TWiV 332: Vanderbilt virology

On episode #332 of the science show This Week in Virology, Vincent visits Vanderbilt University and meets up with Seth, Jim, and Mark to talk about their work on a virus of Wolbachia, anti-viral antibodies, and coronaviruses.

You can find TWiV #332 at

Human infections with influenza H5N1 virus: How many?

The lethality of avian influenza H5N1 infections in humans has been a matter of extensive debate. The >50% case fatality rate established by WHO is high, but the lethality of the virus might be lower if there are many infections accompanied by mild or no disease. One way to answer this question is to determine how many individuals carry antibodies to the virus in populations that are at risk for infection. A number of such studies have been done, and some have concluded that the results imply a low but substantial level of infection (even less than one percent of millions of people is a lot of infections). The conclusion of a new meta-analysis of H5N1 serosurveys is that most of the studies are flawed, and that the frequency of H5 infections appears to be low.

Twenty-nine different H5N1 serological studies were included in this meta-analysis. None of these are particularly satisfactory according to the authors:

None of the 29 serostudies included what we would consider to be optimal, blinded unexposed controls in their published methodologies, i.e., including in the serology runs blinded samples from individuals with essentially no chance of H5N1 infection. Serological assays can easily produce misleading results, especially when paired sera are not available.

Some of the problems identified in the serological surveys include the possibility that many H5N1 positive sera are the result of false positives, that is, cross reaction with antigens from other influenza virus strains. In addition, many studies utilized H5N1 strains that are no longer circulating.

It is clear that most of the H5N1 serosurveys have not been done as well as they should have been. The authors conclude that “it is essential that future serological studies adhere to WHO criteria and include unexposed control groups in their laboratory assays to limit the likelihood of misinterpreting false positive results.”

Let’s not forget that a completely different way of assessing H5N1 infection – by looking for virus-specific T cells – has been reported. The results provide further evidence for subclinical H5N1 infection and are not subject to the caveats noted here for antibody surveys.

I come away from this meta-analysis with an uneasy sense that the authors are not being sufficiently objective, and that they firmly believe that there are no mild or asymptomatic H5N1 infections. One reason is the authors’ use of ‘only’ to describe their findings. For example: “Of studies that used WHO criteria, only [italics mine] 4 found any seropositive results to clades/genotypes of H5N1 that are currently circulating”. The use of ‘only’ in this context implies a judgement, rather than an objective statement of fact. Furthermore, despite the authors stated problems with all H5N1 serosurveys, they nonetheless conclude that there is little evidence for asymptomatic H5N1 infection. If the studies are flawed, how can this conclusion be drawn?

My concern about the authors’ objectivity is further heightened by the fact that they are members of the Center for Biosecurity at the University of Pittsburgh. These are individuals whose job it is to find dangerous viruses that could be used as weapons. On the front page of the website for the Center for Biosecurity is a summary of the meta-analyis article which concludes that “In the article, Assessment of Serosurveys for H5N1, Eric Toner and colleagues discuss their extensive review of past studies and conclude that there is little evidence to suggest that the 60% rate is too high.”

I would argue that if the H5N1 serosurveys are flawed, then do them properly; it is incorrect to simply assume that the H5N1 virus is as lethal as WHO suggests. The World Health Organization should call for and coordinate a study that satisfies criteria established by virologists and epidemiologists for a robust analysis of human H5N1 exposure.

TWiV 151: Dear TWiVers

viral mailHosts: Vincent Racaniello, Alan Dove, and Rich Condit

Vincent, Alan, and Rich review questions and comments from TWiV listeners

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Virology lecture #13: Host defense

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Influenza hemagglutination inhibition assay

Centers for Disease Control and Prevention have determined that some adults have serum cross-reactive antibodies to the new influenza H1N1 virus. One of the techniques used to reach this conclusion is the hemagglutination inhibition (HI) assay. How does this assay work?

To understand the HI assay, we must discuss the hemagglutination assay. Influenza virus particles have an envelope protein called the hemagglutinin, or HA, which binds to sialic acid receptors on cells. The virus will also bind to erythrocytes (red blood cells), causing the formation of a lattice. This property is called hemagglutination, and is the basis of a rapid assay to determine levels of influenza virus present in a sample. To conduct the assay, two-fold serial dilutions of a virus are prepared, mixed with a specific amount of red blood cells, and added to the wells of a plastic tray. The red blood cells that are not bound by influenza virus sink to the bottom of a well and form a button. The red blood cells that are attached to virus particles form a lattice that coats the well. The assay can be performed within 30 minutes, and is therefore a quick indicator of the relative quantities of virus particles.


In the figure above, two-fold dilutions of samples of different influenza viruses (A – H) were prepared, mixed with chicken red blood cells, and added to the wells of a 96-well plate. After 30 minutes the wells were photographed. Sample A causes hemagglutination up to the 1:256 dilution; therefore the HA titer of this virus stock is 256. The sample in row B contains no detectable virus, while that in row D has an HA titer of 512.

The HA assay can be easily modified to determine the level of antibodies to influenza virus present in serum samples. In the CDC study cited below, the authors wished to determine whether stored serum samples contained antibodies to the new influenza H1N1 strain. First they obtained a preparation of one of the new influenza viruses, specifically A/California/04/2009 and determined its HA titer by the method described above. They added a fixed amount of virus to every well of a 96-well plate, equivalent to 32 – 64 HA units. Then they prepared two-fold dilutions of each serum to be tested, and added each dilution series along a row of wells. Finally, they added red blood cells and incubated for 30 minutes.

The basis of the HI assay is that antibodies to influenza virus will prevent attachment of the virus to red blood cells. Therefore hemagglutination is inhibited when antibodies are present. The highest dilution of serum that prevents hemagglutination is called the HI titer of the serum. If the serum contains no antibodies that react with the new H1N1 strain, then hemagglutination will be observed in all wells. Likewise, if antibodies to the virus are present, hemagglutination will not be observed until the antibodies are sufficiently diluted.

The CDC report contains the statement “…serum HI antibody titers of 40 are associated with at least a 50% reduction in risk for influenza infection or disease in populations”. A serum HI antibody titer of 40 means that at a dilution of 1:40, but not higher, the serum blocked hemagglutination. By determining HI titers and comparing them with influenza attack rates in populations, it is possible to calculate the significance of the HI antibody titer with respect to susceptibility to influenza virus infection. When used in this manner, the HI assay is a powerful epidemiological tool.

J Katz, PhD, K Hancock, PhD, V Veguilla, MPH, W Zhong, PhD, XH Lu, MD, H Sun, MD, E Butler, MPH, L Dong, MD, PhD, F Liu, MD, PhD, ZN Li, MD, PhD, J DeVos, MPH, P Gargiullo, PhD, N Cox, PhD (2009). Serum Cross-Reactive Antibody Response to a Novel Influenza A (H1N1) Virus After Vaccination with Seasonal Influenza Vaccine Morbid. Mortal. Weekly Rep., 58 (19), 521-524

Potter, CW, & Oxford, JS (1979). Determinants of immunity to influenza infection in man. Br Med Bull, 35, 69-75