Human cytomegalovirus (HCMV), also referred to as human herpesvirus 5 (HHV-5), is one of the largest human viruses. It contains a linear double-stranded DNA genome of approximately 240 Kbp encoding for roughly 165 genes. HCMV is a herpesvirus that is known to productively infect a wide range of cell types. In addition, it has been suggested to contribute to some proliferative disorders, particularly atherosclerosis. Consistent with this, a number of studies have shown that HCMV profoundly affects normal cell cycle control. Specifically, the virus can stimulate early entry into S phase thus ensuring adequate resources for viral DNA replication. Importantly, however, the virus concomitantly inhibits potentially competing cellular DNA synthesis allowing cellular precursors to be used for viral but not cellular DNA replication. The mechanisms by which HCMV perturbs S phase entry involve interactions between the virus and the cellular replication machinery such that formation of competent pre-replication complexes (Pre-RC) at cellular origins of replication is restricted in infected cells.
Bacillus cereus is widespread in nature and frequently isolated from soil and growing plants, but it is also well adapted for growth in the intestinal tract of insects and mammals. From these habitats it is easily spread to foods, where it may cause an emetic or a diarrhoeal type of food-associated illness that is becoming increasingly important in the industrialized world. The emetic disease is a food intoxication caused by cereulide, a small ring-formed peptide. Similar to the virulence determinants that distinguish Bacillus thuringiensis and Bacillus anthracis from B. cereus, the genetic determinants of cereulide are plasmid-borne.
The diarrhoeal syndrome of B. cereus is an infection caused by vegetative cells, ingested as viable cells or spores, thought to produce protein enterotoxins in the small intestine. Three pore-forming cytotoxins have been associated with diarrhoeal disease. This review focuses on the toxins associated with foodborne diseases frequently caused by B. cereus. The disease characteristics are described, and recent findings regarding the associated toxins are discussed, as well as the present knowledge on virulence regulation.
Two fundamental events in virus replication cycles are the delivery of virus genomes into host cells and the packaging of these genomes into virus protein capsids. In bacteriophages and herpesviruses these processes occur linearly along the genome, base pair after base pair, through a single portal located at a unique site in the viral capsid. New research has addressed the question of whether such a linear translocation through a single portal also takes place for viruses with very large genomes by studying genome delivery and packaging in the amoeba-infecting virus Acanthamoeba polyphaga Mimivirus. With 1.2 million base pairs, this double-stranded DNA genome is the largest documented viral genome. Using electron tomography and cryo-scanning electron microscopy researchers identified a large tunnel in the Mimivirus capsid that is formed shortly after infection, following a large-scale opening of the capsid. The tunnel allows the whole virus genome to exit in a rapid, one-step process. DNA encapsidation is mediated by a transient aperture in the capsid that, they suggest, may promote concomitant entry of multiple segments of the viral DNA molecule. These unprecedented modes of viral genome translocation imply that Mimivirus - and potentially other large viruses - evolved mechanisms that allow them to cope effectively with the exit and entry of particularly large genomes.
Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga mimivirus. 2008 PLoS Biol 6(5): e114
Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the ‘‘stargate’’, allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane–containing viruses.
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.
Human cytomegalovirus (HCMV), also referred to as human herpesvirus 5 (HHV-5), is one of the largest human viruses. It contains a linear double-stranded DNA genome of approximately 240 Kbp encoding for roughly 165 genes. HCMV is a herpesvirus that is known to productively infect a wide range of cell types. In addition, it has been suggested to contribute to some proliferative disorders, particularly atherosclerosis. Consistent with this, a number of studies have shown that HCMV profoundly affects normal cell cycle control. Specifically, the virus can stimulate early entry into S phase thus ensuring adequate resources for viral DNA replication. Importantly, however, the virus concomitantly inhibits potentially competing cellular DNA synthesis allowing cellular precursors to be used for viral but not cellular DNA replication. The mechanisms by which HCMV perturbs S phase entry involve interactions between the virus and the cellular replication machinery such that formation of competent pre-replication complexes (Pre-RC) at cellular origins of replication is restricted in infected cells.
