Genetic signatures coupled with lineage shift characterise endemic evolution of Dengue virus serotype 2 during 2015 outbreak in Delhi, India.

OBJECTIVE: In 2015, New Delhi witnessed a massive outbreak of Dengue virus (DENV) resulting in high morbidity and mortality. We report the molecular characterisation of the dominant circulating DENV strain to understand its evolution and dispersal. MATERIALS AND METHODS: DENV infections were diagnosed by detection of IgM/NS1 antigen, and serotyping was performed by C-PrM PCR. Envelope gene was amplified, and variation(s) in envelope gene were analysed. Phylogenetic tree construction, time-based phylogeny and origin of DENV were analysed. Site-specific selection pressure of envelope gene variants was analysed. RESULTS: Confirmed DENV infection was observed in 11.34% (32 of 282) cases, while PCR positivity for C-PrM region was observed in 54.16% (13 of 24) of NS1 antigen-positive cases. All samples belonged to serotype 2 and cosmopolitan genotype. Phylogenetic analysis using envelope gene revealed segregation of cosmopolitan genotype strains into specific lineages. The Indian strains clustered separately forming a distinct monophyletic lineage (lineage III) with a signature amino acid substitution viz., I162V and R288K. Selection pressure analysis revealed that 215D, 288R and 304K were positively selected sites. The rate of nucleotide substitution was 6.93 × 10-4 substitutions site-1 year-1 with time to most common ancestor was around 10 years with JX475906 (Hyderabad strain) and JN030345 (Singapore strain) as its most probable ancestor. CONCLUSION: We observed evolution of a distinct lineage of DENV-2 strains on the Indian subcontinent with possible changes in endemic circulating dengue strains that might give rise to more pathogenic strains.

Identification and full-length molecular characterization of rare hepatitis C virus genotype 5a from India.

This report presents a molecular characterization of the complete genome of a rare hepatitis C virus (HCV) genotype (GT5a) from India. Sequence homology of full genome revealed that the strain belonged to HCV GT5a. To trace the origin of this virus and to understand its evolutionary pattern, a phylogenetic reconstruction was carried out on full HCV genome sequences using Bayesian coalescent methods. The phylogenetic tree reconstruction revealed genotypic divergence, with formation of distinct clades. This analysis revealed that HCV genotype 5 might have originated from HCV genotype 3, as they have a recent common ancestor.

Guillain-Barré syndrome in a patient of acute Hepatitis E virus infection associated with genotype 1: Case report and literature review.

Hepatitis E is a serious public health problem in developing countries. Most of the patients with Hepatitis E virus (HEV) infection present with typical acute hepatitis symptoms. However, in few patients it may lead to complications such as liver failure and extrahepatic symptoms. One of the rare extrahepatic presentations of this infection is neurological complications such as Guillain-Barré syndrome (GBS) which is observed in 5.5% of HEV infected patients (mainly in developed countries). Moreover, only genotype (gt) 3 HEV was found in association with GBS among patients in developed countries whereas molecular characterisation of HEV cases detected from developing countries have not been reported till now. Here, we are reporting a case of GBS as an extrahepatic complication of HEV associated with gt1 identified by molecular characterization by performing PCR of open-reading frame 2 (ORF2) region of HEV. Phylogenetic analysis by maximum likelihood method revealed that HEV gt1 case reported in this paper rooted closely with other HEV gt1 samples from South-Asian countries with high bootstrap values indicative of fully resolved tree.

Endothelial Cell-Derived TGF-ß Promotes Epithelial-Mesenchymal Transition via CD133 in HBx-Infected Hepatoma Cells.

Background: Hepatitis B-X Protein (HBx) encoded in Hepatitis B virus (HBV) is known to play a critical role in development and progression of HBV induced hepatocellular carcinoma (HCC). HBx interacts with and activates various cells in HCC microenvironment to promote tumor initiation, progression and invasion. In this study, we investigated how surrounding stromal cells interact with HBx-infected hepatoma cells by a series of in vitro co-culture studies. Methods: Huh7 hepatoma cells were cultured and transfected with the mammalian expression vector pGFP-HBx. Co-culture assays were performed between HBx-transfected Huh7 cells and conditioned media (CM) from stromal cells [endothelial cell lines (HUVECs) and hepatic stellate cell lines (LX2 cells)]. The effect of these interactions was studied by a series of functional assays like chemotaxis, invasion, and wound healing scratch assays. Also, quantitative real time (RT)-PCRs of the mesenchymal genes was performed in the hepatoma cells with and without the co-cultures. Hep3B cells with an integrated HBV genome were taken as positive controls. Results: HBx-transfected Huh7 cells cultured in presence of CM from HUVECs illustrated enhanced migration and tube formation as compared to HBx-transfected cells cultured alone or co-cultured with LX2 cells. HBx-transfected hepatoma cells incubated with CM from HUVECs also expressed mesenchymal genes including Thy1, CDH2, TGFßR1, VIM, and CD133. ELISAs revealed increased levels of TGF-ß in CM from HUVECs. In comparison to unstimulated HBx-transfected Huh7 cells, TGF-ß stimulated cells displayed increased invasive properties and mesenchymal gene expression. RT-PCR and flow cytometry analysis further demonstrated that incubation with either CM from HUVECs or TGF-ß significantly increased the expression of a stemness marker, CD133 in HBx-infected hepatoma cells. Gene inhibition experiments with CD133 siRNA showed a downregulation of mesenchymal gene expression and properties in TGF-ß induced HBx-infected hepatoma cells as compared to that observed in control siRNA treated cells, indicating CD133 as one of the key molecules affecting epithelial to mesenchymal transition (EMT) in HBx-infected cells. Conclusion: The study indicates that secretory factors like TGF-ß from neighboring endothelial cells may enhance expression of CD133 and impart an aggressive EMT phenotype to HBx-infected hepatoma cells in HBV induced HCC.

Lower Rates Of Naturally Occurring Resistance-Associated Substitutions (RASs) In Hepatitis C Virus (HCV)-Infected Chronic Kidney Disease (CKD) Patients Than In HCV-Infected Patients With Only Liver Disease.

PURPOSE: Baseline viral load and existence of resistance-associated substitutions (RASs) are associated with direct-acting antiviral agent (DAA) treatment failure in patients with chronic hepatitis C virus (HCV) infection. PATIENTS AND METHODS: This study was done on HCV-infected patients with different clinical conditions, group 1 included HCV-infected patients with only liver disease (n= 24) and group 2 had HCV-infected patients with coexisting chronic kidney disease (CKD) (n =26). Baseline RAS in the viral genome, before treatment initiation, was examined in both the groups to understand the host disease status on their occurrence. RESULTS: Predominant genotype (gt) differed in both the groups, in group 1 it was gt3 while it was gt1 in group 2. Overall, the occurrence of RASs at baseline was seen in 10 patients (20%); in group 1 it was seen in 8 (33.3%) as compared to only 2 (7.6%) in group 2; p < 0.001. RAS in both NS5a and NS5b regions of the virus was seen in group 1 while in group 2, RASs were seen only in the NS5a region of the virus at 30K position. In group 1, multiple RASs were also seen. The existence of RAS at baseline in both the groups did not affect the attainment of post-treatment cure for the virus in terms of sustained virological response (SVR). CONCLUSION: Host disease status influences the occurrence of baseline RAS in the virus.

Absence of chronic hepatitis E virus infection in liver transplant recipients: Report from a hyperendemic region.

Most cases of chronic hepatitis E virus (HEV) infection in solid organ transplant recipients are attributable to genotype 3. Although India is hyperendemic for HEV genotype 1, chronic infection in transplant patients has not been reported. In this study, 30 liver transplant recipients were followed up by systematic testing for various markers of HEV (IgM, IgG, HEV-Ag, and RNA) on blood and stool samples obtained pre-transplant, and then at 3 and 6 months post-transplant to look for HEV exposure and persistence. Evidence of HEV infection was found in 6 (20%) cases post-transplant but none of the recipients demonstrated active viremia or antigenemia. This suggests that the circulating genotype of HEV in our population might have limited potential to cause chronic infections.

Evaluation of dried blood spot as an alternative sample collection method for hepatitis C virus RNA quantitation and genotyping using a commercial system.

Dried blood spot (DBS) is a minimally invasive sampling method suitable for sample collection, storage and transportation in resource limited areas. Aim of this study was to compare the diagnostic utility of DBS with plasma sample for HCV RNA quantitation and genotyping using commercial systems. Plasma and DBS card spotted samples were collected from 95 HCV seropositive patients. Both types of samples were subjected to HCV RNA by real-time PCR (Abbott m2000rt, USA). Genotyping was performed using Abbott HCV genotype II kit (Abbott diagnostics, USA) in samples with viral load > 3 log10 IU/ml. In both plasma and DBS, 14 (14.7%) samples were negative and 81 (85.3%) were positive for HCV RNA. Median viral load in plasma (3.78; range 0-7.43) log10 IU/ml was comparable to DBS (3.93; range 0-7.24) log10 IU/ml. DBS demonstrated sensitivity and specificity of 97.5 and 85.7% respectively, with positive predictive value (PPV) of 97.5% and negative predictive value (NPV) of 85.7%. DBS showed good correlation (r2 = 0.866) and agreement (93.5%) with plasma. Genotyping in 20 patients showed 100% concordance between DBS and plasma samples. DBS showed good sensitivity and specificity as a sampling method for HCV RNA quantitation and genotyping.

Iron-Overload triggers ADAM-17 mediated inflammation in Severe Alcoholic Hepatitis.

Severe alcoholic hepatitis (SAH) is associated with iron accumulation in hepatocytes/macrophages. This possibly correlates with inflammation and stress but the exact mechanism still remains obscure. To understand the role of iron and the mechanisms of systemic iron-overload, a transcriptomic study of liver and Peripheral Blood -Mononuclear-Cells (PBMCs) was undertaken in SAH patients, with and without hepatic iron-overload. Our results show that iron-overload in hepatocytes/macrophages is due to an increased expression of iron-loading receptors and CD163 signaling cascade. Increase in labile iron pool induces expression of iron-loading, oxidative-stress and inflammatory genes along with expression of CD163 and ADAM17. Increased liver iron correlated with circulatory iron, TNF-α, macrophage activation (sCD163) and peroxide-stress in CD163+macrophages in patients who were iron-overloaded and died. Circulatory TNF-α and sCD163 levels were associated with poor outcome. Temporal iron/Fenton stress induced in healthy monocyte-derived-macrophage (MDM)/Tohoku-Hospital-Pediatrics-1(THP1) cells showed higher expression of iron-regulatory, inflammatory and oxidative-stress genes. These genes could be suppressed by iron-chelation. These results suggest that iron mediates inflammation through ADAM17 induction, resulting in macrophage activation and increased shedding of TNF-α and sCD163. These events could be inhibited with iron chelation or with ADAM17-blockade, postulating a therapeutic strategy for SAH patients with iron overload.

Identification of Indian sub-continent as hotspot for HCV genotype 3a origin by Bayesian evolutionary reconstruction.

BACKGROUND: Recent emphasis in Hepatitis C virus (HCV) evolutionary biology has focused on analysis using Core, E1/E2 and/or NS5b regions, with limited appreciation of full length genome. While HCV genotypes have been described as endemic in the Indian subcontinent, there has been no confirmation at the molecular evolutionary level of these genotypes. We have attempted here to determine the status of Indian HCV genotype 3a sequences in relation to similar genotypes from other parts of the world. METHODS: Cloning, sequencing and molecular characterization was performed on 9 Indian sequences and comparative analyses were performed with 46 sequences from other countries. Evolutionary-rate and molecular-clock hypothesis testing was addressed by Bayesian MCMC. RESULTS: Genetic analysis of full length genome revealed two hypervariable regions (HVR) in E2 region - HVR496 and HVR576, with a variable 5-8 amino-acid insertion sequence and a putative N-glycosylation site. Phylogenetic analysis revealed a divergence resulting in 2 distinct clades: clade-1 represented by HCV 3a subtype and clade-2 represented by other 3 subtype genomes. Clade-2 shows earlier divergence than clade-1. Analysis revealed that genotype 3a genomes from India roots out first (∼99 years ago) in clade1. Bayesian skyline plot analysis revealed an increase in effective number of infections from 1940s to 1990s, followed by a gradual decrease after 2000. CONCLUSIONS: Genotype 3a sequences appear to have originated in India and later dispersed to United Kingdom around mid 1940s, most likely around the time of Indian independence and World War II.