RESUMO
Coronaviruses (CoVs) comprise a group of important human and animal pathogens. Despite extensive research in the past 3 years, the host innate immune defense mechanisms against CoVs remain incompletely understood, limiting the development of effective antivirals and non-antibody-based therapeutics. Here, we performed an integrated transcriptomic analysis of porcine jejunal epithelial cells infected with porcine epidemic diarrhea virus (PEDV) and identified cytidine/uridine monophosphate kinase 2 (CMPK2) as a potential host restriction factor. CMPK2 exhibited modest antiviral activity against PEDV infection in multiple cell types. CMPK2 transcription was regulated by interferon-dependent and interferon regulatory factor 1 (IRF1)-dependent pathways post-PEDV infection. We demonstrated that 3'-deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) catalysis by Viperin, another interferon-stimulated protein, was essential for CMPK2's antiviral activity. Both the classical catalytic domain and the newly identified antiviral key domain of CMPK2 played crucial roles in this process. Together, CMPK2, viperin, and ddhCTP suppressed the replication of several other CoVs of different genera through inhibition of the RNA-dependent RNA polymerase activities. Our results revealed a previously unknown function of CMPK2 as a restriction factor for CoVs, implying that CMPK2 might be an alternative target of interfering with the viral polymerase activity.
Assuntos
Infecções por Coronavirus , Coronavirus , Vírus da Diarreia Epidêmica Suína , Humanos , Animais , Suínos , Interferons , Antivirais/farmacologia , Proteínas/genética , Vírus da Diarreia Epidêmica Suína/genéticaRESUMO
Coronaviruses (CoVs) are important pathogens for humans and other vertebrates, causing severe respiratory and intestinal infections that have become a threat to public health because of the potential for interspecies transmission between animals and humans. Therefore, the development of safe, effective vaccines remains a top priority for the control of CoV infection. The unique immunological characteristics of vaccines featuring messenger RNA (mRNA) present an advantageous tool for coronavirus vaccine development. Here, we designed two lipid nanoparticle (LNP)-encapsulated mRNA (mRNA-LNP) vaccines: one encoding full-length spike (S) protein and the other encoding the spike ectodomain (Se) from porcine deltacoronavirus (PDCoV). Fourteen days after primary immunization, both mRNA vaccines induced high levels of immunoglobulin G and neutralizing antibodies in mice, with the S vaccine showing better performance than the Se vaccine. Passive immune protection of the S mRNA vaccine in suckling piglets was confirmed by the induction of robust PDCoV-specific humoral and cellular immune responses. The S mRNA vaccine also showed better protective effects than the inactivated vaccine. Our results suggest that the novel PDCoV-S mRNA-LNP vaccine may have the potential to combat PDCoV infection. IMPORTANCE: As an emerging porcine enteropathogenic coronavirus, porcine deltacoronavirus (PDCoV) has the potential for cross-species transmission, attracting extensive attention. Messenger RNA (mRNA) vaccines are a promising option for combating emerging and re-emerging infectious diseases, as evidenced by the demonstrated efficacy of the COVID-19 mRNA vaccine. Here, we first demonstrated that PDCoV-S mRNA-lipid nanoparticle (LNP) vaccines could induce potent humoral and cellular immune responses in mice. An evaluation of passive immune protection of S mRNA vaccines in suckling piglets confirmed that the protective effect of mRNA vaccine was better than that of inactivated vaccine. This study suggests that the PDCoV-S mRNA-LNP vaccine may serve as a potential and novel vaccine candidate for combating PDCoV infection.
RESUMO
Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, is a serious threat to piglets and has zoonotic potential. Here, we aimed to further explore the role of aminopeptidase N (APN) as a receptor for PDCoV and test the inhibitory effect of a chimeric APN protein strategy on PDCoV infection. PK-15 cells and LLC-PK1 cells expressing chimeric APN were selected and infected with PDCoV. Viral replication was significantly decreased in these chimeric APN cells compared with that in control group cells. To further characterize the effect of the chimeric APN strategy on PDCoV infection in vitro, primary intestinal epithelial cells isolated from chimeric APN pigs were inoculated with PDCoV. Viral challenge of these cells led to decreased PDCoV infection. More importantly, virally challenged chimeric APN neonatal piglets displayed reduced viral load, significantly fewer microscopic lesions in the intestinal tissue, and no diarrhea. Taken together, these findings deepen our understanding of the mechanism of PDCoV infection and provide a valuable model for the production of disease-resistant animals. IMPORTANCE: Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhea in piglets and possesses the potential to infect humans. However, there are currently no effective measures for the prevention or control of PDCoV infection. Here, we have developed PK-15 cells, LLC-PK1 cells, and primary intestinal epithelial cells expressing chimeric APN, and viral challenge of these cells led to decreased PDCoV infection. Furthermore, virally challenged chimeric APN neonatal piglets displayed reduced viral load, significantly fewer microscopic lesions in the intestinal tissue, and no diarrhea. These data show that chimeric APN is a promising strategy to combat PDCoV infection.
Assuntos
Animais Recém-Nascidos , Antígenos CD13 , Infecções por Coronavirus , Deltacoronavirus , Doenças dos Suínos , Replicação Viral , Animais , Suínos , Antígenos CD13/genética , Antígenos CD13/metabolismo , Doenças dos Suínos/virologia , Deltacoronavirus/genética , Infecções por Coronavirus/virologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/prevenção & controle , Carga Viral , Edição de Genes/métodos , Linhagem Celular , Células Epiteliais/virologia , Diarreia/virologiaRESUMO
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.
Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Suínos , Animais , Vírus da Diarreia Epidêmica Suína/genética , Transcriptoma , Intestino Delgado/patologia , Intestinos/patologia , Análise de Sequência de RNARESUMO
BACKGROUND: Methadone maintenance treatment (MMT) is effective for managing opioid use disorder, but adverse effects mean that optimal therapy occurs with the lowest dose that controls opioid craving. OBJECTIVE: To assess the efficacy of acupuncture versus sham acupuncture on methadone dose reduction. DESIGN: Multicenter, 2-group, randomized, sham-controlled trial. (Chinese Clinical Trial Registry: ChiCTR2200058123). SETTING: 6 MMT clinics in China. PARTICIPANTS: Adults aged 65 years or younger with opioid use disorder who attended clinic daily and had been using MMT for at least 6 weeks. INTERVENTION: Acupuncture or sham acupuncture 3 times a week for 8 weeks. MEASUREMENTS: The 2 primary outcomes were the proportion of participants who achieved a reduction in methadone dose of 20% or more compared with baseline and opioid craving, which was measured by the change from baseline on a 100-mm visual analogue scale (VAS). RESULTS: Of 118 eligible participants, 60 were randomly assigned to acupuncture and 58 were randomly assigned to sham acupuncture (2 did not receive acupuncture). At week 8, more patients reduced their methadone dose 20% or more with acupuncture than with sham acupuncture (37 [62%] vs. 16 [29%]; risk difference, 32% [97.5% CI, 13% to 52%]; P < 0.001). In addition, acupuncture was more effective in decreasing opioid craving than sham acupuncture with a mean difference of -11.7 mm VAS (CI, -18.7 to -4.8 mm; P < 0.001). No serious adverse events occurred. There were no notable differences between study groups when participants were asked which type of acupuncture they received. LIMITATION: Fixed acupuncture protocol limited personalization and only 12 weeks of follow-up after stopping acupuncture. CONCLUSION: Eight weeks of acupuncture were superior to sham acupuncture in reducing methadone dose and decreasing opioid craving. PRIMARY FUNDING SOURCE: National Natural Science Foundation of China.
Assuntos
Terapia por Acupuntura , Metadona , Tratamento de Substituição de Opiáceos , Transtornos Relacionados ao Uso de Opioides , Humanos , Metadona/uso terapêutico , Masculino , Terapia por Acupuntura/efeitos adversos , Terapia por Acupuntura/métodos , Feminino , Transtornos Relacionados ao Uso de Opioides/terapia , Adulto , Pessoa de Meia-Idade , Tratamento de Substituição de Opiáceos/métodos , Fissura , Resultado do Tratamento , Analgésicos Opioides/uso terapêutico , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/efeitos adversosRESUMO
With a possible origin from bats, the alphacoronavirus Porcine epidemic diarrhea virus (PEDV) causes significant hazards and widespread epidemics in the swine population. However, the ecology, evolution, and spread of PEDV are still unclear. Here, from 149,869 fecal and intestinal tissue samples of pigs collected in an 11-year survey, we identified PEDV as the most dominant virus in diarrheal animals. Global whole genomic and evolutionary analyses of 672 PEDV strains revealed the fast-evolving PEDV genotype 2 (G2) strains as the main epidemic viruses worldwide, which seems to correlate with the use of G2-targeting vaccines. The evolving pattern of the G2 viruses presents geographic bias as they evolve tachytely in South Korea but undergo the highest recombination in China. Therefore, we clustered six PEDV haplotypes in China, whereas South Korea held five haplotypes, including a unique haplotype G. In addition, an assessment of the spatiotemporal spread route of PEDV indicates Germany and Japan as the primary hubs for PEDV dissemination in Europe and Asia, respectively. Overall, our findings provide novel insights into the epidemiology, evolution, and transmission of PEDV, and thus may lay a foundation for the prevention and control of PEDV and other coronaviruses.
Assuntos
Alphacoronavirus , Infecções por Coronavirus , Coronavirus , Vírus da Diarreia Epidêmica Suína , Animais , Suínos , Vírus da Diarreia Epidêmica Suína/genética , Filogenia , Coronavirus/genética , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/veterináriaRESUMO
IMPORTANCE: Porcine epidemic diarrhea caused by porcine coronaviruses remains a major threat to the global swine industry. Fatty acids are extensively involved in the whole life of the virus. In this study, we found that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) significantly reduced the viral load of porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine delta coronavirus (PDCoV) and acted on the replication of the viruses rather than attachment and entry. We further confirmed that DHA and EPA inhibited PEDV replication by alleviating the endoplasmic reticulum stress. Meanwhile, DHA and EPA alleviate PEDV-induced inflammation and reactive oxygen species (ROS) levels and enhance the cellular antioxidant capacity. These data indicate that DHA and EPA have antiviral effects on porcine coronaviruses and provide a molecular basis for the development of new fatty acid-based therapies to control porcine coronavirus infection and transmission.
Assuntos
Infecções por Coronavirus , Coronavirus , Ácidos Docosa-Hexaenoicos , Ácido Eicosapentaenoico , Doenças dos Suínos , Animais , Coronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/veterinária , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Vírus da Diarreia Epidêmica Suína/fisiologia , Suínos , Doenças dos Suínos/tratamento farmacológico , Vírus da Gastroenterite Transmissível/fisiologia , Replicação Viral/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacosRESUMO
IMPORTANCE: Porcine epidemic diarrhea (PED) caused by PED virus (PEDV) remains a big threat to the swine industry worldwide. Vaccination with live attenuated vaccine is a promising method to prevent and control PED, because it can elicit a more protective immunity than the killed vaccine, subunit vaccine, and so on. In this study, we found two obvious deletions in the genome of a high passage of AH2012/12. We further confirmed the second deletion which contains seven amino acids at the carboxy-terminus of the S2 gene and the start codon of ORF3 can reduce its pathogenicity in vivo. Animal experiments indicated that the recombinant PEDV with deleted carboxy-terminus of S gene showed higher IgG, IgA, neutralization antibodies, and protection effects against virus challenge than the killed vaccine. These data reveal that the engineering of the carboxy-terminus of the S2 gene may be a promising method to develop live attenuated vaccine candidates of PEDV.
Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Diarreia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Suínos , Doenças dos Suínos/virologia , Vacinas Atenuadas/genética , Vacinas de Produtos Inativados , Vacinas Virais/genética , VirulênciaRESUMO
IMPORTANCE: As an emerging porcine enteropathogenic coronavirus that has the potential to infect humans, porcine deltacoronavirus (PDCoV) is receiving increasing attention. However, no effective commercially available vaccines against this virus are available. In this work, we designed a spike (S) protein and receptor-binding domain (RBD) trimer as a candidate PDCoV subunit vaccine. We demonstrated that S protein induced more robust humoral and cellular immune responses than the RBD trimer in mice. Furthermore, the protective efficacy of the S protein was compared with that of inactivated PDCoV vaccines in piglets and sows. Of note, the immunized piglets and suckling pig showed a high level of NAbs and were associated with reduced virus shedding and mild diarrhea, and the high level of NAbs was maintained for at least 4 months. Importantly, we demonstrated that S protein-based subunit vaccines conferred significant protection against PDCoV infection.
Assuntos
Infecções por Coronavirus , Coronavirus , Doenças dos Suínos , Vacinas de Subunidades Antigênicas , Animais , Feminino , Humanos , Camundongos , Coronavirus/genética , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/veterinária , Deltacoronavirus , Suínos , Vacinas de Subunidades Antigênicas/administração & dosagemRESUMO
Porcine deltacoronavirus (PDCoV), a novel enteropathogenic coronavirus, causes diarrhea mainly in suckling piglets and has the potential to infect humans. Whereas, there is no commercially available vaccine which can effectively prevent this disease. In this study, to ascertain the duration of immune protection of inactivated PDCoV vaccine, suckling piglets were injected subcutaneously with inactivated PDCoV vaccine using a prime/boost strategy at 3 and 17-day-old. Neutralizing antibody assay showed that the level of the inactivated PDCoV group was still ≥1:64 at three months after prime vaccination. The three-month-old pigs were orally challenged with PDCoV strain CZ2020. Two pigs in challenge control group showed mild to severe diarrhea at 10-11 day-post-challenge (DPC), while the inactivated PDCoV group had no diarrhea. High levels of viral shedding, substantial intestinal villus atrophy, and positive straining of viral antigens in ileum were detected in challenge control group, while the pigs in inactivated PDCoV group exhibited significantly reduced viral load, minor intestinal villi damage and negative straining of viral antigens. These results demonstrated that PDCoV was pathogenic against three-month-old pigs and inactivated PDCoV vaccine can provide effective protection in pigs lasting for three months.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Infecções por Coronavirus , Diarreia , Doenças dos Suínos , Vacinas de Produtos Inativados , Vacinas Virais , Eliminação de Partículas Virais , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Suínos , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Vacinas Virais/imunologia , Vacinas Virais/administração & dosagem , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/veterinária , Diarreia/prevenção & controle , Diarreia/imunologia , Diarreia/virologia , Vacinação , Coronavirus/imunologia , Carga Viral , Antígenos Virais/imunologiaRESUMO
Rotavirus group A (RVA) is a main pathogen causing diarrheal diseases in humans and animals. Various genotypes are prevalent in the Chinese pig herd. The genetic diversity of RVA lead to distinctly characteristics. In the present study, a porcine RVA strain, named AHFY2022, was successfully isolated from the small intestine tissue of piglets with severe diarrhea. The AHFY2022 strain was identified by cytopathic effects (CPE) observation, indirect immunofluorescence assay (IFA), electron microscopy (EM), high-throughput sequencing, and pathogenesis to piglets. The genomic investigation using NGS data revealed that AHFY2022 exhibited the genotypes G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1, using the online platform the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) (https://www.bv-brc.org/). Moreover, experimental inoculation in 5-day-old and 27-day-old piglets demonstrated that AHFY2022 caused severe diarrhea, fecal shedding, small intestinal villi damage, and colonization in all challenged piglets. Taken together, our results detailed the virological features of the porcine rotavirus G9P[23] from China, including the whole-genome sequences, genotypes, growth kinetics in MA104 cells and the pathogenicity in suckling piglets.
Assuntos
Diarreia , Genoma Viral , Genótipo , Filogenia , Infecções por Rotavirus , Rotavirus , Doenças dos Suínos , Animais , Rotavirus/genética , Rotavirus/isolamento & purificação , Rotavirus/classificação , Rotavirus/patogenicidade , Suínos , Infecções por Rotavirus/virologia , Infecções por Rotavirus/veterinária , China , Doenças dos Suínos/virologia , Diarreia/virologia , Diarreia/veterinária , Intestino Delgado/virologia , Intestino Delgado/patologia , Fezes/virologia , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality in neonatal suckling piglets, leading to significant economic losses to the swine industry. Panax notoginseng saponins (PNS) are bioactive extracts derived from the P. notoginseng plant. In this study, we investigated the anti-PEDV effect of PNS by employing various methodologies to assess their impact on PEDV in Vero cells. Using a CCK-8 (Cell Counting Kit-8) assay, we found that PNS had no significant cytotoxicity below the concentration of 128 µg/mL in Vero cells. Using immunofluorescence assays (IFAs), an enzyme-linked immunosorbent assay (ELISA), and plaque formation assays, we observed a dose-dependent inhibition of PEDV infection by PNS within 24-48 hours postinfection. PNS exerts its anti-PEDV activity specifically at the genome replication stage, and mRNA-seq analysis demonstrated that treatment with PNS resulted in increased expression of various genes, including IFIT1 (interferon-induced protein with tetratricopeptide repeats 1), IFIT3 (interferon-induced protein with tetratricopeptide repeats 3), CFH (complement factor H), IGSF10 (immunoglobulin superfamily member 10), ID2 (inhibitor of DNA binding 2), SPP1 (secreted phosphoprotein 1), PLCB4 (phospholipase C beta 4), and FABP4 (fatty acid binding protein 4), but it resulted in decreased expression of IL1A (interleukin 1 alpha), TNFRSF19 (TNF receptor superfamily member 19), CDH8 (cadherin 8), DDIT3 (DNA damage inducible transcript 3), GADD45A (growth arrest and DNA damage inducible alpha), PTPRG (protein tyrosine phosphatase receptor type G), PCK2 (phosphoenolpyruvate carboxykinase 2), and ADGRA2 (adhesion G protein-coupled receptor A2). This study provides insights into the potential mechanisms underlying the antiviral effects of PNS. Taken together, the results suggest that the PNS might effectively regulate the defense response to the virus and have potential to be used in antiviral therapies.
Assuntos
Infecções por Coronavirus , Panax notoginseng , Vírus da Diarreia Epidêmica Suína , Saponinas , Doenças dos Suínos , Chlorocebus aethiops , Animais , Suínos , Saponinas/farmacologia , Células Vero , Vírus da Diarreia Epidêmica Suína/genética , Interferons , Antivirais/farmacologia , Doenças dos Suínos/tratamento farmacológicoRESUMO
Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that has been the main cause of diarrhea in piglets since 2010 in China. The aim of this study was to investigate sequence variation and recombination events in the spike (S) gene of PEDV isolates from China. Thirty complete S gene sequences were obtained from PEDV-positive samples collected in six provinces in China from 2020 to 2023. Phylogenetic analysis showed that 10% (3/30) belonged to subtype GII-a, 6.67% (2/30) were categorized as subtype GII-b, 66.67% (20/30) were categorized as subtype GII-c, and 16.66% (5/30) were clustered with the S-INDEL strains. Amino acid sequence alignments showed that, when compared to strains of other subtypes, the GII-c strains had two characteristic amino acid substitutions (N139D and I289M). Five S-INDEL subtype strains had a single amino acid deletion (139N) and four amino acid substitutions (N118G, T137S, A138S, and D141G). Recombination analysis allowed six putative recombination events to be identified, one involving recombination between GII-c strains, two involving GII-c and GII-b strains, two involving GII-c and GI-a strains, and one involving GII-a and GI-b strains. These results suggest that recombination between PEDV strains has been common and complex in recent years and is one of the main reasons for the continuous variation of PEDV strains.
Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Recombinação Genética , Glicoproteína da Espícula de Coronavírus , Doenças dos Suínos , Animais , Sequência de Aminoácidos , Substituição de Aminoácidos , China/epidemiologia , Infecções por Coronavirus/virologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/epidemiologia , Diarreia/virologia , Diarreia/veterinária , Diarreia/epidemiologia , Variação Genética , Genótipo , Filogenia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Doenças dos Suínos/virologia , Doenças dos Suínos/epidemiologiaRESUMO
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.
Assuntos
Complemento C3 , Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Proteínas não Estruturais Virais , Replicação Viral , Animais , Antivirais , COVID-19/imunologia , Linhagem Celular Tumoral , Complemento C3/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologiaRESUMO
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.
Assuntos
Doenças dos Suínos , Animais , Técnicas de Cultura de Células , Deltacoronavirus , Diarreia/patologia , Conteúdo Gastrointestinal , Suínos , VirulênciaRESUMO
Online healthcare consultation offers people a convenient way to consult doctors. In this paper, we aim at building a generative dialog system for Chinese healthcare consultation. As the original Seq2seq architecture tends to suffer the issue of generating low-quality responses, the multi-source Seq2seq architecture generating more informative responses is much more preferred in this task. The multi-source Seq2seq architecture takes advantage of retrieval techniques to obtain responses from the database, and then takes these responses alongside the user-issued question as input. However, some of the retrieved responses might be not much related to the user-issued question, resulting in the generation of unsatisfying responses that are not correct in diagnosis or instead provide inappropriate advice on prevention or treatment. Therefore, this paper proposes multi-source Seq2seq guided by knowledge (MSSGK) to handle this problem. MSSGK differs from the multi-source Seq2seq architecture in that domain knowledge, including disease labels and topic labels about prevention and treatment, is introduced into the response generation via a multi-task learning framework. To better exploit the domain knowledge, we propose three attention mechanisms to provide more appropriate guidance for response generation. Experimental results on a dataset of real-world healthcare consultation show the effectiveness of the proposed method.
Assuntos
Aprendizagem , Aprendizado de Máquina , China , Atenção à Saúde , Humanos , Encaminhamento e ConsultaRESUMO
Since late 2010, outbreaks of porcine epidemic diarrhea (PED) have been reported in the swine industry in China. A variant PEDV strain that differs from strain CV777 causes prevalent PEDV infections which commercial vaccines based on CV777 cannot provide complete protection. In this study, we designed a new vaccine based on the epidemic PEDV strain AH2012/12, adjuvanted with flagellin, a mucosal adjuvant that induces mucosal and systemic production of IgA. Three groups of pregnant sows were immunized twice, with a 14-day interval, with PEDV adjuvanted with flagellin, PEDV alone, or PBS before farrowing, and newborn piglets from each group were selected and challenged with PEDV. Immunization with this vaccine elicited high levels of IgG, IgA, and neutralizing antibodies in the serum and colostrum of sows, and newborn piglets were protected against PEDV while suckling. This study should guide the prevention and control strategies for PEDV infection, thereby reducing the losses associated with this virus.
Assuntos
Infecções por Coronavirus/veterinária , Flagelina/administração & dosagem , Vírus da Diarreia Epidêmica Suína/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Animais Recém-Nascidos , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linhagem Celular , Colostro/química , Infecções por Coronavirus/patologia , Infecções por Coronavirus/prevenção & controle , Feminino , Flagelina/imunologia , Imunização , Gravidez , Suínos , Doenças dos Suínos/patologia , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/administração & dosagemRESUMO
BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is mainly responsible for the significant economic losses in pig industry in the world. The adaptive immune responses of the host act as an important source of selective pressure in the evolutionary process of the virus. In the previous study, we confirmed that the amino acid (aa) residues at 102 and 104 sites in GP5 played an important role in escaping from the neutralizing antibodies (NAbs) against highly pathogenic PRRSV (HP-PRRSV). In this study, we further analyzed the aa mutants affecting neutralization susceptibility of NAbs in other structure proteins in NAbs resistant variants. METHODS: Based on the different aa residues of the structural proteins between the resistant virus BB20s and the parent virus BB, 12 recombinant PRRSV strains containing these aa residue substitutions were constructed using reverse genetic techniques. The neutralizing antibody (NA) titers of the recombinant strains were tested on MARC-145 and porcine alveolar macrophages (PAMs). And the NAbs binding abilities of parent and rescued viruses were tested by using ELISA method. RESULTS: By using the neutralization assay, it was revealed that the NA titer of N4 serum with rBB/Ms was significantly lower than that with rBB. Meanwhile, NA titer of the serum with rBB20s/M was significantly higher than that with rBB20s. The ELISA binding results showed that rBB/Ms had higher binding inability to N4 than did rBB. And alignment of M protein revealed that the variant aa residue lysine (K) at 70 was also existed in field type 2 and vaccine PRRSV strains. CONCLUSIONS: The aa residue at 70 in M protein of PRRSV played an important role in regulating neutralization susceptibility to the porcine serum NAbs. It may be helpful for monitoring the antigen variant strains in the field and developing new vaccine against PRRSV in the future.
Assuntos
Aminoácidos/genética , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Soro/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Células Cultivadas , Análise Mutacional de DNA , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Testes de Neutralização , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Genética Reversa , Proteínas da Matriz Viral/genéticaRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) usually establishes a prolonged infection and causes an immunosuppressive state. It has been proposed that IL-10 plays an important role in PRRSV-induced immunosuppression. However, this mechanism has not been completely elucidated. In this study, we found that transfection of 3D4/2 macrophages with the N protein gene of type 2 PRRSV significantly upregulated IL-10 expression at the transcriptional level. Moreover, alanine substitution mutation analysis revealed that the N protein residues 33-37, 65-68 and 112-123 were related to the upregulation of IL-10 promoter activity. Recombinant PRRSV with mutations at residues 33-37 in the N protein (rQ33-5A and rS36A) recovered from corresponding infectious cDNA clones and induced significantly lower levels of IL-10 production in infected monocyte-derived dendritic cells, as compared with their revertants rQ33-5A(R) and rS36A(R), and the wild-type recombinant PRRSV strain rNT/wt. These data indicate that the type 2 PRRSV N protein plays an important role in IL-10 induction and the N-N non-covalent domain is associated with this activity.
Assuntos
Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Interleucina-10/biossíntese , Proteínas do Nucleocapsídeo/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Animais , Linhagem Celular , Macrófagos/imunologia , Macrófagos/virologia , SuínosRESUMO
Porcine epidemic diarrhea (PED) is a highly contagious, enteric disease of swine caused by the porcine epidemic diarrhea virus (PEDV). To find a suitable ELISA method to assess the infection of PEDV and the effectiveness of vaccines, we developed and evaluated an indirect enzyme-linked immunosorbent assay (iELISA) based on a truncated recombinant spike (S) protein expressed in Escherichia coli. The parameters of the iELISA were optimized, and the cutoff value determined as 0.259 by analyzing optical density (OD) values of 80 PEDV negative sera confirmed by western blot. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were both less than 10%. Cross-reactivity assays demonstrated that iELISA was PEDV-specific. A virus neutralization test with sera of 7 different OD values showed a positive correlation between the OD values and virus neutralization. The results suggest this iELISA is specific, sensitive, and repeatable. Further studies should focus on the relationship between OD values of sera and its virus neutralization.