Capturing viruses with bacteria

pvr ecoliWhen my laboratory discovered the cell receptor for poliovirus in 1989, many new research directions were suddenly revealed – such as creating a mouse model for poliomyelitis. One application we did not think of was to use the receptor to screen samples of drinking water for the presence of viruses.

Contamination of the water supply with fecal material can lead to the presence of enteric viruses, which constitute a public health risk. A variety of methods have been developed to screen for viruses in water samples using cell culture or nucleic acid detection techniques. Because the numbers of human viruses in water samples are low, a concentration step must usually be included. These are typically laborious and costly, and innovative improvements are needed. Enter CD155, the cellular receptor for poliovirus, which the authors used as a model for developing a new way to concentrate viruses from water samples.

Viral receptors, which are present on the surface of susceptible cells, are very efficient at capturing viral particles. Why not put these receptors on the surface of bacteria, where they can bind to viruses? Concentrating the viruses would then be a simple matter of centrifuging the bacteria from the water sample. This concept was tested by using poliovirus and the poliovirus receptor.

For this method to work, the viral receptor protein must be present on the surface of bacteria (Figure). To accomplish this goal, an artificial gene was made which codes for the poliovirus receptor protein fused to the ice nucleation protein (INP) gene. This protein is normally present on the surface of the bacterium Pseudomonas syringae.

When the PVR-INP gene was expressed in E. coli, the fusion protein was located to the surface of the bacteria, where it could bind poliovirus. The recovery efficiency was then tested by adding poliovirus to tap water, saline, and samples from several local rivers. The engineered bacteria were added to the poliovirus-laced waters and mixed for 20-60 minutes. The bacteria were then removed by low speed centrifugation, and the viral titers in the cell and in the liquid sample were determined by plaque assay. The recovery of infectious virus ranged from 99% (saline samples) to 75% (river water).

These findings demonstrate that recombinant bacterial cells can be used to capture virus particles in different types of water samples. Compared with other water concentration methods, centrifugation is inexpensive and easy. Whether or not this assay is sensitive enough to detect low levels of viruses in drinking water and other samples must still be determined.

In a way it is fitting that bacteria have been used to capture poliovirus. After all, poliovirus initially replicates in the gastrointestinal tract, where the microbial flora (including E. coli) helps the virus invade the host.

Abbaszadegan M, Alum A, Abbaszadegan H, Stout V. 2011. Cell surface display of poliovirus receptor on Escherichia coli, a novel method for concentrating viral particles in water. Appl Envir Micro 77:5141–5148.

TWiV 168: Super CalTech prophylaxis and ferret runny noses

adeno-associated virusHosts: Vincent Racaniello, Dickson DespommierRich ConditAlan Dove, and Welkin Johnson

Welkin joins the TWiV team for a discussion of HIV prophlaxis using vectored antibodies, and the influenza H5N1 virus studies in ferrets that were not redacted.

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Welkin – Virtual PI (Nature)
DicksonDrain the Ocean
RichNova: To the Moon
Alan – Robert Falcon Scott on Twitter and the Terra Nova expedition
VincentHello, Mr. Chips (I, Cringely)

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CharlotteAnd the Band Played On

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TWiV 166: Breaking and entering

npc1 ebolaHosts: Vincent Racaniello, Dickson DespommierRich Condit, and Alan Dove

Vincent, Dickson, Rich, and Alan review cell proteins essential for entry of hepatitis C, Ebola, and measles viruses.

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Click the arrow above to play, or right-click to download TWiV 166 (59 MB .mp3,  98 minutes).

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Dickson – What are you swimming with?
Rich –
Twelve monkeys
AlanKindle Touch
Vincent – Microbe news (thanks to Dave Winer)

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EricThe Nature of Things with David Suzuki
LanceTrials and Errors (Wired)

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TWiV 105: Finches score again

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, and Rich Condit

On episode #105 of the podcast This Week in Virology, Vincent, Dickson, Alan, and Rich review eradication of rinderpest, endogenous hepatitis B virus in the zebra finch genome, and identification of the cell receptor for an extinct retrovirus.

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Dickson – Winged Migration
Alan – Web-accessible shortwave receivers
Rich – Personal Genome Project
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XVIVO scientific animation

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The D225G change in 2009 H1N1 influenza virus

sialic-acid-2Last year a mutation in the HA gene of the 2009 H1N1 influenza virus was identified in isolates from patients with severe disease. At the time I concluded that the emergence of this change was not a concern. Recently the Norwegian Institute of Public Health reported that the mutation, which causes a change from the amino acid aspartic acid to glycine at position 225 of the viral HA protein (D225G), has been identified in 11 of 61 cases (18%) of severe or fatal influenza, but not in any of 205 mild cases. Have these observations changed my view of the importance of this mutation?

The cell receptor for influenza A virus strains is sialic acid. Human influenza A strains bind preferentially to sialic acids linked to galactose by an alpha(2,6) bond, while avian and equine strains prefer alpha(2,3) linked sialic acids (pictured). Alpha(2,6) linked sialic acids are dominant on epithelial cells in the human nasal mucosa, paranasal sinuses, pharynx, trachea, and bronchi. Alpha(2,3) linked sialic acids are found on nonciliated bronchiolar cells at the junction between the respiratory bronchiole and alveolus, and on type II cells lining the alveolar wall.

The 2009 swine-origin H1N1 influenza virus is known to bind both alpha(2,3) and alpha(2,6) linked sialic acids. This is consistent with the ability of the virus to cause lower respiratory tract disease. The D225G change might be expected to increase affinity for alpha(2,3) linked sialic acids. However, it is not known if increased binding affinity correlates with higher infectivity and pathogenicity. It’s equally likely that high affinity binding might restrict the movement of the virus in lung tissues by causing retention of the virus on nonsusceptible cells.

One view of the D225G mutation is that it is spreading globally and causing more severe disease. However there is no evidence in support of this hypothesis. According to WHO, viruses with the D225G change have been found in 20 countries since April 2009, but there has been no temporal or geographic clustering. As of January, the HA change has been identified in 52 sequences out of more than 2700. Furthermore, the authors of the Norwegian study write, “Our observations are consistent with an epidemiological pattern where the D225G substitution is absent or infrequent in circulating viruses, with the mutation arising sporadically in single cases where it may have contributed to severity of infection”.

One explanation for the sporadic emergence of influenza viruses with the D225G change is that they are selected for in the lower respiratory tract where alpha(2,3) sialic acids are more abundant than in the upper tract. Such selection might be facilitated in individuals with compromised lung function (e.g. asthmatics, smokers) or suboptimal immune responses, in whom the virus more readily reaches the lung. One way to address this hypothesis would be to compare the HA at amino acid 225 of viral isolates obtained early in infection, from the upper tract, with isolates obtained from the lower tract late in disease. However such paired isolates have not yet been obtained. But whether the presence of viruses with D225G increases viral virulence is unknown. Many H1N1 isolates from cases of fatal or severe disease do not contain this amino acid change.

There is an alternative explanation for the isolation of at least some influenza viruses with the D225G change: it is selected by propagation in embryonated chicken eggs. This selection occurs because cells of the allantoic cavity of chicken eggs have only alpha(2,3) linked sialic acids. A change in receptor specificity does not occur when viruses are propagated in MDCK (canine kidney) cells, which possess sialic acids with both alpha(2,3) and alpha(2,6) linkages. Consistent with this hypothesis, WHO reports (pdf) that the D225G substitution in 14 virus isolates occurred after growth in the laboratory.

Studies on the binding of influenza viruses to glycan arrays have shown that attachment is influenced not only by the linkage to the next sugar, but the type of sialic acid as well as the rest of the carbohydrate chain. The distribution of all the possible sialic acid containing sugars in the respiratory tract is unknown, as is the specific molecules that can support productive viral infection. The view that HA preferentially binds to either alpha(2,3) or alpha(2,6) linked sialic acids is likely to be overly simplistic: another casualty of reductionism.

Kilander A, Rykkvin R, Dudman SG, & Hungnes O (2010). Observed association between the HA1 mutation D222G in the 2009 pandemic influenza A(H1N1) virus and severe clinical outcome, Norway 2009-2010. Euro surveillance : bulletin europeen sur les maladies transmissibles = European communicable disease bulletin, 15 (9) PMID: 20214869

Takemae N, Ruttanapumma R, Parchariyanon S, Yoneyama S, Hayashi T, Hiramatsu H, Sriwilaijaroen N, Uchida Y, Kondo S, Yagi H, Kato K, Suzuki Y, & Saito T (2010). Alterations in receptor-binding properties of swine influenza viruses of the H1 subtype after isolation in embryonated chicken eggs. The Journal of general virology, 91 (Pt 4), 938-48 PMID: 20007353

Garcia-Sastre, A. (2010). Influenza Virus Receptor Specificity. Disease and Transmission American Journal Of Pathology DOI: 10.2353/ajpath.2010.100066

Virology lecture #5: Attachment and entry

Download: .wmv (386 MB) | .mp4 (131 MB)

There are some errors in this lecture – I’ll correct them during the next session.

Visit the virology W3310 home page for a complete list of course resources.

TWiV 46: Virus entry into cells

twiv-200Hosts: Vincent Racaniello and Dick Despommier

In episode #46 of the podcast “This Week in Virology”, Vincent and Dick continue virology 101 with a discussion of virus entry into cells, then answer reader email on colony collapse disorder and viruses that confer a benefit to their host.

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Illustrations of virus entry into cells
Nice reference for biological items (thanks Jim!)
Colony collapse disorder: PBS program,  descriptive studymetagenomic study, genetic analysis (thanks Swiss compass!)
Potato virus Y and Alzheimer’s disease (thanks Jennifer!)
virus in a fungus in a plant (thanks Jennifer!)

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Influenza virus attachment to cells

We’ve briefly considered the structure of influenza virions and how the viral RNAs can encode one or more proteins. Now we’ll consider how influenza viruses multiply.

Viruses are obligate intracellular parasites: they cannot reproduce outside of a cell. The production of new infectious particles must take place within a cell. Upon entering cells, viruses parasitize the host machinery to produce new viral progeny. The sum total of all the events that take place in a virus-infected cell is called the infectious cycle, or viral replication. Virologists artificially divide the infectious cycle into steps to make it easier to study. The steps include attachment and entry of the virion, translation of mRNA into protein, genome replication (producing more RNA or DNA), assembly of new particles, and release of particles from the cell. We’ll consider each of these steps, and then move on to a discussion of how influenza virus infects us and causes disease.

Today we’ll focus on the first step, attachment of the virion to cells. Here is a typical cell. I’m sure everyone is familiar with it, but it doesn’t hurt to review.


You can see that there is a substantial barrier to anything getting into this cell – the plasma membrane. Viruses have evolved different ways to get around this. But what they all have in common is that virions must first attach to a receptor on the plasma membrane in order to enter the cell. Every virus has a specific receptor that it attaches to, and in turn there is a particular viral protein that binds this cell receptor. Here is an illustration of an influenza virion binding to its cell receptor.


You can see the individual ‘spikes’ on the virion binding to a structure on the cell. The influenza viral spike that attaches to the cell receptor is the HA protein – hemagglutinin. The cell receptor is sialic acid – a small sugar that is attached to many different proteins on the cell surface. Here’s what sialic acid looks like.

On the left is a drawing of a cell protein embedded in the plasma membrane. The interior of the cell – cytoplasm – is at the bottom. Part of the protein crosses the membrane, and there are also parts on the cytoplasmic and extracellular sides. The spheres are sugars that are attached to many proteins (protein + sugar = glycoprotein). Sialic acid is always the last sugar in a chain that is attached to a protein. On the right is the chemical structure of sialic acid; the next sugar, to the right, is galactose. Influenza virions attach to cells when the HA grabs onto the very small sialic acid.

The sugar is actually quite tiny compared to the HA – it fits into a small pocket on the top of the spike. Here is a molecular model showing the HA bound to an analog of sialic acid. The globular top of the HA is at the top of the image. The tiny red and white spheres show where sialic acid would be bound, in a pocket at the top of the HA.


So far we have docked the influenza virion onto the surface of the cell. It’s sitting there quite firmly, but it’s still on the outside of the cell. How does it get in – or more accurately, how do the viral RNAs get into the cell? Stay tuned.