Over the past week, international news stories have concentrated on the devastating cyclone in Burma (Myanmar), and the almost certain consequence of disease outbreaks in the aftermath. But at the same time, there’s another microbiology story unfolding in East Asia. Beginning in March, a large outbreak of hand, foot and mouth disease (HFMD) was reported from Fuyang city in Anhui Province in China. Note that HFMD is a human disease caused by enteroviruses belonging to the picornavirus family, but is not the same as the animal disease foot and mouth (FMD) caused by a different kind of picornavirus.
HFMD usually affects infants and children, is quite common worldwide and can be caused by a number of different enteroviruses. It is highly contagious and is spread through direct contact with the mucus, saliva, or faeces of an infected person. Like other enterovirus infections (including polio), HFMD typically occurs in small epidemics, usually during the summer and autumn months with an incubation period of 3-7 days.
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Enterovirus infections are common and occur worldwide. Although many infections show no symptoms and often go unnoticed, these viruses are also associated with occasional outbreaks in which a larger than usual number of patients develop clinical disease, sometimes with fatal consequences. The current outbreak is one of these. Initial testing for a variety of respiratory diseases did not reveal any conclusive results, but on April 23, the presence of Enterovirus (EV71) was confirmed. As of May 8th, at least 30 deaths had been reported and the disease had spread to 11 cities and several provinces across China. In all the fatal cases, which represent less than 1% of the thousands of children infected, the victims died with serious complications such as neurogenic pulmonary oedema (breathing difficulties reminiscent to those seen in polio victims).
Enterovirus replication begins in the gastrointestinal or respiratory tract but once the virus is present in the bloodstream may affect various tissues and organs, causing a variety of diseases. Clinically, it is difficult to distinguish the specific cause of most enterovirus infections. Diagnostic testing for non-polio enteroviruses requires specialized laboratory facilities. Diagnosis is made by detecting virus in the throat, in faecal samples or, more convincingly, from specimens collected from the affected part of the body, for example, cerebrospinal fluid (CSF) or biopsy material. A four-fold rise in the level of neutralizing antibody in specimens collected during the acute and convalescent phases of illness provides the best evidence for a recent infection. No specific antiviral agents are currently available for treatment of enterovirus infections, although intravenous administration of immune globulin may have a use in preventing severe disease in immunocompromised individuals or those with life-threatening disease.
EV71 was first isolated in an outbreak of neurological disease in California in 1969. One of the nastier enteroviruses, EV71 has been associated with several epidemics of severe neurological disease in children, mostly in East Asia. An outbreak in Taiwan in 1998 resulted in 129,106 reported cases, 405 children hospitalized and more than 80 deaths. EV71 appears to be emerging as an important virulent neurotropic enterovirus just as poliomyelitis is nearing eradication, but little is known about the molecular mechanisms of host response to EV71 infection.
Transmission of enterovirus infections is increased by poor hygiene and overcrowded living conditions. Improved sanitation and general hygiene are important preventive measures. Measures that can be taken to avoid getting infected with enteroviruses include frequent handwashing, especially after nappy (diaper) changes or going to the toilet, disinfection of contaminated surfaces with bleach, and washing soiled articles of clothing. Enteroviruses are quite resistant to many disinfectants so it is important to use chlorinated (bleach) or iodized disinfectants. During recognised epidemics, it may be advised to close institutions such as schools or child care facilities in order to reduce transmission among young children. Chinese public health experts currently predict that the number of cases will continue to increase and peak around June-July.
With two catalytic activities and many substrates, how does HIV’s reverse transcriptase enzyme know what to do to which substrate? Zooming in on the enzyme’s molecular interactions provides tantalizing clues.
Dynamic binding orientations direct activity of HIV reverse transcriptase. Nature 453, 184-189
The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.
A University of Leicester researcher has discovered how a protein in the blood linked to defence against meningitis plays a more vital role than previously understood in the body’s immune defence system. The published research has helped to advance medical understanding of how the body defends against disease and heals itself. The study also reveals that the same protein, properdin – discovered half a century ago - can also harm internal organs under certain circumstances. Lack of the protein in the human body has previously been linked to susceptibility to meningitis. But the new findings by Cordula Stover of the Department of Infection, Immunity and Inflammation at the University of Leicester assign hitherto unappreciated importance to this protein of the immune defence. Dr Stover, a Lecturer in Immunology, said:
I have a broad interest in immune mechanisms of health and disease, though recently, I have focused on a particular component of the first line immune defence, a protein called properdin. Properdin deficiency in families, though rare, predisposes people to develop meningococcal meningitis, usually with poor outcome of the infection. I hypothesised that the importance of properdin extends beyond this particular infectious disease, and that indeed it is an important player in health generally, and that its importance becomes apparent in conditions involving both acute and chronic states of inflammation.
Now Dr Stover’s paper published in the Journal of Immunology demonstrates that properdin plays a significant role in the survival of conditions relating to surgical perforation of the gut and activation of the immune system by wall components of bacteria. In conditions relating to multi-organ dysfunction, a complication which can occur in response to severe sepsis, properdin however aggravates organ damage.
So far, the system properdin is a part of - the so-called complement system - is classified as a first line, innate, acutely effective immune activation mechanism. This work shows that the activity of properdin extends beyond the acute phase and, importantly, that properdin is stepping onto the stage as an important player in different inflammatory conditions. As the worldwide burden of chronic inflammatory disease increases, it is of practical relevance to understand the contribution of this immune protein.
Trypanosoma brucei is a pathogen which causes epidemics of human and animal sleeping sickness in central and Southern Africa, a fatal disease if untreated. A new paper published this week in PLoS Biology investigates the formation of a trypanosome structure called the Flagellar Pocket (FP). The paper describes a newly identified protein called BILBO1 that is crucial for Flagellar Pocket formation. Experimental inhibition of the protein BILBO1 is fatal to Trypanosoma brucei. The Flagellar Pocket is a part of the single-celled pathogen that has several functions. As well as being the site where the flagellum attaches the flagellum being a structure that enables the bug to propel itself the FP is the site of endo- and exocytosis, processes that transport material in and out of the pathogen from the outside world. The FP also plays an important role in allowing the trypanosome to avoid the immune system of the host. The FP permits the trypanosomes surface proteins (the structures seen by the immune system) to be changed, so that the bug can remain camouflaged from the host’s defences.
The Flagellar Pocket is a multi-tasking organelle but we can now consider it as the parasite’s Achilles heel. How the trypanosome forms this important structure has previously been unknown. The new paper shows that BILBO1 is crucial for the formation of the FP, specifically, it is an important component of a part of the FP called the Flagellar Pocket Collar. BILBO1 is a cytoskeletal protein (the cytoskeleton being a network of proteins that provide internal structure for a cell), thus BILBO1 is involved in creating the cytoskeletal framework that supports the FP. This paper describes the effect of preventing BILBO1 expression in the trypanosome. Without BILBO1, the trypanosome is unable to create a new FP; with the FP disrupted, it is unable to regulate endo- and exocytosis, therefore preventing it from taking up nutrients. These factors prove fatal to the trypanosome. Therefore, this work provides a target for anti-sleeping sickness drugs: knock out BILBO1 function and you will kill the trypanosome.
Viruses are small infectious agents responsible for many human diseases, including acquired immunodeficiency syndrome (AIDS). Like other viruses, the human immunodeficiency virus 1 (HIV-1; the cause of AIDS) enters human cells and uses the cellular machinery to replicate before bursting out of its temporary home. During the initial stage of HIV infection, a particular group of cells in the human immune system, CD8+ T cells, are thought to be important in controlling the level of the virus. These immune system cells recognize pieces of viral protein called antigens displayed on the surface of infected cells; different subsets of CD8+ T cells recognize different antigens. When a CD8+ T cell recognizes its specific antigen (or more accurately, a small part of the antigen called an epitope ), it releases cytotoxins (which kill the infected cells) and cytokines, proteins that stimulate CD8+ T cell proliferation and activate other parts of the immune system. With many viruses, when a person first becomes infected (an acute viral infection), antigen-specific CD8+ T cells completely clear the infection. But with HIV-1 and some other viruses, these cells do not manage to remove all the viruses from the body and a chronic (long-term) infection develops, during which the immune system is constantly exposed to viral antigen.
In HIV-1 infections (and other chronic viral infections), virus-specific CD8+ T cells lose their ability to proliferate, to make cytokines, and to kill infected cells as patients progress to the longterm stages of infection. That is, the virus-specific CD8+ T cells gradually lose their effector functions and become functionally impaired or exhausted. Polyfunctional CD8+ T cells (those that release multiple cytokines in response to antigen) are believed to be essential for an effective CD8+ T cell response, so scientists trying to develop HIV-1 vaccines would like to stimulate the production of this type of cell. To do this they need to understand why these polyfunctional cells are lost during chronic infections. Is their loss the cause or the result of viral persistence? In other words, does the constant presence of viral antigen lead to the exhaustion of CD8+ T cells during chronic HIV infection? In this study, the researchers investigate this question by looking at the polyfunctionality of CD8+ cells responding to several different viral epitopes at various times during HIV-1 infection, starting very early after infection with HIV-1 had occurred.
The researchers enrolled 18 patients recently infected with HIV-1 and analyzed their CD8+ T cell responses to specific epitopes at various times after enrollment using a technique called flow cytometry. They found that the epitope-specific CD8+ cells produced several effector proteins after antigen stimulation during the initial stage of HIV-1 infection, but lost their polyfunctionality in the face of persistent viral infection. The CD8+ T cells also increased their production of programmed death 1 (PD-1), a protein that has been shown to be associated with the functional impairment of CD8+ T cells. Some of the patients began antiretroviral therapy during the study, and the researchers found that this treatment, which reduced the viral load, reversed CD8+ T cell exhaustion. Finally, the appearance in the patients blood of viruses that had made changes in the specific epitopes recognized by the CD8+ T cells to avoid being killed by these cells, also reversed the exhaustion of the T cells recognizing these particular epitopes.
These findings suggest that the constant presence of HIV-1 antigen causes the functional impairment of virus-specific CD8+ T cell responses during chronic HIV-1 infections. Treatment with antiretroviral drugs reversed this functional impairment by reducing the amount of antigen in the patients. Similarly, the appearance of viruses with altered epitopes, which effectively reduced the amount of antigen recognized by those epitope-specific CD8+ T cells without reducing the viral load, also reversed T cell exhaustion. These results would not have been seen if the functional impairment of CD8+ cells were the cause rather than the result of antigen persistence. By providing new insights into how the T cell response to viruses evolves during persistent viral infections, these findings should help in the design of vaccines against HIV and other viruses that cause chronic viral infections.
Humans are hosts to nearly 300 species of parasitic worms and over 70 species of protozoa, some derived from our primate ancestors and some acquired from the animals we have domesticated or come into contact with during our history (History of human parasitology. Clin Microbiol Rev 2002 15: 595-612). The best-documented parasitic disease known from ancient times is caused by the nematode worm Dracunculus medinensis. The earliest description is from an Egyptian papyrus from 1500 BC that refers to both the nature of the infection and to techniques for removing the worm. Confirmation of the presence of this worm in ancient Egypt comes from the finding of a well-preserved worms in Egyptian mummies. Dracunculiasis, or Guinea worm disease, is one of the few diseases unambiguously described in the Bible, and most parasitologists accept that the “fiery serpents” that struck down the Israelites in the region of the Red Sea after the Exodus from Egypt somewhere between 1250 to 1200 BC were actually Guinea worms.
The adult worms live in the subcutaneous connective tissues of their victims, from which the females emerge to release thousands of larvae into water, where they are taken up by intermediate hosts, tiny aquatic crustaceans called Cyclops. In these hosts they mature into infectious larvae that infect humans when the crustaceans are accidentally swallowed in contaminated drinking water. On maturity, the large female worm, up to nearly a metre in length, protrudes from the skin, usually of the leg, and causes intense inflammation and irritation. The effects of the disease are crippling. Its victims develop large ulcers, usually in the lower leg. The ulcers swell, sometimes to the size of a tennis ball, and burst, releasing the spaghetti-like parasitic worm. Victims experience a pain so excruciating that they say it feels as if their leg is on fire. The searing pain compels people to jump into water, often the community’s only source of drinking water, to relieve the pain. When the infected person immerses his or her leg in the water, the worm in the leg releases thousands of larvae. The larvae are then ingested by Cyclops that live in the water. Thus the cycle begins again - when people drink the water, they are in effect drinking in the disease.
The most common way to treat Guinea worm disease involves wrapping the worm around a stick. This treatment has been employed for millennia and may have inspired the Rod of Asclepius which historically has symbolized the medical profession. As the adult worm begins to emerge from the patient’s skin, it is wound around a stick, then further extracted by a few centimeters per day. This slow process can take days or even weeks, but it is required to avoid breakage and leaving behind a portion of the worm. Leaving a portion of the dead worm remain within the host’s body increases the risk of infection, and can trigger immune responses resulting in pain and swelling. In many countries, a broken worm is immediately removed surgically, or the worm can be excised surgically from the very beginning if health care facilities are available. Antihelminthic drugs such as metronidazole or thiabendazole are sometimes used in conjunction with physical extraction. However, one study found that antihelminthic therapy was associated with aberrant migration of worms, resulting in infection in areas other than the lower extremity.
Dracunculiasis is a classic example of a neglected tropical disease, a symptom of poverty and disadvantage. Those most affected are the poorest populations often living in remote, rural areas, urban slums or in conflict zones. With little political voice, neglected tropical diseases have a low profile and status in public health priorities. In 1997 the World Health Assembly pledged to completely eradicate Guinea worm disease. This is no small task, but there are several factors which make eradication a possibility. Dracunculiasis is the first parasitic disease targeted for eradication because:
Diagnosis is easy and unambiguous (presence of an emerging adult worm).
The transmission agent, Cyclops, is not a mobile vector as is a mosquito.
The incubation period in both Cyclops and humans is of limited duration.
Interventions are effective, low cost, and relatively simple to implement.
The disease has a limited geographic distribution and is seasonal in nature.
Success in eliminating the disease has been demonstrated in several countries in Asia and the Middle East.
There is no known animal reservoir.
Is Dracunculiasis eradication close? In 2007 the WHO announced that Guinea worm disease now affects around 25,000 people in nine countries, compared with an estimated 3 million people were infected in over 20 countries in the early 1980s. Twelve countries were declared Guinea worm-free in early March. If progress continues at this rate, the disease could be eradicated in less than two years. It is probable that complete eradication will take quite a few years yet, although it should be possible to eliminate the disease from seven countries in a couple of years, leaving only two endemic countries, Sudan and Ghana (Dracunculiasis eradication by 2009: will endemic countries meet the target? Tropical Medicine & International Health 2007 12: 1403-1408). One lesson to be drawn from the problems of local ownership and the experience of cash rewards is that there are dangers in throwing money at the problem. While the eradication initiative badly needs additional resources, it needs them at such a level and managed in such a way that they do not distort the priorities of the health care system, or exceed the capacity of local staff to manage them. The amounts needed are not large, but their continuity and flexibility is important. Given the highly seasonal transmission of dracunculiasis, the resources must be available at very specific times of the year, which is not always achieved. In spite of the difficulties, complete worldwide eradication of this ancient disease is drawing nearer.
The sociobiology of bacteria, largely unappreciated and ignored by the microbiology research community two decades ago is now a major research area, catalyzed to a significant degree by studies of communication and cooperative behavior among the myxobacteria and in quorum sensing (QS) and biofilm formation by pseudomonads and other microbes. Recently, the topic of multicellular cooperative behaviors among bacteria has been increasingly considered in the context of evolutionary biology. This essay discusses the significance of two recent studies of the phenomenon of “cheating” mutants and their exploitation of cooperating microbial populations of Pseudomonas aeruginosa.
The function of the RNA genome of the human immunodeficiency virus (HIV) is determined both by its sequence and by its ability to fold back on itself to form specific higher-order structures. In order to describe physical structures in a region of the HIV RNA genome known to play multiple, critical roles in viral replication and pathogenesis. In this week’s PLoS Biology scientists from the University of North Carolina show how they have devised a high-throughput, quantitative, and comprehensive structure-mapping approach that locates flexible (unpaired) nucleotides within a folded RNA, assaying hundreds of nucleotides at a time. They find that the first 10% of the HIV-1 genome has a single predominant structure and that regulatory motifs have significantly greater structure than do protein-coding segments. The HIV genome interacts with numerous proteins, including multiple copies of nucleocapsids. They also directly map RNA-protein interactions inside virions and discover that the nucleocapsid interacts with viral RNA in at least three distinct ways, depending on the context within the overall genome structure. The group hopes that further application of the high-throughput RNA-structure analysis tools described will make it possible to address diverse structure-function relationships in intact cellular and viral RNAs.
Replication and pathogenesis of the human immunodeficiency virus (HIV) is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. High-throughput SHAPE (selective 29-hydroxyl acylation analyzed by primer extension) technology uses many of the same tools as DNA sequencing, was used to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further application of this technology will make possible newly informative analysis of any RNA in a cellular transcriptome. High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states. 2008 PLoS Biol 6: e96
A simplified economical method of giving rabies vaccine is just as effective as the expensive standard vaccination method at stimulating anti-rabies antibodies. A clinical trial in healthy volunteers has found that a simpler and cheaper way of using rabies vaccines proved to be just as effective as the current most widely used method at stimulating antibodies against rabies. The trial is published in this week’s PLoS Neglected Tropical Diseases. Dr Mary Warrell (Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford) and colleagues, who conducted the trial with a vaccine in routine use, say that the simplified method has the advantages of requiring fewer clinic visits, being more practicable, and acceptable, and having a wider margin of safety, especially in inexperienced hands. It would therefore be suitable for use anywhere in the world where there are financial constraints, and especially where two or more patients are likely to be treated on the same day.
All human deaths from rabies result from failure to give adequate prophylaxis. After a rabid animal bite, immediate wound cleaning, rabies vaccine and injections of anti-rabies antibody (immunoglobulin) effectively prevent fatal infection. But anti-rabies immunoglobulin is very rarely available in developing countries, and so prevention relies on giving people bitten by rabid animals effective vaccine treatment. The vaccines that are currently approved by the World Health Organization, which are usually injected into the muscle, are prohibitively expensive, and so are unaffordable in developing countries. In Africa, for example, the average cost of an intramuscular course of vaccine is $US 39.6, equivalent to 50 days wages.
Two more economical regimes, involving injecting small amounts of vaccine into the skin (intradermally) at 2 or 8 sites on the first day of the course with subsequent booster doses, are available in a few places. With the 8-site method, a large dose of vaccine is given on the first day only, whereas with the 2-site method the same dose is divided between the first and third days, entailing an extra visit to the clinic. However, practical or perceived difficulties have restricted widespread uptake of these economical methods. Dr Warrell and colleagues set out to test a new, similar simplified regime, involving injections at 4 sites on the first day. They vaccinated healthy volunteers to compare the antibody levels induced by the 4-site intradermal regimen with those induced by the current 2-site and 8-site intradermal regimes and the “gold standard” intramuscular regimen favored internationally. All of the economical intradermal methods worked just as well as the intramuscular method at stimulating anti-rabies antibodies. The authors conclude that the results provide sufficient evidence that the simplified 4-site regimen now meets all the criteria necessary for its recommendation for use wherever the cost of vaccine is prohibitive.
Over the past week, international news stories have concentrated on the devastating cyclone in Burma (Myanmar), and the almost certain consequence of disease outbreaks in the aftermath. But at the same time, there’s another microbiology story unfolding in East Asia. Beginning in March, a large outbreak of hand, foot and mouth disease (HFMD) was reported from Fuyang city in Anhui Province in China. Note that HFMD is a human disease caused by enteroviruses belonging to the picornavirus family, but is not the same as the animal disease foot and mouth (FMD) caused by a different kind of picornavirus.
