The new human retrovirus XMRV, first detected in malignant prostate tissue, was subsequently identified in a high percentage of patients with chronic fatigue syndrome (CFS). The virus was not detected in four independent studies of CFS patients in Europe or the United States. The results of a second American study, whose publication was blocked for two months, provide support for the involvement of murine retroviruses in CFS.

The new study, a collaboration among scientists at the Food and Drug Administration, the National Institutes of Health, and Harvard Medical School, utilized samples from 37 CFS patients obtained in the mid-1990s. A key difference from earlier studies is that some repeat samples were used: four obtained two years later and frozen, and eight taken in 2010 and processed without freezing. The patients were from New England, New York State, and North Carolina and were not part of the previous study in which XMRV was detected. Control samples were obtained from 44 healthy blood donors.

Two kinds of polymerase chain reaction (PCR) assays were used to determine the presence of XMRV. In the first, DNA was extracted from PBMC to search for the presence of proviral DNA (e.g., viral DNA integrated into the host chromosome). In the second, RNA was extracted from plasma, and subjected to reverse-transcriptase PCR to detect viral RNA.

The authors found that 32 of 37 samples from CFS patients (86.5%) were positive in the PCR assay of PBMC DNA, compared with 3 of 44 samples (8.6%) from healthy controls. All four samples taken 2 years later, and 7 of the 8 samples taken 15 years later from CFS patients were also positive in this assay. The latter is an important result because it shows presence of virus in patients for long periods. Viral RNA was detected in plasma of 42% of the samples.

The authors performed additional experiments to rule out false positive results. The nucleotide sequences of all PCR products were determined to ensure that they represented the expected target sequence. Furthermore, contamination with mouse DNA was excluded by assaying for murine mitochondrial DNA – it was not found in any of the samples tested.

Perhaps the most interesting finding, aside from the detection of viral RNA in a large fraction of CFS patients, is the observation that the virus detected is not XMRV. Nucleotide sequence analysis of PCR amplified DNA revealed 96.6% nucleotide identity with XMRV, but they are clearly different viruses. The RNA genome of XMRV is a recombinant between the genomes of xenotropic* and polytropic* murine leukemia viruses (Figure). The genome of the viruses detected in the new study appears to be derived from polytropic MLVs, and could be called PMRV. This observation indicates that both xenotropic and polytropic MLV can infect humans in North America.

What is the significace of the observation that both XMRV and PMRV have been associated with CFS? Some investigators have suggested that variation of XMRV is minimal, based on the close identity of sequences obtained from prostate cancer and CFS patients. The finding of a different virus, PMRV, suggests greater genetic variation than previously thought, and could in part explain previous failures to detect MLV-related viruses in the previous four studies of CFS patients. Other explanations, including geographic differences and patient heterogeneity, are also plausible.

I find it very interesting that the authors readily detected PMRV sequences in both PBMC DNA and plasma. The authors of the 2009 Science paper have suggested that these approaches are not sufficiently sensitive to detect viral nucleic acid, and that culturing with susceptible cells must be done to amplify the virus. That explanation, which was used to explain the failure to detect XMRV in the previous four studies, is not likely to be correct. The authors of those studies might want to repeat their assays using primers more likely to detect PMRV.

Where do these findings lead next? The authors of an accompanying commentary have two appropriate suggestions:

At this juncture, it would seem reasonable to conduct extensive case-control studies in North America…using coded samples from subject with inflammatory disease to determine the frequency of MLV infection in patients with CFS. The potential transmission of MLV-related sequences from human to human should also be epidemiologically evaluated. [...]it would also be appropriate to conduct interventional studies.

It is possible that these studies, which are needed to determine if MLVs cause human disease, might not have been done without a second independent confirmation of the association of MLV-related viruses with CFS.

*Xenotropic murine leukemia viruses infect non-mouse cells, while polytropic murine leukemia viruses can infect the cells of mice and other mammals.

Lo SC, Pripuzova N, Li B, Komaroff AL, Hung GC, Wang R, & Alter HJ (2010). Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors. Proceedings of the National Academy of Sciences of the United States of America, 107 (36), 15874-9 PMID: 20798047

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

Comparing genomes of viral strains can provide very useful insight into evolutionary relationships. Recombination, defined by Posada et al (2001) as the exchange of genetic information between two nucleotide sequences, is quite common in many viruses. Because recombination accounts for much of the genetic diversity observed between viral strains, it is of interest to decipher where the origins of recombinant sequences are, and to know which viral strains are likely to have undergone recombination. Several programs exist to detect recombination among genomes and to identify breakpoints in sequences, which represent recombinant regions. A few are described here, using HIV-1 strains isolated from Uganda, where subtypes A and D are prominent. Recombinant strains have arisen in Eastern Africa, given the co-circulation of different types of strains. Three recombination-detection programs are described here, using HIV-1 strains isolated from Uganda, where subtype A and D are prominent, and recombinant A-D strains have arisen.

Recombination analysis tool (RAT)

RAT is a very simple, easy-to-use cross-platform program that allows for the comparison and detection of recombination of between multiple sequences, in a straightforward graphical user interface. It provides a clear graphical output, depicting recombination crossover points between sequences by plotting the genetic distance between each sequence as a function of its sequence position. A sequence alignment (FASTA format works best, although other alignment files will work) is input, and default parameters may be maintained or changed. The default settings are well optimized for analysis; however, a rule of thumb is that window size should be 10% of the sequence length, and the increment size should be half the window size.

Figure 1. Sequence input window in RAT, where parameters can be adjusted, or left as default values. Here, a FASTA file containing three HIV sequences is input, and the suspected recombinant is selected as the test sequence, to which other sequences will be compared.

RAT is useful in that it allows the user to check for recombination between sequences already thought to be recombinants, as well as to conduct an auto search to find possible recombination spots. By clicking execute, a sequence viewer will display the similarity of all sequences in the alignment as compared to a specified test sequence. Useful in this display is the option to select and unselect different sequences, in order to view all sequences at once to find possible recombination sites, or to view two at a time to decipher specific recombination breakpoints. When conducting an auto search, results can be screened based on customized similarity thresholds. All possible recombination points within the threshold are listed between which sequences they occur.

Figure 2. Output for specified sequence search, showing the genetic distance of two HIV strains of clade A and D from the test sequence (the AD recombinant strain) on the Y-axis, and the sequence position on the X-axis. A possible recombination spot occurs at position 4874, where the recombinant sequence now shares higher similarity with clade D than clade A. This recombinant region appears to end at approximate position 6203.

Finally, the graphical representation of genetic distance between strains can be exported as a JPG file.

RAT works based on a distance method, whereby pairwise comparisons between sequences are performed as a sliding window moves along the length of the sequence. A score is generated based on the similarity between the nucleotides in the current window of the test sequence and the nucleotides in the same window of the other sequences. While useful information is provided in the RAT program, it does not provide statistical support for the results generated. However, this is an advantage as it allows analysis to proceed very quickly, which is extremely useful to get an overview of potential recombinant sites.

SimPlot

Simplot (for Windows) is another useful tool for detecting recombination between sequences, which like RAT, produces similarity plots, but has more features and therefore is slightly more complex. SimPlot allows for the analysis of up to 10 sequences (although the alignment may have more than this), where each can be used as a query sequence with which to compare the rest, or hidden from the analysis. Other useful and unique features of SimPlot are the ability to ignore sites containing gaps in the alignment when generating the similarity plot, as well as being able to identify the sequence position and exact similarity value on any point in the sequence you click on. Furthermore, there is a zoom in feature, as well as options to include titles, legends, grid lines and other useful information as part of the display. SimPlot also allows sequences to be grouped together, and analysis to be performed between groups rather than individual sequences.

Sequences (FASTA format, as well as other common alignment files) are loaded into the program, and those that are desired are selected for analysis. Several options are available for analysis, such as which Distance Model to use, and the number of Bootstrap replicates to use for statistical significance.

Figure 3. Similarity plot generated in SimPlot using one query sequence (recombinantAD) and two other sequences, (HIV-1 strains from Clade A1 and Clade D). Possible recombination sites are identified where sequence crossover occurs. By zooming in to better view, and clicking on crossover regions, breakpoints are determined, such as at regions 4481 and 5681 in the alignment. Other potential recombinant regions are also identified that were not obvious in RAT.

Finally, SimPlot provides the option of finding specific recombinant sites. After identifying potential recombinant sequences on the similarity plot, specific sequences within each group can be chosen for an informative site analysis. Overall, SimPlot provides a very effective means of detecting recombination, in an easy-to-use interface with fast results.

Recombination Detection Program (RDP)

RDP (for Windows) is yet another program that allows for detection of recombination amongst aligned sequences, however it is unique in that it incorporates several detection methods and analysis algorithms into one well laid-out interface, allowing the user to select which method of recombination detection is most suitable and provides the best results.

Figure 4. RDP Overall Display

Furthermore, recombination events that are detected are displayed graphically, with statistical evidence provided, and recombination events are also depicted on phylogenetic trees constructed from proposed recombinant regions. These features allow the user to decipher which events are true recombination events, and discard those that have been incorrectly identified. Importantly, possible recombination events are listed with warnings, to indicate when the program is not confident of the proposed recombination event, its location and sequence breakpoints, or its contributing sequences.

Easy navigation through the sequences is possible, as these are displayed alongside the statistical display of recombinant regions and breakpoints, the schematic display of recombinant sequences, as well as the dendogram display.

Figure 5. Sequence display

Figure 6. Schematic sequence display of recombinant regions; each recombinant block can be selected in order to view the supporting evidence for recombination.

Figure 7. Recombination information; displayed here are any relevant warning suggesting reasons why the recombinant may have been misidentified, as well as statistical evidence from each algorithm used supporting the event.

Figure 8. Graphical representation of recombinant region (shown in pink) as determined by the RDP algorithm. Plotted on the Y axis is Pairwise identity of each pair of sequence, against their position in the alignment on the X axis.

By clicking on the various potential recombinant sequence blocks in the schematic sequence display of recombinant regions, graphical evidence will appear for each on the bottom left display area. The pink region here shows the likely recombinant region. Each potential recombinant region will also have its accompanying statistical evidence displayed in the top right corner in the recombination information display. The tree display is quite useful in determining true recombination events; it provides a dendrogram of non-recombinant regions and recombinant regions which to compare. While the default setting is to create trees using the neighbor joining method (which requires less time), RDP is also able to create trees using UPGMA, least squares, Bayesian and Maximum Likelihood algorithms.

As mentioned, RDP provides statistical evidence for each recombination event, as determined by several different methods. Although the default displays evidence as determined by the method which most strongly suggested recombination in that region, the user can easily see graphical displays of the different methods used to find the breakpoint.

Among the different recombination detection algorithms in the tool, are RDP, Geneconv, Bootscan, MaxChi, Chimaera, SiScan, 3Seq, LARD and TOPAL, all of which are optimized to detect recombination in different ways, thus allowing for detection of recombination in various different alignments. Furthermore, each type of analysis has several customizable options, set to default values that work well. Manual Distance plots, similar to those created by SimPlot and RDP are also possible, where any selected sequence can be queried against all other in the alignment.

With the vast array of options and analysis preferences that are available on RDP, the average run-time for an alignment is longer than for the other programs, however, much more information is provided. Knowing which detection algorithm best suits the alignment allows the user to select which algorithms should be used, allowing the analysis to proceed much faster.

Finally, this program is accompanied by an extremely useful user’s manual, explaining the algorithms that are available and which is best suited to different alignments. The manual also includes a step by step guide, which details the process of detecting recombination in sequences, from preliminary hypothesis, to finding conclusive statistically supported recombinant regions.

Example sequences:

Clade A1: HIV-1 isolate 99UGA07072 from Uganda, partial genome

GenBank: AF484478.1

Clade D: HIV-1 isolate 99UGC06443 from Uganda, partial genome

GenBank: AF484479.1

RecombinantAD: HIV-1 isolate 99UGB21875 from Uganda, partial genome

GenBank: AF484480.1

Hosts: Vincent Racaniello, Peter Sarnow, and Bert Semler

On episode #97 of the podcast This Week in Virology, Vincent visited Peter Sarnow and Bert Semler during a trip to California, and spoke with them about their work on internal ribosome entry, and the requirement for a cellular microRNA for hepatitis C virus replication.

Download TWiV #97 (66 MB .mp3, 91 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• Eukaryotic mRNAs that might contain an IRES (PNAS)
• Modulation of HCV RNA abundance by a liver-specific microRNA (Science)
• Viral small RNAs (PLoS Pathogens)
• Bridging IRES elements to the translation apparatus (Biochim Biophys Acta)
• A nucleo-cytoplasmic SR protein functions in viral IRES mediated translation (EMBO J)
• Nuclear vs cytoplasmic routes to IRES mediated translation (Trends in Microbiology)
• Letter read on TWiV 97

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.

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.

Download TWiV #96 (65 MB .mp3, 90 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• Figures for this episode (pdf)
• RNA silencing as a plant immune system (Trends in Genetics)
• Photos of transgenic petunia (PLoS Biology)
• Letters read on TWiV 96
• Video of this episode – download .mov or .wmv or view below

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.

Hosts: Vincent Racaniello, Dickson Despommier, Alan 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.

Download TWiV #95 (68 MB .mp3, 94 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• WHO declares end of influenza H1N1 pandemic
• CDC’s FluView
• WHO global monitoring of influenza
• Locally acquired dengue in Key West, Florida (MMWR)
• CDC page on dengue
• Vaccinia virus infection in monkeys of the Brazilian Amazon
• Dam site where animals were collected for vaccinia study (Google maps)
• Rich’s article: Whence feral vaccinia?
• Viruses in the faecal microbiota of monozygotic twins and their Mothers (Nature)
• New Yorker article The Treatment (thanks, Jim!)
• Letters read on TWiV 95

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.

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

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.

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.

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

Links for this episode:

• L. braziliensis life cycle (jpg)
• L. donovani life cycle (jpg)
• L. tropica life cycle (jpg)
• L. major lesion (jpg)
• Transgenic mosquito delivers Leishmania vaccine (thanks, Geoffrey)
• Plasmodium falciparum accompanied human expansion out of Africa (thanks, Prasad)
• Who speaks for the guinea worm? (thanks, Michael)
• Letters read on TWiP 14

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

Subscribe to TWiP (free) in iTunes, at the Zune Marketplace, by the RSS feed or by email

Send your questions and comments to twip@twiv.tv

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.

Hosts: Vincent Racaniello, Alan Dove, Rich 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.

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

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• CFIDS Association of America
• Discovery of XMRV (PLoS Pathogens)
• Detection of XMRV in CFS patients (Science)
• Presence of XMRV in malignant prostate (PNAS)
• Inhibition of XMRV by raltegravir (PLoS One)
• Letters read on TWiV 94

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.

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.

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

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.

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

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• Biomedical Advanced Research and Development Authority (BARDA)
• SIGA responds to BARDA request for smallpox antiviral
• BARDA contract for filovirus vaccine
• What is a Ph.D? (pdf)
• HHMI resources for early career scientists
• Pace article on abandoning prokaryote (Nature)
• Three domains of life (Forterre article)
• Mechanoenzymatic cleavage of Von Willebrand’s factor (Science)
• Splitting water with viruses
• WHO coverage on Tajikstan polio outbreak
• Wild type polio infection in immunized Indian children (JID)
• Letters read on TWiV 93

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.

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

First, you may be asking yourself – Why viral bioinformatics? Good question! Although it’s true that much in the world of bioinformatics can be applied to all manner of protein and DNA sequences, there are a number of resources that are specific for viruses and there are a number of analyses that every virologist should be familiar with. So this section of the virology toolbox will highlight database resources, some useful tools and analyses, and some pitfalls you want to avoid.

What do I know about viral bioinformatics? Another good question! Well, all I can say is that although I can’t program my way out of a paper bag – I’ve been analyzing DNA and protein sequences of poxvirus for 20+ years and have been “developing” software for analysis of viral genomes for 10+ years. That is, I say what I want the software to do, and a bunch of talented programmers – including many undergraduate students – figure out how to code it. Over the years, this work has been funded by NSERC and PENCE in Canada, and NIH in the USA.

I’ll also be highlighting many of our own tools – why?

• They were developed precisely for comparative genomics of viruses. That is, genomes from 10-500 kb.
• They were developed for use by the bench virologist – so they’re fairly straightforward to use.
• They’re platform independent – will run on Macs, Windoze and LINUX boxes.
• I know them best and can give good examples of their use.
• PS. The programs also work on any type of DNA and protein sequences.

My first topic is very simple, but equally important. So watch your language.

Homology = common origin

Phrases like “sequence (structural) homology”, “high homology”, “significant homology”, or even “35% homology” are as common, even in top scientific journals, as they are absurd, considering the above definition.

I took this quote from a book (which seems to be online): Sequence – Evolution – Function: Computational Approaches in Comparative Genomics by Eugene V Koonin and Michael Y Galperin: Kluwer Academic; 2003.

So genes/proteins/sequences are either homologous, or they’re not. No fractions or percentages here!

Try writing: 50% identical. But you also have to say whether you mean nucleotides or amino acids.

While we’re on this topic:

Orthology

Homologous sequences are orthologous if they were separated by a speciation event: when a species diverges into two separate species, the divergent copies of a single gene in the resulting species are said to be orthologous. Orthologs, or orthologous genes, are genes in different species that are similar to each other because they originated from a common ancestor. The term “ortholog” was coined in 1970 by Walter Fitch.

Paralogy

Homologous sequences are paralogous if they were separated by a gene duplication event: if a gene in an organism is duplicated to occupy two different positions in the same genome, then the two copies are paralogous. A set of sequences that are paralogous are called paralogs of each other. Paralogs typically have the same or similar function, but sometimes do not: due to lack of the original selective pressure upon one copy of the duplicated gene, this copy is free to mutate and acquire new functions (from Wikipedia).

The figure illustrates homologs, orthologs and paralogs (click for the original link).

Hosts: Vincent Racaniello, Rich Condit, Karla Kirkegaard, and Marilyn Roosinck

On episode #92 of the podcast This Week in Virology, Vincent, Rich, Karla, and Marilyn recorded TWiV at the 29th Annual Meeting of the American Society for Virology in Bozeman, where they discussed plant viruses and how they make plants resistant to adverse conditions, and identification of dominant negative drug targets.

Download TWiV #92 (42 MB .mp3, 57 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• Lifestyles of plant viruses
• Using pyrosequencing to understand virus ecology
• A virus in a fungus in a plant
• Trans-dominant inhibition of RNA viral replication
• Resistance is futile
• Announcement of TWiV at ASV (pdf)
• Photographs of TWiV at ASV
• Letters read on TWiV 92

Weekly Science Picks

Marilyn – Viruses in the faecal microbiota of monozygotic twins and their mothers (Nature)
Rich – The Known Universe by the American Museum of Natural History
Vincent – The Red Queen by Matt Ridley (thanks, Jesper!)

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.

The illustration at left depicts a virion – the infectious particle that is designed for transmission of the nucleic acid genome among hosts or host cells. A virion is not the same as a virus. I define virus as a distinct biological entity with five different characteristics. Others believe that the virus is actually the infected host cell.

The idea that virus and virion are distinct was first proposed by Bandea in 1983. He suggested that a virus is an organism without a cohesive morphological structure, with subsystems that are not in structural continuity:

Viruses are presented as organisms which pass in their ontogenetic cycle through two distinctive phenotypic phases: (1) the vegetative phase and (2) the phase of viral particle or nucleic acid. In the vegetative phase, considered herein to be the ontogenetically mature phase of viruses, their component molecules are dispersed within the host cell. In this phase the virus shows the major physiological properties of other organisms: metabolism, growth, and reproduction.

According to Bandea’s hypothesis, the infected cell is the virus, while the virus particles are ’spores’ or reproductive forms. His theory was largely ignored until the discovery of the giant mimivirus, which replicates its DNA genome and produces new virions in the cytoplasm within complex viral ‘factories’. Claverie suggested that the viral factory corresponds to the organism, whereas the virion is used to spread from cell to cell. He wrote that “to confuse the virion with the virus would be the same as to confuse a sperm cell with a human being”.

If we accept that the virus is the infected cell, then it becomes clear that most virologists have confused the virion and the virus. This is probably a consequence of the fact that modern virology is rooted in the study of bacteriophages that began in the 1940s. These viruses do not induce cellular factories, and disappear (the eclipse phase) early after cell entry. Contemporary examples of such confusion include the production by structural virologists of virus crystals, and the observation that viruses are the most abundant entities in the seas. In both cases it is the virion that is being studied. But virologists are not the only ones at fault – the media writes about the AIDS virus while showing an illustration of the virion.

Those who consider the virus to be the infected cell also believe that viruses are alive.

…one can conclude that infected eukaryotic cells in which viral factories have taken control of the cellular machinery became viruses themselves, the viral factory being in that case the equivalent of the nucleus. By adopting this viewpoint, one should finally consider viruses as cellular organisms. They are of course a particular form of cellular organism, since they do not encode their own ribosomes and cell membranes, but borrow those from the cells in which they live.

This argument leads to the assumption that viruses are living, according to the classical definition of living organisms as cellular organisms. Raoult and Forterre have therefore proposed that the living world should be divided into two major groups of organisms, those that encode ribosomes (archaea, bacteria and eukarya), and capsid-encoding organisms (the viruses).

BANDEA, C. (1983). A new theory on the origin and the nature of viruses Journal of Theoretical Biology, 105 (4), 591-602 DOI: 10.1016/0022-5193(83)90221-7

Forterre, P. (2010). Defining Life: The Virus Viewpoint Origins of Life and Evolution of Biospheres, 40 (2), 151-160 DOI: 10.1007/s11084-010-9194-1

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Welkin Johnson

On episode #91 of the podcast This Week in Virology, Vincent, Dickson, Alan, Rich and Welkin discuss the nature, origin, and evolution of endogenous retroviruses (ERVs), and the recent finding of endogenous filovirus genomes in mammals.

Download TWiV #91 (64 MB .mp3, 89 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• Welkin blogs at Small Things Considered
• Constructing primate phylogenies from ancient retrovirus sequences (PNAS)
• Filoviruses are ancient and integrated into mammalian genomes (BMC Evol Biol)
• Synthetic cells: Momentous breakthrough or ethical morass? (To The Point)
• Creation of a bacterial cell (Science)
• Comments on the synthetic cell (Small Things Considered)
• TWiV rap: T-Number Index by G-Unit (mp3) and Vincent (mp3) (thanks, Darrick and Scott!)
• Letters read on TWiV 91

Weekly Science Picks

Welkin – Advice for a Young Investigator by Santiago Ramon y Cajal
Rich – How microbes define and defend us
Dickson – H1N1 virus lacks 1918 virus killer protein
Alan – The Xtal Set Society
Vincent – Antibodies and the quest for an AIDS vaccine

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.

A more rapid method than Western blot analysis to detect a specific protein in a cell, tissue, organ, or body fluid is enzyme-linked immunosorbent assay, or ELISA. This method, which does not require fractionation of the sample by gel electrophoresisis, is based on the property of proteins to readily bind to a plastic surface.

To detect viral proteins in serum or clinical samples, a capture antibody, directed against the protein, is linked to a solid support such as a plastic 96 well microtiter plate, or a bead. The clinical specimen is added, and if viral antigens are present, they will be captured by the bound antibody. The bound viral antigen is then detected by using a second antibody linked to an enzyme. A chromogenic molecule – one that is converted by the enzyme to an easily detectible product – is then added. The enzyme amplifies the signal because a single catalytic enzyme molecule can generate many product molecules.

To detect antibodies to viruses, viral protein is linked to the plastic support, and then the clinical specimen is added. If antibodies against the virus are present in the specimen, they will bind to the immobilized antigen. The bound antibodies are then detected by using a second antibody that binds to the first antibody.

ELISA is used in both experimental and diagnostic virology. It is a highly sensitive assay that can detect proteins at the picomolar to nanomolar range (10^-12 to 10^-9 moles per liter). It is the mainstay for the diagnosis of infections by many different viruses, including HIV-1, HTLV-1, adenovirus, and cytomegalovirus.

Hosts: Vincent Racaniello and Dickson Despommier

On episode 13 of the podcast This Week in Parasitism, Vincent and Dickson continue their discussion of the obligate intracellular protozoan Toxoplasma gondii with a consideration of the clinical consequences of infection and pathogenesis.

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

Links for this episode:

• Isopod fish parasites (thanks, Kevin!)
• Farmscraper on Word Spy (thanks, Mitchell!)
• Toxoplasma and personalities (thanks, Greg!)
• Science podcast (July 2) on parasites and intelligence (transcript – thanks, Jim!)
• Letters read on TWiP 13

Download TWiP #13 (61 MB .mp3, 84 minutes)

Subscribe to TWiP (free) in iTunes, at the Zune Marketplace, by the RSS feed or by email

Send your questions and comments to twip@twiv.tv

Global fish farming may be the solution to the impending collapse of the commercial fishing industry, but penned fish are susceptible to infectious diseases. Infection with salmon infectious anemia virus, an orthomyxovirus, lead Wal-Mart to stop buying farmed salmon from Chile, the world’s second largest producer of the fish. As a consequence Chilean farmed salmon are being immunized to prevent infection. Heart and skeletal muscle inflammation (HSMI) is another disease of farmed salmon, first detected in Norway – the world’s largest supplier of the fish – in 1999. The results of deep sequencing suggest that HSMI is caused by a novel piscine reovirus.

HSMI was transmitted to salmon by inoculation with tissue extracts of diseased fish or by co-habitation. RNA was extracted from the heart and subjected to high throughput sequence analysis, which revealed the presence of a novel member of the reovirus family. These are are non-enveloped, icosahedral viruses with 10-12 segments of double-stranded RNA that infect a variety of hosts, including humans: rotaviruses are important agents of gastroenteritis.

The presence of piscine reovirus in salmon with HSMI was examined by polymerase chain reaction assays. Included were heart and kidney samples from 29 salmon from three different outbreaks of HSMI, and from 10 healthy fish. All but one of the diseased fish, and none of the healthy fish, contained PRV nucleic acid. To determine the prevalence of PRV in healthy salmon, samples were collected from 9 different Norwegian coastal waters. Sixteen of 66 (24.2%) of these fish were positive for PRV.

Is heart and skeletal muscle inflammation of salmon caused by piscine reovirus? It’s possible, but further work is needed to prove causality, as the authors write:

Formal implication of PRV in HSMI will require isolation in cell culture and fulfillment of Koch’s postulates, or prevention or modification of disease through use of specific drugs or vaccines.

It’s important to identify the etiologic agent of HSMI because it is a threat to both farmed and wild salmon. Farmed fish are kept in pens in the open ocean, facilitating spread of infectious diseases to wild fish. Knowledge of the causative agent will permit preventive measures such as immunization.

Palacios G, Lovoll M, Tengs T, Hornig M, Hutchison S, Hui J, Kongtorp RT, Savji N, Bussetti AV, Solovyov A, Kristoffersen AB, Celone C, Street C, Trifonov V, Hirschberg DL, Rabadan R, Egholm M, Rimstad E, & Lipkin WI (2010). Heart and skeletal muscle inflammation of farmed salmon is associated with infection with a novel reovirus. PloS one, 5 (7) PMID: 20634888

Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and Eric F. Donaldson

On episode #90 of the podcast This Week in Virology, Vincent, Alan, Rich and Eric discuss identification of viruses in Northeastern American bats, vaccinia virus infection after sexual contact with a military vaccinee, and identification of a new flavivirus from an Old World bat in Bangladesh.

Download TWiV #90 (64 MB .mp3, 89 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• Vaccinia virus infection after sexual contact with vaccinee
• Smallpox vaccination overview
• Smallpox vaccine lesions (jpg)
• Smallpox hospital, Roosevelt Island, NY (photo 1, photo 2)
• Isolation of a flavivirus from bats in Bangladesh (PLoS Pathogens)
• Review on hepatitis G virus
• Dickson has been teaching at Singularity University and fishing in Bozeman MT (jpg)
• Letters read on TWiV 90

Weekly Science Picks

Eric – Year of Darwin by Sean Carroll
Rich – March of the Penguins
Alan – Standing-height desks
Vincent – DengueWatch (thanks Richard!)

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.

Readers of virology blog often request explanations of specific experimental techniques. Methods such as complement fixation, deep sequencing, ELISA, PCR and many others are frequently mentioned on this blog without discussion. To do so would interrupt the scientific discourse and make for lengthly posts. To remedy this shortcoming, I have added a new tab to the first page of virology blog called Virology Toolbox. This page will be populated with explanations of experimental techniques used for the study of viruses. Today’s technique is the western blot.

Western blot analysis (also known as immunoblotting) is used to detect a specific protein in a cell, tissue, organ, or body fluid. The technique depends on the reaction of an antibody with a protein that is immobilized on a thin membrane (click the figure for a larger version). The sample is solubilized with detergent, and the proteins are then separated by electrophoresis in a polyacrylamide gel. After electrophoresis, the gel is placed next to a thin, synthetic membrane that has a strong affinity for proteins. In the figure, the gel and membrane are placed between sheets of absorbent paper in a blotting tank. This arrangement allows buffer to flow across the gel and through the thin membrane. As a result, the proteins in the gel are transferred to the membrane by capillary action. Transfer of the proteins to the membrane may also be accomplished by an electrical current.

After the transfer step, the membrane is incubated with an antibody to a specific protein. This antibody may be produced in an experimental animal such as a mouse or rabbit, or in cells as a monoclonal antibody. The antibody may be coupled to an enzyme which can then be used to detect the antibody on the membrane. In the example shown, the antibody is coupled to horseradish peroxidase. The membrane is incubated with a substrate that is converted to a luminescent compound after reaction with this enzyme.  A sheet of X-ray film is then placed next to the membrane, which allows visualization of individual proteins. In a variation of the technique, an unlabeled first antibody is used to bind the protein on the membrane, and a second antibody, directed against the first antibody, is used for detection.

The main advantage of western analysis is that it does not require isotopic labeling of proteins and can be used with tissues and organs, as well as cultured cells.

A variation of the western blot is used to identify antibodies to human immunodeficiency virus in clinical specimens and donated blood. Viral proteins are fractionated by electrophoresis and transferred to a membrane as describe above. The membrane is then incubated with the clinical sample. If antibodies against HIV are present, they will react with one or more of the viral proteins on the membrane. Such an assay is being used to estimate the extent of infection with the retrovirus XMRV in the general population.

Note that the w in western blot is not capitalized. The n of northern analysis (a method in which RNA is detected on a thin membrane) is also lower case. However, Southern analysis deserves a capital S – it’s the last name of Edwin Southern, who in 1975 developed the technique for detecting DNA immobilized on a membrane.

Hosts: Vincent Racaniello and Alan Dove

On episode #89 of the podcast This Week in Virology, Vincent and Alan review recent findings on the association of the retrovirus XMRV with ME/CFS, reassortment of 2009 pandemic H1N1 influenza virus in swine, and where influenza viruses travel in the off-season.

Download TWiV #89 (56 MB .mp3, 78 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• Conflicting XMRV papers on hold
• Leak of PNAS paper
• CDC study on XMRV in CFS patients (Retrovirology) and Science update
• Where influenza viruses travel in the off season (EurekaAlert! and PLoS Pathogens)
• NPR article on Ebola siRNA treatment (thanks, Andreas!)
• Priming mechanism for reovirus entry (thanks, Agyeman-Badu!)
• Wired article on science PR (thanks, Dan!)
• Letters read on TWiV 89

Weekly Science Picks

Alan – Tree of Life graphic
Vincent - TEDx Oil Spill

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.

The findings by the NIH and FDA that XMRV is associated with chronic fatigue syndrome has been accepted for publication by the Proceedings of the National Academy of Sciences (PNAS). Release of the article has been blocked by PNAS due to work carried out by the US Centers for Disease Control and Prevention (CDC). That study, which was submitted to Retrovirology, failed to find a link between XMRV and CFS. Its publication has also been placed on hold. According to ScienceInsider:

The contradiction has caused “nervousness” both at PNAS and among senior officials within the Department of Health and Human Services, of which all three agencies are part, says one scientist with inside knowledge.

It is senseless to block publication because the two papers reach different conclusions. If both manuscripts were subjected to proper peer-review, and were deemed acceptable by the referees, then they should be published. The journal editorial offices must respect the opinions of the reviewers. By overriding their decisions, they have compromised the entire peer reviewer process.

Blocking publication also sends the wrong message to CFS patients, to the public, and scientists. Not only does this action raise suspicions about their motives – are they trying to publish only the result they believe is correct? – but it ignores the very important fact that science is self correcting. Scientists are humans, and they make mistakes. But eventually the right answer will come to the surface. And that is why PNAS and Retrovirology should respect peer review, publish the XMRV papers, and let science correct itself.

Update: As noted in the comments section, the results of the CDC study have been published in Retrovirology.

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 drobostore.com, fill out the questionnaire here.

Download TWiV #88 (68 MB .mp3, 91 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• Controlling the palm rhinoceros beetle with a virus
• The virologist in the Hawaiian shirt
• Information on Orcytes rhinocerus nudivirus (one, two, three)
• RNA based gene therapy for AIDS
• Reconstitution of an infectious human retrovirus (PLoS Pathogens)
• Letters read on TWiV 88

Weekly Science Picks

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

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.

On TWiV 87 a listener asked us to recommend suitable books for children about microbiology. I have since asked for suggestions on Twitter and Facebook, and have begun to compile the following list.

• The Invisible ABC’s by Rodney P. Anderson
• The Magic School Bus #6: The Giant Germ by Anne Capeci
• A World in a Drop of Water by Alvin and Virginia Silverstein
• The Usborne Complete Book of the Microscope by Kirsteen Rogers
• Jig, Jiggle, Sneeze by Joy Vitalis
• Germs Make Me Sick! by Melvin Berger
• Germ Stories by (Nobel prize winner) Arthur Kornberg (reviewed)
• Invisible Allies: Microbes that shape our lives by Jeanette Farrell
• Five Kids & A Monkey Investigate a Vicious Virus by Beth L. Blair
• DNA is Here to Stay by Fran Balkwill

If you know of good microbiology books for children (ages 5-teen) please add them to the comments section, or email them to virology@virology.ws and I’ll add them to this list.

Update: Thanks to the readers who have sent in their suggestions. They are listed above in the order in which I received them.

A press release from the Netherlands indicates that the FDA and NIH have independently confirmed the association of XMRV with chronic fatigue syndrome as published last fall in Science. Apparently two journalists for the Dutch magazine ORTHO obtained a copy of a lecture by Dr. Harvey Alter in Zagreb which confirms these findings. According to Newswire.com:

The ORTHO journalists were able to obtain a pdf document of the lecture given by Harvey Alter at the IPFA/PEI 17th Workshop on ‘Surveillance and screening of Blood Borne Pathogens’ in Zagreb. The International Plasma Fractionation Association (IPFA) represents the not-for-profit organizations around the world involved in plasma fractionation. The IPFA is based in Amsterdam, the Netherlands.

The highly-experienced Dr. Harvey Alter is Clinical Studies Chief at the Infectious Diseases and Immunogenetics Section of the Department of Transfusion Medicine at the NIH Clinical Center in Bethesda. “The data in the Lombardi, et al Science manuscript are extremely strong and likely true, despite the controversy”, was one comment on the XMRV findings reported by Alter in Zagreb. “Although blood transmission to humans has not been proved, it is probable. The association with CFS is very strong, but causality not proved. XMRV and related MLVs are in the donor supply with an early prevalence estimate of 3%‐7%. We (FDA & NIH) have independently confirmed the Lombardi group findings.”

ORTHO contacted Dr. Harvey Alter today for a reaction. He did not want to comment, but confirmed that a paper is soon to be published.

I’m not entirely sure what it means to have confirmed the Lombardi group findings. Did the FDA and NIH use the same clinical specimens, or independently collected ones? We’ll have to wait for the article to appear to find out.

Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and Graham Hatfull

On episode #87 of the podcast This Week in Virology, Vincent, Alan, and Rich hear from Professor Graham Hatfull how students in the Phage Hunters Integrating Research and Education (PHIRE) program learn about scientific inquiry by doing research on bacteriophages.

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 drobostore.com, fill out the questionnaire here.

Download TWiV #87 (62 MB .mp3, 86 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• Bacteriophage Research: Gateway to learning science
• Mycobacterium smegmatis at NCBI
• Prof. Steve Cresawn
• Scientist infected with computer virus (thanks, Jason!)
• The Invisible ABCs
• Letters read on TWiV 87

Weekly Science Picks

Rich - CDC Public Health Image Library
Alan – Great Microbiologists – A Lego Movie
Vincent - March of the Microbes by John L. Ingraham
Graham – Coral Reefs in the Microbial Seas by Forest Rohwer

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.

Hosts: Vincent Racaniello and Dickson Despommier

On episode 12 of the podcast “This Week in Parasitism”, Vincent and Dickson introduce the obligate intracellular protozoan Toxoplasma gondii, one of the most successful parasites on earth.

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

Links for this episode:

• Global clinical burden of malaria (PLoS Medicine)
• T. gondii sporulated oocysts (jpg)
• T. gondii tachyzoites in parasitophorous vacuole (jpg)
• T. gondii life cycle (jpg)
• Insect bioterrorism conference (thanks, Don!)
• Parasite of the Day (thanks, Douglas!)
• World Science Festival
• Letters read on TWiP 12

Download TWiP #12 (70 MB .mp3, 90 minutes)

Subscribe to TWiP (free) in iTunes, at the Zune Marketplace, by the RSS feed or by email

Send your questions and comments to twip@twiv.tv

The influenza virus neuraminidase (NA) protein is required for virus release from the cell, a property exploited by the antiviral drugs oseltamivir (Tamiflu) and zanamavir (Relenza). During clinical testing of oseltamivir in 2001, some individuals shed drug-resistant viruses with an amino acid change from histidine to tyrosine (H274Y) in NA. Such viruses are not inhibited by oseltamivir because the amino acid change leads to  decreased binding of the drug. But these viruses replicated less well in cell culture, and had reduced infectivity in ferrets. It was concluded that oseltamivir resistant influenza virus mutants would not spread in the population. Why was this conclusion wrong?

During the 2008-09 flu season oseltamivir resistant influenza H1N1 viruses with the H274Y change began to spread, and within a year they were found in most seasonal isolates. It was hypothesized that these viruses contained other amino acid changes that masked the deleterious effect of H274Y. The H274Y mutation does not affect the catalytic activity of the NA: the ability to cleave sialic acid from glycoproteins. However it does lead to a decease in the amount of NA protein that is transported to the surface of infected cells.

Computational methods were used to identify amino acids in NA that could potentially compensate for the effect of H274Y. A single amino acid change at position 194 of NA, when present with H274Y, restored NA on the cell surface to normal levels.

Did a similar amino acid change in seasonal H1N1 strains allow the spread of oseltamivir resistant viruses with H274Y? Introduction of this amino acid change into the seasonal H1N1 strains A/Texas/91 and A/New Caledonia/99 causes a decrease in surface NA. However the same change has a lesser effect on surface NA in cells infected with A/Solomon Islands/2006. Two amino acid changes were identified in the NA protein of recent oseltamivir-resistant seasonal H1N1 viruses that restore surface levels of NA in the presence of H274Y: V234M and R222Q.

It seems likely that the amino acid changes V234M and R222Q emerged first in the NA of seasonal H1N1 viruses. Why these changes appeared is unknown, but they could be a consequence of random drift, antigenic selection, or a need to balance HA and NA activities. Once these changes were in place, oseltamivir resistant viruses with the H274Y could be selected, and because they had no defect in fitness, they spread globally.

The conclusion is that H274Y in NA attenuates the fitness of influenza virus by reducing the amount of NA on the cell surface. Spread of such viruses in the population is impossible without secondary amino acid changes that restore adequate levels of surface NA. H274Y probably causes a defect in NA folding or transport that is balanced by the secondary mutations.

These findings are another example of how drug resistance frequently comes with a cost to protein stability or folding, and prevents evolution unless compensated by secondary mutations.

There have been scattered isolations of oseltamivir-resistant, pandemic 2009 H1N1 influenza virus with the H274Y change. Will these viruses spread globally, or are they less fit, evolutionary dead ends? Introduction of the H274Y change into the NA of 2009 pandemic H1N1 virus leads to a large decrease in surface NA. Unless the 2009 swine-origin viruses already produce excess NA, viruses with the H274Y change are not likely to spread without secondary mutations that rescue NA surface expression.

Bloom JD, Gong LI, & Baltimore D (2010). Permissive secondary mutations enable the evolution of influenza oseltamivir resistance. Science (New York, N.Y.), 328 (5983), 1272-5 PMID: 20522774

Hosts: Vincent Racaniello, Rich Condit, and Eric Delwart

In episode #86 of the podcast This Week in Virology, Vincent and Rich travel to the Blood Systems Research Institute in San Francisco to speak with Eric Delwart about his work on virus discovery.

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 drobostore.com, fill out the questionnaire here.

Download TWiV #86 (59 MB .mp3, 81 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• List of Dr. Delwart’s open-access journal articles (pdf) – to find each one, type PMID # into PubMed
• CDC says don’t give rotavirus vaccines to infants with SCID
• The Brighton Collaboration
• Product sheet for RotaTeq (pdf – thanks, Sheldon!)
• Letters read on TWiV 86

Weekly Science Picks

Rich - Google Crisis Response – Gulf of Mexico Oil Spill
Vincent - HHMI resources for teachers and students (thanks, Jim!)
Eric – Vaccine by Arthur Allen

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.

When infection with hepatitis C virus goes from acute to chronic, severe liver disease may occur which requires organ transplantation. Nearly 200 million people are chronically infected with HCV, necessitating approaches to preventing and treating infections. No HCV vaccine is available, and current antiviral therapy consists of administration of interferon plus ribavirin, a combination that is effective about half the time and is associated with undesirable side effects. New antiviral compounds that target a viral protease and RNA polymerase are currently in clinical trials may eventually reach the market. But our experience with HIV-1 has shown that combinations of three drugs are the most effective for derailing the emergences of drug resistant viruses. The third target for HCV could be NS5A, a viral protein without a known function.

To identify new inhibitors of HCV, a chemical library of one million compounds was screened for the ability to inhibit viral replication in cell culture. The active compound were then subjected to a second screen to eliminate inhibitors of known viral enzymes: the viral protease, RNA polymerase, and helicase. One of the remaining inhibitors was further refined chemically until a very potent derivative was obtained. This molecule, called BMS-790052, has a 50% inhibitory concentration in the picomolar range, and inhibits all the viral genotypes tested. It is the most powerful inhibitor of HCV discovered.

The compound was tested for safety and bioavailability in various animal species. After oral administration, the compound was found in plasma and liver, despite a molecular mass of over 700 daltons. Six different levels of the compound were tested in HCV infected individuals. No adverse effects were reported, and the highest amount administered reduced viral levels in the blood 2,000 fold after one day. These results are promising, but larger trials will now be needed to further confirm the safety and efficacy of the drug.

What is the target of BMS-790052? Two lines of evidence suggest that the compound inhibits the viral protein NS5A. The drug appears to bind NS5A, and viruses resistant to the drug have amino acid changes in this protein. Although NS5A is known to be required for viral replication, its precise function is not known. Because NS5A does not have an easily assayable enzymatic function, it has not previously been a target of drug discovery. The identification of a compound that inhibits NS5A function is an important step forward in HCV drug development. The general approach used to discover BMS-790052 should be useful in identifying inhibitors of other viral proteins that do not have well defined and measurable activities.

I discussed this paper on Futures in Biotech episode #60. If you would like to listen only to the conversation about BMS-790052, download this mp3 file, or listen to the discussion below.

Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN, Fridell RA, Serrano-Wu MH, Langley DR, Sun JH, O’Boyle DR 2nd, Lemm JA, Wang C, Knipe JO, Chien C, Colonno RJ, Grasela DM, Meanwell NA, & Hamann LG (2010). Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature, 465 (7294), 96-100 PMID: 20410884

Hosts: Vincent Racaniello and Michael Gale

On episode 85 of the podcast This Week in Virology, Vincent and Michael Gale discuss the origin, pathogenesis, prevention, of hepatitis C virus, and how it evades innate immune responses.

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

Download TWiV #85 (40 MB .mp3, 56 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• The Gale Laboratory at the University of Washington
• Incredible view from the Gale laboratory (jpg)
• Evasion and disruption of innate immune signalling by hepatitis C and West Nile viruses (review)
• New potent HCV inhibitor
• HCV virion and genome structures at ViralZone

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.

Robert H. Silverman, one of the authors on the study implicating the new human retrovirus XMRV as an etiologic agent of chronic fatigue syndrome, has written an excellent review article on the current status of research on the virus. The article is behind a paywall at Nature Reviews Urology, so I’ll provide a summary of the salient points.

The article begins with a description of how XMRV DNA was isolated from surgically removed prostate tumor tissue. Sequence analysis of three strains showed that the virus is most closely related to xenotropic and polytropic murine leukemia viruses and hence was named xenotropic murine leukemia virus-related virus, or XMRV. Five lines of evidence indicate that XMRV is not a laboratory contaminant:

• XMRV was detected in RNA isolated from human prostate tissue
• Mouse sequences were not detected in the human prostate tissues
• Infections were mainly found in tissues from humans with an alteration in the protein RNAse L
• Slightly different viral sequences (polymorphisms) were identified in isolates from different patients
• Both viral RNA and viral proteins were detected in prostate tissues

The article continues with a summary of  subsequent studies in which XMRV has or has not been detected in prostate cancer, then moves to the role of XMRV in CFS. Silverman concludes that “the scientific literature shows that XMRV was detected in the majority, but not all, prostate cancer studies, albeit at different rates, while XMRV was found in CFS in only one study of four published to date.” He offers the the following explanations for the difference in detection of XMRV in prostate cancer and CFS:

• Viral contamination from mouse sources. This cannot explain all positive findings in different laboratories using different experimental techniques.
• Geographical differences in the distribution of XMRV could account for some of the differences.
• Sequence differences could lead to failure to detect viral DNA by polymerase chain reaction.
• There are no standardized, sensitive methods of detection, and no widely available positive control samples.

The remainder of the article entails speculation on how XMRV might cause prostate cancer; the likely origin of the virus from a rodent virus; and antiviral drugs that are known to inhibit replication of the virus. It ends with the suggestion to test for the presence of XMRV in porcine tissues used for human transplantation, and in the blood supply, to avoid additional infections.

Silverman concludes:

Although other retroviruses of the same genus as XMRV (gammaretroviruses) cause cancer and neurological disease in animals, whether XMRV is a cause of either prostate cancer or CFS remains unknown.

Robert H. Silverman, Carvell Nguyen, Christopher J. Weight & Eric A. Klein. The human retrovirus XMRV in prostate cancer and chronic fatigue syndrome. Nature Reviews Urology doi: 10.1038/nrurol.2010.77.

Hosts: Vincent Racaniello and Dickson Despommier

On episode 11 of the podcast “This Week in Parasitism”, Vincent and Dickson continue their discussion of malaria, with emphasis on clinical aspects of the disease.

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

Links for this episode:

• The quest for a malaria vaccine (Meet the Scientist 49)
• Science issue on malaria and tuberculosis
• Malaria pathogenesis (pdf)
• Malaria pathogenesis (jpg)
• Letters read on TWiP 11

Download TWiP #11 (63 MB .mp3, 87 minutes)

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Send your questions and comments to twip@twiv.tv

One property of viruses that is difficult to conceptualize is their small size. I can tell you that viruses can be anywhere from 20 to 750 nanometers in diameter, but that’s not easy to visualize, even for those of us who routinely work with small measurements. One way to demonstrate how small viruses are is to compare them with animal and plant cells, bacteria, proteins, molecules, and atoms, as shown below:

But even comparisons of this type fall short because they do not provide a readily grasped real-life reference. A better way was suggested by my colleague Karla Kirkegaard: Start by multiplying the size of viruses and humans one million times. A supine human would then extend from California to Colorado. At this scale, a cell would be about the size of a lecture hall. Depending on their size, viruses would either be lemons (poliovirus, 30 nanometers), grapefruits (retroviruses, 100 nanometers), or watermelons (poxvirus, 250 nanometers).

Peter Palese has a different way of relating the small size of viruses. If you magnify a virus so it is the size of a human fist, then a cell would be about half the size of the Empire State Building.

Hosts: Vincent Racaniello, Rich Condit, Dave Bloom, and Grant McFadden

On episode #84 of the podcast This Week in Virology, Vincent and Rich spoke with Dave Bloom and Grant McFadden about their work on herpesviruses and poxviruses in this episode recorded before an audience at the University of Florida, Gainesville – home of the Gators.

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

Win a free Drobo S! Contest rules here.

Download TWiV #84 (71 MB .mp3, 99 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• Epigenetic modulation of herpes simplex virus gene expression (thanks, Matthew!)
• The Red Queen and Tierra virtual environment: article one, two, three (thanks, Jesper!)
• Hand-held HIV detector (thanks, Jim!)
• Anti-angiogenic cancer therapy combined with oncolytic virotherapy (thanks, Bill!)
• TWiV at UF Gainesville (jpg)
• Letters read on TWiV 84

Weekly Science Picks

Rich - Charles F. Littlewood photographs
Vincent - Not so humble pie (thanks, Sophie!)
Grant – The Strangest Man by Graham Farmelo
David – Is Parkinson’s Disease a prion disorder?

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.

A serious shortcoming of current influenza virus vaccines is the need to reformulate them every year or two as the virus undergoes antigenic drift. Many virologists have been captivated by the idea of a more universal vaccine that would endure longer, perhaps a decade or more. The identification of a conserved domain in the stalk region of the viral HA protein that gives rise to antibodies that block infection by 10 HA subtypes was a step in this direction. The next phase in the development of a new vaccine, the production of an antigen that confers broader protection, has been achieved using an HA molecule lacking the globular head.

The vast majority of antibodies that block influenza virus infection are directed against the globular head of the HA, the protein essential for attachment to and entry into cells. Unfortunately, the HA head undergoes significant antigenic variation. The HA stalk is more conserved, but this portion of the molecule is likely to be masked by the globular head and therefore not readily recognized receptors on antibody-producing B cells.

To make the HA stalk more accessible, an altered molecule was designed that lacks the globular head. To test whether this protein could give rise to broadly neutralizing antibodies, mice were immunized first with plasmid DNA encoding the truncated protein, and then with virus-like particles bearing the headless HA. When mice were immunized with headless HA from a 1934 H1N1 strain, all survived after intranasal challenge with the homologous virus. The sera from these mice showed broader reactivity against H2N2, H5N1, and 2009 pandemic H1N1 viruses than sera from mice immunized with full-length HA.

While the observations are encouraging, they are not unequivocally positive. For example, it was not possible to demonstrate neutralizing antibodies in sera from mice immunized with headless H1 or H3 HA proteins. Furthermore, the ability of headless H3 HA to protect mice from challenge infection was not determined. Nevertheless, the results show that vaccination with a headless HA confers protection against antigenically diverged influenza virus strains. The authors conclude “Through further development and testing, we predict that a single immunization with a headless HA vaccine will offer effective protection through several influenza epidemics.” Not an influenza vaccine for life, but perhaps more enduring than those available today.

Steel, J. (2010). An Influenza Virus Vaccine Based on the Conserved Hemagglutinin Stalk Domain mBio DOI: 10.1128/mBio.00018-10

I joined Marc Pelletier on episode 60 of Futures in Biotech for a conversation with Dave Brodbeck, George Farr, and Andre Nantel. We talked about primate face recognition, discovery of a new antiviral compound to treat hepatitis C virus infection, changing the length of a codon from three to four bases, and the sequence of the neanderthal genome.

Download FiB #60 (44 MB .mp3, 91 minutes)

Video courtesy of Team ODTV

Download video (179 MB .mp4)

Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and Kirsten Sanford

On episode #83 of the podcast This Week in Virology, Vincent, Alan, Rich, and special guest Dr. Kirsten Sanford talk about her career in science media, then consider whether smallpox eradication led to the AIDS pandemic, high fidelity RNA synthesis, and a new Ebola virus vaccine.

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

Win a free Drobo S! Contest rules here.

Download TWiV #83 (66 MB .mp3, 91 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• Does smallpox vaccine protect against HIV? (thanks, Srinivas; Washington Post and BMC Immunology)
• Was the deltaCRR5 mutation fixed in the human population by smallpox?
• A proofreader in the SARS coronavirus genome
• New ebola vaccine protects monkeys
• House of Numbers trailer, website, Wikipedia article (thanks, Levi!)
• Letters read on TWiV 83

Weekly Science Picks

Alan – Evernote
Rich - The Knife Man by Wendy Moore
Vincent - The Pump Handle

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.

In the final lecture of my virology course, Professor Dickson Despommier weaves a story about the arrival of West Nile virus to the United States in the summer of 1999. This is a special treat that you won’t want to miss, as Prof. Despommier is a wonderful storyteller.

Download: .wmv (422 MB) | .mp4 (108 MB)

Visit the virology W3310 home page for a complete list of course resources.

Hosts: Vincent Racaniello and Dickson Despommier

On episode 10 of the podcast “This Week in Parasitism”, Vincent and Dickson trace the life cycle of Plasmodium in a mosquito and in a human host.

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

Links for this episode:

• Mosquito cycle – sporogany (jpg)
• Plasmodium falciparum cycle (jpg)
• Plasmodium vivax cycle (jpg)
• Plasmodium falciparum ring forms and gametocytes in blood (jpg)
• Plasmodium stages (jpg)
• Letters read on TWiP 10

Download TWiP #10 (62 MB .mp3, 86 minutes)

Subscribe to TWiP (free) in iTunes, at the Zune Marketplace, by the RSS feed or by email

Send your questions and comments to twip@twiv.tv

An important question about the retrovirus XMRV, which has been implicated in prostate cancer and chronic fatigue syndrome, is where the virus replicates in humans. Such information would provide clues about how infection might be transmitted. To date the virus has been detected in malignant prostate cells and in the peripheral blood mononuclear cells and plasma of patients with CFS. A new study reveals that XMRV is present in respiratory secretions.

Polymerase chain reaction was used to detect XMRV in 267 respiratory samples taken from German patients. One group comprised sputum and nasal swab specimens from 75 travelers from Asia who had respiratory tract infections. The second group consisted of 31 bronchoalveolar lavage samples from patients with chronic obstructive pulmonary disease, while samples from the third group were from 161 immunosuppressed patients with severe respiratory tract infections. The study included 62 healthy controls. It should be noted that none of the patients had been diagnosed with CFS.

XMRV sequences were detected in 3 of 75 samples (2.3%) in group 1, 1 of 31 samples (3.2%) in group 2, and 16/161 (9.9%) in group 3. Six of the XMRV-positive samples in the second group also contained rhinovirus, adenovirus, or pathogenic fungi. The higher rate of detection of XMRV and other microbes in immunosuppressed individuals is not unexpected. The control group contained 2 of 62 samples (3.2%) positive for XMRV.

The presence of XMRV in PBMCs and plasma suggests a blood-borne route of transmission of the virus: transfusions, health care associated needle sticks, and intravenous drug use. Does finding XMRV in the respiratory tract prove that the virus can be transmitted by the respiratory route? No, not until we have other information, including the level of virus in respiratory secretions, and the infectivity of XMRV. In this context it is interesting to note that it was not possible to isolate infectious XMRV from the respiratory tract of the German patients.

Reviewing the transmission of another human retrovirus, HIV-1, is instructive in understanding the pathogenesis of XMRV infection. The main modes of transmission of HIV-1 are sexual, parenteral, and from mother to infant. These routes of transmission are consistent with levels of infectious virus in body fluids (shown in this table). Viral RNA can be detected at several levels of the respiratory tract, but respiratory secretions rarely transmit HIV.

FIsher, N., Schulz, C., Stieler, K., Hohn, O., Lange, C., Drosten, C., & Aepfelbacher, M. (2010). Xenotropic murine leukemia virus-related gammaretrovirus in respiratory tract Emerg. Inf. Dis. : 10.3201/eid1606.100066

Google Flu Trends uses analysis of large numbers of search queries to track influenza-like illness in a population. The idea is that the frequency of certain queries correlates with the percentage of physician visits in which a patient presents with influenza-like symptoms. Google claims that it can accurately estimate the level of weekly influenza activity in each region of the United States. But a recent study shows that Google Flu Trends is not as accurate at estimating rates of laboratory-confirmed influenza as surveillance carried out by the CDC.

Google Flu Trends and CDC surveillance results were compared for the period of  2003 – 2008. As reported at the 2010 American Thoracic Society Conference, the greatest deviation of Google Flu Trends from CDC surveillance occurred during the 2003-04 influenza season. That year was characterized by early and frequent influenza activity, many pediatric deaths, and heavy focus by the news media.

The main reason for the discrepancy is likely the fact that influenza may only account for 20-70% of influenza-like illnesses. The remainder are caused by other viruses that produce similar clinical syndromes. The authors of the study concluded that “Internet search behavior is likely different during anomalous seasons such as during 2003-4…during periods of intense media interest or unexpected influenza activity such as the 2009 H1N1 influenza pandemic, Google Flu Trends may be least accurate at estimating influenza activity.”

Google Flu Trends does provide an estimate of influenza activity more quickly and cheaply than can be achieved in a diagnostic laboratory. But in this case, cheaper and quicker means less accurate.

Ginsberg, J., Mohebbi, M., Patel, R., Brammer, L., Smolinski, M., & Brilliant, L. (2008). Detecting influenza epidemics using search engine query data Nature, 457 (7232), 1012-1014 DOI: 10.1038/nature07634

Download: .wmv (350 MB) | .mp4 (96 MB)

Visit the virology W3310 home page for a complete list of course resources.

Hosts: Vincent Racaniello and Rich Condit

On episode #82 of the podcast This Week in Virology (TWiV), Vincent and Rich talk about how thymic selection of T cells might lead to better control of HIV-1 infection, and a mouse model for severe antibody-induced dengue virus disease.

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

Win a free Drobo S! Contest rules here.

Download TWiV #82 (59 MB .mp3, 82 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

• Effect of thymic selection of T-cells on control of AIDS
• Mouse model of antibody-induced severe dengue virus disease
• Natural antibody protects against viral infection
• Kary Mullis idea for fighting infections (thanks, Erik!)
• 40 nm resolution of fluorescence photoactivation localization microscopy (thanks, José)
• Letters read on TWiV 82

Weekly Science Picks

Rich The Mold in Dr. Florey’s Coat: The Story of the Penicillin Miracle by Eric Lax
Vincent Proteopedia (thanks, Erik!)

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.

The US Food and Drug Administration has updated its recommendations on both Rotarix and RotaTeq, vaccines for the prevention of rotavirus disease in infants:

Based on careful evaluation of a variety of scientific information, FDA has determined it is appropriate for clinicians and health care professionals to resume the use of Rotarix and to continue the use of RotaTeq.

In making its recommendation, the FDA considered the strong safety records of both vaccines, including clinical trials in tens of thousands of individuals and the use of the vaccines in millions of recipients. There is no evidence that either porcine circovirus type 1 or type 2 poses a safety risk to humans, and neither virus is known to infect humans or cause disease.

The FDA also noted that the benefits of the rotavirus vaccines are considerable and outweigh the small theoretical risk of the viral contaminant.

The product labels will be updated to reflect the fact that the vaccine contains a PCV contaminant. In addition, GlaxoSmithKline will rederive Rotarix so that it does not contain PCV. Merck has not yet made a decision about whether they should produce a PCV-free rotavirus vaccine. But if my suggestion carries any weight at Merck (I know it does not), they should not hesitate to follow GlaxoSmithKline’s lead.

Download: .wmv (346 MB) | .mp4 (91 MB)

Visit the virology W3310 home page for a complete list of course resources.

Bovine viral diarrhea virus is an economically important animal pathogen that may cause a fatal gastrointestinal disease in beef and dairy herds. Infection of a fetus with this virus during the first trimester leads to the birth of animals that are persistently infected for life. Some animals remain healthy, while others develop severe mucosal disease. The lethal outcome is a consequence of RNA recombination that produces a cytopathic virus.

Pathogenicity of bovine viral diarrhea virus is associated with the synthesis of a the viral protein NS3. This protein is not produced by the noncytopathic virus that persistently infects cows for life. Absence of the protein is due to failure to cleave the precursor of NS3, called NS2-3. In cells infected with the cytopathic, disease-causing virus, NS3 is produced because the virus has acquired an extra cleavage site. This difference is illustrated in the diagram (click for a larger view).

The extra cleavage site in the viral protein is acquired when the viral RNA of the noncytopathic virus recombines with cellular RNA. This exchange of sequence probably occurs when the enzyme copying the viral RNA briefly switches to a cellular RNA, and then back to the viral RNA. The result is a copy of the viral RNA into which a cellular sequence has been inserted.

The cleavage site for NS3 can be created in several ways. One of the most frequent is the insertion of a cellular RNA sequence coding for ubiquitin (UCH in the diagram). This small protein can be cleaved by members of a family of cellular proteases (proteases are enzymes that cut proteins). The insertion of ubiquitin leads to cleavage of NS2-3 and the production of NS3. The recombinant viruses replicate faster than noncytopathic viruses and cause disease in cattle. Why pathogenicity is associated with release of the NS3 protein, which is involved in viral RNA synthesis, is not known.

The production of pathogenic pestiviruses by recombination with cellular RNA is another illustration of the many unexpected pathways of viral evolution.

Meyers, G., Tautz, N., Dubovi, E., & Thiel, H. (1991). Viral cytopathogenicity correlated with integration of ubiquitin-coding sequences. Virology, 180 (2), 602-616 DOI: 10.1016/0042-6822(91)90074-L

Download: .wmv (393 MB) | .mp4 (102 MB)

Visit the virology W3310 home page for a complete list of course resources.