To establish a persistent infection mouse model and study its immunological characteristics, female C57BL/6 mice were infected with Mycobacterium tuberculosis (M. tuberculosis) H37Rv at a dose of 1×10⁵ cfu by tail vein injection. Four weeks post infection, one group of mice was treated with isoniazid and rifampicin in drinking water for 8 weeks. At 4, 8 and 12 weeks after infection, 6 mice were euthanized to examine bacterial burden in the lungs and spleens. The homogenate of each organ was serially diluted and plated on plates for bacterial count after 21-day incubation. Mice sera were separated and anti-M. tuberculosis IgG and subclasses were analyzed by enzyme-linked immunosorbent assay (ELISA). CD4⁺ splenocytes were cultured with purified protein derivative (PPD) stimulation, and the percentage of interferon γ (IFN-γ), interleukin 2 (IL-2), IL-4, and tumor necrosis factor α (TNF-α) expressing cells were then identified by intracellular cytokine staining and flowcytometry. The results showed that the lg CFU of bacterial loads in lungs and spleens reached to 3.67±0.25 and 3.54±0.24, respectively 4 weeks after infection and the level retained for at least 8 weeks. The chemotherapy reduced the bacterial load of organs significantly. 12 weeks after infection, M. tuberculosis specific total IgG level increased significantly in the untreated group (P<0.01), especially IgG1 (P<0.01). The IgG level in the chemotherapy-treated group also increased（P<0.05）but lower than that in the untreated group (P <0.01). Compared with the untreated group, the chemotherapy-treated group had a lower IgG1 level (P <0.01) but a higher IgG2a level (P <0.05). The untreated group had a significantly higher percentage of IFN-γ and IL-2 expressing CD4⁺ splenocytes (P<0.01, P<0.001) and lower percentage of IL-4 expressing CD4⁺ splenocytes compared with the control group (P <0.01). The percentages of IL-2 and IL-4 expressing CD4⁺ splenocytes were both lower after chemotherapy compared with untreated group (P<0.05, P<0.01). A persistent infection mouse model of M. tuberculosis was established, and its immunological characteristics were identified in the study. It may provide certain applications in new vaccine and drug screening study against tuberculosis.

Establishing a Salmonella-zebrafish infection model to study the xenophagy. Zebrafish larvae at 72 h post-fertilization were exposed to different doses of Salmonella typhimurium (S. typhimurium). The survival curve of zebrafish larvae was used to determine the appropriate dose of bacteria. After infection by the above dose, the distribution of green fluorescent protein (GFP)-tagged S. typhimurium in zebrafish larvae was observed under a fluorescence microscope. The bacteria count and expression of autophagy protein Lc3-Ⅱand P62 were used to detect the changes in autophagy, and the autophagosome was observed under the transmission electron microscope. The results revealed that after exposure to S. typhimurium at a final dose of 1×109 cfu/ml, the survival curve was 100%, and the larvae began to die on the third day. Fluorescence microscopy found that bacteria had invaded into the larvae at 4 h after infection, and spreaded around the body in 3 d. The bacteria amount in whole-larvae bacteria was increasing over time, but the amount of intracellular bacteria in the larvae at 10 h after infection was significantly less than that at 8 h. Western blotting analysis showed that Lc3-Ⅱ was significantly increased, but P62 was reduced. Under the transmission electron microscope, autophagosome and autolysosome were observed in larvae at 8 h after infection. According to the above results, Salmonella-zebrafish model can be used to track the dynamic changes in autophagy induced by pathogen infection.

To screen out the standard clinic strains used for animal model of drug-resistant Mycobacterium tuberculosis (M. tuberculosis), the strains were collected from tuberculosis patients in several provinces of China. The drug susceptibility test with first-line and second-line anti-tuberculosis drugs and drug-resistant gene mutant analysis were performed in 98 clinic isolations, the strains with explicit drug resistant phenotype and mutant were selected for in vivo virulence experiment in mice. Among the 40 strains with explicit drug resistant patterns, the half lethal time (LT₅₀) of 35 strains were shorter than that of H37Rv in the virulence experiment in mice. 18 in 35 strains were rifampin-resistant and isoniazid-resistant. 5 strains were rifampin-resistant, 7 strains were isoniazid-resistant, and 5 strains were sensitive to both isoniazid and rifampin. Standard strain with moderate virulence and explicite genotype was screened out for each drug resistant phenotype. For rifampin-resistance combined with isoniazid-resistance, one strain with LT₅₀≤7 d was selected as its standard strong virulence. For rifampin-resistance or isoniazid-resistance, one strain with LT₅₀≤7 d was selected as its standard strong virulence strain respectively. Each standard strain can be used as candidate standard strain used in M. tuberculosis infection model of guinea pig or other animal.

The paper aims to determine the main pathogen causing tuberculous lymphadenitis in infants. A total of 79 lymph node biopsies from 73 infants age 0-3 were conducted Mycobacterium tuberculosis (M. tuberculosis) identification, amplification of RD1, RD9 and RD10, and multiple-locus variable-number tandem repeat analysis (MLVA). The results showed that the main pathogen of tuberculous lymphadenitis in infants was bacillus Calmette-Guérin (BCG) (95.9%), followed by M. tuberculosis (4.1%). There were 4 genotypes including 3 independent genotypes (M. tuberculosis) and 1 genotype cluster analyzed by MLVA. The BCG isolated from clinical samples had no difference.

In order to investigate the performance of two kinds of interferon γ release assays in the diagnosis of childhood pulmonary tuberculosis in China (with a high rate of bacillus Calmette-Guérin vaccination and a high incidence of tuberculosis), a total of 114 children with suspected pulmonary tuberculosis were recruited. Among the cases, 45 received QuantiFERON-Gold In-Tube (QFT-GIT) test while the other 69 were tested with T-SPOT.TB. The clinical information was collected for diagnostic classification. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were compared between the two methods. The sensitivity of QFT-GIT in the diagnosis of childhood pulmonary tuberculosis was 86.4%, the specificity was 81.3%, PPV was 91.7%, and NPV was 76.5%. In contrast, the sensitivity of T-SPOT.TB was72.3%, the specificity was 93.7%, PPV was 97.1%, and NPV was 53.6%. The positive rates of two methods in the cases treated with glucocorticoids decreased significantly compared to those untreated. In conclusion, both QFT-GIT and T-SPOT.TB have high PPV in the diagnosis of childhood pulmonary tuberculosis since the rate of latent tuberculosis infection was low in children.

To study the efficacy and stability of maraviroc gel in vitro, the cytotoxicity of maraviroc in phosphate buffered saline (PBS) or in 0.015% hydroxyethyl cellulose (HEC) were evaluated in TZM-bl and Jurkat-T cells. Meanwhile, two subtypes of human immunodeficiency virus (HIV) pseudovirus (SVPB16 and SVPC12) were used to evaluate the anti-HIV activity in vitro, the half maximal inhibitory concentration (IC₅₀) or 90% maximal inhibitory concentration (IC₉₀) of maraviroc gel were assessed. Furthermore, maraviroc was dissolved in 1.5% HEC with a final concentration of 6 mmol/L, and then maraviroc gel was stored at 4 ℃, room temperature, 30 ℃ and 40 ℃, respectively, in 75% relative humidity for 1 to 8 weeks. The anti-HIV activity was tested weekly. The data showed that maraviroc gel had nearly same cytotoxicity as maraviroc in PBS. HEC did not weaken the ant-HIV activity of maraviroc. Inversely, HEC might slightly enhance the anti-HIV efficacy of maraviroc. Maraviroc gel was stable for at least 8 weeks even at 40 ℃. It is concluded that maraviroc gel has good efficacy and stability as a microbicide candidate.

In order to identify Nocardia quickly and accurately, three primers were designed for detection of rpoB, SecA1 and 16S rRNA genes. The specificity of simplex polymerase chain reaction (PCR) was verified. Then multiple PCR method was established, and the sensitivity and specificity were tested by 44 Nocardia standard strains, 44 clinical isolates and 7 reference strains under the same reaction system and condition. The results showes that 2 strains of Nocardia (DSM 43003 and CDC 51) were amplified by using a single primer pair, and the single bands obtained were consistent with target fragment. Then the target genes were verified by sequencing and Basic Local Alignment Search Tool (BLAST). With the established multiple PCR method, rpoB, SecA1 and 16S rRNA segments were confirmed in 43 (97.7%) of 44 Nocardia standard strains and 42 (95.5%) of 44 clinical isolates, however, there were no bands obtained in 7 reference strains. The specificity met the test requirement, and the detection limit for DNA template was 1×10^-4 ng. It is concluded that multiple PCR method is fast, accurate, specific and sensitive. It can be used for identifying Nocardia strains.

To study cell surface receptors of severe fever with thrombocytopenia syndrome virus (SFTSV), the levels of dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) mRNA and SFTSV infection in different cell lines were identified by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). SFTSV-sensitive cells were inhibited by anti-DC-SIGN monoclonal antibody (mAb) and virus infection levels were detected by real-time RT-PCR. Different cell lines were transfected with DC-SIGN-expressing plasmid and SFTSV infection levels either before or after transfection were detected. The results showed that Vero cells which were sensitive to SFTSV had a relatively higher DC-SIGN level than CHO-K1 cells which were less sensitive to SFTSV. Anti-DC-SIGN mAb was capable to inhibit SFTSV invasion into host cells to a certain extent, showing a dose-response effect. The levels of SFTSV infection in cells transfected with DC-SIGN-expressing plasmid were higher than those in non-transfected cells. It is suggested that DC-SIGN is probable one of the cell surface receptors of SFTSV.

Respiratory virus infections place a great burden on people all over the world, because of the high morbidity and mortality. Within the outbreaks of severe acute respiratory syndrome (SARS) and the more recent avian H5N1 and H7N9 influenza virus, the patient care providers face an enormous challenge in rapidly diagnosing vial respiratory infections. Molecular diagnostic techniques have a great development during the past decade. These techniques have been necessary diagnostic tools in rapid detection of viral pathogens causing respiratory infections for their high sensitivity and specificity. Several molecular techniques leading by nucleic acid amplification techniques provide decrease in turnaround time and the gold standard for diagnosis of respiratory viruses, which has shifted away from classic diagnostic methods to molecular techniques in most clinical laboratories.

Fatty acylation, a posttranslational modification of proteins, is classified into four fundamental forms：palmitoylation, myristoylation, prenylation, and the covalent binding of glycosylphosphatidylinositol (GPI). All forms of fatty acylation may occur on the proteins from a variety of viruses. And the modified proteins are consequently altered in functions. Palmitoylation regulates the intercellular transportation and location of viral transmembrane proteins via enhancing the hydrophobicity, which is involved in the membrane fusion, assembly, and release during virus infection and replication. Through the regulation of the positive charges of protein’s surfaces, myristoylation changes the affinity between the cellular membrane and some viral proteins, for example, the modification of preS1 increases the receptor recognition and infectivity of both hepatitis B virus (HBV) and hepatitis D virus (HDV), and the myristoylation of Nef is necessary for regulation of human immunodeficiency virus (HIV) infection and immunity. The interaction of viral free proteins with the membrane compartments or other proteins is increased after prenylation. For example, prenylation could facilitate L-HDAg’s trafficking to endoplasmic reticulum, in which the proteins are assembled into HDV virions together with HBV surface antigen (HBsAg) and HDV RNA . Additionally, GPI binds to viral proteins covalently, and the GPI moiety would change the membrane structure or cytoplasmic phospholipid components of infected cells. For example, GPI modification induced the cross-linkage of cellular prion protein (PrP^C) and agglutination of scrapie prion protein (PrP^SC), which is involved in the spongiform pathogenesis induced by the prions. It would be greatly beneficial for both design and development of new antiviral drugs when the mechanism of lipid modification of viral proteins is further uncovered.