Tracking MERS-CoV through time: a spikey problem

This morning on Twitter, Helen Branswell (@HelenBranswell) asked this question, with a comment...

How is this so? 16 months after the 1st known #MERS cases, there are only 9 viral sequences in the public domain. So much for collaboration.
� Helen Branswell (@HelenBranswell) August 6, 2013

So I thought a little perspective might be nice. 

The SARS epidemic had its origins around Nov 16th 2002, although the major activity started in Feb of 2003. 

• 64 human SARS-CoV genomes had been produced by September 2003 ([UPDATED:] see Science paper). That is by 317-days later, or 10-months, 13-days (perhaps less given that the genome sequences were possibly sequenced well before the paper was submitted e.g. late phase genome s seem to have been submitted to GenBank by July 2003). 
• For MERS-CoV we currently have 9 genomes at 505-days (give or take), or 1-year, 4-months.

Not that anyone needs to be reminded, but 80% of MERS-CoV cases come from the Kingdom of Saudi Arabia. The world is relying on them, or their collaborators, to turn the nucleic acid extracts used to define these cases (PCR-POSs hopefully kept in a -80'C freezer), into templates for gene or genome sequencing.

I personally don't believe we need to have complete genomes right now in order to fulfil the fairly urgent public health need to monitor the virus and notice if it changes, or is changing, or is not changing. These changes tell us whether the virus is still adapting or has settled in - perhaps having done so prior to this outbreak's indicator, severe disease. 

What else to use to track adaptation?

Perhaps the 4,000nt Spike (S) gene, or some smaller but suitably variable portion of it, could be a target for sequencing? 

Zhang and colleagues have data showing it could be used to track an animal coronavirus's adaptation to humans, through its 3 pandemic phases. This was done using phylogeny (a way to show how one sequence relates to another through time and space) of nucleic acid sequences and alignments of the translated version of these sequences. All we need is primer sequences that could be used to reliably amplify the S gene of the MERS-CoV. If anyone has those already perhaps they could publish them...if they haven't already. A very brief look at the 9 MERS-CoV genomes already shows some variety. Perhaps unsurprisingly, there is very little change among the 4 Al-Ahsa genomes; their collection dates are separated in time by 17-days.

This shows a schematic of the aligned Spike genes. The black lines within the grey boxes represent nucleotides that differ from the consensus. More differences are obvious in the earlier sequences. The oldest MERS-CoV isolate is at the bottom, the most recent, at the top (detailed below). See the full version here at VDU.

Interestingly, the phylogeny of the complete Spike genes looks  similar to that of the complete MERS-CoV genomes. However  its doe snot place the isolates in order of increasing time to the extent that the full genomes do. I also looked at a 900bp fragment of the 3' of the Spike gene - easier to amplify but a very similar tree to that of the complete Spike.

All 9 complete MERS-CoV spike protein genes (nt). Alignment in Geneious Pro, tree in MEGA 5.10.
Full version will be here at VDU.

All 9 complete MERS-CoV genomes (nt). The arrow indicates moving forward in time; the oldest MERS-CoV isolates at the bottom, the most recent at the top. Alignment in Geneious Pro, tree in MEGA 5.10.
Full version will be here at VDU.

So where does that leave us?

Adaptive pressures on the SARS-CoV drove its genome towards settling down in the late stage of the 3-phase outbreak (defined by the Chinese SARS Molecular Epidemiology Consortium), with changes in the Spike gene occurring before that. Complete genomes are clearly the gold standard - so I dial down that personal belief from earlier.

The Spike gene still seems a useful target for MERS-CoV too, although not as accurate at plotting the time of virus isolation as complete MERS-CoV genomes were in my example above. Still, it, or some part of it, is still of use as an early-warning system to alert us to viral change and it will prove easier to amplify by smaller or less genomics-focussed laboratories. Something we need to consider in order to get some information, which is far better than none.

While we've seen predictive modelling for the age of MERS-CoV, we don't actually know when the virus came to be or when it started spilling over to humans. More full genome sequences would certainly help address that question. And finding its origin.

However, perhaps we should make the trade off and use the 3' end of the Spike gene now, in an effort to keep some sort of eye on how the MERS-CoV is travelling? Anyone else have a good region that fits the bill?

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A detailed report of a Jiangsu H7N9 infection family cluster and the probable involvement of human-to-human transmission

Qi and colleagues describe in the British Medical Journal (BMJ), their detailed analysis of a likely human-to-human (h2h) transmission event by influenza A(H7N9) virus. The infection is likely to have traveled a 60-year old father to his 32-year old daughter, but not to any close contacts-1 "passage" of transmission. 

This event was previously defined as a family cluster, but the new BMJ report adds to the information published in the NEJM article by Li et al in April (see "Jiangsu Family Cluster", Figure 3).

The conclusion is that H7N9 likely spread to one other person but no further, in this instance, using these tests to define infection. As the accompanying editorial by Rudge and Coker notes, it's not surprising to see this happen. The media have become somewhat carried away in suggesting this is the first case of h2h transmission. That "honour" probably goes to the family of the first announced case, the 87-year old male from Shanghai and his family cluster. 

To be pedantic, this new BMJ analysis does not actually prove h2h transmission (hence the word "probable" in the title I suspect) highlighting just how hard it is to do that in the "wild" and after the transmission event has occurred. It is probable though. However, we know for certain that H7N9 can be transmitted between the 4-legged furry kind of animal (see related posts here, here and here).

Some key take home messages...

• 2 patients (60M and his daughter 32F) 13 samples of the environment were tested by PCR
• 60M, who had hypertension, developed respiratory disease March 8th and was hospitalized March 11th. He died of multi-organ failure and disseminated coagulation May 4th.
• An endotracheal aspirate (ETA) was H7N9 POS, but a throat swab was NEG
• No diarrhoea was linked to infection 
• 32F was an otherwise healthy developed symptoms March 21st and was hospitalized March 24th. She died of multi-organ and heart attack on April 24th
• Throat swab and ETA were H7N9 POS
• 3 viral isolates were able to be grown in culture and 2 complete genomes (8 segments each) were deposited onto, but not yet released from, GenBank
• The sequences from eh two human cases reportedly differ a little, mostly in the NS and NA segments whereas the environmental isolate's M segment showed the most divergence from the 2 human cases. The genome details are...
• KF034916-KF034923 - 60M (A/Wuxi/2/2013)
• KF034908-KF034915  - 32F (A/Wuxi/1/2013)
• KF150605-KF150612 - (A/Environment/Wuxi/1/2013)
• All samples were negative for other respiratory viruses
• The study did not include interviews with the 2 cases as they were too ill so some avenues of infection could not be excluded
• Both cases were treated with oseltamivir 3-10 days after symptom onset-the US CDC recommends commencement within 48-hours of symptoms, which can be difficult to achieve as patients may present some time after disease is well underway
• Of 43 close contacts, none were RNA positive for H7N9 nor were any positive for antibodies to H7N9 suggesting they had not hosted an infection that was missed by PCR
• The in-house ("home made") antibody test was considered, by the authors, to be insufficiently sensitive and may have missed evidence of exposure in contacts. Prospective screening of contacts was not done.

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Time for the bat signal? The need for an animal model for Middle East respiratory syndrome coronavirus.

Elizabeth Devitt notes in Nature Medicine, that unlike its cousin, the severe acute respiratory syndrome coronavirus (SARS-CoV), some important features of MERS-CoV including its transmission, incubation period, and ability to spread systemically within the host, have not been able to be defined for the MERS-CoV using non-human models, because the virus does not like to infect the same animals. 

When the MERS-CoV infects a larger non-human animal, the rhesus macaque monkey, the disease it produces, while still defined as pneumonia and proving the casual link between MERS-CoV infection and disease, resolved faster and was not as severe as that in humans. These animals are also not easy to work with. I wonder if older monkeys with comorbidities have been looked at in particular? [UPDATE: The macaques above live to about 25-years]. It is this population in which MERS is most severe. Nonetheless, the monkey studies provide an excellent vehicle on which to test the usefulness of 2-drug an antiviral approach (Falzano et al, described earlier) that can clear MERS-CoV infections in vitro.

While cell/tissue culture methods using primary human airway cells have proven extremely useful for looking at cellular biology, antiviral effects, and immunobiology related to MERS-CoV infection, something with legs will be needed for future vaccines and to address the list above. We've seen many examples of how animal models massively improve our understanding of influenza virus pathogenesis, if an example is needed.

Also according to Devitt, Ian Lipkin is still wading through the data from samples collected from a range of animals that may be the natural hosts for the MERS-CoV in Saudi Arabia. Meanwhile, we recently learned of another CoV (PML/2011) found in the fecal pellets from a South African Neoromicia cf. zuluensis bat in 2011. PML/2011's nearest CoV relative was the MERS-CoV - its closest viral relative found to date (at least in the conserved RdRp region used by the authors).

This all begs the question, is there a bat animal model? CoVs, but also studies of other viruses like Hendra and Nipah, would benefit from a well-defined model based on these critters. That is, if they can be worked with and if they show any signs of these diseases - which they may not. My very quick skim of the literature found that bats used for neurological studies and for Hendra virus.

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Memories of H7N9....

The Annals of Internal Medicine has an article by Andrew Pavia that summarizes some of the key events in the influenza A(H7N9) virus outbreak earlier this year, asking "Should we be concerned?". The question is not specifically answered, rather we are reminded that H7N9 has the ingredients with which we could bake a very good pandemic.

1. It faces a population with little or no immunity;
2. H7N9 seems to be a true pathogen, and while we have likely only seen an ice cube's worth of the iceberg of clinical outcomes, we believe it to be an at least partly-adapted pathogen with the potential for fatal infections;
3. While some human-to-human transmission has most likely been seen, it was not sustained and therefore was not, earlier in the year, capable of spreading with pandemic efficiency

Dr Pavia also asks, "Are we fully prepared for a pandemic?" In answer to that question, we find the pantry is somewhat bare of the ingredients we'd need to bake an equally good pandemic response. Perhaps better stocked than when SARS came knocking though.

Perhaps I've worn the cake analogy out.

We have a stressed heath system, we face the potential for our key influenza antiviral, oseltamivir, to be outgrown by its target and we still lack robust tests that can be used on the spot (point-of-care tests) and not just by expert laboratories. While we can make a vaccine faster than ever before, those systems that have suitable approval for global use and rapid deployment may still take too long to stave off the effect of a virus travelling at the speed of global interconnectivity.

Over at the International Journal of Clinical Practice, Richard Stein also recaps H7N9, a virus that some expect to rise again in the northern cold months. 

Dr Stein dwells more on the molecular and the error-prone nature of RNA virus replication, reminding us of the ability of influenza to exchange bits of its multipartite genome with any other influenza in the cellular neighbourhood.

Every infection/co-infection increases the risk that a more lethal, or a more innocuous, viral descendent could emerge; but will those changes produce a virus that likes its lot and Chooses Life? More often, it will burn out before we even notice it because its just not fit enough to survive the hustle and bustle life of being on the lam from the host immune police, seeking new and suitable attachments, engaging with the cellular microenvironment, reproducing, replicating and assembling into new bodies and making it's grand exit, nare left, to the next potential virus factory. Its tough out there for a virus.

Finally, Dr Stein reminds us that we, the potential virus factories, play an active role in the success of viral spread. From those few folks with an unexplained ability to super-spread virus to more people than most other folks do, to our need to hang around in groups (cities I think they're called) that are separated by too little distance to prevent that guy over there's sneeze from impacting on your eyeballs. 

Dr Stein wraps up noting that we must always be concious of seeking to understand the transmission triad that underpins any pandemic: the host, the virus and the environment. 

From what we have seen in 2013 alone, that involves a lot of work, a lot of organising, a lot of clinical and laboratory expertise and capacity...and a whole lot of communication.

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Rhinoviruses (HRVs) in the blood reflect more HRV in the nasopharynx and worse disease...

Of late we have heard quite a it about systemic viruses viruses of the nose, throat and lungs, so-called respiratory viruses. 

In this context, we're taking about the spread of respiratory viruses beyond their expected site of infection and disease...the respiratory tract...and into the blood or extra-respiratory tissues and organs including the kidney, liver, brain etc. 

We tend to see this spread in the severely ill who have been infected by viruses that our body's defences see as particularly "foreign" - not simply a "not us" kinda foreign, but more like a "hey, you usually infect small furry or feathery things, not humans!" kinda foreign.

In a recent article by Esposito and colleagues, we are reminded that this is not limited to influenza and novel coronaviruses but can be a feature of the most numerous of respiratory viruses, the human rhinoviruses (HRVs). An assumption we live by here, based on early work that made these correlations, is that viral RNA detection may approximate the detection of infectious virus at the sampling site. Some key points:

• 12% of children with HRV detected in their nasopharyngeal swabs also had virus RNA detected in their blood plasma (rhinoviraemia)
• Those children with the greatest amount of HRV RNA in their swabs also more often had rhinoviraemia
• Children with rhinoviraemia were more likely to have more severe disease. This included low oxygen saturation, high respiratory rate, white blood cell counts and C-reactive protein levels
• Children with higher viral load did not have a specific type of respiratory disease
• Viral load in the swab or plasma samples was determined by comparing to a dilution series (titration) of in vitro (lab-made) RNA.

A bit of digging in the literature will tell you that this is not a new phenomenon. There other reports of the "common cold virus" (HRVs; I hate that term by the way) in the blood. E. Kathryn Miller and I covered some in a recent HRV-C review (From sneeze to wheeze: What we know about rhinovirus Cs). Rhinoviraemia was reported by  Urquhart et al in 1970 and 1972, by Xatzipsalti et al in children with asthma, bronchiolitis or common colds in 2005. Back in 1964, Cate et al isolated an infectious HRV from faeces. More recently, molecular tools (read: the polymerase chain reaction or PCR) have been used by Tapparel et al (2009), Harvala et al (2012) and Lau et al (2012) to detect HRV genome sequences in the faeces of children with fever of unknown origin, gastroenteritis and pericarditis. In the latter case, Tapparel et al also found HRV RNA (but not infectious virus) in  pericardial and bronchoalveolar lavage fluids and plasma.

So we know that even very widespread viruses, traditionally associated with mild disease, have the capacity to reach high viral loads not just at their historic site of infection, the respiratory tract, but at disseminated sites outside the site of infection. So we're porous!

I don't think it's much of a leap to then presume that all respiratory viruses could do this - whether endemic, zoonotic, or capable of causing mild, severe, or no overt disease at all.

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MERS-CoV: Family cluster, untested case, delayed reporting, missing data

Hat tip to Crof's story for drawing my attention to this article.

Omrani and colleagues describe a family cluster of Middle East Respiratory Coronavirus (MERS-CoV) infections. The study, from the Kingdom of Saudi Arabia Ministry of Health (KSA MOH) is interesting for a few reasons:

1. The first case (51-year old male with type II diabetes; 51M) is believed to have acquired the infection while he was in hospital for investigations into 2 months of back pain, limb weakness and lack of bladder control which lead to diagnosis of multiple myeloma. 14-days after admission to a medical ward he developed fever, cough, shortness of breath (dyspnoea)and hypoxia for which he was treated with antibiotics and oseltamivir (for influenza). He died.
• Because 14.7 days is the suspected latest time between infection and symptoms of infection, the authors believe MERS-CoV was acquired while in the hospital but there is no clear source. The authors note that it could have been another patient or an asymptomatic healthcare worker (HCW). he hospital is not identified.
• There were a number of clinical features shared by previous cases that were known to be MERS-CoV positive: fever and cough are found in 91% of 33 previously published MERS-CoV cases; acute renal failure and requirement for mechanical breathing assistance in 79%; chest X-ray infiltrates in 76%; dyspnoea in 60% etc.
• Numerous friends  relatives and a hospital roommate did not develop symptoms. Two of his brothers, from a household of 10 adults, did.
• No respiratory virus testing of samples from 51M (Patient #1) was undertaken. It's not clear whether any appropriate samples were collected. 
2. Patient 2, (39M; brother of 51M) was admitted to a different hospital on Feb 28^th, 10-days after becoming unwell. He had developed pneumonia. He died March 2^nd.
3. Patient 3 (40M; brother of 51M and 39M) was admitted to the same hospital as 39M, March 4^th. He had developed pneumonia. He had been previously announced by the WHO but without any detail (he seems to be FluTracker's case #16). He developed pneumonia but recovered and was released.
4. Nasopharyngeal swabs from 51M and 39M were negative for influenzavirus, respiratory syncytial virus, adenovirus, rhinovirus and endemic coronaviruses. No parainfluenzavirus, metapneumovirus or bocavirus testing was mentioned. 39M was positive for MERS-CoV in upper and lower respiratory tract samples; 40M was negative from an upper airway sample but positive from a lower respiratory tract sample.
5. The authors note that the high mortality rate appears to be die to under-diagnosis of mild and asymptomatic cases. Presumably, such as the one(s) that lead to infection of 51M.

Its nice to have another data-hole filled (40M). Shame its taken since late March when this case was reported by the WHO, to find out about this case and about the possibility of silent in-hospital transmission events that could explain the strangely disseminated appearances of cases across the KSA, often associated with hospitals. 

Still, this cluster doesn't explain why so few exposures resulted in disease - and why the 2 contacts of 51M, neither having underlying diseases, became ill even though other family members and HCWs had longer or more intimate exposures, some quite early on when viral loads may have been at their highest. Some piece(s) of the puzzle is still missing here. 

• Were more actually infected, but not tested because they were not suitably symptomatic? 
• Could there be underlying levels of cross-protective pre-existing antibodies in the KSA? 
• All 3 cases here were males. Is there a risk factor specific to males in this region?
• Should 51Ms farm be considered more closely? Have those animals been tested? 
• 39M like to climb palm trees to pollinate the dates - could he have been the real index case of the cluster, but 51M,  infected by him, showed signs more rapidly because of his underlying disease?

I'm not a medical doctor and I don't pretend to be but even I am unclear on why a patient with clear signs and symptoms of a respiratory disease, which was in part treated as a respiratory infection (influenza antivirals were given), was not tested for viruses or bacteria to understand what the patient had. 

Further, this cluster started off in February of this year in a country that is on alert for, and living with, MERS-CoV infections. The World Health Organisation noted in its novel CoV update in November of 2012... 

"Until more information is available, it is prudent to consider that the virus is likely more widely distributed than just the two countries which have identified cases. Member States should consider testing of patients with unexplained pneumonias for the new coronavirus even in the absence of travel or other associations with the two affected countries. In addition, any clusters of SARI or SARI in health care workers should be thoroughly investigated regardless of where in the world they occur."

As Omrani et al show, 51M fit the requirement for being a MERS-CoV case very well; an unknown pneumonia. And yet the patient wasn't tested for any virus at all. And the other 2 cases relied on the shipment of samples to the UK (Public Health England, UK Laboratories) for testing before being confirmed. If this level of pneumonia labwork is the norm, then we have very little understanding of the amount of virus-related pneumonias in the KSA. How do we know whether the MERS-CoV is an exceptional disease-causing agent, or just one of many viruses that might be involved in such disease? That very basic information is essential for a world trying to understand how seriously to take this novel virus. 

This publication speaks to a central problem in trying to understand MERS-CoV in its current site of greatest activity, the KSA- too little testing and too heavy a reliance on signs and symptoms. If 51M got his infection from an asymptomatic HCW or another patient, that happened because testing was not conducted on that/those case(s). Of course, we may yet be wrong about the incubation period given that testing of asymptomatics and non-contacts is so limited - perhaps 51M did bring his infection in with him.

If ever there was an example of the need to test prospectively, regardless of signs and symptoms (something I've harped on about before), this paper gives it more than enough credibility.

Test, test, test. You can't test too much.

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