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1.
Emerg Microbes Infect ; 9(1): 20-31, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31859605

RESUMO

Porcine deltacoronavirus (PDCoV) is an emerging swine coronavirus that causes severe diarrhea, resulting in high mortality in neonatal piglets. Despite widespread outbreaks in many countries, no effective PDCoV vaccines are currently available. Here, we generated, for the first time, a full-length infectious cDNA clone of PDCoV. We further manipulated the infectious clone by replacing the NS6 gene with a green fluorescent protein (GFP) to generate rPDCoV-ΔNS6-GFP; likewise, rPDCoV-ΔNS7 was constructed by removing the ATG start codons of the NS7 gene. Growth kinetics studies suggest that rPDCoV-ΔNS7 could replicate similarly to that of the wild-type PDCoV, whereas rPDCoV-ΔNS6-GFP exhibited a substantial reduction of viral titer in vitro and in vivo. Piglets inoculated with rPDCoV-ΔNS7 or wild-type PDCoV showed similar diarrheic scores and pathological injury. In contrast, rPDCoV-ΔNS6-GFP-infected piglets did not show any clinical signs, indicating that the NS6 protein is an important virulence factor of PDCoV and that the NS6-deficient mutant virus might be a promising live-attenuated vaccine candidate. Taken together, the reverse genetics platform described here not only provides more insights into the role of PDCoV accessory proteins in viral replication and pathogenesis, but also allows the development of novel vaccines against PDCoV infection.

2.
BMC Vet Res ; 15(1): 436, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31796026

RESUMO

BACKGROUND: Haemophilus parasuis is a commensal pathogen in the swine upper respiratory tract and causes Glässer's disease. Surveillance, screening for infection, and vaccination response of H. parasuis is hindered by the lack of a rapid antibody detection method. RESULTS: In the present study, a monomeric autotransporter was identified as a novel antigen for developing an indirect ELISA. The autotransporter passenger domain (Apd) was expressed, purified, and demonstrated to be specific in ELISA and western blotting. Mouse antiserum of recombinant Apd (rApd) recognized native Apd in the 15 serotype reference strains and five non-typeable isolate stains, but showed no reaction with seven other bacterial pathogens. The rApd ELISA was optimized and validated using 67 serum samples with known background, including 27 positive sera from experimentally infected and vaccinated pigs along with 40 negative sera that had been screened with H. parasuis whole cell ELISA from clinically healthy herds. The rApd ELISA provided positive and negative percent agreements of 96.4 and 94.9%, respectively, and an AUC value of 0.961, indicating that the assay produced accurate results. CONCLUSION: Apd was a universal antigen component among 15 serotype and non-typeable strains of H. parasuis and was also specific to this pathogen. The rApd ELISA could detect antibodies elicited by H. parasuis infection and vaccination, thereby exhibiting the potential to be applied for Glässer's disease diagnosis, H. parasuis vaccination evaluation, and large-scale serological surveillance.

3.
Viruses ; 11(11)2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31766254

RESUMO

Porcine circovirus type 2 (PCV2) is closely linked to postweaning multisystemic wasting syndrome (PMWS) and other PCV-associated diseases (PCVADs), which influence the global pig industry. MicroRNAs (miRNAs) are evolutionarily conserved classes of endogenous small non-coding RNA that regulate almost every cellular process. According to our previous transcription study, PCV2 infection causes up-regulation of genes related to inflammation. To reveal the function of miRNAs in PCV2 infection and PCV2-encoded miRNAs, next generation sequencing and data analysis was performed to explore miRNA expression in PCV2-infected cells and non-infected cells. Data analysis found some small RNAs matched the PCV2 genome but PCV2 does not express miRNAs in an in vitro infection (PK-15 cells). More than 297 known and 427 novel miRNAs were identified, of which 44 miRNAs were differently expressed (DE). The pathways of inflammation mediated by chemokine and cytokine signaling pathway (P00031), were more perturbed in PCV2-infected cells than in mock controls. DE miRNAs and DE mRNA interaction network clearly revealed that PCV2 regulates the cellular inflammatory response through dysregulating the cellular miRNA-mRNA network. MiRNA overexpression and down-expression results demonstrated that miRNA dysregulation could affect PCV2 infection-induced cellular inflammatory responses. Our study revealed that host miRNA can be dysregulated by PCV2 infection and play an important role in PCV2-modulated inflammation.

4.
Virol J ; 16(1): 95, 2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366370

RESUMO

BACKGROUND: Swine-origin virus infection spreading widely could cause significant economic loss to porcine industry. Novel antiviral agents need to be developed to control this situation. METHODS: In this study, we evaluated the activities of five broad-spectrum antimicrobial peptides (AMPs) against several important swine-origin pathogenic viruses by TCID50 assay. Plaque reduction assay and cell apoptosis assay were also used to test the activity of the peptides. Protection effect of piscidin against pseudorabies virus (PRV) was also examined in mouse model. RESULTS: Piscidin (piscidin 1), caerin (caerin 1.1) and maculatin (maculatin 1.1) could inhibit PRV by direct interaction with the virus particles in a dose-dependent manner and they could also protect the cells from PRV-induced apoptosis. Among the peptides tested, piscidin showed the strongest activity against PRV. Moreover, in vivo assay showed that piscidin can reduce the mortality of mice infected with PRV. CONCLUSION: In vitro and in vivo experiments indicate that piscidin has antiviral activity against PRV.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antivirais/farmacologia , Proteínas de Peixes/farmacologia , Herpesvirus Suídeo 1/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Herpesvirus Suídeo 1/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Pseudorraiva/virologia , Organismos Livres de Patógenos Específicos , Replicação Viral/efeitos dos fármacos
5.
Front Microbiol ; 10: 1513, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31333618

RESUMO

With the increase in commercial pig farming, there is a simultaneous increase in the use of antibiotics for prophylaxis as well as therapeutics in China. In this study, we evaluated the prevalence and resistance diversity of salmonellae isolated from feces of asymptomatic, live and slaughtered pigs. We analyzed 1,732 pig fecal samples collected over 8 months, at Henan province of China. The salmonellae were isolated and identified by PCR. They were serotyped using commercial antisera and assayed for the MIC of 16 antibiotics by broth microdilution method. The average prevalence of Salmonella was 19.4% (95% CI: 17.6-21.4). Large farms (herd size ≥1,000) were found to have a higher prevalence as compared to the small- and medium-scale farms (p < 0.0001). The prevalence of salmonellae in samples collected from the farms [11.77% (95% CI: 10.1-13.6)] and from the slaughterhouse [45.23% (95% CI: 40.3-50.30)] was statistically different (p < 0.0001). Uncommon serovars of Salmonella such as Agama and common serovars such as Derby and Typhimurium were isolated. High resistance (>80%) was recorded toward ciprofloxacin (100%), tetracycline (99.4%), doxycycline (97%), sulfamethoxazole (85.8%), ampicillin (81.6%), and amoxicillin (80.4%). Multidrug resistance (MDR) to four, five, and seven classes of antibiotics was recorded to be approximately 25% in the most prevalent serovar like Derby. We conclude that the presence of alarmingly high resistance, toward the critical antibiotics such as fluoroquinolones and beta-lactams, in large swine farms in China, should draw public attention. These results highlight the need for continued antibiotic stewardship programs for judicious use of critical antibiotics in animal health as well as for producing safe pork.

6.
Sci Rep ; 9(1): 9908, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289289

RESUMO

Bacterial diseases of swine are a kind of multifactorial and uncontrollable diseases that commonly exist in pig farms all over the world and will lead to huge economic losses every year. In this study, a detailed and overall survey was carried out to better understand the prevalence and antimicrobial susceptibilities of bacterial diseases from 2013 to 2017 in China. A total of 19673 bacterial strains were isolated from 44175 samples collected from 9661 pig farms that distributed in 16 Chinese major pig breeding provinces. The results showed that the average isolation rates of Streptococcus suis (SS), Haemophilus parasuis (HPS), Escherichia coli (E. coli), Pasteurella multocida (Pm), Actinobacillus pleuropneumoniae (APP), Brodetella bronchiseptica (Bb), Salmonella enteria (SE), Erysipelothrix rhusiopathiae (E. rhusiopathiae) were 16.9%, 9.7%, 6.3%, 3.4%, 0.3%, 1.5%, 2.3% and 0.9%, respectively. The isolate rates of E. coli, APP and SE showed an increasing trend from 2013 to 2017. The seasonal prevalence characteristics of SS, HPS and Pm were obviously higher from April to August for first two bacteria and higher at February, March, April, and October for Pm. The dominant serotypes for SS, HPS were serotype 2 and serotype 5 (changed from serotype 4), respectively. The SS, HPS, and Pm showed very high antibiotic resistance rates to almost 8 common antibiotics (ß-lactam, aminoglycoside, macrolides, lincomycin, tetracycline, quinolone, polymyxin, and sulfonamide) and an obvious increasing trend of antibiotic resistance rates from 2013 to 2017. In conclusion, the study provides detailed information on the prevalence and antimicrobial susceptibilities of different bacterial pathogens of swine from 2013 to 2017 in China. These data can provide a foundation for monitoring epidemiological patterns of bacterial diseases in the Chinese swine herds, as well as provide insight into potential antibiotic resistance profiles in these pathogens.

7.
J Virol Methods ; 272: 113684, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31288038

RESUMO

Due to outbreaks of porcine epidemic diarrhea (PED) and the wide use of attenuated live vaccine, both wild-type and vaccine strains (CV777) are believed to circulate in Chinese pig farms. Thus, identification of different PEDV strains is of epidemiological importance. In this study, a multiplex RT-PCR method was established based on the sequence features of spike (S) gene and ORF3 gene of PEDVs. The method could identify PEDV variant strains, classical wild-type strains and classical vaccine strains. The limit of detection of the RT-PCR was 1.51 × 104 copies/uL for plasmids and 1 × 101.7 TCID50/100 u L for PEDV, respectively. There were no cross-detections among three different PEDVs and no false detections among six swine pathogens. This assay was used to test 940 samples from China of which 303 samples were PEDV positive, and 289, 5, 10 were positive for variant, classical wild, classical vaccine, respectively. One sample was positive for both variant and classical vaccine PEDV. The variant PEDVs could be detected in samples from 13 provinces, while classical PEDVs were detected from nine provinces, supporting the prevalence of variant PEDV in China. In summary, this multiplex RT-PCR was a useful tool for the clinical detection and epidemiological survey of PEDV.

8.
Artigo em Inglês | MEDLINE | ID: mdl-30941317

RESUMO

Haemophilus parasuis (H. parasuis) is a kind of opportunistic pathogen of the upper respiratory tract of piglets. Under certain circumstances, virulent strains can breach the mucosal barrier and enter the bloodstream, causing severe Glässer's disease. Many virulence factors are found to be related to the pathogenicity of H. parasuis strain, but the pathogenic mechanism remains unclear. LuxS/AI-2, as a kind of very important quorum sensing system, affects the growth characteristics, biofilm formation, antibiotic production, virulence, and metabolism of different strains. In order to investigate the effect of luxS/AI-2 quorum sensing system on the virulence of H. parasuis, a deletion mutant strain (ΔluxS) and complemented strain (C-luxS) were constructed and characterized. The results showed that the luxS gene participated in regulating and controlling stress resistance, biofilm formation and virulence. Compared with wild-type strain, ΔluxS strain decreased the production of AI-2 molecules and the tolerance toward oxidative stress and heat shock, and it reduced the abilities of autoagglutination, hemagglutination, and adherence, whereas it increased the abilities to form biofilm in vitro. In vivo experiments showed that ΔluxS strain attenuated its virulence about 10-folds and significantly decreased its tissue burden of bacteria in mice, compared with the wild-type strain. Taken together, the luxS/AI-2 quorum sensing system in H. parasuis not only plays an important role in growth and biofilm formation, but also affects the pathogenicity of H. parasuis.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Liases de Carbono-Enxofre/metabolismo , Haemophilus parasuis/efeitos dos fármacos , Haemophilus parasuis/crescimento & desenvolvimento , Homosserina/análogos & derivados , Lactonas/metabolismo , Percepção de Quorum , Estruturas Animais/microbiologia , Animais , Carga Bacteriana , Liases de Carbono-Enxofre/deficiência , Modelos Animais de Doenças , Deleção de Genes , Teste de Complementação Genética , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/patologia , Haemophilus parasuis/patogenicidade , Homosserina/metabolismo , Dose Letal Mediana , Camundongos Endogâmicos BALB C , Virulência , Fatores de Virulência/deficiência , Fatores de Virulência/metabolismo
9.
J Vet Pharmacol Ther ; 42(3): 324-335, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30801741

RESUMO

Florfenicol, a structural analog of thiamphenicol, has broad-spectrum antibacterial activity against gram-negative and gram-positive bacteria. This study was conducted to investigate the epidemiological, pharmacokinetic-pharmacodynamic cutoff, and the optimal scheme of florfenicol against Escherichia coli (E. coli) with PK-PD integrated model in the target infectious tissue. 220 E. coli strains were selected to detect the susceptibility to florfenicol, and a virulent strain P190, whose minimum inhibitory concentration (MIC) was similar to the MIC50 (8 µg/ml), was analyzed for PD study in LB and ileum fluid. The MIC of P190 in the ileum fluid was 0.25 times lower than LB. The ratios of MBC/MIC were four both in the ileum and LB. The characteristics of time-killing curves also coincided with the MBC determination. The recommended dosages (30 mg/kg·body weight) were orally administrated in healthy pigs, and both plasma and ileum fluid were collected for PK study. The main pharmacokinetics (PK) parameters including AUC24 hr , AUC0-∞ , Tmax , T1/2 , Cmax , CLb, and Ke were 49.83, 52.33 µg*h/ml, 1.32, 10.58 hr, 9.12 µg/ml, 0.50 L/hr*kg, 0.24 hr-1 and 134.45, 138.71 µg*hr/ml, 2.05, 13.01 hr, 16.57 µg/ml, 0.18 L/hr*kg, 0.14 hr-1 in the serum and ileum fluid, respectively. The optimum doses for bacteriostatic, bactericidal, and elimination activities were 29.81, 34.88, and 36.52 mg/kg for 50% target and 33.95, 39.79, and 42.55 mg/kg for 90% target, respectively. The final sensitive breakpoint was defined as 16 µg/ml. The current data presented provide the optimal regimens (39.79 mg/kg) and susceptible breakpoint (16 µg/ml) for clinical use, but these predicted data should be validated in the clinical practice.


Assuntos
Antibacterianos/uso terapêutico , Infecções por Escherichia coli/veterinária , Escherichia coli/efeitos dos fármacos , Tianfenicol/análogos & derivados , Animais , Antibacterianos/administração & dosagem , Antibacterianos/sangue , Infecções por Escherichia coli/tratamento farmacológico , Feminino , Masculino , Testes de Sensibilidade Microbiana/veterinária , Método de Monte Carlo , Suínos , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/microbiologia , Tianfenicol/administração & dosagem , Tianfenicol/sangue , Tianfenicol/uso terapêutico
10.
Biochim Biophys Acta Biomembr ; 1861(4): 835-844, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30658057

RESUMO

Defensins are a family of cationic antimicrobial peptides of innate immunity with immunomodulatory properties. The prototypic human α-defensins, also known as human neutrophil peptides 1-3 or HNP1-3, are extensively studied for their structure, function and mechanisms of action, yet little is known about HNP4 - the much less abundant "distant cousin" of HNP1-3. Here we report a systematic mutational analysis of HNP4 with respect to its antibacterial activity against E. coli and S. aureus, inhibitory activity against anthrax lethal factor (LF), and binding activity for LF and HIV-1 gp120. Except for nine conserved and structurally important residues (6xCys, 1xArg, 1xGlu and 1xGly), the remaining 24 residues of HNP4 were each individually mutated to Ala. The crystal structures of G23A-HNP4 and T27A-HNP4 were determined, both exhibiting a disulfide-stabilized canonical α-defensin dimer identical to wild-type HNP4. Unlike HNP1-3, HNP4 preferentially killed the Gram-negative bacterium, a property largely attributable to three clustered cationic residues Arg10, Arg11 and Arg15. The cationic cluster was also important for HNP4 killing of S. aureus, inhibition of LF and binding to LF and gp120. However, F26A, while functionally inconsequential for E. coli killing, was far more deleterious than any other mutations. Similarly, N-methylation of Leu20 to destabilize the HNP4 dimer had little effect on E. coli killing, but significantly reduced the ability of HNP4 to kill S. aureus, inhibit LF, and bind to LF and gp120. Our findings unveil the molecular determinants of HNP4 function, completing the atlas of structure and function relationships for all human neutrophil α-defensins.


Assuntos
Antibacterianos , Escherichia coli/crescimento & desenvolvimento , Mutação , Multimerização Proteica , Staphylococcus aureus/crescimento & desenvolvimento , alfa-Defensinas , Substituição de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Antígenos de Bactérias/química , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/química , Humanos , Relação Estrutura-Atividade , alfa-Defensinas/química , alfa-Defensinas/genética , alfa-Defensinas/farmacologia
11.
Arch Virol ; 164(2): 413-425, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30377826

RESUMO

Porcine deltacoronavirus (PDCoV) was first detected in Hong Kong and has recently spread to many countries around the world. PDCoV causes acute diarrhea and vomiting in pigs, resulting in significant economic losses in the global pork industry. In this study, a Chinese PDCoV strain, designated CHN-HG-2017, was isolated from feces of a suckling piglet with severe watery diarrhea on a farm located in central China. Subsequently, the virus was identified by an indirect immunofluorescence assay and electron microscopy. A nucleotide sequence alignment showed that the whole genome of CHN-HG-2017 is 97.6%-99.1% identical to other PDCoV strains. Analysis of potential recombination sites showed that CHN-HG-2017 is a possible recombinant originating from the strains CH/SXD1/2015 and Vietnam/HaNoi6/2015. Furthermore, the pathogenicity of this recombinant PDCoV strain was investigated in 5-day-old piglets by oral inoculation. The challenged piglets developed typical symptoms, such as vomiting, anorexia, diarrhea and lethargy, from 1 to 7 days post-inoculation (DPI). Viral shedding was detected in rectal swabs until 14 DPI in the challenged piglets. Interestingly, high titers of virus-neutralizing antibodies in sera were detected at 21 DPI. Tissues of small intestines from CHN-HG-2017-infected piglets at 4 DPI displayed significant macroscopic and microscopic lesions with clear viral antigen expression. Our analysis of the full genome sequence of a recombinant PDCoV and its virulence in suckling piglets might provide new insights into the pathogenesis of PDCoV and facilitate further investigation of this newly emerged pathogen.


Assuntos
Infecções por Coronavirus/veterinária , Coronavirus/isolamento & purificação , Coronavirus/patogenicidade , Doenças dos Suínos/virologia , Animais , China , Coronavirus/classificação , Coronavirus/genética , Infecções por Coronavirus/virologia , Diarreia/veterinária , Diarreia/virologia , Fezes/virologia , Genoma Viral , Genômica , Filogenia , Suínos , Vietnã , Virulência
13.
Virus Genes ; 54(5): 684-693, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30173363

RESUMO

Porcine bocavirus (PBoV) has a high prevalence in both healthy and diseased swine around the world. It was recently reported that PBoV and porcine circovirus type 2 (PCV2)-which contribute to porcine diarrheal disease-have a high rate of co-infection. To clarify the pathogenesis of PBoV, we examined the co-infection rate and effects of these two pathogens in IPEC-J2 porcine intestinal enterocytes. Both single and co-infection had cytopathic effects in IPEC-J2 cells. The apoptosis and proliferation rates of cells infected with both viruses did not differ significantly from those of cells infected with either one alone. PBoV and PCV2 induced the upregulation of inflammatory cytokines and the downregulation of the tight junction proteins occludin and claudin 1 in the early stage of infection, leading to destruction of epithelial barrier integrity and enhanced cytotoxicity. These findings provide insight into the pathogenic mechanisms of PBoV and PCV2 and a basis for developing effective strategies to prevent the spread of gastrointestinal diseases in pigs and other livestock.


Assuntos
Bocavirus/patogenicidade , Circovirus/patogenicidade , Doenças dos Suínos/virologia , Junções Íntimas/virologia , Animais , Apoptose , Linhagem Celular , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Coinfecção , Citocinas/biossíntese , Efeito Citopatogênico Viral , Infecções por Parvoviridae/virologia , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/prevenção & controle , Replicação Viral
14.
Viruses ; 10(9)2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30231560

RESUMO

Porcine epidemic diarrhea (PED) has re-emerged in recent years and has already caused huge economic losses to the porcine industry all over the world. Therefore, it is urgent for us to find out efficient ways to prevent and control this disease. In this study, the antiviral activity of a cationic amphibian antimicrobial peptide Caerin1.1 against porcine epidemic diarrhea virus (PEDV) was evaluated by an in vitro system using Vero cells. We found that even at a very low concentration, Caerin1.1 has the ability to destroy the integrity of the virus particles to block the release of the viruses, resulting in a considerable decrease in PEDV infections. In addition, Caerin1.1 showed powerful antiviral activity without interfering with the binding progress between PEDV and the receptor of the cells, therefore, it could be used as a potential antiviral drug or as a microbicide compound for prevention and control of PEDV.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antivirais/farmacologia , Vírus da Diarreia Epidêmica Suína/efeitos dos fármacos , Vírus da Diarreia Epidêmica Suína/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antivirais/metabolismo , Sobrevivência Celular , Células Cultivadas , Infecções por Coronavirus/veterinária , Efeito Citopatogênico Viral , Relação Dose-Resposta a Droga , Vírus da Diarreia Epidêmica Suína/ultraestrutura , Suínos , Doenças dos Suínos/virologia , Células Vero , Ensaio de Placa Viral , Replicação Viral/efeitos dos fármacos
15.
Viruses ; 10(8)2018 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-30060558

RESUMO

The porcine epidemic diarrhea virus (PEDV) is an important swine pathogen responsible for severe watery diarrhea, particularly in neonatal piglets. Despite extensive studies performed to elucidate the function of several viral proteins, the contribution of an accessory protein ORF3 in PEDV replication is still largely unknown. Here, we constructed expression plasmids as well as recombinant PEDV carrying myc-tagged ORF3 to assess their expression and subcellular localization in both transfected and infected cells. In PEDV-infected cells, ORF3 was predominantly localized in the cytoplasm, partially in the endoplasmic reticulum (ER) and the Golgi apparatus (Golgi). Interestingly, ORF3 with the N-terminal Flag tag was also detected on the cell surface concomitant with the spike (S) protein as determined by flow cytometry and confocal microscopy. ORF3 and S proteins were also co-localized at perinuclear compartments and in the vesicle-like structures in transfected and infected cells. We also demonstrated that both full-length and naturally truncated ORF3 proteins could interact with the S protein but with different binding affinity, which correlate with the ability of the protein to regulate virus replication in cell culture. Collectively, our results underscore the unprecedented role of the ORF3, which involves the interaction of ORF3 with S and, possibly, other structural protein during PEDV replication.


Assuntos
Vírus da Diarreia Epidêmica Suína/fisiologia , Glicoproteína da Espícula de Coronavírus/fisiologia , Proteínas Virais Reguladoras e Acessórias/fisiologia , Replicação Viral , Animais , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Citoplasma/química , Citoplasma/virologia , Replicação do DNA , Diarreia/veterinária , Diarreia/virologia , Células HEK293 , Humanos , Microscopia Confocal , Plasmídeos/genética , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Doenças dos Suínos/virologia , Transfecção , Células Vero , Proteínas Virais Reguladoras e Acessórias/genética
16.
Front Pharmacol ; 9: 765, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30093860

RESUMO

Pasteurella multocida (PM) can invade the upper respiratory tract of the body and cause death and high morbidity. Tildipirosin, a new 16-membered-ring macrolide antimicrobial, has been recommended for the treatment of respiratory diseases. The objective of this research was to improve the dose regimes of tildipirosin to PM for reducing the macrolides resistance development with the pharmacokinetic/pharmacodynamic (PK/PD) modeling approach and to establish an alternate cutoff for tildipirosin against PM. A single dose (4 mg/kg body weight) of tildipirosin was administered via intramuscular (i.m.) and intravenous (i.v.) injection to the pigs. The minimum inhibitory concentration (MIC) values of clinical isolates (112) were measured in the range of 0.0625-32 µg/ml, and the MIC50 and MIC90 values were 0.5 and 2 µg/ml, respectively. The MIC of the selected PM04 was 2 and 0.5 µg/ml in the tryptic soy broth (TSB) and serum, respectively. The main pharmacokinetic (PK) parameters including the area under the curve at 24 h (AUC24 h), AUC, terminal half-life (T1/2), the time to peak concentration (Tmax), peak concentration (Cmax), relative total systemic clearance (CLb), and the last mean residence time (MRTlast) were calculated to be 7.10, 7.94 µg∗h/ml, 24.02, NA h, NA µg/ml, 0.46 L/h∗kg, 8.06 h and 3.94, 6.79 µg∗h/ml, 44.04, 0.25 h, 0.98 µg/ml, 0.43 L/h∗kg, 22.85 h after i.v. and i.m. induction, respectively. Moreover, the bioavailability of i.m. route was 85.5%, and the unbinding of tildipirosin to serum protein was 78%. The parameters AUC24 h/MIC in serum for bacteriostatic, bactericidal, and elimination activities were calculated as 18.91, 29.13, and 34.03 h based on the inhibitory sigmoid Emax modeling. According to the Monte Carlo simulation, the optimum doses for bacteriostatic, bactericidal, and elimination activities were 6.10, 9.41, and 10.96 mg/kg for 50% target and 7.86, 12.17, and 14.57 mg/kg for 90% target, respectively. The epidemiological cutoff value (ECV) was calculated to be 4 µg/ml which could cover 95% wild-type clinical isolates distribution. The PK-PD cutoff (COPD) was analyzed to be 0.25 µg/ml in vitro for tildipirosin against PM based on the Monte Carlo simulation. Compared with these two cutoff values, the finial susceptible breakpoint was defined as 4 µg/ml. The data presented now provides the optimal regimens (12.17 mg/kg) and susceptible breakpoint (4 µg/ml) for clinical use, but these predicted data should be validated in the clinical practice.

17.
Front Chem ; 6: 244, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29988520

RESUMO

Antimicrobial peptide (Piscidin-1) is an effective natural polypeptide, which has great influence and potential on porcine epidemic diarrhea virus (PEDV) and pseudorabies virus (PRV). As an alternative antibiotic substitute, Piscidin-1 was subjected for pharmacokinetics study with three administration routes (i.v, i.m, and p.o) after a single dose of 2 mg/kg in rats and preliminary pharmacodynamics including antiviral activity in cell against PEDV and PRV. Based on 50 percent tissue culture infective dose (TCID50), there were about 2 and 10% virus survived ratios for Piscidin-1 against PRV and PEDV, respectively. The plaque test showed 1 and 2 µg/ml Piscidin-1 could eliminate 95% PRV and 85% PEDV, respectively. The main pharmacokinetics parameters of Cmax, AUC0-∞, Ke, t1/2, Tmax, MRT, and Clb in plasma were not applicable value, 25.9 µg*h/ml, 0.041 h-1, 16.97 h, not available value, 22.77 h, 0.067 L/h*kg after i.v administration, 2.37 µg/ml, 18.95 µg*h/ml, 0.029 h-1, 23.50 h, 0.33 h, 30.12 h, 0.095 L/h*kg after i.m administration and 0.73 µg/ml, 9.63 µg*h/ml, 0.036 h-1, 19.46 h, 0.50 h, 26.76 h, 0.171 L/h*kg after p.o administration. The bioavailability values after i.m and p.o administrations were calculated as 73.17 and 37.18%, respectively. The i.m administration was selected for pharmacokinetics study in ileum content against PEDV. The main pharmacokinetic parameters of Cmax, AUC0-∞, Ke, t1/2, Tmax, MRT, and Clb in ileum content were 1.67 µg/ml, 78.40 µg*h/ml, 0.034 h-1, 20.16 h, 8.12 h, 36.45 h, 0.026 L/h*kg. The Cmax values in plasma (2.37 µg/ml) and ileum content (1.67 µg/ml) were higher than the effective inhibitory concentration determined in the plaque test, and this indicates that Piscidin-1 might have effective inhibition effect against PRV and PEDV after administration of 2 mg/kg i.m. The results of this study represent the first investigations toward the pharmacokinetic characteristics of piscidin-1 in plasma upon three different administration routes, among which i.m. resulted in the highest bioavailability (73.17%). Furthermore, the pharmacokinetics study of ileum content indicated Piscidin-1 might have good effect against PEDV and could be regarded as an alternative antibiotic in clinical veterinary in the future study.

18.
Front Pharmacol ; 9: 306, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29692725

RESUMO

The current study evaluates a tested marbofloxacin tablet (MBT) (Petsen), in terms of bioavailability and pharmacokinetics (PK) in a comparison of the commercialized and standard tablet (Marbocyl) in beagle dogs. Four different bacterial species were selected for the determination of the minimal inhibitory concentration (MIC) against marbofloxacin (MBF). Target animal safety studies were conducted with a wide spectrum of dosages of Petsen. Pharmacokinetics and bioavailability of Petsen were observed after the oral administration of a recommended dosage of 2 mg/kg. The MIC90 of MBF against Staphylococcus aureus, Escherichia coli, Pasteurella multocida, and Streptococcus were 2.00, 4.00, 0.25, and 0.50 µg/ml, respectively. These results showed that the MBT has an expected antimicrobial activity in vitro. The main parameters of t1/2ß, Clb, AUC0-∞, Cmax, and Ke were 22.14 h, 0.15 L/h, 13.27 µg.h/ml, 0.95 µg/ml, 0.09 h-1, and 16.47 h, 0.14 L/h, 14.10 µg.h/ml, 0.97 µg/ml, 0.11 h-1 after the orally administrated Petsen and Marbocyl, while no biologically significant changes and toxicological significance have been found by their comparison. These findings indicate that the Petsen had a slow elimination, high bioavailability and kinetically similar to the commercialized Marbocyl. Furthermore, no statistically significant differences were distinguished on the continuous gradient dosages of 2, 6, and 10 mg/kg in the term of the clinical presentation. The present study results displayed that the tested MBT (Petsen) was safe, with limited toxicity, which was similar to the commercialized tablet (Marbocyl), could provide an alternative MBT as a veterinary medicine in beagle dogs.

19.
Viruses ; 10(3)2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29534017

RESUMO

The nucleocapsid (N) protein is a major structural component of porcine epidemic diarrhea virus (PEDV), which is predicted to be a multifunctional protein in viral replication. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a cellular protein participating in the splicing of pre-mRNA in the nucleus and translation regulation in the cytoplasm. According to our previous proteomic study about PEDV infection in vivo, hnRNP A1 was thought to be a cellular factor influencing PEDV replication. In this report, PEDV N protein was discovered to colocalize with cellular hnRNP A1 in perinuclear region of PEDV infected cells. Co-immunoprecipitation (CO-IP) results clearly demonstrated that PEDV N protein could bind to human hnRNP A1. Replication of PEDV was inhibited by silencing the expression of hnRNP A1 in CCL-81 cells, suggesting the positive effect of hnRNP A1 on PEDV infection.


Assuntos
Infecções por Coronavirus/veterinária , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Nucleocapsídeo/metabolismo , Vírus da Diarreia Epidêmica Suína/fisiologia , Doenças dos Suínos/metabolismo , Doenças dos Suínos/virologia , Replicação Viral , Animais , Técnicas de Silenciamento de Genes , Ribonucleoproteína Nuclear Heterogênea A1/genética , Humanos , Jejuno/patologia , Jejuno/virologia , Ligação Proteica , Transporte Proteico , Suínos
20.
Front Pharmacol ; 9: 2, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29387013

RESUMO

The aims of the present study were to establish optimal doses and provide an alternate COPD for florfenicol against Streptococcus suis based on pharmacokinetic-pharmacodynamic integration modeling. The recommended dose (30 mg/kg b.w.) were administered in healthy pigs through intramuscular and intravenous routes for pharmacokinetic studies. The main pharmacokinetic parameters of Cmax, AUC0-24h, AUC, Ke, t1/2ke, MRT, Tmax, and Clb, were estimated as 4.44 µg/ml, 88.85 µg⋅h/ml, 158.56 µg⋅h/ml, 0.048 h-1, 14.46 h, 26.11 h, 4 h and 0.185 L/h⋅kg, respectively. The bioavailability of florfenicol was calculated to be 99.14% after I.M administration. A total of 124 Streptococcus suis from most cities of China were isolated to determine the minimum inhibitory concentration (MIC) of florfenicol. The MIC50 and MIC90 were calculated as 1 and 2 µg/ml. A serotype 2 Streptococcus suis (WH-2), with MIC value similar to MIC90, was selected as a representative for an in vitro and ex vivo pharmacodynamics study. The MIC values of WH-2 in TSB and plasma were 2 µg/ml, and the MBC/MIC ratios were 2 in TSB and plasma. The MPC was detected to be 3.2 µg/ml. According to inhibitory sigmoid Emax model, plasma AUC0-24h/MIC values of florfenicol versus Streptococcus suis were 37.89, 44.02, and 46.42 h for the bactericidal, bacteriostatic, and elimination activity, respectively. Monte Carlo simulations the optimal doses for bactericidal, bacteriostatic, and elimination effects were calculated as 16.5, 19.17, and 20.14 mg/kg b.w. for 50% target attainment rates (TAR), and 21.55, 25.02, and 26.85 mg/kg b.w. for 90% TAR, respectively. The PK-PD cutoff value (COPD) analyzed from MCS for florfenicol against Streptococcus suis was 1 µg/ml which could provide a sensitivity cutoff value. These results contributed an optimized alternative to clinical veterinary medicine and showed that the dose of 25.02 mg/kg florfenicol for 24 h could have a bactericidal action against Streptococcus suis after I.M administration. However, it should be validated in clinical practice in the future investigations.

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