Kavita S. Lole

Evaluation of human (genotype 1) and swine (genotype 4)-ORF2-based ELISAs for anti-HEV IgM and IgG detection in an endemic country and search for type 4 human HEV infections

Summary.  Open reading frame 2 proteins (ORF2) from swine (genotype 4, S‐ORF2) and human (genotype 1, H‐ORF2) hepatitis E virus (HEV) having 91.4% identity at amino acid level were expressed using baculovirus expression system. Comparison of ELISAs based on the two proteins yielded identical results when sequential serum samples from monkeys and pigs experimentally infected with genotypes 1 and 4 HEV, respectively, were tested. Samples from patients (n = 258) suffering from non‐A, non‐B hepatitis during outbreaks of the disease and 180 sera from apparently healthy children were screened by H‐ORF2‐, S‐ORF2‐based ELISAs and Genelabs ELISA, a widely used commercial test for HEV diagnosis. Specificity of all three tests in detecting IgM and IgG antibodies in healthy children was comparable. Excellent correlation was noted in detecting both IgM (98.7% concordance) and IgG (97.7% concordance) anti‐HEV antibodies when H‐ORF2 and S‐ORF2 ELISAs were compared. When compared with Genelabs ELISA, both H‐ORF2 and S‐ORF2 ELISAs identified 34 and 18 additional positives, respectively, in IgM and IgG anti‐HEV tests showing comparatively less sensitivity of the commercial assay. The concordance of Genelabs ELISA in IgM detection was 86.4% and 85.6%, respectively, with H‐ORF2 and S‐ORF2 ELISAs. The concordance between Genelabs ELISA and H‐ORF2 decreased further to 73.6% when 129 human samples from recent HEV epidemics (2002–2004) were tested for IgM. Similar results were obtained when sequential samples from 11 hepatitis E patients were examined. Screening of serum samples from 137 sporadic non‐A, non‐B hepatitis cases further confirmed the superiority of the H‐ORF2 and S‐ORF2 ELISAs. All 36/137 HEV‐RNA‐positive samples from sporadic cases belonged to genotype 1 confirming absence/rarity of type 4 human infections. H‐ORF2 and S‐ORF2 antigens were swappable in ELISAs for detecting both genotypes 1 and 4 HEV infections.

Comparison of Hepatitis C Virus Genotyping by 5′ Noncoding Region- and Core-Based Reverse Transcriptase PCR Assay with Sequencing and Use of the Assay for Determining Subtype Distribution in India

ABSTRACT Phylogenetic analysis of the sequences of the 5′ noncoding regions (5′NCR) of 149 samples from hepatitis C virus (HCV) RNA-positive chronic carriers representing northern, southern, eastern, and western India showed that type 3 and type 1 are the predominant genotypes circulating in India, with an overall prevalence of 53.69 and 38.25%, respectively. Type 4 viruses (6.04%) were seen only in southern India. Sequence analysis of the core region of 51 of the above isolates enabled us to classify them further into subtypes as 1b (number of isolates [n] = 10), 1a (n = 6), 3a (n = 9), 3g (n = 14), 3f (n = 1), and 4d (n = 3). Three new subtypes were identified for the first time and designated as 3i (n = 5), 3j (n = 2), and 6l (n = 1). Sequencing the 5′NCR could differentiate HCV types, whereas classification at the level of subtype was possible with sequence analysis of the core region.

Role of host immune response and viral load in the differential outcome of pandemic H1N1 (2009) influenza virus infection in Indian patients.

Background An unusually high number of severe pneumonia cases with considerable mortality is being observed with the pandemic H1N1 2009 virus infections globally. In India, all mild as well as critically ill cases were admitted and treated in the government hospitals during the initial phase of the pandemic. The present study was undertaken during this early phase of the pandemic. Methodology The role of viral load and host factors in the pathogenesis were assessed by examining 26 mild (MP), 15 critically ill patients (CIP) and 20 healthy controls from Pune, India. Sequential blood and lung aspirate samples were collected from CIP. Viral load and cytokines/chemokine levels were determined from the plasma and lung aspirates of the patients. TLR levels were determined by staining and FACS analysis. Gene profiling was done for both cells in the lung aspirates and PBMCs using TaqMan Low Density arrays. Antibody titres and isotyping was done using HA protein based ELISAs. Principal Findings 13/15 critically ill patients expired. All plasma samples were negative for the virus irrespective of the patients category. Sequential lung samples from CIP showed lower viral loads questioning association of viral replication with the severity. Anti-rpH1N1-09-HA-IgG titres were significantly higher in critically ill patients and both categories circulated exclusively IgG1 isotype. Critically ill patients exhibited increase in TLR-3, 4, 7 and decrease in TLR-2 expressions. The disease severity correlated with increased plasma levels of IL1RA, IL2, IL6, CCL3, CCL4 and IL10. Majority of the immune-function genes were down-regulated in the PBMCs and up-regulated in the cells from lung aspirates of critically ill patients. No distinct pattern differentiating fatal and surviving patients was observed when sequential samples were examined for various parameters. Conclusions Disease severity was associated with pronounced impairment of host immune response.

Deubiquitination activity associated with hepatitis E virus putative papain-like cysteine protease.

Hepatitis E virus (HEV) ORF1 protein (pORF1) contains methyltransferase (MetT), papain-like cysteine protease (PCP), RNA helicase (Hel) and RNA-dependent RNA polymerase (RdRp) domains. ORF1 sequence analysis showed two consensus LXGG cleavage sites at 664 and 1205. LXGG sequence is recognized by viral and cellular deubiquitinating enzymes. The protein encompassing the predicted MetT-PCP domains of HEV ORF1 was tested for deubiquitinating activity using fluorogenic substrates - ubiquitin-7-amino-4-methylcoumarin (AMC), IFN-stimulated gene 15 (ISG15)-AMC, Nedd8-AMC and SUMO-AMC. MetT-PCP cleaved all four substrates but processing of ISG15-AMC was more robust. There was no processing of the Hel and RdRp domains having the conserved (1205) LXGG site by the protein. MetT-PCP carried out deISGylation of the ISG15-conjugated cellular proteins, suggesting a possible role in combating cellular antiviral pathways.

NTPase and 5′-RNA Triphosphatase Activities of Chikungunya Virus nsP2 Protein

Chikungunya virus (CHIKV) is an insect borne virus (genus: Alphavirus) which causes acute febrile illness in humans followed by a prolonged arthralgic disease that affects the joints of the extremities. Re-emergence of the virus in the form of outbreaks in last 6–7 years has posed a serious public health problem. CHIKV has a positive sense single stranded RNA genome of about 12,000 nt. Open reading frame 1 of the viral genome encodes a polyprotein precursor, nsP1234, which is processed further into different non structural proteins (nsP1, nsP2, nsP3 and nsP4). Sequence based analyses have shown helicase domain at the N-terminus and protease domain at C-terminus of nsP2. A detailed biochemical analysis of NTPase/RNA helicase and 5′-RNA phosphatase activities of recombinant CHIKV-nsP2T protein (containing conserved NTPase/helicase motifs in the N-terminus and partial papain like protease domain at the C-terminus) was carried out. The protein could hydrolyze all NTPs except dTTP and showed better efficiency for ATP, dATP, GTP and dGTP hydrolysis. ATP was the most preferred substrate by the enzyme. CHIKV-nsP2T also showed 5′-triphosphatase (RTPase) activity that specifically removes the γ-phosphate from the 5′ end of RNA. Both NTPase and RTPase activities of the protein were completely dependent on Mg2+ ions. RTPase activity was inhibited by ATP showing sharing of the binding motif by NTP and RNA. Both enzymatic activities were drastically reduced by mutations in the NTP binding motif (GKT) and co-factor, Mg2+ ion binding motif (DEXX) suggesting that they have a common catalytic site.

Analysis of Antiviral Response in Human Epithelial Cells Infected with Hepatitis E Virus

Hepatitis E virus (HEV) is a major cause of enterically transmitted acute hepatitis in developing nations and occurs in sporadic and epidemic forms. The disease may become severe with high mortality (20%) among pregnant women. Due to lack of efficient cell culture system and small animal model, early molecular events of HEV infection are not yet known. In the present study, human lung epithelial cells, A549, were infected with HEV to monitor expression levels of genes/proteins in antiviral pathways. Both live and UV inactivated virus elicited robust induction of inflammatory cytokines/chemokines such as IL-6, IL-8, TNF-α, and RANTES within 12 h of infection. Cells exposed to soluble capsid protein showed no induction suggesting the capsid structure and not the protein being detected as the pathogen pattern by cells. A delayed up-regulation of type I interferon genes only by the live virus at 48 h post HEV infection indicated the need of virus replication. However, absence of secreted interferons till 96 h suggested possible involvement of post-transcriptional regulation of type I IFN expression. HEV infected cells showed activation of both NF-κB and IRF3 transcription factors when seen at protein levels; however, reporter gene assays showed predominant expression via NF-κB promoter as compared to IRF3 promoter. Knockdown experiments done using siRNAs showed involvement of MyD88 and TRIF adaptors in generating antiviral response thus indicating role of TLR2, TLR4 and TLR3 in sensing viral molecules. MAVS knockdown surprisingly enhanced only proinflammatory cytokines and not type I IFNs. This suggested that HEV not only down-regulates RIG-I helicase like receptor mediated IFN induction but also employs MAVS in curtailing host inflammatory response. Our findings uncover an early cellular response in HEV infection and associated molecular mechanisms suggesting the potential role of inflammatory response triggered by HEV infection in host immune response and pathogenesis.

Challenge studies in Rhesus monkeys immunized with candidate hepatitis E vaccines : DNA, DNA-prime-protein-boost and DNA-protein encapsulated in liposomes

Complete ORF2 gene (1983bp) of hepatitis E virus (HEV) and the 450bp region within ORF2 containing neutralizing epitope (NE) cloned in pVAX1 and corresponding proteins expressed in baculovirus and prokaryotic systems respectively were evaluated as vaccine candidates. Two doses of liposome encapsulated DNA plus corresponding protein with both ORF2 and NE regions (Lipo-ORF2-DP and Lipo-NE-DP) showed 100% seroconversion and comparable anti-HEV titres in Swiss albino mice. These vaccine candidates were further evaluated as DNA, DNA-prime-protein-boost (DPPB) and liposome formulations in Rhesus monkeys. Monkeys receiving ORF2/NE DNA seroconverted after fourth dose while those immunized employing ORF2-DPPB format seroconverted at 7 weeks post third dose. In view of the delayed weak antibody response, these monkeys were not challenged. Though Lipo-ORF2-DP was immunogenic, 2 of the 4 monkeys developed HEV infection following homologous virus challenge of 100 Monkey Infectious Dose(50). Both monkeys immunized with Lipo-NE-DP and 1 of the 2 monkeys immunized with NE-DPPB showed complete protection, the second monkey being protected from hepatitis with limited viral replication. Irrespective of the type of immunogen, all challenged monkeys were protected from hepatitis. The results document Lipo-NE-DP to be a promising vaccine candidate needing further evaluation.

Development of candidate combination vaccine for hepatitis E and hepatitis B: A liposome encapsulation approach

To reduce extra injections, cost and ensure better coverage, use of combination vaccines is preferable. An attempt was made to evaluate the encapsulation of hepatitis E virus neutralizing epitope (NE) region and hepatitis B virus surface antigen (HBsAg) in liposomes as DNAs, proteins and DNA+protein. Mice groups were immunized with different liposome-encapsulated formulations and monitored for anti-HEV and anti-HBs titres, IgG subtypes, antigen-specific lymphocyte proliferation and cytokine levels. The protective levels of anti-HBs and in vitro virus-binding capacity of anti-HEV antibodies were assessed. Liposome-encapsulated DNA either singly or in combination did not elicit antibody response. Anti-HEV and anti-HBs IgG titres of individual component of protein alone (Lipo-E-P/Lipo-B-P) or DNA+protein formulations (Lipo-E-DP/Lipo-B-DP) were comparable to respective titres in combination vaccine of protein (Lipo-BE-P) and DNA+protein formulations (Lipo-BE-DP). IgG1 levels were significantly higher in Lipo-BE-P group whereas, equivalent levels of IgG1 and IgG2a were observed in Lipo-BE-DP group against both components of the vaccine. Combination vaccine group showed mixed Th1/Th2 cytokine profile. Liposome entrapped NE and HBsAg in protein and DNA+protein formats induce excellent immune response to both the components and need to be evaluated in higher animals.

An outbreak of hepatitis B with high mortality in India: association with precore, basal core promoter mutants and improperly sterilized syringes

Summary.  In 2009, an outbreak of hepatitis B with high mortality was observed in Sabarkantha district, Gujarat state, India with 456 cases and 89 deaths. Hospitalized patients with self‐limiting disease (152, AVH)) and fulminant hepatic failure (39, FHF including 27 fatal and 12 survivals) were investigated. These were screened for diagnostic markers for hepatitis viruses, hepatitis B virus (HBV) genotyping and mutant analysis. Complete HBV genomes from 22 FHF and 17 AVH cases were sequenced. Serosurveys were carried out in the most and least affected blocks for the prevalence of HBV and identification of mutants. History of injection from a physician was associated with FHF and AVH cases. Co‐infection with other hepatitis viruses or higher HBV DNA load was not responsible for mortality. Four blocks contributed to 85.7% (391/456) of the cases and 95.5% (85/89) mortality while two adjacent blocks had negligible mortality. Sequence analysis showed the presence of pre‐core and basal core promoter mutants and 4 amino acid substitutions exclusively among FHF cases. None of the self‐limiting patients exhibited these dual mutations. Genotype D was predominant, D1 being present in all FHF cases while D2 was most prevalent in AVH cases. Probably due to violation of accepted infection control procedures by the qualified medical practitioners, HBV prevalence was higher in the affected blocks before the outbreak. Gross and continued use of HBV contaminated (mutant and wild viruses) injection devices led to an explosive outbreak with high mortality with a striking association with pre‐C/BCP mutants and D1 genotype.

Innate immune responses in human hepatocyte-derived cell lines alter genotype 1 hepatitis E virus replication efficiencies.

Hepatitis E virus (HEV) is a significant health problem in developing countries causing sporadic and epidemic forms of acute viral hepatitis. Hepatitis E is a self-limiting disease; however, chronic HEV infections are being reported in immunocompromised individuals. The disease severity is more during pregnancy with high mortality (20–25%), especially in third trimester. Early cellular responses after HEV infection are not completely understood. We analyzed innate immune responses associated with genotype-I HEV replication in human hepatoma cell lines (Huh7, Huh7.5 and HepG2/C3A) using HEV replicon system. These cells supported HEV replication with different efficiencies due to the cell type specific innate immune responses. HepG2/C3A cells were less supportive to HEV replication as compared to Huh7.5 and S10-3 cells. Reconstitution of the defective RIG-I and TLR3 signaling in Huh7.5 cells enabled them to induce higher level antiviral responses and restrict HEV replication, suggesting the involvement of both RIG-I and TLR3 in sensing HEV RNA and downstream activation of interferon regulatory factor 3 (IRF3) to generate antiviral responses. Inhibition of IRF3 mediated downstream responses in HepG2/C3A cells by pharmacological inhibitor BX795 significantly improved HEV replication efficiency implying the importance of this study in establishing a better cell culture system for future HEV studies.