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SARS-CoV-2 Morphology, Genomic Organisation and Lifecycle
Published in Srijan Goswami, Chiranjeeb Dey, COVID-19 and SARS-CoV-2, 2022
Srijan Goswami, Ushmita Gupta Bakshi
But if the translation process while proceeding through OFR1a frameshifts to a region of ORF1b, the resultant protein thus formed is pp1ab. Polyprotein 1ab is the hybrid of ORF1a and ORF1b. These polyproteins are further proteolyzed into numerous smaller proteins and play a critical role in viral replication and transcription. Proteins involved in replication are called replicase complex while the proteins involved in transcription are called transcriptase complex. All these proteins combine with viral genomic RNA (sense strand) and facilitate replication. When the +ssRNA of the coronavirus replicates, an antisense RNA is produced. The conversion of the sense RNA into antisense RNA is very much important for the lifecycle of the virus. The antisense RNA is important because:It can be replicated back to sense RNA, which is essentially the same thing that entered with the original virus during uncoating.This antisense RNA can be transcribed through a method called discontinuous transcription. Discontinuous transcription of antisense RNA generates a diverse range of mRNAs that can then be translated into different proteins.
The Evolution of COVID-19 Diagnostics
Published in Debmalya Barh, Kenneth Lundstrom, COVID-19, 2022
Praveen Rai, Ballamoole Krishna Kumar, Deekshit Vijaya Kumar, Prashant Kumar, Anoop Kumar, Shashi Kumar Shetty, Biswajit Maiti
As per the WHO recommendation, the most widely used nucleic acid–based test for the detection of SARS-CoV-2 is RT-qPCR [19]. Several RT-qPCR assays targeting different genes of the SARS-CoV-2 genome, such as RdRp, N, E, and S genes, and ORF1b or ORF8 regions, have been used for the detection of SARS-CoV-2 from clinical samples. However, the WHO recommends an RT-qPCR-based assay targeting the E gene for screening the RdRp gene to confirm SARS-CoV-2. While the US Centers for Disease Control and Prevention (CDC) recommends an RT-qPCR assay based on two nucleocapsid protein genes (N1, N2) [19], to minimize the chances of false-positive results, CDC has developed a new RT-qPCR diagnostic panel including No Template Control (NTC) and Human Specimen Control (HSC). The method has also undergone tremendous modifications to improve the sensitivity, specificity, and feasibility of the technique. A multiplex RT-qPCR has been developed recently, targeting different regions of SARS-CoV-2 and seasonal influenza virus simultaneously with a limited quantification range between 5 and 10 copies per reaction for influenza and SARS-CoV-2, respectively [20]. Furthermore, a significant improvement has been made in the technique to achieve the results with greater accuracy in a short period of time at a low cost to detect SARS-CoV-2 RNA directly from samples without involving an extraction procedure [21]. The HID-RT-PCR (Heat Inactivated Direct-RT-PCR) developed in this study had an accuracy, sensitivity and specificity of 98.8%, with a limit of detection of 0.009 TCID50/ml for the ORF1 and 0.003 TCID50/ml for E genes.
Other Positive Single-Stranded RNA Viruses
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Figure 30.1 presents a portrait of typical representative and schematic cartoons of an astrovirus. The virions are spherical nonenveloped T = 3 icosahedrons of 28–30 nm in diameter, according to the most recent ICTV report (Bosch et al. 2012). A distinctive five- or six-pointed star is discernible on the surface of about 10% of virions. The virions derived from cell culture are up to 41 nm in diameter, with well-defined surface spikes. The surface projections are small, and the surface appears rough, where spikes are protruding from the 30 vertices. As shown in Figure 30.2, the astrovirus genome is arranged in three ORFs: ORF1a and ORF1b at the 5’ end encoding the nonstructural proteins and ORF2 at the 3’ end encoding the structural proteins (Bosch et al. 2012). The VP90 capsid precursor protein undergoes C-terminal cleavages by host caspases to generate VP70 during virus maturation as 180 copies of VP70 per particle. The infectious particles are generated by further cleavages of VP70 by extracellular proteases resulting in three structural proteins, VP34, VP27, and VP25 (Dryden et al. 2012).
Reply to letter entitled ‘Diagnosis of COVID-19: hemagglutinin-esterase gene is used for molecular assays?’
Published in Expert Review of Molecular Diagnostics, 2022
We heartily thank the authors for reading our paper so meticulously and bringing this point into our notice. We appreciate your effort. The authors of the ‘Diagnosis of COVID-19: hemagglutinin-esterase gene is used for molecular assays?’ are referring to the para “Traditionally, the ideal targets of RT-PCR assays are the conserved and/or copiously expressed genes encoding the structural proteins Spike (S), Envelope (E), and Nucleocapsid (N) genes, the non-structural protein RdRp (RNA-dependent RNA polymerase) gene, replicase open reading frame (ORF) 1a/b genes, ORF1b-nsp14 genes, hemagglutinin-esterase (HE), and helicase genes. Among all, Hel/RdRp assays have the highest sensitivity and specificity. RdRp is used for confirmation following an analysis of the E gene [13,15,17].” The protocols of several RT-PCR assays have recently been made available online. Nucleocapsid (N) protein gene as a molecular target for real qRT-PCR assay is recommended by the CDC USA. Some regions of the ORF1ab gene are highly conserved in the subgenus Sarbecoviruses and hence are considered to be an appropriate target sequence for RT-PCR [15] in our review ‘Recent advances in the diagnosis of COVID-19: a bird’s eye view’ published in “Expert Review of Molecular Diagnostics”.
The immunology of SARS-CoV-2 infection, the potential antibody based treatments and vaccination strategies
Published in Expert Review of Anti-infective Therapy, 2021
Zahra Payandeh, Niloufar Mohammadkhani, Mohsen Nabi Afjadi, Saeed Khalili, Masoumeh Rajabibazl, Zahra Houjaghani, Masoomeh Dadkhah
Four structural genes encode the structural proteins of virus, including spike (S), envelope (E), membrane (M), and nucleocapsid (N) genes. The M, E, and N proteins are a part of the nucleocapsid of viral particles. The ORF1a/b gene is the largest gene in the SARS-CoV-2 genome which is responsible to encode the protein phosphatase 1a (pp1a) and 15 non-structural proteins (nsps). Moreover, the orf1a gene contains 10 nsps which encodes pp1a protein [16]. The spike protein consisted of two main subdomains called S1 and S2. The S1 subunit consists of the receptor-binding domain (RBD), which mediates the virus entry into sensitive cells through the ACE2 receptor. The S1 domain of the SARS-CoV-2 shares high similarity (about 70%) with that of human SARS-CoVs. The amino acids in the RBD have the highest number of variations. Lysine 31 residue on the human-ACE2 receptor critically recognizes the glutamine 394 residue in the RBD [17]. The single N501T mutation in SARS-CoV-2’s spike protein may have a significant role in enhanced binding affinity toward ACE2 [18] (Figure 1).
The laboratory’s role in combating COVID-19
Published in Critical Reviews in Clinical Laboratory Sciences, 2020
The SARS-CoV-2 virus was first identified by quantitative polymerase chain reaction (qPCR) analysis with pan-coronavirus primers on 30 December 2019, in bronchoalveolar lavage samples from a patient with pneumonia of unknown etiology [2,4]. The viral genome was sequenced within a week and found to have 96% homology with the bat SARS-like coronavirus strain BatCov RaTG13 [2,4,57]. Sequence homology of SARS-CoV-2 with SARS-CoV and MERS-CoV is 79% and 50%, respectively [3,4]. More recently, several coronaviruses isolated from pangolins have shown 85–92% sequence similarity to SARS-CoV-2 [58]. Importantly, the receptor binding domain of S protein SARS-CoV-2 has 97% amino acid similarity to the protein isolated from the pangolin [58]. These data suggest that bats might serve as the reservoir for SARS-CoV-2, whereas pangolins may serve as an intermediate host. Whole genome sequencing analysis of 104 strains of the SARS-CoV-2 isolated from patients in different locations in China from December 2019 to mid-February shows that these strains exhibit 99.9% homology, suggesting that these viral genomes are relatively stable during transmission [2]. A similar result was found in 95 full-length genomic sequences of SARS-CoV-2 strains from the NCBI and GISAID databases, although some variation hotspots have been identified, such as nt28144 in ORF8 and nt8782 in ORF1a, both of which may have a mutation rate of up to 30% [59]. Phylogenetic network analyses of complete SARS-CoV-2 genomes revealed three central variants distinguished by amino acid changes and geographically-specific distribution of these variants [60–62].