Viral load, clinical disease severity and cellular immune responses in primary varicella zoster virus infection in Sri Lanka

Background In Sri Lanka, varicella zoster virus (VZV) is typically acquired during adulthood with significant associated disease morbidity and mortality. T cells are believed to be important in the control of VZV replication and in the prevention of reactivation. The relationship between viral load, disease severity and cellular immune responses in primary VZV infection has not been well studied. Methodology We used IFNγ ELISpot assays and MHC class II tetramers based on VZV gE and IE63 epitopes, together with quantitative real time PCR assays to compare the frequency and phenotype of specific T cells with virological and clinical outcomes in 34 adult Sri Lankan individuals with primary VZV infection. Principal Findings Viral loads were found to be significantly higher in patients with moderate to severe infection compared to those with mild infection (p<0.001) and were significantly higher in those over 25 years of age (P<0.01). A significant inverse correlation was seen between the viral loads and the ex vivo IFNγ ELISpot responses of patients (P<0.001, r = −0.85). VZV-specific CD4+ T cells expressed markers of intermediate differentiation and activation. Conclusions Overall, these data show that increased clinical severity in Sri Lankan adults with primary VZV infection associates with higher viral load and reduced viral specific T cell responses

Development and validation of an in house multiplex real time PCR for quantification of all four serotypes of dengue virus

Objectives: In order to determine the infecting dengue virus (DENV) serotype and also to quantify the amount of virus in clinical samples and cell cultures, we proceeded to optimize and validate a quantitative real time RT-PCR assay.Methods: WHO reference strains of the four DENV serotypes were cultured on C6/36 cell lines for 7 days. The supernatants were used to determine plaque forming units (pfus) using BHK cell lines, which is indicative of infecting virus particles. cDNA was synthesized from RNA extracted from the virus culture supernatants. A tenfold dilution series (106 to 101 pfu/ml) of the known pfus of the viruses was prepared using the cDNA for standard curves. Data were analyzed using applied biosystems sequence detection systems. The threshold cycle value (Ct) for each reaction was determined by manually setting the threshold limit. Each standard curve was initially generated individually and later was used in a multiplex assay. All experiments were done in triplicate.Results: The optimized multiplex method detected all four DENV serotypes and generated standard curves with correlation coefficients (R2 values) above 0.95, which suggested that the standard curves and dilutions were of acceptable quality. The slope values of the standard curves were between -3.1 and -3.8 for all assays, implying that the PCR reactions were quite efficient.Conclusions: We have optimized and validated a multiplex quantitative real time RT-PCR assay, which can be effectively used to determine the infecting DENV in samples and also quantify the amount of virus

Role of natural killer T cells in the pathogenesis of dengue infections

Objectives: The dengue virus exploits cellular lipid metabolism pathways and natural killer T cells (iNKT), which recognize glycolipids have been suggested to play a role in mouse models of acute dengue. Therefore, we set out to determine if iNKT cells play a role in acute dengue infectionMethods: The frequency of iNKT cells (CD3+, Vα24+) was determined in 49 acute dengue and 22 healthy individuals. The functionality and phenotype of iNKT cell subsets were defined only in 19 patients and 10 controls by flow cytometry. Clinical disease severity was determined by the WHO 2011 guidelinesResults: The proportion of iNKTs in patients with acute dengue were significantly higher (P=0.03) compared to healthy individuals. We found that the CD4+ iNKTs, which produce inflammatory cytokines and are less cytotoxic, were significantly expanded (p=0.01) in acute dengue. iNKTs of patients were also significantly (p=0.02) more activated (both CD38+ and HLA-DR+), that iNKT cell activation significantly and positively correlated with dengue-specific IgG antibody titres (Spearmans’ r=0.5018, P=0.03). iNKT of patients were also predominantly of the immature phenotype, as the expression of CD161 was significantly more than in healthy individuals (p=0.01).Conclusions: As the iNKT cell population, especially of the CD4+ T cell subset appears to be highly activated and expanded in acute dengue, iNKT cells could be contributing to the pathogenesis of dengue infection

Viral Load, Clinical Disease Severity and Cellular Immune Responses in Primary Varicella Zoster Virus Infection in Sri Lanka

BACKGROUND: In Sri Lanka, varicella zoster virus (VZV) is typically acquired during adulthood with significant associated disease morbidity and mortality. T cells are believed to be important in the control of VZV replication and in the prevention of reactivation. The relationship between viral load, disease severity and cellular immune responses in primary VZV infection has not been well studied. METHODOLOGY: We used IFNgamma ELISpot assays and MHC class II tetramers based on VZV gE and IE63 epitopes, together with quantitative real time PCR assays to compare the frequency and phenotype of specific T cells with virological and clinical outcomes in 34 adult Sri Lankan individuals with primary VZV infection. PRINCIPAL FINDINGS: Viral loads were found to be significantly higher in patients with moderate to severe infection compared to those with mild infection (p&lt;0.001) and were significantly higher in those over 25 years of age (P&lt;0.01). A significant inverse correlation was seen between the viral loads and the ex vivo IFNgamma ELISpot responses of patients (P&lt;0.001, r = -0.85). VZV-specific CD4+ T cells expressed markers of intermediate differentiation and activation. CONCLUSIONS: Overall, these data show that increased clinical severity in Sri Lankan adults with primary VZV infection associates with higher viral load and reduced viral specific T cell responses

Altered monocyte response to the dengue virus in those with varying severity of past dengue infection

Objective We sought to investigate the differences in monocyte immune responses to the dengue virus (DENV) in those who previously had either severe disease (past SD) or non-severe dengue (past NSD) following a secondary dengue infection. Method Monocytes from healthy individuals who had either past SD (n = 6) or past NSD (n = 6) were infected at MOI one with all four DENV serotypes following incubation with autologous serum. 36-hours post infection, levels of inflammatory cytokines and viral loads were measured in the supernatant and expression of genes involved in viral sensing and interferon signaling was determined. Results Monocytes of individuals with past SD produced significantly higher viral loads (p = 0.0426 and cytokines (IL-10 p = 0.008, IL-1β p = 0.008 and IL-6 p = 0.0411) when infected with DENV serotypes they were not immune to, compared to those who has past NSD. Monocytes of individuals with past SD also produced significantly higher viral loads (p = 0.022) and cytokines (IL-10 p &lt; 0.0001, IL-1β &lt; 0.0001 and IL-6 p &lt; 0.0001) when infected with DENV serotypes they were previously exposed to, despite the monocytes being infected in the presence of autologous serum. A significant upregulation of NLRP3 (p = 0.005), RIG-I (0.0004) and IFNB-1 (0.01) genes were observed in those who had past SD compared to past NSD when infected with non-immune DENV serotypes. Conclusion Monocytes from those with past SD appear to show marked differences in viral loads, viral sensing and production of inflammatory mediators in response to the DENV, when compared to those who experienced past NSD, suggesting that initial innate immune responses may influence the disease outcome

Suppression of virus specific immune responses by IL-10 in acute dengue infection

BACKGROUND: Elevated IL-10 has been shown to be associated with severe dengue infection (DI). We proceeded to investigate the role of IL-10 in the pathogenesis of acute DI. MATERIALS AND METHODS: Ex vivo and cultured IFNγ ELISpot assays for dengue virus (DENV) NS3 protein and non dengue viral proteins were carried out in 26 patients with acute DI (16 with dengue haemorrhagic fever) and 12 healthy dengue seropositive individuals from Sri Lanka. DENV serotype specific (SS) responses were determined by using a panel of SS peptides. RESULTS: Serum IL-10 level were significantly higher (p = 0.02) in those who did not have in vitro responses to DENV-SS peptides (mean 144.2 pg/ml) when compared to those who responded (mean 75.7 pg/ml). DENV-NS3 specific ex vivo IFNγ ELISpot responses were also significantly lower (p = 0.0001) in those who did not respond to DENV-SS peptides (mean 42 SFU/million PBMCs) when compared to those who responded to DENV-SS peptides (mean 1024 SFU/million PBMCs). Serum IL-10 levels correlated significantly (p = 0.03) and inversely (Spearmans R = -0.45) with ex vivo DENV-NS3 specific responses but not with ex vivo non DENV specific responses (Spearmans R = -014, p = 0.52). Blockage of IL-10 in vitro significantly increased (p = 0.04) the ex vivo IFNγ ELISpot DENV-NS3 specific responses but had no effect on responses to non DENV proteins. CONCLUSION: IL-10 appears to contribute to the pathogenesis of acute dengue infections by inhibiting DENV-specific T cell responses, which can be restored by blocking IL-10