Infection and dissemination of dengue virus type 2 in Aedes aegypti, Aedes albopictus, and Aedes scutellaris from the Torres Strait, Australia

To determine their relative roles in transmission of dengue virus (DENV) in the Torres Strait region of northern Australia, we examined infection and dissemination of a sympatric strain of dengue virus type 2 (DENV-2) in Aedes scutellaris, Ae. albopictus, and Ae. aegypti. In experiments using membrane feeders for virus exposure, infection rates were 83% and 43% for Ae. scutellaris and Ae. aegypti, respectively. Salivary gland infection rates for both species were 43%. In experiments using pledgets for virus exposure, infection rates for Ae. aegypti, Ae. scutellaris, and Ae. albopictus were 68%, 55%, and 37%, respectively. Aedes albopictus exhibited the greatest barriers to infection with only 7% tested developing a salivary gland infection, compared to 42% and 24% of Ae. aegypti and Ae. scutellaris, respectively. These results suggest that Ae. scutellaris may have been responsible for DENV transmission on Torres Strait islands, where Ae. aegypti does not occur. In contrast, Ae. albopictus may not be an important vector of DENV-2 from the Torres Strait.     

Dengue emergence and adaptation to peridomestic mosquitoes

Phylogenetic evidence suggests that endemic and epidemic dengue viruses (DENV), transmitted among humans by the anthropophilic mosquitoes Aedes aegypti and Ae. albopictus, emerged when ancestral, sylvatic DENV transmitted among nonhuman primates by sylvatic Aedes mosquitoes adapted to these peridomestic vectors. We tested this hypothesis by retrospectively examining evidence for adaptation of epidemic and endemic versus sylvatic strains of DENV-2 to Ae. albopictus and Ae. aegypti. First and second-generation offspring of mosquitoes from different geographic regions in the Americas and Southeast Asia were tested for their susceptibility to epidemic/endemic and sylvatic DENV-2 isolates from West Africa, Southeast Asia, and Oceania. Both Aedes species were highly susceptible (up to 100% infected) to endemic/epidemic DENV-2 strains after ingesting artificial blood meals but significantly less susceptible (as low as 0%) to sylvatic DENV-2 strains. Our findings support the hypothesis that adaptation to peridomestic mosquito vectors mediated dengue emergence from sylvatic progenitor viruses.     

Aedes albopictus in the Solomon and Santa Cruz Islands,South Pacific

Surveys of mosquitoes in the South Pacific up until 1977 have shown the presence of Aedes albopictus only as far east as Papua New Guinea. During the visit to the Solomon Islands in 1978 to collect members of the Aedes (Stegomyia) scutellaris complex, Ae. albopictus was found in large numbers on Guadalcanal and was also present in the Santa Cruz Islands. This species has been incriminated as a vector of dengue haemorrhagic fever.     

Challenges encountered using standard vector control measures for dengue in Boa Vista,Brazil

Problem

In 2010, dengue virus (DENV) serotype–4 was detected during a dengue outbreak in the Amazonian city of Boa Vista. At that time Brazil was already endemic for DENV-1, DENV-2 and DENV-3. This was the first time DENV-4 was observed in the country after it was initially detected and eliminated in 1981.

Approach

To hinder the spread of DENV-4 throughout Brazil, standard vector control measures were intensified. Vector control professionals visited 56 837 households in 22 out of 31 districts of Boa Vista, to eliminate mosquito-breeding sites. Water storage containers were treated with the larvicide diflubenzuron, and deltamethrin was sprayed for adult Aedes aegypti mosquitoes. Fifteen days later, a second larvae survey and additional deltamethrin applications were performed.

Local setting

In Brazil, dengue vector control is managed at all three government levels. Regular surveillance of Aedes aegypti is done four to six times a year to strengthen mosquito control activities in areas with high-vector density. Educational dengue control campaigns in communities are scarce, especially between outbreaks.

Relevant changes

In spite of extensive implementation of all standard control actions recommended by the Brazilian dengue control programme, only a slight decrease in mosquito density was detected.

Lessons learnt

There is a need to redesign all levels of dengue control. Public consultation and engagement, behaviour change and actions that go beyond technical impositions are required. Vector control programme managers need to reflect on what constitutes good practices and whether intermittent information campaigns are effective measures for dengue prevention and control.     

Experimental infection of Aedes aegypti and Aedes albopictus with dengue viruses

The susceptibility of two Aedes (Stegomyia) species, Ae. aegypti and Ae. albopictus, to infection with dengue virus was assessed using both natural feeding on a viraemic gibbon and a membrane feeding technique. Both species were highly susceptible to dengue virus infection.     

Wing shape of dengue vectors from around the world.

Wing shape is increasingly utilized in species identification and characterization. For dengue vectors Aedes aegypti and Aedes albopictus, it could be used as a complement for ensuring accurate diagnostic of damaged specimens. However, the impact of world migration on wing shape is unknown. Has the spread of these invasive species increased shape variation to the extent of producing interspecific overlapping? To answer this question, the geometric patterns of wing venation in Ae. aegypti and Ae. albopictus were compared between natural populations from the Pacific Islands, North and South America and South East Asia. The geometry of 178 female and 174 male wings were described at 13 anatomical landmarks, and processed according to Procrustes superposition, partial warps and subsequent multivariate analyzes. The variation of shape did not produce significant interspecific overlapping. Regardless of geographic origin, Ae. aegypti was recognized as Ae. aegypti and Ae. albopictus as Ae. albopictus. Some significant geographic differentiation was observed in Colombia for Ae. aegypti and in Thailand for Ae. albopictus. Globally, the morphology of these mosquitoes, for both size and shape, appeared well preserved. Strong canalizing mechanisms could account for the observed patterns of relatively uniform morphology, which could also be attributed to sporadic, recurrent mixing of populations, thwarting phenotypic drift.     

Fever versus fever: The role of host and vector susceptibility and interspecific competition in shaping the current and future distributions of the sylvatic cycles of dengue virus and yellow fever virus

Two different species of flaviviruses, dengue virus (DENV) and yellow fever virus (YFV), that originated in sylvatic cycles maintained in non-human primates and forest-dwelling mosquitoes have emerged repeatedly into sustained human-to-human transmission by Aedes aegypti mosquitoes. Sylvatic cycles of both viruses remain active, and where the two viruses overlap in West Africa they utilize similar suites of monkeys and Aedes mosquitoes. These extensive similarities render the differences in the biogeography and epidemiology of the two viruses all the more striking. First, the sylvatic cycle of YFV originated in Africa and was introduced into the New World, probably as a result of the slave trade, but is absent in Asia; in contrast, sylvatic DENV likely originated in Asia and has spread to Africa but not to the New World. Second, while sylvatic YFV can emerge into extensive urban outbreaks in humans, these invariably die out, whereas four different types of DENV have established human transmission cycles that are ecologically and evolutionarily distinct from their sylvatic ancestors. Finally, transmission of YFV among humans has been documented only in Africa and the Americas, whereas DENV is transmitted among humans across most of the range of competent Aedes vectors, which in the last decade has included every continent save Antarctica. This review summarizes current understanding of sylvatic transmission cycles of YFV and DENV, considers possible explanations for their disjunct distributions, and speculates on the potential consequences of future establishment of a sylvatic cycle of DENV in the Americas.     

Emergence of dengue virus serotype 3 on Mayotte Island, Indian Ocean

A serosurvey carried out in 2006 in Mayotte, a French overseas collectivity in the Indian Ocean, confirmed previous circulation of dengue virus (DENV) on the island, but since the set up of a laboratory-based surveillance of dengue-like illness in 2007, no case of DENV has been confirmed. In response to an outbreak of DENV-3 on Comoros Islands in March 2010 surveillance of dengue-like illness in Mayotte was enhanced. By September 15, 76 confirmed and 31 probable cases of DENV have been identified in Mayotte. In urban and periurban settings on the island, Aedes albopictus is the predominant Aedes species, but Ae. aegyptii remains the most common species in rural areas. Given the epidemic potential of dengue virus in Mayotte, adequate monitoring including early detection of cases, timely investigation and sustained mosquito control actions remain essential.     

Rapid blood meal scoring in anthropophilic Aedes albopictus and application of PCR blocking to avoid pseudogenes

Blood meal analysis (BMA) is a useful tool for epidemiologists and vector ecologists to assess which vector species are critical to disease transmission. In most current BMA assays vertebrate primers amplify DNA from a blood meal, commonly an abundant mitochondrial (mtDNA) locus, which is then sequenced and compared to known sequences in GenBank to identify its source. This technique, however, is time consuming and costly as each individual sample must be sequenced for species identification and mixed blood meals cloned prior to sequencing. Further, we found that several standard BMA vertebrate primers match sequences of the mtDNA of the Asian tiger mosquito, Aedes albopictus, making their use for blood meal identification in this species impossible. Because of the importance of Ae. albopictus as a vector of dengue and chikungunya viruses to humans, we designed a rapid assay that allows easy identification of human blood meals as well as mixed meals between human and nonhuman mammals. The assay consists of a nested PCR targeting the cytochrome b (cytb) mtDNA locus with a blocking primer in the internal PCR. The blocking primer has a 3′ inverted dT modification that when used with the Stoffel Taq fragment prevents amplification of nuclear cytochrome b pseudogenes in humans and allows for the continued use of cytb in BMA studies, as it is one of the most species-rich loci in GenBank. We used our assay to examine 164 blooded specimens of Ae. albopictus from suburban coastal New Jersey and found 62% had obtained blood from humans with 7.6% mixes between human and another mammal species. We also confirmed the efficiency of our assay by comparing it with standard BMA primers on a subset of 62 blooded Ae. albopictus. While this assay was designed for use in Ae. albopictus, it will have broader application in other anthropophilic mosquitoes.     

Evolution and epidemiologic dynamics of dengue virus in Nicaragua during the emergence of chikungunya and Zika viruses

Arthropod-borne viruses (arboviruses) comprise a significant and ongoing threat to human health, infecting hundreds of millions annually. Three such arboviruses include circumtropical dengue, Zika, and chikungunya viruses, exhibiting continuous emergence primarily via Aedes mosquito vectors. Nicaragua has experienced endemic dengue virus (DENV) transmission involving multiple serotypes since 1985, with chikungunya virus (CHIKV) reported in 2014–2015, followed by Zika virus (ZIKV) first reported in 2016. In order to identify patterns of genetic variation and selection pressures shaping the evolution of co-circulating DENV serotypes in light of the arrival of CHIKV and ZIKV, we employed whole-genome sequencing on an Illumina MiSeq platform of random-amplified total RNA libraries to characterize 42 DENV low-passage isolates, derived from viremic patients in Nicaragua between 2013 and 2016. Our approach also revealed clinically undetected co-infections with CHIKV. Of the three DENV serotypes (1, 2, and 3) co-circulating during our study, we uncovered distinct patterns of evolution using comparative phylogenetic inference. DENV-1 genetic variation was structured into two distinct co-circulating lineages with no evidence of positive selection in the origins of either lineage, suggesting they are equally fit. In contrast, the evolutionary history of DENV-2 was marked by positive selection, and a unique, divergent lineage correlated with high epidemic potential emerged in 2015 to drive an outbreak in 2016. DENV-3 genetic variation remained unstructured into lineages throughout the period of study. Thus, this study reveals insights into evolutionary and epidemiologic trends exhibited during the circulation of multiple arboviruses in Nicaragua.     

<Emphasis Type="Italic">Aedes albopictus</Emphasis> and the reemergence of Dengue

Dengue is a vector-borne disease that is estimated to affect millions of individuals each year in tropical and subtropical areas, and it is reemerging in areas that have been disease-free for relatively long periods of time. In this issue of the journal, Peng et al. report on a Dengue outbreak in a city in southern China that had been disease-free for more than two decades. The infection, which was due to serotype 1, was introduced by a traveler from South-east Asia and transmitted by Aedes albopictus, the Asian tiger mosquito. Compared to Aedes aegypti, which is the most important vector of Dengue, Ae albopictus is a less competent vector of arboviruses, and the epidemics it causes are milder. However, Ae albopictus is becoming an increasingly important vector because of its rapidly changing global distribution. In particular, the worldwide trade in second hand tires, which often contain water and are an ideal place for eggs and larvae, has been a key factor in the large-scale conquest of Ae albopictus, which easily adapts to new environments, even in a temperate climate. This expansion is creating new opportunities for viruses to circulate in new areas, becoming a common cause of epidemics in Ae aegypti-free countries, from Hawaii to Mauritius. The outbreak in China, like similar events, was mild and short-lived. Because epidemics due to Ae albopictus are milder, the replacement of Ae aegypti with the tiger mosquito could even result in public-health benefits. However, there is no solid evidence of this, and the milder course of the outbreak could be in part explained by the relatively short duration of the hot season in some affected areas. Since it is almost impossible to prevent Ae albopictus from being introduced in a country, mosquito-control measures at local level remain the most effective means of controlling arbovirus outbreaks.     

Amplification of the sylvatic cycle of dengue virus type 2, Senegal, 1999-2000: entomologic findings and epidemiologic considerations

After 8 years of silence, dengue virus serotype 2 (DENV-2) reemerged in southeastern Senegal in 1999. Sixty-four DENV-2 strains were isolated in 1999 and 9 strains in 2000 from mosquitoes captured in the forest gallery and surrounding villages. Isolates were obtained from previously described vectors, Aedes furcifer, Ae. taylori, Ae. luteocephalus, and--for the first time in Senegal--from Ae. aegypti and Ae. vittatus. A retrospective analysis of sylvatic DENV-2 outbreaks in Senegal during the last 28 years of entomologic investigations shows that amplifications are periodic, with intervening, silent intervals of 5-8 years. No correlation was found between sylvatic DENV-2 emergence and rainfall amount. For sylvatic DENV-2 vectors, rainfall seems to particularly affect virus amplification that occurs at the end of the rainy season, from October to November. Data obtained from investigation of preimaginal (i.e., nonadult) mosquitoes suggest a secondary transmission cycle involving mosquitoes other than those identified previously as vectors.     

Co-circulation of Chikungunya and Dengue viruses in Dengue endemic region of New Delhi,India during 2016

Co-circulation of Chikungunya and Dengue viral infections (CHIKV and DENV) have been reported mainly due to transmission by common Aedes vector. The purpose of the study was to identify and characterise the circulating strains of CHIKV and DENV in DENV endemic region of New Delhi during 2016. CHIKV and DENV were identified in the blood samples (n = 130) collected from suspected patients by RT-PCR. CHIKV was identified in 26 of 65 samples (40%). Similarly, DENV was detected in 48 of 120 samples (40%). Co-infection with both the viruses was identified in five (9%) of the samples. Interestingly, concurrent infection with DENV, CHIKV and Plasmodium vivax was detected in two samples. CHIKV strains (n = 11) belonged to the ECSA genotype whereas DENV-3 sequences (n = eight) clustered in Genotype III by phylogenetic analysis. Selection pressure of E1 protein of CHIKV and CprM protein of DENV-3 revealed purifying selection with four and two positive sites, respectively. Four amino acids of the CHIKV were positively selected and had high entropy suggesting probable variations. Co-circulation of both viruses in DENV endemic regions warrants effective monitoring of these emerging pathogens via comprehensive surveillance for implementation of effective control measures.Key words: Chikungunya virus, co-circulation, co-infection, Dengue virus, phylogenetic analysis     

Molecular investigation of the dengue outbreak in Karnataka,South India,reveals co-circulation of all four dengue virus serotypes

The growing incidence of dengue outbreaks in the state of Karnataka prompted us to study the circulating dengue virus (DENV) and their proportion among the suspected cases of dengue patients during the disease outbreak at Mysuru district of Southern India. The presence of the DENV in a patient's serum sample was identified by RT-PCR using previously published primer pairs targeting CprM gene. DENV serotyping was carried out by semi-nested multiplex PCR using serotype-specific primers and nucleotide sequencing. Three hundred fifty-five samples of serum from suspected dengue cases were collected, and 203 samples (57.18%) were found positives. In 2016, DENV-4 (97.87%) was found to be the most dominant DENV serotype either alone or as co-infection, followed by DENV-2 (8.51%) and DENV-3 (4.25%). In 47 positive cases, co-infection with more than one serotype was detected in 4 cases (8.51%). The analysis of the dengue cases in 2017, DENV-4 was dominating serotype (33.97%), followed by the emergence of DENV-2 (32.05%), DENV-3 (25.64%), and DENV-1 (25.00%). Our study also reports the circulation of all four DENV serotypes in the Mysuru district of Southern India, with concurrent infections rate of 16.66% in 2017. The present study provides information regarding the genetic variation among the circulating DENV serotype in an Indian state of Karnataka. The need for the studying genetic diversity of DENV will be useful during the continuous monitoring for disease burden as well as the development of appropriate prophylactic measures to control the spread of dengue infection.     

A novel dengue virus serotype-2 nanovaccine induces robust humoral and cell-mediated immunity in mice

Dengue virus (DENV), a member of the Flaviviridae family, can be transmitted to humans through the bite of infected Aedes mosquitoes. The incidence of dengue has increased worldwide over the past few decades. Inadequate vector control, changing global ecology, increased urbanization, and faster global travel are factors enhancing the rapid spread of the virus and its vector. In the absence of specific antiviral treatments, the search for a safe and effective vaccine grows more imperative. Many strategies have been utilized to develop dengue vaccines. Here, we demonstrate the immunogenic properties of a novel dengue nanovaccine (DNV), composed of ultraviolet radiation (UV)-inactivated DENV-2, which has been loaded into the nanoparticles containing chitosan/Mycobacterium bovis Bacillus Calmette-Guerin cell wall components (CS/BCG-NPs). We investigated the immunogenicity of DNV in a Swiss albino mouse model. Inoculation with various concentrations of vaccine (0.3, 1, 3 and 10 μg/dose) with three doses, 15-day apart, induced strong anti-dengue IgM and IgG antibodies in the mouse serum along with neutralizing antibody against DENV-2 reference strain (16681), a clinical-isolate strain (00745/10) and the mouse-adapted New Guinea-C (NGC) strain. Cytokine and chemokine secretion in the serum of DNV-immunized mice showed elevated levels of IFN-γ, IL-2, IL-5, IL-12p40, IL-12p70, IL-17, eotaxin and RANTES, all of which have varying immune functions. Furthermore, we observed a DNV dose-dependent increase in the frequencies of IFN-γ-producing CD4⁺ and CD8⁺ T cells after in vitro stimulation of nucleated cells. Based on these findings, DNV has the potential to become a candidate dengue vaccine.     

Molecular evolution of dengue viruses: Contributions of phylogenetics to understanding the history and epidemiology of the preeminent arboviral disease

Dengue viruses (DENV) are the most important arboviral pathogens in tropical and subtropical regions throughout the world, putting at risk of infection nearly a third of the global human population. Evidence from the historical record suggests a long association between these viruses and humans. The transmission of DENV includes a sylvatic, enzootic cycle between nonhuman primates and arboreal mosquitoes of the genus Aedes, and an urban, endemic/epidemic cycle between Aedes aegypti, a mosquito with larval development in peridomestic water containers, and human reservoir hosts. DENV are members of the genus Flavivirus in the Family Flaviviridae and comprise of 4 antigenically distinct serotypes (DENV-1–4). Although they are nearly identical epidemiologically, the 4 DENV serotypes are genetically quite distinct. Utilization of phylogenetic analyses based on partial and/or complete genomic sequences has elucidated the origins, epidemiology (genetic diversity, transmission dynamics and epidemic potential), and the forces that shape DENV molecular evolution (rates of evolution, selection pressures, population sizes, putative recombination and evolutionary constraints) in nature. In this review, we examine how phylogenetics have improved understanding of DENV population dynamics and sizes at various stages of infection and transmission, and how this information may influence pathogenesis and improve our ability to understand and predict DENV emergence.     

Evaluation of Dengue Virus strains for human challenge studies

Discordance between the measured levels of dengue virus neutralizing antibody and clinical outcomes in the first-ever efficacy study of a dengue tetravalent vaccine (Lancet, Nov 2012) suggests a need to re-evaluate the process of pre-screening dengue vaccine candidates to better predict clinical benefit prior to large-scale vaccine trials. In the absence of a reliable animal model and established correlates of protection for dengue, a human dengue virus challenge model may provide an approach to down-select vaccine candidates based on their ability to reduce risk of illness following dengue virus challenge. We report here the challenge of flavivirus-naïve adults with cell culture-passaged dengue viruses (DENV) in a controlled setting that resulted in uncomplicated dengue fever (DF). This sets the stage for proof-of-concept efficacy studies that allow the evaluation of dengue vaccine candidates in healthy adult volunteers using qualified DENV challenge strains well before they reach field efficacy trials involving children. Fifteen flavivirus-naïve adult volunteers received 1 of 7 DENV challenge strains (n = 12) or placebo (n = 3). Of the twelve volunteers who received challenge strains, five (two DENV-1 45AZ5 and three DENV-3 CH53489 cl24/28 recipients) developed DF, prospectively defined as ≥2 typical symptoms, ≥48 h of sustained fever (>100.4 °F) and concurrent viremia. Based on our study and historical data, we conclude that the DENV-1 and DENV-3 strains can be advanced as human challenge strains. Both of the DENV-2 strains and one DENV-4 strain failed to meet the protocol case definition of DF. The other two DENV-4 strains require additional testing as the illness approximated but did not satisfy the case definition of DF. Three volunteers exhibited effusions (1 pleural/ascites, 2 pericardial) and 1 volunteer exhibited features of dengue (rash, lymphadenopathy, neutropenia and thrombocytopenia), though in the absence of fever and symptoms. The occurrence of effusions in milder DENV infections counters the long-held belief that plasma leakage syndromes are restricted to dengue hemorrhagic fever/dengue shock syndromes (DHF/DSS). Hence, the human dengue challenge model may be useful not only for predicting the efficacy of vaccine and therapeutic candidates in small adult cohorts, but also for contributing to our further understanding of the mechanisms behind protection and virulence.     

Entomologic and Virologic Investigation of Chikungunya, Singapore

Local transmission of chikungunya, a debilitating mosquito-borne viral disease, was first reported in Singapore in January 2008. After 3 months of absence, locally acquired Chikungunya cases resurfaced in May 2008, causing an outbreak that resulted in a total of 231 cases by September 2008. The circulating viruses were related to East, Central, and South African genotypes that emerged in the Indian Ocean region in 2005. The first local outbreak was due to a wild-type virus (alanine at codon 226 of the envelope 1 gene) and occurred in an area where Aedes aegypti mosquitoes were the primary vector. Strains isolated during subsequent outbreaks showed alanine to valine substitution (A226V) and largely spread in areas predominated by Ae. albopictus mosquitoes. These findings led to a revision of the current vector control strategy in Singapore. This report highlights the use of entomologic and virologic data to assist in the control of chikungunya in disease-endemic areas.