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COVID-19 , SARS-CoV-2 , Humanos , Microscopía Electrónica , Microscopía Electrónica de Transmisión , TemperaturaRESUMEN
We report the development and evaluation of safety and immunogenicity of a whole virion inactivated (WVI) SARS-CoV-2 vaccine (BBV152), adjuvanted with aluminum hydroxide gel (Algel), or TLR7/8 agonist chemisorbed Algel. We used a well-characterized SARS-CoV-2 strain and an established Vero cell platform to produce large-scale GMP-grade highly purified inactivated antigen. Product development and manufacturing process were carried out in a BSL-3 facility. Immunogenicity and safety were determined at two antigen concentrations (3µg and 6µg), with two different adjuvants, in mice, rats, and rabbits. Our results show that BBV152 vaccine formulations generated significantly high antigen-binding and neutralizing antibody titers (NAb), at both concentrations, in all three species with excellent safety profiles. The inactivated vaccine formulation contains TLR7/8 agonist adjuvant-induced Th1-biased antibody responses with elevated IgG2a/IgG1 ratio and increased levels of SARS-CoV-2-specific IFN-γ+ CD4+ T lymphocyte response. Our results support further development for phase I/II clinical trials in humans.
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Betacoronavirus/aislamiento & purificación , Infecciones por Coronavirus/diagnóstico , Neumonía Viral/diagnóstico , Viaje , Adenovirus Humanos/aislamiento & purificación , Adolescente , Adulto , Anciano , COVID-19 , Niño , Preescolar , Coinfección/diagnóstico , Infecciones por Coronavirus/virología , Femenino , Humanos , India , Lactante , Recién Nacido , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza B/aislamiento & purificación , Masculino , Metapneumovirus/aislamiento & purificación , Persona de Mediana Edad , Cavidad Nasal/virología , Pandemias , Faringe/virología , Neumonía Viral/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Respirovirus/aislamiento & purificación , Rhinovirus/aislamiento & purificación , SARS-CoV-2 , Evaluación de Síntomas , Adulto JovenAsunto(s)
Betacoronavirus/aislamiento & purificación , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Animales , COVID-19 , Chlorocebus aethiops , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , India , Microscopía Electrónica de Transmisión , Pandemias , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2 , Células VeroAsunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/virología , Genoma Viral , Neumonía Viral/virología , COVID-19 , China , Humanos , India , Irán , Italia , Pandemias , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2 , Alineación de Secuencia , ViajeRESUMEN
The newly emerged 2019 novel coronavirus (CoV), named as severe acute respiratory syndrome CoV-2 (SARS-CoV-2), like SARS-CoV (now, SARS-CoV-1) and Middle East respiratory syndrome CoV (MERS-CoV), has been associated with high infection rates with over 36,405 deaths. In the absence of approved marketed drugs against coronaviruses, the treatment and management of this novel CoV disease (COVID-19) worldwide is a challenge. Drug repurposing that has emerged as an effective drug discovery approach from earlier approved drugs could reduce the time and cost compared to de novo drug discovery. Direct virus-targeted antiviral agents target specific nucleic acid or proteins of the virus while host-based antivirals target either the host innate immune responses or the cellular machineries that are crucial for viral infection. Both the approaches necessarily interfere with viral pathogenesis. Here we summarize the present status of both virus-based and host-based drug repurposing perspectives for coronaviruses in general and the SARS-CoV-2 in particular.
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Infecciones por Coronavirus/tratamiento farmacológico , Reposicionamiento de Medicamentos , Neumonía Viral/tratamiento farmacológico , Antivirales/uso terapéutico , Betacoronavirus , COVID-19 , Descubrimiento de Drogas , Humanos , Simulación del Acoplamiento Molecular , Pandemias , Inhibidores de Proteasas/uso terapéutico , SARS-CoV-2 , Proteínas Virales/antagonistas & inhibidores , Tratamiento Farmacológico de COVID-19RESUMEN
Background & objectives: Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally affected 195 countries. In India, suspected cases were screened for SARS-CoV-2 as per the advisory of the Ministry of Health and Family Welfare. The objective of this study was to characterize SARS-CoV-2 sequences from three identified positive cases as on February 29, 2020. Methods: Throat swab/nasal swab specimens for a total of 881 suspected cases were screened by E gene and confirmed by RdRp (1), RdRp (2) and N gene real-time reverse transcription-polymerase chain reactions and next-generation sequencing. Phylogenetic analysis, molecular characterization and prediction of B- and T-cell epitopes for Indian SARS-CoV-2 sequences were undertaken. Results: Three cases with a travel history from Wuhan, China, were confirmed positive for SARS-CoV-2. Almost complete (29,851 nucleotides) genomes of case 1, case 3 and a fragmented genome for case 2 were obtained. The sequences of Indian SARS-CoV-2 though not identical showed high (~99.98%) identity with Wuhan seafood market pneumonia virus (accession number: NC 045512). Phylogenetic analysis showed that the Indian sequences belonged to different clusters. Predicted linear B-cell epitopes were found to be concentrated in the S1 domain of spike protein, and a conformational epitope was identified in the receptor-binding domain. The predicted T-cell epitopes showed broad human leucocyte antigen allele coverage of A and B supertypes predominant in the Indian population. Interpretation & conclusions: The two SARS-CoV-2 sequences obtained from India represent two different introductions into the country. The genetic heterogeneity is as noted globally. The identified B- and T-cell epitopes may be considered suitable for future experiments towards the design of vaccines and diagnostics. Continuous monitoring and analysis of the sequences of new cases from India and the other affected countries would be vital to understand the genetic evolution and rates of substitution of the SARS-CoV-2.
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Betacoronavirus/genética , Genoma Viral , COVID-19 , Infecciones por Coronavirus , Epítopos de Linfocito B/genética , Epítopos de Linfocito T/genética , Humanos , India , Modelos Moleculares , Pandemias , Filogenia , Neumonía Viral , Estructura Terciaria de Proteína , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genéticaAsunto(s)
Coronavirus , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Betacoronavirus , COVID-19 , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus , India , Pandemias , Neumonía Viral , ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2RESUMEN
Hematological abnormalities and altered vascular permeability are frequently encountered in Dengue virus infected patients, but the mechanisms that alter platelet-endothelium interactions remain incompletely understood. The DENV NS1 protein has been implicated in adverse disease outcomes. In the present study the role of NS1 protein in affecting the expression of vWF and platelet adhesion properties of endothelial cells was studied in vitro. The results suggest that vWF is down regulated in cultured endothelial cells 6 and 24 h after exposure with increase in vWF levels in culture supernatants at corresponding time points. Ultrastructural studies showed distinct evidence of endothelial cell activation morphology and degranulation of Weibel-Palade bodies in NS1 exposed cells that also showed increased platelet activation physiology. The findings suggest that changes in vWF production and secretion may be induced in endothelial cells exposed to DENV NS1 protein; and play a role in bleeding complications of severe DENV disease.
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We report 3 atypical rubella cases in a family cluster in India. The index case-patient showed only mild febrile illness, whereas the other 2 patients showed acute encephalitis and died of the disease. We confirmed rubella in the index and third cases using next-generation sequencing and IgM.
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Encefalitis Viral/diagnóstico , Fenotipo , Rubéola (Sarampión Alemán)/diagnóstico , Enfermedad Aguda , Anticuerpos Antivirales/inmunología , Biomarcadores , Niño , Preescolar , Encefalitis Viral/inmunología , Encefalitis Viral/virología , Familia , Resultado Fatal , Femenino , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , India , Masculino , ARN Viral , Rubéola (Sarampión Alemán)/inmunología , Rubéola (Sarampión Alemán)/virología , Hermanos , Evaluación de SíntomasRESUMEN
Ebolavirus (EBOV) is the etiology of Ebola hemorrhagic fever (EHF). A major EHF outbreak in 2014-2015 in West Africa claimed >11,000 lives. A licensed vaccine is not available for EHF, although several vaccines have undergone clinical trials. We developed a human adenovirus (Ad) serotype 5-based candidate EHF vaccine based on controlled expression of the EBOV (Makona strain) glycoprotein (GP) as the immunogen. Two clones, AdGP72 and AdGP75, and a control Ad515 vector, were generated and tested for protein expression in vitro and immunogenicity in mice. Eight groups of mice were immunized with three doses of buffer, Ad515, AdGP72, and AdGP75, by two different dose regimens. Three different antigens (AdGP75-infected Vero E6 cell extract and two baculovirus expressed EBOV GP antigens, namely, GP alone or GP with EBOV VP40) were used to evaluate the immune response. Expression studies indicated that full-length GP was cleaved into its component subunits when expressed in mammalian cells through the Ad vectors. Moreover, in coimmunoprecipitation studies, EBOV GP was found to be associated with VP40 when expressed in baculoviruses. The candidate vaccines were immunogenic in mice, as evaluated by enzyme-linked immunosorbent assay using mammalian- or baculovirus-derived antigens. Further characterization and development of the candidate vaccines are warranted.
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Vacunas contra el Virus del Ébola/inmunología , Ebolavirus/inmunología , Glicoproteínas/inmunología , Fiebre Hemorrágica Ebola/terapia , Inmunogenicidad Vacunal/inmunología , Proteínas Virales/inmunología , Adenovirus Humanos/genética , Adenovirus Humanos/inmunología , Animales , Anticuerpos Monoclonales/sangre , Chlorocebus aethiops , Glicoproteínas/genética , Células HEK293 , Fiebre Hemorrágica Ebola/virología , Humanos , Ratones , Ratones Endogámicos BALB C , Células Sf9 , Spodoptera , Vacunas Sintéticas/inmunología , Células Vero , Proteínas Virales/genéticaRESUMEN
BACKGROUND & OBJECTIVES: Differential diagnosis of Crimean-Congo haemorrhagic fever (CCHF) from other acute febrile illnesses with haemorrhagic manifestation is challenging in India. Nosocomial infection is a significant mode of transmission due to exposure of healthcare workers to blood and body fluids of infected patients. Being a risk group 4 virus, laboratory confirmation of infection is not widely available. In such a situation, early identification of potential CCHF patients would be useful in limiting the spread of the disease. The objective of this study was to retrospectively analyse clinical and laboratory findings of CCHF patients that might be useful in early detection of a CCHF case in limited resource settings. METHODS: Retrospective analysis of clinical and laboratory data of patients suspected to have CCHF referred for diagnosis from Gujarat and Rajasthan States of India (2014-2015) was done. Samples were tested using CCHF-specific real time reverse transcription (RT)-PCR and IgM ELISA. RESULTS: Among the 69 patients referred, 21 were laboratory confirmed CCHF cases of whom nine had a history of occupational exposure. No clustering of cases was noted. Platelet count cut-off for detection of positive cases by receiver operating characteristic curve was 21.5×10[9]/l with sensitivity 82.4 per cent and specificity 82.1 per cent. Melaena was a significant clinical presentation in confirmed positive CCHF patients. INTERPRETATION & CONCLUSIONS: The study findings suggest that in endemic areas thrombocytopenia and melaena may be early indicators of CCHF. Further studies are needed to confirm these findings.
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Infección Hospitalaria/diagnóstico , Diagnóstico Diferencial , Virus de la Fiebre Hemorrágica de Crimea-Congo/patogenicidad , Fiebre Hemorrágica de Crimea/diagnóstico , Adolescente , Adulto , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/aislamiento & purificación , Infección Hospitalaria/epidemiología , Infección Hospitalaria/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Personal de Salud , Fiebre Hemorrágica de Crimea/epidemiología , Fiebre Hemorrágica de Crimea/virología , Humanos , India/epidemiología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Acute gastroenteritis outbreak occurred at Pargaon, Maharashtra, India in 1789 cases with an attack rate of 32.5% between November to December 2015. The stool specimens (n = 32) were investigated for different enteric viral agents using conventional methods. Transmission electron microscopy and RNA polyacrylamide gel electrophoresis respectively identified morphologically distinct rotavirus particles in 28% and RNA migration pattern of Group B Rotavirus (GBR) in 72% of the specimens. Reverse transcription polymerase chain reaction and nucleotide sequencing confirmed presence of GBR in 97% of the samples analyzed. The predominance of GBR infections and absence or insignificant presence of other agents confirmed GBR as an etiological agent of the gastroenteritis outbreak occurred in Maharashtra, India.
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Brotes de Enfermedades , Gastroenteritis/etiología , Gastroenteritis/virología , Infecciones por Rotavirus/epidemiología , Rotavirus/aislamiento & purificación , Enfermedad Aguda/epidemiología , Adolescente , Adulto , Niño , Preescolar , Diarrea/virología , Electroforesis en Gel de Poliacrilamida , Heces/virología , Femenino , Gastroenteritis/epidemiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , India/epidemiología , Masculino , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rotavirus/genética , Rotavirus/ultraestructura , Infecciones por Rotavirus/virología , Adulto JovenRESUMEN
Bat-borne viral diseases are a major public health concern among newly emerging infectious diseases which includes severe acute respiratory syndrome, Nipah, Marburg and Ebola virus disease. During the survey for Nipah virus among bats at North-East region of India; Tioman virus (TioV), a new member of the Paramyxoviridae family was isolated from tissues of Pteropus giganteus bats for the first time in India. This isolate was identified and confirmed by RT-PCR, sequence analysis and electron microscopy. A range of vertebrate cell lines were shown to be susceptible to Tioman virus. Negative electron microscopy study revealed the "herringbone" morphology of the nucleocapsid filaments and enveloped particles with distinct envelope projections a characteristic of the Paramyxoviridae family. Sequence analysis of Nucleocapsid gene of TioV demonstrated sequence identity of 99.87% and 99.99% nucleotide and amino acid respectively with of TioV strain isolated in Malaysia, 2001. This report demonstrates the first isolation of Tioman virus from a region where Nipah virus activity has been noticed in the past and recent years. Bat-borne viruses have become serious concern world-wide. A Survey of bats for novel viruses in this region would help in recognizing emerging viruses and combating diseases caused by them.
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Quirópteros/virología , Infecciones por Rubulavirus , Rubulavirus , Animales , Línea Celular , Embrión de Pollo , India , Rubulavirus/clasificación , Rubulavirus/genética , Rubulavirus/aislamiento & purificación , Infecciones por Rubulavirus/veterinaria , Infecciones por Rubulavirus/virologíaRESUMEN
BACKGROUND: Kyasanur Forest disease virus (KFDV) is a tick-borne Flavivirus that causes a severe illness in humans. Disease spectrum can vary from subclinical infection to fatal cases with hemorrhagic complications. The pathology of KFDV remains incompletely understood. METHODS: This study describes the histopathologic and immunohistochemical findings in experimentally infected infant CD-1 mice with an early passage human KFDV isolate. RESULTS: Acute histological changes were primarily seen in the brain. The spectrum of changes included gliosis, inflammatory response, necrosis, neural loss, and syncytium formation in mid and hind brain structures. Microscopic lesions observed in the liver were mainly necrosis and vacuolation of hepatocytes and in small intestine, prominent epithelial cell necrosis. KFDV antigens could be stained by a sensitive immunohistochemical labeling in the same organs. CONCLUSIONS: Findings from this study are suggestive of neuropathology as the main manifestation of an early passaged human KFDV isolate. Importantly, this suggests that KFDV may be causing primarily a neurologic disease and secondary organ damage could be because of disease pathology per se. The use of primary low passage human isolates and neuropathology profile could also be more apt in developing a challenge model for testing potential antivirals and therapeutic agents.
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Infecciones del Sistema Nervioso Central/virología , Virus de la Encefalitis Transmitidos por Garrapatas , Enfermedad del Bosque de Kyasanur/virología , Animales , Animales Recién Nacidos , Encéfalo/patología , Infecciones del Sistema Nervioso Central/patología , Humanos , Enfermedad del Bosque de Kyasanur/patología , RatonesRESUMEN
Survival of Mycobacterium tuberculosis (Mtb) within the host macrophage is mediated through pathogen-dependent inhibition of phagosome-lysosome fusion, which enables bacteria to persist within the immature phagosomal compartment. By employing ultrastructural examination of different field isolates supported by biochemical analysis, we found that some of the Mtb strains were in fact poorly adapted for subsistence within endocytic vesicles of infected macrophages. Instead, through a mechanism involving activation of host cytosolic phospholipase A2, these bacteria rapidly escaped from phagosomes, and established residence in the cytoplasm of the host cell. Interestingly, by facilitating an enhanced suppression of host cellular autophagy, this translocation served as an alternate virulence acquisition mechanism. Thus, our studies reveal plasticity in the adaptation strategies employed by Mtb, for survival in the host macrophage.
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Adaptación Fisiológica/inmunología , Citoplasma/inmunología , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Fagosomas/inmunología , Autofagia/inmunología , Línea Celular Tumoral , Células Cultivadas , Citoplasma/microbiología , Citoplasma/ultraestructura , Interacciones Huésped-Patógeno/inmunología , Humanos , Evasión Inmune/inmunología , Macrófagos/microbiología , Macrófagos/ultraestructura , Microscopía Confocal , Microscopía Electrónica de Transmisión , Mycobacterium tuberculosis/patogenicidad , Mycobacterium tuberculosis/fisiología , Fagocitosis/inmunología , Fagosomas/microbiología , Fagosomas/ultraestructura , Fosfolipasas A2 Citosólicas/inmunología , Fosfolipasas A2 Citosólicas/metabolismo , Vesículas Transportadoras/inmunología , Vesículas Transportadoras/microbiología , Vesículas Transportadoras/ultraestructura , Virulencia/inmunologíaRESUMEN
Staphylococcus aureus is a major human pathogen, first recognized as a leading cause of hospital-acquired infections. Community-associated S. aureus (CA-SA) pose a greater threat due to increase in severity of infection and disease among children and healthy adults. CA-SA strains in India are genetically diverse, among which is the sequence type (ST) 772, which has now spread to Australia, Europe and Japan. Towards understanding the genetic characteristics of ST772, we obtained draft genome sequences of five relevant clinical isolates and studied the properties of their PVL-carrying prophages, whose presence is a defining hallmark of CA-SA. We show that this is a novel prophage, which carries the structural genes of the hlb-carrying prophage and includes the sea enterotoxin. This architecture probably emerged early within the ST772 lineage, at least in India. The sea gene, unique to ST772 PVL, despite having promoter sequence characteristics typical of low expression, appears to be highly expressed during early phase of growth in laboratory conditions. We speculate that this might be a consequence of its novel sequence context. The crippled nature of the hlb-converting prophage in ST772 suggests that widespread mobility of the sea enterotoxin might be a selective force behind its 'transfer' to the PVL prophage. Wild type ST772 strains induced strong proliferative responses as well as high cytotoxic activity against neutrophils, likely mediated by superantigen SEA and the PVL toxin respectively. Both proliferation and cytotoxicity were markedly reduced in a cured ST772 strain indicating the impact of the phage on virulence. The presence of SEA alongside the genes for the immune system-modulating PVL toxin may contribute to the success and virulence of ST772.
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Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Exotoxinas/metabolismo , Genoma Bacteriano/genética , Leucocidinas/metabolismo , Profagos/metabolismo , Análisis de Secuencia de ADN , Staphylococcus aureus/genética , Staphylococcus aureus/virología , Toxinas Bacterianas/genética , Secuencia de Bases , Enterotoxinas/genética , Exotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Humanos , India , Leucocidinas/genética , Datos de Secuencia Molecular , Profagos/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismo , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/ultraestructuraRESUMEN
Human herpesvirus 6B (HHV-6B) primary infections occur in early childhood and establish a life-long latency in the most healthy adults. HHV-6B was detectable in the peripheral blood mononuclear cells (PBMC) and granulocytes by serial genomic DNA dilution PCR till 10 pg of template DNA, in a healthy adult. Epstein Barr virus (EBV) mediated transformation of the PBMC resulted in establishment of a B-cell line. Southern hybridization with the PBMC as well as the cell line DNA showed distinct signals for high copy viral genomes and Gardella gel analysis indicated chromosomal integration of the HHV-6B. Integration site analysis in the PBMC and the cell line indicated an atypical viral integration in non-telomeric region of chromosome 12. Cell free culture medium of the cell line could infect different mononuclear cell lines, naïve or mitogen stimulated PBMC and was found to impart productive infection in a recipient T cell line. An HIV-1 LTR driven luciferase based reporter cell line was made and a single step assay was developed for estimating HHV-6B relative concentration in the culture supernatants. This study thus reports a new infectious HHV-6B isolate with uncommon integration site, spontaneous production from a cell line and also development of a simple relative HHV-6B titer assay.