MicrobiologyBytes

Dengue virus (DENV) is the most common arthropod-borne infection worldwide with 50–100 million cases annually. Despite its high clinical impact, little is known about the infectious cell entry pathway of the virus. Previous studies have shown conflicting evidence about whether the virus fuses directly with the cell plasma membrane or enters cells by receptor-mediated endocytosis.

Entry of DENV into hosts cells is mediated by the virus envelope glycoprotein E, which is organized in 90 homodimers on the surface of the virion. The E glycoprotein is involved in interaction with cellular receptors as well as the subsequent membrane fusion process. In vitro studies show that membrane fusion is triggered on exposure of the virus to low pH, when the E proteins undergo a dramatic re-organization which leads to the formation of trimers. The crystal structure of the E protein has been solved in its dimeric pre-fusion, and trimeric post-fusion configurations. Although much is known about the molecular mechanisms involved in the membrane fusion process, many critical questions regarding the cell entry pathway of flaviviruses remain unanswered.

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A recent paper dissects the cell entry pathway of DENV by tracking single fluorescently-labeled DENV particles in living cells expressing various fluorescent cellular markers, using real-time multi-color fluorescence microscopy (Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells. 2008 PLoS Pathog 4(12): e1000244). It shows that DENV particles are delivered to pre-existing clathrin-coated pits by diffusion along the cell surface. Following clathrin-mediated uptake, the majority of DENV particles are transported to early endosomes, which mature into late endosomes, where membrane fusion occurs. This is the first study that describes the cell entry process of DENV at the single particle level and therefore provides unique mechanistic and kinetic insights into the route of entry, endocytic trafficking behavior, and membrane fusion properties of individual DENV particles in living cells.

This work opens new avenues in flavivirus biology and will lead toward a better understanding of the critical determinants in DENV infection. Single-particle tracking has substantially enriched our knowledge of virus cell entry mechanisms and has revealed previously unknown aspects of virus-host interactions. The mechanistic and kinetic insights offered by this technique provide a better understanding of disease pathogenesis and may lead to a rational design of antiviral drugs and vaccines.

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• First X-Ray Diffraction Image of a Single Virus Particle
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BBC NEWS

Humans could be protected from dengue fever by infecting the mosquitoes carrying it with a parasite which halves their lifespan. Australian scientists, writing in the journal Science, found that Wolbachia bacteria spread well through laboratory-bred mosquitoes. Only older mosquitoes pass on dengue – so killing them could cut disease. It remains to be seen how well the bacteria would spread outside the laboratory.

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• Dengue Virus
• Wolbachia
• Clustering of dengue virus infections
• Malaria Researchers Identify New Mosquito Virus

The BBC is reporting that thousands of people across the UK suffer from Norovirus infection, which causes vomiting and diarrhoea, so this is the time to remind you of the information I posted about these viruses in January 2007:

Hardly a day goes by without another outbreak of Norovirus infection being reported in the news. That’s particularly true at this time of year, and it’s no accident that the consequences of Norovirus infection used to be known as “winter vomiting disease”, although infections occur year round. And yet, in spite of how common they are, these viruses are not household names in the same way as influenza and polio, so MicrobiologyBytes comes to the rescue!

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Historically, noroviruses have been named after the places where outbreaks occurred. By far the most famous Norovirus is Norwalk virus, named after an outbreak of acute gastroenteritis in children at an elementary school in Norwalk, Ohio, in November 1968. These viruses remained poorly understood however, since for the most part, they do not grow in the laboratory. The taxonomy of these viruses became confused and they were previously grouped on the basis of their appearance in the electron microscope, and this led to the name “Small Round-Structured Viruses”. By adding labelled antibodies and performing immune electron microscopy (IEM), it became possible to be a bit more specific which virus was being studied. However, it was not until the genomes of these viruses were cloned and sequenced in the 1990s that the situation became clearer. Based on this molecular information, Norwalk virus and its troublesome little friends became a new genus, Norovirus, in the Calicivirus family, with which they share similarities.

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Microsporidia are single-celled parasites that are capable of causing infections in humans – primarily in people with compromised immune systems, such as those infected with HIV or who have undergone organ transplants. A new article documents a recently discovered species of microsporidian which infects Caenorhabditis elegans, the harmless worm used as a model system by developmental biologists. The work is a breakthrough for public health researchers who had been looking for a suitable laboratory model in which to study microsporidia. Microsporidia are classified as priority pathogens by the U.S. National Institutes of Health, since they have been found in water supplies and because no drugs are available for treating the most common infections they cause in humans. The new species infects C. elegans, a small roundworm that is very easy to study in the lab, and so provides a powerful system in which to study these mysterious microbes, learn how animals respond to infection and develop new drugs to fight infections of microsporidia.

The discovery is particularly exciting because it potentially offers a unique opportunity to identify new drugs for treating human microsporidian infections. The researchers named the new species of microsporidian Nematocida parisii, or “nematode killer from Paris”, because it was discovered in the intestines of roundworms found in Parisian compost pits. Since then, scientists have found closely related naturally-occurring pathogens in Portugal and India, leading them to conclude that they are widespread natural parasites of C. elegans, which has intestinal cells that look almost exactly like human intestinal cells, so researchers are able to tell what the parasites are doing to intestinal cells as they invading and exit the cells. Because the worms are completely transparent, they can take intact worms, put them on slides and see what’s happening. They have already seen some interesting changes in the structure of intestinal cells during different time periods of infection.

Microsporidia are natural intracellular parasites of the nematode Caenorhabditis elegans. 2008 PLoS Biol 6(12): e309
For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wildcaught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.

Researchers have found novel prion infectivity in white and brown fat tissues of mice. Prion diseases, also known as transmissible spongiform encephalopathies, are infectious progressive fatal neurodegenerative diseases which affect humans as well as wild and domestic animals. Distribution of prion infectivity in organs and tissues is important in understanding prion disease pathogenesis and designing strategies to prevent prion infection in animals and humans. Previous studies in animals including sheep, goats, cattle, deer, mink, hamsters and mice, have found prion infectivity mostly in nervous system tissues such as the brain and spinal cord. The tissues studied in a mouse model demonstrate a proof of principle that white and brown fat tissues are sites of prion agent deposition and therefore may play a previously unrecognized role in prion infectivity and transmission of prion disease. The authors state clearly that it will be important to extend their studies to prion-infected large animals, such as cattle, sheep, deer, and elk where they may be potential sources of contamination of human and domestic animal food chains. Results of the current and future studies may merit additional consideration of steps to eliminate from the food chain any fat from ruminants suspected of exposure to or infection with prions.

Detection of Prion Infectivity in Fat Tissues of Scrapie-Infected Mice. 2008 PLoS Pathog 4 (12): e1000232
Distribution of prion infectivity in organs and tissues is important in understanding prion disease pathogenesis and designing strategies to prevent prion infection in animals and humans. Transmission of prion disease from cattle to humans resulted in banning human consumption of ruminant nervous system and certain other tissues. In the present study, we surveyed tissue distribution of prion infectivity in mice with prion disease. We show for the first time detection of infectivity in white and brown fat. Since high amounts of ruminant fat are consumed by humans and also incorporated into animal feed, fat-containing tissues may pose a previously unappreciated hazard for spread of prion infection.

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• MicrobiologyBytes: Prions
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The causative agents of tuberculosis, grouped in the Mycobacterium tuberculosis complex (MTBC), have infected one-third of the present human population and a wide range of other mammals. However, questions, such as why, where and when the disease began and expanded, have largely remained unanswered. A new study provides genetic evidence indicating that the common ancestor of the tuberculosis complex emerged some 40,000 years ago in East Africa, the region from where modern human populations disseminated around the same period. This initial step was followed 10,000 to 20,000 years later by the radiation of two major lineages, one of which spread from human to animals. In more recent years (approximately 180 years ago), coinciding with the human population explosion and the industrial revolution, the human-associated pathogen lineages have strongly expanded. These results thus reveal the strikingly parallel demographic evolution between humans and one of their primary pathogens.

Using mycobacterial tandem repeat sequences as genetic markers, the authors show that the MTBC consists of two independent clades, one composed exclusively of M. tuberculosis lineages from humans and the other composed of both animal and human isolates. The latter also likely derived from a human pathogenic lineage, supporting the hypothesis of an original human host. These findings unveil the dynamic dimension of the association between human host and pathogen populations.

Origin, Spread and Demography of the Mycobacterium tuberculosis Complex. 2008 PLoS Pathog 4(9): e1000160

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Scientists have reported the discovery of a new species of Ebola virus, provisionally named Bundibugyo ebolavirus. This virus was responsible for a hemorrhagic fever outbreak in western Uganda in 2007. Ebola virus infection in humans causes severe disease for which there is presently no vaccine or other treatment. Case fatalities range historically between 53 and 90%. Therefore, research efforts into the Ebola virus genus and potential diagnostics are ongoing, with the discovery of Bundibugyo ebolavirus representing one of the latest pieces added to this puzzle. The new virus is genetically distinct from all other known Ebola virus species, differing by more than 30% at the genetic level. More traditional ELISA-based assays detected the new virus; however, the unique nature of this virus created initial challenges for traditional Ebola virus molecular diagnostic assays and genome sequencing approaches. To determine the genetic signature of this new Ebola virus species, scientists used a recently developed random-primed pyrosequencing approach, quickly determining the genetic sequence of over 70% of the virus genome. Knowledge of this sequence then allowed for the rapid development of a sensitive molecular detection assay which was deployed to the field as part of the outbreak response. This draft sequence also allowed for easy completion of the whole genome sequence using a traditional primer walking approach and prompt confirmation that this virus represented a new Ebola virus species. Current worldwide efforts to design effective diagnostics, antivirals and vaccines will need to take into account the distinct nature of this new member of the Ebola virus genus.

Newly Discovered Ebola Virus Associated with Hemorrhagic Fever Outbreak in Uganda. 2008 PLoS Pathog 4(11): e1000212
Over the past 30 years, Zaire and Sudan ebolaviruses have been responsible for large hemorrhagic fever (HF) outbreaks with case fatalities ranging from 53% to 90%, while a third species, Cote d’Ivoire ebolavirus, caused a single non-fatal HF case. In November 2007, HF cases were reported in Bundibugyo District, Western Uganda. Laboratory investigation of the initial 29 suspect-case blood specimens by classic methods (antigen capture, IgM and IgG ELISA) and a recently developed random primed pyrosequencing approach quickly identified this to be an Ebola HF outbreak associated with a newly discovered ebolavirus species (Bundibugyo ebolavirus) distantly related to the Cote d’Ivoire ebolavirus found in western Africa. Due to the sequence divergence of this new virus relative to all previously recognized ebolaviruses, these findings have important implications for design of future diagnostic assays to monitor Ebola HF disease in humans and animals, and ongoing efforts to develop effective antivirals and vaccines.

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• Congo confirms Ebola outbreak
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It took the entire world – people of all races, countries and religions – to eradicate smallpox. The final naturally occurring cases of “Variola major” in Bangladesh in 1978 and “Variola minor” in Somalia in 1977 marked the end to a chain of suffering and early death dating back to the Biblical plagues, and to Pharoah Ramses, who died from the very same disease. Since then we have continued to face countless pandemics – the Black Death, cholera, and now bird flu, SARS, HIV/AIDS and a new generation of zoonotic diseases – diseases that, often because of changes in population or climate, jump from animals to humans. We can’t be sure where the next smallpox will emerge, but we can be sure that it will take an effort larger than any single person or organization to defeat it.

Google’s Predict and Prevent initiative is working with partners to use digital, genomic and IT technology to identify “hot spots” of emerging threats and provide early warning before they become global crises. When you’re fighting a pandemic, early detection and early response can be the difference between dozens and hundreds of millions infected. What better birthday present could we offer the world after our 20th year, than to say we joined hands with a global movement and helped prevent the next smallpox?

Rapid ecological and social changes are increasing the risk of emerging threats, from infectious diseases to drought and other environmental disasters. This initiative will use information and technology to empower communities to predict and prevent emerging threats before they become local, regional, or global crises. Google.org’s initial focus will be on emerging infectious diseases, which are on the rise worldwide. Climate change, urbanization, and rising international travel and trade all contribute to this threat. Moreover, humans and animals are coming into closer contact because of environmental degradation and increased demand for animal products. Nearly three out of four new diseases in the last three decades have spread from animals to humans. While everyone faces increasing risk from emerging infectious diseases, the world’s poor – who have minimal or no access to health care and may live with and depend on animals for their livelihood – are exceptionally vulnerable and stand to suffer the most. This initiative supports two inter-related pathways from prediction to prevention. The first is vulnerability mapping and identification of “hot spots.” The second, creating systems to better detect threats to provide early warning and enable a rapid response.

Every year, over 50 million people living in tropical and subtropical urban and semi-urban areas become infected with dengue (a mosquito-borne viral infection) and several hundred thousand develop a potentially lethal complication called dengue hemorrhagic fever. Dengue is caused by four closely related viruses that are transmitted to people through the bites of infected female Aedes aegypti mosquitoes. These day-biting insects, which breed in household water containers and in the water that collects in used tires and other discarded containers, acquire dengue virus through feeding on the blood of an infected person. Some people who become infected with dengue virus have no symptoms but others develop high fever, a rash, and severe headache that lasts two to seven days. In dengue hemorrhagic fever, small blood vessels become leaky, which causes nose and gum bleeds, bruising and, in the worst cases, failure of the circulatory system and death. There is no specific treatment for dengue fever or dengue hemorrhagic fever but standard medical care – in particular, replacement of lost blood fluids – helps most people survive the latter condition.

There is no vaccine to prevent dengue. As a result the only way to minimize dengue outbreaks is to control mosquito numbers through environmental management – providing piped water, encouraging people not to store water in open containers, and removing other sources of standing water – and by applying insecticides to areas where mosquitoes breed. During outbreaks, because Ae. aegypti mosquitoes rest in houses, insecticides are also often sprayed in dwellings in the affected areas. However, to improve dengue prevention and surveillance, public-health officials need to know much more about the patterns of dengue virus transmission and about the factors that underlie these patterns. In this study, therefore, the researchers test the idea that dengue virus transmission occurs in localized neighborhood clusters over short periods of time.

In a new study, researchers used “cluster investigations” to examine the pattern of dengue virus transmission among school children in several rural villages in Thailand, a country where dengue is very common (hyperendemic). Primary school children with fever were identified during two seasons of peak dengue virus transmission. Each child was characterized as a dengue-positive index case (by finding dengue virus in their blood) or as a dengue-negative index case. Data on human infection and mosquito infection and density were then collected within 100 meters of the homes of each index case – the “cluster area”. Not all the neighbors of the index cases participated in the study but among the 556 village children who did participate, there were 27 dengue infections, all of which occurred in clusters centered on the homes of the dengue-positive index cases. In the positive clusters, one in eight of the enrolled children became infected within 15 days of the index case becoming ill. Among 1,000 Ae. aegypti mosquitoes collected inside and around the houses in each cluster, only eight were infected with dengue and these were all collected from houses in positive clusters. Finally, there was a greater availability of piped water and fewer Ae. aegypti pupae in the negative clusters than in the positive clusters.

Although this study did not sample all the children or mosquitoes within each cluster area, these findings show that in an area where dengue is hyperendemic, dengue virus transmission among children occurs in localized areas and over short time periods. The findings also suggest that focal transmission is associated with recent dengue virus introductions and that one or a few mosquitoes are likely responsible for all the transmission in each cluster. Although it would be impractical to set up surveillance of all the school children in Thailand for dengue infections, these findings suggest that improved detection of cases within schools combined with local spraying inside the homes in the immediate vicinity of any affected children could help to halt dengue virus transmission. Future cluster studies could explore how human behavior and human immunity affect dengue virus transmission and could also be used to investigate other temporally and spatially clustered infectious diseases, including malaria.

Spatial and temporal clustering of dengue virus transmission in Thai villages. 2008 PLoS Med 5(10): e205
Transmission of dengue viruses (DENV), the leading cause of arboviral disease worldwide, is known to vary through time and space, likely owing to a combination of factors related to the human host, virus, mosquito vector, and environment. An improved understanding of variation in transmission patterns is fundamental to conducting surveillance and implementing disease prevention strategies. To test the hypothesis that DENV transmission is spatially and temporally focal, we compared geographic and temporal characteristics within Thai villages where DENV are and are not being actively transmitted. Cluster investigations were conducted within 100 m of homes where febrile index children with (positive clusters) and without (negative clusters) acute dengue lived during two seasons of peak DENV transmission. Data on human infection and mosquito infection/density were examined to precisely: (1) define the spatial and temporal dimensions of DENV transmission, (2) correlate these factors with variation in DENV transmission, and (3) determine the burden of inapparent and symptomatic infections. Among 556 village children enrolled as neighbors of 12 dengue-positive and 22 dengue-negative index cases, all 27 DENV infections (4.9% of enrollees) occurred in positive clusters. In positive clusters, 12.4% of enrollees became infected in a 15-d period and DENV infections were aggregated centrally near homes of index cases. As only 1 of 217 pairs of serologic specimens tested in positive clusters revealed a recent DENV infection that occurred prior to cluster initiation, we attribute the observed DENV transmission subsequent to cluster investigation to recent DENV transmission activity. Of the 1,022 female adult Ae. aegypti collected, all eight (0.8%) dengue-infected mosquitoes came from houses in positive clusters; none from control clusters or schools. Distinguishing features between positive and negative clusters were greater availability of piped water in negative clusters and greater number of Ae. aegypti pupae per person in positive clusters. During primarily DENV-4 transmission seasons, the ratio of inapparent to symptomatic infections was nearly 1:1 among child enrollees. Study limitations included inability to sample all children and mosquitoes within each cluster and our reliance on serologic rather than virologic evidence of interval infections in enrollees given restrictions on the frequency of blood collections in children. Our data reveal the remarkably focal nature of DENV transmission within a hyperendemic rural area of Thailand. These data suggest that active school-based dengue case detection prompting local spraying could contain recent virus introductions and reduce the longitudinal risk of virus spread within rural areas. Our results should prompt future cluster studies to explore how host immune and behavioral aspects may impact DENV transmission and prevention strategies. Cluster methodology could serve as a useful research tool for investigation of other temporally and spatially clustered infectious diseases.

Related:

• Dengue Virus
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