RESUMO
Several studies have shown that adaptation of various viruses to grow in certain cell lines of vertebrates, leads to the selection of virus variants that bind heparan sulfate (HS) with high affinity. In this study we investigated the susceptibility of strains of dengue virus (DENV) to oversulfated heparin an analogue of HS after passages in BHK-21 cells. Field isolates of the four serotypes of DENV with a limited number of passes in mosquito cells C6/36HT were serially passaged eight times in BHK-21 cells. The adaptation of the DENV to the cell culture selected viral variants with an increased replicative capacity in BHK-21 cells and an increased susceptibility to heparin compared with the original not adapted strains, with a more significant inhibition of the infectivity in DENV-2 and DENV-4.The E protein of the adapted strains showed changes in the amino acid sequence, particularly at the position K204R to DENV-1, N67K to DENV-2, K308R and V452A for DENV-3 and E327G to DENV-4. These substitutions implicated a gain of basic residues that increased the net positive charge of the protein. These results suggest that adaptation of DENV strains to BHK-21 cells implies changes in the envelope protein, changes associated to the protein reactivity with heparin, the inhibitory effectiveness of this compound varying depending on the viral strain. In addition, these results suggest that the HS can play an important role in the infectivity of the DENV strains adapted to vertebrate cell culture, but not in the infectivity of non-adapted DENV isolates.
Assuntos
Vírus da Dengue/efeitos dos fármacos , Heparina/farmacologia , Seleção Genética , Proteínas do Envelope Viral/genética , Aedes/citologia , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Vírus da Dengue/crescimento & desenvolvimento , Rim/citologia , Mesocricetus , Modelos Moleculares , Mutação , Mutação de Sentido Incorreto , Ligação Proteica , Conformação Proteica , RNA Viral/genética , Análise de Sequência de RNA , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/fisiologia , Ensaio de Placa Viral , Cultura de Vírus , Replicação ViralRESUMO
Estudios previos han demostrado que la adaptación de diversos virus a crecer en líneas celulares de vertebrados, conduce a la selección de variantes virales que unen al heparán sulfato (HS) con alta afinidad. En el presente trabajo se determinó la susceptibilidad de cepas del virus dengue (DENV) a la heparina hipersulfatada un análogo al HS, después de pases seriados en células BHK-21. A aislados de campo de los cuatro serotipos de DENV, se les realizaron ocho pases seriados en células BHK-21. La adaptación de los DENV al cultivo celular seleccionó variantes virales con una aumentada capacidad replicativa en células BHK-21 y una incrementada susceptibilidad a la heparina, en relación a las respectivas cepas no adaptadas, obteniéndose una inhibición de la infectividad más significativa en DENV-2 y DENV-4. Las cepas de DENV adaptadas presentaron cambios en la secuencia de aminoácidos de la proteína de envoltura (E), en particular una substitución K204R para DENV-1, N67K para DENV-2, K308R y V452A para DENV-3 y E327G para DENV-4. Estas sustituciones implicaron ganancia de residuos básicos que incrementaron la carga neta positiva de la proteína. Los resultados sugieren, que la adaptación de cepas de DENV a células BHK-21 selecciona variantes virales sensibles a la heparina y que la efectividad de este compuesto varía dependiendo de la cepa viral. Además sugieren que el HS puede jugar un papel importante en la infectividad de las cepas de DENV adaptadas al cultivo celular, a diferencia de los aislados de DENV no adaptados.
Several studies have shown that adaptation of various viruses to grow in certain cell lines of vertebrates, leads to the selection of virus variants that bind heparan sulfate (HS) with high affinity. In this study we investigated the susceptibility of strains of dengue virus (DENV) to oversulfated heparin an analogue of HS after passages in BHK-21 cells. Field isolates of the four serotypes of DENV with a limited number of passes in mosquito cells C6/36HT were serially passaged eight times in BHK-21 cells. The adaptation of the DENV to the cell culture selected viral variants with an increased replicative capacity in BHK-21 cells and an increased susceptibility to heparin compared with the original not adapted strains, with a more significant inhibition of the infectivity in DENV-2 and DENV-4.The E protein of the adapted strains showed changes in the amino acid sequence, particularly at the position K204R to DENV-1, N67K to DENV-2, K308R and V452A for DENV-3 and E327G to DENV-4. These substitutions implicated a gain of basic residues that increased the net positive charge of the protein. These results suggest that adaptation of DENV strains to BHK-21 cells implies changes in the envelope protein, changes associated to the protein reactivity with heparin, the inhibitory effectiveness of this compound varying depending on the viral strain. In addition, these results suggest that the HS can play an important role in the infectivity of the DENV strains adapted to vertebrate cell culture, but not in the infectivity of non-adapted DENV isolates.