Activation of the Innate Immune Response against DENV in Normal Non-Transformed Human Fibroblasts

In this work, we demonstrate that that both human whole skin and freshly isolated skin fibroblasts are productively infected with Dengue virus (DENV). In addition, primary skin fibroblast cultures were established and subsequently infected with DENV-2; we showed in these cells the presence of the viral antigen NS3, and we found productive viral infection by a conventional plaque assay. Of note, the infectivity rate was almost the same in all the primary cultures analyzed from different donors. The skin fibroblasts infected with DENV-2 underwent signaling through both TLR3 and RIG-1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 IRF7, when compared with mock-infected fibroblasts. Our data suggest that fibroblasts might even participate producing mediators involved in innate immunity that activate and contribute to the orchestration of the local innate responses. This work is the first evaluating primary skin fibroblast cultures obtained from different humans, assessing both their susceptibility to DENV infection as well as their ability to produce molecules crucial for innate immunity

Evolution of subgenomic RNA shapes dengue virus adaptation and epidemiological fitness

Changes in dengue virus (DENV) genome affect viral fitness both clinically and epidemiologically. Even in the 3' untranslated region (3' UTR), mutations could affect subgenomic flaviviral RNA (sfRNA) production and its affinity for host proteins, which are necessary for successful viral replication. Indeed, we recently showed that mutations in DENV2 3' UTR of epidemic strains increased sfRNA ability to bind host proteins and reduce interferon expression. However, whether 3' UTR differences shape the overall DENV evolution remains incompletely understood. Herein, we combined RNA phylogeny with phylogenetics to gain insights on sfRNA evolution. We found that sfRNA structures are under purifying selection and highly conserved despite sequence divergence. Only the second flaviviral nuclease-resistant RNA (fNR2) structure of DENV2 sfRNA has undergone strong positive selection. Epidemiological reports suggest that substitutions in fNR2 may drive DENV2 epidemiological fitness, possibly through sfRNA-protein interactions. Collectively, our findings indicate that 3' UTRs are important determinants of DENV fitness in human-mosquito cycles

Construct and expression of recombinant domains I/II of dengue virus- 2 and its efficacy to evaluate immune response in endemic area: possible use in prognosis

The envelope (E) protein from DENV, contain three functional and structural domains (DI, DII and DIII). Some studies suggest that neutralizing antibodies during natural DENV infection are predominantly against DI and DII, in contrast, low proportion of the antibodies were against DIII. Thus it is necessary to establish the proportion of human antibodies against DENV E protein that bind to DI and DII during the normal course of infection; as an indicator of the quality of the antibody response and to further design new vaccine candidates for DENV. The aim of this study was to express recombinant proteins harboring a 240-aminoacid fragment of the E protein from DI and DII of DENV serotypes 2 and 3 in a eukaryotic S2 system. Further, we evaluate the antibodies against these antigens in samples from patients in acute phase of DF or DHF and compare it with the response of samples from healthy individuals from the same endemic areas and samples from healthy individuals from a non-endemic area (EA and NEA, respectively). These results suggest that the presence of antibodies against rEDI/DII might be used to identify patients at risk for severe disease

Dual miRNA Targeting Restricts Host Range and Attenuates Neurovirulence of Flaviviruses

<div><p>Mosquito-borne flaviviruses are among the most significant arboviral pathogens worldwide. Vaccinations and mosquito population control programs remain the most reliable means for flavivirus disease prevention, and live attenuated viruses remain one of the most attractive flavivirus vaccine platforms. Some live attenuated viruses are capable of infecting principle mosquito vectors, as demonstrated in the laboratory, which in combination with their intrinsic genetic instability could potentially lead to a vaccine virus reversion back to wild-type in nature, followed by introduction and dissemination of potentially dangerous viral strains into new geographic locations. To mitigate this risk we developed a microRNA-targeting approach that selectively restricts replication of flavivirus in the mosquito host. Introduction of sequences complementary to a mosquito-specific mir-184 and mir-275 miRNAs individually or in combination into the 3’NCR and/or ORF region resulted in selective restriction of dengue type 4 virus (DEN4) replication in mosquito cell lines and adult <i>Aedes</i> mosquitos. Moreover a combined targeting of DEN4 genome with mosquito-specific and vertebrate CNS-specific mir-124 miRNA can silence viral replication in two evolutionally distant biological systems: mosquitoes and mouse brains. Thus, this approach can reinforce the safety of newly developed or existing vaccines for use in humans and could provide an additional level of biosafety for laboratories using viruses with altered pathogenic or transmissibility characteristics.</p></div

miRNA targeting of the DEN4 genome within the ORF and 3’NCR greatly attenuates virus replication in Aag2 and C6/36 cells but not in Vero cells.

<p>Confluent monolayers of Aag2 (<b>A</b>), C6/36 (<b>B</b>) and Vero (<b>C</b>) cells were infected in duplicate with either D4, D4-E, D4-E*, D4-275-184, D4-E-NCR1, and D4-E-NCR2 viruses at an MOI of 0.01. Each time point represents an average titer for two replicates ± standard deviation (shown as brackets). The dashed line indicates the limit of virus detection [0.7 log₁₀ pfu/ml].</p

Effect of combined mir-184 and mir-275 co-targeting of DEN4 genome in the 3’NCR on virus fitness in A. aegypti <i>and</i> A. albopictus.

<p>A. aegypti <i>(</i><b><i>A</i></b><i>and</i><b><i>C</i></b><i>) and</i> A. albopictus <i>(</i><b><i>B</i></b><i>and</i><b><i>D</i></b><i>) were</i> orally infected with blood meals (BM) containing 7.1 to 7.4 log₁₀ pfu/ml of recombinant D4 or D4-275-184 virus, respectively, and processed at 14 dpi. (<b>A</b> and <b>B</b>) Each point represents virus titer in individual mosquito (n = 18 for <i>A</i>. <i>aegypti</i> exposed to D4, and n = 24 mosquitoes for each other virus). Horizontal line represents an average titer for all mosquitoes. The dashed line indicates the limit of the assay detection [0.2 log₁₀ pfu/mosquito]. P-values comparing mean titers of D4 and miRNA targeted viruses were calculated using unpaired one-tailed Student's t-test and adjusted using Bonferroni correction method to account for multiple comparisons. (<b>C</b> and <b>D</b>) Percentage of mosquitoes that became infected (blue) or developed a disseminated infection (orange) with each virus. Differences in infection and dissemination frequencies were compared between D4 and miRNA targeted viruses using one-tailed Fisher’s exact test. P-values were adjusted using Bonferroni correction method to account for multiple comparisons.</p

Effect of multiple mosquito- and brain-specific miRNA target insertions on DEN4 neurovirulence and replication in the brain of sucking mice.

<p>(<b>A</b>) Replication kinetics of D4, D4-E, D4-E**, D4-184, D4-E-NCR1, and D4-E-NCR2 viruses in the brains of mice. Three-day-old mice were inoculated IC with 3 log₁₀ pfu of the indicated virus, and brains were collected from three mice in each group following the course of infection. Each time point represents an average titer for two replicates ± standard deviation (shown as brackets). The dashed line indicates the limit of virus detection [1.7 log₁₀ pfu/ml]. Brain collection of mice infected with D4 and D4-184 was not performed at 14 dpi due to earlier paralysis of the animals. (<b>B</b>) Survival curves of sucking mice infected IC with 10³ pfu of recombinant DEN4 viruses (litter of eight 3-day-old mice per virus). Mice were monitored daily for morbidity for 21 days. We selected a 3 log₁₀ pfu dose of virus inoculation since parental DEN4 strain 814669 has an IC LD₅₀ of 407 pfu as estimated previously for this age of mice [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004852#ppat.1004852.ref048" target="_blank">48</a>].</p

Effect of a single copy of miRNA target in the 3’NCR on DEN4 replication in mosquito cells and live mosquitoes.

<p><b>(A</b> and <b>B)</b> Growth kinetics of D4s, D4-1s, D4-184s and D4-275s viruses in mosquito Aag2 (<b>A</b>) and C₇10 (<b>B</b>) cells. Cells were infected at an MOI of 0.01. Each time point represents an average of two replicates ± standard deviation (shown as brackets). The dashed line indicates the limit of virus detection [0.7 log₁₀ pfu/ml]. (<b>C)</b> A. aegypti <i>mosquitoes were infected</i> intrathoracically with 100 pfu of the indicated viruses and incubated for 7 days. Virus titer in each whole mosquito body suspension was determined in Vero cells. Each point represents the virus titer of an individual mosquito. Horizontal line represents the mean virus titer for all mosquitoes. The dashed line indicates the limit of virus detection [0.2 log₁₀ pfu/mosquito]. P-values were calculated using unpaired one-tailed Student's t-test and adjusted using Bonferroni correction method to account for multiple comparisons.</p

Effect of combined mir-184 and mir-275 co-targeting of DEN4 genome in the 3’NCR on virus replication in mosquito and Vero cells.

<p>Confluent monolayers of Aag2 (<b>A</b>), C6/36 (<b>B</b>) or Vero (<b>C</b>) cells were infected with D4, D4-184, D4-275, D4-275x2 and D4-275-184 viruses at an MOI of 0.01. Each time point represents an average of two replicates ± standard deviation (shown as brackets). The dashed line indicates the limit of virus detection [0.7 log₁₀ pfu/ml].</p

Schematic representation of viral genomes used in this study.

<p>Positions of miRNA targets for brain-expressed mir-124 and mosquito-specific mir-1, mir-184, or mir-275 in the ORF and 3’NCR of DEN4 genome are indicated by blue and red boxes, respectively. Gray area represents duplicated, codon optimized DEN4 E/NS1 sequence (nts from 2130 to 2451 of DEN4 genome) encoding 98 amino acids from the C-terminal end of the DEN4 E protein and 7 amino acids from the N-terminal end of the NS1 protein. TM1 and TM2 are transmembrane helixes in the C-terminal anchor region of protein E. Red arrows indicate signalase cleavage sites. Asterisk in the table indicates that miRNA target sequence has been altered using synonymous codons.</p