ABSTRACT
The RNA dependent RNA polymerase (RdRp) plays crucial role in virus life cycle by replicating the viral genome. The SARS-CoV-2 is an RNA virus that rapidly spread worldwide and acquired mutations. This study was carried out to identify mutations in RdRp as the SARS-CoV-2 spread in India. We compared 50217 RdRp sequences reported from India with the first reported RdRp sequence from Wuhan, China to identify 223 mutations acquired among Indian isolates. Our protein modelling study revealed that several mutants can potentially alter stability and flexibility of RdRp. We predicted the potential B cell epitopes contributed by RdRp and identified thirty-six linear continuous and twenty-five discontinuous epitopes. Among 223 RdRp mutants, 44% of them localises in the B cell epitopes region. Altogether, this study highlights the need to identify and characterize the variations in RdRp to understand the impact of these mutations on SARS-CoV-2.
Subject(s)
COVID-19/immunology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/immunology , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Mutation , SARS-CoV-2/enzymology , COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Enzyme Stability/genetics , Humans , India , SARS-CoV-2/genetics , SARS-CoV-2/immunologyABSTRACT
Cross-reactivity and direct killing of target cells remain underexplored for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific CD8+ T cells. Isolation of T cell receptors (TCRs) and overexpression in allogeneic cells allows for extensive T cell reactivity profiling. We identify SARS-CoV-2 RNA-dependent RNA polymerase (RdRp/NSP12) as highly conserved, likely due to its critical role in the virus life cycle. We perform single-cell TCRαß sequencing in human leukocyte antigen (HLA)-A∗02:01-restricted, RdRp-specific T cells from SARS-CoV-2-unexposed individuals. Human T cells expressing these TCRαß constructs kill target cell lines engineered to express full-length RdRp. Three TCR constructs recognize homologous epitopes from common cold coronaviruses, indicating CD8+ T cells can recognize evolutionarily diverse coronaviruses. Analysis of individual TCR clones may help define vaccine epitopes that can induce long-term immunity against SARS-CoV-2 and other coronaviruses.
Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/immunology , HLA-A2 Antigen/immunology , SARS-CoV-2/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , COVID-19/therapy , Cell Culture Techniques , Cross Reactions/immunology , Epitopes, T-Lymphocyte/immunology , HLA-A Antigens/immunology , HLA-A2 Antigen/genetics , Humans , Immunodominant Epitopes/immunology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/virology , RNA, Viral/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
Reverse transcription fluorescence resonance energy transfer-polymerase chain reaction (FRET-PCRs) were designed against the two most common mutations in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (A23403G in the spike protein; C14408T in the RNA-dependent RNA polymerase). Based on high-resolution melting curve analysis, the reverse transcription (RT) FRET-PCRs identified the mutations in american type culture collection control viruses, and feline and human clinical samples. All major makes of PCR machines can perform melting curve analysis and thus further specifically designed FRET-PCRs could enable active surveillance for mutations and variants in countries where genome sequencing is not readily available.
Subject(s)
COVID-19 Serological Testing/methods , Polymerase Chain Reaction , RNA-Dependent RNA Polymerase , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Animals , COVID-19/diagnosis , COVID-19/virology , Cats , Coronavirus RNA-Dependent RNA Polymerase/analysis , Coronavirus RNA-Dependent RNA Polymerase/immunology , Humans , Mutation , RNA, Viral/genetics , SARS-CoV-2/immunology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/immunology , TemperatureABSTRACT
Con el avance de la pandemia por COVID-19, la aparición de pacientes con un segundo episodio confirmado por reacción en cadena de la polimerasa, con transcripción inversa (rt-PCR) compatible con reinfección, puso de manifiesto la falta de recomendaciones para su abordaje.Presentamos un estudio descriptivo multicéntrico retro-prospectivo de una serie de doce casos atendidos entre el 01/06/2020 y el 20/10/2020. En la misma, diez casos presentaron el segundo episodio en un período de tiempo menor a 90 días.Por su complejidad, la confirmación de una reinfección no está al alcance en la práctica diaria. Esto requiere de estudios que incluyan comparaciones genómicas de cepas virales involucradas en ambos episodios, determinación de la infectividad del ARN por cultivo viral y análisis molecular.Es necesario establecer definiciones operativas y algoritmos clínicos para la atención de los segundos episodios
As COVID-19 pandemic progresses, patients with a second confirmed episode by reverse transcription-polymerase chain reaction (RT PCR) compatible with reinfection reveals the lack of recommendations for its approach.A multicenter retro-prospective descriptive work was done of a series of 12 cases evaluated between June 1, 2020 and October 20, 2020. In this study, 10 out of 12 cases presented the second episode occurred in less than 90 days.Due to the diagnosing reinfection complexity, its confirmation is not available in the daily practice, this requires studies, which include viral strains genomic comparisons involved in both episodes, ARN determination infectivity by viral culture and molecular analysis.It is necessary to establish operational and clinical algorithms definitions to assist second episodes
