The use of insect cells to identify potent and selective inhibitors of the replication of the Dengue and Chikungunya viruses and unravel their molecular mechanism of action

Dengue and Chikungunya viruses cause the most important arthropod-borne viral infections for humans. These viruses are predominant in tropical and subtropical regions. In addition, these viruses are predominant in tropical and subtropical regions. Dengue mortality rate is around 1.2 to 3.5% and deaths due to chikungunya fever are around 1 in 1000; however, half of chikungunya-infected patients evolve into a chronic state that can persist for months up to years. There are no antiviral drugs available for DENV and CHIKV treatment and prevention. Moreover, vector control strategies have failed so far. Thus, the development of potent inhibitors for a broad spectrum of RNA viruses is urgently needed.\ud We established and characterized a new embryonic insect cell line from Culex quinquefasciatus mosquito. Also we established the flaviviruses and alphavirus replication, both in C6/36 and Lulo insect cell lines, as well as in Vero cell line. In addition we carried out a reference compound library and reference panel of assays and data for DENV, which provides a benchmark for further studies. During this study, a panel of 9 antiviral molecules, with proven in vitro anti-dengue virus activity and that act at different stages of the DENV life cycle, was selected. Finally, Favipiravir or T-705, was identified as inhibitor in vitro and in vivo of alphaviruses and the mutation K291R in nsP4, which is responsible of the polymerase activity, was found as the mode of action in CHIKV. Interestingly, lysine in motif F1 is also highly conserved in positive-stranded RNA viruses and this might explain the broad spectrum of T-705 antiviral activity.ColcienciasErasmus MundusKU Leuve

Comparative assessment of the replication efficiency of dengue, yellow fever, and chikungunya arboviruses in some insect and mammalian cell lines

Abstract INTRODUCTION: Insect cell cultures play an essential role in understanding arboviral replication. However, the replicative efficiency of some of these viruses such as dengue (DENV), yellow fever (YFV), and chikungunya (CHIKV) in a new cellular substrate (Lulo) and in the other two recognized cell lines has not been comparatively assessed. METHODS: Vero, C6/36, and Lulo cell lines were infected with DENV, YFV, and CHIKV. The viral progeny was quantified through plaque assays and quantitative reverse transcription-polymerase chain reaction, while for DENV2, the findings were confirmed by immunofluorescence antibody assay. RESULTS: The higher DENV2 titer (from multiplicity of infection 0.001) was obtained on day four post-infection in C6/36 and on day six in Vero cells, while the Lulo cell line was almost impossible to infect under the same conditions. However, C6/36 showed the highest values of viral RNA production compared to Vero cells, while the quantification of the viral RNA in Lulo cells showed high levels of viral genomes, which had no correlation to the infectious viral particles. CONCLUSIONS: C6/36 was the most efficient cell line in the alpha and flavivirus production, followed by Vero cells. Thus, Lulo cells may be a useful substrate to study the mechanisms by which cells evade viral replication

Comparative assessment of the replication efficiency of dengue, yellow fever, and chikungunya arboviruses in some insect and mammalian cell lines

Introduction: Insect cell cultures play an essential role in understanding arboviral replication. However, the replicative efficiency of some of these viruses such as dengue (DENV), yellow fever (YFV), and chikungunya (CHIKV) in a new cellular substrate (Lulo) and in the other two recognized cell lines has not been comparatively assessed. Methods: Vero, C6/36, and Lulo cell lines were infected with DENV, YFV, and CHIKV. The viral progeny was quantified through plaque assays and quantitative reverse transcription-polymerase chain reaction, while for DENV2, the findings were confirmed by immunofluorescence antibody assay. Results: The higher DENV2 titer (from multiplicity of infection 0.001) was obtained on day four post-infection in C6/36 and on day six in Vero cells, while the Lulo cell line was almost impossible to infect under the same conditions. However, C6/36 showed the highest values of viral RNA production compared to Vero cells, while the quantification of the viral RNA in Lulo cells showed high levels of viral genomes, which had no correlation to the infectious viral particles. Conclusions: C6/36 was the most efficient cell line in the alpha and flavivirus production, followed by Vero cells. Thus, Lulo cells may be a useful substrate to study the mechanisms by which cells evade viral replication

Growth and development curves of the first colonizing insects (Diptera: Calliphoridae) on pig carcasses (sus scrofa) in Bogotá DC, Colombia

El objetivo del presente trabajo fue establecercurvas de crecimiento y desarrollo de las primerasespecies de dípteros colonizadores de un cadáverde cerdo doméstico (Sus scrofa), expuesto alproceso de descomposición en la finca San José deGuaúsa, Bogotá. Este conocimiento tiene aplicaciónen la determinación del intervalo postmortem. Seempleó como biomodelo el cadáver del cerdodoméstico, debido a la similitud en hábitos alimenticios,cantidad de vello y procesos de descomposición conlos cadáveres humanos. La colonización de los insectosse inició el mismo día del sacrificio del animal. Lasmasas de huevos encontradas en el cadáver en estadofresco, se recolectaron desde el primero hasta elquinto día y fueron criadas en condiciones delaboratorio hasta el estado adulto. Las especies queemergieron fueron Sarconesia magellanica (LeGuillou) (Diptera: Calliphoridae) y Compsomyiopsverena (Walker) (Diptera: Calliphoridae). La duraciónpromedio del estado de huevo hasta la emergenciadel adulto de la especie dominante, S. magellanica,fue de 40 días. Con base en los datos de variablesmorfométricas, estadío, tiempo de desarrollo decada estadío y variables ambientales se aplicó unageneralización del análisis multivariado de varianza,a partir del cual se elaboraron las curvas decrecimiento y desarrollo. Estos resultados son desingular importancia en la determinación del intervalopostmortem y pueden ser extrapolados a cadávereshumanos expuestos a similares condicionesambientales, para ayudar a resolver casos donde sedesconoce el tiempo de muerte de las víctimas enprocesos de investigación legalThe objective to this work was to make the growing anddevelopmental curves of the first species of dipteracollected from a infested carcass of domestic pig (Susscrofa), exposed to decomposition in the San José deGuaúsa farm, from Bogotá. These curves allow us toestimate precisely the postmortem interval. Domesticpig was the biomodel used because its alimentary habits,villous density and decomposing processes, similar tohuman. Insect colonization began the same day ofsacrifice of the pig. Egg masses found in the carcass infresh stage were collected from day first to fifth andthen sowed and reared in laboratory conditions untiladulthood. The emerged species were Sarconesiamagellanica (Le Guillou) (Diptera: Calliphoridae) andCompsomyiops verena (Walker) (Diptera: Calliphoridae).The mean time elapsed from egg to adult in dominantspecie, Sarconesia magellanica, was 40 days. Onmorphometric, instar time, and environmental data wasapplied a generalization of multivariate analysis ofvariance, from which growing and developmental curveswere made. These results are of singular importance todetermine the postmortem interval and may beextrapolated to human carcasses exposed to similarenvironmental conditions, to help resolve legal caseswhere time between finding the infested carcass andthe real time of death is unknow