Influenza HA cleavage is required for infectivity

The influenza virus hemagglutinin (HA) is the viral protein that attaches to cell receptors. The HA also plays an important role in the release of the viral RNA into the cell, by causing fusion of viral and cellular membranes. HA must be cleaved by cellular proteases to be active as a fusion protein.

The HA on the influenza virion is a trimer: it is made up of three copies of the HA polypeptide. The cleavage site for cell proteases on the HA protein is located near the viral membrane.


In the diagram, the globular head of the HA protein, which attaches to cell receptors, is at the top, and the viral membrane is at the bottom. For clarity, only one HA cleavage site is labeled. The uncleaved form of the protein is called HA0; after cleavage by a cellular enzyme, two proteins are produced, called HA1 (blue) and HA2 (red). The two subunits remain together at the surface of the virus particle. The new amino(N)-terminal end of HA2 that is produced by cleavage contains a sequence of hydrophobic amino acids called a fusion peptide. During entry of influenza virus into cells, the fusion peptide inserts into the endosomal membrane and causes fusion of the viral and cell membranes. Consequently, the influenza viral RNAs can enter the cytoplasm. The fusion process is described in a previous post.

If the HA protein is not cleaved to form HA1 and HA2, fusion cannot occur. Therefore influenza viruses with uncleaved HA are not infectious. Cleavage of the viral HA occurs after newly synthesized virions are released from cells. Influenza viruses replicate efficiently in eggs because of the presence of a protease in allantoic fluid that can cleave HA. However, replication of many influenza virus strains in cell cultures requires addition of the appropriate protease (often trypsin) to the medium.

In humans, influenza virus replication is restricted to the respiratory tract, because that is the only location where the protease that cleaves HA is produced. However, the HA protein of highly pathogenic H5 and H7 avian influenza virus strains can be cleaved by proteases that are produced in many different tissues. As a result, these viruses can replicate in many organs of the bird, including the spleen, liver, lungs, kidneys, and brain. This property may explain the ability of avian H5N1 influenza virus strains to replicate outside of the human respiratory tract.

Like the HA proteins of highly pathogenic H5 and H7 viruses, the HA of the 1918 influenza virus strain can also be cleaved by ubiquitous cellular proteases. Consequently, the virus can replicate in cell cultures in the absence of added trypsin.

The H5 and H7 HA proteins have multiple basic amino acid residues at the HA1-HA2 cleavage site which allows cleavage by widely expressed proteases. But the 1918 H1 HA does not have this feature. Nor does the 1918 N1 help recruit proteases that cleave the HA, a mechanism that allows the A/WSN/33 influenza virus strain to multiply in cells without trypsin. An understanding of how the 1918 H1 HA protein can be cleaved by ubiquitous proteases is essential for understanding the high pathogenicity of this strain.

Chaipan, C., Kobasa, D., Bertram, S., Glowacka, I., Steffen, I., Solomon Tsegaye, T., Takeda, M., Bugge, T., Kim, S., Park, Y., Marzi, A., & Pohlmann, S. (2009). Proteolytic Activation of the 1918 Influenza Virus Hemagglutinin Journal of Virology, 83 (7), 3200-3211 DOI: 10.1128/JVI.02205-08

Brazilian influenza H1N1 isolate is not novel

3184877559_e8bbb242ba_mReaders of virology blog have no doubt seen news reports that a Brazilian influenza isolate called A/Sao Paulo/1454/2009 (H1N1) is novel strain with mutations that enable it to infect new hosts. Here is one example of such a report, from The Australian:

Brazilian scientists have identified a new strain of the H1N1 virus after examining samples from a patient in Sao Paulo, a research institute says. The variant has been called A/Sao Paulo/1454/H1N1 by the Adolfo Lutz Bacteriological Institute, which compared it with samples of the A(H1N1) swine flu from California. The genetic sequence of the new sub-type of the H1N1 virus was isolated by a virology team led by one of its researchers, Terezinha Maria de Paiva. The mutation was comprised of alterations in the Hemagglutinin protein which allows the virus to infect new hosts, it said.

There is nothing novel about this Brazilian isolate. Comparison of the amino acid sequence of the HA protein of A/Sao Paulo/1454/H1N1 with those of other isolates of the current pandemic strain reveals no alterations in the HA protein which would allow the virus to infect new hosts. The HA protein of this virus and many other 2009 H1N1 isolates are identical. The few amino acid differences with other 2009 H1N1 isolates are in areas that would not be expected to influence antigenicity or host range.

I’ll give the journalists the benefit of doubt: perhaps they misinterpreted the statements of the Brazilian scientist. But they should have confirmed their story with a virologist. They would have learned within a few minutes that there is nothing novel about A/Sao Paulo/1454/2009 (H1N1).

Was the swine influenza threat underestimated?

h1n1-ha-treeMany virologists, including myself, believe that the threat of avian H5N1 influenza is not only overestimated, but also distracts from serious consideration of other potential pandemic strains. In early 2005 I wrote about this problem (“Should we worry about avian influenza?) and suggested that we should pay attention to H2N2 strains as a potential pandemic threat. Although I was wrong in my choice of the next pandemic strain, the lesson is that we should keep a broad perspective when attempting to predict which virus will cause the next global epidemic.  The authors of an article in Eurosurveillance chide us for ignoring the swine flu threat:

Although the role of swine as “mixing vessels” for influenza A(H1N1) viruses was established more than a decade ago, it appears that the policy makers and scientific community have underestimated it. In fact, in 1998 influenza experts proposed the establishment of surveillance in swine populations as a major part of an integrated early warning system to detect pandemic threats for humans but, to some extent, this task was overlooked.

In support of their contention, the authors compare the number of swine influenza A sequences (4,648) with those of human (46,911) and avian (41,142) influenza A viruses. They also point out that some countries, such as the United States, have devoted all of their pandemic preparedness budget ($3.8 billion for the US) towards the prevention and control of avian A(H5N1) influenza. They believe that

…in this plan, a substantial effort was dedicated to prevent and contain the foreign threat of Asian avian flu, neglecting the influenza threat that the North American swine population presents. Specifically, we believe that the aforementioned strategy ignores the swine farm and industry workers which constitute the population at higher risk of contracting and spreading the hypothetical pandemic influenza virus.

This strategy is based on the authors’ conclusions that the new influenza H1N1 viruses are genetically distinct from other H1N1 viruses that have been circulating in humans for the past 20 years. The new swine derived viruses are more similar to viruses that were transmitted from pigs to humans in Iowa, Maryland, and Wisconsin between 1991 and 2006. This conclusion is illustrated by the phylogenetic tree of HA protein sequences from H1N1 influenza viruses. Included in the analysis are H1 HA proteins from the 2009 influenza A(H1N1) virus (red triangles), earlier human (red and pink circles), swine (navy blue and purple circles), and avian (green circles) viruses. The viruses involved in pig-human interspecies transmission in Iowa, Maryland and Wisconsin, shown as orange squares, cluster with the swine viruses and the 2009 H1N1 virus.

An extensive surveillance of the genetic evolution of influenza A viruses that circulate in Mexican poultry farms has been carried out since 2002. A similar system will now be put into place on swine farms. The goal is to identify genetically distinct influenza viruses that could lead to pandemics.

G M Nava, M S Attene-Ramos, J K Ang, & M Escorcia (2009). Origins of the new influenza A(H1N1) virus: time to take action Eurosurveillance, 14 (22)

Influenza microneutralization assay

The microneutralization assay is another technique used by the Centers for Disease Control and Prevention to determine that some adults have serum cross-reactive antibodies to the new influenza H1N1 virus. Let’s explore how this assay works.

Viral replication is often studied in the laboratory by infecting cells that are grown in plastic dishes or flasks, commonly called cell cultures. Many viruses kill such cells. Here is an example of HeLa cells being killed by poliovirus:


The upper left panel shows uninfected cells, and the other panels show the cells at the indicated times after infection. As the virus replicates, infected cells round up and detach from the cell culture plate. These visible changes are called cytopathic effects.

There is another way to visualize viral cell killing without using a microscope: by staining the cells with a dye. In the example shown below, cells have been plated in the small wells of a 96 well plate. One well was infected with virus, the other was not. After a period of incubation, the cells were stained with the dye crystal violet, which stains only living cells. It is obvious which cells were infected with virus and which were not.


We can use this visual assay to determine whether a serum sample contains antibodies that block virus infection. A serum sample is mixed with virus before infecting the cells. If the serum contains antibodies that block viral infection, then the cells will survive, as determined by staining with crystal violet. If no antiviral antibodies are present in the serum, the cells will die.

In its present form, this assay tells us only whether or not there are antiviral antibodies in a serum sample. To make the assay quantitative, two-fold dilutions of the serum are prepared, and each is mixed with virus and used to infect cells. At the lower dilutions, antibodies will block infection, but at higher dilutions, there will be too few antibodies to have an effect. The simple process of dilution provides a way to compare the virus-neutralizing abilities of different sera. The neutralization titer is expressed as the reciprocal of the highest dilution at which virus infection is blocked.

neutralizationIn the example shown here, the serum blocks virus infection at the 1:2 and 1:4 dilutions, but less at 1:8 and not at all at 1:16. Each serum dilution was tested in triplicate, which allows for more accuracy. In this sample, the neutralization titer would be 4, the reciprocal of the last dilution at which infection was completely blocked.

This explanation should clarify how the neutralization titers were obtained that are reported in the CDC study cited below. By the way, microneutralization simply means that the neutralization assay is done in a small format, such as a 96 well plate, instead of larger cell culture dishes.

The authors of the CDC study note that “although serum hemagglutination inhibition (HI) antibody titers of 40 are associated with at least a 50% reduction in risk for influenza infection or disease in populations, no such correlate of protection exists for microneutralization antibody titers”. They used mathematical analysis to determine the relationship between HI and microneutralization titers. They found that in sera from children, an HI titer of 40 corresponded to a microneutralization titer of 40. However, in adults, an HI titer of 40 corresponded to a microneutralization titer of 160 or more. I don’t know the reason for this difference, but one possibility is that not all neutralizing antibodies in adult sera are able to inhibit hemagglutination. Understanding why this situation might occur will require a discussion of how antibodies block viral infection.

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

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

Adults have cross-reactive antibodies to A/California/04/2009 (H1N1)

hemagglutinationDoes previous exposure to influenza H1N1 viruses, either by infection or vaccination, provide any protection against infection with the new H1N1 influenza virus strains? The answer to this question might provide insight as to why over 60% of confirmed cases of influenza caused by the swine-like H1N1 viruses in the US are in 5- to 24-year-olds, as reported at a CDC press conference.

To answer this question, CDC has analyzed serum specimens that were collected during previous vaccine studies. These sera were collected from children and adults before and after they received influenza vaccine in the 2005-06, 2006-07, 2007-08, or 2008-09 influenza seasons. Virus neutralization and hemagglutination-inhibition assays were done to determine whether these sera contain antibodies that cross-react with the new H1N1 strain. The authors of the study used the A/California/04/2009 as a representative of the new H1N1 virus isolates.

The results show that previous immunization of children (age 6 months to 9 years, total of 79 specimens) with either seasonal trivalent inactivated vaccine or infectious, attenuated influenza vaccine of the previous four years did not induce cross-reactive antibody to the new influenza A H1N1 strain. Previous immunization did induce a low cross-reactive antibody response to A/California/04/2009 in adults. Among 18-64 year olds, there was a twofold increase in cross reactivity antibody to the virus, compared with a 12-19 fold increase in antibody titers against the seasonal strains. There was no increase in cross reactive antibodies in adults over 60 years of age. These data indicate that immunization with seasonal influenza vaccines containing previous H1N1 strains (years 2005-2009) is not likely to confer protection against infection with the new H1N1 strains.

An important question is whether the sera obtained before administration of vaccine contain cross-reactive antibody titers against A/California/04/2009. Such analyses would indicate whether natural infection with H1N1 strains confers some protection agains the new isolates. There were no pre-vaccination cross-reactive antibodies to A/California/04/2009 in sera of any of the 79 children of ages 6 months to 9 years. However, 6% of adults 18-40 years old, 9% of adults 18-64 years old, and 33% of adults over 60 years of age had pre-vaccination neutralizing antibody titers to A/California/04/2009 greater than or equal to 160. These antibodies were likely acquired by infection with an H1N1 virus that is antigenically more similar to the A/California/04/2009 than other seasonal H1N1 strains. Whether such antibodies would confer protection against infection is unknown, but they could reduce the severity of disease symptoms.

I suspect that not all readers of virology blog are familiar with the microneutralization and hemagglutination-inhibition assays used in this study. In a separate post, I will explain how the assays work, and the significance of the test results.

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

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.

Influenza vaccine for life?

influenza-HA-antibodiesThe best way to prevent influenza is by immunization. Unlike vaccines for polio and measles, which confer life-long immunity, the influenza vaccine protects for only one year. Influenza virus undergoes antigenic variation, necessitating annual production of a new vaccine. Is it possible to formulate an influenza vaccine that protects against all virus strains for life?

Two studies of newly isolated monoclonal antibodies against influenza virus suggest that the answer could be yes. The authors of one study identified human antibodies against influenza virus by phage display. In this technique, recombinant HA protein (the H5 subtype) was used to bind bacteriophage particles that bear on their surfaces variable chains of human antibodies. Ten antibodies were identified that neutralized the infectivity of H5 influenza viruses in cell culture. The antibodies also protected mice against lethal H5N1 influenza even when administered after infection.

The key result is that the monoclonal antibodies neutralize infectivity not only of H5 viruses, but also viruses of 9 other HA subtypes. There are 16 known HA subtypes, divided into two groups. The 10 subtypes neutralized by the monoclonal antibodies comprise group 1, which includes H1, H2, and H5. If another epitope can be identified that elicits neutralizing antibodies against group 2 HA subtypes, then a universal vaccine that confers life-long protection might be feasible.

Such broadly-reacting neutralizing monoclonal antibodies have been reported previously, but they are rare. When animals are immunized with influenza virus, most of the antibodies that are produced are directed against the membrane-distal, globular head of the HA molecule (top of image). Resolution of the X-ray structure of one of the monclonal antibodies bound the the H5 HA protein revealed that the antibody binding site is a hydrophobic pocket on the stem of the HA molecule. This membrane-proximal site  is probably not readily recognized by B cell receptors and therefore rarely gives rise to antibodies. The authors circumvented this problem by selecting antibodies using a soluble form of the HA protein, which is not subject to such steric constraints.

A number of significant hurdles remain to be overcome before these findings translate into an influenza vaccine. As noted above, another epitope must still be identified that elicits neutralizing antibodies against viruses of the 6 other HA types. Additional screening of human antibodies with soluble HA protein will presumably address this issue. A more difficult is how to formulate a vaccine with these epitopes. The most effective influenza vaccines are whole or split virus preparations, but how can these be prepared so that the membrane-proximal HA epitope is immunodominant? If the globular head of the HA is removed, the virus will not be infectious and cannot be propagated for vaccine production. One solution might be to produce defective particles in producer cell lines. The problem is not insurmountable, but will require some new and innovative approaches in influenza vaccine development.

These are very exciting findings, and in my opinion, bode well for a universal influenza vaccine within the next decade.

Jianhua Sui, William C Hwang, Sandra Perez, Ge Wei, Daniel Aird, Li-mei Chen, Eugenio Santelli, Boguslaw Stec, Greg Cadwell, Maryam Ali, Hongquan Wan, Akikazu Murakami, Anuradha Yammanuru, Thomas Han, Nancy J Cox, Laurie A Bankston, Ruben O Donis, Robert C Liddington, Wayne A Marasco (2009). Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses Nature Structural & Molecular Biology DOI: 10.1038/nsmb.1566

Mark Throsby, Edward van den Brink, Mandy Jongeneelen, Leo L. M. Poon, Philippe Alard, Lisette Cornelissen, Arjen Bakker, Freek Cox, Els van Deventer, Yi Guan, Jindrich Cinatl, Jan ter Meulen, Ignace Lasters, Rita Carsetti, Malik Peiris, John de Kruif, Jaap Goudsmit (2008). Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells PLoS ONE, 3 (12) DOI: 10.1371/journal.pone.0003942