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characterization of genotypes for
BURKHOLDERIA CEPACIA CoMPLEx strains
isolated from patients in hospitals of Russian federation
O.L. Voronina', M.Yu. Chernukha', I.A. Shaginyan', M.S. Kunda',
L.R. Avetisyan', AA. Orlova', V.G. Lunin', L.V. Avakyan2, N.I. Kapranov3, E.L. Amelina4, A.G. Chuchalin 4, A.L. Gintsburg '
1 Gamaleya Scientifc Research Institute of Epidemiology and Microbiology, Ministry of Health and Social Development of the Russian Federation, Moscow, Russia; 2 Russian Children's Clinical Hospital Ministry of Health and Social Development of the Russian Federation, Moscow, Russia; 3 Research Centre of Medical Genetics RAMS, Moscow, Russia; 4 Research Institute of Pulmonology FMBA of Russia, Moscow, Russia
88 cultures of microorganisms referred to the Burkholderia cepacia complex (Bcc) during initial identification were analyzed by multilocus sequencing (Multilocus Sequence Typing, MLST). 13 genotypes (sequence type, ST) were detected, 9 of them (708, 709, 710, 711, 712, 714, 727, 728, 729) were identified for the first time. Two new alleles for the gene trpB (357, 358), one of the genes atpD (306) and gltB (352) were detected and registered. It was found that strains of 2 genotypes (711, 712) belong to the species B. multivorans, 1 (ST102)
- B. contaminans, 1 (ST51) - B. stabilis, 1 (ST729) - B. vietnamiensis. Most strains of the sample, representing 8 genotypes (208, 241, 728, 727, 708, 709, 710, 714), belong to the species B. cenocepacia. Identified genotypes differ in the global spread of the world: 4 genotype (51, 102, 208, 241) have intercontinental distribution, 1 (712) - intra. It is shown that strains causing nosocomial infections, in most cases refer to genotypes 728 and 708. Epidemiologically significant in respect of patients with cystic fibrosis should recognize genotype 709, detected in strains isolated from patients in seven federal districts (FD) of Russia. The Bcc strains of genotypes 241 (B. cenocepacia) and 729 (B. vietnamiensis) were isolated from the patients of the Far Eastern FD. They are not typical for other FD Russia. The possibility of concomitant infection in cystic fibrosis patient with two genotypes 709
- epidemiologically significant and 708 - nosocomial, was indicated. The long-term persistence of a single genotype strain in the organism of patients with cystic fibrosis was demonstrated as for Bcc species B. cenocepacia (ST 709), so for B. multivorans (ST712). The possibility of transferring the strain Bcc, typical for nosocomial environment to patient with cystic fibrosis at surgery was observed.
© КОЛЛЕКТИВ АВТОРОВ, 2013
Aylin DOGEN1, Engin Kaplan1, Mehmet Sami Serin1, Zehra Oksuz1, Seda Tezcan2, Gonul Asian2,
Orhan Sezgin3, Engin Altintas3, Gurol Emekdas2
detection of hepatitis B virus x GENE AND pREC promoter MUTATioNs from chronic hepatitis b patients in the south of turkey
1Mersin University, Faculty of Pharmacy, Dept. Pharmaceutical Microbiology, Mersin, TURKIYE; 2Mersin University, Faculty of Medicine, Dept. Medical Microbiology, Mersin, TURKIYE; 3Mersin University, Faculty of Medicine, Dept. Gastroenterology,
Mersin, TURKIYE
Hepatitis B virus (HBV) infection is a global health problem with more than 2 billion infected individuals. HBV infection leads to diverse outcomes ranging from acute to chronic hepatitis, which may result in severe complications as liver cirrhosis and hepatocellular carcinoma (HCC). HBV is one of the most important human DNA viruses having strong oncogenic potential. Recently, many studies have reported on HBV X gene and PreC promoter mutations associated with HCC.
In order to detect the prevalence of HBx gene and PreC promoter mutations possibly related to HCC, we have analyzed sera samples collected from 61 patients with chronic hepatitis B. We have detected T1653 mutation in 1 of 61 (1,63%), A1896 mutation in 10 of 61 (16,39%), and T1762 - A1764 dual mutation in 4 of 61 (6,55%). T1653 and T1762 - A1764 dual mutations were suggested significantly related to HCC in earlier reported studies. Our findings demonstrate that HBx gene and PreC promoter mutations related to HCC are present in our region and prospective clinical chord studies would be useful for better patient management and of early diagnosis of possible HCC cases.
Key words: Hepatitis B virus, Chronic Hepatitis B, X gene mutations, PreC promoter mutations, Hepatocellular Carcinoma
Hepatitis B virus (HBV) infection is a health problem threatening people living all over the world. HBV infection leads to a wide spectrum of liver disease ranging from acute to chronic hepatitis, cirrhosis, and hepatocellular carcinoma [1]. More than two billion people are infected with HBV around the world. Of these 350 million people are chronically infected with HBV and one million deaths occur each year due to active hepatitis, cirrhosis or primary liver cancer [2]. HBV, liver cirrhosis, exposure to aflatoxin Bl, alcohol consumption, and diabetes have etiological roles in development of hepatocellu-lar carcinoma (HCC) [3]. Among these, HBV is the major risk factor for HCC and associated with more than 50% of cases of HCC [4].
HBV genome contains overlapping open reading frames
(C, S, P, X) regulated with 4 promoter (Basal core promoter-BCP, Pre-S1, PreS2/S and X), two enhancers (Enh I and Enh II) and negative regulatory elements [5].
From the acute to chronic phase of HBV infection different structural, non-structural or regulatory region based mutation profiles may occur [6]. In fact, the main reason of the mutations is the lack of proofreading activity of HBV polymerase. All possible single base changes can be produced per day because the HBV genome is only ~3200 base pairs. However, mutated variants can be selected and mutations arise increasingly under the pressure of host defense and antivirals [7].
There has been increasing evidence of an association between molecular alteration and the development of HCC in patients with HBV infection [6]. Mutations in the basal core promoter (BCP) region at nucleotides (nt) 1762 and 1764 (T1762/A1764) and in the precore region at nt 1896 (A1896) are associated with HBV e antigen (HBeAg) seroconversion and persistent viral replication. Double mutations in the BCP at nt 1762 and nt 1764, resulting in T-1762, A-1764 (termed the BCP mutant), have been described in patients with chronic HBV, fulminant hepatitis, or who are immunosuppressed. This double mutation results in a decrease in HBeAg production by 70% and may increase viral replication [8].
It is noteworthy that both BCP and precore mutations are often found in patients with advanced liver disease such as HCC [6]. The T1762/A1764 mutations alter HBeAg production at the transcriptional level, and the A1896 mutation in the precore region terminates the translation of precursor protein, abrogates HBeAg production, and results in seroconversion. At the stage of chronic HBV infection, T1653 mutation in EnhII in X gene region is important to the contribution of HCC risk [9]. T1762 - A1764 dual mutation in BCP region [10, 11] T1753C/A/G mutations in C promoter [12] could also be important to contribute to HCC risk [13, 14, 15].
In our study, we aimed to detect X/PreC (EnhII, BCP, Precore) mutation patterns in chronic HBV patients without HCC in Mersin (a southern costal of Turkey).
2. Materials and Methods
2.1. Sera Samples
Sera samples were provided from the stored samples of 61 patients, priory diagnosed as chronic hepatitis B in Mersin university hospital, department of gastroenterology. All sera samples were HBsAg (+) and HBV DNA (+).
2.2. Extraction of HBV DNA
One hundred microliters of serum was mixed with 300 ^L of lysis buffer (13.3 mmol/^L Tris_HCl, pH 8.0, 6.7 mmol/^L ethylene-diaminetetraacetic acid, 0.67% sodium dodecyl sulfate, 133 ^g/mL proteinase K and incubated at 55°C for 4 h. Two phenol-chloroform extractions were followed by one chloroform extraction, and DNA was precipitated with ethanol. The precipitate was dissolved in 20
¡L of TE buffer (10 mmol/^L Tris-HCl, pH 8.0, 1 mmol ethylenedi-aminetetraacetic acid).
2.3. Semi-Nested PCR Amplification
The first round of PCR was performed with 5 ¡¡L of DNA, 1.25 U of Taq polymerase (Promega, Madison, WI), 0.2 mM dNTP (each), 1 mM MgCl2, 0.25 mM of each forward and reverse primers, and 1x PCR buffer (20 mM Tris HCl, pH 8.4, 50 mM KCl). The primers; HBV-X1 (Nt. 1177-1190) and HBV-X2 (Nt. 1961-1979) were used for the first round of PCR. The reaction was allowed to produce at 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute in each cycle. We have also used a hot start (at 94°C for 5 minutes) and a post elongation step (at 72°C for 5 minutes) before and after the reaction). The amplification was performed for 25 cycles in a thermal cycler (Boeco Germany, TC-PRO). In the second round of PCR, we have used 1 ¡L of the first PCR product and internal primers (at the same conditions and same thermal cycles described above). The primers; HBX-IS (Nt. 1400-1423) and HBV-X2 (Nt. 1961-1979) were used for the second round of PCR. The primer sequences and PCR product lengths are demonstrated at table 1.
2.4. DNA Cycle-Sequencing of HBV isolates
The second-round PCR products were 579 bp. These products included X gene region and significant portion of PreC promoter. The PCR products were purified by Wizard PCR preps DNA purification system (Promega). HBX-IS (Nt. 1400-1423) was used as sequencing primer. Purified PCR products were cycle-sequenced by SILVER SEQUENCE(tm) DNA Sequencing System (Promega) on a thermal cycler (Boeco Germany, TC-S) according to the manufacturer's instructions. The cycle-sequencing reaction was performed for 95°C for 2 minutes, then 95°C for 30 seconds (denaturation), 42°C for 30 seconds (annealing), 70°C for 1 minute (extension) for a total of 45 cycles, then 4°C soak. The cycle-sequencing products were separated by electrophoresis through a polyacrylamide gel with urea. The gel was visualized by silver staining according to the recommendation of the manufacturer.
2.5. Analysis of Sequences
The sequences of these products were determined and compared with previously reported HBV complete genome (AY721609).
We have compared the isolated sequences with 1 previously reported HBV complete genome sequence; AY721609 which was isolated in Turkey [16]. Nucleotide sequence was obtained from the GenBank databases. The comparative analysis of the HBV sequences was performed with Vector NTI 9.0 software (InforMax Invitrogen Life Science Software, Frederick, MD).
3. RESULTS
After the second round of PCR amplification, we had a 579 bp segment of HBV DNA includes X gene region and significant portion of PreC promoter (Fig. 1). The sequences of this product were determined by DNA cycle-sequencing. These sequences were compared with 1 reported HBV complete genome sequence (AY721609).
In our cases, T1653 mutation in 1 of 61 (1.63%), A1896 mutation in 10 of 61 (16.39%), and T1762 - A1764 dual mutation in 4 of 61 (6.55%) were detected (Figure 2). All obtained data is summarized in table 2.
T a b l e 1
Primer sequences and PCR product lengths of HBx gene Core promoter/Precore gene region
X Gene Region
Primer position
Sequences (5'-3')
PCR product lenghts (bp)
1st Round HBV Xlsense external (Nt. 1177-01190)
HBV X2 (Nt. 1961-1979) 2nd Round HBX IS (Nt. 1400-1423) HBV X2 (Nt. 1961-1979)
5'-TGC CAA GTG TTT GCT GAC GC-3' 5'- AAG GAA AGA AGT CAG AAG G - 3' 5'- CTG GAT CCT ACG CGG GAC GTC CTT-3' 5'- AAG GAA AGA AGT CAG AAG G - 3'
802
579
4. discussion
Although HBV is a DNA virus, its replication strategy is through a reverse transcription process with a RNA-replica-tive intermediate requiring a viral reverse transcriptase. The reverse transcriptase is believed to lack a proofreading function that is common to other polymerases. This error prone viral reverse transcriptase resulting in a large pool of qua-sispecies with mutations spread throughout the genome [8, 17]. Therefore, HBV exhibits a mutation rate more than 10fold higher than other DNA viruses and all possible single base changes can be produced every day because the small genome size of HBV (~3200 nt). [7, 8]. However, mutated variants can be selected and mutations arise. The pressure of host defense and antiviral treatment result the increases of this selection [7]. This means that many mutations are naturally occurring. However, there has been increasing evidence of an association between molecular alterations and the development of severe liver diseases such as chronic HBV, fulminant hepatitis and HCC as reported earlier [8].
In our study, we have investigate the mutations in EnhII, BCP, Pre-core within the X gene and overlapping C gene region because of these mutations are often found in patients with advanced liver disease such as HCC [6]. However, our sera samples were obtained from chronic hepatitis B patients without HCC.
In our study we have detected T1653 mutation in EnhII in 1 of 61 (1.63%). Nucleotide C1653 is located in the alpha box elements (Nt. 1648-1668) which is a strong activation element of EnhII and core promoter [18]. It is reported that T1653 mutation might upregulate EnhII/core promoter activity thought alpha box binding site affinity alterations [19]. Therefore, T1653 mutation could have potential to influence the HBe production and viral replication through BCP activity [20]. Shinkai et al. had found T1653 mutation in 20 of 80 (30%) and 45 of 80 (56%) in patients without and with HCC, respectively [6]. Ito et al., had also found T1653 in 10 of 40 (25%), 14 of 40 (35%) and 28 of 40 (70%) in carriers chronic hepatitis B patients and patients with HCC, respectively [20]. There are several studies reporting T1652 mutation in patients without or with HCC. Most of them reporting T1653 mutation is higher in HCC group. However, they also report the presence of this mutation in patient without HCC group. This data is in agreement with our results, nevertheless our T1653 rate is low (1.63%).
In our study, we have detected T1762/A1764 dual mutations in 4 of 61 (6.55%) samples in the BCP region and A1896 mutation in 10 of 61 (16.39%) sample in the preC region. The BCP plays a central role in HBV replication and morphogenesis, controlling the transcription of both pregenomic RNA and precore mRNA and also enhance transactivating ability of X sequence with its regulatory elements. Since predominant role in viral replication of this overlapping region, mutations could be limited T1762 and A1764 mutations in the BCP region and A1896 mutation in the preC region are associated with seroconversion and persistent viral replication. Both BCP and preC mutations are often found in patients with advanced liver disease [21, 22, 23,]. T1762/A1764 double mutation is one of the common mutations associated with increased inflammatory response in the progression of liver damage thorough chronic hepatitis and cirrhosis stage [23].
1 2 3 4 5 6 7 8
: i i
Figure 1. HBV X gene PCR products. Lane 1: 100 bp step ladder; Lane 2, 3, 4, 5, 6, 7 positive samples (579 bp); Lane 8 negative sample.
А С G T А С G T
a b
Wild type Nt. 1762 A-T,
Nt. 1764 G-A
a b
Wild type Nt. 1653 C-T
Figure 2. Wild type DNA sequences and mutation patterns at HBx
gene Core promoter/Precore gene region. I-a: G to A single base substitution at nucleotide 1896; I-b: Wild type DNA sequences; II-a: Wild type DNA sequences; II-b: A to T and G to A single base substitutions at nucleotides 1762 and 1764; III-a: Wild type DNA sequences; III-b: C to T single base substitution at nucleotide 1653.
T a b l e 2
Mutation patterns and proportion rates
Group Mutations
T1653 n (%) A1896 n (%) T1762 n (%) T1762-A1764 n (%)
Chronic Hepatitis B (n = 61) 1 (1.63) 10 (16.39) 3 (4.91) 4 (6.55)
In a prospective cohort study, 15 HCC cases selected from a prospective cohort of 1,638 high-risk individuals on the basis of available plasma samples spanning the years before and after diagnosis. In this study, T1762/A1764 double mutation was detected in 8 of the 15 cases (53.3%) before cancer. It is suggested T1762/A1764 double mutation in plasma could be a valuable predictive biomarker for HCC development [24]. Shinkai et al., had found T1762/A1764 double mutation in 58 of 80 (73%) and 73 of 80 (91%) in patients without and with HCC, respectively [6]. This workers had also found A1896 mutation in 41 of 80 (51%) and 43 of 80 (54%) in patients without and with HCC, respectively [6]. Ito et al., had found T1762/A1764 double mutation in 31 of 40 (77.5%) and 36 of 40 (90%) in patients without and with HCC, respectively [20]. This workers had also found A1896 mutation in 26 of 40 (65%) and 25 of 40 (62.5%) in patients without and with HCC, respectively.
T1762/A1764 double mutation rate has been detected in 4 of 61 (6.55%) samples without HCC in our study. All of this data are also in agreement with our results, nevertheless our T1762/A1764 double mutation and A1896 mutation rates are also low (6.55%, 16.39% respectively).
Our low mutation rates may be due to our sequencing protocol. Automated sequencing protocol use probably will increase revealing of mutation rates.
In the literature the assosication between HBV mutations and HCC remains controversial because of conflicting data based on different populations. In a meta analysis, 43 studies evaluated a total of 11552 HBV-infected participants of whom 2801 had HCC. According to the study, A1896 mutation was not statistically associated with HCC risk. However, T1653 and T1762/A1764 double mutation are associated with the development of HCC with an increased risk of 2.76 and 3.79 fold, respectively. The authors suggest that these mutations alone and in combination may be predictive for HCC [3].
In conclusion, due to the statistically signicifant association between HBV mutations and HCC, underlying mechanisms are still unclear. However, the associated mutations might be reveal the HCC risks for people in a population and could be used in early detection of HcC. Our findings demonstrated that mutations might be related to HCC such as T1653 and T1762/A1764 double mutation are present in our region. Prospective clinical chord studies should be planned in the
future for better patient management to follow HCC thorough mutational examination of HBV.
ACKNOWLEDGMENTS
This study was supported by the University of Mersin research grants BAP-SBE FMA (EK) 2010-1 YL.
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Поступила 06.09.12
© КОЛЛЕКТИВ АВТОРОВ, 2013
L. V. Ozerova", M. S. Krasnikovab, A. V. Troitskyb, A. G. Solovyevb, and S. Y. MorozoV
TAS3 GENES FOR SMALL TA-SIARF RNAS IN PLANTS BELONGING TO SUBTRIBE SENECIONINAE: occurrence of prematurely terminated RNA
precursors
a Tsytsin Main Botanical Garden, Russian Academy of Sciences, Botanicheskaya 4, 127276 Moscow, Russia;b A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119992, Russia e-mail: [email protected]
Abstract
The various classes of plant 21- to 24-nt siRNAs derive from long dsRNA precursors that are processed by the ribonuclease Dicer-like (DCL). The species of ta-siRNA were originally discovered in Arabidopsis thaliana. Four gene families have been identified in Arabidopsis that each produces a number of ta-siRNAs: TAS1, TAS2, TAS3 and TAS4. The TAS3 genes encode tasiR-ARF species which target the mRNA of three Auxin Response Factor (ARF) genes (ARF2, ARF3/ETT and ARF4) for subsequent degradation. The function of TAS3 precursor RNA is controlled by two miR390 target sites flanking tandem of ta-siARF sequences. In this paper, we have studied the presence of ta-siARF RNA genes in the representatives of subtribe Senecioninae. Senecioneae is the largest tribe of Asteraceae, comprised of ca. 150 genera and 3,000 species which include many common succulents of greenhouses.
Approximately one-third of species are placed in genus Senecio, making it one of the largest genera of flowering plants. However, there was no information on the structure of TAS genes in these plants. We revealed that the TAS3 species (TAS3-Sen1) in Senecio representatives was actively transcribed, and its homologues are distributed among many Asteracea plants and found to be similar to Arabidopsis AtTAS3a gene. We revealed several prematurely terminated transcripts of TAS3-Sen1. Finding the alternative shortened transcripts of TAS3-Sen1 lacking the 3' -terminal site cleaved by miR390 and retaining the 5 '-terminal miR3 90 non-cleaved site suggested their using as decoys for the modulation of miR390 activity to regulate synthesis of ta-siARF RNAs in different Senecioninae species.
Keywords: micro RNA, trans-acting siRNA, cDNA, plant, succulent, phylogeny.
