TWiV 361: Zombie viruses on the loose

On episode #361 of the science show This Week in Virology, the TWiVsters discuss Frederick Novy’s return from retirement to recover a lost rat virus, and evidence for persistence of Ebolavirus in semen.

You can find TWiV #361 at

TWiV 315: Must be something in the water

On episode #315 of the science show This Week in Virology, Vincent, Alan, Rich and Kathy discuss the association of a virus with sea star melting disease, and the finding of a phycodnavirus in the oropharynx of humans with altered cognitive functions.

You can find TWiV #315 at

A virus that melts sea stars

Sun flower sea starSea stars are lovely marine invertebrates with a round central body connected to multiple radiating legs (photo credit). In the past year millions of sea stars in the west coast waters of North America have melted into piles of slime and ossicles. Sea star associated densovirus might be the cause of this lethal disease.

Sea star wasting disease (SSWD) is characterized by lesions, limb curling and deflation, and death as the animals rapidly degrade or ‘melt’. The current outbreak began in June 2013 and has killed sea stars from Baja California, Mexico, to Southern Alaska. SSWD might be the biggest marine wildlife epizootic ever observed.

Evidence that SSWD is caused by a virus came from experiments in which extracts of diseased sea stars were passed through a filter with pore sizes small enough to allow passage of viruses but not bacteria or other microbes. When injected into healthy sea stars, these filtrates induced sea star wasting disease. Extracts of diseased sea stars collected in Vancouver, CA contained 25 nanometer virus particles, as determined by electron microscopy.

Nucleic acid sequencing was to identify the viral agent of SSWD. Virus particles were purified from diseased animals, and both DNA and RNA was extracted. Analysis of the nucleotide sequences revealed the presence of giant DNA viruses such as mimiviruses and phycodnaviruses (link to algal virus paper), and among RNA viruses, retroviruses, dicistroviruses, and parvoviruses. With few exceptions, all samples containing parvoviruses were from symptomatic asteroids, and so the authors decided to pursue the study of this virus.

Analysis of the DNA sequence data revealed the presence of a densovirus (a parvovirus) related to viruses found in Hawaiian sea urchins. The authors called this virus sea star-associated densovirus, SSaDV. Like other members of the parvovirus family, these are small (25 nm diameter), naked icosahedral viruses with a ~6 kb single stranded DNA genome. When sea stars were infected in the laboratory with filtrates from diseased animals, virus loads, determined by PCR, increased with time together with disease progression. Field surveys revealed that the virus was more abundant in diseased than in healthy sea stars. The virus was found in marine sediments, plankton, and sea urchins. Viral nucleic acid was also found in sea stars preserved in museums since 1942.

While retrovirus sequences were found in sea star tissues, the authors believe that such ‘Retroviral annotations are likely spurious because they were detected in DNA libraries (and have RNA as nucleic acids).’ The detection of retrovirus sequences in sea stars is not at all spurious! DNA copies of retrovirus genomes are produced during infection and integrated into the host genome, explaining why these sequences were detected in DNA libraries. Their absence in RNA libraries means that virus particles are not produced, as is the case in many other organisms that contain endogenous retroviruses.

The evidence that SSaDV causes sea star wasting disease is strong but not yet complete. The crucial experiment that remains to be done is to isolate infectious virus in cell culture, inoculate it into sea stars, and show that it causes wasting disease.

While the authors’ work reveals that sea star wasting disease virus has been present on the North American Pacific Coast for over 70 years, the disease is not always observed. The current outbreak has been ongoing since just June 2013. Perhaps other environmental conditions have lead to the increased susceptibility of sea stars to disease. It is important to determine if a human activity is involved in precipitating the disease, so that we can prevent future loss of sea stars. The authors suggest one possibility, that increased populations of adult sea stars in small bays and inlets have produced increased virus levels which lead to more infections. It will also be important to determine if the virus has undergone any changes in transmissibility or virulence.

Sea star associated densovirus was also found in non-asteroids, including ophiuroids (brittle stars and basket stars) and echinoids (sea urchins and sand dollars). We need to know the host range of this virus, how it is transmitted, and whether it can cause disease in other species. A troubling scenario is that the recent amplification of the virus in sea stars could lead to infection, and perhaps death of, many other marine species.

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 242: I want my MMTV

On episode #242 of the science show This Week in Virology, the complete TWiV team talks about how two different viruses shape the evolution of an essential housekeeping protein.

You can find TWiV #242 at

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 106: Making viral DNA II

the 5prime end problemHosts: Vincent Racaniello, Dickson Despommier, and Rich Condit

On episode #106 of the podcast This Week in Virology, Vincent, Dickson, and Rich continue Virology 101 with a second installment of their discussion of how viruses with DNA genomes replicate their genetic information.

Click the arrow above to play, or right-click to download TWiV #106 (69 MB .mp3, 95 minutes)

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Links for this episode:

  • Figures for this episode (pdf)
  • Letters read on TWiV 106
  • Video of this episode – download .mov or .wmv or view below

Weekly Science Picks

Rich – Google Health
Dickson – The Neandertal genome
Vincent – Lab techniques videos (thanks, Erik!)

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

TWiV 96: Making viral DNA

Hosts: Vincent Racaniello, Dickson Despommier, and Rich Condit

On episode #96 of the podcast This Week in Virology, Vincent, Dickson, and Rich continue Virology 101 with a discussion of how viruses with DNA genomes replicate their genetic information.

Click the arrow above to play, or right-click to download TWiV #96 (65 MB .mp3, 90 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 – Breast milk sugars give infants a protective coat (NY Times and PNAS article)
Vincent – The Great American University by Jonathan R. Cole

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

TWiV 44: No hysteria

twiv_aa_2001Hosts: Vincent Racaniello, Dick Despommier, Alan Dove, and Jennifer Drahos

In episode #44 of the podcast “This Week in Virology”, Vincent, Dick, Alan, and Jennifer Drahos consider Marburg virus in Egyptian fruit bats, bacterial citrus pathogen found in shipping facility, canine parvovirus in Michigan, Relenza-resistant influenza virus, new HIV from gorillas, and public engagement on H1N1 immunization program.

Click the arrow above to play, or right-click to download TWiV #44 (54 MB .mp3, 78 minutes)

Subscribe to TWiV in iTunes, by the RSS feed, or by email

Links for this episode:
Isolation of Marburg virus from Egyptian fruit bats
Inspectors find bacterial citrus pathogen in California
Parvovirus killing hundreds of dogs in Michigan
Relenza-resistant H1N1 identified in Australia (press and journal article)
New HIV from gorilla
CDC wants public comment on H1N1 vaccination
Original antigenic sin (article 1 and article 2)
Dr. Stanley Plotkin on Meet the Scientist (thanks Peter!)
audioBoo (iPhone app – thanks Jim!)
Audio clips (first and second) from the podcast No Agenda (thanks peripatetic apoplectic!)

Weekly Science Picks
Jennifer Piled Higher and Deeper (PhD Comics)
Vincent Giant Microbes (thanks Stephen!)
Dick Virology in the 21st Century
Alan Annals of the Former World by John McPhee

Send your virology questions and comments (email or mp3 file) to or leave voicemail at Skype: twivpodcast

TWiV 31: Fever plus cough or sore throat

twiv_aa_2001On episode #31 of the podcast “This Week in Virology”, Vincent, Dick, Alan, and Marc Pelletier summarize the past week’s influenza H1N1 activity, and discuss parvovirus infection of dogs and humans.

Click the arrow above to play, or right-click to download TWiV #31