Objective To sequence and analyze the complete genome of two human coronavirus NL63 (HCoV-NL63) strains collected from Beijing Children Hospital .Methods Eighteen pairs of primers were designed according to the gene sequences of HCoV-NL63 reference strain ( HCoV-NL63_Amsterdam 1) and used to amplify the target fragments covering the complete genome of HCoV-NL63 strains.Rapid ampli-fication of cDNA ends ( RACE) and RT-PCR assays were used to amplify the full length genome of HCoV-NL63 strains.Phylogenetic analysis was conducted by using Mega 5.0 software.Results The complete ge-nome sequences of the two HCoV-NL63 strains were 27 538 bp in length, showing a homology of 99.1%in nucleotide sequences .There were 15 consecutive bases deleted from 1a region.The systematic phylogenetic analysis demonstrated that four genotypes of NL 63 virus including A , B, C and D have been identified , and two domestic strains were belonged to the new genotype D .Conclusion The complete genome sequences of two domestic HCoV-NL63 isolates were identified for the first time .This study provided evidence for further investigation on molecular epidemiology of HCoV-NL63 in China .

Objective To investigate the effectiveness of the affinity adsorption material developed by our team for the specific removal of exogenous endotoxin in the blood circulation. Methods Fifteen beagle dogs were intravenously injected with endotoxin to establish a dog model of endotoxemia, and then they were randomly divided into the treatment group(n=10)and the control group(n=5). The treatment group received an extracorporeal perfusion to remove the endotoxin using the self-made disposable hemoperfusion device,while the control group using routine perfusion device. The levels of endotoxin, tumor necrosis factor α(TNF-α), interleukin 1β(IL-1β), interleukin 6(IL-6)and interleukin 8 (IL-8)in the blood of the dogs were measured at the beginning and 120 min after hemoperfusion for 120 minutes. The vital signs of the dogs were monitored during the hemoperfusion. Results After successful establishment of the endotoxemia model,the level of endotoxin at the beginning of hemoperfusion in the treatment group and control group was 118.63 ± 27.98 EU/mL and 117.16 ± 22.95 EU/mL,respectively. After hemoperfusion for 120 min,it was 0.039 ± 0.01 EU/mL and 131.98 ± 7.01 EU/mL, showing a significant difference(P﹤0.05). The clearance rate of hemoperfusion in the treatment group was 94.07%. At the beginning of hemoperfusion, the levels of TNF-α, IL-1β, IL-6 and IL-8 in the treatment group were 1.53 ± 0.27 ng/mL,12.82 ± 1.66 ng/mL,54.77 ± 3.98 ng/mL and 0.25 ± 0.32 ng/mL, and the levels in the control group were 1.53 ± 0.06 ng/mL,13.05 ± 0.18 ng/mL,54.58 ± 0.19 ng/mL and 0.28 ± 0.06 ng/mL, respectively. After hemoperfusion for 120 min, the levels of TNF-α, IL-1β, IL-6 and IL-8 in the treatment group were 0.13 ± 0.06 ng/mL, 0.70 ± 0.36 ng/mL, 1.62 ± 0.80 ng/mL and 0.01 ± 0.00 ng/mL, respectively, and as for the control group,the levels were 2.26 ± 0.15 ng/mL,15.12 ± 0.18 ng/mL,62.54 ± 0.93 ng/mL and 0.73 ± 0.93 ng/mL. There were significant differences between the beginning and after perfusion for 120 min in those two groups(P< 0.05). Conclusions This affinity adsorption material can effectively remove endotoxin and the inflammatory mediators in the blood of experimental dogs,with a clearance rate of 94.07%.

OBJECTIVE To optimize the water extraction process of Flos Lonicerae-Fructus Forsythuae and determine the range of water extraction process parameters.METHODS The active components were screened by network pharmacology,and the indica-tor ingredients were determined in combination with the quality markers under the relevant terms of Chinese Pharmacopoeia 2020 edition and the literature.Take extraction yield and the extraction rate of the indicative component as the critical quality attributes of the water extraction process to screen critical process parameters.The mathematical model was established by Box-Behnken experimental design to investigate the interaction between CQAs and CPPs and build the design space of the water extraction process of Flos Lonicerae-Fruc-tus Forsythuae.RESULTS The extraction percentages of phenolic acids,forsythoside A and forsythin were screened as the index components;specifications of medicinal slices,extraction time and water addition were the key process parameters.Based on the estab-lishment and optimization of the design space,the optimum water extraction process was obtained as follows:the medicinal slice of Lian-Qiao was broken into 0.8-1.2 cm,adding 12 times the amount of water in the first and extract for 30-50 min,10 times the a-mount of water in the second and extract for 25-30 min.CONCLUSION The verification results show that the measured value ob-tained by using the design space method to optimize the water extraction process is close to the predicted value,indicating that the method is stable and reliable,which can provide ideas for its further process development and quality control for the couple medicines of Flos Lonicerae-Fructus Forsythuae.