Transgenic pigs resistant to foot-and-mouth disease

Foot-and-mouth disease virus (FMDV) infects cloven-hoofed animals such as cattle, pigs, sheep, goats, and many wild species. The disease caused by this virus is a substantial problem for farmers because infected animals cannot be sold. Transgenic pigs have now been produced which express a short interfering RNA (siRNA) and consequently have reduced susceptibility to infection with FMDV.

FMDV is classified in the picornavirus family which also contains poliovirus and rhinoviruses. The virus is highly contagious and readily spreads long distances via wind currents, and among animals by aerosols and contact with farm equipment. Infection causes a high fever and blisters in the mouth and on the feet – hence the name of the disease. When outbreaks occur, they are economically devastating. The 2001 FMDV outbreak in the United Kingdom was stopped by mass slaughter of all animals surrounding the affected areas – an estimated 6,131,440 – in less than a year.

Vaccines against the virus can be protective but they are not an optimal solution. One problem is that antigenic variation of the virus may thwart protection. In addition, countries free of FMDV generally do not vaccinate because this practice would make the animals seropositive and prevent their export (it is not possible to differentiate between antibodies produced by natural infection versus immunization). Furthermore, if there were an outbreak of foot-and-mouth disease in such countries, the rapid replication and spread of the virus would make vaccination ineffective – hence culling of animals as described above is required. Clearly other means of protecting animals against FMDV are needed.

Synthetic short interfering RNAs (siRNA) have been shown to block viral replication in cell culture and in animals. To achieve such inhibition, short synthetic RNAs complementary to viral sequences are produced in cells. Upon infection, these siRNAs combine with the cellular RNA-induced silencing complex (RISC) which then targets the viral RNA for degradation.

To determine if siRNA could be used to protect pigs from foot-and-mouth disease, a complementary viral sequence was first identified that blocks FMDV replication in cell culture by ~97%. A vector containing this siRNA sequence was then used to produce transgenic pigs. Such animals not only express the antiviral siRNA, but as the encoding vector is present in germ cells, it is passed on to progeny pigs.

Expression of the siRNA was confirmed in a variety of transgenic pig tissues, including heart, lung, spleen, liver, kidney, and muscle. In fibroblasts produced from transgenic pigs, virus replication was reduced 30 fold. When transgenic pigs were inoculated intramuscularly with FMDV, none of the animals developed signs of disease such as fever or blisters of the feet and nose. In contrast, control non-transgenic pigs developed high fever and lesions. Viral RNA levels in the blood of transgenic pigs were 100-fold lower than in control animals. At 10 days post-infection no viral RNA was detected in heart, lung, spleen, liver, kidney, and muscle, while high levels were observed in these organs from non-transgenic controls.

These results show that siRNAs can protect transgenic pigs from FMDV induced disease. An important question that must be answered is whether transgenic pigs still contain enough virus to transmit infection to other animals. In addition, siRNAs are short – 21 nucleotides – and a mutation in the viral genome can block their inhibitory activity. Therefore it would be important to determine if mutations arise in the FMDV genome that lead to resistance to siRNAs.

Even if transgenic siRNA pigs do not transmit infection, and viral resistance does not arise, I am not sure that consumers are ready to accept such genetically modified animals.

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 140: An aptitude for microbicides

cd4 aptamer

Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and Judy Lieberman

Vincent, Alan, Rich, and Judy Lieberman review the use of CD4 aptamer-siRNA chimeras to inhibit HIV transmission.

Click the arrow above to play, or right-click to download TWiV #140 (110 MB .mp3, 92 minutes).

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Weekly Science Picks

Alan – Inside insides
Rich – Rock stars of science
Vincent – A new open-access journal, and Francis Collins on NIH budget

Listener Pick of the Week

Kathy – Scientists and musicians compare notes (NPR)

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TWiV 138: RISCy business with Raul Andino

Drosophila melanogasterHosts: Vincent Racaniello and Raul Andino

Vincent meets up with Raul Andino in San Francisco to discuss the RNAi-based antiviral defense system of Drosophila, the fruit fly, and how it is antagonized by viruses.

Click the arrow above to play, or right-click to download TWiV #138 (53 MB .mp3, 73 minutes).

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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).

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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

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TWiV 89: Where do viruses vacation?

Hosts: Vincent Racaniello and Alan Dove

On episode #89 of the podcast This Week in Virology, Vincent and Alan review recent findings on the association of the retrovirus XMRV with ME/CFS, reassortment of 2009 pandemic H1N1 influenza virus in swine, and where influenza viruses travel in the off-season.

Click the arrow above to play, or right-click to download TWiV #89 (56 MB .mp3, 78 minutes)

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Weekly Science Picks

AlanTree of Life graphic
Vincent
TEDx Oil Spill

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.