TWiV 285: Hokies go viral

On episode #285 of the science show This Week in Virology, Vincent meets up with XJ Meng and Sarah McDonald at Virginia Tech to talk about their work on viruses of swine and rotaviruses.

You can find TWiV #285 at www.microbe.tv/twiv.

TWiV 271: To bee, or not to bee, that is the infection

On episode #271 of the science show This Week in Virology, the TWiV crew discusses two reports on viruses that might have crossed kingdoms, from plants to honeybees and from plants to vertebrates.

You can find TWiV #271 at www.microbe.tv/twiv.

Why do viruses cause disease?

EvolutionVirulence, the capacity to cause disease, varies markedly among viruses. Some viruses cause lethal disease while others do not. For example, nearly all humans infected with rabies virus develop a disease of the central nervous system which ultimately leads to death. In contrast, most humans are infected with circoviruses with no apparent consequence. Is there a benefit for a virus to be virulent?

One explanation for viral virulence is that it facilitates transmission. However, a comparison of infections caused by two enteric viruses, poliovirus and norovirus, does not support this general view. Both viruses infect the gastrointestinal tract and are spread efficiently among humans by fecal contamination. However, norovirus infection causes vomiting and diarrhea, while poliovirus infection of the intestine is without symptoms (the rare invasion of the nervous system, and subsequent paralysis, is an accidental dead end). Both viruses have successfully colonized humans for many years, so why does only one of them cause gastrointestinal tract disease?

Two recent studies of bacterial virulence provide some clues about the evolution of virulence. In one a commensal strain of Escherichia coli was serially propagated in the presence of macrophages, which are cells of the immune system that take up and destroy the bacteria. After many such passages, bacterial clones were isolated that escape phagocytosis and killing by macrophages. These clones had also acquired increased pathogenicity in mice. In other words, the genetic changes that allowed the bacteria to evade the immune response also lead to increased virulence.

In another example of evolution to virulence, it was found the the bacterium Pseudomonas aeruginosa can sense the presence of competing gram-positive bacteria because the latter shed the cell wall component peptidoglycan. In response to this molecule, P. aeruginosa secretes proteins that kill the other bacteria. These secreted proteins also make the bacterium more virulent in a host – in their absence, the bacteria are less virulent. In other words, P. aeruginosa damages its host in an attempt to remove nearby bacterial competitors.

In both bacterial examples, virulence can be viewed as collateral damage: the consequence of evading the immune response, or killing off competitors. Being virulent was not the primary goal. This explanation for bacterial virulence is straightforward and compelling: virulence is not directly selected for during evolution but comes along for the ride. Can it be applied to viruses?

All eukaryotic viruses must encode at least one protein that antagonizes host immune responses, otherwise they would be eliminated. These immune evasion proteins are certainly virulence factors: in general, when they are deleted or altered, the capacity of the virus to cause disease in a host is reduced. Like bacterial virulence, viral virulence might be collateral damage incurred by having to evade immune responses. This hypothesis is attractive but seems overly simplistic. If the ubiquitous and benign circoviruses did not evade host responses, then they would be eliminated from the human population.

The reasons why some viruses are virulent and others are not remain elusive. It is possible to reduce viral virulence by mutation, but this type of experiment does not reveal why viruses cause disease. The inverse experiment would be more informative: to select from a population of avirulent virus those that can cause disease. The results of such an experiment would help to identify the selection pressures that allow viruses to evolve to virulence.

De-discovering pathogens: Viral contamination strikes again

Spin column

Qiagen spin column at right. The silica layer is white. The spin column is placed in the microcentrifuge tube, left, to remove liquids and elute nucleic acids.

Do you remember the retrovirus XMRV, initially implicated as the cause of chronic fatigue syndrome, and later shown to be a murine virus that contaminated human cells grown in mice? Another virus thought to be associated with human disease has recently been shown to be a contaminant, derived from a piece of laboratory plasticware that is commonly used to purify nucleic acids from clinical samples.

During a search for the causative agent of seronegative hepatitis (disease not caused by hepatitis A, B, C, D, or E virus) in Chinese patients, a novel virus was discovered in sera by next generation sequencing. This virus, provisionally called NIH-CQV, has a single-stranded DNA genome that is a hybrid between parvoviruses and circoviruses. When human sera were screened by polymerase chain reaction (PCR), 63 of 90 patient samples (70%) were positive for the virus, while sera from 45 healthy controls were negative. Furthermore, 84% of patients were positive for IgG antibodies against the virus, and 31% were positive for IgM antibodies (suggesting a recent infection). Among healthy controls, 78% were positive for IgG and all were negative for IgM. The authors concluded that this virus was highly prevalent in some patients with seronegative hepatitis.

A second independent laboratory also identified the same virus (which they called PHV-1) in sera from patients in the United States with non-A-E hepatitis, while a third group identified the virus in diarrheal stool samples from Nigeria.

The first clue that something was amiss was the observation that the novel virus identified in all three laboratories shared 99% nucleotide and amino acid identity. This would not be expected in virus samples from such geographically, temporally, and clinically diverse samples. Another problem was that in the US non-A-E study, all patient sample pools were positive for viral sequences. These observations suggested the possibility of viral contamination.

When nucleic acids were re-purified from the US non-A-E samples using a different method, none of the samples were positive for the novel virus. Presence of the virus was ultimately traced to the use of column-based purification kits manufactured by Qiagen, Inc. Nearly the entire novel viral genome could be detected by deep sequencing in water that was passed through these columns.

The nucleic acid purification columns contaminated with the novel virus were used to purify nucleic acid from patient samples. These columns (pictured), produced by a number of manufacturers, are typically a few inches in length and contain a silica gel membrane that binds nucleic acids. The clinical samples are added to the column, which is then centrifuged briefly to remove liquids (hence the name ‘spin’ columns). The nucleic acid adheres to the silica gel membrane. Contaminants are washed away, and then the nucleic acids are released from the silica by the addition of a buffer.

Why were the Qiagen spin columns contaminated with the parvovirus-circovirus hybrid? A search of the publicly available environmental metagenomic datasets revealed the presence of sequences highly related to PHV-1 (87-99% nucleotide identity). The datasets containing PHV-1 sequences were obtained from sampled seawater off the Pacific coast of North America, and coastal regions of Oregon and Chile. Silica, a component of spin columns, may be produced from diatoms. If the silica in the Qiagen spin columns was produced from diatoms, and if PHV-1 is a virus of ocean-dwelling diatoms, this could explain the source of contamination.

In retrospect it was easy to be fooled into believing that NIH-CQV might be a human pathogen because it was only detected in sick, and not healthy patients. Why antibodies to the virus were detected in samples from sick and healthy patients remains to be explained. However NIH-CQV/PHV-1 is likely not associated with any human illness: when non-Qiagen spin columns were used, PHV-1 was not found in any patient sample.

The lesson to be learned from this story is clear: deep sequencing is a very powerful and sensitive method and must be applied with great care. Every step of the virus discovery process must be carefully controlled, from the water used to the plastic reagents. Most importantly, laboratories involved in pathogen discovery must share their sequence data, something that took place during this study.

Trust science, not scientists.

TWiV 225: Transcripts from the inbox

On episode #225 of the science show This Week in Virology, Vincent, Rich, and Kathy read listener comments and questions on viral oncotherapy, science communication, a functional HIV cure in an infant, and much more.

You can find TWiV #225 at www.microbe.tv/twiv.

TWiV 195: They did it in the hot tub

On episode #195 of the science show This Week in Virology, the complete TWiV team meets with Ken Stedman to discuss the discovery in Boiling Spring Lake of a DNA virus with the capsid of an RNA virus.

You can find TWiV #195 at www.microbe.tv/twiv.

TWiV 155: XXII Brazilian National Virology Meeting

sbv_logoenv2011Hosts: Vincent Racaniello, Grant McFadden, Eurico de Arruda Neto, Paulo Eduardo Brandão, Francisco Murilo Zerbini, and Janice Reis Ciacci Zanella

Vincent, Grant, Eurico, Paulo, Francisco and Janice discuss their work on bocavirus, infectious bronchitis virus, begomoviruses, and circoviruses at the Brazilian Virology Society meeting in Atibaia, São Paulo, Brazil.

Click the arrow above to play, or right-click to download TWiV 155 (56 MB .mp3, 93 minutes).

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, by email, or listen on your mobile device with the Microbeworld app.

Links for this episode:

Weekly Science Picks

Vincent – AAM Colloquium Program
Grant – The Disappearing Spoon by Sam Kean

Listener Pick of the Week

Antonio2011 Nobel Laureates Lectures at Lindau

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

TWiV 114: Ten out of ’10

vaccinia plaqueHosts: Vincent RacanielloAlan Dove, and Rich Condit

On episode #114 of the podcast This Week in Virology, Vincent, Alan, and Rich revisit ten compelling virology stories of 2010.

Click the arrow above to play, or right-click to download TWiV #114 (64 MB .mp3, 88 minutes).

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Ten virology stories of 2010:

  1. XMRV, CFS, and prostate cancer (TWiV 113, 99, 98, 94, 89, 76, 70, 65)
  2. The ongoing saga of polio eradication (TWiV 110, 79)
  3. Viruses interact with the miRNA/siRNA system (TWiV 108, 72)
  4. Endogenous viruses – retro and beyond (TWiV 105, 91, 88, 65)
  5. Dengue virus progress and new outbreak (TWiV 111, 95, 82)
  6. Colony collapse disorder (TWiV 104)
  7. David Baltimore (TWiV 100)
  8. Ode to a plaque (TWiV 68)
  9. Vaccine contamination with circovirus (TWiV 86, 77, 75)
  10. Universal influenza vaccines (TWiV 107)

Weekly Science Picks

Rich – Elementary schoolchildren publish a science paper (original article and editorial with video) – thanks Kathy!
Alan – White-nose syndrome blog
Vincent – Headway, headlines and healthy skepticism

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

Unexpected endogenous viruses

circovirus parvovirus genomeDuring the replication of retroviruses, a double-stranded DNA copy of the viral RNA genome is synthesized by reverse transcription and integrated into the genomes of the infected cell. When retroviral DNA is integrated into the DNA of germ line cells, it is passed on to future generations in Mendelian fashion as an endogenous provirus. Until very recently, retroviruses were the only known endogenous viruses. This honor has now been extended to other RNA viruses, and to circoviruses and parvoviruses, which possess single-stranded DNA genomes. Such integration events constitute a fossil record from which it is possible to determine the age of viruses.

The first non-retroviral endogenous virus described was bornavirus, a virus with a negative-stranded RNA genome. Bornaviral sequences were found in the genomes of humans, non-human primates, rodents, and elephants. Phylogenetic analyses revealed that these sequences entered the primate genome over 40 million years ago. Endogenous filovirus (ebolavirus, marburgvirus) sequences were subsequently identified in the genomes of bats, rodents, shrews, tenrecs and marsupials. Based on these analyses it was estimated that filoviruses are at least tens of millions of years old. The presence of endogenous bornavirus and filovirus sequences were subsequently confirmed and extended to 19 different vertebrate species. Endogenous hepadnaviruses probably entered the genome of the zebra finch 19 million years ago.

Recent additions to the endogenous virus catalog are the circoviruses and parvoviruses. The genome of circoviruses are composed of single-stranded DNA, while those of parvoviruses are linear single-stranded DNAs with base-paired ends (figure). Phylogenetic analyses of these endogenous viral sequences reveal that both virus families are 40 to 50 million years old. Examination of insect genomes has revealed endogenous viral sequences from members of the Bunyaviridae, Rhabdoviridae, Orthomyxoviridae, Reoviridae, and Flaviviridae.

With the exception of retroviruses, these endogenous viral sequences have no role in viral replication – they are accidentally integrated into host DNA. Such sequences are highly mutated and typically comprise only fragments of the viral genome, and therefore cannot give rise to infectious virus. Whether these sequences confer any biological advantage to the host is an interesting question. It is possible that some of the endogenous viral sequences are copied into RNA, or translated into protein, and could have consequences for the host. For example, it has been suggested that synthesis of the bornaviral N protein from endogenous sequences might render the host resistant to infection with bornaviruses.

How are non-retroviral genomes integrated into the host DNA? For viruses with an RNA genome, the nucleic acid must enter the nucleus (perhaps accidentally for viruses without a nuclear phase) and be converted to a DNA copy by reverse transcriptase encoded by endogenous retroviruses. Hepadnaviruses encode a reverse transcriptase which produces the genomic DNA from an RNA template. In all cases, recombination could lead to integration of viral DNA into the host chromosome.

Almost half of the human genome is made up of mobile genetic elements, which includes endogenous proviruses and other sequences derived from retroviruses such as retrotransposons, retroposons, and processed pseudogenes. It seems likely that even more diverse viral sequences lurk in cellular genomes, awaiting discovery.

Horie M, Honda T, Suzuki Y, Kobayashi Y, Daito T, Oshida T, Ikuta K, Jern P, Gojobori T, Coffin JM, & Tomonaga K (2010). Endogenous non-retroviral RNA virus elements in mammalian genomes. Nature, 463 (7277), 84-7 PMID: 20054395

Taylor DJ, Leach RW, & Bruenn J (2010). Filoviruses are ancient and integrated into mammalian genomes. BMC evolutionary biology, 10 PMID: 20569424

Belyi VA, Levine AJ, & Skalka AM (2010). Unexpected inheritance: multiple integrations of ancient bornavirus and ebolavirus/marburgvirus sequences in vertebrate genomes. PLoS pathogens, 6 (7) PMID: 20686665

Gilbert C, & Feschotte C (2010). Genomic fossils calibrate the long-term evolution of hepadnaviruses. PLoS biology, 8 (9) PMID: 20927357

Katzourakis A, & Gifford RJ (2010). Endogenous viral elements in animal genomes. PLoS genetics, 6 (11) PMID: 21124940

Belyi VA, Levine AJ, & Skalka AM (2010). Sequences from ancestral single-stranded DNA viruses in vertebrate genomes: the parvoviridae and circoviridae are more than 40 to 50 million years old. Journal of virology, 84 (23), 12458-62 PMID: 20861255

TWiV 86: Dark matter with Dr. Eric Delwart

Hosts: Vincent Racaniello, Rich Condit, and Eric Delwart

In episode #86 of the podcast This Week in Virology, Vincent and Rich travel to the Blood Systems Research Institute in San Francisco to speak with Eric Delwart about his work on virus discovery.

This episode is sponsored by Data Robotics Inc. Use the promotion code TWIVPOD to receive $75-$500 off a Drobo.

To enter a drawing to receive 50% off the manufacturers suggested retail price of a Drobo S or FS at drobostore.com, fill out the questionnaire here.

Click the arrow above to play, or right-click to download TWiV #86 (59 MB .mp3, 81 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

Weekly Science Picks

Rich – Google Crisis Response – Gulf of Mexico Oil Spill
Vincent
HHMI resources for teachers and students (thanks, Jim!)
EricVaccine by Arthur Allen

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.