TWiV 348: Chicken shift

2 August 2015

On episode #348 of the science show This Week in Virology, Vincent and Rich discuss fruit fly viruses, one year without polio in Nigeria, and a permissive Marek’s disease viral vaccine that allows transmission of virulent viruses.

You can find TWiV #348 at www.twiv.tv.

chicken farmA permissive vaccine prevents disease in the immunized host, but does not block virus infection. Would a permissive vaccine lead to the emergence of more virulent viruses?

This hypothesis is based on the notion that viruses which kill their hosts too quickly are not efficiently transmitted, and are therefore removed by selection. However a vaccine that prevents disease, but not viral replication in the host, would allow virulent viruses to be maintained in the host population. It has been suggested that in this scenario, viruses with increased virulence would be selected if such a property aids transmission between hosts.

On the surface this hypothesis seems reasonable, but in my opinion it is flawed. One problem is that increased transmission might not always be associated with increased virulence. The more serious flaw lies in making anthropomorphic assessments of what we think viruses require, such as concluding that increased viral transmission is a desired trait. Our assumptions fail to recognize the main goal of evolution: survival. Evolution does not move a virus along a trajectory aimed at perfection. Change comes about by eliminating those viruses that are not well adapted for the current conditions, not by building a virus that will fare better tomorrow. All the viruses on Earth today transmit well enough, or they would not be here; yet some kill their hosts clearly much faster than others. The fact is that humans have little understanding of what drives virus evolution in large populations. Our assumptions of what constitute the selective forces are usually tainted by anthropomorphism.

This long preamble is an introduction to a series of findings which are purported to support the idea that permissive vaccines (the authors call them ‘leaky’ and ‘imperfect’ vaccines but I dislike both names because they imply defects) can lead to the selection of more virulent viruses. The subject of the paper is Marek’s disease virus (MDV), a herpesvirus that infects chickens. MDV is shed from feather follicles of infected chickens and is spread to other birds when then inhale contaminated dust. Vaccines have been used to prevent MDV infection since the early 1970s. These vaccines prevent disease, but do not block viral replication, and vaccinated, infected birds can shed wild type virus. The virulence of MDV has been increasing since the 1950s, initially from a paralytic disease, to paralysis and death. The authors wonder if the use of permissive Marek’s vaccines has lead to the selection of more virulent viruses.

To address their hypothesis, the authors inoculate vaccinated or unvaccinated chickens with a series of MDV isolates that range from low to high virulence. Unvaccinated chickens inoculated with the most virulent MDV died within a week and shed little virus. In contrast, most vaccinated birds survived infection with virulent viruses, and shed virus for the length of the experiment, 56 days.

A transmission experiment was done to determine if shed virus could infect other birds. The authors infected vaccinated or unvaccinated birds and asked if sentinel, unvaccinated chickens became infected. Unvaccinated birds died within 10 days after infection with virulent MDV, and did not transmit infection. In contrast, vaccinated birds survived at least 30 days, and co-housed sentinel animals became infected and died.

The experiments are well done and the conclusions are clear: more virulent Marek’s disease viruses replicate longer in vaccinated than unvaccinated chickens, and can be readily transmitted to other chickens. But these results do not prove that more virulent MDV arose because of permissive vaccines. Nor do the results prove in general that leaky vaccines lead to selection of more virulent viruses. The results simply show that a vaccine that does not prevent replication will allow transmission of virulent viruses.

To prove that vaccinated chickens can allow the selection of more virulent viruses, vaccinated chickens could be infected with an avirulent virus, and the shed virus collected and used to infect additional, vaccinated birds. This process could be repeated to determine if more virulent viruses arise. While the results of this gain-of-function experiment would be informative, they would be done in a controlled laboratory setting which would not duplicate all the selective forces present on a poultry farm.

The authors note that most human vaccines do prevent replication of infecting virus. They do not mention the one important exception: the Salk poliovirus vaccines. People who are immunized with the Salk vaccine can be infected with poliovirus, which will then replicate in the intestines, be shed in the feces, and transmitted to others. This behavior has been well documented in human populations, yet the virulence of poliovirus has not increased for the 60 years during which the Salk vaccine has been used.

I do not feel that these experimental results have general implications for the use of any animal vaccine. It is unfortunate that the work has been covered in many news sources with the incorrect implication that vaccines may be responsible for the emergence of more virulent viruses.

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Principles of Virology 4th EditionI am pleased to announce the publication by ASM Press of the fourth edition of our virology textbook, Principles of Virology. Two years in the making, this new edition is fully updated to represent the rapidly changing field of virology.

Principles of Virology has been written according to the authors’ philosophy that the best approach to teaching introductory virology is by emphasizing shared principles. Studying the phases of the viral reproductive cycle, illustrated with a set of representative viruses, provides an overview of the steps required to maintain these infectious agents in nature. Such knowledge cannot be acquired by learning a collection of facts about individual viruses. Consequently, the major goal of this book is to define and illustrate the basic principles of animal virus biology.

This edition is marked by a change in the author team. Our new member, Glenn Rall, has brought expertise in viral immunology and pathogenesis, pedagogical clarity, and down-to-earth humor to our work. Although no longer a coauthor, our colleague Lynn Enquist has continued to provide insight, advice, and comments on the chapters.

A major new feature is the inclusion of 26 video interviews with leading scientists who have made significant contributions to the field of virology. These in-depth interviews provide the background and thinking that went into the discoveries or observations connected to the concepts being taught in this text. Students will discover the personal stories and twists of fate that led the scientists to work with viruses and make their seminal discoveries.

Principles of Virology is ideal for teaching the strategies by which all viruses reproduce, spread within a host, and are maintained within populations. It is appropriate for undergraduate courses in virology and microbiology as well as graduate courses in virology and infectious diseases. I have used previous editions of this textbook to build my Columbia University virology course. Volume I: Molecular Biology covers the molecular biology of viral reproduction. Volume II: Pathogenesis & Control addresses the interplay between viruses and their host organisms. The two volumes can be used for separate courses or together in a single course. Each includes a unique appendix, glossary, and links to Internet resources such as websites, podcasts, and blogs.

PoV4 goes on sale the week of 24 August 2015. If you are thinking about using the book for your course, reserve your review copy today at http://www.asm.org/pov.

Watch the video below to hear authors Jane Flint, Vincent Racaniello, Glenn Rall, and Ann Skalka talk about the making of PoV 4.

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On episode #347 of the science show This Week in Virology, Vincent, Alan, and Rich discuss the virus behind rose rosette disease, and fatal human encephalitis caused by a variegated squirrel bornavirus.

You can find TWiV #347 at www.twiv.tv.

Foot-and-mouth disease virus (FMDV) infects cloven-hoofed animals such as cattle, pigs, sheep, goats, and many wild species. The disease caused by this virus is a substantial problem for farmers because infected animals cannot be sold. Transgenic pigs have now been produced which express a short interfering RNA (siRNA) and consequently have reduced susceptibility to infection with FMDV.

FMDV is classified in the picornavirus family which also contains poliovirus and rhinoviruses. The virus is highly contagious and readily spreads long distances via wind currents, and among animals by aerosols and contact with farm equipment. Infection causes a high fever and blisters in the mouth and on the feet – hence the name of the disease. When outbreaks occur, they are economically devastating. The 2001 FMDV outbreak in the United Kingdom was stopped by mass slaughter of all animals surrounding the affected areas – an estimated 6,131,440 – in less than a year.

Vaccines against the virus can be protective but they are not an optimal solution. One problem is that antigenic variation of the virus may thwart protection. In addition, countries free of FMDV generally do not vaccinate because this practice would make the animals seropositive and prevent their export (it is not possible to differentiate between antibodies produced by natural infection versus immunization). Furthermore, if there were an outbreak of foot-and-mouth disease in such countries, the rapid replication and spread of the virus would make vaccination ineffective – hence culling of animals as described above is required. Clearly other means of protecting animals against FMDV are needed.

Synthetic short interfering RNAs (siRNA) have been shown to block viral replication in cell culture and in animals. To achieve such inhibition, short synthetic RNAs complementary to viral sequences are produced in cells. Upon infection, these siRNAs combine with the cellular RNA-induced silencing complex (RISC) which then targets the viral RNA for degradation.

To determine if siRNA could be used to protect pigs from foot-and-mouth disease, a complementary viral sequence was first identified that blocks FMDV replication in cell culture by ~97%. A vector containing this siRNA sequence was then used to produce transgenic pigs. Such animals not only express the antiviral siRNA, but as the encoding vector is present in germ cells, it is passed on to progeny pigs.

Expression of the siRNA was confirmed in a variety of transgenic pig tissues, including heart, lung, spleen, liver, kidney, and muscle. In fibroblasts produced from transgenic pigs, virus replication was reduced 30 fold. When transgenic pigs were inoculated intramuscularly with FMDV, none of the animals developed signs of disease such as fever or blisters of the feet and nose. In contrast, control non-transgenic pigs developed high fever and lesions. Viral RNA levels in the blood of transgenic pigs were 100-fold lower than in control animals. At 10 days post-infection no viral RNA was detected in heart, lung, spleen, liver, kidney, and muscle, while high levels were observed in these organs from non-transgenic controls.

These results show that siRNAs can protect transgenic pigs from FMDV induced disease. An important question that must be answered is whether transgenic pigs still contain enough virus to transmit infection to other animals. In addition, siRNAs are short – 21 nucleotides – and a mutation in the viral genome can block their inhibitory activity. Therefore it would be important to determine if mutations arise in the FMDV genome that lead to resistance to siRNAs.

Even if transgenic siRNA pigs do not transmit infection, and viral resistance does not arise, I am not sure that consumers are ready to accept such genetically modified animals.

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Episode #346 of the science show This Week in Virology was recorded at the 34th Annual Meeting of the American Society for Virology, where Vincent, Rich, and Kathy spoke with Joan Steitz, a tireless promoter of women in science and one of the greatest scientists of our generation.

You can find TWiV #346 at www.twiv.tv.

SpitsbergenAlthough we now understand that viruses are the most abundant organisms on Earth, there are gaps in our knowledge about their distribution in different environments. Results of a new study reveal the diversity and distribution of viruses in Arctic fresh waters.

Fresh waters in high latitudes such as the Arctic and Antarctic have low levels of nutrients (e.g. are oligotrophic) and support the growth mainly of microorganisms. They are good model systems for understanding how viruses affect microbial communities and the entire ecosystem. It is known that diverse viral communities, comprising novel families of single-stranded (ss) DNA viruses, dominate the fresh waters of the Antarctic Lake Limnopolar. However no large scale studies of the Arctic fresh water virome have been done.

Fresh water was collected in three different years from six lakes in Spitsbergen, Norway (red symbol on map). Viral particles were purified from the water samples and their genome sequences were determined. Only about 10% of the viral sequences could be assigned to a previously known virus family. Most (86%) of the recognizable sequences were from ssDNA viruses, and similar viruses were found in all six lakes.

Comparisons with viromes from other freshwater locations revealed similar taxonomic distributions in Antarctic freshwater but not elsewhere. As these locations are at opposite ends of the global poles, the results suggest that some viruses may be dispersed over long distances. The Arctic and Antarctic fresh water viromes do contain different viral species, despite being quite similar environments. On the other hand, the Arctic fresh water virome is very different from the Arctic Ocean virome. The finding of diverse viral communities in Arctic and Antarctic fresh waters indicates that, unlike larger organisms, viral richness might not decrease with distance from the equator.

The authors of this study did not characterize the RNA virome of Arctic fresh water lakes, but they did find sequences of single-stranded RNA viruses in their data sets. Because the authors sequenced DNA only (their protocol did not include a step to convert RNA to DNA before amplification), these RNA viral sequences likely represent DNA-RNA hybrid viruses. These viruses probably were produced by recombination of a DNA virus with DNA produced by reverse transcription of an RNA virus.

When Lake Limnopolar thaws in the spring, its viral community changes from ssDNA viruses to dsDNA viruses, perhaps as the hosts also change. Whether similar changes take place in Spitsbergen should be determined to help illuminate how viruses control high latitude microbial communities.

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On episode #345 of the science show This Week in Virology, the TWiVonauts review how the weather affects West Nile virus disease in the US, benefit of B cell depletion for ME/CFS patients, and an autoimmune reaction induced by influenza virus vaccine that leads to narcolepsy.

You can find TWiV #345 at www.twiv.tv.

B cellPatients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) showed clinical improvement after extended treatment with the anti-B-cell monoclonal antibody rituximab. This result suggests that in a subset of patients, ME/CFS might be an autoimmune disease.

Rituximab is a monoclonal antibody against a protein on the surface of B cells known as CD20. When the antibody is given to patients, it leads to destruction of B cells, which are the producers of antibodies, proteins that are made by the immune system to counter infections. The drug has been approved by the US Food and Drug administration to treat diseases of B cells such as lymphomas, leukemias, and autoimmune conditions.

ME/CFS is a disease of unknown etiology and mechanism that includes symptoms of severe fatigue, post-exertional malaise, pain, cognitive and sleep problems that affects 0.1-0.2% of the population. A previous randomized, phase II trial of rituximab treatment showed clinical benefit in 20 of 30 patients. The improvements were evident 2-8 months after treatment, leading the study authors to suggest that remission requires elimination of long-lived antibodies after depletion of B cells.

The current study was done to determine the effects of sustained treatment with rituximab. Included patients (29) were 18-66 years of age and diagnosed with ME/CFS according to Fukuda 1994 criteria. All were given rutiximab infusions two weeks apart, then at 3, 6, 10, and 15 months, and followed up for 36 months. Self-reported symptoms were recorded every second week and used to calculate scores for fatigue (comprising post-exertional malaise, need for rest, daily functioning), pain (muscle, joint, and cutaneous pain and headache) and cognitive scores (concentration ability, memory disturbance, mental tiredness).

Clinically significant responses were found in 18/29 patients (64%), with a lag of 8-66 weeks. After 36 weeks 11 of 18 responding patients were still in clinical remission. Nine patients from the placebo group in the previous study were included in this trial; of these, six had clinical improvement.

These results show that some ME/CFS patients benefit from ablating B cells. The delayed response, coupled with the relapse after cessation of treatment and B cell regeneration, suggests that antibodies are involved in the pathogenesis of the disease. Because onset of ME/CFS in many patients correlates with a viral infection, it is possible that antibodies to viral proteins may cross-react with self proteins, leading to autoimmune reactions that cause disease. Treatment with rituximab would lead to reduced levels of such antibodies, thereby reducing symptoms.

These results warrant trials of larger numbers of ME/CFS patients in other countries (this study was carried out in Norway) to determine if ablation of B cells would have a similar effects elsewhere. It would also be useful to determine the total repertoire of antiviral antibodies produced by ME/CFS patients. Such antibodies can be identified using the newly developed VirScan assay, which requires a small amount of blood and is relatively inexpensive. The results will indicate whether certain viral infections in a large population of ME/CFS patients predispose to the illness. Furthermore, the results may also be used to guide efforts to determine whether such antibodies react with human cellular proteins. A similar approach was used to determine that antibodies to an influenza virus protein cross react with a neuropeptide receptor, leading to narcolepsy.

While these findings are promising, they also show that not all ME/CFS may involve autoimmune pathogenesis. Other creative approaches will be needed to determine the cause of disease in individuals who do not respond to rituximab.

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Episode #344 of the science show This Week in Virology was recorded at the Glasgow Science Festival microTALKS, where Vincent spoke with Ruth, Glen, and Esther about their research on viruses and Hodgkin lymphoma, adenovirus structure and entry into cells, and interactions between arthropod borne viruses and their hosts.

You can find TWiV #344 at www.twiv.tv.