TWiV 96: Making viral DNA

22 August 2010

Hosts: Vincent Racaniello, Dickson Despommier, and Rich Condit

On episode #96 of the podcast This Week in Virology, Vincent, Dickson, and Rich continue Virology 101 with a discussion of how viruses with DNA genomes replicate their genetic information.

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Weekly Science Picks

Rich – Breast milk sugars give infants a protective coat (NY Times and PNAS article)
Vincent – The Great American University by Jonathan R. Cole

Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

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Hosts: Vincent Racaniello, Dickson DespommierAlan Dove, and Rich Condit

On episode #95 of the podcast This Week in Virology, Vincent, Dickson, Alan, and Rich consider the end of the influenza H1N1 pandemic, dengue in Florida, vaccinia virus infection in Brazilian monkeys, and viruses in the faecal microbiota.

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Weekly Science Picks

Alan – Families Fighting Flu
Rich –
Food, Inc.
Dickson –  Fuel
Vincent – MIT Open Courseware
Michael –  Waiting for Superman and Can Science Feed the World? (Nature)

Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

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How XMRV, the new human retrovirus associated with prostate cancer and chronic fatigue syndrome, might be transmitted among humans is unknown. The finding that the virus can be detected in prostate cancer cells, and in prostatic secretions of men with prostate cancer suggests that it could be sexually transmitted. To address this question, the presence of XMRV in seminal plasma of men with HIV-1 was examined. Although the virus was not detected in 93 samples from 54 HIV-1 infected men, the study provides little information on possible transmission mechanisms of XMRV.

This study involved two groups of HIV-1 infected men from the Netherlands: 29 who have sex with men, and 25 heterosexual men. The rationale for examining HIV-1 infected men for XMRV was that “they have a higher chance of contracting sexually transmitted pathogens than non-HIV-1 infected men”. For 39 men a second sample was also available from another time point, bringing the total samples to 93.

To detect XMRV, semen samples were diluted 1:1 with buffer and centrifuged to remove cells, yielding seminal plasma. Total nucleic acid was then extracted and subjected to reverse-transcription and then polymerase chain reaction. This procedure assays for the presence of XMRV viral RNA. At the same time, the samples were also tested for the presence of HIV-1 RNA. The positive control for XMRV was total nucleic acid extracted from a prostate cancer cell line known to produce viral RNA.

The results show that HIV-1 was detected in 25% of the seminal plasma samples, while none contained XMRV nucleic acid. The authors conclude:

Although HIV-1 was amplified from 25% of the seminal plasma samples, no XMRV was detected, suggesting that either the prevalence of XMRV is very low in The Netherlands, or that XMRV is not naturally present in the seminal plasma.

In my opinion, these conclusions are not supported by the data obtained in this study. Here are my reasons:

  • The semen samples were subjected to centrifugation, which removes all cells, including spermatozoa, epithelial cells, and lymphocytes. Such cells could harbor virions.
  • The study was designed only to search for XMRV virions or viral RNA, not proviral DNA, which is integrated into cellular DNA.
  • No attempt was made to determine if the 54 men were infected with XMRV. This could have been done by taking blood samples and co-culturing them with LNCaP cells, then performing PCR. If none of the men were infected with the virus, then absence of the virus in their semen is meaningless.

To determine if XMRV could be transmitted in semen, I would obtain semen samples from patients known to be infected with the virus. Then I would co-culture total semen and seminal plasma with LNCaP cells to amplify any virus present, followed by PCR to detect either virions or proviral DNA. I realize it may be difficult to conduct the study in this way, but I don’t see the value of doing it any other way.

Marion Cornelissen, Fokla Zorgdrager, Petra Blom, Suzanne Jurriaans, Sjoerd Repping, Elisabeth van Leeuwen, Margreet Bakker, Ben Berkhout, & Antoinette C. van der Kuyl (2010). Lack of Detection of XMRV in Seminal Plasma from HIV-1 Infected Men in The Netherlands PLoS One : 10.1371/journal.pone.0012040

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H1N1 pandemic is over

12 August 2010

The World Health Organization has declared the end of the pandemic caused by H1N1 influenza virus. According to Director-General Margaret Chan,

The world is no longer in phase 6 of influenza pandemic alert. We are now moving into the post-pandemic period. The new H1N1 virus has largely run its course.

As we enter the post-pandemic period, this does not mean that the H1N1 virus has gone away. Based on experience with past pandemics, we expect the H1N1 virus to take on the behaviour of a seasonal influenza virus and continue to circulate for some years to come.

According to the Director-General, levels and patterns of H1N1 transmission are now different from those observed during the pandemic. Out-of-season outbreaks are no longer being reported, and their intensity is similar to that seen during seasonal epidemics. In addition, multiple influenza viruses are being isolated in many countries, a pattern typical of many recent seasonal epidemics.

I take particular interest in what the Director-General believes did not happen:

This time around, we have been aided by pure good luck. The virus did not mutate during the pandemic to a more lethal form. Widespread resistance to oseltamivir did not develop. The vaccine proved to be a good match with circulating viruses and showed an excellent safety profile.

I continue to wonder why the Director-General, and many others, feel that influenza virus must change to a more lethal form. Although the four previous influenza pandemics occurred in multiple waves of increasing lethality, there is no evidence that they are a consequence of viral mutation. For example, the only virus available from the 1918 pandemic was rescued from an Alaskan influenza victim who was buried in permafrost in November of that year, when higher mortality was already evident. This makes it impossible to correlate any genetic changes in the virus with increased virulence. Viruses are available from different stages of the pandemics of 1957 and 1968, which also occurred in waves of increasing lethality, but to my knowledge the virulence studies have not been done.

I believe that a major selective force for viral evolution is the need to maintain efficient transmission among hosts. This may be achieved by any number of phenotypic changes, such as increases in stability and virion production. Changes in lethality might also lead to more effective transmission – for example, by inducing more severe coughing, the virus could be better transmitted among humans. But there is no genetic evidence that such changes have occurred during influenza virus pandemics.

How has the idea that influenza virus mutates to greater lethality permeated our popular culture? I don’t know the answer, but John Barry’s The Great Influenza is a prime suspect.

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TWiP 14: Leishmania

11 August 2010

Hosts: Vincent Racaniello and Dickson Despommier

On episode 14 of the podcast This Week in Parasitism, Vincent and Dickson consider the life cycle and pathogenesis of the protozoan parasite Leishmania.

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TWiP is brought to you by the American Society for Microbiology at Microbeworld.org.

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Download TWiP #14 (61 MB .mp3, 85 minutes)

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Send your questions and comments to twip@twiv.tv

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This week’s addition to the virology toolbox was written by Chris Upton

Dotplots are an extremely useful way of visualizing comparisons of small and large DNA sequences (as well as protein sequences), providing insight into the degree of similarity, deletions, insertions and direct and indirect repeats. In a dotplot, each nucleotide, or small window of nucleotides, of one sequence is compared with every nucleotide of a second sequence. Dotplots can quickly provide an overview of the relationship between sequences.

The Dotter program [1] has several very useful features including the ability to save and reload dotplots, the ability to zoom into particular regions of the plot, an option to create a multi-dotplot by aligning more than two DNA (or protein) sequences and permitting users to adjust the stringency of the matrix being displayed in real-time by changing the greyscale of the dots.

JDotter [2] provides an easy to use Java (platform independent) interface to Dotter giving all the benefits of Dotter in a single web-accessible tool. You can access JDotter here.

Additional background information on nucleic acid dotplots is available.

The first figure is a dotplot of three poxvirus interferon gamma binding proteins plotted against each other. Genes are displayed along the axes. This plot takes a few seconds to calculate.

Here is a dotplot of vaccinia CVA and MVA genomes (~170 kb). Large deletions are present in MVA, a result of >500 passages in chicken embryo fibroblasts.  Terminal inverted repeat sequences are obvious in the bottom-left and top-right corners of the plot. A plot for these sequences takes ~ 10 min to calculate.

Next is a self plot of the Molluscum contagiosum virus genome. Enhancing “background” shows that it’s not totally random. The “stripes” are caused by segments of DNA with different nucleotide composition. The region that creates the area in the red box has a higher A+T%, and appears to be derived from host sequences: it contains virulence genes.

Another view of the Molluscum contagiosum virus genome self plot – a zoomed-in view of the red box shown in the previous figure. Three of the genes in the “pale stripe” appear to be paralogs, probably resulting from duplications of an ancestral gene acquired from the host [3].

1. Sonnhammer EL, Durbin R: A dot-matrix program with dynamic threshold control suited for genomic DNA and protein sequence analysis. Gene 1995, 167:GC1-10.

2. Brodie R, Roper RL, Upton C: JDotter: a Java interface to multiple dotplots generated by dotter. Bioinformatics 2004, 20:279-281.

3. Da Silva M, Upton C: Host-derived pathogenicity islands in poxviruses. Virol. J 2005, 2:30.

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Hosts: Vincent Racaniello, Alan DoveRich Condit, and Ila Singh

On episode #94 of the podcast This Week in Virology, Vincent, Alan, and Rich speak with Ila Singh about the new human retrovirus XMRV, and how her laboratory is studying its association with prostate cancer and chronic fatigue syndrome.

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Download TWiV #94 (56 MB .mp3, 77 minutes)

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Weekly Science Picks

Alan – The new Federal Register site (see also regulations.gov)
Rich –
The Florida Museum of Nautural History Butterfly Rainforest
Vincent – JoVE, the Journal of Visualized Experiments

Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

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Inside the outbreaks

5 August 2010

If there’s something strange in your neighborhood, who you gonna call? EIS!

In the early 1950s, Alexander Langmuir, an epidemiologist for the Communicable Disease Center (CDC) in Atlanta, Georgia, warned that pathogenic microbes could be used as agents of biological warfare. To counter the threat, he advised the federal government to establish a ready response team at CDC. This advice was prescient: when Korean hemorrhagic fever virus infected 25,000 American troops in June 1951, killing 3,000, funding was provided to establish the Epidemic Intelligence Service (EIS). The two-year program trained young epidemiologists not only to look out for biological warfare, but to respond quickly to unintentional epidemics.

Despite the success of EIS in producing the world’s disease detectives, the history of the organization has never been told. Neither does Mark Pendergrast tell the history of EIS in Inside the Outbreaks — although it is a compelling collection of dozens of vignettes that cover many of the most interesting disease outbreaks of the past 60 years. If you are a microbe geek like I am, you will love reading about how EIS officers travel the world to quell lethal threats to global health.

All of the well-known infectious disease stories are here: pandemic influenza, the eradication of smallpox, the “Cutter incident” involving contaminated polio vaccine, and the first outbreak of Legionnaires’ disease in Philadelphia, to name just a few. But there are many other less well-known incidents that established disease etiologies. An example is the finding by the EIS in 1955 of the importance of Staphylococcus aureus in hospital-acquired infections.

Inside the Outbreaks is divided into three sections: “The Grand Adventures of Dr. Langmuir’s Boys” covers 1951–1970; “The Golden Age of Epi” continues to 1982; and “Complex Challenges” takes us to the present. Each section is composed of individual chapters that are further broken down into outbreak stories, such as “Mystery in Tuba City,” “Profuse Diaphoresis in Infants,” and “An Exhausting Disease.” While I found this approach appealing, it does have weak points. Because of the focus on outbreaks, there is no overall view of the history of the EIS. Furthermore, character development is minimal: there are few memorable individuals, with the exception of Dr. Langmuir. This book is about outbreaks, not people. While EIS officers obviously play important roles in each story, we quickly forget them as we move on to the next problem.

There are so many riveting stories in Inside the Outbreaks that I had difficulty identifying one that conveyed the book’s atmosphere. One of my favorites is “Health-Conscious Sprout Eaters,” which describes outbreaks with Salmonella or E. coli O157:H7 caused by alfalfa sprouts. The sprouts, consumed uncooked, are difficult to sterilize because the bacteria may be internalized in the inner plant tissues. Sprouts contaminated with E. coli O157:H7 were tracked to Idaho farms, where deer droppings may have been the source of the bacteria. EIS officer Roger Shapiro concluded, “Raw sprouts are inherently dangerous. They are the only food I stopped eating as a result of my EIS experience.”

I finished Inside the Outbreaks while traveling, and as I looked for a snack in the airport, I had difficulty identifying food that would be safe. The yogurt looked terrific, but it contained berries, and I had just read about outbreaks of infections in Texas and Florida with the parasite Cyclospora, caused by raspberries from Guatemala. There were also lovely sandwiches, but who knew what lurked in the salad greens — perhaps E. coli O157:H7, which caused gastroenteritis in Illinois when it contaminated mesclun from California. Reading Inside the Outbreaks will cause you to suspect nearly every food or food supplement, as well you should. The global economy and the demand for fresh food throughout the year have led to many opportunities for traveling microbes.

The list of former EIS officers is a Who’s Who of significant figures in science and medicine. Some individuals I was surprised to find in this program include D.A. Henderson and William Foege, architects of the smallpox eradication program; Neal Nathanson, a prominent virologist; current CDC Director Tom Frieden; former CDC Director Julie Gerberding*; and WHO Assistant Director-General Keiji Fukuda. Neither had I known that Lawrence Altman, the well-known New York Times science writer, had been an EIS officer.

You’ll have to read Inside the Outbreaks to learn how an EIS trainee learns the craft of disease epidemiology. Perhaps Alexander Langmuir’s approach is the most informative: he would send only one or two EIS officers to an outbreak. “We’ll get them on an epidemic as fast as we can. Throw them overboard. See if they can swim, and if they can’t, throw them a life ring; pull them out and throw them in again.”

Originally published in the Journal of Clinical Investigation.

*Author Mark Pendergrast and former EIS officer Patrick Moore have reminded me that Gerberding was not a member of this training program. My apologies for the error.

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From the Washington Post:

Dr. ROBERT M. CHANOCK (Age 86) On July 30, 2010 of Bethesda, MD. He was a resident in the Washington area for over 50 years, a distinguished scientist at the National Institute of Health. He received many awards and was a member of the National Academy of Sciences. He received his undergrad and medical degrees at the University of Chicago where he also received an honorary doctorate degree.

Chanock received his MD in 1947 from the University of Chicago, and after clinical training in pediatrics (note the bowtie), joined Albert Sabin at the University of Cincinnati where he studied arthropod-borne viruses. After a stint in the US Army, he rejoined Sabin’s laboratory in 1954 as an independent investigator. Sabin advised him to work on something other than poliomyelitis, to establish his own scientific identity. He decided to study an ongoing outbreak of croup in Cincinnati children and isolated a new virus, subsequently called human parainfluenza virus type 2. This discovery ensured that he would study respiratory viruses for the rest of his career.

His move to the Laboratory of Infectious Diseases, National Institutes of Health in 1957 was the last of his career but lead to his most productive years. Together with Robert Huebner he developed an effective adenovirus vaccine which was used by the military. He discovered four additional human parainfluenza viruses, but his most important finding was the isolation of respiratory syncytial virus, the most common viral cause of serious lower respiratory tract disease in infants and young children. Under his leadership, the Laboratory of Infectious Diseases began to study other important human viruses, including gastroenteritis viruses (e.g. Norwalk virus) and hepatitis viruses.

I was fortunate to interact with Dr. Chanock early in my career, at scientific meetings and during visits to the NIH. My main recollection was that he was always enthusiastic and supportive. His first question upon seeing me was always ‘how’s the work with polio?’ Since his early years with Albert Sabin he had always followed basic research on poliovirus with great interest. Sabin had a significant positive influence on Chanock’s career and his view of viruses – in fact, Sabin considered Chanock his ’scientific son’. It is therefore fitting that the last award bestowed upon Chanock was the Albert B. Sabin Gold Medal in 1995, for his work in the field of vaccinology, particularly the control of respiratory diseases.

Update: Washington Post story

LIGON, B. (1998). Robert M. Chanock, MD: A living legend in the war against viruses Seminars in Pediatric Infectious Diseases, 9 (3), 258-269 DOI: 10.1016/S1045-1870(98)80040-X

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Hosts: Vincent Racaniello, Alan Dove, and Rich Condit

On episode #93 of the podcast This Week in Virology, Vincent, Alan, and Rich answer listener questions about lab procedures, prokaryotes, endogenous retroviruses, the iPad and teaching, prions, mimivirus, splitting water with viruses, and the polio outbreak in Tajikistan.

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Download TWiV #93 (76 MB .mp3, 105 minutes)

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Links for this episode:

Weekly Science Picks

Alan – Southern Fried Science
Rich –
Tree of Life web project
Vincent – Dickson Despommier at Big Think

Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

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