On episode #322 of the science show This Week in Virology, the TWiVodes answer listener email about hantaviruses, antivirals, H1N1 vaccine and narcolepsy, credibility of peer review, Bourbon virus, influenza vaccine, careers in virology, and much more.

You can find TWiV #322 at www.twiv.tv.

prion conversionIf the reader does not believe that viroids and satellites are distinctive, then surely prions, infectious agents composed only of protein, must impress.

The question of whether infectious agents exist without genomes arose with the discovery and characterization of infectious agents associated with a group of diseases called transmissible spongiform encephalopathies (TSEs). These diseases are rare, but always fatal, neurodegenerative disorders that afflict humans and other mammals. They are characterized by long incubation periods, spongiform changes in the brain associated with loss of neurons, and the absence of host responses. TSEs are caused by infectious proteins called prions.

The first TSE recognized was scrapie, so called because infected sheep tend to scrape their bodies on fences so much that they rub themselves raw. Scrapie has been recognized as a disease of European sheep for more than 250 years. It is endemic in some countries, for example, the United Kingdom, where it affects 0.5 to 1% of the sheep population each year.

Sheep farmers discovered that animals from affected herds could pass the disease to a scrapie-free herd, implicating an infectious agent. Infectivity from extracts of scrapie-affected sheep brains was shown to pass through filters with pores small enough to retain everything but viruses. As early as 1966, scrapie infectivity was shown to be considerably more resistant than that of most viruses to ultraviolet (UV) and ionizing radiation. Other TSE agents exhibit similar UV resistance. On the basis of this relative resistance to UV irradiation, some investigators argued that TSE agents are viruses well shielded from irradiation, whereas others claimed that TSE agents have little or no nucleic acids.

Several lines of evidence indicated that human spongiform encephalopathies might be caused by an infectious agent. Carleton Gajdusek and colleagues studied the disease kuru, found in the Fore people of New Guinea. This disease is characterized by cerebellar ataxia (defective motion or gait) without loss of cognitive functions. Kuru spread among women and children as a result of ritual cannibalism of the brains of deceased relatives. When cannibalism stopped in the late 1950s, kuru disappeared. Others observed that lesions in the brains of humans with kuru were similar to lesions in the brains of animals with scrapie. It was soon demonstrated that kuru and other human TSEs can be transmitted to chimpanzees and laboratory animals.

Human spongiform encephalopathies are placed into three groups: infectious, familial or genetic, and sporadic, distinguished by how the disease is acquired initially. An infectious (or transmissible) spongiform encephalopathy is exemplified by kuru and iatrogenic spread of disease to healthy individuals by transplantation of infected corneas, the use of purified hormones, or transfusion with blood from patients with the TSE Creutzfeldt-Jakob disease (CJD). Over 400 cases of iatrogenic Creutzfeldt-Jakob disease have been reported worldwide. The epidemic spread of bovine spongiform encephalopathy (mad cow disease, see below) among cattle in Britain can be ascribed to the practice of feeding processed animal by-products to cattle as a protein supplement. Similarly, the new human disease, variant CJD, arose after consumption of beef from diseased cattle. Sporadic CJD is a disease affecting one to five per million annually, usually late in life (with a peak at 68 years). As the name indicates, the disease appears with no warning or epidemiological indications. Kuru may have been originally established in the small population of Fore people in New Guinea when the brain of an individual with sporadic CJD was eaten. Familial spongiform encephalopathy is associated with an autosomal dominant mutation in the prnp gene. Together familial and sporadic forms of prion disease account for ~99% of all cases.

Clinical signs of infection commonly include cerebellar ataxia, memory loss, visual changes, dementia, and akinetic mutism, with death occurring after months or years. Once the infectious agent is in the central nervous system, the characteristic pathology includes severe astrocytosis, vacuolization (hence the term spongiform), and loss of neurons. There are no inflammatory, antibody, or cellular immune responses.

The unconventional physical attributes and slow infection pattern originally prompted many to argue that TSE agents are not viruses at all. In 1967 it was suggested that scrapie could be caused by a host protein, not by a nucleic acid-carrying virus.

An important breakthrough occurred in 1981, when characteristic fibrillar protein aggregates were visualized in infected brains. These aggregates could be concentrated by centrifugation and remained infectious. Stanley Prusiner and colleagues isolated a protein with unusual properties from scrapie-infected tissue. This protein is insoluble and relatively resistant to proteases. He named the scrapie infectious agent a prion, from the words protein and infectious.

Prusiner’s unconventional proposal was that an altered form of a normal cellular protein, called PrPC, causes the fatal encephalopathy characteristic of scrapie. This controversial protein-only hypothesis caused a firestorm among those who study infectious disease. The hypothesis was that the essential pathogenic component is the host-encoded PrPC protein with an altered conformation, called PrPsc (“PrP-scrapie”). Furthermore, in the simplest case, PrPSc was proposed to have the property of converting normal PrPC protein into more copies of the pathogenic form (illustrated). In recognition of his work on prions, Prusiner was awarded the Nobel Prize in physiology or medicine in 1997.

Sequence analysis of this protein led to the identification of the prnp gene, which is highly conserved in the genomes of many mammals, including humans. Expression of the prnp gene is now known to be essential for the pathogenesis of TSEs. The prnp gene encodes a 35-kDa membrane-associated neuronal glycoprotein, PrPC. The function of this protein has been difficult to determine because mice lacking both copies of the prnp gene develop normally and have few obvious defects. However, these mice are resistant to TSE infection, showing that PrPC is essential for prion propagation.

The discovery of the prnp gene has helped explain the basis of familial TSE diseases such as Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, and fatal familial insomnia. Gerstmann-Straussler-Scheinker disease is associated with a change at PrPC amino acid 102 from proline to leucine. Introduction of this amino acid change into mice gives rise to a spontaneous neurodegenerative disease characteristic of a TSE. Familial Creutzfeldt-Jakob disease may be associated with an insertion of 144 base pairs at codon 53, or changes at amino acids 129, 178, or 200. In fatal familial insomnia, adults develop a progressive sleep disorder and typically die within one year. Development of this disease is strongly linked to the D178N amino acid change.

In the mid 1980s, a new disease appeared in cows in the United Kingdom: bovine spongiform encephalopathy, also called mad cow disease. It is believed to have been transmitted to cows by feeding them meat and bone meal, a high protein supplement prepared from the offal of sheep, cattle, pigs, and chicken. In the late 1970s the method of preparation of meat and bone meal was changed, resulting in material with a higher fat content. It is believed that this change allowed prions, from either a diseased sheep or cow, to retain infectivity and pass on to cattle. Before the disease was recognized in 1985, it was amplified by feeding cows the remains of infected bovine tissues: the incubation period for bovine spongiform encephalopathy is 5 years, but disease was not observed because most cattle are slaughtered between 2-3 years of age. Three years later, as the number of cases of mad cow disease increased, a ban on the use of meat and bone meal was put in place, a practice that together with culling infected cattle has stopped the epidemic. Over 180,000 cattle, mostly dairy cows, died of bovine spongiform encephalopathy from 1986-2000.

Cases of variant Creutzfeld-Jakob disease, a new TSE of humans, began to appear in 1994 in Great Britain. These were characterized by a lower mean age of the patients (26 years), longer duration of illness, and differences in other clinical and pathological characteristics. The results of epidemiologic and experimental studies indicate that variant Creutzfeld-Jakob disease is caused by prions transmitted by the consumption of cattle with bovine spongiform encephalopathy. As of 2011 there had been 175 cases of variant Creutzfeld-Jakob disease in the United Kingdom, and 215 globally.

Bovine spongiform encephalopathy continues to be detected in cattle. As of April 2012, 4 cases have been identified in the United States and 19 in Canada. These cases may arise sporadically, or through consumption of contaminated feed. Because cattle are slaughtered before disease symptoms are evident, there is concern that variant Creutzfeldt-Jakob might increase as contaminated meat enters the food supply. These concerns are being addressed by imposing bans on animal protein-containing feed, and increased surveillance of cows for the disease, for which diagnostic tests are being developed.

Chronic wasting disease is a transmissible spongiform encephalopathy of cervids such as deer, elk, and moose. It is the only known TSE to occur in free-ranging animals. The disease has been reported in the United States, Canada, and South Korea. In captive herds in the US and Canada up to 90% of mule deer and 60% of elk are infected, and the incidence in wild cervids is as high as 15%. Hunters are advised not to shoot or consume an elk or deer that is acting abnormally or appears to be sick, to avoid the brain and spinal cord when field dressing game, and not to consume brain, spinal cord, eyes, spleen, or lymph nodes. No case of transmission of chronic wasting disease prions to deer hunters has yet been reported.

It is not known how the disease is spread among cervids, but transmission by grass contaminated with saliva and feces is one possibility. 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. A concern is that prions of chronic wasting disease could be transmitted to cows grazing in pastures contaminated by cervids.

Since prions were discovered it has become clear that they cause a wider spectrum of neurodegenerative diseases. For example, the amyloid fibrils in Alzheimer’s disease contain the amyloid-beta peptide that is processed from the amyloid precursor protein; familial disease is caused by mutations in the gene for this protein. Mutations in the tau gene are responsible for heritable tauopathies including familial frontotemporal dementia and inherited progressive supranuclear palsy. Self-propagating tau aggregates pass from cell to cell. The prion-like spread of misfolded alpha-synuclein is believed to be involved in Parkinson’s disease. In these cases there is good evidence that the causative protein, like PrPSc, adopts a conformation that becomes self-propagating.

Despite the involvement of prions in human neurological diseases, in other organisms such proteins are not pathogenic but rather impart diverse functions through templated conformational change of a normal cellular protein. Such prions have been described in fungi where they do not form infectious particles and do not spread from cell to cell. These proteins change conformation in response to an environmental stimulus and acquire a new, beneficial function. An example is the Saccharomyces cerevisiae Ure2p protein, which normally is a nitrogen catabolite repressor when cells are grown in the presence of a rich source of nitrogen. In the aggregated prion state, called [URE3], the protein allows growth on poor nitrogen sources. These findings prompt the question of whether the conversion of PrPC to PrPSC once had a beneficial function that became pathogenic. If so, identifying that function, and how it was usurped, will be important for understanding the pathogenesis of transmissible spongiform encephalopathies.

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TWiV 321: aTRIP and a pause

25 January 2015

On episode #321 of the science show This Week in Virology, Paul Duprex joins the TWiV team to discuss the current moratorium on viral research to alter transmission, range and resistance, infectivity and immunity, and pathogenesis.

You can find TWiV #321 at www.twiv.tv.

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.

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On episode #320 of the science show This Week in Virology, Vincent speaks with John Coffin about his career studying retroviruses, including working with Howard Temin, endogenous retroviruses, XMRV, chronic fatigue syndrome and prostate cancer, HIV/AIDS, and his interest in growing cranberries.

You can find TWiV #320 at www.twiv.tv.

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.

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On episode #319 of the science show This Week in Virology, the TWiVers review the outcomes of two recent phase 3 clinical trials of a quadrivalent dengue virus vaccine in Asia and Latin America.

You can find TWiV #319 at www.twiv.tv.

Poliovirus by Jason Roberts

Poliovirus by Jason Roberts

The polio eradication and endgame strategic plan announced by the World Health Organization in 2014 includes at least one dose of inactivated poliovirus vaccine (IPV). Since 1988, when WHO announced the polio eradication plan, it had relied exclusively on the use of oral poliovirus vaccine (OPV). The rationale for including a dose of IPV was to avoid outbreaks of vaccine-derived type 2 poliovirus. This serotype had been eradicated in 1999 and had consequently been removed from OPV. However IPV, which is injected intramuscularly and induces highly protective humoral immunity, is less effective in producing intestinal immunity than OPV. This property was underscored by the finding that wild poliovirus circulated in Israel during 2013, a country which had high coverage with IPV. Furthermore, in countries that use only IPV, over 90% of immunized children shed poliovirus after oral challenge. I have always viewed this shortcoming of IPV as problematic, in view of the recommendation of the World Health Organization to gradually shift from OPV to IPV. Even if the shift to IPV occurs after eradication of wild type polioviruses, vaccine-derived polioviruses will continue to circulate because they cannot be eradicated by IPV. My concerns are now mitigated by new results from a study in India which indicate that IPV can boost intestinal immunity in individuals who have already received OPV.

To assess the ability of IPV to boost mucosal immunity, 954 children in three age groups (6-11 months, 5 and 10 years) were immunized with IPV, bivalent OPV (bOPV, containing types 1 and 3 only), or no vaccine. Four weeks later all children were challenged with bOPV, and virus shedding in the feces was determined 0, 3, 7, and 14 days later. The results show that 8.8, 9.1, and 13.5% of children in the 6-11 month, 5-year and 10-year old groups shed type 1 poliovirus in feces, compared with 14.4, 24.1, and 52.4% in the control group. Immunization with IPV reduced fecal shedding of poliovirus types 1 (39-74%) and 3 (53-76%). The reduction of shedding was greater after immunization with IPV compared with bOPV.

This study shows that a dose of IPV is more effective than OPV at boosting intestinal immunity in children who have previously been immunized with OPV. Both IPV and OPV should be used together in the polio eradication program. WHO therefore recommends the following vaccine regimens:

  • In all countries using OPV only, at least 1 dose of type 2 IPV should be added to the schedule.
  • In polio-endemic countries and in countries with a high risk for wild poliovirus importation and spread: one OPV birth dose, followed by 3 OPV and at least 1 IPV doses.
  • In countries with high immunization coverage (90-95%) and low wild poliovirus importation risk: an IPV-OPV sequential schedule when VAPP is a concern, comprising 1-2 doses of IPV followed by 2 or mores doses of OPV.
  • In countries with both sustained high immunization coverage and low risk of wild poliovirus importation and transmission: an IPV only schedule.

Type 2 OPV will be gradually removed from the global immunization schedules. There have been no reported cases of type 3 poliovirus since November 2012. If this wild type virus is declared eradicated later this year, presumably WHO will recommend withdrawal of type 3 OPV and replacement with type 3 IPV.

All 342 confirmed cases of poliomyelitis in 2014 were caused by type 1 poliovirus in 9 countries, mainly Pakistan and Afghanistan. Given the social and political barriers to immunization, it will likely take many years to eradicate this serotype.

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On episode #318 of the science show This Week in Virology, the TWiV gang reviews ten fascinating, compelling, and riveting virology stories from 2014.

You can find TWiV #318 at www.twiv.tv.

TWiV 317: Brazil goes viral

28 December 2014

On episode #317 of the science show This Week in Virology, Vincent travels to Brazil and joins Eurico to speak with three four young virologists, Gustavo, Cintia, Tatiana, and Suellen, about their work and their prospects for careers in science.

You can find TWiV #317 at www.twiv.tv.