Emergence of new genotypes and lineages of dengue viruses during the 2012–15 epidemics in southern India

Objectives: To genotypically characterize dengue virus (DENV) isolates among dengue-infected children from 2012–13/2014–15 outbreaks in southern India. Methods: Children hospitalized with suspected dengue were tested for dengue RT-PCR targeting Capsid-preMembrane (C-prM) and Envelope (Env) regions. Following virologic confirmation (n = 612), a representative selection of DENV isolates (n = 99) were sequenced for C-prM, aligned using ClustalW and subjected to phylogenetic analysis by maximum-likelihood method in MEGA6. Results: In 2012–13 (n = 113), DENV-3 (44, 38.9%) and DENV-2 (43, 38.1%) predominated; DENV-1 (22, 19.5%) and DENV-4 (1, 0.9%) were less common. The pattern changed in 2014–15 (n = 499), when DENV-1 (329, 65.7%) predominated, followed by DENV-2 (97, 21.2%), DENV-3 (36, 6.7%) and DENV-4 (10, 2.0%). Multiple-serotype co-infections occurred in 2.7% and 5.4% in 2012–13 and 2014–15, respectively. Genotype III (GIII) of DENV-1 predominated (85.7%) in 2012–13, ceding to GI predominance (80.8%) in 2014–15. Among DENV-2, 71.9% (23/32) showed distinct clustering suggesting a new lineage, 'GIVc'. All tested DENV-4 were GIC, whose clustering pattern showed the emergence of two distinct clades. Conclusions: New genotypic/lineage variations in DENV-1 and DENV-2 may have influenced the magnitude and severity of dengue epidemics in southern India during this period. These findings emphasize the role of active surveillance of DENV serotypes/genotypes in aiding outbreak control and vaccine studies. Keywords: Dengue virus, Serotyping, Sequencing, Phylogenetics, Genotypes, Lineage

Molecular surveillance of Dengue Virus (DENV) and its co-infection with Chikungunya Virus (CHIKV) among febrile patients: A comparative study from South Delhi, India

Dengue and Chikungunya are two major arboviral infections transmitted worldwide by the mosquitoes, Aedes aegypti and Ae. albopictus. India suffers enormously with both Dengue and Chikungunya as they pose a great public health challenge. The present study aims to evaluate the prevalence of Dengue Virus (DENV), Chikungunya Virus (CHIKV) and DENV/CHIKV co-infection (by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)/Enzyme Linked Immunosorbent Assay (ELISA), their clinical features, DENV serotypes and CHIKV specific Immunoglobulin G (IgG) within a 7 years gap in the Delhi population. The study sample included clinically suspected febrile patients (?7 days) sera collected during 2017-2018 (n=87) and during 2008-2010 (n=623) from Delhi. Captured ELISA was performed for CHIKV IgG screening and nested PCR was done for DENV serotyping. The percentage prevalence for DENV was significantly higher than CHIKV with 41.38% (n=87) and 16.1% (n=87), respectively; interestingly, DENV/CHIKV co-infection was detected in 10.34% (n=9/87) cases during 2017-2018. Similarly, a high DENV prevalence was observed during 2008-2010 with the prevalence rate of 38.3% (69/180),  34.65% (35/101) and 47.07% (161/342), respectively. DENV 1 and DENV 3 were dominant serotype during 2008-2010 and 2017-2018 respectively. We have noticed a high prevalence (36.67%, 22/60) of the CHIKV IgG antibody in the 2017-2018 samples. Joint pain was more preferential to CHIKV mono-infection and DENV/CHIKV co-infection compared to DENV mono-infection. The present study highlights the need for active surveillance simultaneously for both DENV and CHIKV and to evaluate the role of CHIKV/DENV co-infections in disease severity in the endemic regions.

Malaria parasite tyrosyl-tRNA synthetase secretion triggers pro-inflammatory responses

Malaria infection triggers pro-inflammatory responses in humans that are detrimental to host health. Parasite-induced enhancement in cytokine levels correlate with malaria-associated pathologies. Here we show that parasite tyrosyl-tRNA synthetase (PfTyrRS), a housekeeping protein translation enzyme, induces pro-inflammatory responses from host immune cells. PfTyrRS exits from the parasite cytoplasm into the infected red blood cell (iRBC) cytoplasm, from where it is released into the extracellular medium on iRBC lysis. Using its ELR peptide motif, PfTyrRS specifically binds to and internalizes into host macrophages, leading to enhanced secretion of the pro-inflammatory cytokines TNF-α ± and IL-6. PfTyrRS-macrophage interaction also augments expression of adherence-linked host endothelial receptors ICAM-1 and VCAM-1. Our description of PfTyrRS as a parasite-secreted protein that triggers pro-inflammatory host responses, along with its atomic resolution crystal structure in complex with tyrosyl-adenylate, provides a novel platform for targeting PfTyrRS in anti-parasitic strategies

Upregulation of Survivin in G2/M Cells and Inhibition of Caspase 9 Activity Enhances Resistance in Staurosporine-Induced Apoptosis

Survivin, a member of the inhibitor of apoptosis (IAP) gene family, plays an important role in both the regulation of cell cycle and the inhibition of apoptosis, and is frequently overexpressed in many tumor types. In neuroblastomas, the expression of survivin correlates with a more aggressive and histologically unfavorable disease. Survivin is predominantly a cytoplasmic protein that is expressed in a cell cycle-dependent manner, increasing in the G2/M phase of the cell cycle followed by a rapid decline in the G1 phase. Recently, the role of survivin in resistance to chemotherapy has become an area of intensive investigation. In this study, we demonstrate a phase-specific resistance due to survivin in staurosporine (STS)-induced apoptosis in the human neuroblastoma cell line SK-N-MC. G2/M-arrested cultures show an upregulation of survivin expression and are more resistant, whereas G1-phase cells that show decreased levels of survivin are more sensitive to apoptosis. Localization studies revealed differences in the distribution of survivin in two synchronized populations, with G1 cells having weakly positive staining confined to the nucleus, in contrast to G2/M cells that depicted a more uniform and intense expression of survivin throughout the cell. In our experimental system, STS induced apoptosis through the mitochondrial-caspase 9-mediated pathway. Retention of survivin in G1 cells by inhibition of the ubiquitin-proteosome pathway or inhibition of caspase 9 protected the cells against apoptosis. Our data suggest that survivin exerts its antiapoptotic effect by inhibiting caspase 9 activity, an important event in STS-mediated apoptosis. In context with cell cycle-dependent responses to chemotherapy, the data from this study suggest the possibility of exploiting the survivin pathway for inducing apoptosis in tumor cells

Optimization of Flow-Cytometry Based Assay for Measuring Neutralizing Antibody Responses against Each of the Four Dengue Virus Serotypes

Dengue is an important public health problem worldwide, with India contributing nearly a third of global dengue disease burden. The measurement of neutralizing antibody responses is critical for understanding dengue pathophysiology, vaccine development and evaluation. Historically, dengue virus neutralization titers were measured using plaque reduction neutralization tests (PRNTs), which were later adapted to focus reduction neutralization tests (FRNTs). Given the slow and laborious nature of both these assays, there has been interest in adapting a high-throughput flow cytometry based neutralization assay. However, flow cytometry based assays typically underestimate neutralization titers, and in situations where the titers are low they can even fail to detect neutralization activity. In this study, by evaluating graded numbers of input Vero cell numbers and viral inoculum, we optimized the flow cytometry based neutralization assay in such a way that it is sensitive and scores titers that are in concordance with focus reduction neutralization tests for each of the four dengue virus serotypes (p < 0.0001). Given that dengue is a global public health concern, and several research groups are making efforts to understand its pathophysiology and accelerate vaccine development and evaluation both in India and worldwide, our findings have timely significance for facilitating these efforts

Optimization of Flow-Cytometry Based Assay for Measuring Neutralizing Antibody Responses against Each of the Four Dengue Virus Serotypes

Dengue is an important public health problem worldwide, with India contributing nearly a third of global dengue disease burden. The measurement of neutralizing antibody responses is critical for understanding dengue pathophysiology, vaccine development and evaluation. Historically, dengue virus neutralization titers were measured using plaque reduction neutralization tests (PRNTs), which were later adapted to focus reduction neutralization tests (FRNTs). Given the slow and laborious nature of both these assays, there has been interest in adapting a high-throughput flow cytometry based neutralization assay. However, flow cytometry based assays typically underestimate neutralization titers, and in situations where the titers are low they can even fail to detect neutralization activity. In this study, by evaluating graded numbers of input Vero cell numbers and viral inoculum, we optimized the flow cytometry based neutralization assay in such a way that it is sensitive and scores titers that are in concordance with focus reduction neutralization tests for each of the four dengue virus serotypes (p < 0.0001). Given that dengue is a global public health concern, and several research groups are making efforts to understand its pathophysiology and accelerate vaccine development and evaluation both in India and worldwide, our findings have timely significance for facilitating these efforts