Rapid displacement of dengue virus type 1 by type 4, Pacific region, 2007-2009

Since 2000-2001, dengue virus type 1 has circulated in the Pacific region. However, in 2007, type 4 reemerged and has almost completely displaced the strains of type 1. If only 1 serotype circulates at any time and is replaced approximately every 5 years, DENV-3 may reappear in 2012.

Identification of a cryptic Prokaryotic promoter within the cDNA encoding the 5′ End of Dengue Virus RNA Genome

Infectious cDNA clones of RNA viruses are important research tools, but flavivirus cDNA clones have proven difficult to assemble and propagate in bacteria. This has been attributed to genetic instability and/or host cell toxicity, however the mechanism leading to these difficulties has not been fully elucidated. Here we identify and characterize an efficient cryptic bacterial promoter in the cDNA encoding the dengue virus (DENV) 5′ UTR. Following cryptic transcription in E. coli, protein expression initiated at a conserved in-frame AUG that is downstream from the authentic DENV initiation codon, yielding a DENV polyprotein fragment that was truncated at the N-terminus. A more complete understanding of constitutive viral protein expression in E. coli might help explain the cloning and propagation difficulties generally observed with flavivirus cDNA.

A portable approach for the surveillance of dengue virus-infected mosquitoes

Dengue virus is the most significant human viral pathogen spread by the bite of an infected mosquito. With no vaccine or antiviral therapy currently available, disease prevention relies largely on surveillance and mosquito control. Preventing the onset of dengue outbreaks and effective vector management would be considerably enhanced through surveillance of dengue virus prevalence in natural mosquito populations. However, current approaches to the identification of virus in field-caught mosquitoes require relatively slow and labor intensive techniques such as virus isolation or RT-PCR involving specialized facilities and personnel. A rapid and portable method for detecting dengue virus-infected mosquitoes is described. Using a hand held battery operated homogenizer and a dengue diagnostic rapid strip the viral protein NS1 was detected as a marker of dengue virus infection. This method could be performed in less than 30 min in the field, requiring no downstream processing, and is able to detect a single infected mosquito in a pool of at least 50 uninfected mosquitoes. The method described in this study allows rapid, real-time monitoring of dengue virus presence in mosquito populations and could be a useful addition to effective monitoring and vector control responses.

Wolbachia-mediated resistance to dengue virus infection and death at the cellular level

Dengue is currently the most important arthropod-borne viral disease of humans. Recent work has shown dengue virus displays limited replication in its primary vector, the mosquito Aedes aegypti, when the insect harbors the endosymbiotic bacterium Wolbachia pipientis. Wolbachia-mediated inhibition of virus replication may lead to novel methods of arboviral control, yet the functional and cellular mechanisms that underpin it are unknown.

Recent emergence of dengue virus serotype 4 in French Polynesia results from multiple introductions from other south pacific islands

BACKGROUND: Infection by dengue virus (DENV) is a major public health concern in hundreds of tropical and subtropical countries. French Polynesia (FP) regularly experiences epidemics that initiate, or are consecutive to, DENV circulation in other South Pacific Island Countries (SPICs). In January 2009, after a decade of serotype 1 (DENV-1) circulation, the first cases of DENV-4 infection were reported in FP. Two months later a new epidemic emerged, occurring about 20 years after the previous circulation of DENV-4 in FP. In this study, we investigated the epidemiological and molecular characteristics of the introduction, spread and genetic microevolution of DENV-4 in FP. METHODOLOGY/PRINCIPAL FINDINGS: Epidemiological data suggested that recent transmission of DENV-4 in FP started in the Leeward Islands and this serotype quickly displaced DENV-1 throughout FP. Phylogenetic analyses of the nucleotide sequences of the envelope (E) gene of 64 DENV-4 strains collected in FP in the 1980s and in 2009-2010, and some additional strains from other SPICs showed that DENV-4 strains from the SPICs were distributed into genotypes IIa and IIb. Recent FP strains were distributed into two clusters, each comprising viruses from other but distinct SPICs, suggesting that emergence of DENV-4 in FP in 2009 resulted from multiple introductions. Otherwise, we observed that almost all strains collected in the SPICs in the 1980s exhibit an amino acid (aa) substitution V287I within domain I of the E protein, and all recent South Pacific strains exhibit a T365I substitution within domain III. CONCLUSIONS/SIGNIFICANCE: This study confirmed the cyclic re-emergence and displacement of DENV serotypes in FP. Otherwise, our results showed that specific aa substitutions on the E protein were present on all DENV-4 strains circulating in SPICs. These substitutions probably acquired and subsequently conserved could reflect a founder effect to be associated with epidemiological, geographical, eco-biological and social specificities in SPICs.

Comparative susceptibility of mosquito populations in North Queensland, Australia to oral infection with dengue virus

Dengue is the most prevalent arthropod-borne virus, with at least 40% of the world’s population at risk of infection each year. In Australia, dengue is not endemic, but viremic travelers trigger outbreaks involving hundreds of cases. We compared the susceptibility of Aedes aegypti mosquitoes from two geographically isolated populations with two strains of dengue virus serotype 2. We found, interestingly, that mosquitoes from a city with no history of dengue were more susceptible to virus than mosquitoes from an outbreak-prone region, particularly with respect to one dengue strain. These findings suggest recent evolution of population-based differences in vector competence or different historical origins. Future genomic comparisons of these populations could reveal the genetic basis of vector competence and the relative role of selection and stochastic processes in shaping their differences. Lastly, we show the novel finding of a correlation between midgut dengue titer and titer in tissues colonized after dissemination.

Limited dengue virus replication in field-collected Aedes aegypti mosquitoes infected with Wolbachia

Introduction Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus. Methodology/Principal Findings Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes. Conclusions/Significance Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.

Epidemiological and molecular features of dengue virus type-1 in New Caledonia, South Pacific, 2001–2013

Background The epidemiology of dengue in the South Pacific has been characterized by transmission of a single dominant serotype for 3–5 years, with subsequent replacement by another serotype. From 2001 to 2008 only DENV-1 was reported in the Pacific. In 2008, DENV-4 emerged and quickly displaced DENV-1 in the Pacific, except in New Caledonia (NC) where DENV-1 and DENV-4 co-circulated in 2008–2009. During 2012–2013, another DENV-1 outbreak occurred in NC, the third DENV-1 outbreak in a decade. Given that dengue is a serotype-specific immunizing infection, the recurrent outbreaks of a single serotype within a 10-year period was unexpected. Findings This study aimed to inform this phenomenon by examining the phylogenetic characteristics of the DENV-1 viruses in NC and other Pacific islands between 2001 and 2013. As a result, we have demonstrated that NC experienced introductions of viruses from both the Pacific (genotype IV) and South-east Asia (genotype I). Moreover, whereas genotype IV and I were co-circulating at the beginning of 2012, we observed that from the second half of 2012, i.e. during the major DENV-1 outbreak, all analyzed viruses were genotype I suggesting that a genotype switch occurred. Conclusions Repeated outbreaks of the same dengue serotype, as observed in NC, is uncommon in the Pacific islands. Why the earlier DENV-1 outbreaks did not induce sufficient herd immunity is unclear, and likely multifactorial, but the robust vector control program may have played a role by limiting transmission and thus maintaining a large susceptible pool in the population. Keywords: Dengue; Phylogeny; Genotype; Epidemics; New Caledonia

A native Wolbachia Endosymbiont does not limit Dengue virus infection in the mosquito Aedes notoscriptus (Diptera: Culicidae)

The endosymbiotic bacterium Wolbachia pipientis infects many species of insects and has been transinfected into the mosquito Aedes aegypti (L.), the primary vector of dengue virus (DENV). Recently, it has been shown that Wolbachia blocks the replication and transmission of RNA viruses, such as DENV, in a number of mosquito species including Ae. aegypti and Aedes albopictus (Skuse), which is naturally infected with Wolbachia and considered a secondary vector for DENV. The mosquito species Aedes notoscriptus (Skuse) is highly prevalent in Australia, including in areas where DENV outbreaks have been recorded. The mosquito has been implicated in the transmission of Ross River and Barmah Forest viruses, but not DENV. We investigated whether Wolbachia naturally infects this mosquito species and whether it has an impact on the ability of Ae. notoscriptus to transmit DENV. We show, for the first time, that Ae. notoscriptus is naturally infected with a strain of Wolbachia that belongs to supergroup B and is localized only in the ovaries. However, Wolbachia infection in Ae. notoscriptus did not induce resistance to DENV and had no effect on overall DENV infection rate or titer. The presence of a native Wolbachia in Ae. notoscriptus cannot explain why this mosquito is an ineffective vector of DENV.

Dengue virus infection : Diagnostics and molecular epidemiology

Dengue is a mosquito-borne viral disease caused by the four dengue virus serotypes (DENV-1-4) and is currently considered as the most important arthropod-borne viral disease in the world. Nearly half of the human population lives in risk areas, and 50-100 million infections occur yearly according to World Health Organization. The disease can vary from a mild febrile disease to severe haemorrhagic fever and shock. A secondary infection with heterologous serotype increases the risk for severe disease outcome. During the last three decades the impact of dengue has dramatically increased in the endemic areas including the tropics and subtropics of the world. The current situation with massive epidemics of severe disease forms has been associated with socio-ecological changes that have increased the transmission and enabled the co-circulation of different serotypes. Consequently, an increase of dengue has also been observed in travelers visiting these areas. Currently approximately 30 cases are diagnosed yearly in Finnish travelers. In travelers dengue is rarely a life-threatening disease, however in the current study, a fatality was documented in a young Finnish patient who experienced a prolonged primary dengue infection. To improve particularly early laboratory diagnostics, a novel real-time RT-PCR method was developed for the detection of DENV-1-4 RNA based on TaqMan chemistry. The method was shown to be sensitive and specific for detecting DENV RNA and suitable for diagnostic use. The newly developed real-time RT-PCR was compared to other available early diagnostic methods including IgM and NS1 antigen detection using a panel of selected patient samples. The results suggest that the best diagnostic rates are achieved by a combination of IgM with RNA or NS1 detection. The dengue virus strains studied here included the first DENV strains isolated from serum samples of Finnish travelers collected in 2000-2005. The results of sequence analysis demonstrated that the 11 isolates included all four DENV serotypes and presented a global sample of DENV strains from different geographical areas including Asia, Africa and South America. In the present study sequence analysis was also carried out for a collection of 23 novel DENV-2 isolates from Venezuelan patients collected in 1999-2005. The Venezuelan DENV-2 exclusively represented the American-Asian genotype, suggesting that no foreign DENV-2 lineages have recently been introduced to the country. The results also suggest that the DENV-2 viruses detected earlier from Venezuela have been maintained in the area where they have evolved into several lineages. This is in contrast to the pattern observed in some other dengue endemic areas, where introductions of novel virus types and lineages are frequently detected.

Prediction of protein-protein interactions in dengue virus coat proteins guided by low resolution cryoEM structures

Background: Dengue virus along with the other members of the flaviviridae family has reemerged as deadly human pathogens. Understanding the mechanistic details of these infections can be highly rewarding in developing effective antivirals. During maturation of the virus inside the host cell, the coat proteins E and M undergo conformational changes, altering the morphology of the viral coat. However, due to low resolution nature of the available 3-D structures of viral assemblies, the atomic details of these changes are still elusive. Results: In the present analysis, starting from C alpha positions of low resolution cryo electron microscopic structures the residue level details of protein-protein interaction interfaces of dengue virus coat proteins have been predicted. By comparing the preexisting structures of virus in different phases of life cycle, the changes taking place in these predicted protein-protein interaction interfaces were followed as a function of maturation process of the virus. Besides changing the current notion about the presence of only homodimers in the mature viral coat, the present analysis indicated presence of a proline-rich motif at the protein-protein interaction interface of the coat protein. Investigating the conservation status of these seemingly functionally crucial residues across other members of flaviviridae family enabled dissecting common mechanisms used for infections by these viruses. Conclusions: Thus, using computational approach the present analysis has provided better insights into the preexisting low resolution structures of virus assemblies, the findings of which can be made use of in designing effective antivirals against these deadly human pathogens.