Molecular evolution and genomic characteristics of genotype H1 of measles virus.

Measles is one of the most infectious diseases of humans. It is caused by the measles virus (MeV) and can lead to serious illness, lifelong complications, and even death. Whole-genome sequencing (WGS) is now available to study molecular epidemiology and identify MeV transmission pathways. In the present study, WGS of 23 MeV strains of genotype H1, collected in Mainland China between 2006 and 2018, were generated and compared to 31 WGSs from the public domain to analyze genomic characteristics, evolutionary rates and date of emergence of H1 genotype. The noncoding region between M and F protein genes (M/F NCR) was the most variable region throughout the genome. Although the nucleotide substitution rate of H1 WGS was around 0.75 × 10-3 substitution per site per year, the M/F NCR had an evolutionary rate three times higher, with 2.44 × 10-3 substitution per site per year. Phylogenetic analysis identified three distinct genetic groups. The Time of the Most Recent Common Ancestor (TMRCA) of H1 genotype was estimated at approximately 1988, while the first genetic group appeared around 1995 followed by two other genetic groups in 1999-2002. Bayesian skyline plot showed that the genetic diversity of the H1 genotype remained stable even though the number of MeV cases decreased 50 times between 2014 (52 628) and 2020 (993). The current coronavirus disease 2019 (COVID-19) pandemic might have some effect on the measles epidemic and further studies will be necessary to assess the genetic diversity of the H1 genotype in a post-COVID area.

Diagnostic evaluation of qRT-PCR-based kit and dPCR-based kit for COVID-19.

BACKGROUND: Coronavirus disease of 2019 (COVID-19) is well known as a fatal disease, first discovered at Wuhan in China, ranging from mild to death, such as shortness of breath and fever. Early diagnosis of COVID-19 is a crucial point in preventing global prevalence. OBJECTIVE: We aimed to evaluate the diagnostic competency and efficiency with the Allplex™ 2019-nCoV Assay kit and the Dr. PCR 20 K COVID-19 Detection kit, designed based on the qRT-PCR and dPCR technologies, respectively. METHODS: A total of 30 negative and 20 COVID-19 positive specimens were assigned to the diagnostic test by using different COVID-19 diagnosis kits. Diagnostic accuracy was measured by statistical testing with sensitivity, specificity, and co-efficiency calculations. RESULTS: Comparing both diagnostic kits, we confirmed that the diagnostic results of 30 negative and 20 positive cases were the same pre-diagnostic results. The diagnostic statistics test results were perfectly matched with value (1). Cohen's Kappa coefficient was demonstrated that the given kits in two different ways were "almost perfect" with value (1). In evaluating the detection capability, the dilutional linearity experiments substantiate that the Dr. PCR 20 K COVID-19 Detection kit could detect SARS-CoV-2 viral load at a concentration ten times lower than that of the Allplex™ 2019-nCoV Assay kit. CONCLUSIONS: In this study, we propose that the dPCR diagnosis using LOAA dPCR could be a powerful method for COVID-19 point-of-care tests requiring immediate diagnosis in a limited time and space through the advantages of relatively low sample concentration and small equipment size compared to conventional qRT-PCR.

Comparative genetic analyses of Korean bat coronaviruses with SARS-CoV and the newly emerged SARS-CoV-2.

BACKGROUND: Bats have been considered natural reservoirs for several pathogenic human coronaviruses (CoVs) in the last two decades. Recently, a bat CoV was detected in the Republic of Korea; its entire genome was sequenced and reported to be genetically similar to that of the severe acute respiratory syndrome CoV (SARS-CoV). OBJECTIVES: The objective of this study was to compare the genetic sequences of SARS-CoV, SARS-CoV-2, and the two Korean bat CoV strains 16BO133 and B15-21, to estimate the likelihood of an interaction between the Korean bat CoVs and the human angiotensin-converting enzyme 2 (ACE2) receptor. METHODS: The phylogenetic analysis was conducted with the maximum-likelihood (ML) method using MEGA 7 software. The Korean bat CoVs receptor binding domain (RBD) of the spike protein was analyzed by comparative homology modeling using the SWISS-MODEL server. The binding energies of the complexes were calculated using PRODIGY and MM/GBGA. RESULTS: Phylogenetic analyses of the entire RNA-dependent RNA polymerase, spike regions, and the complete genome revealed that the Korean CoVs, along with SARS-CoV and SARS-CoV-2, belong to the subgenus Sarbecovirus, within BetaCoVs. However, the two Korean CoVs were distinct from SARS-CoV-2. Specifically, the spike gene of the Korean CoVs, which is involved in host infection, differed from that of SARS-CoV-2, showing only 66.8%-67.0% nucleotide homology and presented deletions within the RBD, particularly within regions critical for cross-species transmission and that mediate interaction with ACE2. Binding free energy calculation revealed that the binding affinity of Korean bat CoV RBD to hACE2 was drastically lower than that of SARS-CoV and SARS-CoV-2. CONCLUSIONS: These results suggest that Korean bat CoVs are unlikely to bind to the human ACE2 receptor.

Recombination events are concentrated in the spike protein region of Betacoronaviruses.

The Betacoronaviruses comprise multiple subgenera whose members have been implicated in human disease. As with SARS, MERS and now SARS-CoV-2, the origin and emergence of new variants are often attributed to events of recombination that alter host tropism or disease severity. In most cases, recombination has been detected by searches for excessively similar genomic regions in divergent strains; however, such analyses are complicated by the high mutation rates of RNA viruses, which can produce sequence similarities in distant strains by convergent mutations. By applying a genome-wide approach that examines the source of individual polymorphisms and that can be tested against null models in which recombination is absent and homoplasies can arise only by convergent mutations, we examine the extent and limits of recombination in Betacoronaviruses. We find that recombination accounts for nearly 40% of the polymorphisms circulating in populations and that gene exchange occurs almost exclusively among strains belonging to the same subgenus. Although experimental studies have shown that recombinational exchanges occur at random along the coronaviral genome, in nature, they are vastly overrepresented in regions controlling viral interaction with host cells.

CRISPR/Cas12a-mediated gold nanoparticle aggregation for colorimetric detection of SARS-CoV-2.

The trans-cleavage activity of the target-activated CRISPR/Cas12a liberated an RNA crosslinker from a molecular transducer, which facilitated the assembly of gold nanoparticles. Integration of the molecular transducer with isothermal amplification and CRISPR/Cas12a resulted in visual detection of the N gene and E gene of SARS-CoV-2 in 45 min.

Clinical performance of Roche cobas 6800, Luminex ARIES, MiRXES Fortitude Kit 2.1, Altona RealStar, and Applied Biosystems TaqPath for SARS-CoV-2 detection in nasopharyngeal swabs.

We compared the performance of five assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection on nasopharyngeal swab samples: Roche "cobas," Luminex "ARIES," MiRXES "Fortitude," Altona "RealStar," and Thermo Fisher Scientific "TaqPath." A total of 94 nasopharyngeal swab samples were obtained from 80 confirmed coronavirus disease 2019 cases in the first 2 weeks of illness (median, 7 days; range, 2-14 days) and 14 healthy controls. After collection, all samples were transported to the hospital clinical laboratory within 24 h. These samples were tested on all five assays within 3 days of sample receipt. Of the 94 samples, 69 yielded the same result on all platforms, resulting in an agreement of 73.4% (69 of 94). Of these, 14 were the healthy control swabs which all tested negative, demonstrating good specificity across all platforms. The ARIES assay had the lowest detection rate (68.8%), followed by Fortitude (85.0%), RealStar (86.3%), cobas (95.0%), and TaqPath (100%). Statistically significant differences were observed for ARIES, Fortitude, and RealStar when compared against the best performing TaqPath using McNemar's χ2 test. A consensus result was established based on the results obtained by the cobas, Fortitude, RealStar, and TaqPath. Six discrepancies had failed to reach a consensus and were adjudicated using the Cepheid Xpert Xpress SARS-CoV-2. Overall, the TaqPath and cobas assays were the most sensitive at detecting their designated SARS-CoV-2 gene targets. On the other hand, the ARIES assay was the least sensitive, thus warranting the need for assay re-optimization before go-live at the testing laboratory.

A rapid, accurate, scalable, and portable testing system for COVID-19 diagnosis.

The need for rapid, accurate, and scalable testing systems for COVID-19 diagnosis is clear and urgent. Here, we report a rapid Scalable and Portable Testing (SPOT) system consisting of a rapid, highly sensitive, and accurate assay and a battery-powered portable device for COVID-19 diagnosis. The SPOT assay comprises a one-pot reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) followed by PfAgo-based target sequence detection. It is capable of detecting the N gene and E gene in a multiplexed reaction with the limit of detection (LoD) of 0.44 copies/µL and 1.09 copies/µL, respectively, in SARS-CoV-2 virus-spiked saliva samples within 30 min. Moreover, the SPOT system is used to analyze 104 clinical saliva samples and identified 28/30 (93.3% sensitivity) SARS-CoV-2 positive samples (100% sensitivity if LoD is considered) and 73/74 (98.6% specificity) SARS-CoV-2 negative samples. This combination of speed, accuracy, sensitivity, and portability will enable high-volume, low-cost access to areas in need of urgent COVID-19 testing capabilities.

Saliva for diagnosis of SARS-CoV-2: First report from India.

There are very few studies in search of an alternate and convenient diagnostic tool which can substitute nasopharyngeal swab (NPS) specimen for detection of SARS-CoV-2. In the study we analyzed, the comparison and agreement between the feasibility of using the saliva in comparison to NPS for diagnosis of SARS-CoV-2. A total number of 74 patients were enrolled for this study. We analyzed and compared the NPS and saliva specimen collected within 48 h after the symptom onset. We carried out real-time quantitative polymerase chain reaction, gene sequencing for the detection and determination SARS-CoV-2 specific genes. Phylogenetic tree was constructed to establish the isolation of viral RNA from saliva. We used the Bland-Altman model to identify the agreement between two specimens. This study showed a lower cycle threshold (CT ) mean value for the detection of SARS-CoV-2 ORF1 gene (mean, 27.07; 95% confidence interval [CI], 25.62 to 28.52) in saliva methods than that of NPS (mean 28.24; 95% CI, 26.62 to 29.85) specimen although the difference is statistically nonsignificant (p > .05). Bland-Altman analysis produced relatively smaller bias and high agreement between these two clinical specimens. Phylogenetic analysis with the RdRp and S gene confirmed the presence of SARS-CoV-2 in the saliva samples. Saliva represented a promising tool in COVID-19 diagnosis and the collection method would reduce the exposure risk of frontline health workers which is one of the major concerns in primary healthcare settings.

Novel Negative Sense Genes in the RNA Genome of Coronaviruses.

The coronavirus family consists of lipid-containing envelope viruses that have a single-stranded RNA genome that encodes 25-30 proteins in different viruses by the mechanism of positive-polarity strategy. In addition, extended open reading trnslation frames (ORFs, genes) located in a negative-sense orientation were found in the genomes of coronaviruses. The size of negative-sense genes varies in the range of 150-450 nt, which corresponds to polypeptides encoded by negative-polarity genes (negative gene proteins, NGP) with m. m. 5-30 × 103 kDa. Coronaviruses show marked differences from virus to virus in the number of negative genes detected. These negative-sense genes in the coronavirus genome allow this family to be considered as viruses developing an ambisense genome strategy.

SARS-CoV-2 variants combining spike mutations and the absence of ORF8 may be more transmissible and require close monitoring.

The SARS-CoV-2 Variant of Concern 202012/01 (VOC-202012/01) emerged in southeast England and rapidly spread worldwide. This variant is believed to be more transmissible, with all attention being given to its spike mutations. However, VOC-202012/01 has also a mutation (Q27stop) that truncates the ORF8, a likely immune evasion protein. Removal of ORF8 changes the clinical outset of the disease, which may affect the virus transmissibility. Here I provide a detailed analysis of all reported ORF8-deficient lineages found in the background of relevant spike mutations, identified among 231,433 SARS-CoV-2 genomes. I found 19 ORF8 nonsense mutations, most of them occurring in the 5' half of the gene. The ORF8-deficient lineages were rare, representing 0.67% of sequenced genomes. Nevertheless, I identified two clusters of related sequences that emerged recently and spread in different countries. The widespread D614G spike mutation was found in most ORF-deficient lineages. Although less frequent, HV69-70del and L5F spike mutations occurred in the background of six different ORF8 nonsense mutations. I also confirmed that VOC-202012/01 is the ORF8-deficient variant with more spike mutations reported to date, although other variants could have up to six spike mutations, some of putative biological relevance. Overall, these results suggest that monitoring ORF8-deficient lineages is important for the progression of the COVID-19 pandemic, particularly when associated with relevant spike mutations.

Validation of SARS CoV-2 detection by real-time PCR in matched pooled and deconvoluted clinical samples before and after nucleic acid extraction: a study in tertiary care hospital of North India.

The diagnosis of coronavirus disease-19 (COVID-19) relies on the detection of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) RNA by real-time reverse-transcription polymerase chain reaction in respiratory samples. Rapid increase in the COVID-19 cases across the world requires fast and efficient testing as testing capacity is a bottleneck in diagnosis. In this context, pooling strategy can be opted for rapid testing in a cost-effective manner. In this study, the authors have optimized and compared the effect of pooling (5 and 10 samples) before and after nucleic acid extraction. It was concluded that there was no significant difference in the SARS CoV-2 RNA detection in the pools prepared at sample or RNA level. Even after pooling, 10-fold dilution was detectable with 3-cycle threshold value change in both type of pools when compared with individual samples. Hence, sample pool size of 10 can be used in low-prevalent areas, and testing capacity can be substantially increased.

CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19.

Accurate, reliable and rapid detection of SARS-CoV-2 is essential not only for correct diagnosis of individual COVID-19 disease but also for the development of a rational strategy aimed at lifting confinement restrictions and preparing for possible recurrent waves of viral infections. We have used the MIQE guidelines to develop two versions of a unique five plex RT-qPCR test, termed CoV2-ID, that allows the detection of three viral target genes, a human internal control for confirming the presence of human cells in a sample and a control artificial RNA for quality assessment and potential quantification. Viral targets can be detected either individually with separate fluorophores or jointly using the same fluorophore, thus increasing the test's reliability and sensitivity. It is robust, can consistently detect two copies of viral RNA, with a limit of detection of a single copy and can be completed in around 15 min. It was 100% sensitive and 100% specific when tested on 23 RNA samples extracted from COVID-19 positive patients and five COVID-19 negative patients. We also propose using multiple cycle fluorescence detection, rather than real-time PCR to reduce significantly the time taken to complete the assay as well as assuage the misunderstandings underlying the use of quantification cycles (Cq). Finally, we have designed an assay for the detection of the D614G mutation and show that all of the samples isolated in the Chelmsford, Essex area between mid-April and June 2020, have the mutant genotype whereas a sample originating in Australia was infected with the wild type genotype.

Global analysis of more than 50,000 SARS-CoV-2 genomes reveals epistasis between eight viral genes.

Genome-wide epistasis analysis is a powerful tool to infer gene interactions, which can guide drug and vaccine development and lead to deeper understanding of microbial pathogenesis. We have considered all complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes deposited in the Global Initiative on Sharing All Influenza Data (GISAID) repository until four different cutoff dates, and used direct coupling analysis together with an assumption of quasi-linkage equilibrium to infer epistatic contributions to fitness from polymorphic loci. We find eight interactions, of which three are between pairs where one locus lies in gene ORF3a, both loci holding nonsynonymous mutations. We also find interactions between two loci in gene nsp13, both holding nonsynonymous mutations, and four interactions involving one locus holding a synonymous mutation. Altogether, we infer interactions between loci in viral genes ORF3a and nsp2, nsp12, and nsp6, between ORF8 and nsp4, and between loci in genes nsp2, nsp13, and nsp14. The paper opens the prospect to use prominent epistatically linked pairs as a starting point to search for combinatorial weaknesses of recombinant viral pathogens.

SARS-CoV-2: Comparative analysis of different RNA extraction methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.

High-resolution structures of the SARS-CoV-2 2'-O-methyltransferase reveal strategies for structure-based inhibitor design.

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.

Chemosensory Dysfunction in COVID-19: Integration of Genetic and Epidemiological Data Points to D614G Spike Protein Variant as a Contributing Factor.

After several months of rapid pandemic expansion, it is now apparent that the SARS-CoV-2 coronavirus interferes with smell and taste sensation in a substantial proportion of COVID-19 patients. Recent epidemiological data documented intriguing differences in prevalence of chemosensory dysfunctions between different world regions. Viral genetic factors as well as host genetic factors appear to be relevant; however, it is not yet known which mutations or polymorphisms actually contribute to such phenotypic differences between populations. Here, we discuss recent genetic and epidemiological data on the D614G spike protein variant and assess whether current evidence is consistent with the notion that this single nucleotide polymorphism augments chemosensory impairments in COVID-19 patients. We hypothesize that this spike variant is an important viral genetic factor that facilitates infection of chemosensory epithelia, possibly acting together with yet to be identified host factors, and thereby increases smell and taste impairment. We suggest that the prevalence of chemosensory deficits may reflect the pandemic potential for transmissibility and spread which differs between populations.

Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19.

The recent outbreak of human infections caused by SARS-CoV-2, the third zoonotic coronavirus has raised great public health concern globally. Rapid and accurate diagnosis of this novel pathogen posts great challenges not only clinically but also technologically. Metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methodologies. However, each has their own limitations. In this study, we developed an isothermal, CRISPR-based diagnostic for COVID-19 with near single-copy sensitivity. The diagnostic performances of all three technology platforms were also compared. Our study aimed to provide more insights into the molecular detection of SARS-CoV-2, and also to present a novel diagnostic option for this new emerging virus.

Network pharmacology and molecular docking analysis on molecular targets and mechanisms of Huashi Baidu formula in the treatment of COVID-19.

PURPOSE: Huashi Baidu formula (HSBDF) was developed to treat the patients with severe COVID-19 in China. The purpose of this study was to explore its active compounds and demonstrate its mechanisms against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through network pharmacology and molecular docking. METHODS: All the components of HSBDF were retrieved from the pharmacology database of TCM system. The genes corresponding to the targets were retrieved using UniProt and GeneCards database. The herb-compound-target network was constructed by Cytoscape. The target protein-protein interaction network was built using STRING database. The core targets of HSBDF were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The main active compounds of HSBDF were docked with SARS-CoV-2 and angiotensin converting enzyme II (ACE2). RESULTS: Compound-target network mainly contained 178 compounds and 272 corresponding targets. Key targets contained MAPK3, MAPK8, TP53, CASP3, IL6, TNF, MAPK1, CCL2, PTGS2, etc. There were 522 GO items in GO enrichment analysis (p < .05) and 168 signaling pathways (p < .05) in KEGG, mainly including TNF signaling pathway, PI3K-Akt signaling pathway, NOD-like receptor signaling pathway, MAPK signaling pathway, and HIF-1 signaling pathway. The results of molecular docking showed that baicalein and quercetin were the top two compounds of HSBDF, which had high affinity with ACE2. CONCLUSION: Baicalein and quercetin in HSBDF may regulate multiple signaling pathways through ACE2, which might play a therapeutic role on COVID-19.