Giant dsDNA Virus Origins: Megaviridae Evolutionary Analysis

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

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?

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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