Journal: bioRxiv

Article Title: Modeling herpes simplex virus type 2 and Zika virus replication in vaginal organoids and spheroids

doi: 10.64898/2026.03.02.709097

Figure Lengend Snippet: NS1 concentration in the supernatant (A) and NS5 transcript expression (B) in mouse vaginal organoids infected with ZIKV at MOI of 1. Values normalized to uninfected samples (Ni) and reported as fold change. (C) NS4B staining in organoids sections from 48 hours post-infection. ZIKV titer of mouse vaginal organoids (D) and VK2 spheroids (E) infected with ZIKV in the presence of 10 µM RDV. Values are mean ± SEM of 3 biological replicates. One-way ANOVA or two-way ANOVA with Tukey’s test for multiple comparisons was performed for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Magnification 63X; scale bars represent 50 µm. LD, limit of detection (5 PFU/ml). h, hours post-infection.

Article Snippet: ZIKV titer from infection experiments was quantified using an ELISA to detect NS1 concentration (Sino Biological, Cat No. KIT40544).

Techniques: Concentration Assay, Expressing, Infection, Staining

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation

doi: 10.1590/0074-02760250272

Figure Lengend Snippet: Aedes aegypti salivary gland extract (SGE) enhances Zika virus (ZIKV) replication in Vero cells. (A) Dose-response curve showing plaque-forming units per millilitre (PFU/mL) recovered from Vero cells infected with ZIKV [multiplicity of infection (MOI) = 0.1] in the presence of increasing concentrations of SGE (0.5, 1, 2.5, and 5 µg/mL). Data are expressed as mean ± standard error of the mean (SEM) from three independent experiments (n = 3). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Asterisks indicate statistical significance compared with ZIKV alone (p < 0.05). (B) Representative plaque assay images of Vero cell monolayers infected with ZIKV (MOI 0.1) and treated with SGE at the indicated concentrations, illustrating the dose-dependent increase in cytopathic effects.

Article Snippet: Quantification of ZIKV RNA was performed using the AllplexTM Zika Virus Assay (Seegene®), a multiplex Real-Time PCR kit designed for specific detection of ZIKV RNA.

Techniques: Virus, Infection, Plaque Assay

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation

doi: 10.1590/0074-02760250272

Figure Lengend Snippet: leukocyte death profiles in peripheral blood mononuclear cells (PBMCs) following Zika virus (ZIKV) infection and Aedes aegypti salivary gland extract (SGE) exposure. (A) Absolute number of dead leukocytes after 72 h of culture with medium only, SGE (5 µg/mL), ZIKV, or ZIKV+SGE (5 µg/mL). (B) Absolute counts of Annexin V⁺ (early apoptotic), PI⁺ (late apoptotic/necrotic), and Annexin V⁺/PI⁺ (late apoptotic/secondary necrotic) leukocytes across groups. (C) Cell death dynamics representation (Annexin V⁺, PI⁺, Annexin V⁺/PI⁺) illustrating the redistribution of death profiles under different experimental conditions. Data represent PBMCs from five independent donors, each tested in duplicate (individual points shown), with median ± interquartile range (IQR). Statistical comparisons were performed using Friedman test with post-hoc analysis (Panel A: p = 0.0129 overall; ZIKV+SGE vs SGE, p = 0.0109; Panel B: PI⁺ ZIKV+SGE vs SGE, p = 0.0335; Annexin V⁺/PI⁺ ZIKV+SGE vs SGE, p = 0.0327; Annexin V⁺ frequencies, p < 0.0001). Two-way analysis of variance (ANOVA) for the Cell death dynamics (Panel C) detected significant main effects of condition and marker type, and their interaction (p < 0.0001, p = 0.0216, p = 0.0257, respectively).

Article Snippet: Quantification of ZIKV RNA was performed using the AllplexTM Zika Virus Assay (Seegene®), a multiplex Real-Time PCR kit designed for specific detection of ZIKV RNA.

Techniques: Virus, Infection, Marker

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation

doi: 10.1590/0074-02760250272

Figure Lengend Snippet: Zika virus (ZIKV) and salivary gland extract (SGE) co-exposure reduces late apoptotic CD4⁺ T cells while sparing CD8⁺ populations. (A) CD4⁺ T cells: absolute numbers of Annexin V⁺ (early apoptosis), PI⁺ (late apoptosis/necrosis), and Annexin V⁺/PI⁺ (late apoptosis/secondary necrosis) populations after 72 h under the indicated conditions (Media, SGE (5 µg/mL), ZIKV, ZIKV+SGE (5 µg/mL)). A significant difference was detected for the Annexin V⁺/PI⁺ population, with reductions in ZIKV+SGE compared with ZIKV (p = 0.0423). (B) CD8⁺ T cells: no significant differences were observed across groups for any marker in absolute numbers (Friedman test, p > 0.05). Data represent peripheral blood mononuclear cells (PBMCs) from five independent donors shown as individual values with median ± interquartile range (IQR).

Article Snippet: Quantification of ZIKV RNA was performed using the AllplexTM Zika Virus Assay (Seegene®), a multiplex Real-Time PCR kit designed for specific detection of ZIKV RNA.

Techniques: Virus, Marker

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation

doi: 10.1590/0074-02760250272

Figure Lengend Snippet: Zika virus (ZIKV)-induced dendritic cell death is attenuated by Aedes aegypti salivary gland extract (SGE). Absolute numbers of dendritic cells positive for Annexin V⁺ (early apoptosis), PI⁺ (late apoptosis/necrosis), and Annexin V⁺/PI⁺ (late apoptosis/secondary necrosis) were quantified after 72 h of culture under the indicated conditions (Media, SGE (5 µg/mL), ZIKV, ZIKV+SGE (5 µg/mL)). Significant overall differences were observed for early apoptosis (p < 0.0001), late apoptosis/necrosis (p = 0.0087), and late apoptosis/secondary necrosis (p = 0.0023). Post-hoc analyses showed reduced early apoptosis counts in ZIKV+SGE compared to Media (p = 0.0014), fewer late apoptosis/necrosis cells in ZIKV+SGE versus ZIKV (p = 0.0197), and lower late apoptosis/secondary necrosis counts in ZIKV+SGE compared to ZIKV (p = 0.0087). Percentage analyses yielded consistent trends (Annexin V⁺, p < 0.0001; PI⁺, p = 0.0226; Annexin V⁺/PI⁺, p = 0.0167). Data represent peripheral blood mononuclear cells (PBMCs)-derived dendritic cells from five independent donors (no technical replicates), shown as individual values with median ± interquartile range (IQR).

Article Snippet: Quantification of ZIKV RNA was performed using the AllplexTM Zika Virus Assay (Seegene®), a multiplex Real-Time PCR kit designed for specific detection of ZIKV RNA.

Techniques: Virus, Derivative Assay

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation

doi: 10.1590/0074-02760250272

Figure Lengend Snippet: salivary gland extract (SGE) enhances Zika virus (ZIKV) replication in human peripheral blood mononuclear cells (PBMCs). (A) Absolute viral RNA copy numbers in PBMCs cultured under the indicated conditions (Media, SGE, ZIKV, ZIKV+SGE) for 72 h. Each symbol represents one culture replicate; two replicates were performed per donor, resulting in 10 points per group from five independent donors. Quantitative polymerase chain reaction (qPCR) was performed in technical triplicates and averaged per well. (B) Δ viral RNA copies (ZIKV+SGE - ZIKV) calculated for each donor, showing consistent increases across all individuals. (C) Assessment of normality of viral RNA values by Shapiro-Wilk test (p > 0.05), supporting the use of paired analysis. (D) Paired comparison of viral RNA levels between ZIKV and ZIKV+SGE conditions across donors, shown as both absolute copy numbers and log10-transformed values. Wilcoxon matched-pairs test confirmed significantly higher viral loads in ZIKV+SGE (p = 0.002), with a median increase of 1,779 copies per donor (log10 median difference = 0.3367). Data represent PBMCs from five independent donors; each condition was cultured in duplicate, and qPCR performed in technical triplicates. Points depict culture-level replicates, while statistical analyses were performed on donor-level aggregates (duplicates averaged per donor).

Article Snippet: Quantification of ZIKV RNA was performed using the AllplexTM Zika Virus Assay (Seegene®), a multiplex Real-Time PCR kit designed for specific detection of ZIKV RNA.

Techniques: Virus, Cell Culture, Real-time Polymerase Chain Reaction, Comparison, Transformation Assay

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation

doi: 10.1590/0074-02760250272

Figure Lengend Snippet: lipid peroxidation during Zika virus (ZIKV) infection is reduced by Aedes aegypti salivary gland extract (SGE). Thiobarbituric acid reactive substance (TBARS) levels (nmol/mg of protein) were quantified in peripheral blood mononuclear cells (PBMCs) cultures after 72 h under the following conditions: control medium, SGE (1 µg/mL), SGE (5 µg/mL), ZIKV, and ZIKV+SGE (1 µg/mL). ZIKV infection significantly increased TBARS relative to controls (**p < 0.001), while co-treatment with SGE (1 µg/mL) significantly reduced TBARS compared with ZIKV alone (**p < 0.001). Data represent PBMCs from six independent donors (individual values shown) with mean ± standard error of the mean (SEM). Statistical analysis: Kruskal-Wallis with Dunn’s post-hoc test; asterisks denote significant pairwise comparisons as indicated.

Article Snippet: Quantification of ZIKV RNA was performed using the AllplexTM Zika Virus Assay (Seegene®), a multiplex Real-Time PCR kit designed for specific detection of ZIKV RNA.

Techniques: Virus, Infection, Control

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation

doi: 10.1590/0074-02760250272

Figure Lengend Snippet: Aedes aegypti salivary gland extract (SGE) modulates oxidative stress markers in Zika virus (ZIKV)-infected peripheral blood mononuclear cells (PBMCs). (A) Total glutathione (GSH) content, (B) advanced oxidised protein products (AOPP), and (C) nitric oxide (NO) levels in PBMC cultures after 72 h under the indicated conditions (Medium, SGE 1 µg/mL, ZIKV, ZIKV+SGE 1 µg/mL, ZIKV+SGE 5 µg/mL). ZIKV infection increased AOPP and decreased GSH compared with controls, consistent with oxidative imbalance. Co-treatment with SGE reduced AOPP levels and restored GSH content. NO production was significantly higher in ZIKV+SGE compared with ZIKV alone (*p < 0.01). Data represent PBMCs from five independent donors (individual values shown) with mean ± standard error of the mean (SEM). Statistical analysis: Kruskal-Wallis with Dunn’s post-hoc test; asterisks denote significant pairwise differences.

Article Snippet: Quantification of ZIKV RNA was performed using the AllplexTM Zika Virus Assay (Seegene®), a multiplex Real-Time PCR kit designed for specific detection of ZIKV RNA.

Techniques: Virus, Infection

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation

doi: 10.1590/0074-02760250272

Figure Lengend Snippet: characterisation of CD4⁺ T cell frequency, proliferation, and cytotoxic profile following exposure to Zika virus (ZIKV) and/or Aedes aegypti salivary gland extract (SGE). (A) Frequency (%) and (B) absolute number of CD4⁺ T cells in peripheral blood mononuclear cells (PBMCs) cultures after 72 h. (C) Frequency (%) and (D) absolute number of Ki-67⁺ CD4⁺ T cells. (E) Frequency (%) and (F) absolute number of granzyme B⁺ CD4⁺ T cells. Data were analysed by Friedman test followed by Dunn’s multiple comparisons. *p < 0.05; ns: not significant.

Article Snippet: Quantification of ZIKV RNA was performed using the AllplexTM Zika Virus Assay (Seegene®), a multiplex Real-Time PCR kit designed for specific detection of ZIKV RNA.

Techniques: Virus

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation

doi: 10.1590/0074-02760250272

Figure Lengend Snippet: modulation of CD8⁺ T cell frequency, absolute number, proliferation, and cytotoxic profile following exposure to Zika virus (ZIKV) and Aedes aegypti salivary gland extract (SGE). (A) Frequency of CD8⁺ T cells among peripheral blood mononuclear cells (PBMCs) after 72 h of treatment. (B) Absolute number of CD8⁺ T cells in culture. (C) Frequency of granzyme B⁺ CD8⁺ T cells. (D) Absolute number of granzyme B⁺ CD8⁺ T cells. (E) Frequency of Ki-67⁺ CD8⁺ T cells. (F) Absolute number of Ki-67⁺ CD8⁺ T cells. Data are presented as individual donors with medians. Statistical analysis was performed using the Friedman test with Dunn’s multiple comparisons post hoc test. *p < 0.05.

Article Snippet: Quantification of ZIKV RNA was performed using the AllplexTM Zika Virus Assay (Seegene®), a multiplex Real-Time PCR kit designed for specific detection of ZIKV RNA.

Techniques: Virus

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation

doi: 10.1590/0074-02760250272

Figure Lengend Snippet: expansion of dendritic cells and subsets in peripheral blood mononuclear cells (PBMCs) exposed to Zika virus (ZIKV) and Aedes aegypti salivary gland extract (SGE). (A) Absolute numbers of total dendritic cells (DCs) and (B-D) subsets (DC1, mDCs, pDCs) after 72 h of culture under the indicated conditions (Only Media, SGE, ZIKV, ZIKV+SGE). Absolute counts of total DCs, DC1, mDCs, and pDCs were significantly increased in the ZIKV+SGE group compared to controls (p < 0.05). (E) Mean fluorescence intensity (MFI) of CD11c (mDCs), CD141 (DC1), and CD303 (pDCs). No significant differences were observed in CD11c or CD141 expression, while CD303 showed a trend toward reduction under ZIKV+SGE exposure. Data represent PBMCs from five independent donors. Individual values are shown with bars indicating median and interquartile range. Statistical analysis: Friedman test with Dunn’s post-hoc comparisons; p < 0.05 considered significant.

Article Snippet: Quantification of ZIKV RNA was performed using the AllplexTM Zika Virus Assay (Seegene®), a multiplex Real-Time PCR kit designed for specific detection of ZIKV RNA.

Techniques: Virus, Fluorescence, Expressing

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation

doi: 10.1590/0074-02760250272

Figure Lengend Snippet: Aedes aegypti salivary gland extract (SGE) skews cytokine responses toward a Th2 profile during Zika virus (ZIKV) infection. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured for 72 h with medium alone (Only Media), SGE, ZIKV, or ZIKV+SGE. (A) Interferon-y (IFN-γ) levels increased after ZIKV infection but were significantly reduced by SGE treatment. (B) Interleukin-4 (IL-4) production was significantly elevated in the presence of SGE, both with and without ZIKV infection. (C) The IFN-γ: IL-4 ratio decreased significantly following SGE treatment, indicating a shift toward a Th2-skewed cytokine profile. Data were analysed using the Friedman test followed by Dunn’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Quantification of ZIKV RNA was performed using the AllplexTM Zika Virus Assay (Seegene®), a multiplex Real-Time PCR kit designed for specific detection of ZIKV RNA.

Techniques: Virus, Infection, Cell Culture

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation

doi: 10.1590/0074-02760250272

Figure Lengend Snippet: Aedes aegypti salivary gland extract (SGE) modulates cytokine production by antigen-presenting cell lines during Zika virus (ZIKV) infection. JAWS II dendritic cells and RAW 264.7 macrophages were cultured for 72 h under the indicated conditions (Only Media, SGE, ZIKV, or ZIKV+SGE at different concentrations). Cytokine concentrations [tumour necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), IL-10, IL-12] were quantified in culture supernatants by enzyme-linked immunosorbent assay (ELISA). In JAWS II cells (A-D), significant differences were observed for TNF-α, IL-1β, and IL-12, while IL-10 levels remained stable. In RAW 264.7 cells (E-H), SGE enhanced TNF-α, IL-1β, and IL-10 secretion, whereas IL-12 showed no significant modulation. Data represent individual values from independent experiments with bars indicating mean ± standard error of the mean (SEM). Statistical analysis was performed using Kruskal-Wallis with Dunn’s post-hoc test; p < 0.05 was considered significant.

Article Snippet: Quantification of ZIKV RNA was performed using the AllplexTM Zika Virus Assay (Seegene®), a multiplex Real-Time PCR kit designed for specific detection of ZIKV RNA.

Techniques: Virus, Infection, Cell Culture, Enzyme-linked Immunosorbent Assay