Objective To explore the effect of cryopreserved canine kidney cells (MDCK)single -layer on the isolation and proliferation of influenza virus B.Methods Revived P17 MDCK cells were passage for 2 -4 gener-ations,and subsequently preserved in 4℃ refrigerator for 3,6 and 9 days,respectively.Under same experimental con-ditions,the 4℃ refigerater preserved cells were co -incubated with influenza -like illness(ILI)throat swab speci-mens.Cytopathic effect (CPE)was observed,and the proliferation of virus was determined using real -time PCR and the hemagglutinin titers were determined by serological test.Results (1)CPE:The CPE of the MDCK cells pre-served in 4℃ refrigerator for 3 or 6 days had no significant differences compared with that in the control group,while the cell preserved in 4℃ refrigerator for 9 days showed CPE fastly and maintained for a short time.(2)Real -time PCR:the proliferation of influenza virus B in the MDCK cells preserved with 4℃refrigerator for 3 or 6 days (25.86 × 105 -30.25 ×106 ,26.31 ×105 -30.54 ×106 )had on difference compared with that of the control group (24.82 × 105 -29.86 ×106 ),with the proliferation rate of 105 to 106 times,while the proliferation cell with 4℃ cryopreserved for 9 days(19.72 ×104 -28.34 ×105 )the proliferation in cells preserved for 9 days was sharply decreased,with pro-liferation rate of 104 to 105 times.(3)The HA titer:The virus strains with hemagglutination titer above or equal to 116 (P >0.05)isolated with MDCK cells preserved in 4℃ refrigerator 3 or 6 days were not significantly different from that of the control group (10.92 ±0.79).And the cells with 4℃ cryopreserved for 9 days were significantly dicreased (P <0.01).Conclusion No significant effects on the isolation and proliferation of influenza virus B using MDCK cell preserved in 4℃ frigerator for near one week were observed in the present study.

Objective:To understand the genetic variation and the prevalence of H9N2 subtype avian influenza virus in Guizhou province, and to provide the scientific evidence for the prevention and control of avian influenza virus.Methods:The results of AIV detection in live poultry market(LPM) environment in Guizhou province from October 2018 to March 2019 were statistically analyzed, RNAs were extracted and sequenced from the HA genes of 13 samples of H9N2 positive screened by real-time PCR. Then the homology, the genetic evolution and the mutations of important amino acid were analyzed by bioinformation softwares. Results:The positive rate of AIV was 52.2% and the positive rate of H9N2 was 83.7% in LPM environment. The homology between nucleotides of the HA gene of 21 strains ranged from 91.6% to 100.0%, and the homology between amino acids of the HA gene ranged from 91.0% to 100.0%. All strains belonged to Y280 sublineage and G57 genotype. Key sites analysis showed that they had a common motif PSRSSRGLF and LSRSSRGLF at the cleavage site, which indicated that they were lentogenic and low pathogenic strains. Mutations H191N, E198T/A and Q234L at the receptor binding sites in the HA was found in 21 strains, while indicated the viruses had the potential to bind human-like receptor. The analysis results of glycosylation motifs showed that all 21 strains had 7 glycosylation sites, but had a site deletion at amino acid site 218 and an addition at 313.There was no significant mutation in the key site compared with the human infected strains. Conclusions:The detection rate of AIV in LPM environment in Guizhou province was high, and the pollution was very serious, and H9N2 subtype is the main subtype, All H9N2 subtype AIVs belonged to Y280 sublineage and G57 genotype, and they were low pathogenic avian influenza viruses in Guizhou province, but the genetic gap were widening and mutations of key amino acid site might enhance susceptibility and pathogenicity to human beings. Hence, It is necessary to strengthen the surveillance of molecular characteristic variation of H9N2 subtype AIV.

Objective To understand the epidemic characteristics and regularity of influenza B virus in Guizhou Province, and provide scientific evidence for the control and prevention of influenza.Methods Results of reverse transcription polymerase chain reaction(PT-PCR) of influenza B virus in Guizhou Province from April 1, 2013 to March 31, 2016 were statistically analyzed.Results A total of 1 904 samples were detected influenza B virus by RT-PCR, B/Yamagata (By) lineage and B/Victoria (Bv) lineage were 1 215 and 642 respectively.In April 2013-March 2014 and April 2014-March 2015, the predominant strains of influenza B were both By lineage, in April 2015-March 2016, the predominant strains of influenza B were Bv and By lineages, the epidemic peaks were in winter and spring;there's a higher positive percentage of influenza B in male, accounting for 56.83%;the highest detection rate of influenza B virus was found in population aged <15 years(70.80%),Bv and By lineages were the highest in the 0~ (42.37%) and 5~ age groups (35.56%) respectively;the main pathogen causing mixed infection was By+Bv (67.65%),mixed infection with influenza B virus accounted for 95.59%.Conclusion There are two lineages By and Bv epidemic in Guizhou Province, the epidemic peaks of influenza B are in winter and spring, male cases are higher than female, people under 15 years old are the high-risk group for influenza B, it is of great significance to strengthen the vaccination and surveillance of influenza in low age population.

Objective To understand the genetic variations of influenza B virus outbreaks in Guizhou province in 2016,and to compare the matching situation of outbreak epidemic strains with the vaccine strains recommended by WHO and representative strains in China.Methods The haemagglutinin HA1 gene of 8 strains isolated from two episodes of influenza B virus outbreaks in Tongren area was amplified and sequenced.The sequencing products were analyzed by bioinformatics software DNAStar. Results The two episodes of influenza outbreaks were both caused by influenza B Victoria lineage virus (BV).The homologies of the isolated strains were 99.8%—100.0% in nucleotide and 99.5 %—100.0%in amino acid.Mutation was only detected at 274 site in some strains.Compared with reference strain B/Victoria/2/87,the homologies were 91 .8%—92.0% and 91 .5 %—92.0%,respectively.Mutations developed at 17 amino acid sites,among which,I143V,V163I and V201I site were associated with the main antigenic determinant area B,C and D.Compared with previous vaccine strain B/Brisbane/60/2008, the homologies were 98.2%—98.3% and 98.5 %—99.0%,respectively,and mutations were detected at 3 sites.Mutations at I143V and N155D were detected in all 8 strains and at T247I in some strains.The mutation of I143V was associated with antigenic determinant area B.Compared with the representative strain B/Chongqing-Yuzhong/1384/2010,the homologies were 96.7%—96.8% and 97.0%—97.5 %, respectively.A total of 6 sites developed mutations,among which,5 sites were P84L,I143V,N155D, V172I and T223N mutations.The mutation of T247I was detected in some strains,and I143V was associated with area B.Compared with the epidemic strain in Guizhou in 2016,the homologies were 99.8%—100.0% and 99.5 %—100.0%,respectively.Mutation was only detected at site 247 in some strains and was not associated with the main antigenic determinant area.Conclusions The two episodes of influenza outbreaks in Guizhou are caused by the same BV lineage epidemic virus strain.Haemagglutinin gene of BV lineage virus is constantly changing.However,there is no new mutation emerged at important site.Compared with previous influenza vaccine strain B/Brisbane/60/2008 recommended by WHO,BV lineage virus is well matched and could provide a positive protection effect.

Objective: To investigate the molecular characteristics and tracing of the hemagglutinin (HA) gene, and to analyze the risk of human infection with influenza virus A (H7N9) in Guizhou Province, so that to provide evidence for the prevention and control of highly pathogenic avian influenza A (H7N9). Methods: Nucleic acids of 5 strains of H7N9 including 1 sample of the patient′s nasopharyngeal swab and 4 samples of the live poultry market (LPM) environment were extracted and HA genes were amplified and sequenced. Then the homology, genetic evolution and the pivotal sites related to receptor binding regions, pathogenicity and potential glycosylation of the avian influenza A (H7N9) viruses were analyzed by a series of bioinformatics softwares. Results: Homology analysis revealed that the homologies of nucleotide and amino-acid of the HA gene of H7N9 strains from the patient and LPM in Weining County, Guizhou Province were 99.8% and 99.6%, respectively, while those of 4 strains from LPM were both 100%. The homologies of nucleotide and amino-acid of the HA gene of H7N9 strains were the highest with the strain of A/Guangxi/5/2017 isolated from a Guangxi infected patient (99.7%-99.9% and 99.4%-99.8%, respectively), while those with the strain isolated from LPMs environment at the end of 2016 (A/Environment/Guangdong/C16283222/2016) were 99.0%-99.2% and 98.9%-99.2%, respectively. However, the homologies of nucleotide and amino-acid of the HA gene of H7N9 strains with A/Shanghai/2/2013 recommended by world health organization and the candidate vaccine strain A/Anhui/1/2013 were 96.8%-97.0% and 95.8%-96.2%, respectively. Phylogenetic analysis showed that the 5 strains had the nearest genetic distance to the strain A/Guangxi/5/2017. All the 5 strains cleavage site sequences of HA protein showed mutation of PEVPKRKRTAR↓GLF, and they were highly pathogenic avian influenza viruses mutant strains, which all had mutation of G186V at the receptor binding sites of HA gene, while no Q226L mutation was found. All 5 strains had new mutation of A363S, and new mutations of R56K and I297V were only found in the strain isolated from the patient. Among the five potential glycosylation motifs in the HA, only 421NWT and 493NNT had variation of the position post shift. Conclusions All the 5 H7N9 strains isolated in Weining County, Guizhou Province are highly pathogenic avian influenza mutative viruses. The current candidate vaccine may not provide a very good protection. The mutations of cleavage site of HA protein, G186V as well as other new mutation sites of HA may enhance the susceptibility and pathogenicity to human beings.

Objective:To analyze the genetic variation characteristics of the HA gene of influenza A virus (H3N2) in Guizhou province from 2017 to 2019. Methods:Twenty strains of influenza A virus (H3N2) were randomly selected from 10 network laboratories in Guizhou province for RNA extraction. Reverse transcriptase-polymerase chain reaction and sequencing were performed. The products were analyzed using bioinformatics software.Results:The nucleotide homology of the HA gene of the 20 strains was 97.7%-100%, which was highly homologous to the vaccine strains A/Hong-Kong/4801/2014 recommended by WHO in 2017 and A/Singapore-INFIMH/16-0019/2016 recommended by WHO in 2018, but they were significantly different from the vaccine strain A/Kansas/14/2017 recommended by WHO in 2019. Genetic analysis showed that the 20 strains were divided into two branches, and the strains that were prevalent in 2019 were located in different branches, with marked genetic differences. Key site analysis showed mutations in antigenic determinants A, B, C, and E and mutations in the anterior and posterior walls of receptor binding sites. Key site analysis also showed that there was an increase in the number of glycosylation sites compared with the vaccine strains prevalent in the same year. Genetic distance, antigen sites, and glycosylation sites were slightly different between virus strains prevalent in 2017-2018 and virus strains prevalent in 2019. Conclusion:The HA gene of the influenza A virus subtype H3N2 in Guizhou province from 2017 to 2019 showed heterogeneity and gene mutation, especially in 2019. Therefore, close monitoring of the genetic evolution of the influenza A virus subtype H3N2 is necessary.

Objective:To analyze the molecular evolution characteristics of HA and NA genes of influenza B/Yamagata (BY) and influenza B/Victoria (BV) lineage viruses in Guizhou Province, aiming to provide reference for scientific prevention and control of influenza. Methods:The prevalence of various types of influenza viruses in Guizhou Province from 2017 to 2021 was analyzed. The nucleic acid of influenza B viruses was extracted, and then the HA and NA genes were amplified by RT-PCR. Fourteen strains were sequenced and the sequences of 83 strains were obtained from GISAID. Homologies between the 97 influenza B viruses as well as the phylogenetic characteristics and amino acid site variations were analyzed. Results:Influenza A, BY and BV lineage viruses co-circulated in Guizhou Province and BV lineage was the predominant type. The homologies of HA and NA genes were 98.7%-99.4% and 98.4%-99.6% between BY lineage viruses and the reference vaccine strain B/PHUKET/3073/2013. BV lineage viruses shared 98.3%-99.3% and 98.9%-99.6% homologies with the reference vaccine strain B/Colorado/06/2017. The BY lineage strains in Guizhou Province mainly belonged to Y3 genetic group with HA gene in two branches of Y3-H1-2 and NA gene in three branches of Y3-N1-3. Three reassortant strains were found in Y3 clade. The isolated BV lineage strains mainly belonged to V1A-2 genetic group with HA gene in four branches of V1A-2 H1-4 and NA gene in five branches of V1A-2 N1-5. Twenty reassortant strains were found in V1A-2 clade and no inter-lineage reassortants were found. Analysis of variations at key amino acid sites showed that there was no mutation at epitopes in Y3 genetic group. However, there were point mutations at four main epitopes and a shift mutation in 190 helix in V1A-2 genetic group. There was no mutation in drug resistance sites. Conclusions:Various types of influenza viruses circulated in Guizhou Province. The homology between influenza B viruses and vaccine strains was decreasing. Different branches of HA and NA genes had been evolved and various forms of mutations were detected in the sequences. Intra-lineage reassortant strains and new varieties emerged. Surveillance of influenza B viruses should be strengthened.

Insomnia is the most common sleep disorder in clinical practice， and prolonged insomnia can significantly impact daily life and work. Currently， the clinical treatment of insomnia primarily relies on sedative and hypnotic drugs， which， although fast-acting， come with numerous adverse reactions. Traditional Chinese medicine （TCM） approaches the treatment of insomnia by employing syndrome differentiation and focusing on addressing the root cause of the disease. This approach is characterized by fewer adverse reactions， stable long-term effects， and better patient compliance. Huanglian Ejiaotang was first recorded in Zhang Zhongjing's Treatise on Febrile and Miscellaneous Diseases（《伤寒杂病论》） during the Han dynasty. It has the effects of nourishing Yin， reducing fire， eliminating irritability， and calming the nerves. Clinical studies have shown that Huanglian Ejiaotang is frequently used to treat insomnia and， when combined with other classic prescriptions， Western medicine， or other therapies， it can have a synergistic effect， thereby enhancing therapeutic outcomes. Research indicates that the mechanism of action of Huanglian Ejiaotang in treating insomnia may be related to the regulation of the hypothalamic-pituitary-adrenal （HPA） axis， 5-hydroxytryptamine （5-HT）， norepinephrine （NE）， dopamine （DA）， central neurotransmitters， gut microbiota， and the inhibition of inflammatory factors. The active components of Huanglian Ejiaotang that are effective in treating insomnia include berberine， baicalin， baicalein， total glucosides of paeony， and collagen. This article discussed the treatment of insomnia with Huanglian Ejiaotang from several perspectives， including its theoretical foundation， clinical research， and mechanisms of action， and also summarized the latest research progress on Huanglian Ejiaotang for insomnia， with the aim of providing more comprehensive ideas for the clinical diagnosis and treatment of insomnia.