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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On episode #276 of the science show This Week in Virology, Vincent meets up with Susan Baker and Tom Gallagher at Loyola University to talk about their work on coronaviruses.

You can find TWiV #276 at www.twiv.tv.

Can a virus be revived?

14 March 2014

PithovirusIn Carl Zimmer’s New York Times article describing the recovery of the giant virus Pithovirus sibericum from the Siberian permafrost, he used the words revive and resurrect. Can a virus be restored to life?

The headline of the article read ‘Out of Siberian ice, a virus revived‘. Within the body of the article, Zimmer wrote ‘From Siberian permafrost more than 30,000 years old, they have revived a virus that’s new to science’, and later considered the ‘risk of an outbreak of resurrected viruses’. Both words mean ‘restore to life’.

When most people say ‘virus’ they usually mean the very small virus particle that infects cells. Virus particles are not living: they are assemblies of protein, nucleic acid, and sometimes lipids that do nothing until they infect a cell. That is why they are called obligate intracellular parasites. In the case of Pithovirus, infectious virus particles were present in the frozen sample that were able to infect amoeba in the laboratory.

To say that a virus was revived or resurrected is wrong, although I understand that the idea of bringing anything back to life has a great deal of general appeal. The key fact in this story is that the infectivity of the virus particle was maintained for over 30,000 years in the Siberian permafrost. I realize that this does not make for compelling headlines, but mine would have been: ‘Infectious virus recovered from Siberian ice after 30,000 years’. I suspect that Zimmer might understand this, but as he’s told me before, sometimes it’s much easier (and requires fewer words) to write something for the non-scientist that is not quite right.

Even virologists confuse the living with the non-living. When Paul Bieniasz and his laboratory reported that they had reconstituted an infectious retrovirus from viral sequences in the human genome, they used the phrase ‘the resurrection of this extinct infectious agent’.

A virus particle is not alive, but a virus infected cell certainly is living. A virus can be viewed as an organism with two phases, a non-living virus particle, or virion; and an infected cell, which is alive.  This definition solves the problem of whether a virus is alive or not, a subject of much debate here and elsewhere. Even if we use this terminology, the use of resurrect and revive to describe viral infectivity is still wrong, because virus particles cannot be brought back to life – they are not alive to begin with.

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attenuated influenzaInfection with influenza virus is known to increase susceptibility to bacterial infections of the respiratory tract. In a mouse model of influenza, increased bacterial colonization was also observed after administration of an infectious, attenuated influenza virus vaccine.

Primary influenza virus infection increases colonization of the human upper and lower respiratory tract with bacteria, including Streptococcus pneumoniae and Staphylococcus aureus. Such infections may lead to complications of influenza, including pneumonia, bacteria in the blood, sinusitis, and ear infections.

One of the vaccines available to prevent influenza is an infectious, attenuated preparation called Flumist. To determine if a vaccine such as Flumist increases susceptibility to bacterial infection, the authors created their own version of the vaccine (illustrated) in which the six RNA segments encoding internal proteins were derived from the A/Puerto Rico/8/34 (H1N1) strain (allowing replication in mice), and the HA and NA proteins were derived from A/Hong Kong/1/68 (H3N2). In addition, mutations were introduced into the viral genome that are important for the safe and protective properties of Flumist. For simplicity we’ll call this virus ‘live attenuated influenza virus’, or LAIV.

Mice were inoculated intranasally with a strain of S. pneumoniae known to colonize the nasopharynx, followed 7 days later by LAIV or wild type influenza virus. Inoculation with either virus similarly increased the bacterial levels in the nasopharynx, and extended the time of colonization from 35 to 57 days. In mice that were given only bacteria and no influenza virus, the inoculated bacteria were cleared beginning 4 days after administration. The more extensive and extended colonization of virus-infected mice was not associated with overt disease.

Administration of LAIV or wild type virus 7 days before bacteria also resulted in excess bacterial growth in mice. Similar results were obtained using S. aureus. Administration of S. pneumoniae up to 28 days after virus also lead to excess bacterial growth, despite clearance of the viruses around 7 days after vaccination.

All mice died when they were vaccinated with wild type influenza virus followed 7 days later by a sublethal dose of a highly invasive strain of S. pneumoniae. In contrast, pretreatment with LAIV lead to no disease or death of any mice.

It is not known if these findings in a mouse model directly apply to humans. However, because Flumist reduces influenza virus replication, it is associated with a decrease in secondary bacterial infections. It is possible that, after administration of LAIV to humans, there is an increase in bacterial colonization of the respiratory tract. Upper respiratory tract symptoms are a known adverse effect of LAIV, and it is possible that these might be related to increased bacterial loads. It is important to emphasize that use of LAIV is not associated with severe upper or lower tract disease.

These findings are important because they show that a mouse model could be used to understand why influenza virus infection leads to increased bacterial colonization of the respiratory tract. It will be important to determine the precise mechanisms by which influenza virus infection, and the associated virus and immune-mediated alteration to the respiratory tract, allows enhanced bacterial colonization. At least one mechanism, which we discussed on episode #62 of This Week in Microbiology, involves the disruption of biofilms, allowing bacteria to enter the bloodstream.

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On episode #275 of the science show This Week in Virology, Vincent and Rich meet up with Eugene Koonin to talk about the central role of viruses in the evolution of all life.

You can find TWiV #275 at www.twiv.tv.

PithovirusA new virus called Pithovirus sibericum has been isolated from 30,000 year old Siberian permafrost. It is the oldest DNA virus of eukaryotes ever isolated, showing that viruses can retain infectivity in nature for very long periods of time.

Pithovirus was isolated by inoculating cultures of the amoeba Acanthamoeba castellani with samples taken in the year 2000 from 30 meters below the surface of a late Pleistocene sediment in the Kolyma lowland region. This amoeba had been previously used to propagate other giant viruses, such as Mimivirus and Pandoravirus. Light microscopy of the cultures revealed the presence of ovoid particles which were subsequently shown by electron microscopy to resemble those of Pandoravirus. Pithovirus particles are flask-shaped and slightly larger than Pandoravirus – 1.5 microns long, 500 nm in diameter, encased by a 60 nm thick membrane. One end of the virus particle appears to be sealed with what the authors call a cork (photo). This feature, along with the shape of the virus particle,  inspired the authors to name the new isolate Pithovirus, from the Greek word pithos which refers to the amphora given to Pandora. The name therefore refers both to the morphology of the virus particle and its similarity to Pandoravirus.

Although the Pithovirus particle is larger than Pandoravirus, the viral genome – which is a double-stranded molecule of DNA – is smaller, a ‘mere 610,033 base pairs’, to use the authors’ words (the Pandoravirus genome is 2.8 million base pairs in length). There are other viruses with genomes of this size packed into much smaller particles – so why is the Pithovirus particle so large? Might it have recently lost a good deal of its genome and the particle size has not yet caught up? One theory of the origin of viruses is that they originated from cells and then lost genes on their way to becoming parasitic.

We now know of viruses from two different families that have similar morphology: an amphora-like shape, an apex, and a thick electron-dense tegument covered by a lipid membrane enclosing an internal compartment. This finding should not be surprising: similar viral architectures are known to span families. The icosahedral architecture for building a particle, for example, can be found in highly diverse viral families. The question is how many viruses are built with the pithovirus/pandoravirus structure. My guess would be many, and they could contain either DNA genomes. We just need to look for them, a process, as the authors say that ‘will remain a challenging and serendipitous process’.

Despite the physical similarity with Pandoravirus, the Pithovirus genome sequence reveals that it is barely related to that virus, but more closely resembles members of the Marseillviridae, Megaviridae, and Iridoviridae. These families all contain large icosahedral viruses with DNA genomes.  Only 32% of the 467 predicted Pithovirus proteins have homologs in protein databases (this number was 61% for Mimivirus and 16% for Pandoravirus). In contrast to other giant DNA viruses, the genome of Pithovirus does not encode any component of the protein synthesis machinery. However the viral genome does encode the complete machinery needed to produce mRNAs. These proteins are present in the purified Pithovirus particle. Pithovirus therefore undergoes its entire replication cycle in the cytoplasm, much like other large DNA viruses such as poxviruses.

Pithovirus is an amazing virus that hints about the yet undiscovered viral diversity that awaits discovery. Its preservation in a permafrost layer suggests that these regions might harbor a vast array of infectious organisms that could be released as these regions thaw or are subjected to exploration for mineral and oil recovery. A detailed analysis of the microbes present in these regions is clearly needed, both by the culture technique used in this paper and by metagenomic analysis, to assess whether any constitute a threat to animals.

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TWiV 274: Data dump

2 March 2014

On episode #274 of the science show This Week in Virology, the TWiV team discusses recent cases of polio-like paralysis in California, and the virome of 14th century paleofeces.

You can find TWiV #274 at www.twiv.tv.

Enterovirus

Image credit: Jason Roberts

Recently a number of children in California have developed a poliomyelitis-like paralysis. The cause of this paralysis is not yet known, and information about the outbreak is scarce. Here is what we know so far:

  • At least 5, and perhaps as many as 20 children have suffered weakness or paralysis in one or more limbs. The median age of the patients is 12 years and the cases have been reported since 2012.
  • One group of 5 patients recently presented at the American Academy of Neurology Annual meeting developed full paralysis within 2 days, and have not recovered limb function in 6 months.
  • The cases are all located within a 100-mile radius.
  • A mild respiratory illness preceded paralysis in some of the children.
  • Enterovirus type 68 has been recovered from the stool of some of the patients.

I do not have any more information on this outbreak other than what I’ve obtained from ProMedMail. I have worked on enteroviruses, including poliovirus, for over 30 years, so I thought I might speculate on what might be transpiring.

What is a polio-like illness? Acute flaccid paralysis (AFP) is the term used to describe the sudden onset of weakness in limbs. AFP can have many etiologies, including viruses, bacteria, toxins, and systemic disease. It is used by the World Health Organization to maximize the ability to detect all cases of poliovirus. Confirmation that AFP is caused by poliovirus requires demonstration that the virus is present in the infected individual.

Is poliovirus the cause? I do not believe that poliovirus is causing the paralysis of children in California. I understand that they have all been immunized against poliovirus. In addition, should immunization have failed in any of these children, it seems unlikely that wild type polioviruses would be circulating in this area. Vaccine-derived polioviruses can cause paralysis but the US has not used this type of vaccine since 2000.

What might be causing the paralysis? AFP has both infectious and non-infectious etiologies. One possibility is that  a non-polio enterovirus is involved. Poliovirus is classified within the genus Enterovirus in the family Picornaviridae. Other enteroviruses besides poliovirus are known to cause paralytic disease, such as Coxsackieviruses, echoviruses, and many enteroviruses including types 70, 71, 89, 90, 91,96, 99, 102, and 114.

Most enterovirus infections can be associated with different clinical syndromes besides paralysis (such as respiratory disease), and therefore diagnosis is difficult. Stool is generally the most sensitive specimen for establishing an enterovirus infection. However, the virus may no longer be present at onset of symptoms. Polio is much easier to diagnose in individuals with AFP from whom virus can be identified: paralysis is the main serious symptom caused by infection. However note that 99 out of 100 poliovirus infections are asymptomatic or present with undifferentiated viral illness. The incidence of paralytic disease caused by other enteroviruses is even lower – for example 1 in 10,000 EV71 infections are paralytic. If all of the 20 California cases are caused by enteroviruses, this means that there have been many more infections without symptoms.

In one study of non-polio AFP in India, no virus could be isolated in 70% of the cases. Enterovirus 71 was the single most prevalent serotype associated with non-polio AFP. This virus currently causes large outbreaks of hand, foot, and mouth disease throughout Asia, with many fatalities and cases of acute flaccid paralysis. EV71 is known to circulate within the United States.

What about enterovirus 68? It has been reported that EV68 has been isolated from some of the paralyzed children. This isolation does not mean that the virus has caused the paralysis. Enterovirus infections of the respiratory and gastrointestinal tracts are very common and often do not result in any signs of disease. Random samplings of healthy individuals frequently demonstrate substantial rates of enterovirus infections.

Enterovirus type 68 was first isolated in California from an individual with respiratory illness. The virus is known to cause clusters of acute respiratory disease, and there is at least one report of its association with central nervous system disease. I believe it is an unlikely cause of the paralytic cases in California based solely on the past history of the virus and the fact that other enteroviruses are more likely to cause paralysis. It is not clear to me why enterovirus 68 would evolve to become substantially more neurotropic: entering the central nervous system is a dead end because the infection cannot be transmitted to a new host.

All of the above is pure speculation based on very little data. The paralysis might not even be caused by an infection. At this point a great deal of basic epidemiology needs to be done to solve the problem – if indeed it can be solved at all. Based on its population, California would be expected to have about 75 cases of acute flaccid paralysis each year of various etiologies, suggesting that the current number of cases is not unusual or unexpected.

Update: N. Gopal Raj wrote a story last year about acute flaccid paralysis in India, which has the highest rate of non-polio AFP in the world, with 60,000 cases reported in 2011.

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