Selection Pressure on Haemagglutinin Genes of H9N2 Influenza Viruses from Different Hosts

Positive selection and differential selective pressure analyses were carried out to study Haemagglutinin (HA) genes of H9N2 influenza viruses from different hosts in this paper. Results showed that, although most positions in HAs were under neutral or purifying evolution, a few positions located in the antigenic regions and receptor binding sites were subject to positive selection and some of them were even positively selected at the population level. In addition, there were always some positions differentially selected for viruses from different hosts. Both selection pressure working on HA codons and positions differentially selected might account for the extension of the host range and adaptations to different hosts of H9N2 influenza viruses.

Cloning, Characterization and Chromosome Localization of Two Powdery Mildew Resistance-Related Gene Sequences from Wheat

Reverse_transcription Polymerase Chain Reaction (RT_PCR) was performed using cDNAs as templates from wheat_ Haynaldia villosa 6VS/6AL translocation line and 'Yangmai 5' induced with fungus Erysiphe graminis , and degenerate primers designed based on the conserved amino acid sequences of known plant disease_resistance genes. The cDNA sequences encoding cyclophilin_like and H +_ATPase_like genes were first isolated and characterized in wheat. The putative amino acid sequences of the two clones showed that they were highly homologous to those of cyclophilin proteins and H +_ATPases isolated from other plants. Thus they were designated as Ta_Cyp and Ta_MAH . The obvious expression differences could be observed between wheat_ H. villosa 6VS/6AL translocation line and susceptible wheat cultivar 'Yangmai 5', implying that the two genes may be related with the resistance of wheat_ H. villosa 6VS/6AL translocation line to disease. Southern blot indicated that the wheat genome contained 2-3 copies of Ta_Cyp gene and one copy of the Ta_MAH gene. Chinese Spring nulli_tetrasomic line analysis located the Ta_Cyp homologous genes on wheat chromosome 6A, 6B and 6D. Southern blot using Ta_Cyp clone as a probe showed that the polymorphic bands existed among the H. villosa , amphiploid of Triticum durum _ H. villosa , wheat_ H. villosa 6VS/6AL translocation line and 'Yangmai 5', suggesting that Ta_Cyp homologies exist in wheat genome as well as on the short arm of chromosome 6V in H. villosa .

Construction of Recombinant Pseudorabies Virus Expressing Canine Distemper Virus H Gene and Analysis on Its Biological Characters

[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and investigate its biological characters.[Method] H gene of canine distemper virus（CDV）strain Onderstepoort was produced by RT-PCR,inserted into pcDNA3.1（＋）vector to construct a expression cassette,which was then subcloned into transfer vector p8AA,prior to the insertion of LacZ expression cassette.The resulting new transfer vector was named as p8AAZH.Subsequently,p8AAZH was co-transfected with the genome of pseudorabies virus（PRV）Bartha-K61 into BHK-21 cells to enable gene recombination and virus package,and the virus solution was collected as cytopathic effect occurring.A series of procedures including blue plaque purification,PCR identification,observation under electron microscope and Western blot were carried out to screen the recombinant pseudorabies virus and identify the protein expression of target gene.Meanwhile,growth curve of the recombinant virus was determined in BHK-21 cells.[Result] The H gene had been inserted into the genome of Bartha-K61 strain,and RPRV-H was the same as Bartha-K61 in the one-step growth curve and cytopathic effect in BHK-21 cells.[Conclusion] The recombinant pseudorabies virus was constructed,and the insertion of H gene did not influence proliferation of recombinant virus,which laid a foundation for development of recombinant canine distemper virus vaccine.

Construction and Identification of a Goat Pox Virus Transfer Vector to Express Peste des Petits Ruminants H gene

[Objective] This study was to develop a live vector vaccine of goat pox virus of Peste des petits ruminants(PPR). [Method] Using PCR amplification technique, PPR H gene was obtained, then ligated into pGEM-T easy vector; the recombinants were digested by Nhe Ⅰ and Hind Ⅲ, and ligated into pEGFP-N1-P7.5, yielding the recombinant vector pEGFP-N1-P7.5-H; next the expression cassette EGFP-N1-P7.5-H was first released from recombinant vector pEGFP-N1-P7.5-H by double digestion of Hind Ⅲ and Nhe Ⅰ and ligated into pUC119-TK that was digested by Kpn Ⅰ, yielding the transfer vector pUC119-TK-EGFP-P7.5-H. [Result] Identification and double enzyme digestion showed that the transfer vector pUC119-TK-EGFP-P7.5-H was correctly constructed. From the transfer vector transfected BHK-21 cells which infected GTPV AV41, specific fluorescence was observed at 48th h of transfection. [Conclusion] The construction of goat poxvirus live vector laid a foundation for the live vector vaccine of PPR vaccine.

Screening an Na^+/H^+ Antiporter Gene from the Halophiles Colonizing in the Dagong Ancient Brine Well of Zigong City,China

[Objective] This study aimed to screen an Na＋/H＋ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City, China, and then analyze the gene structure and properties of the protein encoded by this gene. [Method] Metagenomic DNA libraries of halophiles from the Dagong Ancient Brine Well were used for screening genes with Na＋/H＋ antiporter activity in antiporter-defi- cient E. coil KNabc strain by functional complementation. Then the start codon, stop codon, ORF, -35 region, -10 region and SD sequence of Na~/H＋ antiporter gene, as well as the molecular weight, isoelectric point, hydrophobic region, transmembrane domain, phyletic evolution and salt resistance of protein encoded by the gene were investigated. [Result] A new Na＋/H＋ antiporter gene m-nha was obtained, which ,ren- dered the antiporter-negative mutant E. coil KNabc cells with both the resistance to Na＋ and the ability to grow under alkaline conditions. [Conclusion] The structure and amino acid sequence of M-Nha was different from the previously reported Na＋/H~ antiporters, and the m-nha gene disclosed from the Dagong Ancient Brine Well was identified as a novel Na＋/H＋ antiporter gene. This study was significant not only in helping us understand the salt tolerance of halophiles in ancient brine wells and develop and utilize the genes resource, but also in exploring new salt-tolerant genes.

Sequence Analysis of HA Genes from Three H9N2 Subtype Avian Influenza Viruses

[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% （ Dk/HK/Y439/97 ） -98.0% （ Ck/GX17/00 ）, 85.1% （Dk/HK/Y439/97） - 99.1% （ Ck/GXl 7/00）, 90.7% （ Ck/BJ/3/01 ） - 99.1% （Ck/GX17/00） with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L （ Leu）, suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin.

Sequence Comparison and Analysis of HA Gene of Four H9N2 Avian Influenza Virus Isolates

[ Objective] To determine the HA gene sequences of four H9N2 Avian influenza virus （AIV） strains and carry out comparative analysis so as to understand the difference and variation pattern of each strain from the angle of molecular biology and to know the distribution and epidemic law of H9N2 AIV. [Method] One pair of primers was designed referring to HA gene sequences of H9N2 AIV. The HA genes of A/Chicken/Hebei/WD/98 （H9N2; WD98 for short）, A/Chicken/Hebei/ZD/04 （H9N2; ZD04 for short））, A/Chicken/Beijing/MY/06 （H9N2; MY06 for short） ）, and A/Chicken/Beijing/PG/08 （H9N2; PG08 for short）） were amplified, cloned and sequenced. Then the HA gene sequences of these strains were compared with that of 10 H9N2 AIV stains in GenBank. [Result] The ORF of HA genes of the four strains was 1 683 bp in size, encoding 516 amino acids. The HA gene sequences of the four strains, WD98, MY06, PG08, and ZD04, were 82.6% -95.1%, 83.0% -99.0%, 82.7% -95.5%, and 81.3% -95.7% homologous to that of the 10 H9N2 AIV stains, respectively. And the homology of amino acid was respectively 86.6% -96.3%, 86.6% -97.9%, 87.0% -97.1%, and 86.9% -97.3%. [ Conclusion] The HA gene has greatly high homology among different strains.

Cloning and Phylogenetic Analysis of NS1 Genes from Different Isolates of H9N2 Subtype Duck Influenza Virus

[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007（H9N2） （A3 for short）, A/Duck/FJ/301/2007 （H9N2） （C1 for short）, A/Duck/NH/306/2007（H9N2） （ D6 for short）, A/duck/SS/402/2007（H9N2） （ E2 for short）, and a strain named A/duck/ZC/2007（H9N2） （L1 for short） from a muscovy duck died of avian influenza virus （AIV）, were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 （chicken, 2003）. By comparison with the NS1 gene sequences of L1, AF523514 （duck）, AY664743 （chicken） and EF155262.1 （quail） using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 （ R→Q）, 70, 71 （ KE→EG）, 86 （ A→S）, 124 （V→M） and 225 （ S→N）, and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase （PKR） binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.

Sequence Analysis of Type III Effector tccP and tccP2 Genes in Escherichia coli O157:H7 from Chinese Water-chestnut

[Objective] This study aimed to analyze the type III effector tccP and tccP2 genes in Escherichia coli O157：H7 from Chinese water-chestnut. [Method] Gene-specific and locus-specific primers were utilized to amplify tccP/tccP2 and their flanking regions for sequence analysis. [Result] E. coli O157：H7 CWN11 harbored intact tccP and tccP2 genes, however, the number of proline-rich repeats in tccP gene was only one that probably resulted in biological incapability, whereas, the tccP2 gene consisted of five and half proline-rich repeats and could encode functional protein. [Conclusion] Here, we reported the first sequence of tccP gene that consisted of only one proline-rich repeat and tccP2 was assumed to play a crucial role in colonization and subsequent signaling cascades.

Cloning and Sequence of Glycoprotein H Gene of Duck Plague Virus

The glycoprotein H （gH） gene homologue of duck plague virus （DPV） was cloned by degenerate polymerase chain reaction （PCR） and sequenced. It was located immediately downstream from the thymidine kinase gene （TK）. In addition, the 3＇-end of the gene homologue to herpesvirus UL21 was located downstream from the gH gene. DPV gH gene open reading frame （ORF） was 2 505 bp in length and its primary translation product was a polypeptide of 834 amino acids long. It possessed several characteristics of membrane glycoproteins, including an N-terminal hydrophobic signal sequence, an external domain containing eight putative N-linked glycosylation sites, a C-terminal transmembrane domain, and a charged cytoplasmic tail. Comparison with other herpesvirus revealed identities of 20.2, 25.1, 23.0, 23.0, 26.5 and 26.0% with the gH counterparts of the human herpesvirus virus 1 （HSV1）, equine herpesvirus 4 （EHV4）, bovine herpesvirus 1 （BHV1）, pseudorabies virus （PRV）, gallid herpesvirus 2 （GHV2） and gallid herpesvirus 3 （GHV3）, respectively.

Research of AtNHX1 Gene Transformation in Brassica napus L. by Agrobacterium tumefaciens

[Objective] The aim was to investigate AtNHX1 gene transformation in Brassica napus L. mediated by Agrobacterium tumefaciens. [Method] By using Agrobacterium-mediated method and cre/lox plant expression vector,the transformation of AtNHX1 gene of Na＋/H＋ antiporter in Brassica napus was studied. [Result] The regeneration rate of cotyledon with petiole was much higher than that of hypocotyl,thus,the cotyledon with petiole was selected as the recipient for transformation. After the cotyledon with petiole was soaked in bacterial solution （OD600=0.4） for 8-10 min,kanamycin-resistant green seeding percentage could reach 3.75%. [Conclusion] The PCR detection of kanamycin-resistant plants proved that NHX1 gene had been inserted into Brassica napus genome. And this research could provide a new way to improve the salt tolerance of Brassica napus.

Sequence and phylogenetic analysis of hemagglutinin genes of H9N2 influenza viruses isolated from chicken in China from 2013 to 2015

H9N2 avian influenza virus（AIV） infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin（HA） gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.

Screening for metronidazole-resistance associated gene fragments of H pylori by suppression subtractive hybridization

AIM： To screen for metronidazole （MTZ）-resistance associated gene fragments of H pylori by suppression subtractive hybridization （SSH）. METHODS： Five MTZ-resistant （tester, T） and 1 MTZ- susceptible （driver, D） clinical H pylori isolates were selected. Genomic DNAs were prepared and submitted to Rsa I digestion. Then two different adaptors were ligated respectively to the 5＇-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNAs. The specific inserts of tester strains were screened and MTZ-resistance related gene fragments were identified by dot blotting. RESULTS： Among the randomly selected 120 subtractive colonies, 37 DNA fragments had a different number of DNA copies （≥ 2 times） in resistant and susceptible strains and 17 of them had a significantly different number of DNA copies （≥ 3 times）. Among the sequences obtained from the 17 DNA fragments, new sequences were found in 10 DNA fragments and duplicated sequences in 7 DNA fragments, representing respectively the sequences of depeptide ABC transporter periplasmic dipeptide-binding protein （dppA）, permease protein （dppB）, ribosomal protein S4 （rps4）, ribonuclease Ⅲ （rnc）, protease （pqqE）, diaminopimelate epimerase （dapF）, acetatekinase （ackA）, H pylori plasmid pHP51 and Hpylori gene 1334. CONCLUSION： Gene fragments specific to MTZ-resistant H pylori strains can be screened by SSH and may be associated with MTZ-resistant Hpylori.

Frequency of primary iron overload and HFE gene mutations (C282Y,H63D and S65C) in chronic liver disease patients in north India

AIM:To identify the frequency of iron overload and study the three mutations in the HFE gene (C282Y,H63D,and S65C) in patients with chronic liver disorders (CLD) and controls. METHODS:To identify patients with iron overload (transferrin saturation > 45% in females and > 50% in males and serum ferritin > 1000 ng/mL) we evaluated 236 patients with CLD,including 59 with non-alcoholic steatohepatitis (NASH),22 with alcoholic liver disease (ALD),19 of cirrhosis due to viruses (HBV,HCV),and 136 with cryptogenic cirrhosis. Mutations of the HFE gene were analyzed by PCR-RE. hundred controls were screened for iron status and the mutations. RESULTS:Seventeen patients with CLD showed evidence of iron overload. Fifteen cases of iron overload had cryptogenic cirrhosis and two had ALD. None of the controls showed iron overload. We did not find any individual with 282Y or 65C either in the cases or in the controls. The prevalence of H63D heterozygosity was 12% in normal individuals,14.8% in 236 patients (16.9% in NASH,13.6% in ALD,26.3% in viral and 12.5% in cryptogenic cirrhosis) and the overall prevalence was 13.98%. Only two of the 17 patients with primary iron overload were heterozygous for H63D. One patient with NASH and one normal individual who were homozygous for H63D showed no iron overload.CONCLUSION:Primary iron overload in Indians is nonHFE type,which is different from that in Europeans and further molecular studies are required to determine the defect in various iron regulatory genes.

Reference Gene Selection for Normalization of PCR Analysis in Chicken Embryo Fibroblast Infected with H5N1 AIV

Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.

Expression analysis of a Na^+/H^+ antiporter gene PeNHX1 from Populus euphratica

Na+/H+ antiporters play an important role in the salt tolerance of a wide variety of plants.Using the rapid amplification of cDNA ends method,a Na+/H+ antiporter gene (PeNHX1) was isolated from Populus euphratica.The deduced amino acid sequence contained 528 amino acid residues with a conserved amiloride-binding domain (77LFFIYLLPPI86) and shared more than 68% identity with that of AtNHX1 from Arabidopsis thaliana.PeNHX1 can confer resistance to Na+,as well as Li+,to (EP432) an Escherichia coli strain deficient in both nhaA and nhaB,thus proving that it is a functional Na+/H+ antiporter.PeNHX1 expression profile in EP432 reflected pH independent manner.PeNHX1 expression was regulated by salt at the transcriptional level.Meanwhile,results demonstrated that transcripts of PeNHX1 in P.euphratica calli showed a salt dependent response,and thus provide a valuable tool for studying signaling and biochemical pathways involved in salt recognition and response in P.euphratica.

Prevalence of shiga toxins(stx_1,stx_2),eaeA and hly genes of Escherichia coli O157:H7 strains among children with acute gastroenteritis in southern of Iran

Objective:To survey the prevalence severe diarrhea arising from these bacteria in children under 5 years old in Marvdasht.Methods:In this study faecal sample from 615 children aged <5years old who were hospitalized lor gastroenteritis in Fars hospitals in Iran were collected and then enriched in Escherichia coli(E.coli) broth and modified tryplone soy broth with novobiocin media,fermentation of sorbitol,lactose and β— glucoronidase activity of isolated strains was examined by CT—SMAC,VRBA and chromogenic media respectively.Then isolation of E.coli O157:H7 have been confirmed with the use of specific antisera and with multiplex PCR method presence of virulence genes including:xtx_1.stx_2,eae.A.hly has been analyzed.Results:E.coli O157:H7 was detected in 7(1.14%) stool specimens.A significanl difference was seen between detection rale of isolated bacteria from age groups 18-23 months and other age groups(P=0.004).Out of considered virulence genes.only 1 of the isolated strains(0.16%)he stx,and eaeA genes were seen and also all isolated hacleria had resistance to penicillin,ampicillin and erythromycin antibiotics.Conclusions:We found thai children < 2 years of age were at highest risk of infection with E.coli O157:H7.Regarding severity of E.coli O157:H7 pathogenesis,low infectious dose and lack of routine assay for detection ol these bacleria in clinical laboratory,further and completed studies on diagnosis and genolyping of this E.coli O157:H7 strain has been recommended.

Host markers and correlated mutations in the overlapping genes of influenza viruses: M1, M2;NS1, NS2;and PB1, PB1-F2

The influenza A viruses have three gene segments, M, NS, and PB1, which code for more than one protein. The overlapping genes from the same segment entail their interdependence, which could be reflected in the evolutionary constraints, host distinction, and co-mutations of influenza. Most previous studies of overlapping genes focused on their unique evolutionary constraints, and very little was achieved to assess the potential impact of the overlap on other biological aspects of influenza. In this study, our aim was to explore the mutual dependence in host differentiation and co-mutations in M, NS, and PB1 of avian, human, 2009 H1N1, and swine viruses, with Random Forests, information entropy, and mutual information. The host markers and highly co-mutated individual sites and site pairs (P values < 0.035) in the three gene segments were identified with their relative significance between the overlapping genes calculated. Further, Random Forests predicted that among the three stop codons in the current PB1-F2 gene of 2009 H1N1, the significance of a mutation at these sites for host differentiation was, in order from most to least, that at 12, 58, and 88, i.e., the closer to the start of the gene the more important the mutation was. Finally, our sequence analysis surprisingly revealed that the full-length PB1-F2, if the three stop codons were all mutated, would function more as a swine protein than a human protein, although the PB1 of 2009 H1N1 was derived from human H3N2.

High frequency of the c.3207C>A (p.H1069Q) mutation in ATP7B gene of Lithuanian patients with hepatic presentation of Wilson's disease

AIM： To investigate the prevalence of the ATP7B gene mutation in patients with hepatic presentation of Wilson＇s disease （WD） in Lithuania. METHODS： Eleven unrelated Lithuanian families, including 13 WD patients were tested. Clinically WD diagnosis was established in accordance to the Leipzig scoring system. Genomic DNA was extracted from whole venous blood using a salt precipitation method. Firstly, the semi-nested polymerase chain reaction （PCR） technique was used to detect the c.3207C〉A （p.H1069Q） mutation. Patients not homozygous for the c.3207C〉A （p.H1069Q） mutation were further analyzed. The 21 exons of the WD gene were amplified in a thermal cycler （Biometra T3 Thermocycler, G0ttingen, Germany）. Direct sequencing of the amplified PCR products was performed by cycle sequencing using fluorescent dye terminators in an automatic sequencer （Applied Biosystems, Darmstadt, Germany）. RESULTS： Total of 13 WD patients （mean age 26.4 years; range 17-40; male/female 3/10） presented with hepatic disorders and 16 their first degree relatives （including 12 siblings） were studied. Some of WD patients, in addition to hepatic symptoms, have had extrahepatic disorders （hemolytic anemia 3; Fanconi syndrome 1; neurophsychiatric and behavioural disorder 2）. Liver biopsy specimens were available in all of 13 WD patients （8 had cirrhosis; 1-chronic hepatitis; 3-acute liver failure, 1-1iver steatosis）. Twelve of 13 （92.3%） WD patients had the c.3207C〉A （p.HI069Q） mutation, 6 of them in both chromosomes, 6 were presented as compound heterozygotes with additional c.3472-82delGGTTTAACCAT, c.3402delC, c.3121C〉T （p.RI041W） or unknown mutations. For one patient with liver cirrhosis and psychiatric disorder （Leipzig score 6）, no mutations were found. Out of 16 first degree WD relatives, 11 （68.7%） were heterozygous for the c.3207C〉A （p.H1069Q） mutation. Two patients with fulminant WD died from acute liver failure and ii are in full remission under peniciilamine or zinc acetate treatment. Three women with WD successfully delivered healthy babies. CONCLUSION： The c.3207C〉A （p.HI069Q） missense mutation is the most characteristic mutation for Lithuanian patients with WD. Even 92.3% of WD patients with hepatic presentation of the disease are homozygous or compound heterozygotes for the p.H1069Q mutation.

Restriction fragment length polymorphism of adhesin gene hpaA from different Helicobacter pylori strains of Chongqing, China

AIM:To assess the variability of adhesin gene hpaA between different Helicobacter pylori (H pylori) strains with PCR-restriction fragment length polymorphism (RFLP). METHODS: Twelve different H pylori strains were chosen to amplify the 710-bp segments of gene hpaA. These strains were NCTC11637, SS1; Chongqing clinical isolates CCS9801, CCS9802, CCS9803, CCS9806, CCS9809, CCS9810, CCS9813, which were gained from patients of gastritis; Mongolia gerbil adapted H pylori strains (abbreviation MG), which were gained from the following steps: gastric mucosal specimens of Mongolia gerbils infected by clinical isolate CCS9803 were cultured and detected, the positive H pylori strains were named as the first generation of Mongolia gerbil adapted H pylori strains (abbreviation MG1) and then were subcultured with healthy Mongolia gerbil to generate MG2, in turn to gain the ninth generation (abbreviation MG9). All hpaA segments, obtained from 12 different H pylori strains, were digested by Hhal and HaeIll individually and analyzed by agarose gel electrophoresis. RESULTS: In all 12 strains, the 710-bp PCR products were successfully amplified and products were cloned to pMD18-T vector respectively, then the recombinant plasmids were digested simultaneously with Ncol and Xhol to recover the small fragments. The objective fragments from 12 different H pylori strains digested with Haelll could be seen as 4 types of bands and 5 types with Hha I. According to the hpaA RFLP patterns, the 12 H pylori strains could be divided into 5 groups: group I, NCTC11637 and SS1; group II, CCS9809, which RFLP type digested with HaeIll was the same as strains of group I, but Hhal RFLP showed difference compared with the other groups; group III, CCS9810; group IV, CCS9803; group V: CCS9801, CCS9802, CCS9806, CCS9813, MGl, MG3 and MG9. The sequence data of 12 hpaA segments were analyzed by DNAsis software and it was observed that: (1) The homologies of base pair and amino acid sequence between strains NCTC11637, SS1, CCS9809 were 99.6% and 98.9%, respectively; (2) The homology of base pair and amino acid sequence between CCS9803 and CCS9810 was 97.7% and 99.1%; (3) That of the rest strains, CCS9801, CCS9802, CCS9806, CCS9813, MGl, MG3, MG9 reached 99.4% and 98.4%; (4) The base pair homologies between all hpaA fragments of different sources were higher than 94.6%, therefore the correspondence of deduced amino acid sequence was higher than 96.8% between each other. CONCLUSION: The gene hpaA from different H pylori strains revealed variation, and this might provide an effective method for molecular epidemiological survey of H pylori.