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1.
Transbound Emerg Dis ; 69(5): e2443-e2455, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-2053020

ABSTRACT

The porcine deltacoronavirus (PDCoV) is a newly discovered pig enteric coronavirus that can infect cells from various species. In Haiti, PDCoV infections in children with acute undifferentiated febrile fever were recently reported. Considering the great potential of inter-species transmission of PDCoV, we performed a comprehensive analysis of codon usage patterns and host adaptation profiles of 54 representative PDCoV strains with the spike (S) gene. Phylogenetic analysis of the PDCoV S gene indicates that the PDCoV strains can be divided into five genogroups. We found a certain codon usage bias existed in the S gene, in which the synonymous codons are often ended with U or A. Heat map analysis revealed that all the PDCoV strains shared a similar codon usage trend. The PDCoV S gene with a dN/dS ratio lower than 1 reveals a negative selection on the PDCoV S gene. Neutrality analysis showed that natural selection is the dominant force in shaping the codon usage bias of the PDCoV S gene. Unexpectedly, host adaptation analysis reveals a higher adaptation level of PDCoV to Homo sapiens and Gallus gallus than to Sus scrofa. Compared to the USA lineage, the PDCoV strains in the Early China lineage and Thailand lineage were less adapted to their hosts, which indicates that the evolutionary process plays an important role in the adaptation ability of PDCoV. These findings of this study add to our understanding of PDCoV's evolution, adaptability, and inter-species transmission.


Subject(s)
Coronavirus Infections , Swine Diseases , Animals , Codon/genetics , Codon Usage , Coronavirus Infections/epidemiology , Coronavirus Infections/veterinary , Deltacoronavirus , Genome, Viral/genetics , Phylogeny , Swine , Swine Diseases/epidemiology
3.
J Virol ; 96(18): e0102422, 2022 09 28.
Article in English | MEDLINE | ID: covidwho-2008764

ABSTRACT

Zoonotic coronaviruses represent an ongoing threat to public health. The classical porcine epidemic diarrhea virus (PEDV) first appeared in the early 1970s. Since 2010, outbreaks of highly virulent PEDV variants have caused great economic losses to the swine industry worldwide. However, the strategies by which PEDV variants escape host immune responses are not fully understood. Complement component 3 (C3) is considered a central component of the three complement activation pathways and plays a crucial role in preventing viral infection. In this study, we found that C3 significantly inhibited PEDV replication in vitro, and both variant and classical PEDV strains induced high levels of interleukin-1ß (IL-1ß) in Huh7 cells. However, the PEDV variant strain reduces C3 transcript and protein levels induced by IL-1ß compared with the PEDV classical strain. Examination of key molecules of the C3 transcriptional signaling pathway revealed that variant PEDV reduced C3 by inhibiting CCAAT/enhancer-binding protein ß (C/EBP-ß) phosphorylation. Mechanistically, PEDV nonstructural protein 1 (NSP1) inhibited C/EBP-ß phosphorylation via amino acid residue 50. Finally, we constructed recombinant PEDVs to verify the critical role of amino acid 50 of NSP1 in the regulation of C3 expression. In summary, we identified a novel antiviral role of C3 in inhibiting PEDV replication and the viral immune evasion strategies of PEDV variants. Our study reveals new information on PEDV-host interactions and furthers our understanding of the pathogenic mechanism of this virus. IMPORTANCE The complement system acts as a vital link between the innate and the adaptive immunity and has the ability to recognize and neutralize various pathogens. Activation of the complement system acts as a double-edged sword, as appropriate levels of activation protect against pathogenic infections, but excessive responses can provoke a dramatic inflammatory response and cause tissue damage, leading to pathological processes, which often appear in COVID-19 patients. However, how PEDV, as the most severe coronavirus causing diarrhea in piglets, regulates the complement system has not been previously reported. In this study, for the first time, we identified a novel mechanism of a PEDV variant in the suppression of C3 expression, showing that different coronaviruses and even different subtype strains differ in regulation of C3 expression. In addition, this study provides a deeper understanding of the mechanism of the PEDV variant in immune escape and enhanced virulence.


Subject(s)
Complement C3 , Coronavirus Infections , Porcine epidemic diarrhea virus , Swine Diseases , Viral Nonstructural Proteins , Virus Replication , Animals , Antiviral Agents , COVID-19/immunology , Cell Line, Tumor , Complement C3/immunology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Humans , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Swine , Swine Diseases/immunology , Swine Diseases/virology , Viral Nonstructural Proteins/metabolism , Virus Replication/physiology
4.
Nat Commun ; 13(1): 4958, 2022 08 24.
Article in English | MEDLINE | ID: covidwho-2000890

ABSTRACT

Omicron SARS-CoV-2 is rapidly spreading worldwide. To delineate the impact of emerging mutations on spike's properties, we performed systematic structural analyses on apo Omicron spike and its complexes with human ACE2 or S309 neutralizing antibody (NAb) by cryo-EM. The Omicron spike preferentially adopts the one-RBD-up conformation both before and after ACE2 binding, which is in sharp contrast to the orchestrated conformational changes to create more up-RBDs upon ACE2 binding as observed in the prototype and other four variants of concern (VOCs). Furthermore, we found that S371L, S373P and S375F substitutions enhance the stability of the one-RBD-up conformation to prevent exposing more up-RBDs triggered by ACE2 binding. The increased stability of the one-RBD-up conformation restricts the accessibility of S304 NAb, which targets a cryptic epitope in the closed conformation, thus facilitating the immune evasion by Omicron. These results expand our understanding of Omicron spike's conformation, receptor binding and antibody evasion mechanism.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Antibodies, Monoclonal/genetics , Antibodies, Neutralizing/genetics , Humans , Mutation , Receptors, Virus/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus
5.
Microb Pathog ; 170: 105723, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-1983663

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an emenging swine enteropathogenic coronavirus that can cause high mortality rate. It affects pigs of all ages, but most several in neonatal piglets. Little is known regarding the pathogenicity of PDCoV against 27-day-old piglets. In this study, 27-day-old piglets were experimentally infected with PDCoV CZ2020 from cell culture, the challenged piglets do not have obvious symptoms from 1 to 7 days post-challenge (DPC), while viral shedding was detected in rectal swab at 1 DPC. Tissues of small intestines displayed slight macroscopic and microscopic lesions with no viral antigen detection. On the other hand, 27-day-old piglets were infected with PDCoV from intestinal contents, the piglets developed mild to severe diarrhea, shedding increasing from 2 to 7 DPC, and developed macroscopic and microscopic lesions in small intestines with clear viral antigen confirmed by immunohistochemistry staining. Indicating the small intestine was still the major target organ in PDCoV-challenged pigs at the age of 27-day-old. Diarrhea caused by PDCoV from intestinal contents in 27-day-old piglets is less reported. Thus, our results might provide new insights into the pathogenesis of PDCoV.


Subject(s)
Swine Diseases , Animals , Cell Culture Techniques , Deltacoronavirus , Diarrhea/pathology , Gastrointestinal Contents , Swine , Virulence
6.
J Virol ; 96(9): e0040022, 2022 05 11.
Article in English | MEDLINE | ID: covidwho-1807320

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus that causes high mortality in piglets. Interferon (IFN) responses are the primary defense mechanism against viral infection; however, viruses always evolve elaborate strategies to antagonize the antiviral action of IFN. Previous study showed that PEDV nonstructural protein 7 (nsp7), a component of the viral replicase polyprotein, can antagonize ploy(I:C)-induced type I IFN production. Here, we found that PEDV nsp7 also antagonized IFN-α-induced JAK-STAT signaling and the production of IFN-stimulated genes. PEDV nsp7 did not affect the protein and phosphorylation levels of JAK1, Tyk2, STAT1, and STAT2 or the formation of the interferon-stimulated gene factor 3 (ISGF3) complex. However, PEDV nsp7 prevented the nuclear translocation of STAT1 and STAT2. Mechanistically, PEDV nsp7 interacted with the DNA binding domain of STAT1/STAT2, which sequestered the interaction between karyopherin α1 (KPNA1) and STAT1, thereby blocking the nuclear transport of ISGF3. Collectively, these data reveal a new mechanism developed by PEDV to inhibit type I IFN signaling pathway. IMPORTANCE In recent years, an emerging porcine epidemic diarrhea virus (PEDV) variant has gained attention because of serious outbreaks of piglet diarrhea in China and the United States. Coronavirus nonstructural protein 7 (nsp7) has been proposed to act with nsp8 as part of an RNA primase to generate RNA primers for viral RNA synthesis. However, accumulating evidence indicates that coronavirus nsp7 can also antagonize type I IFN production. Our present study extends previous findings and demonstrates that PEDV nsp7 also antagonizes IFN-α-induced IFN signaling by competing with KPNA1 for binding to STAT1, thereby enriching the immune regulation function of coronavirus nsp7.


Subject(s)
Janus Kinase 1 , Porcine epidemic diarrhea virus , STAT1 Transcription Factor , Signal Transduction , Viral Nonstructural Proteins , alpha Karyopherins , Animals , Cell Line , Interferons/metabolism , Janus Kinase 1/metabolism , Porcine epidemic diarrhea virus/genetics , STAT1 Transcription Factor/metabolism , Swine , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , alpha Karyopherins/metabolism
7.
Nat Commun ; 13(1): 1467, 2022 03 18.
Article in English | MEDLINE | ID: covidwho-1751714

ABSTRACT

Porcine deltacoronavirus (PDCoV) can experimentally infect a variety of animals. Human infection by PDCoV has also been reported. Consistently, PDCoV can use aminopeptidase N (APN) from different host species as receptors to enter cells. To understand this broad receptor usage and interspecies transmission of PDCoV, we determined the crystal structures of the receptor binding domain (RBD) of PDCoV spike protein bound to human APN (hAPN) and porcine APN (pAPN), respectively. The structures of the two complexes exhibit high similarity. PDCoV RBD binds to common regions on hAPN and pAPN, which are different from the sites engaged by two alphacoronaviruses: HCoV-229E and porcine respiratory coronavirus (PRCoV). Based on structure guided mutagenesis, we identified conserved residues on hAPN and pAPN that are essential for PDCoV binding and infection. We report the detailed mechanism for how a deltacoronavirus recognizes homologous receptors and provide insights into the cross-species transmission of PDCoV.


Subject(s)
Coronavirus 229E, Human , Coronavirus Infections , Coronavirus , Animals , Deltacoronavirus , Humans , Spike Glycoprotein, Coronavirus/metabolism , Swine
8.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-323657

ABSTRACT

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously designated as 2019-nCoV) outbreak has caused global concern1. Currently, there are no clinically approved specific drugs or vaccines available for this virus. The viral polymerase is a promising target for developing broad- spectrum antiviral drugs. Here, based on the highly similar structure of SARS- CoV non-structural protein 12 (nsp12) polymerase subunit2, we applied virtual screen for the available compounds, including both the FDA-approved and under- clinic drugs, to identify potential antiviral molecules against SARS-CoV-2. We found two drugs, the clinically approved anti-fungi drug Caspofungin Acetate (Cancidas) and the oncolytic peptide LTX-315, can bind SARS-CoV-2 nsp12 protein to block the polymerase activity in vitro . Further live virus assay revealed that both Caspofungin Acetate and LTX-315 can effectively inhibit SARS-CoV-2 replication in vero cells. These findings present promising drug candidates for treatment of related diseases and would also stimulate the development of pan- coronavirus antiviral agents.Authors Min Wang, Fei Ye, Jiaqi Su, Jingru Zhao, and Bin Yuan contributed equally to this work.

9.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-315877

ABSTRACT

This study sought to identify the most important clinical variables that can be used to determine which COVID-19 patients will need escalated care early on using deep-learning neural networks. Analysis was performed on hospitalized COVID-19 patients between February 7, 2020 and May 4, 2020 in Stony Brook Hospital. Demographics, comorbidities, laboratory tests, vital signs, and blood gases were collected. We compared data obtained at the time in emergency department and the time of intensive care unit (ICU) upgrade of: i) COVID-19 patients admitted to the general floor (N=1203) versus those directly admitted to ICU (N=104), and ii) patients not upgraded to ICU (N=979) versus those upgraded to the ICU (N=224) from the general floor. A deep neural network algorithm was used to predict ICU admission, with 80% training and 20% testing. Prediction performance used area under the curve (AUC) of the receiver operating characteristic analysis (ROC). We found that C-reactive protein, lactate dehydrogenase, creatinine, white-blood cell count, D-dimer, and lymphocyte count showed temporal divergence between patients were upgraded to ICU compared to those were not. The deep learning predictive model ranked essentially the same set of laboratory variables to be important predictors of needing ICU care. The AUC for predicting ICU admission was 0.782±0.013 for the test dataset. Adding vital sign and blood-gas data improved AUC (0.861±0.018). This study identified a few laboratory tests that were predictive of escalated care. This work could help frontline physicians to anticipate downstream ICU needs to more effectively allocate healthcare resources.

10.
Cell ; 185(4): 630-640.e10, 2022 02 17.
Article in English | MEDLINE | ID: covidwho-1611650

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic continues worldwide with many variants arising, some of which are variants of concern (VOCs). A recent VOC, omicron (B.1.1.529), which obtains a large number of mutations in the receptor-binding domain (RBD) of the spike protein, has risen to intense scientific and public attention. Here, we studied the binding properties between the human receptor ACE2 (hACE2) and the VOC RBDs and resolved the crystal and cryoelectron microscopy structures of the omicron RBD-hACE2 complex as well as the crystal structure of the delta RBD-hACE2 complex. We found that, unlike alpha, beta, and gamma, omicron RBD binds to hACE2 at a similar affinity to that of the prototype RBD, which might be due to compensation of multiple mutations for both immune escape and transmissibility. The complex structures of omicron RBD-hACE2 and delta RBD-hACE2 reveal the structural basis of how RBD-specific mutations bind to hACE2.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Receptors, Virus/chemistry , SARS-CoV-2/chemistry , Amino Acid Sequence , Cryoelectron Microscopy , Humans , Models, Molecular , Mutation/genetics , Phylogeny , Protein Binding , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/ultrastructure , Static Electricity , Structural Homology, Protein
11.
PeerJ ; 9: e11205, 2021.
Article in English | MEDLINE | ID: covidwho-1194810

ABSTRACT

This study sought to identify the most important clinical variables that can be used to determine which COVID-19 patients hospitalized in the general floor will need escalated care early on using neural networks (NNs). Analysis was performed on hospitalized COVID-19 patients between 7 February 2020 and 4 May 2020 in Stony Brook Hospital. Demographics, comorbidities, laboratory tests, vital signs and blood gases were collected. We compared those data obtained at the time in emergency department and the time of intensive care unit (ICU) upgrade of: (i) COVID-19 patients admitted to the general floor (N = 1203) vs. those directly admitted to ICU (N = 104), and (ii) patients not upgraded to ICU (N = 979) vs. those upgraded to the ICU (N = 224) from the general floor. A NN algorithm was used to predict ICU admission, with 80% training and 20% testing. Prediction performance used area under the curve (AUC) of the receiver operating characteristic analysis (ROC). We found that C-reactive protein, lactate dehydrogenase, creatinine, white-blood cell count, D-dimer and lymphocyte count showed temporal divergence between COVID-19 patients hospitalized in the general floor that were upgraded to ICU compared to those that were not. The NN predictive model essentially ranked the same laboratory variables to be important predictors of needing ICU care. The AUC for predicting ICU admission was 0.782 ± 0.013 for the test dataset. Adding vital sign and blood-gas data improved AUC (0.822 ± 0.018). This work could help frontline physicians to anticipate downstream ICU need to more effectively allocate healthcare resources.

12.
Innovation (N Y) ; 2(1): 100080, 2021 Feb 28.
Article in English | MEDLINE | ID: covidwho-1033608

ABSTRACT

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has developed into an unprecedented global pandemic. Nucleoside analogs, such as Remdesivir and Favipiravir, can serve as the first-line broad-spectrum antiviral drugs by targeting the viral polymerases. However, the underlying mechanisms for the antiviral efficacies of these drugs are far from well understood. Here, we reveal that Favipiravir, as a pyrazine derivative, could be incorporated into the viral RNA products by mimicking both adenine and guanine nucleotides. This drug thus inhibits viral replication mainly by inducing mutations in progeny RNAs, different from Remdesivir or other RNA-terminating nucleoside analogs that impair the elongation of RNA products. We further determined the cryo-EM structure of Favipiravir bound to the replicating polymerase complex of SARS-CoV-2 in the pre-catalytic state. This structure provides a missing snapshot for visualizing the catalysis dynamics of coronavirus polymerase, and reveals an unexpected base-pairing pattern between Favipiravir and pyrimidine residues that may explain its capacity for mimicking both adenine and guanine nucleotides. These findings shed light on the mechanism of coronavirus polymerase catalysis and provide a rational basis for developing antiviral drugs to combat the SARS-CoV-2 pandemic.

14.
J Virol Methods ; 276: 113772, 2020 02.
Article in English | MEDLINE | ID: covidwho-829838

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. Reverse genetics is a valuable tool to study the functions of viral genes and to generate vaccine candidates. In this study, a full-length infectious cDNA clone of the highly virulent PEDV strain AJ1102 was assembled in a bacterial artificial chromosome (BAC). The rescued virus (rAJ1102) exhibited similar proliferation characteristics in vitro to the wildtype AJ1102. Using CRISPR/Cas9 technology, a recombinant virus rAJ1102-ΔORF3-EGFP in which the ORF3 gene was replaced with an EGFP gene, was successfully generated, and its proliferation characteristics were compared with the parental rAJ1102. Importantly, it just took one week to construct the recombinant PEDV rAJ1102-ΔORF3-EGFP using this method, providing a more efficient platform for PEDV genome manipulation, which could also be applied to other RNA viruses.


Subject(s)
CRISPR-Cas Systems , Coronavirus Infections/virology , Porcine epidemic diarrhea virus/genetics , Swine Diseases/virology , Animals , Coronavirus Infections/prevention & control , Genome, Viral , Recombination, Genetic , Swine , Viral Vaccines/genetics
15.
Cell Rep ; 31(11): 107774, 2020 06 16.
Article in English | MEDLINE | ID: covidwho-594914

ABSTRACT

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) has caused a huge number of human deaths. Currently, there are no specific drugs or vaccines available for this virus (SARS-CoV-2). The viral polymerase is a promising antiviral target. Here, we describe the near-atomic-resolution structure of the SARS-CoV-2 polymerase complex consisting of the nsp12 catalytic subunit and nsp7-nsp8 cofactors. This structure highly resembles the counterpart of SARS-CoV with conserved motifs for all viral RNA-dependent RNA polymerases and suggests a mechanism of activation by cofactors. Biochemical studies reveal reduced activity of the core polymerase complex and lower thermostability of individual subunits of SARS-CoV-2 compared with SARS-CoV. These findings provide important insights into RNA synthesis by coronavirus polymerase and indicate adaptation of SARS-CoV-2 toward humans with a relatively lower body temperature than the natural bat hosts.


Subject(s)
Betacoronavirus/enzymology , Cryoelectron Microscopy , RNA-Dependent RNA Polymerase/chemistry , Viral Nonstructural Proteins/chemistry , Amino Acid Substitution , Coronavirus RNA-Dependent RNA Polymerase , Escherichia coli/genetics , Evolution, Molecular , Models, Molecular , Multiprotein Complexes/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS Virus/enzymology , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism
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