MicrobiologyBytes

It was my priveledge to work with Brian Mahy many years ago. Brian has just retired as long-serving Editor of Virus Research, and his swansong is an excellent special issue on negative strand RNA viruses – an important read for all virologists and an even more impirtant one for all aspiring virologists.

Virus Research: Negative Strand RNA Viruses Special Issue

• Insights on influenza pathogenesis from the grave
• Taming influenza viruses
• Induction and evasion of type I interferon responses by influenza viruses
• Immune responses to influenza virus infection
• Novel vaccines against influenza viruses
• Prospects for controlling future pandemics of influenza
• New concepts in measles virus replication: Getting in and out in vivo and modulating the host cell environment
• Recombinant vaccines against the mononegaviruses—What we have learned from animal disease controls
• Biological feasibility of measles eradication
• Progress in understanding and controlling respiratory syncytial virus: Still crazy after all these years
• An unconventional pathway of mRNA cap formation by vesiculoviruses
• Rhabdovirus accessory genes
• Structural insights into the rhabdovirus transcription/replication complex
• Hantavirus pulmonary syndrome
• Progress in recombinant DNA-derived vaccines for Lassa virus and filoviruses
• Borna disease virus – Fact and fantasy
• A review of Nipah and Hendra viruses with an historical aside
• Negative-strand RNA viruses: The plant-infecting counterparts
• Quasispecies as a matter of fact: Viruses and beyond

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Although it has not been in the news much recently, the highly pathogenic avian influenza A virus subtype H5N1 has been endemic in some bird species since its emergence in 1996 and its ecology, genetics and antigenic properties continue to evolve. This has allowed diverse virus strains to emerge in endemic areas with altered receptor specificity, including a new H5 sublineage with enhanced binding affinity to the human-type receptor. The pandemic potential of H5N1 viruses is alarming and may be increasing. This article reviews the complex and changing nature of the H5N1 virus that may contribute to the emergence of pandemic strains – with really serious consequences.

The changing nature of avian influenza A virus (H5N1). Tresnd in Microbiology, 5 December 2011

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To deliver their genomes into host cells during entry, enveloped viruses contain glycoproteins that bind to cellular receptors and cause fusion of viral and cellular membranes. The influenza virus Hemagglutinin (HA) protein is the archetypal viral fusion glycoprotein, promoting entry by undergoing irreversible structural changes that drive membrane merger. HA trimers on the surfaces of infectious influenza virions are trapped in a metastable, high-energy conformation and are triggered to refold and cause membrane fusion after the virus is internalized and exposed to low pH.

This paper provides biochemical and x-ray crystallographic evidence that naturally occurring amino-acid variations at the interface of the esterase and fusogenic domains alter HA acid stability for highly pathogenic H5N1 influenza, resulting in a shift in the threshold pH required to activate HA protein structural changes that cause membrane fusion. Furthermore, the data reveals that an increased HA activation pH correlates with increased H5N1 virulence in chickens. Overall, the acid stability of the HA protein is identified as a novel virulence factor for emerging H5N1 influenza viruses. A major implication of this work is that the fitness of enveloped viruses may be fine-tuned by mutations that alter the activation energy thresholds of their fusion glycoproteins.

Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity. (2011) PLoS Pathog 7(12): e1002398. doi:10.1371/journal.ppat.1002398
Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 Å resolution and two structures of HP HA at 2.95 and 3.10 Å resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.

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New research is beginning to crack the problem of which strain of flu will be prevalent in a given year, with major implications for global public health preparedness. A computational study of 40 years of flu genomes offers a new way of looking at mutations: by cataloging pairs of genetic changes that have occurred in rapid succession, observing that a mutation in one half of the pair can act as an early warning sign of a mutation about to occur in the other.

Tracking single mutations in a vacuum is not always enough to understand how the flu virus evolves. Sometimes a mutation is functional or adaptive only if it’s in the context of a certain genetic background – that is, if the protein already has some other mutation. The influence such combinations have on an organism’s adaptive fitness is known as epistasis. If you see a mutation occur in Site A and then very soon after you see a mutation in Site B, and this pattern happens repeatedly, then you have some evidence that A and B influence fitness epistatically. The first mutation might be useless on its own, but it might be a prerequisite for the second mutation to be useful. The first mutation is like giving you a nail, and the second one is like giving you a hammer.

Because the studied mutations generally affect the surface proteins that determine whether the virus can enter and infect human cells, being able to predict what mutations are likely to happen in the near future has lifesaving applications. Tens of thousands of Americans, and hundreds of thousands worldwide, die of seasonal flu complications every year. Flu vaccine production is labor intensive and time consuming; to have enough supplies ready for the flu season, public health groups like the Centers for Disease Control and the World Health Organization must make an educated guess as to which strain is likely to be the most active several months in advance. Observing the leading site of an epistatic pair could give them a head start.

Prevalence of Epistasis in the Evolution of Influenza A Surface Proteins. (2011) PLoS Genet 7(2): e1001301. doi:10.1371/journal.pgen.1001301
The surface proteins of human influenza A viruses experience positive selection to escape both human immunity and, more recently, antiviral drug treatments. In bacteria and viruses, immune-escape and drug-resistant phenotypes often appear through a combination of several mutations that have epistatic effects on pathogen fitness. However, the extent and structure of epistasis in influenza viral proteins have not been systematically investigated. Here, we develop a novel statistical method to detect positive epistasis between pairs of sites in a protein, based on the observed temporal patterns of sequence evolution. The method rests on the simple idea that a substitution at one site should rapidly follow a substitution at another site if the sites are positively epistatic. We apply this method to the surface proteins hemagglutinin and neuraminidase of influenza A virus subtypes H3N2 and H1N1. Compared to a non-epistatic null distribution, we detect substantial amounts of epistasis and determine the identities of putatively epistatic pairs of sites. In particular, using sequence data alone, our method identifies epistatic interactions between specific sites in neuraminidase that have recently been demonstrated, in vitro, to confer resistance to the drug oseltamivir; these epistatic interactions are responsible for widespread drug resistance among H1N1 viruses circulating today. This experimental validation demonstrates the predictive power of our method to identify epistatic sites of importance for viral adaptation and public health. We conclude that epistasis plays a large role in shaping the molecular evolution of influenza viruses. In particular, sites with dN=dSv1, which would normally not be identified as positively selected, can facilitate viral adaptation through epistatic interactions with their partner sites. The knowledge of specific interactions among sites in influenza proteins may help us to predict the course of antigenic evolution and, consequently, to select more appropriate vaccines and drugs.

Related:

• Spotting pandemic influenza viruses
• How can vaccines against influenza and other virus diseases be made more effective?
• Protective immunity against influenza

The current UK National Framework for Pandemic Influenza states that during a pandemic, domestic travel should continue to operate normally but users should adopt good hygiene measures, stagger journeys where possible to reduce overcrowding; and stay at home altogether if symptomatic with pandemic influenza. This advice reflects the need to maintain, as far as possible, business continuity and near normal functioning of society, but acknowledges that some data exist about the transmission of influenza on board public transport, notably commercial airliners. Until very recently, there were no data that directly supported or refuted an association between the use of public ground transportation and the risk of acute respiratory infection. The risk posed by large numbers of transient casual human contacts has not been adequately defined. The current uncertainty makes the formulation of pandemic transport policies difficult. So what’s the risk?

Is public transport a risk factor for acute respiratory infection? BMC Infectious Diseases 2011, 11:16doi:10.1186/1471-2334-11-16
Background: The relationship between public transport use and acquisition of acute respiratory infection (ARI) is not well understood but potentially important during epidemics and pandemics.
Methods: A case-control study performed during the 2008/09 influenza season. Cases (n=72) consulted a General Practitioner with ARI, and controls with another non-respiratory acute condition (n=66). Data were obtained on bus or tram usage in the five days preceding illness onset (cases) or the five days before consultation (controls) alongside demographic details. Multiple logistic regression modelling was used to investigate the association between bus or tram use and ARI, adjusting for potential confounders.
Results: Recent bus or tram use within five days of symptom onset was associated with an almost six-fold increased risk of consulting for ARI (adjusted OR=5.94 95% CI 1.33-26.5). The risk of ARI appeared to be modified according to the degree of habitual bus and tram use, but this was not statistically significant (1-3 times/week: adjusted OR=0.54 (95% CI 0.15-1.95; >3 times/week: 0.37 (95% CI 0.13-1.06).
Conclusions: We found a statistically significant association between ARI and bus or tram use in the five days before symptom onset. The risk appeared greatest among occasional bus or tram users, but this trend was not statistically significant. However, these data are plausible in relation to the greater likelihood of developing protective antibodies to common respiratory viruses if repeatedly exposed. The findings have differing implications for the control of seasonal acute respiratory infections and for pandemic influenza.

Related:

• Dirty Money
• Influenza virus transmission is dependent on humidity and temperature
• Global migration of influenza viruses

A large fraction of the world’s most widespread and problematic pathogens, such as the influenza virus, seem to persist in nature by evading host immune responses by inducing immunity to genetically and phenotypically plastic epitopes (aka antigenic variation). The more recent re-emergence of pandemic influenza A/H1N1 and avian H5N1 viruses has called attention to the urgent need for more effective influenza vaccines. Developing such vaccines will require more than just moving from an egg-based to a tissue-culture–based manufacturing process. It will also require a new conceptual understanding of pathogen–host interactions, as well as new approaches and technologies to circumvent immune evasion by pathogens capable of more genetic variation. This paper discusses these challenges, focusing on some potentially fruitful directions for future research.

Vaccines often take between 16 and 20 years to develop, and the challenge now is to understand deceptive imprinting better and to systematically identify and characterize deceptive epitopes and low-efficiency, interfering epitopes in influenza and other viruses. Progress would enable targeting of both immunodominant deceptive epitopes and low-efficiency epitopes for genetic modification. In addition, more studies are needed to determine whether such genetic modifications can actually lead to significantly greater vaccine efficacy, but there is great promise in these understanding-driven approaches.

How Can Vaccines Against Influenza and Other Viral Diseases Be Made More Effective? 2010 PLoS Biol 8(12): e1000571. doi:10.1371/journal.pbio.1000571

Related:

• The 2009 H1N1 pandemic – what went right and what went wrong?
• RNA replicons – a new approach to influenza virus vaccines
• Protective immunity against influenza

Given the critical role of interferons (IFNs) as a first line of defense against infection, it is not surprising that many viruses have evolved strategies to block an IFN response as a means to increase their replication efficiency. Virus-mediated inhibition of IFNs can be generalized into three categories, including disruption of IFN induction, disruption of IFN-inducible signaling and disruption of IFN-mediated effector functions.

The non-structural protein 1 (NS1) of influenza A viruses exerts its inhibitory effects on IFN predominately by interfering with IFN productionType I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β.

Expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. However, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections.

Influenza Virus Non-Structural Protein 1 (NS1) Disrupts Interferon Signaling. (2010) PLoS ONE 5(11): e13927. doi:10.1371/journal.pone.0013927

Related:

• Virus tricks to grid-lock the type I interferon system
• Interferons and Viruses
• The Interferon Antiviral Response

In this week’s PLoS Medicine, Gabriel Leung from the Government of the Hong Kong SAR and Angus Nicoll from the European Centre for Disease Prevention and Control offer their reflections on the international response to the 2009 H1N1 influenza pandemic, including what went well and what changes need to be made on the part of global and national authorities in anticipation of future flu pandemics.

Summary points:

• Many of the initial responses to the 2009 H1N1 pandemic went well but there are many lessons to learn for future pandemic planning.
• Clear communication of public health messages is crucial, and should not confuse what could happen (and should be prepared for) with what is most likely to happen.
• Decisions regarding pandemic response during the exigencies of a public health emergency must be judged according to the best evidence available at the time.
• Revising pandemic plans – to be more flexible and more detailed – should wait for WHO leadership if national plans are not to diverge. Surveillance beyond influenza should be stepped up, and contingencies drawn up for the emergence or re-emergence of other novel and known pathogens.
• Data collection and sharing are paramount, and include epidemiological and immunological data. Clinical management of severe influenza disease should not be limited to the current antiviral regimen, and include the development of other therapeutics (e.g., novel antivirals and immunotherapy).
• Greater and more timely access to antivirals and influenza vaccines worldwide remains an ongoing challenge.

Reflections on Pandemic (H1N1) 2009 and the International Response. (2010) PLoS Med 7(10): e1000346. doi:10.1371/journal.pmed.1000346

Related:

• The Biology of Influenza
• 10 things you should know about H1N1 (swineflu)

Infection with influenza virus affects a substantial proportion of the worldwide population each year. In 2009, the perceived global threat of influenza reached an exceptional level after the emergence of a novel swine-origin influenza A H1N1 (so-called swine flu), which was first isolated in local outbreaks in Mexico, Canada, and the USA. The subsequent rapid global spread of this strain and concerns about its possible virulence led national and global health authorities to initiate countermeasures in early 2009, by means of mass immunisation programmes in several countries, including the USA and countries of the European Union. These vaccination campaigns were the cornerstone of public health measures to prevent the undesired consequences of a pandemic. They also served as a reminder (at least to the neurological community) of the 1976 national influenza immunisation programme against swine flu subtype A/NJ/76 in the USA, which was stopped because of the emergence of Guillain-Barré syndrome (GBS) in vaccine recipients.

GBS after vaccination is rare, and most studies have concluded that it is a chance event except in the 1976 programme. However, the small size of vaccine safety trials before licensing, the testing and licensing of vaccines for potential pandemic diseases before the start of the pandemic (so-called mock-up licensing), and the very low incidence of GBS mean that data could be insufficient to assess the risk reliably. Conventional vaccine safety monitoring after licensing does not entirely eradicate this concern.

By contrast with vaccination, evidence is increasing that influenza infection and influenza-like illnesses can act as triggers for GBS. This important fact, which has been highlighted in epidemiological studies, and seems to be underappreciated in public and professional advisory interpretations of influenza vaccine adverse event data and subsequent risk–benefit assessments. The establishment of background rates for GBS will be very useful in this regard; this information has already been provided for several countries.

In addition to the prevention of multiple non-neurological diseases by influenza vaccination, awareness and correct interpretation of all available data about the relation of GBS, influenza infection, and influenza vaccination are a prerequisite for an objective risk–benefit analysis of current and future influenza vaccination campaigns. This paper reviews the existing data derived from studies about GBS after influenza infection and GBS after exposure to influenza vaccine and summarises current information about the plausibility of influenza immunisation as a biological cause of GBS.

Guillain-Barré syndrome after exposure to influenza virus. Lancet Infectious Diseases (2010) 10(9) 643- 651 doi:10.1016/S1473-3099(10)70140-7
Guillain-Barré syndrome (GBS) is an acute, acquired, monophasic autoimmune disorder of peripheral nerves that develops in susceptible individuals after infection and, in rare cases, after immunisation. Exposure to influenza via infection or vaccination has been associated with GBS. We review the relation between GBS and these routes of exposure. Epidemiological studies have shown that, except for the 1976 US national immunisation programme against swine-origin influenza A H1N1 subtype A/NJ/76, influenza vaccine has probably not caused GBS or, if it has, rates have been extremely low (less than one case per million vaccine recipients). By contrast, influenza-like illnesses seem to be relevant triggering events for GBS. The concerns about the risk of inducing GBS in mass immunisation programmes against H1N1 2009 do not, therefore, seem justified by the available epidemiological data. However, the experiences from the 1976 swine flu vaccination programme emphasise the importance for active and passive surveillance to monitor vaccine safety.

Related:

• Fact Sheet on Guillain-Barré syndrome
• MicrobiologyBytes: Influenza
• Campylobacter jejuni