MicrobiologyBytes

Dengue is one of the most rapidly spreading vector-borne diseases in the world, with the incidence increasing 30-fold in the past 50 years. There are currently no licensed treatments or vaccines for dengue. This review covers the recent advances in our understanding of dengue pathogenesis, including host and viral determinants.

The pathogenesis of severe dengue is thought to be immune-mediated due to the timing of the clinical manifestations and higher incidence in secondary infections with a heterologous serotype. Recent evidence has provided further information of neutralizing versus enhancing monoclonal antibodies and their target epitopes on the dengue virion, which has major implications for vaccine design. The role of T-cell immunopathology has also been advanced with recent evidence of cross-reactive high pro-inflammatory cytokine producing T cells predominating in severe dengue. Recent large genome-wide association studies have identified specific susceptibility loci associated with severe disease. Epidemiological studies have served to define certain at-risk groups and specific viral virulence factors have recently been described.

The pathogenesis of dengue is likely to be a complex interplay of host immunity and genetic predisposition combined with certain viral virulence factors. Better understanding of the underlying mechanisms leading to severe dengue is crucial if we are to develop prognostic markers, novel diagnostics and therapeutics and ultimately a balanced and safe vaccine.

The pathogenesis of dengue. Curr Opin Infect Dis. 27 Feb 2013

The outbreak of dengue fever in Madeira has evolved rapidly since its onset on 3 October 2012. The Portuguese Ministry of Public Health has reported 1,148 cases of dengue infection, 517 of which have been laboratory confirmed. During the outbreak, 57 people have been hospitalised for observation but there have been no deaths or severe cases of dengue fever. http://goo.gl/WXvi6

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To put it bluntly, dengue virus is winning. We have so far failed in our attempts to make a safe and effective dengue virus vaccine, so we need alternatives. Recently, there has been emphasis on alternatives to vasccines (Should Science Pull the Trigger on Antiviral Drugs). A new paper looks at the biochemisty of dengue virus infection, knowledge that might lead us towards alternative strategies to fight this nasty virus.

Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells. (2012) PLoS Pathog 8(3): e1002584. doi:10.1371/journal.ppat.1002584
Dengue virus causes ~50–100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture.

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A number of flaviviruses constitute a significant threat to global health. Dengue virus (DENV) infection causes around 21,000 human deaths annually, and it is estimated that at least 120 countries have endemic DENV transmission, whilst in recent years, West Nile virus (WNV) has become more prominent as a zoonotic agent, particularly in North America where the virus first emerged in 1999 and rapidly spread across the continent. WNV has now emerged in a number of European countries, particularly around the Mediterranean basin, where infections in humans, horses and birds have been reported.

Cross-reactivity of sera raised against one flavivirus recognising another flavivirus has been well documented. One consequence of flavivirus cross-reactivity is the occurrence of false-positive results, yet cross-reactivity can lead to cross-protection. Understanding and manipulating the cross-reactive properties of flaviviruses has the potential to assist the development of effective broad-spectrum human vaccines against WNV and other existing and emerging flaviviruses.

Flavivirus-induced antibody cross-reactivity. J Gen Virol. Sep 7 2011
Dengue viruses (DENV) cause countless human deaths each year, whilst West Nile virus (WNV) has re-emerged as an important human pathogen. There are currently no WNV or DENV vaccines licensed for human use, yet vaccines exist against other flaviviruses. To investigate flavivirus cross-reactivity, sera from a human cohort with a history of vaccination against tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and yellow fever virus (YFV) were tested for antibodies by plaque reduction neutralisation test. Neutralisation of Louping ill virus (LIV) occurred, but no significant neutralisation of Murray Valley encephalitis virus (MVEV) was observed. Sera from some individuals vaccinated against TBEV and JEV neutralised WNV, which was enhanced by YFV vaccination in some recipients. Similarly, some individuals neutralised DENV-2, but this was not significantly influenced by YFV vaccination. Antigenic cartography techniques were used to generate a geometric illustration of the neutralisation titres of selected sera against WNV, TBEV, JEV, LIV, YFV and DENV-2. This demonstrated the individual variation in antibody responses. Most sera had detectable titres against LIV and some had titres against WNV and DENV-2. Generally, LIV titres were similar to titres against TBEV, confirming the close antigenic relationship between TBEV and LIV. JEV was also antigenically closer to TBEV than WNV, using these sera. The use of sera from individuals vaccinated against multiple pathogens is unique relative to previous applications of antigenic cartography techniques. It is evident from these data that notable differences exists between amino acid sequence identity and mapped antigenic relationships within the family Flaviviridae.

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Dengue virus infection is a growing public health problem with up to 100 million cases annually, and neither vaccines nor effective therapies are available. To search for the ways of preventing and treating dengue infections we need to better understand their molecular mechanisms. As with many other viruses, dengue virus enters cells by fusion between the viral membrane and the membrane of intracellular vesicles (endosomes). In this paper the authors explore the fusion stage of dengue virus entry in different experimental systems ranging from virus fusion to artificial lipid membranes to fusion inside the cells. While earlier work on dengue virus entry has focused on the virus protein that mediates fusion, they found that effective action of this protein requires specific lipid composition of the membrane the virus fuses to. In effect, this lipid dependence allows virus to control intracellular location of the fusion event and, thus, the place of its RNA release by exploiting cell-controlled differences between lipid compositions of different organelles the virus travels through. The essential role of the interactions between dengue virus and its lipid cofactors during virus entry suggests that these interactions may be targeted in drug design.

Dengue Virus Ensures Its Fusion in Late Endosomes Using Compartment-Specific Lipids. PLoS Pathog 6(10): e1001131. doi:10.1371/journal.ppat.1001131
Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN), the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycero)phosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycero)phosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes, but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN, and the essential role of the newly identified DEN-bis(monoacylglycero)phosphate interactions in viral genome escape from the endosome suggests a novel target for drug design.

Related:

• Dengue Virus
• Pathogenesis and treatment of dengue fever

The incidence and geographic range of dengue and dengue hemorrhagic fever has increased dramatically in recent decades. With 2.5 billion people now living in areas at risk for epidemic transmission, dengue has become the most important mosquito-borne viral disease affecting humans. Dengue virus (DENV) is a positive-strand RNA virus of the family Flaviviridae. It exists as four closely related but antigenically distinct serotypes, all of which have Aedes aegypti mosquitoes as their primary vector, with A. albopictus as a secondary vector.

Mosquitoes, like all insects, are exposed to a variety of microbes in their natural habitats, and possess an innate immune system that is capable of mounting a potent response against microbial challenge. The insect innate immune response is largely regulated by three main immune signaling pathways: the Toll, immune deficiency (IMD) and Janus kinase signal transducer and activator of transcription (JAK-STAT) pathways. The Toll pathway is involved in defense against fungi, Gram-positive bacteria, and viruses, and has been found to be specifically involved in the A. aegypti anti-DENV response.

In order to study the interaction of DENV with the A. aegypti immune response, researchers have characterized the DENV infection-responsive transcriptome of the immune-competent A. aegypti cell line. As in mosquitoes, DENV infection transcriptionally activated the cell line Toll pathway and a variety of cellular physiological systems. Most notably, however, DENV infection down-regulated the expression levels of numerous immune signaling molecules and antimicrobial peptides (AMPs). Functional assays showed that transcriptional induction of AMPs from the Toll and IMD pathways in response to bacterial challenge is impaired in DENV-infected cells. In addition, Escherichia coli, a Gram-negative bacteria species, grew better when co-cultured with DENV-infected cells than with uninfected cells, suggesting a decreased production of AMPs from the IMD pathway in virus-infected cells. Pre-stimulation of the cell line with Gram-positive bacteria prior to DENV infection had no effect on DENV titers, while pre-stimulation with Gram-negative bacteria resulted in an increase in DENV titers. These results indicate that DENV is capable of actively suppressing immune responses in the cells it infects, a phenomenon that may have important consequences for virus transmission and insect physiology.

Dengue Virus Inhibits Immune Responses in Aedes aegypti Cells. 2010 PLoS ONE 5(5): e10678. doi:10.1371/journal.pone.0010678

Related:

• The dengue vector Aedes aegypti
• Dengue Virus
• Rethinking dengue hemorrhagic fever

Aedes aegypti is the urban vector of dengue viruses worldwide. While climate influences the geographical distribution of this mosquito species, other factors also determine the suitability of the physical environment for this mosquito. Importantly, the close association of Ae. aegypti with humans and the domestic environment allows this species to persist in regions that may otherwise be unsuitable based on climatic factors alone. This review highlights the need to incorporate the impact of the urban environment in attempts to model the potential distribution of Ae. aegypti and briefly discuss the potential for future technology to aid management and control of this widespread vector species.

The dengue vector Aedes aegypti: What comes next. Microbes Infect. Jan 20 2010. doi:10.1016/j.micinf.2009.12.011

Related:

• Population movement a critical factor in dengue virus spread
• Dengue Virus
• Rethinking dengue hemorrhagic fever
• Pathogenic Flaviviruses

Dengue fever is a major public health problem in many tropical regions of the world. It is a vector-borne disease, transmitted most often by the mosquito Aedes aegypti. According to the WHO, the prevalence of dengue is highest in tropical areas of Asia and the Americas, with 50-100 million estimated cases of dengue fever and 250,000-500,000 cases of dengue hemorrhagic fever occurring annually worldwide as explosive outbreaks in urban areas.

In Brazil, three dengue virus serotypes (DENV) have been introduced in the past three decades. In 2007-2008, a dengue fever epidemic in Rio de Janeiro led to 240 deaths registered (100 deaths due to dengue hemorrhagic fever and 140 due to other dengue-related complications). This populous city presents highly favorable conditions for transmission of dengue. Dengue surveillance and control in large urban areas with high levels of dengue transmission pose important challenges. Therefore, consistent knowledge of the dynamics of this disease that integrates epidemiological and entomological data is essential.

Human movement is a key factor of dengue virus inflow in Rio de Janeiro, Brazil. The results published in a new paper, based on data from a severe epidemic in 2007-2008, contribute to new understanding on the dynamics of dengue fever in the second largest city in Brazil. This research combines data on dengue fever seroprevalence, recent dengue infection, and vector density in three neighborhoods of Rio de Janeiro: an urban, a suburban, and a slum area. Serological surveys were conducted before and during the epidemic period. Entomological surveys consisted of weekly collections of A. aegypti eggs and adults from traps. This integrated entomological-serological survey showed evidence of silent transmission even during a severe epidemic. No association was observed between household infestation index and risk of dengue infection in these areas, raising new questions about where transmission occurs – in the household, at work or elsewhere. When combined, the neighborhood-specific seroprevalence maps correlated significantly higher risk with areas of intense people traffic. These results add to previous epidemiological studies of dengue virus infections and contribute to the understanding of A. aegypti habits. The conclusions may provide a basis for new studies that could further identify the higher seroprevalence risk areas and help to develop and implement dengue-control programs.

Spatial Evaluation and Modeling of Dengue Seroprevalence and Vector Density in Rio de Janeiro, Brazil. PLoS Negl Trop Dis 3(11): e545. doi:10.1371/journal.pntd.0000545
Rio de Janeiro, Brazil, experienced a severe dengue fever epidemic in 2008. This was the worst epidemic ever, characterized by a sharp increase in case-fatality rate, mainly among younger individuals. A combination of factors, such as climate, mosquito abundance, buildup of the susceptible population, or viral evolution, could explain the severity of this epidemic. The main objective of this study is to model the spatial patterns of dengue seroprevalence in three neighborhoods with different socioeconomic profiles in Rio de Janeiro. As blood sampling coincided with the peak of dengue transmission, we were also able to identify recent dengue infections and visually relate them to Aedes aegypti spatial distribution abundance. We analyzed individual and spatial factors associated with seroprevalence using Generalized Additive Model (GAM). Three neighborhoods were investigated: a central urban neighborhood, and two isolated areas characterized as a slum and a suburban area. Weekly mosquito collections started in September 2006 and continued until March 2008. In each study area, 40 adult traps and 40 egg traps were installed in a random sample of premises, and two infestation indexes calculated: mean adult density and mean egg density. Sera from individuals living in the three neighborhoods were collected before the 2008 epidemic (July through November 2007) and during the epidemic (February through April 2008). Sera were tested for DENV-reactive IgM, IgG, Nested RT-PCR, and Real Time RT-PCR. From the before– after epidemics paired data, we described seroprevalence, recent dengue infections (asymptomatic or not), and seroconversion. Recent dengue infection varied from 1.3% to 14.1% among study areas. The highest IgM seropositivity occurred in the slum, where mosquito abundance was the lowest, but household conditions were the best for promoting contact between hosts and vectors. By fitting spatial GAM we found dengue seroprevalence hotspots located at the entrances of the two isolated communities, which are commercial activity areas with high human movement. No association between recent dengue infection and household’s high mosquito abundance was observed in this sample. This study contributes to better understanding the dynamics of dengue in Rio de Janeiro by assessing the relationship between dengue seroprevalence, recent dengue infection, and vector density. In conclusion, the variation in spatial seroprevalence patterns inside the neighborhoods, with significantly higher risk patches close to the areas with large human movement, suggests that humans may be responsible for virus inflow to small neighborhoods in Rio de Janeiro. Surveillance guidelines should be further discussed, considering these findings, particularly the spatial patterns for both human and mosquito populations.

Related:

• Dengue Virus
• Rethinking dengue hemorrhagic fever
• Pathogenic Flaviviruses

Dengue virus infection usually causes a severe flu like illness, although symptoms may be mild in young children. DHF, however, is a severe and sometimes fatal complication of dengue virus infection that affects about half a million people every year after infection with any one of the four dengue virus (DENV) serotypes. DHF patients usually fall into two groups; children and adults who become infected with a second dengue virus serotype after an initial primary dengue virus infection with a different serotype, and infants with primary dengue virus infections born to mothers who have some dengue virus immunity. The widely accepted explanation for the pathogenesis of DHF in these settings, particularly during infancy, is antibody-dependent enhancement (ADE) of DENV infection.

Researchers conducted a prospective nested case-control study of DENV infections during infancy. Clinical data and blood samples were collected from 4,441 mothers and infants in up to two pre-illness study visits, and surveillance was performed for symptomatic and inapparent DENV infections. Pre-illness plasma samples were used to measure the associations between maternally derived anti-DENV3 antibody-neutralizing and enhancing capacities at the time of DENV3 infection and development of infant DHF. The study examined 60 infants with DENV infections across a wide spectrum of disease severity. DENV3 was the predominant serotype among the infants with symptomatic (35/40) and inapparent (15/20) DENV infections, and 59/60 infants had a primary DENV infection. The estimated in vitro anti-DENV3 neutralizing capacity at birth positively correlated with the age of symptomatic primary DENV3 illness in infants. At the time of symptomatic DENV3 infection, essentially all infants had low anti-DENV3 neutralizing activity and measurable DENV3 ADE activity. The infants who developed DHF did not have significantly higher frequencies or levels of DENV3 ADE activity compared to symptomatic infants without DHF. A higher weight-for-age in the first 3 mo of life and at illness presentation was associated with a greater risk for DHF from a primary DENV infection during infancy. This prospective nested case-control study of primarily DENV3 infections during infancy has shown that infants exhibit a full range of disease severity after primary DENV infections.

The current model for development of DHF in infants around 6 months old is that anti-dengue virus antibodies transferred from a dengue-immune mother to her child somehow enhance dengue virus infection, resulting in more severe symptoms (the  antibody-dependent enhancement  model). These results support an initial in vivo protective role for maternally derived antibody. There was no significant association between DENV3 ADE activity at illness onset and the development of DHF compared with less severe symptomatic illness. The results of this study should encourage rethinking or refinement of the current ADE pathogenesis model for infant DHF and stimulate new directions of research into mechanisms responsible for the development of DHF during infancy.

A Prospective Nested Case-Control Study of Dengue in Infants: Rethinking and Refining the Antibody-Dependent Enhancement Dengue Hemorrhagic Fever Model. PLoS Med 6(10): e1000171 doi:10.1371/journal.pmed.1000171

Related:

• Dengue Virus
• Dengue virus infection activates the unfolded protein response
• Pathogenic Flaviviruses