Detection of suid herpesvirus 1 infectivity in pigs by propidium monoazide-qPCR.

At present, there is no effective experimental method for detecting whether the suid herpesvirus 1 (SHV-1) detected in pigs is infectious. Although the technique of quantitative polymerase chain reaction (qPCR) has significantly improved the detection rate and accuracy of the disease, it does not differentiate between infective and non-infective status of the virus. Propidium monoazide (PMA) is a dye that can be combined with DNA molecules. The decomposition of PMA produces an azene compound covalently crosslinked with DNA molecules, thereby inhibiting PCR amplification of DNA. In this study, the combination of PMA and qPCR was used to determine the infectivity of SHV-1. We optimized the method from the selection of primers, the working concentration of PMA, and the method of inactivation using UV or heat inactivation. We found that when specific primer 1 was used and a PMA working concentration was 50-100 µM, heat inactivation was able to distinguish whether SHV-1 was infectious or not. We also showed that UV prevented the virus from replicating, it did not destroy the capsid of the virus, and therefore, PMA cannot enter the virus and bind to the nucleic acid of the virus. Consequently, there is no way to identify the infectivity of the virus using UV inactivation. The study showed that the method was stable and the detection rate reached 96%. In conclusion, this method exhibited strong specificity and high sensitivity and can identify the infectivity of SHV-1. This method has practical significance for clinical virus isolation and the effects of disinfection of farms.

Propidium Monoazide Combined With RT-qPCR Detects Infectivity of Porcine Epidemic Diarrhea Virus.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) allows sensitive detection of viral particles and viruses in epidemic samples but it cannot discriminate noninfectious viruses from infectious ones. Propidium monoazide (PMA) coupled with quantitative polymerase chain reaction (qPCR) was assessed to detect infectious viruses. Currently, there is no established test method to detect the infection of the porcine epidemic diarrhea virus (PEDV). In this study, propidium monoazide coupled with qPCR detects infectivity of PEDV. We optimized the method from the selection of primers, the working concentration of PMA, and the inactivation method using heat or ultraviolet (UV). The viruses which were treated with PMA before qPCR were inactivated using heat or UV. However, the addition of PMA alone did not affect the detection of live viruses, which indicates that a viral capsid break may be essential for PMA to bind to the genome. A repetition of the method on naked PEDV RNA suggests that it can be used to detect potentially infectious PEDV. The results indicated that an optimal plan of PMA could be extremely useful for evaluating infectious and noninfectious viruses.

Development and Validation of a Clinical Score for Predicting the Risk of Tympanosclerosis Before Surgery.

OBJECTIVE: To develop and validate a clinical score to predict the risk of tympanosclerosis before surgery. METHODS: A sample of 404 patients who underwent middle ear microsurgery for otitis media was enrolled. These patients were randomly divided into 2 cohorts: the training cohort (n = 243, 60%) and the validation cohort (n = 161, 40%). The preoperative predictors of tympanosclerosis were determined by multivariate logistic regression analysis and implemented using a clinical score tool. The predictive accuracy and discriminative ability of the clinical score were determined by the area under the curve (AUC) and the calibration curve. RESULTS: The multivariate analysis in the training cohort (n = 243, 60%) identified independent factors for tympanosclerosis as the female sex (odds ratio [OR]: 3.83; 95% CI: 1.66-9.37), the frequency-specific air-bone gap at 250 Hz ≥ 45 dB HL (OR: 3.68; 95% CI: 1.68-8.57), aditus ad antrum blockage (OR: 3.29; 95% CI: 1.38-8.43), type I eardrum calcification (OR: 25.37; 95% CI: 8.41-88.91) or type II eardrum calcification (OR: 18.86; 95% CI: 6.89-58.77), and a history of otitis media ≥ 10 years (OR: 4.10; 95% CI: 1.58-11.83), which were all included in the clinical score tool. The AUC of the clinical score for predicting tympanosclerosis was 0.89 (95% CI: 0.85-0.93) in the training cohort and 0.89 (95% CI: 0.84-0.95) in the validation cohort. The calibration curve also showed good agreement between the predicted and observed probability. CONCLUSIONS: The clinical score achieved an optimal prediction of tympanosclerosis before surgery. The presence of calcification pearls on the promontorium tympani is a strong predictor of tympanosclerosis with stapes fixation.

LINC01433 targets miR-506-3p to promote the biological progress of nasopharyngeal carcinoma cells.

PURPOSE: The current study aimed to investigate the role of long intergenic noncoding 01433 (LINC01433) in the proliferation, migration and invasion of nasopharyngeal carcinoma (NPC). METHODS: Real-time quantitative PCR (RT-qPCR) was performed to determine the expressions of LINC01433 and miR-506-3p in NPC samples and cell lines. The effects of LINC01433 on cell proliferation, migration and invasion were measured by CCK-8, wound healing assay and Transwell, respectively. In addition, Pearson correlation analysis, starBase, RNA immunoprecipitation, luciferase assay, Western blot and functional experiments were conducted to detect and confirm the relationship between LINC01433 and miR-506-3p. RESULTS: LINC01433 level was noticeably elevated in NPC tissues and cell lines. As the expression of LINC01433 in 5-8F cells was the highest in NPC cell lines and the expression of LINC01433 in SUNE1 cells was the lowest, 5-8F and SUNE1 cells were therefore selected as the target cells for following experiments. Furthermore, miR-506-3p was predicted as the target of LINC01433, and the two were negatively correlated with each other. Interestingly, overexpression of LINC01433 promoted proliferation, migration and invasion of NPC cells, while miR-506-3p reversed such effects of LINC01433. Moreover, LINC01433 silencing had the opposite effects to LINC01433 overexpression. Furthermore, miR-506-3p overexpression inhibited the expressions of MMP2, N-cadherin, p-PI3K and p-Akt, and promoted the expressions of E-cadherin and TIMP-2, and partially reversed the role of LINC01433 in promoting cancer development. CONCLUSION: The current findings reveal that LINC01433 regulates NPC cell biological progress through miR-506-3p.

A novel negative pressure drainage treatment of auricular pseudocyst.

OBJECTIVE: Pseudocyst of the auricle is a benign cystic lesions of the ear. A variety of methods have been proposed priorly with different treatment effects. Although aggressive treatments may have good results, we aimed to introduce a less invasive method that also yield optimal cosmetic outcome. PATIENTS AND METHODS: From August 2019 to April 2020, a total of 32 patients with pseudocyst of the auricle were treated with a novel negative pressure drainage method. RESULTS: The treatment has performed successfully in all patients. Only 2 patients experienced negative pressure drainage device detachment, and recovered smoothly after reinstallment. Patients were followed up for an average period of 3 months. The appearance of the auricle was recovered excellent in all patients. There were no postoperative complications or episodes of recurrence during the follow-up period. CONCLUSION: Considering the excellent cosmetic outcome and free of recurrence, we would recommend our negative pressure drainage method as a first-line treatment for all patients with auricular pseudocyst.

Tanshinone IIA suppress the proliferation of HNE-1 nasopharyngeal carcinoma an in vitro study.

Nasopharyngeal carcinoma (NPC) at present is considered to be one of the fatal diseases detected commonly in the people belonging to Southeast Asia and southern China. According to the WHO reports among the detected cases of NPC worldwide, 80% are from China. The present study investigates the effect of tanshinone IIA on the migration and invasion potential of HNE-1NPC cells and studied the detailed mechanism involved. Effect of the tanshinone IIA on viability of the HNE-1NPC cells was analyzed by MTS assay. Cell matrigel invasion and wound-healing motility assays, respectively were used for the analysis of invasion and migration potential of HNE-1 cells. Tanshinone IIA inhibited the viability of HNE-1cells in a dose dependent manner. Migration and invasion potential of the tanshinone IIA treated cells was reduced significantly (P < 0.05) compared to the control cells after 48 h. Analysis of the proteins involved in migration and invasion revealed a significant decrease in the expression of matrix metalloproteinase (MMP)-2 and MMP-9 on treatment with tanshinone IIA. It also inhibited the p65 and p50 expression in the nuclear fractions of HNE-1 cells after 48 h. Thus, tanshinone IIA inhibits migration and invasion potential of the HNE-1NPC cells through reduction in the expression of matrix metalloproteinases. Therefore, tanshinone IIA can be used for the treatment of NPC.

Molecular characterisation of resistance to fluoroquinolones in Haemophilus parasuis isolated from China.

There are many studies on fluoroquinolone (FQ) resistance in the literature, but little is known regarding the molecular characterisation of FQ-resistant Haemophilus parasuis. In this study, a total of 138 H. parasuis isolates were examined, among which 83 (60.1%) were resistant to enrofloxacin (EFX) and 8 (5.8%) were resistant to levofloxacin (LFX) as determined by Etest. Ten point mutations in the quinolone resistance-determining regions of gyrA, gyrB, parC and parE were detected by PCR and DNA sequencing. Interestingly, 100% of the resistant isolates contained mutations at 87D of gyrA, but other mutations occurred less frequently. Furthermore, it was found that there was synergy between 73S→R/I in parC and other point mutations with respect to FQ resistance. To examine the effect of different point mutations on FQ resistance, the minimum inhibitory concentrations of EFX and LFX were determined for strains generated by site-directed mutagenesis, and three point mutations (gyrA 87D→N, parC 73S→R and parE 551T→A) were shown to be involved in FQ resistance.

Haemophilus parasuis encodes two functional cytolethal distending toxins: CdtC contains an atypical cholesterol recognition/interaction region.

Haemophilus parasuis is the causative agent of Glässer's disease of pigs, a disease associated with fibrinous polyserositis, polyarthritis and meningitis. We report here H. parasuis encodes two copies of cytolethal distending toxins (Cdts), which these two Cdts showed the uniform toxin activity in vitro. We demonstrate that three Cdt peptides can form an active tripartite holotoxin that exhibits maximum cellular toxicity, and CdtA and CdtB form a more active toxin than CdtB and CdtC. Moreover, the cellular toxicity is associated with the binding of Cdt subunits to cells. Further analysis indicates that CdtC subunit contains an atypical cholesterol recognition/interaction amino acid consensus (CRAC) region. The mutation of CRAC site resulted in decreased cell toxicity. Finally, western blot analysis show all the 15 H. parasuis reference strains and 109 clinical isolates expressed CdtB subunit, indicating that Cdt is a conservative putative virulence factor for H. parasuis. This is the first report of the molecular and cellular basis of Cdt host interactions in H. parasuis.

Proteomics analysis of differential expression of chicken brain tissue proteins in response to the neurovirulent H5N1 avian influenza virus infection.

A certain H5N1 avian influenza virus has gained the ability to cause the classic central nervous system dysfunction in poultry and migratory birds. This study presents the proteomics analysis on the change of proteins to H5N1 avian influenza virus with neurovirulence infection in chicken brain tissue. By using 2-DE, coupled with MALDI-TOF MS/MS, we identified a set of differentially expressed cellular proteins, including 18 up-regulated proteins and 13 down-regulated proteins. The most significant changes were found in cytoskeleton proteins, proteins associated with the ubiquitin-proteasome pathway, and neural signal transduction proteins. Some identified proteins such as CRMP and SEP5 were found to participate in the pathogenesis progress of Parkinson's and Huntington's diseases, which also developed encephalitis accompanied with CNS dysfunction. The obtained data can provide insight into the virus-chicken brain tissue interaction and reveal the potential mechanism of the neuropathogenesis when the host was infected by the neurovirulent avian influenza virus.

Identification of a cell wall-associated subtilisin-like serine protease involved in the pathogenesis of Streptococcus suis serotype 2.

Streptococcus suis is an important swine and human pathogen, and also an emerging zoonotic agent. A surface-associated subtilisin-like serine protease (SspA) of S. suis was identified by screening a genomic expression library as fragments of this protein reacted most strongly with convalescent-phase pig sera. The sspA gene is present in 29 of 33 S. suis serotypes reference strains and is expressed on the surface of S. suis. Relative real-time quantitative PCR assay demonstrated that sspA mRNA expression in vivo was several thousand fold of that in vitro. A sspA(-) mutant was generated from a S. suis serotype 2 strain SC19 by allelic exchange. The mutant was not different from the wild type strain in subcellular structures and in hemolytic phenotype. However, the virulence of the sspA(-) mutant was markedly lower than the wild type in pigs as demonstrated in experimental infections. These data indicated that the surface-associated protein SspA is a conserved virulence factor of S. suis and is involved in the pathogenesis of S. suis.

Immunoproteomic analysis of outer membrane proteins and extracellular proteins of Actinobacillus pleuropneumoniae JL03 serotype 3.

BACKGROUND: Actinobacillus pleuropneumoniae is the causative agent of porcine contagious pleuropneumonia, a highly contagious respiratory infection in pigs, and all the 15 serotypes are able to cause disease. Current vaccines including subunit vaccines could not provide satisfactory protection against A. pleuropneumoniae. In this study, the immunoproteomic approach was applied to the analysis of extracellular and outer membrane proteins of A. pleuropneumoniae JL03 serotype 3 for the identification of novel immunogenic proteins for A. pleuropneumoniae. RESULTS: A total of 30 immunogenic proteins were identified from outer membrane and extracellular proteins of JL03 serotype 3, of which 6 were known antigens and 24 were novel immunogenic proteins for A. pleuropneumoniae. CONCLUSION: These data provide information about novel immunogenic proteins for A. pleuropneumoniae serotype 3, and are expected to aid in development of novel vaccines against A. pleuropneumoniae.

Identification and characterization of novel immunogenic outer membrane proteins of Haemophilus parasuis serovar 5.

Haemophilus parasuis is the aetiological agent of Glässer's disease, which is responsible for cases of fibrinous polyserositis, polyarthritis and meningitis in young pigs. To develop more effective vaccines, an immunoproteome-based approach was used to analyze the outer membrane proteins of H. parasuis serovar 5. A total of 15 proteins with high immunogenicity were identified and all were showed to be immunogens for the first time in H. parasuis. Further analyses of 8 selected proteins revealed that (1) significantly higher level of serum antibodies against 6 proteins was detected with convalescent sera and immunized sera; (2) antisera against 5 of the selected proteins could effectively inhibit H. parasuis growth in mouse blood; and (3) 4 proteins could induce protective response of the vaccinated mice against H. parasuis. The results suggest these 4 proteins (PalA, Omp2, D15 and HPS_06257) have strong potential to be vaccine candidates.

A comprehensive proteome map of the Haemophilus parasuis serovar 5.

Haemophilus parasuis is the causative agent of Glässer's disease of pigs, a disease associated with fibrinous polyserositis, polyarthritis and meningitis. Systematic reference maps of outer membrane, intracellular and extracellular proteome fractions of the clinical isolate H. parasuis SH0165 were examined by 2-DE coupled with MALDI-TOF MS. A total of 539 proteins spots were successfully identified, corresponding to 317 different proteins that were classified into functional categories. The majority of these proteins were linked to housekeeping functions in amino acid transport and metabolism, secondary metabolites biosynthesis, transport and catabolism and post-translational modification, protein turnover and chaperones. A significant number of outer membrane proteins were identified, such as Wza, Omp2, Omp5, D15 and PalA, which were supposed to play important roles in basic physiology of H. parasuis. In addition, several virulence-associated proteins involved in type I (TolC), type III (DsbA and DsbC) and type V (Autotransporter adhesins) secretion systems, and solute-binding proteins participating in iron-uptake systems were also identified in the present study.