Genus Coronavirus Research Articles

Prediction of conformational states in a coronavirus channel using Alphafold-2 and DeepMSA2: Strengths and limitations

The envelope (E) protein is present in all coronavirus genera. This protein can form pentameric oligomers with ion channel activity which have been proposed as a possible therapeutic target. However, high resolution structures of E channels are limited to those of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), responsible for the recent COVID-19 pandemic. In the present work, we used Alphafold-2 (AF2), in ColabFold without templates, to predict the transmembrane domain (TMD) structure of six E-channels representative of genera alpha-, beta- and gamma-coronaviruses in the Coronaviridae family. High-confidence models were produced in all cases when combining multiple sequence alignments (MSAs) obtained from DeepMSA2. Overall, AF2 predicted at least two possible orientations of the α-helices in E-TMD channels: one where a conserved polar residue (Asn-15 in the SARS sequence) is oriented towards the center of the channel, ‘polar-in’, and one where this residue is in an interhelical orientation ‘polar-inter’. For the SARS models, the comparison with the two experimental models ‘closed’ (PDB: 7K3G) and ‘open’ (PDB: 8SUZ) is described, and suggests a ∼60˚ α-helix rotation mechanism involving either the full TMD or only its N-terminal half, to allow the passage of ions. While the results obtained are not identical to the two high resolution models available, they suggest various conformational states with striking similarities to those models. We believe these results can be further optimized by means of MSA subsampling, and guide future high resolution structural studies in these and other viral channels.

Construction of pseudotyped human coronaviruses and detection of pre-existing antibodies in the human population

In order to clarify the pre-exist immunity background of different human coronaviruses (HCoV), this study investigated the positive rate of spike (S) protein antibodies of HCoV, including HCoV- severe acute respiratory syndrome (SARS) −associated coronavirus (SARS-CoV-1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-229E, HCoV-NL63, HCoV-HKU1 and HCoV-OC43, before and after the Coronavirus Disease 2019 (COVID-19) outbreak. We utilized pseudotyped virus-based neutralization assays (PBNA) or enzyme-linked immunosorbent assays (ELISA) to detect antibody levels against HCoV in serum samples collected in 2009–2010 and 2023. The PBNA results showed that neutralizing antibodies against SARS-CoV-1 and the MERS-CoV were negative. In the serum samples from 2009 to 2010, neutralizing antibodies against SARS-CoV-2 (D614G) were negative, whereas in the serum samples from 2023, 73 samples (73 %) showed neutralizing reactions with the SARS-CoV-2 D614G strain, 96 samples (96 %) with the BA.5 strain, and 91 samples (91 %) with the BF.7 strain. Among pre-COVID-19 samples, 33 % (33/100) showed neutralizing reactions with HCoV-229E and 63 % (63/100) with HCoV-NL63. Among post-COVID-19 samples, 50 % (50/100) showed neutralizing reactions with HCoV-229E and 49 % (49/100) with HCoV-NL63. Due to the different receptors of alpha coronavirus genus compared to other beta coronavirus genus, neutralizing antibodies against HCoV-OC43 and HCoV-HKU1 virus cannot be detected by constructing corresponding pseudotyped virus. Binding antibodies against HCoV-OC43 and HCoV-HKU1 virus were detected using ELISA. The results revealed that among pre-COVID-19 samples, 83 % (83/100) and 45 % (45/100) had binding activity with HCoV-OC43 and HCoV-HKU1, respectively. Among post-COVID-19 samples, 100 % (100/100) and 81 % (81/100) had binding activity with HCoV-OC43 and HCoV-HKU1, respectively.

ESCRT Protein VPS4A Is Required for the Formation of Replication Centers and Replication of Human Coronavirus 229E (HCoV-229E)

Human coronavirus 229E (HCoV-229E) is an alpha coronavirus that infects humans and bats. In common with all positive-strand RNA viruses, 229E infection causes rearrangements of the host’s intracellular membranes to form replication organelles, a highly conserved and vital step in the viral replication cycle. Here, we investigated the role of the ESCRT protein VPS4A in 229E infection. We found that functional VPS4A was required for the formation of replication organelles and localizing viral RNA to these structures in host cells to facilitate viral genome replication. We validated this effect using small molecule inhibitors to VPS4A, significantly reducing virus replication. We also showed that other ESCRTS, like CHMP4B, were required for the virus replication step, whereas VPS37A was involved in the post-replication stages. The absence of a functional VPS4A prevented the remodeling of membranes to form viral replication centers and, therefore, exposed the viral RNA, triggering an inflammatory immune response as indicated by elevated levels of IL-6. Interestingly, we observed the role of VPS4A to be similar for the OC43 coronavirus, indicating it could be conserved across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of coronaviruses is imperative to finding more effective ways to control them.

Comparative Performance in the Detection of Four Coronavirus Genera from Human, Animal, and Environmental Specimens.

Emerging coronaviruses (CoVs) are understood to cause critical human and domestic animal diseases; the spillover from wildlife reservoirs can result in mild and severe respiratory illness in humans and domestic animals and can spread more readily in these naïve hosts. A low-cost CoV molecular method that can detect a variety of CoVs from humans, animals, and environmental specimens is an initial step to ensure the early identification of known and new viruses. We examine a collection of 50 human, 46 wastewater, 28 bat, and 17 avian archived specimens using 3 published pan-CoV PCR assays called Q-, W-, and X-CoV PCR, to compare the performance of each assay against four CoV genera. X-CoV PCR can detect all four CoV genera, but Q- and W-CoV PCR failed to detect δ-CoV. In total, 21 (42.0%), 9 (18.0%), and 21 (42.0%) of 50 human specimens and 30 (65.22%), 6 (13.04%), and 27 (58.70%) of 46 wastewater specimens were detected using Q-, W-, and X-CoV PCR assays, respectively. The X-CoV PCR assay has a comparable sensitivity to Q-CoV PCR in bat CoV detection. Combining Q- and X-CoV PCR assays can increase sensitivity and avoid false negative results in the early detection of novel CoVs.

A Mini-Review on the Common Antiviral Drug Targets of Coronavirus.

Coronaviruses in general are a zoonotic pathogen with significant cross-species transmission. They are widely distributed in nature and have recently become a major threat to global public health. Vaccines are the preferred strategy for the prevention of coronaviruses. However, the rapid rate of virus mutation, large number of prevalent strains, and lag in vaccine development contribute to the continuing frequent occurrence of coronavirus diseases. There is an urgent need for new antiviral strategies to address coronavirus infections effectively. Antiviral drugs are important in the prevention and control of viral diseases. Members of the genus coronavirus are highly similar in life-cycle processes such as viral invasion and replication. These, together with the high degree of similarity in the protein sequences and structures of viruses in the same genus, provide common targets for antiviral drug screening of coronaviruses and have led to important advances in recent years. In this review, we summarize the pathogenic mechanisms of coronavirus, common drugs targeting coronavirus entry into host cells, and common drug targets against coronaviruses based on biosynthesis and on viral assembly and release. We also describe the common targets of antiviral drugs against coronaviruses and the progress of antiviral drug research. Our aim is to provide a theoretical basis for the development of antiviral drugs and to accelerate the development and utilization of commonly used antiviral drugs in China.

Synergistic inhibition effects of andrographolide and baicalin on coronavirus mechanisms by downregulation of ACE2 protein level

The SARS-CoV-2 virus, belonging to the Coronavirus genus, which poses a threat to human health worldwide. Current therapies focus on inhibiting viral replication or using anti-inflammatory/immunomodulatory compounds to enhance host immunity. This makes the active ingredients of traditional Chinese medicine compounds ideal therapies due to their proven safety and minimal toxicity. Previous research suggests that andrographolide and baicalin inhibit coronaviruses; however, their synergistic effects remain unclear. Here, we studied the antiviral mechanisms of their synergistic use in vitro and in vivo. We selected the SARS-CoV-2 pseudovirus for viral studies and found that synergistic andrographolide and baicalein significantly reduced angiotensin-converting enzyme 2 protein level and viral entry of SARS-CoV-2 into cells compared to singal compound individually and inhibited the major protease activity of SARS-CoV-2. This mechanism is essential to reduce the pathogenesis of SARS-CoV-2. In addition, their synergistic use in vivo also inhibited the elevation of pro-inflammatory cytokines, including IL-6 and TNF-α—the primary cytokines in the development of acute respiratory distress syndrome (the main cause of COVID-19 deaths). In conclusion, this study shows that synergistic andrographolide and baicalein treatment acts as potent inhibitors of coronavirus mechanisms in vitro and in vivo—and is more effective together than in isolation.

Current Update of Severe Acute Respiratory Syndrome Coronavirus 2

Coronaviruses are group viruses belong to Corona viridae family the order Nidovirales, and the genus Coronavirus. They are the largest group of viruses causing respiratory infections. There are four genera under this family; Alpha coronavirus, Beta coronavirus, Gamma coronavirus, and Delta coronavirus. However, most of the human coronaviruses produce mild illness in the upper respiratory tract while some strains are lethal and can cause severe acute respiratory syndrome (SARS), middle East respiratory syndrome (MERS) and coronavirus disease 2019 (COVID-19).

Water Extract of Portulaca Oleracea Inhibits PEDV Infection-Induced Pyrolysis by Caspase-1/GSDMD.

Porcine epidemic diarrhea virus (PEDV) belongs to the coronavirus family and the coronavirus genus, causing contact enteric infection in pigs. It is one of the most serious diseases that threatens the pig industry. However, there is currently no specific drug to prevent and treat the disease, indicating that we need to be vigilant about the spread of the disease and the development of anti-PEDV drugs. The dried aerial parts of the plant Portulaca oleracea in the family Portulacaceous, whose decoction can be used to treat acute enteritis, dysentery, diarrhea, and other diseases. This study explored the potential mechanism of water extract of Portulaca oleracea (WEPO) in PEDV-induced pyroptosis in Vero cells. PEDV decreased the viability of Vero cells in a dose- and time-dependent manner, causing cell damage, upregulating the level of intracellular Nlrp3, and inhibiting the level of Gasdermin D (GSDMD) and the activation of Caspase-1. WEPO can inhibit PEDV-induced pyroptosis, reduce the elevation of inflammatory factors caused by infection, and exhibit a dose-dependent effect. Knockdown of Caspase-1 and GSDMD separately can induce the production of the inflammatory factor IL-1β to significantly decrease and increase, respectively. These results suggest that WEPO can inhibit cell pyroptosis caused by PEDV and that the Caspase-1 and GSDMD pathways play an important role in this process.

Whole-Genome, Recombinant, and Phylogenetic Analysis of Porcine Epidemic Diarrhea Virus Strain CH/JSXZ/2015

Background. Porcine epidemic diarrhea virus (PEDV) is an important pathogen causing highly contact infectious intestinal infections in pigs belonging to the family Coronaviridae, genus Coronavirus, which can cause porcine epidemic diarrhea (PED). Since 2010, outbreaks of PEDV variants have caused great economic losses to the swine industry worldwide. Our study will provide the basis for discovering the key points of PEDV variation and help in understanding the trend of popularity and evolution of PEDV in China. Methods. We amplify the complete PEDV CH/JSXZ/2015 genome sequence from naturally infected piglets in Xuzhou, Jiangsu Province, China, by RT-PCR. The comparative genome circle graph, characterization and phylogenetic analysis, and recombination in the PEDV CH/JSXZ/2015 and other PEDVs are analyzed by bioinformatics analysis software. Results. At the whole-genome level, CH/JSXZ/2015 showed the highest nucleotide identity (99.5%) with CH/GDZHDM/1401 strain (KR153326.1 and KX016034.1) and the lowest similarity (93.5%) to 85-7-mutant1 strain. CH/JSXZ/2015 S amino acid identity was 90.8%–99.8% compared with other strains. CH/JSXZ/2015 ORF3 amino acid identity was 91.2%–100.0% compared with other PEDV strains. Phylogenetic analysis based on the whole genome, S, and ORF3 revealed that CH/JSXZ /2015 is most genetically close to the CH/GDZHDM/1401 strain (KR153326.1 and KX016034.1) isolated in China in 2014. In addition, we revealed that the CH/GDZHDM/1401 (KX016034.1) strain, PEDV 1842/2016 ITA, and CH/JSXZ /2015 had recombination, and the position of recombination was 1–5530 and 28185–28544 bp. 5531–28184 bp of JSXZ are highly homologous to CH/GDZHDM/1401 (KX016034.1), while 1–5530 and 28185–28544 bp of JSXZ are highly homologous to 1842/2016 ITA. These results helped discover PEDV variation rules and provided a basis for preventing and controlling PEDV. Conclusions. Our study revealed that PEDV CH/JSXZ/2015 was a variant strain, the current epidemic strain in China. The CH/GDZHDM/1401 (KX016034.1) strain, PEDV 1842/2016 ITA (KY111278) and CH/JSXZ /2015 had recombination.

Identification of subgenomic mRNAs derived from the coronavirus 1a/1b protein gene: Implications for coronavirus transcription

Synthesis of coronavirus subgenomic mRNA (sgmRNA) is guided by the transcription regulatory sequence (TRS). sgmRNA derived from the body TRS (TRS-B) located at the 1a/1b protein gene is designated 1ab/sgmRNA. In the current study, we comprehensively identified the 1ab/sgmRNAs synthesized from TRS-Bs located at the 1a/1b protein genes of different coronavirus genera both in vitro and in vivo by RT‒PCR and sequencing. The results suggested that the degree of sequence homology between the leader TRS (TRS-L) and TRS-B may not be a decisive factor for 1ab/sgmRNA synthesis. This observation led us to revisit the coronavirus transcription mechanism and to propose that the disassociation of coronavirus polymerase from the viral genome may be a prerequisite for sgmRNA synthesis. Once the polymerase can disassociate at TRS-B, the sequence homology between TRS-L and TRS-B is important for sgmRNA synthesis. The study therefore extends our understanding of transcription mechanisms.

Nicotinamide Efficiently Suppresses Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus Replication.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV), members of the genus Coronavirus, mainly cause acute diarrhea, vomiting and dehydration in piglets, and thus lead to serious economic losses. In this study, we investigated the effects of nicotinamide (NAM) on PEDV and PDCoV replication and found that NAM treatment significantly inhibited PEDV and PDCoV reproduction. Moreover, NAM plays an important role in replication processes. NAM primarily inhibited PEDV and PDCoV RNA and protein synthesis rather than other processes. Furthermore, we discovered that NAM treatment likely inhibits the replication of PEDV and PDCoV by downregulating the expression of transcription factors through activation of the ERK1/2/MAPK pathway. Overall, this study is the first to suggest that NAM might be not only an important antiviral factor for swine intestinal coronavirus, but also a potential candidate to be evaluated in the context of other human and animal coronaviruses.

Choice of Target in the Genomes of Prototypic Strains to Recognize Subgenus of Coronaviruses

Targeted approach to recognition of coronavirus subgenus on the base of codon frequency distribution in the N-gene of nucleocapsid protein was proposed in the work. Deviation of codon frequency distribution in the N-gene of coronavirus genome analyzed from the same distributions for the 67 prototypic strains, which characterize the 23 subgenera in the four coronavirus genera, is calculated on the base of statistics in the approach proposed. The smallest value of such a deviation from certain prototypic strain points at subgenus to which this strain belongs. The approach proposed appeared to be effective and supports significance for recognizing coronavirus subgenus at least 99 %. Populations of the 38 and 7 codons providing for needed efficiency level were selected out of all codons of the genetic code in accordance with their frequency distribution. The codons from the populations outlined fix taxonomic structure of coronavirus subgenus.

Adaptive evolution of the Spike protein in coronaviruses.

Coronaviruses are single-stranded, positive-sense RNA viruses that can infect many mammal and avian species. The Spike (S) protein of coronaviruses binds to a receptor on the host cell surface to promote viral entry. The interactions between the S proteins of coronaviruses and receptors of host cells are extraordinarily complex, with coronaviruses from different genera being able to recognize the same receptor and coronaviruses from the same genus able to bind distinct receptors. As the COVID-19 pandemic has developed, many changes in the S protein have been under positive selection by altering the receptor binding affinity, reducing antibody neutralization activities, or affecting T-cell responses. It is intriguing to determine whether the selection pressure on the S gene differs between SARS-CoV-2 and other coronaviruses due to the host shift from nonhuman animals to humans. Here, we show that the S gene, particularly the S1 region, has experienced positive selection in both SARS-CoV-2 and other coronaviruses. While the S1-NTD domain exhibits signals of positive selection in the pairwise comparisons in all four coronavirus genera, positive selection is primarily detected in the S1-CTD domain (the receptor-binding domain, RBD) in the ongoing evolution of SARS-CoV-2, possibly owing to the change in host settings and the widespread natural infection and SARS-CoV-2 vaccination in humans.

Stabilization of the Dimeric State of SARS-CoV-2 Main Protease by GC376 and Nirmatrelvir

The main protease (Mpro or 3CLpro) is an enzyme that is evolutionarily conserved among different genera of coronaviruses. As it is essential for processing and maturing viral polyproteins, Mpro has been identified as a promising target for the development of broad-spectrum drugs against coronaviruses. Like SARS-CoV and MERS-CoV, the mature and active form of SARS-CoV-2 Mpro is a dimer composed of identical subunits, each with a single active site. Individual monomers, however, have very low or no catalytic activity. As such, inhibition of Mpro can be achieved by molecules that target the substrate binding pocket to block catalytic activity or target the dimerization process. In this study, we investigated GC376, a transition-state analog inhibitor of the main protease of feline infectious peritonitis coronavirus, and Nirmatrelvir (NMV), an oral, bioavailable SARS-CoV-2 Mpro inhibitor with pan-human coronavirus antiviral activity. Our results show that both GC376 and NMV are capable of strongly binding to SARS-CoV-2 Mpro and altering the monomer-dimer equilibrium by stabilizing the dimeric state. This behavior is proposed to be related to a structured hydrogen-bond network established at the Mpro active site, where hydrogen bonds between Ser1' and Glu166/Phe140 are formed in addition to those achieved by the latter residues with GC376 or NMV.

The main protease of SARS-CoV-2 cleaves histone deacetylases and DCP1A, attenuating the immune defense of the interferon-stimulated genes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019, constitutes an emerging human pathogen of zoonotic origin. A critical role in protecting the host against invading pathogens is carried out by interferon-stimulated genes (ISGs), the primary effectors of the type I interferon (IFN) response. All coronaviruses studied thus far have to first overcome the inhibitory effects of the IFN/ISG system before establishing efficient viral replication. However, whether SARS-CoV-2 evades IFN antiviral immunity by manipulating ISG activation remains to be elucidated. Here, we show that the SARS-CoV-2 main protease (Mpro) significantly suppresses the expression and transcription of downstream ISGs driven by IFN-stimulated response elements in a dose-dependent manner, and similar negative regulations were observed in two mammalian epithelial cell lines (simian Vero E6 and human A549). Our analysis shows that to inhibit the ISG production, Mpro cleaves histone deacetylases (HDACs) rather than directly targeting IFN signal transducers. Interestingly, Mpro also abolishes the activity of ISG effector mRNA-decapping enzyme 1a (DCP1A) by cleaving it at residue Q343. In addition, Mpro from different genera of coronaviruses has the protease activity to cleave both HDAC2 and DCP1A, even though the alphacoronaviruse Mpro exhibits weaker catalytic activity in cleaving HDAC2. In conclusion, our findings clearly demonstrate that SARS-CoV-2 Mpro constitutes a critical anti-immune effector that modulates the IFN/ISG system at multiple levels, thus providing a novel molecular explanation for viral immune evasion and allowing for new therapeutic approaches against coronavirus disease 2019 infection.

Chicken or Porcine Aminopeptidase N Mediates Cellular Entry of Pseudoviruses Carrying Spike Glycoprotein from the Avian Deltacoronaviruses HKU11, HKU13, and HKU17.

Members of deltacoronavirus (DCoV) have mostly been identified in diverse avian species as natural reservoirs, though the porcine DCoV (PDCoV) is a major swine enteropathogenic virus with global spread. The important role of aminopeptidase N (APN) orthologues from various mammalian and avian species in PDCoV cellular entry and interspecies transmission has been revealed recently. In this study, comparative analysis indicated that three avian DCoVs, bulbul DCoV HKU11, munia DCoV HKU13, and sparrow DCoV HKU17 (Chinese strain), and PDCoV in the subgenera Buldecovirus are grouped together at whole-genome levels; however, the spike (S) glycoprotein and its S1 subunit of HKU17 are more closely related to night heron DCoV HKU19 in Herdecovirus. Nevertheless, the S1 protein of HKU11, HKU13, or HKU17 bound to or interacted with chicken APN (chAPN) or porcine APN (pAPN) by flow cytometry analysis of cell surface expression of APN and by coimmunoprecipitation in APN-overexpressing cells. Expression of chAPN or pAPN allowed entry of pseudotyped lentiviruses with the S proteins from HKU11, HKU13 and HKU17 into nonsusceptible cells and natural avian and porcine cells, which could be inhibited by the antibody against APN or anti-PDCoV-S1. APN knockdown by siRNA or knockout by CRISPR/Cas9 in chicken or swine cell lines significantly or almost completely blocked infection of these pseudoviruses. Hence, we demonstrate that HKU11, HKU13, and HKU17 with divergent S genes likely engage chAPN or pAPN to enter the cells, suggesting a potential interspecies transmission from wild birds to poultry and from birds to mammals by certain avian DCoVs. IMPORTANCE The receptor usage of avian deltacoronaviruses (DCoVs) has not been investigated thus far, though porcine deltacoronavirus (PDCoV) has been shown to utilize aminopeptidase N (APN) as a cell receptor. We report here that chicken or porcine APN also mediates cellular entry by three avian DCoV (HKU11, HKU13, and HKU17) spike pseudoviruses, and the S1 subunit of three avian DCoVs binds to APN in vitro and in the surface of avian and porcine cells. The results fill the gaps in knowledge about the avian DCoV receptor and elucidate important insights for the monitoring and prevention of potential interspecies transmission of certain avian DCoVs. In view of the diversity of DCoVs, whether this coronavirus genus will cause novel virus to emerge in other mammals from birds, are worthy of further surveillance and investigation.

Optimization of a coronavirus genus recognition procedure based on the n-gene of prototypic strains

The article offers a solution to the problem of fast and efficient recognition of the coronavirus genus. For this purpose, the authors apply a virus genome targeting method based on the use of a sufficiently short and conserved N-gene of the nucleocapsid protein. Comparison of the codon frequency distributions in the N-gene of the analyzed genome and a set of 67 prototypical strains corresponding to the coronavirus subgenus allows us to recognize the genus of the coronavirus. This paper proposes optimization of the genus recognition of coronavirus by eliminating a significant number of codons from the 64 codons of the genetic code (26 in one case and 57 in the other). The authors achieved 100% genus recognition efficiency in a sample of 2,051 coronavirus genomes from the GenBank database with annotated subgenus in the optimized procedure. The authors also achieved 99% confidence when using the optimized coronavirus genus recognition procedure in a total sample of 3,242 genomes.

Plant MicroRNA Potential in Targeting Sars-CoV-2 Genome Offering Efficient Antiviral MiRNA-Based Therapies.

In 2019, severe acute respiratory coronavirus II (or SARS-COV-2) emerged in Wuhan, China, rapidly becoming a global pandemic. Coronavirus genus (Coronaviridae) has the largest single-stranded positive-sense RNA genome (~30 kb) among the human infected single-stranded RNA viruses. For the study of active therapeutic plant-derived miRNA(s), it may be possible to uptake the miRNAs and their biological role in the host cell. In this study, we bioinformatically searched plant miRNAs that can potentially interact with the Sars-CoV-2 genome within the 3'- UTR region and have prompt antiviral activity. We searched the plant miRNAs that target the 3'-UTR flanking region of the Sars-CoV-2 genome by employing the RNAHybrid, RNA22, and STarMir miRNA/target prediction tools. The RNAHybrid algorithm found 63 plant miRNAs having hybridization energy with less or equal to -25 kcal.mol-1. Besides, RNA22 and STarMir tools identified eight interactions between the plant miRNAs and the targeted RNA sequence. pvu-miR159a. 2 and sbi-miR5387b were predicted as the most effectively interacting miRNAs in targeting the 3'-UTR sequence, not only by the RNA22 tool but also by the STarMir tool at the same position. However, the GC content of the pvumiR159a. 2 is 55% instead of sbi-miR5387b, which is a GC enriched sequence (71.43%) that may activate TLR receptors. In our opinion, they are potent plant-derived miRNA candidates that have a great chance of targeting the Sars-CoV-2 genome in the 3'-UTR region in vitro. Therefore, we propose pvu-miR159a.2 for studying antiviral miRNA-based therapies without any essential side effects in vivo.

Coronaviruses: modern taxonomy and research chronology

The classification of coronaviruses began in 1968 when five viruses (IBV, MHV, B814, 229E, and OC43) were united into an independent group, “coronaviruses”, based on the characteristic morphology of the virions. In 1971, the genus Coronavirus was formed, and in 1975, the family Coronaviridae, which in 1996 was included in the order Nidovirales. In 2009, the Coronaviridae family was divided into subfamilies. In 2018, new taxa of viruses were created – region, suborder, and subgenus, and in 2019 – kingdom, type, and class. According to the International Committee on Taxonomy of Viruses, issue No. 37 of 07.2021 (ratification of 03.2022), the family Coronaviridae belongs to the region Riboviria, kingdom Orthornavirae, phylum Pisuviricota, class Pisoniviricetes, order Nidovirales, suborder Cornidovirineae. The Coronaviridae family includes 54 viruses, which are grouped into three subfamilies (Letovirinae, Orthocoronavirinae, Pitovirinae), six genera (Alphaletovirus, Alphacoronavirus, Betacoronavirus, Deltacoronavirus, Gammacoronavirus, Alphapironavirus) and 28 subgenera. Representatives of the most numerous subfamily Orthocoronavirinae infect various species of mammals and birds, causing various pathologies: respiratory and intestinal infections, polyserositis, myocarditis, hepatitis, nephritis, neuroinfections, immunopathology. In particular, viruses that cause infectious bronchitis in chickens, transmissible gastroenteritis in pigs, epidemic diarrhea in pigs, encephalomyelitis in pigs, coronavirus infection in cattle, and epizootic catarrhal gastroenteritis in minks are relevant for veterinary practice. The natural reservoirs of coronaviruses from the genera Alphacoronavirus and Betacoronavirus are bats and birds from the genera Gammacoronavirus and Deltacoronavirus. Particularly dangerous human coronaviruses are SARS-CoV, MERS-CoV and SARS-CoV-2, which cause emergent infections. The circulation of SARS-CoV, MERS-CoV, and SARS-CoV-2 among animals is shown. The natural reservoir of these viruses is bats, and the intermediate hosts for SARS-CoV are Himalayan civets, one-humped camels for MERS-CoV, and pangolins for SARS-CoV-2. The circulation of SARS-CoV-2 among different species of domestic and wild animals leads to the accumulation of mutations, which causes the adaptation of the virus to new hosts and ecological niches and its subsequent introduction into the human population.

Structural Basis of Main Proteases of Coronavirus Bound to Drug Candidate PF-07304814

New variants of the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) emerged and spread rapidly all over the world, which strongly supports the need for pharmacological options to complement vaccine strategies. Main protease (Mpro or 3CLpro) is a critical enzyme in the life cycle of SARS-CoV-2 and appears to be highly conserved among different genera of coronaviruses, making it an ideal target for the development of drugs with broad-spectrum property. PF-07304814 developed by Pfizer is an intravenously administered inhibitor targeting SARS-CoV-2 Mpro. Here we showed that PF-07304814 displays broad-spectrum inhibitory activity against Mpros from multiple coronaviruses. Crystal structures of Mpros of SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-NL63 bound to the inhibitor PF-07304814 revealed a conserved ligand-binding site, providing new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures complemented by comprehensive comparison defined the key structural determinants essential for inhibition and illustrated the binding mode of action of Mpros from different coronaviruses. In view of the importance of Mpro for the medications of SARS-CoV-2 infection, insights derived from the present study should accelerate the design of pan-coronaviral main protease inhibitors that are safer and more effective.