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1.
BMC Immunol ; 22(1): 71, 2021 11 03.
Article in English | MEDLINE | ID: mdl-34732126

ABSTRACT

BACKGROUND: The natural antibody responses to B-cell epitopes from dengue structural proteins were assessed using immune sera from people having well-defined past dengue infections with one of the four serotypes. METHOD: Based on an immune-computational analysis previously conducted, nineteen epitopes from the envelope (E) and eight epitopes from pre-membrane (prM), which were more than 50% conserved across all the four DENV serotypes, were selected. Peptides to represent these B-cell epitopes were obtained from commercially available arrays, and were subjected to enzyme linked immunosorbent assay with sera obtained from dengue seropositive healthy volunteers (DENV1 n = 12: DENV2 n = 12: DENV3 n = 12 and DENV4 n = 12), and 10 dengue seronegative healthy volunteers from Sri Lanka. The cut-off value for the positive antibody response was set by taking the mean response of a peptide to the negative sera plus three standard deviations. The peptides (N = 7) showing the broad immune responses were used to generate antibodies in three mice (Balb/c) batches. The mice antisera were then subjected to microneutralization assays against all the four DENV serotypes. An EC50 viral neutralization ≥ 40 times the serum dilution was considered as neutralizing. RESULTS: Five of the E-peptide and two prM peptides were recognised by most individuls exposed to infections with each of the four serotypes, showing a serotype cross-reactive broad antibody response. The mice immune sera against the peptides representing the five E protein epitopes neutralized all the four DENV serotypes. Two of these five epitopes are from the Domain II, whereas one of them includes the whole bc-loop region. CONCLUSION: The antibody responses of highly conserved epitopes across the serotypes, were broadly responsive with sera of all four DENV serotypes collected from individuals infected with only one DENV serotype. Weakly conserved epitopes showed rather specific antibody responses dominated by one or few serotypes.


Subject(s)
Computational Biology/methods , Dengue Virus/physiology , Dengue/immunology , Epitopes, B-Lymphocyte/genetics , Viral Envelope Proteins/genetics , Viral Proteins/genetics , Animals , Antibodies, Neutralizing/metabolism , Conserved Sequence/genetics , Cross Reactions , Epitope Mapping , Epitopes, B-Lymphocyte/immunology , Healthy Volunteers , Humans , Immunization , Mice , Mice, Inbred BALB C , Viral Envelope Proteins/immunology , Viral Proteins/immunology
2.
Diagnostics (Basel) ; 11(11)2021 Nov 10.
Article in English | MEDLINE | ID: mdl-34829432

ABSTRACT

Dengue is a significant health concern in Sri Lanka, but diagnosis of the infecting dengue virus (DENV) serotype has hitherto been largely restricted to the Colombo district in the western province. Salinity tolerant Aedes vectors are present in the island's northern Jaffna peninsula, which is undergoing rapid groundwater salinization. Virus serotypes were determined by RT-qPCR in 107 and 112 patients diagnosed by NS1 antigen positivity from the Jaffna district in 2018 and 2019, respectively, and related to clinical characteristics. DENV1 and DENV2 were the most common serotypes in both years. Infections with multiple serotypes were not detected. DENV1 was significantly more prevalent in 2019 than 2018, while DENV3 was significantly more prevalent in 2018 than 2019 among the Jaffna patients. Limited genomic sequencing identified DENV1 genotype-I and DENV3 genotype-I in Jaffna patients in 2018. Dengue was more prevalent in working age persons and males among the serotyped Jaffna patients. DENV1 and DENV2 were the predominant serotypes in 2019 in the Colombo district. However, DENV1 and DENV3 were significantly more prevalent in Colombo compared with Jaffna in 2019. The differences in the prevalence of DENV1 and DENV3 between the Jaffna and Colombo districts in 2019 have implications for dengue epidemiology and vaccination. Salinity-tolerant Aedes vector strains, widespread in the Jaffna peninsula, may have contributed to differences in serotype prevalence compared with the Colombo district in 2019. Significant associations were not identified between virus serotypes and clinical characteristics among Jaffna patients.

3.
Cell ; 181(7): 1489-1501.e15, 2020 06 25.
Article in English | MEDLINE | ID: mdl-32473127

ABSTRACT

Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using HLA class I and II predicted peptide "megapools," circulating SARS-CoV-2-specific CD8+ and CD4+ T cells were identified in ∼70% and 100% of COVID-19 convalescent patients, respectively. CD4+ T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike, and N proteins each accounted for 11%-27% of the total CD4+ response, with additional responses commonly targeting nsp3, nsp4, ORF3a, and ORF8, among others. For CD8+ T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2-reactive CD4+ T cells in ∼40%-60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating "common cold" coronaviruses and SARS-CoV-2.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/immunology , Epitopes, T-Lymphocyte , Pneumonia, Viral/immunology , Betacoronavirus/genetics , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19 , COVID-19 Vaccines , Convalescence , Coronavirus Infections/blood , Coronavirus Infections/metabolism , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Cross Reactions , Humans , Leukocytes, Mononuclear/immunology , Pandemics , Pneumonia, Viral/blood , Pneumonia, Viral/metabolism , Pneumonia, Viral/virology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Viral Proteins/metabolism , Viral Vaccines/immunology
4.
JCI Insight ; 4(8)2019 04 18.
Article in English | MEDLINE | ID: mdl-30996133

ABSTRACT

The recent Zika virus (ZIKV) epidemic in the Americas has revealed rare but serious manifestations of infection. ZIKV has emerged in regions endemic for dengue virus (DENV), a closely related mosquito-borne flavivirus. Cross-reactive antibodies confound studies of ZIKV epidemiology and pathogenesis. The immune responses to ZIKV may be different in people, depending on their DENV immune status. Here, we focus on the human B cell and antibody response to ZIKV as a primary flavivirus infection to define the properties of neutralizing and protective antibodies generated in the absence of preexisting immunity to DENV. The plasma antibody and memory B cell response is highly ZIKV type-specific, and ZIKV-neutralizing antibodies mainly target quaternary structure epitopes on the viral envelope. To map viral epitopes targeted by protective antibodies, we isolated 2 type-specific monoclonal antibodies (mAbs) from a ZIKV case. Both mAbs were strongly neutralizing in vitro and protective in vivo. The mAbs recognize distinct epitopes centered on domains I and II of the envelope protein. We also demonstrate that the epitopes of these mAbs define antigenic regions commonly targeted by plasma antibodies in individuals from endemic and nonendemic regions who have recovered from ZIKV infections.


Subject(s)
Antibodies, Viral/immunology , Antigens, Viral/chemistry , Epitopes, B-Lymphocyte/chemistry , Zika Virus Infection/immunology , Zika Virus/immunology , Animals , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/isolation & purification , Antigens, Viral/immunology , Cross Protection/immunology , Cross Reactions/immunology , Dengue/epidemiology , Dengue/immunology , Dengue/prevention & control , Dengue/virology , Dengue Virus/immunology , Disease Models, Animal , Endemic Diseases/prevention & control , Epidemics/prevention & control , Epitopes, B-Lymphocyte/immunology , Female , Host-Pathogen Interactions/immunology , Humans , Immunologic Memory , Male , Mice , Protein Structure, Quaternary , Viral Vaccines/therapeutic use , Zika Virus Infection/epidemiology , Zika Virus Infection/prevention & control , Zika Virus Infection/virology
5.
PLoS Negl Trop Dis ; 10(10): e0005026, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27792777

ABSTRACT

BACKGROUND: Dengue is an emerging infectious disease of global significance. Suspected dengue, especially in children in Nicaragua's heavily-urbanized capital of Managua, has been well documented, but unsuspected dengue among children and adults with undifferentitated fever has not. METHODOLOGY/PRINCIPAL FINDINGS: To prospectively study dengue in semi-urban and rural western Nicaragua, we obtained epidemiologic and clinical data as well as acute and convalescent sera (2 to 4 weeks after onset of illness) from a convenience sample (enrollment Monday to Saturday daytime to early evening) of consecutively enrolled patients (n = 740) aged ≥ 1 years presenting with acute febrile illness. We tested paired sera for dengue IgG and IgM and serotyped dengue virus using reverse transcriptase-PCR. Among 740 febrile patients enrolled, 90% had paired sera. We found 470 (63.5%) were seropositive for dengue at enrollment. The dengue seroprevalance increased with age and reached >90% in people over the age of 20 years. We identified acute dengue (serotypes 1 and 2) in 38 (5.1%) patients. Only 8.1% (3/37) of confirmed cases were suspected clinically. CONCLUSIONS/SIGNIFICANCE: Dengue is an important and largely unrecognized cause of fever in rural western Nicaragua. Since Zika virus is transmitted by the same vector and has been associated with severe congenital infections, the population we studied is at particular risk for being devastated by the Zika epidemic that has now reached Central America.


Subject(s)
Dengue/diagnosis , Fever/diagnosis , Adolescent , Adult , Antibodies, Viral/blood , Child , Child, Preschool , Dengue/blood , Dengue/epidemiology , Dengue/virology , Dengue Virus/immunology , Dengue Virus/isolation & purification , Female , Fever/blood , Fever/epidemiology , Fever/virology , Humans , Male , Middle Aged , Nicaragua/epidemiology , Prospective Studies , Young Adult
6.
Vaccine ; 34(17): 2008-14, 2016 Apr 12.
Article in English | MEDLINE | ID: mdl-26954467

ABSTRACT

Severe acute respiratory syndrome (SARS) is a highly contagious infectious disease which first emerged in late 2002, caused by a then novel human coronavirus, SARS coronavirus (SARS-CoV). The virus is believed to have originated from bats and transmitted to human through intermediate animals such as civet cats. The re-emergence of SARS-CoV remains a valid concern due to the continual persistence of zoonotic SARS-CoVs and SARS-like CoVs (SL-CoVs) in bat reservoirs. In this study, the screening for the presence of SARS-specific T cells in a cohort of three SARS-recovered individuals at 9 and 11 years post-infection was carried out, and all memory T cell responses detected target the SARS-CoV structural proteins. Two CD8(+) T cell responses targeting the SARS-CoV membrane (M) and nucleocapsid (N) proteins were characterized by determining their HLA restriction and minimal T cell epitope regions. Furthermore, these responses were found to persist up to 11 years post-infection. An absence of cross-reactivity of these CD8(+) T cell responses against the newly-emerged Middle East respiratory syndrome coronavirus (MERS-CoV) was also demonstrated. The knowledge of the persistence of SARS-specific celullar immunity targeting the viral structural proteins in SARS-recovered individuals is important in the design and development of SARS vaccines, which are currently unavailable.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunity, Cellular , Immunologic Memory , Severe Acute Respiratory Syndrome/immunology , Coronavirus M Proteins , Coronavirus Nucleocapsid Proteins , Cross Reactions , Epitopes, T-Lymphocyte/immunology , Histocompatibility Antigens Class I/immunology , Humans , Middle East Respiratory Syndrome Coronavirus , Nucleocapsid Proteins/immunology , Severe acute respiratory syndrome-related coronavirus , Severe Acute Respiratory Syndrome/prevention & control , Viral Matrix Proteins/immunology
7.
N Engl J Med ; 372(16): 1519-29, 2015 Apr 16.
Article in English | MEDLINE | ID: mdl-25875257

ABSTRACT

BACKGROUND: Aerosolized vaccine can be used as a needle-free method of immunization against measles, a disease that remains a major cause of illness and death. Data on the immunogenicity of aerosolized vaccine against measles in children are inconsistent. METHODS: We conducted an open-label noninferiority trial involving children 9.0 to 11.9 months of age in India who were eligible to receive a first dose of measles vaccine. Children were randomly assigned to receive a single dose of vaccine by means of either aerosol inhalation or a subcutaneous injection. The primary end points were seropositivity for antibodies against measles and adverse events 91 days after vaccination. The noninferiority margin was 5 percentage points. RESULTS: A total of 1001 children were assigned to receive aerosolized vaccine, and 1003 children were assigned to receive subcutaneous vaccine; 1956 of all the children (97.6%) were followed to day 91, but outcome data were missing for 331 children because of thawed specimens. In the per-protocol population, data on 1560 of 2004 children (77.8%) could be evaluated. At day 91, a total of 662 of 775 children (85.4%; 95% confidence interval [CI], 82.5 to 88.0) in the aerosol group, as compared with 743 of 785 children (94.6%; 95% CI, 92.7 to 96.1) in the subcutaneous group, were seropositive, a difference of -9.2 percentage points (95% CI, -12.2 to -6.3). Findings were similar in the full-analysis set (673 of 788 children in the aerosol group [85.4%] and 754 of 796 children in the subcutaneous group [94.7%] were seropositive at day 91, a difference of -9.3 percentage points [95% CI, -12.3 to -6.4]) and after multiple imputation of missing results. No serious adverse events were attributable to measles vaccination. Adverse-event profiles were similar in the two groups. CONCLUSIONS: Aerosolized vaccine against measles was immunogenic, but, at the prespecified margin, the aerosolized vaccine was inferior to the subcutaneous vaccine with respect to the rate of seropositivity. (Funded by the Bill and Melinda Gates Foundation; Measles Aerosol Vaccine Project Clinical Trials Registry-India number, CTRI/2009/091/000673.).


Subject(s)
Measles Vaccine/administration & dosage , Measles virus/immunology , Measles/prevention & control , Administration, Inhalation , Aerosols , Antibodies, Viral/blood , Female , Humans , India , Infant , Injections, Subcutaneous , Male , Measles/immunology , Measles Vaccine/adverse effects , Measles Vaccine/immunology
8.
Nat Commun ; 6: 6341, 2015 Feb 20.
Article in English | MEDLINE | ID: mdl-25698059

ABSTRACT

Dengue virus (DENV) infects ~400 million people annually. There is no licensed vaccine or therapeutic drug. Only a small fraction of the total DENV-specific antibodies in a naturally occurring dengue infection consists of highly neutralizing antibodies. Here we show that the DENV-specific human monoclonal antibody 5J7 is exceptionally potent, neutralizing 50% of virus at nanogram-range antibody concentration. The 9 Å resolution cryo-electron microscopy structure of the Fab 5J7-DENV complex shows that a single Fab molecule binds across three envelope proteins and engages three functionally important domains, each from a different envelope protein. These domains are critical for receptor binding and fusion to the endosomal membrane. The ability to bind to multiple domains allows the antibody to fully coat the virus surface with only 60 copies of Fab, that is, half the amount compared with other potent antibodies. Our study reveals a highly efficient and unusual mechanism of molecular recognition by an antibody.


Subject(s)
Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Dengue Virus/metabolism , Dengue/immunology , Immunoglobulin Fab Fragments/chemistry , Membrane Proteins/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Cell Membrane/chemistry , Chlorocebus aethiops , Cryoelectron Microscopy , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Genotype , Humans , Mice , Molecular Sequence Data , Neutralization Tests , Protein Binding , Protein Structure, Quaternary , Sequence Homology, Amino Acid , Serogroup , Vero Cells
9.
PLoS One ; 9(10): e108786, 2014.
Article in English | MEDLINE | ID: mdl-25290339

ABSTRACT

BACKGROUND: According to WHO estimates, 35% of global measles deaths in 2011 occurred in India. In 2013, India committed to a goal of measles elimination by 2020. Laboratory supported case based measles surveillance is an essential component of measles elimination strategies. Results from a case-based measles surveillance system in Pune district (November 2009 through December 2011) are reported here with wider implications for measles elimination efforts in India. METHODS: Standard protocols were followed for case identification, investigation and classification. Suspected measles cases were confirmed through serology (IgM) or epidemiological linkage or clinical presentation. Data regarding age, sex, vaccination status were collected and annualized incidence rates for measles and rubella cases calculated. RESULTS: Of the 1011 suspected measles cases reported to the surveillance system, 76% were confirmed measles, 6% were confirmed rubella, and 17% were non-measles, non-rubella cases. Of the confirmed measles cases, 95% were less than 15 years of age. Annual measles incidence rate was more than 250 per million persons and nearly half were associated with outbreaks. Thirty-nine per cent of the confirmed measles cases were vaccinated with one dose of measles vaccine (MCV1). CONCLUSION: Surveillance demonstrated high measles incidence and frequent outbreaks in Pune where MCV1 coverage in infants was above 90%. Results indicate that even high coverage with a single dose of measles vaccine was insufficient to provide population protection and prevent measles outbreaks. An effective measles and rubella surveillance system provides essential information to plan, implement and evaluate measles immunization strategies and monitor progress towards measles elimination.


Subject(s)
Measles/epidemiology , Population Surveillance , Adolescent , Age Factors , Child , Child, Preschool , Disease Outbreaks , Female , Geography, Medical , Humans , Incidence , India/epidemiology , Infant , Infant, Newborn , Male , Measles/prevention & control , Measles Vaccine , Mortality , Seasons , Sex Factors , Young Adult
10.
J Virol ; 88(21): 12233-41, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25100837

ABSTRACT

UNLABELLED: Natural dengue virus (DENV) infection in humans induces antibodies (Abs) that neutralize the serotype of infection in a potent and type-specific manner; however, most Abs generated in response to infection are serotype cross-reactive and poorly neutralizing. Such cross-reactive Abs may enhance disease during subsequent infection with a virus of a different DENV serotype. Previous screening assays for DENV-specific human B cells and antibodies, using viral and recombinant antigens, mainly led to the isolation of dominant nonneutralizing B cell clones. To improve upon our ability to recover and study rare but durable and potently neutralizing DENV-specific Abs, we isolated human DENV-specific B cells by using a primary screen of binding to live virus, followed by a secondary screen with a high-throughput, flow cytometry-based neutralization assay to identify DENV-specific B cell lines prior to generation of hybridomas. Using this strategy, we identified several new classes of serotype-specific and serotype-cross-neutralizing anti-DENV monoclonal Abs (MAbs), including ultrapotent inhibitory antibodies with neutralizing activity concentrations of <10 ng/ml. We isolated serotype-specific neutralizing Abs that target diverse regions of the E protein, including epitopes present only on the intact, fully assembled viral particle. We also isolated a number of serotype-cross-neutralizing MAbs, most of which recognized a region in E protein domain I/II containing the fusion loop. These data provide insights into targets of the protective Ab-mediated immune response to natural DENV infection, which will prove valuable in the design and testing of new experimental DENV vaccines. IMPORTANCE: Dengue virus infection is one of the most common mosquito-borne diseases and occurs in most countries of the world. Infection of humans with dengue virus induces a small number of antibodies that inhibit the infecting strain but also induces a large number of antibodies that can bind but do not inhibit dengue virus strains of other serotypes. We used a focused screening strategy to discover a large number of rare potently inhibiting antibodies, and we mapped the regions on the virus that were recognized by such antibodies. Our studies revealed that humans have the potential to generate very potent antibodies directed to diverse regions of the dengue virus surface protein. These studies provide important new information about protection from dengue virus infection that will be useful in the design and testing of new experimental dengue vaccines for humans.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , B-Lymphocytes/immunology , Dengue Virus/immunology , Dengue/virology , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/isolation & purification , Cross Reactions , Humans , Neutralization Tests , Viral Envelope Proteins/immunology
11.
PLoS Negl Trop Dis ; 6(5): e1653, 2012.
Article in English | MEDLINE | ID: mdl-22616022

ABSTRACT

BACKGROUND: In January 2011, human cases with hemorrhagic manifestations in the hospital staff were reported from a tertiary care hospital in Ahmadabad, India. This paper reports a detailed epidemiological investigation of nosocomial outbreak from the affected area of Ahmadabad, Gujarat, India. PRINCIPAL FINDINGS: Samples from 3 suspected cases, 83 contacts, Hyalomma ticks and livestock were screened for Crimean-Congo hemorrhagic fever (CCHF) virus by qRT-PCR of which samples of two medical professionals (case C and E) and the husband of the index case (case D) were positive for CCHFV. The sensitivity and specificity of indigenous developed IgM ELISA to screen CCHFV specific antibodies in human serum was 75.0% and 97.5% respectively as compared to commercial kit. About 17.0% domestic animals from Kolat, Ahmadabad were positive for IgG antibodies while only two cattle and a goat showed positivity by qRT-PCR. Surprisingly, 43.0% domestic animals (Buffalo, cattle, sheep and goat) showed IgG antibodies in the adjoining village Jivanpara but only one of the buffalo was positive for CCHFV. The Hyalomma anatolicum anatolicum ticks were positive in PCR and virus isolation. CCHFV was isolated from the blood sample of case C, E in Vero E-6 cells and Swiss albino mice. In partial nucleocapsid gene phylogeny from CCHFV positive human samples of the years 2010 and 2011, livestock and ticks showed this virus was similar to Tajikistan (strain TAJ/H08966), which belongs in the Asian/middle east genetic lineage IV. CONCLUSIONS: The likely source of CCHFV was identified as virus infected Hyalomma ticks and livestock at the rural village residence of the primary case (case A). In addition, retrospective sample analysis revealed the existence of CCHFV in Gujarat and Rajasthan states before this outbreak. An indigenous developed IgM ELISA kit will be of great use for screening this virus in India.


Subject(s)
Cross Infection/epidemiology , Disease Outbreaks , Hemorrhagic Fever Virus, Crimean-Congo/isolation & purification , Hemorrhagic Fever, Crimean/epidemiology , Livestock/virology , Ticks/virology , Adult , Animals , Antibodies, Viral/blood , Cross Infection/virology , Enzyme-Linked Immunosorbent Assay/methods , Female , Hemorrhagic Fever, Crimean/virology , Humans , Immunoglobulin M/blood , India/epidemiology , Male , RNA, Viral/genetics , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity
12.
Indian J Med Res ; 132: 395-9, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20966517

ABSTRACT

BACKGROUND & 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<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<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 & CONCLUSIONS: Chandipura virus was confirmed as the aetiological agent of this acute encephalitis outbreak with high case-fatality among children.


Subject(s)
Disease Outbreaks , Encephalitis, Viral/epidemiology , Insect Vectors/virology , Phylogeny , Psychodidae/virology , Rhabdoviridae Infections/epidemiology , Vesiculovirus/genetics , Animals , Antibodies, Viral/blood , Antibodies, Viral/cerebrospinal fluid , Base Sequence , Cell Line, Tumor , Child , Cluster Analysis , DNA Primers/genetics , Encephalitis, Viral/pathology , Enzyme-Linked Immunosorbent Assay , Female , Humans , India/epidemiology , Male , Molecular Sequence Data , Nucleocapsid Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Rhabdoviridae Infections/pathology , Sequence Analysis, DNA
13.
In Vitro Cell Dev Biol Anim ; 45(9): 491-5, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19533252

ABSTRACT

A new cell line from the neonate larvae of Aedes aegypti (L) mosquito was established and characterized. The cell line at the 50th passage (P) level consisted of three prominent cell types, i.e., epithelial-like cells (92%), fibroblast-like cells (7%), and giant cells ( approximately 1%). Karyological analysis showed diploid (2n = 6) number of chromosomes in >75% cells at P-50. The growth kinetics studied at 52nd passage level showed approximately tenfold increase in cell number over a 10-d study period. The species specificity studies using DNA amplification fingerprinting profile analysis using RAPD primers demonstrated 100% homology with the host profile showing the integrity of the cell line. Electron microscopy revealed the absence of mycoplasma or other adventitious agents. The cell line supported the multiplication of seven arboviruses, i.e., Chikungunya (CHIK), Japanese encephalitis, West Nile, dengue 2 (DEN-2), Chandipura, vesicular stomatitis, and Chittoor viruses. The cell line did not replicate Ganjam and Kaisodi viruses. CHIK virus yield in the new cell line was approximately 3log and 0.5log 50% tissue culture infective dose (TCID(50))/mL higher than Vero E6 and C6/36 cell lines, respectively. In the case of DEN-2 virus, it yielded 1log TCID(50)/mL higher than Vero E6, but lesser than C6/36 cell line. Due to its high susceptibility to a broad spectrum of viruses, the new cell line may find application in virus isolation during epidemics and in antigen production.


Subject(s)
Aedes/cytology , Aedes/virology , Arboviruses/physiology , Cell Culture Techniques/methods , Cell Line/virology , Animals , Arboviruses/growth & development , Virus Replication
14.
BMC Infect Dis ; 8: 168, 2008 Dec 17.
Article in English | MEDLINE | ID: mdl-19091082

ABSTRACT

BACKGROUND: Chandipura virus (CHPV), a member of family Rhabdoviridae 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. METHODS: 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 [in vivo (mice) in ovo (eggs), in vitro (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. RESULTS: Real-time one step RT-PCR was optimized using in vitro 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 x 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 x 102 PFU/ml). Vero and PS cell-lines (1.2 x 103 PFU/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. CONCLUSION: 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.


Subject(s)
Reverse Transcriptase Polymerase Chain Reaction/methods , Rhabdoviridae Infections/diagnosis , Vesiculovirus/isolation & purification , Animals , Child , Clinical Laboratory Techniques , Computer Systems , DNA Primers , Humans , India , Infant , Mice , RNA, Viral/analysis , RNA-Directed DNA Polymerase/metabolism , Sensitivity and Specificity , Vesiculovirus/genetics
15.
Am J Trop Med Hyg ; 73(3): 566-70, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16172482

ABSTRACT

An outbreak of encephalitis with a case fatality rate of 78.3% was investigated among children in Gujarat State, India. Twenty-six cases were reported. Three patients had IgM antibodies to Chandipura virus. Virus was isolated from one patient with rhabdomyosarcoma in porcine stable cell lines and in suckling mice. Chandipura virus RNA was present in 9 of 20 acute-phase serum samples, and virus sequences from the present outbreak were closely related to prototype strain (1965) and Andhra Pradesh, India (2003) isolates. Serologic and molecular assays documented the absence of Japanese encephalitis virus, West Nile virus, dengue virus, and paramyxoviruses in clinical samples. The etiologic agent was Chandipura virus, which has become an important encephalitis-causing virus in India.


Subject(s)
Rhabdoviridae Infections/epidemiology , Vesiculovirus , Adolescent , Antibodies, Viral/blood , Child , Child, Preschool , Disease Outbreaks , Female , Humans , Immunoglobulin M/blood , India/epidemiology , Infant , Male , Phylogeny , RNA, Viral/blood , Vesiculovirus/genetics , Vesiculovirus/isolation & purification
16.
Lancet ; 364(9437): 869-74, 2004.
Article in English | MEDLINE | ID: mdl-15351194

ABSTRACT

BACKGROUND: An outbreak of acute encephalitis of unknown origin with high case fatality (183 of 329 cases) was reported in children from Andhra Pradesh state in southern India during 2003. We investigated the causative agent. METHODS: Cell lines and peripheral blood lymphocyte co-cultures were used to isolate the causative agent from clinical samples. Identity of the agent was established by electron microscopy and serological and molecular assays. FINDINGS: Clinical samples tested negative for IgM antibodies to Japanese encephalitis, West Nile, dengue, and measles viruses, and for RNA of coronavirus, paramyxovirus, enterovirus, and influenza viruses. Virus was isolated from six patients with encephalitis and was identified as Chandipura virus by electron microscopy, complement fixation, and neutralisation tests. Chandipura virus RNA was detected in clinical samples from nine patients. Sequencing of five of these RNA samples showed 96.7-97.5% identity with the reference strain of 1965. Chandipura viral antigen and RNA were detected in brain tissue of a deceased child by immunofluorescent antibody test and PCR. Neutralising, IgG, and IgM antibodies to Chandipura virus were present in some patients' serum samples. Serum samples obtained after 4 days of illness were more frequently positive for IgM to Chandipura virus than were those obtained earlier (p<0.001). A similar trend was noted for neutralising antibodies. INTERPRETATION: Our findings suggest that this outbreak of acute encephalitis in Andhra Pradesh was associated with Chandipura virus, adding to the evidence suggesting that this virus should be considered as an important emerging pathogen.


Subject(s)
Disease Outbreaks , Encephalitis, Viral/epidemiology , Rhabdoviridae Infections/epidemiology , Vesiculovirus , Acute Disease , Adolescent , Antibodies, Viral/blood , Brain/virology , Child , Child, Preschool , Communicable Diseases, Emerging , Encephalitis, Viral/diagnosis , Encephalitis, Viral/mortality , Encephalitis, Viral/virology , Enzyme-Linked Immunosorbent Assay , Female , Humans , India/epidemiology , Infant , Male , Polymerase Chain Reaction , Rhabdoviridae Infections/diagnosis , Rhabdoviridae Infections/mortality , Serologic Tests , Survival Rate , Vesiculovirus/isolation & purification
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