Sunday, 6 March 2016

Detecting Zika viruses using PCR: a look at published assays...

[NOTE: I'll update this post with other assays and information as I find time]

You may be helped - hopefully anyway - by reading the earlier post in this series - Primers prime PCR unless past their prime... [1]

Also hopefully helpful - the Zika virus (ZIKV) lineages I'll be referring too can be seen in this (very basic) tree. The viruses currently circulating in the Americas fall into the "Asian lineage".
Basic tree showing the majority genome sequences of ZIKV variants form discovery to 2015.
PCR and ZIKV...

Because flaviviruses are "RNA viruses" and because ZIKV is a flavivirus, we'll be talking about the range of RT-PCRs that have been described in the literature for ZIKV. This will include both RT-rtPCR (Reverse Transcript real-time Polymerase Chain Reaction) but also the non-real-time versions which I'll be calling "conventional" RT-PCRs or RT-conPCRs.

This will not be all the variants and home brew concoction of RT-PCR out there for ZIKV, but it is the ones that have been published and some of the articles that reference them.

I'll be counting nucleotide positions from 5' to 3' (or "left" to "right") so nucleotide #1 will be furthest form the pointy end of the green arrow-no, not that Green Arrow).

I'm using Spondweni virus, a related but different virus (usually) at the top of the alignment and in the tree. This is to provide something that shows how different a very near cousin is, even when "zoomed in" and compared to the brothers and sister variants of ZIKV. 

In some alignments I have marked, in the left most panel, some - but not all - examples of Asian or African lineage variants. See if you can spot patterns among the nucleotides that may be specific to each. 
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One-Step RT-PCR for detection of Zika virus
Faye et al. J Clin Virol 2008 43:96-101 [2]
PCR Aim: To detect ZIKV in human serum
Subsequent publications using this assay:
Faye et al assay. (A) The forward primer and (B) the reverse primer aligned against a range of African and Asian lineage ZIKV variant sequences.
  • Both primers have degenerate positions and look as though they should work well against African and Asian lineages on paper
  • There is a mismatch in the reverse primer at position #16 that is not accounted for and may be destabilising for a number of Asian lineage variant templates
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Genetic and Serologic Properties of Zika Virus Associated with an Epidemic, Yap State,Micronesia, 2007
Lanciotti et al. Emerg Infect Dis 14(8):1232-9 [3]
PCR Aim: To rescreen sera from the 2007 Yap Island epidemic
Subsequent publications using this assay:[3]

Assay 1...

Lanciotti et al assay #1. (A) The forward primer (853), (B) the real-time PCR probe (860-FAM) and (C) the reverse primer (911c) aligned against a range of African and Asian lineage ZIKV variant sequences.
  • The forward primer must interact with a 2-3 mutations in some of the the African variants, but is a good match with all Asian lineages variants.
  • The probe also has 3 mismatches with the same variant as above and 1-2 with African lineage variants but is again a good  match for most Asian lineage variants
  • The reverse primer is a great match to Asian variants but not very good if faced with trying to detect an African variant, especially KF383117

Assay 2...

Lanciotti et al assay #2. (A) The forward primer (1086), (B) the real-time PCR probe (1107-FAM) and (C) the reverse primer (1162c) aligned against a range of African and Asian lineage ZIKV variant sequences.
  • The forward primer works well against most variants and the mismatches it does face are at the less destabilising 5' end, against some African lineage variants.
  • The probe is a good match for most variants of either lineage, having a couple of issues with 2 Central African Republic variants and KF383118 poses a problem at the 5' end of the probe which could be a issue for the polymerase which comes charging down the same strand as the which the forward primer is hybridised to, looking to chop the probe up.
  • The reverse primer is a great match to Asian variants except for KF383117 and will suffer between 1 to 5 mismatches with African lineage variants - also impacting across the primer landing site. 
Generally these 2 assays should be good for detecting the Asian variants, will suffer varying degrees of performance issues against African variants.

Imported Zika Virus Infection from the Cook Islands into Australia, 2014
Pyke et al...PLoS Curr. 2014 Jun 2;6. pii

PCR Aim:
Subsequent publications using this assay

E gene assay...



Coming up...
Quantitative real-time PCR detection of Zika virus and evaluation with field-caught Mosquitoes
Faye et al. Virol J 2013 10:311


References...

  1. Primers prime PCR unless past their prime...
    http://virologydownunder.blogspot.com.au/2016/03/primers-prime-pcr-unless-past-their.html
  2. One-step RT-PCR for detection of Zika virus
    http://www.ncbi.nlm.nih.gov/pubmed/18674965
  3. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007
    http://dx.doi.org/10.3201/eid1408.080287
  4. Zika Virus Infection in Pregnant Women in Rio de Janeiro — Preliminary Report
    http://www.nejm.org/doi/pdf/10.1056/NEJMoa1602412
  5. Quantitative real-time PCR detection of Zika virus and evaluation with field-caught Mosquitoes
    http://www.virologyj.com/content/10/1/311
  6. Imported Zika Virus Infection from the Cook Islands into Australia, 2014
    http://currents.plos.org/outbreaks/article/imported-zika-virus-infection-from-the-cook-islands-into-australia-2014/