Developing and implementing biosafety standards for pathogenic microbiology laboratories is essential to achieving scientific, efficient, and standardized management and operation. This article analyzes the current standardization construction in biosafety in pathogenic microbiology laboratories domestically and internationally. It proposes a framework for the biosafety standard system of pathogenic microbiology laboratories, which mainly includes four parts: basic standards, management standards, technical standards, and industry applications. It provides a reference for the standardization work of pathogenic microbiology laboratories and helps to standardize the biosafety industry in China.

Objective:To investigate the in vitro viability of rabies virus in tissues and body fluid samples. Methods:The viability of rabies virus in tissues and suspensions was analyzed by virus titer determination method, direct immunofluorescence, RT-PCR and laboratory techniques for virus isolation.Results:With the increase of temperature, the viability of rabies virus in brain tissues and suspensions decreased gradually. Rabies virus lost infectivity after 30 min at 56℃, but remained viable in tissues for 7 d at 37℃. The virus showed no viability after 1 h at pH9.6. The rabies virus in suspensions could be completely inactivated after the stimulation with ethanol at a final concentration above 30%, sodium hypochlorite above 500 mg/L or benzalkonium bromide above 100 mg/L for 3 min. It was found that 80% acetone had the strongest inactivation effect on rabies virus in tissues, and no virus could be isolated after soaking for 4 h.Conclusions:Rabies virus was not tolerant to high temperature and relatively stable in the environment with pH6.8-7.4. Common disinfectants could kill the virus. This study provided detailed data about the viability of rabies virus in vitro, which would be conducive to the prevention and control of rabies.

Objective:To analyze the in vitro inhibitory effects of resveratrol on rabies virus. Methods:The challenge virus standard (CVS)-11 strain of rabies virus and BHK-21 cells were used to establish the infection model. In vitro inhibitory effects of resveratrol on rabies virus were analyzed at different stages of infection by direct immunofluorescence and cell fluorescence focus unit assay. Results:Without affecting cell growth, resveratrol could block the adsorption of virus, interfere with the entry of virus into cells and inhibit virus proliferation in a concentration-dependent manner. The inhibition rate could reach up to about 95%. The results of co-incubation experiment showed that 40 μmol/L resveratrol could directly kill the virus.Conclusions:This study indicated that resveratrol inhibited the activity of rabies virus in a concentration-dependent manner.

Objective:Objective The inhibitory effect of Niuhuang Qinggan Capsule on 2019-nCoV virus was studied to provide experimental evidence for drug screening and treatment of 2019-nCoV acute respiratory disease.Methods:The 2019-nCoV virus GS048 strain was isolated and cultured by Vero-E6 cells. The solution of Niuhuang Qinggan capsule was incubated with virus in vitro to determine the change of virus titer. The effect of Niuhuang Qinggan capsule on the proliferation of 2019-nCoV virus was determined by cell culture medium method.Results:It was found that the concentration of 274.3 μg/ml was not toxic to the cells when the concentration of drugs was lower. When the drug was incubated with virus, the titer of virus decreased from 10 4.4TCID 50/ml to 10 2.8TCID 50/ml. In vitro 2019-nCoV virus culture experiment showed that the addition of the drugs reduced the cytopathy, and the logarithmic value of virus titer decreased by 40% -50%. Conclusion:In this study, in vitro inhibition experiments suggest that Niuhuang Qinggan capsule has the potential to inhibit the proliferation of 2019-nCoV. It can be used as a candidate drug to further verify the therapeutic effect of the 2019-nCoV acute respiratory disease.

Strain-resource engineering is often considered as an important infrastructure of microbiology related research and industry. The western developed countries took the lead in establishing the classical microbial resource utilization method, and continuously improved the preservation system, species annotation technology and global sharing mechanism, which realized the expansion and reserve of biological resources since end of the 19th century. The rich and diversified germplasm resources, standard strains and production strains not only have important economic values, but also maintain the advantages of scientific research, bioeconomy (such as antimicrobial agents, vaccines, detection reagent development and standard development, etc.) and national security. Although there has been a lot of progress in related research in recent years, compared with developed countries, there is still a big gap in related fields in China. The investment and top-level design in this area lag far behind the western developed countries, and it is not commensurate with the current level of economic and social development in my country. Drawing lessons from the practice of WFCC and WDCM (World Data Center for Microorganisms, Global microbial data Center, affiliated to WFCC), for the purpose of collecting new clinical species/strains, this paper puts forward some suggestions on the identification, preservation and upload system of isolates.

Objective:To obtain the high-efficiency expression of the biological active rabies virus nucleoprotein in the prokaryotic expression system.Methods:This experiment uses codon optimization technology to re-encode the nucleoprotein gene of rabies virus CTN-1 strain, artificially synthesize the full-length gene and clone it into pET-43.1a prokaryotic expression vector, induced expression in BL21 (DE3) strain of Escherichia coli( E. coli), and used Western blot to detect its reactogenicity. Results:The results showed that after induction, SDS-PAGE electrophoresis analysis showed that an obvious expression band appeared at a molecular weight of 50×10 3, which was consistent with the expected protein band size. Among them, the E. coli concentration A600 is about 0.5, and the expression yield is the highest (about 32.3%) when induced at 37℃ for 5 h. Nucleoprotein expression product is mainly inclusion body when it is expressed in large quantities. After purification by Ni 2+ chelating chromatography, the purity of the target protein can reach over 95%. The purified product was identified by Western blot and positively reacted with the sera of mice immunized with rabies vaccine, indicating that the prokaryotic expression of the CTN-1 strain nucleoprotein has biological activity. Conclusions:This experiment successfully established a high-efficiency expression method for the nucleoprotein of the CTN-1 strain in the prokaryotic expression system, and obtained high-purity target protein, which provides a basis for further clinical diagnosis and preparation of new vaccines.

Objective:The recombinant shuttle peptide-single chain antibody with neutralizing activity is expected to be obtained by combining the shuttle peptide sequence with rabies single chain antibody and optimizing the expression conditions in E. coli.Methods:In this study, the SO57 single-stranded antibody sequence was recoded and the shuttle peptide of Arg 12 was fused at the N-terminal to construct pET22 (b)-rSO57 expression vector, which was then expressed in E. coli. The growth density OD 600, induction time, induction temperature and inducer concentration were optimized to obtain a higher expression effect. The recombinant protein was purified by immobilized metal chelating chromatography, and the relative affinity of the recombinant single-chain variable fragment (scFv) was determined. The neutralization effect of the recombinant scFv was verified by rSO57/CVS-11 virus strain mixed with infected cells and rapid fluorescent focus inhibition test (RFFIT). Results:The recombinant rSO57 was successfully constructed and induced in BL21 (de3). The optimal expression condition was that when the cell density of OD 600 was 0.8, IPTG of 0.4 mmol/ml was used for induction. After induction at 16 ℃ for 24 hours, rSO57 was expressed in soluble form, accounting for 19.8% of the soluble protein in the cell. After chromatography purification, the recombinant rSO57 with a purity of 84% was obtained. ELISA showed that the relative affinity coefficient Ka of rSO57 was 4.3×10 5. When rSO57 was mixed with CVS-11 strain, the infection rate of cells increased with the increase of dilution, indicating the neutralization activity of recombinant antibodies. Using the RFFIT, 50 μg rSO57 was equivalent to 2.17 IU of rabies neutralizing serum. Conclusions:In this study, the recombinant scFv fusion with shuttle peptide was expressed, which could neutralize rabies virus, in order to prepare specific and targeted antiviral drug carriers for rabies treatment.

Objective:To investigate the availability, prices and affordability of essential medicines in pediatric population across China, in the hope of improving rational use of medicines.Methods:A multicenter cross-sectional survey of medicine prices, availability and affordability was conducted in 17 provinces, municipalities and autonomous region across east, south-central part, west and north of China. Data on 42 medicines used in pediatric population, both original and generic, were collected in 55 public hospitals from May 26 to June 2, 2017. Availability was expressed as the percentage of hospitals with stock of the target medicine on the day of data collection,and median price ratio (MPR) was the ratio of price upon investigation to international reference. Based on national minimum daily wage, affordability represents the number of working days needed to earn the expense which covers a standard course using the target medicine. Statistical software SPSS 13.0 was applied for descriptive analysis of availability, MPR and affordability.Results:Mean Availability of original and generic medicine was 33% and 32%, with median MPR being 5.43 and 1.55. Among the 19 medicines with price information for both original and generic product, the median MPR was 7.73 and 2.04 respectively. Regarding the five medicines used to treat four common pediatric diseases (pneumonia,peptic ulcer, congenital hypothyroidism, refractory nephrotic syndrome), the affordability was 0.63 (0.16-6.17) d for generic medicine, and 1.03 (0.16-11.53) d for its original counterpart.Conclusions:The availability to both original and generic products of the 42 medicines used in pediatric population was low in China. The prices of generic medicines seem to be lower and affordability higher than those of original medicines. There is an urgent need to improve the availability and affordability of pediatric medicines.

We constructed prokaryotic recombinant expression vector of P61 gene from Nocardia brasiliensis,expressing P61 protein with biological activity in E.coli,and lay a foundation for further studies related to P61.P61 gene was synthesized and cloned into an expression vector pET-30a(+).The recombinant vector was transformed into Escherichia coli BL21 and induced with IPTG.The production was analyzed with Western blot and the catalase activity of P61 was tested with Catalase Assay Kit.The protein of P61was successfully expressed in E.coli with solubility and high catalase activity,and could be identified by anti-N.brasiliensis sera from mice.The prokaryotic expression plasmid of protein P61 was constructed successfully and can be expressed efficiently in E.coli BL21 cells with higher catalase.

Objective: To investigate the inactivating effect of soap solution on rabies virus and to explore the concentration of soap solutions which could be effective in post-exposure prophylaxis (PEP) of rabies virus infection. Methods: The BSR and N2a cells were respectively infected by the mixture of different concentrations of soap solution and rabies virus CVS-11. Based on the direct immunofluorescent method (DFA) and reverse transcription PCR (RT-PCR), the inactivating effects of soap solutions on rabies viruse in BSR and N2a cells were detected, respectively. Results: This experiment established the BSR or N2a cells model in 24 well cell culture plates, and we found that the upper limit of soap solution concentration which BSR or N2a cells could tolerate was 1%. The inhibitory effect test of different soap solution on rabies virus showed that the 0.5% concentration of soap solution could completely inhibit the survival of CVS-11 strain in both the BSR and N2a cells, but the 0.1% concentration of soap solution could not inhibit the rabies viruses completely. Conclusions The 0.5%-1% concentration of soap solutions could inhibit the survival of CVS-11 strain in three minutes in vivo experiment.