Shaofu Zhuyu Decoction attenuates fibrosis in endometriosis through regulating PTEN/Akt/mTOR signaling pathway / 中国中药杂志

The present study aimed to investigate the protective role of Shaofu Zhuyu Decoction(SFZY) against endometriosis fibrosis in mice, and decipher the underlying mechanism through the phosphatase and tensin homolog deleted on chromosome ten(PTEN)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) pathway. Eighty-five BALB/c female mice were randomly assigned into a blank group, a model group, high-, medium, and low-dose SFZY(SFZY-H, SFZY-M, and SFZY-L, respectively) groups, and a gestrinone suspension(YT) group. The model of endometriosis was induced by intraperitoneal injection of uterine fragments. The mice in different groups were administrated with corresponding groups by gavage 14 days after modeling, and the blank group and model group with equal volume of distilled water by gavage. The treatment lasted for 14 days. The body weight, paw withdrawal latency caused by heat stimuli, and total weight of dissected ectopic focus were compared between different groups. The pathological changes of the ectopic tissue were observed via hematoxylin-eosin(HE) and Masson staining. Real-time PCR was employed to measure the mRNA levels of α-smooth muscle actin(α-SMA) and collagen type Ⅰ(collagen-Ⅰ) in the ectopic tissue. The protein levels of PTEN, Akt, mTOR, p-Akt, and p-mTOR in the ectopic tissue were determined by Western blot. Compared with the blank group, the modeling first decreased and then increased the body weight of mice, increased the total weight of ectopic focus, and shortened the paw withdrawal latency. Compared with the model group, SFZY and YT increased the body weight, prolonged the paw withdrawal latency, and decreased the weight of ectopic focus. Furthermore, the drug administration, especially SFZY-H and YT(P<0.01), recovered the pathological and reduced the area of collagen deposition. Compared with the blank group, the modeling up-regulated the mRNA levels of α-SMA and collagen-Ⅰ in the ectopic focus, and such up-regulation was attenuated after drug intervention, especially in the SFZY-H and YT groups(P<0.05,P<0.01). Compared with the blank group, the modeling down-regulated the protein level of PTEN and up-regulated the protein levels of Akt, mTOR, p-Akt, and p-mTOR(P<0.01, P<0.001). Drug administration, especially SFZY-H and YT, restored such changes(P<0.01). SFZY may significantly attenuate the focal fibrosis in the mouse model of endometriosis by regulating the PTEN/Akt/mTOR signaling pathway.

Effect of Modified Shaofu Zhuyutang on Neural Angiogenesis and Pelvic Pain of Endometriosis with Syndrome of Cold Congeal and Blood Stasis / 中国实验方剂学杂志

ObjectiveTo evaluate the clinical curative effect of modified Shaofu Zhuyutang on pelvic pain of endometriosis （EMT） with syndrome of cold congeal and blood stasis and the influence on neural angiogenesis. MethodA total of 110 cases were divided into a control group （54 cases） and an observation group （56 cases） by the random number table method. Patients in the control group took Aifu Nuangong pills with 6 g/time and 2 times/day. Patients in the observation group took modified Shaofu Zhuyutang with 1 dose/day. Course of treatment continued for 3 menstrual cycles. Dysmenorrhea， other symptoms and signs of pelvic pain， and the EMT health profile-5 （EHP-5） for patients with syndrome of cold congeal and blood stasis and EMT were scored before and after treatment. The levels of peripheral blood nerve growth factor （NGF）， substance P （SP）， calcitonin gene-related peptide （CGRP）， brain-derived neurotrophic factor （BDNF）， soluble Fms-like tyrosine kinase（sFlt-1）， vascular endothelial growth factor （VEGF）， matrix metalloproteinase-9 （MMP-9）， and insulin growth factor-1 （IGF-1） were detected before and after treatment. The levels of prostaglandin E2 （PGE2）， cyclooxygenase-2 （COX-2）， tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and other inflammatory factors were detected before and after treatment. The pain medication usage and recurrence were recorded. ResultThe scores of dysmenorrhea， symptoms of pelvic pain symptoms （non-menstrual pelvic pain， dyspareunia， anal falling pain， and defecation pain， etc.）， and signs of pelvic pain （pelvic tenderness and sacral ligament tender nodules） in the observation group were lower than those in the control group （P<0.01）. The disappearance rates of dysmenorrhea， pelvic tenderness， and sacral ligament tender nodules were 67.35% （33/49）， 73.33% （33/45）， and 77.27% （34/44） in the observation group， which were all higher than 45.83% （22/48）， 48.84% （21/43）， and 52.27% （23/44） in the control group. The difference was statistically significant （χ2=4.571， χ2=5.565， χ2=6.026， P<0.05）. The scores of EHP-5 and syndrome of cold congeal and blood stasis in the observation group were lower than those in the control group （P<0.01）. The levels of VEGF， MMP-9， IGF-1， NGF， SP， CGRP， and BDNF in the observation group were lower than those in the control group （P<0.01）， while the level of sFlt-1 was higher than that in the control group （P<0.01）. The levels of PGE2， COX-2， TNF-α， and IL-6 in the observation group were lower than those in the control group （P<0.01）. The total effective rate of the observation group was 92.45% （49/53）， which was higher than 76.00% （38/50） of the control group （χ2=5.307， P<0.05）. After 6 months of follow-up， the recurrence rate in the observation group was 30.61% （15/49）， which was lower than 52.63% （20/38） in the control group （χ2=4.315， P<0.05）. The average of taking ibuprofen sustained-release capsules in the control group was higher than that in the observation group per menstrual period （P<0.01）. ConclusionModified Shaofu Zhuyutang treated pelvic pain of EMT with syndrome of cold congeal and blood stasis by regulating the mechanism of neural angiogenesis， reducing pain， and promoting the disappearance of related pains， thus improving the quality of life. Shaofu Zhuyutang has a better clinical effect than Aifu Nuangong pills and has a low recurrence rate.

Mechanism of Shaofu Zhuyu Decoction in treatment of EMT induced dysmenorrhea based on network pharmacology and molecular docking / 中国中药杂志

Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN-TCM) were searched for the effective components and targets of Shaofu Zhuyu Decoction. The relevant targets for endometriosis(EMT) and dysmenorrhea were retrieved from the Comparative Toxicogenomics Database(CTD), Therapeutic Target Database(TTD), GeneCards, and DisGeNET with the terms of "endometriosis" and "dysmenorrhea". Cytoscape 3.8.0 was employed to construct the drug-active component-therapeutic target network. A protein-protein interaction(PPI) network was established by STRING 11.0. Analyze Network, the plug-in in the Cytoscape 3.8.0, was used to calculate the topological parameters of the nodes and screen out the critical proteins in the network. The potential therapeutic targets were imported into RStudio and subjected to Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses with clusterProfiler package. Finally, the AutoDock Vina(Vina) platform was used for molecular docking to predict the binding degree of the main active components of Shaofu Zhuyu Decoction to key targets. As revealed by the screening results, 136 active components and 380 targets of Shaofu Zhuyu Decoction were obtained. Additionally, there were 1 627 targets related to EMT and 142 targets related to dysmenorrhea with 107 common targets, and 42 potential therapeutic targets of Shaofu Zhuyu Decoction for the treatment of EMT-induced dysmenorrhea. The targets such as interleukin 6(IL6) and prostaglandi-nendoperoxide synthase-2(PTGS2) were pivotal in the biological network of Shaofu Zhuyu Decoction intervention in EMT-induced dysmenorrhea, which involved multiple signaling pathways, including inflammation, hormones, and those promoted cell proliferation [e.g., mitogen-activated protein kinase(MAPK) and phosphatidylinositol-3 kinase(PI3 K)-protein kinase B(AKT)]. The results of molecular docking showed that the active components of Shaofu Zhuyu Decoction had good binding capacities to key targets such as IL6 and PTGS2. The findings of this study demonstrated that Shaofu Zhuyu Decoction could treat EMT-induced dysmenorrhea through multiple targets and multiple pathways, which could provide new ideas for investigating the underlying mechanism of Shaofu Zhuyu Decoction in the treatment of EMT-induced dysmenorrhea.

Analysis of genetic variation of hemagglutinin and three internal genes of influenza B virus isolated in Zhejiang province from 1999 to 2012 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the characteristics of the genetic variation of hemagglutinin( HA) and three internal genes coding for the nucleoprotein ( NP) , matrix protein ( M) and nonstructural protein ( NS) of influenza B virus.</p><p><b>METHODS</b>A total of 31 strains of influenza B virus were isolated in Zhejiang province from 1999 to 2012, and then were amplified and sequenced the genes of HAl , NP, M and NS. The phylogenetic tree was constructed, the nucleotide substitution rate of the above individual gene was estimated and the variation sites of amino acids were analyzed.</p><p><b>RESULTS</b>The 31 isolated strains of influenza B virus were divided into two distinct lineages Victoria and Yamagata in the phylogenetic tree of HAl gene,represented by B/Victoria/2/87 and B/Yamagata/16/88. Phylogenetic analysis of the NP gene showed that the NP gene of Victoria-like influenza B strains which were isolated after 2010 was highly homologous with Yamagata-like isolates, and thereby they were found to be on the same branch of the phylogenetic tree of the NP gene. Nucleotide substitution rates of HAl , NP, M and NS genes were estimated to be 2. 29 x 10 -3 ,1. 39 X 10-3 ,1. 78 X 10-3 ,1. 30 X 10-3 /site per year, respectively. Variations of amino acid of HAl domain of Victoria-like isolates mainly included K48E ,L58P ,N75K,K80R,K129N/S,N165K,S172P ,Sl97N/D and A202V; while those in Yamagata-like isolates were R48K, S1501, N166Y, N203S, G230D and D233N. Determined amino acid sequences of NP of Victoria-like influenza B isolates were similar to Yamagata-like isolates after 2010 and variations happened on four characteristic amino acid sites, naming A60D, I233V, N513S and V5341, compared with previous Victoria-like influenza B isolates.</p><p><b>CONCLUSION</b>Significant variation was found among influenza B strains isolated in Zhejiang province from 1999 to 2012. The surface HAl gene evolved more rapidly than internal genes. Gene reassortment and gene mutation were the main evolutionary mechanism of influenza B virus.</p>

Molecular evolution of two lineages related to influenza B virus based on HA1 gene / 中华流行病学杂志

Objective To study the evolutionary characteristics and rules of two lineages on influenza B virus.Methods A total of 126 HA1 sequences of strains isolated during 1940 to 2012were downloaded from the GenBank.Time of the most recent common ancestor (TMRCA) and divergence of the two lineages were calculated based on the data from phylogenetic analysis of HA1gene,using Bayesian Markov Chain Monte Carlo (Bayesian-MCMC) and molecular clock method.Results The average amino acid variant ratios were ranged from 5.4％ to 10.2％ within the strains of influenza B virus isolated during 1978 to 2010.Compared with the Victoria-like strains,all Yamagatalike strains showed an amino acid deletion at 163th site,while some of them showing a deletion at position 166.HA1 gene of influenza B virus seemed not have been affected by positive selection except a few sites.The evolutionary average rate on HA1 gene was 2.138 × 10-3 substitutions/site/year (95％HPD:1.833 × 10-3-2.437 × 10-3 substitutions/site/year).The estimated dates for TMRCA of the two lineages of influenza B virus could be dated back to 1971 (95％ HPD:1969-1972),while the divergence times of the two lineages were 1973 (95％ HPD:1971-1974) and 1977 (95％ HPD:1975-1978) respectively.Conclusion Significant differences were found on HA1 gene between earlier and recent identified strains of Victoria and Yamagata lineage.Differences between the two lineages increased and showing the potential of dividing themselves into different subtypes in the future.More attention should be paid to these trends and the related epidemiological significance.

Genomic sequences of human infection of avian-origin influenza A(H7N9)virus in Zhejiang province / 中华流行病学杂志

<p><b>OBJECTIVE</b>To analyze the etiology and genomic sequences of human infection of avian-origin influenza A(H7N9)virus from Zhejiang province.</p><p><b>METHODS</b>Viral RNA was extracted from patients of suspected H7N9 influenza virus infection and real-time RT-PCR was conducted for detection of viral RNA. All 8 segments of influenza virus were amplified by one-step RT-PCR and genomic sequences were assembled using the sequencing data. All the currently available HA and NA genes of the novel H7N9 virus, some other HAs from H7 subtype and NAs from N9 subtype were downloaded from public database for phylogenetic analysis, using the Mega 5.1 software. Mutations and variations were analyzed, using the genomic sequence data.</p><p><b>RESULTS</b>Reactions for influenza type A, subtype H7 and subtype N9 were all positive and all the genomic fragments were amplified for sequencing. After alignment, sequences were subjected for phylogenetic analysis. The results revealed highest homology with A/duck/Zhejiang/12/2011(H7N3)in HA gene and with A/wild bird/Korea/ A14/2011(H7N9)in NA gene of the H7N9 influenza virus. All 6 genes coding for internal proteins shared highest identities with H9N2 avian influenza which were circulated in the Chinese mainland, in the last two years. The sequenced virus showed Q226L mutation in HA protein, but E627K was not presented in PB2 protein of this virus. The E627K mutation was shared by all the other novel H7N9 viruses resulted in human infections through analysis on the currently available sequences.</p><p><b>CONCLUSION</b>Using the clinical samples, both detection of the viral genes and amplification of all 8 segments of the novel H7N9 influenza virus were accomplished. High homology of the novel H7N9 influenza viruses was observed by phylogenetic test, using the currently available sequences. The virus showed Q226L mutation on HA protein but E627K did not present on PB2 protein of this virus.</p>

Genetic variation of the hemagglutinin and neuraminidase of influenza B viruses isolated in Zhejiang province during 1999-2010 / 中华流行病学杂志

Objective To Characterize the genetic diversity of hemagglutinin(HA)and neuraminidase(NA)of influenza B viruses isolated in Zhejiang province during 1999-2010.Methods Respiratory specimens were collected from patients with flu-like syndrome during the influenza outbreaks or from the hospitals which carrying out influenza surveillance project in Zhejiang province.Samples were detected by real-time RT-PCR and isolated for influenza virus.HA1 and NA genes of influenza B virus isolates were amplified and sequenced.Phylogenetic comparison and genetic diversity analysis were performed using the bioinformation software.Results A total of 34 influenza B viruses were evolved in this study including Victoria-like and Yamagata-like strains according to the results of the HI test.The mutation rate of Victoria-like HA1 gene was 4.5% and Yamagata-like HA1 gene was 3.4%,respectively.The Victoria-like influenza B isolates had appeared to be all re-assortants having a Victoria lincage HA and Yamagata lineage NA since 2004.The predominant type of influenza virus isolates in 2010 was also influenza B virus after the H1N1 flu pandemic in Zhejiang province.The isolated strains were antigenicaily and genetically similar to B/Brisbane/60/2008--the vaccine strain proposed for 2009-2010.Many difierences of HA1 and NA amino acids existed in the current isolates when compared to previous influenza B strains.Conclusion Significant diversity was generated among influcnza B virus isolated from 1999 to 2010 in Zhejiang province.Genetic re-assortment and antigenic drift seemed the main evolutionary mechanism on influenza B virus.

c.359T>C mutation of the MYH14 gene in two autosomal dominant non-syndromic hearing impairment families with common ancestor / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the gene mutation for two Chinese families with autosomal dominant non-syndromic hearing impairment(NSHI).</p><p><b>METHODS</b>Two NSHI pedigrees with common ancestor were identified by clinical examination and family investigation. Linkage analysis was performed for all known NSHI loci, and all exons and exon-intron boundaries of the non-muscle myosin heavy chain 14 (MYH14) gene were amplified by PCR and sequenced.</p><p><b>RESULTS</b>The disease-causing gene of these 2 pedigrees was fine mapped to the DFNA4 locus on 19q13.33. A heterozygous transition of c. 359T>C (p.S120L) in MYH14 gene was identified. The mutation was detected in all patients but not in normal members in the two families.</p><p><b>CONCLUSION</b>It is the first report that mutation in MYH14 gene can cause dominant non-syndromic hearing impairment in Asian population, suggesting that MYH14 gene can be a disease-causing gene of Chinese patients with hearing impairment.</p>

Diagnosis of a dengue fever outbreak in Yiwu city, Zhejiang province in 2009 and its molecular tracing of the pathogen / 中华预防医学杂志

<p><b>OBJECTIVE</b>To determine a dengue fever outbreak in Yiwu city, Zhejiang Province in 2009 and to trace the origin of the pathogen.</p><p><b>METHODS</b>The dengue virus IgM, IgG antibodies and viral nucleic acid were detected and virus was isolated using 40 serum samples from the suspected patients. The viral RNA of the isolated virus strains was extracted and the E gene was amplified by RT-PCR. The amplicons were sequenced and the phylogenetic and homological analyses were also constructed.</p><p><b>RESULTS</b>Among 40 serum samples from dengue fever suspected patients, 17 were positive from for dengue IgM (42.5%); 4 were IgG positive (10.0%); 34 samples were dengue virus RNA positive (85.0%), 28 dengue virus type 3 (D3) strains were isolated (70.0%). The complete coding region of envelope genes (E) from 13 D3 strains was all 1479 nt without any insertion or deletion, which encoded with 493 amino acids (aa). E gene from the 13 D3 strains from Zhejiang in 2009 (D3/ZJ/2009) was 100.0% identical. The strain from Saudi Arabia shared the highest similarity with the D3 strain, 99.3% and 100.0% of their E genes and deduced amino acids were identical, respectively, whereas they were 93.4% and 97.4% between D3/ZJ/2009 strain and its prototype strain (D3/H87/1956), and 93.6% and 97.4% between D3/ZJ/2009 and a D3 strain isolated in Guangxi Province in 1980. The phylogenetic tree of E genes also indicated that D3/ZJ/2009 had maximum similarity with the D3/Saudi Arabia/2004. They all belonged to the D3/GIII branch, which was originated from Indian Subcontinent.</p><p><b>CONCLUSION</b>The outbreak of dengue fever in Zhejiang in 2009 was caused by type 3 dengue virus III genotype. The virus was most likely originated from Saudi Arabia.</p>

Epidemiological and etiologic characteristics of ocular fungal infection in Haikou / 南方医科大学学报

<p><b>OBJECTIVE</b>To analyze the epidemiological and etiologic characteristics of ocular fungal infection in Haikou.</p><p><b>METHODS</b>The samples of corneal, conjunctivae, anterior chambers, lacrimal sacs were taken from the outpatients and inpatients in the Department of Ophthalmology for fungal culture. The positivity rates of fungal culture were statistically analyzed to describe the seasonal and genus distribution of the fungal infections.</p><p><b>RESULTS</b>A total of 683 patients were involved in this study, and the total positivity rate of fungal culture was 27.96%. The positivity rate was 33.42% in male patients and 21.52% in female patients (P<0.01). The positivity rates were 31.42%, 11.32%, 10.81%, 21.28% and 29.17% in the cornea, conjunctivae, anterior chambers, lacrimal sacs and other locations (P<0.01), and were 34.88%, 22.58%, 11.76%, 4.11%, 21.43% and 13.33% in farmers, workers, teachers, government officers, students and personnel of other occupations (Chi2=39.550, P=0.001), respectively. In terms of age, the rate was 32.30% in 21-40 years group, 31.72% in 41-60 years group, 17.12% in over 60 years group and 26.61% in below 20 years group, showing significant differences (P<0.01). The rates were higher in August, September, October, December, November, all above 30%, but lower in March. Nine genera were identified, including Candida (49.21%), Aspergillus (19.37%), Fusarium (9.42%), mucor (5.76%), Actinomyces (5.24%), Penicillium (3.66%), Saccharomyces (2.62%), non-spore group (1.57%), Alternaria (1.57%), and other genera (1.57%). Among the 94 strains of Candida, 63 (67.02%) belonged to Candida albicans and 14 to Candida tropicalis (14.89%).</p><p><b>CONCLUSION</b>Ocular fungal infection occurs more often in male patients and in the cornea. The common pathogen of ocular fungal infection is Candida, which is different from the etiological characteristics in other areas possibly in association with the tropical climate.</p>

Study on the genome sequence of influenza virus subtype A/H3N2 strains circulated in Zhejiang province during 1998 to 2009 / 中华流行病学杂志

Objective To analyze the relationship between influenza epidemic and genetic characteristic on the whole genome of influenza virus subtype A/H3N2 strains isolated in Zhejiang province during 1998 to 2009. Methods All of the eight genes from the 19 Zhejiang influenza virus isolates, circulated during 1998 to 2009, were amplified by RT-PCR and sequenced. The obtained sequences were aligned and analyzed with the vaccine strains being used in the last 10 years.Results The highest mutation happened within HA and NA genes and the amino acid divergent ratios were 13.98% and 10.00%. Amongst the six internal proteins, the amino acid divergent ratios of NP, M2 and NS1 were 6.43%, 6.19% and 3.48% respectively, and the others were lower than 3%.Other than the HA and NA genes, mutations were also observed on six internal genes of the strains isolated in those years when the influenza virus subtype A/H3N2 was widely circulating.Additionally, there had been an obvious genetic lag between vaccine strains recommended by WHO and the contemporary Zhejiang epidemic strains for many years. Conclusion Besides on HA and NA genes, surveillance programs should also be covered mutations regarding the internal genes of influenza virus subtype A/H3N2 strains, in order to provide important information for forecasting and warning of a new round of influenza epidemic.

Effects of radicular dentin treatments and luting materials on the bond of quartz fiber posts: scanning electron microscope study / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of radicular dentin treatments of sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA) on the regional root canal bonding interface of quartz fiber posts using 2 luting materials with SEM analysis.</p><p><b>METHODS</b>Nine intact maxillary central incisors were sectioned and endodontically treated. Standardized post space preparations and acid etch were performed. All specimens were randomly divided into 3 groups (n = 3). D.T.LIGHT posts were placed into the root canal using one of three radicular dentin treatments (0.9% NaCl for 60 s, 10% NaOCl for 60 s, 17% EDTA for 60 s followed by 5.25% NaOCl for 60 s) in combination of one of two luting materials (DuoLink, LuxaCore) respectively (factorial design). Cervical, middle, apical sections of each teeth are used for SEM study and spectroscopy of dispersion energy (EDS) microanalysis.</p><p><b>RESULTS</b>With the radicular dentin treatment with 10% NaOCl alone or with 17% EDTA followed by 5.25% NaOCl, longer and increased number of penetration of resin tags into the dentinal tubules were observed at the resin-dentin interfaces, and adhesive lateral branches could be found easily. EDS microanalysis showed increase in the infiltration behavior of the luting cement.</p><p><b>CONCLUSIONS</b>Radicular dentin treatments provide good resin infiltration, which can produce a three-dimensional interlocking micronetwork of resin tags in the dentin tubules with multiple lateral branches that penetrate the intertubular dentin, thus positively influence the adhesion between dentin and the luting materials.</p>

Comparative analysis of measles genome between vaccine strain and wild-type strain in Zhejiang province of China / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the distinction between wild-type strain MVi/Zhejiang, CHN/7.05/4 and vaccine strain Shanghai-191 at genome level.</p><p><b>METHODS</b>After sequencing of measles wild-stain MVi/Zhejiang. CHN/7.05/4, the distinction between the wild-type strain and the vaccine strain was analysed by MEGA 3.1 software at genome level, and the antigen variation was studied by means of combining the epidemiological data.</p><p><b>RESULTS</b>There were 822 nucleotide differences (5.17%) and 161 amino acid differences between these two strains, including three glycosylation sites variation found. Meanwhile, the antigen ratio between wild-type strain and vaccine strain was found to be 5.66.</p><p><b>CONCLUSION</b>There should be certain differences between the contemporary wild-type strain MVi/Zhejiang, CHN/7.05/4 and vaccine strain Shanghai-191 at genome level, and the protective effects of measles vaccine should be studied further.</p>

Study on the genetic characteristics of the avian influenza virus strain A/Zhejiang/16/2006 / 中华流行病学杂志

Objective To study the genetic characteristics of avian influenza virus strain A/Zhejiang/16/2006 which was isolated from the case first reported in Zhejiang province.Methods Complete genome of A/Zhejiang/16/2006 including eight segments were sequenced and compared on the genetic homogeneity with sequences of the similar strains provided through domestic and overseas sources.Results There were 11 amino acids showing differences on HA between A/Zhejiang/16/2006 and the H5N1 isolates of neighboring countries,but these differences had not affected the stability of glycosylation sites.In the NA region,20 amino acids located in the 49th to 68th position were found absent in the isolates obtained after 1997.Eight segments of H5N1 isolates,circulating in the mainland of China in the recent years,formed a separate branch compared to the strains in neighboring countries and there was also obviously different from the strains isolated in Hong Kong and Guangzhou in 1996 and 1997.However,several Chinese strains were close to the Hong Kong strains isolated in 1997 but diffferent from the current strains in the phylogenetic tree.Conclusion The influenza virus strain A/Zhejiang/16/2006 formed a separate branch with the strains isolated in the mainland of China in the past years but it presented an obvious difference with the isolates from the neighboring countries.

Molecular characteristics and its evolution of the complete genome of avian influenza H5N1 virus isolated in Zhejiang province from 2002 to 2006 / 中华流行病学杂志

Objective To analyze the molecular characteristics and evolution reassortment of the complete genome of avian influenza H5N1 virus isolated in Zhejiang province in recent years. Methods Complete genomes of avian influenza H5N1 viruses isolated in Zhejiang province from 2002 to 2006 were sequenced. Molecular and evolution reassortment characterization of these virus strains were analyzed by Mega 3.0 bioinformatics software. Results Through study on the HA genes from all these strains,our data revealed that there were multiple basic amino acids at the cleavage site, which was typical for HPAIV.Compared with Gs/Guangdong/1/96,all these strains had a 20 amino acid deletion in the stalk of the NA, except for Dk/Zhejiang/2/02 and Ck/Zhejiang/8/03. Results from phylogenetic analysis showed that from2002 to 2003 the H5N1 viruses belonged to the genotype of B,W,X,Y,Z, and other genotypes were prevailed in corresponding year. However, different gene fragments of several strains belonged to different genotypes. Conclusion Recombinant might be widespread among the poultry in Zhejiang province. The genetic genes of viruses isolated after 2005, the Ck/Zhejiang/24/05 and Zhejiang/16/06 strains were almost all originated from the FJ-like genotype stably.

Genetic analysis on HA1 and NA regions of influenza virus subtype A3 isolates of Zhejiang province during 1998-2005 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the relationship between influenza epidemic and genetic characteristic on HA and NA regions of influenza virus subtype A3 isolates of Zhejiang province in the recent years.</p><p><b>METHODS</b>RNA of 25 influenza virus subtype A3 isolates, circulated in Zhejiang province during 1998 to 2005, was extracted. HA1 and NA regions were amplified and sequenced. All the sequence data were analyzed using BioEdit.</p><p><b>RESULTS</b>HA1 and NA regions of all the isolates belonged to 987nt and 1362nt, encoding protein of 329 and 454 amino acids respectively. Isolates shared amino acid homology of 90.9%-99.3% and 95.2%-99.5% on HA1 and NA regions, while divergence on HA1 was greater than that on NA region. During a period of 8 years, 30 amino acids on HA1 region were substituted and 14 of which refer to 4 antigenic determinant sites. Meanwhile,21 amino acids on NA region were substituted and 5 of which referred to 3 antigenic determinant sites. Significant divergences, both in HA1 and NA, were observed among isolates in 1998 and 2002, showing that they belonged to absolutely different branches. Additionally, influenza virus subtype A3 isolates identified in recent years, with 11 N-linked glyeosylation sites in HA1 region, had 5 sites more than early A/Aichi/2/68 strain. Since 1998,3 sites had been inserted in epidemic strains, indicating the accelerated trend of glyeosylation sites were increasing.</p><p><b>CONCLUSION</b>There is a correlation between antigenic drift of influenza virus subtype A3 and the two epidemics in Zhejiang province in 1998 and 2002.</p>

Analysis on the genetic characteristics of the wide-type measles virus circulating in Zhejiang province in 2005 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the genetic characteristics of measles viruses circulating in Zhejiang province in 2005.</p><p><b>METHODS</b>4 groups of measles viruses isolated in outbreaks and the H and N gene were amplified by reverse transcription polymerase chain reaction (RT-PCR). PCR products were purified, sequenced and data was analyzed.</p><p><b>RESULTS</b>All of the 4 measles isolates belonged to genotype H1 which had been a main genotype containing all of the isolates in China. The isolates shared 99.2% -99.7% identity of amino acid sequence on H and 99.8% identity of amino acid sequence on N gene. When comparing to the China vaccine strain (Shanghai 191), there were 95.2%-95.5% homogeneties and 95.5% homogeneties on H and N gene respectively.</p><p><b>CONCLUSION</b>Data from phylogenic trees of H and N gene revealed that the wide-type measles viruses circulating in Zhejiang province in 2005 all belonged to genotype H1. There were obvious differences on genetic characteristics between the isolates and the genotype A (Shanghai 191).</p>

Multiplex RT-PCR for the rapid detection of influenza virus types and subtypes / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a sensitive and specific multiplex RT-PCR(MRT-PCR) for the simultaneous detection of influenza virus types and subtypes.</p><p><b>METHODS</b>Primers were designed from highly conserved region of the hemagglutinin(HA) gene of influenza H1N1H3N2 and B virus and MRT-PCR was performed. Additional two pairs of primers were designed to determine the N1 and N2 subtypes of neuramidinase NA of influenza H1N1 and H3N2 virus.</p><p><b>RESULTS</b>The fragments of HA gene of all types of influenza virus were amplified and there was no cross reaction. The sensitivity of detection of influenza H1N1 and H3N2 virus was 0.10 TCID50/50 microliter by the second PCR and that was 0.01 TCID50/50 microliter for influenza B virus. The NA gene of influenza H1N1 and H3N2 virus was also amplified by this method.</p><p><b>CONCLUSION</b>The sensitivity of detection of influenza virus from clinical patients' throat washing specimens by MRT-PCR was higher than that by MDCK cell culture or egg embryo isolation. This method was highly sensitive and timely for detection of influenza virus types and subtypes.</p>

Isolation and sequencing of VP1 region of enterovirus 71 strains in Zhejiang, China / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the gene characterization of enterovirus 71 (EV71) virus strains isolated from clinical specimens of children with hand-foot-and-mouth disease (HFMD) in Zhejiang province.</p><p><b>METHODS</b>Virus were isolated from clinical samples including stool, throat swab and vesicle from patients with HFMD. The EV71 isolates were identified by microneutralization assay and reverse transcriptase PCR (RT-PCR) with specific primer pair for VP1 genes of EV71. Complete VP1 gene sequences (891 nucleotides) for recent 6 EV71 isolates were determined and compared with that of A, B, C genotype reference EV71 strains and 11 EV71 China isolates available from GeneBank by homogeneity and phylogenetic tree analyses.</p><p><b>RESULTS</b>9 strains of EV were isolated from 14 clinical specimens. Data from microneutralization and RT-PCR results indicated that all the strains belong to EV71. The nucleotide and amino acid homogeneity of these 6 Zhejiang strains with the representative isolates of A and B genotypes were 82.9%-85.5% and 94.9%-98.0% respectively; with the representative isolates of C were 89.2%-94.1% and 97.0%-99.0% respectively. There were 91.0%-92.2%, 90.2%-90.3%, 89.2%-89.5%, 96.7%-96.9% nucleotide, homology with representative strains of C1, C2, C3,C4 subgenotypes of EV71. The nucleotide homogeneity of these 6 EV71 isolated strains with 9 previously isolated Chinese strains appeared to be 93.8%-97.1%. These 6 EV71 isolated strains were within genotype C subgenogroup C4 in the phylogenetic tree.</p><p><b>CONCLUSION</b>The recently identified EV71 isolates in Zhejiang province belonged to subgenogroup C4.</p>

Analysis of babA2 cagA and vacA genotypes of Helicobacter pylori in chronic gastritis and peptic ulcer / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the distribution of babA2 cagA and vacA genotypes of Helicobacter pylori (Hp) in chronic gastritis and peptic ulcer and to discuss the relationship between babA2, cagA and vacA genotypes of Hp and chronic gastritis and peptic ulcer.</p><p><b>METHODS</b>babA2, cagA genotypes and vacA subtype of 58 Hp strains isolated from patients of Zhejiang province with chronic gastritis or peptic ulcer were tested by polymerase chain reaction.</p><p><b>RESULTS</b>The positive rates of babA2, cagA, vacAs1a, vacA m1 and vacA m2 of 58 Hp strains were 87.9%, 100%, 93.1%, 1.7% and 65.5%, respectively. There were no significant differences between the positive rates of babA2, vacA s1a and vacA m2 genes of Hp strains isolated from patients with chronic gastritis and peptic ulcer.</p><p><b>CONCLUSION</b>The genotypes of Hp isolated from patients of Zhejiang province were predominatly babA2 positive, cagA positive and vacA s1a/m2. The relationships between babA2, cagA and vacA genotypes of Hp and chronic gastritis and peptic ulcer can not be identified.</p>