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Objective:To investigate the phylogenetic and antigenic characteristics of hemagglutinin (HA) gene of influenza B/Victoria lineage (BV) viruses in Beijing during the 2021-2022 influenza surveillance season, and to analyze whether the circulating BV viruses match the vaccine strain.Methods:Pharyngeal swab specimens from influenza like-illness (ILI) cases in the 2021-2022 influenza surveillance season were collected from surveillance network labs in Beijing and cultured in MDCK cells and chicken embryo to isolate BV viruses. Nucleic acids of the viruses were extracted, and the HA gene was amplified and sequenced. The nucleotide and amino acid sequence identity of the HA gene was analyzed using MEGA5.0 software. A phylogenetic tree of HA gene was constructed using the maximum likelihood method. The N-glycosylation sites in HA were predicted online. Three-dimensional structure of HA was constructed using SWISS-MODEL homologous modeling. Hemagglutination inhibition (HI) test was performed to analyze the antigenicity of BV viruses.Results:A total of 402 BV viruses were collected and 58 strains with full-length HA gene sequences were chosen for further analysis. Compared with the HA gene of this year′s vaccine strain (B/Washington/02/2019), there were 27 amino acid mutations, 11 of which were located in four different antigenic determinants. The phylogenetic analysis revealed that three subgroups of 1A.3, 1A.3a1, and 1A.3a2 co-circulated in Beijing with 54 strains (54/58, 93.10%) clustered to the Clade 1A.3a2, two strains (2/58, 3.45%) clustered to the Clade 1A.3a1, and two strains (2/58, 3.45%) in the same subgroup (Clade 1A.3) as the vaccine component BV strain in 2021-2022. Compared with the vaccine strain (B/Washington/02/2019), two BV strains had an additional N-glycosylation site at residue 197, while the other 56 strains showed no change in N-glycosylation sites. Antigenic analysis showed that 35 BV strains (35/58, 60.34%) were antigenically similar to the vaccine strain and 23 strains (23/58, 39.66%) were low-response strains.Conclusions:Three subgroups of BV viruses co-circulated in Beijing during the 2021-2022 influenza surveillance season. The predominant subgroup was Clade 1A.3a2 (93.10%), showing a certain genetic distance with the vaccine strain (B/Washington/02/2019). Nearly 40% (39.66%) of the viruses were low-response strains. This study indicated that continuous monitoring of the variations of influenza epidemic strains and timely providing laboratory basis for screening vaccine component strains were the basic technical guarantee for coping with influenza pandemic.
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Objective:To clarify the M protein ( emm gene) types and drug susceptibility characteristic variations of Group A Streptococcus (GAS) in children in Beijing. Methods:The GAS strains isolated from throat swab samples of children diagnosed with scarlet fever and pharyngeal infection in scarlet fever etiology surveillance sentinel hospitals in 16 districts of Beijing in 2018, 2019 and 2021 were analyzed retrospectively.PCR amplification and sequencing were used for emm genotyping, and the minimum inhibitory concentrations (MIC) of 10 antibiotics were determined by the broth microdilution method.The data were analyzed using χ2 test and Fisher′ s exact method between groups. Results:A total of 557 GAS strains were collected, and 11 emm genotypes ( emm1, emm3, emm4, emm6, emm11, emm12, emm22, emm75, emm89, emm128, and emm212) were detected.Of 557 strains, 238 trains were of emm1 type (42.73%), 271 strains were of emm12 type (48.65%) and 48 strains were of other emm types (8.62%). The detection rates of emm1, emm12 and other emm type genes in 2018, 2019, and 2021 were [37.50% (105/280 strains), 57.14% (160/280 strains), 5.36% (15/280 strains)], [49.05% (129/263 strains), 39.54% (104/263 strains), 11.41% (30/263 strains)], and [28.57% (4/14 strains), 50.00% (7/14 strains), 21.43% (3/14 strains)], respectively.In children infected with emm12 in 2018 and 2019, there were more children under 6 years old than children over 6 years old (62.50% vs.46.88%, 46.36% vs.30.36%) (χ 2=7.182, 6.973; all P<0.05). Drug susceptibility testing results suggested that 225 randomly selected GAS strains were all 100.00% sensitive to 7 antibiotics including Penicillin, Levofloxacin, Meropenem, Linezolid, Cefotaxime, Cefepime and Vancomycin.The rates of resistance to Erythromycin, Tetracycline and Clindamycin were [88.57% (93/105 strains), 87.62% (92/105 strains), 86.67% (91/105 strains)], and [94.34% (100/106 strains), 94.34% (100/106 strains), 87.74% (93/106 strains)] in 2018 and 2019, respectively.The test strains were 100.00% (14/14 strains) resistant to the above 3 antibiotics in 2021.MIC 50 and MIC 90 values of Penicillin in 2018, 2019, and 2021 were (0.03 mg/L, 0.03 mg/L), (0.03 mg/L, 0.06 mg/L), and (0.06 mg/L, 0.06 mg/L), respectively.Among 225 GAS strains, 207 strains had drug resistance and were resistant to more than one drug.Specifically, 94.69% (196/207 strains) were resistant to Erythromycin, Tetracycline and Clindamycin.About 4.35% (9/207 strains) were resistant to both Erythromycin and Clindamycin.A total of 0.97% (2/207 strains) were resistant to Erythromycin and Tetracycline. Conclusions:The emm genotypes of GAS in children in Beijing are diverse in 2018, 2019 and 2021.The dominant genotypes are emm12 and emm1, and emm12 is the main epidemiological type.GAS strains maintain highly resistant to Erythromycin, Clindamycin and Tetracycline, and sensitive to Penicillin and other antibiotics.However, MIC 50 and MIC 90 of Penicillin shows an ascending trend.
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Objective@#To understand the antigenicity and genetic characterization of influenza B virus HA gene in B/Victoria-lineage virus (BV) in Beijing during 2017-2018.@*Methods@#Thirty BV virus strains isolated from MDCK cell culture by 17 laboratories in Beijing were collected. The antigenicity was analyzed by comparing with the vaccine strain recommended by WHO. The total viral nucleic acid was extracted and HA gene was amplified by RT-PCR and sequenced. The phylogenetic tree was constructed by HA and mutant sites were analyzed.@*Results@#Among 30 strains of BV, 23 strains (76.7%) were low-reactive strains, other 7 strains (23.3%) were related to the vaccine. The phylogenetic analysis showed that the HA gene of all 30 strains located in Clade 1A branch. In addition, amino acid mutations occurred in 8 sites, and 6 of them located in the antigen determining region.@*Conclusions@#There was a correlation between the high proportion of low-reactive antigenicity and 6 aa variation in antigenic determinants involved in HA region of BV influenza virus between 2017-2018, which provides an important laboratory basis for the recommendation of BV influenza vaccine.
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Objective To analyze the characteristics of super-antigen (SAg) of group A Streptococcus pyogenes (GAS),isolated from patients with scarlet fever or pharyngeal infections in Beijing between 2015-2017.Methods Throat swab specimens from patients with scarlet fever or pharyngeal infections were collected and tested for GAS.Eleven currently known SAg genes including SpeA,speC,speG,speH,speI,speJ,speK,speL,speM,smeZ and ssa were tested by real-time PCR while M protein genes (emm genes) were amplified and sequenced by PCR.Results A total of 377 GAS were isolated from 6 801 throat swab specimens,with the positive rate as 5.5％.There were obvious changes noticed among speC,speG,speH and speK in three years.A total of 45 SAg genes profiles were observed,according to the SAgs inclusion.There were significant differences appeared in the frequencies among two of the highest SAg genes profiles between emml and emml2 strains (x2=38.196,P＜0.001;x 2=72.310,P＜0.001).There also appeared significant differences in the frequencies of speA,speH,speI and speJ between emm 1 and emm 12 strains (x2 =146.154,P＜ 0.001;x2=52.31,P＜0.001;x2=58.43,P＜0.001;x2=144.70,P＜0.001).Conclusions Obvious changes were noticed among SAg genes including speC,speG,speH and speK from patients with scarlet fever or pharyngeal infections in Beijing between 2015-2017.SAg genes including speA,speH,speI and speJ appeared to be associated with the emm 1 and emm 12 strains.More kinds of SAg genes profiles were isolated form GAS but with no significant differences seen in the main SAg genes profiles,during the epidemic period.
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Objective To analyze the characteristics of super-antigen (SAg) of group A Streptococcus pyogenes (GAS),isolated from patients with scarlet fever or pharyngeal infections in Beijing between 2015-2017.Methods Throat swab specimens from patients with scarlet fever or pharyngeal infections were collected and tested for GAS.Eleven currently known SAg genes including SpeA,speC,speG,speH,speI,speJ,speK,speL,speM,smeZ and ssa were tested by real-time PCR while M protein genes (emm genes) were amplified and sequenced by PCR.Results A total of 377 GAS were isolated from 6 801 throat swab specimens,with the positive rate as 5.5％.There were obvious changes noticed among speC,speG,speH and speK in three years.A total of 45 SAg genes profiles were observed,according to the SAgs inclusion.There were significant differences appeared in the frequencies among two of the highest SAg genes profiles between emml and emml2 strains (x2=38.196,P＜0.001;x 2=72.310,P＜0.001).There also appeared significant differences in the frequencies of speA,speH,speI and speJ between emm 1 and emm 12 strains (x2 =146.154,P＜ 0.001;x2=52.31,P＜0.001;x2=58.43,P＜0.001;x2=144.70,P＜0.001).Conclusions Obvious changes were noticed among SAg genes including speC,speG,speH and speK from patients with scarlet fever or pharyngeal infections in Beijing between 2015-2017.SAg genes including speA,speH,speI and speJ appeared to be associated with the emm 1 and emm 12 strains.More kinds of SAg genes profiles were isolated form GAS but with no significant differences seen in the main SAg genes profiles,during the epidemic period.
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Objective To reveal the genetic distribution of group A streptococcus progeny(GAS)isolated from children with re-spiratory tract infection in Beijing in 2015.Methods The throat swabs were collected from children with clinical diagnosis of scarlet fever and pharyngeal infection during May to July,2015.16 pediatric outpatient departments or pediatric emergency department of hospitals located in 16 districts of Beijing were enrolled in this study.PCR amplification and gene sequencing were employed.The encoding mature M protein gene(emm)distribution in different condition was compared and analyzed.Results A total of 247 GAS were isolated,including 73(29.6%,73/247)isolates from cases with scarlet fever and 174(70.4%,174/247)isolates from cases with pharyngeal infection.Among all isolates,six genotypes of emm were identified,including emm12(53.4%,132/247),emm1(44. 1%,109/247)and others(emm22,emm75,emm131 and emm241,2.5%,6/247).The significant difference of emm distribution with age was observed in this study:emm1 isolates were dominant in children aged 6-12,whereas emm12 isolates were dominant in chil-dren aged 1-5(P<0.05).While the emm distribution showed no significant difference in groups with different location,gender,and disease pattern(P>0.05).Conclusion Emm1 and emm12 were the circulating genotype of GAS in Beijing children,2015.Moreo-ver,the difference of distribution between emm1 and emm12 was proved in this study.
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Objective:To screen the new candidate molecules interacting with protein kinase Wee1B by yeast two hybrid system, and to analyze their interaction with Wee1B in the early stage of mouse fertilized eggs by bioinformatics.Methods:The plasmid pcDNA3.1/V5-His-TOPO-Wee1B wild type encoding mouse Wee1B gene was used as template to construct bait plasmid pGBKT7 Wee1B and the bait plasmid pGBKT7-Wee1B was transformed into yeast competent cells at SD/Trp (SDO),SD/Trp/X-α-Gal (SDO/X)and SD/Trp/X α Gal/AbA plates (SDO/X/A)plates to detect the toxicity and self-activation ability of yeast and its expression in yeast using Western blotting method.The yeast cells containing pGBKT7-Wee1B were fused with human ovary cDNA library, the yeast plasmid transformation of Escherichia coli positive clones were sequenced after identified by yeast transformation.BLAST analysis was carried out in GenBank,and its effect on the development of mouse fertilized eggs was deduced according to the gene annotation.Results:The double enzyme digestion analysis and sequencing analysis results showed that the pGBKT7-Wee1B bait plasmid was successfully constructed.The plasmid was transformed into the yeast,and there were no clones in the SDO/X/A plates.The pGBKT7-Wee1B and pGBKT7 empty vectors were transformed into the yeast,the bacteria were inoculated on the SDO plates,and the clones were uniformly grown on the two SDO plates.The positive clones were picked and expanded in culture,the protein was extracted and Western blotting showed that pGBKT7 Wee1B was expressed in the yeast.The bait plasmids were fused with human ovary cDNA library and the positive clones inserted into the fragment were identified by PCR. Nine proteins which interacted with Wee1B protein kinase were screened out by sequencing and blast analysis,and the proteins which could be closely related to the development of mouse oocytes and the development of fertilized eggs were analyzed by bioinformatics analysis.Conclusion:Using the yeast two hybrid system from human ovary cDNA library,nine interacting proteins with Wee1B protein kinase are screened and these screened proteins may regulate mouse oocyte maturation and early embryo development through interacting with Wee1B.
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Phenylalanine hydroxylase (PAH) is a member of aromatic amino acid hydroxylase (AAAHs) family, and catalyze phenylalanine (Phe) into tyrosine (Tyr). Using immunological and RT-PCR methods to prove the existence of phenylalanine hydroxylase (PAH) gene in the brain of Neanthes japonica in protein and nucleic acid level. Using Western blotting to detect the pah immunogenicity of Neanthes japonica. Making paraffin sections and using immunohistochemical technique to identify the presence and distribution of the phenylalanine hydroxylase gene in the brain of Neanthes japonica. Clone pah gene from the brain of Neanthes japonica by RT-PCR, constructing plasmid and transferring into E. coli to amplification, picking a single homogeneous colony, double digesting then making sequence and comparing homology. Western blotting results showed that the expression of the protein is present in Neanthes japonica brain, immunohistochemistry technique results showed that phenylalanine hydroxylase mainly expressed in abdominal of forebrain, dorsal and sides of midbrain. RT-PCR technique results showed that the phenylalanine hydroxylase exist in the brain of Neanthes japonica and has a high homology with others animals. PAH is present in the lower organisms Neanthes japonica, in protein and nucleic acid level. Which provide the foundation for further study the evolution of aromatic amino acid hydroxylase genes in invertebrate.
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Animals , Blotting, Western , Brain , Escherichia coli , Metabolism , Phenylalanine Hydroxylase , Genetics , Metabolism , Polychaeta , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate superantigen gene profiles of group A Streptococcus (GAS) isolated in Beijing pediatric patients in 2014, and to explore the the relationship between superantigen gene profiles with emm types, and GAS infections with diseases.</p><p><b>METHODS</b>A total of 259 GAS strains were isolated from pediatric patients clinically who diagnosed with scarlet fever and pharyngitis from 36 hospitals in Beijing from May to July, 2014.The Superantigens genes of strains were performed by Real-time PCR (speA, speB, speC, speF, speG, speH, speI, speJ, speK, speL, speM, smeZ, ssa). PCR amplification of GAS strain M protein N gene segments were carried ort; products after sequencing comparison were analyzed to determine the GAS types of emm. The differences in distributions of superantigen genes and emm types of GAS isolates were compared between subgroups.</p><p><b>RESULTS</b>Among the 259 GAS strains, the detection rates of 13 superantigens were as the following: speA 48.6% (126), speB 99.2% (257), speC 99.2% (257), speF 98.8% (256), speG 98.5% (255), speH 43.6% (113), speI 46.3% (120), speJ 49.0% (127), smeZ 99.2% (257) and ssa 98.5% (255), respectively, however, speK, speL, and speM were not found. Eleven superantigen gene profiles in all were observed (A-K). The percentage of emm1 strains harbored spe A and speJ were 94.2% (113/120), 95.0% (114/120), respectively, which were significantly higher than those of emm12 strains (5.6% (7/124), 5.6% (7/124), respectively; χ(2) = 191.20, 194.80, P < 0.001). The percentage of emm12 strains harbored speH and speI were 83.9% (104/124), 88.7% (110/124), respectively, which were significantly higher than those of emm1 strains (3.3% (4/120), 4.2% (5/120), respectively; χ(2) = 160.30, 174.90, P < 0.001).The superantigen genotypes of GAS strains and emm types, which were isolated from scarlet fever and pharyngitis cases, were not significant different (P > 0.05).</p><p><b>CONCLUSION</b>The GSA strains isolated in Beijing pediatric patients in 2014, the relevance ratio of speB, speC, speF, smeZ, speG, ssa were higher than others, while speK, speL, and speM were no detected in any GAS strains. The superantigen genes appeared to be associated with the emm type. Furthermore, emm type distribution and superantigen genes were not different between scalet fever and pharyngitis.</p>
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Child , Humans , Antigens, Bacterial , Genetics , Beijing , Genotype , Real-Time Polymerase Chain Reaction , Streptococcal Infections , Microbiology , Streptococcus pyogenes , Genetics , Allergy and Immunology , Superantigens , GeneticsABSTRACT
Objective To study the expressions of casein kinase 2α(Ck2α),β-catenin, survivin in ovarian cancer tissue and analyze the relationships between them, and to explore the clinical application value. Methods Immunohistochemistry staining was used to detect the expressions of Ck2α,β-catenin and survivin in 8 cases of simple ovarian cysts tissue,8 cases of ovarian benign tumor tissue and 29 cases of ovarian cancer tissue;the correlations of the expressions of Ck2α,β-catenin,survivin in ovarian cancer tissue and their associations with the clinicopathologic characteristics were analyzed.Results The expression levels of Ck2α,β-catenin and survivin in ovarian cancer tissue were significantly higher than those in simple ovarian cysts tissue and ovarian benign tumor tissue(P<0.05);the Ck2αexpression level in ovarian cancer tissue was positively corelated with the expression levels ofβ-catenin and survivin(r=0.438,r=0.479,P<0.05);the expression levels of Ck2α,β-catenin and survivin in low-medium differentiation group were significantly higher than those in high differentiation group (P<0.05);the expression levels of Ck2α,β-catenin and survivin in stagesⅡandⅢ group were significantly higher than those in stage Ⅰ group(P<0.05).Conclusion The protein expression levels of Ck2α,β-catenin and survivin are increased in ovarian cancer tissue, and three are positive corelations between them;Ck2α,β-catenin and survivin may play an important role in the occurrence and development of ovarian cancer;the combined detection of them has important clinical value.
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<p><b>OBJECTIVE</b>To analyze the prevalence of super-antigens (SAgs) of group A streptococcus (GAS)isolated from Beijing pediatric patients in 2011, and to explore the relationship between emm types, characteristics of patients and SAgs.</p><p><b>METHODS</b>A total of 635 isolates of GAS were collected from children in 36 hospitals in Beijing from May to July, 2011. Thirteen currently known SAg genes were tested by real-time PCR, and emm gene was performed by PCR and sequencing of N-terminal gene fragments of M protein.</p><p><b>RESULTS</b>Prevalence rates of 13 SAg genes speA, speB, speC, speF, speG, speH, speI, speJ, speK, speL, speM, smeZ, ssa were 22.4%, 100.0%, 99.4%, 99.7%, 99.7%, 76.4%, 76.2%, 21.7%,0.6%, 1.1%, 2.2%, 99.7% and 98.0%, respectively. A total of 26 SAgs profiles were observed according to the SAgs inclusion. There were significant differences in frequencies of speA, speH, speI and speJ between emm1 and emm12 strains (P < 0.05). In isolates from patients with pharyngeal infection, the prevalence rates for speK and speL were higher while the frequency for ssa was lower than that from scarlet fever cases (P < 0.05).</p><p><b>CONCLUSION</b>The frequencies of speB, speF, smeZ, speG, speC, and ssa were high among strains isolated while speM, speL and speK were relatively low from children in Beijing, 2011. SAg genes appeared to be associated with the emm types. In this study, differences of frequency for speA and speC from strains collected from patients with scarlet fever and pharyngeal infection had not been found.</p>