Giant dsDNA Virus Origins: Megaviridae Evolutionary Analysis

Posted on May 16, 2012 by David Esteban

Contributed by guest blogger: Katy Hwang ’12

The discovery of the double stranded DNA (dsDNA) virus, Mimivirus, and the subsequent discovery of related Megavirus confounded the size limits of viral particles and the complexity of viral genomes. They are larger or just as large as some bacteria. Megavirus has a 1,259,197-bp genome.  Megavirus contains a genome 6.5% larger than that of Mimivirus. Each of these viruses, classified as Megaviridae, have approximately 979+ proteins, including the first aminoacyl tRNA synthetases (AARS), enzymes that promote translation, found outside of cellular organisms.

Mimivirus infects amoeba; Megavirus natural host is still unknown as it was found in a sea water sample through a campaign on random aquatic environmental sampling off of the coast of Las Cruces, Chile. Megavirus research is conducted in A. castellanii. The virus even reopened the debate on whether or not viruses are alive as Megaviridae have traits that overlap with unicellular organisms such as parasitic bacteria. Phylogenic evidence does not cluster either virus into a prokaryotic clade, but more deeply into a eukaryotic clade. Megavirus is of the archeal type, so it branched out before the radiation of eukaryotes. This provides some insight into the Megaviridae ancestor.  So now the question is: from what did these giant viruses originate and  how did they evolve?

Megavirus and Mimivirus are similar enough to have unambiguous homologous features, but have also diverged enough on the evolutionarily tree to provide more information on the features of a common ancestor. 23% of the Megavirus genome does not have a counterpart in Mimivirus, but it shares about 77% of its 1120 putative coding sequences with Mimivius. Megavirus and Mimivrius use the same motif to specify early gene expression, the expression pattern of the orthologous genes is conserved globally. Not only do these giant viruses have their own AARS, but they also code for their own DNA repair enzymes that correct damage caused by UV light, ionizing radiation and chemical mutagens. Megavirus also has a DNA photolyase, which is an enzyme that uses light energy to make repairs to DNA and increases resistance to UV radiation.

The author’s hypothesis is that the last Megaviridae common ancestor originated from a cellular organism, where the now current Megaviridae have undergone specific genome reduction events. Mimivirus codes for four AARS and Megavirus codes for seven, four being orthologs to those found in Mimivirus and one of the other AARS that Megavirus codes for is a class-II AARS. This observation that viral AARS are not limited to type-I suggests that independent acquisition of these genes by horizontal gene transfer is unlikely.  The ancestor of Megaviridae and Mimivirus most likely started with 20 AARS from a cellular ancestor. It is very unlikely that these seven coding sequences were added, and more likely that there were various lineage specific gene family deletions.

The discovery of the AARS substantiate the claims that the Megaviridae ancestor originated from a cellular organism.  What were the driving factors of these genome reduction events?  More etiological data is required for further analysis of the evolutionary process of the development into these giant dsDNA viruses. What other giant viruses are still out in the world waiting to be discovered and what can we learn from them?

Link:

http://www.pnas.org/content/early/2011/10/04/1110889108

 

Katy Hwang is a senior at Vassar College, majoring in biology.

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H5N1 Ferret Transmission Experiment Published

Posted on May 3, 2012 by David Esteban

At last, one of the papers investigating H5N1 influenza transmission in ferrets has been published in the journal Nature yesterday.  To recap the controversy briefly:  news of experimental studies investigating transmissibility of avian H5N1 influenza hit the news this fall, igniting a fierce debate about biosecurity, “dual use” research, and the damage that censorship can have on scientific advancement.  An advisory group, the NSABB, recommended partial censorship of the data, perhaps believing that redacting specific data from the publications would prevent information on how to generate a highly pathogenic mammalian transmissible virus from getting into the hands of bioterrorists or others incapable of handling such viruses safely.  However, after some new data or clarification of data presented in a revised version of the submitted manuscript, the NSABB recommended publication in full.

Avian H5N1 influenza virus has caused sporadic infections in humans who have close contact with infected animals.  Human-to-human transmission has not been observed.  But could an H5 virus mutate or reassort, allowing human-to-human transmission?

One thing that has been lost in this whole controversy is that this study is actually a great demonstration and application of evolution.  How does a virus switch to a new host and transmit efficiently between individuals?  Start with a diverse population that varies in a particular trait (in this case, ability to bind the human receptor).  Put it through selective pressure. This occurred in several steps: first, select for viruses that can bind the human receptor in vitro.  From those that bind, select ones that can efficiently replicate in the respiratory tract of the animal.  Finally, take the efficient replicators and allow for transmission.  At each step of selection, mutations that naturally occur during viral replication further diversify the population resulting in variants that possess the desired property.  Those variants get selected for the next experiment.

This study focused on one particular influenza virus protein, hemagglutinin (HA).  HA is on the surface of the virion and is what the virus uses to attach to the host cell, the first step in viral infection.  HA of human influenza viruses bind a sugar on the surface of human cells, which is slightly different from that found in the avian respiratory tract.  Avian viruses, of course, bind to the form found in birds.  H5 shows a strong preference for binding the avian receptor, so Karaoka et al were interested in finding out if H5 could change to recognize the human receptor, allowing more efficient transmission between mammals.

To address this, they began with a mutagenesis technique to introduce random mutations in the globular head (the receptor binding part) of HA, then used an in vitro approach to select for mutants that bound the human receptor (selection step 1).  Through this process they identified three H5 variants that gained the ability to bind the human receptor while maintaining the ability to bind the avian receptor, and one that switched specificity completely to the human receptor.  Several of the mutations identified in this study had already been shown in previous studies to be important in receptor specificity.

To test if the variant H5s conferred binding to human receptors in vivo, sections of human tracheal tissue were exposed to the viruses and only two were able to infect (selection step 2).  This suggests the virus can infect human epithelium of the upper respiratory tract.

Next, the two remaining variants were used to infect ferrets.  Both replicated in ferret respiratory tracts, but one replicated to higher levels.  When they sequenced the virus that they isolated from that ferret, it was different from what they had put in: a new mutation had appeared.  This new mutation presumably confers the property of better replication in the ferret respiratory tract, so it outgrew the original input virus (selection step 3).

Using this new virus (now with a total of 3 mutations in H5), transmission was tested in ferrets.  Compared to the original H5, which did not transmit via aerosol, the 3-mutation variant did transmit, although between only 2 of 6 animal pairs.  Again, they sequenced the virus that was present in the contact animals and found that it was different than what had gone in to the inoculated animals.  Yet another mutation had appeared (selection step 4).  This additional mutation appears to enhance transmission:  the new virus, now with 4 mutations, transmitted more quickly and between more pairs of animals than the 3-mutation virus.  Although the virus can transmit, none of the infected animals died, but they did show pathology at the site of infection.

So what does this all mean? The best model available for influenza transmission studies is ferrets.  Ferrets aren’t humans, so its important to keep in mind that this is a model that helps us understand what viral or host factors are involved in aerosol transmission in these mammals, and maybe, but not necessarily, in humans.  Since we don’t know what is necessary for human-to-human transmission, it is valuable to have an animal model to give us some ideas of what to look for.  It can provide some good hypotheses on what mediates transmission in humans, which would then have to be further tested.  Obviously, specificity for the human receptor is necessary, but the mutations identified tell us more than that.  Mutations that change specificity are not sufficient for transmission.  It turns out that those mutations also decrease the stability of the HA protein.  The additional mutations acquired through the selection steps compensate for that, and enhance stability.   So now we know that HA stability is important in influenza transmission.  Between ferrets.  That’s probably true for humans too, but it would need to be tested.

If you are still reading, you are obviously procrastinating, and are probably avoiding studying for your final exam.  But here are some more thoughts on the controversy overall.  This is a really interesting paper, with nothing particularly frightening or worrisome about it.  Certainly not any more so than other papers doing similar work that were published without so much controversy.  If “dual use” research needs to be regulated, it needs to be done before the work is done, not after.  If the NSABB was concerned about this kind of research, why only express concern once the experiment succeeds?  In my intro biology class, we read another paper, published in 2005, which was addressing the exact same question, in an almost identical way.  The difference is that they failed to make a transmissible virus.  If there is a concern about this kind of research, a concern that it is too risky to do these kinds of experiments, shouldn’t the alarm have been raised regardless of the outcome?  It just doesn’t make sense to me why it suddenly became so concerning.  If anything good has come of this controversy, it is the widespread discussion that this has stimulated on the importance of open communication of scientific data, the importance of not censoring in science.  Ironically, had we all been given access to the data, like through a journal publication, it would have been apparent that there wasn’t anything to be concerned about.

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Mouse Pneumonia: Are We to Blame?

Posted on May 2, 2012 by David Esteban

Contributed by guest blogger: Alix Zongrone ’12

Pneumonia virus of mice, or PVM, is the leading cause of pneumonia in laboratory mice; however, lack of evidence of PVM in wild rodents has left scientists in the dark with regards to the history and natural host of the virus. Because PVM is mostly found in captive settings (i.e. laboratories, pet shops, etc.) and PVM-neutralizing behavior has been observed in human cells, it has been suggested that human contact may play a pivotal role in the virus’s spread. Several studies have sought to investigate the prevalence of PVM in humans and its role in human respiratory infection; however, since PVM is closely related to human respiratory syncytial virus, or RSV, it is difficult to make sound conclusions based on this evidence alone.

Due to the evidence of PVM in humans, researchers inoculated two different non-human primate species with PVM to investigate replication activity of PVM in these mammals. They found that, over the course of twelve days, most of the samples exhibited viral replication as well as viral shedding. Although not all of the animals showed virus replication and shedding behavior, PVM antibodies were found in all test animals, suggesting that infection did take place, but replication was highly restricted. Though PVM was observed to not replicate well in non-human primates, human lung epithelial cells exhibited similar permissiveness of both PVM and RSV in vitro.
Controlling the interferon (IFN) immune response is a known mechanism of successful viral replication in the host. Researchers investigated the ability of PVM to block IFN response to further explore PVM host range restriction. The virus demonstrated an ability to block IFN response in these human epithelial cells thanks to the NS2 protein. However, a Western blot was used to compare proteins made from PVM and RSV and  PVM-neutralizing activity specificity was also determined. Humans were tested for PVM antibodies to examine whether an immune response was triggered. No PVM antibodies were found in the human sera, and no reactivity between PVM proteins and observed PVM-neutralizing behavior was recorded. This demonstrates a lack of immune response in the human cells.
Although PVM was observed to replicate in vitro in human epithelial cells, the results remain inconclusive as to whether or not the virus should be considered a human pathogen. The lack of permissiveness in non-human primates suggests that the virus may not actually cause infection in humans. This is supported by the lack of reaction shown between PVM proteins and PVM-neutralizing activity in the Western Blot.
Questions remain as to the nature of the PVM-neutralizing activity in human serum as well the origin of PVM and its natural host. Is that which is categorized as PVM-neutralizing behavior not actually PVM-specific? What is causing PVM in captive laboratory mice but not in wild rodent species?  Finally, what could possibly be the natural host of pneumonia virus of mice, if not mice?

Link: http://jvi.asm.org/content/86/10/5829.abstract?etoc

Alix Zongrone is a senior at Vassar College, majoring in biology.

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After many setbacks, cross-presentation provides new hope for a Herpes Simplex Virus 1 vaccine

Posted on April 27, 2012 by David Esteban

Contributed by guest blogger: Stephanie Mischell ’12

Herpes simplex virus type 1 (HSV-1) is making news due to a paper by Jing et al identifying two promising new candidate antigens for a vaccine. HSV-1 is a widespread public health issue, infecting approximately 60% of Americans and causing symptoms, most likely cold sores or genital sores but on rare occasion blindness or fatal brain damage. Furthermore, finding a vaccine for HSV-1 has proved difficult, in part because of the vital but elusive role of CD8+ T-cells in the HSV-1 immune response. Mice studies suggest that a CD8-response could facilitate memory cell formation and ameliorate chronic disease caused by HSV-1, but human blood does not have many HSV-1 specific CD8+ T-cells and very few CD8 epitopes have been identified.  Previous attempts at vaccines most recently using the HSV glycoprotein D (gD2), have focused on CD4+ T-cell specific epitopes. These attempts were unable to stimulate a CD8+ T-cell response, and the vaccine failed during clinical trials. A way to stimulate both CD4+ and CD8+ T-cell responses seems necessary to create an effective vaccine.

Jing et al’s work is significant because it harnesses properties originally used to study HSV-2 to identify HSV-1 epitopes recognized by CD8+ T-cells. An epitope, or antigenic determinant, is the part of an antigen that is recognized by the immune system; this interaction is what triggers a host immune response. Jing et al demonstrated previously that in vitro monocyte-derived dendritic cells (moDC’s), or antigen-presenting cells, can cross-present HSV-2  epitopes to create  HSV-2 specific memory T-cells. In this paper, they harnessed this cross-reactivity of moDC’s and applied it to HSV-1, stimulating and identifying HSV-1 specific CD8+ T-cells. 45 distinct CD8+ T-cell epitopes were identified. Furthermore, the genomes of host responder cells were cloned, and HSV-1 epitopes were analyzed for HLA restriction. Proteins from two genes, UL39 and UL46, were identified as most highly restricted, suggesting that they are most involved in the immunogenic response. PMBC assays confirmed these results quantitatively.

Jing et al conclude that the viral proteins coded by UL39 and UL46 are good candidate antigens for an HSV-1 vaccine because of their CD4+ and CD8+ T-cell  immunogenicity. However, they also acknowledge that their sample size is small and that subunit vaccines have not been successful vaccines for HSV-1. In fact, the large number of CD8+ T-cell   epitopes identified led the authors to conclude that a whole-virus vaccine may be more successful than subunits. Most of the failed vaccines showed similar promise until phase II or phase III of clinical trials, suggesting that the small amount of data from this study is just a start. This discovery is important but not a guaranteed vaccine.

While the identification of UL39 and UL46 are important steps in solving the public health issue posed by HSV-1, as is the identification of other CD8+ T-cell   epitopes, perhaps the most significant part of the study is the implications of their novel research methods on the study of viral vaccines. The enrichment techniques used could potentially make studying T-cell responses easier. The authors confirmed the applicability of their methods by using the same techniques to study the vaccinia virus, a microbe with a large genome of over 200 genes. This paper demonstrates a small advancement in HSV-1 research and control, but may have larger implications for this and other large viruses.

Link to original article: http://www.jci.org/articles/view/60556

Stephanie Mischell is a senior at Vassar College, majoring in biology.

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Posted in Guest Blogger, Immunology, Vaccines | Tagged , , , , | 3 Comments | Edit

Luring HIV out of its latency may be the secret to developing an effective HIV cure

Posted on April 19, 2012 by David Esteban

Contributed by guest blogger: Steven Chan ‘12

The emergence of highly active antiretroviral therapy (HAART) in the treatment of HIV-infected individuals has certainly changed the outlook of an HIV diagnosis today, compared to what such an outlook looked like in the earliest years of the epidemic. Such a treatment regimen, if strictly adhered to, has the potential to suppress the levels of active circulating HIV in the infected individual to a level that is manageable, essentially halting the progression of the disease. It soon became clear however, that these treatments could not effectively clear the body of all HIV particles—the virus manages to stow itself away within the cellular genome of the memory CD4+ T-cells, and remain transcriptionally silent indefinitely. These latent reservoirs of HIV-infected cells prove to be undetectable for these antiretroviral therapies, since antiretroviral drugs can only target HIV-infected cells when they are replicating. And so, memory cells, which replicate infrequently, cannot be effectively targeted, making it impossible to clear HIV-infected bodies of all HIV-particles. “We’re never going to cure anybody unless we go for this latent pool,” says Robert Siliciano, the researcher at Johns Hopkins University that first identified the latent HIV memory-T cells.

A great deal of HIV-therapy research over the past decade has focused on finding a way to coax these infected cells out of their latency to make them detectable by antiretroviral drugs. The problem that has been persistently hounding researchers has been the difficulty in luring these cells out of their latency without triggering the immune system in an inflammation response that would end up doing more harm than good. David Margolis, MD, and his research team at UNC Chapel Hill, who have been working on this problem for a while now, have found success with a set of histone deacetylase inhibitors called Zolinza (vorinostat), a chemotherapeutic cancer drug that has been found to stimulate gene expression within the latent HIV-infected cells without inducing an overwhelming immune response. HDAC inhibitors accomplish this by inhibiting the activity of histone deacetylase, which removes the acetyl groups from the lysine residues in the core histones, resulting in the formation of a condensed and transcriptionally silenced chromatin. By inhibiting this activity, the core histones become less compact, and the chromatin becomes more transcriptionally active. After initial success with in vitro tests in cell cultures and in blood tissues, six HIV-positive men were recruited in a clinical trial pairing this treatment alongside consistent antiretroviral therapy. Each of the study volunteers had already been taking part in a robust antiviral regimen for an average of four years, and displayed undetectable viral loads and stable CD4+ T-cell counts. Post-exposure to Zolinza, HIV-RNA levels—a marker of viral activity—in these patients increased by an average of 4.8 times, ranging from a 1.5-fold increase in one patient to a 10.0-fold increase in another. The drug took effect in as little as 8 hours, inducing a two-fold increase in cellular and chromatin-bound histone acetylation within that time span. Increased expression made these cells susceptible to detection and eradication by the antiretroviral drugs, which proceeds just as efficiently as usual.

Margolis addresses the significance of this advancement, “This study provides first proof of concept, demonstrating disruption of latency, a significant step toward eradication.” Just how effective this drug is in teasing out the latent cells still remains to be seen—with nearly a ten-fold difference in one trial participant compared to the other, the efficacy of such a drug remains questionable. The limited sample size in this initial trial also doesn’t give us too much to go on. There are also concerns that the drug could induce some serious side effects such as blood clots in the legs and lungs, diabetes, fewer platelets and RBC count, as well as dehydration from nausea and vomiting, but at least in this trial, there were only mild adverse effects at worst. Little is known about the potential adverse effects of long-term use of the drug. Margolis et al.’s study design made use of a single dose of Vorinostat, but it is likely that repeated intermittent doses would yield the most optimal effects. “Vorinostat may not be the magic bullet, but this success shows us a new way to test drugs to target latency and suggests that we can build a path that may lead to a cure,” says Margolis. Further studies to assess Vorinostat’s safety and effectiveness, and the way it interacts with other HAART treatments, would certainly be crucial before it can be deployed as a component in future HIV treatment regimen.

 

Links:

Archin N, Liberty A, Kashuba A, Choudhary S, Kuruc J, Hudgens M, Kearney M, Eron J, Hazuda D, and Margolis D. “Administration of Vorinostat Disrupts HIV-1 Latency in Patients on ART,” HIV Persistence, Latency, and Eradication at 19th Conference on Retroviruses and Opportunistic Infections, March 8, 2012,              http://www.retroconference.org/2012b/Abstracts/45315.htm

Contreras X, Schwenwker M, Chen CS, McCune JM, Deeks SG, Martin J, Peterlin BM. Suberoylanilide Hydroxamic Acid Reactivates HIV from Latently Infected Cells, J. Biol. Chem., January 9, 2009, http://www.jbc.org/content/284/11/6782.full

Horn T. “Pathway to a Cure: Cancer Drug Helps Purge HIV From Resting Cells,”  AidsMeds, March 9, 2012, http://www.aidsmeds.com/articles/hiv_vorinostat_ cure_1667_22059.shtml

“Lymphoma Drug Wakes Up Dormant HIV,” AidsMeds, March 17, 2009,     http://www.aidsmeds.com/articles/hiv_zolinza_latent_1667_16307.shtml

Steven Chan is a senior at Vassar College, majoring in Science, Technology, and Society

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Posted in Antiviral Drugs, Epidemics, Immunology, Molecular Virology | Tagged , , , , | 7 Comments | Edit

The Viral Theory of Schizophrenia

Posted on April 17, 2012 by David Esteban

Contributed by guest blogger: Hannah Ziobrowski ’12

Schizophrenia is a severely debilitating mental illness with no known cause or cure, although there is a strong genetic correlation.  Interestingly, there is additionally a significant relationship between season of birth and the development of schizophrenia, as individuals born during late winter and spring have a significantly increased risk for developing schizophrenia.  One hypothesis to explain this phenomenon is that this is due to prenatal viral infection, which is more likely to occur in the winter months.  It is hypothesized that viral infections occurring during the third trimester of pregnancy result in the increased risk for developing schizophrenia.  However, there is currently debate as to how this happens- is it due to a direct viral infection of the fetus, or due to maternal cytokines in response to infection?

A study by Faterni et al (2012) found that the placenta may be a site of pathology in viral infections.  Using pregnant mice infected with a sublethal dose of influenza on the seventh day of pregnancy (E7), they found that viral infection resulted in many histological abnormalities in the placentae.  These abnormalities included an absence of the labyrinth zone, the region of the maternal placenta in which nutrients and oxygen are exchanged between the maternal and fetal blood, the presence of thrombi, and an increased number of inflammatory cells.  Additionally, microarray analyses revealed a significant upregulation of 77 genes and downregulaton of 93 genes in the placentae of infected mothers, compared to sham-infected mothers.  20% of these altered genes were involved in apoptotic or anti-apoptotic pathways, 10% were associated with immune response, 11% were involved with hypoxia, and about 11% were involved with inflammation.  9.4% were associated with major mental disorders including schizophrenia, bipolar disorder, major depression, and autism.  All of these changes could potentially affect developing embryos.  The deletion of a labyrinth zone could result in a reduction of oxygen delivered to the developing fetus and result in neural abnormalities, which may be ultimately caused by an inflammatory immune response.

The authors also analyzed gene expression in the hippocampus and prefrontal cortex of the offspring of infected mothers.  Compared to offspring of sham-infected mothers, they found 6 upregulated and 24 downregulated genes in the prefrontal cortex at the first day after birth (P0), and 4 upregulated and 13 downregulated genes in the hippocampus at P0.  Genes in the prefrontal cortex that showed a significant alteration in expression included glutamate receptor interacting protein I, platelet factor 4, contactin 1, and neurotrophic tyrosine kinase receptor type 3.  Important genes in the hippocampus that showed altered levels of expression included paralemmin 2, and protein tyrosine kinase 2 beta.  In total, 40 different genes showed altered expression in the two areas at P0 after infection at E7 (first trimester), compared to 39 at E9, 676 at E16, and 247 at E18 (as found in previous studies).   These altered expression levels most likely reflect altered neural organization.

Importantly, HINI viral genes were not detected in either the placenta or brains of offspring whose mothers were infected at E7, suggesting that the virus did not cross the placenta to directly infect the offspring.  This consequently implicates that the changes found in gene expression as well as the structural abnormalities of the placenta were most likely due to the production of maternal or fetal cytokines, most likely due to an increase in inflammatory cells in infected placenta.

These data overall illustrate that viral infections during pregnancy can lead to an inflammatory response and structural abnormalities in the placenta.  These structural abnormalities may cause significant alterations in oxygen and nutrients delivered to the fetus, causing abnormalities in the overall development of the fetus, including the brain.  Could these organizational neural abnormalities lead to an increased risk for developing schizophrenia later in life?

A next step for the authors would be to directly check the levels of cytokines in the placenta in order to assess the inflammatory response. Furthermore, are these results also found with infection of other viruses?  Or are they more or less significant depending on the virus and time of infection?

Link: http://www.sciencedirect.com/science/article/pii/S0028390811000141

Hannah Ziobrowski is a senior at Vassar College, majoring in Neuroscience and Behavior.

 

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Posted in Guest Blogger, Immunology | Tagged , , , | 19 Comments | Edit

Herpes-Family Viruses are Associated with Stressed Out Corals

Posted on April 13, 2012 by David Esteban

Contributed by guest blogger: Ian Heller ‘12

A new review out in the Journal of Experimental Marine Biology and Ecology is causing a rash of media attention regarding the presence of viruses in stressed out coral. However, this media attention, with catchy titles playing at old stigmas against herpes infection in humans, misses the true story told being uncovered in the new field of coral virology. What has the science actually shown?

Coral reefs are hotspots of biodiversity and essential components of the ocean ecosystem. Corals themselves contain an amazingly diverse assembly of different organisms. Tiny organisms like symbiotic algae, fungi bacteria, and archaea are all necessary for healthy coral. Unfortunately, coral reefs are threatened world wide due to rising sea temperatures, acidifying ocean water, and pollution in the form of sewage and fertilizer runoff. These stressors seem linked to an increased incidence of disease in coral, but what pathogens are actually making corals sick?

To investigate whether any viruses were associated with stressed coral, researchers compared the metagenomes of healthy corals and corals grown in water that was too hot, too acidic, too polluted with organic carbon (to simulate sewage stress) or too polluted with plant fertilizer nutrients. Within this “metagenome” is all of the DNA sequences from all of the different algae, bacteria, virus, etc., that are part of each sample, in this case, a coral fragment.

The first step in making such a comparison is sequencing as many of the genes as possible each sample, a feat made feasible by the increasing accessibility of gene sequencing. Next, researchers identify all of the sequences in their samples’ “library” of genes that correspond to viral genes. This means sifting through over 51,000 sequences! To figure out the identify of these sequences, the researchers use a computer algorithm known as BLAST to compared their unknown sequences with known sequences in National Center for Biotechnology Information’s public database of nucleic acid sequences. Then, to find their “viral needles” in the “metagenome haystack”, they use various computational approaches to eliminate non-viral sequences and identify viral sequences. In their results, the researchers found viral sequence from 19 different virus families. Then, when the metagenomes from healthy corals were compared to stressed corals, it was found that the stressed corals had an increased frequency of herpes-virus family sequences.

To confirm that this frequency shift actually corresponded to more herpes genes in stressed corals, the researchers used Real-Time PCR (also know as quatitative PCR) to measure the concentrations of a specific nucleic acid sequence in different corals.  The nucleic acid sequence that was focused on was a herpes virus sequence similar (62% identical) to the thymidylate synthase gene from Saimiriine herpesvirus 2. This experiment showed that indeed, stressed corals tended to have more of this gene in their metagenome than their healthy counterparts.

This study provides an excellent first step into the world of coral virology; it identifies possible candidate viruses that may be contributing to coral illness. However many more questions need to be answered to understand viruses’ role in coral health. For example, few studies have actually observed virus actively hosted by a coral, and none have yet shown that herpes-like viruses can make healthy, unstressed corals sick. The ecological role of viruses may turn out to be surprisingly complex. Some researchers have even proposed that viruses may be necessary for coral survival. Corals host symbiotic algae within their cells, in a mutualism that is a requirement for corals to survive. In order to live inside coral cells, the algae must somehow evade or suppress the corals innate immune response, just as many viruses must do. Will future studies discover a link between the algae infection and virus infection?

Links:

http://dx.doi.org/10.1016/j.jembe.2011.07.030

http://www.pnas.org/content/105/47/18413.full

Ian Heller is a senior at Vassar, majoring in biology.  He is also good at making puns, and had a hard time choosing a title for this article.  Rejected titles included: Catching herpes from coral sex, Viruses and corals: friends or anemonies?, and Virus in the O.K. Coral. 

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Zinc ionophores block the replication of nidovirus

Posted on April 10, 2012 by David Esteban

Contributed by guest blogger: Brian Lu ’13

Zinc ions function in many different cellular processes and have been shown to play important roles in the proper folding and activity of various cellular enzymes and transcription factors, but zinc ion concentrations are kept relatively low in the cell by metallothioneins. High concentration of zinc ions and compounds that stimulate cellular import of zinc ions have been shown to inhibit the replication of various RNA viruses, including influenza virus, respiratory syncytial virus, and several picornaviruses, but details of the effects of zinc ions on nidoviruses are not well understood. Nidoviruses include major pathogens of both human and livestock, including severe acute respiratory syndrome coronavirus (SARS-CoV), the arterivirus equine arteritis virus (EAV), and porcine reproductive and respiratory syndrome virus (PRRSV). A recent study suggests that zinc ions also inhibit nidovirus replication by blocking RNA synthesis.

 

As high concentrations of zinc is known to inhibit cellular translation, the researchers tested if high concentrations of zinc would also inhibit viral translation. After determining the concentration of pyrithione (PT) cells will tolerate without negative effects, cells were incubated with non-toxic concentrations of PT and zinc ions. PT, functioning as an ionophore, stimulated the cellular import of zinc ions and increased the cellular concentration of zinc. The results showed a dose-dependent inhibition of viral gene expression of both SARS-CoV and EAV by the addition of PT. The inhibition of viral gene expression appears to be the result of direct inhibition of RNA-dependent RNA polymerase (RdRp) activity. The researchers also observed a dose-dependent decrease in RNA synthesis for SARS-CoV and EAV by testing the effect of zinc on the virus’s replication/transcription complex (RTC). RNA synthesis, separate from mRNA synthesis for gene expression, is an integral part of viral replication, and a decrease in RNA synthesis would imply a decrease in viral replication as well. Interestingly, the zinc ion’s effects on RNA synthesis are reversible. The addition of magnesium-saturated ethylenediaminetetraacetic acid (MgEDTA) restored RTC activity in both EAV and SARS-CoV. MgEDTA ionizes to magnesium ions and EDTA in solution, which binds to the zinc ions and prevents them from interacting with viral RTC. Adding zinc ions at different stages of RNA synthesis showed that zinc inhibits synthesis at the initiation stage for EAV but inhibits synthesis at the initiation and elongation stages for SARS-CoV.

 

The use of zinc ions and PT as inhibitors of nidovirus replication in cell culture can be further investigated for use as antiviral compounds, and a better understanding of the inhibition mechanism may yield future antiviral drugs against SARS and other nidovirus-related diseases. But before zinc can be used as an antiviral compound, several questions need to be answered. What is the exact mechanism for RdRp inhibition? What are the systemic effects of PT? What levels of zinc and PT would be safe for an organism? The U.S. Food and Drug Administration has approved pyrithione zinc less than 2 percent in concentration for topical use in treating dandruff, but there are no guidelines for internal uses of pyrithione zinc. It is known, however, that industrial concentrations of PT zinc is highly toxic. Additionally, in depth structural analysis and mutational studies of nidovirus RdRps is needed to determine a structural mechanism for zinc-induced inhibition of RdRp activity. Unfortunately, zinc ion binding is very fleeting and not detectable with currently available methods.  As such, more sensitive methods of detecting zinc binding may be needed before the mechanism for zinc-induced inhibition of RdRp activity can be determined. Water-soluble zinc-ionophore may be better suited as the compound appears to be non-toxic even at concentrations that were effective against tumors in a mouse model. The reversible property of zinc-induced inhibition can be used in future research to gain a better understanding of nidoviral RNA synthesis. If pyrithione zinc is shown to be safe and effective in animal models, it still has to go through clinical trials before it can be used as an antiviral treatment.

Link:

http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1001176

Brian Lu is a junior at Vassar College, majoring in biochemistry.

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Posted in Guest Blogger, Molecular Virology | Tagged , | 2 Comments | Edit

Natural Resistance: How Your Genes Can Determine The Severity of Your Influenza Infection

Posted on April 6, 2012 by David Esteban

Contributed by guest blogger: Jared Saunders ’13

Every winter, the general public frantically agonizes over influenza prevention and protection. But is the purchase of hand sanitizer in bulk and tissue boxes by the dozen really necessary? After all, many people don’t even get sick during the winter months, and some just feel a little down for a couple of days. Why do some people catch “the flu” and end up in the hospital, fighting lung infections and plowing through boxes of tissues, while others just end up with a cough or runny nose? The answer may be come down to three letters. DNA.

Recent research by Everitt et al. at the Wellcome Trust Sanger Institute (WTSI) has revealed that a single gene found in humans can determine your fate when infected with a variety of the most common strains of the influenza virus. The gene encodes the important protein referred to as IFITM3, a member of the interferon-inducible transmembrane protein family. These IFITM proteins have been shown to potently restrict the replication of a variety of pathogenic viruses, and IFITM3 has been shown to greatly alter the course of influenza infection in both mice and humans.

Brass et al. previously identified IFITM3 through a functional genetic screen that indicated it mediated resistance to influenza A, dengue virus, and West Nile virus infection in vitro. This supported the hypothesis  of the WTSI group (more than 30 authors!), that IFITM proteins are critical for intrinsic resistance to these viruses, and allowed them to proceed with determining the effects of IFITM3 in vivo using mice. IFITM3 knockout mice showed severe signs of clinical illness, including massive body weight loss, rapid breathing, and piloerection (also known as “goosebumps”) when infected with low-pathogenecity strains of influenza that do not usually cause such intense symptoms. Their presentation of infection was more consistent with high-virulence strains of influenza. Contrary to the knockout mice, the wild-type mice shed significantly less of their body weight before fully recovering.

With this significant data now being collected, the group moved on to testing their hypothesis that individuals who are homozygous dominant for the IFITM3 gene develop less virulent influenza infections. They sequenced the IFITM3 gene from 53 people who were hospitalized by the H1N1 or seasonal influenza infection during 2009 to 2010 to determine if they carried the wild-type gene or one with some polymorphism. Genetic analysis of a subset of these individuals showed no evidence of hidden population structure differences with respect to a 1000 genome control group from WTSI. In the hospitalized patients, the group found significant over-representation of a specific single nucleotide polymorphism (or SNP), referred to as SNP rs12252, that has a recessive C allele substituted for a normal dominant T allele. This leads to an ineffective IFITM3 variant lacking the first 21 amino acids of the protein. This recessive C variant leads to lower IFITM3 expression in the host and consequent increased susceptibility of the host to influenza infection, and is correlated with lower levels of IFITM3 protein expression.

The group’s work has shown conclusively that IFITM3 expression can act as a barrier to influenza A virus infection both in vitro and in vivo, and that in vivo it can lower the mortality and morbidity associated with infection by a variety of human influenza viruses. Discovery of this innate resistance factor in humans may explain why encounters with a novel strain that may cause severe infections in others that do not affect you or your family.

But can the IFITM3 gene be used to help develop treatments or vaccines for future influenza strain outbreaks? Is it possible to recover this gene, if an individual has an ineffective variant, through gene therapy so as to make someone more resistant to influenza? With more research being done on the genetic aspects of disease infection, many more questions will arise, and many more answers will as well!

Links:

http://www.nature.com/nature/journal/vaop/ncurrent/full/nature10921.html

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824905/

Jared Saunders is a junior at Vassar College, majoring in biochemistry.

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Posted in Guest Blogger, Immunology, Molecular Virology | Tagged , , , , , | 5 Comments | Edit

Highly Active Antiretroviral Therapy and Tenofovir: Lowering HIV Viral Loads, Raising the Risk of Renal Failure

Posted on April 4, 2012 by David Esteban

Contributed by guest blogger: Michael McManus ‘12

People undergoing anti-retroviral therapies, which target and interrupt the replicative processes of HIV, are living longer due to the relative success of treatments. Those with HIV are using these drug cocktails for longer periods of time, an important characteristic with results that could not be observed in short-term clinical studies.

Mortality for patients with HIV who are able to undergo highly active antiretroviral therapy (HAART) has shifted from higher rates, during the initial HIV scare, to relatively lower rates. HAART has been incredibly successful, increasing the quality of life for those who have access to it. For some, however, the effects of an HAART regimen, which combines up to four medications, can lead to renal failure within two weeks of regimen administration due to the toxic nature of some of the medications.

Renal failure, or loss of kidney function, can lead to organ failure and eventually death. The kidneys are normally responsible for filtering the blood. They maintain homeostasis by regulating electrolytes, regulating blood pressure, maintaining pH balances, and by removing and diverting waste from the blood into the urinary bladder, producing urine. The kidneys filter many things from the blood in order to retain them in the body, ranging from proteins to glucose. When the kidneys fail, proteins, glucose, and even blood become detectable in the urine. Glycosuria, proteinuria, and hematuria are all biological indicators of a lack of reabsorption and therefore renal failure.

In a recently published paper, Juliette Pavie et al. describe a case of renal failure in an HIV-positive patient after only two weeks of tenofovir therapy. Tenofovir is a nucleotide reverse transcriptase inhibitor associated with low risk of severe renal adverse events in clinical trials. However, tenofovir is in the same class of drugs such as adefovir and cidofovir, which have well-documented nephrotoxicity1,2.

The patient followed was a 46-year-old homosexual male of Scottish descent. His weight, CD4 cell count, plasma HIV RNA level, serum creatine, and urea levels were all taken before HAART regimen, which consisted of tenofovir, emtricitabine, atazanavir, and ritonavir, was started. During treatment, the patient did not take any nonsteroidal anti-inflammatory drugs (NSAIDs), which are known to lead to kidney dysfunction.

Fifteen days after treatment began levels were tested again: serum creatine and urea had increased 3-fold and 2.5-fold respectively. Five days later, the patient became unable to pass urine and levels were immediately measured again: serum creatine showed a 15-fold increase from the original amount, and urea a 12-fold increase. Urinalysis showed glycosuria and proteinuria, indicating loss of kidney function. Renal biopsy indicated necrosis of the kidneys and other abnormalities. After these observations, HAART was halted and hemodialysis was started to rescue lost kidney function. After three months of hemodialysis, HAART was resumed, but tenofovir was excluded from the treatment.

After one year of treatment, the patient showed signs of recovery. CD4 cell count increased to relatively normal levels, and serum creatine and plasma HIV RNA levels dropped. To this day, the patient is still undergoing the same HAART, with serum creatine and plasma HIV RNA levels remaining stable. However, the patient still suffers from moderate glycosuria and proteinuria, indicating that kidney function has not fully recovered.

Although this case only followed one individual undergoing a HAART regimen containing tenofovir, the observations and results are still crucial to studying renal failure resulting from HAART. The novel form of nephrotoxicity observed may serve as a model for other forms of nephrotoxicity caused by reverse transcriptase inhibitors. Although the nephrotoxicity studies1,2 only reported findings in one individual each, their findings should not be discredited, as this is the nature HIV symptom studies. For example, the emergence of Kaposi’s sarcoma as a symptom of HIV began with isolated incidents. The nature of HIV and its rapid mutation also obfuscates the relationship between HAART effectiveness and strain type. From this observation, one question out of many must be addressed: Did the combination of drugs used in the patient’s HAART regimen have an effect on nephrotoxicity?

Despite the emergence of renal failure as a threat, great strides have been made in the fight against HIV. The quality of life for those who are suffering from HIV and who have access to HAART is drastically improved compared to those who are unable to undergo HAART. However, now that HIV patients are living longer, research must switch from just targeting HIV to focusing on HIV and the complications created by decreased mortality. Nephropathy, or disease of the kidney, and subsequent nephrectomy, removal of a kidney, now contribute to the decrease in quality of life associated with the aging HIV population. When developing future treatments, scientists and doctors must analyze the nephrotoxicity of the products they are synthesizing, as renal failure is a clear and present danger for those undergoing HAART.

Article Links:

http://online.liebertpub.com/doi/abs/10.1089/apc.2011.0056

1Tanji, N., Tanji, K., Kambham, N., Markowitz, G. S., Bell, A., and D’Agati, V. D. (2001) Adefovir nephrotoxicity: Possible role of mitochondrial DNA depletion. Human Pathology. 32, 732-740.

2 Meier, P., Dautheville-Guibal, S., Ronco, P. M., and Rossert, P. (2001) Cidofovir-induced end-stage renal failure. Nephrology Dialysis Transplantation. 17, 148-149.

Michael McManus is a senior at Vassar College, majoring in Biochemistry

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Posted in Antiviral Drugs, Epidemics | Tagged , , , , , , | 3 Comments | Edit