Archive for June, 2008


25 June, 2008

Hot on the heels of the popular term genomics – defined by Wikipedia as “the study of an organism’s entire genome” – comes one for virologists: viromics.  There have been a number of articles in recent years on “viromes”, or the total viral genome content of the DNA found in certain biological sampling areas such as entire oceans or just in humans, so one might be forgiven for assuming that viromics was the study of entire viral genomes.

 But you might be wrong…for example, Applied Viromics of California have this to say:

“The term “viromics” was introduced on a biochemical pharmacology discussion conference in 2001 (Lotze MT and Kost TA. Cancer Gene Therapy. 2002 Aug; 9(8):692-9). It refers to “the use of viruses and viral gene transfer to explore the complexity arising from the vast array of new targets available from the human and murine genomes”.”

So: just about using viruses to study animals, then?  The Katze lab at the University of Washington – who I note have the word in their Web address – focus on:

…the use of genomic and proteomic technologies to study virus-host interactions and the varied strategies used by viruses to evade cellular defense mechanisms. We study a wide range of viral pathogens, including hepatitis C, influenza, Ebola, West Nile, SARS-associated coronavirus, herpes simplex virus, and human and simian immunodeficiency viruses.”

 So it’s studying human-virus interactions, then?  However, the Marine Microbiology Group at the Universrsity of South Florida has as its viromics interest

“…the interaction of viral genomes with their environments by sequencing several temperate/pseudotemperate marine phage genomes”.

 Much more like what I thought it was!  However, possibly the best definition I have seen is from a Nature Biotechnology Commentary on “Systems biology and the host response to viral infection“, which pops up the term “Systems virology” as a synonym for viromics.  the article states:

“There is increasing evidence from functional genomics experiments that the patterns of cellular response to a variety of viral infections may reflect the pathogenic properties of the viruses. We contend that dissection of the critical, and often subtly different, cellular pathways will eventually unveil opportunities for manipulating the host immune response to fight off viral infection, control pathogenesis or both.” 

And in a side box on viromics: 

“Innovations in sequencing technologies, particularly the rapid, high-throughput pyrosequencing platforms, continue to transform large-scale biology. … The fields of viral metagenomics and viral diagnostics are therefore poised for rapid expansion.”

Again – unsurprisingly, given that the Katze lab mentioned above is involved – a rather pathocentric approach, but all in all, a nice summary of current thinking.

So it is not the “what” so much as the “what it does” with viruses – at least, with the human virome.  Of course, for those of us with other interests, the new viromics can largely be concerned with discovery: there is a largely undescribed universe of viruses out there, in everything from seawater to duck ponds, biofilms to beehives, colons to parrots.  Cataloguing some of that diversity can only lead to new insights – which will lead to the kinds of questions that can be answered by “systems virology” approaches.

For instance, in my PhD thesis in 1984, I published an virus-like particles in plant extractselectron micrograph of a collection of unidentified particles found in preparations of known viruses, concentrated and partially purified from plant extracts.  there is a veritable zoo of things present, from a variety of fialments – some probably flagellae – to a motley collection of spheres and even some recognisable phages or parts thereof.  It has also been stuck on my office wall for the last ten years, since I found it kicking about in a drawer….

The point is, we generally find what we are looking for – and presume that what is present in the greatest abundance is just that, and then ignore everything else. 

Now we don’t have to do that any more: a metagenomic study of a particle-enriched preparation such as that seen on the right could yield fabulous riches, and unveil hitherto-unsuspected minor populations of plant viruses, unknown fungal and bacterial viruses, and even viruses infecting insects, which may be passively “vectored” by plants.

In a dramatic application of the worth of this sort of discovery, a major metagenomic project involving colony decline disorder of honeybees was described in MicrobiologyBytes recently: this involved sequencing all of the DNA and cDNA from many beehives, and doing a subtractive analysis to determine which possible pathogen was involved.

Other efforts under way in our laboratories in Cape Town include a study of the single-stranded circular DNA virome of grasses in Africa: this has resulted in six publications to date, and over 300 full mastrevirus genomic sequences in less than two years, and has radically altered our perception of both the diversity and the recombination potential of these commercially-important viruses.  This phenomenal progress has been enabled by recent technological breakthroughs – in this case, the use of bacteriophage phi29 DNA polymerase for isothermal “rolling circle” genome amplification, and cheap commercial sequencing.  This is rapidly diversifying into similar work on Beak and feather disease circoviruses in parrots, and even dsDNA viruses.

We are at the threshold of an era of significant discovery potential in virology, much as happened in the early 1990s with the advent of PCR – only more so.  The nice thing is that rapidly-developing sequencing techniques will allow ever-cheaper large-scale sequencing – so that finances will not be the limiting factor they may have been up to now, and developing countries can share in the viromic bounty.

And every now and then, the young people around will tell me what is going on – for which I am grateful.

West Nile virus vaccine: almost a replicant

2 June, 2008

West Nile virus – a member of the family Flaviviridae – has insidiously spread halfway around the world from its origins in Africa, in just a few years.  It invaded the east coast of the USA, probably from the Middle East,  via either infected birds, mosquitoes, humans, or another vertebrate host in around 1999; since then it has spread all the way across the continent to the west coast, and has become truly endemic. 

Virions have a regular icosahedral-type structure, despite being enveloped, as a result of a structured nucleocapsid and a highly-structured array of envelope glycoprotein.  They contain a positive strand RNA genome of ~11 kb with a single long open reading frame that is translated as a polyprotein of about 3400 amino acids, which is then processed into individual regulatory and structural proteins.

The virus subtype spreading in North America – lineage 1 – causes encephalitis in humans, unlike the enzootic variant circulating in birds and animals in Africa.  It also cause severe mortality – near 100% in experimentally infected animals – among American Crows and other corvids: a feature of the spread of the disease has been dead crows found in and around towns in the USA.  A feature of lineage 1 viruses is their infection of horses and other equines as well – with up to one in three clinically-infected horses dying.  The human impact, however, is seen as a major problem: systemic febrile illness develops in ~20% of those infected with WNV, while severe neurologic illness developes in <1% of persons infected – with mortality rates of 5 -14% among persons with neurologic symptoms in recent US, Romanian, Russian, and Israeli outbreaks.

There has been a concentrated effort to develop a human vaccine or vaccines since the onset of the US epidemic – horse vaccines are already commercially available – and our knowledge of the virus has benefitted greatly as a result.  This includes a detailed structure for the virus, obtained by cryoelectron microscopy image reconstruction.

Purdue team solves structure of West Nile virus via kwout

 Now a team led by Alexander Khromykh from Brisbane in Queensland, Australia, writing in the May issue of Nature Biotechnology, have described a novel “single-round infectious particle” DNA vaccine against WNV which significantly increases protection in mice to lethal challenge with the live virus.   In the words of the authors:

“We augment the protective capacity of a capsid-deleted flavivirus DNA vaccine by co-expressing the capsid protein from a separate promoter. In transfected cells, the capsid-deleted RNA transcript is replicated and translated to produce secreted virus-like particles lacking the nucleocapsid. This RNA is also packaged with the help of co-expressed capsid protein to form secreted single-round infectious particles (SRIPs) that deliver the RNA into neighboring cells. In SRIP-infected cells, the RNA is replicated again and produces additional virus-like particles, but in the absence of capsid RNA no SRIPs are formed and no further spread occurs. Compared with an otherwise identical construct that does not encode capsid, our vaccine offers better protection to mice after lethal West Nile virus infection. It also elicits virus-neutralizing antibodies in horses. This approach may enable vaccination against pathogenic flaviviruses other than West Nile virus.”

Adapted by permission from Macmillan Publishers Ltd: Nature Biotechnology 26, 571 – 577, 20 April 2008 doi:10.1038/nbt1400 Single-round infectious particles enhance immunogenicity of a DNA vaccine against West Nile virus, David C Chang et al., copyright 2008

This is a very clever use of fundamental knowledge of virus structure and assembly: the virus envelope proteins – E and prM – can form budded particles if expressed in isolation; if expressed with the capsid protein, the particles encapsidate RNA with the appropriate encapsidation signal to form virions.  The DNA vaccine encodes a transcriptional unit corresponding to a viral genome which lacks only the capsid protein gene, as well as a separate capsid gene under back-to-back cytomegalovirus (CMV) promoters.  Thus, cells transfected with the DNA vaccine can produce both virus-like prM and E protein and membrane particles (VLPs), or pseudovirions which in addition contain a capsid and the engineered (=lacking capsid protein gene) genome.  While both are highly immunogenic, the pseudovirions can additionally infect other cells to release replicative genomic RNA, which can produce VLPs but not pseudovirions, as the capsid protein-encoding RNA is not encapsidated.  Thus, initial transfection leads to release of particles which allow a single subsequent round of VLP production, but no further spread of the replicative RNA.

A very clever trick – and worthy of being repeated for a number of related pathogenic flaviruses, including dengue and yellow fever viruses.

Even if the particles can’t pass the Voight-Kampff test…B-)