RESUMEN
BACKGROUND: Measuring dengue virus transmission in endemic areas is a difficult task as many variables drive transmission, and often are not independent of one another. OBJECTIVES: We aimed to determine the utility of vectorial capacity to explain the observed dengue infection rates in three hyperendemic cities in Colombia, and tested hypotheses related to three variables: mosquito density, effective vector competence, and biting rate. METHODS: We estimated two of the most influential entomological variables related to cumulative vectorial capacity, which is a modification of the traditional vectorial capacity equation, of three Colombian mosquito populations. Laboratory studies were undertaken to measure vector competence and man biting rate of local mosquito populations. In addition, the assessment of cumulative vectorial capacity also incorporated site-specific estimations of mosquito density and the probability of daily survival from previous studies conducted in those cities. FINDINGS: We found that the biting rates and mosquito infection rates differed among populations of mosquitoes from these three cities, resulting in differences in the site-specific measures of transmission potential. Specifically, we found that using site-specific entomological measures to populate the cumulative vectorial capacity equation was best at recapitulating observed mosquito infection rates when mosquito density was discounted compared to when we incorporated site-specific density measures. CONCLUSIONS: Specific mosquito-biting rate is likely sufficient to explain transmission differences in these three cities, confirming that this parameter is a critical parameter when predicting and assessing dengue transmission in three Colombian cities with different field observed transmission patterns.
Asunto(s)
Aedes/microbiología , Dengue/transmisión , Entomología/métodos , Conducta Alimentaria , Mosquitos Vectores , Animales , Ciudades/epidemiología , Colombia/epidemiología , Dengue/epidemiología , Femenino , Humanos , Conceptos Matemáticos , Mosquitos Vectores/microbiología , Densidad de Población , ProbabilidadRESUMEN
Flaviviruses include a diverse group of medically important viruses that cycle between mosquitoes and humans. During this natural process of switching hosts, each species imposes different selective forces on the viral population. Using dengue virus (DENV) as model, we found that paralogous RNA structures originating from duplications in the viral 3' untranslated region (UTR) are under different selective pressures in the two hosts. These RNA structures, known as dumbbells (DB1 and DB2), were originally proposed to be enhancers of viral replication. Analysis of viruses obtained from infected mosquitoes showed selection of mutations that mapped in DB2. Recombinant viruses carrying the identified variations confirmed that these mutations greatly increase viral replication in mosquito cells, with low or no impact in human cells. Use of viruses lacking each of the DB structures revealed opposite viral phenotypes. While deletion of DB1 reduced viral replication about 10-fold, viruses lacking DB2 displayed a great increase of fitness in mosquitoes, confirming a functional diversification of these similar RNA elements. Mechanistic analysis indicated that DB1 and DB2 differentially modulate viral genome cyclization and RNA replication. We found that a pseudoknot formed within DB2 competes with long-range RNA-RNA interactions that are necessary for minus-strand RNA synthesis. Our results support a model in which a functional diversification of duplicated RNA elements in the viral 3' UTR is driven by host-specific requirements. This study provides new ideas for understanding molecular aspects of the evolution of RNA viruses that naturally jump between different species.IMPORTANCE Flaviviruses constitute the most relevant group of arthropod-transmitted viruses, including important human pathogens such as the dengue, Zika, yellow fever, and West Nile viruses. The natural alternation of these viruses between vertebrate and invertebrate hosts shapes the viral genome population, which leads to selection of different viral variants with potential implications for epidemiological fitness and pathogenesis. However, the selective forces and mechanisms acting on the viral RNA during host adaptation are still largely unknown. Here, we found that two almost identical tandem RNA structures present at the viral 3' untranslated region are under different selective pressures in the two hosts. Mechanistic studies indicated that the two RNA elements, known as dumbbells, contain sequences that overlap essential RNA cyclization elements involved in viral RNA synthesis. The data support a model in which the duplicated RNA structures differentially evolved to accommodate distinct functions for viral replication in the two hosts.
Asunto(s)
Regiones no Traducidas 3' , Virus del Dengue/genética , Conformación de Ácido Nucleico , ARN Viral/genética , Animales , Culicidae , Virus del Dengue/crecimiento & desarrollo , Especificidad del Huésped , Humanos , Secuencias Repetitivas de Ácidos Nucleicos , Selección Genética , Replicación ViralRESUMEN
The Flavivirus genus includes a large number of medically relevant pathogens that cycle between humans and arthropods. This host alternation imposes a selective pressure on the viral population. Here, we found that dengue virus, the most important viral human pathogen transmitted by insects, evolved a mechanism to differentially regulate the production of viral non-coding RNAs in mosquitos and humans, with a significant impact on viral fitness in each host. Flavivirus infections accumulate non-coding RNAs derived from the viral 3'UTRs (known as sfRNAs), relevant in viral pathogenesis and immune evasion. We found that dengue virus host adaptation leads to the accumulation of different species of sfRNAs in vertebrate and invertebrate cells. This process does not depend on differences in the host machinery; but it was found to be dependent on the selection of specific mutations in the viral 3'UTR. Dissecting the viral population and studying phenotypes of cloned variants, the molecular determinants for the switch in the sfRNA pattern during host change were mapped to a single RNA structure. Point mutations selected in mosquito cells were sufficient to change the pattern of sfRNAs, induce higher type I interferon responses and reduce viral fitness in human cells, explaining the rapid clearance of certain viral variants after host change. In addition, using epidemic and pre-epidemic Zika viruses, similar patterns of sfRNAs were observed in mosquito and human infected cells, but they were different from those observed during dengue virus infections, indicating that distinct selective pressures act on the 3'UTR of these closely related viruses. In summary, we present a novel mechanism by which dengue virus evolved an RNA structure that is under strong selective pressure in the two hosts, as regulator of non-coding RNA accumulation and viral fitness. This work provides new ideas about the impact of host adaptation on the variability and evolution of flavivirus 3'UTRs with possible implications in virulence and viral transmission.
Asunto(s)
Adaptación Biológica/genética , Culicidae/virología , Virus del Dengue/genética , Aptitud Genética/genética , ARN Viral/genética , Regiones no Traducidas 3'/genética , Animales , Northern Blotting , Dengue/genética , Variación Genética , Genoma Viral , Interacciones Huésped-Patógeno/genética , Humanos , Insectos Vectores/virología , Filogenia , Reacción en Cadena de la Polimerasa , TransfecciónRESUMEN
Many viral pathogens cycle between humans and insects. These viruses must have evolved strategies for rapid adaptation to different host environments. However, the mechanistic basis for the adaptation process remains poorly understood. To study the mosquito-human adaptation cycle, we examined changes in RNA structures of the dengue virus genome during host adaptation. Deep sequencing and RNA structure analysis, together with fitness evaluation, revealed a process of host specialization of RNA elements of the viral 3'UTR. Adaptation to mosquito or mammalian cells involved selection of different viral populations harvesting mutations in a single stem-loop structure. The host specialization of the identified RNA structure resulted in a significant viral fitness cost in the non-specialized host, posing a constraint during host switching. Sequence conservation analysis indicated that the identified host adaptable stem loop structure is duplicated in dengue and other mosquito-borne viruses. Interestingly, functional studies using recombinant viruses with single or double stem loops revealed that duplication of the RNA structure allows the virus to accommodate mutations beneficial in one host and deleterious in the other. Our findings reveal new concepts in adaptation of RNA viruses, in which host specialization of RNA structures results in high fitness in the adapted host, while RNA duplication confers robustness during host switching.
Asunto(s)
Virus del Dengue/genética , Interacciones Huésped-Patógeno/genética , Conformación de Ácido Nucleico , ARN Viral/química , Regiones no Traducidas 3' , Adaptación Biológica/genética , Animales , Células Cultivadas , Cricetinae , Culicidae , Especificidad del Huésped/genética , Humanos , Mutación , ARN Viral/genéticaRESUMEN
BACKGROUND: To be transmitted by its mosquito vector, dengue virus (DENV) must infect midgut epithelial cells, replicate and disseminate into the hemocoel, and finally infect the salivary glands, which is essential for transmission. The extrinsic incubation period (EIP) is very relevant epidemiologically and is the time required from the ingestion of virus until it can be transmitted to the next vertebrate host. The EIP is conditioned by the kinetics and tropisms of virus replication in its vector. Here we document the virogenesis of DENV-2 in newly-colonized Aedes aegypti mosquitoes from Chetumal, Mexico in order to understand better the effect of vector-virus interactions on dengue transmission. RESULTS: After ingestion of DENV-2, midgut infections in Chetumal mosquitoes were characterized by a peak in virus titers between 7 and 10 days post-infection (dpi). The amount of viral antigen and viral titers in the midgut then declined, but viral RNA levels remained stable. The presence of DENV-2 antigen in the trachea was positively correlated with virus dissemination from the midgut. DENV-2 antigen was found in salivary gland tissue in more than a third of mosquitoes at 4 dpi. Unlike in the midgut, the amount of viral antigen (as well as the percent of infected salivary glands) increased with time. DENV-2 antigen also accumulated and increased in neural tissue throughout the EIP. DENV-2 antigen was detected in multiple tissues of the vector, but unlike some other arboviruses, was not detected in muscle. CONCLUSION: Our results suggest that the EIP of DENV-2 in its vector may be shorter that the previously reported and that the tracheal system may facilitate DENV-2 dissemination from the midgut. Mosquito organs (e.g. midgut, neural tissue, and salivary glands) differed in their response to DENV-2 infection.