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The Porcine reproductive and respiratory syndrome (PRRS) causes severe financial losses to the global swine industry. Due to continuous virus evolution, the protection against the PRRS provided by current vaccines is limited. In order to find new antiviral strategies, this study investigated the antiviral potential of antimicrobial peptides (AMPs) against PRRSV. Given the diversity of PRRSV strains and the limited effectiveness of existing vaccines in controlling PRRSV, this study evaluated the inhibitory effects of KLAK, Cecropin B, Piscidin1, and Caerin1.1 on 3 strains of PRRSV (lineage 5 classical strain, lineage 8 highly pathogenic strain, and lineage 1 NADC30-like strain). Caerin1.1 exhibited significant dose-dependent antiviral activity, with an effective concentration (EC50) of 7.5⯵M. Caerin1.1 effectively inhibited PRRSV replication when added before or in early infection but showed reduced effectiveness when added in late infection, indicating its potential involvement in targeting early transcription mechanisms of viral RNA polymerase and significantly upregulating cytokine gene expression. In the NADC30 strain-based animal infection model, Caerin1.1 treatment significantly reduced lung viral loads and inflammation in the lungs of PRRSV-infected pigs, with a mortality rate of 0â¯% (0/5) in the treated group compared to 66.67â¯% (4/6) in the untreated group, indicating a reduction in the mortality rate. Additionally, compared with the untreated group, the Caerin1.1-treated group showed significant improvements, such as lighter fever, more daily weight gain, less clinical symptoms, less viral load in blood, and less virus oral shedding (P < 0.05). These findings reveal the potential of antimicrobial peptides as PRRSV therapeutic agents and suggest that Caerin1.1 is a promising candidate for a novel anti-PRRSV drug.
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Proteins serve as the primary executors of cellular activities in organisms, and thus investigating the subcellular localization and interactions of proteins is crucial for understanding protein functions and elucidating the molecular mechanisms in organisms. Proximity labeling is a recently developed effective method for detecting protein-protein interactions in live cells. Compared with the conventional methods for studying protein-protein interactions, proximity labeling demonstrates high sensitivity, strong specificity, and low background and is widely employed in the research of protein-protein interactions between pathogens and hosts. This article reviews the recent progress in the development and applications of the biotin ligase BirA and its mutants and elucidates the functioning principles of several classical biotin ligases. This review aims to clarify the role of proximity labeling based on BirA and its mutants in identifying protein-protein interactions between pathogens and hosts.
Assuntos
Carbono-Nitrogênio Ligases , Interações Hospedeiro-Patógeno , Mutação , Carbono-Nitrogênio Ligases/metabolismo , Carbono-Nitrogênio Ligases/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Biotina/metabolismo , Humanos , Mapeamento de Interação de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
Identification of a conserved G-quadruplex in E165R of ASFVAfrican swine fever virus (ASFV) is a double-stranded DNA arbovirus with high transmissibility and mortality rates. It has caused immense economic losses to the global pig industry. Currently, no effective vaccines or medications are to combat ASFV infection. G-quadruplex (G4) structures have attracted increasing interest because of their regulatory role in vital biological processes. In this study, we identified a conserved G-rich sequence within the E165R gene of ASFV. Subsequently, using various methods, we verified that this sequence could fold into a parallel G4. In addition, the G4-stabilizers pyridostatin and 5,10,15,20-tetrakis-(N-methyl-4-pyridyl) porphin (TMPyP4) can bind and stabilize this G4 structure, thereby inhibiting E165R gene expression, and the inhibitory effect is associated with G4 formation. Moreover, the G4 ligand pyridostatin substantially impeded ASFV proliferation in Vero cells by reducing gene copy number and viral protein expression. These compelling findings suggest that G4 structures may represent a promising and novel antiviral target against ASFV.
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Vírus da Febre Suína Africana , Antivirais , Quadruplex G , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/metabolismo , Animais , Chlorocebus aethiops , Células Vero , Antivirais/farmacologia , Antivirais/química , Suínos , Febre Suína Africana/virologia , Febre Suína Africana/metabolismo , Porfirinas/química , Porfirinas/farmacologia , Ácidos Picolínicos/química , Ácidos Picolínicos/farmacologia , Ácidos Picolínicos/metabolismo , Replicação Viral/efeitos dos fármacos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/química , AminoquinolinasRESUMO
Group A rotaviruses (RVAs) are major causes of severe gastroenteritis in infants and young animals. To enhance our understanding of the relationship between human and animals RVAs, complete genome data are necessary. We screened 92 intestinal and stool samples from diarrheic piglets by RTâPCR targeting the VP6 gene, revealing a prevalence of 10.9%. RVA was confirmed in two out of 5 calf samples. We successfully isolated two porcine samples using MA104 cell line. The full-length genetic constellation of the two isolates were determined to be G9-P[23]-I5-R1-C1-M1-A8-N1-T7-E1-H1, with close similarity to human Wa-like and porcine strains. Sequence analysis revealed the majority of genes were closely related to porcine and human RVAs. Phylogenetic analysis revealed that these isolates might have their ancestral origin from pigs, although some of their gene segments were related to human strains. This study reveals evidence of reassortment and possible interspecies transmission between pigs and humans in China.
Assuntos
Genoma Viral , Filogenia , Infecções por Rotavirus , Rotavirus , Doenças dos Suínos , Animais , Rotavirus/genética , Rotavirus/isolamento & purificação , Rotavirus/classificação , Suínos , Infecções por Rotavirus/virologia , Infecções por Rotavirus/veterinária , Infecções por Rotavirus/transmissão , Infecções por Rotavirus/epidemiologia , Humanos , China/epidemiologia , Doenças dos Suínos/virologia , Doenças dos Suínos/transmissão , Doenças dos Suínos/epidemiologia , Bovinos , Fezes/virologia , Sequenciamento Completo do Genoma , Genótipo , Diarreia/virologia , Diarreia/veterinária , Diarreia/epidemiologia , Linhagem Celular , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Vírus Reordenados/classificaçãoRESUMO
Widespread distribution of porcine epidemic diarrhea virus (PEDV) has led to catastrophic losses to the global pig farming industry. As a result, there is an urgent need for rapid, sensitive and accurate tests for PEDV to enable timely and effective interventions. In the present study, we develop and validate a floating gate carbon nanotubes field-effect transistor (FG CNT-FET)-based portable immunosensor for rapid identification of PEDV in a sensitive and accurate manner. To improve the affinity, a unique PEDV spike protein-specific monoclonal antibody is prepared by purification, and subsequently modified on FG CNT-FET sensor to recognize PEDV. The developed FET biosensor enables highly sensitive detection (LoD: 8.1 fg/mL and 100.14 TCID50/mL for recombinant spike proteins and PEDV, respectively), as well as satisfactory specificity. Notably, an integrated portable platform consisting of a pluggable FG CNT-FET chip and a portable device can discriminate PEDV positive from negative samples and even identify PEDV and porcine deltacoronavirus within 1 min with 100% accuracy. The portable sensing platform offers the capability to quickly, sensitively and accurately identify PEDV, which further points to a possibility of point of care (POC) applications of large-scale surveillance in pig breeding facilities.
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Técnicas Biossensoriais , Nanotubos de Carbono , Vírus da Diarreia Epidêmica Suína , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Animais , Suínos , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Nanotubos de Carbono/química , Limite de Detecção , Imunoensaio/métodos , Imunoensaio/instrumentação , Anticorpos Monoclonais/imunologia , Transistores Eletrônicos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/análise , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Anticorpos Antivirais/imunologia , Desenho de EquipamentoRESUMO
African Swine Fever virus (ASFV), the causative agent of African swine fever, is a highly lethal hemorrhagic virus affecting domestic pigs and wild boars. The primary target cells for ASFV infection are porcine alveolar macrophages (PAMs), which are difficult to obtain and maintain in vitro, and less subjective to genetic editing. To overcome these issues and facilitate ASFV research, we obtained a subclonal cell line PK1-C5 by subcloning LLC-PK1 cells that support stable ASFV proliferation. This consequential cell line exhibited high ASFV infection levels and similar viral growth characteristics to PAMs, while also allowing high-efficiency genomic editing through transfection or lentivirus transduction of Cas9. Taken together, our study provided a valuable tool for research aspects including ASFV-host interactions, pathogenicity, and vaccine development.
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Vírus da Febre Suína Africana , Febre Suína Africana , Doenças dos Suínos , Suínos , Animais , Vírus da Febre Suína Africana/genética , Sus scrofa , Linhagem Celular , RimRESUMO
Streptococcus suis (S. suis) is a significant zoonotic microorganism that causes a severe illness in both pigs and humans and is characterized by severe meningitis and septicemia. Suilysin (SLY), which is secreted by S. suis, plays a crucial role as a virulence factor in the disease. To date, the interaction between SLY and host cells is not fully understood. In this study, we identified the interacting proteins between SLY and human brain microvascular endothelial cells (HBMECs) using the TurboID-mediated proximity labeling method. 251 unique proteins were identified in TurboID-SLY treated group, of which six plasma membrane proteins including ARF6, GRK6, EPB41L5, DSC1, TJP2, and PNN were identified. We found that the proteins capable of interacting with SLY are ARF6 and PNN. Subsequent investigations revealed that ARF6 substantially increased the invasive ability of S. suis in HBMECs. Furthermore, ARF6 promoted SLY-induced the activation of p38 MAPK signaling pathway in HBMECs. Moreover, ARF6 promoted the apoptosis in HBMECs through the activation of p38 MAPK signaling pathway induced by SLY. Finally, we confirmed that ARF6 could increase the virulence of SLY in C57BL/6 mice. These findings offer valuable insights that contribute to a deeper understanding of the pathogenic mechanism of SLY.
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Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Apoptose , Células Endoteliais , Proteínas Hemolisinas , Streptococcus suis , Streptococcus suis/patogenicidade , Streptococcus suis/metabolismo , Humanos , Animais , Apoptose/efeitos dos fármacos , Camundongos , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/microbiologia , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/metabolismo , Virulência , Encéfalo/metabolismoRESUMO
Swine acute diarrhea syndrome coronavirus (SADS-CoV), a member of the family Coronaviridae and the genus Alphacoronavirus, primarily affects piglets under 7 days old, causing symptoms such as diarrhea, vomiting, and dehydration. It has the potential to infect human primary and passaged cells in vitro, indicating a potential risk of zoonotic transmission. In this study, we successfully generated and purified six monoclonal antibodies (mAbs) specifically targeting the spike protein of SADS-CoV, whose epitope were demonstrated specificity to the S1A or S1B region by immunofluorescence assay and enzyme-linked immunosorbent assay. Three of these mAbs were capable of neutralizing SADS-CoV infection on HeLa-R19 and A549. Furthermore, we observed that SADS-CoV induced the agglutination of erythrocytes from both humans and rats, and the hemagglutination inhibition capacity and antigen-antibody binding capacity of the antibodies were assessed. Our study reveals that mAbs specifically targeting the S1A domain demonstrated notable efficacy in suppressing the hemagglutination phenomenon induced by SADS-CoV. This finding represents the first instance of narrowing down the protein region responsible for SADS-CoV-mediated hemagglutination to the S1A domain, and reveals that the cell attachment domains S1A and S1B are the main targets of neutralizing antibodies.
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Alphacoronavirus , Doenças dos Suínos , Ratos , Animais , Humanos , Suínos , Glicoproteína da Espícula de Coronavírus/química , Anticorpos Monoclonais , Anticorpos Neutralizantes/metabolismoRESUMO
IMPORTANCE: The key bacterial pathogen Glaesserella parasuis, which can cause Glässer's disease, has caused significant financial losses to the swine industry worldwide. Capsular polysaccharide (CPS) is an important virulence factor for bacteria, providing the ability to avoid recognition and killing by the host immune system. Exploring the alteration of CPS synthesis in G. parasuis in response to epinephrine stimulation can lay the groundwork for revealing the pathogenic mechanism of G. parasuis as well as providing ideas for Glässer's disease control.
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Infecções por Haemophilus , Haemophilus parasuis , Doenças dos Suínos , Animais , Suínos , Fatores de Virulência , Haemophilus parasuis/genética , Infecções por Haemophilus/veterinária , Infecções por Haemophilus/microbiologia , Doenças dos Suínos/microbiologiaRESUMO
BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), leading to abortion in sows and respiratory distress in breeding pigs. In China, PRRSV1 and PRRSV2 are the two circulating genotypes in swine herds, with distinct virulence. PRRSV2 further consists of classical (C-PRRSV2), highly pathogenic (HP-PRRSV2), and NADC30-Like (N-PRRSV2) subtypes. The diversity of PRRSV poses challenges for control and eradication, necessitating reliable detection assays for differentiating PRRSV genotypes. METHODS: A new TaqMan-based RT-qPCR assay with four sets of primers and probes targeting conserved regions of the ORF7 and NSP2 genes of PRRSV was developed, optimized, and evaluated by us. Reaction conditions such as annealing temperature, primer concentration, and probe concentration were optimized for the assay. Specificity, sensitivity, repeatability, stability, limit of detection (LOD), concordance with the reference method were evaluated for the assay. RESULTS: The assay could detect and type PRRSV1, C-PRRSV2, HP-PRRSV2, and N-PRRSV2 simultaneously with 97.33% specificity, 96.00% sensitivity, 12 copies/µL LOD, 97.00% concordance with reference assays. We applied the assay to 321 clinical samples from swine farms in China. The assay successfully detected and typed 230 PRRSV-positive samples, with 24.78% (57/230) of them further confirmed by ORF5 gene sequencing. The prevalence of PRRSV subtypes among the positive samples was as follows: C-PRRSV2 (15.22%), HP-PRRSV2 (23.48%), and N-PRRSV2 (61.30%). Two samples showed coinfection with different PRRSV subtypes. CONCLUSION: The quadruple RT-qPCR assay is a powerful tool for detecting and typing the currently circulating PRRSV strains in Chinese swine populations. It can assist in the surveillance of PRRSV prevalence and the implementation of prevention and control strategies.
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Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Animais , Feminino , Gravidez , China/epidemiologia , Síndrome Respiratória e Reprodutiva Suína/diagnóstico , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Sus scrofa , Suínos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterináriaRESUMO
Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.
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Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Doenças dos Suínos , Suínos , Animais , Actinobacillus pleuropneumoniae/genética , Macrófagos Alveolares/metabolismo , Exotoxinas/farmacologia , Apoptose , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/genética , Infecções por Actinobacillus/veterinária , Infecções por Actinobacillus/microbiologia , Proteínas Hemolisinas/toxicidade , Doenças dos Suínos/microbiologiaRESUMO
Glaesserella parasuis is an important pathogen that causes fibrinous polyserositis, peritonitis and meningitis in pigs, leading to considerable economic losses to the swine industry worldwide. It is well established that the serine protease HtrA is closely associated with bacterial virulence, but the role of HtrA in G. parasuis pathogenesis remains largely unknown. To characterize the function of the htrA gene in G. parasuis, a ΔhtrA mutant was constructed. We found that the ΔhtrA mutant showed significant growth inhibition under heat shock and alkaline stress conditions, indicating HtrA is involved in stress tolerance and survival of G. parasuis. In addition, deletion of htrA gene resulted in decreased adherence to PIEC and PK-15 cells and increased phagocytic resistance to 3D4/2 macrophages, suggesting that htrA is essential for adherence of G. parasuis. Scanning electron microscopy revealed morphological surface changes of the ΔhtrA mutant, and transcription analysis confirmed that a number of adhesion-associated genes are downregulated, which corroborated the aforementioned phenomenon. Furthermore, G. parasuis HtrA induced a potent antibody response in piglets with Glässer's disease. These observations confirmed that the htrA gene is related to the survival and pathogenicity of G. parasuis.
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Infecções por Haemophilus , Haemophilus parasuis , Doenças dos Suínos , Animais , Suínos , Sorogrupo , Virulência/genética , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/veterinária , Doenças dos Suínos/microbiologiaRESUMO
Porcine epidemic diarrhea virus (PEDV), a member of the α-coronavirus genus, can cause vomiting, diarrhea, and dehydration in piglets. Neonatal piglets infected with PEDV have a mortality rate as high as 100%. PEDV has caused substantial economic losses to the pork industry. Endoplasmic reticulum (ER) stress, which can alleviate the accumulation of unfolded or misfolded proteins in ER, involves in coronavirus infection. Previous studies have indicated that ER stress could inhibit the replication of human coronaviruses, and some human coronaviruses in turn could suppress ER stress-related factors. In this study, we demonstrated that PEDV could interact with ER stress. We determined that ER stress could potently inhibit the replication of Gâ , Gâ ¡-a, and Gâ ¡-b PEDV strains. Moreover, we found that these PEDV strains can dampen the expression of the 78 kDa glucose-regulated protein (GRP78), an ER stress marker, while GRP78 overexpression showed antiviral activity against PEDV. Among different PEDV proteins, PEDV non-structural protein 14 (nsp14) was revealed to play an essential role in the inhibition of GRP78 by PEDV, and its guanine-N7-methyltransferase domain is necessary for this role. Further studies show that both PEDV and its nsp14 negatively regulated host translation, which could account for their inhibitory effects against GRP78. In addition, we found that PEDV nsp14 could inhibit the activity of GRP78 promotor, helping suppress GRP78 transcription. Our results reveal that PEDV possesses the potential to antagonize ER stress, and suggest that ER stress and PEDV nsp14 could be the targets for developing anti-PEDV drugs.
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Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Antivirais/farmacologia , Infecções por Coronavirus/veterinária , Chaperona BiP do Retículo Endoplasmático , Vírus da Diarreia Epidêmica Suína/fisiologia , Proteínas/farmacologia , Suínos , Doenças dos Suínos/virologiaRESUMO
Rotaviruses are rising as zoonotic viruses worldwide, causing the lethal dehydrating diarrhea in children, piglets, and other livestock of economic importance. A simple, swift, cost-effective, highly specific, and sensitive antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for detection of porcine rotavirus-A (PoRVA) by employing rabbit (capture antibody) and murine polyclonal antibodies (detector antibody) produced against VP6 of PoRVA (RVA/Pig-tc/CHN/TM-a/2009/G9P23). Reactivity of the both polyclonal antibodies was confirmed by using an indirect ELISA, western-blot analysis and indirect fluorescence assay against rVP6 protein and PoRVA. The detection limit of AC-ELISA was found 50 ng/ml of PoRVA protein. The relative sensitivity and specificity of this in-house AC-ELISA were evaluated for detection of PoRVA from 295 porcine diarrhea samples, and results were compared with that of RT-PCR and TaqMan RT-qPCR. The relative sensitivity and specificity of AC-ELISA compared with those of TaqMan RT-qPCR were found as 94.4 and 99.2%, respectively, with the strong agreement (κ -0.58) between these two techniques. Furthermore, AC-ELISA could not detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pseudo rabies virus and porcine circovirus-2. This in-house AC-ELISA efficiently detected PoRVA from clinical samples, which suggests that this technique can be used for large-scale surveillance and timely detection of rotavirus infection in the porcine farms.
In this study, we used a Chinese porcine rotavirus-A (PoRVA) strain containing the I5, a dominant VP6-genotype in pigs, for production of VP6 (most conserved) protein based polyclonal antibodies (pAb) in rabbits (as capture Ab) and mouse (as detector Ab) for development of simple, cost effective, highly specific and sensitive AC-ELISA for detection of PoRVA. Furthermore, there is no any previous published report on application of rabbit and mouse pAb against VP6 for developing an AC-ELISA against PoRVA.
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Infecções por Rotavirus , Rotavirus , Doenças dos Suínos , Animais , Suínos , Coelhos , Camundongos , Infecções por Rotavirus/diagnóstico , Infecções por Rotavirus/veterinária , Diarreia , Ensaio de Imunoadsorção Enzimática/veterinária , Anticorpos Antivirais , Sensibilidade e Especificidade , Doenças dos Suínos/diagnósticoRESUMO
Bovine viral diarrhea virus (BVDV) is a ubiquitous immunosuppressive etiological agent which is economically important for a wide host range in the livestock industry. Lactobacillus spp. has widely been using in the field of management and treatment of gastro-enteric disease for both humans and animals. The ability of Lacticaseibacillus casei MCJ protein-based metabolized to suppress BVDV infection in Madin-Darby Bovine Kidney cell line was demonstrated in this study. The protein-based metabolites were extracted from the cultured L. casei to obtain the safest and beneficial form of the probiotic bacteria. It is revealed that LPM have no cytotoxic effect and the cell viability remain more than 80% even after the cells are treated with 3000 µg/mL of LPM. The results of the plaque formation assay showed that LPM can reduce the viral infection rate. To know the mechanism of LPM for anti-BVDV activity, MDBK cells were exposed to LPM before, after and co-incubation of virus infection. The co-treatment of LPM with BVDV revealed the best results. The results suggest that the LPM has a potential anti-BVDV activity which could be a prospective candidate for the prevention and control of BVDV infection in an animal.
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Vírus da Diarreia Viral Bovina , Lacticaseibacillus casei , Humanos , Animais , Antivirais , Lacticaseibacillus , Vírus da Diarreia Viral Bovina/metabolismo , DiarreiaRESUMO
The emergence and dissemination of Escherichia coli (E. coli) strains that produce extended-spectrum beta-lactamases (ESBLs) represents a major public health threat. The present study was designed to evaluate the prevalence and characteristics of ESBL-producing Escherichia coli isolates from chickens in central China during 2016-2019. A total of 407 E. coli strains isolated from 581 chicken swabs were identified conventionally and analyzed for various cephalosporin susceptibility by disk-diffusion assay. ESBL-producing strains were screened using the double=disk synergy test and ESBL-encoding genes were carried out by PCR/sequencing. A total of 402 E. coli isolates exhibited strong resistance to first- to fourth-generation cephalosporins and monobactam antibiotics, especially cefazolin (60.69%), cefuroxime (54.05%), cefepime (35.14%), ceftriaxone (54.30%), and aztreonam (40.29%). Piperacillin/tazobactam (1.72%) was the most effective drug against the strains, but the resistance rates increased each year. Among the isolates, 262 were identified as ESBL producers and the isolation rates for the ESBL producers increased from 63.37% to 67.35% over the four years. CTX-M (97.33%) was the most prevalent type, followed by TEM (76.72%) and SHV (3.05%). The most common ESBL genotype combination was blaTEM + blaCTX-M (74.46%), in which the frequency of carriers increased steadily, followed by blaCTX-M + blaSHV (3.05%). In addition, the most predominant specific CTX-M subtypes were CTX-M-55 (48.47%) and CTX-M-1 (17.94%), followed by CTX-M-14 (11.01%), CTX-M-15 (8.02%), CTX-M-9 (6.11%), CTX-M-65 (4.58%), and CTX-M-3 (1.15%). Moreover, a novel multiplex qPCR assay was developed to detect blaCTX-M, blaTEM, and blaSHV, with limits of detection of 2.06 × 101 copies/µL, 1.10 × 101 copies/µL, and 1.86 × 101 copies/µL, respectively, and no cross-reactivity with other ESBL genes and avian pathogens. The assays exhibited 100% sensitivity and specificities of 85%, 100%, and 100% for blaCTX-M, blaTEM, and blaSHV, respectively. In conclusion, our findings indicated that ESBL-producing E.coli strains isolated from chickens in central China were highly resistant to cephalosporins and frequently harbored diversity in ESBL-encoding genes. These isolates can pose a significant public health risk. The novel multiplex qPCR method developed in this study may be a useful tool for molecular epidemiology and surveillance studies of ESBL genes.
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In China, the duck industry has been severely impacted by the newly emerging duck Tembusu virus (DTMUV). For DTMUV to successfully infect host cells, it employs several strategies that subvert the host's innate immune response. It has been found that several viral proteins encoded by DTMUV have strategically targeted the crucial molecules of the RIG-I-like Receptor (RLR) signaling pathway to antagonize host antiviral responses. However, it is not well known how the host proteins manipulated by DTMUV contribute to innate immune evasion. The present study reports that duck TRIM35 (duTRIM35) antagonizes DTMUV-induced innate immune responses by targeting duck RIG-I (duRIG-I) in duck embryo fibroblasts. A significant increase in duTRIM35 expression occurred during DTMUV infection. DuTRIM35 overexpression suppressed DTMUV-triggered expression of interferon beta (IFN-ß) and interferon-stimulated genes (ISGs), promoting viral replication, whereas knockdown of duTRIM35 augments the innate immune response, reducing viral replication. Furthermore, duTRIM35 significantly impaired the IFN-ß expression mediated by duRIG-I but not by other RLR signaling molecules. Mechanistically, duTRIM35 interfered with duRIG-I-duTRIM25 interaction and impeded duTRIM25-mediated duRIG-I ubiquitination by interacting with both duRIG-I and duTRIM25. Our findings indicate that duTRIM35 expression induced by DTMUV infection interfered with the duRIG-I-mediated antiviral response, illustrating a novel strategy in which DTMUV can evade the host's innate immunity. IMPORTANCE Duck Tembusu virus (DTMUV), an emerging flavivirus pathogen causing a substantial drop in egg production and severe neurological disorders in duck populations, has led to massive economic losses in the global duck industry. DTMUV has employed various strategies to subvert the host's innate immune response to establish a productive infection in host cells. In this study, we report that duck TRIM35 (duTRIM35) expression was upregulated upon DTMUV infection in vitro and in vivo, and its expression antagonized DTMUV-induced innate immune responses by targeting duck RIG-I (duRIG-I) in duck embryo fibroblasts. Further studies suggest that duTRIM35 interfered with duRIG-I-duTRIM25 interaction and impeded duTRIM25-mediated duRIG-I ubiquitination by interacting with both duRIG-I and duTRIM25. Together, these results revealed that duTRIM35 expression induced by DTMUV infection downregulated duRIG-I-mediated host antiviral response, which elucidated a novel strategy of DTMUV for innate immune evasion.
Assuntos
Infecções por Flavivirus , Flavivirus , Doenças das Aves Domésticas , Animais , Antivirais , Patos/metabolismo , Infecções por Flavivirus/veterinária , Proteína DEAD-box 58/metabolismo , Flavivirus/genética , Interferon beta , Transdução de Sinais , Imunidade Inata , Replicação Viral , Fibroblastos/metabolismoRESUMO
Porcine circovirus 3 (PCV3) is a newly emerging virus and has been found associated with porcine dermatitis and nephropathy syndrome in pigs. Compared with PCV2, research into PCV3 cap gene sequencing is deficient. To investigate the prevalence and genotype distribution of PCV3, we collected 1291 samples from 211 pig farms throughout 15 provinces and municipalities. 312 out of 1291 samples were tested positive by PCR. We further sequenced and analyzed 164 PCR-positive samples. The majority (61.8%) of isolates we sequenced belong to genotype PCV3c. PCV3c is also the dominant genotype in Hubei, Hunan, Hebei province and Chongqing city. We found 3 sites under positive selection and located in predicted epitope peptide, revealing that the pig's immunity may be a reason those sites are undergoing highly positive selection.
Assuntos
Infecções por Circoviridae , Circovirus , Doenças dos Suínos , Suínos , Animais , Circovirus/genética , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/veterinária , Cidades , Doenças dos Suínos/epidemiologia , Filogenia , China/epidemiologiaRESUMO
Porcine respiratory diseases complex (PRDC) is a highly serious threat to the pig industry. In the present study, we investigated and analyzed the etiology associated with PRDC and explored the role of viruses in respiratory bacterial infections. From 2017 to 2021, clinical samples were collected from 1,307 pigs with typical respiratory symptoms in 269 farms in China and screened for pathogens related to PRDC by PCR and bacterial isolation. The results indicated that PRRSV (41.16%, 95%CI: 38.49~43.83%), PCV2 (21.58%,95%CI: 19.35~23.81%), S. suis (63.50%, 95%CI: 60.89~66.11%), and G. parasuis (28.54%, 95%CI: 26.09~30.99%) were the most commonly detected pathogens in pigs with PRDC in China. The dominant epidemic serotypes (serogroups) of S. suis, G. parasuis, and P. multocida were serotype 2, serotype 1, and capsular serogroups D, respectively. Pigs of different ages exhibited different susceptibilities to these pathogens, e.g., PRRSV, PCV2, and G. parasuis had the highest detection rates in nursery pigs, whereas fattening pigs had the highest detection rates of P. multocida and A. pleuropneumoniae. Among the 1,307 pigs, the detection rates of S. suis, G. parasuis, P. multocida, and B. bronchiseptica were higher in virus-positive pigs, especially G. parasuis and P. multocida were significantly (p < 0.01) higher than in virus-negative pigs. In addition, a strong positive correlation was found between coinfection by PRRSV and G. parasuis (OR = 2.33, 95%CI: 1.12~2.14), PRRSV and P. multocida (OR = 1.55, 95%CI: 1.12~2.14), PCV2 and P. multocida (OR = 2.27, 95%CI: 1.33~3.87), PRRSV-PCV2 and S. suis (OR = 1.83, 95%CI: 1.29~2.60), PRRSV-PCV2 and G. parasuis (OR = 3.39, 95%CI: 2.42~4.74), and PRRSV-PCV2 and P. multocida (OR = 2.09, 95%CI: 1.46~3.00). In summary, PRRSV, PCV2, S. suis, and G. parasuis were the major pathogens in pigs with PRDC, and coinfections of two or more PRDC-related pathogens with strong positive correlations were common in China, such as PRRSV and G. parasuis, PRRSV and P. multocida, PCV2 and P. multocida, and also PRRSV-PCV2 and G. parasuis and PRRSV-PCV2 and P. multocida.