Identification of 5' capped structure and 3' terminal sequence of hepatitis E virus isolated from Morocco

AIM:To examine 5' and 3' terminal sequences of hepatitisE virus (HEV) isolated from Morocco,to confirm 5' methylatedcap structure of the genome,and to investigate whetherthe 3' UTR can be used to distinguish HEV genotypes insteadof HEV complete genome sequence.METHODS:RNA ligase-mediated rapid amplification ofcDNA ends (RLM-RACE) was employed to obtain the 5' and3' terminal sequences of HEV Morocco strain.The 3' UTRsequence of the Morocco strain was compared with that ofthe other 29 HEV strains using the DNAStar software.RESULTS:The 5' PCR product was obtained only from theRLM-RACE based on the capped RNA template.The 5' UTRof the Morocco strain had 26 nucleotides,and the 3' UTRhad 65 nucleotides upstream to the polyA.The 5' UTRbetween HEV strains had only point mutations of nucleotides.The phylogenetic tree based on the sequences of 3' UTRwas not the same as that based on the complete sequences.CONCLUSION:The genome of HEV Morocco strain wasmethylated cap structure.The 3' terminal sequence can notbe used for distinguishing HEV genotype for all HEV strainsin place of the whole HEV genome sequence.     

A new subtype of 3′ region of cagA gene in Helicobacter pyloristrains isolated from Zhejiang Province in China

AIM: To isolate the subtypes of 3′ region of cagA gene in Helicobacter pylori (H pylon) strains from Zhejiang Province in China and to investigate their relations to H pylori-associated gastroduodenal diseases. METHODS: One hundred and thirty-seven H pylori clinical strains were isolated from the gastric mucosa specimens of 74 patients with chronic gastritis, 61 with peptic ulceration, and 2 with gastric cancer. Bacterial genomic DNA was extracted and 3′ region of cagA gene was amplified by polymerase chain reaction (PCR). Subtypes of 3′ region of cagA gene were determined by the size of PCR amplified segments. The sequences of the subtypes were analyzed by PCR-based sequencing. RESULTS: Of the 137 H pyloriisolates from Zhejiang Province, 132 (96.4%) yielded PCR products that could be classified into three groups of subtypes, named as subtypes Ⅰ, Ⅱ, and Ⅲ according to their sizes. The sizes of subtypes Ⅰ, Ⅱ, and Ⅲ were 648-650bp, 705-707bp, and 815bp, respectively. Among the 132 cagA-positive H pylori strains, 123(93.2%) belonged to the group of subtype Ⅰ, 6 (4.5%) presented subtype Ⅱ, 1(0.8%) was subtype Ⅲ, and 2(1.5%) presented subtypes Ⅰ and Ⅲ both. The primary structure of subtype Ⅰ was composed of 3 repeats of R1, 1 repeat of R2 and 1 repeat of R3. Subtype Ⅱ possessing 4 repeats of R1, 2 repeats of R2 and 1 repeat of R3 was a newly found type of 3′ region of cagA gene which had not been reported before. The primary structure of subtype Ⅲ consisted of 4 repeats of R1, Ⅰ repeat of R2 and 2 repeats of R3. Comparison of the sequences of subtype Ⅰ strains with the corresponding sequences deposited in GenBank, showed a similarity of 95.0% (94.0-96.1%) for nucleotide sequences and 95.9% (94.9-97.4%) for deduced amino acid sequences. Comparison of the sequences of subtype Ⅲ strains with the corresponding sequences deposited in GenBank, showed a similarity of 93.9% (90.8-96.9%) for nucleotide sequences and 93.2% (90.2-96.2%) for deduced amino acid sequences. Among subtype Ⅱ strains, the nucleotide and deduced amino acid sequences showed a similarity of 95.2% (94.1-96.5%) and 96.4% (93.8-97.9%),respectively. There were no statistical differences in the distribution of subtypes of 3′ region of cagA gene among different H pylori-associated gastroduodenal diseases (x^2=11.544, P>0.05). CONCLUSION: There are three subtypes (Ⅰ,Ⅱ, and Ⅲ) of 3′ region of cagA gene in Hpylori strains isolated from Zhejiang Province, and subtype Ⅰ is predominant. Subtype Ⅱ is a newly found subtype of 3′ region of cagA gene. The result of this study does not support the view that the subtypes of 3′ region of cagA gene in H pylori isolated from Zhejiang Province are correlated with the clinical outcomes of H pylori infection.     

Identification of H. pylori strain specific DNA sequences between two clinical isolates from NUD and gastric ulcer by SSH

AIM: The genomes of Helicobacter pylori (H. pylon) from different individuals are different. This project was to identify the strain specific DNA sequences between two clinical H. pylori isolates by suppression subtractive hybridization (SSH).METHODS: Two clinical H. pyloriisolates, one from gastric ulcer (GU, tester) and the other from non-ulcer dyspepsia (NUD, driver), were cultured and the genomic DNA was prepared and submitted to A/uI 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 DNA. The un-hybridized tester DNA sequences were amplified by two sequential PCR and cloned into pGEM-T-Easy Vector. The tester strain specific inserts were screened and disease related DNA sequences were identified by dot blotting.RESULTS: Among the 240 colonies randomly chosen, 50 contained the tester strain specific DNA sequences. Twenty three inserts were sequenced and the sizes ranged from 261 bp to 1 036 bp. Fifteen inserts belonged to the H.pylorl plasmid pHPO100 that is about 3.5 kb and codes a replication protein A. Other inserts had patches of homologous to the genes of H.py/or/in GenBank. Various patterns of dot blots were given and no GU strain unique DNA sequences were found when 4 inserts were used as probes to screen the genomic DNA from 27 clinical isolates, 8 from GU, 12 from duodenum ulcer (DU), 4 from GU-DU, 2 from NUD and 1 from gastric cancer (GO. But a 670 bp DNA fragment (GU198) that was a bit homologous to the 3‘-end of the gene of thymidylate kinase was positive in 7 GU strains (7/8), 3 GU-DU strains (3/4) and 3 DU strains (3/12). A 384 bp fragment (GU79) of the replication gene A (repA) was positive only in 4 H.pylori isolates, 2 from GU and 2 from GU-DU.CONCLUSION: Differences exist in the genes of different H.pylori isolates. SSH is very effective to screen H.pylori strain specific DNA sequences between two clinical isolates,and some of these sequences may have clinical significance.     

左氧氟沙星体外诱导马耳他布鲁杆菌耐药后gyrA基因的变异

Objective To study on the drug-resistance mechanism of Brucella resistance to Quinolone antibiotics to guide the selection and use of antimicrobial agents in clinical practice. Methods Six strains of Brucella melitensis(Bru1, Bru2, Bru3, Bru4, Bru5, Bru6) were selected to be induced resistance to levofloxacin in vitro respectively. The MICs of the 6 strains of Brucella melitensis and induced resistant strains were measured by agar dilution method. The sensitivity to Quinolone antibiotics (Levofloxacin, Ciprofloxacin, Lomefloxacin, Norfloxacin, Fleroxacin, Ofloxaein) of 6 strains of Brucella melitensis and induced resistant strains was measured by K-B method. The gyrA of the 6 strains of Brucella melitensis and induced resistant strains was amplified by PCR, then the nucleotide sequence of the genes were analyzed. Results The MICs of Bru1,Bru2,Bru3,Bru4, Bru6 were 0.50 μg/ml and Bru5 was 0.25 μg/ml. The strains Bin3, Bru4 were induced into drug-resistant strains by Levofloxacin, then were named LEVR3 and LEVR4 respectively. The MICs of LEVR3 and LEVR4 were 64,128 μg/ml with 128 and 256 times higher than that of the parental strains. The 6 strains of Brucella melitensis were sensitive to Quinolone antibiotics, LEVR3 and LEVR4 were resistant to Quinolone antibiotics. Neucleotide sequence analysis and comparison of the derived amino acid sequence revealed that Quinolone resistance-determing region of GyrA had a substitution at position Ala87 and Asp91 in laboratory resistant strains. Conclusion The results of in vitro experiments show that acquired resistance of Brucella melitensis strains to Levofloxacin could beinduced when exposed to high level of some antibacterial agents for short term. Two drug-resistant strains occur mutations in gyrA and have cross-resistance to other Quinolones.     

Correlation between rpoB gene mutation in Mycobacterium avium subspecies paratubercu/osis and clinical rifabutin and rifampicin resistance for treatment of Crohn＇s disease

AIM： To investigate overlapping regions of the rpoB gene previously involved with rifamycin resistance in M. tuberculosis and seek correlation between rpoB mutations in dinical MAP strains with susceptibility to RIF and RFB.
METHODS： We designed a molecular-based PCR method for the evaluation of rifabutin （RFB） and rifampicin （RIF） resistance based on probable determinant regions within the rpoB gene of MAP, including the 81 bp variable site located between nucleotides 1363 and 1443. The minimum inhibitory concentration （MIC） for RIF was also determined against 11 MAP isolates in attempt to seek correlation with rpoB sequences.
RESULTS： We determined that MAP strain 18 had an MIC of 〉 30 mg/L and ≤ 5 mg/L for RIF and RFB respectively, and a significant and novel rpoB mutation C1367T, compared to an MIC of ≤ 1.0 mg/L for both drugs in the wild type MAP. The 30-fold increase in the MIC was a direct result of the rpoB mutation C1367T, which caused an amino acid change Thr456 to Ile456 in the drug＇s binding site. In addition, MAP strain 185 contained five silent rpoB mutations and exhibited an MIC comparable to the wild-type. Moreover, our in vitro selected mutation in MAP strain UCF5 resulted in the generation of a new resistant strain （UCF5-RIF16r） that possessed T1442C rpoB mutation and an MIC 〉 30 mg/L and 〉 10 mg/L for RIF and RFB respectively. Sequencing of the entire rpoB gene in MAP strains UCF4, 18, and UCF5-RIF16r revealed an rpoB mutation A2284C further downstream of the 81 bp variable region in UCF4, accounting for observed slight increase in MIC. In addition, no other significant mutations were found in strains 18 and UCF-RIF16r.
CONCLUSION： The data clearly illustrates that clinical and in vitro-selected MAP mutants with rpoB mutations result in resistance to RIF and RFB, and that a single amino acid change in the beta subunit may have a significant impact on RIF resistance. Unconventional drug susceptibility testing such as our molecular approach will be benefi     

Predicting a novel pathogenicity island in Helicobacter pylori by genomic barcoding

AIM:To apply a new,integrated technique for visualizing bacterial genomes to identify novel pathogenicity islands in Helicobacter pylori(H.pylori).METHODS:A genomic barcode imaging method(converting frequency matrices to grey-scale levels)was designed to visually distinguish origin-specific genomic regions in H.pylori.The complete genome sequences of the six H.pylori strains published in the National Center for Biotechnological Information prokaryotic genome database were scanned,and compared to the genome barcodes of Escherichia coli(E.coli)O157:H7 strain EDL933 and a random nucleotide sequence.The following criteria were applied to identify potential pathogenicity islands(PAIs):(1)barcode distance distinct from that of the general background;(2)length greater than 10000 continuous base pairs;and(3)containing genes with known virulence-related functions(as determined by PfamScan and Blast2GO).RESULTS:Comparison of the barcode images generated for the 26695,HPAG1,J99,Shi470,G27 and P12 H.pylori genomes with those for the E.coli and random sequence controls revealed that H.pylori genomes contained fewer anomalous regions.Among the H.pylorispecific continuous anomalous regions(longer than 20 kbp in each strain’s genome),two fit the criteria for identifying candidate PAIs.The bioinformatic-based functional analyses revealed that one of the two anomalous regions was the known pathogenicity island cag PAI,this finding also served as proof-of-principle for the utility of the genomic barcoding approach for identifying PAIs,and characterized the other as a novel PAI,which was designated as tfs3-PAI.Furthermore,the cag-PAI and tfs3-PAI harbored genes encoding type IV secretion system proteins and were predicted to have potential for functional synergy.CONCLUSION:Genomic barcode imaging represents an effective bioinformatic-based approach for scanning bacterial genomes,such as H.pylori,to identify candidate PAIs.     

Mutational characteristics in consecutive passage of rapidly replicating variants of hepatitis A virus strain H2 during cell culture adaptation

AIM：To investigate the molecular mechanism of cell adaptation and rapid replication of hepatitis A virus strain H2 in KBM17cells.METHODS:Virus of strain H2at passage7was consecutively passaged in KBM17cells for 22passages,every passage was incubated for 14days,Antigenic and infectious titers of every passage and one-step growth dynamics of passage22were determined with ELISA.Genomes of passage6.passage12,passage18and passage22were sequenced and compared withK2K7,RESULTS:During continuous passage of vaccine strain H2at passageK7 in KMB17cells,infectious and antigenic titers increased with the increase of passages,infectious titers at day 14reached6.77LgCCID50ml^-1for passage6(P6).7.0LgCCID50ml^-1for passage12(P12),7.33LgCCID50ml^-1for passage18(P18)and7.83LgCCID50ml^-1for passage 22(P22).respectively.The one-step growth dynamics showed that replicating peak of P22 appeared at day 14with infectious titers of 7.83LgCCID50ml^-1and antigenic titer of 1:1024,After passage22a new cell-adapted variant(P22)of H2K7 with rapid and shoirtened replication cycle from28days to 14days was obtained.Sequencing and comparison of genomes of P6,P12,P18andP22showed that mutational numbers in genomes of different passages increased with adaptive passages,and mutations scattered overthe genome,In comparison with that of K7,P6had only6 nucleotides(nt)mutations,P12had7mutational changes,in addition to 6same mutations with p6,there appeared a new mutatopm om 5′NTRat nucleotide position 591resulting in a nucleotide exchange from Ato G.P18had10nt mutations.among the 10mutations,7mutational changes were same as with P12,three new mutational changes appeared in the genome,one in 5′NTR,one in 3Ccoding region.one in 3Dcoding region.atP22 there appeared 18nucleotide changes in the genome,on the basis of P18,there occured additional8nucleotide mutations,two in 5′NTR,three in 2C,one in3A,one in 3Cand one in3D,The results suggested that although H2K7 was already an attenuated strain,the mutations of genome is not sufficient to completely adapt the KMB17,futher mutations caused rapid replication adaptation.CONCLUSION:18-nt changes scattering over the genome are cooperatively responsible for further adaptation characterized by rapid and shortened replication cycle from 28days to14days in KMB17 cells.The mutations in 2Ccoding region play more important role in increase of infectious titer than other mutations,the mutations in 2Bcoding region show less important role than it usually does in cell adaptation,nucleotide changes in5′NTRseem to be not relevant to cell adaptation during initial stages(before P6),but do in late stages.     

The major 5' end of HIV type 1 RNA corresponds to G456

In the course of studies on HIV-1 RNA structure, we determined that the main 5' end of viral RNA from virions and virus producer cells corresponds to G456 in the proviral DNA sequence, one or two nucleotides down-stream from the reported ends that correspond to G454 and G455. We mapped 5' ends using the highly accurate RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) method. The reactivity of the 5' ends indicates that they are mainly capped, although the presence of some uncapped (5'-triphosphorylated) RNA cannot formally be excluded. When we used a 5' mapping method susceptible to incorporating a cytosine at the 3' end of cDNA first strands, at a position templated by the 7-methylguanosine cap, 50% of clones derived from virion RNA had incorporated the additional cytosine. Reassignment of the 5' end has consequences for the design of short RNAs used to study HIV-1 RNA structural dynamics.     

Masking the 5' terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex

Hepatitis C virus subverts liver-specific microRNA, miR-122, to upregulate viral RNA abundance in both infected cultured cells and in the liver of infected chimpanzees. These findings have identified miR-122 as an attractive antiviral target. Thus, it is imperative to know whether a distinct functional complex exists between miR-122 and the viral RNA versus its normal cellular target mRNAs. Toward this goal, effects on viral RNA abundance of mutated miR-122 duplex molecules, bound at each of the two target sites in the viral genome, were compared to effects on microRNA- or siRNA-mediated regulation of reporter target mRNAs. It was found that miR-122 formed an unusual microRNA complex with the viral RNA that is distinct from miR-122 complexes with reporter mRNAs. Notably, miR-122 forms an oligomeric complex in which one miR-122 molecule binds to the 5' terminus of the hepatitis C virus (HCV) RNA with 3' overhanging nucleotides, masking the 5' terminal sequences of the HCV genome. Furthermore, specific internal nucleotides as well as the 3' terminal nucleotides in miR-122 were absolutely required for maintaining HCV RNA abundance but not for microRNA function. Both miR-122 molecules utilize similar internal nucleotides to interact with the viral genome, creating a bulge and tail in the miR-122 molecules, revealing tandemly oriented oligomeric RNA complexes. These findings suggest that miR-122 protects the 5' terminal viral sequences from nucleolytic degradation or from inducing innate immune responses to the RNA terminus. Finally, this remarkable microRNA-mRNA complex could be targeted with compounds that inactivate miR-122 or interfere with this unique RNA structure.     

山东省肠道病毒71型分离株SDLY11全基因组序列分析

目的了解2009年山东省临沂市分离的肠道病毒71型(EV71)分离株SDLY11的基因组序列特征,探讨病毒的神经毒力候选位点。[HTH]方法自手足口病患者的粪便标本中分离EV71,采用一步法RT PCR对病毒株基因组进行全序列扩增,运用DNAstar 和MEGA 4软件进行序列分析。[HTH]结果SDLY 11基因组全长为7 405 nt,其中5′非编码区（UTR） 742 nt,3′UTR 84nt,中间为6 579 nt的开放阅读框架（ORF）,编码2 193个氨基酸的多聚蛋白。VP1区及全基因组序列系统进化分析表明SDLY 11位于EV71病毒C4亚型的分支,同源性分析显示SDLY 11与安徽阜阳株同源性最高。序列比对结果发现,CNS累及HFMD患者病毒株在5[WTBX]′[WTBZ]UTR区出现了两处核苷酸位点的突变（T40C、C575T）,在VP2区第144位出现了氨基酸的突变（T144S）。[HTH]结论SDLY 11分离株属EV71病毒C4亚型。5′UTR区的两处突变（T40C、C575T）及VP2区的一处突变（T144S）可能与病毒的致神经毒力作用有关。     

MicroRNA-122 antagonism against hepatitis C virus genotypes 1-6 and reduced efficacy by host RNA insertion or mutations in the HCV 5' UTR

MicroRNA-122 (miR-122) is believed to stimulate hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5' untranslated region (5' UTR). Recently, it was demonstrated that locked nucleic acid SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a and 1b infection in vivo. However, virus-producing culture systems with 5' UTR of different HCV genotypes have not been available for testing 5' UTR-based treatment approaches. Using JFH1-based Core-NS2 genotype recombinants, we developed 5' UTR-NS2 recombinants of HCV genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a with efficient growth in Huh7.5 cells. Deletion mutagenesis studies demonstrated that the 5' UTR SLI was essential for genotypes 1-6 infection. However, lack of SLI could be compensated for by insertion of other structured HCV or host RNA sequences, including U3 small nucleolar RNA. We demonstrated that SPC3649-induced miR-122 antagonism had a potent antiviral effect against HCV genotypes 1-6 5' UTR-NS2 viruses. Strikingly, HCV recombinant virus with substitution of SLI and miR-122 binding site 1 (S1) by the U3 RNA sequence was not affected by miR-122 antagonism; this was attributed to the lack of an intact S1 by reverse genetics studies. Therefore, we engineered the corresponding U3 RNA sequences into S1 and demonstrated that HCV recombinants with wild-type SLI and single or combined mutations at four of eight nucleotides of S1 were viable in Huh7.5 cells. These mutations reduced the efficacy of SPC3649 treatment, indicating that escape variants to miR-122 antagonism-based HCV therapy could potentially occur.     

5''-Terminal sequences and coding region of late simian virus 40 mRNAs are derived from noncontiguous segments of the viral genome.

The region of the simian virus 40 genome complementary to the 5' end of the most abundant poly(A)-containing 19S and 16S mRNAs was mapped by hybridization of double-labeled RNA ([3H]methyl group and [14C]uridine) to specific DNA fragments. Chemical identification of methylated residues indicated that a common "leader" sequence adjacent to the 5' terminus of both 19S and 16S mRNA is transcribed from DNA sequences located between 0.67 and 0.76 map units. The estimated size of this "leader" RNA, which does not code for any known viral protein, is 170-200 nucleotides. Our results indicate that sequences complementary to the "leader" region and coding portion of 16S mRNA are located in separate parts of the simian virus 40 genome.     

Phylogenetic analysis of the 5' subterminal region of isolates of Leishmania RNA virus-1

Leishmania RNA virus-1 (LRV1) is a double-stranded RNA virus present in some Leishmania species. The virus genome consists of a 450-nucleotide, 5' untranslated region (UTR) followed by the coat gene and the RNA-dependent RNA polymerase (RDRP). It has been shown that the 5' end UTR of the genome promotes internal initiation of translation in an in-vitro assay, indicating the presence of an internal ribosomal entry site (IRES) element upstream of the coat gene. The nucleotide sequences of the 5' subterminal regions of six new isolates of LRV1, of different geographical origins, have now been determined. The RNA folding of the 5' subterminal region of LRV1 has been predicted, using a combination of thermodynamic parameters and folding constraints based on nucleotide substitutions. Furthermore, a putative pyrimidine-rich region (a feature unique to all IRES elements), which is complementary to the Leishmania 18S rRNA, has been identified. The significance and relevance of these findings in the context of the function of the 5' UTR of LRV1 as an IRES element are discussed.     

Cell proteins bind to multiple sites within the 5'' untranslated region of poliovirus RNA.

The 5' noncoding region of poliovirus RNA contains sequences necessary for translation and replication. These functions are probably carried out by recognition of poliovirus RNA by cellular and/or viral proteins. Using a mobility-shift electrophoresis assay and 1,10-phenanthroline/Cu+ footprinting, we demonstrate specific binding of cytoplasmic factors with a sequence from nucleotides 510-629 within the 5' untranslated region (UTR). Complex formation was also observed with a second sequence (nucleotides 97-182) within the 5' UTR. These two regions of the 5' UTR appear to be recognized by distinct cell factors as determined by competition analysis and the effects of ionic strength on complex formation. However, both complexes contain eukaryotic initiation factor 2 alpha, as revealed by their reaction with specific antibody.