonia with real-time polymerase chain reaction. Clin Infect Dis 41, 345-351 (2005).
2. van Kraaij,M.G.J. et al. Frequent detection of respiratory viruses in adult recipients of stem cell transplants with the use of real-time polymerase chain reaction, compared with viral culture. Clin Infect Dis 40, (2005).
3. Garbino,J. et al. Lower respiratory viral illnesses: Improved diagnosis by molecular methods and clinical impact. Am J Resp Crit Care Med 170, (2004).
4. Gunson,R.N., Collins,T.C., & Carman,W.F. Real-time RT-PCR detection of 12 respiratory viral infections in four triplex reactions. Journal of Clinical Virology(2005).

Kenyan camel coronaviruses...

Two studies have now found antibodies from Middle East respiratory syndrome coronavirus (MERS-CoV)-like coronaviruses in dromedary camels (DCs). The "like" bit reflects that unless we have some sequence, we can't say for certain that the virus that infected those camels in the past, causing them to respond with these antibodies, was a MERS-CoV variant. The virus(es) may have been a different camel CoV that just so happens to share some antigens and is detected by MERS-CoV-"specific" antibody detection tests. The old story of "we don't know what we don't know" can perhaps be extended here to "we can't validate a test against viruses we haven't found yet". Or that may just be too nerdy.

Anyhoo, we have two papers to look at here. 

Antibodies against MERS coronavirus in dromedary camels, Kenya, 1992-2013

This paper went into the August edition of Emerging Infectious Diseases, authored by Corman and team (online much earlier but no way to track that thanks to no date of ePub ahead of print - loud sigh!) from Germany, Kenya, the Netherlands and Sweden.[1]

The introduction sets the scene for a paper seeking to know about where the MERS-CoV we know and love today, may have come from to be so common amongst camels. We suspect that this could be from another animal in its current form, or by recombination and mutation from a different ancestral form that has yet to be discovered in an animal (or human). This study seeks out MERS-CoV or a MERS-CoV like virus, or an ancestor, from camels in Kenya using their blood to look for footprints of previous infection - in this case, antibodies.

774 DC blood and stored sera collected from three regions of Kenya between 1992 and 2013 were subjected to a multi-step testing process:

1. All samples, diluted 1:100, were screened using MERS-CoV spike protein subunit 1–based ELISA (rELISA; described before at [2])
   .228 of 774 (29%) were positive
2. The 228, diluted 1:40, were next examined using a recombinant immunofluorescence assay using Vero cells expressing MERS-CoV spike protein (rIFA; described before at [3]
   .213 of 228 (93%; 28% of the 774) were still positive in the second tier of testing
3. The third tier of testing of samples diluted between 1:80 and 1:800 used a highly specific MERS-CoV microneutralization assay (MNT assay; also previously described in [3])
   .119 of 213 (56%; 15% of the 774) had titres (dilutable levels) greater than or equal to 1:80 and 14 had titres above 800
   .Some counties of Kenya had 60-100% of samples test positive 

Figure 1. From Corman et al, Emerg Infect
Dis. 2014 Vol 20, No 8. 1319:1322.[1]
Click on image to enlarge.

North-eastern and northern regions generally had higher titres (Fig.1).These are regions closer to other countries with known antibody-positive camels (Egypt, Sudan, Somalia and Ethiopia). Further, nomadic camels from the East had higher antibody titres than those farmed in the north-west of the Rift Valley. Nomadic camels are taken across borders for trade.[2] DCs that had been kept isolated since 1998 were negative signs of past MERS-CoV virus. 

Adults had higher antibody levels than juveniles - presumably because infections happen when the DCs are young, producing the antibodies we detect in adult DCs.

Figure 2. Quote from [1]
Click on image to enlarge.

Camel density was also important. More camels were antibody positive in areas with higher densities of camels - also presumably because virus can spread better from one infected DC to others when more DC contacts are around. Similar story for humans, a contributing factor for those super-spreading conditions. The authors also made a comment that is very important to the answer the question of why human cases have not been found in areas with animal infections (see Figure 2).

Moving on to the next publication from Kenya.

Serological Evidence of MERS-CoV Antibodies in Dromedary Camels (Camelus dromedaries) in Laikipia County, Kenya

This one just came out on PLOS|ONE authored by Deem and colleagues from the United Stets of America, Kenya, New Zealand and the Netherlands.[4]

The introduction also reminds us that understanding MERS-CoV in camels in countries with herds, can help us assess and manage the risk for humans in those countries. In this case, Kenya has over 3 million DCs and mean and milk is worth $USD 11 million a year. These figures that may help you understand why DC interests don't want to have a significant human pathogen harboured by their animals.

This study is based in Laikipia County, almost in the centre of Kenya (Fig 1), which has a growing camel population. 

335 camels were sample from 9 easily accessed herds.

1. All samples, diluted 1:20, were screened using a MERS-CoV, severe acute respiratory syndrome (SARS)-CoV, human CoV (HCoV)-OC43 spike domain S1 antigen protein-microarray method used previously by this group [5,6,7,8,9]
   _46.9% of DCs were seropositive (had antibodies) including at least 1 animal per herd
   _60.8% of adult DCs were seropositive and 21.3% of the juvenile animals
   _bovine CoV (tested for by including the HCoV-OC43 antigens) seroprevalence was high, as it often is in DCs
   _this study did not see a significant difference in seroprevalence between nomadic herds or those managed in more commercial ways and no differences between different degrees of herd isolation

Figure 3. Quote from [4]
Click on image to enlarge.

The authors concluded that these herds were being exposed to MERS-CoV (or a similar virus) on an ongoing basis, even though they were not near borders and at lower densities that the more northern sites reported by the Corman et al. study above. They did not feel these disparities were due to diagnostic differences and that the DC densities in Lakipia County were sufficient to maintain virus circulation. 

The conclusion noted the need to get sequence from this virus or these viruses n order to see whether they are the MERS-CoV we know, a different clade of MERS-CoV variants or another virus entirely. That sort of information can't be gleaned from antibody studies and so RT-PCR methods are needed.

The report wrapped up with a comment about a lack of reporting of human cases (Fig.3).

Clearly, camels are commonly infected by MERS-CoV or a close relative in parts of Africa and the Arabian Peninsula which receives camel imports from Africa. 

Also very clearly, DCs survive the experience apparently fine and unharmed lending more support for MERS-CoV in DCs being just a "camel cold". The camels do not need to be culled the way we do to other ill virus-infected animals (I'm looking at you chooks with high pathogenicity influenza A(H5N1) virus..or other flu viruses). We just need to remove camels from humans - or better manage the interactions we have to have. It's not rocket science but it will take thoughtful, considered and collaborative discussions.

References...

1. Antibodies against MERS coronavirus in dromedary camels, Kenya, 1992-2013
   Corman VM, Jores J, Meyer B, Younan M, Liljander A, Said MY, Gluecks I, Lattwein E, Bosch BJ, Drexler JF, Bornstein S, Drosten C, Müller MA.
   http://www.ncbi.nlm.nih.gov/pubmed/25075637
2. http://wwwnc.cdc.gov/eid/article/20/6/14-0402_article
3. http://wwwnc.cdc.gov/eid/article/20/4/13-1746_article
4. Serological Evidence of MERS-CoV Antibodies in Dromedary Camels (Camelus dromedaries) in Laikipia County, Kenya
   Sharon L. Deem , Eric M. Fèvre, Margaret Kinnaird, A. Springer Browne, Dishon Muloi, Gert-Jan Godeke, Marion Koopmans, Chantal B. Reusken
   http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140125
5. http://virologydownunder.blogspot.com.au/2013/08/camels-carry-signs-of-coronavirus.html
6. http://virologydownunder.blogspot.com.au/2013/12/middle-east-respiratory-syndrome.html
7. http://virologydownunder.blogspot.com.au/2014/01/antibodies-in-10-year-old-uae-camel.html
8. http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(13)70164-6/abstract
9. http://wwwnc.cdc.gov/eid/article/20/8/14-0590_article

MERS and the media in Saudi Arabia - a match that fuels confusion....

I'm interrupting my reviews on MERS and camels to briefly critique a recent media article published in the Arab News, Wednesday 14th of October. This was also on camels, but a view into the other side of this story.

The title of the article: ‘No conclusive proof’ camels spread MERS: Expert.[1]

There are two trains of thought here - and perhaps I have not clarified them so far. The first train doesn't believe that camels have a role in human cases of MERS-I disagree completely with the sentiment here. The second train of thought wants more testing of more and different animal species. I agree with this wholeheartedly. And those who can do this should be getting on with the job of doing it or organising those who can do it instead of wishing the data we absolutely do have, were different.

Let's keep in mind that seeking out other sources is a research endeavour. You cannot write public health messaging around things for which you have absolutely no supporting evidence. You can't protect your population, especially those most at risk, if you don't have proof to support how they are at risk. Looking after the public's health requires data. Research gets those data. Support the research. Look at those data instead of ignoring them because they scare you, point a finger at your favourite animal or conjure fears of an animal-driven negative economic impact. 

Distancing humans from infected camel vectors is a here and now action. It is not the result of a future research study. Finding ways to act on the data may have an impact on cases. 

Rather than guess at what people's concerns are - let's have a read of some key sections of this article, and comment as we go.

"There is no conclusive evidence that shows camels are responsible for the spread of the deadly Middle East Respiratory Syndrome coronavirus (MERS-CoV), according to research conducted by a Saudi expert at King Saud University in Riyadh."

• This is plain wrong. There is considerable body of peer reviewed scientific literature providing evidence both for spread among camels and between camels and humans, even data strongly suggesting that direction (camel>human). A review I co-authored last year goes into a lot of that detail - it has a huge table on the camel related literature.[2]

“All the studies published in scientific journals do not at this stage show that the blood samples taken from camels have the virus present."

• This is an English language article so I am going to take that at face value. Virus in the blood, or viraemia, is not considered a major concern for spread human or camel MERS-CoV infection - the real and larger concern is virus, in high amounts, that is regularly identified in the nose of infected camels. This is quote is not evidence of an expert comment.

"The reality is that more than 80 percent of the tested samples prove that camels’ blood carry protective antibodies against the virus,” he said."

• Again, this comment highlights a lack of expertise in virology or immunology. Fields important for this discussion. The camels have antibodies because they have been previously infected. Research has found that camels seem capable of being reinfected - infected again even when they already have antibody from a previous infection. These camel antibodies may not be protective. Cell mediated immunity (h/t @MarionKoopmans) may be an important study subject here to better understand what happens in camels.

"He said some studies have found that 5 to 6 percent of shepherds and persons dealing with camels carry antibodies against MERS, and do not have the virus itself."

• This is where a science reporter would have been really helpful to Arab News. Shepherds (camel herders?) don't have the virus by the time they develop antibodies because, like many viruses, MERS-CoV causes a short-lived, or acute, infection in humans and camels. Those few percent of shepherds with antibodies were previously infected and the virus was subsequently cleared by their immune system, usually near to or before those antibodies develop.

“In our previous studies we found out that Heavy Chain Antibodies are present in the blood of camels and are carried out with its milk. This research was published in the Journal of Proteomics. This in itself proves that immunity is transferred from camels to humans,” he said."

• This does no such thing at all. This shows that antibodies are in camel blood and milk - if indeed that was what was found. What are the previous studies that showed these antibodies were ingested by humans and survived the digestive tract to remain effective against MERS-CoV? There are none that I have read but I'd be interested in seeing them.

Science tests and measures, it calculates and concludes. Very little of that process is evident here, but a sense of the confusion around this topic is. These stories should be great starting points for the Saudi Ministry of Health to work up local, relevant and specific answers, (more) factsheets or Ministry-involved media interviews and internet posts to help educate those with concerns about there camels. Listen, communicate, take feedback, re-tune, communicate, listen....

References...

1. ‘No conclusive proof’ camels spread MERS: Expert
   http://www.arabnews.com/featured/news/820181
2. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd.
   http://www.ncbi.nlm.nih.gov/pubmed/25656066

What's hard to understand about camels carrying MERS-CoV and rarely infecting humans...?

What is the hard part to understanding that camels harbour the same Middle East respiratory syndrome coronavirus (MERS-CoV) that infects humans, but only does so rarely? Not a different strain or species - variants of the same virus.

It may be that this is not a simple 1 + 1 = 2 kind of equation and sometimes that can be a tough camel to ride. 

There is a need to understand a few things where camels are concerned. For example...

1. Camels definitely get infected by MERS-CoV - they can get mildly ill or not. Infection effectively results in a camel "common cold" illness
2. When a camel in a herd is infected, that doesn't mean that every camel in that herd is infected
3. When a camel is infected it may not be very ill, or show no sign of illness at all
4. MERS is a respiratory disease - while there is no evidence for exactly how humans acquire MERS-CoV, it has been considered, by medical experts in the Kingdom of Saudi Arabia, to be most likely acquired via droplets or other modes of transport of virus contaminated material and the upper or lower respiratory tract epithelium (lining). Ingestion is not considered to be a likely route of infection to date. This may be why those who drink fresh camel milk do not all get infected by MERS-CoV. But frothy bowls of milk have lots of popping bubbles that could create droplets that can be inhaled. And MERS-CoV can survive in milk and in the cold and on surfaces (which can be contacted and then self-inoculated via eye rubbing, nose picking etc). But the distinction between ingestion and inhalation can be confusing.
5. Most human MERS cases have not reported camel contact. Most cases have acquired their infection in association with a small or uncontrolled hospital outbreak of disease
6. In the 185 cases of MERS-CoV infection acquired in South Korea - none were infected by or had any, camel contact. Those cases were due to human-to-human infections. Camel contact is a sporadic cause of infection despite most camels in the Arabian Peninsula showings signs of past infection
7. Because camel contact is rare does not mean it never
   occurs - just that it is rare. A single camel-to-human
   
   infection may trigger dozens or more human cases if the hospital which that first case attends does not have effective infection prevention and control procedures. We have seen this again and again and again since 2012
8. Those in close contact with camels, who are otherwise healthy, may have been infected but not developed more than a cold or flu-like illness (who does anything about those - or remembers when they had them?). 
9. We do not yet know whether those who are in frequent close contact with camels and who have underlying disease, may also have some cross-protective immunity due to infection by a closely or perhaps even distantly related camel virus that does not cause lethal infections, as MERS-CoV does, in those with a comorbidity 
10. MERS-CoV may move around Africa and the Arabian Peninsula via infected camel imports and exports but no surveys of camels in Africa for MERS-CoV, or other coronavirus ancestors, have been reported to date
    
11. No MERS-CoV PCR-based diagnostic surveys of respiratory disease cases - mild, moderate or severe - have been conducted in countries harbouring camels known to have been infected by MERS-CoV (or an antigenically similar virus) in the past. Ethiopia the Sudan and Somalia are such countries
12. Camels do not need to be culled to prevent infection - they just need to be approached with more awareness and appropriate care to reduce the risk of infection and disease. Plenty of animals that we co-exist with carry viruses that can infect us and seriously harm us - we don't seek out and kill them all to stop getting infected, we need to address the activities by which we humans get infected.

MERS-CoV on the farm...

I'm going to spend a few posts catching up on some excellent papers showing the role of camels in harbouring and transmitting the Middle East respiratory syndrome coronavirus (MERS-CoV). There also seems to be some confusion remaining about what we know, what we don't know, and also how bats fit in to the picture. I'll finish the next few posts with a summary.

Please do check out my previous listing of the literature around MERS-CoV and camels.[1]

I'll republish an updated literature list in the summary post as well.

First up, an article from the scientific literature which was published by Hemida et al. in the July 2014 edition of Emerging Infectious Diseases, but would have gone online much earlier (CDC don't list that date for some reason I cannot fathom).[2]

This authors first remind us that MERS-CoV RNA has been detected in humans and dromedary camels (DCs) before and that DC infection has been shown to precede human infection in one study (well, two but they both analyse the same camels and humans).[3]

This study collected and froze nasal, oral or rectal swabs and blood samples from DCs on 2 farms in Al-Ahsa in the Kingdom of Saudi Arabia (KSA). The authors then looked for MERS-CoV RNA and antibodies.

• Farm A:
• 70 DCs
• 4: 1 month of age
• 8: approximately 1 year of age
• 58: adults
• Sampled 5 times between NOV2013 & FEB2014
• Herd never grazed in the desert (so wasn't exposed to other camels)
• November 30th 2013 results
• 10 DCs were MERS-CoV RNA positive; 8 of 9 DCs that had both nasal and faecal samples tested were only positive in the nasal swab, 1 DC only in the faecal swab
• December 2013 results
• No positive DCs December 4th; the following resulted from December 30th
• 7 of 8 calves and 2 of 3 adults
• 12 adults with sera collected before this testing were seropositive - this include 2 that were MERS-CoV RNA positive suggesting DCs can be reinfected
• 2 seronegative 1-year old calves had the highest nasal loads of MERS-CoV RNA suggesting maternal antibody may not be protective
• 4 DCs had the equivalent of a human cold - cough, sneeze, discharge, elevated temperature and were off their food
• February 14th 2014 results
• No MERS-CoV RNA was detected in DCs
• All 3 MERS-CoV RNA-positive DC calves who had sera collected on December 30th and February 14th, were MERS-CoV RNA negative in the February sample (thus an acute not chronic infection in camels) and all had a four-fold or great rise in antibody titer
• Farm B:
• 20 DCs
• 3: calves
• 17: adults
• Sampled once, FEB-2014
• Herd sometimes grazed in the desert
• No MERS-CoV RNA was detected in DCs

Samples were tested by 2 MERS-CoV specific real-time RT-PCRs and a broadly reactive coronavirus conventional RT-PCR. MERS-CoV positive samples were re-extracted (nucleic acids were purified from another aliquot of the original sample) and re-tested to confirm.

Conventional (Sanger) full genome sequencing was also conducted generating 3 genomes from Farm A, KFU-HKU 13, KFU-HKU 19Dam (faecal swab) and KFU-HKU 1. These were identical in sequence across the full 30,100 nucleotide genome and across the spike gene of 4 more viruses.

Virus isolation using the Vero E6 cell line was successful from 2 nasal swabs and 1 faecal swab - all with high amounts of viral RNA (culture is nowhere near as sensitive as PCR-based detection methods) - collected on December 30, 2013.

• A genome sequence from the faecal swab and the 2nd passage of culture isolate from the same faecal swab were directly compared - 3 nucleotide changes were identified, 2 of which led to an amino acid change (spike and membrane proteins)

So we learned from this study that DC MERS-CoV (genetically near identical to virus found in humans) doesn't mutate within a given DC herd (genetically stable in DCs), but does change a little upon cell culture in the laboratory. That change is not unexpected as cell lines in a flask are not camel/human cells in a complex microenvironment in the body. It's also not the first time such mutation has been seen.

We can also see that not all farms in a region of KSA have MERS-CoV when one does but that infections spreads within and around the herd - not persisting once it has moved through. However this herd and others in the region is one from which DCs can be moved to the via Buraidah in the KSA to the United Arab Emirates. Imports and exports and movement to shows and festivals being a problem when your animal is carrying an infectious agent - just as it is when an infected human hops on a plane and travels to Nigeria, or South Korea or the United States...or anywhere. We saw that adult DCs could probably be reinfected despite a pre-existing antibody response. But we learned nothing about the cell-mediated immune response - a gap in our knowledge that extends to the human immune response to MERS-CoV infection also.

While the peak of infection at Farm A occurred in late December in this study, only a limited time periods was sampled and too few farms to know if this is the pattern throughout the Arabian Peninsula, or just chance in Al Ahsa in 2013/2014. But there is another study that has looked a little longer and I'll review that soon. 

Sadly, there were no human farmers involved. The study would have been made more valuable if it had also followed any and all humans in contact with these camels over this period as well. More examples of camel-to-human infection would be great to have since there are still those who don't "believe" camels play a role in MERS. Of course, it's not belief that's needed, it's the willingness to sit down and listen to the scientific facts we have at hand. And that comes down to finding a way to pitch the facts in a way that works for each type of audience.

References...

1. http://virologydownunder.blogspot.com.au/2014/05/camels-and-mers-links-to-peer-reviewed_27.html
2. MERS Coronavirus in Dromedary Camel Herd, Saudi Arabia
   Hemida MG, Chu DK, Poon LL, Perera RA, Alhammadi MA, Ng HY, Siu LY, Guan Y, Alnaeem A, Peiris M.
   Emerg Infect Dis. 2014 Jul;20(7):1231-4
   http://www.ncbi.nlm.nih.gov/pubmed/24964193
3. http://virologydownunder.blogspot.com.au/2014/06/1-of-these-papers-is-pretty-much.html

Jon Snow is remarkably well informed compared to us...

...because we clearly know almost nothing when it comes to the specifics of where Ebola virus (EBOV) can, in some portion of survivors, hide. 

What triggers EBOV to come out of hiding? If indeed that is what it has done within PC, the United Kingdom nurse who seems to have recently become ill while (or due to) hosting a reappearance of EBOV in her blood - 9 months after her blood was defined as containing no detectable EBOV. 

This is not a new infection - but a return of a virus that had not been cleared from its hidey hole(s).

There are obvious concerns to be discussed around this, including:

1. What triggered its re-emergence
2. Is this a strange and rare event or a more common one?
3. Was the virus hiding in PC's central nervous system (she has meningitis-like symptoms reportedly) or in another place or places? 
4. Is the thyroid a site of persistence? 
5. Did the mysterious antiviral treatment she was given alongside a plasma treatment in 2014/15 act to push the virus into hiding? 
6. Has the activated virus mutated in any significant way during its holiday out of the bloodstream? 
7. Can EBOV become truly latent or at least dormant (still producing proteins, but not infectious virus particles) when off the beaten track, or is it constantly replicating?
8. Is PC suffering from full-blown Ebola virus disease (EVD) now, or a disease due to tissue or organ damage from her earlier infection - although if she has detectable EBOV in her blood, one would assume it is EVD. 
9. What the hell are a "lucky set of genes" in the context of a return to systemic EBOV infection? 

Below are some known and some proposed/possible/unproven sites at which EBOV may linger, avoiding or not exposed to clearance by the full force of our immune response.

In the future, perhaps tomorrow, in a non-rich nation, will others become infected from a convalescent patient who experience a return of EBOV long after they were declared virus-free based on a their blood test results. Has that happened already? Will they be our colleagues, friends or family members? The sooner we hear more about what is going on with PC, the better prepared those on the ground will be to intervene. Also, those tending to convalescent health workers in countries around the globe. 

Communication has always been a key issue for EVD and other emerging diseases. Have we been listening?

We know nothing but we can learn. Will we?

Is the next Ebola virus revelation...reactivating infection?

Update #1 11OCT2015 AEST
Update #2 11OCT2015 AEST
Update #3 22OCT2015 AEST

Great. Are members of the Zaire ebolavirus (EBOV) species the most educational viruses of modern times or what? I mean, we've "known" about EBOV since 1976, but the West Africa Ebola virus disease (EVD) epidemic is the epidemic that keeps on giving - we seem to learn a brand new thing every few months.

And the latest is a doozy although we don't know many of the details yet.

So what do we know about this new finding of a seeming return of infection in a former EVD case? Or is this new disease because of damage from the old infection?

1. A 39 year old nurse, PC, was originally infected with EBOV while working for 3 weeks in the Save the Children’s Kerry Town Treatment Centre in Sierra Leone. She did not show signs of illness until after arriving home in Scotland [1,11]
2. PC was believed to have become infected while treating EVD patients in some way related to her use of a visor as part of her personal protective equipment, rather than goggles, [10]
3. PC entered a Gartnavel Hospital isolation unit on 29-DEC-2014, and was subsequently flown to the Royal Free Hospital (RFH) in Hampstead, North London on 30-DEC-2015. She stayed there for around a month [2,4]
4. During her time in the RFH, PC was treated with convalescent blood plasma and an experimental antiviral drug
5. PC was declared free of EBOV and discharged from the RFH on 24-JAN-2015 but continued to report thyroid problems afterwards as she described just a week ago [3,4,12]
6. On Monday evening, PC went to a GP service at Victoria Hospital with a temperature, headache, sore neck and sensitivity to light (photophobia). [15] She was sent home.
7. On Wednesday 07-OCT-2015, PC was admitted to the Queen Elizabeth University Hospital (QEUH) in Glasgow, Scotland. Tests revealed that EBOV (presumably) RNA was present.[13] 
8. On Friday 09-OCT-2015, PC was admitted to the RFH, 8 months and 15 days after being declared free of Ebola virus and discharged.[16]
9. She is described as being in serious condition. However, it is unclear what her signs and symptoms were at presentation, or have become since.[4]
10. On Wednesday 14-Oct-2015, PC's conditions was described as having deteriorated and was now classified as in critical.[16,19,20]
11. On Monday 19-Oct-2015, PC's condition was described as improved.[16,21]
12. On Thursday 22-Oct-2015, at a press conference made possible because PC had given permission for her case to be publicly discussed, PC was described as having significantly improved. She had meningitis and has received a "highly experimental" drug, GS-5734, under development by Gilead Sciences, and proven highly effective in the lab and in monkeys post-infection.[22,23,25,26]

Descriptions note that PC is "not thought to be contagious". Presumably this means she is not symptomatic with EVD and if so the testing that must have identified EBOV somewhere in her system must have does so from a part of her system that is not readily in contact with the environment. In one report, laboratory staff in Glasgow who handled PC's samples, were not wearing the most basic of personal protective equipment (PPE) for lab staff-gloves.[24] All of that side, she is once again isolated at one of the world's best infectious diseases hospitals.[4] 

There are also recent reports of PC having had thyroid problems after recovering-perhaps virus has been replicating in this tissue. PC's "condition is a complication of a previous infection with the Ebola virus".[4] Which leaves a lot of room for idle speculation but could just be that she is ill because of what the fallout from what EBOV previously did to a tissue/organ rather than because of EVD itself. Perhaps follicles in the scalp have been a site for virus replication, relating to her earlier hair loss. Another site may be the central nervous system...

@amymaxmen I've heard she has a meningitis like syndrome, probably CSF is where #Ebola virus is replicating

— Ahmed Tejan-Sie MD (@AhmedTejanSie) October 9, 2015

@MackayIM @amymaxmen CSF and blood (later) positive

— Ahmed Tejan-Sie MD (@AhmedTejanSie) October 15, 2015

All speculation. Again, nothing is known about PC's signs and symptoms of disease when she presented herself to the QEUH, what tissue(s) are involved in her current illness, which samples tested positive first, whether viral culture has been conducted or just RT-PCR and where the virus may have been replicating all this time. While we understand that some tissues are sites for EBOV persistence, there is clearly much more to learn about the frequency and full range of tissues that harbour infectious EBOV once it becomes undetectable in the blood.

Apart from how shocking and scary this must be for PC herself, another issue is how this will impact on the fragile processes of accepting of EVD survivors back into their West African communities. Extending the length of time that some male survivors are known to harbour EBOV already put pressure on their acceptance by some, but the potential for virus to return to the blood or other tissues - if indeed that is what has happened here - even after that time frame, will require a lot of communication to explain. It will be vitally important for this process that the facts underpinning what's happened here are deduced soon and communicated in ways that can be understood in West Africa. This is a chance for the World Health Organization to show off their shiny new intent to do better at communicating and reacting. 

This is not the first time EBOV has been found to persist in a convalescent former EVD case.[5,6,7,8,9] But this may be the first documented time that the virus has re-emerged from an immune privileged site and returned to the blood, possibly causing EVD symptoms in the same former EVD case (recent media article mentions that this is the 2nd such case[15]).

The comments about PC's photophobia are similar to those from Dr Ian Crozier - who was found to have persistent infectious virus in the aqueous humor collected by needle from the anterior chamber of his eye causing uveitis-14 weeks after his EVD diagnosis.[6,17] His blood did not become EBOV positive again.[17] The virus found in his blood earlier and his ocular fluid 14 weeks later were nearly identical-just five mutations differentiating the genomes. Could the eye be a site of PC's hidden virus also? Perhaps the central nervous system the reservoir given the meningitis-like symptoms PC's family mentioned early and which was confirmed 22OCT.[15,18] Again, lots of speculation. 

Shingles has been thrown up as an example of a similar disease that results from a virus recurring but it's not the same thing at all. Although, we don't know that with absolute certainty. The viruses are very different - that we know for certain. Varicella zoster virus (VZV) is the herpesvirus that first causes chickenpox (doctors call it 'varicella'), usually in childhood. The virus then goes dormant in your nerves. In this state, the virus is not producing full virus particles and so VZV no longer excites our (cellular) immune system, which can eventually "forget" it. Decades later (again, usually) after lying dormant and because of triggers and a lack of suitable immune memory VZV may arise from dormancy (reactivate) to produce lots of whole virus and cause shingles (doctors call this 'herpes zoster' - still the VZV though).[14] As far as we know, EBOV does not go dormant or become latent, but remains active at some sites, like the testes and the eyeball,[5,7] where our immune system is programmed not to venture in full force, so as to protect those sites from unwanted inflammation (in a nutshell). There may well be other sites.

Hopefully, more official key information will be made clear soon (as opposed to in the scientific literature weeks or months from now) as it will be vitally important for the continued management and support of EVD survivors in West Africa. It is also important knowledge for communicating real risks, and informing and toning down perceived but unrealistic ones. What falls into which category is however becoming harder and harder to discern.

I'll update this blog post as more information comes to hand.

References...

1. http://www.bmj.com/content/350/bmj.h36.long
2. http://www.bbc.com/news/uk-scotland-30629397
3. http://www.independent.co.uk/news/uk/pauline-cafferkey-ebola-nurse-back-in-hospital-virus-can-live-on-in-tissue-for-months-after-recovery-a6687571.html
4. http://www.bbc.com/news/uk-scotland-34483584
5. http://virologydownunder.blogspot.com.au/2015/08/post-ebola-syndrome-or-just-chronic.html
6. http://virologydownunder.blogspot.com.au/2015/05/ibola-and-speed-of-research-reporting.html
7. http://virologydownunder.blogspot.com.au/2014/08/ebola-virus-in-semen-is-real-deal.html
8. http://virologydownunder.blogspot.com.au/2015/03/liberia-enters-next-phase-of-ebola.html
9. http://virologydownunder.blogspot.com.au/2015/03/catching-ebola-mistakes-messages-and.html
10. http://www.theguardian.com/world/2015/oct/09/scottish-ebola-nurse-pauline-cafferkey-royal-free-hospital-london
11. http://america.aljazeera.com/articles/2015/1/3/scottish-nurse-ebola.html
12. http://metro.co.uk/2015/01/24/british-nurse-with-ebola-pauline-cafferkey-makes-complete-recovery-and-leaves-hospital-5035112/
13. http://www.bbc.com/news/uk-scotland-34495250
14. http://www.nytimes.com/ref/health/healthguide/esn-shingles-expert.html?pagewanted=all
15. http://www.dailyrecord.co.uk/news/scottish-news/family-condemns-hospital-failures-after-6612236
16. https://www.royalfree.nhs.uk/news-media/news/update-on-pauline-cafferkey/
17. Persistence of Ebola Virus in Ocular Fluid during Convalescence
    http://www.nejm.org/doi/full/10.1056/NEJMoa1500306
18. http://www.theguardian.com/world/2015/oct/21/ebola-nurse-pauline-cafferkey-condition-serious-but-stable-royal-free-hospital
19. http://www.bbc.com/news/uk-scotland-34529575
20. http://www.theguardian.com/world/2015/oct/14/ebola-nurse-pauline-cafferkey-critically-ill
21. http://www.bbc.com/news/uk-34574899
22. https://www.royalfree.nhs.uk/news-media/news/pauline-cafferkey-makes-significant-improvement/
23. http://www.bbc.com/news/uk-scotland-glasgow-west-34592132
24. http://m.eveningtimes.co.uk/news/13842874.Lab_staff_at_Glasgow_hospital_told_NOT_to_leave_the_country_over_Ebola_fears/
25. http://uk.advfn.com/news/DJN/2015/article/68969936
26. http://www.telegraph.co.uk/news/health/news/11945802/Ebola-caused-meningitis-in-nurse-Pauline-Cafferkey.html

Updates...

1. Added dates for PC being initially released from the RFH and that she was tested at the QEUH; described "dormant" and qualified that chickenpox and shingles can occur at any times but usually as a child and adult respectively; provided references about chickenpox
2. Updated information from Ref 15 including dates, new calculation of time between RFH 1st discharge and admission and some symptom information from family
3.  Added CSF+Blood Tweet; some tiny bits of new info from the RFH press conference; links to GS-5734 info