Targeted metagenomics using next generation sequencing in laboratory diagnosis of culture negative endophthalmitis.

To study the feasibility of 16S rRNA metagenomics using next generation sequencing (NGS) along with broad range PCR assay for 762 bp region of 16S rRNA gene with Sanger's sequencing, in microbial diagnosis of culture negative endophthalmitis. Vitreous fluid from 16 culture negative and one culture positive endophthalmitis patients, admitted to a tertiary care hospital were processed for targeted metagenomics. NGS of 7 variable regions of 16S rRNA gene was done using Ion Torrent Personal Genome Machine (PGM). Sequence data were analyzed using Ion Reporter software using QIIME and BLSATN tools and Greengenes and NCBI-Genbank databases. Bacterial genome sequences were detected in 15 culture negative and culture positive vitreous specimens. The sequence reads varied between 25,245-540,916 with read length between 142bp-228bp and coverage depth was 41.0X and 81.2X. Operational taxonomic unit (OTUs) of multiple bacterial genera and species were detected in 13 culture negative vitreous specimens and OTUs of a single bacterial species were detected in 2 culture negative and 1 culture positive specimens; one negative specimen had no bacterial DNA. Maximum numbers of OTUs detected by NGS for a bacterial species from any vitreous specimen was the one which was detected and identified by Sanger's sequencing in broad range PCR. All the bacteria were belonging to clinically relevant species. Broad range PCR with sequencing failed to identify bacteria from 5 of the 16 (31.25%) culture negative vitreous specimens. Metagenomics could detect and identify bacterial pathogens in 15 of the 16 culture negative vitreous specimen's up to species level. With rapidly decreasing cost, metagenomics has a potential to be used widely in endophthalmitis diagnosis, in which culture negativity is usually high.

Impact of physical activity on mitochondrial enzymes, muscle stem cell and anti-oxidant protein Sestrins in Sarcopenic mice.

INTRODUCTION: Sarcopenia is the loss of skeletal muscle mass and function. It is a major health issue in old age due to lack of understanding of the origin and molecular mechanism. Altered dietary pattern, sedentary lifestyle and physical inactivity have shown adverse effect of skeletal muscle function. Sedentary behaviour and low protein intake have been well associated with sarcopenia. Here, we aim to develop Sarcopenia mimicking murine model to observe the physiological and biochemical changes with physical activity intervention. We also intended to find the association of muscle stem cells and stress induced protein Sestrins in the developed sarcopenic model. METHODS: Male C57BL/6 mice were categorized into 4 groups: young-control (Y-Cntrl), aged-matched control (A-Cntrl), Sarcopenic-model (SAR-model) and Sarcopenic intervention group (SAR-INT) with physical exercises. SAR-model group was kept in a retrofitted confined cage for sedentary lifestyle and was fed with protein-restricted diet. Phenotypic assessment for body mass, grip strength and functional endurance was analysed to confirm the sarcopenic state. Mitochondrial enzymatic assessment, muscle stem cell (MuSCs) proliferation potential and protein quantification of Sestrins expression were performed by enzyme histochemistry, flow cytometry and surface plasmon resonance (SPR), respectively. SAR-model group was given 10 weeks physical activity intervention to assess the physiological and biochemical changes. RESULTS: Simultaneous implementation of physical inactivity by sedentary confinement and protein restricted diet led the animals to exhibit the features of sarcopenia. SAR-model group showed a decline of 8.6% (p < 0.0001) in the body weight assessment, 32% decline (p < 0.0001) in the grip strength, 28% increase in time elapsed (p < 0.0001) indicating decline in functional performance. Mitochondrial enzymes (ATPase, NADH-TR and SDH/COX) assessment exhibited low expression in SAR-model group. Ki67 positive muscle stem cell declines around 50% in the model group. SPR quantification of Sestrin 2 showed a decline of 14% which significantly improved to 28% upon physical activity intervention (p = 0.0025) in SAR-INT group. CONCLUSION: It can be summarized that the mouse model generated in the present study mimics the feature of human Sarcopenia. Physical activity intervention may improve the sarcopenic status via modulation of Sestrin 2 which can serve as potential molecule for therapeutic implication.

Evaluation of 16S rRNA broad range PCR assay for microbial detection in serum specimens in sepsis patients.

BACKGROUND: Early and accurate laboratory diagnosis and appropriate management of infection improves the survival rate in sepsis. In this study we evaluated broad range 16S rRNA and 16 S-23 S intergenic spacer region (ISR) PCR assays followed by nucleotide sequencing directly from patients' serum and automated blood culture for laboratory diagnosis in admitted sepsis patients. METHODS: A broad range 16S rRNA PCR and 16 S-23 S ISR PCR assay followed by nucleotide sequencing was used directly from patients' serum in hospital admitted patients in 62 sepsis and 16 suspected blood stream infection (sBSI) patients. Automated blood culture was also used in the same patients. Nucleotide sequences were analyzed against NCBI Genbank database and organisms were identified using CLSI MM18A guidelines. RESULTS: Bacterial culture were positive in 10/62 (16.12%) sepsis and 3/16 (18.75%) suspected BSI patients along with 3 detected fungi (2 in sepsis and 1 in suspected BSI group). PCR assay was positive in 36/62 (58.06%) sepsis and 6/16 (37.5%) suspected BSI patients respectively. All but 2 bacteria (both from culture negative patients) detected by PCR assay could be identified from nucleotide sequencing. Survival in sepsis patients was 77%. PCR assay could detect bacteria in 9/14 (64.28%) of sepsis patients with death. CONCLUSION: Broad range PCR assay was far superior for early diagnosis of infection. The bacteria which could not be detected by culture and were not commonly reported from this centre, were detected by the broad range PCR assays. Detection of these rare bacteria/fungi had significant clinical correlation with patient's underlying clinical conditions, immune status and prognosis. The tests could provide definitive diagnosis of infection in >58% of sepsis patients, which helped in patient management and better survival.

Utility of broad-range 16S rRNA PCR assay versus conventional methods for laboratory diagnosis of bacterial endophthalmitis in a tertiary care hospital.

BACKGROUND: Endophthalmitis, a sight-threatening intraocular infection, can be of postsurgical, post-traumatic or endogenous origin. Laboratory diagnosis-based appropriate therapy can be vision-saving. Conventional culture-based laboratory diagnosis takes time and lacks sensitivity. In this study a broad-range PCR assay was assessed against conventional and automated culture methods in vitreous specimens for accurate microbiological diagnosis. AIMS: To use broad-range PCR assay targeting 16S ribosomal RNA (rRNA) region of bacteria and to assess its performance vis-à-vis conventional and automated culture methods in the laboratory diagnosis of endophthalmitis. METHODS: Vitreous specimens from 195 patients with clinically diagnosed endophthalmitis were processed for classical and automated culture methods, antimicrobial sensitivity and broad-range PCR assay targeting 762 bp region of 16S rRNA followed by nucleotide sequencing by Sanger's method. Causative agents were identified from the nucleotide sequences analysed against the GenBank database, and organisms were identified using the Clinical and Laboratory Standards Institute (CLSI) MM18A guidelines. RESULTS: Bacteria could be detected from 127 (65.13%) of the 195 vitreous specimens by broad-range PCR assay; bacterial isolation was possible from 17 (8.7%) and 60 (30.76%) of these specimens by conventional and automated culture methods, respectively (p<0.0001). PCR assay could detect two uncultured bacterium, and in five cases the bacterial identity could not be determined from NCBI database matching. CONCLUSION: Broad-range PCR assay could provide definitive microbial diagnosis within 24 hours in significantly more patients (p<0.0001). Some rare organisms could be detected, useful in treatment modalities. Automated culture was significantly more sensitive than conventional culture.

Hepatitis E Virus (HEV) egress: Role of BST2 (Tetherin) and interferon induced long non- coding RNA (lncRNA) BISPR.

BACKGROUND: The biology of Hepatitis E Virus (HEV), a common cause of epidemic and sporadic hepatitis, is still being explored. HEV exits liver through bile, a process which is essential for its natural transmission by feco-oral route. Though the process of this polarised HEV egress is not known in detail, HEV pORF3 and hepatocyte actin cytoskeleton have been shown to play a role. METHODS: Our transcriptome analysis in Hepatitis E virus (HEV) replicon transfected Huh7 cells at 24 and 72 hrs indicated that at 24hrs, both LncBISPR and BST2, expressed by a bidirectional promoter were highly upregulated whereas at 72 hrs, BST2 expression was comparatively reduced accompanied by normal levels of BISPR. These findings were confirmed by qPCR analysis. Co-localisation of BST2 and HEV pORF2 was confirmed in HEV transfected Huh7 by confocal microscopy. To investigate the role of BISPR/BST2 in HEV life cycle, particularly virus egress, we generated Huh7 cells with ~8kb deletion in BISPR gene using Crispr-Cas9 system. The deletion was confirmed by PCR screening, Sanger sequencing and Real time PCR. Virus egress in ΔBISPR Huh7 and Huh7 cells was compared by measuring HEV positive strand RNA copy numbers in cell lysates and culture supernatants at 24 and 72 hrs post HEV replicon transfection and further validated by western blot for HEV pORF2 capsid protein. RESULTS: ΔBISPR Huh7 cells showed ~8 fold increase in virus egress at 24 hrs compared to Huh7 cells. No significant difference in virus egress was observed at 72hrs. Immunohistochemistry in histologically normal liver and HEV associated acute liver failure revealed BST2 overexpression in HEV infected hepatocytes and a dominant canalicular BST2 distribution in normal liver in addition to the cytoplasmic localisation reported in literature. CONCLUSIONS: These findings lead us to believe that BISPR and BST2 may regulate egress of HEV virions into bile in vivo. This system may also be used to scale up virus production in vitro.

Demographic profile, host, disease & viral predictive factors of response in patients with chronic hepatitis C virus infection at a tertiary care hospital in north India.

BACKGROUND & OBJECTIVES: Standard of care for chronic hepatitis C (CHC) in India is peginterferon and ribavirin (RBV). The response to treatment in real life stetting is unclear. The objectives of this study were to evaluate the demographic profile and assess the virological response and predictors of response in CHC patients. METHODS: Consecutive patients with CHC were included in this study. Detailed clinical history, risk factors, and predictive factors of response were noted. Patients were treated with peginterferon α2b (1.5 µg/kg/wk) and RBV (12 mg/kg/day) for 6 to 18 months based on response. RESULTS: A total of 211 patients were included in the analysis, mean age 40.6±12.3 yr, 144 (68%) were males and 71 (34%) had compensated cirrhosis. Commonest risk factor for acquiring CHC was previous transfusion and surgery (51%). Genotype 3 (72%) was most common followed by genotype 1 (23%). Overall sustained virologic response (SVR) was 64 per cent [95% CI 57.1%-70.4%]. The SVR was 66.5 per cent [95% CI 58.34-73.89%] for genotype 3 and 61.2 per cent [95% CI 46.23 to 74.80%] for genotype 1. Non-cirrhotics had better SVR rates compared to cirrhotics (76 vs 41%, p<0.001). On multivariate analysis, BMI ≥23 kg/m2, HOMA-IR ≥2, compliance (≤80%), and fibrosis >2 were predictors of low SVR. INTERPRETATION & CONCLUSIONS: Genotype 3 was the commonest HCV genotype. The commonest source of infection was previous transfusion and surgery. SVR rates for genotypes 3 were better than genotype 1 patients. Predictors of non-response were high BMI, insulin resistance, significant fibrosis and inadequate compliance.

Acute on chronic liver failure because of acute hepatic insults: Etiologies, course, extrahepatic organ failure and predictors of mortality.

BACKGROUND AND AIM: Acute on chronic liver failure (ACLF) because of precipitating factors (variceal bleed/infections) identifies cirrhotics at risk for high short-term mortality. Information on ACLF because of acute hepatic insults is lacking. The aim of the study was to evaluate acute hepatic insults in ACLF and their effect on the course and outcome. METHODS: In a prospective study, 213 consecutive patients of ACLF because of acute hepatic insults were included. Etiology of acute hepatic insult, frequency of silent, and overt chronic liver disease (CLD), organ failure (OF), and outcomes were assessed. Prognostic models such as model for endstage liver disease (MELD), acute physiology and chronic health evaluation (APACHE II), and chronic liver failure-sequential organ failure (CLIF-SOFA) were evaluated. RESULTS: Etiologies of acute hepatic insult were hepatitis virus(es)- 81 (38%; HBV-42, HEV-39), continuous alcohol consumption-77 (33.3%), antituberculosis drugs-11 (5.2%), autoimmune hepatitis flare-5(2.3%), cryptogenic-44 (20.7%). The common causes of CLD were alcohol (n = 85/40%), HBV(n = 52/24%), and cryptogenic(n = 50/20%). The MELD, APACHE II, and CLIF-SOFA scores were similar among silent and overt CLD and did not influence outcome. Predominant etiologies of ACLF were hepatitis virus(es) reactivation or superinfection in silent CLD(52/112, 46.4%) and alcohol among overt CLD(43/101, 43%). Independent predictors of mortality included hepatic-encephalopathy (early, HR: 4.01; advanced, HR: 6.10), serum creatinine ≥1.5 mg/dl (HR: 4.53), CLIF-SOFA ≥8(HR: 1.69), and etiology of acute hepatic insult (alcohol, HR: 4.08; cryptogenic, HR: 3.18). HEV-ACLF had lower mortality (12.8% vs. 33-54% in other etiologies;P < 0.001). OF was major determinant of mortality. With increasing number of OF, mortality increased linearly(P = 0.001). CONCLUSIONS: Hepatitis virus(es) and continuous alcohol consumption are important causes of ACLF caused by acute hepatic insults. HEV-ACLF has lower mortality. OF is an important prognostic predictor.

Recombinant Hepatitis E virus like particles can function as RNA nanocarriers.

BACKGROUND: Assembled virus-like particles (VLPs) without genetic material, with structure similar to infectious virions, have been successfully used as vaccines. We earlier described in vitro assembly, characterisation and tissue specific receptor dependent Clathrin mediated entry of empty HEV VLPs, produced from Escherichia coli expressed HEV capsid protein (pORF2). Similar VLP's have been described as a potential candidate vaccine (Hecolin) against HEV. FINDINGS: We have attempted to use such recombinant assembled Hepatitis E virus (HEV) VLPs as a carrier for heterologous RNA with protein coding sequence fused in-frame with HEV 5' region (containing cap and encapsidation signal) and investigated, if the relevant protein could be expressed and elicit an immune response in vivo. In vitro transcribed red fluorescent protein (RFP)/Hepatitis B virus surface antigen (HBsAg) RNA, fused to 5'-HEV sequence with cap and encapsidation signal (1-249 nt), was packaged into the recombinant HEV-VLPs and incubated with five different cell lines (Huh7, A549, Vero, HeLa and SiHa). The pORF2-VLPs could specifically transfer exogenous coding RNA into Huh7 and A549 cells. In vivo, Balb/c mice were immunized (intramuscular injections) with 100 µg pORF2-VLP encapsidated with 5'-methyl-G-HEV (1-249 nt)-HBsAg RNA, blood samples were collected and screened by ELISA for anti-pORF2 and anti-HBsAg antibodies. Humoral immune response could be elicited in Balb/c mice against both HEV capsid protein and cargo RNA encoded HBsAg protein. CONCLUSIONS: These findings suggest that other than being a possible vaccine, HEV pORF2-VLPs can be used as a promising non-replicative tissue specific gene delivery system.

Hepatitis E virus (HEV) protease: a chymotrypsin-like enzyme that processes both non-structural (pORF1) and capsid (pORF2) protein.

Hepatitis E virus (HEV), a major cause of acute viral hepatitis across the world, is a non-enveloped, plus-strand RNA virus. Its genome codes three proteins, pORF1 (multifunctional polyprotein), pORF2 (capsid protein) and pORF3 (multi-regulatory protein). pORF1 encodes methyltransferase, putative papain-like cysteine protease, helicase and replicase enzymes. Of these, the protease domain has not been characterized. On the basis of sequence analysis, we cloned and expressed a protein covering aa 440-610 of pORF1, expression of which led to cell death in Escherichia coli BL-21 and Huh7 hepatoma cells. Finally, we expressed and purified this protein from E. coli C43 cells (resistant to toxic proteins). The refolded form of this protein showed protease activity in gelatin zymography. Digestion assays showed cleavage of both pORF1 and pORF2 as observed previously. MS revealed digestion of capsid protein at both the N and C termini. N-terminal sequencing of the ~35 kDa methyltransferase, ~35 kDa replicase and ~56 kDa pORF2 proteins released by protease digestion revealed that the cleavage sites were alanine15/isoleucine16, alanine1364/valine1365 in pORF1 and leucine197/valine198 in pORF2. Specificity of these cleavage sites was validated by site-directed mutagenesis. Further characterization of the HEV protease, carried out using twelve inhibitors, showed chymostatin and PMSF to be the most efficient inhibitors, indicating this protein as a chymotrypsin-like protease. The specificity was further confirmed by cleavage of the chymotrypsin-specific fluorogenic peptide N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Mutational analysis of the conserved serine/cysteine/histidine residues suggested that H443 and C472/C481/C483 are possibly the active site residues. To our knowledge, this is the first direct demonstration of HEV protease and its function.

RNA-seq based transcriptome analysis of hepatitis E virus (HEV) and hepatitis B virus (HBV) replicon transfected Huh-7 cells.

Pathogenesis of hepatitis B virus (HBV) and hepatitis E virus (HEV) infection is as varied as they appear similar; while HBV causes an acute and/or chronic liver disease and hepatocellular carcinoma, HEV mostly causes an acute self-limiting disease. In both infections, host responses are crucial in disease establishment and/or virus clearance. In the wake of worsening prognosis described during HEV super-infection over chronic HBV hepatitis, we investigated the host responses by studying alterations in gene expression in liver cells (Huh-7 cell line) by transfection with HEV replicon only (HEV-only), HBV replicon only (HBV-only) and both HBV and HEV replicons (HBV+HEV). Virus replication was validated by strand-specific real-time RT-PCR for HEV and HBsAg ELISA of the culture supernatants for HBV. Indirect immunofluorescence for the respective viral proteins confirmed infection. Transcription profiling was carried out by RNA Sequencing (RNA-Seq) analysis of the poly-A enriched RNA from the transfected cells. Averages of 600 million bases within 5.6 million reads were sequenced in each sample and ∼15,800 genes were mapped with at least one or more reads. A total of 461 genes in HBV+HEV, 408 in HBV-only and 306 in HEV-only groups were differentially expressed as compared to mock transfection control by two folds (p<0.05) or more. Majority of the significant genes with altered expression clustered into immune-associated, signal transduction, and metabolic process categories. Differential gene expression of functionally important genes in these categories was also validated by real-time RT-PCR based relative gene-expression analysis. To our knowledge, this is the first report of in vitro replicon transfected RNA-Seq based transcriptome analysis to understand the host responses against HEV and HBV.

Role of hepatitis B virus genotype D & its mutants in occult hepatitis B infection.

BACKGROUND & OBJECTIVES: Non-detection of hepatitis B virus (HBV) envelope protein (hepatitis B surface antigen, HBsAg) in a chronically HBV infected individual has been described as occult infection. One possible reason for this phenotype is alteration in large (L-HBsAg) to small (S-HBsAg) envelope protein ratio associated with reduced or non secretion of HBsAg. This results in quantitative levels of serum HBsAg below the detection limit of enzyme immunoassays. Genotype D of HBV has a characteristic 33 nucleotide (nt) deletion upstream of the pre-S2/S promoter. This deletion may reduce HBsAg secretion in occult infection patients infected with genotype D HBV. Additional deletions in the pre-S2/S promoter may further aggravate reduced HBsAg secretion in patients infected with genotype D HBV. Thus, the aim of the present study was to determine the role of genotype D specific 33nt deletion and additional pre-S2/S promoter deletions in causing reduced or no secretion of HBsAg, in occult infection. Since these deletions overlap virus polymerase, their effect on virus replication was also investigated. METHODS: We examined the in vitro expression of HBsAg, ratio of cure and 'e' antigen (HBcAg/HBeAg), their secretion and virus replication, using overlength 1.3 mer/1.86 mer genotype A replicons, and genotype D replicons with and without additional pre-S2/S promoter deletions from cases of occult infection. RESULTS: Genotype D replicon showed a decrease in HBsAg secretion compared to the wild-type genotype A. Genotype D replicons carrying additional pre-S2/S promoter deletions, showed further reduction in HBsAg secretion, demonstrated presence of intracellular HBcAg/HBeAg, virus replication intermediates and 'e' antigen secretion. INTERPRETATION & CONCLUSIONS: The characteristic 33 nt deletion of genotype D HBV reduces HBsAg secretion. Additional pre-S2/S promoter deletions may further diminish HBsAg secretion, leading to occult infection. Pre-S2/S promoter deletions do not affect HBV replication.

Persistent hyperammonemia is associated with complications and poor outcomes in patients with acute liver failure.

BACKGROUND & AIMS: Patients admitted to the hospital with acute liver failure (ALF) and high arterial levels of ammonia are more likely to have complications and poor outcomes than patients with lower levels of ammonia. ALF is a dynamic process; ammonia levels can change over time. We investigated whether early changes (first 3 days after admission) in arterial levels of ammonia were associated with complications and outcomes and identified factors associated with persistent hyperammonemia. METHODS: We performed a prospective observational study that measured arterial ammonia levels each day for 5 days in 295 consecutive patients with ALF. We analyzed associations of changes in ammonia levels during the first 3 days with complications and outcomes. RESULTS: Patients with persistent arterial hyperammonemia (≥122 µmol/L for 3 consecutive days), compared with those with decreasing levels, had lower rates of survival (23% vs 72%; P < .001) and higher percentages of cerebral edema (71% vs 37%; P < .001), infection (67% vs 28%; P = .003), and seizures (41% vs 7.7%; P < .001). Patients with persistent hyperammonemia had greater mortality, with an odds ratio (OR) of 10.7, compared with patients with baseline levels of ammonia ≥122 µmol/L (OR, 2.4). Patients with persistent hyperammonemia were more likely to progress to and maintain advanced hepatic encephalopathy than those with decreasing levels. Patients with persistent, mild hyperammonemia (≥85 µmol/L for 3 days) were also more likely to have complications or die (P < .001) than patients with serial ammonia levels <85 µmol/L. Infections (OR, 4.17), renal failure (OR, 2.20), and decreased arterial pH (OR, 0.003) were independent predictors of persistent hyperammonemia. CONCLUSIONS: Patients with ALF and persistent arterial hyperammonemia for 3 days after admission are more likely to develop complications and have greater mortality than patients with decreasing levels or high baseline levels. Infection, renal failure, and decreased arterial pH are independent predictors of persistent hyperammonemia.

Prospective derivation and validation of early dynamic model for predicting outcome in patients with acute liver failure.

OBJECTIVE: It is difficult to predict the outcome in patients with acute liver failure (ALF) using existing prognostic models. This study investigated whether early changes in the levels of dynamic variables can predict outcome better than models based on static baseline variables. DESIGN: 380 patients with ALF (derivation cohort n=244, validation cohort n=136) participated in a prospective observational study. The derivation cohort was used to identify predictors of mortality. The ALF early dynamic (ALFED) model was constructed based on whether the levels of predictive variables remained persistently high or increased over 3 days above the discriminatory cut-off values identified in this study. The model had four variables: arterial ammonia, serum bilirubin, international normalised ratio and hepatic encephalopathy >grade II. The model was validated in a cohort of 136 patients with ALF. RESULTS: The ALFED model demonstrated excellent discrimination with an area under the receiver operator characteristic curve of 0.91 in the derivation cohort and of 0.92 in the validation cohort. The model was well calibrated in both cohorts and showed a similar increase in mortality with increasing risk scores from 0 to 6. The performance of the ALFED model was superior to the King's College Hospital criteria and the Model for End stage Liver Disease score, even when their 3-day serial values were taken into consideration. An ALFED score of ≥4 had a high positive predictive value (85%) and negative predictive value (87%) in the validation cohort. CONCLUSION: The ALFED model accurately predicted outcome in patients with ALF, which may be useful in clinical decision-making.

Hepatitis E virus replication involves alternating negative- and positive-sense RNA synthesis.

Hepatitis E virus (HEV) is the major cause of epidemic hepatitis and many outbreaks of sporadic hepatitis. The virus responsible has a single-stranded, positive-sense RNA. Its replication and the regulatory process involved therein are poorly understood. Much of the HEV biology studied has been done by using full-length capped RNA transcripts (replicons) and transient transfections in cell cultures. We investigated replicon replication using negative-sense strand-specific molecular beacons in live cell imaging, and quantifying intracellular viral RNA using strand-specific real-time PCR every 2 h until 24 h post-transfection. A graph of the copy numbers of both positive- and negative-sense RNA at the different time points was plotted. This showed a temporal separation and alternating cycles of negative- and positive-sense RNA formation. As a control, a dysfunctional replicase mutant (GDD→GAA) was used, which showed no increase in copy number. The live cell imaging corroborated the quantitative data, in that the maximal amount of negative-sense RNA was observed at 8 h post-transfection. The real-time-PCR copy-number analysis of the subgenome showed the presence of a single subgenomic RNA. Using fluorescent protein genes mCherry and EGFP fused in-frame to ORF2 and ORF3 in separate constructs and immunofluorescence, we showed the formation of both proteins pORF2 and pORF3 from a single subgenomic RNA. Our study demonstrated cyclical bursts of virus replication and the role of subgenomic RNA in the HEV life cycle.

Inhibition of Hepatitis E virus replication using short hairpin RNA (shRNA).

Hepatitis E virus (HEV) is a non-enveloped, single-stranded, positive sense RNA virus, which is a major cause of water-borne hepatitis. RNA interference (RNAi) is a sequence-specific cellular antiviral defence mechanism, induced by double-stranded RNA, which we used to investigate knockdown of several genes and the 3' cis-acting element (CAE) of HEV. In the present report, shRNAs were developed against the putative helicase and replicase domains and the 3'CAE region of HEV. Production of siRNA was confirmed by northern hybridization. The possible innate response induction due to shRNA expressions was verified by transcript analysis for interferon-beta and 2',5'-oligoadenylate synthetase genes and was found to be absent. Initially, the selected shRNAs were tested for their efficiency against the respective genes/3'CAE using inhibition of fused viral subgenomic target domain-renilla luciferase reporter constructs. The effective shRNAs were studied for their inhibitory effects on HEV replication in HepG2 cells using HEV replicon and reporter replicon. RNAi mediated silencing was demonstrated by reduction of luciferase activity in subgenomic target-reporter constructs and reporter replicon. The real time PCR was used to demonstrate inhibition of native replicon replication in transfected cells. Designed shRNAs were found to be effective in inhibiting virus replication to a variable extent (45-93%).

Development of highly sensitive Bicistronic vector based non-radioactive antigen-specific cytotoxicity assay.

In the absence of a better alternate, (51)Cr release assay, with its several disadvantages is still the most common method for detection of MHC class I restricted T-cell mediated cytotoxicity. We describe a system in which the T-cell mediated cytotoxicity can be assessed using host-derived cells transfected with a bicistronic vector expressing the specific antigen and a quantifiable reporter as target cells. This overcomes the problems associated with use of radioactivity, pre-loading of target cells with reporter/antigen and the MHC restriction. We used HBV core antigen to prove the concept, as it is an established CTL target. Bicistronic vectors containing HBV core and reporter (EGFP/Fluc) gene were generated and further checked for antigen/reporter expression in human HepG2, mouse fibroblast BALB/c.3T3 and mouse lymphoma A20 cell lines. The effector cells to study the cytolytic activity were generated in vivo using BALB/c mice immunized with antigen expressing DNA clone or protein. The target cells (BALB/c.3T3 and A20) transiently transfected with bicistronic constructs were incubated with effector cells (splenocytes) from immunized mice at a different effector to target ratio. Following incubation the CTL activity was calculated by measuring the reporter luciferase in the remaining viable target cells that inversely correlates with the cytolysis of susceptible cells. The percent specific lysis measured in our assay was compared with conventional (51)Cr release assay to validate this approach. This novel bicistronic vector based cytotoxicity assay demonstrated an easy to perform, antigen-specific and non-radioactive method of determining T-cell mediated cytotoxicity.

Efficacy of L-ornithine L-aspartate in acute liver failure: a double-blind, randomized, placebo-controlled study.

BACKGROUND & AIMS: In acute liver failure (ALF), high blood ammonia levels have been documented that correlate with mortality and complications. L-ornithine L-aspartate (LOLA) reduces ammonia levels by increasing hepatic ammonia disposal and its peripheral metabolism. Present study evaluated efficacy and ammonia lowering effect of LOLA in ALF. METHODS: This study was placebo-controlled and blinded. We randomized 201 patients with ALF between January 2005 and October 2007 to either placebo or LOLA infusions (30 g daily) for 3 days. Arterial ammonia was measured at baseline and daily for 6 days. The primary end point was improvement in survival. The study followed CONSORT guidelines and was registered at the ClinicalTrials.gov (Identifier: NCT00470314). RESULTS: There was no reduction in mortality with LOLA treatment (mortality: 33.3% in placebo and 42.4% in LOLA; relative risk of death 1.27; 95% CI: 0.88-1.85; P = .204). By multivariate analysis, ammonia levels were an independent predictor of survival. There was significant decrease in ammonia levels in both groups with time (P < .001), but the levels of ammonia between the randomized groups at any time point, either during the 72 hours of LOLA infusion or during the follow-up were similar (P = .492). There was no difference between the 2 groups in the improvement in encephalopathy grade (P = .418), consciousness recovery time (P = .347), survival time (P = .612), or complications like seizures (P = .058) and renal failure (P = .615). The fetal outcome was also similar (P = .172). No adverse drug effect was noted. CONCLUSIONS: LOLA infusion did not lower the ammonia or improved survival in ALF.

A 20-year single-center experience with acute liver failure during pregnancy: is the prognosis really worse?

UNLABELLED: Pregnant patients with acute liver failure (ALF) are believed to have a worse outcome than nonpregnant women and men with ALF. However objective data supporting this supposition are scant. Therefore, the current study compared the outcome, complications, and causes of ALF among pregnant women and girls with age-matched nonpregnant women and girls and men and boys with ALF. One thousand fifteen consecutive ALF patients in the reproductive age group, admitted at the All India Institute of Medical Sciences, New Delhi, from January 1986 to December 2006, were included in the study. A total of 249 (38.5%) women were pregnant. They were compared with 341 nonpregnant women and girls and 425 men and boys, aged 15 to 45 years. The mortality rate of pregnant women and girls (53.8%) was similar to age-matched nonpregnant women and girls (57.2%), and men and boys (57.9%); P = 0.572. The clinical and biochemical features, disease severity, and complications were also similar in the three groups. A significantly higher proportion of ALF was attributable to hepatitis E virus (HEV) among women and girls who were pregnant (59.4%), as compared with both nonpregnant women and girls (30.4%), and men and boys (23.1%); P < 0.001. However, the outcome of HEV-related ALF was independent of the sex and pregnancy status of the patients (P = 0.103). Mortality in HEV-ALF and non-HEV-ALF patients in pregnant women and girls was 51% (74/145) and 54.7% (52/95)(P > 0.1), respectively. The outcome of pregnant ALF patients was also unrelated to the trimester of pregnancy. The mortality of non-HEV-related ALF among the pregnant women and girls (54.7%), age-matched nonpregnant women and girls (61.7%), and men and boys (62.8%) were also similar (P > 0.1). CONCLUSION: The mortality of pregnant patients with ALF is similar to that of nonpregnant women and girls and men and boys and is independent of the cause or trimester. Pregnancy per se should not be regarded as a poor prognostic factor for a patient with ALF.

Subcellular localization of hepatitis E virus (HEV) replicase.

Hepatitis E virus (HEV) is a hepatotropic virus with a single sense-strand RNA genome of approximately 7.2 kb in length. Details of the intracellular site of HEV replication can pave further understanding of HEV biology. In-frame fusion construct of functionally active replicase-enhanced green fluorescent protein (EGFP) gene was made in eukaryotic expression vector. The functionality of replicase-EGFP fusion protein was established by its ability to synthesize negative-strand viral RNA in vivo, by strand-specific anchored RT-PCR and molecular beacon binding. Subcellular co-localization was carried out using organelle specific fluorophores and by immuno-electron microscopy. Fluorescence Resonance Energy Transfer (FRET) demonstrated the interaction of this protein with the 3' end of HEV genome. The results show localization of replicase on the endoplasmic reticulum membranes. The protein regions responsible for membrane localization was predicted and identified by use of deletion mutants. Endoplasmic reticulum was identified as the site of replicase localization and possible site of replication.