On episode #342 of the science show This Week in Virology, the TWiVniks discuss the structure of a virus that reproduces in an extreme environment, long-term consequences of Ebolavirus infection, and VirScan, a method to identify the different virus infections you have had in your lifetime.

You can find TWiV #342 at www.twiv.tv.

filovirusThe thousands of survivors of the Ebolavirus outbreak in western Africa – more than at any other time – are fortunate to have survived the disease. However, their health problems are not behind them. A new study shows that the survivors of Ebolavirus have long-term sequelae more than 2 years after infection.

Acute infections caused by viruses such as Ebolavirus are characterized by rapid production of infectious virus particles, followed by resolution and elimination of infection by the host. However, chronic symptoms may persist for a long time after the infection is cleared. There have been long-term health consequences identified in survivors of previous Ebolavirus outbreaks. These include joint and muscle pain, vision and hearing loss, abdominal pain, bleeding, malaise, and psychological problems. Some patients were unable to perform their previous jobs for up to one year.

The long term health consequences of infection was studied in detail for the 2007 outbreak of Bundibugyo ebolavirus in Uganda. Survivors (49) were contacted 29 months after the outbreak and provided information about health status and their ability to function, and blood samples were obtained for further study. Controls for the study were seronegative contacts.

The results show that survivors of Bundibugyo ebolavirus infection are at significantly greater risk than controls for long term health problems. These include ocular problems (pain, blurred vision), loss of hearing, sleep difficulty, and joint pain. Other issues are abdominal and back pain, fatigue, impotence, severe headaches, memory problems and confusion. No differences in results of blood analyses were observed between the two groups. This study only included adults; children who have recovered should also be examined as their health care needs may be different.

These results confirm that there are long-term sequelae of Ebolavirus infection. The basis for the complications is not known, but is likely a consequence of tissue damage due to viral replication and the immune response. Whether or not virus was present in the patients was not determined. However it is known that Ebolavirus can persist in the testicles and eye long after it is absent from serum.

Other serious viral infections are also accompanied by long term health effects. For example, 29% of Lassa virus survivors have hearing loss, while joint pain persists for 3-5 years in about 10% of those infected with chikungunya virus.

Ebolavirus is a highly lethal virus, and those who survive infection are fortunate. Despite recovering, their health problems are not over. The size of the recent outbreak provided impetus for clinical trials of vaccines and antivirals; now research is needed to determine how to best care for the many survivors.

Update: The NIH has announced a study in Liberia to examine the long-term consequences of Ebolavirus infection.

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Vincent and Richard ElliottVirologist Richard Elliott passed away on 5 June 2015. I have known Richard since 1979 and I would like to provide some personal recollections of this outstanding virologist. A summary of his work can be found at the MRC-University of Glasgow Centre for Virus Research science blog.

I first met Richard in 1979 when he joined Peter Palese’s laboratory at Mt. Sinai School of Medicine in New York City. We overlapped for only about a year but it was enough to get to know him: he was a hard-working, enthusiastic virologists and a good friend. We shared many beers in New York City. At the end of 1979 I went off to David Baltimore’s laboratory where in 1981 I produced an infectious DNA clone of poliovirus. It proved very difficult to make infectious DNAs of negative strand RNA viruses, and it was Richard who was the first to accomplish this feat in 1996 for a virus with a segmented genome. This work was very important as it showed that infectious DNA clones were not limited to RNA viruses with monopartite genomes.

I remained in contact with Richard over the years but I did not see him in person until the 2010 meeting in Edinburgh of the Society for General Microbiology. The following year he joined me, Connor Bamford, Wendy Barclay, and Ron Fouchier for TWiV #177 recorded in Dublin. Schmallenberg virus had just emerged as a new pathogen of livestock, and he discussed his work on this virus.

I next saw Richard in a 2011 meeting of the Brazilian Society for Virology. When I arrived in Brazil it was quite hot, and I found Richard sitting by the pool, reviewing manuscripts in his bathing suit. I snapped a few photos of him and put them on Facebook. Later that evening he said his laboratory had asked why pornographic photos of him were on the internet – he was shirtless in my pictures (with Grant McFadden in the photo).

Richard Elliott and Grant McFadden

Richard visited New York in the summer of 2014 but we were unable to connect. Early this year Richard had agreed to join TWiV again for an episode from Glasgow. Sadly he became too ill to participate and died on the Friday before I traveled to Scotland. While there I briefly visited the Elliott lab at the University of Glasgow MRC-Centre for Virus Research, nearly a week after his death. I’m happy that I made the lab members smile:

Elliott lab

I can still remember Richard telling me how to spell his name: two ls, two ts. Richard was an excellent virologist, mentor, and friend. I will miss him.

Update: Corrected to reflect the fact that Richard produced the first infectious DNA of a segmented (-) strand RNA virus.

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On episode #341 of the science show This Week in Virology, Vincent returns to the University of Glasgow MRC-Center for Virus Research and speaks with Emma, Gillian, and Adam about their ebolavirus experiences: caring for an infected patient, working in an Ebola treatment center in Sierra Leone, and making epidemiological predictions about the outbreak in west Africa.

You can find TWiV #341 at www.twiv.tv.

prion conversionTransmissible spongiform encephalopathies (TSEs) are rare human neurodegenerative disorders that are caused by infectious proteins called prions. A naturally occurring variant of the human prion has been found that completely protects against the disease.

A protective variant of the prion protein was discovered in the Fore people of Papua New Guinea. Beginning in the early 1900s, the prion disease kuru spread among Fore women and children as a result of ritual cannibalism of the brains of deceased relatives. When cannibalism stopped in the late 1950s, kuru disappeared.

Survivors of the kuru epidemic are heterozygous for a prion protein gene (prnp) with a unique amino acid change not seen in other populations, a change at position 127 from glycine to valine (G127V). The G127V change was always seen together with methionine at 129. Heterozygosity for M and V at amino acid 129, which is protective against prion disease, is found in humans all over the world.

Transgenic mice were used to determine if the G127V change in prion protein protects against disease. These mice lack the murine prnp gene (which encodes the normal prion protein) and contain a copy of either the wild type human prnp gene, or one with changes at amino acids 127 and 129. The mice were then inoculated intracerebrally with brain extracts from individuals who died of kuru. Mice with wild type human prnp were susceptible to infection. In contrast, transgenic mice heterozygous for the variant prnp (G127M129/V127M129) were completely resistant to infection. The mice were also resistant to infection with prions from cases of another human TSE, Creutzfeldt-Jacob disease.

Prnp transgenic mice were also challenged with variant Creutzfeldt-Jacob disease prions. This novel TSE arose after consumption of beef from animals with the prion disease bovine spongiform encephalopathy (BSE). These mice were susceptible to infection with vCJD prions, not a surprising result given that the Fore people were never exposed to BSE prions. However, mice homogygous for the altered prnp (V127M129/V127M129) were completely resistant to infection with vCJD prions – as resistant as mice with no prnp genes.

The protective effect of the M129V polymorphism is thought to be a consequence of inhibition of protein-protein interactions during prion propagation (i.e. the conversion of normal prion to pathogenic prion). How the G127V change confers protection is unknown.

These results show that the G127V change confers resistance to kuru and was likely selected as a consequence of the epidemic. If kuru had not been stopped by the abolition of cannibalism, it likely would have been self-limiting, as individuals with resistance to the disease, caused by the G127V change, repopulated the Fore people.

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On episode #340 of the science show This Week in Virology, the TWiV teams reviews a MERS-coronavirus serosurvey and an outbreak in South Korea, and constraints on measles virus antigenic variation.

You can find TWiV #340 at www.twiv.tv.

Your viral past

4 June 2015

virusesDid you ever wonder what different virus infections you have had in your lifetime? Now you can find out with just a drop of your blood and about $25.

Immune defense systems of many hosts produce antibodies in response to virus infections. These large proteins, which are generally virus specific, can block or inhibit virus infection, and persist at low levels for many years after the initial infection. Hence it is possible to determine whether an individual has had a virus infection by looking for anti-viral antibodies in the blood. Up to now the process of identifying such antibodies has been slow and limited to one or a few viruses. A new assay called VirScan allows unbiased searches for all the virus antibodies in your blood, providing a picture of all your past infections.

To identify the human antivirome, DNAs were synthesized encoding proteins from all viruses known to infect humans – 206 species and over 1000 strains. These DNAs were inserted into the genome of a bacteriophage, so that upon infecting bacteria, the viral peptides are displayed on the phage capsid. These ‘display’ phages were then mixed with human serum, and those that were bound by antibodies were isolated. The DNA sequence of the phage genomes were then determined to identify the human virus bound by the antibodies.

This method was used to assay samples from 569 humans. The results show that each person had been exposed to an average of 10 viruses, with a range from a few to over 20 (two individuals had antibodies to 84 different virus species!). The most frequently identified viruses included herpesviruses, rhinoviruses, adenoviruses, influenza viruses, respiratory syncytial virus, and enteroviruses. The overall winner, found in 88% of samples, is Epstein-Barr virus.

These results are not unexpected: all of us are infected with at least a dozen viruses at any time, and the viruses identified in this study known to infect much of the human population. What was surprising is the absence of some common viruses, such as rotaviruses, and the ubiquitous polyomaviruses. According to serological surveys, the most common human viruses are the small, single-stranded DNA containing anelloviruses. Yet the related torque teno virus was only found in 1.7% of samples. These differences are likely due to a combination of technical and biological issues (e.g., failure of antibodies to certain viruses to persist in serum).

This new assay may one day become a routine diagnostic tool that is used along with complete blood counts and chemistries to know if a patient’s signs and symptoms might be attributable to a past virus infection. VirScan technology is not limited to virus infections – it can be used to provide a history of bouts with bacteria, fungi, and parasites.

VirScan might also allow us to determine which virus infections are beneficial, and which contribute to chronic diseases such as autoimmune or neurodevelopmental disorders or cancer. The assay can be used to conduct unbiased population-based studies of the prevalence of virus infections and their possible association with these diseases. Such connections were not previously possible with antibody assays that search for one virus at a time. This approach was not only inefficient, but required guessing the responsible virus.

Some other findings of this study are noteworthy. As expected, children had fewer virus infections than adults. HIV-positive individuals had antibodies to more viruses than HIV-negative individuals, also expected given the damage done by this virus to the immune system. Frequencies of anti-viral antibodies were higher outside of the United States, possible due to differences in genetics, sanitation, or population density. In most samples, there was a single dominant peptide per virus, although there were occasional differences among populations. This information might be useful for improving vaccines, or tailoring them to specific countries or regions.

Update: It would be very informative to use VirScan to search for antibodies against viruses that are not known to infect humans. Other animal viruses, plant viruses, insect viruses: to which do a significant fraction of humans respond? The information might identify other viruses that replicate in humans and which might constitute future threats (or present benefits).

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On episode #339 of the science show This Week in Virology, tre TWiV amici present three snippets and a side of sashimi: how herpesvirus inhibits host cell gene expression by disrupting transcription termination.

You can find TWiV #339 at www.twiv.tv.

RudivirusMany microbes live in extreme environments, encountering conditions that are very hot, very cold, highly acidic, or very salty. The viruses that infect such microbes must also be able to retain infectivity in extreme conditions. How do they do it?

Clues come from the observations that the genomes of viruses that infect Archaea in extreme geothermal environments encode proteins that have never been seen before. The idea is that such unusual proteins must endow these viruses with the ability to maintain infectivity under extreme conditions.

The hosts of Rudiviruses (rudi=small rod in Latin), the Archea Sulfobolus islandicus, live at high temperatures (80° C) and low pH (3.0). These non-enveloped viruses consist of double-stranded DNA wrapped in a helical manner with thousands of copies of a 134 amino acid protein (illustrated; image credit). The three-dimensional structure of Sulfobolus islandicus rod-shaped virus 2 (SIRV2) reveals a new type of organization of virus particles, and provides clues about how it retains infectivity in extreme environments.

Resolution of the SIRV2 structure reveals that it consists of dimers of a single protein which forms helices that are tightly wrapped around the DNA genome. The result is a coiled DNA protected by a coat of protein that stabilizes and protects the genome. Without DNA, over half of the capsid protein is unstructured. Only in the presence of DNA does the viral protein form an alpha helix that wraps around the nucleic acid.

The DNA genome of SIRV2 is in the A-form, in contrast to B-form DNA which is found in most other organisms. The two types of DNA differ in their geometry and dimensions. It was previously thought that A-DNA occurs only when the nucleic acid is dehydrated.

These two usual properties of SIRV2 are also found in gram positive bacteria which form desiccation and heat resistant spores when starved of nutrients. Sporulation is accompanied by a change in the bacterial genome from B-DNA to A-DNA, which is caused by the binding of small acid-soluble proteins. Like the SIRV2 capsid protein, small acid-soluble proteins of spore-forming bacteria are unstructured in solution, and become alpha helices when bound to DNA. These observations suggest that binding of the SIRV2 capsid protein changes the viral DNA to the A-form, conferring stability in extreme environments.

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mosquito brainAs far as I know, mosquitoes do not eat sushi. But mosquito cells have proteins with sushi repeat domains, and these proteins protect the brain from lethal virus infections.

Mosquitoes are vectors for the transmission of many human viral diseases, including yellow fever, West Nile disease, Japanese encephalitis, and dengue hemorrhagic fever. Many mosquito-borne viruses enter the human central nervous system and cause neurological disease. In contrast, these viruses replicate in many tissues of the mosquito, including the central nervous system, with little pathological effect and no alteration of behavior or lifespan. The defenses that allow such persistent infection of mosquitoes are slowly being unraveled.

A protein called Hikaru genki, or Hig, is crucial for controlling viral infections of the mosquito brain. Originally discovered in the fruit fly Drosophila, Hig is produced mainly in the brain of Aedes aegyptii, the natural vector for dengue and yellow fever viruses. Experimental reduction of Hig mRNA or protein in the mosquito leads to increased replication of dengue virus and Japanese encephalitis virus. This increase in viral replication is accompanied by more cell death in the mosquito brain, and decreased survival.

How does Hig protein impair virus replication? The Hig protein of A. aegyptii binds dengue virus particles via the E membrane glycoprotein. As Hig protein is located on the cell surface, binding to virus particles prevents virus entry into cells. Impairment of endocytosis is limited to insect cells – introduction of Hig into mammalian cells had no effect on virus replication. Clearly other components of insect cells must participate in the Hig-mediated antiviral mechanism.

The antiviral activity of Hig protein depends on the presence of sushi repeat domains, also known as complement control protein (CCP) domains. These consist of 60 amino acid repeats with four conserved cysteines and a tryptophan. The CCP domain is found in many proteins of the complement system, a collection of blood and cell surface proteins that is a major primary defense and a clearance component of innate and adaptive immune responses. The sushi domain mediates protein-protein interactions among complement components. Capturing the dengue and Japanese encephalitis viruses by the A. aegyptii Hig protein is just one example of the virus-binding ability of proteins with CCP domains. An insect scavenger receptor with two CCP domains is a pattern recognition receptor that recognizes dengue virus and recruits mosquito complement to limit viral replication. Some CCP containing proteins are virus receptors (complement receptor 2 binds Epstein-Barr virus, and membrane cofactor protein is a receptor tor measles virus).

Because the Hig antiviral machinery is largely limited to the mosquito brain, it is possible that it prolongs mosquito life to allow virus transmission to other hosts. Transmission of virus to other hosts requires replication in the salivary gland, which cannot take if the mosquito dies of neural infection. I wonder why humans do not have have similar mechanisms to protect their neural tissues from virus infections. Is neuroinvasion a less frequent event in humans, compared with mosquitoes, thereby providing less selective pressure for protective mechanisms to evolve?

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