Molecular Characteristics and Potent Immunomodulatory Activity of Fasciola hepatica Cystatin

Cystatin, a cysteine protease inhibitor found in many parasites, plays important roles in immune evasion. This study analyzed the molecular characteristics of a cystatin from Fasciola hepatica (FhCystatin) and expressed recombinant FhCystatin (rFhcystatin) to investigate the immune modulatory effects on lipopolysaccharide-induced proliferation, migration, cytokine secretion, nitric oxide (NO) production, and apoptosis in mouse macrophages. The FhCystatin gene encoded 116 amino acids and contained a conserved cystatin-like domain. rFhCystatin significantly inhibited the activity of cathepsin B. rFhCystatin bound to the surface of mouse RAW264.7 cells, significantly inhibited cell proliferation and promoted apoptosis. Moreover, rFhCystatin inhibited the expression of cellular nitric oxide, interleukin-6, and tumor necrosis factor-α, and promoted the expression of transforming growth factor-β and interleukin-10. These results showed that FhCystatin played an important role in regulating the activity of mouse macrophages. Our findings provide new insights into mechanisms underlying the immune evasion and contribute to the exploration of potential targets for the development of new drug to control F. hepatica infection.

Packaging of Rift Valley fever virus pseudoviruses and establishment of a neutralization assay method

Rift Valley fever (RVF) is an acute, febrile zoonotic disease that is caused by the RVF virus (RVFV). RVF is mainly prevalent on the Arabian Peninsula, the African continent, and several islands in the Indian Ocean near southeast Africa. RVFV has been classified by the World Organisation for Animal Health (OIE) as a category A pathogen. To avoid biological safety concerns associated with use of the pathogen in RVFV neutralization assays, the present study investigated and established an RVFV pseudovirus-based neutralization assay. This study used the human immunodeficiency virus (HIV) lentiviral packaging system and RVFV structural proteins to successfully construct RVFV pseudoviruses. Electron microscopy observation and western blotting indicated that the size, structure, and shape of the packaged pseudoviruses were notably similar to those of HIV lentiviral vectors. Infection inhibition assay results showed that an antibody against RVFV inhibited the infective ability of the RVFV pseudoviruses, and an antibody neutralization assay for RVFV detection was then established. This study has successfully established a neutralization assay based on RVFV pseudoviruses and demonstrated that this method can be used to effectively evaluate antibody neutralization.

Effect of Deletion of the Carboxyl Terminal of the NS1 Protein on Pathogenicity of the Influenza B Virus / 病毒学报

To analyze the molecular basis of the variation of the pathogenicity of the influenza B virus, we rescued a recombinant virus with a deletion in the carboxyl terminal of the NS1 protein using reverse genetics based on the parental virus B-S9 of B/Yamagata/16/88. A mutant strain with a deletion of 171 amino acids in the carboxyl terminal of the NS1 protein was named "B-L5". BALB/c mice were inoculated with 3 X 105 EID50 of B-L5 and the parental virus B-S9, respectively. Then, weight changes, survival, and viral titers were documented. During 3 days post-inoculation (dpi) to 7 dpi, the weight of mice infected with B-S9 decreased. However, the weight of mice infected with B-L5 showed weight decreases only at 2 dpi, and quickly recovered at 3 dpi. B-S9 and B-L5 could replicate in the lungs of BALB/c mice. However, viral titers in the lungs of mice infected with B-L5 were 7900-times lower than those of mice infected with B-S9 at 3 dpi. Viral titers in the lungs of mice infected with B-L5 were not detected at 6 dpi. These results showed that, compared with the parent virus B-S9, the mutant virus B-L5 showed lower pathogenicity in BALB/c mice. Our study suggests that deletion of the carboxyl terminal of the NS1 protein decreases the pathogenicity of the influenza B virus. Establishment of a reverse-genetics system for the B influenza virus will provide a platform for studying its pathogenesis, and mechanism of transmission, and for developing live-attenuated influenza B virus vaccines.

Aptamers: characteristics and applications in pathogenic microorganism / 生物工程学报

Aptamers are a group of artificial oligonucleotides identified by exponential enrichment system evolution technology (Selective expansion of ligands by exponential enrichment, SELEX). Aptamers have been widely used in basic research, clinical diagnostics, and nano-technology. In this article we will introduce the technology of aptamer and summarize its applications in medical microbiology.

Preparation and application of a colloidal gold strip to detect the rabies antibody / 生物工程学报

To develop a specific, rapid, and convenient immunochromatography assay (ICA) to detect the rabies antibody in clinical sample from immuned dogs by rabies vaccine. Colloidal gold particles labeled with purified rabies virus (CVS11) were used as the detector reagent. The staphylococcal protein A (SPA) and pured rabbit anti-rabies virus IgG were blotted on the test and control regions of nitrocellulose membrane. Then the strip was assembled with sample pad, absorbing pad, and dorsal shield. The assay samples (261 dog's serum) were collected from Wildlife Rabies Disease Diagnostic Laboratories of Ministry of Agriculture in China, Institute of Military Veterinary, Academy of Military Medical Sciences and other six provinces, including rabies virus positive and negative serum. The performance of the strip was compared to fluorescent antibody virus neutralization test. The neutralizing antibody titer could be detected above 0.5 IU. The strip did not change of performance when stored at room temperature for 12 months. It may offer reference of neutralizing antibody titer level after dogs immuned rabies vaccine and determin whether the dogs need to be immuned again.