TWiV 343: The silence of the turnips

On episode #343 of the science show This Week in Virology, the TWiVerinoes discuss the potential for prion spread by plants, global circulation patterns of influenza virus, and the roles of Argonautes and a viral protein in RNA silencing in plants.

You can find TWiV #343 at www.microbe.tv/twiv.

Friday flu shot

Yesterday many US newspapers carried front-page stories on the severity of influenza so far this season. The New York Times story began with “It is not your imagination — more people you know are sick this winter, even people who have had flu shots.” Is this really a bad flu season?

Before we answer that question, I would like to complain about what the Times wrote: ‘more people you know are sick this winter, even people who have had flu shots”.  A similar sentiment appeared in a recent Forbes column “Influenza-like-illness is sweeping the country with the Centers for Disease Control & Prevention reporting that most areas of the country experiencing high rates. I should know, my family is in the midst of it despite having been vaccinated.”

Remember that having a respiratory illness does not mean that you have influenza – it could be caused by a number of other viruses which of course would not be blocked by influenza vaccine. Furthermore, the influenza vaccine is not 100% effective – it’s about 60-70% effective in individuals younger than 65 years. That’s not great, but it is better than having no vaccine. The point that I want to make is that it is not useful for anyone to relay anecdotal information about immunization and infection unless you know for sure that you had influenza virus. It only further discourages widespread immunization, which is already isn’t where it should be (~40%).

To answer my question  – is this a bad flu season? – I looked at data from the Centers for Disease Control and Prevention, which receives thousands of respiratory specimens from laboratories throughout the US and determines if they contain influenza virus, and if so, which subtype. Here are the results through week 1 of January 2013:

influenza 2013 week 1

According to these data, there was a peak of influenza activity in week 51 (December 2012). This is early compared with recent influenza seasons. In the 2011-12 season, influenza activity peaked in week 11 (March) of 2012:

influenza 2011-12

During the 2010-11 season, the peak of influenza activity was week 8 (February) of 2011:

influenza 2010-11

During the 2009-10 season, the peak of influenza activity was quite early, in week 42 (October) 2009:

influenza 2009-10

The number of diagnosed infections each year is also indicative of the extent of the influenza season. So far in the 2012-12 season there have been 28,747 influenza positive specimens. Numbers in the previous years: 157,449 in the 2009-10 season, and 55,403 in the 2010-11 season (I was not able to locate totals for 2011-12).

Pneumonia and influenza mortality has so far not substantially exceeded the epidemic threshold as it has in previous years:

pneumonia and influenza mortality

Pediatric deaths from influenza are on track to exceed last year’s total but not the previous two years:

influenza pediatric deaths

The percentage of outpatient visits for influenza-like illness (based on symptoms, not virus isolation) is following a pattern that resembles recent moderately severe influenza seasons:

influenza like illness

The New York City Department of Health and Mental Hygiene also monitors influenza and produces weekly summaries during the season. One metric they report is the percentage of visits for outpatient influenza-like illness. The curve resembles that for 2010-11:

NYC influenza-like illness

New York City also identifies what type of respiratory virus is associated with influenza-like illness, including influenza viruses, adenovirus, respiratory syncytial virus, parainfluenza virus, and metapneumovirus. For week 1 of 2013 all isolates (n=45) were either influenza A/H3N2 or B, although analysis of about a third of them is pending. In previous weeks a mix of other viruses were found in addition to influenza.

Note also the prevalence of influenza H3N2 this season, which has largely displaced the 2009 pandemic H1N1 strain. The H3N2 subtype is generally associated with more severe influenza seasons.

In summary: the data so far suggest that influenza activity is peaking early than in the past two years, but not at an unprecedented time. Numbers of infections, pneumonia, and mortality, are not off the charts. I would agree with Jean Weinberg, a city health department spokeswoman, who said “This is not a season that is out of the ordinary, though H3 seasons tend to be worse than H1 seasons”.

TWiV 212: Apocalypse TWiV 122112 212

On episode #212 of the science show This Week in Virology, the TWiVerers answer listener email about genetically modified chickens, a hendra vaccine for horses, online education, curing color blindness, Roosevelt and polio, Th cells, and much more.

You can find TWiV #212 at www.microbe.tv/twiv.

Influenza is on the rise

flu week 47December 2-8 is National Influenza Vaccination Week. It was established in 2005 by the Centers for Disease Control and Prevention to highlight the importance of continuing immunization throughout the holiday season. This year the push to immunize against flu comes as the disease has begun to increase substantially throughout the United States, as shown in the figure (click the figure for a larger version).

During week 47, 812 of 5,342 (15.2%) of respiratory samples tested positive for influenza virus. Of those isolates that were subtyped, most were either H3N2 or an influenza B virus strain. The 2009 swine-origin H1N1 strain has only been found in one sample so far. Fortunately the H3N2 component of the influenza vaccine for 2012-13 is a good match for the circulating H3N2 strain.

A substantial rise in the number of influenza cases typically does not occur until the end of December in the US. The last time that the disease incidence rose so early was in 2003-04. That was one of the most lethal seasons in 35 years, with 48,000 deaths. This year two children have already died of influenza in the US.

The good news is that 112 million doses of influenza vaccine have already been administered this year, and there is still time to be immunized. The CDC recommends that everyone over 6 months of age be immunized against influenza. Vaccination is especially important for individuals who are at risk for developing serious influenza-related complications: pregnant women, children younger than 5 years old (but those less than 6 months of age should not be immunized), people with with chronic medical conditions such as asthma, diabetes, and heart disease, and those over 65 years old.

The influenza vaccine is available in two types: an injected, inactivated preparation, and an infectious version that is sprayed in the nose. After immunization, approximately two weeks are required to be fully protected against infection.

Derek Smith on antigenic cartography

Derek Smith, Professor of Infectious Disease Informatics, University of Cambridge, U.K., has developed a method for visualizing antigenic evolution by creating two-dimensional maps in a process called antigenic cartography. These maps are made with data that provide information on the antigenic properties of the pathogen. In the case of influenza virus, the data come from measuring the ability of an antiviral antibody to inhibit hemagglutination – binding of virions to red blood cells. Such maps show how amino acid changes can affect antibody binding to virus particles, which cannot be done by comparing nucleotide sequences of different virus isolates. By charting influenza virus strains in this way, it should be possible to better understand genetic and antigenic evolution.

I discussed antigenic cartography with Dr. Smith during ICAAC Boston 2010, as part of TWiV 99. View the video below, or right click to download the 292 MB .mp4 file.

Thoughts on this season’s influenza vaccine

After my lecture on influenza pathogenesis and evolution at the Northeast Laboratory Conference 2010 in Portland, Maine, I was asked if it is necessary to receive the influenza vaccine every year. This question was precipitated by my statement that the 2010-11 trivalent influenza vaccine contains the same swine-origin H1N1 strain as last year’s monovalent vaccine. That virus has not undergone sufficient antigenic drift to necessitate the formulation of a new vaccine.

There are two main considerations* when deciding whether to be immunized yearly against influenza: the nature of the vaccine and age of the recipient. Last year’s seasonal influenza vaccine for the northern hemisphere contained the following strains:

  • A/Brisbane/59/2007 (H1N1)
  • A/Brisbane/10/2007 (H3N2)
  • B/Brisbane/60/2008

Also made available last year was a monovalent vaccine comprising the pandemic strain, A/California/7/2009.

Strains included in the 2010-11 vaccine are:

  • A/California/7/2009 (H1N1)
  • A/Perth/16/2009 (H3N2)
  • B/Brisbane/60/2008

The seasonal H1N1 strains of previous years are no longer circulating and have been eliminated from the vaccine. If you received last year’s seasonal vaccine and the monovalent vaccine, is it necessary to receive this year’s vaccine? The answer is yes, because the H3N2 strain is different – last year the vaccine contained a Brisbane strain while this year’s H3N2 isolate is from Perth. The full WHO report on strain selection is available as a pdf document.

What would be the answer if this year’s trivalent vaccine were identical to that used last year? The answer would still be to receive the vaccine, because the duration of immunity provided by the inactivated influenza vaccine has always been an issue. In elderly recipients (>65 years of age) immunity barely lasts for a single influenza season. Younger recipients will likely be protected from disease for one influenza season, and perhaps a second season as well, although in the latter case a milder respiratory disease may result. For these reasons the CDC recommends annual immunization against influenza virus for all individuals 6 months of age and older.

The current inactivated influenza vaccines were developed during World War II, and although they have since been refined and purified, there are still deficiencies, including brief duration of protective immunity. Development of new influenza vaccines that not only overcome this problem, but also provide broader protection, is clearly needed.

*This post, like everything else on virology blog, does not contain medical advice. It comprises scientific information about the vaccines which can be used, together with your health care provider, to reach a decision about immunization.

Whither 2009 H1N1?

When will the 2009 swine-origin influenza virus become a seasonal strain? While prediction is very hard, especially of the future (at least according to Yogi Berra), examining past pandemics can be informative.

h3n2

The 1968 pandemic began with the emergence of a novel H3N2 influenza virus in Hong Kong in July 1968. First isolates (stars) were obtained globally throughout the summer. The previous seasonal H2N2 strain was last isolated in August 1968 in Australia and was subsequently not seen again. There were sporadic H3N2 outbreaks for several months (hatched lines). Epidemic spread (solid lines) ensued in the northern hemisphere throughout the winter, and then ceased in the spring of 1969. In the southern hemisphere the first epidemic occurred from January through October. There were second seasons of epidemic spread in both hemispheres, ending in the summer of 1970. After that time the H3N2 strain became a seasonal strain, causing local epidemics each year as the virus underwent antigenic drift.

If the pattern of H3N2 serves as a guide, we might predict that the 2009 swine-origin H1N1 virus will display pandemic spread for at least one more season. In the northern hemisphere, the second season would comprise November 2010 – April 2011. However, immunity in older individuals might blunt the spread of the virus beyond the current first season. It also remains to be seen how much immunization will impact pandemic spread.

At one point the 2009 H1N1 swine-origin influenza virus will become a seasonal strain, and the monovalent vaccine will no longer be produced. At that time the season influenza vaccine will likely comprise a ‘drifted’ version of the 2009 H1N1 strain and an influenza B virus. By all current indications the previous seasonal H1N1 and H3N2 strains will soon disappear from humans – although not from the globe.

Viboud, C., Grais, R., Lafont, B., Miller, M., Simonsen, L., & , . (2005). Multinational Impact of the 1968 Hong Kong Influenza Pandemic: Evidence for a Smoldering Pandemic The Journal of Infectious Diseases, 192 (2), 233-248 DOI: 10.1086/431150

Influenza virus reassortment, then and now

influenza-rna-gelIn a recent study of influenza virus reassortment in ferrets, the authors used polymerase chain reaction (PCR) to search for viruses with RNA segments from the 2009 pandemic H1N1 strain and seasonal H1N1 and H3N2 strains. I thought you might like to see how I did a similar experiment in 1979 – a very different era for laboratory techniques.

For my Ph.D. thesis project, I wanted to isolate reassortants of two influenza B virus strains, B/Lee and B/Maryland. The goal was to obtain viruses with a genome consisting of one RNA segment from one parent, and 7 RNA segments from the other parent. These viruses would then be used to identify the protein product of each viral RNA.

To isolate these reassortants, I co-infected cells in culture with both viruses, allowed them to replicate, and then harvested the newly synthesized viruses. I then did a plaque assay with the viruses produced by the co-infected cells, and isolated individual clones by plaque purification. I prepared virus stocks from each plaque-purified clone, and then asked if any of these viruses were reassortants.

In 1979, we identified viral reassortants by a tedious method. Each virus to be examined was used to infect cultured cells in the presence of radioactive phosphate. The viruses were purified by centrifugation, and the viral RNA was extracted, concentrated, and fractionated by gel electrophoresis. The gel was dried and exposed to X-ray film. Because the viruses were propagated in the presence of radioactive phosphate, which was incorporated into the viral RNAs, it was possible to visualize each viral segment as a band on the film. The X-ray is shown above.

The RNAs of three different viruses are included: B/Lee on the left, B/Maryland in the middle, and a plaque-purified virus called R3. You can see that each lane contains 8 RNAs, as expected. It is also evident that the migration pattern of the RNAs is different for B/Lee and B/Maryland. This property allowed us to determine that the plaque purified virus R3 is a reassortant that inherits RNA 2 from B/Lee, and the remaining 7 RNAs from B/Maryland. Just what I wanted!

In fact, in just one infection, I isolated all the different reassortant viruses that I needed. I remember showing the gel to my thesis advisor: he told me I didn’t deserve such luck.

The whole procedure – from infecting cells in the presence of radioactive phosphate, to producing the X-ray, took about a week. And to get all the right viral reassortants required a great deal of luck.

Today, the process of identifying influenza viral reassortants is far simpler and faster. The process begins in a similar way – co-infect cells (or animals) with two different viruses. Once the infection is complete, a small sample of the cell culture medium is taken, heated to disrupt the virions, and the viral RNA is converted to DNA using reverse transcriptase. The DNA is amplified by PCR, in eight separate reactions, using primer pairs specific for the individuals segments. The products are then fractionated by gel electrophoresis, as shown here.

Total time to identify reassortants by PCR – less than a day. That’s progress.

In a few years, we’ll skip the gel electrophoresis and simply determine the sequence of the RNAs using a small, inexpensive machine that will be on most laboratory benches. And who knows what will be next? That’s one of the beauties of science: it is driven forward by technological innovation.

Racaniello VR, & Palese P (1979). Influenza B virus genome: assignment of viral polypeptides to RNA segments. Journal of virology, 29 (1), 361-73 PMID: 430594

Influenza PB1-F2 protein and viral fitness

influenza-rna-2The second RNA segment of the influenza virus genome encodes the PB1 protein – part of the viral RNA polymerase – and, in some strains, a second protein called PB1-F2. The latter protein is believed to be an important determinant of influenza virus virulence. The absence of a full-length PB1-F2 protein has been suggested as one possible determinant for the low pathogenicity of the 2009 influenza H1N1 pandemic strain. Analysis of the evolutionary history of PB1-F2 suggests that it does not contribute significantly to viral fitness – the ability of the virus to replicate.

PB1-F2 binds to mitochondria, leading to a release of cytochrome c and induction of apoptosis in CD8 T-cells and alveolar macrophages. The protein increases the severity of primary viral and secondary bacterial infections in mice, and is associated with the high pathogenicity of avian H5N1 and the 1918 H1N1 pandemic virus.

The PB1-F2 protein is not produced in cells infected with the 2009 H1N1 strain because there are three stop codons at nucleotide positions 12, 58, and 88.  The PB1 segment of the 2009 H1N1 strain is related to PB1 of H1N2 and H3N2 swine viruses from 1998 and human H3N2 viruses. Curiously, all the relatives of the 2009 H1N1 strain in swine and in humans encode a complete PB1-F2 protein. A truncated PB1-F2 is encoded by the genome of classical swine H1N1 viruses and human H1N1 viruses since 1947. But 96% of the avian influenza virus sequences deposited in NCBI as of 2007 encode the full length version of the protein.

Because the full-length PB1-F2 protein is not encoded in the genome of many influenza viruses, its evolutionary role and contribution to the fitness of the virus is unclear. To answer these questions, the evolution of PB1-F2 was compared with PB1 and two other open reading frames of similar size within the same RNA segment that are not translated into protein.

PB1-F2 is complete in all H1N1 human isolates before 1947, when a stop codon appeared which leads to production of a shorter version of the protein – 57 amino acids. If the complete protein conferred a functional advantage to the virus, a change in the evolutionary rates of the human H1N1 PB1-F2 proteins should have occurred in 1947. No such change is observed.

Results of sequence analysis reveal that the PB1-F2 open reading frame is as conserved, and maintained as a full-length protein, as other non-coding regions of the same RNA segment and of a randomly generated PB1 segment. These observations, and the fact that PB1-F2 is truncated in many virus isolates, suggest that the evolutionary role of PB1-F2 in animal hosts is minimal. Why the full length protein is produced by some viruses – and unfortunately leads to higher virulence – remains a puzzle.

Trifonov, V., Racaniello, V., & Rabadan, R. (2009). The Contribution of the PB1-F2 Protein to the Fitness of Influenza A Viruses and its Recent Evolution in the 2009 Influenza A (H1N1) Pandemic Virus PLoS Currents: Influenza

No tetravalent influenza vaccine

influenza-vaccineTwo different influenza vaccines will be available in the fall of 2009. One will be a trivalent preparation comprising seasonal influenza H3N2 and H1N1 strains, plus an influenza B virus strain. A monovalent vaccine will also be available against the 2009 pandemic H1N1 strain. Many readers of virology blog have asked why these four virus strains will not be combined into a single, tetravalent formulation. I posed this question to Dr. Ruben Donis, chief of the molecular virology and vaccines branch at the U.S. Centers for Disease Control and Prevention. Here is his answer.

Some of the reasons that make tetravalent influenza vaccine less attractive at this time:

  • Regulatory considerations: there have been no clinical trials with tetravalent influenza vaccines, and there is little time to do them now. Licensing without data is not a favored option. However, clinical trials for a monovalent have been started and will help establish appropriate doses, formulations, in different population groups.
  • Public health considerations: the populations at greater risk of severe outcomes are not the same for the seasonal viruses and the H1N1 – pregnant women are high on the list for receiving H1N1 vaccine.
  • Timing considerations: Seasonal vaccine will be available in September whereas pandemic H1N1 vaccine will become available later.
  • Vaccine effectiveness, adverse events, pharmacovigilance: it will be easier to monitor vaccine performance characteristics using a monovalent

He also mentioned the possiblity of making a trivalent preparation with the pandemic H1N1 strain. But he said that

…a trivalent with pandemic H1N1 would entail eliminating one of the current seasonal viruses – we don’t have evidence that any of the three seasonal viruses is becoming extinct.

I thank Dr. Donis for responding.