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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TWiV 396: Influenza viruses with Peter Palese

TWiVVincent speaks with Peter Palese about his illustrious career in virology, from early work on neuraminidases to universal influenza virus vaccines, on episode #396 of the science show This Week in Virology.

You can find TWiV #396 at microbe.tv/twiv, or listen below.

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Moving beyond metagenomics to find the next pandemic virus

I was asked to write a commentary for the Proceedings of the National Academy of Sciences to accompany an article entitled SARS-like WIV1-CoV poised for human emergence. I’d like to explain why I wrote it and why I spent the last five paragraphs railing against regulating gain-of-function experiments.

Towards the end of 2014 the US government announced a pause of gain-of-function research involving research on influenza virus, SARS virus, and MERS virus that “may be reasonably anticipated to confer attributes to influenza, MERS, or SARS viruses such that the virus would have enhanced pathogenicity and/or transmissibility in mammals via the respiratory route.”

From the start I have opposed the gain-of-function pause. It’s a bad idea fostered by individuals who continue to believe, among other things, that influenza H5N1 virus adapted to transmit by aerosol among ferrets can also infect humans by the same route. Instead of stopping important research, a debate on the merits and risks of gain-of-function experiments should have been conducted while experiments were allowed to proceed.

Towards the end of last year a paper was published a paper on the potential of SARS-virus-like bat coronaviruses to cause human disease. The paper reawakened the debate on the risks and benefits of engineering viruses. Opponents of gain-of-function research began to make incorrect statements about this work. Richard Ebright said that ‘The only impact of this work is the creation, in a lab, of a new, non-natural risk”. Simon Wain-Hobson wrote that a novel virus was created that “grows remarkably well” in human cells; “if the virus escaped, nobody could predict the trajectory”. I have written extensively about why these are other similar statements ignore the value of the work. In my opinion these critics either did not read the paper, or if they did, did not understand it.

Several months later I was asked to write the commentary on a second paper examining the potential of SARS like viruses in bats to cause human disease. I agreed to write it because the science is excellent, the conclusions are important, and it would provide me with another venue for criticizing the gain-of-function pause.

In the PNAS paper, Menachery et al. describe a platform comprising metagenomics data, synthetic virology, transgenic mouse models, and monoclonal antibody therapy to assess the ability of SARS-CoV–like viruses to infect human cells and cause disease in mouse models. The results indicate that a bat SARS-like virus, WIV1-CoV, can infect human cells but is attenuated in mice. Additional changes in the WIV1-CoV genome are likely required to increase the pathogenesis of the virus for mice. The same experimental approaches could be used to examine the potential to infect humans of other animal viruses identified by metagenomics surveys. Unfortunately my commentary is behind a paywall, so for those who cannot read it, I’d like to quote from my final paragraphs on the gain-of-function issue:

The current government pause on these gain-of-function experiments was brought about in part by several vocal critics who feel that the risks of this work outweigh potential benefits. On multiple occasions these individuals have indicated that some of the SARS-CoV work discussed in the Menachery et al. article is of no merit. … These findings provide clear experimental paths for developing monoclonal antibodies and vaccines that could be used should another CoV begin to infect humans. The critics of gain-of-function experiments frequently cite apocalyptic scenarios involving the release of altered viruses and subsequent catastrophic effects on humans. Such statements represent personal opinions that are simply meant to scare the public and push us toward unneeded regulation. Virologists have been manipulating viruses for years—this author was the first to produce, 35 y ago, an infectious DNA clone of an animal virus—and no altered virus has gone on to cause an epidemic in humans. Although there have been recent lapses in high-containment biological facilities, none have resulted in harm, and work has gone on for years in many other facilities without incident. I understand that none of these arguments tell us what will happen in the future, but these are the data that we have to calculate risk, and it appears to be very low. As shown by Menacherry et al. in PNAS, the benefits are considerable.

A major goal of life science research is to improve human health, and prohibiting experiments because they may have some risk is contrary to this goal. Being overly cautious is not without its own risks, as we may not develop the advances needed to not only identify future pandemic viruses and develop methods to prevent and control disease, but to develop a basic understand- ing of pathogenesis that guides prevention. These are just some of the beneficial outcomes that we can predict. There are many examples of how science has progressed in areas that were never anticipated, the so-called serendipity of science. Examples abound, including the discovery of restriction enzymes that helped fuel the biotechnology revolution, and the development of the powerful CRISPR/Cas9 gene-editing technology from its obscure origins as a bacterial defense system.

Banning certain types of potentially risky experiments is short sighted and impedes the potential of science to improve human health. Rather than banning experiments, such as those described by Menachery et al., measures should be put in place to allow their safe conduct. In this way science’s full benefits for society can be realized, unfettered by artificial boundaries.

TWiV 363: Eat flu and dyad

On episode #363 of the science show This Week in Virology, The TWiVers reveal influenza virus replication in the ferret mammary gland and spread to a nursing infant, and selection of transmissible influenza viruses in the soft palate.

You can find TWiV #363 at www.microbe.tv/twiv.

Influenza virus in breast milk

Ferret mother-infantDuring breastfeeding, mothers provide the infant with nutrients, beneficial bacteria, and immune protection. Fluids from the infant may also enter the mammary gland through retrograde flux of the nipple. Studies in a ferret model reveal that influenza virus replicates in the mammary gland, is shed in breast milk and transmitted to the infant. Virus may also travel in the opposite direction, from infant to mother.

The role of the mammary gland in influenza virus transmission was studied using a ferret model comprising lactating mothers and nursing infants. Intranasal inoculation of nursing mother ferrets with the 2009 H1N1 influenza virus lead to viral replication and development of influenza in both mother and infant. When the study design was reversed, and 4 week old nursing ferrets were inoculated intranasally with the same virus, viral replication and disease ensued first in the infants, and then in the mothers. Infectious virus was recovered both in the mammary glands and in the nipples at day 4 post infant inoculation, and in mother’s milk from 3-5 days post infant inoculation. Histopathological examination of sections of mammary glands from infected mothers revealed destruction of the mammary architecture.

These results show that nursing infants may pass influenza virus to mothers. It seems clear that influenza virus replicates in the mammary gland and that infectious virus is present in milk. How does this virus infect the mother? One possibility is that infection is transmitted by respiratory contact with virus-containing milk, or by inhalation of aerosols produced by nursing. How influenza virus in the mammary gland would reach the mother’s lung via the blood to cause respiratory disease is more difficult to envision and seems unlikely.

When influenza virus was inoculated into the mammary gland of lactating mothers via the lactiferous ducts, both mother and breast feeding infant developed serious influenza. Infectious virus was detected first in the nasal wash of infants, then later in the nasal wash of mothers. Breast milk contained infectious virus starting on day 2 after inoculation. Histopathological examination of sections from infected mammary glands revealed destruction of glandular architecture and cessation of milk production. This observation is consistent with the results of gene expression analysis of RNA from virus infected mammary glands, which revealed reduction in transcripts of genes associated with milk production.

To determine if human breast cells can be infected with influenza virus, three different human epithelial breast cell lines were infected with the 2009 H1N1 virus strain. Virus-induced cell killing was observed and infectious virus was produced.

Even if we assume that influenza virus can replicate in the human breast, the implications for influenza transmission and disease severity are not clear. Transmission of HIV-1 from mother to infant by breast milk has been well documented. In contrast to influenza virus, HIV-1 is present in the blood from where it spreads to the breast. Most human influenza virus strains do not enter the blood so it seems unlikely that virus would spread to the breast of a mother infected via the respiratory route. However, viral RNA has been detected in the blood of humans infected with the 2009 H1N1 strain, the virus used in these ferret studies. Therefore we cannot rule out the possibility that some strains of influenza virus spread from lung via the blood to the breast, allowing infection of a nursing infant. Some answers might be provided by determining if influenza virus can be detected in the breast milk of humans with influenza.

What would be the implication of a nursing infant infecting the mother’s breast with influenza virus? As I mentioned above, it seems unlikely that this virus would enter the blood, and even if it could, how would the virus infect the apical side of the respiratory epithelium? What does seem clear is that viral replication in the breast could lead to a decrease in milk production which could be detrimental to the infant. If the mother had multiple births, then influenza virus might be transmitted to siblings nursing on the infected mother.

Are you wondering how an infant drinking influenza virus-laded breast milk acquires a respiratory infection? Recently it has been shown that influenza virus replicates in the soft palate of ferrets. The soft palate has mucosal surfaces that face both the oral cavity and the nasopharynx. Ingested virus could first replicate in the soft palate, then spread to the nasopharynx and the lung. A simpler explanation is that nursing produces virus-containing aerosols which are inhaled by the infant.