Evaluation of the immunogenicity of liposome encapsulated HVR1 and NS3 regions of genotype 3 HCV, either singly or in combination

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus displays a high rate of mutation and exists as a quasispecies in infected patients. In the absence of an effective universal vaccine, genotype-specific vaccine development represents an alternative. We have attempted to develop a genotype 3 based, liposome encapsulated HCV vaccine with hypervariable region-1 (HVR1) and non-structural region-3 (NS3) components.</p> <p>Results</p> <p>HCV RNA extracted from serum samples of 49 chronically infected patients was PCR amplified to obtain HVR1 region. These amplified products were cloned to obtain 20 clones per sample in order to identify the quasispecies pattern. The HVR1 consensus sequence, along with three variants was reverse transcribed to obtain peptides. The peptides were checked for immunoreactivity individually, as a pool or as a single peptide tetramer interspersed with four glycine residues. Anti-HCV positivity varied from 42.6% (tetramer) to 92.2% (variant-4) when 115 anti-HCV positive sera representing genotypes 1, 3, 4 and 6 were screened. All the 95 anti-HCV negatives were scored negative by all antigens. Mice were immunized with different liposome encapsulated or Al(OH)₃adjuvanted formulations of HVR1 variants and recombinant NS3 protein, and monitored for anti-HVR1 and anti-NS3 antibody titres, IgG isotypes and antigen specific cytokine levels. A balanced Th1/Th2 isotyping response with high antibody titres was observed in most of the liposome encapsulated antigen groups. The effect of liposomes and aluminium hydroxide on the expression of immune response genes was studied using Taqman Low Density Array. Both Th1 (IFN-gamma, Il18) and Th2 (Il4) genes were up regulated in the liposome encapsulated HVR1 variant pool-NS3 combination group. In-vitro binding of the virus to anti-HVR1 antibodies was demonstrated.</p> <p>Conclusion</p> <p>The optimum immunogen was identified to be combination of peptides of HVR1 consensus sequence and its variants along with pNS3 encapsulated in liposomes, which could generate both cellular and humoral immune responses in mice deserving further evaluation in a suitable cell culture system/non-human primate model.</p

Comparison of etiology of sporadic acute and fulminant viral hepatitis in hospitalized patients in Pune, India during 1978-81 and 1994-97

Objective: To determine and compare the etiology of sporadic acute and fulminant viral hepatitis in two groups of patients 16 years apart. Methods: Serologic diagnostic tests for hepatitis A, B, C, D and E, and cytomegalovirus infection were carried out in 276 patients during 1994-1997 (Group A) and 206 patients during 1978-1981 (Group B). Results: Among children, hepatitis A virus was the major etiologic agent (81.6% in Group A and 51.4% in Group B), followed by hepatitis E virus (12.2%, 46.4%) and hepatitis B virus (5.4%, none). Among adults, hepatitis E virus was the main causative agent (42.4% in Group A and 71.2% in Group B) followed by HBV (28%, 25.5%) and hepatitis A virus (10.6%, 3.5%). Delta hepatitis was found only in Group A. No viral cause was found in 25% of patients in Group A and 13.5% patients in Group B. Conclusions: Hepatitis E virus is a major cause of sporadic acute and fulminant hepatitis. There has been an increase in hepatitis A in adults who developed fulminant hepatic failure. Our data points to the emergence of hepatitis A in adults and emergence of delta virus infection. Hepatitis C virus was unimportant in causing sporadic hepatitis

Genetic divergence of Chikungunya viruses in India (1963-2006) with special reference to the 2005-2006 explosive epidemic

Re-emergence of Chikungunya (CHIK), caused by CHIK virus, was recorded in India during 2005-2006 after a gap of 32 years, causing 1.3 million cases in 13 states. Several islands of the Indian Ocean reported similar outbreaks in the same period. These outbreaks were attributed to the African genotype of CHIK virus. To examine relatedness of the Indian isolates (IND-06) with Reunion Island isolates (RU), full-genome sequences of five CHIK virus isolates representative of different Indian states were determined. In addition, an isolate obtained from mosquitoes in the year 2000 (Yawat-2000), identified as being of the African genotype, and two older strains isolated in 1963 and 1973 (of the Asian genotype), were sequenced. The IND-06 isolates shared 99.9 % nucleotide identity with RU isolates, confirming involvement of the same strain in these outbreaks. The IND-06 isolates shared 98.2 % identity with the Yawat-2000 isolate. Of two crucial substitutions reported for RU isolates in the E1 region, M269V was noted in the Yawat-2000 and IND-06 isolates, whereas D284E was seen only in the IND-06 isolates. The A226V shift observed with the progression of the epidemic in Reunion Island, probably associated with adaptation to the mosquito vector, was absent in all of the Indian isolates. Three unique substitutions were noted in the IND-06 isolates: two (T128K and T376M) in the Nsp1 region and one (P23S) in the capsid protein. The two Asian strains showed 99.4 % nucleotide identity to each other, indicating relative stability of the virus. No evidence of recombination of the Asian and African genotypes, or of positive selection was observed. The results may help in understanding the association, if any, of the unique mutations with the explosive nature of the CHIK outbreak

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 patient's 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

Chandipura virus: a major cause of acute encephalitis in children in North Telangana, Andhra Pradesh, India

A hospital-based surveillance was undertaken between May 2005 and April 2006 to elucidate the contribution of Chandipura virus (CHPV) to acute viral encephalitis cases in children, seroconversion in recovered cases and to compare the seroprevalences of anti-CHPV IgM and N antibodies in areas reporting cases with those without any case of acute viral encephalitis. During this period, 90 cases of acute encephalitis were hospitalized in the pediatric wards of Mahatma Gandhi Memorial (MGM) Hospital, Warangal. There were 49 deaths (Case Fatality Rate, i.e., CFR of 54.4%). Clinical samples and records were obtained from 52 suspected cases. The cases were below 15 years, majority in 0-4 years (35/52, 67.3%). Computerized tomography (CT) scans and cerebro-spinal fluid (CSF) picture favored viral etiology. No neurological sequelae were observed. CHPV etiology was detected in 25 cases (48.1%, n = 52; RNA in 20, IgM in 3 and N antibody seroconversion in 2). JEV etiology was detected in 5 cases (IgM in 4 cases and seroconversion in 1 case). Anti-CHPV IgM seroprevalence in contacts (26/167, 15.6%) was significantly higher (P &lt; 0.05) than in non-contacts (11/430, 2.6%); which was also observed in children &lt; 15 years (19/90, 21.1% vs. 3/109, 2.7%). Anti-CHPV N antibody seroprevalence in &lt;15 years contacts (66/90, 73.3%) and non-contacts (77/109, 70.6%) was significantly lower (P &lt; 0.05) than in contacts (75/77, 97.4%) and non-contacts (302/321, 94.1%) more than 15 years respectively. CHPV appears to be the major cause of acute viral encephalitis in children in endemic areas during early monsoon months

Immunogenicity and safety of live attenuated hepatitis A Vaccine: a multicentric study

Objective: To evaluate immunogenicity and tolerability of single dose live attenuated injectable hepatitis A vaccine in four metropolitan cities of India. Methods: Live attenuated hepatitis A vaccine was administered to 505 children aged 18-60 months in four centers across India. Immunogenicity of the vaccine was assessed by estimation of anti-HAV antibody titer at 6 weeks and 6 months following administration of the vaccine. Safety evaluation of the vaccine was also done during the visits. Results: At 6 weeks, 480 subjects (95%) came for the follow-up and 411 (81.4%) subjects reported at the end of 6 months. The geometric mean titer (GMT) of anti-HAV antibody of the subjects who did not have the seroprotective titer at the baseline were assessed at 6 weeks and 6 months which was 81.04 mIU/ml and 150.66 mIU/ml respectively. At 6 weeks, 95.1% seroconverted and at the end of 6 months, 97.9% had seroconverted. Both solicited and unsolicited vaccine-induced local and systemic adverse events were insignificant at all the centers, except swelling and induration in a few. Conclusion: Live attenuated injectable hepatitis A vaccine was immunogenic and tolerable with minimal reactogenecity, in this study of single dose schedule. Safety profile was also satisfactory in the study population

Chandipura virus encephalitis outbreak among children in Nagpur division, Maharashtra, 2007

Background &amp; objectives: An outbreak of acute encephalitis syndrome (AES) among children from Nagpur division, Maharashtra was investigated to confirm the aetiology and to describe clinico-epidemiological features. Methods: AES cases among children &lt; 15 yr, from Nagpur division, hospitalized between June-September 2007, were investigated. Serum and cerebrospinal fluid (CSF) were tested for IgM antibodies against Chandipura virus (CHPV) and Japanese encephalitis virus (JEV) and for CHPV RNA by RT-PCR. Partial N gene sequences were used for phylogenetic analysis. Virus isolations were attempted in rhabdomyosarcoma (RD) cell line. Sandflies were collected, pooled and tested for CHPV RNA by RT-PCR. Results: A total of 78 AES cases were recorded in children &lt; 15 yr of age. Case fatality ratio was 43.6 per cent. Male to female ratio was 1:1.2. Chandipura (CHP) was confirmed in 39 cases. CHPV RNA was detected in both CSF and serum specimens of 2 cases and in serum of 22 cases. Phylogenetic analysis showed 99.98-100 per cent nucleotide identity in the sequences studied. Anti-CHPV IgM antibodies were detected in CSF of 2 cases and in serum of 8 cases. Seroconversion to anti-CHPV IgM antibodies was observed in 5 cases. Clinical manifestations of CHP cases (n=38) were fever (100%), convulsion (76.3%), altered sensorium (34.2%), headache (23.7%), vomiting (44.7%) and diarrhoea (23.7%). CHPV RNA was detected in one of two pools of sandflies from affected locality. Interpretation &amp; conclusions: Chandipura virus was confirmed as the aetiological agent of this acute encephalitis outbreak with high case-fatality among children

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

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

Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

<p>Abstract</p> <p>Background</p> <p>Chandipura virus (CHPV), a member of family <it>Rhabdoviridae </it>was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR) using TaqMan technology was developed for rapid diagnosis.</p> <p>Methods</p> <p>Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [<it>in vivo </it>(mice) <it>in ovo </it>(eggs), <it>in vitro </it>(Vero E6, PS, RD and Sand fly cell line)] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard.</p> <p>Results</p> <p>Real-time one step RT-PCR was optimized using <it>in vitro </it>transcribed (IVT) RNA. Standard curve showed linear relationship for wide range of 10²-10¹⁰(r²= 0.99) with maximum Coefficient of variation (CV = 5.91%) for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice) used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 10⁰PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 10²PFU/ml). Vero and PS cell-lines (1.2 × 10³PFU/ml) were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy individual was used.</p> <p>Conclusion</p> <p>On account of the high sensitivity, reproducibility and specificity, the assay can be used for the rapid detection and quantitation of CHPV RNA from clinical samples during epidemics and from endemic areas. The assay may also find application in screening of antiviral compounds, understanding of pathogenesis as well as evaluation of vaccine.</p

Intracranial Administration of P Gene siRNA Protects Mice from Lethal Chandipura Virus Encephalitis

Background: In parts of India, Chandipura Virus (CHPV) has emerged as an encephalitis causing pathogen in both epidemic and sporadic forms. This pediatric disease follows rapid course leading to 55–75 % mortality. In the absence of specific treatment, effectiveness of RNA interference (RNAi) was evaluated. Methods and Findings: Efficacy of synthetic short interfering RNA (siRNA) or short hairpin RNA (shRNA) in protecting mice from CHPV infection was assessed. The target genes were P and M genes primarily because important role of the former in viral replication and lethal nature of the latter. Real time one step RT-PCR and plaque assay were used for the assessment of gene silencing. Using pAcGFP1N1-CHPV-P, we showed that P-2 siRNA was most efficient in reducing the expression of P gene in-vitro. Both quantitative assays documented 2logs reduction in the virus titer when P-2, M-5 or M-6 siRNAs were transfected 2hr post infection (PI). Use of these siRNAs in combination did not result in enhanced efficiency. P-2 siRNA was found to tolerate four mismatches in the center. As compared to five different shRNAs, P-2 siRNA was most effective in inhibiting CHPV replication. An extended survival was noted when mice infected intracranially with 100 LD 50 CHPV were treated with cationic lipid complexed 5 mg P-2 siRNA simultaneously. Infection with 10LD 50 and treatment with two doses of siRNA first, simultaneously and second 24 hr PI, resulted in 70 % survival. Surviving mice showed 4logs less CHPV titers in brain without histopathological changes or antibody response. Gene expression profiles of P-2 siRNA treated mice showed no interferon response. First dose of siRNA at 2h