Editor's Note #10: VDU takes a break...

Hi All,

Just to let you know that Virology Down Under is having a rest for a few weeks. 

I'll be away from my desk for some of that time so posts will be rare or absent.

Please keep an eye on on my fellow infectious disease watchers. Each of these sites has its own "personality" and all are worth checking out regularly. You can also follow them via Twitter which is a great way to see new post announcements and gain other insightful information gems. They Big3 are:

• Crawford Kilian (who recently made his own DIY blogging list which I highly recommend)
• Mike Coston's Avian Flu Diary
• FluTrackers
• CIDRAP

Since MERS-CoV has been in focus on VDU in recent weeks, if for no other reason than because it needs as many hands helping you to wade through the data disaster as possible, here are my Hypotheses for the next few weeks:

• No MERS-CoV pandemic, although cases numbers will rise as the hajj pilgrim numbers peak
• Prospective studies to find out what's happening with MERS in the wider community of the Kingdom of Saudi Arabia will not be reported
• Transparent and comprehensive coronavirus communication will have become the norm. Well, hypotheses are for testing aren't they?

Apart from lots of other posts that have not yet passed their use-by date (there are 250 now, many with at least the 350+ words of this one), there is the main Virology Down Under site which is slowly being overhauled to read more clearly. I've also got a bunch of new post ideas queuing up and I expect that my pile of H7N9-papers-to-be-read (like my pile of papers-to-be-written) will be even more daunting in a few weeks, than it is now. 

Since more of you seem to visit with each passing week, the viral views, news and...something that rhymes with news, will keep being posted in the future.

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Molecular epidemiology of Middle East respiratory syndrome coronavirus (MERS-CoV)

And a newcomer to the MERS-CoV birthday celebrations! What great timing to have this released today.

The Lancet paper accompanying those recent partial and full genome sequences has been released form its cage. It's a collaborative effort by authors affiliated with the Global Centre for Mass Gatherings Medicine (Ministry of Health Saudi Arabia), Welcome Trust Sanger Institute (United Kingdom) and many other locations.

A few highlights of the largest MERS-CoV molecular epidemiology study to date, which includes some great transmission figures and trees (hat tip to the graphics people at Lancet):

• Genetic diversity analyses 3 distinct genotypes were identified from human cases in Riyadh 
• The Al-Ahsa hospital cluster may have had more than 1 viral introduction
• Other clusters and standalnone cases can be representd as distinct genoytpes of MERS-CoV, posisbly indicating multiple different virus acquisitions from different sources
• Predictive evolutionary analysis suggests an evolutionary rate of 6.3x10^-4 substitutions per nucleotide site per year suggesting a time to the most recent common viral ancestor was July 2011 (ranging from July 2007- June 2012). So we can rule out my harebrained "What If.." MERS-CoV was an endemic virus that we had only just discovered
• This evolutionary rate of change suggest more than 1 jump from animal to human was the cause of the outbreak. Unlikely to be just a single introduction followed by human-to-human transmission across Saudi Arabia and beyond. This also reduced any possible R₀ value (the number of cases that 1 case generates, on average, over it's period of infectivity) since transmission events were not a continuous chain but likely to be multiple different spillovers
• The rates also suggest it's been substantial period since these viruses shared a common ancestor - so an intermediate host is still a likely culprit for spillover into humans (ongoing studies are examining camels, bats, goats, sheep, dogs, cats, rodents and others - no baboons?)
• Contact with goats and camels has been reported in some cases and we know that camels from Oman and Egypt have antibodies to a MERS-CoV-like virus
• A particular change in the Spike protein that may impact on its role as a site for enzymatic cleavage (by endosomal furin or trypsin-like proteases) should be further examined (codon 1020; all recent MERS-CoV S protein differ here from the EMC/2012 strain of MERS-CoV exported to the Erasmus Medical Center researchers).

The authors conclude it is imperative that a better understanding of the exposures causing these spillover events be identified.

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In a nutshell: Why MERS-CoV data from Saudi Arabia is often limited....

From a Q&A with Dr Ziad Memish, Deputy Health Minister for the Kingdom of Saudi Arabia written up by Ellen Knickmeyer of the Wall Street Journal...

�I know that there are some newspapers and news agencies requesting more detailed information. As a public-health officer, I feel strongly this is not acceptable. The news media is not the place to detail the critical information about patients or how many people in the same family got infected, or where they live.

Speaks for itself really.

It does somewhat miss the point of what many have been asking for (including me). The data would obviously have to be deidentified. A standard practice in for research epidemiology publications and a frequent (usual?) requirement by ethical panels that approve your projects. At least in some States. That would mean leaving out patient and family names (as has been happening to date with MERS, but not so much with H7N9 where too much private information was shared), hospital names and any household addresses.

His subsequent comments outline Dr Memish's view of a minimal publicly available dataset...

What needs to be given to the public is positive case, the age, the sex, the location and if there�s anything unusual about increased spread or a new event that has not been reported in the past.

This doesn't explain why it take so long to hear of a MERS-CoV death when the KSA has a daily-updated (at 5pm!), coronavirus-specific, public health announcement website. Presumably testing is slower than we thought or samples are not being collected for MERS-CoV testing often enough? Who knows? It also doesn't explain why data content varies from post-to-post.

Despite this, and I agree that patient details should be kept private, my list of details to help out global public health officials, amended from an posted earlier, is...

1. A unique, continuous identifying code specific to this emerging virus
2. Sex
3. Age
4. Possible exposures Occupation
5. Co-morbidities
6. Date of illness onset
7. Town of illness onset [for internal and collaborative investigation]
8. Town of acquisition acquisition [for internal and collaborative investigation]
9. Date of hospitalisation
10. Type of laboratory testing
11. Date of laboratory confirmation
12. Date of death
13. Date of release from hospital
14. Treatments/management
15. Town of treatment [for internal and collaborative investigation]
16. Relationships to any other cases

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Happy 1st birthday Middle East respiratory syndrome coronavirus (MERS-CoV)

A coronavirus schematic. The spiky bits give the virus
its name(corona=crown) and represent the
receptor binding, antigenic Spike protein. 

...I can remember when you were just a novel little thing.
How you have grown young prince and how clever of you to emerge in a Kingdom of all places (corona=crown, named for it's spikey appearance). You've certainly garnered attention worthy of a King given the relatively few cases of disease you gave been associated with in the first year we've known of you.

It was September 20th when Dr Zaki 1st alerted the world to the death of a Saudi man due to what looked to be a new coronavirus (CoV). Today we have over 135 cases 58 deaths (43%).

I've previously covered Zaki's disocvery and the problems posed for the Kingdom of Saudi Arabia (KSA) by the way in which he announced that discovery, apparently without the Ministry of Health's (MOH) foreknowledge. The way in which the sample was exported from the KSA without their prior consent was also problematic for them.
Soon after we heard of it, we had virus-detection assays with which we could seek out new cases. Were they used as they might have been in the days of the SARS-CoV? Nope. And there still seems to be only a single laboratory in KSA testing for MERS-CoV (despite reports of 3), with Dr Abdullah Al-Aeeri (a director of hospital infection control) claiming a 72-hour reporting turnaround time.

Is there an antibody detection assay that has been validated using a panel of known positive sera? Nope. There are some innovative antibody-detection methods around but why do they only include a single positive control? Is there no collaboration at all? Why is the KSA not leading the charge to develop these diagnostics and to hunt for an animal host? Why wait on advice from external organizations to screen samples? Why has the necessary testing capacity not been built well before now? Is it to do with that pesky material transfer agreement? I hope not because there is little evidence for that being a real block to anything from a public health standpoint.

At least we have some new MERS-CoV sequences to celebrate the birthday with. Although they and the 9 preceding them represent less than half of the relatively small number of cases described to date. Why can't the typing region sequences be released? That should really be part of the diagnostic process. Okay, those may not inform us about the evolution of key regions of the virus but they do confirm it is the strain we know. Why not focus on full or subgenomic Spike gene sequences? They might be a better sentinel for keeping tabs on MERS-CoV change over time.

Most of the detail about MERS-CoV and cases of MERS has come through the peer-reviewed scientific literature. That is pretty normal for respiratory viruses that are not notifiable. But it's generally a slow medium. Is MERS infection a notifiable disease? It is in some countries (e.g. the US and New Zealand), but is it at the epicenter of the outbreak, the KSA? I'm not sure. It's not obviously stated as such anywhere I looked on the KSA MOH website.

The World Health Organization politely notes:

WHO encourages all Member States to enhance their surveillance for severe acute respiratory infections (SARI) and to carefully review any unusual patterns of SARI or pneumonia cases. WHO urges Member States to notify or verify to WHO any probable or confirmed case of infection with MERS-CoV.

How's that been working out? In a nice summary of the lack of communication, Helen Branswell and Declan Butler highlight that, as usual, everyone  who was asked agreed that it's not working out well at all. In fact it's pretty woeful. And to add to matters, the latest WHO Disease Outbreak News (DON) takes the form of a summary of 18 "new" cases; no extra or confirmatory detail to be had from it. SO the KSA MOH is now the source for detail.

If we were talking about wanting more data on the monthly proportion of rhinovirus infections, the KSA would be justified in saying that the world doesn't need to know (I'd like to but that's my thing). 

If we were talking about influenza, then there are plenty of international public health sites publishing these notifiable data on the internet; here's Queensland, Australia's for example.

But we're talking about an emerging disease which kills half of the people it infects, is caused by a novel virus for which no host is known, which transmits between people in a way we don't yet understand, which is shed from ill (or well) people for an undefined period of time (if at all), which remains infectious in the environment for who knows how long, which jumps to other countries, which may only cause severe disease in those who are already ill with another disease, which may be endemically spreading within the community as mild or asymptomatic infections, for which there is no vaccine or proven antiviral therapy available..I'd say it's a no-brainer that at the very least the WHO deserves regular and detailed updates of what's going on. Reading between the lines, that does not seem to be happening even behind closed doors.

The mass gathering of pilgrims known as the Hajj is fast approaching. This may trigger a large increase in MERS cases or, in the worst case, a pandemic. I personally believe it won't go that far. We shouldn't forget is the 2nd Hajj for MERS. But perhaps the virus is much more widespread than it was in October 2012. But without testing data, we can only guess.

So, it's your 1st birthday MERS-CoV. But instead of wishing you a happy birthday you opportunistic, spiky little killer, I'm wishing Dr Zaki well and congratulating him on co-parenting the birth of this novel coronavirus. Going by what we've seen to date, his actions may have been the only way we would have ever heard of this virus otherwise.

And, as noted previously, but not given much air to in the above rant (thanks to @MicorbeLover for straightening me out)...

It's very sad that there are real people in these numbers who have died from MERS. You may have noticed that I try and stick with the cold number-crunching aspect of these outbreaks. It's not because I'm a heartless b&^$# but because that is not what this blog is about. That and my editorialisation and expositionary writing consume what little time I have spare. But I don't feel that I have enough information to make any other comments about these or any other lives lost to infectious disease. I personally feel that any unexpected and acute loss of life (if I had to scale loss of life) is the worst kind of loss; it's a waste of potential, a source of great sorrow for all involved and it's something we should all strive to prevent, if we can. I know that's not much to convey, but it's all I can offer from my kinda comfy chair in Brisbane. 

The MOH says it better in anyway; May Allah have mercy upon the deceased.

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MERS-CoV detections over the past 6-weeks: 38 cases, 13 deaths.

Click to enlarge.
Laboratory confirmed MERS-CoV
cases (including deaths; green) and
deaths (red) by day (bottom, x-axis), per
week. Number of cases on
the left hand (y) axis peak at 8/week

Updating the Middle East respiratory syndrome coronavirus (MERS-CoV) graphs from just over 2-weeks ago and adding in recent weeks, we see how the cases have been accruing. 

Last week was a big week; 42% of cases from the past 6-weeks occurred then.

There are some differences in some charts when comparing to the earlier post with some of these; put that down to updated dates due to extra data being released and some cases being reported the week(s) after they occurred. I'll keep updating this figure. Those changes may keep happening.

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3 new MERS-CoV cases, 2 deaths tally at 135

Three new cases were reported from the (Arabic, not English yet) Kingdom of Saudi Arabia (KSA) Ministry of Health (MOH) website and details of the entire list can be found on FluTracker's website of confirmed cases. The tally sits at 135 confirmed cases. There may, or may not, be a chunk of other cases ("probables") but they have not been confirmed.

Today's cases all had comorbidities and were (FT-FluTracker's case numbering system):

• FT#133: 75-year female (75F), contact of a case in Medina, death
• FT#134: 35M contact (Mkhalt=male contact; hat-tip Helen Branswell) in intensive care
• FT#135: 83M, Riyadh, death

Declan Butler has a nice article preempting the MERS-CoV birthday "celebrations" that is good summary, as was Helen Branswell's article earlier in the week, These show just how obvious the poor progress on some key aspects of MERS-CoV research have been. Is the slow progress due to a system that does not utilize its own Universities and research infrastructure  does the KSA not have such capacity? I don't know. But the lion's share of the MERS-CoV work does seem to await off-shore advice and direction. Principal research direction does not seem to originate from within the hot-zone of viral emergence and this may be a key difference between the rate of early progress in understanding the emergence of the MERS-CoV compared to that of SARS-CoV, H1N1pmd09, H7N9 or many other viral outbreaks.

Thanks to @HelenBranswell for input on sex of 83[M]

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17 new MERS-CoV sequences bind perfectly to frontline screening PCR assay for MERS...

Click to enlarge. The primers/probe are depicted as grey boxes.
If mismatches existed they would show up as horizontal black
lines within the grey box. No mismatches are evident.
The GenBank accession numbers are
shown on the left of this alignment of 17 MERS-CoV
sequences.

Only 17 of the 45 sequences seem to include the region covered by the upE laboratory assay I just posted about in the WHO laboratory testing update but of those, the forward and reverse oligonucleotide primers and the probe all bind without any mismatch.

While that may sound like an obvious statement considering that these viruses were probably detected using that assay it isn't.

The new MERS-CoV sequences were determined using using unbiased 2nd generation high-throughput sequencing technologies that did not rely on these primers to generate them. So we are now able to check and see if there are any nucleotide changes at the target sites for the primers and probe, that would reduce the efficiency the assay.

There are no such oligonucleotide mismatches between primer and viral genes among those 17 sequences, which is good news for that assay's continued usefulness.

Built to last eh?

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MERS-CoV WHO testing guidelines: September update

The World Health Organization has updates its laboratory testing guidelines. They can be accessed here.

Some key points:

• Lower respiratory tract samples are recommended since there are data to support higher viral loads (better detectability using PCR) from these samples
• The upE real-time reverse transcriptase polymerase chain reaction (RT-rtPCR) assay of Corman et al is considered highly sensitive and is recommended for screening
• The ORF1a screening assay is of equal sensitivity and the ORF1b slightly less sensitive than upE
• RdRp and N gene assay are suitable for genotyping
• Antibody testing assays are described and some seem to be very sensitive. none have been validated against a large panel of known MERS-CoV POS sera and result interpretation is still without consensus
• Laboratories should notify their local public health authorities as soon as they receive a specimen for MERS-CoV testing and all results should also be passed along to these authorities.
• Member States are asked to immediately notify WHO of initially POS laboratory results even before confirmatory testing is complete.

Pretty clear and straightforward really.

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MERS-CoV genomes on GenBank...[UPDATE]

Click to enlarge. A scale schematic of the first
MERS-CoV genome, EMC/2012.

45 subgenomic (the smallest is 361 nucleotides [nt]) to full length genome (only 13; >30,000nt) sequences of the MERS-CoV have been released onto GenBank ahead of a Lancet Infectious Diseases paper arriving in days. The GenBank accession numbers range from KF600612 - KF600656 and repsenst human cases form 2012 & 2013. 

Usually (and hopefully soon), I would get the entire batch using a search of KF600612:KF600656[ACCN] at http://www.ncbi.nlm.nih.gov/nuccore. They seem to have to be downloaded manually for now.

The list of Middle East respiratory syndrome coronavirus sequences with their date of collection (DOC) includes:

• KF600612, Riyadh_1_2012 ; 30,063nt
  DOC: 23-Oct-2012 
• KF600613, Riyadh_3_2013; 30,064nt
  DOC: 05-Feb-2013
• KF600614, Al-Hasa_10e_2013; 2,151nt
  DOC:02-May-2013
• KF600615, Al-Hasa_14a_2013; 6,673nt
  DOC: 08-May-2013
• KF600616, Al-Hasa_13b_2013; 3,787nt
  DOC: 07-May-2013
• KF600617, Al-Hasa_22a_2013; 2,102nt
  DOC: 09-May-2013

Most are from cases in Al-Ahsa (adding to the previous 4), as well as from Riyadh, Buraidah and Hafr-Al-Batin

In case you read otherwise, there are no obvious signs of recombination among these viral sequences.

Thanks to @Sarah_E_Smith1 for announcing location on GenBank ahead of the Lancet paper.

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The Rubik's cube of influenza A genes spins up a new lineage of H7N7

Click to enlarge. A (very) summary view of the latest
contributing influenza viruses that precedes the emergence of
human infections with influenza A(H7N9) virus
in south-east China in 2013.

Lam and a global host of collaborators, writing in Nature on the 21st of August, have identified a previously unknown influenza A(H7N7) virus line circulating in chickens. The authors indicate that more influenza viruses lurk among poultry and that active surveillance is needed. This report comes from testing 1,341 pairs of oropharyngeal and cloacal swabs and 1,006 faecal and waters samples from live bird markets (LBMs) in Wenzhou and Rizhao of Zhejiang province, as well as Shenzhen from Guangdong province.


In a complex alphabet soup of influenza A virus findings, the authors, sequenced 34 H7N7, 4 H7N9 and 19 H9N2 egg-isolated viruses but also found H7N2 and H7N3 in ducks. Animals tested were chickens, ducks, geese, pigeons  partridges and quail.

The authors note that rather than wild birds from Europe and Korea, the neuraminidase (NA or N) gene segment from H7N9 is more temporally related to those from H11N9 and H2N9 found in wild birds (wild water fowl, Northern shoveller and common teal) in Hong Kong during 2010-11 with links to domestic ducks in China prior to the H7N9 outbreak this year. Overall, domestic ducks proved to be an important mixing pot between wild birds and chickens.

And it's not just H7N9; the H7N7 found in chickens reminds us that the colours on the cubes are many and are in constant motion. These virus may become/may already be enzootic (endemic in non-humans) and so continuing exposures to live poultry in markets and backyards remains a continuing source of risk for new zoonoses.

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Age and sex morbidity and mortality from avian influenza A(H7N9) virus

Click to enlarge. The majority of cases of H7N9 that occurred
worldwide earlier in 2013. Taken from Virology Down Under's
H7N9 page.

In a study co-written by yours truly using a lot of data collected for Virology Down Under, Dr Joseph Dudley and I have just described, in the Journal of Clinical Virology, the age-specific and sex-specific morbidity and mortality from the avian influenza A(H7N9) virus outbreak earlier in the year.

We sought to highlight differences between H7N9 and another zoonotic influenza A virus, H5N1. The distribution of age and sex is notably different between cases of each virus in more distant countries (Saudi Arabia vs Egypt) as it is within the same country (see Cowling et al reference in the article's discussion). Such differences and patterns may be instructive for identifying specific risk factors for an outbreak and also serve to highlight that there are differences between outbreaks which, on the surface, might be expected to have very similar courses. 

Intriguingly, there were marked similarities between H7N9 and Middle East respiratory syndrome coronavirus age and sex case distribution.

We also published the term created here on VDU, the Proportion of Fatal Cases (PFC). A percentage defined as the number of currently known fatalities divided by the number of total lab-confirmed cases including fatalities, regardless of whether they are inpatients (hospitalized) or outpatients. It was created to avoid the need for a gauge of recovered cases (released from hospital) which is linked with use of the term Case Fatality Ratio.

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