Pseudorabies virus(PRV)is the etiological agent of pseudorabies in pigs,which is char-acterized by dyspnea,reproductive disorders,and neurological diseases,and it spreads widely a-round the world.Since 2011,the newly emerged PRV variants have resulted in poor immunity pro-tection of traditional vaccine strains,and the original method of vaccine strain preparation is time-consuming and labor-intensive.Therefore,it is urgently needed to develop an efficient screening method of the vaccine strain at present.Using CRISPR/Cas9 gene editing technology in this study,two single guide RNAs(sgRNA)were designed targeting the virulence gene TK of PRV variant strain CH/JX/2016,and then the enhanced green fluorescent protein the reporter(EGFP)gene was inserted at the TK locus by a homologous repair plasmid.After multiple rounds of plaque puri-fication,the rPRV-ΔTK/EGFP strain was obtained.The results showed the cleavage efficiency of the two sgRNAs was extremely high.The preparation of rPRV-ΔTK/EGFP strain was succeed af-ter only three rounds of purification,and the EGFP expressed normally.The CRISPR/Cas9 system can edit the PRV gene simply,rapidly,and efficiently,and exhibits great potential in the construction of vaccine candidate strains.Meanwhile,the rescued rPRV-ΔTK/EGFP strain not only could be used as a tracer strain in PRV variant infection progresses,but also for subsequent antivi-ral drug screening.

Objective:To evaluate the status of T, B and NK lymphocytes in peripheral blood of patients with chronic hepatitis B virus(HBV) infection and low-level viremia after nucleos(t)ide analogue (NA) treatment and to provide ideas for solving low-level viremia.Methods:This retrospective study involved 344 patients with chronic HBV infection who had been treated with NAs. They were divided into two groups: low-level viremia group (LLV group) and complete virological response group (CVR group). Clinical data including basic information, biochemistry and coagulation test results, HBV DNA, peripheral blood lymphocyte counts, PD1 and CD28 expression by T lymphocytes, and perforin and granzyme B expression by NK lymphocytes were collected and compared between the two groups. Propensity matching analysis was performed to verify the accuracy of the results.Results:Among the 344 cases, 162 were in the LLV group and 182 in the CVR group. There were no significant differences in disease diagnosis, alanine aminotransferase (ALT), aspartate aminotransferase (AST) or albumin (ALB) level between the two groups ( P>0.05), but the differences in gender and age were statistically significant ( P<0.05). The differences in the counts and percentages of peripheral blood CD3 +, CD4 + and CD8 + T lymphocyte and CD4 + /CD8 + ratios between the two groups were not statistically significant ( P>0.05), but the expression of PD1 and CD28 by peripheral blood CD3 +, CD4 + and CD8 + T lymphocytes was higher in the LLV group than in the CVR group ( P<0.05). The count of peripheral blood CD19 + B lymphocytes in the LLV group was higher than that in the CVR group ( P>0.05), and the percentage of peripheral blood CD19 + B lymphocytes was also higher in the LLV group ( P<0.05). The count of peripheral blood CD16 + CD56 + NK lymphocytes and the expression of perforin in the LLV group were lower than those in the CVR group ( P>0.05). The percentage of peripheral blood CD16 + CD56 + NK lymphocytes and the expression of granzyme B in the LLV group were lower than those in the CVR group ( P<0.05). After propensity score matching, 108 cases in the LLV group and 108 cases in the CVR group showed no significant differences in basic information ( P>0.05); the percentage of CD4 + T lymphocytes and CD4 + /CD8 + ratio in peripheral blood T lymphocyte subsets were higher in the LLV group than in the CVR group, while the percentage of CD8 + lymphocytes was lower in the LLV group ( P<0.05); the expression of PD1 and CD28 by CD3 +, CD4 + and CD8 + T lymphocytes remained higher in the LLV group ( P<0.05); the differences in the counts and percentages of peripheral blood CD19 + B lymphocytes as well as CD16 + CD56 + NK lymphocytes between the two groups were not statistically significant ( P>0.05); no significant difference in the expression of perforin by CD16 + CD56 + NK lymphocytes was found between the two groups ( P>0.05), and the expression of granzyme B remained lower in the LLV group ( P<0.05). Conclusions:Abnormal number and function of T lymphocytes and decreased function of NK lymphocytes might be related to the development of LLV in patients with chronic HBV infection after treatment. Therefore, in addition to adjusting NAs, targeting of T and NK lymphocytes might also be a feasible measure for future LLV treatment.

Objective:To explore the expression of caveolin-1 (Cav-1) and Chitinase-40 (YKL-40) in acute cerebral infarction and the value of combined detection in prognosis evaluation.Methods:118 patients with acute cerebral infarction admitted to the First Affiliated Hospital of Hebei Northern University from January 2016 to June 2019 were selected as the research objects. According to the cerebral infarction volume, the patients were divided into small infarction group (<5 cm 3), middle infarction group (5-10 cm 3) and large infarction group (>10 cm 3). 108 healthy people were selected as the healthy control group. The serum levels of Cav-1 and YKL-40 were compared in the 3 groups, and the correlation between the degree of cerebral infarction and serum levels of Cav-1 and YKL-40 was analyzed. Receiver operating characteristic curve (ROC) was used to analyze the diagnostic value of the expression levels of Cav-1 and YKL-40 in patients with acute cerebral infarction; the patients were followed up for one year and the prognosis was evaluated by modified Rankin Scale (mRS); the correlation between serum Cav-1 and YKL-40 and prognosis was analyzed. Results:The expression levels of serum Cav-1 and YKL-40 in patients with acute cerebral infarction were significantly higher than those in healthy group ( P<0.001). The serum levels of Cav-1 and YKL-40 were positively correlated with the infarct volume of acute cerebral infarction ( r=0.854, P=0.004; r=0.867, P=0.002). ROC curve analysis showed that the sensitivity, Youden index and area under ROC curve of Cav-1 (21.78 μg/L) combined with YKL-40 (158.69 ng/ml) in the diagnosis of acute cerebral infarction were 85.59%, 0.532 and 0.896 (95% CI: 0.741-0.932), respectively, which were significantly higher than those of single index ( P<0.05). At 8 and 12 months of follow-up, the proportion of death and mRS score in the positive group were significantly higher than those in the negative group ( P<0.05). Conclusions:The serum Cav-1 and YKL-40 levels are significantly higher in patients with acute cerebral infarction. The combined examination of Cav-1 and YKL-40 can improve the diagnostic efficiency and has potential application value for early diagnosis and prognosis prediction of patients.

Several putative transcription factor binding sites (TFBSs) exist in the PCV2 rep gene promoter. To explore if porcine circovirus type 2 (PCV2) could regulate the viral replication by using these TFBSs, we conducted electrophoretic mobility shift assay (EMSA), DNA-pull down and liquid chromatography-tandem mass spectrometric (LC-MS/MS) assays. EMSA confirmed the binding activity of the rep gene promoter with nuclear proteins of host cells. DNA-pull down and LC-MS/MS identified the porcine transcription factor AP-2δ (poTFAP2δ) could bind the PCV2 rep gene promoter. Dual-luciferase reporter assay, quantitative real-time PCR, Western blotting and indirect immunofluorescent assay demonstrated that poTFAP2δ could not only promote the activity of the rep gene promoter, but also enhance the transcription/translation activity of the rep/cap gene and the virus titer of PCV2 during the entire life cycle of PCV2 infection. This study revealed the molecular mechanism of PCV2 using host proteins to enhance the viral replication, provided a new perspective for studying the pathogenic mechanism of PCV2 from virus and host interactions, and provided a theoretical basis for developing highly effective PCV2 vaccines.
Animals ; Cell Line ; Chromatography, Liquid ; Circoviridae Infections ; Circovirus ; DNA Helicases ; Diabetes Mellitus, Type 2 ; Promoter Regions, Genetic ; Swine ; Tandem Mass Spectrometry ; Transcription Factor AP-2 ; Virus Replication

Objective To establish a quick electrochemical biosensor for the detection of nucleic acid of Ebola virus . Methods The DNA tetrahedral nanostructure was self-assembled on gold surface by strong Au-S chemical bonds , leaving the target probe at the top .A biotinylated-ssDNA was introduced as the detection probe by specific binding of the captured target sequence , before avidin-horseradish peroxidase ( HRP) was used as a signal amplifier to transduce amperometric sig-nal through interactions with TMB substrate .Results The results indicated that the nucleotide sequence of Ebola virus could be recognized and detected by the sensor .The linear range for the detection of target DNA was from 1.0 ×10 -9 to 5.0 ×10 -6 mol/L,and the detection limit was 5.2 ×10 -10 mol/L.Conclusion The fabricated sensor is demonstrated to be sensitive and specific for the detection of Ebola virus nucleotide .

Rapid detection of infection pathogens is of great importance to the prevention and control of infectious diseases.Compared with traditional approaches,point-of-care testing (POCT) technologies promise great advantages in simple, rapid and portable detection of pathogens.In this review, the technologies, categories, developments and applications of POCT in detection of infectious pathogens are elaborated.Furthermore, the future developments of POCT detection of infectious pathogen are also discussed.This review focuses on loop-mediated isothermal amplification ( LAMP) technology, microfluidic chip and biosensor technology in the POCT detection of infectious pathogens while elaborating on the application of these new technologies associated with POCT detection.

Background:As the empirical studies on human body are restricted extremely,the establishment and selection of suitable animal models are important for researches on ulcerative colitis( UC ). Aims:To compare the symptoms and colonic pathology of rat models with experimental colitis induced by dextran sulfate sodium( DSS ) and trinitrobenzene sulfonic acid( TNBS),so as to provide a reference for selecting animal models in UC-related studies. Methods:Drinking 4% DSS freely for 7 days or intrarectal administration of single dose 100 mg/kg TNBS-50% ethanol were used to establish experimental colitis model in Sprague-Dawley rats. The disease activity index( DAI)was assessed dynamically during the course of experiment. The whole colon was removed in batches for measurements of colonic damage score and activity of myeloperoxidase(MPO)at different time points. Results:The DAI score reached the peak at the 7th day and the 2nd day in DSS group and TNBS group,respectively,and decreased gradually afterwards. Six and one deaths occurred during the experimental course in DSS and TNBS groups,respectively. In DSS group,the duration of inflammation was short,the colonic injury was moderate and recovered after drug withdrawal. At the 18th day,the colonic damage score and MPO activity was 0. 25 ± 0. 50 and(0. 80 ± 0. 33)U/g,respectively,and no significant differences were seen between DSS group and normal control group. In TNBS group,the duration of inflammation was longer and the colonic injury was more severe. At the 21st day,the colonic damage score and MPO activity was 3. 60 ± 0. 55 and( 1. 60 ± 0. 39 ) U/g, respectively,and chronic inflammation was observed histologically. Conclusions:Both DSS and TNBS can induce experimental colitis model in rats. The course of TNBS-induced colitis model presents a transformation of acute to chronic inflammation,and may be more suitable for treatment-related studies of UC.

Objective To detect the positive rates of antibodies against avian influenza virus (AIV) subtypes H5, H6, H7 and H9 among people in poultry occupations in Guangdong province and to analyze the transmission of various subtypes of AIV from poultry to human contacts for the prevention and control of novel AIV infection in human beings.Methods Serum specimens were collected from 1066 peo-ple in poultry occupations ( occupational group) and 205 people not in poultry occupations ( non-occupational group) in 10 cities of Guangdong province.The inactivated AIV strains, isolated from poultry or environment of Guangdong province, were used as antigens to detect antibodies against AIV subtypes H5, H6, H7 and H9 by using the hemagglutination inhibition ( HI) assay.Results The positive rates of antibodies against AIV subtypes H5, H6, H7 and H9 carried by people from the occupational group were respectively 0.44%, 0%, 0.30%and 0.30%in 2013 and 1.08%, 0.0%, 0.0%and 0.27%in 2014.Only the anti-H9 anti-bodies were detected in serum samples collected form people in the non-occupational group in 2013 with a positive rate of 0.95%.No significant differences with the positive rates of anti-AIV antibodies were found between the occupational group and the non-occupational group.However, the geometric mean titer ( GMT) of anti-AVI antibodies in people from the occupational group was higher than that of the non-occupational group.Conclusion Although a grand spread of AIV from avian to human is not likely to happen yet, con-tacting with poultry is the risk factor for AIV infection in Guangdong population.A long-term surveillance of anti-AIV antibodies in serum should be strengthened among people in poultry occupations for the timely pre-vention and control of novel AIV outbreak.

China ; Humans ; Industry ; Pneumoconiosis

Objective To construct eukaryotic enhanced green fluorescent protein (EGFP) expressing recombinant plasmid, pEGFP-C1-hnRNP A1, which contains coding sequence of human hnRNP A1 (heterogeneous nuclear ribonucleo-protein A1), and to perform cellular localization analysis of EGFP tagged hnRNP A1 under stress. Methods Total RNA was isolated from HeLa cell used for synthesis of first-strand cDNAs using reverse primers that are specific for the 3′-un-translated region of hnRNP A1. hnRNP A1 gene fragments were then amplified by touch-down PCR from those cDNAs and inserted into pEGFP-C1 fluorescent bearing vector through EcoRⅠ/BamHⅡdouble enzyme digestion and T4 DNA Ligase connection. The recombinant pEGFP-C1-hnRNP A1 plasmid was transfected into HeLa cells and green fluorescent tagged fusion proteins was examined by Western blot and confocal fluorescence microscopy. Co-localization of EGFP-hnRNP A 1 with poly (A)+mRNA (the marker of the stress granules), or DCP1a (the marker of processome) were detected by RNA fluores-cence in situ hybridization and immunofluorescence. Results The pEGFP-C1-hnRNP A1 was sequenced and digested cor-rectly by restriction single/double enzyme. The green fluorescent fusion protein was also detected in transfected HeLa cell by Western blot and confocal fluorescence microscopy. EGFP-hnRNP A1 co-localizes with poly(A)+mRNA, but not DCP1a. Conclusion Recombinant eukaryotic plasmid of pEGFP-C1-hnRNP A1 was constructed successfully and expressed effec-tively. EGFP tagged hnRNP A1 takes part in forming stress granules.