MicrobiologyBytes

Malaria has been a major selective force on the human population, and several erythrocyte polymorphisms have evolved that confer resistance to severe malaria. Plasmodium falciparum rosetting, a parasite virulence phenotype associated with severe malaria, is reduced in blood group O erythrocytes compared with groups A, B, and AB, but the contribution of the ABO blood group system to protection against severe malaria has received little attention. We hypothesized that blood group O may confer resistance to severe falciparum malaria through the mechanism of reduced rosetting. In a matched case-control study of 567 Malian children, we found that group O was present in only 21% of severe malaria cases compared with 44-45% of uncomplicated malaria controls and healthy controls. Group O was associated with a 66% reduction in the odds of developing severe malaria compared with the non-O blood groups. In the same sample set, P. falciparum rosetting was reduced in parasite isolates from group O children compared with isolates from the non-O blood groups. Statistical analysis indicated a significant interaction between host ABO blood group and parasite rosette frequency that supports the hypothesis that the protective effect of group O operates through the mechanism of reduced P. falciparum rosetting. This work provides insights into malaria pathogenesis and suggests that the selective pressure imposed by malaria may contribute to the variable global distribution of ABO blood groups in the human population.

Blood group O protects against severe Plasmodium falciparum malaria through the mechanism of reduced rosetting. PNAS 2007 104: 17471-17476

Related:

• Tough Choices – DDT or Malaria?
• Lean and Mean Invasion Machines
• Malaria is a continuing danger to UK travellers
• Malaria: now you see me, now you don’t

Despite its early promise, interferon has not turned out to be a miracle cure for virus infections. In this article in Microbiology Today, Rick Randall and Steve Goodbourn explain what is known now and what we still need to find out.

By understanding at the molecular level how viruses counteract the IFN response, new medicines and new ways of combating infections may be developed. For example, it is clear that if a virus fails to circumvent the IFN response it will be attenuated in vivo. Consequently, attenuated virus vaccines may be developed by specifically isolating viruses that are unable to circumvent the IFN response. This may be achieved either by genetically engineering viruses to knockout their IFN antagonists, or by selecting mutants that are sensitive to IFN. The fact that most viruses encode specific IFN antagonists also raises the possibility that novel antiviral drugs may be developed which block the activity of the viral antagonists.

Also in Microbiology Today, Derek Burke recalls his time as a researcher during the “steam age of virology”:

My first project was to determine the nucleic acid content of influenza virus, known to be an RNA virus, but how much RNA? Near its end, Alick suggested that I should help him “with something interesting that we are doing on interference”. “We” was Jean Lindenmann and himself, it was March 1957, and interferon was only a few weeks old. The name was new Alick once explained that it was “time that biologists had a fundamental particle, for the physicists have so many: electron, neutron, proton, etc.” That did not stop Lord Hailsham, then Chairman of the MRC, objecting to such a nasty hybrid word with both Latin and Greek roots! By then, though, the name had stuck.

RNA interference, RNAi, is a natural antiviral mechanism in plants and invertebrates. Based on the results obtained in plants, Drosophila and worms and because the RNAi machinery is present in all animals from nematodes to mammals, RNAi has often been proposed to be involved in the response to viral infection in vertebrates. In mammals the interferon (IFN) system is a central innate antiviral defence mechanism, while the involvement of RNA interference (RNAi) in antiviral response against RNA viruses is uncertain. Here, we tested whether RNAi is involved in the antiviral response in mammalian cells. To investigate the role of RNAi in influenza A virus-infected cells in the absence of IFN, we used Vero cells that lack IFN-alpha and IFN-beta genes. Our results demonstrate that knockdown of a key RNAi component, Dicer, led to a modest increase of virus production and accelerated apoptosis of influenza A virus-infected cells. These effects were much weaker in the presence of IFN. The results also show that in both Vero cells and the IFN-producing alveolar epithelial A549 cell line influenza A virus targets Dicer at mRNA and protein levels. Thus, RNAi is involved in antiviral response, and Dicer is important for protection against influenza A virus infection.

Dicer is involved in protection against influenza A virus infection
J Gen Virol. 2007 88: 2627-2635

Dengue fever and Dengue hemorrhagic fever have emerged as some of the most important mosquito-borne virus diseases in the tropics. No effective vaccine or antiviral drug therapy is currently available against Dengue viruses and the mechanisms of pathogenesis in Dengue infections remain elusive. Recently, virus-induced apoptosis mediated by the Unfolded Protein Response (UPR, or ER stress response) has been hypothesised to represent a crucial pathogenic event in virus infection. In Dengue virus infection, cells elicit an UPR which is observed at the level of translation attenuation (seen by phosphorylation of eIF2alpha) and activation of other specific pathways. Interestingly, specific serotypes of Dengue virus modulate the UPR with different selectivity. Perturbation of the UPR by preventing the dephosphorylation of the translation initiation factor eIF2alpha considerably alters virus infectivity. This report provides evidence that Dengue infection induces and regulates the three branches of the UPR signaling cascades. This is a basis for understanding of Dengue virus regulation and conditions beneficial to virus infection. Furthermore, modulators of UPR such as Salubrinal that inhibit Dengue replication may open up avenues for anti-viral therapy.

Dengue virus serotype infection specifies the activation of the unfolded protein response
Virology Journal 2007 4: 91

Opening Doors – Scientific Workshops for Young Researchers
An initiative aimed at promoting links between young scientists from the UK and Spain
Workshop on innate anti-viral immunity and virus evasion strategies
Sigenza (Guadalajara) 17 21 February 2008

The British Council in Spain in collaboration with the Spanish Council for Scientific Research (CSIC) is organising a series of scientific seminars to provide opportunities for young researchers from the UK and Spain to meet face-to-face for the exchange of ideas, knowledge and information on priority topics and to explore future areas of research and collaboration. The next seminar of this series will take place in the Parador de Sigenza (Guadalajara), Spain, from 17th to 21st February 2008. The meeting will involve ample opportunities for discussion in structured and informal sessions with keynote speakers and young researchers. Keynote speakers will give 30 min talks and participants will give a 15 min presentation of their work.

We are starting to understand at the molecular level the battle between viruses and the host defence systems, and it is becoming evident that the initial virus-cell interaction and the activation of innate immunity are critical to control viral diseases. The Meeting will discuss the initial molecular events occurring upon infection that may restrict virus replication, and will include intracellular signalling, the interferon system, cytokine networks, natural killer cells and other innate immunity pathways. Viral evasion strategies to counteract the host anti-viral responses will also be considered. Viral systems will include emerging viruses, human pathogens and virus-host models that are providing insights into mechanisms of viral pathogenesis.

Keynote speakers:

Richard Randall (University of St. Andrews) Modulation of interferon by paramyxovirus
Gavin Wilkinson (Cardiff University) Human cytomegalovirus evasion of natural killer cells
Greg Towers (University College London) Cellular factors restricting the replication of retroviruses
John Sinclair (University of Cambridge) Factors controlling human cytomegalovirus replication
Richard Elliot (University of St. Andrews) Immune evasion by viruses causing hemorragic diseases
Adolfo Garca Sastre (Mount Sinai School of Medicine) Pathogenesis of Spanish flu
Luis Enjuanes (Centro Nacional de Biotecnologa) Pathogenesis of SARS coronavirus
Jos Alcami (Instituto de Salud Carlos III) Factors controlling HIV infection of immune cells
Amelia Nieto (Centro Nacional de Biotecnologa) Interaction of influenza virus with cell factors
Antonio Alcami (Centro de Biologa Molecular) Chemokine modulation by poxvirus and herpesvirus

The seminar is limited to about 30 participants and participation is by invitation. The organisers of this scientific workshop will cover participants travel and full board accommodation costs. Early to mid career researchers wishing to participate should send their CV and the attached application form to belen.fortea@britishcouncil.es before 30th November 2007.
Further details: Antonio Alcami (aalcami@cbm.uam.es) or Richard Randall (rer@st-and.ac.uk)

Campylobacter jejuni is a curved, rod-shaped, Gram-negative bacterium commonly found in animal faeces. This genus is closely related to Helicobacter and like it has a relatively small genome (1.6-2.0 megabases) and can establish long-term associations with its hosts. Campylobacter is one of the most common causes of bacterial diarrhoea in developed countries and it is only relatively recently that we have realized the importance of this pathogen.

Many aspects of Campylobacter biology remain slightly mysterious. C. jejuni naturally colonises many bird species and is commonly associated with chickens. It is considered to be a commensal of chickens and other birds because although they commonly carry the organism, it does not cause them any disease. The reason why the organism causes disease in humans but not in chickens is not completely understood.

Contaminated food is a major source of human infections, with incorrectly prepared poultry meat the major route of infection. The organism is microaerophilic which means that it requires oxygen to survive, but that optimum growth occurs at lower levels of oxygen than are present in the atmosphere. This reflects the relatively low oxygen concentrations found in the gut.

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Infection with C. jejuni usually results in enteritis, characterised by abdominal pain, diarrhoea, fever, and sickness. The symptoms usually persist for between 24 hours and a week, but sometimes longer. The diarrhoea caused can vary in severity from loose stools to bloody diarrhoea. Although food poisoning caused by Campylobacter species can be severely debilitating, it is rarely life-threatening except in very frail patients. Antibiotics are not usually given as Campylobacter disease is usually self-limiting. However, severe or prolonged cases may require ciprofloxacin, erythromycin or norfloxacin. As with other types of diarrhoea, fluid and electrolyte replacement may be required for serious cases.

C. jejuni shows extensive genetic variation, with hypervariable sequences present in the genome. Most of these are in regions which encode proteins that are involved in the synthesis or modification of cell surface structures such as the capsule, lipooligosaccharide and flagellum. The organism is naturally competent, meaning that it can take up DNA from the environment. This allows recombination between strains, generating even more genetic diversity. Horizontal gene transfer involving both plasmid and chromosomal DNA occurs both in vitro and in vivo, and this could have an important role in rapid genome evolution and in the spread of antibiotic resistance.

The lipooligosaccharide capsule of C. jejuni is highly variable which is probably important in immune avoidance and allowing the organism to establish persistent infections. Some capsular structures resemble human neuronal gangliosides and this molecular mimicry is thought to lead to autoimmune disorders, including Guillain-Barre syndrome (GBS), a neurological complication which occurs in approximately 1 in every 1,000 cases of campylobacterosis.

C. jejuni also produces a toxin known as cytolethal distending toxin (CDT), which causes arrest at the G1/S or G2/M transition of the cell cycle, depending on the cell type. However, the role of CDT in C. jejuni pathogenesis remains unclear. At least some strains of Campylobacter can invade intestinal epithelial cells, as intracellular bacteria have been observed in patient tissues and invasion can be reproduced in cell lines in vitro.

As with many other pathogens, Toll-like receptors (TLRs) represent the first immunological challenge that C. jejuni must overcome during infection. The major adaptive immune response needed to clear C. jejuni infection primarily involves cell-mediated immunity as opposed to antibody-mediated mechanisms. This is presumably related to cellular invasion.

There is a lot we still don’t understand about the pathogenesis of C. jejuni, and our knowledge of its pathogenic mechanisms still lags considerably behind that of pathogenic bacteria such as Salmonella, Shigella, Vibrio and Listeria. Campylobacter is only distantly related to these other enteropathogens, and so has probably evolved distinct infection and virulence mechanisms which have not been observed before. More widespread understanding of the role of C. jejuni in the burden of human illness has brought this organism into the spotlight in recent years, but much work remains to be done.

Links:

• Campylobacter jejuni: molecular biology and pathogenesis. Nature Reviews Microbiology 5, 665-679 (September 2007)
• US Center for Food Safety & Applied Nutrition Online Bacteriological Analytical Manual, Chapter 7: Campylobacter
• UK Health Protection Agency: Campylobacter

Interferon discovery and ferret flu. Jean Lindenmann, who discovered how inactivated viruses help to protect cells, talks about his career.

My 50 years with interferon – Jan Vilcek:
It was undoubtedly a coincidence that I graduated from medical school in 1957 – the same year that Alick Isaacs and Jean Lindenmann published their first papers on interferon. That I graduated from medical school at all was a coincidence, too; had I not been growing up in the then communist Czechoslovakia, I very likely would have chosen a different field of study. In high school (called gymnasium in central Europe), I liked humanities-oriented subjects better than chemistry and math. Besides, my mother was an ophthalmologist, my parents wanted me to become a physician, and I was not inclined to choose a career that would please my parents! I enjoyed creative writing, and for many years, I had been thinking of becoming a journalist. When during my senior year at the gymnasium I had to make a final decision about what studies I would choose, however, my resolve not to become a physician began to weaken …
My fifty years with interferon. Journal of Interferon and Cytokine Research. 2007 27: 535-542

PLoS Pathogens is two years old, and to celebrate, they’ve just published a list of the top ten papers downloaded from September 2005 to July 2007, so if you need to catch up on your reading:

1. Carrageenan Is a Potent Inhibitor of Papillomavirus Infection
   Sexually transmitted human papillomavirus (HPV) infections are very common. Although most HPV infections don’t cause noticeable symptoms, persistent infection with some genital HPV types can lead to cervical cancer or other anal/genital cancers. Another subset of HPV types can cause genital warts. Recent studies have suggested that condoms are not highly effective in preventing HPV infection. Although HPV vaccines will soon become available, they probably will not protect against all genital HPV types and will be too expensive for use in the developing world. Inexpensive HPV-inhibitory compounds (known as topical microbicides) might be useful for blocking the spread of HPV. Using a newly developed cell culture–based HPV inhibition test, we have discovered that an inexpensive gelling agent called carrageenan is an unexpectedly potent HPV infection inhibitor. Carrageenan is also under investigation as a topical microbicide targeting HIV and herpes viruses, but it is a thousand times more effective against HPV in cell culture tests. Interestingly, carrageenan is used as a thickener in some commercially available sexual lubricants and lubricated condoms. Several of these commercial lubricant products are potent HPV inhibitors in our cell culture–infection system. Clinical trials are needed to determine the effectiveness of carrageenan as a topical microbicide against HPV.
2. Modulation of Tumor Necrosis Factor by Microbial Pathogens
   In response to invasion by microbial pathogens, host defense mechanisms get activated by both the innate and adaptive arms of the immune responses. TNF (tumor necrosis factor) is a potent proinflammatory cytokine expressed by activated macrophages and lymphocytes that induces diverse cellular responses that can vary from apoptosis to the expression of genes involved in both early inflammatory and acquired immune responses. A wide spectrum of microbes has acquired elegant mechanisms to overcome or deflect the host responses mediated by TNF. For example, modulatory proteins encoded by multiple families of viruses can block TNF and TNF-mediated responses at multiple levels, such as the inhibition of the TNF ligand or its receptors, or by modulating key transduction molecules of the TNF signaling pathway. Bacteria, on the other hand, tend to modify TNF-mediated responses specifically by regulating components of the TNF signaling pathway. Investigation of these diverse strategies employed by viral and bacterial pathogens has significantly advanced our understanding of both host TNF responses and microbial pathogenesis. This review summarizes the diverse microbial strategies to regulate TNF and how such insights into TNF modulation could benefit the treatment of inflammatory or autoimmune diseases.
3. Identification of a Novel Gammaretrovirus in Prostate Tumors of Patients Homozygous for R462Q RNASEL Variant
   Prostate cancer is the most frequent cancer and the second leading cause of cancer deaths in US men over the age of 50. Several genetic factors have been proposed as potential risk factors for the development of prostate cancer, including a viral defense gene called RNASEL. A common genetic variant in this gene, R462Q, was recently implicated in up to 13% of prostate cancer cases. Given the antiviral role of RNASEL, the authors sought to examine if a virus might be present in prostate cancers associated with the R462Q variant. Using a DNA microarray designed to detect all known viral families, the authors identified a novel virus, named XMRV, in a subset of prostate tumor samples. Polymerase chain reaction testing of 86 prostate tumors for the presence of XMRV revealed a strong association between the presence of the virus and being homozygous for the R462Q variant. Cloning and sequencing of the virus showed that XMRV is a close relative of several known xenotropic murine leukemia viruses. This report presents the first documented cases of human infection with a xenotropic retrovirus. Future work will address the potential connection between XMRV infection and the increased prostate cancer risk in patients with the R462Q RNASEL variant.
4. Human Neutrophils Kill Bacillus anthracis
   Bacillus anthracis is the bacterium that causes anthrax, a disease that can occur through natural infections and also through intentional release. B. anthracis makes spores, which are in a dormant state, similar to seeds of a plant, and are extremely resistant to the environment. B. anthracis spores can infect through the skin or the lung. Lung infections disseminate through the body and are lethal. In contrast, skin infections often remain localized, and patients survive even without treatment. It is not well understood why these bacteria cause a localized infection through the skin and a lethal disease through the lung. Little is known about how B. anthracis is controlled. Neutrophils are the first white blood cells recruited to a site of infection and are specialized in killing microbes. Previous studies show that neutrophils are abundant in the skin form, but not in the lung form of anthrax. The researchers report that human neutrophils can take up B. anthracis spores. Once inside, the spores germinate to form vegetative bacteria. The vegetative bacteria are extremely susceptible to neutrophil-killing mechanisms. The B. anthracis virulence factors (molecules that make bacteria cause diseases) manipulate other human cells but do not deter neutrophils. B. anthracis is indeed exquisitely sensitive to the neutrophil protein α-defensin. These data support a new model where B. anthracis skin, but not lung, infections are controlled by the antimicrobial activity of neutrophils.
5. A Novel Bacterium Associated with Lymphadenitis in a Patient with Chronic Granulomatous Disease
   As new bacteria continue to be discovered every year, it is inevitable that some of them will be found to cause human disease. The authors describe the isolation and characterization of a new bacterium, grown from a patient with chronic granulomatous disease (CGD). In this genetic disease, one of the main lines of defense against infection, the neutrophil, has a discrete defect in the generation of superoxide, leading to recurrent infections with a narrow spectrum of bacteria and fungi. This new organism was cultured from lymph nodes that had been inflamed for several months. To prove that this new bacterium was indeed a pathogen, Greenberg and colleagues measured specific antibody response in the patient: they inoculated CGD mice with this organism and reproduced the appearance of the human infection; they recovered the organism in pure growth from infected mouse spleens. This new bacterium belongs to the family Acetobacteraceae, bacteria that are found widely in the environment. They have a variety of industrial uses, such as the production of vinegar, but have never been reported to cause invasive human disease. Disease-causing organisms remain to be discovered. The researchers outline some of the steps that can be taken to verify the pathogenicity of novel organisms.
6. Gene-Specific Countermeasures against Ebola Virus Based on Antisense Phosphorodiamidate Morpholino Oligomers
   Ebola virus (EBOV) causes a highly lethal hemorrhagic fever that results in up to 50%–90% mortality in humans. There are currently no available vaccines or therapeutics to treat EBOV infection. To date, multiple pre- and post-exposure therapeutic strategies, primarily focused on bolstering the host immune response or inhibiting viral replication, have been undertaken with limited success. Here, Bavari and colleagues report the development of a successful therapeutic regimen for EBOV infection based on antisense phosphorodiamidate morpholino oligomers (PMOs). PMOs are a subclass of chemically modified antisense oligonucleotides that interfere with the translation of viral mRNA, thus inhibiting viral amplification. Using a cell-free translation system, a cell-based assay, and survival studies in rodents, we identified several efficacious EBOV-specific PMOs. Further, prophylactic administration of a combination of three EBOV-specific PMOs specifically targeting VP24, VP35, and the viral polymerase L protected rhesus macaques from lethal EBOV infection. This is the first successful antiviral intervention against filoviruses in nonhuman primates. These findings may serve as the basis for a new strategy to quickly develop virus-specific therapies in defense against known, emerging, and genetically engineered bioterrorism threats.
7. The Role of Innate Immune Responses in the Outcome of Interspecies Competition for Colonization of Mucosal Surfaces
   Bacterial infection commonly begins with organisms that colonize and proliferate on mucosal surfaces. These microenvironments may be occupied by multiple microbial species, suggesting that successful colonizers are distinguished by their capacity to prevail over their competitors. This study examines interactions between two bacterial species that both colonize and infect the human upper respiratory tract. In a mouse model, strains of both Haemophilus influenzae and Streptococcus pneumoniae efficiently colonize the nasal mucosa when tested individually. In contrast, following co-inoculation, H. influenzae rapidly and completely outcompetes S. pneumoniae. This competitive effect is dependent on the local responses from the host in the form of a specific type of white blood cell (neutrophil) that acts to engulf and kill microorganisms that have been labeled by proteins that bind to microbial surfaces (complement). The results of this study show that recognition of microbial products from one species may activate inflammatory responses that promote the clearance of another competing species. This study also demonstrates how manipulations such as antibiotics or vaccines, which are meant to diminish the presence of a single pathogen, may inadvertently alter the competitive interactions of complex microbial communities.
8. Prions Adhere to Soil Minerals and Remain Infectious
   Transmissible spongiform encephalopathies (TSEs) are a group of incurable diseases likely caused by a misfolded form of the prion protein (PrP^Sc). TSEs include scrapie in sheep, bovine spongiform encephalopathy (“mad cow” disease) in cattle, chronic wasting disease (CWD) in deer and elk, and Creutzfeldt-Jakob disease in humans. Scrapie and CWD are unique among TSEs because they can be transmitted between animals, and the disease agents appear to persist in environments previously inhabited by infected animals. Soil has been hypothesized to act as a reservoir of infectivity, because PrP^Sc likely enters soil environments through urinary or alimentary shedding and decomposition of infected animals. In this manuscript, the authors test the potential for soil to serve as a reservoir for PrP^Sc and TSE infectivity. They demonstrate that PrP^Sc binds to a variety of soil minerals and to whole soils. They also quantitate the levels of protein binding to three common soil minerals and show that the interaction of PrP^Sc with montmorillonite, a common clay mineral, is remarkably strong. PrP^Sc bound to Mte remained infectious to laboratory animals, suggesting that soil can serve as a reservoir of TSE infectivity.
9. The Expanding Universe of Prion Diseases
   Prions cause fatal and transmissible neurodegenerative disease. These etiological infectious agents are formed in greater part from a misfolded cell-surface protein called PrP^C. Several mammalian species are affected by the diseases, and in the case of “mad cow disease” (BSE) the agent has a tropism for humans, with negative consequences for agribusiness and public health. Unfortunately, the known universe of prion diseases is expanding. At least four novel prion diseases—including human diseases variant Creutzfeldt-Jakob disease (vCJD) and sporadic fatal insomnia (sFI), bovine amyloidotic spongiform encephalopathy (BASE), and Nor98 of sheep—have been identified in the last ten years, and chronic wasting disease (CWD) of North American deer (Odocoileus Specis) and Rocky Mountain elk (Cervus elaphus nelsoni) is undergoing a dramatic spread across North America. While amplification (BSE) and dissemination (CWD, commercial sourcing of cervids from the wild and movement of farmed elk) can be attributed to human activity, the origins of emergent prion diseases cannot always be laid at the door of humankind. Instead, the continued appearance of new outbreaks in the form of “sporadic” disease may be an inevitable outcome in a situation where the replicating pathogen is host-encoded.
10. Crossing the Line: Selection and Evolution of Virulence Traits
    The evolution of pathogens presents a paradox. Pathogenic species are often absolutely dependent on their host species for their propagation through evolutionary time, yet the pathogenic lifestyle requires that the host be damaged during this dependence. It is clear that pathogenic strategies are successful in evolutionary terms because a diverse array of pathogens exists in nature. Pathogens also evolve using a broad range of molecular mechanisms to acquire and modulate existing virulence traits in order to achieve this success. Detailing the benefit of enhanced selection derived through virulence and understanding the mechanisms through which virulence evolves are important to understanding the natural world and both have implications for human health.

Nice work. Open access publishing has come of age.

“Multiple sclerosis is an autoimmune disease,” is heard so often that it is widely accepted as fact by the current generation of students and physicians. Yet, although it is undisputed that multiple sclerosis (MS) is immune mediated, an autoimmune mechanism remains unproven. Immune-mediated tissue damage can also result from viral infections in which the host immune response is directed to viral rather than self proteins, or as a consequence of nonspecific or bystander immune responses that change the local cytokine environment. Increasing evidence suggests that poorly controlled host immune responses account for much of the tissue damage in chronic infections, and it has been postulated that a similar mechanism may underlie many chronic diseases with features suggestive of an infectious causative factor, including MS. A recent study suggesting that oligodendrocyte death accompanied by microglial activation is the primary event in new MS lesion formation, rather than lymphocyte infiltration, could change the current mindset almost exclusively focused on autoimmunity. This review presents the rationale for considering MS a single disease caused by one virus, as well as the anticipated pattern of a persistent central nervous system infection, the application of Koch’s postulates to viral discovery in MS as the causative agent, and tissue culture-independent genotypic approaches to viral discovery in MS.

A specific viral cause of multiple sclerosis: one virus, one disease.
Ann Neurol. 2007 61: 514-523