TWiV 455: Pork and genes

Erin Garcia joins the TWiVirions to discuss a computer exploit encoded in DNA, creation of pigs free of endogenous retroviruses, and mutations in the gene encoding an innate sensor of RNA in children with severe viral respiratory disease.


Click arrow to play
Download TWiV 455 (64 MB .mp3, 105 min)
Subscribe (free): iTunesRSSemail

Become a patron of TWiV!

Show notes at

TWiV 374: Discordance in B

TWiVOn episode #374 of the science show This Week in Virology, the TWiVniks consider the role of a cell enzyme that removes a protein linked to the 5′-end of the picornavirus genome, and the connection between malaria, Epstein-Barr virus, and endemic Burkitt’s lymphoma.

You can find TWiV #374 at

A new cell receptor for rhinovirus

rhinovirus receptorsRhinovirus is the most frequent cause of the common cold, and the virus itself is quite common: there are over 160 types, classified into 3 species. The cell receptor has just been identified for the rhinovirus C species, which can cause more severe illness than members of the A or B species: it is cadherin-related family member 3.

Because viruses are obligate intracellular parasites, the genome must enter a cell before new particles can be made. The first step in this process is binding of the virus particle to a receptor on the plasma membrane. Two different membrane proteins serve as receptors for members of rhinovirus A and B species: intracellular adhesion molecule 1, and low-density lipoprotein receptor (illustrated).

It has not been possible to propagate species C rhinoviruses in conventional cell cultures, which has hampered research on how the virus replicates. The lack of a cell culture system required a different approach to identifying a cell receptor for this virus. It was known that the virus replicates in primary organ or cell cultures derived from sinus tissue, but not in a variety of epithelial and transformed cell lines (e.g. HeLa cells). In silico comparison of gene expression profiles revealed 400 genes that are preferentially expressed in virus-susceptible cells. This list was narrowed down to 12 genes that encode plasma membrane proteins. A subset of these genes were introduced into cells and tested for the ability to serve as a rhinovirus C receptor. Introduction of the gene encoding cadherin-related family member 3 (CDHR3) into HeLa cells allowed rhinovirus C binding and infection.

The cadherin family comprises cell surface proteins that are involved in cell-cell communication. The exact cell function of CDHR3 is not known, but the protein is found in human lung, bronchial epithelium, and cultured airway epithelial cells. A mutation in the gene encoding this protein is associated with wheezing illness and asthma in children. This mutation, which causes a change from cysteine to tyrosine at amino acid 529, was found to increase virus binding and virus replication in HeLa cells that synthesize CDHR3. It will be important to determine if this amino acid change increases rhinovirus C replication in humans, thereby leading to more serious respiratory illness.

The CDHR3 gene was used to establish a stable HeLa cell line that produces the receptor and which can be infected with species C rhinoviruses. This cell line will be useful for illuminating the details of viral replication in cells, which has so far been elusive due to lack of a susceptible and permissive cell line. It may also be possible to produce transgenic mice with the human CDHR3 gene, which could serve as a model for studying rhinovirus C pathogenesis. Transgenic mice that produce the receptor for the related polioviruses, CD155, are a model for poliomyelitis.

TWiV 328: Lariat tricks in 3D

On episode #328 of the science show This Week in Virology, the TWiVocateurs discuss how the RNA polymerase of enteroviruses binds a component of the splicing machinery and inhibits mRNA processing.

You can find TWiV #328 at

TWiV 318: Last year in virology

On episode #318 of the science show This Week in Virology, the TWiV gang reviews ten fascinating, compelling, and riveting virology stories from 2014.

You can find TWiV #318 at

Acute flaccid paralysis of unknown etiology in California


Enterovirus D68 by Jason Roberts

In February 2014 I wrote about children in California who developed a poliomyelitis-like paralysis, also called acute flaccid paralysis or AFP. However, the cause of this paralysis was not known. The CDC has released its study of these cases and concludes “The etiology of AFP with anterior myelitis in the cases described in this report remains undetermined.”

A total of 23 cases of AFP* in California were reported to CDC during the period June 2012 through June 2014. These cases were from diverse geographic regions of the state. Specimens from 19 of the patients were available and tested for poliovirus, aroboviruses, herpesviruses, parechoviruses, adenoviruses, rabies virus, influenza virus, metapneumovirus, respiratory syncytial virus, parainfluenza viruses, Mycoplasma pneumoniae, Rickettsia, and amoebas. Rhinovirus was detected in one patient, and enterovirus D68 in two patients; all others were negative for potential etiologic agents.

All 23 patients with AFP also had anterior myelitis, inflammation of the grey matter of the spinal cord, which is characteristic of poliomyelitis. While the rate of AFP in California betweeen 1992-1998 was 1.4 cases per 100,000 children per year,  anterior myelitis was not described in any of 245 cases reviewed by CDC. However, poliovirus was ruled out as a cause in the 19 individuals who could be tested.

The cause of AFP is often difficult to determine because there infectious and non-infectious etiologies. Only 2 of the 19 clinical specimens met CDC guidelines for poliovirus detection (two stool specimens collected ≥24 hours apart and <14 days after symptom onset) and the others were likely taken too late to detect the presence of virus. The finding of enterovirus D68 in two of the samples is difficult to interpret, as the virus was detected in respiratory specimens and could have been a coincidental infection.

This investigation began with a request from a San Francisco area physician to the California State Department of Public Health to determine whether poliovirus was present in a 29 year old male with AFP and anterior myelitis. Subsequently this department posted alerts for AFP with anterior myelitis to  local health departments, and it is from the cases submitted that the 23 were drawn. Therefore the number of cases of AFP with anterior myelitis might be a consequence of this surveillance.

We are left with the unsatisfying conclusion that these 23 cases of AFP with anterior myelitis were either caused by an undetected infectious agent, or by something else.

*Defined by CDC as “at least one limb consistent with anterior myelitis, as indicated by neuroimaging of the spine or electrodiagnostic studies (e.g., nerve conduction studies and electromyography), and with no known alternative etiology”.

TWiV 267: Snow in the headlights

On episode #267 of the science show This Week in Virology, Vincent, Alan, Rich and Kathy review a protease essential for influenza pathogenesis in mice, and directionality of rhinovirus RNA exit from the capsid.

You can find TWiV #267 at

A saga of HeLa cells

HeLa spinnerWe have been using HeLa cells in my laboratory since 1982, when I arrived at Columbia University Medical Center fresh from postdoctoral work with David Baltimore at MIT. I brought with me a line of HeLa cells and used them for 30 years for our research on viruses. Here is a story of how we lost the cells and then found them ten months later.

As everyone knows, the continuous HeLa cell line was derived from a cervical tumor taken from Henrietta Lacks in 1951 (if you don’t know the story, you should read Rebecca Skloot’s The Immortal Life of Henrietta Lacks, or my shorter summary). When I arrived at the Baltimore lab in 1979, they were using cells derived from the S3 clone of HeLa cells that had been produced by Philip Marcus in the 1950s. I write ‘derived from’ because someone at MIT had further  cloned the S3 line and selected one that was particularly susceptible and permissive to poliovirus infection. This was the cell line that I took with me to Columbia in 1982.

Because we use so many HeLa cells each week, we grow them in spinner cultures (pictured). The cells are suspended in a glass bottle in nutrient medium and continuously stirred by a magnetic bar. The spinner bottle is placed on top of a stir plate, which contains a motor that drives a rotating magnet that in turn spins the bar in the bottle. When we need to produce monolayers of cells for experiments, we remove cells from suspension and plate them on plastic dishes. The HeLa S3 clone that we use grows very well in suspension and also forms excellent monolayers on plastic dishes.

Over the years we used the HeLa S3 subclone to conduct experiments with poliovirus, echoviruses, Coxsackieviruses, enteroviruses, rhinoviruses, and encephalomyocarditis virus. The cells could be infected with all these viruses, develop cytopathic effects, and form plaques, allowing titration of virus titers. They have been an essential part of my laboratory. The Wall of Polio is just one example of how important these cells have been for our work.

In December 2012, the spinner went down. The drive belt that turns the magnet in the spinner platform broke overnight; the cells settled out and died. Normally we would simply go to our stock of cells frozen in liquid nitrogen, thaw them out, and be up and working again within a week. Unfortunately, our liquid nitrogen tank had run dry one week in the summer of 2012, and all the cells had died. We tried recovering some of the HeLa cells that were frozen, but what grew out were not the same as our S3 subclone.

In the course of the next 9 months we tried HeLa cells from many different sources – laboratories here at Columbia, the American Type Culture Collection, and our colleagues elsewhere. None of the HeLa cells performed like our S3 subclone. Some HeLa lines did not grow well in spinner; others did, but formed poor monolayers. Still others did not support replication or plaque formation when infected with viruses we work on now – poliovirus, rhinoviruses, and encephalomyocarditis virus. I located former members of the Baltimore laboratory, hoping that they had taken the special HeLa cells with them, but I came up empty handed.

A few weeks ago I received an email from a former student who had heard my pleas for HeLa cell help on This Week in Virology. She remembered bringing some of our HeLa cells to her postdoctoral laboratory in Canada, and freezing them down when she left. I contacted the laboratory and found to my delight that our HeLa cells were indeed frozen there; a kind member of the laboratory grew up a stock of the cells, froze them, and shipped them off to us. I received them a week ago and put them into culture. They were the HeLa cells that I used to know: I recognized their morphology immediately. They grew beautifully as monolayers, and just today I set up a spinner culture. We are all looking forward to using the cells in our virology experiments.

Meanwhile, I have a large stock of belts for the magnetic spinner plate (I had to buy seventeen of them, to meet the $50 minimum order); I have placed 6 vials of the cells in our liquid nitrogen tank; and I plan to freeze additional vials in my colleagues’ freezers in case ours goes down again.

There are several lessons to be learned from this saga. First, because virologists are completely dependent on cells, they must take care that they have a reliable stock. This means having someone in the lab checking the level of liquid nitrogen every day, and ordering a new tank when the level is low. Second, it’s important to keep stocks of cells frozen elsewhere. We were very lucky to find them in Canada. Third, HeLa cell lines are very different. Finally, HeLa cells are special. I don’t know of any other cell line that grows well in spinner, makes beautiful monolayers, and allows us to work with so many different viruses. Thank you, Henrietta Lacks.

TWiM 64: URI and UTI at ICAAC

This episode of TWiM was recorded at the 53rd ICAAC in Denver, Colorado, where Michael Schmidt and I spoke with James Gern about rhinoviruses, and James Johnson about extra-intestinal pathogenic E. coli.

You can find TWiM #64 at, or view the video below.

Thirty years in my laboratory at Columbia University

Racaniello labThirty years ago this month I arrived in the Department of Microbiology at Columbia University’s College of Physicians and Surgeons (P&S) to start my own laboratory. Thirty is not only a multiple of ten (which we tend to celebrate), but also a long time to be at one place. It’s clearly time to reminisce!

After studying influenza viruses with Peter Palese in New York City, in 1979 I headed to David Baltimore‘s laboratory at MIT. It was not long after Baltimore had received the Nobel Prize in Physiology or Medicine for discovering retroviral reverse transcriptase. In his laboratory I first encountered poliovirus, which would hold my interest for many years to come. The moratorium on recombinant DNA research had just been lifted, and it was now possible to clone complete viral DNA genomes. My first project was to make a DNA copy of poliovirus RNA, clone it into a bacterial plasmid, and determine its sequence. The result gave us the first glimpse of the viral genome. I then found that a DNA copy of poliovirus RNA is infectious in mammalian cells, a story that I have documented elsewhere.

The next step in my career was to have my own laboratory. With these two papers in hand I was able to obtain several respectable job offers, including one in the Microbiology Department at P&S. The department chair was Harold S. Ginsberg, an adenovirologist. My decision to accept his offer was influenced by the strong virology component of the department, which included Saul Silverstein and Hamish Young. I moved back to New York City in September 1982 with a DNA copy of the poliovirus genome in hand. In the last few weeks in the Baltimore laboratory, I had cloned a DNA copy of another poliovirus strain – type 2 Lansing – which had the interesting ability to infect mice. I spent the first few years in my new laboratory studying this virus and how it caused paralysis in mice. We found that the type 2 Lansing viral capsid was important for the ability to infect mice. Later, we narrowed this down to 8 amino acids. The type 1 Mahoney strain of poliovirus – which I had studied in the Baltimore laboratory – cannot infect mice. However, if we substituted 8 amino acids of the Mahoney capsid with the corresponding sequence from the Lansing genome, the recombinant virus could infect mice.

Our sequence analysis of poliovirus RNA had revealed an unusually long 5′-noncoding region. We began to carry out experiments to understand how such a viral RNA could be translated, and found that this long sequence enabled translation in the absence of a cap protein. This observation lead to the discovery by others that the poliovirus 5′-noncoding region contains an internal ribosome entry site (IRES). In the ensuing years our interest in translation continued. Examples include our finding that cell proteins bind to the poliovirus 5′-noncoding region, now known to participate in regulation of translation and genome replication, and understanding the inhibition of cell translation by poliovirus. Years later we developed a functional assay for the IRES in yeast, allowing identification of cell proteins needed for internal ribosome binding.

There is one area of research that has received the most attention in my laboratory, and on which we have published most extensively: the interaction of viruses with cell receptors. Towards the end of my stay in the Baltimore laboratory I became interested in how poliovirus attaches to and enters cells.  I came to Columbia with a strong interest in identifying the cell receptor for poliovirus, which we subsequently achieved. This finding lead to a series of studies on virus attachment to cells and virus entry. We produced transgenic mice susceptible to poliovirus, and used them to study aspects of poliovirus replication and pathogenesis, including how the virus attaches to its cellular receptor, regulation of viral tissue tropism, and the basis for attenuation of the Sabin vaccine strains.

The finding that poliovirus tropism is regulated by the interferon response lead to a change in the direction of our research. Beginning in the early 2000s we began studying how poliovirus interacted with the innate immune response. We found that poliovirus is relatively resistant to the antiviral effects of interferon, a property conferred by the viral 2Apro proteinase. How poliovirus is sensed by the innate immune system has also become a focus of our work. With the looming prospect of poliovirus eradication, and subsequent prohibition of work on the virus, we have also turned our attention to rhinoviruses, agents of respiratory illness. One focus has been to establish a mouse model for rhinovirus infection.

This story would not be complete without mentioning my foray into science communication. I have written a virology textbook and a blog about viruses, and began three science podcasts, including This Week in Virology, This Week in Parasitism, and This Week in Microbiology. My use of social media to teach microbiology to the world has been documented in a Social Media and Microbiology Education and in my Peter Wildy Prize Address.

None of this work would have been possible without the participation of 24 Ph.D. students (Nicola La Monica, Cathy Mendelsohn, Eric Moss, Robert O’Neill, Mary Morrison, Ruibao Ren, Elizabeth Colston, Michael Bouchard, Suhua Zhang, Alan Dove, Sa Liao, Yanzhang Dong, Yi Lin, Brian McDermott, Melissa Stewart Kim, Steven Kauder, Julie Harris, Amy Rosenfeld, Juliet Morrison, Angela Rasmussen, Jennifer Drahos, and Esther Francisco), 7 postdoctoral fellows (Gerardo Kaplan, Marion Freistadt, Michael Shepley, Ornella Flore, Juan Salas-Benito, and Scott Hughes), and many technicians and undergraduate students. My laboratory currently consists of postdoctoral scientist Rea Dabelic, and graduate students Ashlee Bennett and Michael Schreiber (pictured above).

Counting my time here, together with my Ph.D. and postdoctoral years, I’ve been working on viruses for 37 years. I do not know how much longer I will be doing the same, but it’s safe to say that it won’t be for another 37 years. But whenever I stop directing virology research, I will continue to write, podcast, and teach – you can expect nothing less from Earth’s virology professor.