TWiV 389: Alphabet hepatitis with Stan Lemon

TWiVVincent speaks with Stan Lemon about his career in virology, from early work on Epstein Barr virus, through making essential discoveries about hepatitis A virus, hepatitis C virus, and rhinoviruses, on episode #389 of the science show This Week in Virology.

You can find TWiV #389 at microbe.tv/twiv, or listen below.

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TWiV 365: Blood, feuds, and a foodborne disease

On episode #365 of the science show This Week in Virology, Vincent, Alan, and Kathy trace the feud over genome editing, a new virus discovered in human blood, and the origins of hepatitis A virus.

You can find TWiV #365 at www.microbe.tv/twiv.

A protein platform for priming

Priming RNA synthesisThe enzymes that make copies of the DNA or RNA genomes of viruses – nucleic acid polymerases – can be placed into two broad categories depending on whether or not they require a primer, a short piece of DNA or RNA, to get going. The structure of the primer-independent RNA polymerase of hepatitis C virus reveals how a priming platform is built into the enzyme.

The requirement for a primer in the initiation step of nucleic acid synthesis varies among the different classes of polymerases. All DNA polymerases are primer-dependent enzymes, while DNA-dependent RNA polymerases initiate RNA synthesis de novo – without a primer. Some RNA-dependent RNA polymerases can also initiate RNA synthesis without a primer: the enzyme begins by adding the first base complementary to the template RNA (illustrated). Other RNA-dependent RNA polymerases require a primer to initiate synthesis. Examples shown on the illustration include the protein-linked primer of picornaviruses, which consists of the protein VPg covalently attached to two U residues. The primer for influenza virus mRNA synthesis is a capped oligonucleotide 12-14 bases in length that is cleaved from the 5′ end of cellular mRNA.

The structure of the RNA-dependent RNA polymerase of hepatitis C virus reveals how a primer-independent RNA polymerase positions the first nucleotide on the RNA template. This process is illustrated below. With the RNA template (dark green) in the active site of the enzyme (panel A), a short beta-loop (brown) provides a platform on which the first complementary nucleotide (light green) is added to the template. The second nucleotide is then added (panel B), producing a dinucleotide primer for RNA synthesis. At this point nothing further can happen because  the priming platform blocks the exit of the RNA product from the enzyme (panel B). The solution to this problem is that the polymerase undergoes a conformational change that moves the priming platform out of the way and allows the newly synthesized complementary RNA (panel C, light green) to exit as the enzyme moves along the template strand.

 HCV priming of RNA synthesis

The structure of the RNA polymerase of hepatitis C virus reveals that it is not really a primer-independent enzyme: a dinucleotide primer is synthesized by the polymerase using a protein platform in the active site. Such protein platforms also appear to be involved in the priming of RNA synthesis by other flaviviruses (dengue and West Nile viruses), influenza virus (genome RNA synthesis is primer independent), reovirus, and bacteriophage phi6. Perhaps all viral RNA-dependent RNA polymerases are dependent on such priming platforms to initiate RNA synthesis.

Covering up a naked virus

Sabin type 2 poliovirusViruses can be broadly classified according to whether or not the particle is enveloped – surrounded by a membrane taken from the host cell – or naked. Some naked viruses apparently are more modest than we believed.

Members of the family Picornaviridae, which include Hepatitis A virus, poliovirus, and Coxsackieviruses, have non-enveloped particles that consist of a protein shell surrounding the viral RNA genome (poliovirus is illustrated). Examples of viruses that are enveloped include dengue virus, influenza virus, and measles virus.

Recently it was discovered that hepatitis A virus (HAV) particles are released from cells in membrane vesicles containing 1-4 virus particles. These membranous structures resemble exosomes, which are also released from uninfected cells and play roles in various biological processes. Enveloped hepatitis A virus particles are present in the blood of infected humans. However virus in the feces, which is transmitted to other hosts, is not enveloped.

Viral envelopes typically contain viral glycoproteins, such as the HA protein of influenza viruses, which serve important functions during replication, such as attachment to cell receptors. Envelope glycoproteins are also the target of antibodies that block viral infection. The presence of an envelope makes HAV resistant to neutralization with antibodies, because the membrane contains no viral proteins that can be blocked by antibodies.

Two other non-enveloped picornaviruses, Coxsackievirus B and poliovirus, are also released from cells within membrane vesicles. These virus particles are in vesicles derived from the autophagy pathway, which captures and recycles cytoplasmic contents by ejecting them from the cell.

What is the function of the membrane acquired by these naked viruses? Perhaps immune evasion: the presence of the cell membrane makes HAV and Coxsackievirus B virus particles resistant to neutralization with antibody. The ability to deliver multiple virus particles to a single cell might help to overcome genetic defects in the viral genome that are a consequence of the high mutation rates of these viruses.

An interesting problem is how these cloaked viruses enter cells, because there is no evidence that the membranes contain any viral proteins that could interact with a cell receptor. Nevertheless, entry of enveloped HAV and poliovirus into cells requires the known viral receptor. Perhaps the vesicles are taken into the cell by endocytosis, where viral particles are released from the vesicles, and then bind receptors to initiate escape of the genome.

Should HAV, poliovirus, and Coxsackievirus B be reclassified as enveloped viruses? Probably not, in part because the membranes surrounding these virus particles are not needed for infectivity. In contrast, removal of the membrane from influenza virus, dengue virus, or measles virus destroys their infectivity. Enveloped viruses acquire a membrane after the internal components have been assembled, whether they are helical or icosahedral nucleocapsids. In contrast, HAV, poliovirus, and Coxsackievirus B become fully infectious particles before they acquire an envelope.

Another argument against calling picornaviruses enveloped is that viral membranes contain viral glycoproteins that allow attachment to cell receptors and release of the viral genome into the cell. There is no evidence that the membranes of picornaviruses contain viral proteins.

The acquisition of a membrane may have taken place later in the evolution of picornaviruses, to allow more efficient infection or evasion of host responses. Alternatively, the membrane may simply be a by-product acquired when these viruses exit the cell by a non-lytic mechanism.

While the finding of membranes around picornavirus particles is intriguing, I am not yet convinced that these viruses should be considered to be enveloped. I would like to know if other non-enveloped viruses are similarly released from cells in membranous cloaks, and the function of this addition for viral replication in the host.

TWiV 274: Data dump

On episode #274 of the science show This Week in Virology, the TWiV team discusses recent cases of polio-like paralysis in California, and the virome of 14th century paleofeces.

You can find TWiV #274 at www.microbe.tv/twiv.

Hepatitis A virus infections associated with berry and pomegranate mix

HAV epi-curveAn outbreak of hepatitis A virus (HAV) infections in eight US states has been attributed to consumption of Townsend Farms Organic Anti-Oxidant Blend frozen berry and pomegranate mix purchased from Costco markets. Since March 2013, 118 individuals have acquired the infection and 80% report having eaten this fruit product. Townsend Farms has recalled some lots of this product because, according to the producer, one of the ingredients of the blend, “pomegranate seeds processed in Turkey, may be linked to an illness outbreak outside of the United States”. The Food and Drug Administration will soon begin testing for the presence of hepatitis A virus in the berry mix.

Hepatitis A virus is a member of the picornavirus family, which also contains poliovirus and rhinovirus. The virion is a naked, icosahedral particle containing a single strand of positive-sense RNA. Infection is typically acquired by ingestion of food contaminated by feces containing the virus. In one scenario, food is contaminated by an HAV-infected food handler who does not practice good hand hygiene. After ingestion, the virus enters the gastrointestinal tract and then passes to the blood. It then replicates in the liver leading to jaundice and elevated serum levels of liver enzymes. After replicating in the liver, HAV passes into the intestine via the bile canaliculi and is then shed in feces. The incubation period of the disease is on average 4 weeks, but infectious particles are present in feces about two weeks before the onset of clinical symptoms. The virus is most likely to be transmitted during this period as the infected individual does not display clinical symptoms.

There are six genotypes of HAV that circulate worldwide. According to the CDC, the virus strain causing this outbreak is HAV genotype 1B, which is often found in North Africa and the Middle East but is rarely isolated in the Americas. Genotype 1b caused a 2013 European outbreak linked to frozen berries, and a 2012 British Columbia outbreak in which a frozen berry blend was implicated. In the BC outbreak the berry blend contained pomegranate seeds from Egypt. This is likely why Townsend Farms recalled the berry mix when they found that it contained pomegranate seeds processed in Turkey.

A well-known outbreak of hepatitis A infection took place in November 2003 among the patrons of a single Pennsylvania restaurant. In this outbreak of 601 patients, 3 died and 124 were hospitalized. Infection was caused by consumption of salsa which had been prepared with green onions grown in northern Mexico that were contaminated with hepatitis A virus.

Transmission of HAV infection can be prevented by proper hand hygiene, avoiding hand contamination with fecal matter, or by using available vaccines.

PS if you buy whole pomegranates, don’t miss this video by the Produce Picker on how to cut them.

Happy as a clam? Maybe not.

oystersThis article was written for extra credit by a student in my recently concluded virology course.

by Adriana Lopez

The expression “Happy as a Clam” comes with new meaning as hepatitis A virus has been detected in clams, mussels, and oysters in markets for human consumption. As bivalve shellfish are excellent bio-accumulators of contaminants and chemicals, it is no surprise that they also harbor waterborne viruses such as hepatitis A in areas with poor sanitation. Since hepatitis A virus is spread via the fecal-oral route, food-borne outbreaks due to ingestion of shellfish harvested from polluted waters have not been uncommon.

Despite development of an effective vaccine against hepatitis A virus, it continues to be a serious disease worldwide. In developing countries, access to healthcare and vaccination may not be available and many remain susceptible to infection. Eating raw or undercooked shellfish may pose a serious food safety threat to those unvaccinated in the event the mollusks are infected. Though many people have inapparent infections at a young age and acquire immune memory to hepatitis A, infections in adults can be quite severe and have led to death in some instances. While hepatitis A vaccination has been routinely administered to children in developed countries, anti-vaccine sentiments and public complacency have led to decreased childhood vaccination of hepatitis A. Though herd immunity* will likely protect susceptible individuals in developed nations at present, potential for outbreaks in the future is greatly increased if people continue to refuse vaccination and shellfish suspect to contamination are imported/shipped to market. As such, the U.S. Department of Agriculture (USDA) is considering different approaches to ensure the safety of human health in shellfish consumption.

One of the most promising techniques being studied by the USDA in regard to hepatitis A contaminated shellfish is known as high pressure processing (HPP). This commercial technology is already used for processing of several products in the food industry and demonstrates potential for inactivation of hepatitis A virus in shellfish. In laboratory tests, HPP treatment of 60,000 pounds per square inch of pressure for five minutes exhibited inactivation of 99.9% of hepatitis A in oysters subjected to the pathogen. Since human hepatitis A virus strains are unable to replicate in the tissues of contaminated shellfish, virions damaged or inactivated by HPP processing are unable to replicate and repair themselves to restore infectivity. However, since shellfish osmoregulate – meaning the osmotic pressure of the organism’s body fluids are kept the same as the surrounding water – different pressures may need to be applied to inactivate virus for shellfish found at various depths as mutants may have been selected for pressure sensitivity. While there are some concerns for how this technique may affect the texture and taste of the shellfish, HPP is a promising technique for treatment of shellfish potentially infected with hepatitis A virus.

*herd immunity: the concept that if you immunize ‘enough people,’ (a complicated variable that differs according to the virus, social issues/beliefs, population, and environment) virus spread stops when the probability of infection drops below a critical threshold. Not everyone needs to be immunized to protect the population.

 Kingsley, D. (2004). Inactivation of selected picornaviruses by high hydrostatic pressure Virus Research, 102 (2), 221-224 DOI: 10.1016/j.virusres.2004.01.030
 Terio, V., Tantillo, G., Martella, V., Pinto, P., Buonavoglia, C., & Kingsley, D. (2010). High Pressure Inactivation of HAV Within Mussels Food and Environmental Virology, 2 (2), 83-88 DOI: 10.1007/s12560-010-9032-7
 Kingsley, D., Calci, K., Holliman, S., Dancho, B., & Flick, G. (2009). High Pressure Inactivation of HAV Within Oysters: Comparison of Shucked Oysters with Whole-In-Shell Meats Food and Environmental Virology, 1 (3-4), 137-140 DOI: 10.1007/s12560-009-9018-5