All posts by David Esteban

Teaching and Research

Microbiology can be dirty

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Students are starting to repopulate the campus and the relaxed pace of the summer is being quickly replaced by frenzied preparation for the start of the semester. This semester, I will be teaching the intro to microbiology course that I have taught every year. In the lab, we do some bioinformatics analysis of metagenomic data from a Winogradsky column. A Winogradsky column is a clear cylinder full of pond mud that is used as an enrichment culture to grow bacteria that cant be grown under normal laboratory conditions. To make a Winogradsky column, you collect mud from a pond or riverbank. (For those of us that are used to the cleanliness of working in a sterile hood, that means you have to take off your lab coat, get down on your hands and knees, and scoop up handfuls of goopy stinky slimy stuff from the the edge of a pond. I wear latex gloves.) You then add it to a plexiglass cylinder along with a source of cellulose (I use leaf litter) and additional sulfate to promote enrichment for microorganisms involved in the sulfur cycle. Over a period of months, layers of microorganisms requiring a range of environmental conditions develop in distinct niches with distinct populations participating in diverse metabolic activities. As various metabolites in the column are used, byproducts are produced, and the environment in the column changes. As a result of changing concentrations of oxygen, hydrogen sulfide, and variations in metabolites, different microbes will thrive and create their own niche.

Although you can see some changes occurring in the first few days, it takes several weeks or months for it to develop so I always set it up before the semester starts. This year, I had the help of my daughter (age 5) who was eager to get her hands into the gooey muck. We took the mud from the edge of Vassar Lake, a pond on campus. In the pictures you can see the changes that take place over time. The column starts out as grey silt, while the column on the left is 1 year old. The patches of colours are the different communities of bacteria.

(OK, so there is no virology in this post but it sure would be interesting to analyze the viral population of the column in addition to the bacterial population. What is the role of bacteriophages in the community dynamics and nutrient cycling in the Winogradsky column?)

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Antiviral Drugs, Molecular Virology

Broad Spectrum Antiviral is an Effective Inhibitor of Viruses but Not Media Hype

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There have been a few important successes in antiviral drug development, but for the most part it is extremely difficult to find drugs to treat viral infections. Viruses are highly effective hijackers of cellular processes, and since drugs that target cellular processes are likely to be toxic, that leaves only a few potential virus specific targets. Further, because of the diversity of viruses most antiviral drugs are very specific for their target virus and are not effective against others. The holy grail of antiviral drugs would be something that inhibits replication of many viruses – a broad spectrum antiviral.

A new paper in PLOS One describes a creative approach to the problem of broadly targeting viral infections. The approach uses a recombinant protein that combines the properties of two natural antiviral response pathways in the cell. Almost all viruses generate long double stranded RNA (dsRNA) at some point in their replication cycle. But long dsRNA is not found in normal, healthy cells. So the presence of dsRNA triggers a response in the cell that shuts down gene expression, which can effectively shut down viral replication. Another response is apoptosis: an infected cell can trigger a cellular suicide pathway, destroying the infected cell and the virus along with it. In this new study, researchers fused parts of proteins from key mediators of both pathways to generate a new protein called DRACO that triggers apoptosis when it binds to dsRNA.

In cell culture, DRACOs induce apoptosis in cells only when dsRNA is present. The study goes on to test whether DRACOs can protect cells from infection. Cells pre-treated with DRACOs survive viral infection, while untreated cells don’t. And it protects against a large variety of different viruses; viruses from 7 different families were tested and DRACOs appear to be effective against all of them. But for an antiviral to be useful it has to also work in vivo, and DRACOs show some promise here too. 60-70% of mice pre-treated with DRACOs survive influenza virus challenge, while only 10% of untreated mice survive.

The results of this study are exciting and show promise but we must be cautious about over-extending the findings. It is a long and challenging process to go from an early stage development like this one to a clinically useful anti-viral drug. There will be some very significant hurdles to overcome to develop this further. In all the experiments, the cells (or animals) are pre-treated with DRACOs, but its not known if there is any post-exposure protection. The animal studies show that interperitoneal injection can protect animals, but the distribution of the drug in different tissues varies and in some tissues disappears before 24 hours, which could be a problem if it can’t reach the required concentrations in the target tissues. Also, being a protein, there will be challenges for effective delivery (since interperitoneal injection is not likely to be a favored route!). Many years of further testing, development, and clinical trials (if it gets that far) are needed. Despite the many challenges that lay ahead for further development of this broad spectrum anti-viral, it appears promising and worth pursuing.

As usual though, news reports describing this as a major breakthrough have missed the point that this is an interesting new development but that it is very much in its infancy. Since nearly every discovery is hailed as a breakthrough, the public gets a distorted idea of the way science proceeds. Media reports have also been misleading, including promoting it as a potential treatment for HIV or Hepatitis, which weren’t even among the viruses tested, or touting it as a possible cure for nearly all viral infections. Slow down a little, folks. There is a long way to go before those claims can be made. Perhaps someone can develop a treatment for the disease that makes headline writers distort information.

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Evolution, Teaching and Research

Variola virus evolution

Why do some people get severely ill with an infection while others catch the same virus but don’t get sick? There are many factors that can influence the progression and outcome of disease, but they can be lumped into four basic categories: host, agent, transmission and environment. For example, infection with Variola virus results in smallpox, but case fatality rates in different outbreaks range from very low to as high as 30%. Its likely that many factors contribute to this variability, but it’s likely that differences in viral strains is one of them.

Summers are quiet here at Vassar. There are no summer classes but we do have a program to support undergraduate research (“URSI”) so that students can gain some research experience and professors can get cheap labor. This summer I had a student working with me who is a Biochemistry major and Computer Science minor (called a “correlate” here). She can write code and I cant, so I had her working on a bioinformatics project. We were interested in investigating the difference between poxviruses that cause high mortality rates in humans (like some strains of Monkeypox virus and most strains of Variola virus) and those that dont. I recently published a paper along with another undergraduate student showing that certain genes in poxviruses are under Darwinian selective pressure. We wanted to test the hypothesis that the selective pressure differs between virulent and avirulent strains. She used several approaches involving analysis of synonymous and non-synonymous mutation rates to see if amino acid altering mutations were fixed at different rates in virulent and avirulent viruses.

As she crunched away at code writing and data analysis and discovered one of the joys(?) of science: failing to support your hypothesis. We could not find evidence that selective pressure differed between virulent and avirulent strains. Although no Vassar student wants to fail, failing to support a hypothesis is not actually failure. Rather, its an integral part of the scientific process, something that comes from the successful execution of an experiment that tests your hypothesis. The scientific method is actually quite humbling: you set up experiments that will tell you if you are wrong, and as a scientist, you have to get used to proving yourself wrong.

So now we must ask a new question: since some poxvirus genes show evidence of selective pressure, and that selection is not related to virulence, what is the cause of that pressure?

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Teaching and Research

Virology Research and Teaching in the Liberal Arts

I work at a small liberal arts college, where I teach and do research. The balance can be difficult; the teaching is quite demanding so getting research done is challenging. During the school year my efforts are focused on teaching and the summers are when I can get more research done. With any job there are always things that could be better, but I have enjoyed being at Vassar since I got here in 2007. I just spent several days at the ASV (American Society for Virology) meeting in Minneapolis, where I reunited with several friends, met new people, and gained a greater appreciation for the great job that I have!

First, just having a job is good, let alone one that is exactly what I wanted. I met many post docs seeking and failing to find positions. I met researchers that want to teach but can’t due to the lack of opportunities at their institutions, or simply aren’t allowed to. I met researchers who are completely dependent on grant money for the continued existence of their position. With the current state of funding, that is certainly not a position I would want to find myself in.

The nature of my position is very different from most other virologists. I imagine there are other virologists at liberal arts colleges but I only met one at ASV. In addition to posts on cool things in virology, and posts by students, I will start adding posts on my experiences doing virology research and teaching at a liberal arts college

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Epidemics, Uncategorized, Vaccines

Goodbye Rinderpest, Hello Measles

Variola virus, the agent of smallpox, once held a lonely spot on the list of globally eradicated diseases. Now it is joined by rinderpest, the cattle plague. The OIE (Organization for Animal Health) declared the disease eradicated and the UN’s Food and Agriculture Organization (FAO) is expected to adopt a resolution in June declaring it eradicated. The disease affects cloven-hoofed animals and can have an extremely high mortality rate in some cattle and buffalo. It is also extremely contagious, so its rapid spread through livestock can have an obviously large impact on animal health, food production and livelihood of cattle farmers. Thanks to an intensive word-wide vaccination effort, rinderpest virus can now be added to the list of organisms we actually intended to make extinct.

Rinderpest is caused by rinderpest virus, a member of the Morbillivirus genus. Another member of that genus is measles virus. Oh, and there is a vaccine for that too. In fact North America was free of measles in 2002 and perhaps it was on track for global eradication. But not anymore.

My son recently turned one, so I took him to get his measles, mumps and rubella (MMR) vaccine, feeling confident that Im helping protect him from three pretty nasty viruses, and not giving him Autism. In fact, before he turned one, I’d been feeling a little anxious about getting him the vaccine soon enough. My email inbox keeps filling up with notices from ProMED mail (Program to Monitor Emerging Diseases) with news of various measles outbreaks across the globe.

There are outbreaks all over Europe, especially in France, the UK, Spain and Switzerland. The epidemic in France that started in 2008 has now reached over 14,000 people, with 9000 of those infections reported in the last 6 months. (France has a vaccination rate of about 60%. Vaccination rates in the UK bottomed out at 80% and are slowly on the rise again). Outbreaks in the USA and Canada have been small, the vaccination rates are higher but not high enough. Many of these can be tracked to travel of unvaccinated individuals to areas where measles is still endemic or flaring up. In Minnesota, an outbreak counting 21 people has sent 13 people to the hospital (an unusually high number). Of the 21 people, 8 were old enough to be vaccinated but weren’t, 7 were too young to be vaccinated, 1 was vaccinated and the status of the others is unknown.

The Minnesota outbreak emphasizes an important point: it is necessary to maintain a sufficiently high level of herd immunity to prevent outbreaks and protect those who can’t be vaccinated. For most diseases, vaccination rates need to be at or above 95% to prevent outbreaks, and may need to be even higher for measles. Virus transmission depends on the virus finding a susceptible host. If a population is primarily made up of immune individuals, the virus has a hard time maintaining a chain of transmission.

Vaccination is therefore not just a matter of personal health, but community health. Maintaining high herd immunity helps protect babies too young to be vaccinated by limiting the chances that they ever encounter the virus. Don’t just get your kids vaccinated to protect themselves, do it to protect us all.

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Epidemics, Evolution, Guest Blogger, Vaccines

Swine Flu: New and Improved!

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Contributed by guest blogger: Marni Hershbain ’11

Flu season is never enjoyable, but some seasons are certainly worse than others. The 2009 swine flu outbreak was particularly serious because the 2009 H1N1 strain was a novel virus, formed via the reassortment of swine, avian and human flu viruses. There were over 600,000 confirmed cases of H1N1 and over 18,449 deaths during the course of the pandemic. While this sounds pretty bad, it could have been much worse. The transmission efficiency of H1N1 was actually much lower than those of other pandemic strains, such as the 1918 H1N1 strain. Unfortunately, recent research demonstrates that this could change.

Flu strains are characterized by the hemagglutinin and neuraminidase found on their surfaces, hence names like H1N1. In order for the virus to infect a cell, hemagglutinin on the surface of the virus must bind to glycan receptors on the cell. Therefore, to explain the low transmission efficiency of 2009 H1N1, researchers looked to its hemagglutinin.
In most flu strains, the amino acids at positions 219 and 227 within the hemagglutinin are both hydrophobic or both charged. In 1918 H1N1 both are hydrophobic. However, the 2009 H1N1 strain has isolucine, a hydrophobic molecule, in position 219 and glutamic acid, a charged molecule, in position 227. Researchers hypothesized that lacking either hydrophobic or ionic interactions at these positions would disrupt the positioning of neighboring residues and decrease the hemagglutinin’s binding affinity. They further hypothesized that if they replaced isolucine with the charged amino acid lysine, stable inter-residue interactions would occur and binding affinity would increase.

When researchers compared the ability of wild type and isolucine→lysine mutant strains to bind to an array of glycans representing human binding sites, they found the binding ability of the mutant strain was 30 times greater. The mutant version also bound more intensely to receptors in human tracheal tissue. Researchers also infected ferrets (commonly used as models in human influenza studies) with either wild type or mutant virus. Only the ferrets infected with mutant virus spread the infection to all of the previously uninfected ferrets placed in close proximity to them.

The mutation of just one amino acid could greatly impact the transmission efficiency of 2009 H1N1. Flu viruses tend to mutate frequently, which is why a new vaccine needs to be developed every year. Predicting what these mutations will be is not an easy task, but mutations at the positions in this study will certainly be monitored closely.

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Immunology, Vaccines

Can adenovirus be used to help cure a cocaine addiction?

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Contributed by guest blogger: Jessica Hughes ’11

It is well known that drug addiction is a worldwide problem, and so finding a therapy or cure for this issue would be extremely valuable. Scientists have been trying to create a vaccine for people with drug addictions that would allow them to be rid of their chemical dependence, but there are several challenges they face in trying to do so. First, addictive drugs are small molecules that do not cause an immune response on their own. Furthermore, because of the extremely high level of drugs often found in the blood of a systemic drug user, there needs to be a way to create high-titer, high-affinity antidrug antibodies to address that extremely high drug concentration. This second challenge has limited the effectiveness of many attempts at anti-addiction active immunization strategies.

In a 2010 study, researchers looked at creating an anticocaine vaccine with the help of adenovirus. With the knowledge that inhaled cocaine could not reach its target receptors in the brain when exposed to anticocaine antibodies, researchers looked into the possibility that cocaine addiction could possibly be reversed with an anticocaine vaccine. Here’s where adenovirus came in. Researchers knew that adenovirus gene transfer vectors act as potent immunogens, which provoke adaptive immune responses. They predicted that if they coupled the adenovirus with a cocaine analog, they could elicit high-titer antibodies against cocaine and successfully prevent this drug’s access to the brain. Specifically, they used a disrupted E1-E3- adenovirus gene transfer vector, which means they were able to avoid viral gene products that would pose a risk of infection to the vaccine receiver but still have the benefit of the immunogenic property of the vectors. E1-E3- has been used many times in gene transfer applications, proving to be very safe.

In their experiment, once they created the vaccine (called dAd5GNC), they used mice to test its effects. Both naïve mice and vaccinated mice were given cocaine intravenously, and subsequently their locomotor activity was observed. The administration of cocaine caused hyperlocomotor activity in mice. These effects were completely and consistently reversed for the vaccinated mice. This is a promising result, and further studies obviously need to be done to continue looking into the possibility of using anti-addictive drug vaccines. Some questions to think about: Would an anticocaine vaccine work in the real-life scenario of preventing an addict from relapsing? Could there be dangers with taking these vaccines, such as accidental overdoses of someone trying to obtain the feeling he/she is used to getting from the drug?

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Vaccines

Dear Hudson Valley Parent Magazine

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Updated April 20

A local parenting magazine just published a story by Robert Lachman on vaccines and autism perpetuating the false link between them. An online version is available, which is slightly different from the paper version. In the past, Ive ignored these kinds of articles because I just get so frustrated. But I recently decided that if someone like me doesn’t speak up and start contributing accurate information to the public, who will?

As such, I am drafting a letter to the magazine and am seeking your editorial input. I want to write a clear, strong letter but also dont want to scare readers away with too much “science-y” stuff. The target audience is parents with, most likely, no science background. Please read what I have written and let me know what you think.

The article also refers to a study presented at a 2010 Pediatrics Academic Society Conference in Vancouver which he claims confirms the link. Thanks to a reader, we may have found the studies to which he refers, which do not support his claim (see comments section).

Anyway, here is a draft of my letter:

“Dear Hudson Valley Parent Magazine:

In your most recent issue, a story titled “Vaccines and Autism: The Controversy Continues” was published. The only thing that is continuing the controversy is continued publication of articles such as these that present misleading information. Scientifically, the issue is settled: there is no link between autism and vaccines.

The story downplays the fraud committed by Andrew Wakefield. It is clear that Wakefield falsified his data. He has been stripped of his positions, degrees, and license to practice. Brian Deer, the reporter who uncovered the fraud, is now under attack. Shooting the messenger is just a last ditch attempt to save a movement based on falsehoods. All you have to do is look at the data and the findings, the large body of independent scientific research that clearly shows there is no link.

It is also clear that he had financial interests in seeing the MMR vaccine discredited, since he had developed his own vaccine. Interestingly, the anti-vaccine movement is quick to blame vaccine manufacturers as being influenced by profit motive or finding conspiracy in discrediting Wakefield’s findings, but Wakefiled’s clear financial conflict of interest is conveniently overlooked.

However, I realize that evidence of fraud and financial conflict of interest isn’t going to resonate with many in the anti-vacccine movement. The fact is, scientifically, it doesn’t really matter that his data was fraudulent. The Wakefiled study involved only 12 children, a sample size much too small to draw strong conclusions. Further, the study was poorly designed: to connect vaccines to autism, the study depended entirely on parental or physician recall of events in the past, a method known to be highly ineffective. In fact, it’s not much different from anecdotal data. Since Wakefield’s study, there have been many subsequent studies involving larger number of participants that have been rigorously designed and appropriately controlled and NONE have supported a link between vaccines and autism. The reason the scientific method works, and the reason humanity has gained great knowledge and understanding of the natural world around us is that the scientific method is self-correcting. Any finding must be verified independently, by other researchers, using a combination of different approaches. Only after a significant body of work is developed can a strong conclusion be made. Wakefield’s study triggered such a body of research, which has consistently shown that that there is no link between vaccines and autism. That is to say, Wakefield’s conclusions have not withstood the test of the scientific method. The fact that his data was fraudulent only confirms what scientist already knew: vaccines do not cause autism.

The article describes the personal experience of specific parents who believe their children are autistic as a result of vaccination. Anecdotal data can be used to support any position. Where are the interviews with parents of vaccinated children who don’t have autism? The anecdotal data doesn’t sand up when you consider the many anecdotes I have about parents who vaccinated and don’t have autistic kids. Whose anecdotes should we believe? The only information we should consider is from well designed and controlled studies.

The article also describes a study presented at the Pediatrics Academic Society conference in Vancouver in 2010, which he claims confirms the link between autism and vaccines. I, and others, have carefully searched the abstracts of the work presented at the conference and found no such study. Two abstracts addressed gastrointestinal symptoms associated with autism, the closest I could find to the topic. However, the issue of vaccination is not addressed in these studies. It would be appreciated if the author provided a specific citation so that readers can look at the study themselves. It is concerning to me that either Mr. Lachman completely misunderstood the research or is being intentionally misleading. In fact, in the article, he writes that Wakefield’s study connecting vaccines to autism has been vindicated, and in support states that the PAS study links gastrointestinal disease in autistic children. The issue in question is not regarding a connection between gastrointestinal symptoms in autism but a connection between vaccines and autism. There appears to be a vast leap, making conclusions that simply can not be made from the data. Implying that these studies support a link to vaccination is entirely misleading.

Finally, Wakefield’s study, the actions of the anti-vaccine movement and the perpetuation of misleading information is troubling on a very deep level. Children are dying from preventable diseases, directly attributable to decreased vaccination rates. It is a crime that this should happen. The other victims in this whole scandal are kids with autism. Rather than focusing on finding the actual cause of autism, the distraction of the vaccine link has driven the focus away from valuable research that needs to be done. With so much data to support the absence of a link between vaccines and autism, and so much reason to find the real cause (or causes) of autism, I seriously wonder whether the driving force behind this movement is now the desire to be right, rather than the desire to protect our kids.

Sincerely,
David Esteban
Assistant Professor of Virology and Microbiology
Vassar College

Im adding some links:
Here is Wakefield’s paper. You might find it hard to read due to the big red RETRACTED written over each page.
One of the articles in BMJ regarding the fraud.
An Editorial from BMJ

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Epidemics, Guest Blogger, Immunology, Vaccines

A possible new HIV vaccine target?

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Contributed by guest blogger: Lydia Mendoza ’11

In 2009, it was estimated that 33.3 million people in the world were living with HIV/AIDS. Since the discovery of HIV, more than two decades ago, money has poured into research in the hopes that an effective vaccine might be developed. As of yet a vaccine remains elusive. One reason why it is so difficult to create a vaccine is because HIV is highly mutable and genetically diverse subtypes, or clades, have evolved. A vaccine needs to be able to offer protection from a range of HIV clades.

Normally viral vaccines are based upon neutralizing antibodies, which prevent infection of the host cell. The first attempts to develop neutralizing antibodies against HIV targeted gp120, which is known to play a role in HIV’s ability to enter and infect CD4 t-cells. These attempts have not been successful as of yet because of the gene’s high rate of mutations. However a recent paper has shown that the V3 loop of gp120 is a potential vaccine target.

The strand of protein known as the V3 loop was never thought to be an attractive vaccine target because it is not highly conserved. However, it appears to have conserved structural elements that are involved in interactions with coreceptors. To study whether V3 was a viable vaccine target, a human monoclonal antibody, HGN194 was used. HGN194 was isolated from memory B cells of a person infected with HIV-1 clade AG circulating recombianant form (CRF). HGN194 targets the V3 loop and has been previously shown to neutralize a broad range of neutralization-sensitive and resistant strains of HIV.

The study evaluated whether HGN194 was able to protect rhesus monkeys from an HIV model system. One group of monkeys was injected with HGN194 then they were challenged with a high dose of a clade C SHIV, which is a chimeric simian-human imunodeficiency virus encoding HIV envelope genes in a SIV backbone. The second group of monkeys was also given a high dose of SHIV but was not given the HGN194. The monkeys given the antibody were protected from SHIV infection, and those not given the antibody were infected. The researchers concluded that HGN194, isolated from an HIV-positive individual harboring a clade AG CFR, was able to confer complete cross-clade protection against clade C SHIV.

The antibody apparently latches onto the virus’s V3 loop and prevents the virus from invading cells. This does not mean that this antibody treatment technique is a vaccine for HIV. It does not create long-term protection because the antibodies do not remain active in the body for very long. This is only a first step. A vaccine target has been identified but now scientists must create an antigen that induces formation of an antibody similar in structure to HGN194. There is a lot of work left to be done but this finding hopefully brings researchers much closer to the development of a vaccine.

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Evolution, Guest Blogger

The strangest family reunions

Contributed by guest blogger: Amelia McKitterick ’11

Next time you are sick from a viral infection, you should ask yourself if you’re just hosting a visit from distant relatives. Although “relative” might not seem like the most appropriate term for a virus, there has been evidence of a history viral influences and insertions into animal genomes, including that of humans!

A crucial step in the replication of RNA retroviruses is the integration of the viral genome into the host genome. Fragments of a viral genome in the genome of a non-viral cell are called endogenous viral elements (EVEs), and they can either be phased-out of the host genome or be passed on to become fixed within a population. A recent study examined genomes of a variety of mammals, birds, and insects for EVEs with matching amino acid sequences to extant, non RNA retroviruses. The genomes of 44 animals were converted into amino acid sequences and checked via tBLASTn (a BLAST that matches amino acid sequences with nucleotide sequences) for alignment with a library of currently known mammalian viruses with genomes larger than 100 kb in length. Matches were found to viruses with all types of RNA genomes (ss/ds, +/-, segmented, un-segmented) in all three of the major phyla tested, matches to DNA genomes (ss/ds, rt) were only found in mammals and birds, and even unclassifiable viral proteins were found in mammals that could represent extinct or undiscovered lineages.

But what is the use of all this new information? First, the data can be used to determine the minimum evolutionary divergence dates of different viral families based on host divergence dates. This study of paleovirology estimated the minimum ages of virus fossils Parvo-, Circo-, Filo- and Bornaviridae within the mammalian samples and found the oldest (Borna-) to be about 93 million years old, where it was originally infecting the distant relatives of the Afrotheria clade (Elephants, hyrax, tenrec, etc. For reference, the common ancestor of the primates evolved about 85 million years ago). A second use of the data is to illustrate the variety of viruses, and to give a better indication of the types of viruses that infect different hosts. The presence of the EVEs in a host genome can provide new insight about the replication process of non-reverse transcription viruses, and show patterns of host vulnerability. Similarly, new viruses, such as the unclassifiable EVE in mammals, could lead to new routes of investigation into the types of viruses and cures to infections.

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