MicrobiologyBytes

Mimivirus is the largest virus known to scientists, about half of a micrometre (0.0005 millimeter) in diameter. It is more than 10 times larger than the virus that causes the common cold and – unlike other viruses – is large enough to be seen with a light microscope. An international team of researchers have now determined key structural features of Mimivirus, findings that could help scientists study how the simplest life forms evolved and whether this unusual virus causes any human diseases. Mimivirus infects amoebae, but it is also thought that it may act as a human pathogen, because antibodies to the virus have been discovered in people with pneumonia. However, many details about the virus remain unknown. Now researchers have determined the basic design of the virus’s outer shell, or capsid, and also of the hundreds of smaller units – called capsomeres – making up this outer shell. Their findings confirmed the existence of a starfish-shaped structure that covers a ‘special vertex’ – an opening in the capsid where the genetic material leaves the virus to infect its host; an indentation in the virus’s genetic material itself is positioned opposite this opening.

The findings are important in terms of studying the evolution of cells, bacteria and viruses. Mimivirus is like an intermediate between a cell and a virus. We usually think of cells as being alive and a virus is thought of as being non-living because it needs a host cell to complete its life cycle. Mimivirus straddles a middle ground between viruses and living cells, perhaps redefining what a virus is. Scientists had previously been unable to determine the virus’s structure because they had assumed that, like many other viruses, it’s capsid had a design known as icosahedral symmetry. These authors discovered the true structure when they tried reconstructing the virus, assuming it had not the standard icosahedral symmetry but another configuration called five-fold symmetry. If you start out thinking the object has icosahedral symmetry, then you assume there are 60 identical pieces, and that influences how you reconstruct the virus’s structure.

The researchers took images of the virus using an atomic force microscope, revealing a pattern of holes regularly spaced throughout the virus’s outer shell. The capsids of most other large, pseudo-icosahedral viruses do not contain such holes, and their function is unknown. The researchers used cryo-electron microscopy reconstruction to determine the structural details. This reconstruction method enabled them to reassemble three-dimensional images from two-dimensional pictures, much as a complete architectural drawing of a house can be assembled with two-dimensional drawings of the sides, the roof and other elements. An icosahedron has a roughly spherical shape containing 20 triangular facets and 60 identical subunits. Like an icosahedron, the mimivirus capsid also has 20 facets. However, unlike an icosohedron, five facets of the capsid are slightly different than the others and surround the special vertex. Icosohedra contain 12 similar vertices, whereas the mimivirus contains eleven such vertices, with the 12th being different than the others.

Structural studies of the giant Mimivirus. 2009 PLoS Biol 7(4): e1000092
Mimivirus is the largest known virus whose genome and physical size are comparable to some small bacteria, blurring the boundary between a virus and a cell. Structural studies of Mimivirus have been difficult because of its size and long surface fibers. Here we report the use of enzymatic digestions to remove the surface fibers of Mimivirus in order to expose the surface of the viral capsid. Cryo-electron microscopy (cryoEM) and atomic force microscopy were able to show that the 20 icosahedral faces of Mimivirus capsids have hexagonal arrays of depressions. Each depression is surrounded by six trimeric capsomers that are similar in structure to those in many other large, icosahedral doublestranded DNA viruses. Whereas in most viruses these capsomers are hexagonally close-packed with the same orientation in each face, in Mimivirus there are vacancies at the systematic depressions with neighboring capsomers differing in orientation by 608. The previously observed starfish-shaped feature is well-resolved and found to be on each virus particle and is associated with a special pentameric vertex. The arms of the starfish fit into the gaps between the five faces surrounding the unique vertex, acting as a seal. Furthermore, the enveloped nucleocapsid is accurately positioned and oriented within the capsid with a concave surface facing the unique vertex. Thus, the starfish-shaped feature and the organization of the nucleocapsid might regulate the delivery of the genome to the host. The structure of Mimivirus, as well as the various fiber components observed in the virus, suggests that the Mimivirus genome includes genes derived from both eukaryotic and prokaryotic organisms. The three-dimensional cryoEM reconstruction reported here is of a virus with a volume that is one order of magnitude larger than any previously reported molecular assembly studied at a resolution of equal to or better than 65 A°.

Related:

• Marvellous mimivirus
• Mimivirus and the Stargate
• Marine mimivirus relatives are large algal viruses

Originally, this darn virus infected pigs, and it was reasonable to call it swine flu (even though it appears that some people don’t know that “swine” means “pig” :-)

But after it jumped from pigs to humans – the World Health Organization agrees that extensive human-to-human transmission is now happening – it’s no longer reasonable to go on calling it swine flu, and we have to find a new name.

So I’m following President Obama’s example, and from now on, I’ll be calling it H1N1. This virus has a longer scientific name (to distinguish it from other H1N1 viruses), but everyone’s going to know what you mean, so H1N1 is OK. (By the way, it is formally H1N1, not h1n1, but no-one at MicrobiologyBytes is going to worry about that).

So if you want to stay up to date with all the latest news about the H1N1 pandemic, follow MicroBytes on Twitter, where you’ll get the latest and most accurate H1N1 news.

Now where’s that bacon sandwich? :-)

Because of its central position in the microbial research community, the Gram-negative bacterium Escherichia coli plays a leading role in investigations of the fundamental molecular biology of bacteria. This experimentally tractable microbe is a workhorse in basic and applied research aimed at elucidating the mechanistic basis of prokaryotic processes and traits, including those of pathogens. The ever-expanding availability of genomic resources makes E. coli particularly well-suited to systematic investigations of microbial protein components and functional relationships on a global scale. These include a genome-wide collection of single-gene deletion strains along with extensive knowledge of regulatory circuits and metabolic pathways. Yet despite being the most highly studied model bacterium, a recent comprehensive community annotation effort for the fully sequenced reference K-12 laboratory strains indicated that only half (54%) of the protein-coding gene products of E. coli currently have experimental evidence indicative of a biological role. The remaining genes have either only generic, homology-derived functional attributes (e.g. “predicted DNA-binding”) or no discernable physiological significance. Some of these functional “orphans” may have eluded characterization in part because they exhibit mild mutant phenotypes, are expressed at low or undetectable levels, or have limited homology to annotated genes.

One goal of modern biology is to chart groups of proteins that act together to perform biological processes via direct and indirect interactions. Such groupings are sometimes called functional modules. The types of protein interaction within modules include physical interactions that generate protein complexes and biochemical associations that make up metabolic pathways. Researchers have combined proteomic and bioinformatic tools and used them to decipher a large number of protein interactions, complexes and functional modules with high confidence. In addition, exploring the topology of the resulting interaction networks, they successfully predicted specific biological roles for a number of proteins with previously unknown functions, and identified some potential drug targets. Although their work is focused on E. coli, their phylogenetic projections suggest that a considerable fraction of observations and predictions can be extrapolated to many other bacterial taxa. As all the data derived from this study are publicly available (at eNet), others may build on this work for further hypothesis-driven studies of gene function discovery.

Global functional atlas of Escherichia coli encompassing previously uncharacterized proteins. PLoS Biol 7(4): e1000096
One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans’ biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a ‘‘systems-wide’’ functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.

Related:

• An introduction to genomics (video)
• Digitizing Life (video)
• The end of the world? Dr Franken-Venter? Nope

New research published yesterday (Monday April 27) from the University of Leicester and University Hospitals of Leicester NHS Trust warns of a six-month time lag before effective vaccines can be manufactured in the event of an influenza pandemic. By that time, the first wave of pandemic flu may be over before people are vaccinated, says Dr Iain Stephenson, Consultant in Infectious Diseases at the Leicester Royal Infirmary and a Clinical Senior Lecturer at the University of Leicester.

Pandemic preparedness plans show that vaccination is critical for controlling pandemics. Some authorities have invested in vaccine stockpiles, but these resources are small in comparison to global demand. The use of stockpiled vaccine is challenged by the need for two doses and secondary manufacturing constraints. MF59, a proprietary adjuvant, was licensed in seasonal influenza vaccines in 1997, and more than 30 million doses have been administered safely so far. These new findings suggest that consideration could be given to advance priming to induce memory responses that enable cross-reactive antibodies to be generated rapidly after infection with the pandemic virus or by a single low-dose vaccination when required at the onset of future pandemic.

Fast rise of broadly cross-reactive antibodies after boosting long-lived human memory B cells primed by an MF59 adjuvanted prepandemic vaccine. PNAS USA April 27, 2009
Proactive priming before the next pandemic could induce immune memory responses to novel influenza antigens. In an open-label study, we analyzed B cell memory and antibody responses of 54 adults who received 2 7.5-μg doses of MF59-adjuvanted A/Vietnam/1194/2004 clade 1 (H5N1) vaccine. Twenty-four subjects had been previously primed with MF59-adjuvanted or plain clade 0-like A/duck/Singapore/1997 (H5N3) vaccine during 1999–2001. The prevaccination frequency of circulating memory B cells reactive to A/Vietnam/1194/2004 was low in both primed and unprimed individuals. However, at day 21 after boosting, MF59-adjuvanted primed subjects displayed a higher frequency of H5N1-specific memory B cells than plain-primed or unprimed subjects. The immune memory was rapidly mobilized by a single vaccine administration and resulted in high titers of neutralizing antibodies to antigenically diverse clade 0, 1, and 2 H5N1 viruses already at day 7. In general, postvaccination antibody titers were significantly higher in primed subjects than in unprimed subjects. Subjects primed with MF59-adjuvanted vaccine responded significantly better than those primed with plain vaccine, most notably in early induction and duration of cross-reacting antibody responses. After 6 months, high titers of cross-reactive antibody remained detectable among MF59-primed subjects. We conclude that distant priming with clade 0-like H5N3 induces a pool of cross-reactive memory B cells that can be boosted rapidly years afterward by a mismatched MF59-adjuvanted vaccine to generate high titers of cross-reactive neutralizing antibodies rapidly. These results suggest that pre-pandemic vaccination strategies should be considered.

Related:

• 10 things you should know about H1N1 (swineflu)
• 10 more things you should know about H1N1 (swineflu)

Acquired immunodeficiency syndrome (AIDS) has killed more than 25 million people since 1981 and more than 30 million people (22 million in sub-Saharan Africa alone) are now infected with the human immunodeficiency virus (HIV), which causes AIDS. HIV destroys immune system cells (including CD4 cells, a type of lymphocyte), leaving infected individuals susceptible to other infections. Early in the AIDS epidemic, most HIV-positive people died within ten years of infection. Then, in 1996, highly active antiretroviral therapy (ART) – combinations of powerful antiretroviral drugs – was developed and the life expectancy of HIV-infected people living in affluent countries improved dramatically. Now, in industrialized countries, all-cause mortality (death from any cause) among HIV-infected patients treated successfully with ART is similar to that of the general population and the mortality rate (the number of deaths in a population per year) among patients with HIV/ AIDS is comparable to that among patients with diabetes and other chronic conditions.

Unfortunately, combination ART is costly, so although HIV/AIDS quickly became a chronic disease in industrialized countries, AIDS deaths continued unabated among the millions of HIV-infected people living in low- and middle-income countries. Then, in 2003, governments, international agencies and funding bodies began to implement plans to increase ART coverage in developing countries. By the end of 2007, nearly three million people living with HIV/AIDS in these countries were receiving ART – nearly a third of the people who urgently need ART. In sub-Saharan Africa more than 2 million people now receive ART and mortality in HIV-infected patients who have access to ART is declining. However, no-one knows how mortality among HIV-infected people starting ART compares with non-HIV related mortality in sub-Saharan Africa. This information is needed to ensure that appropriate health services (including access to ART) are provided in this region. In a new study, researchers compared mortality rates among HIV-infected patients starting ART with non-HIV related mortality in the general population of four sub-Saharan countries.

The researchers obtained estimates of the number of HIV-unrelated deaths and information about patients during their first two years on ART at five antiretroviral treatment programs in the Cote d’Ivoire, Malawi, South Africa, and Zimbabwe from the World Health Organization Global Burden of Disease project and the International epidemiological Databases to Evaluate AIDS initiative. They then calculated the excess mortality rates among the HIV-infected patients (the death rates in HIV-infected patients minus the national HIV-unrelated death rates) and the standardized mortality rate (SMR; the number of deaths among HIV-infected patients divided by the number of HIV-unrelated deaths in the general population). The excess mortality rate among HIV-infected people who started ART when they had a low CD4 cell count and clinically advanced disease was 17.5 per 100 person-years of follow-up. For HIV-infected people who started ART with a high CD4 cell count and early disease, the excess mortality rate was 1.0 per 100 person-years. The SMRs over two years of ART for these two groups of HIV-infected patients were 47.1 and 3.4, respectively. Finally, patients who started ART with a high CD4 cell count and early disease who survived the first year of ART had an excess mortality of only 0.27 per 100 person-years and an SMR over two years followup of only 1.14.

These findings indicate that mortality among HIV-infected people during the first two years of ART is higher than in the general population in these four sub-Saharan countries. However, for patients who start ART when they have a high CD4 count and clinically early disease, the excess mortality is moderate and similar to that associated with diabetes. Because the researchers compared the death rates among HIV-infected patients with estimates of national death rates rather than with estimates of death rates for the areas where the ART programs were located, these findings may not be completely accurate. Nevertheless, these findings support further expansion of strategies that increase access to ART in sub-Saharan Africa and suggest the excess mortality among HIV-infected patients in this region might be largely prevented by starting ART before an individual’s HIV infection has progressed to advanced stages.

Mortality of HIV-Infected Patients Starting Antiretroviral Therapy in Sub-Saharan Africa: Comparison with HIV-Unrelated Mortality. 2009 PLoS Med 6(4): e1000066

Mortality in HIV-infected patients who have access to highly active antiretroviral therapy (ART) has declined in sub-Saharan Africa, but it is unclear how mortality compares to the non-HIV–infected population. We compared mortality rates observed in HIV-1–infected patients starting ART with non-HIV–related background mortality in four countries in sub- Saharan Africa. Patients enrolled in antiretroviral treatment programmes in Cote d’Ivoire, Malawi, South Africa, and Zimbabwe were included. We calculated excess mortality rates and standardised mortality ratios (SMRs) with 95% confidence intervals (CIs). Expected numbers of deaths were obtained using estimates of age-, sex-, and country-specific, HIV-unrelated, mortality rates from the Global Burden of Disease project. Among 13,249 eligible patients 1,177 deaths were recorded during 14,695 person-years of follow-up. The median age was 34 y, 8,831 (67%) patients were female, and 10,811 of 12,720 patients (85%) with information on clinical stage had advanced disease when starting ART. The excess mortality rate was 17.5 (95% CI 14.5–21.1) per 100 person-years SMR in patients who started ART with a CD4 cell count of less than 25 cells/ml and World Health Organization (WHO) stage III/IV, compared to 1.00 (0.55–1.81) per 100 person-years in patients who started with 200 cells/ml or above with WHO stage I/II. The corresponding SMRs were 47.1 (39.1–56.6) and 3.44 (1.91– 6.17). Among patients who started ART with 200 cells/ml or above in WHO stage I/II and survived the first year of ART, the excess mortality rate was 0.27 (0.08–0.94) per 100 person-years and the SMR was 1.14 (0.47–2.77). Mortality of HIV-infected patients treated with combination ART in sub-Saharan Africa continues to be higher than in the general population, but for some patients excess mortality is moderate and reaches that of the general population in the second year of ART. Much of the excess mortality might be prevented by timely initiation of ART.

Related:

• HIV treatment in Africa as successful as in Europe, if started in time
• HIV gene therapy trial promising
• HIV “can never be cured”
• Retroviruses
• HIV mutation and the immune response
• Immune exhaustion in HIV infection
• How does HIV cause AIDS?

Under the proposed new EU rules, broadband providers will be legally able to limit the number of websites you can look at, and to tell you whether or not you are allowed to use particular services. It will be dressed up as “new consumer options” which people can choose from. People will be offered TV-like packages – with a limited number of options for you to access.

Sounds scary? If so, then you need to find out more about the proposed EU Telecoms Package that will destroy a key part of internet openness by allowing telecoms companies to discriminate in the way that they handle IP packets according to their type. This will be voted on on May 5th 2009.

Find your MEP now (other EU countries) and send them this suggested letter (or your own version). You might like to remind them that the European elections take place on 4th June 2009 and they’ll be looking for your vote ;-)
You can also join the Blackout Europe Facebook group to stay up to date on the issue.

Ebola and Marburg virus are filoviruses that cause outbreaks of highly lethal haemorrhagic fever. Mortality rates in these diseases average more than 50%, with the highest recorded rates seen for Ebola Zaire virus (88%) and Marburg Angola virus (90%). Infection with these filoviruses produces a very high fever followed by interference with blood coagulation and vascular permeability, causing internal bleeding, bruising and skin rashes. After an asymptomatic incubation period, which can last days to weeks, symptoms of a typical filovirus infection emerge; headache, nausea, fever and malaise followed by more serious haemorrhagic symptoms and, in fatal cases, death results from multi-organ failure owing to shock.

Present treatments for filovirus infection are palliative, and consist primarily of supportive care, including hydration and pain management. There is no effective treatment or cure for these diseases. Therefore, vaccine development is crucially important as a strategy for fighting filovirus outbreaks. However, vaccine efficacy testing for Ebola virus is very difficult. There is no readily identifiable high-risk human population that can be targeted for a placebo-controlled clinical trials because disease outbreaks are unpredictable and sporadic, both geographically and temporally. Normally, clinical trials of medicines and vaccines intended for human use follow a lengthy but predictable sequence of safety and efficacy testing.

Because of its sporadic nature, the incidence of Ebola virus infection in human populations is not predictable and does not allow for adequate testing. Moreover, the immune correlates of protection from filovirus disease in humans remain unknown and therefore cannot be used to assess candidate vaccine efficacy. To facilitate the licensing of medicines when efficacy cannot be evaluated in the setting of natural infection, the U.S. Food and Drug Administration (FDA) introduced a new regulation in 2002 as an alternative licensing pathway for pharmaceutical products that target highly lethal pathogens. The FDA’s “animal rule” allows approval based on animal efficacy data. The animal rule is intended to be used as a pathway for regulatory approval only when there is no other way to licence a vaccine (Correlates of protective immunity for Ebola vaccines: implications for regulatory approval by the animal rule. 2009 Nature Reviews Microbiology 7: 393-400).

In the case of Ebola virus, the relevant animal models are non-human primates and mice. The immune correlates of Ebola virus infection consist of immunoglobulin G responses, although other factors, such as T cells, are also likely to be important in a successful immune response. Current vaccine candidates against Ebola virus include the virus glycoprotein and nucleocapsid proteins. Initial animal testing of Ebola vaccines has shown a protective effect in non-human primates and positive antibody titres in humans.

To date, no vaccines have received regulatory approval and been licensed using the FDA animal rule. This pathway does not diminish the level of regulatory contol required for vaccine approval; extensive human testing is still required to demonstrate safety and immunogenicity. The predictive relationship between animals and humans for protective efficacy is unknown, and therefore an immune correlate is used to bridge the gap between animal efficacy studies and human immunogenicity trials. It has not yet been determined what level of efficacy in animals will be required for vaccine approval, but other vaccines currently administered to the U.S. population have shown efficacies in human trials that are as low as 18%. Even this level of efficacy will provide a benefit against pathogens such as filoviruses with high mortality rates, and therefore may be acceptable against emerging natural infections or bioterrorism threats.

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Related:

• Congo confirms Ebola outbreak
• New species of Ebola virus discovered
• Marburg haemorrhagic fever in Uganda
• Deadly Marburg virus linked to fruit bat

Latest News (unfiltered) | Latest news (filtered) (via Twitter)

1. What is swine flu?
Swine flu is a type of influenza virus. Influenza viruses are named after the proteins on the outside which are recognized by the body, H and N. There are dozens of combinations of these two proteins, each one giving a different type of influenza virus. Swine flu virus is H1N1 influenza. The original swine flu virus was first isolated from a pig in 1930.

2. Can it hurt me?
Influenza viruses infect pigs (swine), birds, humans and a few other species. Most strains of influenza are quite restricted in the host they will infect but occasionally jump from one species to another. Swine flu infects pigs but is also capable of infecting humans.

3. Will there be a swine flu pandemic?
It’s too early to say. Scientists are carefully recording the spread of the current epidemic to see how easily this virus is capable of spreading from person to person. World Health Organization (WHO) Director-General Margaret Chan says the present outbreak “has pandemic potential” but that “it is too early to say whether a pandemic will actually occur”.
Update: This outbreak is now officially a pandemic.

4. How many people have been affected by swine flu?
The number is growing – click here for the latest news.

5. Is there any treatment for swine flu?
Vaccines are available against H1N1 influenza but it is not known how effective they are against this strain. WHO says the virus appears to be susceptible to the influenza drug Tamiflu (oseltamivir), and Relenza (zanamivir). It is not known if resistance to these drugs will occur.

6. How does swine flu spread?
Influenza viruses are transmitted through coughing or sneezing by people infected with the virus. People may become infected by touching something with the virus on it and then touching their mouth or nose, so frequent hand washing is a good idea. You cannot get swine influenza from eating cooked pork or pork products.

7. Has swine flu infected humans before?
Sporadic human infections with swine flu occur regularly but not frequently, e.g. one or two a year in the USA. Most commonly, these cases occur in persons with direct exposure to pigs. There are a few previous cases of one person transmitting swine flu to others.

8. What are the symptoms of swine flu?
The symptoms of swine flu in people are similar to the symptoms of regular influenza, including fever, lethargy, lack of appetite and coughing. Some people with swine flu also have reported runny nose, sore throat, nausea, vomiting and diarrhea.

9. Should I travel to Mexico / the USA?
The World Health Organization (WHO) is not presently advising against travel to Mexico or the USA. National governments may be offering different advice (check locally). Travellers to affected areas are advised to consult a doctor immediately if they show signs of flu-like symptoms.

10. More information:

• CDC
• WHO
• UK HPA
  • UK HPA pandemic influenza FAQ
  • UK national framework for responding to an influenza pandemic
• MicrobiologyBytes: Influenza

11. Are we all going to die?
Probably not. Every year many thousands of people around the world die as a result of influenza, a fact which goes largely unreported. The number of deaths increases in epidemic years. Pandemics (worldwide epidemics) occur unpredictably every 10-30 years. Millions of people die, billions survive.

Update: 10 more things you should know about H1N1 (swineflu)

Related:

• Dissecting the Cell Entry Pathway of Dengue Virus
• Dengue Virus
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• Clustering of dengue virus infections
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