Where do these 'primary' MERS cases come from?

I was just dropping by the General Directorate for Infection Prevention and Control's Middle East respiratory syndrome (MERS) statistics website ([1]; Kingdom of Saudi Arabia) and noticed this graph which I've snipped and labelled below.

What strikes me as very strange is the listing of so many "primary" MERS cases (green). Keep in mind that the Command and Control Center of the KSA Ministry of Health has a case definition that does not allow for a person who has no symptoms as a Case-even if they are laboratory confirmed as being infected [2]. A strict clinical-based definition. In a more real-world definition for reporting a MERS Case, the World Health Organization (WHO) has, since at least July 2014, included any laboratory confirmed person (which includes suitably serologically confirmed) as a Case. 

In other words, the green bars represent KSA-defined Cases that have not acquired MERS-CoV infection from family, friends or house mates (yellow), not from healthcare workers or the healthcare facility (nosocomial; red and purple) and that are not unclassified (magenta). 

Excerpted figure from the General Directorate for Infection Prevention and
Control website.[1]
Blue labelling by me using Photoshop.

Doesn't that just leave camels? 

But camel exposures are not being listed in WHO Disease Outbreak News (DONs) in anything like that number. It's very rare to see a camel contact listed in the current Ar Riyad province outbreak at all (just one, early on).

So where do these primary cases acquire their infection from?

Reference...

1. http://www.gdipc.org/mers-stat.html
2. http://nebula.wsimg.com/9e5c08742ca99231959e266fe5e46ee0?AccessKeyId=56AE5CCCF0799F235A2E&disposition=0&alloworigin=1

Ebola: Blood, sweat and tears...

This post follows up the recent one on convalescent semen being able to harbour infectious Ebola virus (EBOV; although I am not aware of any infection resulting from this route of transmission there has been at least one report for Marburg virus [4]).

I thought I'd give the same treatment to tears and sweat which are also fluids intermittently listed as possible sources of EBOV infection for humans. Some examples of the scientific literature which support the risk messaging, follow.

Blood...

I think we are all pretty clear that the blood of an advanced case of Ebola virus disease (EVD) is heavily laden with virus and is the most serious of the risk factors for acquiring infection by an ebolavirus.

Viral loads (amount of virus in the sample) in blood can be above 10⁶-10⁸ plaque forming units or copies (pfu; a measure of infectious virus present using a lab test that measure the impact of virus on infected cells; copies measure viral genome and cannot prove infectious virus is present) per millilitre of blood in acute phase disease patients and non-human primates.[5,6]

Sweat...

There is not a lot on sweat containing signs of an ebolavirus.

• Bausch and colleagues found no trace of EBOV in a single acute sweat sample.[1]
• Jaax and colleagues found that in experimentally infected non-human primates (NHPs; rhesus macaques), that connective tissues next to hair follicles and sweat glands in the skin as well as the cells lining ducts of glands in the skin were sometimes positive for EBOV antigens (proteins).[2]
• Davis and colleagues infected NHPs (African green monkeys) and found signs of EBOV antigens in the cells lining the sweat gland ducts and in cells in the connective tissues next to hair follicles, but no virus particles by electron microscopy.[3]
• Zaki and colleagues found heavy signs of EBOV antigens (proteins) in the tissues around the sweat glands, but rarely also within sweat glands and ducts.[8] No virus particles were seen in the sweat glands or ducts when examined by electron microscopy.

Tears...

There is also very little I have found on this one.

• Bausch and colleagues found EBOV RNA in tears from 1 sample, but no infectious virus could be isolated.[1]
• Jaax and colleagues found some signs of virus in macrophages in the ciliary body of the eye of experimentally infected NHPs

Spit...

• Bausch and colleagues found infectious EBOV in 1 of 12 acute saliva samples (from 10 patients; none from 4 convalescent samples) and EBOV RNA in 8 of 12 (67%) of acute samples (none from convalescent samples).[1] RT-PCR positivity was significantly associated with fatal outcome.
• Formenty and colleagues found EBOV antigens and EBOV RNA in oral fluids from fatal cases and those who survived infection.[7]

References...

1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2395567/pdf/bullwho00439-0113.pdf
2. http://www.ncbi.nlm.nih.gov/pubmed/8712894
3. http://www.ncbi.nlm.nih.gov/pubmed/9278608
4. http://link.springer.com/article/10.1007/BF01734141
5. http://www.sciencemag.org/content/suppl/2014/08/27/science.1259657.DC1/Gire.SM.pdf
6. http://www.nature.com/nature/journal/vnfv/ncurrent/pdf/nature13777.pdf
7. http://www.ncbi.nlm.nih.gov/pubmed/16652308
8. http://www.ncbi.nlm.nih.gov/pubmed/9988163

Snapdate: MERS-CoV detections by where they were probably acquired...

...yeah its "probably" because sometimes it's just not clear to anyone. I've about 14 (554 in total) more cases attributed to a likely source in the Kingdom of Saudi Arabia (KSA) compared to the Ministry of Health tally on their website (540), but such is life.

Anyway, I haven't updated this chart for 17-days (woah - sorry about that). The latest version now has the two major hospital-related outbreaks shown in pink - I'm still liking the Janadriyah festival as the possible source of community cases that then filter back throughout the country as visitors and tourists return to different areas or travel afterwards for other reasons. That would be on my 2015's "festivals that need to be tweaked to avoid camel contact" list.

As per yesterday's post, these accumulation curves highlight that new detections of MERS-CoV have slowed right down in the KSA and in the United Arab Emirates (UAE). However, the UAE are now the reporting slow pokes, if the last batch posted by the World Health Organization is anything to go by, so we should stay tuned to what's happening there (and also stay tuned to the extent of upper respiratory tract signs and symptoms in those cases which continue to bug me).

Click on image to enlarge.

More camel mentions among MERS-CoV cases...

Click on image to enlarge.

Special thanks to Professor Andrew Rambaut ([4]; @arambaut) for keeping such great track of the number of human cases in which camels has been mentioned.
Also thanks to Prof Rambaut and Ellen Knickmeyer  (@EllenKnickMeyer) for putting up with my stupid questions.

The current tally is now 11 human cases with a link to camels; 3 more than my earlier post on this topic.

The charts still show that cases outside of the Kingdom of Saudi Arabia (KSA) are proportionately more likely to identify human contact with camels than are MERS cases acquired within the KSA. 

The first case from Qatar with a camel link was from Sept-2012; from the United Arab Emirates (UAE) on Oct-2012; from Oman 20-Dec-2013. The very first (index) case of MERS-CoV to be announced to the world on Sept-2012, that from a 60-year old man living in Bisha in the KSA, also had contact with his 4 pet camels which we learned of in an article in the New York Times ([1] and later in an article late February 2014 [3]). 

I have not added the case of a Qatari male who owned a camel and goat farm [5], because the report in Eurosurveillance notes he claimed no direct contact with sick animals. I do wonder about contact with healthy or asymptomatic animals though. 

References...

1. New York Times article on Prof Memish et al's mBio paper.
   http://www.nytimes.com/2013/08/22/health/mystery-virus-thats-killed-47-is-tied-to-bats-in-saudi-arabia.html
2. Prof Memish et al's mBio paper (does not mention camels though)
   http://wwwnc.cdc.gov/eid/article/19/11/13-1172_article.htm
3. Alagaili et al's paper noting camels were a contact of 60M index MERS case
   http://mbio.asm.org/content/5/2/e00884-14.full.pdf+html
4. Professor Andrew Rambaut's MERS-CoV case list
   http://epidemic.bio.ed.ac.uk/coronavirus_background
5. Eurosurveillance contact study of 45-year old male from Qatar (FluTracker's Case #6)
   http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20406

The decline of H7N9 Wave 2: some thoughts on why it may be different from Wave 1...

Influenza virus and influenza the disease certainly give scientists a run for their limited money when it comes to predicting what either will do from year-to-year, country-to-country or outbreak-to-outbreak. 

And just when you think you know enough, things change. 

This morning my Twitter stream was fed by a sparkling rivulet of informed comment by @influenza_bio ("A biologist"; follow him if you don't already) on the subject of why H7N9 cases are falling. @influenza_bio groups together a few great points:

1. H7N9 cases are declining.
   Agreed, I think Wave 2 ended almost a month ago.
2. Overall, influenza-like illness (ILI) visits in China have declined.
   A clear parallel, but is it causal? ILIs provide a general guide to influenza circulation (general, because other viruses cause ILI which is basically fever + upper and or lower respiratory signs and symptoms - so a very broad but useful good guide

Percentage of Visits for ILI at Sentinel Hospitals in South China through 2/23/14. http://t.co/yKlqgzOkLg pic.twitter.com/IXKlWlltWX

— A biologist (@influenza_bio) March 12, 2014

3. Is the drop in human H7N9 cases linked to the end of a (silent) outbreak in birds (poultry, waterbirds, songbirds, both...)?
   Finding data on specific bird migration dates in the region is difficult. See here and here for some generalizations. Seems very reasonable.
4. Live bird market closures cannot be the only cause of a drop in H7N9 cases otherwise we'd expect to see cases in other areas continue to rise (presumably areas where markets are not closed).
   If we compare Zhejiang to Guangdong, then we can see that the delay in closing Guangdong's bird markets seems to have manifested as a delay in slowing of human cases; most recent H7N9 case acquisitions have indeed been in Guangdong (a major poultry producing area in southern China) whereas cases in Zhejiang which, like other eastern coastal regions shut their markets earlier and "permanently", generally speaking, have dried up.
5. If H7N9 human case decreases were linked solely to weather, then how could we explain the peak in 2013 which extended into late April whereas it looks to have peaked well before that, in early Feb, in 2014?
   Given that the seasons have not differed between the years (or have they?), I'd suggest we look more at the start of the 2 Waves; Wave 2 commenced earlier in 2014 than did Wave 1 in 2013, but the precipitous decline of both outbreaks of human notifications seemed to have been more closely tied to market closures than dates on a calendar. Of course markets are stocked with H7N9 infected birds and that which links to outbreaks at the supply end unless poultry acquired their infections at markets and then spread that between markets by bird movements which can extend right across southeast China. Why did it start earlier is a key question for me.

@influenza_bio finishes with the comment that...

Thus, wld seem possible that decline we're seeing in human #H7N9 may mostly be due to fall in bird #H7N9 cases. Need more bird surveillance.
— A biologist (@influenza_bio) March 12, 2014

As I've learned from @influenza_bio, many factors go into humans acquiring a particular influenza virus at a particular time/season, and probably no single thing is responsible for all events for any given outbreak. Phew. But that's why we don't have influenza infections all the time and it underpins why they peak at a certain time.

Human acquisition of influenza virus is related to:

• How a person is exposed to the virus (aerosol from upper respiratory tract coughs and sneezes or self-inoculation from contact with contaminated surfaces)
• Whether the virus survives long enough to be inhaled/self inoculated which is in turn linked to virus subtype and strain and environmental temperature and humidity (see some more on that in a guinea pig model here)
• The host and their immune state and general health, smoking, underlying diseases etc
• How much virus enters the host and where it "lands" and makes a footing in the host's respiratory tract
• The spaces we share with infected people and how fast and well the air is filtered/exchanged in those spaces
• The virus subtype in terms of what receptor it prefers and where those might be located throughout the respiratory tract.
• For avian influenza in humans there is also the type and length of exposure to the animal hosts and their environment

Not an all inclusive list I'm sure, but you get the point. Influenza viruses are a complex beast, made more so by the fact that any given subtype could be represented by a range of strains indicating a variety of stabilities, preferences for receptors, antiviral susceptibilities etc. 

So I complete agree with @influenza_bio, more bird surveillance would indeed be a very important step in understanding what is happening in and perhaps predicting the risk of, human outbreaks of this and other avian influenza viruses.

H7N9 and human infections: not just a paltry matter

Jones and an all-star cast of colleagues from Hong Kong, Shenzen, Beijing and Tennessee have looked at songbirds and their susceptibility to a human isolate (infectious virus recovered from a human case of H7N9 influenza) H7N9 infection (1).

But before I note the good bits of their study, this paper is one of importance for adding a lot to our understanding of how H7N9 is jumping to people from poultry/live bird/wet markets. It's also a great reference if you want to better understand influenza and birds overall. 

We've read much about human cases of this avian virus having had contact with "poultry" and bird markets and  from that we assume that poultry are the pool in which H7N9 is swimming (reservoir); but where did the poultry get it from (source/natural host)? Its also interesting to note that: 

1. Very few poultry test positive for the virus; may simply be because of a test-based problem including using a test that is insensitive or sampling from the wrong end of the bird (testing the cloaca instead of throat as was discussed ). 
2. Looking at the sequences of the H7N9 gene segments suggested that wild birds (bramblings) played a part in the evolution of the virus currently infecting humans in south east China (4)
3. Pigeons have tested H7N9-positive (2,3)

So it might be that at least some of the human exposures are not from poultry but from other birds.

The authors of this latest article note the songbirds are common pets and so are in close contact with their owners. In the wild, such birds are likely to interact with farm birds.

Some key findings of this new study are:

• A/Anhui/1/2013 was the strain used for H7N9 studies; it was an isolate from a human but early on and similar to bird (6) strains. H5N1 (A/Vietnam/1203/05) and H3N8 (A/songbird/Hong Kong/SV102/2001) were also used for comparisons
• Zebra finches (Taeniopygia guttata), society finches (Lonchura striata domestica), parakeets (Melopsittacus undulates) and wild-caught house sparrows (Passer domesticus) were kept isolated for 3-weeks prior to experiments to let any naturally acquired infections burn out; none of the birds had antibodies suggestive of previous infection by an H3, H5, H7 influenza A virus (is that low prevalence normal?)
• Birds were inoculated with 10⁵ 50% egg infectious doses of virus via nose, eye and mouth (that should do it) and then put in the same cages, sharing water and food, with uninfected birds
• Virus testing was by growth using eggs (3/sample collected)
• All inoculated birds shed virus (only) from the oropharynx; finches shed most virus at 2-days post inoculation (dpi); parakeet viral shedding could be detected by culture for 2-days and from finches for 6-days
• Communal water troughs yielded culturable virus; zebra finches shed most virus but water consumption and drinking frequency were not measured and may have differed among bird species
• No virus could be detected at 8dpi
• 1 sparrow showed signs of disease and died; 1 zebra finch died without signs of disease (some loss of appetite)
• Birds in contact with infected birds did not often acquire infection but when they did, they also shed via the oropharynx
• In finches that were killed for organ testing, virus was mostly found in the trachea; some was isolated from brain and eye tissues of 1 society finch and in the small and large intestine and a high titre form the lung of the other. H7N9 was grown from the brain, lung and intestines of zebra finch. H7N9 was not found in surviving sparrow organ tissues; in the dead sparrow, some H7N9 was found only in the lungs
• Nearly all inoculated birds mounted a specific antibody response to H7N9 after inoculation. Among the contact birds, 3/3 zebra finches, 1/3 society finches (had highest amount of antibody), 2/3 sparrows and 0/2 parakeets mounted a response to virus indicating that they were infected but did not show signs of illness nor did they shed virus, at least at culture-detectable levels

So songbirds, can be infected by a human H7N9 isolate, they can shed the virus into the environment, they can die (presumably) due to H7N9 infection, 33-66% of songbirds in contact with an experimentally infected songbird acquire aninfection (even if it was rare to grow infectious virus from that contact which may be a sensitivity issue of the testing) and they mount an immune response to the infection. Given that H7N9 acquisition seems to be a numbers among humans, this degree of transmission among birds fits well.

It was also very interesting that water troughs often contained lots of shed H7N9 virus. This is not new in the world of influenza virus but its nice to cross the 't' for H7N9. The authors note that studies of transmission from songbirds to poultry via communal water sources are yet to be conducted. Seems like this would be a very important piece of the influenza puzzle and with broad application to future outbreaks and seasonality in birds via migration. 

Add to all of this that songbirds are present in many markets (thanks to @Crof, @Laurie_Garrett and @debmackenzie1 for supporting info via Twitter this morning; also see refs from Jones et al (1) and a related story from New Scientist (8)) and that older males are a key demographic for keeping songbirds as luck-enticing (and cute) pets. They are also over-represented among H7N9 cases (see adjacent chart). A good fit.

Another recent study (7) shows chickens and quail (a possible amplification host helping bridge the gap between wild birds and poultry) shed a lot of H7N9 after experimental inoculation via an intranasal route. Also, quail (but not pigeons) shed enough H7N9, for long enough, to pass it along to their contacts; less so ducks.

None of this may be very new to some of you, but it's nice to see data that confirm it all for H7N9. After all, as someone reminded me recently on Twitter, data is just how we roll.

It's not hard to see the circle of life for influenza viruses is there for the interpreting and that non-poultry birds may be important intermediate hosts of H7N9 and act as a source of other influenza A viruses. 

Just how many human cases of H7N9 are acquired by songbirds vs chickens/ducks/quail/geese remains unquantified....perhaps unquantifiable.

References...

1. Possible Role of Songbirds and Parakeets in Transmission of Influenza A(H7N9) Virus to humans.
   http://wwwnc.cdc.gov/eid/article/20/3/pdfs/13-1271.pdf
2. A summary of Influenza A(H7N9) virus findings in birds and humans
   http://virologydownunder.blogspot.com.au/2013/10/a-summary-of-influenza-ah7n9-virus.html
3. Emergence of avian influenza A(H7N9) virus causing severe human illness - China, February-April 2013.
   http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6218a6.htm
4. Sunny summer or birds on the wing?
   http://virologydownunder.blogspot.com.au/2013/05/sunny-summer-or-birds-on-wing.html
5. Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses
   http://press.thelancet.com/H7N9genetics.pdf
6. Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20453
7. Role of poultry in spread of novel H7N9 influenza virus in Chinahttp://jvi.asm.org/content/early/2014/02/20/JVI.03689-13.long
8. Budgies may be behind latest spread of H7N9 bird flu
   http://www.newscientist.com/article/mg22129542.000-budgies-may-be-behind-latest-spread-of-h7n9-bird-flu.html#.Ux5CnvmSx8F

Dromedary camels are a host of MERS-CoV...

Yes. Not a "MERS-CoV-like" virus or "something very closely related to but slightly different" from MERS-CoV. Camels. Are. A. Host. 

There was already plenty of evidence to suggest this (see some of my previous posts on this linked below), and none to really dissuade me from thinking otherwise. And yesterday we saw a new paper by Ian Lipkin and his collaborating crew from King Saud University in Saudi Arabia that make this issue more obvious than ever. 

So let's stop messing around. There is an elephant in the MERS-room...and its a camel! 

The Middle East respiratory syndrome coronavirus, does in fact look to be a camel virus that causes few symptoms in that host, is acquired by young camels and has been for at least 22-years, and then people somehow get infected, probably from proximity to camels or due to habits involving camels. the keeping of camels or at gatherings in which grumpy slavering camels are congregating. 

Yes, there is little evidence for any contact with camels among the 186 human cases as Dr Ziad Memish, deputy health minister of the Kingdom of Saudi Arabia (KSA) points out, but as Prof Marion Koopmans noted to NPR, "few people [with MERS] have had the kind of follow-up you would want". So we take denial of any contact with a grain of salt.

There is definitely some contact however; some that is pretty solid. For example, the owner of a camel in Jeddah (4) that both tested positive for MERS-CoV or the Qatari farm camels and owner and an employee that were all MERS-CoV positive (5,7). 

And there is far less evidence against camels as a source of  some/many/most human cases and for anything else. 

So in the new paper in mBio published online 25-Feb, we read of the most comprehensive KSA camel study to date. Camels from 2013 were sampled and camel sera collected and frozen since 1992-2010 were tested and many were found to be MERS-CoV antibody positive. From the more recent dromedary camels, nasal swabs were also found to be viral RNA positive; RNA that is definitely from MERS-CoV.

Some key findings...

• No sheep or goats were MERS-CoV antibody or RNA positive; a routine finding now. Bovine CoV antibody reactivity was identified in these animals however, and in 17% of camels
• Antibody was detected using infected cells and also using a method that employs a specific portion of a MERS-CoV protein (part of the nucleoprotein)
• 150/203 (74%) of camels from all over the KSA had MERS-CoV antibodies in a pattern reminiscent of any endemic human respiratory virus
• 95% in camels older then 2-years (adults)
• 55% in those ≤2-years (juveniles)
• The south west had the lowest proportion of positive camels (5%)
• Higher proportions were found in central KSA (Riyadh)
• 3 rectal swabs were positive for MERS-CoV RNA using real-time reverse transcriptase polymerase chain reaction (RT-rtPCR). 2/3 camels were also nasal swab POS
• 36/104 juvenile camels were nasal swab POS
• 15/98 adult camels were POS
• 66% of samples from the west (Taif) were POS but none from the south west
• no RNA was detected in a sampling of camel blood/sera and so the archived samples from earlier years could not be sequenced to verify that they had MERS-CoV sequences in them
• Amplification and sequencing of a 1,044nt portion of the Spike gene, 2,004nt ORF1ab region found that less than 1% difference from previous published MERS-CoV sequences and the nucleocapsid gene region was identical. Great to see a move away from recent reliance on complete genome sequences and a more practical and rapid subgenomic, multi-target molecular epidemiology approach used. 
• 11/13 higher viral load samples could be amplified and sequenced

It was interesting hear the TWiV (this week in virology) podcast interview with Prof Lipkin [IL] and Assoc. Prof Thomas Briese (TB) in which they noted:

• MERS-CoV is a "puny" virus causing little overt disease on camels [IL]
• The MERS-CoV genome seems to be fairly stable; its not influenza virus and does not seem likely to evolve rapidly
• Baboons[!], dogs, cats, rodents are on the list to test when the team return to KSA
• There has been a previous report on limited human antibody levels to MERS-CoV in at least the east of the KSA

Sources and previous posts on camels and MERS-CoV...

1. mBio paper by Alagaili and colleagues
   http://mbio.asm.org/content/5/2/e00884-14.full.pdf+html
2. NPR's Richard Knox: excellent story
   http://www.npr.org/blogs/health/2014/02/25/282136478/deadly-mers-virus-circulates-among-arabian-camels
3. CIDRAP Story
   http://www.cidrap.umn.edu/news-perspective/2014/02/study-mers-cov-may-have-been-saudi-camels-22-years-ago
4. Camels owner in Jeddah
   http://virologydownunder.blogspot.com.au/2013/11/camel-cough-coronavirus-caught.html
5. Two Eurosurveillance studies reporting MERS-CoV antibodies in camels
   http://virologydownunder.blogspot.com.au/2013/12/middle-east-respiratory-syndrome.html
6. MERS-CoV antibodies in 10-year old UAE camel sera
   http://virologydownunder.blogspot.com.au/2014/01/antibodies-in-10-year-old-uae-camel.html
7. More on the Qatari camels and some MERS-CoV sequencing and social media chatting
   http://virologydownunder.blogspot.com.au/2013/11/dutch-researchers-in-collaboraion-with.html
8. MERS-CoV antibodies in camels from the Canary islands and Oman
   http://virologydownunder.blogspot.com.au/2013/08/camels-carry-signs-of-coronavirus.html
9. Early cautionary thoughts from the WHO
   http://virologydownunder.blogspot.com.au/2013/08/who-urges-dont-put-camel-before-cart.html
10. Thoughts about MERS-CoV acquisition
    http://virologydownunder.blogspot.com.au/2013/09/most-mers-may-not-have-met-camel-but.html
11. Querying whether there is a better possible source for human cases
    http://virologydownunder.blogspot.com.au/2013/09/is-there-better-smoking-bat-or-camel.html
12. Summing up the first 100-days of (human) MERS-CoV infections
    http://virologydownunder.blogspot.com.au/2014/02/middle-east-respiratory-syndrome.html
13. MERS-CoV antibodies in camel sera dating back to 2005 in the UAE
    http://virologydownunder.blogspot.com.au/2014/01/mers-cov-antibodies-in-dromedary-camels.html
14. Gatherings and acquisition/transmission of MERS-CoV between animals and humans
    http://virologydownunder.blogspot.com.au/2014/01/a-date-with-middle-east-respiratory.html
15. Qatari camels clear the MERS-CoV from their systems
    http://virologydownunder.blogspot.com.au/2013/12/qatari-camels-clear-coronavirus.html
16. TWiV podcast
    http://www.twiv.tv/2014/02/25/twiv-special-mers-coronavirus-in-dromedary-camels/

H7N9 snapdate: latest hotzone map

Avian influenza A(H7N9) virus human cases by region of acquisition.
Click on image to enlarge.

H7N9 snapdate: hotspot map

Click on image to enlarge.
Modified from here.  

Guangdong province starts to see red while Guangxi very quickly moved from 1 case to 3.

Activity in China's north east remains low.

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

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

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

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

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

Neither market nor farm poultry all that positive for H7N9; songbirds the culprit...?

Following on from yesterday's post, "If not poultry then what?", I thought it worth noting the impressive numbers from the Chinese Ministry of Agriculture.

From 2013:

• 1,630,000 poultry and environmental samples tested
• 88 POS; all from live bird markets
• None from poultry farms

From 2014, to date:

• 33,400 poultry and environmental samples
• 8 H7N9 POS; all from live bird markets
• None from poultry farms

The other alternative to answer the question in my heading; the testing methods are at fault. 

No detail of what approach has been used to obtain these numbers in the links below. Viral culture and serology with some PCR have been noted before. I'd wager culture yields chicken scratchings compared to PCR for detecting virus in he wild; but serology has successfully been a pillar upon which animal testing rests. So that's why the numbers above are such a quandary for the epidemiologist who reads about the high frequency of links between human disease and exposure to poultry.

It would be nice to see some technical papers on antibody test testing (development and validation) at some point. If only to reassure everyone that the testing methods are doing what testing methods should be doing.

See #3 below for influenza PCR discussion at WHO.

Sources..

1. http://english.agri.gov.cn/news/dqnf/201304/t20130427_19537.htm
2. http://english.cntv.cn/program/china24/20140130/100709.shtml
3. http://www.who.int/influenza/gisrs_laboratory/report_2012pcrwg5thmeeting.pdf

If not poultry then what? [UPDATED]

Maybe China needs to look at other
animals, both within market 
environments and at the source farms 
(using sensitive molecular tools as they 
have been), to find the "smoking chicken" 
(shamelessly stolen from Mike Coston)...
which may not be a chicken at all.

Mike Coston has written a nice post about the Chinese MOA denying that there is any proof of direct transmission of H7N9 from poultry to humans. 

Technically, they are of course correct. 

We have yet to see a human put in a cage downwind, but separated from, a flock of infected chickens or duck or geese to see if the human acquires H7N9 infection and disease. Nor have we seen any card-playing lockdown transmission scenarios to investigate aerosol, droplet and direct transmission routes. 

Nonetheless - if you take a look at a snippet of my list of H7N9 cases compiled from various public sources, "poultry" contact is mentioned...a lot; 72% of those rows (if you exclude the 19 awaiting WHO notification data; see below for update). 

Of course it may be a "poultry" euphemism or a translation error (I'm asking @WHO) for contact with any feathery creatures, so song birds and wild birds may be the source in the markets....or another animal altogether of course but I think something non-feathery would have shown up as a pattern by now.

[UPDATE] Gregory Härtl notes..

.@MackayIM @WHO "Domestic" birds are also being looked at,but so far wild birds r not being looked at as a source of human infection. #H7N9
— Gregory Härtl (@HaertlG) January 31, 2014

Only one way to tell what's happening now; get more samples and RT-PCR them. Serology is historically solid and well relied upon, particularly in the world of influenza surveillance. See expert comments on this in a recent CIDRAP article here. I'm just not convinced its providing enough sensitivity at a suitable speed to feed the needs of rapid response and control measures for an emerging virus like an influenza. A virus which is spilling into humans from somewhere not yet convincingly defined to everyone's satisfaction. 

But I'm new to flu and am coming at this from an endemic human virus detection and characterization angle. I may be waaay off point.

H7N9 snapdate: new charts for sex and age distribution and region of acquisition...

Two new charts.

Click on image to enlarge.

Firstly, the "age pyramid", a revised and combined version of the age and sex distribution charts for 265/267 H7N9 cases to date. This one comes with many thanks to Shane Granger for helping me learn a new trick. Please follow him @gmggranger or visit his chart-tacular blog, Random Analytics at http://gmggranger.wordpress.com/.

Secondly, we have the latest map of the H7N9 hotzone; adjusted to account for Zhejiang tipping over the 100 cases mark (new colours!), and for the addition of a new province into the world of H7N9 human infections; Guangxi. This marks the first new region and 13th to generate cases overall, since 9-Aug-2013. 

H7N9 has thus crept sideways towards the west and as FluTrackers noted, Guangxi shares a border with Vietnam. An entire other country. The first shared land border since we learned of H7N9 I think. I'm no expert in this topic, but trade in poultry and tourists between these two regions seems commonplace, as noted here, here and to the extent that research such as this study (and its references) indicate H5N1 sequences are shared between the regions.

Do we know if Vietnam actively employs laboratory methods to screen poultry for H7N9, H9N2, H10N8 screening of their birds? I suspect we'll learn soon if not.

A date with Middle East respiratory syndrome coronavirus (MERS-CoV)..

Click on image to enlarge.
Do regions that host transient high concentrations
of MERS-CoV (or a related virus)-positive animals play a
key role in the sporadic and geographically widespread
human infections?

This article in the Saudi Gazette was referred to me by a kindly commenter to a recent MERS-CoV post I wrote 24-Jan here.

It notes that August (through to December - see Ref #5 below for a lot of info on dates) is the date-harvesting season. When I received this comment, new MERS-CoV case announcements had ceased; a lull which, as I also wrote on the 24-Jan, I could not understand given that there had been no publicized steps to interrupt any type of transmission chain. 

Now we are seeing some publicized cases again, but it's clearly out of sync with date picking season. Nonetheless, I thought it might be worth looking at regional activities that may gather potential animal sources/vectors and humans, together , possibly addressing links in my disease acquisition scheme above; in particular links between dates, bats, camels and humans. No baboons this time around (I'm expecting a Tweet).

Click on image to enlarge.
Buraidah is located slightly north of 
central Saudi Arabia.

Buraidah (Buraydah) hosts the world's largest date festival in August/September and the Qassim date markets are a feature of the region as is agricultural in general.

Buraidah (population >600,000) is the well connected capital of Qassim Region (see map to the left). Qassim region is described as having plentiful water and, clearly, lots of palm trees as well as other fruit trees and wheat. 

Something else Buraidah has going for it? The world's largest camel market. Those are the beauties that seem to frequently have antibodies to the MERS-CoV (or a very similar virus that probably isn't any known coronavirus but reacts really specifically in MERS-CoV antibody-detection assays designed to detect the MERS-CoV but mainly in animals localized to the Arabian peninsula where most human MERS-CoV cases have been documented). Antibodies indicate past (about 1-2 weeks or more usually) exposure to replicating virus, with or without overt signs of disease in the host.

Recently there was also the the "Palm and Tree Date Festival" in Riyadh (15-16 Jan) and the "King Abdulaziz Award for Camels Beauty Contest" (26-Nov to 4-Jan) in Hafar Al-Batin.

I've made brief mention of the possibility of dates having a role in transmission (ingestion, self-inoculation or perhaps aerosolizing virus off bat-contaminated dates during their preparation?) previously here and here.

While this is all speculative, these latter events coincide a little with a small uptick in noted, and "social media suggested", MERS-CoV human cases.

So here's a speculative story:

• A nexus point, like Buraidah, with its central, well-connected location (transportation-wise) serves or once served as an "inoculation station" for susceptible camels exposed to infected bats
• Bats may be more reproductively active or in greater numbers because of higher concentrations of flowering insect-attracting date palms and other fruiting orchards in this region/at certain overlapping times (not actually sure if there is overlap)
• Camels are brought in, sold and then return to herds all over the region. 
• During their time in the markets, some camels become infected with the bat MERS-CoV and go on to infect their herd
• Rarely, humans in close contact with their camels also get infected (it does happen - Ref#7) 
• Rarely, some infected humans infect other humans
• Rarely/frequently (unknown proportion) infected humans become severely ill and "show up" as hospitalized cases who get tested for MERS-CoV. 

As I said, twice, its all speculative and as also I've said, infection events are pretty rare. If nothing else, that bulleted list may address the geographically widespread and rare nature of human case distribution to date.

End of speculation. For now.

Sources..

1. Saudi Gazette story
   http://www.saudigazette.com.sa/index.cfm?method=home.regcon&contentid=20130819177328
2. Date markets
   http://www.youtube.com/watch?v=QJDBDmziikY
3. Tourist information
   http://sauditourism.sa/en/About/Pages/k-Cities.aspx
4. Events and festivals
   http://sauditourism.sa/en/Events/Pages/default.aspx
5. 2012 Al-Rasub article on date festival
   http://www.alrasub.com/ksa-qassim-hosts-worlds-largest-date-market/
6. Many, many date details
   http://postharvest.ucdavis.edu/files/71533.pdf
7. Clinical course and outcomes of critically ill patients with Middle East respiratory syndrome coronvirus infection
   http://annals.org/article.aspx?articleid=1817260

Middle East respiratory syndrome coronavirus (MERS-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 MERS-CoV, or MERS-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 MERS-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 MERS-CoV ppNT test. No other animal sera reacted in this test
• 96% of camels had MERS-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 MERS-CoV microneutralization test (MNT). High levels of calf antibody specific to BCoV did not block MERS-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 MERS-CoV (or a close relative)
• Cows did not have any sign of MERS-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 MERS-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 MERS-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 MERS-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 MERS-CoV but, in additional testing, the sheep sera were not able to neutralize infection by MERS-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 MERS-CoV RNA in the faeces hinting that there was not an active infection at the time of sampling. This last point assumes that MERS-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 MERS-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 MERS-CoV acquisition at or very close to birth, or simply remaining protective maternal antibody. Perhaps camel farms and farmers 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 MERS-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 MERS-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 MERS-CoV
• MERS-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 MERS-CoV PCR positive using upE, N and ORF1a assays
• Sequencing of a fragment of Spike gene yielded 100% identity with other Saudi MERS-CoV sequences; sequence only differing by 1 base from the original isolate, MERS-CoV/EMC.
• All camel sera were antibody positive using an immunofluorescence test on MERS-CoV/EMC-infected cells
• There was no "direction" to the acquisition of MERS-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 MERS-CoV infection in camels. No positivity was found from gastrointestinal samples. Despite no isolation of infectious MERS-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 MERS-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 MERS-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 MERS-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.