TWiV 481: And biles to go before I delete

The TWiVodrome considers the intestinal tract as an alternative infection route for MERS coronavirus, and how reduced accumulation of defective viral RNAs might lead to severe influenza.

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Show notes at microbe.tv/twiv

Defective viral genomes and severe influenza

Defective influenza virus RNAsThe virulence of a virus – its capacity to cause disease – is determined by both viral and host factors. Even among healthy individuals, infection with a particular virus may have different outcomes ranging from benign to lethal. The study of influenza viruses that cause mild or fatal outcomes reveals that defective viral genomes play a role in determining viral virulence.

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TWiV 480: The PFU in your achoo

Scott Hensley joins the TWiVites to review the current influenza season and presence of the virus in exhaled breath of symptomatic cases.

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Scott Hensley of the University of Pennsylvania helps us summarize the current influenza season so far. Bottom line: it’s a very serious season with a great deal of morbidity and mortality. As usually, serious disease and deaths occur in unvaccinated individuals. Although there is a mismatch between the influenza vaccine and this year’s H3N2 strain, which is causing most of the infections, vaccination still reduces disease severity cause by this strain, as well as by the circulating H1N1 and influenza B viruses. Next we discuss an interesting study of symptomatic college students with influenza. The goal was to determine which mode of influenza transmission is likely to be important: contact, or aerosols produced by speaking, coughing, or sneezing. The results show that simple breathing leads to virus in the breath – small aerosols that can travel distances to infect others. Coughing, but not sneezing, was a major observed sign of infection, but neither was needed for infectious aerosol generation.

A breath of fresh influenza virus

Gesundheit-II bio aerosol samplerInfluenza virus may be transmitted among humans in three ways: by direct contact with infected individuals; by contact with contaminated objects (called fomites, such as toys, doorknobs); and by inhalation of virus-laden aerosols. A recent study suggests that normal tidal breathing plays a substantial role in aerosol transmission.

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TWiV 441: Don’t ChrY for me influenza

The Beacons of Viral Education (aka the TWiVoners) reveal a cost of being a male mouse – the Y chromosome regulates their susceptibility to influenza virus infection.

You can find TWiV #441 at microbe.tv/twiv, or listen below.

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Flu and the Y chromosome

X and Y chromosomesDisease and death caused by influenza virus are greater in human females than in males. But disease is more common in males from birth through age 15, after which more females are affected. In mice, genetic variation in the Y chromosome controls susceptibility to influenza virus infection (link to paper). Increased susceptibility does not correlate with increased viral replication, but an expanded pathogenic immune response in the lungs.

A panel of mice (strain B6) with the Y chromosome from eleven different strains were used to determine the effect of infection. The mice fell into two groups with distinct high and low survival after intranasal infection with the mouse-adapted PR8 strain of influenza virus. These results show that variation in the Y chromosome influences survival after infection. Furthermore, the previously reported greater susceptibility of female B6 mice to influenza virus infection compared with male mice is due to the presence of the Y chromosome.

Viral replication in the lung does not differ between mouse strains with high and low mortality. Increased mortality is associated with an increase in a type of T lymphocyte called gamma-delta T cells which produce interleukin 17. The latter is known to provoke lung-damaging inflammation.

Differences in susceptibility of mice with different Y chromosomes has nothing to do with the immunosuppressive effects of testosterone. Exactly which genes on the Y chromosome affect influenza virus susceptibility are unknown. Analysis of RNA expression revealed differences in levels of small RNAs in mice of higher versus lower susceptibility. This observation raises the possibility that the Y chromosome might have global effects on gene expression from other chromosomes, which in turn influences susceptibility to infection.

Others have found that the Y chromosome regulates the speed of progression to AIDS in HIV-1 infected men. It seems likely that the Y chromosome has an important general role in modulating the pathogenesis of infectious diseases. A better understanding of how the Y chromosome regulates the expression of other genes will be needed to understand these effects.

Paradoxical vaccines

gene stops hereA new breed of vaccines is on the horizon: they replicate in one type of cell, allowing for their production, but will not replicate in humans. Two different examples have recently been described for influenza and chikungunya viruses.

The influenza virus vaccine is produced by introducing multiple amber (UAG) translation stop codons in multiple viral genes. Cloned DNA copies of the mutated viral RNAs are not infectious in normal cells. However, when introduced into specially engineered ‘suppressor’ cells that can insert an amino acid at each amber stop codon, infectious viruses can be produced. These viruses will only replicate in the suppressor cells, not in normal cells, because the stop codons lead to the production of short proteins which do not function properly.

When inoculated into mice, the stop-codon containing influenza viruses infect cells, and although they do not replicate, a strong and protective immune response is induced. Because the viral genomes contain multiple mutations, the viruses are far less likely than traditional infectious, attenuated vaccines to sustain mutations that allow them to replicate in normal cells. It’s a clever approach to designing an infectious, but replication-incompetent vaccine (for more discussion, listen to TWiV #420).

Another approach is exemplified by an experimental vaccine against chikungunya virus. The authors utilize Eilat virus, a virus that only replicates in insects. The genes encoding the structural proteins of Eilat virus were replaced with those of chikungunya virus. The recombinant virus replicates in insect cells, but not in mammalian cells. The virus enters the latter cells, and some viral proteins are produced, but genome replication does not take place.

When the Eilat-Chikungunya recombinant virus in inoculated into mice, there is no genome replication, but a strong and protective immune response is induced. The block to replication – viral RNA synthesis does not occur – is not overcome by multiple passages in mice. Like the stop-codon containing influenza viruses, the Eilat recombinant virus is a replication-incompetent vaccine.

These are two different approaches to making viruses that replicate in specific cells in culture – the suppressor cells for influenza virus, and insect cells for Eilat virus. When inoculated into non-suppressor cells (influenza virus) or non-insect cells (Eilat virus), a strong immune response is initiated. Neither virus should replicate in humans, but clinical trials have to be done to determine if they are immunogenic and protective.

The advantage of these vaccine candidates compared with inactivated vaccines is that they enter cells and produce some viral proteins, likely resulting in a stronger immune response. Compared with infectious, attenuated vaccines, they are far less likely to revert to virulence, and are easier to isolate.

These two potential vaccine technologies have been demonstrated with influenza and chikungunya viruses, but they can be used for other virus. The stop-codon approach is more universally applicable, because the mutations can be introduced into the genome of any virus. The Eilat virus approach can only be used with viruses whose structural proteins are compatible with the vector – probably only togaviruses and flaviviruses. A similar approach might be used with insect-specific viruses in other virus families.

Why do I call these vaccines ‘paradoxical’? Because they are infectious and non-infectious, depending on the host cell that is used.

Note: The illustration is from a t-shirt, and the single letter code of the protein spells out a message. However the title, ‘the gene stops here’, is wrong. It should be ‘the protein stops here. The 3’-untranslated region, which continues beyond the stop codon, is considered part of the gene.

TWiV 420: Orthogonal vectors

The TWiV gurus describe how to use an orthogonal translation system to produce infectious but replication-incompetent influenza vaccines.

You can find TWiV #420 at microbe.tv/twiv, or listen below.

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TWiV 413: Partnerships not parachutes

From the EIDA2Z conference at Boston University, Vincent, Alan and Paul meet up with Ralph Baric, Felix Drexler, Marion Koopmans, and Stacey Schultz-Cherry to talk about discovering, understanding, protecting, and collaborating on emerging infectious diseases.

You can find TWiV #413 at microbe.tv/twiv, or watch or listen here.

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TWiV 403: It’s not easy being vaccine

The TWiV team takes on an experimental plant-based poliovirus vaccine, contradictory findings on the efficacy of Flumist, waning protection conferred by Zostavax, and a new adjuvanted subunit zoster vaccine.

You can find TWiV #403 at microbe.tv/twiv, or listen below.

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