Emergence and spreading of the largest SARS-CoV-2 deletion in the Delta AY.20 lineage from Uruguay.

The genetic variability of SARS-CoV-2 (genus Betacoronavirus, family Coronaviridae) has been scrutinized since its first detection in December 2019. Although the role of structural variants, particularly deletions, in virus evolution is little explored, these genome changes are extremely frequent. They are associated with relevant processes, including immune escape and attenuation. Deletions commonly occur in accessory ORFs and might even lead to the complete loss of one or more ORFs. This scenario poses an interesting question about the origin and spreading of extreme structural rearrangements that persist without compromising virus viability. Here, we analyze the genome of SARS-CoV-2 in late 2021 in Uruguay and identify a Delta lineage (AY.20) that experienced a large deletion (872 nucleotides according to the reference Wuhan strain) that removes the 7a, 7b, and 8 ORFs. Deleted viruses coexist with wild-type (without deletion) AY.20 and AY.43 strains. The Uruguayan deletion is like those identified in Delta strains from Poland and Japan but occurs in a different Delta clade. Besides providing proof of the circulation of this large deletion in America, we infer that the 872-deletion arises by the consecutive occurrence of a 6-nucleotide deletion, characteristic of delta strains, and an 866-nucleotide deletion that arose independently in the AY.20 Uruguayan lineage. The largest deletion occurs adjacent to transcription regulatory sequences needed to synthesize the nested set of subgenomic mRNAs that serve as templates for transcription. Our findings support the role of transcription sequences as a hotspot for copy-choice recombination and highlight the remarkable dynamic of SARS-CoV-2 genomes.

Expediting the drug discovery for ideal leads against SARS-CoV-2 via molecular docking of repurposed drugs.

SARS-CoV-2, the novel coronavirus spreading worldwide urges the need to repurpose drugs that can quickly enter clinical trials to combat the on-going global pandemic. A cluster of proteins are encoded for by the viral genome, each assuming a critical role in pathogen endurance inside the host. To handle the adverse circumstances, robust virtual strategies such as repurposing are coming to the fore due to being economical, efficient and rapid. Five FDA approved repurposed drugs proposed to act as inhibitors by targeting SARS-CoV-2 were used for initial evaluation via molecular docking. Moreover, a comparative analysis of the selected SARS-CoV-2 proteins against five ligands (Clemizole hydrochloride, Exemestane, Nafamostat, Pregnenolone and Umifenovir) was designed. In this regard, non-structural proteins (nsp3, nsp5, nsp10, nsp12 and nsp15), structural proteins (Spike, Nucleocapsid protein) and accessory proteins (ORF 3a, ORF 7a and ORF 9 b) were selected. Here, we aim to expedite the search for a potential drug from the five FDA approved repurposing drugs already in use for treatment of multiple diseases. Based on docking analysis, Umifenovir and Pregnenolone are suggested to show potential inhibitory effects against most of the SARS-CoV-2 proteins. These drugs are noteworthy since they exhibit high binding towards target proteins and should be used as lead compounds towards in vitro and in vivo studies. Communicated by Ramaswamy H. Sarma.

Establishment and preliminary application of a TaqMan real-time quantitative PCR method for PastV3 detection

In order to build a specific, sensitive and rapid detection method for PAstV3 detection, the PAstVB gene sequences in Genbank were used and the conserved region in ORFlb was selected to design specific primers and TaqMan probe. Clinical stool samples were collected and preliminary detected by this newly established real-time RT-PCR method after reaction systems and conditions optimization. This detection method established in this study has a good linear relationship with the standard curve, with R2 value up to 0.9971. The sensitivity is 100 times higher than conventional PCR method, The variation co-efficient of in-batch and inter-batch repeatability test is less than 2.0%, indicating good repeatability. The detection results of Clinical samples showed that the positive rate of this method is higher than conventional PCR method. The establishment of this method provides a rapid detection means for PAstV3 laboratory diagnosis and epidemiological investigation.

Therapeutic approaches and vaccination in fighting COVID-19 infections: A review.

Coronavirus disease 2019 (COVID-19) is a remarkably contagious and pathogenic viral infection arising from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which first appeared in Wuhan, China. For the time being, COVID-19 is not treated with a specific therapy. The Food and Drug Administration (FDA) has approved Remdesivir as the first drug to treat COVID-19. However, many other therapeutic approaches are being investigated as possible treatments for COVID-19. As part of this review, we discussed the development of various drugs, their mechanism of action, and how they might be applied to different cases of COVID-19 patients. Furthermore, this review highlights an update in the emergence of new prophylactic or therapeutic vaccines against COVID-19. In addition to FDA or The World Health Organization (WHO) approved vaccines, we intended to incorporate the latest published data from phase III trials about different COVID-19 vaccines and provide clinical data released on the networks or peer-review journals.

The status and analysis of common mutations found in the SARS-CoV-2 whole genome sequences from Bangladesh.

Rapid emergence of covid-19 variants by continuous mutation made the world experience continuous waves of infections and as a result, a huge number of death-toll recorded so far. It is, therefore, very important to investigate the diversity and nature of the mutations in the SARS-CoV-2 genomes. In this study, the common mutations occurred in the whole genome sequences of SARS-CoV-2 variants of Bangladesh in a certain timeline were analyzed to better understand its status. Hence, a total of 78 complete genome sequences available in the NCBI database were obtained, aligned and further analyzed. Scattered Single Nucleotide Polymorphisms (SNPs) were identified throughout the genome of variants and common SNPs such as: 241:C>T in the 5'UTR of Open Reading Frame 1A (ORF1A), 3037: C>T in Non-structural Protein 3 (NSP3), 14,408: C>T in ORF6 and 23,402: A>G, 23,403: A>G in Spike Protein (S) were observed, but all of them were synonymous mutations. About 97% of the studied genomes showed a block of tri-nucleotide alteration (GGG>AAC), the most common non-synonymous mutation in the 28,881-28,883 location of the genome. This block results in two amino acid changes (203-204: RG>KR) in the SR rich motif of the nucleocapsid (N) protein of SARS-CoV-2, introducing a lysine in between serine and arginine. The N protein structure of the mutant was predicted through protein modeling. However, no observable difference was found between the mutant and the reference (Wuhan) protein. Further, the protein stability changes upon mutations were analyzed using the I-Mutant2.0 tool. The alteration of the arginine to lysine at the amino acid position 203, showed reduction of entropy, suggesting a possible impact on the overall stability of the N protein. The estimation of the non-synonymous to synonymous substitution ratio (dN/dS) were analyzed for the common mutations and the results showed that the overall mean distance among the N-protein variants were statistically significant, supporting the non-synonymous nature of the mutations. The phylogenetic analysis of the selected 78 genomes, compared with the most common genomic variants of this virus across the globe showed a distinct cluster for the analyzed Bangladeshi sequences. Further studies are warranted for conferring any plausible association of these mutations with the clinical manifestation.

Unheeded SARS-CoV-2 proteins? A deep look into negative-sense RNA.

SARS-CoV-2 is a novel positive-sense single-stranded RNA virus from the Coronaviridae family (genus Betacoronavirus), which has been established as causing the COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest among known RNA viruses, comprising of at least 26 known protein-coding loci. Studies thus far have outlined the coding capacity of the positive-sense strand of the SARS-CoV-2 genome, which can be used directly for protein translation. However, it has been recently shown that transcribed negative-sense viral RNA intermediates that arise during viral genome replication from positive-sense viruses can also code for proteins. No studies have yet explored the potential for negative-sense SARS-CoV-2 RNA intermediates to contain protein-coding loci. Thus, using sequence and structure-based bioinformatics methodologies, we have investigated the presence and validity of putative negative-sense ORFs (nsORFs) in the SARS-CoV-2 genome. Nine nsORFs were discovered to contain strong eukaryotic translation initiation signals and high codon adaptability scores, and several of the nsORFs were predicted to interact with RNA-binding proteins. Evolutionary conservation analyses indicated that some of the nsORFs are deeply conserved among related coronaviruses. Three-dimensional protein modeling revealed the presence of higher order folding among all putative SARS-CoV-2 nsORFs, and subsequent structural mimicry analyses suggest similarity of the nsORFs to DNA/RNA-binding proteins and proteins involved in immune signaling pathways. Altogether, these results suggest the potential existence of still undescribed SARS-CoV-2 proteins, which may play an important role in the viral lifecycle and COVID-19 pathogenesis.

The Omic Insights on Unfolding Saga of COVID-19.

The year 2019 has seen an emergence of the novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease of 2019 (COVID-19). Since the onset of the pandemic, biological and interdisciplinary research is being carried out across the world at a rapid pace to beat the pandemic. There is an increased need to comprehensively understand various aspects of the virus from detection to treatment options including drugs and vaccines for effective global management of the disease. In this review, we summarize the salient findings pertaining to SARS-CoV-2 biology, including symptoms, hosts, epidemiology, SARS-CoV-2 genome, and its emerging variants, viral diagnostics, host-pathogen interactions, alternative antiviral strategies and application of machine learning heuristics and artificial intelligence for effective management of COVID-19 and future pandemics.

Identification of presented SARS-CoV-2 HLA class I and HLA class II peptides using HLA peptidomics.

The human leukocyte antigen (HLA)-bound viral antigens serve as an immunological signature that can be selectively recognized by T cells. As viruses evolve by acquiring mutations, it is essential to identify a range of presented viral antigens. Using HLA peptidomics, we are able to identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived peptides presented by highly prevalent HLA class I (HLA-I) molecules by using infected cells as well as overexpression of SARS-CoV-2 genes. We find 26 HLA-I peptides and 36 HLA class II (HLA-II) peptides. Among the identified peptides, some are shared between different cells and some are derived from out-of-frame open reading frames (ORFs). Seven of these peptides were previously shown to be immunogenic, and we identify two additional immunoreactive peptides by using HLA multimer staining. These results may aid the development of the next generation of SARS-CoV-2 vaccines based on presented viral-specific antigens that span several of the viral genes.

Development of in-house ELISAs for the detection of anti-SARS­CoV­2 RBD and N IgG and IgM antibodies in biological samples.

By the end of year 2019, the new virus SARS-CoV-2 appeared, causing the Coronavirus Disease 2019 (COVID-19), and spread very fast globally. A continuing need for diagnostic tools is a must to contain its spread. Till now, the gold standard method, the reverse transcription polymerase chain reaction (RT-PCR), is the precise procedure to detect the virus. However, SARS-CoV-2 may escape RT-PCR detection for several reasons. The development of well-designed, specific and sensitive serological test like enzyme immunoassay (EIA) is needed. This EIA can stand alone or work side by side with RT-PCR. In this study, we developed several EIAs including plates that are coated with either specially designed SARS-CoV-2 nucleocapsid or surface recombinant proteins. Each protein type can separately detect anti-SARS-CoV-2 IgM or IgG antibodies. For each EIAs, the cut-off value, specificity and sensitivity were determined utilizing RT-PCR confirmed Covid-19 and pre-pandemic healthy and other viruses-infected sera. Also, the receiver operator characteristic (ROC) analysis was performed to define the specificities and sensitivities of the optimized assay. The in-house EIAs were validated by comparing against commercial EIA kits. All in-house EIAs showed high specificity (98-99%) and sensitivity (97.8-98.9%) for the detection of IgG/IgM against RBD and N proteins of SARS-CoV-2. From these results, the developed Anti-RBD and anti-N IgG and IgM antibodies EIAs can be used as a specific and sensitive tool to detect SARS-CoV-2 infection, calculate the burden of disease and case fatality rates.

Clinical and molecular aspects of veterinary coronaviruses.

Coronaviruses are a large group of RNA viruses that infect a wide range of animal species. The replication strategy of coronaviruses involves recombination and mutation events that lead to the possibility of cross-species transmission. The high plasticity of the viral receptor due to a continuous modification of the host species habitat may be the cause of cross-species transmission that can turn into a threat to other species including the human population. The successive emergence of highly pathogenic coronaviruses such as the Severe Acute Respiratory Syndrome (SARS) in 2003, the Middle East Respiratory Syndrome Coronavirus in 2012, and the recent SARS-CoV-2 has incentivized a number of studies on the molecular basis of the coronavirus and its pathogenesis. The high degree of interrelatedness between humans and wild and domestic animals and the modification of animal habitats by human urbanization, has favored new viral spreads. Hence, knowledge on the main clinical signs of coronavirus infection in the different hosts and the distinctive molecular characteristics of each coronavirus is essential to prevent the emergence of new coronavirus diseases. The coronavirus infections routinely studied in veterinary medicine must be properly recognized and diagnosed not only to prevent animal disease but also to promote public health.

Insights into forsythia honeysuckle (Lianhuaqingwen) capsules: A Chinese herbal medicine repurposed for COVID-19 pandemic.

Background: In December 2019, a novel coronavirus, SARS-CoV-2 caused a series of acute atypical respiratory diseases worldwide. However, there is still a lack of drugs with clear curative effects, and the clinical trial research of vaccines has not been completely finished. Purpose: LH capsules are approved TCM patent medicine that are widely used for the treatment of respiratory tract infectious diseases caused by colds and flu. On April 12, 2020, LH capsules and granules were officially repurposed by the China Food and Drug Administration (CFDA) for patients with mild COVID-19 based on their safety and efficacy demonstrated through multicentre, randomized, controlled clinical trials. We hope to conduct a comprehensive review of it through modern pharmacy methods, and try to explain its possible mechanism. Methods: Using the full names of LH capsules Lianhuaqingwen, Lianhua Qingwen andSARS-COV-2, COVID-19 as the keywords of the search terms, systemically search for existing related papers in various databases such as Web of Science and PubMed. And completed the collection of clinical data in ClinicalTrials.gov and Chinese Clinical Trial Registry. Last but not least, we have sorted out the anti-inflammatory and antiviral mechanisms of LH capsules through literature and Selleck. Results: This review systematically sorted out the active ingredients in LH capsules. Furthermore, the related pharmacological and clinical trials of LH capsule on SARS-CoV-2, IAV and IBV were discussed in detail. Moreover, the present review provides the first summary of the potential molecular mechanism of specific substances in LH capsules involved in resistance to SARS-COV-2 infection and the inhibition of cytokine storm syndrome (CSS) caused by IL-6. Conclusion: This review summarizes the available reports and evidence that support the use of LH capsules as potential drug candidates for the prevention and treatment of COVID-19. However, TCM exerts its effects through multiple targets and multiple pathways, and LH capsules are not an exception. Therefore, the relevant mechanisms need to be further improved and experimentally verified.

Current approaches used in treating COVID-19 from a molecular mechanisms and immune response perspective.

Coronavirus disease 2019 (COVID-19), which is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared by the World Health Organization (WHO) as a global pandemic on March 11, 2020. SARS-CoV-2 targets the respiratory system, resulting in symptoms such as fever, headache, dry cough, dyspnea, and dizziness. These symptoms vary from person to person, ranging from mild to hypoxia with acute respiratory distress syndrome (ARDS) and sometimes death. Although not confirmed, phylogenetic analysis suggests that SARS-CoV-2 may have originated from bats; the intermediary facilitating its transfer from bats to humans is unknown. Owing to the rapid spread of infection and high number of deaths caused by SARS-CoV-2, most countries have enacted strict curfews and the practice of social distancing while awaiting the availability of effective U.S. Food and Drug Administration (FDA)-approved medications and/or vaccines. This review offers an overview of the various types of coronaviruses (CoVs), their targeted hosts and cellular receptors, a timeline of their emergence, and the roles of key elements of the immune system in fighting pathogen attacks, while focusing on SARS-CoV-2 and its genomic structure and pathogenesis. Furthermore, we review drugs targeting COVID-19 that are under investigation and in clinical trials, in addition to progress using mesenchymal stem cells to treat COVID-19. We conclude by reviewing the latest updates on COVID-19 vaccine development. Understanding the molecular mechanisms of how SARS-CoV-2 interacts with host cells and stimulates the immune response is extremely important, especially as scientists look for new strategies to guide their development of specific COVID-19 therapies and vaccines.