Transportin-3 Facilitates Uncoating of Influenza A Virus.

Influenza A viruses (IAVs) are a major global health threat and in the future, may cause the next pandemic. Although studies have partly uncovered the molecular mechanism of IAV-host interaction, it requires further research. In this study, we explored the roles of transportin-3 (TNPO3) in IAV infection. We found that TNPO3-deficient cells inhibited infection with four different IAV strains, whereas restoration of TNPO3 expression in knockout (KO) cells restored IAV infection. TNPO3 overexpression in wild-type (WT) cells promoted IAV infection, suggesting that TNPO3 is involved in the IAV replication. Furthermore, we found that TNPO3 depletion restrained the uncoating in the IAV life cycle, thereby inhibiting the process of viral ribonucleoprotein (vRNP) entry into the nucleus. However, KO of TNPO3 did not affect the virus attachment, endocytosis, or endosomal acidification processes. Subsequently, we found that TNPO3 can colocalize and interact with viral proteins M1 and M2. Taken together, the depletion of TNPO3 inhibits IAV uncoating, thereby inhibiting IAV replication. Our study provides new insights and potential therapeutic targets for unraveling the mechanism of IAV replication and treating influenza disease.

Influenza A virus protein PB1-F2 impairs innate immunity by inducing mitophagy.

Influenza A virus (IAV) infection induces mitophagy, which is essential for the clearance of damaged mitochondria. Dysfunctional mitochondria can be selectively targeted by PINK1, which recruits PRKN/PARK2 and leads to subsequent mitochondrial sequestration within autophagosomes. The IAV PB1-F2 protein translocates to mitochondria, accelerates the mitochondrial fragmentation and impairs the innate immunity. However, whether PB1-F2 mediates IAV-induced mitophagy and the relation between mitophagy and PB1-F2-attenuated innate immunity remain obscure. Here, we showed that PB1-F2 translocated to mitochondria by interacting and colocalizing with TUFM (Tu translation elongation factor, mitochondrial). Further studies revealed that PB1-F2 induced complete mitophagy, which required the interactions of PB1-F2 with both TUFM and MAP1LC3B/LC3B that mediated the autophagosome formation. PB1-F2-induced mitophagy was critical for the MAVS (mitochondrial antiviral signaling protein) degradation and led to its suppression of the type I IFN production. Importantly, the C-terminal LIR motif of PB1-F2 protein was demonstrated to be essential for its mitophagy induction and attenuated innate immunity. In conclusion, PB1-F2-induced mitophagy strongly correlates with impaired cellular innate immunity, revealing it is a potential therapeutic target.Abbreviations: BCL2L13: BCL2 like 13; BECN1: beclin 1; BNIP3L/Nix: BCL2 interacting protein 3 like; CQ: chloroquine; DDX58: DExD/H-box helicase 58; eGFP: enhanced green fluorescent protein; hpi: hours post infection; IAV: influenza A virus; IFN: interferon; IP: immunoprecipitation; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOI, multiplicity of infection; mRFP: monomeric red fluorescent protein; NBR1: NBR1 autophagy cargo receptor; NC: negative control; NLRP3: NLR family pyrin domain containing 3; PINK1: PTEN induced kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; RLR: RIG-I-like-receptor; ROS: reactive oxygen species; SEV: sendai virus; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TM: transmembrane; TOMM20/40: translocase of outer mitochondrial membrane 20/40; TUFM: Tu translation elongation factor, mitochondrial.

Tilmicosin enteric granules and premix to pigs: Antimicrobial susceptibility testing and comparative pharmacokinetics.

The purpose of this study was to compare the pharmacokinetics and relative bioavailability of tilmicosin enteric granules and premix after oral administration at a dose of 40 mg/kg in pigs. Three kinds of different respiratory pathogens were selected for determination of minimal inhibitory concentration (MIC) to tilmicosin. Eight healthy pigs were assigned to a two-period, randomized crossover design. A modified rapid, sensitive HPLC method was used for determining the concentrations of tilmicosin in plasma. Pharmacokinetic parameters were calculated by using WinNonlin 5.2 software. The MIC90 of tilmicosin against Haemophilus parasuis, Actinbacillus pleuropneumoniae, and Pasteurella multocida were all 8 µg/ml. These results indicated that these common pig respiratory bacteria are sensitive to tilmicosin. The main parameters of time to reach maximum plasma concentration (Tmax ), elimination half-life (t1/2ß ), mean residence time (MRT), and apparent volume of distribution (VF ) were 2.03 ± 0.37 hr, 29.31 ± 5.56 hr, 25.22 ± 2.57 hr, 4.06 ± 1.04 L/kg, and 3.05 ± 0.08 hr, 17.06 ± 1.77 hr, 15.55 ± 1.37 hr, 2.95 ± 0.62 L/kg after the orally administrated tilmicosin enteric granules and premix. The relative bioavailability of tilmicosin enteric granules to premix was 114.97 ± 7.19%, according to the AUC0-t values. These results demonstrated that tilmicosin enteric granules produced faster tilmicosin absorption, slower elimination, larger tissue distribution, and higher bioavailability compared to the tilmicosin premix. The present study results manifest that tilmicosin enteric granules can be used as a therapeutic alternative to premix in clinical treatment.

Bioequivalence evaluation of two 5% ceftiofur hydrochloride sterile suspension in pigs.

The purpose of this study was to evaluate the bioequivalence of 5% ceftiofur hydrochloride sterile suspension in two formulations, a test formulation (Saifukang 5% CEF, Hvsen) and a reference formulation (Excenel®RTU 5% CEF, Pfizer). Twenty-four healthy pigs were assigned to a two-period, two-treatment crossover parallel trial, and both formulations were administered at a single intramuscular dose of 5 mg/kg weight, with a 7-day washout period. Blood samples were collected consecutively for up to 144 hr after administration. The concentrations of ceftiofur- and desfuroylceftiofur-related metabolites in the plasma were determined by high-performance liquid chromatography. In addition, the major pharmacokinetic parameters (Cmax, AUC0-t and AUC0-∞) were computed and compared via analysis of variance, with 90% confidence intervals. Bioequivalence evaluation of Tmax was statistically analyzed with the nonparametric test. The comparison values between test and reference formulation for AUC0-t, AUC0-∞, Cmax, and Tmax were 376.7 ± 75.3 µg·hr/ml, 390.5 ± 78.6 µg·hr/ml, 385.9 ± 79.2 µg·hr/ml, 402.7 ± 80.4 µg·hr/ml, 34.6 ± 5.5 µg/ml, 36.1 ± 6.2 µg/ml, 1.27 ± 0.18 hr, and 1.26 ± 0.21 hr, respectively, and we observed no significant differences between the two formulations. The 90% CI values were within the recommended range of 80-125% (P>0.05), and the relative bioavailability of the test product was 96.47 ± 10.92% according to AUC0-t values. Based on our results, the two formulations exhibit comparable pharmacokinetic profiles, and the test product is bioequivalent to the reference formulation.

PK-PD Integration Modeling and Cutoff Value of Florfenicol against Streptococcus suis in Pigs.

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.

Pharmacokinetic and Pharmacodynamic Evaluation of Marbofloxacin and PK/PD Modeling against Escherichia coli in Pigs.

The aim of this study was to evaluate the activity of marbofloxacin and establish the optimal dose regimens for decreasing the development of fluoroquinolone resistance in pigs against Escherichia coli with ex vivo pharmacokinetic/pharmacodynamic (PK/PD) modeling. The recommended dose (2 mg/kg body weight) of marbofloxacin was orally administered in healthy pigs. The ileum content and plasma were both collected for the determination of marbofloxacin. The main parameters of Cmax, AUC0-24 h, AUC, Ke, t1/2ke, MRT and Clb were 11.28 µg/g, 46.15, 77.81 µg⋅h/g, 0.001 h-1, 69.97 h, 52.45 h, 0.026 kg/h in ileum content, and 0.55 µg/ml, 8.15, 14.67 µg⋅h/ml, 0.023 h-1, 30.67 h, 34.83 h, 0.14 L/h in plasma, respectively In total, 218 E. coli strains were isolated from most cities of China. The antibacterial activity in vitro and ex vivo of marbofloxacin against E. coli was determined following CLSI guidance. The MIC90 of sensitive strains (142) was calculated as 2 µg/ml. The minimum inhibitory concentration (MIC) of HB197 was 2 and 4 µg/ml in broth and ileum fluids, respectively. In vitro mutant prevention concentration, growth and killing-time in vitro and ex vivo of marbofloxacin against selected HB197 were assayed for pharmacodynamic studies. According to the inhibitory sigmoid Emax modeling, the value of AUC0-24 h/MIC produced in ileum content was achieved, and bacteriostatic, bactericidal activity, and elimination were calculated as 16.26, 23.54, and 27.18 h, respectively. Based on Monte Carlo simulations to obtain 90% target attainment rate, the optimal doses to achieve bacteriostatic, bactericidal, and elimination effects were 0.85, 1.22, and 1.41 mg/kg.bw for 50% target, respectively, and 0.92, 1.33, and 1.53 mg/kg.bw for 90% target, respectively, after oral administration. The results in this study provided a more optimized alternative for clinical use and demonstrated that the dosage 2 mg/kg of marbofloxacin by oral administration could have an effect on bactericidal activity against E. coli.