Temporal differences in culturable severe acute respiratory coronavirus virus 2 (SARS-CoV-2) from the respiratory and gastrointestinal tracts in a patient with moderate coronavirus disease 2019 (COVID-19)
Jennifer AudsleyNatasha E. HolmesFrancesca L. MordantCelia DourosSara ZufanThi H. O. NguyenŁukasz KedzierskiLouise C. RowntreeLuca HensenKanta SubbaraoKatherine KedzierskaSuellen NicholsonNorelle L. SherryIrani ThevarajanThomas TranJulian Druce
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Temporal differences in culturable severe acute respiratory coronavirus virus 2 (SARS-CoV-2) from the respiratory and gastrointestinal tracts in a patient with moderate coronavirus disease 2019 (COVID-19) - Volume 43 Issue 9Keywords:
Coronavirus
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2019-20 coronavirus outbreak
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reignited global interest in animal coronaviruses and their potential for human transmission. While bats are thought to be the wildlife reservoir of SARS-CoV and SARS-CoV-2, the widespread human coronavirus OC43 is thought to have originated in rodents. Here, we sampled 297 rodents and shrews, representing eight species, from three municipalities of southern China. We report coronavirus prevalences of 23.3% and 0.7% in Guangzhou and Guilin, respectively, with samples from urban areas having significantly higher coronavirus prevalences than those from rural areas. We obtained three coronavirus genome sequences from Rattus norvegicus, including a Betacoronavirus (rat coronavirus [RCoV] GCCDC3), an Alphacoronavirus (RCoV-GCCDC5), and a novel Betacoronavirus (RCoV-GCCDC4). Recombination analysis suggests that there was a potential recombination event involving RCoV-GCCDC4, murine hepatitis virus (MHV), and Longquan Rl rat coronavirus (LRLV). Furthermore, we uncovered a polybasic cleavage site, RARR, in the spike (S) protein of RCoV-GCCDC4, which is dominant in RCoV. These findings provide further information on the potential for interspecies transmission of coronaviruses and demonstrate the value of a One Health approach to virus discovery. IMPORTANCE Surveillance of viruses among rodents in rural and urban areas of South China identified three rodent coronaviruses, RCoV-GCCDC3, RCoV-GCCDC4, and RCoV-GCCDC5, one of which was identified as a novel potentially recombinant coronavirus with a polybasic cleavage site in the spike (S) protein. Through reverse transcription-PCR (RT-PCR) screening of coronaviruses, we found that coronavirus prevalence in urban areas is much higher than that in rural areas. Subsequently, we obtained three coronavirus genome sequences by deep sequencing. After different method-based analyses, we found that RCoV-GCCDC4 was a novel potentially recombinant coronavirus with a polybasic cleavage site in the S protein, dominant in RCoV. This newly identified coronavirus RCoV-GCCDC4 with its potentially recombinant genome and polybasic cleavage site provides a new insight into the evolution of coronaviruses. Furthermore, our results provide further information on the potential for interspecies transmission of coronaviruses and demonstrate the necessity of a One Health approach for zoonotic disease surveillance.
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The 2019 novel Coronavirus (2019-nCoV) has caused the pneumonia outbreak in Wuhan (a city of China) In our previous study, the analytical results showed that both 2019-nCoV and SARS coronavirus belong to Betacoronavirus subgroup B (BB coronavirus), but have large differences, which are consistent with the differences in the clinical symptoms of two related diseases The most important finding was that the alternative translation of Nankai CDS could produce more than 17 putative proteins, which may be responsible for the host adaption The genotyping of 13 viruses using the 17 putative proteins revealed the high mutation rate and diversity of BB coronavirus The present study for the first time (on January 21st, 2020) reported a very important mutation in the Spike (S) proteins of Betacoronavirus By this mutation, 2019-nCoV acquired a cleavage site for furin enzyme in its S protein, which is not present in the S proteins of most other Betacoronavirus (e g SARS coronavirus) This cleavage site may increase the efficiency of virus infection into cells, making 2019-nCoV has significantly stronger transmissibility than SARS coronavirus The infection mechanism of 2019-nCoV may be changed to being more similar to those of MHV, HIV, Ebola virus (EBoV) and some avian influenza viruses, other than those of most other Betacoronavirus (e g SARS coronavirus) In addition, we unexpectedly found that some avian influenza viruses acquired a cleavage site for furin enzyme by the similar mutation as 2019-nCoV Therefore, the natural mutation can result in a short insertion to form a cleavage site for furin enzyme The cleavage site for furin enzyme in 2019-nCoV contains the CGGCGG sequence encoding two arginine (R) residues CGG, however, is a rare codon for human So we concluded that these two codons were present in the 2019-nCoV -like Betacoronavirus before they transmitted into human and the intermediate host (s) are mammals with a high relative frequency of usage We provide a relative frequency table of CGG usage in mammals to help identify the intermediate hosts of 2019-nCoV Future studies of this mutation will help to reveal the stronger transmissibility of 2019-nCoV and lay foundations for vaccine development and drug design of, but not limited to 2019-nCoV
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The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 19 (COVID-19) pandemic. Despite unprecedented research and developmental efforts, SARS-CoV-2-specific antivirals are still unavailable for the treatment of COVID-19. In most instances, SARS-CoV-2 infection initiates with the binding of Spike glycoprotein to the host cell ACE2 receptor. Utilizing the crystal structure of the ACE2/Spike receptor-binding domain (S-RBD) complex (PDB file 6M0J) in a computer-aided drug design approach, we identified and validated five potential inhibitors of S-RBD and ACE-2 interaction. Two of the five compounds, MU-UNMC-1 and MU-UNMC-2, blocked the entry of pseudovirus particles expressing SARS-CoV-2 Spike glycoprotein. In live SARS-CoV-2 infection assays, both compounds showed antiviral activity with IC
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ABSTRACT We discovered a novel Betacoronavirus lineage A coronavirus, China Rattus coronavirus (ChRCoV) HKU24, from Norway rats in China. ChRCoV HKU24 occupied a deep branch at the root of members of Betacoronavirus 1 , being distinct from murine coronavirus and human coronavirus HKU1. Its unique putative cleavage sites between nonstructural proteins 1 and 2 and in the spike (S) protein and low sequence identities to other lineage A betacoronaviruses (βCoVs) in conserved replicase domains support ChRCoV HKU24 as a separate species. ChRCoV HKU24 possessed genome features that resemble those of both Betacoronavirus 1 and murine coronavirus, being closer to Betacoronavirus 1 in most predicted proteins but closer to murine coronavirus by G+C content, the presence of a single nonstructural protein (NS4), and an absent transcription regulatory sequence for the envelope (E) protein. Its N-terminal domain (NTD) demonstrated higher sequence identity to the bovine coronavirus (BCoV) NTD than to the mouse hepatitis virus (MHV) NTD, with 3 of 4 critical sugar-binding residues in BCoV and 2 of 14 contact residues at the MHV NTD/murine CEACAM1a interface being conserved. Molecular clock analysis dated the time of the most recent common ancestor of ChRCoV HKU24, Betacoronavirus 1 , and rabbit coronavirus HKU14 to about the year 1400. Cross-reactivities between other lineage A and B βCoVs and ChRCoV HKU24 nucleocapsid but not spike polypeptide were demonstrated. Using the spike polypeptide-based Western blot assay, we showed that only Norway rats and two oriental house rats from Guangzhou, China, were infected by ChRCoV HKU24. Other rats, including Norway rats from Hong Kong, possessed antibodies only against N protein and not against the spike polypeptide, suggesting infection by βCoVs different from ChRCoV HKU24. ChRCoV HKU24 may represent the murine origin of Betacoronavirus 1 , and rodents are likely an important reservoir for ancestors of lineage A βCoVs. IMPORTANCE While bats and birds are hosts for ancestors of most coronaviruses (CoVs), lineage A βCoVs have never been found in these animals and the origin of Betacoronavirus lineage A remains obscure. We discovered a novel lineage A βCoV, China Rattus coronavirus HKU24 (ChRCoV HKU24), from Norway rats in China with a high seroprevalence. The unique genome features and phylogenetic analysis supported the suggestion that ChRCoV HKU24 represents a novel CoV species, occupying a deep branch at the root of members of Betacoronavirus 1 and being distinct from murine coronavirus. Nevertheless, ChRCoV HKU24 possessed genome characteristics that resemble those of both Betacoronavirus 1 and murine coronavirus. Our data suggest that ChRCoV HKU24 represents the murine origin of Betacoronavirus 1 , with interspecies transmission from rodents to other mammals having occurred centuries ago, before the emergence of human coronavirus (HCoV) OC43 in the late 1800s. Rodents are likely an important reservoir for ancestors of lineage A βCoVs.
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In 2019, a new pandemic virus belonging to the betacoronavirus family emerged, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This new coronavirus appeared in Wuhan, China, and is responsible for severe respiratory pneumonia in humans, namely, coronavirus disease 2019 (COVID-19). Having infected almost 200 million people worldwide and caused more than 4.1 million deaths as of today, this new disease has raised a significant number of questions about its molecular mechanism of replication and, in particular, how infectious viral particles are produced. Although viral entry is well characterized, the full assembly steps of SARS-CoV-2 have still not been fully described. Coronaviruses, including SARS-CoV-2, have four main structural proteins, namely, the spike glycoprotein (S), the membrane glycoprotein (M), the envelope protein (E), and the nucleocapsid protein (N). All these proteins have key roles in the process of coronavirus assembly and budding. In this review, we gathered the current knowledge about betacoronavirus structural proteins involved in viral particle assembly, membrane curvature and scission, and then egress in order to suggest and question a coherent model for SARS-CoV-2 particle production and release.
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Since the identification of a new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV]) as the causative agent of the SARS epidemic in the winter of 2002-2003, the origin of the novel agent has remained a hotly debated topic.Which virus was the immediate ancestor of SARS-CoV, and what are the relationships between SARS-CoV and other previously described coronaviruses?Correct answers to these two questions are vital, as substantiated below, for designing strategies to detect, contain, and combat new outbreaks and for dissecting the fundamentals of the SARS-CoV life cycle.Major efforts have been invested in a thus far unsuccessful search for a natural SARS-CoV reservoir.In the meantime, and more outside the spotlight, SARS-CoV genome sequences have been used to define the phylogenetic position of SARS-CoV among coronaviruses.These studies have resulted in a lot of controversy whose intricacies may not be very clear to outsiders.Our purpose is to clarify the situation from an insider's point of view.Originally, coronaviruses were classified on the basis of antigenic cross-reactivity, and in this manner three antigenic groups (1 to 3) were recognized (14).When coronavirus genome sequences began to accumulate, the same groups were evident from phylogenetic analyses of the four structural proteins, N, M, E, and S (19), and of different regions of the giant replicase (3,22).Group boundaries were also supported by the diversity of small open reading frames (ORFs) encoding accessory proteins, which are dispersed among the structural protein genes in the 3Ј-proximal region of the genome (Fig. 1).In the middle of the nineties, a first discord between the antigenicity-based and phylogenetic classifications emerged upon the characterization of the coronavirus porcine epidemic diarrhea virus (PEDV) and human coronavirus 229E (HCoV-229E), one of the common cold viruses.These viruses proved not to have antigenic cross-reactivity with members of the established groups ( 18), yet on the basis of sequence comparisons it was concluded that they segregate into group 1, although they are somewhat separated from porcine transmissible gastroenteritis virus and closely related viruses (subgroup 1b and subgroup 1a, respectively, in Fig. 2) (9).The PEDV and HCoV-229E genomes also share an ORF specific for group 1
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A novel coronavirus has been identified as the causative agent of severe acute respiratory syndrome (SARS). The viral main proteinase (M pro , also called 3CL pro ), which controls the activities of the coronavirus replication complex, is an attractive target for therapy. We determined crystal structures for human coronavirus (strain 229E) M pro and for an inhibitor complex of porcine coronavirus [transmissible gastroenteritis virus (TGEV)] M pro , and we constructed a homology model for SARS coronavirus (SARS-CoV) M pro . The structures reveal a remarkable degree of conservation of the substrate-binding sites, which is further supported by recombinant SARS-CoV M pro -mediated cleavage of a TGEV M pro substrate. Molecular modeling suggests that available rhinovirus 3C pro inhibitors may be modified to make them useful for treating SARS.
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Design of hACE2-based small peptide inhibitors against spike protein of SARS-CoV-2: a computational approach,
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Bats have been identified as the natural reservoir of severe acute respiratory syndrome (SARS)-like and SARS coronaviruses (SLCoV and SCoV). However, previous studies suggested that none of the currently sampled bat SLCoVs is the descendant of the direct ancestor of SCoV, based on their relatively distant phylogenetic relationship. In this study, evidence of the recombinant origin of the genome of a bat SLCoV is demonstrated. We identified a potential recombination breakpoint immediately after the consensus intergenic sequence between open reading frame 1 and the S coding region, suggesting the replication intermediates may participate in the recombination event, as previously speculated for other CoVs. Phylogenetic analysis of its parental regions suggests the presence of an uncharacterized SLCoV lineage that is phylogenetically closer to SCoVs than any of the currently sampled bat SLCoVs. Using various Bayesian molecular-clock models, interspecies transfer of this SLCoV lineage from bats to the amplifying host (e.g., civets) was estimated to have happened a median of 4.08 years before the SARS outbreak. Based on this relatively short window period, we speculate that this uncharacterized SLCoV lineage may contain the direct ancestor of SCoV. This study sheds light on the possible host bat species of the direct ancestor of SCoV, providing valuable information on the scope and focus of surveillance for the origin of SCoV.
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