Systematic Review and Geospatial Modeling of Molecular Markers of Resistance to Artemisinins and Sulfadoxine-Pyrimethamine in Plasmodium falciparum in India.

Surveillance for genetic markers of resistance can provide valuable information on the likely efficacy of antimalarials but needs to be targeted to ensure optimal use of resources. We conducted a systematic search and review of publications in seven databases to compile resistance marker data from studies in India. The sample collection from the studies identified from this search was conducted between 1994 and 2020, and these studies were published between 1994 and 2022. In all, Plasmodium falciparum Kelch13 (PfK13), P. falciparum dihydropteroate synthase, and P. falciparum dihydrofolate reductase (PfDHPS) genotype data from 2,953, 4,148, and 4,222 blood samples from patients with laboratory-confirmed malaria, respectively, were extracted from these publications and uploaded onto the WorldWide Antimalarial Resistance Network molecular surveyors. These data were fed into hierarchical geostatistical models to produce maps with a predicted prevalence of the PfK13 and PfDHPS markers, and of the associated uncertainty. Zones with a predicted PfDHPS 540E prevalence of >15% were identified in central, eastern, and northeastern India. The predicted prevalence of PfK13 mutants was nonzero at only a few locations, but were within or adjacent to the zones with >15% prevalence of PfDHPS 540E. There may be a greater probability of artesunate-sulfadoxine-pyrimethamine failures in these regions, but these predictions need confirmation. This work can be applied in India and elsewhere to help identify the treatments most likely to be effective for malaria elimination.

Role of Human Twin Studies to Identify Genetic Linkage of Malaria Pathogenesis and Outcomes.

Malaria remains a major public health challenge that needs attention, especially when the world is aiming at malaria elimination in the near future. It is crucial to understand the underlying genetic factors and epigenetics involved in malaria susceptibility and the dynamics of host immune responses that affect disease outcomes and relapses in Plasmodium vivax and Plasmodium ovale. Studies in newborn and adult twins can help in understanding the comparative roles of environmental and genetic factors on disease pathogenesis and outcome. These studies can help in providing insights into the factors responsible for malaria susceptibility, clinical presentation, responsiveness toward existing as well as candidate antimalarials, and even identification of novel therapeutic targets. The results and outcomes from twin studies can be further applied to the entire population. In the present manuscript, we analyze the available literature on malaria and human twins and discuss the significance and benefits of twin studies to help in better understanding malaria.

Dried blood spots: a robust tool for malaria surveillance in countries targeting elimination.

With the advancements in analytical and molecular techniques, Dried Blood Spots (DBS) are re-emerging as attractive and cost-effective alternatives for global health surveillance. The use of DBS has been well-characterized in the neonatal screening of metabolic diseases, therapeutic screening as well as in epidemiological studies for biomonitoring. Malaria is one such infectious disease where DBS use can expedite molecular surveillance for assessing drug resistance and for refining drug usage policies. In India, malaria cases have reduced significantly over the past decade but to achieve malaria elimination by 2030, country-wide DBS-based screening should be conducted to identify the presence of molecular markers of artemisinin resistance and to study parasite reservoirs in asymptomatic populations. DBS has wide applications in genomics, proteomics, and metabolomic studies concerning both host and pathogen factors. Hence, it is a comprehensive tool for malaria surveillance that can capture both host and parasite information. In this review, we elucidate the current and prospective role of DBS in malaria surveillance and its applications in studies ranging from genetic epidemiology, parasite and vector surveillance, drug development and polymorphisms to ultimately how they can pave the roadmap for countries aiming malaria elimination.

Single-Nucleotide Polymorphisms in Glucose-6-Phosphate Dehydrogenase and their Relevance for the Deployment of Primaquine as a Radical Cure for Malaria.

Malaria remains an important public health problem despite efforts to control it. Besides active transmission, relapsing malaria caused by dormant liver stages of Plasmodium vivax and Plasmodium ovale hypnozoites is a major hurdle in malaria control and elimination programs. Primaquine (PQ) is the most widely used drug for radical cure of malaria. Due to its anti-hypnozoite and gametocidal activity, PQ plays a key role in malaria relapse and transmission. The human enzyme glucose-6-phosphate dehydrogenase (G6PD) is crucial in determining the safety of PQ because G6PD-deficient individuals are prone to hemolysis if treated with PQ. Therefore, there is a need to study the prevalence of G6PD-deficient genetic variants in endemic populations to assess the risk of PQ treatment and the necessity to develop alternative treatments. In this work, we discuss the common G6PD variants, their varying enzymatic activity, and their distribution on the three-dimensional structure of G6PD. Our work highlights the important G6PD variants and the need for large-scale G6PD gene polymorphism studies to predict populations at risk of PQ-induced toxicity.

Linkages between blood groups and malaria susceptibility.

Blood typing has revolutionized the field of medical science since its discovery about a century ago. Besides its established role in life-saving blood transfusions, researchers have always been curious about the relationship between blood groups and human ailments. The effect of blood groups on disease outcomes, susceptibility, and mortality has been widely explored. According to a particular school of thought, the endemicity of diseases shapes the distribution of blood group frequency in human populations and exert selection pressure favoring one blood type over another. Here we discuss the scope and association of different blood groups in the context of malaria.

High-density lipoproteins may play a crucial role in COVID-19.

BACKGROUND: Lipids play a central role in the virus life cycle and are a crucial target to develop antiviral therapeutics. Importantly, among the other lipoproteins, the 'good cholesterol' high-density lipoprotein (HDL) has been widely studied for its role in not only cardiovascular but several infectious diseases as well. Studies have suggested a role of serum lipids and lipoproteins including HDL, total cholesterol (TC), triglycerides (TG), and low-density lipoproteins (LDL) in several viral infections including COVID-19. This disease is currently a major public health problem and there is a need to explore the role of these host lipids/lipoproteins in virus pathogenesis. METHODOLOGY: A total of 75 retrospective COVID-19 positive serum samples and 10 COVID-19 negative controls were studied for their lipid profiles including TC, HDL, LDL, and very-low-density lipoproteins (VLDL), and TG. RESULTS: Systematic literature search on dyslipidemia status in India shows that low HDL is the most common dyslipidemia. In this cohort, 65% (49) of COVID-19 patients had severely low HDL levels whereas 35% (26) had moderately low HDL and none had normal HDL levels. On the other hand, ~ 96% of samples had normal TC (72) and LDL (72) levels. VLDL and TG levels were also variable. In the controls, 100% of samples had moderately low HDL but none severely low HDL levels. CONCLUSION: HDL likely plays a crucial role in COVID-19 infection and outcomes. The causal relationships between HDL levels and COVID-19 need to be studied extensively for an understanding of disease pathogenesis and management.

Effects of Host Genetic Polymorphisms on the Efficacy of the Radical Cure Malaria Drug Primaquine.

Malaria is a major cause of death in low-income countries. Malaria relapses are caused by Plasmodium vivax-induced latent liver stage hypnozoites, and relapses contribute significantly to the total disease burden. The goal of malaria elimination is threatened in countries where P. vivax is endemic and relapses remain a key aspect of concern. Targeting of the hypnozoites is crucial for radical cure and this is achieved by primaquine (PQ). In addition to its anti-hypnozoite effects, PQ also possesses gametocidal activity against all malaria causing Plasmodium species and is hence a useful tool to curtail malaria transmission. It is well known that host glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with hemolysis after treatment with PQ. Multiple other host polymorphisms impact on PQ metabolism, potentially affecting drug efficacy. Being a prodrug, PQ requires host factors cytochrome P450 2D6 (CYP2D6), cytochrome P450 NADPH: oxidoreductase (CPR) and monoamine oxidase (MAO) for its metabolism and conversion to active form. The efficacy of PQ in the host is therefore dependent on genetic polymorphisms of these three host genes. The efficacy of PQ is important for clearing reservoirs of P. vivax infection. Here, we have analyzed the known spectrum of genetic polymorphisms for host genes that enable PQ metabolism. It is vital to delineate the polymorphisms that determine the ultimate efficacy of PQ for formulating better malaria elimination strategies in countries with severe malaria burden. Thus population-based studies of these gene variants will provide new insights into the role of host genetics on PQ treatment outcomes.

GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target.IMPORTANCE Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric population with a high mortality rate. This study is focused on identification of crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective antivirals. We have identified a cellular protein, GRP78, that plays a dual role in virus entry and virus replication, two crucial steps of the virus life cycle, and thus is a novel host factor that could be a potential therapeutic target.

Japanese encephalitis virus invasion of cell: allies and alleys.

The mosquito-borne flavivirus, Japanese encephalitis virus (JEV), is the leading cause of virus-induced encephalitis globally and a major public health concern of several countries in Southeast Asia, with the potential to become a global pathogen. The virus is neurotropic, and the disease ranges from mild fever to severe hemorrhagic and encephalitic manifestations and death. The early steps of the virus life cycle, binding, and entry into the cell are crucial determinants of infection and are potential targets for the development of antiviral therapies. JEV can infect multiple cell types; however, the key receptor molecule(s) still remains elusive. JEV also has the capacity to utilize multiple endocytic pathways for entry into cells of different lineages. This review not only gives a comprehensive update on what is known about the virus attachment and receptor system (allies) and the endocytic pathways (alleys) exploited by the virus to gain entry into the cell and establish infection but also discusses crucial unresolved issues. We also highlight common themes and key differences between JEV and other flaviviruses in these contexts.

Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes.

Autophagy is a lysosomal degradative pathway that has diverse physiological functions and plays crucial roles in several viral infections. Here we examine the role of autophagy in the life cycle of JEV, a neurotropic flavivirus. JEV infection leads to induction of autophagy in several cell types. JEV replication was significantly enhanced in neuronal cells where autophagy was rendered dysfunctional by ATG7 depletion, and in Atg5-deficient mouse embryonic fibroblasts (MEFs), resulting in higher viral titers. Autophagy was functional during early stages of infection however it becomes dysfunctional as infection progressed resulting in accumulation of misfolded proteins. Autophagy-deficient cells were highly susceptible to virus-induced cell death. We also observed JEV replication complexes that are marked by nonstructural protein 1 (NS1) and dsRNA colocalized with endogenous LC3 but not with GFP-LC3. Colocalization of NS1 and LC3 was also observed in Atg5 deficient MEFs, which contain only the nonlipidated form of LC3. Viral replication complexes furthermore show association with a marker of the ER-associated degradation (ERAD) pathway, EDEM1 (ER degradation enhancer, mannosidase α-like 1). Our data suggest that virus replication occurs on ERAD-derived EDEM1 and LC3-I-positive structures referred to as EDEMosomes. While silencing of ERAD regulators EDEM1 and SEL1L suppressed JEV replication, LC3 depletion exerted a profound inhibition with significantly reduced RNA levels and virus titers. Our study suggests that while autophagy is primarily antiviral for JEV and might have implications for disease progression and pathogenesis of JEV, nonlipidated LC3 plays an important autophagy independent function in the virus life cycle.

Japanese encephalitis virus expands regulatory T cells by increasing the expression of PD-L1 on dendritic cells.

The mechanisms underlying Japanese encephalitis virus (JEV) pathogenesis need to be thoroughly explored to delineate therapeutic approaches. It is believed that JEV manipulates the innate and adaptive compartments of the host's immune system to evade immune response and cross the blood-brain barrier. The present study was thus designed to investigate the functional modulation of DCs after exposure to JEV and to assess the consequences on CD4(+) T-lymphocyte functions. Human monocyte-derived DCs were either infected with 1 MOI of live virus, UV-inactivated virus, or were mock-infected. Replication-competent JEV induced a significant increase in the expression of maturation markers 48 h postinfection, along with that of programmed cell death 1 ligand 1 (PD-L1; also called B7-H1 and CD274). JEV-infected DCs expanded the Treg cells in allogenic mixed lymphocyte reactions. The expansion of Treg cells by JEV-infected DCs was significantly reduced upon blocking PD-L1 using an antagonist. In addition, JEV-infected DCs significantly altered the proliferation and reduced the polarization of Th cells toward the Th1-cell phenotype. The results, for the first time, suggest that JEV evades the host's immune system by modulating the crosstalk between DCs and T lymphocytes via the PD-L1 axis.

Japanese encephalitis virus infects neuronal cells through a clathrin-independent endocytic mechanism.

Japanese encephalitis virus (JEV) is a mosquito-borne pathogenic flavivirus responsible for acute viral encephalitis in humans. The cellular entry of JEV is poorly characterized in terms of molecular requirements and pathways. Here we present a systematic study of the internalization mechanism of JEV in fibroblasts and neuroblastoma cells. To verify the roles of distinct pathways of cell entry, we used fluorescently labeled virus particles, a combination of pharmacological inhibitors, RNA interference (RNAi), and dominant-negative (DN) mutants of regulatory proteins involved in endocytosis. Our study demonstrates that JEV infects fibroblasts in a clathrin-dependent manner, but it deploys a clathrin-independent mechanism to infect neuronal cells. The clathrin-independent pathway requires dynamin and plasma membrane cholesterol. Virus binding to neuronal cells leads to rapid actin rearrangements and an intact and dynamic actin cytoskeleton, and the small GTPase RhoA plays an important role in viral entry. Immunofluorescence analysis of viral colocalization with endocytic markers showed that JEV traffics through Rab5-positive early endosomes and that release of the viral nucleocapsid occurs at the level of the early and not the late endosomes.