Enhanced hepatitis B virus (HBV) pre-genomic RNA levels and higher transcription efficiency of defective HBV genomes.

Defective hepatitis B virus (dHBV) particles contain genomes corresponding to singly spliced HBV RNA. A limited number of studies show that dHBV is present in all chronically HBV-infected patients. Clinical studies have linked dHBV and dHBV gene products to high virus loads and liver damage. The replication characteristics of dHBV genomes remain poorly understood. We found that the splice donor/acceptor sites critical for the formation of dHBV genomes are conserved across HBV genotypes. We report a novel method to create dHBV constructs from corresponding wild-type (WT) HBV constructs. We assessed the transcriptional characteristics of the dHBV constructs with those of the corresponding WT construct using a cell culture model. Interestingly, dHBV constructs had higher pre-genomic RNA levels, transcription efficiency, HBV e antigen levels and intracellular HBV core antigen levels compared with the corresponding WT HBV constructs. Our findings highlight previously unrecognized fundamental molecular characteristics of dHBV genomes and their potential role in the pathogenesis of HBV infection.

CpG dinucleotide frequencies reveal the role of host methylation capabilities in parvovirus evolution.

Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to "fractional" methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.

Molecular mechanisms underlying occult hepatitis B virus infection.

Chronic hepatitis B virus (HBV) infection is a complex clinical entity frequently associated with cirrhosis and hepatocellular carcinoma (HCC). The persistence of HBV genomes in the absence of detectable surface antigenemia is termed occult HBV infection. Mutations in the surface gene rendering HBsAg undetectable by commercial assays and inhibition of HBV by suppression of viral replication and viral proteins represent two fundamentally different mechanisms that lead to occult HBV infections. The molecular mechanisms underlying occult HBV infections, including recently identified mechanisms associated with the suppression of HBV replication and inhibition of HBV proteins, are reviewed in detail. The availability of highly sensitive molecular methods has led to increased detection of occult HBV infections in various clinical settings. The clinical relevance of occult HBV infection and the utility of appropriate diagnostic methods to detect occult HBV infection are discussed. The need for specific guidelines on the diagnosis and management of occult HBV infection is being increasingly recognized; the aspects of mechanistic studies that warrant further investigation are discussed in the final section.

Performance Evaluation of Determine HBsAg 2 Rapid Card Test for the Detection of Hepatitis B Surface Antigen in Clinical Samples.

Aim and background: Hepatitis B virus is one of the leading underlying causes of chronic liver disease. Rapid diagnostic tests with improved sensitivity and specificity for detecting hepatitis B infection could aid in large-scale community screening in resource-limited settings. This study was designed to assess the clinical performance of a rapid card test to detect HBsAg. Materials and methods: In this study, archived once-thawed serum samples were tested on the Determine HBsAg 2 card and their performance was evaluated in reference to a chemiluminescence-based assay (HBsAg qualitative assay, Abbott Diagnostics, US). Results: A total of 120 patient samples (46 confirmed HBsAg-positive and 74 confirmed HBsAg-negative) were used in this study. The overall median age of the study population was 44 years (IQR: 36-51 years), with a male gender predominance (90%). A specificity of 100% (74/74) and sensitivity of 84.7% (39/46) was observed for the Determine HBsAg 2 assay compared with the reference assay. The samples that showed false-negative results (n = 7) by the card test had HBsAg levels below the limit-of-detection of the card assay. Conclusion: The Determine HBsAg 2 assay gives rapid results in 15 minutes with good sensitivity and specificity. This makes it a good, affordable tool for large-scale screening and public health surveillance programs. Clinical significance: Accurate and cost-effective rapid card tests for early detection of Hepatitis B infection would enable quick isolation of infected cases, thus reducing transmission in the community. How to cite this article: Samal J, Soni A, Pandey A, et al. Performance Evaluation of Determine HBsAg 2 Rapid Card Test for the Detection of Hepatitis B Surface Antigen in Clinical Samples. Euroasian J Hepato-Gastroenterol 2024;14(1):9-11.

Expression of a unique M. tuberculosis DNA MTase Rv1509 in M. smegmatis alters the gene expression pattern and enhances virulence.

Mycobacterium tuberculosis (M. tb) genome encompasses 4,173 genes, about a quarter of which remain uncharacterized and hypothetical. Considering the current limitations associated with the diagnosis and treatment of tuberculosis, it is imperative to comprehend the pathomechanism of the disease and host-pathogen interactions to identify new drug targets for intervention strategies. Using in-silico comparative genome analysis, we identified one of the M. tb genes, Rv1509, as a signature protein exclusively present in M. tb. To explore the role of Rv1509, a likely methyl transferase, we constructed a knock-in Mycobacterium smegmatis (M. smegmatis) constitutively expressing Rv1509 (Ms_Rv1509). The Ms_Rv1509 led to differential expression of many transcriptional regulator genes as assessed by RNA-seq analysis. Further, in-vitro and in-vivo studies demonstrated an enhanced survival of Ms_Rv1509 inside the host macrophages. Ms_Rv1509 also promoted phagolysosomal escape inside macrophages to boost bacterial replication and dissemination. In-vivo infection studies revealed that Ms_Rv1509 survives better than BCG and causes pathological manifestations in the pancreas after intraperitoneal infection. Long-time survival of Ms_Rv1509 resulted in lymphocyte migration, increased T regulatory cells, giant cell formation, and likely granuloma formation in the pancreas, pointing toward the role of Rv1509 in M. tb pathogenesis.

Rising trend of symptomatic infections due to Hepatitis A virus infection in adolescent and adult age group: An observational study from a tertiary care liver institute in India.

BACKGROUND: Hepatitis A Virus (HAV) is the most common cause of Acute Viral Hepatitis (AVH) in children. It causes self-limiting illness and rarely acute liver failure. The shifting pattern in HAV endemicity is rendering adolescents and adults vulnerable to infection. METHODS: In this retrospective study, samples received from 14,807 patients with acute onset icteric illness from January 2014-December 2022 were analyzed. HAV infection was detected by anti-HAV IgM positivity. The cases were divided into 3 age groups, pediatric, adolescents and adults, and clinical presentations were compared. RESULTS: Overall, 7.72%(1144) were positive for anti-HAV IgM. Of these, 60%(690) were finally included in the study. The positive cases were divided into adults, ≥18 years (44%, 304); pediatric, <12 years (31%, 212) and adolescents (25%,174) age groups. Overall males were predominant [72.4%(500)], with a median age of 16 (IQR:9-21) years. Cases were characterised into AVH (68.1%, 470/690), Acute Liver Failure (ALF) (31.4%, 217/690) and Acute-on-Chronic Liver Failure (0.43%, 3/690). AVH in the pediatric age group was 69%(146/212), adolescents was 67%(117/174), and adults was 68%(207/304). ALF cases among the 3 groups were 30%(65/212), 33%(57/174), and 31%(95/304) respectively. Overall mortality was seen in 6.52%(45/690), maximum in adolescents with ALF presentation [10.3%(18/174)]. On molecular characterization of infection, viremia was seen in 28.9%(200/690) and all the isolates were Genotype IIIA. CONCLUSIONS: The number of adults experiencing symptomatic HAV infection was seen to increase over the years in the present study. Infection in adolescents was associated with higher mortality and ALF as the clinical presentation.

Respiratory tract viral infections associated sepsis in patients with underlying liver disease: Viral sepsis an entity to look forward!

BACKGROUND: Sepsis remains a global health burden associated with significant morbidity and mortality. Bacteria are known to be the predominant pathogens in sepsis; however, viral etiologies in sepsis are still under diagnosed. Respiratory viral pathogens have been previously linked to sepsis, but the knowledge of incidence, disease burden and mortality of viral-induced sepsis remains limited. This study aimed at understanding the role of respiratory viral infections in the causation of sepsis in liver disease patients. METHODS: In this retrospective study, the clinical records of liver disease patients with influenza-like illness, whose requests for respiratory viral testing were received from January 2019 to December 2022, were reviewed. Respiratory viruses were identified using FilmArray 2.0 respiratory panel (BioFire Diagnostics, Utah, USA). RESULTS: Of 1391 patients tested, a respiratory viral etiology was detected in 23%. The occurrence of sepsis was seen in 35%. Among these, isolated viral etiology with no other bacterial/fungal coinfection was found in 55% of patients. Rhinovirus/Enterovirus was found as the most common underlying viral etiology (23.4%). The sepsis prevalence was higher among patients with associated comorbidities (45%) and decompensated cirrhosis (84%). On multi-variable analysis, no factor was found independently associated with sepsis-related mortality. CONCLUSION: This study underlines the importance of isolated viral etiology in causation of sepsis among liver disease patients. Patients with comorbidities, older age and decompensated cirrhosis are at an increased risk of developing sepsis and are associated with poorer outcomes. Accurate and timely identification of the viral etiology in sepsis would prevent the misuse of antibiotics and improve overall patient care.

Nef defect attenuates HIV viremia and immune dysregulation in the bone marrow-liver-thymus-spleen (BLTS) humanized mouse model.

In vitro studies have shown that deletion of nef and deleterious mutation in the Nef dimerization interface attenuates HIV replication and associated pathogenesis. Humanized rodents with human immune cells and lymphoid tissues are robust in vivo models for investigating the interactions between HIV and the human immune system. Here, we demonstrate that nef deletion impairs HIV replication and HIV-induced immune dysregulation in the blood and human secondary lymphoid tissue (human spleen) in bone marrow-liver-thymus-spleen (BLTS) humanized mice. Furthermore, we also show that nef defects (via deleterious mutations in the dimerization interface) impair HIV replication and HIV-induced immune dysregulation in the blood and human spleen in BLTS-humanized mice. We demonstrate that the reduced replication of nef-deleted and nef-defective HIV is associated with robust antiviral innate immune response, and T helper 1 response. Our results support the proposition that Nef may be a therapeutic target for adjuvants in HIV cure strategies.

Current surge of COVID-19 infection in China and its impact on India.

BACKGROUND: COVID-19, the pandemic caused by the SARS-CoV-2, is a global health calamity and one of the greatest challenges faced by the humankind across the globe. The virus originated in Wuhan, China and spread rapidly to more than 200 countries/nations, affected more than 600 billion individuals and caused around 65 lakh deaths worldwide. Since the start of the pandemic, SARS-CoV-2 mutates and accumulates genetic variations which constantly resulted in the emergence of new variants. OBJECTIVE: The current article discusses about the new omicron sub variant BF.7, and how this BF.7 variant may pose risk in India, if it overrides the current COVID-19 circulating variants. CONTENT: The emergence and potential consequences of the circulating SARS-CoV-2 variants usually augment virus transmissibility and host immune evasion. The current spurt in COVID-19 infections in China which has alarmed people around the world, is believed to be driven by an omicron sub variant BF.7. Although India has been reporting a "steady decline" in COVID-19 cases, we need constant surveillance to keep a track of the new emerging variants in circulation. Keeping in mind, the new surge in COVID-19 cases across many nations, we discuss about the new variant and its possible impact on India.

Performance Evaluation of an Improved HBsAg Assay (HBsAg NEXT) for the Detection of HBsAg Levels.

Objectives Detection of hepatitis B surface antigen (HBsAg) plays an important role in the screening and diagnosis of hepatitis B virus (HBV) infections. There is a need of highly sensitive assays with an improved lower limit of detection (LoD). Here, we evaluate the performance characteristics of the HBsAg NEXT (HBsAg new) assay in the detection of HBsAg in clinical samples. Materials and Methods This was a cross-sectional study conducted at a tertiary care liver center in North India. The study included 439 clinical samples. The HBsAg new assay was compared to the conventional chemiluminescence-based assay (HBsAg old assay, Architect, Abbott Diagnostics, United States). The analytical sensitivity of the HBsAg new assay was evaluated by checking its performance with the second World Health Organization (WHO) international standards for HBsAg. Results Out of 439 blood samples that were retrieved from the departmental repository stored at -80°C, 100 samples were positive and 339 samples were negative for HBsAg as per the HBsAg old assay. The HBsAg new assay showed incremental detection of HBsAg in 11 additional samples. Out of these, 5 samples were confirmed as occult HBV infection and the remaining 6 were classified as "exposed-to-virus" samples (HBV core total antibody-positive). The HBsAg new assay demonstrated a high positive significant correlation with the HBsAg old assay ( r = 0.881, p -value < 0.001). The HBsAg new assay could effectively detect the second WHO international standards to the level of 0.0033 IU/mL. Conclusion The HBsAg NEXT assay is a highly sensitive assay with an improved lower LoD.

Performance evaluation of NeuMoDx 96 system for hepatitis B and C viral load.

BACKGROUND: Hepatitis B virus (HBV) and hepatitis C virus (HCV) viral load (VL) estimation is essential for the management of both HBV and HCV infections. Due to a longer turnaround time for VL estimation, many patients drop out from the cascade of care. To achieve the global goals of reducing morbidity and mortality due to HBV/HCV and moving towards their elimination by 2030, molecular diagnostic platforms with faster and random (i.e. single sample) access are needed. AIM: To evaluate the performance of the recently launched NeuMoDx 96 random access system with the conventional COBAS®AmpliPrep/COBAS TaqMan system for HBV and HCV VL estimation. METHODS: Archived once-thawed plasma samples were retrieved and tested on both platforms. Correlation between the assays was determined by linear regression and Bland-Altman analysis. The study included samples from 186 patients, 99 for HBV of which 49 were true infected HBV cases (hepatitis B surface antigen, anti-hepatitis B core antibody, and HBV DNA-positive) and 87 for HCV assay in which 39 were true positives for HCV infection (anti-HCV and HCV RNA-positive). RESULTS: The median VL detected by NeuMoDx for HBV was 2.9 (interquartile range [IQR]: 2.0-4.3) log10 IU/mL and by COBAS it was 3.70 (IQR: 2.28-4.56) log10 IU/mL, with excellent correlation (R2 = 0.98). In HCV, the median VL detected by NeuMoDx was 4.9 (IQR: 4.2-5.4) log10 IU/mL and by COBAS it was 5.10 (IQR: 4.07-5.80) log10 IU/mL with good correlation (R2 = 0.96). CONCLUSION: The overall concordance between both the systems was 100% for both HBV and HCV VL estimation. Moreover, no genotype-specific bias for HBV/HCV VL quantification was seen in both the systems. Our findings reveal that NeuMoDx HBV and HCV quantitative assays have shown overall good clinical performance and provide faster results with 100% sensitivity and specificity compared to the COBAS AmpliPrep/COBAS TaqMan system.

The exploitation of host autophagy and ubiquitin machinery by Mycobacterium tuberculosis in shaping immune responses and host defense during infection.

Intracellular pathogens have evolved various efficient molecular armaments to subvert innate defenses. Cellular ubiquitination, a normal physiological process to maintain homeostasis, is emerging one such exploited mechanism. Ubiquitin (Ub), a small protein modifier, is conjugated to diverse protein substrates to regulate many functions. Structurally diverse linkages of poly-Ub to target proteins allow enormous functional diversity with specificity being governed by evolutionarily conserved enzymes (E3-Ub ligases). The Ub-binding domain (UBD) and LC3-interacting region (LIR) are critical features of macroautophagy/autophagy receptors that recognize Ub-conjugated on protein substrates. Emerging evidence suggests that E3-Ub ligases unexpectedly protect against intracellular pathogens by tagging poly-Ub on their surfaces and targeting them to phagophores. Two E3-Ub ligases, PRKN and SMURF1, provide immunity against Mycobacterium tuberculosis (M. tb). Both enzymes conjugate K63 and K48-linked poly-Ub to M. tb for successful delivery to phagophores. Intriguingly, M. tb exploits virulence factors to effectively dampen host-directed autophagy utilizing diverse mechanisms. Autophagy receptors contain LIR-motifs that interact with conserved Atg8-family proteins to modulate phagophore biogenesis and fusion to the lysosome. Intracellular pathogens have evolved a vast repertoire of virulence effectors to subdue host-immunity via hijacking the host ubiquitination process. This review highlights the xenophagy-mediated clearance of M. tb involving host E3-Ub ligases and counter-strategy of autophagy inhibition by M. tb using virulence factors. The role of Ub-binding receptors and their mode of autophagy regulation is also explained. We also discuss the co-opting and utilization of the host Ub system by M. tb for its survival and virulence.Abbreviations: APC: anaphase promoting complex/cyclosome; ATG5: autophagy related 5; BCG: bacille Calmette-Guerin; C2: Ca2+-binding motif; CALCOCO2: calcium binding and coiled-coil domain 2; CUE: coupling of ubiquitin conjugation to ER degradation domains; DUB: deubiquitinating enzyme; GABARAP: GABA type A receptor-associated protein; HECT: homologous to the E6-AP carboxyl terminus; IBR: in-between-ring fingers; IFN: interferon; IL1B: interleukin 1 beta; KEAP1: kelch like ECH associated protein 1; LAMP1: lysosomal associated membrane protein 1; LGALS: galectin; LIR: LC3-interacting region; MAPK11/p38: mitogen-activated protein kinase 11; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MAPK8/JNK: mitogen-activated protein kinase 8; MHC-II: major histocompatibility complex-II; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; NFKB1/p50: nuclear factor kappa B subunit 1; OPTN: optineurin; PB1: phox and bem 1; PE/PPE: proline-glutamic acid/proline-proline-glutamic acid; PknG: serine/threonine-protein kinase PknG; PRKN: parkin RBR E3 ubiquitin protein ligase; RBR: RING-in between RING; RING: really interesting new gene; RNF166: RING finger protein 166; ROS: reactive oxygen species; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6; Ub: ubiquitin; UBA: ubiquitin-associated; UBAN: ubiquitin-binding domain in ABIN proteins and NEMO; UBD: ubiquitin-binding domain; UBL: ubiquitin-like; ULK1: unc-51 like autophagy activating kinase 1.

Prevalence and Predictors for Respiratory Viral Infections among Liver Disease Patients.

Aim and background: Respiratory viral infections (RVIs) cause significant hospitalizations every year. Also, RVIs caused by either influenza or noninfluenza group of viruses can have adverse outcomes, especially among immunosuppressed patients. Regular and timely supervision is needed for accurate etiological identification, to prevent inappropriate use of antibiotics in patients with nonbacterial etiology. This study aimed to identify the spectrum of RVIs and clinical characteristics among liver disease patients with influenza-like illness (ILI). Materials and methods: In this study, medical records of patients with ILI, whose requests for respiratory viral testing came from September 2016 to December 2022 were retrospectively reviewed. Respiratory viruses were identified using FilmArray 2.0 respiratory panel (BioFire Diagnostics, USA). Results: Of the 1,577 liver disease patients with ILI, the overall prevalence of RVI was 28% (n = 449). Infection by noninfluenza viruses (NIVs) was detected in 329 patients (73%), higher than those infected with influenza viruses. In multivariable logistic regression analysis, female gender [odds ratio (OR): 2.5, 95% confidence interval (CI): 1.5-4.2], infection with influenza B (OR: 3.3, 95% CI: 1.09-9.9) and decompensated cirrhosis (OR: 3.9, 95% CI: 1.7-8.5) were independent risk factors for mortality. Regarding seasonality, influenza peaked in monsoons and winters, whereas NIVs circulated throughout the year. Conclusion: Overall, this study adds new knowledge regarding the incidence of RVI and the distribution of respiratory viral etiologies among liver disease patients with ILI. The findings highlight that female gender, decompensated cirrhosis, and influenza B infection are independently associated with poor clinical outcomes. Early etiological identification of viral causes of ILI could aid in an enhanced understanding of the prevalence of ILI and the timely management of the patients. Clinical significance: Respiratory viral infections can cause severe illness in individuals with underlying liver disease. Accurate diagnosis and risk stratification is crucial in mitigating the adverse health effects. How to cite this article: Samal J, Prabhakar T, Prasad M, et al. Prevalence and Predictors for Respiratory Viral Infections among Liver Disease Patients. Euroasian J Hepato-Gastroenterol 2023;13(2):108-114.

Co-infection of SARS-CoV-2 with other respiratory pathogens in patients with liver disease.

Respiratory illness caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) was first documented in Wuhan, China, in December 2019, followed by its rapid spread across the globe. Accumulating evidence has demonstrated viral/bacterial co-infection in the respiratory tract could modulate disease severity and its outcome in COVID-19 infection. In this retrospective study, 300 chronic liver disease patients with radiologically confirmed lower respiratory tract infection were enrolled from September 2020 to December 2021. In all of them, along with SARS-CoV-2, other respiratory viral/bacterial pathogens were studied. In total, 23.7 % (n=71) patients were positive for SARS-CoV-2. Among the positive patients, 23.9 % (n=17) had co-infection with other respiratory pathogens, bacterial co-infections being dominant. The SARS-CoV-2 negative cohort had 39.7  % positivity (n=91) for other respiratory pathogens, the most common being those of the rhinovirus/enterovirus family. Ground glass opacity (GGO) with consolidation was found to be the most common radiological finding among SARS-CoV-2 positive co-infected patients, as compared to only GGO among SARS-CoV-2 mono-infected patients. Accurate diagnosis of co-infections, especially during pandemics including COVID-19, can ameliorate the treatment and management of suspected cases.

Selective Depletion of ZAP-Binding CpG Motifs in HCV Evolution.

Hepatitis C virus (HCV) is a bloodborne pathogen that can cause chronic liver disease and hepatocellular carcinoma. The loss of CpGs from virus genomes allows escape from restriction by the host zinc-finger antiviral protein (ZAP). The evolution of HCV in the human host has not been explored in the context of CpG depletion. We analysed 2616 full-length HCV genomes from 1977 to 2021. During the four decades of evolution in humans, we found that HCV genomes have become significantly depleted in (a) CpG numbers, (b) CpG O/E ratios (i.e., relative abundance of CpGs), and (c) the number of ZAP-binding motifs. Interestingly, our data suggests that the loss of CpGs in HCV genomes over time is primarily driven by the loss of ZAP-binding motifs; thus suggesting a yet unknown role for ZAP-mediated selection pressures in HCV evolution. The HCV core gene is significantly enriched for the number of CpGs and ZAP-binding motifs. In contrast to the rest of the HCV genome, the loss of CpGs from the core gene does not appear to be driven by ZAP-mediated selection. This work highlights CpG depletion in HCV genomes during their evolution in humans and the role of ZAP-mediated selection in HCV evolution.

Treatment Response and Drug Resistance Profiling of Genotype 6 of Hepatitis C Virus in HCV/HIV Co-Infected Patients: A Pilot Study from INDIA.

Hepatitis C Virus (HCV) genotype (GT) 6 demonstrates maximum genomic diversity out of all the known genotypes of HCV, attributable to its inherent intra-genotype and inter-genotype recombination property. This is the most common genotype seen in HCV/HIV co-infected cases. HIV/HCV co-infection is linked with increased genetic diversity in HCV structural genes. The detailed information on the distribution of HCV GT6, its subtypes, and resistance to currently available antiviral drugs is limited in the Indian subcontinent. Therefore, in this single-center retrospective cross-sectional study, we aimed to map the occurrence of HCV GT6, its subtypes and resistance-associated substitution (RAS), and its correlation with antiviral treatment response in HCV-infected patients. From a cohort of 2052 HCV-infected patients, the overall prevalence of GT6 was 2.5% (n = 53), with a maximum of 81.1% (n = 43) seen in HCV/HIV co-infected patients. Nine different subtypes, 6a, 6b, 6f, 6i, 6n, 6u, 6v, 6w, and 6xa, were detected in the Indian population for the first time, with a predominance of 6xa (41.5%), a rare subtype, followed by 6n (39.6%). The phylogenetic analysis by the neighbor-joining method revealed three prominent viral clades, 6v, 6n, and 6xa-6u. The baseline (before treatment initiation) plasma samples of all GT6-infected patients were retrieved from -80 °C and a part of the NS5a and NS5b region of the viral genome was analyzed for the presence of RAS. No RASs were seen in the NS5b region, while in two patients (3.7%) RASs were seen at baseline in the NS5a region of the virus. Sustained viral response (SVR) was attained in 81% (n = 43) of patients. No difference in GT6 subtype distribution or occurrence of RAS was seen between mono-infected HCV and HIV/HCV co-infected cases. Our study revealed that RAS at baseline did not influence the attainment of SVR and the currently available antiviral therapy is effective against GT6 mono-infected and HIV/HCV co-infected patients.

Role of multiple factors likely contributing to severity-mortality of COVID-19.

COVID-19 stalled the world in 2020 and continues to be the greatest health crisis of this generation. While the apparent case fatality rates across fluctuates around ~2% globally, associated mortality/death rate (deaths per million population) varies distinctly across regions from the global average of ~600 per million population. Heterogeneous factors have been linked with COVID-19 associated mortalities and these include age, share of geriatric population, comorbidities, trained immunity and climatic conditions. Apart from direct or indirect role of endemic diseases, dietary factors and host immunity in regulating COVID-19 severity, human behaviour will inevitably control outcome of this pandemic. Comprehensive understanding of these factors will have a bearing on management of future health crises.

Mycobacterium tuberculosis Specific Protein Rv1509 Evokes Efficient Innate and Adaptive Immune Response Indicative of Protective Th1 Immune Signature.

Dissecting the function(s) of proteins present exclusively in Mycobacterium tuberculosis (M.tb) will provide important clues regarding the role of these proteins in mycobacterial pathogenesis. Using extensive computational approaches, we shortlisted ORFs/proteins unique to M.tb among 13 different species of mycobacteria and identified a hypothetical protein Rv1509 as a 'signature protein' of M.tb. This unique protein was found to be present only in M.tb and absent in all other mycobacterial species, including BCG. In silico analysis identified numerous putative T cell and B cell epitopes in Rv1509. Initial in vitro experiments using innate immune cells demonstrated Rv1509 to be immunogenic with potential to modulate innate immune responses. Macrophages treated with Rv1509 exhibited higher activation status along with substantial release of pro-inflammatory cytokines. Besides, Rv1509 protein boosts dendritic cell maturation by increasing the expression of activation markers such as CD80, HLA-DR and decreasing DC-SIGN expression and this interaction was mediated by innate immune receptor TLR2. Further, in vivo experiments in mice demonstrated that Rv1509 protein promotes the expansion of multifunctional CD4+ and CD8+T cells and induces effector memory response along with evoking a canonical Th1 type of immune response. Rv1509 also induces substantial B cell response as revealed by increased IgG reactivity in sera of immunized animals. This allowed us to demonstrate the diagnostic efficacy of this protein in sera of human TB patients compared to the healthy controls. Taken together, our results reveal that Rv1509 signature protein has immunomodulatory functions evoking immunological memory response with possible implications in serodiagnosis and TB vaccine development.

Structure-Function Analyses of New SARS-CoV-2 Variants B.1.1.7, B.1.351 and B.1.1.28.1: Clinical, Diagnostic, Therapeutic and Public Health Implications.

SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus 2) has accumulated multiple mutations during its global circulation. Recently, three SARS-CoV-2 lineages, B.1.1.7 (501Y.V1), B.1.351 (501Y.V2) and B.1.1.28.1 (P.1), have emerged in the United Kingdom, South Africa and Brazil, respectively. Here, we have presented global viewpoint on implications of emerging SARS-CoV-2 variants based on structural-function impact of crucial mutations occurring in its spike (S), ORF8 and nucleocapsid (N) proteins. While the N501Y mutation was observed in all three lineages, the 501Y.V1 and P.1 accumulated a different set of mutations in the S protein. The missense mutational effects were predicted through a COVID-19 dedicated resource followed by atomistic molecular dynamics simulations. Current findings indicate that some mutations in the S protein might lead to higher affinity with host receptors and resistance against antibodies, but not all are due to different antibody binding (epitope) regions. Mutations may, however, result in diagnostic tests failures and possible interference with binding of newly identified anti-viral candidates against SARS-CoV-2, likely necessitating roll out of recurring "flu-like shots" annually for tackling COVID-19. The functional relevance of these mutations has been described in terms of modulation of host tropism, antibody resistance, diagnostic sensitivity and therapeutic candidates. Besides global economic losses, post-vaccine reinfections with emerging variants can have significant clinical, therapeutic and public health impacts.