TWiV #356: Got viruses?

On episode #356 of the science show This Week in Virology, Stephanie joins the super professors to discuss the gut virome of children with serious malnutrition, caterpillar genes acquired from parasitic wasps, and the effect of adding chemokines to a simian immunodeficiency virus DNA vaccine.

You can find TWiV #356 at

Wasps do a gain-of-function experiment in caterpillars

parasitic waspParasitic wasps (in the order Hymenoptera) inject their eggs into lepidopteran hosts, where the eggs go through their developmental stages. Along with the eggs, the wasps also deliver viruses carrying genes encoding proteins that inhibit caterpillar immune defenses. Some of these genes are permanently transferred to the lepidopteran host where they have assumed new defensive functions against other viruses.

The viruses that parasitic wasps inject with their eggs, called Bracoviruses, are encoded in the wasp genome. About 100 million years ago a nudivirus genome integrated into the genome of a common wasp ancestor. With time the viral genes became dispersed in the wasp genome. The viruses produced by these wasps today no longer carry capsid coding genes – they are found only in the wasp genome – but only carry genes whose products can modulate lepidopteran defenses. Once in the lepidopteran host, these viruses deliver their genes but no longer form new particles.

An important question is whether wasp Bracoviruses can contribute genes to Lepidoptera – a process called horizontal gene transfer. This possibility would seem remote because the lepidopteran hosts for wasp larvae are dead ends – they die after serving as hosts for wasp development. However, it is possible that some hosts resist killing, or that wasps occasionally inject their eggs and viruses into the wrong host, one that can resist killing.

To answer this question, the genome sequence of Cotesia congregata bracovirus was compared with the genomes of a regular host as well as non-host Lepidoptera. Bracovirus DNA insertions were identified in genomes of the monarch, the silkworm, the beet armyworm and the fall armyworm, but not in the genome of the tobacco hornworm, the usual host of the wasp (C. congregata).

Not only were the Bracovirus sequences found in these varied Lepidoptera, but some appeared to be functional. Two such genes encode a protein that interferes with the replication of baculovirus, a known pathogen of Lepidoptera. This discovery was made in the process of producing the encoded proteins using baculovirus vectors! In other words, viral genes delivered by Hymenopteran wasps were appropriated by the Lepidoptera and used for their defense against a pathogen.

To put it another way, nature has carried out a gain-of-function experiment. Should we impose a moratorium?

The delivery of immunosuppressive viruses by wasps along with their eggs is by all accounts a remarkable story. The appropriation of some of these genes by the wrong hosts should not come as a surprise, yet the finding is nevertheless simply amazing. As long as we keep looking, we will find that the biological world is always full of new revelations.

TWiV 325: Wildcats go viral

On episode #325 of the science show This Week in Virology, Vincent visits the ‘Little Apple’ and speaks with Rollie and Lorena about their work on mosquito-born viruses and baculoviruses.

You can find TWiV #325 at

Influenza vaccines for individuals with egg allergy

baculovirusA CDC (US) advisory committee has recommended the use of FluBlok for individuals with egg allergy:

The Advisory Committee on Immunization Practices (ACIP) voted today, 13 to 0, in favor of recommending FluBlok during the 2013-2014 influenza season for vaccination of persons 18 through 49 years of age with egg allergy of any severity.

FluBlok is an influenza virus vaccine that is produced by expressing the influenza virus hemagglutinin (HA) protein in insect cells using a baculovirus vector. Baculoviruses are rod-shaped viruses (see photograph) that infect insects and other arthropods. The baculovirus virion contains a double-stranded DNA genome. To express the influenza virus HA proteins, recombinant baculoviruses are produced in which DNA encoding the HA protein is inserted into the baculovirus DNA genome. When insect cells are infected with the recombinant baculoviruses, the influenza HA protein is produced.

To manufacture FluBlok, three recombinant baculoviruses were isolated  that contain the HA gene from A/Panama/2007/99 (H3N2), A/New Caledonia/20/99 (H1N1), and B/Hong Kong/330/2001. After infection of insect cells with these recombinant baculoviruses, the HA proteins were purified to greater than 95% purity. In a randomized, placebo-controlled clinical trial, FluBlok was shown to be 44.6% effective in preventing culture-confirmed influenza. The low efficacy might in part be due to antigenic mismatch between the HA proteins used in the vaccine and circulating viruses.

Because FluBlok is not produced in eggs it may be used in individuals with egg allergies. Another alternative for such individuals is Flucelvax, an influenza vaccine produced in cell culture, which was approved by the Food and Drug Administration in November 2012.

The viruses in your food

cole slawAt the recent Brazilian Virology Society meeting George Rohrmann gave me a copy of his book, Baculovirus Molecular Biology. Baculoviruses are double-stranded DNA containing viruses that infect insects and other arthropods. Early in the first chapter of George’s book I learned that baculoviruses may be present on uncooked cabbage leaves:

…in one study it was found that cabbage purchased from 5 different supermarkets in the Washington D.C. area were all contaminated with baculoviruses to such an extent that each serving (about 100 cm2 of leaf material) would contain up to 108 polyhedra of an NPV pathogenic for the cabbage looper, Trichoplusia ni!

We ingest many other non-animal viruses regularly with foods. In TWiV #79, Red hot chili viruses, we discussed the finding of pepper mild mottle virus, one of the major pathogens of chili peppers, in human feces as well as in pepper or spice-containing food products. Metagenomic analysis of the RNA viruses present in human feces revealed that most viral sequences are similar to plant viruses.  Of 36,769 sequences obtained, 25,040 (91%) resembled plant viruses. In this study, the most abundant human fecal virus was pepper mild mottle virus, present in concentrations of up to 109 virions per gram of dry fecal matter.

The plant (and perhaps insect) viruses that we ingest on a regular basis do not appear to replicate or cause disease in humans. Might they play important roles in development of the immune system, as do the commensal bacteria in our gut?

Zhang, T., Breitbart, M., Lee, W., Run, J., Wei, C., Soh, S., Hibberd, M., Liu, E., Rohwer, F., & Ruan, Y. (2006). RNA Viral Community in Human Feces: Prevalence of Plant Pathogenic Viruses PLoS Biology, 4 (1) DOI: 10.1371/journal.pbio.0040003

TWiV 152: Viromes in the effluence of society

shinolaHosts: Vincent Racaniello, Alan Dove, and Rich Condit

Vincent, Alan, and Rich cover the virome of raw sewage, and a baculovirus gene that causes caterpillars to climb to their doom.

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Weekly Science Picks

Alan – xkcd tribute to Steve Jobs
Vincent –
Steve Jobs’ 2005 Stanford commencement address (YouTube)
Rich –
1984 Apple Macintosh commercial (YouTube)

Listener Pick of the Week

David Your Inner Fish by Neil Shubin

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TWiV 150: Contaminated

pXMRVHosts: Vincent RacanielloRich Condit, and Dickson Despommier

Vincent, Dickson, and Rich meant to do an all-email episode, but first they review results of the Blood XMRV Scientific Research Working Group, and partial retraction of the paper associating XMRV with chronic fatigue syndrome.

With this episode TWiV is three years old.

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Weekly Science Picks

Dickson – The Tree of Life
Vincent –
When do you fact-check article content with sources? (take as directed)
Rich –

Listener Pick of the Week

LuisNIH videocasting and podcasting

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TWiV 88: A bug fix, an AIDS treatment, and an undead retrovirus

Hosts: Vincent Racaniello, Alan Dove, and Marc Pelletier

On episode #88 of the podcast This Week in Virology, Vincent, Alan, and Marc discuss using a virus for beetle control, RNA based gene therapy for AIDS, and reconstitution of a endogenous human retrovirus.

This episode is sponsored by Data Robotics Inc. Use the promotion code TWIVPOD to receive $75-$500 off a Drobo.

To enter a drawing to receive 50% off the manufacturers suggested retail price of a Drobo S or FS at, fill out the questionnaire here.

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Weekly Science Picks

Marc Apple iPad as a tool for writing, with Papers, Pages, and GoodReader
AlanThe Bacterium and the Bacteriophage
Naturally Obsessed (thanks, Sharon!)

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Influenza virus-like particle vaccine

influenza-vlpA new type of vaccine against influenza, made with virus-like particles, has been shown to protect ferrets from infection with the 2009 H1N1 swine-origin strain. What is a virus-like particle, and how is it produced?

If you have been taking influenza 101, you know that new virus particles are produced in infected cells by budding. During this process, the membrane bulges from the cell and is eventually pinched off to form a free particle. These virus particles contain the viral RNA segments, and an assortment of viral proteins including PA, PB1, PB2, NP, M1, M2, HA, and NA. But not all of those viral proteins are needed to produce an influenza virus particle. When only the viral HA, NA, and M1 proteins are synthesized in cells, particles are released from cells that look very much like influenza virions (illustrated). These are called ‘virus-like particles’ because they resemble influenza viruses, but lack the viral genome and many viral proteins.

Influenza virus-like particles are not infectious, but they are immunogenic: when injected into animals, they induce the production of anti-viral antibodies that can block infection. In one study, virus-like particles were produced in cultured insect cells by using an insect virus vector – a baculovirus – to deliver genes encoding the influenza HA, NA, and M1 proteins. Mice inoculated with these virus-like particles were protected after challenge with infectious virus. More recently, mice vaccinated with virus-like particles produced with proteins from an H5N1 avian strain were protected against challenge with lethal H5N1 viruses.

These findings suggest that virus-like particles could be used in humans to protect against influenza infection. They offer a number of advantages over the current influenza virus vaccines, most of which are prepared by growing virus in embryonated chicken eggs. Viral infectivity is destroyed with formalin, and the virions are then disrupted with detergents. Virus-like particle vaccines would not require these treatments, and would be available to individuals with egg allergies. Some influenza virus vaccines are produced in cell culture, but these are also treated to eliminate infectivity.

Another important advantage of the virus-like particle vaccine is that it can be produced relatively rapidly: within weeks, compared to months for egg-produced vaccines. This property would be especially useful when new pandemic strains emerge. For example, the swine-origin H1N1 influenza virus emerged in the spring of 2009, and vaccine manufacturers are scrambling to have a product ready for the fall.

Because an influenza virus-like particle vaccine is a new type of vaccine, many years of testing in animals and in humans will be required before it can be used. Some of the questions that must be addressed include the safety of the vaccine in humans, whether the anti-viral antibody repertoire induced by the vaccine is sufficiently broad, and of course whether immunization confers efficient protection against challenge in the majority of recipients.

I am particularly curious about how an influenza virus-like particle vaccine would compare with the infectious, attenuated influenza vaccine, Flumist. This intranasally-administered vaccine mimics a natural infection and has been shown to be more effective in preventing influenza than inactivated vaccine. While virus-like particle vaccines are an attractive option, they are not infectious and therefore might not induce the same antibody repertoire as would an infectious virus. A disadvantage of Flumist is that it is produced in eggs. If influenza virus-like particles prove safe and efficacious in humans, they could replace the egg-grown, inactivated vaccines.

Pushko, P., Tumpey, T., Bu, F., Knell, J., Robinson, R., & Smith, G. (2005). Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice Vaccine, 23 (50), 5751-5759 DOI: 10.1016/j.vaccine.2005.07.098

Bright, R., Carter, D., Crevar, C., Toapanta, F., Steckbeck, J., Cole, K., Kumar, N., Pushko, P., Smith, G., Tumpey, T., & Ross, T. (2008). Cross-Clade Protective Immune Responses to Influenza Viruses with H5N1 HA and NA Elicited by an Influenza Virus-Like Particle PLoS ONE, 3 (1) DOI: 10.1371/journal.pone.0001501

Virology pop quiz: Answers

BaculovirusA few weeks ago I asked readers to find the errors in the following statement concerning an experimental influenza vaccine produced by Protein Sciences which involves synthesis of the viral HA protein in insect cells.

They warned that the virus could mutate during the southern hemisphere’s flu season before returning north in a more lethal form in autumn, in a pattern similar to that seen in the deadly 1918 flu pandemic, which claimed an estimated 20 to 50 million lives around the globe.

The CDC (Centers for Disease Control and Prevention) sent us a dead virus, which is perfectly safe, and then we extracted genetic information from that virus.

The statement ‘in a pattern similar to that seen in the deadly 1918 flu pandemic’ is wrong. There is no evidence that mutation led to the emergence of a ‘more virulent’ virus that caused more severe disease in the fall of 1918. The only virus available to study was reconstructed from material obtained in November 1918. The first influenza virus was not isolated until 1933. The idea that a more virulent virus emerged in the fall has nevertheless become firmly established – without any scientific evidence to support the hypothesis. See “Riding the influenza pandemic wave” for more information.

The second problem is the statement that CDC sent the company a dead virus. Viruses are not living, so they cannot be killed. What the company received is an inactivated virus which cannot replicate in cells. There are many ways to inactivate viral infectivity, including heat, ultraviolet radiation, or treatment with chemicals such as formalin.

For extra credit I asked readers to critique the following statement:

Protein Sciences’ technology is also safer “because these caterpillars don’t have any association with man or other animals, so there’s no chance for their cells to learn how to propagate human viruses,” Adams told AFP.

What exactly was the spokesman trying to say? That there is no chance that influenza virus will replicate in insect cells? That’s impossible to say. The fact that ‘caterpillars don’t have any association with man’ is irrelevant (I’m not an entomologist, but I don’t believe that statement is correct). It’s possible that the virus could be adapted to grow in insect cells in the laboratory. And of course, cells don’t ‘learn’ how to propagate viruses. When viruses are selected for growth in new cells – a process that we call expanding the tropism of the virus – changes in the viral genome are usually responsible.

Parenthetically, there is a better way to make an influenza virus vaccine in insect cells – by synthesizing virus-like particles. When the influenza viral HA, NA, and M1 proteins are made in insect cells, virus-like particles are produced that lack the viral genome. These have been shown to be immunogenic in ferrets, and are capable of inducing a protective immune response.

Ross, T., Mahmood, K., Crevar, C., Schneider-Ohrum, K., Heaton, P., & Bright, R. (2009). A Trivalent Virus-Like Particle Vaccine Elicits Protective Immune Responses against Seasonal Influenza Strains in Mice and Ferrets PLoS ONE, 4 (6) DOI: 10.1371/journal.pone.0006032