Emergence of two distinct variants of SARS-CoV-2 and an explosive second wave of COVID-19: the experience of a tertiary care hospital in Pune, India.

The emergence of novel variants of SARS-CoV-2 in several countries has been associated with increased transmissibility or reduced neutralization potential of antibodies against the Wuhan virus (wild type). From August 2021 onwards, India experienced a progressive decline in the number of active SARS-CoV-2 infections, indicative of a downward trend in the explosive second wave. This prospective study was conducted quarterly for one year (May 2020 to June 2021) at a tertiary care hospital in the city of Pune in western India. Receptor-binding domain (RBD, n = 319) and full genome (n = 20) sequences from viral-RNA-positive nasopharyngeal swabs of COVID-19 patients representing the first and second waves were used for analysis. No Brazilian, South African, or California variants were detected in this study. Until December 2020, only the wild-type strain was prevalent. Concurrent with the upsurge of the second wave in March 2021, 73% (33/45) of RBD sequences harboured L452R/E484Q mutations characteristic of the Kappa variant. In April 2021, co-circulation of Kappa (37%) and Delta (L452R/T478K, 59%) variants was recorded. During May and June 2021, the Delta variant became the predominant circulating variant, and this coincided with a significant decline in the number of COVID-19 cases. Of the 20 full genome sequences, six isolates each exhibited signature mutations of the Kappa and Delta variant. With several states witnessing a reduction in the number of COVID-19 cases, continuous monitoring of newer mutations and assessment of their effect on virus transmissibility and their impact on vaccinated or previously exposed individuals is necessary.

Isolation and genetic characterization of SARS-CoV-2 from Indian patients in a single family without H/O travel abroad.

In view of the rapidly progressing COVID-19 pandemic, our aim was to isolate and characterize SARS-CoV-2 from Indian patients. SARS-CoV-2 was isolated from nasopharyngeal swabs collected from the two members of a family without any history of (H/O) travel abroad. Both the virus isolates (8003 and 8004) showed CPE on day 3 post-inoculation, viral antigens by immunofluorescence assay and produced distinct, clear and uniform plaques. Infectious virus titers were 5 × 106 and 4 × 106 Pfu/ml by plaque assay and 107.5 and 107 by CPE-based TCID50/ml, respectively. Phylogenetic analysis grouped our isolates with the Italian strains. On comparison with Wuhan strain, 3 unique mutations were identified in nsp3 (A1812D), exonuclease (P1821S) of Orf1ab and spike protein (Q677H) regions, respectively. Both the viruses grouped with Italian strains of SARS-CoV-2 suggesting possible source being the virus imported from Italy. These fully characterized virus isolates will be useful in developing neutralization/virological assays for the evaluation of vaccines/antivirals.

Performance assessment of SARS-CoV-2 IgM & IgG ELISAs in comparison with plaque reduction neutralization test.

Background & objectives: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) continues to be a devastating pandemic. This study was aimed at performance assessment of SARS-CoV-2 IgM and IgG ELISAs, and investigation of their utility for patient diagnosis and sero-epidemiologic investigations. Methods: Serum/plasma samples from COVID-19 patients or asymptomatic contacts (n=180) and healthy donors (n=90) were tested in parallel using two commercial IgM ELISAs (Erbalisa and Inbios), and four IgG ELISAs (Kavach, Euroimmun, Erbalisa and Inbios) along with an indigenous ß-propiolactone inactivated virus-based ELISA (IRSHA-IgG-ELISA). Plaque reduction neutralization test (PRNT) was used as reference test. Results: Among 180 COVID-19 patients, 125 tested positive by PRNT. Inbios-IgM-ELISA showed sensitivity (Se)/specificity (Sp)/positive predictive value (PPV)/negative predictive value (NPV) of 93.6/97.8/98.4/94.4 per cent in relation to PRNT, and performed better than Erbalisa-IgM-ELISA (Se: 48%, Sp: 95.6%, PPV: 95.2%, NPV: 65.2%). During the first week of disease, only 47.4 per cent of the COVID-19 patients tested IgM positive by Inbios-IgM-ELISA, detection improving at two weeks and beyond (~86-100%). Among IgG tests, Inbios-IgG-ELISA ranked first in terms of sensitivity (83.2%), followed by IRSHA (64.8%), Euroimmun (64%), Erbalisa (57.6%) and Kavach (56%) tests. For all IgG tests, sensitivity improved during the third (73.9-95.7%) and fourth week (100%) of illness. The specificity (96.7-100%) and PPV (96.2-100%) of all IgG tests were high; NPV ranged between 71.9 and 87.1 per cent with Inbios-IgG-ELISA scoring highest. Interpretation & conclusions: Our results show that IgM detection by the current, most sensitive ELISAs cannot replace molecular diagnosis, but may aid as a supplement test. The available IgG tests are suitable for serosurveys for the assessment of previous virus exposure.

Risk of Stroke Outcomes in Atrial Fibrillation Patients Treated with Rivaroxaban and Warfarin.

OBJECTIVES: In a previous real-world study, rivaroxaban reduced the risk of stroke overall and severe stroke compared with warfarin in patients with nonvalvular atrial fibrillation (NVAF). The aim of this study was to assess the reproducibility in a different database of our previously observed results (Alberts M, et al. Stroke. 2020;51:549-555) on the risk of severe stroke among NVAF patients in a different population treated with rivaroxaban or warfarin. MATERIAL AND METHODS: This retrospective cohort study included patients from the IBM® MarketScan® Commercial and Medicare databases (2011-2019) who initiated rivaroxaban or warfarin after a diagnosis of NVAF, had ≥6 months of continuous health plan enrollment, had a CHA2DS2-VASc score ≥2, and had no history of stroke or anticoagulant use. Patient data were assessed until the earliest occurrence of a primary inpatient diagnosis of stroke, death, end of health plan enrollment, or end of study. Stroke severity was defined by National Institutes of Health Stroke Scale (NIHSS) score, imputed by random forest model. Cox proportional hazard regression was used to compare risk of stroke between cohorts, balanced by inverse probability of treatment weighting. RESULTS: The mean observation period from index date to either stroke, or end of eligibility or end of data was 28 months. Data from 13,599 rivaroxaban and 39,861 warfarin patients were included. Stroke occurred in 272 rivaroxaban-treated patients (0.97/100 person-years [PY]) and 1,303 warfarin-treated patients (1.32/100 PY). Rivaroxaban patients had lower risk for stroke overall (hazard ratio [HR], 0.82; 95% confidence interval [CI], 0.76-0.88) and for minor (NIHSS 1 to <5; HR, 0.83; 95% CI, 0.74-0.93), moderate (NIHSS 5 to <16; HR, 0.88; 95% CI, 0.78-0.99), and severe stroke (NIHSS 16 to 42; HR, 0.44; 95% CI, 0.22-0.91). CONCLUSIONS: The results of this study in a larger population of NVAF patients align with previous real-world findings and the ROCKET-AF trial by showing improved stroke prevention with rivaroxaban versus warfarin across all stroke severities.

Rivaroxaban versus warfarin in patients with nonvalvular atrial fibrillation and stage IV-V chronic kidney disease.

BACKGROUND: There is limited evidence on the effectiveness and safety of direct-acting oral anticoagulants in patients with nonvalvular atrial fibrillation (NVAF) and advanced chronic kidney disease (CKD). This study compared the risks of ischemic stroke/systemic embolism (ISSE) and major bleeding in patients with NVAF and stage IV-V CKD treated with rivaroxaban or warfarin. METHODS: Patients with NVAF and stage IV-V CKD who initiated rivaroxaban or warfarin treatment between November 2011 and June 2018 were selected from the Optum® Deidentified Electronic Health Record Database. Propensity score matching was used to balance rivaroxaban and warfarin patients on 112 measured baseline covariates. ISSE and major bleeding events over 2 years following treatment initiation were ascertained with validated end point definitions. Outcomes were analyzed as time-to-event data using Kaplan-Meier survival estimators and Cox regression. RESULTS: A total of 781 eligible rivaroxaban-treated patients were propensity score-matched to 1,536 warfarin-treated patients; baseline covariates were well balanced after matching (absolute standardized differences <0.1). The average patient age was 80 years; 60.5% were female; 81.3% and 18.7% had CKD stage IV and V, respectively. Hazard ratios for rivaroxaban compared to warfarin were 0.93 (95% CI 0.46-1.90, P = .85) for the risk of ISSE and 0.91 (95% CI 0.65-1.28, P = .60) for major bleeding. CONCLUSIONS: No statistically significant difference in the risk of ISSE or major bleeding was found between rivaroxaban- and warfarin-treated patients. Although further study is needed, rivaroxaban appears to be a reasonable alternative to warfarin for ISSE prevention in the setting of NVAF and stage IV-V CKD.

Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells.

Fibrogenic pathways in the liver are principally regulated by activation of hepatic stellate cells (HSC). Fibrosis is associated with chronic hepatitis C virus (HCV) infection, although the mechanism is poorly understood. HSC comprise the major population of nonparenchymal cells in the liver. Since HCV does not replicate in HSC, we hypothesized that exosomes secreted from HCV-infected hepatocytes activate HSC. Primary or immortalized human hepatic stellate (LX2) cells were exposed to exosomes derived from HCV-infected hepatocytes (HCV-exo), and the expression of fibrosis-related genes was examined. Our results demonstrated that HCV-exo internalized to HSC and increased the expression of profibrotic markers. Further analysis suggested that HCV-exo carry miR-19a and target SOCS3 in HSC, which in turn activates the STAT3-mediated transforming growth factor ß (TGF-ß) signaling pathway and enhances fibrosis marker genes. The higher expression of miR-19a in exosomes was also observed from HCV-infected hepatocytes and in sera of chronic HCV patients with fibrosis compared to healthy volunteers and non-HCV-related liver disease patients with fibrosis. Together, our results demonstrated that miR-19a carried through the exosomes from HCV-infected hepatocytes activates HSC by modulating the SOCS-STAT3 axis. Our results implicated a novel mechanism of exosome-mediated intercellular communication in the activation of HSC for liver fibrosis in HCV infection.IMPORTANCE HCV-associated liver fibrosis is a critical step for end-stage liver disease progression. However, the molecular mechanisms for hepatic stellate-cell activation by HCV-infected hepatocytes are underexplored. Here, we provide a role for miR-19a carried through the exosomes in intercellular communication between HCV-infected hepatocytes and HSC in fibrogenic activation. Furthermore, we demonstrate the role of exosomal miR-19a in activation of the STAT3-TGF-ß pathway in HSC. This study contributes to the understanding of intercellular communication in the pathogenesis of liver disease during HCV infection.

Knockdown of Autophagy Inhibits Infectious Hepatitis C Virus Release by the Exosomal Pathway.

UNLABELLED: Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. We showed previously that HCV induces autophagy for viral persistence by preventing the innate immune response. Knockdown of autophagy reduces extracellular HCV release, although the precise mechanism remains unknown. In this study, we observed that knockdown of autophagy genes enhances intracellular HCV RNA and accumulates infectious virus particles in cells. Since HCV release is linked with the exosomal pathway, we examined whether autophagy proteins associate with exosomes in HCV-infected cells. We observed an association between HCV and the exosomal marker CD63 in autophagy knockdown cells. Subsequently, we observed that levels of extracellular infectious HCV were significantly lower in exosomes released from autophagy knockdown cells. To understand the mechanism for reduced extracellular infectious HCV in the exosome, we observed that an interferon (IFN)-stimulated BST-2 gene is upregulated in autophagy knockdown cells and associated with the exosome marker CD63, which may inhibit HCV assembly or release. Taken together, our results suggest a novel mechanism involving autophagy and exosome-mediated HCV release from infected hepatocytes. IMPORTANCE: Autophagy plays an important role in HCV pathogenesis. Autophagy suppresses the innate immune response and promotes survival of virus-infected hepatocytes. The present study examined the role of autophagy in secretion of infectious HCV from hepatocytes. Autophagy promoted HCV trafficking from late endosomes to lysosomes, thus providing a link with the exosome. Inhibition of HCV-induced autophagy could be used as a strategy to block exosome-mediated virus transmission.

Serum miR-30e and miR-223 as Novel Noninvasive Biomarkers for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers and is the third leading cause of all cancer-related death. Limited noninvasive biomarkers are available for HCC detection. Early detection is the key in improving the survival of HCC patients. In this study, we tested the hypothesis that serum miRNAs can be used as a potential biomarker for HCC. Quantitative RT-PCR for miRNA analysis was performed using 70 serum samples. Receiver operating characteristic analysis was performed to measure the prognostic power of the miRNAs. The miRNA expression level was also measured from liver biopsy samples. Our study revealed that two miRNAs, miR-30e and miR-223, were expressed at significantly lower levels (P < 0.003) in the sera of HCC patients compared with healthy volunteers. Furthermore, expression of these miRNAs was compared between sera from chronic liver disease and sera from HCC patients. miR-30e and miR-223 expression was significantly lower in HCC sera compared with sera from chronic liver disease patients. Both miRNA expression levels were lower in HCC liver biopsy specimens compared with normal liver RNA. Taken together, our results suggested that serum miR-30e and miR-223 are useful biomarkers of HCC, irrespective of etiology, and deserve further study for their diagnostic value.

Transcriptional suppression of miR-181c by hepatitis C virus enhances homeobox A1 expression.

Hepatitis C virus (HCV)-induced chronic liver disease is one of the leading causes of hepatocellular carcinoma (HCC). The molecular events leading to HCC following chronic HCV infection remain poorly defined. MicroRNAs (miRNAs) have been implicated in the control of many biological processes, and their deregulation is associated with different viral infections. In this study, we observed that HCV infection of hepatocytes transcriptionally downregulates miR-181c expression by modulating CCAAT/enhancer binding protein ß (C/EBP-ß). Reduced expression of the pri-miR-181c transcript was noted following HCV infection. In silico prediction suggests that homeobox A1 (HOXA1) is a direct target of miR-181c. HOXA1 is a member of the homeodomain-containing transcription factor family and possesses pivotal roles in normal growth, development, and differentiation of mammalian tissues. Our results demonstrated that HOXA1 expression is enhanced in HCV-infected hepatocytes. Exogenous expression of the miR-181c mimic inhibits HOXA1 and its downstream molecules STAT3 and STAT5, which are involved in cell growth regulation. Interestingly, overexpression of miR-181c inhibited HCV replication by direct binding with E1 and NS5A sequences. Furthermore, accumulation of HCV genotype 2a RNA with miR-181c was observed in an RNA-induced silencing complex in Huh7.5 cells. Our results provide new mechanistic insights into the role of miR-181c in HCV-hepatocyte interactions, and miR-181c may act as a target for therapeutic intervention. Importance: Chronic HCV infection is one of the major causes of end-stage liver disease, including hepatocellular carcinoma. An understanding of the molecular mechanisms of HCV-mediated hepatocyte growth promotion is necessary for therapeutic intervention against HCC. In this study, we have provided evidence of HCV-mediated transcriptional downregulation of miR-181c. HCV-infected liver biopsy specimens also displayed lower expression levels of miR-181c. We have further demonstrated that inhibition of miR-181c upregulates homeobox A1 (HOXA1), which is important for hepatocyte growth promotion. Exogenous expression of miR-181c inhibited HCV replication by directly binding with HCV E1 and NS5A sequences. Taken together, our results provided new mechanistic insights for an understanding of the role of miR-181c in HCV-hepatocyte interactions and revealed miR-181c as a potential target for therapeutic intervention.

Hepatitis C virus induces interleukin-1ß (IL-1ß)/IL-18 in circulatory and resident liver macrophages.

Hepatitis C virus (HCV)-mediated chronic liver disease is a global health problem, and inflammation is believed to be an important player in disease pathogenesis. HCV infection often leads to severe fibrosis/cirrhosis and hepatocellular carcinoma, although the mechanisms for advancement of disease are not fully understood. The proinflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 have critical roles in establishment of inflammation. In this study, we examined induction of IL-1ß/IL-18 secretion following HCV infection. Our results demonstrated that monocyte-derived human macrophages (THP-1) incubated with cell culture-grown HCV enhance the secretion of IL-1ß/IL-18 into culture supernatants. A similar cytokine release was also observed for peripheral blood mononuclear cell (PBMC)-derived primary human macrophages and Kupffer cells (liver-resident macrophages) upon incubation with HCV. THP-1 cells incubated with HCV led to caspase-1 activation and release of proinflammatory cytokines. Subsequent studies demonstrated that HCV induces pro-IL-1ß and pro-IL-18 synthesis via the NF-κB signaling pathway in macrophages. Furthermore, introduction of HCV viroporin p7 RNA into THP-1 cells was sufficient to cause IL-1ß secretion. Together, our results suggested that human macrophages exposed to HCV induce IL-1ß and IL-18 secretion, which may play a role in hepatic inflammation.

Up-regulation of circulating miR-20a is correlated with hepatitis C virus-mediated liver disease progression.

UNLABELLED: Chronic hepatitis C virus (HCV) infection is one of the major causes of liver fibrosis and liver transplantation in the United States. Circulating microRNAs (miRNAs) in the blood are emerging as biomarkers for pathological conditions. In the present study we performed a systematic screening approach to identify up-regulated miRNAs in the plasma/serum of HCV-infected patients with different stages of hepatic histological disease severity. We initially screened serum samples of HCV-infected patients with fibrosis and compared them with sera of healthy volunteers using serum miRNA array profiling and identified a group of modulated miRNAs. Subsequent study demonstrated that miR-20a and miR-92a in HCV-infected fibrosis patients sera were significantly up-regulated when compared with that of healthy volunteers or non-HCV-associated liver disease. We have also observed an increase of plasma miR-20a and miR-92a in acute and chronic HCV-infected patients as compared to that of healthy volunteers. However, there was no correlation between the plasma/serum levels of any of these miRNAs with HCV viral loads. We next investigated longitudinal plasma samples from HCV-infected patients. Our results suggested that miR-20a and miR-92a remained unaltered in HCV-infected patients who progressed from acute to chronic infection. On the other hand, miR-92a expression was reduced in acute to resolved individuals. These data provide evidence that plasma/serum levels of miR-20a and miR-92a have potential as sensitive and cost-effective biomarkers for early detection of HCV infection. CONCLUSION: Circulating miR-20a may serve as a potential for predictive biomarker in HCV-mediated fibrosis.

Hepatitis C virus upregulates Beclin1 for induction of autophagy and activates mTOR signaling.

Hepatitis C virus (HCV) induces autophagosome formation in infected human hepatocytes. We have previously reported that HCV exploits autophagic machinery in favor of virus growth and survival in host cells (S. Shrivastava et al., Hepatology 53:406-414, 2011); however, the mechanisms for autophagy induction is poorly understood. In the present study, we observed that HCV infection transcriptionally upregulates Beclin1, which forms complex with Vps34, the class III phosphatidylinositol 3-kinase, as a first step for autophagy initiation. Although Bcl-2 has an anti-autophagy effect by its association with Beclin1 in nutrient-deprived cells, our studies revealed that HCV-mediated autophagy occurs independent of Beclin1-Bcl-2 dissociation. Mammalian target of rapamycin (mTOR) is a positive regulator of cell growth and is recognized as an inhibitor of autophagy induction. Our results demonstrated that HCV infection enhances phospho-mTOR expression and its downstream target 4EBP1 activation, suggesting that mTOR is not a negative regulator of HCV-induced autophagy. On the other hand, HCV infection in autophagy-impaired cells reduced phospho-mTOR, mTOR, and phospho-4EBP1 expression. Together, these results suggested that HCV induces autophagy by upregulating Beclin1 and activates mTOR signaling pathway, which in turn may promote hepatocyte growth.

Hepatitis C virus activates the mTOR/S6K1 signaling pathway in inhibiting IRS-1 function for insulin resistance.

Hepatitis C virus (HCV) infection significantly increases the prevalence of type 2 diabetes mellitus (T2DM). Insulin receptor substrate 1 (IRS-1) plays a key role in insulin signaling, thus enabling metabolic regulation in mammalian cells. We have previously shown that HCV infection modulates phosphorylation of Akt, a downstream target of IRS-1. In this study, we further examined the status of total IRS-1 and the downstream regulation of the Akt pathway in understanding mTOR/S6K1 signaling using HCV genotype 2a (clone JFH1)-infected hepatocytes. Inhibition of IRS-1 expression was observed in HCV-infected hepatocytes compared to that in a mock-infected control. The status of the tuberous sclerosis complex (TSC-1/TSC-2) was significantly decreased after HCV infection of human hepatocytes, showing a modulation of the downstream Akt pathway. Subsequent study indicated an increased level of Rheb and mTOR expression in HCV-infected hepatocytes. Interestingly, the phosphoS6K1 level was higher in HCV-infected hepatocytes, suggesting a novel mechanism for IRS-1 inhibition. Ectopic expression of TSC-1/TSC-2 significantly recovered the IRS-1 protein expression level in HCV-infected hepatocytes. Further analyses indicated that HCV core protein plays a significant role in modulating the mTOR/S6K1 signaling pathway. Proteasome inhibitor MG 132 recovered IRS-1 and TSC1/2 expression, suggesting that degradation occurred via the ubiquitin proteasome pathway. A functional consequence of IRS-1 inhibition was reflected in a decrease in GLUT4 protein expression and upregulation of the gluconeogenic enzyme PCK2 in HCV-infected hepatocytes. Together, these observations suggested that HCV infection activates the mTOR/S6K1 pathway in inhibiting IRS-1 function and perturbs glucose metabolism via downregulation of GLUT4 and upregulation of PCK2 for insulin resistance.

Hepatitis C virus infection modulates expression of interferon stimulatory gene IFITM1 by upregulating miR-130A.

We have examined the underlying mechanism of hepatitis C virus (HCV)-mediated IFITM1 regulation. IFITM1 is a potential target of miR-130a. Our results demonstrated that miR-130a expression was significantly higher in HCV-infected hepatocytes and liver biopsy specimens than in controls. Introduction of anti-miR-130a in hepatocytes increased IFITM1 expression. Hepatocytes stably expressing IFITM1 reduced HCV replication. Together, these results suggested that HCV infection of hepatocytes upregulates miR-130a and that use of anti-miR-130a may have potential for restriction of HCV replication.

Segmentation of mine overburden dump particles from images using Mask R CNN.

The stability of mine overburden dumps is crucial for the efficient operation of mining industries. The size distribution of particles affects the shear strength of dump slopes. Identification of dump particles from images is challenging as they vary in size, shape, color, granularity, and texture. In this paper, a unique way of identifying the particles from dump images using Artificial Intelligence is presented that can be used to determine the particle size distribution of dump. Mask R CNN with ResNet50 plus an FPN as a backbone network which is the current state of the art for instance segmentation has been implemented to segment the particles from dump images at detailed pixel level and to obtain their boundary. Experimental results showed promising results to delineate the particles and obtain masks over them. Our model has achieved a training accuracy of 97.2% for the dataset containing 31,505 particles. The model predicted the areas of dump particles with a mean percentage error of 0.39% and a standard deviation of 0.25 when compared to the ground truth values. The calculation of coordinates of the detected boundaries using the model significantly reduces the time and effort that are generally put in rock mechanics laboratories.

Platelet derived exosomes disrupt endothelial cell monolayer integrity and enhance vascular inflammation in dengue patients.

Background: Thrombocytopenia is the most notable phenomenon in dengue. Activation status of platelets and interaction of platelets with endothelium contribute towards dengue disease pathogenesis. Platelets are the major cell types known to release extracellular vesicles, especially exosomes in circulation. However, the role of platelet derived exosomes (PLT-EXOs) in endothelial dysfunction during dengue infection remains unknown. Methods: In this study, we recruited 28 healthy subjects and 69 dengue patients categorized as WS- (n=31), WS+ (n=29) and SD (n=9). Platelets were isolated from platelet rich plasma of dengue patients and their activation was assessed by flow cytometry. PLT-EXOs were isolated by ultracentrifugation method. Western blot analyses were performed to characterize the exosomes. Exosome uptake experiment was carried out to see the internalization of exosomes inside endothelial cells (HUVECs). To observe the effect of exosomes on endothelial cells, exosomes were added on HUVECs and expression of adherens and tight junctional proteins were examined by immunofluorescence assay and western blot. Expression levels of vascular injury markers were measured in the culture supernatants of Exosome-HUVEC coculture and sera of dengue patients by MSD-multiplex assay. Results: As compared to healthy subjects, CD41/CD61 expression was significantly reduced (p<0.0001) and CD62p expression was significantly increased (p<0.0001) on platelets in dengue patients. PLT-EXOs isolated from the dengue patients showed higher expression of CD63 and CD9 proteins than the healthy subjects. With in-vitro immunofluorescence assays, we illustrated the internalization of PLT-EXOs by the HUVECs and observed disruption of endothelial cell monolayer integrity in the presence of PLT-EXOs from WS+ and SD patients. Furthermore, the significant reduction in the expressions of ZO-2, VE-Cadherin and CD31 in endothelial cells following exposure to PLT-EXOs from the dengue patients provide direct evidence of PLT-EXOs mediated vascular permeability. PLT-EXOs stimulated the release of inflammatory markers CRP, SAA, sVCAM-1 and sICAM-1 in the supernatants of HUVEC cells. Importantly, significantly higher levels of CRP, sVCAM-1 and sICAM-1 in the sera of severe than mild dengue patients (p<0.0001) suggest their role in disease severity. Conclusions: In summary, our data suggest that PLT-EXOs promote vascular leakage via release of proinflammatory mediators and compromise vascular barrier integrity in dengue patients.

A Phase 1, double-blind, randomized, placebo-controlled study to evaluate the safety and immunogenicity of a tetravalent live attenuated dengue vaccine in adults.

BACKGROUND: Dengue fever is an important public health problem, especially in Asia and South America. A tetravalent live attenuated dengue vaccine was manufactured in India after receipt of vaccine strains from NIAID, NIH, USA. METHODS: This was a Phase 1, double-blind, randomized, placebo-controlled study performed in 60 healthy adults of 18 to 45 years. Participants were randomized 2:1 to receive a single subcutaneous injection of either a tetravalent live attenuated dengue vaccine or placebo. Safety was assessed by unsolicited adverse events (AEs) and solicited reactions through 21 days after vaccination and serious adverse events (SAEs) through the entire study period of 180 days. Dengue viremia was assessed at baseline and on day 9, 11 and 13 post-vaccination using a plaque assay. Immunogenicity was assessed using the plaque reduction neutralization test (PRNT) assay using vaccine-matched wild virus serotypes (DENV 1, DENV 2, DENV 3 and DENV 4) at baseline and on 56-, 84- and 180-days post-vaccination. PRNT assay using circulating wild type DENV 1, DENV 2, DENV 3 and DENV 4 were done on day 1 and day 85 for a subset of 31 participants. RESULTS: 60 participants were randomized to receive dengue vaccine (n = 40) or placebo (n = 20). 23 participants (59 %) showed DENV vaccine viremia post- vaccination for any of the four serotypes with majority on day 9 and day 11. At baseline, all participants were naïve by dengue PRNT50 for all four serotypes in both the study groups except for four in the dengue vaccine group and two in the placebo group. On day 57, the GMTs of neutralizing antibodies ranged from 66.76 (95 % CI 36.63, 121.69) to 293.84 (95 % CI 192.25, 449.11) for all four serotypes in the dengue vaccine group. On day 181 though the titers declined, they still remained much higher than the baseline. The titers in the placebo group did not change after vaccination. Seroconversion through day 85 ranged from 79.5 % for DENV 1 to 100 % for DENV2 while in the placebo group, no participant showed seroconversion through day 85. Similar trends were noted when PRNT was done using wild DENV serotypes in both vaccine and placebo groups. Among solicited reactions, injection site erythema, rash, headache, fatigue, myalgia and arthralgia were reported more frequently in the vaccine group than placebo group. All solicited reactions were of grade 1 or grade 2 severity and completely resolved. One unrelated serious adverse event was reported in the vaccine group. CONCLUSION: A single dose of dengue vaccine was safe and well tolerated in adults. The vaccine was highly immunogenic with trivalent or tetravalent seroconversion and seropositivity in most of the participants. The study was funded by Serum Institute of India Pvt. Ltd., Pune, India. CLINICALTRIALS: gov: NCT04035278.

ISG56 and IFITM1 proteins inhibit hepatitis C virus replication.

Hepatitis C virus (HCV) often leads to persistent infection. Interferon (IFN) and IFN-stimulated genes (ISGs) are amplified during HCV infection but fail to eliminate virus from the liver in a large number of infected patients. We have observed previously that HCV infection induces IFN-ß production in immortalized human hepatocytes (IHH) as early as 24 h after infection, although virus replication is not inhibited. To gain insights on possible countermeasures of virus for the suppression of host antiviral response, the cellular transcriptional profiles of ISGs were examined after various treatments of IHH. The majority of ISGs were upregulated in IFN-treated IHH from the level for mock-treated cells. However, the comparison of ISG expression in IFN-treated IHH and IFN-pretreated, HCV genotype 2a-infected IHH indicated that virus infection suppresses the upregulation of a subset of effector molecules, including ISG56 and IFITM1. Similar results were observed for HCV-infected Huh7 cells. Subsequent study suggested that the exogenous expression of ISG56 or IFITM1 inhibits HCV replication in IHH or Huh7 cells, and the knockdown of these genes enhanced HCV replication. Further characterization revealed that the overexpression of these ISGs does not block HCV pseudotype entry into Huh7 cells. Taken together, our results demonstrated that ISG56 and IFITM1 serve as important molecules to restrict HCV infection, and they may have implications in the development of therapeutic modalities.