Unraveling the NEIDL

11 April 2014

Threading the NEIDLThe NEIDL (National Emerging Infectious Diseases Laboratory) at Boston University is a newly constructed biosafety level 4 facility which can be used to study the most dangerous human pathogens. The facility is amazingly safe, as we documented in our film about the facility, Threading the NEIDL. Some members of the Boston City Council think otherwise and have moved to stop the facility from opening.

In January 2014 a draft ordinance was introduced to the Boston City Council that would prohibit BSL-4 research within the city limits. The first public hearing on the proposed ordinance is scheduled for Wednesday, April 16, 2014 at Boston City Hall. Members of the public are invited to attend and testify. I encourage you to read the proposed ordinance which is available online. If you live in the Boston area and have a view on this ordinance, you might consider attending the public hearing. The Boston City Council Calendar of Meetings should be consulted for scheduling changes.

The American Society for Microbiology has provided this statement on the ordinance:

As background information you should be aware that In 2007, the ASM filed an Amicus brief with the Supreme Court of Massachusetts on a case involving a BSL-4 laboratory affirming the importance and safety of BSL-4 research laboratories. That same year, the Society also provided similar testimony before the U.S. House of Representatives.

We believe that scientists should be aware of events that can impact science in their region and have the opportunity to voice their opinions on actions that could affect them, directly or indirectly. We urge any members who are inclined to attend.

The Boston City Council Ordinance seems ill-advised, especially since the NEIDL has gone through a supplemental risk assessment process, and has been blessed by two independent scientific groups (the National Research Council and the NIH Blue Ribbon Panel), and prevailed in Federal Court.

While it might seem frightening to have a BSL-4 facility within a major city, after having toured the NEIDL I can say with confidence that the precautions taken to prevent release of pathogens, both physical and operational, are second to none. As Elke Muhlberger noted during our tour, it is the safest place to work on Earth. I strongly recommend that the Boston City Council members view Threading the NEIDL to learn why it does not make sense to prevent opening of this facility.

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HIV binding CD4 and ccrOn the science show This Week in Virology we receive many questions and comments, which are read every week. I also get many questions here on virology blog, which I tend to answer by email. However I think that everyone could benefit from these questions, so I’ve decided to post one here each week along with my answer.

This week’s question is from Joseph, who wrote:

I’m relatively new to virology or anything biology-related. Hell, I’m studying computer science as an undergrad at the moment; however, there’s something about virology that fascinates me – the simplistic fact that we can’t cure viruses, which are less complex than bacterium (in which we can treat, and they’ll eventually pack their bags and leave).

I’ll get to my question … since most, if not all, cells in the body replicate and reproduce and none of them merge, why do our cells let virions in? You would think after years of viral/immune system encounters, our bodies would have adapted to repelling these viruses off. I understand it’s probably much more complicated than that, but I would love to hear your answer. Does it have anything to do with virions’ size being so small?

This is a great question. In fact, I had a similar question on a midterm examination in my virology course. I phrased it this way: Could cells evolve to not have receptors for binding viruses?

I sent this answer to Joseph:

Viruses get into cells by binding to proteins on the cell surface – viruses have evolved to do this: they are safecrackers.

You would think that the cells would evolve to change these proteins – and you would be right. Over thousands of years, the cell proteins change, so the viruses can’t bind anymore.

But guess what? The viruses change right back so that they can bind to the cell protein once more.

Now you might ask: why doesn’t the cell get rid of that surface protein? The answer there is that they are needed for the cell, so they can’t be removed.

There seems to be one exception to the last statement: about 4-16% of people of Northern European descent don’t make one of the receptors for HIV. They are resistant to infection. But this doesn’t happen for most other viruses.

Joseph wrote back:

Hmm. I thought by definition virions weren’t living organisms, yet they “adapt” to bind to living cells. Sounds like those emotional virions just can’t deal with rejection – that and our cells just aren’t as smart as we need them to be. I’m not sure if you are a Trekkie; however, it reminds me of the Borg and The Enterprise’s encounter – The Enterprise adapting to The Borg’s every frequency of their phasers, bypassing their bruteforce.

That does make sense that our cells do need that protein surface for energy; however, I never thought it would actually be the surface itself. Interesting.

I did read about that somewhere – because of the Bubonic Plague causing some genetic mutation, if I’m not mistaken.

To which I responded:

Virus particles are not alive – but once they infect a living cell they can evolve.

Both cells and viruses are smart – they both have managed to be around for a long time. We have great immune systems; virus infected cells can evolve very quickly. It’s an arms race.

Correct, one idea is that the mutation conferring resistance to HIV was acquired in the Plague, but that’s hard to prove.

The mutation we are discussing is of course ccr5delta32, which confers resistance to infection with HIV-1 (the illustration shows the HIV-1 glycoprotein binding CD4 and ccr, a chemokine receptor). You can read more about ccr5delta32 here or listen to us discuss it on TWiV #278. We also talked about virus-receptor arms races on TWiV #242, and I wrote about it here.

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On episode #279 of the science show This Week in Virology, Vincent, Alan, and Kathy reveal how a retrovirus in the human genome keeps embryonic stem cells in a pluripotent state, from where they can differentiate into all cells of the body.

You can find TWiV #279 at www.twiv.tv.

Retroviruses R us

3 April 2014

HERV-HAbout eight percent of human DNA is viral – remnants of ancestral infections with retroviruses. These endogenous retroviral sequences do not produce infectious viruses, and most are considered to be junk DNA. But some of them provide important functions. The protein called syncytin, which is essential for formation of the placenta, originally came to the genome of our ancestors, and those of other mammals, via a retrovirus infection. Another amazing role of endogenous retroviruses is that they regulate the stem cells that are the precursors of all the cells in our body.

The genetic material of retroviruses is RNA, but during infection it is converted to DNA which then integrates into the chromosome of the cell.  If the infected cell happens to be a germ cell, then the viral DNA, now called called an endogenous retrovirus, becomes a permanent part of the animal and its offspring. One of our endogenous retroviruses, called HERV-H, infected human ancestors about 25 million years ago. HERV-H has been found to be important for the properties of human embryonic stem cells.

Embryonic stem cells (ES cells), which are derived from the inner cell mass of a blastocyst (which forms 4-5 days after implantation), are pluripotent – they can differentiate into every cell type in the human body. Being pluripotent means expressing a very different set of genes compared with somatic cells – the cells of skin, muscle, organs, to name a few. The genes that are expressed in ES cells are controlled by a small number of key proteins that regulate mRNA synthesis. If these proteins – just four – are produced in a differentiated cell, it will turn into an ES cell – an induced, pluripotent embryonic stem cell, or iPSC. This observation garnered Shinya Yamanaka the Nobel Prize in 2012.

The first clue that HERV-H might be important for the pluripotency of ES cells was the finding that this DNA is preferentially expressed in human ES cells (the figure [credit] shows the expression of HERV-H in ES and two other cell types). When the levels of HERV-H RNAs are reduced (by RNA interference) in ES cells, the morphology of the cells changes – they become fibroblast-like, a sign of differentiation. In contrast, when fibroblasts are reprogrammed to become iPSCs, the levels of HERV-H RNAs rise. These findings suggest that HERV-H is essential for keeping ES cells pluripotent, and for making somatic cells pluripotent.

The HERV-H DNA in our genome is flanked by viral sequences called long terminal repeats, or LTRs. These provide initiation sites for the synthesis of viral mRNAs. In human ES cells the HERV-H LTRs appear to be enhancing the transcription of nearby human genes that are important for maintaing pluripotency. In an interesting twist, the HERV-H viral RNA is important for this activity: it appears to bind proteins involved in the regulation of mRNAs important for pluripotency. This observation explains why reducing HERV-H viral RNA leads to loss of pluripotency.

The HERV-H RNA made in human ES cells is not translated into protein because it contains many mutations that have accumulated over the past 25 million years. Therefore HERV-H is a long, non-coding RNA (lncRNA), a relatively recently discovered class of regulatory RNAs. There are about 35,000 lncRNAs in human cells that are involved in controlling a variety of processes such as splicing, translation and epigenetic modifications. Now we know that endogenous retroviruses can also produce lncRNAs.

Without endogenous retroviruses, humans might not be recognizable as the Homo sapiens that today walk the Earth. They might also be egg-layers – but the eggs would be white. Viruses don’t just make us sick.

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On episode #278 of the science show This Week in Virology, Vincent, Dickson, Alan, and Kathy discuss disruption of the ccr5 gene in lymphocytes of patients infected with HIV-1.

You can find TWiV #278 at www.twiv.tv.

Cross-stitched viruses

29 March 2014

The latest addition to the Microbe Art gallery here at virology blog is Watty’s Wall Stuff, where you will find beautiful cross-stitched viruses such as influenza virus, rabies virus, human immunodeficiency virus, herpesvirus and more. Here are some examples of Alicia Watkin’s delicate and creative work.

influenza virus

 

bacteriophage T4

There are even bacteria, fungi, and parasites:

E. coli

Aspergillus

Naegleria

And one of my favorite cells of the immune system, dendritic cells:

Dendritic cell

You can even ask Alicia to make a cross-stitch of your favorite microbe. Mine, of course, is poliovirus. You can also purchase the patterns if you’d like to engage in some synthetic virology.

Thanks to Stephen and Jon for making Watty’s Wall Stuff a Listener Pick of the Week on TWiV #278.

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Heartland virus disease

28 March 2014

Amblyomma americanum

Six new cases of Heartland virus disease have been identified in residents of Missouri and Tennessee. The cause of this disease appears to be a member of the Phlebovirus genus in the Bunyaviridae family that was first identified in 2009 and appears to be transmitted by the Lone Star tick (Amblyomma americanum, pictured).

Heartland virus was first identified in two Missouri farmers who were hospitalized with fever, leukopenia (low numbers of white blood cells), and thrombocytopenia (low numbers of platelets). Both were males over 55 years old who reported being bitten by ticks in the week before disease onset. The novel virus was identified by electron microscopy, viral culture, and genome sequencing. For a complete discussion of this case, listen to This Week in Virology #199.

The tick A. americanum was implicated in transmission of Heartland virus because this species is extremely abundant in central and southern Missouri. However, virus was not isolated from ticks.

Since the description of the first two cases of Heartland virus disease, the Centers for Disease Control and Prevention have worked with state and local partners to develop diagnostic tests and identify additional cases. The six new cases were identified in 2012-13 in men over 50 years of age with fever, leukopenia, and thrombocytopenia. Presence of Heartland virus was determined by polymerase chain reaction using blood or tissue specimens, and by detecting a rise in antibodies against the virus in serum samples taken during and after illness. All six patients spent hours outdoors each day, and five reported tick bites in the two weeks preceding illness onset.

These studies strongly suggest but do not prove that Heartland virus was transmitted by ticks to these patients. One important piece of information will be finding Heartland virus in ticks. A first step is the identification of viral RNA sequences by polymerase chain reaction from ticks captured on the farms of the first two Missouri patients.

These findings raise several interesting questions. Why have the 8 patients with Heartland virus disease all been elderly males? Has this virus been present in the US for some time, and we have just detected it, or was it introduced from elsewhere? Does the virus circulate only in Missouri and Tennessee, and might it be found in other species of tick? Is there an animal reservoir as has been suggested? And what is the relationship of Heartland virus with the phlebovirus that causes severe fever with thrombocytopenia syndrome (SFTSV), an emerging disease in China that has also been detected in Japan and South Korea? Nucleotide sequence analysis reveals that Heartland viruses and SFTSV are highly related.

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Virology class 2014In the spring of each year I teach a virology course to undergraduates and masters students at Columbia University. I produce video recordings of all my lectures not only for students in the course, but for anyone else who is interested in learning about viruses.

You can find my virology lectures in several locations: at this blog and at iTunes University, where lecture slides are also available as pdf files, or at YouTube.

This is the fifth year that I have taught my virology course (current class is in the photo), and every version is different. This year, in addition to updating the material, I’ve added a new lecture on viral gene therapy, and include new lectures on immune defenses, viral virulence, acute and persistent infections.

The goal of my virology course is to provide an understanding of how viruses are built, how they replicate and evolve, how they cause disease, and how to prevent infection. The first half of the course explores the viral replication cycle, including attachment and entry, genome replication, protein synthesis, and assembly. In the second half of the course we explore viral pathogenesis: how viruses cause disease, defenses against infection, antivirals, vaccines, and much more. After taking the course, some students might want to become virologists. The course will also provide the knowledge required to make informed decisions about health issues such as immunization against viral infections.

If you have read this blog in the past you know that it is my goal to be Earth’s virology professor. I also teach two virology courses at Coursera (these are completed but the material is still accessible), and my colleagues in Mexico have translated my 2012 lectures into Spanish. Next year I plan to each a new virology course, focused on individual viruses, which will build upon knowledge obtained in my first offering – and of course you will be able to find the lectures online.

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On episode #277 of the science show This Week in Virology, Glenn Rall and Ann Skalka meet up with Vincent to talk about his career in science and science communication.

You can find TWiV #277 at www.twiv.tv.

HIV gets the zinc finger

19 March 2014

zinc finger nucleaseBecause all animal viruses initiate infection by binding to a receptor on the cell surface, this step has long been considered a prime target for antiviral therapy. Unfortunately, drugs that block virus attachment to cells have never shown much promise. Another approach, which is to ablate the receptor from the cell surface, is also problematic because these molecules have essential cellular functions. Removing one of the receptors for human immunodeficiency virus type 1 might be an exception.

HIV-1 must interact with two cell surface proteins to initiate infection: a T lymphocyte protein called CD4, and a second receptor, which can be one of two molecules called CCR5 or CXCR4. For many years it has been known that humans can survive without the CCR5 protein: from 4-16% of people of European descent carry the ccr5-delta32 mutation, that prevents the protein from reaching the cell surface. Individuals who are homozygous for ccr5-delta32 (the mutation is present in both copies of the gene) are resistant to HIV infection. Because the vast majority of HIV viruses that are transmitted are those that require CCR5 for cell entry, absence of the protein on the cell surface confers resistance to infection.

The key role of CCR5 in HIV infection in humans was further confirmed when an AIDS patient was given a bone marrow transplant from a donor with the ccr5-delta32 mutation. The patient has been free of HIV for years despite not taking anti-retroviral drugs.

These findings suggest that one possible therapy for AIDS would be to disrupt the ccr5 gene in patient lymphocytes. The development of gene-targeting technologies has brought this approach closer to reality. One approach uses zinc finger nucleases, which are artificial proteins made by joining a protein that can specifically bind DNA with an enzyme that can cleave DNA. A zinc finger nuclease can be designed, for example, to specifically cut within the ccr5 gene. When the cell tries to repair the cut, the gene may be damaged so that the CCR5 protein is no longer made (illustrated).

This approach works: when CD4 T lymphocytes are removed from humans, cultured, and treated with a ccr5 zinc finger nuclease, they become resistant to HIV infection. We discussed this experiment on episode #144 of This Week in Virology.

The next step has now been done: to remove CD4 T lymphocytes from HIV positive donors, treat the cells with the ccr5 zinc finger nuclease (delivered using an adenovirus vector), and infuse the cells back into the patients (each person receives his or her own modified cells). Half of the donors were removed from anti-retroviral therapy, and then the levels of HIV, and CD4 lymphocytes, were measured over the next 250 days.

The result were encouraging: not only were the infusions safe, but the overall levels of CD4 lymphocytes increased, and a good fraction of these had modified ccr5 genes. The initial rise of HIV viremia after interruption of treatment was followed by a decline in virus load. These results show that the CD4 T lymphocytes with modified ccr5 were able to expand in the recipients, and survived better than the unaltered lymphocytes, probably because they were at least partially resistant to HIV infection.

This important clinical trial is only the beginning of a new approach to HIV therapy, and several substantial problems still remain to be solved. Both copies of the ccr5 gene were modified in only 33% of the CD4 T lymphocytes; the remaining cells can still be infected by HIV, albeit less efficiently. New approaches are needed to disrupt both copies of the ccr5 gene in most of the T lymphocytes.

Another issue is that the modified T cells can proliferate for a long time, but not indefinitely. As these cells divide from a limited number of infused cells, they will not have the broad repertoire needed to fight pathogens. T cells are also known to become “exhausted”: they eventually lose their protective functions. Patients given modified lymphocytes still harbor a pool of long-lived T cells which contain HIV DNA. These cells will likely always be present and could give rise to viremia. CD4 T lymphocytes with normal levels of ccr5 protein will always be produced, serving as potential hosts for HIV replication. Modifying stem cells so that they do not produce CCR5 is one long-term solution, but more difficult and dangerous for the patient.

Despite these drawbacks, it is amazing that we can now remove cells from patients, modify their genes, and place them back in patients with little harm and some clear benefit. This is a complicated set of procedures, made even more difficult because humans are involved. It’s truly a landmark clinical trial.

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