Phage synergy with the immune system

bacteriophage modelNot long after their discovery, viruses that infect bacteria – bacteriophages – were considered as therapeutic agents for treating infections. Despite many years of research on so-called phage therapy, clinical trials have produced conflicting results. They might be explained in part by the results of a new study which show that the host innate immune system is crucial for the efficacy of phage therapy.

When mice are infected intranasally with Pseudomonas aeruginosa (which causes pneumonia in patients with weak immune systems), the bacterium multiplies in the lungs and kills the animals in less than two days. When a P. aeruginosa lytic phage (i.e. that kills the bacteria) is instilled in the nose of the mice two hours after bacterial infection, all the mice survive and there are no detectable bacteria in the lungs. The phage can even be used prophylactically: it can prevent pneumonia when given up to four days before bacterial challenge.

The ability of phage to clear P. aeruginosa infection in the mouse lungs depends on the innate immune response. When bacteria infect a host, they are rapidly detected by pattern recognition receptors such as toll-like receptors. These receptors detect pathogen-specific molecular patterns and initiate a signaling cascade that leads to the production of cytokines, which may stop the infection. Phage cannot clear P. aeruginosa infection in mice lacking the myd88 gene, which is central to the activity of toll like receptors. This result shows that the innate immune response is crucial for the ability of phages to clear bacterial infections. In contrast, neither T cells, B cells, or innate lymphoid cells such as NK cells are needed for phage therapy to work.

The neutrophil is a cell of the immune system that is important in curtailing bacterial infections. Phage therapy does not work in mice depleted of neutrophils. This result suggests that humans with neutropenia, or low neutrophil counts, might not respond well to phage therapy.

A concern with phage therapy is that bacterial mutants resistant to infection might arise, leading to treatment failure. In silico modeling indicated that phage-resistant bacteria are eliminated by the innate immune response. In contrast, phage resistant bacteria dominate the population in mice lacking the myd88 gene.

These results demonstrate that in mice, successful phage therapy depends on a both the innate immune response of the host, which the authors call ‘immunophage synergy’. Whether such synergy also occurs in humans is not known, but should be studied. Even if observed in humans, immunophage synergy might not be a feature of infections in other anatomical locations, or those caused by other bacteria. Nevertheless, should immunophage synergy occur in people, then clearly only those with appropriate host immunity – which needs to be defined – should be given phage therapy.

The largest viral genome from a human

Mimivirus LBA111The biggest known viruses are Mimivirus (750 nanometer capsid, 1.2 million base pair DNA) and Megavirus (680 nanometer capsid, 1.3 million base pair DNA). These giant viruses have all been isolated from environmental samples, and many infect amoebae. A new Mimivirus has now been isolated from a human patient with pneumonia.

To search for giant viruses in humans, respiratory samples from 196 Tunisian patients with community-acquired pneumonia were co-cultured with the amoebae Acanthamoeba polyphaga. One sample, a bronchial aspirate, yielded a new Megavirus that was given the catchy name LBA111. The patient from which LBA111 was isolated had been hospitalized with fever, cough, shortness of breath, and bloody sputum. Serum from this patient, but not from those of 50 healthy blood donors, reacted with LBA111 proteins, indicating suggesting that this individual had been infected with the virus.

Mimivirus LBA111 is not likely to be a contaminant introduced from the laboratory in which it was isolated, because its genome sequence is original. The LBA111 genome is double-stranded DNA 1,230,522 base pairs in length, mostly closely related to the genome of Megavirus chilensis, a giant virus isolated off the coast of Chile. In addition to other differences, the LBA111 genome encodes two  tRNAs (histidine and cysteine) not present in the genome of M. chilensis.

While these findings indicate an association of Mimivirus LBA111 with human pneumonia, they do not prove that this virus is the causative agent of human disease. However, the possibility that Mimivirus causes human disease makes sense. Mimiviruses are present in soil and water where they multiply in amoebae. A known agent of human respiratory disease, Legionella pneumophila, also colonizes amoebae. Antibodies to Mimivirus, as well as Mimivirus DNA, have been found in patients with pneumoniaMimivirus should therefore be added to the list of agents that should be considered in patients with pneumonia.

TWiV 239 – Filterable camels

On episode #239 of the science show This Week in Virology, Matt joins Vincent, Alan, and Rich to summarize what we know and what we do not know about the MERS coronavirus.

You can find TWiV #239 at

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 204: M m m my corona

On episode #204 of the science show This Week in Virology, Vincent, Alan, Matt and Kathy review isolation of a new coronavirus from two patients in the Middle East, and expansion of the enteric virome during simian AIDS.

You can find TWiV #204 at

TWiV 59: Dog bites virus

TWiV_AA_200Hosts: Vincent Racaniello, Alan Dove, Rich Condit, Gustavo Palacios, and Mady Hornig

A TWiV panel of five considers the finding of Streptococcus pneumoniae in fatal H1N1 cases in Argentina, hysteria in the Ukraine over pandemic influenza, and human vaccinia infection after contact with a raccoon rabies vaccine bait.

[powerpress url=”″]

Click the arrow above to play, or right-click to download TWiV #59 (58 MB .mp3, 80 minutes)

Subscribe to TWiV in iTunes, by the RSS feed, or by email

Links for this episode:

Weekly Science Picks
Rich Longitude by Dava Sobel
Alan Heil Pro Set Media Headset (for a good price, order from a ham radio store)
MicrobeWorld app for iPhone and iTouch (iTunes or MicrobeWorld)

Send your virology questions and comments (email or mp3 file) to or leave voicemail at Skype: twivpodcast. You can also send articles that you would like us to discuss to delicious and tag them with to:twivpodcast.

How many people die from influenza?

WHO reports that as of 15 June 2009, 76 countries have officially reported 35, 928 cases of influenza A(H1N1) infection, including 163 deaths. These numbers can be used to calculate a case fatality ratio (CFR) of 0.45%. Is this number an accurate indication of the lethality of influenza?

Determining how many people die from influenza is a tricky business. The main problem is that not every influenza virus infection is confirmed by laboratory testing. For example, early in the Mexico H1N1 outbreak, the apparent CFR was much higher because the total number of infections had not been established. Even with the intense surveillance being conducted at the onset of this pandemic, many infections are still not diagnosed. Virologic surveillance is likely become even more incomplete as health systems become overburdened:

The size of Victoria’s outbreak is now so great that only those most at risk – the elderly, pregnant women and those with other underlying medical conditions – are being tested, resulting in 199 new cases last week. “At the moment cases confirmed in the laboratory signify only a small fraction of the cases,” Dr Lester said. “It could be three or four times the laboratory confirmed number, but it’s very hard to estimate, given the mild nature of the virus. It is not anywhere near the one in three some have suggested.

So how do we determine how many people are killed by influenza virus?

In fact, the Centers for Disease Control and Prevention of the US does not know exactly how many people die from flu each year. The number has to be estimated using statistical procedures.

There are several reasons why influenza mortality in the US is estimated. States are not required to report to the CDC individual influenza cases, or deaths of people older than the age of 18. Influenza is rarely listed as a cause of death on death certificates, even when people die from influenza-related complications. Many flu-related deaths occur one or two weeks after the initial infection, when influenza can no longer be detected from respiratory samples. Most people who die from influenza-related complications are not given diagnostic tests to detect influenza.

To determine the level of influenza-related mortality, each week, from October to mid-May, the vital statistics offices of 122 cities report the number of death certificates which list pneumonia or influenza as the underlying or cause of death. The percentage of deaths due to pneumonia and influenza are compared with a seasonal baseline and epidemic threshold value determined each week. The seasonal baseline is calculated using statistical procedures using data from the previous five years, and the epidemic threshold is calculated as 1.645 standard deviations above the seasonal baseline. This is the point at which the observed proportion of deaths attributed to pneumonia or influenza becomes significantly higher than would be expected without substantial influenza-related mortality.

For the 2007–08 influenza season, the percentage of deaths attributed to pneumonia and influenza exceeded the epidemic threshold for 8 consecutive weeks from January 12–May 17, 2008, with a peak at 9.1% at week 11, as shown below. In contrast, pneumonia and influenza deaths remained below the epidemic threshold in the relatively mild 2008-2009 season:


This method clearly is not perfect. The rationale is that the ‘excess mortality’ (over the epidemic theshold) is likely to be caused by influenza, but so could at least some of the deaths between the baseline and excess threshold. For example, the pneumonia and influenza deaths are below the epidemic threshold this season, yet we know that people have died from influenza. It also misses deaths caused by influenza, but for one reason or another influenza or pneumonia were not entered on the death certificate.

The answer to this dilemma is more statistics – methods that use the CDC data to estimate the number of deaths caused by influenza. In the paper cited below, the authors calculated an average of 41,400 deaths each year , for the years 1979 – 2001, in the US due to influenza. Remember that this is an average, and the actual numbers may vary substantially each year.

To answer the question posed at the beginning of this post: except in well-contained outbreaks in which the number of infected individuals can be determined with precision, the case-fatality ratio is bound to be inaccurate. The use of serological assays to determine the extent of infection, coupled with statistical estimates of influenza mortality, are likely to provide more reliable data.

Dushoff, J. (2005). Mortality due to Influenza in the United States–An Annualized Regression Approach Using Multiple-Cause Mortality Data American Journal of Epidemiology, 163 (2), 181-187 DOI: 10.1093/aje/kwj024

Interferons and secondary pneumonia after influenza


Now that we have discussed influenza pathogenesis in humans and the innate immune defenses, we can tackle the conclusion that type I IFN mediates the development of secondary bacterial pneumonia in mice.

Secondary bacterial pneumonia occurs after the patient has begun to recover from influenza infection, and often influenza virus can no longer be isolated. The reasons why influenza virus infections may lead to pneumonia are not understood. One group studied this problem by using mice inoculated in the trachea with a mouse-adapted strain of influenza virus, A/PR/8/1934 (H1N1). Five days later,  Streptococcus pneumoniae bacteria are administered by the same route. The bacteria multiplied to high levels in mice that had been previously infected with influenza virus, but not in saline-treated control mice. Furthermore, significant mortality was observed in the doubly-infected mice but not in mice infected with virus or bacteria alone.

The same experiment was then repeated, using mice that lack the genes encoding cell surface receptors for type I IFNs (IFN-α and IFN-β). These mice can produce IFN, but they cannot synthesize the hundreds of antiviral proteins that are made in response to IFN, because the receptor for this cytokine (illustrated) is absent from cell surfaces. These mice were resistant to secondary bacterial pneumonia. When infected with influenza virus and then S. pneumoniae, the mice had no higher bacterial burden in the lung, and no more mortality, than mice infected only with bacteria.

Why does type I IFN predispose mice to secondary bacterial pneumonia? Two cytokines, called KC and Mip2, appear to be the culprits. After secondary challenge with S. pneumoniae, these cytokines were detected at higher levels in the lungs of type I IFN receptor deficient mice than in the lungs of wild type mice.  These observations indicate that type I IFNs appear to inhibit the production of KC and Mip2 chemokines.

The chemokines KC and Mip 2 are believed to be essential for recruiting neutrophils, the most abundant type of white blood cell in the blood. Neutrophils are attracted to sites of bacterial infection, where they engulf and destroy the microbes. As expected, more immune cells were detected in the lungs of mice lacking type I IFN receptors than in the lungs of wild type mice.

These observations may explain why secondary bacterial infections occur after influenza in humans, according to the following model. During infection with influenza virus, type I IFN is produced, as the host innate defenses attempt to clear infection. Type I IFNs inhibit the production of the chemokines KC and Mip 2. Because these chemokines are essential for recruiting bacteria-destroying neutrophils into the lung, bacteria that enter the lung cannot be effectively cleared, and pneumonia occurs.

This is an interesting hypothesis, but it fails to explain two important observations. First, it does not explain why secondary bacterial pneumonia only occurs in a subset of influenza virus infected humans. And if the influenza virus NS1 protein inhibits the production of IFN, as we discussed yesterday, there should be no defect in the recruitment of neutrophils to the influenza virus-infected lung.

An intriguing observation is that influenza virus does not replicate any better in the lungs of mice lacking the IFN receptor than in the lungs of wild type mice. Perhaps the type I IFN system is redundant for limiting viral replication: when it is missing, other systems take its place. I don’t understand this finding, given the role of the viral NS1 protein in blocking IFN production. Nevertheless, it might be possible to avoid secondary bacterial pneumonia by treating influenza patients with drugs that inhibit type I IFN.

Shahangian A, Chow EK, Tian X, Kang JR, Ghaffari A, Liu SY, Belperio JA, Cheng G, & Deng JC (2009). Type I IFNs mediate development of postinfluenza bacterial pneumonia in mice J Clin Inves.

Pathogenesis of influenza in humans

When influenza virus is introduced into the respiratory tract, by aerosol or by contact with saliva or other respiratory secretions from an infected individual, it attaches to and replicates in epithelial cells. The virus replicates in cells of both the upper and lower respiratory tract. Viral replication combined with the immune response to infection (which we’ll discuss in later posts) lead to destruction and loss of cells lining the respiratory tract. As infection subsides, the epithelium is regenerated, a process that can take up to a month. Cough and weakness may persist for up to 2 weeks after infection.

A recent paper compiled data from a number of studies in which human volunteers were given influenza virus, and the production of virus and flu-like symptoms were recorded. The results are summarized in this graph:


Volunteers were infected with influenza virus by intranasal instillation, and virus titers were determined in daily nasal washes. Symptoms monitored included nasal stuffiness, runny nose, sore throat, sneezing, hoarseness, ear pressure, earache, cough, breathing difficulty, chest discomfort, and fever. The results indicate that viral shedding precedes illness by one day, but the curves are otherwise very similar. Most infections are mild and complete in 5 days but some continued for a week. Interestingly, 1 in 3 volunteers did not develop clinical illness but nevertheless shed virus.

These experimental findings most likely do not completely duplicate what occurs in natural influenza infections. First, the study did not involve children or older individuals, in whom the disease course is likely to be different. Furthermore, the pattern of infection will vary depending on the strain of influenza and the immunological status of the host.

Influenza complications of the upper and lower respiratory tract are common. These include otitis media, sinusitis, bronchitis, and croup. Pneumonia is among the more severe complications of influenza infection, an event most frequently observed in children or adults. In primary viral pneumonia, the virus replicates in alveolar epithelial cells, leading to rupture of walls of alveoli and bronchioles. Influenza H5N1 viruses frequently cause primary viral pneumonia characterized by diffuse alveolar damage and interstitial fibrosis. Primary viral pneumonia occurs mostly in individuals at high risk for influenza complications (e.g. elderly patients) but a quarter of the cases occur in those not at risk, including pregnant women.

Combined viral-bacterial pneumonia is common. In secondary bacterial pneumonia, the patient appears to be recovering from uncomplicated influenza but then develops shaking chills, pleuritic chest pain, and coughs up bloody or purulent sputum. Often influenza virus can no longer be isolated from such cases. The most common bacteria causing influenza associated pneumonia are Streptococcus pneumoniae, Staphylococcus aureus, and Hemophilus influenzae. These cases can be treated with antibiotics but the case fatality rate is still about 7%. Secondary bacterial pneumonia was a major cause of death during the 1918-19 influenza pandemic, during which antibiotics were not available.

The reasons why influenza virus infections may lead to pneumonia are not understood. Several hypotheses have been proposed and disproved over the years, including one in which reduced numbers of lymphocytes allow increased susceptibility to superinfection.

Carrat, F., Vergu, E., Ferguson, N., Lemaitre, M., Cauchemez, S., Leach, S., & Valleron, A. (2008). Time Lines of Infection and Disease in Human Influenza: A Review of Volunteer Challenge Studies American Journal of Epidemiology, 167 (7), 775-785 DOI: 10.1093/aje/kwm375

Stegemann, S., Dahlberg, S., Kröger, A., Gereke, M., Bruder, D., Henriques-Normark, B., & Gunzer, M. (2009). Increased Susceptibility for Superinfection with Streptococcus pneumoniae during Influenza Virus Infection Is Not Caused by TLR7-Mediated Lymphopenia PLoS ONE, 4 (3) DOI: 10.1371/journal.pone.0004840