Posts Tagged ‘circovirus’

DNA Preparation Tubes Contaminated with Novel ssDNA Virus

21 September, 2013

See on Scoop.it – Virology News

A novel virus thought to have come from human samples appears to have been derived from seawater during the manufacture of tubes used to extract DNA.  

Ed Rybicki‘s insight:

I have a problem with the original report, in J Virol (http://jvi.asm.org/content/early/2013/09/05/JVI.02323-13.abstract?related-urls=yes&legid=jvi;JVI.02323-13v1): not that they discovered it, because that was done well.  However, they essentially REdiscovered something that was ±100% identical to a virus already sequenced and named by Chinese researchers – who did not use the Qiagen kits, apparently – and then gave it a new name!

 

Sorry, that is simply bad practice!  It also smacks of scientific imperialism of a sort that characterised early discovery work on HTLVs and on HIV, when US researchers calmly treated earlier characterisations as if they had never happened.

 

There is another leap that I do not think is justified: the authors claim that

 

"Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture".

Really?  On the basis of presence of a sequence in a metagenomic trawl?  No resampling with specific primers on a fresh sample?   And surely the generation of the silica matrix is done under conditions that would totally destroy adventitiious DNA?

So – an interesting paper, and a valuable notification (although it might have been nicer if they’d shared their findings informally, to save other people like our Virology Diagnostics lab time and money).  But flawed, in my opinion.

See on www.the-scientist.com

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Posted in biotechnology, General Virology, techniques, Uncategorized | Leave a Comment »

Doctor, there’s a…pig virus in my vaccine??

6 June, 2010

rotavirus particle

I have for some time taught my third year students about how one must weigh relative risk vs. relative benefit when it comes to vaccination – with the Wyeth live rotavirus vaccine that was withdrawn in 2000 or so due to isolated incidents of intussusception (=telescoping of the bowel) as an object example.

Consider: the vaccine MAY have caused a couple of incidents (which granted, were serious) – but on the whole was protective, and well tolerated.  The publication referred to has this as the relative risk:

“…epidemiologic evidence supports a causal relationship, with a population attributable risk of ~1 per 10 000 (range of 1 in 5000 to 1 in 12 000) vaccine recipients.”

While this may be an unacceptable risk in a North American community – which is where it was tested, where children mostly just get sick from rotavirus infection – what about in a developing country, where the risk of an infant dying from rotavirus diarrhoea is far higher than 1 / 10 000?  Indeed, the same article says:

“Because perceptions of vaccine safety derive from the relative disease burdens of the illness prevented and adverse events induced, the acceptance of rare adverse events may vary substantially in different settings. [ my emphasis]”

Yes – like the vaccine may well have done a great deal of good, and very little harm, in a developing country setting where rehydration therapy is not the norm.  But it was pulled, and the world had to wait for Merck’s Rotateq pentavalent live vaccine and GSK’s Rotarix tetravalent live vaccine, YEARS later, and probably a lot of children died that may not have needed to.

I note that the Merck product has this as a warning, too:

“In post-marketing experience, intussusception (including death) and Kawasaki disease have been reported in infants who have received RotaTeq”.

So the vaccine has the same risk profile as Wyeth’s – yet it has been widely distributed, and is apparently highly effective – as is Rotarix.  In fact, in 2009 the WHO issued a recommendation “…that health authorities in all nations routinely vaccinate young children against rotavirus…”.

And then…news that must have made many a heart sink, in March 2010:

Pig virus contamination halts GSK Rotarix use

“GlaxoSmithKline’s oral rotavirus vaccine Rotarix has been temporarily shelved in the U.S. due to a pig-virus contamination. Researchers stumbled on DNA from porcine circovirus type 1–believed nonthreatening to humans–while using new molecular detection techniques. More work is being done to determine whether the whole virus or just DNA pieces are present.

Additional testing has confirmed presence of the matter in the cell bank and seed from which the vaccine is derived, in addition to the vaccine itself. So the vaccine has been contaminated since its early stages of development.”

The finding of the porcine circovirus type 1 (PCV-1) DNA in the vaccine was due to what seems to have been publication of an academic investigation in February 2010 of “Viral Nucleic Acids in Live-Attenuated Vaccines” by Eri L Delwart and team, mainly from Blood Systems Research Institute and University of California, San Francisco.  They used deep sequencing and microbial array technology to:

“…examine eight live-attenuated viral vaccines. Viral nucleic acids in trivalent oral poliovirus (OPV), rubella, measles, yellow fever, varicella-zoster, multivalent measles/mumps/rubella, and two rotavirus live vaccines were partially purified, randomly amplified, and pyrosequenced. Over half a million sequence reads were generated covering from 20 to 99% of the attenuated viral genomes at depths reaching up to 8,000 reads per nucleotides.”

And they found:

“Mutations and minority variants, relative to vaccine strains, not known to affect attenuation were detected in OPV, mumps virus, and varicella-zoster virus. The anticipated detection of endogenous retroviral sequences from the producer avian and primate cells was confirmed. Avian leukosis virus (ALV), previously shown to be noninfectious for humans, was present as RNA in viral particles [!!], while simian retrovirus (SRV) was present as genetically defective DNA.”

Whooooo…possibly live animal retroviruses in human vaccines??  But most importantly for our purposes:

“Rotarix, an orally administered rotavirus vaccine, contained porcine circovirus-1 (PCV1), a highly prevalent nonpathogenic pig virus, which has not been shown to be infectious in humans. Hybridization of vaccine nucleic acids to a panmicrobial microarray confirmed the presence of endogenous retroviral and PCV1 nucleic acids.”

I don’t know about you, but I’d be more worried about the retroviruses!  The authors concluded [my emphases in bold and red]:

“Given that live-attenuated viral vaccines are safe, effective, and relatively inexpensive, their use against human and animal pathogens should be encouraged. The application of high-throughput sequencing and microarrays provides effective means to interrogate current and future vaccines for genetic variants of the attenuated viruses and the presence of adventitious viruses. The wide range of sequences detectable by these methods (endogenous retroviruses, bacterial and other nucleic acids whose taxonomic origin cannot be determined, and adventitious viruses, such as PCV1) is an expected outcome of closer scrutiny to the nucleic acids present in vaccines and not necessarily a reflection of unsafe products. In view of the demonstrated benefit and safety of Rotarix, the >implications (if any) for current immunization policies of the detection of PCV1 DNA of unknown infectivity for humans need to be carefully considered.”

So they find all these bits of adventitious nucleic acids in a live human vaccine, then tell us it’s all right?  They go to say, however:

“As an added note, recent testing by GSK indicates that PCV1 was also present in the working cell bank and viral seed used for the generation of Rotarix used in the extensive clinical trials that demonstrated the safety and efficacy of this vaccine. These trials indicate a lack of detectable pathogenic effects from PCV1 DNA on vaccinees.”

So: a clinical trial in retrospect, then??  Interesting idea, that – it’s OK because they inadvertently tested it already and no obvious harm came of it!  Mind you, the same thing happened with SV40 in poliovirus vaccines over a lot longer period and on a much larger scale – and while the jury is still out on long-term effects, it appears as though there were none.

The first outcome of the finding, though, was that the FDA recommended in March that use of Rotarix be suspended, pending further investigations.

The same GSK press release reminds us that:

“Rotavirus is the leading cause of severe gastroenteritis among children below five years of age and it is estimated that more than half a million children die of rotavirus gastroenteritis each year – a child a minute [my bold – Ed]. It is predicted that rotavirus vaccination could prevent more than 2 million rotavirus deaths over the next decade. The continued availability of rotavirus vaccines around the world remains critical from a public health perspective to protect children from rotavirus disease. “

Cementing the risk/benefit argument very firmly and pre-emptively, then!

The next development was that Merck’s Rotateq, initially thought to be free of PCVs, was found to contain both PCV-1 AND PCV-2 DNA.  From their press release:

“In March 2010, an independent research team and the FDA tested for PCV DNA in rotavirus vaccines; at that time, PCV DNA was not detected in ROTATEQ by the assays that were used initially. Subsequently, Merck initiated PCV testing of ROTATEQ using highly sensitive assays. Merck’s testing detected low levels of DNA from PCV1 and PCV2 in ROTATEQ. Merck immediately shared these results with the FDA and other regulatory agencies.”

Alarming at first sight – but a variety of someones had done their relative risk calculations, and by mid-May, both vaccines had been cleared by the FDA – much to Merck and GSK stockholder relief, one imagines.

“The agency’s decision follows a May 7 recommendation from an FDA advisory panel, which said the PCV contamination didn’t appear to be harmful to humans and the vaccines’ benefits outweighed any “theoretical” risk the products might pose.

In announcing its decision, FDA said that both vaccines have strong safety records, including clinical trials of the vaccines in tens of thousands of patients, plus clinical experience with their administration in millions more. PCV isn’t known to cause illness in humans, whereas the rotavirus these vaccines ward off can cause severe illness and even death.”

All in all, what appears to be a sensible, logical decision, based on evidence – whether collected in retrospect or not – and common sense.  After all, as GSK points out in a press release,

“[PCV] is found in everyday meat products and is frequently eaten with no resulting disease or illness.”

Like plant viruses in vegetables, retroviruses in undercooked chicken and other meat, and a myriad other viruses and bacteria that live in, on, and with us – you really can’t keep away from everything.

But there’s still a good case to be made for killed vaccines….

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Posted in Vaccines: General, Viruses | 2 Comments »

Virus origins: from what did viruses evolve or how did they initially arise?

19 March, 2008

This was originally written as an Answer to a Question posted to Scientific American Online; however, as what they published was considerably shorter and simpler than what I wrote, I shall post the [now updated] original here.

The answer to this question is not simple, because, while viruses all share the characteristics of being obligate intracellular parasites which use host cell machinery to make their components which then self-assemble to make particles which contain their genomes, they most definitely do not have a single origin, and indeed their origins may be spread out over a considerable period of geological and evolutionary time.

Viruses infect all types of cellular organisms, from Bacteria through Archaea to Eukarya; from E. coli to mushrooms; from amoebae to human beings – and virus particles may even be the single most abundant and varied organisms on the planet, given their abundance in all the waters of all the seas of planet Earth.  Given this diversity and abundance, and the propensity of viruses to swap and share successful modules between very different lineages and to pick up bits of genome from their hosts, it is very difficult to speculate sensibly on their deep origins – but I shall outline some of the probable evolutionary scenarios.

The graphic depicts a possible scenario for the evolution of viruses: “wild” genetic elements could have escaped, or even been the agents for transfer of genetic information between, both RNA-containing and DNA-containing “protocells”, to provide the precursors of retroelements and of RNA and DNA viruses.  Later escapes from Bacteria, Archaea and their progeny Eukarya would complete the virus zoo.

virus descent

It is generally accepted that many viruses have their origins as “escapees” from cells; rogue bits of nucleic acid that have taken the autonomy already characteristic of certain cellular genome components to a new level.  Simple RNA viruses are a good example of these: their genetic structure is far too simple for them to be degenerate cells; indeed, many resemble renegade messenger RNAs in their simplicity.

RdRp cassettes and virus evolution

RNA virus supergroups and RdRp and CP cassettes

What they have in common is a strategy which involves use of a virus-encoded RNA-dependent RNA polymerase (RdRp) or replicase to replicate RNA genomes – a process which does not occur in cells, although most eukaryotes so far investigated do have RdRp-like enzymes involved in regulation of gene expression and resistance to viruses.  The surmise is that in some instances, an RdRp-encoding element could have became autonomous – or independent of DNA – by encoding its own replicase, and then acquired structural protein-encoding sequences by recombination, to become wholly autonomous and potentially infectious.

A useful example is the viruses sometimes referred to as the “Picornavirus-like” and “Sindbis virus-like” supergroups of ssRNA+ viruses, respectively.  These two sets of viruses can be neatly divided into two groups according to their RdRp affinities, which determine how they replicate.  However, they can also be divided according to their capsid protein affinities, which is where it is obvious that the phenomenon the late Rob Goldbach termed “cassette evolution” has occurred: some viruses that are relatively closely related in terms of RdRp and other non-structural protein sequences have completely different capsid proteins and particle morphologies, due to acquisition by the same RdRp module of different structural protein modules.

Given the very significant diversity in these sorts of viruses, it is quite possible that this has happened a number of times in the evolution of cellular organisms on this planet – and that some single-stranded RNA viruses like bacterial RNA viruses or bacteriophages and some plant viruses (like Tobacco mosaic virus, TMV) may be very ancient indeed.

However, other ssRNA viruses – such as the negative sense mononegaviruses, Order Mononegavirales, which includes the families Bornaviridae, Rhabdoviridae, Filoviridae and Paramyxoviridae, represented by Borna disease virus, rabies virus, Zaire Ebola virus, and measles and mumps viruses respectively – may be evolutionarily much younger.  In this latter case, the viruses all have the same basic genome with genes in the same order and helical nucleocapsids within differently-shaped enveloped particles.

Their host ranges also indicate that they originated in insects: the ones with more than one phylum of host either infect vertebrates and insects or plants and insects, while some infect insects only, or only vertebrates – indicating an evolutionary origin in insects, and a subsequent evolutionary divergence in them and in their feeding targets.

Slide1

HIV: a retrovirus

The Retroid Cycle

The ssRNA retroviruses – like HIV – are another good example of possible cell-derived viruses, as many of these have a very similar genetic structure to elements which appear to be integral parts of cell genomes – termed retrotransposons –  and share the peculiar property of replicating their genomes via a pathway which goes from single-stranded RNA through double-stranded DNA (reverse transcription) and back again, and yet have become infectious.  They can go full circle, incidentally, by permanently becoming part of the cell genome by insertion into germ-line cells – so that they are then inherited as “endogenous retroviruses“, which can be used as evolutionary markers for species divergence.

The Retroid Cycle

Indeed, there is a whole extended family of reverse-transcribing mobile genetic elements in organisms ranging from bacteria all the way through to plants, insects and vertebrates, indicating a very ancient evolutionary origin indeed – and which includes two completely different groups of double-standed DNA viruses, the vertebrate-infecting hepadnaviruses or hepatitis B virus-like group, and the plant-infecting badna- and caulimoviruses.

Metaviruses and pseudoviruses

These are two families of long terminal repeat-containing (LTR) retrotransposons, with different genetic organisations. 

Members of family Pseudoviridae, also known as Ty1/copia elements,  have polygenic genomes of 5-9 kb ssRNA which encode a retrovirus-like Gag-type protein, and a polyprotein with protease (PR), integrase (IN) and reverse transcriptase / RNAse H  (RT) domains, in that order.  While some members also encode an env-like ORF, the 30-40 nm particles that are an essential replication intermediate have no envelope or Env protein.  They are not infectious.  Host species include yeasts, insects, plants and algae.

Metaviruses – family Metaviridae – are also known as Ty3-gypsy elements, and have ssRNA genomes of 4-10 kb in length.  They replicate via particles 45-100 nm in diameter composed of Gag-type protein, and some species have envelopes and associated Env proteins.  Gene order in the genomes is Gag-PR-RT-IN-(Env), as for retroviruses.  One virus – Drosophila melanogaster Gypsy virus – is infectious; however, as for pseudoviruses, most are not.  The genomes have been found in all lineages of eukaryotes so far studied in sufficient detail.

Both pseudovirus and metavirus genomes are clearly related to classic retroviruses; moreover, RT sequences point to metavirus RTs being most closely related to plant DNA pararetrovirus lineage of caulimoviruses.  This gives rise to the speculation that pseudoviruses and metaviruses have a common and ancient ancestor – and that two different metavirus lineages gave rise to retroviruses and caulimoviruses respectively.

All of these cellular elements and viruses have in common a “reverse transcriptase” or RNA-dependent DNA polymerase, which may in fact be an evolutionary link back to the postulated “RNA world” at the dawn of evolutionary history, when the only extant genomes were composed of RNA, and probably double-stranded RNA.  Thus, a part of what could be a very primitive machinery indeed has survived into very different nucleic acid lineages, some viral and many wholly cellular in nature, from bacteria through to higher eukaryotes.

The possibility that certain non-retro RNA viruses can actually insert bits of themselves by obscure mechanisms into host cell genomes – and afford them protection against future infection – complicates the issue rather, by reversing the canonical flow of genetic material.  This may have been happening over aeons of evolutionary time, and to have involved hosts and viruses as diverse as plants (integrated poty– and geminivirus sequences), honeybees (integrated Israeli bee paralysis virus) – and the recent discovery of “…integrated filovirus-like elements in the genomes of bats, rodents, shrews, tenrecs and marsupials…” which, in the case of mammals, transcribed fragments “…homologous to a fragment of the filovirus genome whose expression is known to interfere with the assembly of Ebolavirus”.

Rolling circle replication

There are also obvious similarities in mode of replication between a family of elements which include bacterial plasmids, bacterial single-strand DNA viruses, and viruses of eukaryotes which include geminiviruses and nanoviruses of plants, parvoviruses of insects and vertebrates, and circoviruses and anelloviruses of vertebrates.

Geminivirus particle

These agents all share a “rolling circle” DNA replication mechanism, with replication-associated proteins and DNA sequence motifs that appear similar enough to be evolutionarily related – and again demonstrate a continuum from the cell-associated and cell-dependent plasmids through to the completely autonomous agents such as relatively simple but ancient bacterial and eukaryote viruses.

geminivirus rolling circle replication

Big DNA viruses

Mimivirus particle, showing basic structure

However, there are a significant number of viruses with large DNA genomes for which an origin as cell-derived subcomponents is not as obvious.  In fact, one of the largest viruses yet discovered – mimivirus, with a genome size of greater than 1 million base pairs of DNA – have genomes which are larger and more complex than those of obligately parasitic bacteria such as Mycoplasma genitalium (around 0.5 million), despite their sharing the life habits of tiny viruses like canine parvovirus (0.005 million, or 5000 bases).

Mimivirus has been joined, since its discovery in 2003, by Megavirus (2011; 1.2 Mbp) and now Pandoravirus (2013; 1.9 -2.5 Mbp). 

The nucleocytoplasmic large DNA viruses or NCLDVs – including pox-, irido-, asfar-, phyco-, mimi-, mega- and pandoraviruses, among others – have been grouped as the proposed Order Megavirales, and it is proposed that they evolved, and started to diverge, before the evolutionary separation of eukaryotes into their present groupings.

It is a striking fact that the largest viral DNA genomes so far characterised seem to infect primitive eukaryotes such as amoebae and simple marine algae – and they and other large DNA viruses like pox- and herpesviruses seem to be related to cellular DNA sequences only at a level close to the base of the “tree of life”.

Variola virus, the agent of smallpox. Image courtesy Russell Kightley Media.

This indicates a very ancient origin or set of origins for these viruses, which may conceivably have been as obligately parasitic cellular lifeforms which then made the final adaptation to the “virus lifestyle”.

However, their actual origin could be in an even more complex interaction with early cellular lifeforms, given that viruses may well be responsible for very significant episodes of evolutionary change in cellular life, all the way from the origin of eukaryotes through to the much more recent evolution of placental mammals.  In fact, there is informed speculation as to the possibility of viruses having significantly influenced the evolution of eukaryotes as a cognate group of organisms, including the possibility that a large DNA virus may have been the first cellular nucleus.

In summary, viruses are as much a concept as a unitary entity: all viruses have in common, given their polyphyletic origins, is a base-level strategy for replicating their genomes.  Otherwise, their origins are possibly as varied as their genomes, and may remain forever obscure.

I am indebted to Russell Kightley for use of his excellent virus images.

Updated 12th August 2015

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MicrobiologyBytes Archive

14 December, 2007

Before I established this site, I posted a number of guest blogs to do with viruses on Alan Cann’s very wonderful MicrobiologyBytes site. Here are links to all the virus-related ones.

Maybe Not Quite The End

Posted on January 15, 2008
Review of a paper describing the receptor for the H5N1 HA protein

Given the current scare over H5N1 influenza virus in swans in the UK, it is possibly timely to recall that I wrote a little while ago in MicrobiologyBytes about how easy it appeared to be for […]

Bandicoot Blues

Posted on November 30, 2007
Description of a unique newly-described virus that looks like a chimaera of a papillomavirus and a polyomavirus

Now that the dust has begun to settle after the launch of Merck’s much-hyped Gardasil genital papillomavirus vaccine – discussed in MicrobiologyBytes here and here – people are turning again to looking at the natural history […]

Hurting rather than helping?

Posted on November 21, 2007
Some news on the failure of the Merck Adenovirus 5-vectored HIV vaccine

It should not have escaped the eye of the interested bystander that there has been a most unfortunate and premature end to a HIV vaccine trial recently – and that something that had been tested as […]

A Deeper Meaning

Posted on November 10, 2007
Some microbiology-related poetry….

I inadvertently became a published literary critic a little while ago. A long-time English Department colleague asked me for some help interpreting the collected works of possibly the most important modern poet from South Africa, and […]

Don’t look now, they’re in your genes

Posted on September 14, 2007
Description of natural insertions of virus gene fragments into a variety of organisms and how they elicit pathogen-derived resistance

And they’re protecting you! If you’re an insect, that is. Or possibly a plant.
In a remarkable convergence of news, an Israeli group led by Ilan Sela described how Israeli acute paralysis virus, which is implicated in […]

To bee or not to bee

Posted on September 11, 2007
News of how a single virus is suspected in the causation of “colony collapse disorder” of bee hives in the USA

A major recent mystery in US agriculture has been the phenomenon of “colony collapse disorder” (CCD) in honey bees. […]

This is the End

Posted on August 29, 2007
H5N1 highly pathogenic avian influenza virus mutates…

This is the End. Or the beginning of the end. Or possibly, the end of the beginning?
To misquote the immortal Bill Shankly: “It’s not a matter of life and death: it’s much more important than that”.
Having […]

Rolling down the road

Posted on August 27, 2007
Musings on rolling circle replication in viruses

In my idle moments (alas, too few these days!) I often try to think up lists of rock songs with a virus theme: you know, like “Cucumo” by the Beech Boys… “I got them ol’ burnin’, […]

Rooting the tree

Posted on August 3, 2007
News on inferring “ancestor sequences” for HIV to help make broadly effective vaccines

While fossilized viruses have never been found, we can often infer probable lines of evolutionary descent by analysis of extant genomic sequences. This sort of molecular phylogenetic approach has thrown up all sorts of interesting […]

It’s Life, Jim, but not as we know it…

Posted on July 24, 2007
Exploring what it means to be “alive”

Which could well apply to viruses, my very own favourite organisms – after all, they don’t respire, grow, excrete or any of those other good things […]

A feeling for the molechism*

Posted on June 26, 2007
Musings on what viruses are.

I think it’s permissible, after working on your favourite virus for over 20 years, to develop some sort of feeling for it: you know, the kind of insight that isn’t […]

Plus ça change, plus c’est … le same Web, only better?

Posted on June 8, 2007
A personal history of teaching Virology via the Web.

My, how things do change… I found myself reflecting, while I was looking over the detritus on our Web server of some 13 years of posting pages on the Web. “Orphan” pages, unconnected […]

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