TWiV 374: Discordance in B

TWiVOn episode #374 of the science show This Week in Virology, the TWiVniks consider the role of a cell enzyme that removes a protein linked to the 5′-end of the picornavirus genome, and the connection between malaria, Epstein-Barr virus, and endemic Burkitt’s lymphoma.

You can find TWiV #374 at

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

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.

A new cell receptor for rhinovirus

rhinovirus receptorsRhinovirus is the most frequent cause of the common cold, and the virus itself is quite common: there are over 160 types, classified into 3 species. The cell receptor has just been identified for the rhinovirus C species, which can cause more severe illness than members of the A or B species: it is cadherin-related family member 3.

Because viruses are obligate intracellular parasites, the genome must enter a cell before new particles can be made. The first step in this process is binding of the virus particle to a receptor on the plasma membrane. Two different membrane proteins serve as receptors for members of rhinovirus A and B species: intracellular adhesion molecule 1, and low-density lipoprotein receptor (illustrated).

It has not been possible to propagate species C rhinoviruses in conventional cell cultures, which has hampered research on how the virus replicates. The lack of a cell culture system required a different approach to identifying a cell receptor for this virus. It was known that the virus replicates in primary organ or cell cultures derived from sinus tissue, but not in a variety of epithelial and transformed cell lines (e.g. HeLa cells). In silico comparison of gene expression profiles revealed 400 genes that are preferentially expressed in virus-susceptible cells. This list was narrowed down to 12 genes that encode plasma membrane proteins. A subset of these genes were introduced into cells and tested for the ability to serve as a rhinovirus C receptor. Introduction of the gene encoding cadherin-related family member 3 (CDHR3) into HeLa cells allowed rhinovirus C binding and infection.

The cadherin family comprises cell surface proteins that are involved in cell-cell communication. The exact cell function of CDHR3 is not known, but the protein is found in human lung, bronchial epithelium, and cultured airway epithelial cells. A mutation in the gene encoding this protein is associated with wheezing illness and asthma in children. This mutation, which causes a change from cysteine to tyrosine at amino acid 529, was found to increase virus binding and virus replication in HeLa cells that synthesize CDHR3. It will be important to determine if this amino acid change increases rhinovirus C replication in humans, thereby leading to more serious respiratory illness.

The CDHR3 gene was used to establish a stable HeLa cell line that produces the receptor and which can be infected with species C rhinoviruses. This cell line will be useful for illuminating the details of viral replication in cells, which has so far been elusive due to lack of a susceptible and permissive cell line. It may also be possible to produce transgenic mice with the human CDHR3 gene, which could serve as a model for studying rhinovirus C pathogenesis. Transgenic mice that produce the receptor for the related polioviruses, CD155, are a model for poliomyelitis.

TWiV 331: Why is this outbreak different from all other outbreaks?

On episode #331 of the science show This Week in Virology, the TWiV team discusses the possible association of the respiratory pathogen enterovirus D68 with neurological disease.

You can find TWiV #331 at

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 328: Lariat tricks in 3D

On episode #328 of the science show This Week in Virology, the TWiVocateurs discuss how the RNA polymerase of enteroviruses binds a component of the splicing machinery and inhibits mRNA processing.

You can find TWiV #328 at

Viral genomes in 700 year old caribou scat

CaribouRecovering viral genomes from ancient specimens can provide information about viral evolution, but not many old nucleic acids have been identified. A study of 700 year old caribou feces reveals that viruses can be protected for long periods of time – under the right conditions.

The oldest virus recovered so far is the giant Pithovirus sibericum, which was isolated from 30,000 year old Siberian permafrost. Other attempts have yielded fragments of viral genomes. It was possible to reconstruct the 1918 influenza virus from small RNAs recovered from formalin fixed and frozen human tissues. However this feat was not achieved for viral RNA in 140,000 year old Greenland ice cores, 900 year old North African barley grains, or 7,000 year old Black Sea Sediments.

Caribou feces have been frozen for the past 5,000 years in ice patches in the Selwyn Mountains of the Canadian Northwest territories. To determine if viruses could be recovered from this material, the frozen feces were thawed, resuspended in buffer, filtered, and treated with nucleases to destroy any nucleic acids not contained within a viral capsid. Sequence analysis of the remaining nucleic acids revealed two different viruses.

Ancient caribou feces associated virus (aCFV) has a single stranded, circular DNA genome distantly related to plant-infecting geminiviruses and gemycircularviruses. The entire 2.2 kb genome of aCFV was amplified from the caribou feces specimen. This reconstructed viral DNA replicated upon introduction into tobacco plant leaves.

Sequences of an RNA virus distantly related to picornaviruses of insects (such as Drosophila C virus) were also identified in the caribou feces. These viral genomes exceed 7.4 kb, but it was only possible to recover a 1.8 kb fragment of this virus, ancient Northwest Territories cripavirus (aNCV).

Neither virus was isolated from contemporary Caribou feces collected from an animal living in the same region. The authors also went to great pains to demonstrate that the two 700 year old viral genomes were not contaminants. The isolation was repeated in a different laboratory, and was not to be a consequence of contamination from any laboratory reagent or apparatus used for purification of nucleic acids.

It is not likely that aCFV or aNCV infected a caribou 700 years ago. The viruses were probably acquired when a caribou ingested plant material infected with the plant virus; perhaps insects harboring aNCV were also present on these plants. The exact hosts for both viruses are unknown.

The fact that two relatively large fragments of viral DNA and RNA were identified suggests that intact capsids were present in the caribou feces. Their preservation is probably a consequence of the low temperature of the arctic ice, and the stable icosahedral capsids characteristic of members of geminiviruses, gemycircularviruses, and cripaviruses.

We already know that viruses have been around for a long time, more than hundreds of millions of years, so what is the value of this work? Studying ancient viruses can provide insight into viral diversity and evolution. However, the value of two viral genome sequences is limited, and additional work should be done to acquire additional specimens spanning a long period of time. Similar sampling of other environments would also be desirable, but it is unlikely that large fragments of viral genomes can be recovered from specimens that are not frozen. And as the ice caps melt away, we will lose our ability to decode this important viral record.

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TWiV 302: The sky is falling

On episode #302 of the science show This Week in Virology, the TWiVers discuss the growing Ebola virus outbreak in West Africa, and an epidemic of respiratory disease in the US caused by enterovirus D68.

You can find TWiV #302 at

An outbreak of enterovirus 68


EV-A71 by Jason Roberts

During the winter of 1962 in California, a new virus was isolated from the oropharynx of 4 children who had been hospitalized with respiratory disease that included pneumonia and bronchiolitis. On the basis of its physical, chemical, and biological properties, the virus was classified as an enterovirus in the picornavirus family. Subsequently named enterovirus D68, it has been rarely reported in the United States (there were 79 isolations from 2009-2013). Towards the end of August 2014, an outbreak of severe respiratory disease associated with EV-D68 emerged in Kansas and Illinois.

Hospitals in Kansas City, Missouri, and Chicago, Illinois reported to the CDC an increase in the number of patients hospitalized with severe respiratory illness. EV-D68 was subsequently identified by polymerase chain reaction and nucleotide sequencing in 19/22 and 11/14 nasopharyngeal specimens from Kansas City and Chicago, respectively. Median ages of the patients were 4 and 5 years in the two cities, and most were admitted to the pediatric intensive care units due to respiratory distress. Other states have reported increases in cases of severe respiratory illness, and these are being investigated at CDC to determine if they are also associated with EV-D68.

There is no vaccine to prevent EV-D68 infection, nor is antiviral therapy available to treat infected patients. Current treatment is supportive to assist breathing; in a healthy individual the infection will resolve within a week. In the current outbreak no fatalities have been reported.

EV-D68 has been previously associated with mild to severe respiratory illness and is known to cause clusters of infections. It is not clear why there has been a sudden increase in the number of cases in the US. According to Mark Pallansch, Director of the Division of Viral Diseases at CDC, “our ability to find and detect the virus has improved to the point where we may now be recognizing more frequently what has always occurred in the past. So a lot of these techniques are now being applied more routinely both at the CDC but also at state health departments.” (Source: NPR).

I am sure that the nucleotide sequence of the EV-D68 virus isolated from these patients will reveal differences with previous strains. However whether or not those changes have anything to do with the increased number of isolations in the US will be very difficult to determine, especially as there is no animal model for EV-D68 respiratory disease.

Although how EV-D68 is transmitted has not been well studied, the virus can be detected in respiratory secretions (saliva, nasal mucus, sputum) and is therefore likely to spread from person to person by coughing, sneezing, or touching contaminated surfaces. The virus has been isolated from some of the children in California with acute flaccid paralysis, and there is at least one report of its association with central nervous system disease. In this case viral nucleic acids were detected in the cerebrospinal fluid. EV-D68 probably does not replicate in the human intestinal tract because the virus is inactivated by low pH.

Readers might wonder why a virus that causes respiratory illness is called an enterovirus. This nomenclature is largely historical: poliovirus, which replicates in the enteric tract, was the prototype member of this genus. Other viruses, including Coxsackieviruses and echoviruses, were added to the genus based on their physical and chemical properties. However soon it became apparent that many of these viruses could also replicate in the respiratory tract. Years later the rhinoviruses, which do not replicate in the enteric tract, were added to the enterovirus genus based on nucleotide sequence comparisons. While it was decided to keep the name ‘enterovirus’ for this group of viruses, it is certainly confusing and I would argue that it should be replaced by a more descriptive name.