Friday, 23 May 2014

The impact of cell culture on virus as highlighted by deep sequencing..[UPDATED]

Alignment of complete or near complete MERS-CoV
genomes made using Geneious v6.1.7.
The Neighbor-Joining tree was made using MEGA with
1000 bootstraps. Red stars indicate virus from same patient. 
Vertical bars to the right indicate Clade A (dark blue)
and B (pale blue). Sequences from the the 2013 Al-Ahsa
hospital outbreak are boxed in pink. GenBank
accession numbers are indicated at the end of
each sequence name which also includes region
of detection, host and date of sample collection.
Click on image to enlarge.
Just a quick post to note the difference that "passage" in cell culture (isolation of an infectious virus using lab cells inoculated with the original virus-positive patient material, some of which is taken off and added to a new flask of cells and this process repeated as needed)  can do to the virus as it changes to grow most effectively in the new environment...this is akin to the adaptive changes seen when a virus first jumps to a an entirely different species.

Apparently the 2 starred (red stars in the figure) virus genomes are from the same 60-year old male patient [1,2,3; ] but the original variant, EMC/2012, was sequenced from material after 6 rounds of cell culture [3] while the Bisha_1 variant was not [1]; it was subjected to deep sequencing directly after nucleic acid preparation using an original respiratory sample aliquot (nasopharyngeal swab)[1]. 

Given that cell culture passage seems to be related to the positioning of EMC/2012 in Clade A versus Bisha_1 in Clade B (indicated by a pale blue line), does this mean there is no Clade A (dark blue vertical bar) and that it's just an artefact??

Probably not. Why? Because the Jordan-N3/2012 virus that is also found in Clade A and it also originates from a human specimen collected in 2012. It is listed as having been sequenced from a bronchial sample. There is no mention of cell culture on its GenBank record - which does not mean there was no culture. But when that sample was passaged through culture and sequenced (N3/2012 MG167; sequence not shown in this tree) it remained 99.95% identical to the original sequence; just 2 nucleotide differences out of 30,028nt. 1 difference in the spike gene and 1 Open Reading Frame 1a). These are unlikely to be enough to switch its clade  but I'm realigning with this sequence included just to be sure about that!

I thought that was kinda interesting.

NB. There may be some concern over the specimen labelling used to identify samples for sequencing of EMC/2012 or Bisha_1. I'm attempting to sort our by following this up with the lead author.[1]

Reference...
  1. Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study. Cotten et al. Lancet 2012;382:1993-2002
  2. 60-year old man from Bisha who died in a Jeddah hospital (EMC/2012 variant)
    Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia. Zaki et al. N Engl J Med 2012; 367:1814-1820
  3. Genomic workup on EMC/2012
    Genomic Characterization of a Newly Discovered Coronavirus Associated with Acute Respiratory Distress Syndrome in Humans. van Boheemen. mBio 2002; 3(6): e00473-12