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1.
Proc Natl Acad Sci U S A ; 119(16): e2117142119, 2022 Apr 19.
Article in English | MEDLINE | ID: covidwho-1774040

ABSTRACT

SignificanceCOVID-19 is a deadly rampaging infectious disease with over 480 million cases worldwide. Unfortunately, effective therapies remain very limited. Novel antiviral agents are urgently needed to combat this global healthcare crisis. Here, we elucidate the structural basis for replicase polyprotein cleavage and substrate specificity of SARS-CoV-2 main protease (Mpro). Through analyzing a series of high-resolution structures of SARS-CoV-2 Mpro throughout the proteolytic process, we demonstrate the molecular mechanism of Mpro in proteolytic processing that confers substrate specificity. Substrate selectivity is revealed using structures of the H41A mutant in complex with six individual native cleavage substrates. Our study underscores the mechanistic function of Mpro in the viral life cycle, which provides structural insights to develop effective inhibitors against this essential target of SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/chemistry , Endopeptidases , Humans , Peptide Hydrolases/genetics , Polyproteins/genetics , Protease Inhibitors/chemistry , SARS-CoV-2/genetics , Substrate Specificity
2.
J Med Virol ; 94(4): 1606-1616, 2022 04.
Article in English | MEDLINE | ID: covidwho-1718406

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has sparked the rapid development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS-CoV-2 Panel combines reverse-transcription polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified data set of 1262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 to April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly specific for the Alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , COVID-19/epidemiology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Genetic Variation , Genome, Viral/genetics , Humans , New York City/epidemiology , Phosphoproteins/genetics , Polyproteins/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics
3.
Antiviral Res ; 198: 105247, 2022 02.
Article in English | MEDLINE | ID: covidwho-1632314

ABSTRACT

Massive usage of antiviral compounds during a pandemic creates an ideal ground for emergence of resistant strains. Remdesivir, a broad-spectrum inhibitor of the viral RNA-dependent RNA polymerase (RdRp), was extensively prescribed under emergency use authorization during the first 18 months of the COVID19 pandemic, before randomized controlled trials showed poor efficacy in hospitalized patients. RdRp mutations conferring resistance to remdesivir are well known from in vitro studies, and the huge SARS-CoV-2 sequencing effort during the ongoing COVID19 pandemic represents an unprecedented opportunity to assess emergence and fitness of antiviral resistance in vivo. We mined the GISAID database to extrapolate the frequency of remdesivir escape mutations. Our analysis reveals very low levels of remdesivir resistance worldwide despite massive usage.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Drug Resistance, Viral/genetics , SARS-CoV-2/genetics , Adenosine Monophosphate/therapeutic use , Alanine/therapeutic use , Drug Repositioning , Genome, Viral/genetics , Humans , Polyproteins/genetics , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/drug effects , Viral Proteins/genetics
4.
Microbiol Spectr ; 9(3): e0165921, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1598527

ABSTRACT

COVID-19 vaccines are currently being administered worldwide and playing a critical role in controlling the pandemic. They have been designed to elicit neutralizing antibodies against Spike protein of the original SARS-CoV-2, and hence they are less effective against SARS-CoV-2 variants with mutated Spike than the original virus. It is possible that novel variants with abilities of enhanced transmissibility and/or immunoevasion will appear in the near future and perfectly escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Several lines of evidence suggest the contribution of CTLs on the viral control in COVID-19, and CTLs target a wide range of proteins involving comparatively conserved nonstructural proteins. Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2 using computational algorithms, HLA-A*24:02 transgenic mice and the peptide-encapsulated liposomes. We focused on pp1a and HLA-A*24:02 because pp1a is relatively conserved and HLA-A*24:02 is predominant in East Asians such as Japanese. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by a number of mutations in the Sequence Read Archive database of SARS-CoV-2 variants. The information of such conserved epitopes might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any SARS-CoV-2 variants by the induction of both anti-Spike neutralizing antibodies and CTLs specific for conserved epitopes. IMPORTANCE COVID-19 vaccines have been designed to elicit neutralizing antibodies against the Spike protein of the original SARS-CoV-2, and hence they are less effective against variants. It is possible that novel variants will appear and escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2. We focused on pp1a and HLA-A*24:02 because pp1a is conserved and HLA-A*24:02 is predominant in East Asians. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by mutations in the database of SARS-CoV-2 variants. The information might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any variants.


Subject(s)
COVID-19/immunology , Epitopes/immunology , HLA-A24 Antigen/genetics , HLA-A24 Antigen/immunology , Mutation , Polyproteins/genetics , SARS-CoV-2/genetics , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/immunology , Epitopes/genetics , HLA-A24 Antigen/isolation & purification , Humans , Mice , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology
5.
J Med Virol ; 94(4): 1606-1616, 2022 04.
Article in English | MEDLINE | ID: covidwho-1589045

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has sparked the rapid development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS-CoV-2 Panel combines reverse-transcription polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified data set of 1262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 to April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly specific for the Alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , COVID-19/epidemiology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Genetic Variation , Genome, Viral/genetics , Humans , New York City/epidemiology , Phosphoproteins/genetics , Polyproteins/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics
6.
Infect Genet Evol ; 97: 105192, 2022 01.
Article in English | MEDLINE | ID: covidwho-1586991

ABSTRACT

The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is the etiopathogenic agent of COVID-19, a condition that has led to a formally recognized pandemic by March 2020 (World Health Organization -WHO). The SARS-CoV-2 genome is constituted of 29,903 base pairs, that code for four structural proteins (N, M, S, and E) and more than 20 non-structural proteins. Mutations in any of these regions, especially in those that encode for the structural proteins, have allowed the identification of diverse lineages around the world, some of them named as Variants of Concern (VOC) and Variants of Interest (VOI), according to the WHO and CDC. In this study, by using Next Generation Sequencing (NGS) technology, we sequenced the SARS-CoV-2 genome of 422 samples from Colombian residents, all of them collected between April 2020 and January 2021. We obtained genetic information from 386 samples, leading us to the identification of 14 new lineages circulating in Colombia, 13 of which were identified for the first time in South America. GH was the predominant GISAID clade in our sample. Most mutations were either missense (53.6%) or synonymous mutations (37.4%), and most genetic changes were located in the ORF1ab gene (63.9%), followed by the S gene (12.9%). In the latter, we identified mutations E484K, L18F, and D614G. Recent evidence suggests that these mutations concede important particularities to the virus, compromising host immunity, the diagnostic test performance, and the effectiveness of some vaccines. Some important lineages containing these mutations are the Alpha, Beta, and Gamma (WHO Label). Further genomic surveillance is important for the understanding of emerging genomic variants and their correlation with disease severity.


Subject(s)
COVID-19/epidemiology , Genome, Viral , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics , COVID-19/transmission , COVID-19/virology , Colombia/epidemiology , Epidemiological Monitoring , Evolution, Molecular , Gene Expression , Humans , Phylogeny , Polyproteins/genetics , Polyproteins/metabolism , SARS-CoV-2/classification , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Time Factors , Viral Proteins/metabolism , Whole Genome Sequencing
7.
Viruses ; 14(1)2022 01 01.
Article in English | MEDLINE | ID: covidwho-1580395

ABSTRACT

In recent months, several SARS-CoV-2 variants have emerged that enhance transmissibility and escape host humoral immunity. Hence, the tracking of viral evolutionary trajectories is clearly of great importance. Little is known about SARS-CoV-2 evolution in nonhuman primate models used to test vaccines and therapies and to model human disease. Viral RNA was sequenced from rectal swabs from Chlorocebus aethiops (African green monkeys) after experimental respiratory SARS-CoV-2 infection. Two distinct patterns of viral evolution were identified that were shared between all collected samples. First, mutations in the furin cleavage site that were initially present in the virus as a consequence of VeroE6 cell culture adaptation were not detected in viral RNA recovered in rectal swabs, confirming the necessity of this motif for viral infection in vivo. Three amino acid changes were also identified; ORF 1a S2103F, and spike D215G and H655Y, which were detected in rectal swabs from all sampled animals. These findings are demonstrative of intra-host SARS-CoV-2 evolution and may identify a host-adapted variant of SARS-CoV-2 that would be useful in future primate models involving SARS-CoV-2 infection.


Subject(s)
COVID-19/virology , SARS-CoV-2/genetics , Animals , Chlorocebus aethiops , Disease Models, Animal , Evolution, Molecular , Mutation , Polyproteins/genetics , RNA, Viral/genetics , Rectum/virology , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Viral Proteins/genetics
8.
J Korean Med Sci ; 36(48): e328, 2021 Dec 13.
Article in English | MEDLINE | ID: covidwho-1572278

ABSTRACT

BACKGROUND: In the coronavirus disease 2019 (COVID-19) pandemic era, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus (Flu), and respiratory syncytial virus (RSV) is important in the rapid differential diagnosis in patients with respiratory symptoms. Three multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been recently developed commercially in Korea: PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech); STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor); and Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene). We evaluated the analytical and clinical performances of these kits. METHODS: A limit of detection tests were performed and cross-reactivity analysis was executed using clinical respiratory samples. Ninety-seven SARS-CoV-2-positive, 201 SARS-CoV-2-negative, 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive nasopharyngeal swabs were tested using the three assays. The AdvanSure™ respiratory viruses rRT-PCR assay (AdvanSure; LG Life Sciences) was used as a comparator assay for RSV. RESULTS: Except in influenza B, in SARS-CoV-2 and influenza A, there were no significant differences in detecting specific genes of the viruses among the three assays. All three kits did not cross-react with common respiratory viruses. All three kits had greater than 92% positive percent agreement and negative percent agreement and ≥ 0.95 kappa value in the detection of SARS-CoV-2 and flu A/B. Allplex detected RSV more sensitively than AdvanSure. CONCLUSION: The overall performance of three multiplex rRT-PCR assays for the concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. These kits will promote prompt differential diagnosis of COVID-19, influenza, and RSV infection in the COVID-19 pandemic era.


Subject(s)
COVID-19/diagnosis , Influenza, Human/diagnosis , Multiplex Polymerase Chain Reaction/methods , Nasopharynx/virology , RNA, Viral/analysis , Respiratory Syncytial Virus Infections/diagnosis , COVID-19/virology , Cross Reactions , Humans , Influenza A virus/genetics , Influenza A virus/isolation & purification , Influenza B virus/genetics , Influenza B virus/isolation & purification , Influenza, Human/virology , Limit of Detection , Nucleocapsid Proteins/genetics , Polyproteins/genetics , RNA, Viral/metabolism , Reagent Kits, Diagnostic , Republic of Korea , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/genetics , Respiratory Syncytial Virus, Human/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/genetics , Viral Proteins/genetics
9.
J Med Virol ; 93(12): 6525-6534, 2021 12.
Article in English | MEDLINE | ID: covidwho-1544299

ABSTRACT

By analyzing newly collected SARS-CoV-2 genomes and comparing them with our previous study about SARS-CoV-2 single nucleotide variants (SNVs) before June 2020, we found that the SNV clustering had changed remarkably since June 2020. Apart from that the group of SNVs became dominant, which is represented by two nonsynonymous mutations A23403G (S:D614G) and C14408T (ORF1ab:P4715L), a few emerging groups of SNVs were recognized with sharply increased monthly incidence ratios of up to 70% in November 2020. Further investigation revealed sets of SNVs specific to patients' ages and/or gender, or strongly associated with mortality. Our logistic regression model explored features contributing to mortality status, including three critical SNVs, G25088T(S:V1176F), T27484C (ORF7a:L31L), and T25A (upstream of ORF1ab), ages above 40 years old, and the male gender. The protein structure analysis indicated that the emerging subgroups of nonsynonymous SNVs and the mortality-related ones were located on the protein surface area. The clashes in protein structure introduced by these mutations might in turn affect the viral pathogenesis through the alteration of protein conformation, leading to a difference in transmission and virulence. Particularly, we explored the fact that nonsynonymous SNVs tended to occur in intrinsic disordered regions of Spike and ORF1ab to significantly increase hydrophobicity, suggesting a potential role in the change of protein folding related to immune evasion.


Subject(s)
COVID-19/mortality , Genome, Viral/genetics , Polymorphism, Single Nucleotide/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Adult , Aged , Aged, 80 and over , COVID-19/pathology , Female , Humans , Male , Middle Aged , Mutation , Polyproteins/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics , Virulence/genetics , Young Adult
10.
Sci Rep ; 11(1): 20471, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1469980

ABSTRACT

Dual-labeled PNA probe used RT-LAMP molecular rapid assay targeting SARS-CoV-2 ORF1ab and N genes was developed, and the analytical, clinical performances for detection of SARS-CoV-2 RNA extracted from clinical nasopharyngeal swab specimens were evaluated in this study. Data showed that this assay is highly specific for SARS-CoV-2, and the absolute detection limit is 1 genomic copy per microliter of viral RNA which can be considered to be comparable to gold-standard molecular diagnostic method real-time reverse transcriptase PCR. Both clinical sensitivity and specificity against a commercial real-time RT-PCR assay were determined as identical. In conclusion, the PNA RT-LAMP assay showed high analytical and clinical accuracy which are identical to real-time RT-PCR which has been routinely used for the detection of SARS-CoV-2.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Coronavirus Nucleocapsid Proteins/genetics , Genes, Viral , Humans , Limit of Detection , Phosphoproteins/genetics , Polyproteins/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral Proteins/genetics
11.
PLoS One ; 16(7): e0255096, 2021.
Article in English | MEDLINE | ID: covidwho-1325440

ABSTRACT

The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19 Serological Testing/methods , COVID-19/diagnosis , SARS-CoV-2/immunology , Single Molecule Imaging/methods , Viral Proteins/genetics , Antibodies, Viral/blood , Base Sequence , COVID-19/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Nucleic Acid Testing/standards , COVID-19 Serological Testing/standards , Enzyme-Linked Immunosorbent Assay , Humans , Immune Sera/chemistry , Immunoglobulin G/blood , Immunoglobulin M/blood , Nasopharynx/virology , Polyproteins/blood , Polyproteins/genetics , RNA, Viral/blood , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Single Molecule Imaging/instrumentation , Viral Proteins/blood
12.
Genes Genomics ; 43(11): 1351-1359, 2021 11.
Article in English | MEDLINE | ID: covidwho-1296973

ABSTRACT

BACKGROUND: COVID-19, as a novel coronavirus disease caused by new coronavirus SARS-CoV-2, spreads all over the world, and brings harm to human in many countries. Humans suffered a lot from both SARS-CoV-2 now and by SARS-CoV in the year 2003. It is important to understand the differences and the relationships between these two types of viruses. OBJECTIVE: To compare relative synonymous codon usage of ORF1ab gene in SARS-CoV-2 and SARS-CoV, relative synonymous codon usage of their genomes are studied in this paper from the bioinformatics perspective. METHODS: The ORF1ab gene, which is an important non-structural polyprotein coding gene and now used for nucleic acid detection markers in many measurement method, in both SARS-CoV-2 (30 strains) and SARS-CoV (20 strains) are considered to be the research object in the present paper. The relative synonymous codon usage values of the ORF1ab gene are calculated to characterize the differences and the evolutionary characteristics among 50 strains. RESULTS: There is a significant difference between SARS-CoV and SARS-CoV-2 when the relative synonymous codon usage value of ORF1ab genes is concerned. The results suggest that codon usage pattern of SARS-CoV is more similar to human than that of the SARS-CoV-2, and that the inner difference in SARS-CoV-2 strains is larger than that of SARS-CoV, which denote the larger diversity exits in the SARS-CoV-2 virus. CONCLUSION: These results show that the relative synonymous codon usage values in the coronavirus could be used for further research on their evolutionary phenomenon.


Subject(s)
Codon Usage/genetics , Polyproteins/genetics , SARS Virus/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics , COVID-19 , Computational Biology , Evolution, Molecular , Genome, Viral , Humans , Open Reading Frames , Phylogeny , SARS-CoV-2/classification
13.
PLoS One ; 16(6): e0249568, 2021.
Article in English | MEDLINE | ID: covidwho-1286866

ABSTRACT

The ongoing COVID-19 pandemic is caused by SARs-CoV-2. The virus is transmitted from person to person through droplet infections i.e. when infected person is in close contact with another person. In January 2020, first report of detection of SARS-CoV-2 in faeces, has made it clear that human wastewater might contain this virus. This may illustrate the probability of environmentally facilitated transmission, mainly the sewage, however, environmental conditions that could facilitate faecal oral transmission is not yet clear. We used existing Pakistan polio environment surveillance network to investigate presence of SARs-CoV-2 using three commercially available kits and E-Gene detection published assay for surety and confirmatory of positivity. A Two-phase separation method is used for sample clarification and concentration. An additional high-speed centrifugation (14000Xg for 30 min) step was introduced, prior RNA extraction, to increase viral RNA yield resulting a decrease in Cq value. A total of 78 wastewater samples collected from 38 districts across Pakistan, 74 wastewater samples from existing polio environment surveillance sites, 3 from drains of COVID-19 infected areas and 1 from COVID 19 quarantine center drainage, were tested for presence of SARs-CoV-2. 21 wastewater samples (27%) from 13 districts turned to be positive on RT-qPCR. SARs-COV-2 RNA positive samples from areas with COVID 19 patients and quarantine center strengthen the findings and use of wastewater surveillance in future. Furthermore, sequence data of partial ORF 1a generated from COVID 19 patient quarantine center drainage sample also reinforce our findings that SARs-CoV-2 can be detected in wastewater. This study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an epidemiologic potential that can be used as supplementary system to monitor viral tracking and circulation in cities with lower COVID-19 testing capacity or heavily populated areas where door-to-door tracing may not be possible. However, attention is needed on virus concentration and detection assay to increase the sensitivity. Development of highly sensitive assay will be an indicator for virus monitoring and to provide early warning signs.


Subject(s)
Environmental Monitoring , RNA, Viral/analysis , SARS-CoV-2/genetics , Waste Water/virology , COVID-19/pathology , COVID-19/transmission , COVID-19/virology , Humans , Pakistan , Polyproteins/genetics , Quarantine , Real-Time Polymerase Chain Reaction , SARS-CoV-2/isolation & purification , Viral Proteins/genetics
14.
Int J Infect Dis ; 106: 395-400, 2021 May.
Article in English | MEDLINE | ID: covidwho-1279604

ABSTRACT

BACKGROUND: India bears the second largest burden of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. A multitude of reverse transcription polymerase chain reaction (RT-PCR) detection assays with disparate gene targets, including automated high-throughput platforms, are available. Varying concordance and interpretation of diagnostic results in this setting can result in significant reporting delays, leading to suboptimal disease management. This article reports the development of a novel ORF1a-based SARS-CoV-2 RT-PCR assay - Viroselect - that shows high concordance with conventional assays and the ability to resolve inconclusive results generated during the peak of the epidemic in Mumbai, India. METHODS: A unique target region within SARS-CoV-2 ORF1a - the non-structural protein 3 (nsp3) region - was used to design and develop the assay. This hypervariable region (1923-3956) between SARS-CoV-2, SARS-CoV-1 and Middle East respiratory syndrome coronavirus was utilized to design the primers and probes for the RT-PCR assay. The concordance of this assay with commonly used emergency use authorization (US Food and Drug Administration) manual kits and an automated high-throughput testing platform was evaluated. Further, a retrospective analysis was carried out using Viroselect on samples reported as 'inconclusive' between April and October 2020. RESULTS: In total, 701 samples were tested. Concordance analysis of 477 samples demonstrated high overall agreement of Viroselect with both manual (87.6%) and automated (84.7%) assays. Also, in the retrospective analysis of 224 additional samples reported as 'inconclusive', Viroselect was able to resolve 100% (19/19) and 93.7% (192/205) of samples which had inconclusive results on manual and automated high-throughput platforms, respectively. CONCLUSION: Viroselect had high concordance with conventional assays, both manual and automated, and has potential to resolve inconclusive samples.


Subject(s)
COVID-19 Testing/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Viral Proteins/genetics , Humans , Limit of Detection , Polyproteins/genetics , Retrospective Studies , SARS-CoV-2/isolation & purification
15.
Virus Res ; 302: 198472, 2021 09.
Article in English | MEDLINE | ID: covidwho-1263387

ABSTRACT

The human ß-coronavirus SARS-CoV-2 epidemic started in late December 2019 in Wuhan, China. It causes Covid-19 disease which has become pandemic. Each of the five-known human ß-coronaviruses has four major structural proteins (E, M, N and S) and 16 non-structural proteins encoded by ORF1a and ORF1b together (ORF1ab) that are involved in virus pathogenicity and infectivity. Here, we performed detailed positive selection analyses for those six genes among the four previously known human ß-coronaviruses and within 38 SARS-CoV-2 genomes to assess signatures of adaptive evolution using maximum likelihood approaches. Our results suggest that three genes (E, S and ORF1ab genes) are under strong signatures of positive selection among human ß-coronavirus, influencing codons that are located in functional important protein domains. The E protein-coding gene showed signatures of positive selection in two sites, Asp 66 and Ser 68, located inside a putative transmembrane α-helical domain C-terminal part, which is preferentially composed by hydrophilic residues. Such Asp and Ser sites substitutions (hydrophilic residues) increase the stability of the transmembrane domain in SARS-CoV-2. Moreover, substitutions in the spike (S) protein S1 N-terminal domain have been found, all of them were located on the S protein surface, suggesting their importance in viral transmissibility and survival. Furthermore, evidence of strong positive selection was detected in three of the SARS-CoV-2 nonstructural proteins (NSP1, NSP3, NSP16), which are encoded by ORF1ab and play vital roles in suppressing host translation machinery, viral replication and transcription and inhibiting the host immune response. These results are insightful to assess the role of positive selection in the SARS-CoV-2 encoded proteins, which will allow to better understand the virulent pathogenicity of the virus and potentially identifying targets for drug or vaccine strategy design.


Subject(s)
COVID-19/virology , Coronavirus Envelope Proteins/genetics , Pandemics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics , Amino Acid Substitution , COVID-19/epidemiology , Humans , Polyproteins/genetics , Protein Domains , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Virulence/genetics , Virus Replication
16.
Virol J ; 18(1): 110, 2021 06 02.
Article in English | MEDLINE | ID: covidwho-1255943

ABSTRACT

BACKGROUND: The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply. AIM: Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol). METHODS: Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen. RESULTS: Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits. CONCLUSION: The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Coronavirus Envelope Proteins/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Limit of Detection , Polyproteins/genetics , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Viral Proteins/genetics
17.
Carbohydr Polym ; 268: 118259, 2021 Sep 15.
Article in English | MEDLINE | ID: covidwho-1242891

ABSTRACT

Nitrocellulose (NC) membrane can have value-added applications for lateral flow assay (LFA)-based diagnostic tools, which has great potential for the detection of pathogens, such as COVID-19, in different environments. However, poor sensitivity of the NC membrane based LFA limits its further application in many cases. Herein, we developed a facile method for LFA sensitivity enhancement, by incorporating two-sugar barrier into LFAs: one between the conjugation pad and the test line, and the other between the test line and the control line. ORF1ab nucleic acid of COVID-19 was used as the model target to demonstrate the concept on the HF120 membrane. Results show that at optimum conditions, the two sugar barrier LFAs have a detection limit of 0.5 nM, which is compared to that of 2.5 nM for the control LFA, achieving a 5-fold sensitivity increase. This low cost, easy-to-fabricate and easy-to-integrate LFA method may have potential applications in other cellulose paper-based platforms.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Collodion/chemistry , RNA, Messenger/analysis , Sugars/chemistry , Viral Proteins/genetics , COVID-19 Nucleic Acid Testing/instrumentation , DNA/chemistry , DNA Probes/chemistry , Gold/chemistry , Limit of Detection , Metal Nanoparticles/chemistry , Polyproteins/genetics , SARS-CoV-2/chemistry , Sensitivity and Specificity
18.
PLoS Biol ; 19(5): e3001236, 2021 05.
Article in English | MEDLINE | ID: covidwho-1220158

ABSTRACT

With the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants that may increase transmissibility and/or cause escape from immune responses, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant, first detected in the United Kingdom, could be serendipitously detected by the Thermo Fisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern (VOC) that lack spike Δ69-70, such as B.1.351 (also 501Y.V2), detected in South Africa, and P.1 (also 501Y.V3), recently detected in Brazil. We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all 3 variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open-source PCR assay to detect SARS-CoV-2 VOC. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, and P.1.


Subject(s)
COVID-19/virology , SARS-CoV-2/genetics , COVID-19/diagnosis , COVID-19/genetics , DNA Primers , Humans , Multiplex Polymerase Chain Reaction/methods , Mutation , Polyproteins/genetics , Viral Proteins/genetics
19.
Virol Sin ; 35(6): 758-767, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-1217478

ABSTRACT

Reverse transcription-polymerase chain reaction (RT-PCR) is an essential method for specific diagnosis of SARS-CoV-2 infection. Unfortunately, false negative test results are often reported. In this study, we attempted to determine the principal causes leading to false negative results of RT-PCR detection of SARS-CoV-2 RNAs in respiratory tract specimens. Multiple sputum and throat swab specimens from 161 confirmed COVID-19 patients were tested with a commercial fluorescent RT-PCR kit targeting the ORF1ab and N regions of SARS-CoV-2 genome. The RNA level of a cellular housekeeping gene ribonuclease P/MRP subunit p30 (RPP30) in these specimens was also assessed by RT-PCR. Data for a total of 1052 samples were retrospectively re-analyzed and a strong association between positive results in SARS-CoV-2 RNA tests and high level of RPP30 RNA in respiratory tract specimens was revealed. By using the ROC-AUC analysis, we identified Ct cutoff values for RPP30 RT-PCR which predicted false negative results for SARS-CoV-2 RT-PCR with high sensitivity (95.03%-95.26%) and specificity (83.72%-98.55%) for respective combination of specimen type and amplification reaction. Using these Ct cutoff values, false negative results could be reliably identified. Therefore, the presence of cellular materials, likely infected host cells, are essential for correct SARS-CoV-2 RNA detection by RT-PCR in patient specimens. RPP30 could serve as an indicator for cellular content, or a surrogate indicator for specimen quality. In addition, our results demonstrated that false negativity accounted for a vast majority of contradicting results in SARS-CoV-2 RNA test by RT-PCR.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , RNA, Viral/genetics , SARS-CoV-2/genetics , Autoantigens/genetics , COVID-19/epidemiology , COVID-19/virology , China/epidemiology , Humans , Negative Results , Polyproteins/genetics , RNA, Viral/isolation & purification , Reference Standards , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction/methods , Ribonuclease P/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Viral Proteins/genetics
20.
FASEB J ; 35(5): e21573, 2021 05.
Article in English | MEDLINE | ID: covidwho-1207324

ABSTRACT

Coronavirus (CoV) 3-chymotrypsin (C)-like cysteine protease (3CLpro ) is a target for anti-CoV drug development and drug repurposing because along with papain-like protease, it cleaves CoV-encoded polyproteins (pp1a and pp1ab) into nonstructural proteins (nsps) for viral replication. However, the cleavage sites of 3CLpro and their relevant nsps remain unclear, which is the subject of this perspective. Here, we address the subject from three standpoints. First, we explore the inconsistency in the cleavage sites and relevant nsps across CoVs, and investigate the function of nsp11. Second, we consider the nsp16 mRNA overlapping of the spike protein mRNA, and analyze the effect of this overlapping on mRNA vaccines. Finally, we study nsp12, whose existence depends on ribosomal frameshifting, and investigate whether 3CLpro requires a large number of inhibitors to achieve full inhibition. This perspective helps us to clarify viral replication and is useful for developing anti-CoV drugs with 3CLpro as a target in the current coronavirus disease 2019 (COVID-19) pandemic.


Subject(s)
Coronavirus 3C Proteases/metabolism , SARS-CoV-2/metabolism , Viral Proteins/metabolism , Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Drug Development/methods , Polyproteins/chemistry , Polyproteins/genetics , Polyproteins/metabolism , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Vaccines, Synthetic/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics
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