Development of a colloidal gold immunochromatographic assay strip using monoclonal antibody for rapid detection of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) cause diarrhea and dehydration in newborn piglets and has the potential for cross-species transmission. Rapid and early diagnosis is important for preventing and controlling infectious disease. In this study, two monoclonal antibodies (mAbs) were generated, which could specifically recognize recombinant PDCoV nucleocapsid (rPDCoV-N) protein. A colloidal gold immunochromatographic assay (GICA) strip using these mAbs was developed to detect PDCoV antigens within 15 min. Results showed that the detection limit of the GICA strip developed in this study was 103 TCID50/ml for the suspension of virus-infected cell culture and 0.125 µg/ml for rPDCoV-N protein, respectively. Besides, the GICA strip showed high specificity with no cross-reactivity with other porcine pathogenic viruses. Three hundred and twenty-five fecal samples were detected for PDCoV using the GICA strip and reverse transcription-quantitative real-time PCR (RT-qPCR). The coincidence rate of the GICA strip and RT-qPCR was 96.9%. The GICA strip had a diagnostic sensitivity of 88.9% and diagnostic specificity of 98.5%. The specific and efficient detection by the strip provides a convenient, rapid, easy to use and valuable diagnostic tool for PDCoV under laboratory and field conditions.

Attenuation of a Highly Pathogenic Porcine Deltacoronavirus Strain CZ2020 by a Serial Passage In Vitro

Porcine deltacoronavirus (PDCoV) is an emerging swine coronavirus that causes severe diarrhea to pigs of all ages, especially the suckling piglets under one-week-old. We previously isolated a highly pathogenic PDCoV strain, CZ2020, from a diarrheal piglet and have passaged it for over 100 passages. The adaptability of the CZ2020 increased gradually in vitro as the passage increased. Amino acid mutations were observed in pp1a, pp1ab, spike, envelop, and membrane proteins, and the spike protein accounts for 66.7% of all amino acid mutations. Then, the high passage strains, CZ2020-F80 and CZ2020-F100, were selected for evaluation of the pathogenicity in three-day-old piglets to examine whether these amino acid changes affected their virulence. At 2 days postchallenge (DPC), 2/5 piglets started to show typical diarrhea, and at 4 DPC, severe diarrhea was observed in the CZ2020-challenged piglets. Viral RNA could be detected at 1 DPC in rectal swabs and reached its highest at 4 DPC in the CZ2020-challenged group. CZ2020-F80- and CZ2020-F100-challenged groups have one piglet exhibiting mild diarrhea at 4 and 6 DPC, respectively. Compared with the CZ2020-challenged group, the piglets in CZ2020-F80- and F100-challenged groups had lower viral loads in rectal swabs, intestines, and other organs. No obvious histopathological lesions were observed in the intestines of CZ2020-F80- and F100-challenged piglets. Virulent PDCoV infection could also induce strong interferons and proinflammatory cytokines in vitro and in vivo. These data indicate that the strains, CZ2020-F80 and CZ2020-F100, were significantly attenuated via serial passaging in vitro and have the potential for developing attenuated vaccine candidates.

Isolation,identification, and genetic evolution analysis of variant strain CH-HK-2021 of G2a porcine epidemic diarrhea virus

Object: To explore genetic evolution relationship of variant porcine epidemic diarrhea virus(PEDV)and antigenic differential sites among variant strain subtypes,so as to lay a foundation for the development of novel vaccines and diagnostic kits. Method: Three PEDV-positive porcine intestinal samples were inoculated on to confluent Vero cells to isolate PEDV. Virus identification was performed by indirect fluorescence assay(IFA), Western blotting,RT-PCR and whole genome sequencing and electron microscopic observation;virus titer was determined by TCID50and the in vitvo proliferation dynamin curve of the virus was drawn. The genome of the isolated strain was divided into 33 segments for RT-PCR amplification, and the SeqMan of Lasergene was used to splice sequences. Then the genetic evolution analysis was performed with MEGA 7.0, and the antigenicity analysis was performed with Jameson-Wolf algorithm in Protean. Result: Typical cytopathic effect appeared in one PEDV-positive porcine intestinal sample in Vero cells when it was blindly passaged to the 6thgeneration and the sample was designated as CH-HK-2021. IFA and Western blotting results showed that the strain CH-HK-2021 could react with PEDV N monoclonal antibody and expected reads were obtained through RT-PCR amplification, which demonstrated this virus was PEDV. Diameter of strain CH-HK-2021 was 80-120 nm and the surface of the virus particles were in spike-like shape, indicating it was coronavirus. The strain could be stably propagated in Vero cells, and it has been passaged to 100thgeneration. After 24 h of infecting the Vero cells, virus titer of strain CH-HK-2021 reached the highest,105.6TCID50/mL. The size whole genome of strain CH-HK-2021 not including poly(A)tail was 28034 bp, with a similarity of 96.0%-98.9% with nucleotide sequence of the PEDV reference strain and a similarity of 93.1%-99.0% with S-base nucleotide sequence of the reference strain. The strain had the highest similarity with nucleotide sequence of variant strain CH/JX/01(KX058031)and the lowest similarity with nucleotide sequence of classical strain AVCT12(LC053455). Strain CH-HK-2021 was a subtype of G2a and it is spreading in China. Strain G2a and variant strain G2b had 42 nucleotide differential sites in S gene and 6 antigenic differential sites;and main differential sites located in subunit S2.

Identification of Cell Types and Transcriptome Landscapes of Porcine Epidemic Diarrhea Virus-Infected Porcine Small Intestine Using Single-Cell RNA Sequencing.

Swine coronavirus-porcine epidemic diarrhea virus (PEDV) with specific susceptibility to pigs has existed for decades, and recurrent epidemics caused by mutant strains have swept the world again since 2010. In this study, single-cell RNA sequencing was used to perform for the first time, to our knowledge, a systematic analysis of pig jejunum infected with PEDV. Pig intestinal cell types were identified by representative markers and identified a new tuft cell marker, DNAH11. Excepting enterocyte cells, the goblet and tuft cells confirmed susceptibility to PEDV. Enrichment analyses showed that PEDV infection resulted in upregulation of cell apoptosis, junctions, and the MAPK signaling pathway and downregulation of oxidative phosphorylation in intestinal epithelial cell types. The T cell differentiation and IgA production were decreased in T and B cells, respectively. Cytokine gene analyses revealed that PEDV infection downregulated CXCL8, CXCL16, and IL34 in tuft cells and upregulated IL22 in Th17 cells. Further studies found that infection of goblet cells with PEDV decreased the expression of MUC2, as well as other mucin components. Moreover, the antimicrobial peptide REG3G was obviously upregulated through the IL33-STAT3 signaling pathway in enterocyte cells in the PEDV-infected group, and REG3G inhibited the PEDV replication. Finally, enterocyte cells expressed almost all coronavirus entry factors, and PEDV infection caused significant upregulation of the coronavirus receptor ACE2 in enterocyte cells. In summary, this study systematically investigated the responses of different cell types in the jejunum of piglets after PEDV infection, which deepened the understanding of viral pathogenesis.

Comprehensive analysis of codon usage patterns of porcine deltacoronavirus and its host adaptability.

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.

Nonstructural Protein 1 of Variant PEDV Plays a Key Role in Escaping Replication Restriction by Complement C3.

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.

Omicron SARS-CoV-2 mutations stabilize spike up-RBD conformation and lead to a non-RBM-binding monoclonal antibody escape.

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.

Comparison of pathogenicity of porcine deltacoronavirus CZ2020 from cell culture and intestinal contents in 27-day-old piglets.

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.

Porcine Epidemic Diarrhea Virus nsp7 Inhibits Interferon-Induced JAK-STAT Signaling through Sequestering the Interaction between KPNA1 and STAT1.

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.

Structures of a deltacoronavirus spike protein bound to porcine and human receptors.

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.

Receptor binding and complex structures of human ACE2 to spike RBD from omicron and delta SARS-CoV-2.

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.

Neural network analysis of clinical variables predicts escalated care in COVID-19 patients: a retrospective study.

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.

Structural Basis of SARS-CoV-2 Polymerase Inhibition by Favipiravir.

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.

Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology.

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.

Structural and Biochemical Characterization of the nsp12-nsp7-nsp8 Core Polymerase Complex from SARS-CoV-2.

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.