American genotype structures decrease dengue virus output from human monocytes and dendritic cells

Abstract

The dengue virus type 2 structures probably involved in human virulence were previously defined by sequencing the complete genome of both American and Southeast (SE) Asian genotype templates in patient serum (K. C. Leitmeyer et al., J. Virol. 73:4738-4747, 1999). We have now evaluated the effects of introducing a mutation in the envelope glycoprotein (E) gene and/or replacement of 5'- and 3'-nontranslated regions on dengue virus replication in human primary cell cultures. A series of chimeric infectious clones were generated containing different combinations of American and SE Asian genotype sequences. Some of the chimeric viruses had altered plaque morphology in mammalian cells; however, they replicated at similar rates in mosquito cells as measured by quantitative reverse transcription-PCR and plaque assay. Although susceptibility to virus infection varied from donor to donor in experiments using human macrophage and dendritic cells, we were able to measure consistent differences in viral RNA output per infected cell. Using this measurement, we demonstrated that the chimeric virus containing the E mutation had a lower virus output compared to the parental infectious clone. A larger reduction in virus output was observed for the triple mutant and the wild-type, American genotype virus from which chimeric inserts were derived. It appears that the three changes function synergistically, although the E mutation alone gives a lower output compared to the 5'- and 3'-terminal mutations. The data suggest that these changes may be responsible for decreased dengue virus replication in human target cells and for virulence characteristics during infection.

FIG. 1.

Schematic of chimeric dengue type 2 infectious clones. The D2/IC-30P-A SE Asian infectious clone is depicted showing viral proteins, ntr's (5′ ntr and 3′ ntr), and two restriction sites (SalI and HpaI) used for the cloning of the chimeric viruses. Chimeric viruses are depicted with parental sequences shown as open boxes, with inserts in gray or black. The SE Asian insert from K0008 is depicted as a gray box for virus D2/IC-3ntrK. American genotype inserts from IQT2913 are depicted as black boxes for the viruses D2/IC-3ntrIQT, D2/IC-5ntrIQT, D2/IC-5+3ntrIQT, D2/IC-Em, and D2/IC-5E3IQT.

FIG. 2.

Plaque morphology for infectious clones. Vero cells were infected and overlaid with 1× MEM-2% FCS containing 1% agarose. At 9 days postinfection the plaque assay was developed and the wells were photographed.

FIG. 3.

Growth kinetics of chimeric viruses. Mosquito cells were infected at an MOI of 1,000 genome Eq/cell, and supernatants were collected every day for 6 days. Virus output was determined by quantitative RT-PCR from two experiments, and the graph represents the mean (with the standard deviations indicated in panel A). For one of these experiments, virus output for four viruses was determined by plaque assay and is shown in panel B.

FIG. 4.

Virus output for MDM and DC cultures. MDM (A and C) and DC (B and D) cultures were infected at MOIs of 500 (circles), 1,000 (squares), or 1,500 (triangles) genome Eq/cell. The graphs in panels A and B represent the mean virus output per infected cell, with the standard deviations indicated, for duplicate wells infected with D2/IC 30P-A (solid symbols, solid lines) or D2/IC-5E3IQT (open symbols, dashed lines). The graphs in panels C and D represent the mean virus output per infected cell, with the standard deviations indicated, for duplicate wells infected with 16681 (solid symbols, solid lines) or IQT2913 (open symbols, dashed lines).

FIG. 5.

Comparison of virus output in DC cultures. Data are from infected DC cultures at 48 h postinfection from three different donors with a total of nine samples for each virus. The bars represent the mean virus output per infected cell, and the error bars represent the standard deviation of the means. Statistical significance was determined by using the Kruskal-Wallis test, and pairwise comparisons with D2/IC-30P-A were used to calculate the P values for D2/IC-5E3IQT and IQT2913.