Parechovirus infections in babies in New South Wales, Australia

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Rhinovirus (RV) transmission by aerosol: does it happen or is transmission solely by hand-contact and self-inoculation?

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Stuff from the literature: very SARS-like coronavirus in Chinese horsehoe bats...

The smoking bat for SARS-CoV?

Xing-Yi Ge and colleagues from China, USA, Australia and Singapore described some new severe acute respiratory syndrome (SARS)-like coronaviruses (SL-CoVs) in bats, publishing in Nature last month.

These discoveries were especially notable (not that any new virus discovery isn't) because they displayed more "SARS-like" properties than many earlier so-called SARS-like CoVs. One could grow in the same line of lab cells and also in human cells, it could be visualized by electron microscopy and it could use the same receptor as the SARS-CoV (angiotensin converting enzyme II; ACE2) . Plus, they were genetically very similar.

The bat species was confirmed by gene sequencing to be Rhinolophus sinicus, family Rhinolophidae; the Chinese rufous horseshoe bat.

Throat and faecal samples (anal swabs and faeces) were screened using RT-PCR with primers towards the conserved RNA-dependent RNA polymerase region (RdRp) and new primers were designed to detect other regions of any discoveries. 27 of 117 samples were CoV POS and had sequences determined.

Two novel (and 5 previously identified) SL-CoVs, each with a 29,787+ base pair RNA genome and sharing 95% nucleotide identity with the Tor2 strain of the SARS-CoV which is higher than previous SL-CoVs from China. The receptor binding domain (RBD) of the new CoVs shared 85-96% amino acid identify with the SARS-CoV. These were called:

1. RsSHC014
2. Rs3367

Vero cells were used to attempt growth of SL-CoV virions that were first concentrated from samples. This succeeded for one sample, a variant of Rs2267 (99.9% nucleotide identity with Rs3367) and they named this isolate SL-CoV-WIV1. This success is something that hasn't been achieved with the majority of recently identified bat CoVs.

WIV1 also grew, although less efficiently, in:

• human alveolar basal epithelial (A549) cells
• pig kidney (PK-15) cells
• R.sinicus kidney (RSKT) cells

...but not in...

• Human cervix (HeLa) cells
• Syrian golden hamster kidney (BHK21) cells
• Myotis davidii kidney (BK) cells
• Myotis chinensis kidney (MCKT) cells
• Rousettus leschenaulti kidney (RLK) cells
• Pteropus alecto kidney (PaKi) cells

So we have much more convincing evidence that the SARS-CoV is likely to have originated from a bat.

h/t to @MERS_inSAUDI

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

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

The three camels which have tested positive with #MERS in #Qatar were detected in a barn linked to two confirmed human infections
— WHO (@WHO) November 28, 2013

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

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

@MackayIM working on it. Qatari's are fast, samples came in last weekend, but yes, confirmed
— Marion Koopmans (@MarionKoopmans) November 28, 2013

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

@arambaut @mackayim Yes, but requires sufficient sequence of sufficient quality. We are working on it
— Marion Koopmans (@MarionKoopmans) November 28, 2013

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

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

@arambaut @mackayim Yes, but we DO need some time. We are 4 days into the labwork. ;>)
— Marion Koopmans (@MarionKoopmans) November 28, 2013

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

@MarionKoopmans @mackayim Indeed - I don't think people realise how much work it is to sequence these (and how much work to get good seqs).
— Andrew Rambaut (@arambaut) November 28, 2013

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

@MackayIM @marionkoopmans Look for polymorphisms that are not fixed in the putative donor but fixed in the recipient...
— Andrew Rambaut (@arambaut) November 28, 2013

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

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

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

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

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

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

New influenza A(H7N9) virus case in

Chinanews reports a new infection by H7N9 today.

A 57-year old male was confirmed as positive 27-Nov and is in hospital in Hangzhou, the largest city in Zhejiang province. Zhejiang province was the hot spot for H7N9 earlier in the year.

This is the 141st case of H7N9 which emerged in 2013 in south east China. Most of the recent cases have been in Zhejiang.

Clustered camel coronavirus cases...

Adding significant weight to the camel-as-vector hypothesis, stories here,  and here are being reported of a collaborative study between the Qatari Supreme Council of Health, the Qatari Ministry of Environment, the Netherlands’ Health Ministry’s National Public Health Institute, the Erasmus Medical College and the World Health Organization (WHO) that have identified 3 camels that are positive for the MERS-CoV.

The camels were part of a a farm herd of 14 asymptotic animals that have been linked to 2 previous MERS-CoV cases in Qatar. 

h/t @Crof, @MERS_inSAUDI, @HelenBranswell

MERS by the numbers: monthly MERS

Click on image to enlarge.Cases (numbers on the y-axis; left) including
deaths (green) and fatal cases (red) are
shown for each month (x-axis, bottom) of
2012 and 2013. This includes 68 deaths
and 161 total cases. The Hajj occurred
in October of each year.

These two charts show the number of cases (including deaths) and the number of deaths by month, split between the 2-years we've known the Middle East respiratory syndrome coronavirus (MERS-CoV) to have existed.
The charts are only as good as the public data they are based on but they give a good idea of what's been happening and what is happening. Cases are not declining [a slow moving epidemic ;) ]

Numbers are ridiculously small in 2012 (overall really) to conclude anything much but it does look like more deaths happen toward April while more cases occurs around September. I Doubt that would be statistically significant though - just something to watch over time.

MERS-CoV by the numbers: recent weekly case activity...

Click on image to enlarge.

Confirmed MERS-CoV cases including 

fatal infections (green) and deaths (red) 

each week. Case numbers are listed on the 

y-axis (side), days of each week  along 

the x-axis (bottom). Case dates are 

derived from announced date of onset 

but if absent, on the date of reporting. 

Deaths are listed by date of death.

This follows on from my previous post (you can track links to earlier weekly charts) about lab-confirmed Middle East respiratory syndrome coronavirus (MERS-CoV) cases, plotted by week.

Approximately 85% of all cases have come from the Kingdom of Saudi Arabia. Around 63% of all cases with sex data are male (1:1.73, M:F). Among the fatal cases with data it's 75% male (1:3.06, M:F).

I've added in some previous charts because as the new cases and case details appear, so do the placement of the cases alter slightly. Hopefully, with WHO doing such a good job in providing details, these graphs will solidify and we can move on in the next post. 

I've marked in the Hajj week and the 14-day outer limit of the incubation period. Nothing much to see from that; no spike in cases, even after time has passed to allow the case data to catch up.

My tally suggests 161 cases (still awaiting the Spanish case to be confirmed or not) with 68 deaths, a proportion of fatal cases sitting at 42%.

 

A couple of things stand out to me from these charts...

1. What is the lag between illness onset and MERS-CoV case announcement like? For example the recent 37-year old man who died was reported on 20-Nov, but became ill 9-Nov. Obviously there is time required to reach hospital (13-Nov) and then be tested and re-tested [confirmed] but this case died 18-Nov and was not reported as MERS-CoV case for 2-days. The case before that, a 65-year old man became ill 4-Nov, was in hospital 14-Nov and was reported 19-Nov. Before that the 73-year old woman became ill 13-Nov, hospitalized 14-Nov, died 19-Nov and was also reported 19-Nov.
   I presume that this indicates there is no active MERS-CoV PCR screening of influenza-like illness, but rather for  in the Kingdom of Saudi Arabia?
2. There have been 5-8 fatal cases per month since June and 16-25 cases per month in total. However, with that lag, there may be more to come from November. 

In particular, point #2 makes me wonder if the KSA is settling in to stable (albeit very small numbers of total cases) transmission or acquisitions of MERS-CoV?

The MERS-CoV case slience has fallen lifted thanks to the WHO

Well, apart from a blatant Dr Who references, this post is dedicated to providing a huge portion of back-patting for the great job the World Health Organisation (WHO) have done on their recent Disease Outbreak News reports (see yesterday's here). I now have a new colour code in my Excel sheet that indicates "confirmed by WHO" - because its become worthwhile doing that. 

The current approach to detailed MERS-CoV News posts continue to hold the sort of detail I'd hoped for. 

Also, congratulations must go to the Kingdom of Saudi Arabia's Ministry of Health (KSA MOH; and at other times, other MOHs from the region) for providing the WHO with these details. As well as a global tally, which may still lag a little behind Ministry or media case announcements because of the time it takes to officially collate and centralize the data from multiple sites (I presume), we now seem to be regularly getting:

1. Sex
2. Age
3. Occurrence of animal exposure
4. Presence of comorbidities
5. Date of illness onset
6. Date of hospitalization
7. Date of death if a fatal case
8. Region the case occurred in (still a bit patchy)

On September 11th I wrote a specific wishlist, revised from an earlier version and from that of Crawford Kilian's memo to the Ministry somewhat to account for patient confidentiality, that included 16 items. The WHO's efforts address most of the items on that list. Well done and keep up the good work!

My full wishlist, with some amendments, is below. I still feel these extra few bits (in blue) of information would be useful, especially the unique code to globally track cases and some detail on what may have worked to help support the infected patients course. 

1. A unique, continuous identifying code of KSA cases
2. Sex of case
3. Age of case
4. Possible exposures to animals and other human contacts
5. Occurrence of comorbidities
6. Date of illness onset
7. Date of hospitalization
8. Date and type of laboratory testing
9. Date of death if a fatal case
10. Region the case occurred in 
11. Date of release from hospital
12. Treatments or management

In the meantime, FluTrackers curates the world's best, and most rigorously checked, MERS-CoV case key. Such a stable Rosetta stone of MERS-CoV cases is essential. It provides the world with a solid, unchanging and reliable set point for each case around which our discussions and ideas can revolve. Not the kind of stone out of which a Weeping Angel is made - one that shifts menacingly every time you blink or look away - but one with the dependability of a Dalek's determination to "Exterminate", or of a Sontaran's desire for a good fight.

Research papers come and go and their conclusions change like a shape-shifting Zygon, but a permanent list of public information on MERS-CoV cases over time is the gold standard against which they can be given important context. 

The more information we can rely on during times of emerging viral outbreaks (or slow-moving epidemics), the better prepared we are to get in front of them, contain them and not be unnecessarily scared by them.

No symptoms but still shedding virus?

Click on image to enlarge.
A stylized trace of the temperatures during a PCR cycle.
D-denaturation, when primers and double-stranded
DNA (dsDNA) are reverted to single strands of DNA;
A-annealing, when primers bind to their complementary
target and DNA re anneals to form dsDNA; E-extension,
when the DNA-dependent DNA polymerase enzyme
finds a primer, binds to it attached to a strand of
template  and makes the complementary strand.
Feel free to use. Please cite this website and
Dr I M Mackay as illustrator.

One of the many questions that remain unresolved for MERS-CoV is whether a human who is PCR-positive for the virus, but does not show signs or symptoms of being sick, can spread that infection on to other humans - or animals for that matter.

Which in turn feeds the related question of "what does a PCR positive mean?"

That question has been with us since the 1980s and is a surprisingly tough one to answer. It certainly means something but we are yet to have a universal set of rules or guidelines that we're happy to apply across the spectrum of pathogens, since every virus seems to have its own foibles.

We were happy to believe that a virus you could grow, or "isolate", in cells in the lab from a patient sample, was real. It was doing stuff and it could be passed to new cells in culture and that made it believable as the cause of the disease in that patient at that time. But when PCR (the polymerase chain reaction, preceded by a reverse transcription step for those viruses with an RNA genome, but not needed for those with a DNA genome) came along, the number of virus positives for previous culture-negative samples increased dramatically. This was due to:

• Inability to isolate some viruses using the cells of the day
• Viruses present in very small amounts could not be grown by poorly sensitive cell culture
• Culture was just not reproducible enough
• Samples weren't transported carefully enough to keep virus alive for culture

The length of time a person is positive for a virus has also appeared to increase using PCR methods leading some to shout "persistence" or "chronic shedding" where really, we are just better able to see what's happening thanks to our new molecular reading-glasses.

Click on image to enlarge.
Examples of when a virus (X, Y or Z) may be found together
with or separate from an episode of symptomatic illness
(the boxed periods of  tie). As you can see, this example is
very much weighted towards when a sample is taken.
3 testing scenarios are shown. (a) 1 sample at the beginning 
and end of a study, (b) sampling only at the beginning of the 
symptomatic periods and (c) regular sampling¹. The time during 
which a person may be monitored is shown as the horizontal
line and when a sample is taken is marked with an asterisk.

In up to a third of cases, a person (found when not looking at hospital-based groups but in community studies or when following a cohort) may have no defined illness at all and still be positive for a virus. Heresy!!

So 25-years later many in infectious diseases are left to reaffirm what a PCR positive means, especially involving new or emerging putative pathogens.

For the Middle East respiratory syndrome coronavirus (MERS-CoV) we may be able to draw some conclusions from a viral relative; the severe acute respiratory syndrome (SARS) CoV, did during its short time in humans back in 2002-2003.

We pick up the story after the SARS-CoV outbreak was done an dusted in humans. Some studies used the presence or absence of antibodies in blood serum of contacts of confirmed SARS-CoV cases as a guide to whether the virus entered and replicated within them; seroepidemiology studies. The contacts do not appear to have been screened using RT-PCR; also the current situation with MERS. 

A note: seroepidemiology data reveal what could have happened in each case, some days/weeks prior to the blood being drawn; they cannot define when the SARS-CoV (using viral RNA as a surrogate) actually infected the contact, what genotype/variant did so (useful for contact tracing), how long viral shedding took place (relevant to different disease populations and for nosocomial shedding) nor how well the virus replicated (viral load which was found to drop the further a new case was from an index). 

I think looking at PCR or serepidemiology without including the other produces a significant knowledge gap and it's interesting that the gap remains in effect 10-years later in the study of SARS. Perhaps MERS-CoV is just like SARS-CoV and, as we see below, no symptoms=no infection=no onward transmission. Gut feelings don't really tick the box in science though.

Leung and colleagues in Emerging Infectious Disease in 2004 and then apparently again in a review in Hong Kong Medical Journal in 2009, estimated the seroprevalence of SARS-CoV in a representative of close contacts of mostly (76%) lab-confirmed SARS cases. 

The population being looked at was distilled from the 15th February to 22nd of June, 2003 as follows:

• 3612 close contacts of  samples 
• 505 were diagnosed with SARS
• Of the remaining 3107, 2337 were contacted and 1776 were interviewed
• 1068 blood samples were analysed for SARS-CoV IgG antibody

Only 2 of the 1068 (0.19%) had an antibody titre of 1:25 to 1:50. Most recovered SARS cases had titres of ≥1:100. Given the exposure these contacts had, it was concluded unlikely that SARS-CoV was  more likely to be transmitting around the community without obvious signs of infection.

Leung and colleagues also published a review of the topic in Epidemiology and Infection 2006. They concluded an overall SARS-CoV seroprevalence of 0.1% overall with 0.23% in healthcare workers and contacts and 0.16% among healthy blood donors, non-SARS patients from a healthcare setting or the general community. Other interesting bits of information from this review include:

• 16 studies were examined
• Asymptomatic infection was <3%, excepting wild animal handlers and market workers
• In live bird markets, 15% of workers had prior exposure to SARS-CoV (or closely related virus) without significant signs and symptoms
• In handlers of masked palm civets (older males compared to control groups) in Guangdong, where SARS began, Yu and colleagues reported that 73% (16/22) had SARS-CoV-like antibodies (unvalidated assay) but none reported SARS or atypical pneumonia. Which leaves room for milder illness, and larger studies.
• Prevailing SARS-CoV strains almost always led to symptomatic illness

So what has been done for MERS-CoV? We have some camel seroepidemiology studies which I've previously described here and here. Human studies?

1. In the study that found MERS-CoV-like neutralizing antibodies in Egyptian camels, no human sera from Egypt (815 from 2012-13 as part of an influenza-like illness study in Cairo and the Nile delta region) nor any from China (528 archived samples from Hong Kong) were MERS-CoV neutralizing-antibody positive.
2. No sera or plasma from 158 children admitted to hospital with lower respiratory tract disease or healthy adult blood donors were MERS-CoV neutralizing-antibody positive. Small sample and the ill children may not yet have mounted a relevant antibody response if they had been infected by MERS-CoV.

Work like that mentioned for SARS largely remains to be done for MERS. The SARS-CoV studies provide a useful model on which to base such studies and the World Health Organisation recently provided a detailed approach for seroepidemiology studies seeking to test contacts of laboratory confirmed MERS-CoV cases. 

What does a positive PCR result mean in an asymptomatic MERS-CoV case? Still can't answer that. Are contacts seroconverting as an indication of MERS-CoV infection? Still can't answer that. How many mild or asymptomatic MERS-CoV infections are there beyond contacts of lab-confirmed cases? Still can't answer that.

Once we can rule out occult community transmission - we can tick another concern off the MERS-list.

Further reading...

1. Observational Research in Childhood Infectious Diseases (ORChID): a dynamic birth cohort study
   http://bmjopen.bmj.com/cgi/pmidlookup?view=long&pmid=23117571
2. Middle East respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility
   http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(13)70304-
   9/abstract
3. Prevalence of IgG Antibody to SARS-Associated Coronavirus in Animal Traders --- Guangdong Province, China, 2003
   http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5241a2.htm
5. Viral Load Distribution in
6. SARS Outbreak
http://wwwnc.cdc.gov/eid/article/11/12/pdfs/04-0949.pdf