Friday, March 15, 2013

EID Journal: Deep Sequencing and Phylogenetic Analysis of NCoV



Coronavirus – Credit CDC PHIL

# 7008


Like police detectives on the trail of a shadowy killer, epidemiologists, virologists, and microbiologists are using every scientific tool in their arsenal to profile, identify, and (with luck) halt the transmission of the novel coronavirus (NCoV) that has recently emerged in a handful of patients in the Middle East.


Some of these efforts involve old fashioned shoe-leather detective work; conducting patient interviews and contact tracing.


Other disease detectives concentrate primarily on the physical evidence, conducting experiments in the lab.


The result is, practically every day, new clues are revealed, and new avenues of investigation are opened.  


This week alone we’ve seen reports on NCoV’s receptor binding (see Nature: Receptor For NCoV Found) and a detailed epidemiological investigation into last month’s family cluster (see Eurosurveillance: H2H Transmission of NCoV In UK Family Cluster).


Yesterday, the CDC’s EID Journal added a fairly technical report to NCoV’s dossier entitled:


Full-Genome Deep Sequencing and Phylogenetic Analysis of Novel Human Betacoronavirus

 Matthew Cotten, Tommy T. Lam, Simon J. Watson, Anne L. Palser, Velislava Petrova, Paul Grant, Oliver G. Pybus, Andrew Rambaut, Yi Guan, Deenan Pillay, Paul KellamComments to Author , and Eleni


A novel betacoronavirus associated with lethal respiratory and renal complications was recently identified in patients from several countries in the Middle East. We report the deep genome sequencing of the virus directly from a patient’s sputum sample.


Our high-throughput sequencing yielded a substantial depth of genome sequence assembly and showed the minority viral variants in the specimen. Detailed phylogenetic analysis of the virus genome (England/Qatar/2012) revealed its close relationship to European bat coronaviruses circulating among the bat species of the Vespertilionidae family.


Molecular clock analysis showed that the 2 human infections of this betacoronavirus in June 2012 (EMC/2012) and September 2012 (England/Qatar/2012) share a common virus ancestor most likely considerably before early 2012, suggesting the human diversity is the result of multiple zoonotic events.



Since it was first proposed by Pauling and Zuckerkandl in 1962 ("Molecular disease, evolution, and genetic heterogeneity"), molecular biologists have been refining the Molecular Clock Hypothesis (MCH). 


One that proposes the speed of evolutionary change in different organisms is reasonably constant, can be measured, and can be used to extrapolate (roughly) how long it has been since two or more related organisms diverged from a common ancestor.


What is called their tMRCA  (Time To Most Recent Common Ancestor).


You can read a nice history and explanation of this hypothesis in:


The Molecular Clock and Estimating Species Divergence

By: Simon Ho, Ph.D. (Australia National University) © 2008 Nature Education



The problem is calibration, as individual species mutate at vastly different rates.


Fortunately, scientists are getting much better at determining how fast many different organism’s genetic clock runs, and research since the 2003 SARS outbreak has helped to quantify the approximate speed of change among human coronaviruses.


Using this information, and comparing the whole genome of two novel human NCoVs samples taken from patients last summer (England/Qatar/2012 & EMC/2012), their analysis has led to two possible scenarios.


From the discussion portion of this paper:


In the interest of public health, it is critical to determine whether these CoV infections in humans are the consequence of a single zoonotic event followed by ongoing human-to-human transmissions or whether the 3 geographic sites of infection (Jordan, Saudi Arabia, and Qatar) represent independent transmissions from a common nonhuman reservoir.


The large genetic diversity of CoV maintained in animal reservoirs suggests that viruses that independently moved to humans from animals at different times and places are likely to be reasonably dissimilar in their genomes, possibly making the multiple transmission events model less likely.

Further information is needed to confirm this point because the currently available data are limited.


If we calibrate our molecular clock analysis using the evolutionary rate of Zhao et al. (20) estimated for SARS-CoV, we dated the tMRCA of EMC/2012 and England/Qatar/2012 viruses to early 2011.


Therefore, if both sequenced viruses and the other cases descended from a single zoonotic event, then this tMRCA suggests that the novel virus has been circulating in human population for >1 year without detection and would suggest most infections were mild or asymptomatic.The rate would have to be considerably faster, of a magnitude observed for human influenza A virus, for the tMRCA to be compatible with the earliest known cases in April 2012.


Perhaps more probable, therefore, is that the 13 known cases of this disease represent >1 independent zoonotic transmission from an unknown source.


Viral sequence data from other patients infected with this novel human betaCoV will help to more accurately estimate the estimate a genomic evolutionary rate specific to this virus, which will then yield a tMRCA estimate closer to the actual time.



So while the authors admit it is possible that these two cases are linked by a largely mild or asymptomatic chain of undetected human transmission, they believe it is more probable that NCoV has spilled over from more than one zoonotic sources over the past year in the Middle East.


Probabilities aside, determining which of these two scenarios is correct remains the top priority of epidemiologists investigating this virus.


The authors conclude by writing:


Precise identification of the origin of this virus, defining its mode of evolution, and determining the mechanisms of viral pathogenesis will require full-genome sequences from all cases of human infection and substantially more sampling and sequencing from Vespertilionidae bats and other related animals.


The sequencing method reported here markedly shortens the time required to process the clinical sample to genome assembly to 1 week and will provide a useful tool to study this novel virus.

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