TWiV 403: It’s not easy being vaccine

The TWiV team takes on an experimental plant-based poliovirus vaccine, contradictory findings on the efficacy of Flumist, waning protection conferred by Zostavax, and a new adjuvanted subunit zoster vaccine.

You can find TWiV #403 at, or listen below.

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TWiV 343: The silence of the turnips

On episode #343 of the science show This Week in Virology, the TWiVerinoes discuss the potential for prion spread by plants, global circulation patterns of influenza virus, and the roles of Argonautes and a viral protein in RNA silencing in plants.

You can find TWiV #343 at

Prions in plants

prions in plants

Chronic wasting disease is a prion disease of cervids (deer, elk, moose) that is potentially a threat to human health. A role for environmental prion contamination in transmission is supported by the finding that plants can take up prions from the soil and transmit them to animals.

A concern is that prions of chronic wasting disease could be transmitted to cows grazing in pastures contaminated by cervids. Consumption of infected cows would then pass the disease on to humans. When deer are fed prions they excrete them in the feces before developing clinical signs of infection, and prions can also be detected in deer saliva. In the laboratory, brain homogenates from infected deer can transmit the disease to cows.

To determine whether prions can enter plants, wheat grass roots and leaves were exposed to brain homogenates from hamsters that had died of prion disease. The plant materials were then washed and amounts of prions were determined by protein misfolding cyclic amplification. Prions readily bound these plant tissues, at low concentrations and after as little as 2 minutes of incubation. Mouse, cervid, and human prions also bound to plant roots and leaves. When living wheat grass leaves were sprayed with a 1% hamster brain homogenate, prions could attach to the leaves and be detected for 49 days.

To determine if prions in plants could infect animals, plants were exposed to brain homogenates, washed thoroughly, and then fed to hamsters. The positive control for this experiment was to feed hamsters the brain homogenates. All animals fed infected plants or brain homogenates succumbed to prion disease.

Plants can also take up prions from animal waste. This conclusion was reached by incubating leaves and roots for 1 hour with urine or feces obtained from prion-infected hamsters or cervids. Prions were readily detected in these samples, even after extensive washing.

Experiments were also done to examine whether plants could take up prions from the soil. Barley grass plants were grown on soil that had been mixed with hamster brain homogenate, and then 1-3 weeks later, stem and leaves were assayed for the presence of prions. Small amounts of prions were detected in stems from all plants, while 1 in 4 plants contained prions in leaves, at levels that should be able to infect an animal.

These results show that prions can bind to plants and be taken into the roots, where they may travel to the stem and leaves. Therefore it is possible that prions excreted by deer could pass on to other animals, such as grazing cows, or even humans consuming contaminated plants (illustrated – image credit). Cooking plants will not eliminate infectivity, just as cooking contaminated beef did not halt the spread of bovine spongiform encephalopathy. Keeping cervids out of grazing or growing fields should be considered as a way to manage the risk of prions entering the human food chain.

Satellites – the viral kind

Hepatitis delta satellite genomeSatellites are subviral agents that differ from viroids because they depend on the presence of a helper virus for their propagation. Satellite viruses are particles that contain nucleic acid genomes encoding a structural protein that encapsidates the satellite genome. Satellite RNAs do not encode capsid protein, but are packaged by a protein encoded in the helper virus genome. Satellite genomes may be single-stranded RNA or DNA or circular RNA, and are replicated by enzymes provided by the helper virus. The origin of satellites remains obscure, but they are not derived from the helper virus.

Satellite viruses may infect plants, animals, or bacteria. An example of a satellite virus is satellite tobacco necrosis virus, which encodes a capsid protein that forms an icosahedral capsid that packages only the 1,260 nucleotide satellite RNA. The helper virus, tobacco necrosis virus, encodes an RNA polymerase that replicates its genome and that of the satellite.

Satellite RNAs do not encode a capsid protein and therefore require helper virus proteins for both genome encapsidation and replication. Satellite RNA genomes range in length from 220-1500 nucleotides, and have been placed into one of three classes. Class 1 satellite RNAs are 800-1500 nucleotide linear molecules with a single open reading frame encoding at least one non-structural protein. Class 2 satellite RNAs are linear, less than 700 nucleotides long and do not encode protein. Class 3 satellite RNAs are 350-400 nucleotide long circles without an open reading frame.

In plants, satellites and satellite viruses may attenuate or exacerbate disease caused by the helper virus. Examples of disease include necrosis and systemic chlorosis, or reduced chlorophyll production leading to leaves that are pale, yellow, or yellow-white. The symptoms induced by satellite RNAs are thought to be a consequence of silencing of host genes. For example, the Y-satellite RNA of cucumber mosaic virus causes systemic chlorosis in tobacco. This syndrome is caused by production of a small RNA from the Y-satellite RNA that has homology to a gene needed for chlorophyll biosynthesis. Production of this small RNA leads to degradation of the corresponding mRNA, causing the bright yellow leaves.

The giant DNA viruses including Acanthamoeba polyophaga mimivirus, Cafeteria roenbergensis virus, and others are associated with much smaller viruses (sputnik and mavirus, respectively) that depend upon the larger viruses for reproduction. For example, sputnik virus can only replicate in cells infected with mimivirus, and does so within viral factories. Whether these are satellite viruses or something new (they have been called virophages) has been a matter of controversy.

Like satellite viruses, sputnik and others have similar relationships with their helper viruses: they require their helper for their propagation, but their genomes are not derived from the helper, and they negatively impact helper reproduction. Others argue that the definition of satellite viruses as sub-viral agents cannot apply to these very large viruses. For example, sputnik virophage contains a circular dsDNA genome of 18,343 bp encoding 21 proteins encased in a 75 nm t=27 icosahedral capsid. Sputnik is dependent upon mimivirus not for DNA polymerase – it encodes its own – but probably for the transcriptional machinery of the helper virus. Those who favor the name virophage argue that dependence upon the cellular transcriptional machinery is a property of many autonomous viruses – the only difference is that Sputnik depends upon the machinery provided by another virus. It seems likely that a redefinition of what constitutes a satellite virus will be required to solve this disagreement.

Most known satellites are associated with plant viruses, but hepatitis delta satellite virus is associated with a human helper virus, hepatitis B virus. The genome (illustrated) is 1.7 kb – the smallest of any known animal virus – of circular single-stranded RNA that is 70% base paired and folds upon itself in a tight rod-like structure. The RNA molecule is replicated by cellular RNA polymerase II. These properties resemble those of viroid genomes. On the other hand, the genome encodes a protein (delta) that encapsidates the RNA, a property shared with satellite nucleic acids. The hepatitis delta satellite virus particle comprises the satellite nucleocapsid packaged within an envelope that contains the surface protein of the helper, hepatitis B virus.

Infection with hepatitis delta satellite virus only occurs in individuals infected with hepatitis B virus: it is globally distributed, present in about 5% of the 350 million carriers of hepatitis B virus. Acute co-infections of the two viruses can be more severe than infection with hepatitis B virus alone, leading to more cases of liver failure. In chronic hepatitis B virus infections, hepatitis delta satellite virus aggravates pre-existing liver disease, and may lead to more rapid progression to cirrhosis and death than monoinfections. Why co-infection with both viruses leads to more serious outcomes is not known.

Viroids, infectious agents that encode no proteins

potato spindle tuber viroidGenomes of non-defective viruses range in size from 2,400,000 bp of dsDNA (Pandoravirus salinus) to 1,759 bp of ssDNA (porcine circovirus). Are even smaller viral genomes possible? The subviral agents called viroids provide an answer to this question.

Viroids, the smallest known pathogens, are naked, circular, single-stranded RNA molecules that do not encode protein yet replicate autonomously when introduced into host plants. Potato spindle tuber viroid, discovered in 1971, is the prototype; 29 other viroids have since been discovered ranging in length from 120 to 475 nucleotides. Viroids only infect plants; some cause economically important diseases of crop plants, while others appear to be benign. Two examples of economically important viroids are coconut cadang-cadang viroid (which causes a lethal infection of coconut palms) and apple scar skin viroid (which causes an infection that results in visually unappealing apples).

The 30 known viroids have been classified in two families. Members of the Pospiviroidae, named for potato spindle tuber viroid, have a rod-like secondary structure with small single stranded regions, a central conserved region, and replicate in the nucleus (illustrated; click to enlarge; figure credit). The Avsunviroidae, named for avocado sunblotch viroid, have both rod-like and branched regions, but lack a central conserved region and replicate in chloroplasts. In contrast to the Pospiviroidae, the latter RNA molecules are functional ribozymes, and this activity is essential for replication.

There is no evidence that viroids encode proteins or mRNA. Unlike viruses, which are parasites of host translation machinery, viroids are parasites of cellular transcription proteins: they depend on cellular RNA polymerase for replication. Such polymerases normally recognize DNA templates, but can copy viroid RNAs.

In plants infected with members of the Pospiviroidae, viroid RNA is imported into the nucleus, and copied by plant DNA-dependent RNA polymerase II. The viroid is copied by a rolling circle mechanism that produces complementary linear, concatameric, RNAs. These are copied again to produce concatameric, linear molecules, which are cleaved by the host enzyme RNAse III. Their ends are joined by a host enzyme to form circles.

In plants infected with members of the Avsunviroidae, viroid RNA is imported into the chloroplast, and complementary concatameric RNAs are produced by chloroplast DNA-dependent RNA polymerase. Cleavage of these molecules is carried out by a ribozyme, an enzyme encoded in the viroid RNA.

After replication, viroid progeny exit the nucleus or chloroplast and move to adjacent cells through plasmodesmata, and can travel systemically via the phloem to infect other cells. Viroids enter the pollen and ovule, from where they are transmitted to the seed. When the seed germinates, the new plant becomes infected. Viroids can also be transmitted among plants by contaminated farm machinery and insects.

Symptoms of viroid infection in plants include stunting of growth, deformation of leaves and fruit, stem necrosis, and death. Because viroids do not produce mRNAs, it was first proposed that disease must be a consequence of viroid RNA binding to host proteins or nucleic acids.  The discovery of RNA silencing in plants lead to the hypothesis that small interfering RNAs derived from viroid RNAs guide silencing of host genes, leading to induction of disease. In support of this hypothesis, peach latent mosaic viroid small RNAs have been identified that silence chloroplast heat shock protein 90, which correlates with disease symptoms. The different disease patterns caused by viroids in their hosts might all have in common an origin in RNA silencing.

Our current understanding is that the disease-causing viroids were transferred from wild plants used for breeding modern crops. The widespread prevalence of these agents can be traced to the use of genetically identical plants (monoculture), worldwide distribution of breeding lines, and mechanical transmission by contaminated farm machinery. As a consequence, these unusual pathogens now occupy niches around the planet that never before were available to them.

The origin of viroids remains an enigma, but it has been proposed that they are relics from the RNA world, which is thought to have been populated only by non-coding RNA molecules that catalysed their own synthesis. Viroids have properties that make them candidates for survivors of the RNA world: small genome size (to avoid error catastrophe caused by error-prone replication), high G+C content (for greater thermodynamic stability), circular genomes (to avoid the need for mechanisms to prevent loss of information at the ends of linear genomes), no protein content, and the presence of a ribozyme, a fingerprint of the RNA world. Today’s viroids can no longer self-replicate, possibly having lost that function when they became parasites of plants. What began as a search for virus-like agents that cause disease in plants has lead to new insights into the evolution of life.

TWiV 272: Give peas a chance

On episode #272 of the science show This Week in Virology, the TWiV team describes aphid control by using a viral capsid protein to deliver a spider toxin to plants, and a human endogenous retrovirus that enhances expression of a neuronal gene.

You can find TWiV #272 at

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

Tulips broken by viruses

three broken tulipsA consequence of the recent warm weather in the northeastern United States is the emergence of crocuses, an event that I documented at the TWiV Facebook page. A reader replied that it reminded her of the highly valued tulips with beautiful variegations produced by viruses.

In 17th-century Holland patterned tulips such as the Semper Augustus (image) were of enormous value, with single bulbs selling for 3000 guilders or more (about $1600 US today). The intricate lines and flame-like streaks produced stunning effects. We now know that these colorful patterns are caused by infection with potyviruses, which are filamentous plant viruses with positive-strand RNA genomes. The specific viruses involved are tulip-breaking virus, tulip top-breaking virus, tulip bandbreaking virus, and Rembrandt tulip-breaking virus. Lilies may also be patterned by infection with Lily mottle virus. These viruses infect the bulb and cause the single color to break, leading to bars, stripes, streaks, featherings or flame-like effects of different colors on the petals. These effects are caused by altered distribution of pigments in the petal caused by virus replication.

Unfortunately, infection with tulip-breaking viruses is not benign: with successive generations the bulb shrinks until it can no longer flower. For this reason most of the lines of broken tulips, including Semper Augustus, no longer exist. These viruses still circulate globally, transmitted by aphids. Because infection can cause costly damage to tulips, precautions must be taken to minimize spread. Contemporary variegated tulips such as Rem’s Sensation are produced by breeding, not virus infection.

TWiV 48: Outbreaks near you

twiv_aa_2001Hosts: Vincent Racaniello, Dick DespommierAlan Dove, and Rich Condit

On episode 48 of the podcast “This Week in Virology”, Vincent, Dick, Alan and Rich revisit a vaccinia virus lab accident and viral vaccines produced in plants, then talk about an iPhone app to track infectious diseases, flying foxes, and an inhaled measles vaccine.

Click the arrow above to play, or right-click to download TWiV #48 (58 MB .mp3, 79 minutes)

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

Links for this episode:
Laboratory acquired vaccinia infection
Medicago (investor presentation, pdf) and Fraunhofer produce vaccines in plants
iPhone app Outbreaks near me
No culling of flying foxes in Australia
Inhaled powdered measles virus vaccine
Herpesvirus latency confers symbiotic protection from bacterial infection (thanks Juliet!)
Porcine circovirus vaccine
Swine flu vaccine and Guillain-Barré (thanks Tom!)
The Great Flu and blog post (thanks Swiss compass and Allison!)
Building semiconductors with DNA (thanks Duncan!)
Email on viral classification (thanks Rodney and Eliot!)

Weekly Science Picks
Rich Stumbling on Happiness by Daniel Gilbert
Dick Eliot Porter at the Carter Museum and bookstore at Amazon
Alan Dr. Clarke’s H1N1 rap at the HHS sponsored YouTube contest
Vincent Coast to Coast Bio Podcast

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

TWiV 47: Vertical vaccine farm

twiv-200Hosts: Vincent Racaniello and Dick Despommier

On episode #47 of the podcast “This Week in Virology”, Vincent and Dick discuss influenza virus-like particle vaccines produced in insect and plant cells, rapid sharing of influenza research, and answer listener questions about cytomegalovirus, viral evolution and symbiosis and much more.

Click the arrow above to play, or right-click to download TWiV #47 (51 MB .mp3, 71 minutes)

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

Links for this episode:
A Farm on Every Floor
Influenza virus-like particles in insect and plant cells
PLoS Currents: Influenza
Transmission of 2009 H1N1 influenza virus to turkeys [Thanks Debbie!]
Baxter produces Vero cell H1N1 vaccine [Thanks Peter!]
Boundaries of Darwinism podcast [Thanks David!]
Phages in human intestine: papers onetwothree [Thanks Terry!]
Post-exposure varicella vaccine [Thanks Patricia!]
Open science movement hereherehere, and here [Thanks Jim!]
Graduate programs in virology [Thanks Greggory and Blake!]
Post-exposure Marburg and Ebola vaccines [Thanks John!]
Vaccinia infection in the laboratory [Thanks Russ!]
Animations of bacteriophage T4 life cycle [Thanks Jim!]

Weekly Science Picks
Vincent Bionumbers
Dick Ocean: An Illustrated Atlas by Sylvia A. Earle, Linda K. Glover

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