Evolutionary processes among sylvatic dengue type 2 viruses

Sylvatic dengue viruses (DENV) are transmitted in an enzootic cycle between nonhuman primates and arboreal Aedes mosquitoes in Southeast Asia and West Africa. Although previous analyses have revealed the evolutionary processes among endemic (human) DENV, little is known about viral evolution in the sylvatic cycle. Through an analysis of 14 complete coding regions of sylvatic Dengue type 2 virus sampled over a 33-year period, we show that both the rate of evolutionary change and the pattern of natural selection are similar among endemic and sylvatic DENV, although the latter have a uniquely high frequency of positive selection in the NS4B protein gene. Our findings support a recent cross-species transmission event and suggest the possibility of future DENV reemergence from the sylvatic cycle.     

Evolution of endemic and sylvatic lineages of dengue virus

Recent disease outbreaks have raised awareness of tropical pathogens, especially mosquito-borne viruses. Dengue virus (DENV) is a widely studied mammalian pathogen transmitted by various species of mosquito in the genus Aedes, especially Aedes aegypti and Aedes albopictus. The prevailing view of the research community is that endemic viral lineages that cause epidemics of DENV in humans have emerged over time from sylvatic viral lineages, which persist in wild, non-human primates. These notions have been examined by researchers through phylogenetic analyses of the envelope gene (E) from the four serotypes of DENV (serotypes DENV-1 to DENV-4). In these previous reports, researchers used visual inspection of a phylogeny in order to assert that sylvatic lineages lead to endemic clades. In making this assertion, these researchers also reasserted the model of periodic sylvatic to endemic disease outbreaks. Since that study, there has been a significant increase in data both in terms of metadata (e.g., place and host of isolation) and genetic sequences of DENV. Here, we re-examine the model of sylvatic to endemic shifts in viral lineages through a phylogenetic tree search and character evolution study of metadata on the tree. We built a dataset of nucleotide sequences for 188 isolates of DENV that have metadata on sylvatic or endemic sampling along with three orthologous sequences from West Nile virus as the outgroup for the phylogenetic analysis. In contrast to previous research, we find that there are several shifts from endemic to sylvatic lineages as well as sylvatic to endemic lineages, indicating a much more dynamic model of evolution. We propose that a model that allows oscillation between sylvatic and endemic hosts better captures the dynamics of DENV transmission.     

Transplacental Zika virus transmission in ex vivo perfused human placentas

A Zika virus (ZIKV) infection during pregnancy can result in severe birth defects such as microcephaly. To date, it is incompletely understood how ZIKV can cross the human placenta. Furthermore, results from studies in pregnant mice and non-human primates are conflicting regarding the role of cross-reactive dengue virus (DENV) antibodies on transplacental ZIKV transmission. Elucidating how ZIKV can cross the placenta and which risk factors contribute to this is important for risk assessment and for potential intervention strategies for transplacental ZIKV transmission. In this study we use an ex vivo human placental perfusion model to study transplacental ZIKV transmission and the effect that cross-reactive DENV antibodies have on this transmission. By using this model, we demonstrate that DENV antibodies significantly increase ZIKV uptake in perfused human placentas and that this increased uptake is neonatal Fc-receptor-dependent. Furthermore, we show that cross-reactive DENV antibodies enhance ZIKV infection in term human placental explants and in primary fetal macrophages but not in primary trophoblasts. Our data supports the hypothesis that presence of cross-reactive DENV antibodies could be an important risk factor for transplacental ZIKV transmission. Furthermore, we demonstrate that the ex vivo placental perfusion model is a relevant and animal friendly model to study transplacental pathogen transmission.     

Preliminary lack of evidence for simian odour preferences of savanna populations of Anopheles gambiae and other malaria vectors

The behavioural response to several culicine and anopheline mosquitoes to the odour of alternative hosts (human vs monkey) arranged in a choice set-up using odour-baited entry traps (OBETs) was assessed in a field experiment in south-eastern Senegal. The experimental protocol followed procedures analogous to those adopted in olfactometer laboratory tests. Two adult Cercopithecus aethiops and a child of similar mass slept inside separate tents and their odours were drawn to each one of two paired OBETs so that approaching mosquitoes could experience both odour-laden streams before "choosing" to fly against one of the two air currents and into the trap. The traps were set up in a riverine forest clearing near the town of Kedougou, where primates (Papio papio, Cercopithecus aethiops, and Erythrocebus patas) are common. A total of 192 mosquitoes belonging to 4 genera was captured during 8 trap nights. All major human malaria vectors including Anopheles gambiae sensu lato, An. funestus, and An. nili, which constituted the bulk of the trap catch (N = 153), clearly expressed a preference for human odour, with > 90% of captured mosquitoes caught in the human-baited trap. A sub-sample of specimens belonging to the An. gambiae complex caught in both traps was identified by rDNA-PCR and RFLP as An. gambiae sensu stricto molecular form S (7/10), and An. arabiensis (3/10). The only species that did not show a preference for the alternative odour-laden air streams, among those caught in significant numbers, were mosquitoes of the genus Mansonia, with both Ma. uniformis and Ma. africana weakly preferring human odour, but not at a statistically significant level. These results are in accordance with the hypothesis that the strongly anthropophilic feeding preferences of An. gambiae did not evolve from an ancestral association with non-human primates.     

A mathematical model for zoonotic transmission of malaria in the Atlantic Forest: Exploring the effects of variations in vector abundance and acrodendrophily

Transmission foci of autochthonous malaria caused by Plasmodium vivax-like parasites have frequently been reported in the Atlantic Forest in Southeastern and Southern Brazil. Evidence suggests that malaria is a zoonosis in these areas as human infections by simian Plasmodium species have been detected, and the main vector of malaria in the Atlantic Forest, Anopheles (Kerteszia) cruzii, can blood feed on human and simian hosts. In view of the lack of models that seek to predict the dynamics of zoonotic transmission in this part of the Atlantic Forest, the present study proposes a new deterministic mathematical model that includes a transmission compartment for non-human primates and parameters that take into account vector displacement between the upper and lower forest strata. The effects of variations in the abundance and acrodendrophily of An. cruzii on the prevalence of infected humans in the study area and the basic reproduction number (R₀) for malaria were analyzed. The model parameters are based on the literature and fitting of the empirical data. Simulations performed with the model indicate that (1) an increase in the abundance of the vector in relation to the total number of blood-seeking mosquitoes leads to an asymptotic increase in both the proportion of infected individuals at steady state and R₀; (2) the proportion of infected humans at steady state is higher when displacement of the vector mosquito between the forest strata increases; and (3) in most scenarios, Plasmodium transmission cannot be sustained only between mosquitoes and humans, which implies that non-human primates play an important role in maintaining the transmission cycle. The proposed model contributes to a better understanding of the dynamics of malaria transmission in the Atlantic Forest.     

Multiplex detection of antibodies to Chikungunya,O’nyong-nyong,Zika, Dengue,West Nile and Usutu viruses in diverse non-human primate species from Cameroon and the Democratic Republic of Congo

BackgroundEpidemic arbovirus transmission occurs among humans by mosquito bites and the sylvatic transmission cycles involving non-human primates (NHPs) still exists. However, limited data are available on the extent in NHPs infections and their role. In this study, we have developed and validated a high-throughput serological screening tool to study the circulation of multiple arboviruses that represent a significant threat to human health, in NHPs in Central Africa.Methodology/Principal findingsRecombinant proteins NS1, envelope domain-3 (DIII) for the dengue (DENV), yellow fever (YFV), usutu (USUV), west nile (WNV) and zika (ZIKV) and envelope 2 for the chikungunya (CHIKV) and o''nyong-nyong (ONNV) were coupled to Luminex beads to detect IgG directed against these viruses. Evaluation of test performance was made using 161 human sera of known arboviral status (66 negative and 95 positive). The sensitivity and specificity of each antigen were determined by statistical methods and ROC curves (except for ONNV and USUV). All NS1 antigens (except NS1-YFV), CHIKV-E2 and WNV-DIII had sensitivities and specificities > 95%. For the other DIII antigens, the sensitivity was low, limiting the interest of their use for seroprevalence studies. Few simultaneous reactions were observed between the CHIKV+ samples and the NS1 antigens to the non-CHIKV arboviruses. On the other hand, the DENV+ samples crossed-reacted with NS1 of all the DENV serotypes (1 to 4), as well as with ZIKV, USUV and to a lesser extent with YFV. A total of 3,518 samples of 29 species of NHPs from Cameroon and the Democratic Republic of Congo (DRC) were tested against NS1 (except YFV), E2 (CHIKV/ONNV) and DIII (WNV) antigens. In monkeys (n = 2,100), the global prevalence varied between 2 and 5% for the ten antigens tested. When we stratified by monkey’s biotope, the arboreal species showed the highest reactivity. In monkeys from Cameroon, the highest IgG prevalence were observed against ONNV-E2 and DENV2-NS1 with 3.95% and 3.40% respectively and in DRC, ONNV-E2 (6.63%) and WNV-NS1 (4.42%). Overall prevalence was low in apes (n = 1,418): ranging from 0% for USUV-NS1 to 2.6% for CHIKV-E2. However, a very large disparity was observed among collection site and ape species, e.g. 18% (9/40) and 8.2% (4/49) of gorillas were reactive with CHIKV-E2 or WNV-NS1, respectively in two different sites in Cameroon.Conclusions/SignificanceWe have developed a serological assay based on Luminex technology, with high specificity and sensitivity for simultaneous detection of antibodies to 10 antigens from 6 different arboviruses. This is the first study that evaluated on a large scale the presence of antibodies to arboviruses in NHPs to evaluate their role in sylvatic cycles. The overall low prevalence (<5%) in more than 3,500 NHPs samples from Cameroon and the DRC does not allow us to affirm that NHP are reservoirs, but rather, intermediate hosts of these viruses.     

Evolutionary relationships of endemic/epidemic and sylvatic dengue viruses

Endemic/epidemic dengue viruses (DEN) that are transmitted among humans by the mosquito vectors Aedes aegypti and Aedes albopictus are hypothesized to have evolved from sylvatic DEN strains that are transmitted among nonhuman primates in West Africa and Malaysia by other Aedes mosquitoes. We tested this hypothesis with phylogenetic studies using envelope protein gene sequences of both endemic/epidemic and sylvatic strains. The basal position of sylvatic lineages of DEN-1, -2, and -4 suggested that the endemic/epidemic lineages of these three DEN serotypes evolved independently from sylvatic progenitors. Time estimates for evolution of the endemic/epidemic forms ranged from 100 to 1,500 years ago, and the evolution of endemic/epidemic forms represents relatively recent events in the history of DEN evolution. Analysis of envelope protein amino acid changes predicted to have accompanied endemic/epidemic emergence suggested a role for domain III in adaptation to new mosquito and/or human hosts.     

Allelic lineages of the ficolin genes (FCNs) are passed from ancestral to descendant primates

The ficolins recognize carbohydrates and acetylated compounds on microorganisms and dying host cells and are able to activate the lectin pathway of the complement system. In humans, three ficolin genes have been identified: FCN1, FCN2 and FCN3, which encode ficolin-1, ficolin-2 and ficolin-3, respectively. Rodents have only two ficolins designated ficolin-A and ficolin-B that are closely related to human ficolin-1, while the rodent FCN3 orthologue is a pseudogene. Ficolin-2 and ficolin-3 have so far only been observed in humans. Thus, we performed a systematic investigation of the FCN genes in non-human primates. The exons and intron-exon boundaries of the FCN1-3 genes were sequenced in the following primate species: chimpanzee, gorilla, orangutan, rhesus macaque, cynomolgus macaque, baboon and common marmoset. We found that the exon organisation of the FCN genes was very similar between all the non-human primates and the human FCN genes. Several variations in the FCN genes were found in more than one primate specie suggesting that they were carried from one species to another including humans. The amino acid diversity of the ficolins among human and non-human primate species was estimated by calculating the Shannon entropy revealing that all three proteins are generally highly conserved. Ficolin-1 and ficolin-2 showed the highest diversity, whereas ficolin-3 was more conserved. Ficolin-2 and ficolin-3 were present in non-human primate sera with the same characteristic oligomeric structures as seen in human serum. Taken together all the FCN genes show the same characteristics in lower and higher primates. The existence of trans-species polymorphisms suggests that different FCN allelic lineages may be passed from ancestral to descendant species.     

Origin of the human hand

A means of determining the phylogenetic implications of similarities between the hands of man and non-human primates is proposed. The only traits shared by man and non-human primates accepted as evidence of our ancestral hand structure and functions are those which are incompatible or out of keeping with current behavior of the human hand. They may be assumed to remain only as relics of adaptations to former habits of locomotion and feeding. The relics found through this analysis are all traits that are present in the apes (some of them only in African apes) and probably related to functions of the hand in suspension of the body, fist-walking, and knuckle-walking. The presence of these traits in man implies that human ancestors similarly used the hand to suspend the body in the trees and to support it on the back of the flexed fingers.     

Comparison of Vector Competence of Aedes mediovittatus and Aedes aegypti for Dengue Virus: Implications for Dengue Control in the Caribbean

Background

Aedes mediovittatus mosquitoes are found throughout the Greater Antilles in the Caribbean and often share the same larval habitats with Ae. Aegypti, the primary vector for dengue virus (DENV). Implementation of vector control measures to control dengue that specifically target Ae. Aegypti may not control DENV transmission in Puerto Rico (PR). Even if Ae. Aegypti is eliminated or DENV refractory mosquitoes are released, DENV transmission may not cease when other competent mosquito species like Ae. Mediovittatus are present. To compare vector competence of Ae. Mediovittatus and Ae. Aegypti mosquitoes, we studied relative infection and transmission rates for all four DENV serotypes.

Methods

To compare the vector competence of Ae. Mediovittatus and Ae. Aegypti, mosquitoes were exposed to DENV 1–4 per os at viral titers of 5–6 logs plaque-forming unit (pfu) equivalents. At 14 days post infectious bloodmeal, viral RNA was extracted and tested by qRT-PCR to determine infection and transmission rates. Infection and transmission rates were analyzed with a generalized linear model assuming a binomial distribution.

Results

Ae. Aegypti had significantly higher DENV-4 infection and transmission rates than Ae. mediovittatus.

Conclusions

This study determined that Ae. Mediovittatus is a competent DENV vector. Therefore dengue prevention programs in PR and the Caribbean should consider both Ae. Mediovittatus and Ae. Aegypti mosquitoes in their vector control programs.     

Evolutionary potential of the extrinsic incubation period of dengue virus in Aedes aegypti

Dengue fever is the most common arboviral disease worldwide. It is caused by dengue viruses (DENV) and the mosquito Aedes aegypti is its primary vector. One of the most powerful determinants of a mosquito's ability to transmit DENV is the length of the extrinsic incubation period (EIP), the time it takes for a virus to be transmitted by a mosquito after consuming an infected blood meal. Here, we repeatedly measured DENV load in the saliva of individual mosquitoes over their lifetime and used this in combination with a breeding design to determine the extent to which EIP might respond to the evolutionary forces of drift and selection. We demonstrated that genetic variation among mosquitoes contributes significantly to transmission potential and length of EIP. We reveal that shorter EIP is genetically correlated with reduced mosquito lifespan, highlighting negative life‐history consequences for virus‐infected mosquitoes. This work highlights the capacity for local genetic variation in mosquito populations to evolve and to dramatically affect the nature of human outbreaks. It also provides the impetus for isolating mosquito genes that determine EIP. More broadly, our dual experimental approach offers new opportunities for studying the evolutionary potential of transmission traits in other vector/pathogen systems.     

Failure to detect simian immunodeficiency virus infection in a large Cameroonian cohort with high non-human primate exposure

Hunting and butchering of wildlife in Central Africa are known risk factors for a variety of human diseases, including HIV/AIDS. Due to the high incidence of human exposure to body fluids of non-human primates, the significant prevalence of simian immunodeficiency virus (SIV) in non-human primates, and hunting/butchering associated cross-species transmission of other retroviruses in Central Africa, it is possible that SIV is actively transmitted to humans from primate species other than mangabeys, chimpanzees, and/or gorillas. We evaluated SIV transmission to humans by screening 2,436 individuals that hunt and butcher non-human primates, a population in which simian foamy virus and simian T-lymphotropic virus were previously detected. We identified 23 individuals with high seroreactivity to SIV. Nucleic acid sequences of SIV genes could not be detected, suggesting that SIV infection in humans could occur at a lower frequency than infections with other retroviruses, including simian foamy virus and simian T-lymphotropic virus. Additional studies on human populations at risk for non-human primate zoonosis are necessary to determine whether these results are due to viral/host characteristics or are indicative of low SIV prevalence in primate species consumed as bushmeat as compared to other retroviruses in Cameroon.     

Involvement of Tetraspanin C189 in Cell-to-Cell Spreading of the Dengue Virus in C6/36 Cells

Dengue virus (DENV) is naturally transmitted by mosquitoes to humans, infecting cells of both hosts. Unlike in mammalian cells, DENV usually does not cause extremely deleterious effects on cells of mosquitoes. Despite this, clustered progeny virions were found to form infection foci in a high density cell culture. It is thus interesting to know how the virus spreads among cells in tissues such as the midgut within live mosquitoes. This report demonstrates that cell-to-cell spread is one way for DENV to infect neighboring cells without depending on the “release and entry” mode. In the meantime, a membrane-bound vacuole incorporating tetraspanin C189 was formed in response to DENV infection in the C6/36 cell and was subsequently transported along with the contained virus from one cell to another. Knockdown of C189 in DENV-infected C6/36 cells is shown herein to reduce cell-to-cell transmission of the virus, which may be recovered by co-transfection with a C189-expressing vector in DENV-infected C6/36 cells. Moreover, cell-to-cell transmission usually occurred at the site where the donor cell directly contacts the recipient cell. It suggested that C189 is crucially involved in the intercellular spread of progeny viral particles between mosquito cells. This novel finding presumably accounts for the rapid and efficient infection of DENV after its initial replication within tissues of the mosquito.     

Using RNA interference to develop dengue virus resistance in genetically modified Aedes aegypti

Diseases caused by arthropod-borne viruses are significant public health problems, and novel methods are needed to control pathogen transmission. We hypothesize that genetic manipulation of Aedes aegypti mosquitoes can profoundly and permanently reduce vector competence and subsequent transmission of dengue viruses (DENV) to human hosts. We have identified RNA interference (RNAi) as a potential anti-viral, intracellular pathway in the vector that can be triggered by expression of virus-specific, double stranded RNAs (dsRNAs) to reduce vector competence to DENV. We identified DENV-derived RNA segments using recombinant Sindbis viruses to trigger RNAi, that when expressed in mosquitoes ablate homologous DENV replication and transmission. We also demonstrated that heritable expression of DENV-derived dsRNA in cultured mosquito cells can silence virus replication. We now have developed a number of transgenic mosquito lines that transcribe the effector dsRNA from constitutive promoters such as immediate early 1 (baculovirus) and polyubiquitin (Drosophila melanogaster). We have detected DENV-specific small interfering RNAs, the hallmark of RNAi, in at least one of these lines. Surprisingly, none of these lines expressed dsRNA in relevant tissues (e.g., midguts) that will ultimately affect transmission. A major challenge now is to express the effector dsRNA from tissue-specific promoters to allow RNAi to silence virus replication at critical sites in the vector such as midguts and salivary glands. If successful, this strategy has the advantage of harnessing a naturally occurring vector response to block DENV infection in a mosquito vector and profoundly affect virus transmission.     

Synchrony of Sylvatic Dengue Isolations: A Multi-Host,Multi-Vector SIR Model of Dengue Virus Transmission in Senegal

Isolations of sylvatic dengue-2 virus from mosquitoes, humans and non-human primates in Senegal show synchronized multi-annual dynamics over the past 50 years. Host demography has been shown to directly affect the period between epidemics in other pathogen systems, therefore, one might expect unsynchronized multi-annual cycles occurring in hosts with dramatically different birth rates and life spans. However, in Senegal, we observe a single synchronized eight-year cycle across all vector species, suggesting synchronized dynamics in all vertebrate hosts. In the current study, we aim to explore two specific hypotheses: 1) primates with different demographics will experience outbreaks of dengue at different periodicities when observed as isolated systems, and that coupling of these subsystems through mosquito biting will act to synchronize incidence; and 2) the eight-year periodicity of isolations observed across multiple primate species is the result of long-term cycling in population immunity in the host populations. To test these hypotheses, we develop a multi-host, multi-vector Susceptible, Infected, Removed (SIR) model to explore the effects of coupling multiple host-vector systems of dengue virus transmission through cross-species biting rates. We find that under small amounts of coupling, incidence in the host species synchronize. Long-period multi-annual dynamics are observed only when prevalence in troughs reaches vanishingly small levels (), suggesting that these dynamics are inconsistent with sustained transmission in this setting, but are consistent with local dengue virus extinctions followed by reintroductions. Inclusion of a constant introduction of infectious individuals into the system causes the multi-annual periods to shrink, while the effects of coupling remain the same. Inclusion of a stochastic rate of introduction allows for multi-annual periods at a cost of reduced synchrony. Thus, we conclude that the eight-year period separating amplifications of dengue may be explained by cycling in immunity with stochastic introductions.     

Fine scale spatiotemporal clustering of dengue virus transmission in children and Aedes aegypti in rural Thai villages

Background

Based on spatiotemporal clustering of human dengue virus (DENV) infections, transmission is thought to occur at fine spatiotemporal scales by horizontal transfer of virus between humans and mosquito vectors. To define the dimensions of local transmission and quantify the factors that support it, we examined relationships between infected humans and Aedes aegypti in Thai villages.

Methodology/Principal Findings

Geographic cluster investigations of 100-meter radius were conducted around DENV-positive and DENV-negative febrile “index” cases (positive and negative clusters, respectively) from a longitudinal cohort study in rural Thailand. Child contacts and Ae. aegypti from cluster houses were assessed for DENV infection. Spatiotemporal, demographic, and entomological parameters were evaluated. In positive clusters, the DENV infection rate among child contacts was 35.3% in index houses, 29.9% in houses within 20 meters, and decreased with distance from the index house to 6.2% in houses 80–100 meters away (p<0.001). Significantly more Ae. aegypti were DENV-infectious (i.e., DENV-positive in head/thorax) in positive clusters (23/1755; 1.3%) than negative clusters (1/1548; 0.1%). In positive clusters, 8.2% of mosquitoes were DENV-infectious in index houses, 4.2% in other houses with DENV-infected children, and 0.4% in houses without infected children (p<0.001). The DENV infection rate in contacts was 47.4% in houses with infectious mosquitoes, 28.7% in other houses in the same cluster, and 10.8% in positive clusters without infectious mosquitoes (p<0.001). Ae. aegypti pupae and adult females were more numerous only in houses containing infectious mosquitoes.

Conclusions/Significance

Human and mosquito infections are positively associated at the level of individual houses and neighboring residences. Certain houses with high transmission risk contribute disproportionately to DENV spread to neighboring houses. Small groups of houses with elevated transmission risk are consistent with over-dispersion of transmission (i.e., at a given point in time, people/mosquitoes from a small portion of houses are responsible for the majority of transmission).     

Spatial and temporal clustering of dengue virus transmission in Thai villages

Background

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.

Methods and Findings

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 (p < 0.01; attributable risk [AR] = 10.4 per 100; 95% confidence interval 1–19.8 per 100]. 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 (p < 0.01) and greater number of Ae. aegypti pupae per person in positive clusters (p = 0.04). 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.

Conclusions

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.     

Natural vertical transmission of dengue viruses in Aedes aegypti in selected sites in Cebu City,Philippines

We attempted to determine the vertical transmission of dengue virus (DENV) in Aedes aegypti in selected sites in Cebu City, Philippines. Mosquito sub‐adults were collected monthly from households and the field during the wet‐dry‐wet season from November, 2011 to July, 2012 and were laboratory‐reared to adults. Viral RNA extracts in mosquitoes were assayed by hemi‐nested RT‐PCR. Results showed that 62 (36.26%; n=679) out of 171 mosquito pools (n=2,871) were DENV+. The minimum infection rate (MIR) of DENV ranged from 0 in wet months to 48.22/1,000 mosquitoes in April, 2012 (mid‐dry). DENVs were detected in larvae, pupae, and male and female adults, with DENV‐4, DENV‐3, and DENV‐1, in that rank of prevalence. DENV‐1 co‐infected with either DENV‐3 or ?4 or with both in April, 2012; DENV‐3 and ?4 were present in both seasons. More DENV+ mosquitoes were collected from households than in field premises (p<0.001) and in the dry than in the wet season (p<0.05), with significant interaction (p<0.05) between sites and premises but no interaction between sites and seasons (p>0.05). By Generalized Linear Mixed models, the type of premises nested in sites and monthly total rainfall were significant predictors of monthly dengue cases (p<0.05) and not MIR, season, temperature, and relative humidity. Surveillance of DENV prevalence in Ae. aegypti and detecting their natural foci in the dry season provide an early warning signal of dengue outbreak.