Posts Tagged ‘metagenomics’

Papillomavirus and HIV: a nasty combination

17 August, 2012

I started working on human papillomaviruses (HPVs) some 22 years ago, back at the dawn of PCR: I helped my then-new major collaborator (and wife of 2 years), Anna-Lise Williamson, design some degenerate primers for amplifying as wide a range as possible of high-risk HPVs from cervical biopsy samples.  These worked pretty well, and are still highly useful for the purpose, despite the many novel types found since then.

We went on to do another two papers together on looking at variation and typing of HPVs via PCR and and sequencing, then took a deviation into making candidate vaccines for HPV and HIV.  Anna-Lise carried on with surveilling for HPVs, however, and has ended up with a WHO Regional Laboratory for HPV work.  She also started working on HPV infections in HIV-infected women: work on a study cohort showed that while HIV-free women usually had only 1 HPV type, the 109 HIV-infected often were infected with multiple HPV types.  In association with Anna Salimo in my lab, we started a deep sequencing pilot study on the sample with the most HPVs.  This turned this into a regional study, with help on assembling and interpreting sequence data from Prof Johan Burger’s lab at the University of Stellenbosch, and it was revelatory: while a commercial kit could detect 12 HPV types in one sample, next-gen sequencing found 16.

We went on to do PCR on all 109 samples in the cohort with specific primers for the types not found by the kit, and showed prevalences up to 15% in the HIV-infected group.  This is an important result, because otherwise-innocuous HPV types that do not show up in normal women, may well be associated with disease in the HIV-infected – and will probably not be protected against by the current HPV vaccines.

We continue to do work on these samples, and it will be very interesting to see what the new methodologies show up.  Especially as sequencing becomes cheaper, and we can do more samples…!  Meantime, we have published the pilot study:

Next-generation sequencing of cervical DNA detects human papillomavirus types not detected by commercial kits

Tracy L MeiringAnna T SalimoBeatrix CoetzeeHans J MareeJennifer MoodleyInga I HitzerothMichael-John FreeboroughEd P Rybicki and Anna-Lise Williamson

Virology Journal 2012, 9:164 doi:10.1186/1743-422X-9-164

Published: 16 August 2012

Abstract (provisional)


Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV + women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women.


Total DNA was isolated from 109 cervical specimens from South African HIV + women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR.


Illumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence’s of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6 %, 12.8 %, 4.6 % and 8.3 % respectively.


Our results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.

I thank Russell Kightley Media for use of the HPV and cervical cancer graphic.

Venter can do WHAT for influenza??

5 June, 2010

I have kept out of commenting on what J Craig Venter and others have done recently, given that many others have done so, and done so well – however, there is recurring mention of what “this technology” could do for influenza vaccines specifically, which has both puzzled and intrigued me, given a distinct lack of obviousness.

So I will comment, if only to clarify this issue for me and anyone else who cares.

To give some background, the New Scientist issue of the 29th of May has a guest editorial by J Craig Venter, Clyde Hutchison and Hamilton Smith, where they discuss some of the implications of their having made a totally synthetic and viable Mycoplasma mycoides genome (see also Science, DOI: 10.1126/science.1190719).

So what, exactly, is it they did?  OK, so they spent US$40 million or so constructing a genome, in segments, from sequence information housed in an electronic database, via chemical synthesis of long stretches of DNA.  They then assembled these segments into a singular genome in yeast, and then inserted this into cells of the closely-related Mycoplasma capricolum which had been stripped of their genomes – and incidentally, rendered unable to destroy the incoming genome as “foreign”, by a process which is now proprietary.  These cells then expressed the new DNA, which allowed them to multiply, and to take on all of the characteristics of the synthetic M mycoides, given that all of the original cell constituents from the original bug (proteins, mRNA, membranes, etc) would be turned over in time, and become those specified by the new genomes.

This is a big deal – a really, really big deal – but at the same time, they themselves recognise it is an incremental step in a long series of steps that started with Arthur Kornberg’s lab making the first complete synthetic and viable genome of a virus (phiX174) by in vitro synthesis from virion DNA, polymerase and nucleotides.  In fact they modestly point out that this is not even the first  complete cell genome that has been synthesised; it is the largest, however, and the one that worked.  They were not so modest in missing out a few other landmarks before their own complete synthesis of phiX174 in 2003, however: for example, the first synthesis of a functional plasmid, and the first generation of a RNA virus genome from a cDNA copy, and the complete synthesis of an infectious poliovirus genome, are not mentioned.

So what is it they did not do?

Well, they did not “create life”, however much even relatively respectable publications might claim they did: life is a lot more complex than chemicals, and people have “rebooted” cells before with exogenous genomes; what they did is not really qualitatively different to infecting a cell with a synthetic virus.

They have also not done anything that is immediately useful: their new organism differs from the original only in having a few genes missing, and a long literary message and ownership-encoding “watermark” inserted.

More positively, they have also most emphatically not opened the floodgates for bioterrorists to mail order complete poxvirus or anthrax genomes: as I have noted here previously,

“…There are more than enough nasty agents out there that are relatively easy to obtain, and do simple kitchen-based microbiology with, to keep entire cave complexes and Montana libertarian enclaves busy for years, without resorting to complicated molecular biology”.

Or spending $40 million dollars.  And I will say it again….

So aside from the details, what have they done?  In the NS editorial, Venter et al. say this:

“We now have the means to design and build a cell that will define the minimal set of instructions necessary for life, and to begin the design of cells with commercial potential, such as fuel production from carbon dioxide. We can assemble genome-sized stretches of DNA that can also be used to mix and match natural and synthetic pieces to make genomes with new capabilities.

Synthesising DNA in this way is still expensive, but we expect the cost to fall dramatically. This may make the complete synthesis of genomes competitive with the alteration of natural genomes to add new capabilities to bacterial cells. It should also be practical to synthesise simple eukaryotes, such as yeast, to which it is already possible to add extra chromosomes. The construction of large pieces of synthetic DNA and their introduction into a receptive cytoplasm is no longer a barrier. The limits to progress in synthetic biology are now set by our ability to design genomes with particular properties.”

Right: so what they have done is set the benchmark for what is possible – rather than what should be done.

Because it is a lot easier to do things such as they propose by other ways – as is pointed out in the companion article to the NS editorial.  For that matter, I am sure one could more easily end up with a completely synthetic and much larger cellular genome by incrementally replacing genome chunks by homologous recombination or transposon-mediated insertion / Cre-Lox deletion, and have it cost far less and be less subject to error, than by synthesising it de novo.

Influenza virus - Copyright Russell Kightley Media

And how does any of this relate to influenza vaccines?

The only comment I can find in the NS article that sheds light on this is the comment:

“As soon as next year, the flu vaccine you get could be made synthetically,” he [Venter] says.

Except that this has been possible for years already, after the poliovirus synthesis…?  I think it was rather a badly-chosen example rather than any actual plan; however, there is not a lot of point in making a synthetic influenza virus genome, given that the attenuated originals already work quite well as vaccines – and we don’t yet understand how to specify avirulence in influenza, so any synthetic version would necessarily be a copy of an extant version.

So hype rather than fact for ‘flu; promise rather than substance for carbon dioxide sequestration and biofuels – but still the coolest thing since sequencing your own dog…B-)

Oxygen from viruses??

7 April, 2008

I thank my colleague Suhail Rafudeen for alerting me to this:

 “Some Of Our Oxygen Is Produced By Viruses Infecting Micro-organisms In The Oceans

ScienceDaily (Apr. 6, 2008) – Some of the oxygen we breathe today is being produced because of viruses infecting micro-organisms in the world’s oceans, scientists heard April 2, 2008 at the Society for General Microbiology’s 162nd meeting.

About half the world’s oxygen is being produced by tiny photosynthesising creatures called phytoplankton in the major oceans. These organisms are also responsible for removing carbon dioxide from our atmosphere and locking it away in their bodies, which sink to the bottom of the ocean when they die, removing it forever and limiting global warming.

“In major parts of the oceans, the micro-organisms responsible for providing oxygen and locking away carbon dioxide are actually single celled bacteria called cyanobacteria,” says Professor Nicholas Mann of the University of Warwick. “These organisms, which are so important for making our planet inhabitable, are attacked and infected by a range of different types of viruses.”

The researchers have identified the genetic codes of these viruses using molecular techniques and discovered that some of them are responsible for providing the genetic material that codes for key components of photosynthesis machinery.

“It is beginning to become to clear to us that at least a proportion of the oxygen we breathe is a by-product of the bacteria suffering from a virus infection,” says Professor Mann. “Instead of being viewed solely as evolutionary bad guys, causing diseases, viruses appear to be of central importance in the planetary process. In fact they may be essential to our survival.”

Viruses may also help to spread useful genes for photosynthesis from one strain of bacteria to another.

Adapted from materials provided by Society for General Microbiology, via EurekAlert!, a service of AAAS”

Fascinating concept: viruses as an essential link in the circle of life?!  Not so far-fetched, though: just because we know them largely because of their propensity to cause, and our fascination with, diseases that affect us and our livestock and crops…doesn’t mean that is all there is.

Viruses have been around as long as any other form of life, and it would be strange indeed if some form(s) of commensalism and/or symbiosis had not evolved.

…and see here for some fascinating speculations on the possible involvement of viruses with the origin of eukaryotes.