Differential replicative fitness of the four dengue virus serotypes circulating in Colombia in human liver Huh7 cells

ABSTRACT Dengue has been a significant public health problem in Colombia since the simultaneous circulation of the four dengue virus serotypes. The replicative fitness of dengue is a biological feature important for virus evolution and contributes to elucidating the behavior of virus populations and viral pathogenesis. However, it has not yet been studied in Colombian isolates. This study aimed to compare the replicative fitness of the four dengue virus serotypes and understand the association between the serotypes, their in vitro infection ability, and their replication in target cells. We used three isolates of each DENV serotype to infect Huh-7 cells at an MOI of 0.5. The percentage of infected cells was evaluated by flow cytometry, cell viability was evaluated by MTT assay, and the pathogenicity index was calculated as a ratio of both parameters. The replicative fitness was measured by the number of viral genome copies produced using quantitative PCR and the production of infectious viral progeny was measured by plaque assay. We showed that Huh-7 cells were susceptible to infection with all the different strain isolates. Nevertheless, the biological characteristics, such as infectious ability and cell viability, were strain-dependent. We also found different degrees of pathogenicity between strains of the four serotypes, representative of the heterogeneity displayed in the circulating population. When we analyzed the replicative fitness using the mean values obtained from RT-qPCR and plaque assay for the different strains, we found serotype-dependent behavior. The highest mean values of replicative fitness were obtained for DENV-1 (log 4.9 PFU/ml) and DENV-4 (log 5.28 PFU/ml), followed by DENV-2 (log 3.9 PFU/ml) and DENV-3 (log 4.31 PFU/ml). The internal heterogeneity of the replicative fitness within each serotype could explain the simultaneous circulation of the four DENV serotypes in Colombia.

LÍNEA CELULAR DE Aedes aegypti (DIPTERA: CULICIDAE) AEGY28 REFRACTARIA A LA INFECCIÓN CON LOS VIRUS DENGUE 2 Y FIEBRE AMARILLA / AEGY28 Cell Line of Aedes aegypti (Diptera Culicidae) is Infection Refractory to Dengue 2 and Yellow Fever Virus

Los cultivos celulares de mosquitos son frecuentemente utilizados para el aislamiento, identificación y caracterización de arbovirus. Para el estudio de los virus dengue (VDEN) y virus de fiebre amarilla (VFA) se emplean, principalmente, la línea celular C6/36 de Aedes albopictus y la línea celular AP61, obtenida de Aedes pseudoscutellaris. La línea celular de A. aegypti AEGY28, previamente obtenida a partir de tejidos embrionarios del vector, se utilizó en el presente trabajo para evaluar la susceptibilidad a la infección por VDEN y VFA. Para ello, los cultivos celulares se ensayaron a diferente multiplicidad de infección con los aislados clínicos de virus dengue tipo 2 (COL789, MOI: 1 y 5) y VFA (V341, MOI 0,02). Posteriormente se realizó la detección de antígenos virales por la técnica de inmunocitoquímica y su cuantificación por la técnica de CellELISA fluorométrica. Se usaron como controles positivos de infección, tanto células C6/36 como células VERO. Inesperadamente, no se observó inmunoreactividad en las células de A. aegypti infectadas con ambos tipos de virus en ninguno de los MOI o tiempos estudiados. Tampoco se evidenció antígeno por la técnica fluorométrica ni fue posible detectar RNA viral por RTPCR a partir de células infectadas. Por lo tanto, se puede concluir que la línea celular de A. aegypti no es susceptible a la infección por VDEN ni por VFA. Ello podría estar relacionado con características propias de la membrana celular o de la maquinaria enzimática necesaria para la replicación viral.

Mosquito cell derived cultures are useful tools for arbovirus isolation, identification or characterization. For studying dengue (DENV) and yellow fever viruses (YFV) Aedes albopictus C6/36 or Aedes pseudoscutellaris AP61 cell lines, are normally used. The Aedes aegypti AEGY28 cell line was obtained from embryonic tissues and characterized previously by one of us. In order to evaluate its susceptibility to two Flavivirus, AEGY28 cells were inoculated with different multiplicity of infection (MOI) with type 2 DENV (COL789, MOI: 1 and 5) and YFV clinical isolates (V341, MOI 0,02) then processed at different times post infection (p.i.). Immunostaining and fluorometric cellELISA were carried out to identify and quantify viral antigens. C6/36 and Vero cells were used as positive controls. Unexpectedly, immunoreactivity was not found in inoculated AEGY28 cells, even in higher MOI or late times p.i., therefore antigen quantification using fluorometric cellELISA were not plausible. Reverse transcriptase PCR with specific primers did not detect viral RNA in AEGY28 inoculated cells. We can conclude that Aedes aegypti AEGY28 cell line is not susceptible to dengue and yellow fever Flavivirus, a finding possibly related with the lacking of specific molecules at the plasma membrane or absence of cell machinery necessary for viral replication.