乙肝病毒基因多态性检测芯片临床应用价值

目的:探讨DNA芯片检测乙型肝炎病毒基因多态性的临床应用价值。方法:设计多条寡核苷酸探针,用于针对DNA、HBV的多个产检点突变位点,将硅烷化芯片固定,让HBV基因与其特定区段杂交,并在硝酸纤维素膜上影印杂交结果,经过避光显色,判断杂交信号的有无及探针序列实际情况。结果:通过杂交反应检测HBV前C/C区(ntl895/1816)、BCP区(ntl767/1766)和P区(aa524/551)等多个位点的变异,与测序分析结果符合率96.83%。检测重复性和灵敏度反应很好。结论:将酶呈色法DNA芯片用于检测乙肝病毒基因多态性,检测费用少,操作便捷,值得临床应用。

Polymorphism Detection of the Twelfth Exon of Equine MxA Gene

[Objective] The study aimed to establish a fast and accurate method to detect the polymorphism of the 12^th exon of equine MxA gene. [Method] The 12^th exon of MxA gene was amplified by mismatch PCR and the products were analyzed by restriction fragment length polymorphism （RFLP） to determine the point mutation at the 1 790 nt of MxA cDNA. The sequence of the PCR products was also analyzed. [Result] There were three genotypes （AA, AB and BB） in the 12^th exon of equine MxA gene; the 2 081 nt of MxA cDNA mutated from G to C, correspondingly changing the 562^th amino acid of the coding region of MxA protein from tryptophan to cysteine; the specific sequence of the PCR products amplified by mismatch PCR-RFLP was consistent with the analysis results of RFLP. [ Conclusion] The mismatch PCR-RFLP was an easy method with accurate results to detect the polymorphism of the 12^th exon of equine MxA gene.

Polymorphisms of some cytokines and chronic hepatitis B and C virus infection

AIM： To study the relationship between the polymorphisms in some cytokines and the outcome of hepatitis B virus （HBV） and hepatitis C virus （HCV） infection. METHODS： Samples were obtained from 203 patients infected with HBV and/or HCV while donating plasma in 1987, and 74 controls were obtained from a rural area of North China. Antibodies to HBV or HCV antigens were detected by enzyme-linked imrnunoassay. The presence of viral particles in the serum was determined by nested reverse-transcriptase polymerase chain reaction （PCR）. Hepatocellular injury, as revealed by alanine aminotransferase （ALT） and aspartate aminotransferase level, was detected by a Beckman LX-20 analyzer. DNA was extracted from blood cells. Then, the single nucleotide polymorphisms of IL-2-330, IFN-γ＋874, IL-10-1082/-592 and IL-4-589 were investigated by restriction fragment length polymorphism-PCR or sequence specific primer-PCR.RESULTS： Persistent infection with HBV, HCV, and HBV/HCV coinfection was associated with IL-2-330 TT genotype and T allele, IFN-γ＋874 AA genotype, and IL-10-1082 AA genotype. The clinical outcome of HBV and/or HCV infection was associated with IL-2-330 TT genotype and T allele, IFN-γ＋874 AA genotype, and IL-10-1082 AA genotype. IL-2-330 GG genotype frequency showed a negative correlation with clinical progression, IL-10-1082 AA genotype frequency showed a positive correlation and IL-10-1082 AG genotype frequency showed a negative correlation with clinical progression. HCV RNA positive expression was associated with IL-10-1082 AA genotype and the A allele frequency. Abnormal serum ALT level was associated with IL-10-592 AC genotype frequency and IL-4-589 CC genotype, CT genotype, and the C allele. CONCLUSION： These results suggest that polymorphisms in some cytokine genes influence persistent HBV and HCV infection, clinical outcome, HCV replication, and liver damage.

Effect of cytokine gene polymorphism on histological activity index, viral load and response to treatment in patients with chronic hepatitis C genotype 3

AIM： To investigate the association between cytokine gene polymorphism and disease status in chronic hepatitis C genotype 3 by liver biopsy, ALl, HCV RNA levels and response to treatment. METHODS： Patients with chronic hepatitis C genotype 3 were analyzed for single nucleotide polymorphisms of interleukin （IL）-10, IL-1 beta, interferon-gamma （IFN-y）, tumor necrosis factor-alpha （TNF-y） and transforming growth factor-beta （TGF-β） by polymerase chain reaction using sequence-specific oligonucleotide primers. Liver biopsies were assessed by modified histological activity index （HAI） scoring system using a scale of 0-18 for grading the necro-inflammatory activity and 0-6 for staging the fibrosis. HCV RNA levels were determined by bDNA assay. The patients were treated with interferon alpha and ribavirin for 6 mo. Sustained virological response was assessed 6 mo after the completion of the treatment. RESULTS： Out of the 40 patients analyzed, 26 were males. Mean age was 40.5±12.5 years （range 18- 65 years）. The frequencies of different dimorphic polymorphisms based on single nucleotide substitution were as follows： IL-10-1082 G/A 85%, A/A 12.5%, G/ G 2.5%; IL-10-819 A/C 87.5%, C/C 10%, A/A 2.5%; IL-10-592 C/A 72.5%, C/C 27.5%; IL-1 C 90%, U 10%; IFN-874 T/A 50%, T/T 27.5%, A/A 22.5%; TNF-308 A/G 95%, G/G 5%; TGF-10 T/C 52.5%, C/C 35%, T/T 12.5%. The mean grades of necroinflammatory activity of different genotypes of IL-10 at promoter site -1082 were A/A = 3.6, A/G = 5.0, and G/G = 10.0 and the difference was significant （P = 0.029）. The difference in the stage of disease at a scale of 0-6 was A/A 0.8, A/G 2.3, and G/G 4.0 （P = 0.079）. The difference in the HAI seemed to be related to the presence of allele -1082G.For IL-10 -819 genotypes, mean scores of fibrosis were A/A = 6.0, A/C = 2.2, and C/C = 1.0 （P = 0.020） though the inflammatory activity was not much different. No significant differences in HAI were noted among polymorphisms of other cytokines. Moreover, ALT and HCV RNA levels were not significantly different among different cytokine polymorphisms. There was a significant correlation of HAI and HCV RNA levels with the duration of disease. TGFI3 -10 genotype CC patients had a better end of treatment response than those with other genotypes （P = 0：020）. Sustained virological response to the treatment was not influenced by the cytokine polymorphism. No effect of other factors like viral load, degree of fibrosis, gender, steatosis, was observed on sustained virological response in this population infected with genotype 3. CONCLUSION： There is no significant correlation between cytokine polymorphisms and HAI except for the polymorphisms of anti-inflammatory cytokine IL-10, which may influence hepatic inflammatory activity and fibrosis in patients with chronic hepatitis C genotype 3. Sustained virological response in this genotype does not seem to be influenced by cytokine gene polymorphisms.

Genetic diversity of the hepatitis C virus: Impact and issues inthe antiviral therapy

The hepatitis C Virus (HCV) presents a high degree of genetic variability which is explained by the combination of a lack of proof reading by the RNA dependant RNA polymerase and a high level of viral replication. The re- sulting genetic polymorphism defines a classification in clades, genotypes, subtypes, isolates and quasispecies. This diversity is known to reflect the range of responses to Interferon therapy. The genotype is one of the pre- dictive parameters currently used to define the antiviral treatment strategy and the chance of therapeutic suc- cess. Studies have also reported the potential impact of the viral genetic polymorphism in the outcome of antivi- ral therapy in patients infected by the same HCV geno- type. Both structural and non structural genomic regions of HCV have been suggested to be involved in the Inter- feron pathway and the resistance to antiviral therapy. In this review, we first detail the viral basis of HCV diversity. Then, the HCV genetic regions that may be implicated in resistance to therapy are described, with a focus on the structural region encoded by the E2 gene and the non- structural genes NS3, NS5A and NS5B. Both mechanisms of the Interferon resistance and of the new antiviral drugs are described in this review.

Relationship between IFN-γ gene polymorphism and susceptibility to intrauterine HBV infection

AIM： To explore the susceptibility of children to intrauterine HBV infection by studying the relationship between IFN-γ gene polymorphism, including IFN-γ＋874A/T single nucleotide polymorphism（SNP） and CA repeat microsatellite polymorphism and intrauterine HBV infection. METHODS： A TaqMan fluorescence polymerase chain reaction in the IFN-γ＋874A/T single nucleotide polymorphism was tested in the intrauterine HBV infection group（group Ⅰ） and the normal immune children group（group Ⅱ）. Capillary electrophoresis was performed in the above two groups to assay the IFN-γ, CA repeat microsatellite polymorphism. RESULTS： Frequencies of AA, AT and TT genotypes were 67.4%, 19.6% and 13.0% in the intrauterine HBV infection group, and 45.2%, 30.1% and 24.7% in the normal immune children group, respectively. A significant difference was found in the frequency distribution of IFN-γ＋874 genotype between the two groups （x^2 = 5.102, P = 0.02389）. In the intrauterine HBV infection group the AA genotype was more common than in the normal immune group. Frequency of IFN-γ＋874A allele was 77.17% in the intrauterine HBV infection group, and 60.27% in the normal immune children group. In the intrauterine HBV infection group the IFN-γ＋874A allele was more common than in normal immune group. A significant difference was found in the frequency distribution between the two groups （x^2= 7.238, P= 0.02389, OR = 2.228, 95% CI = 1.244-3.992）. （CA12）^＋/（CA12）^＋ of IFN-γ CA microsatellite polymorphism was 11.90% in the intrauterine HBV infection group and 26.47% in the normal immune children group. A significant difference was found in the frequency distribution between the two groups （x^2 = 5.64, P = 0.0176）. Frequency of IFN-γ CA repeat was 25% in the intrauterine HBV infection group and 43.38% in the normal immune children group. The frequency of IFN-γ CA repeat was less in the intrauterine HBV infection group than in normal immune group. A significant difference was found in the frequency distribution between the two groups （x^2 = 7.548, P= 0.0060）. CONCLUSION： There is a relationship between IFN-γ＋874A/T SNP and intrauterine HBV infection as well as between IFN-γ CA microsatellite polymorphism and intrauterine HBV infection. IFN-γ gene polymorphism might be important in determining individual＇s susceptibility to intrauterine HBV infection.

Relationship between HLA-DR gene polymorphisms and outcomes of hepatitis B viral infections:A meta-analysis

AIM:To assess the rigorous relationship between human leukocyte antigens(HLA)-DR alleles and outcomes of hepatitis B virus(HBV) infections by means of metaanalysis.METHODS:Medline/PubMed,EMBASE,CNKI and VIP were searched to identify relevant studies.Study quality was evaluated using the Newcastle-Ottawa Scale.Odds ratios(OR) and 95% confidence interval(95% CI) were pooled using Stata 11.0.Subgroup analyses were performed by ethnicity.Heterogeneity and publication bias analyses were performed to validate the credibility.RESULTS:A total of 2609 patients with chronic hepatitis B and 2606 controls spontaneously recovering from prior HBV infection were included.Meta-analysis showed that HLA-DR*04(OR = 0.72,95% CI:0.60-0.85) and DR*13(OR = 0.27,95% CI:0.19-0.37) alleles were significantly associated with HBV clearance while patients carrying HLA-DR*03(OR = 1.47,95% CI:1.16-1.87) or DR*07(OR = 1.59,95% CI:1.24-2.03) alleles had a significantly increased risk of chronic HBV persistence.For the HLA-DR*01 polymorphism,a significantly association with HBV clearance was found in Chinese Han group(OR = 0.48,95% CI:0.26-0.86),but not found in other ethnic groups(P = 0.191).For other polymorphisms,no association with the HBV infection outcome was found.CONCLUSION:HLA-DR*04 and DR*13 alleles may be the protective factors for HBV clearance and HLADR*03,and DR*07 alleles may be the risk factors for HBV persistence.

Different effects of a CD14 gene polymorphism on disease outcome in patients with alcoholic liver disease and chronic hepatitis C infection

AIM： Clinical and experimental data suggest that gut-derived endotoxins are an important pathogenic factors for progression of chronic liver disease. Recently, a C-T （-159） polymorphism in the promoter region of the CD14 gene was detected and found to confer increased CD14 expression and to be associated with advanced alcoholic liver damage. Here, we investigated this polymorphism in patients with less advanced alcoholic liver disease （ALD） and chronic hepatitis C virus （HCV） infection. METHODS： CD14 genotyping was performed by PCR-RFLP analysis in （a） 121 HCV patients, （b） 62 patients with alcohol-associated cirrhosis （AIc-Ci）, （c） 118 individuals with heavy alcohol abuse without evidence of advanced liver damage （Alc-w/o Ci）, and （d） 247 healthy controls. Furthermore, serum levels of soluble CD14 （sCD14） and transaminases were determined. RESULTS： The TT genotype was significantly more frequent in Alc-Ci compared to Alc-w/o Ci or controls （40.3% vs 23.7% or 24.0%, respectively）. In Alc-w/o Ci, serum levels of transaminases did not differ significantly between patients with different CD14 genotypes. In HCV patients, TT-homozygotes had significantly higher sCD14 levels and sCD14 serum levels were significantly higher in patients with advanced fibrosis or cirrhosis. However, no association was found between CD14 genotypes and histological staging or grading. CONCLUSION： Considering serum transaminases as surrogate markers for alcoholic liver damage, the CD14 polymorphism seems to exhibit different effects during the course of ALD. Differences in genotype distribution between cirrhotic HCV patients and alcoholics and the known functional impact of this polymorphism on CD14 expression levels further indicate differences in the pathophysiological role of CD14 and CD14-mediated lipopolysaccharides signal transduction with regard to the stage as well as the type of the underlying liver disease.

Torque teno virus:Its prevalence and isotypes in North India

AIM： To investigate the prevalence and genotype distribution of Torque teno virus （TTV） in patients with different liver diseases and chronic renal failure treated at a referral hospital in North India. METHODS： Whereas prevalence of TFV was based on amplification of conserved region of ORF2 of TTV genome, the genotyping of TFV was carried out using restriction fragment length polymorphism （RFLP） procedure on the N22 region of ORFI. RESULTS： TTV-DNA was detected in 137 of 513 （26.7%） patients with liver diseases and 38 of 65 （58.5%） patients with chronic renal failure. Trv was also detected in 2/7% of healthy controls. The sequence analysis of the PCR product from 10 randomly selected cases failed to show a significant sequence divergence when compared with that of the TRM1 isolate of TTV genotype 1. The results of genotyping in 55 randomly selected patients showed the presence of genotype 1 （G1） in 53 （96.4%） and genotype 2 （G2） in 2 cases （3.6%）, respectively. Other genotypes were not identified in this patient subgroup, suggesting that G1 is predominant in this area. The results of genotyping by RFLP were also supported by phylogenetic tree analysis, where G1 was found to be the major genotype. CONCLUSION： These results indicate that TTV is moderately present in Indian patients, with G1 to be the major genotype in North India. The pathogenicity and etiological role of TTV in different diseases is still a question mark and warrant further studies.

Indinavir Resistance Evolution in One Human Immunodeficiency Virus Type 1 Infected Patient Revealed by Single-Genome Amplification

Human Immunodeficiency Virus Type 1 exists in vivo as quasispecies, and one of the genome's characteristics is its diversity. During the antiretroviral therapy, drug resistance is the main obstacle to effective viral prevention. Understanding the molecular evolution process is fundamental to analyze the mechanism of drug resistance and develop a strategy to minimize resistance. Objective: The molecular evolution of drug resistance of one patient who had received reverse transcriptase inhibitors for a long time and had treatment which replaced Nevirapine with Indinavir was analyzed, with the aim of observing the drug resistance evolution pathway. Methods: The patient, XLF, was followed-up for six successive times. The viral populations were amplified and sequenced by single-genome amplification. All the sequences were submitted to the Stanford HIV Drug Resistance Database for the analysis of genotypic drug resistance. Results: 149 entire protease and 171 entire reverse transcriptase sequences were obtained from these samples, and all sequences were identified as subtype B. Before the patient received Indinavir, the viral population only had some polymorphisms in the protease sequences. After the patient began Indinavir treatment, the variants carrying polymorphisms declined while variants carrying the secondary mutation G73S gained the advantage. As therapy was prolonged, G73S was combined with M46I/L90M to form a resistance pattern M46I/G73S/L90M, which then became the dominant population. 97.9% of variants had the M46I/G73S/L90M pattern at XLF6. During the emergence of protease inhibitors resistance, reverse transcriptase inhibitors resistance maintained high levels. Conclusion: Indinavirresistance evolution was observed by single-genome amplification. During the course of changing the regimen to incorporate Indinavir, the G73S mutation occurred and was combined with M46I/L90M.

Distribution and effects of polymorphic RANTES gene alleles in HIV/HCV coinfection - A prospective cross-sectional study

AIM: Chemokines and their receptors are crucial for immune responses in HCV and HIV infection. RANTES gene polymorphisms lead to altered gene expression and influence the natural course of HIV infection. Therefore,these mutations may also affect the course of HIV/HCV coinfection.METHODS: We determined allele frequencies of RANTES-403 (G→A), RANTES-28 (C→G) and RANTESIN1.1 (T→C) polymorphisms using real-time PCR and hybridization probes in patients with HIV (n = 85), HCV (n= 112), HIV/HCV coinfection (n = 121), and 109 healthy controls. Furthermore, HIV and HCV loads as well as CD4+ and CD8+ cell counts were compared between different RANTES genotypes.RESULTS: Frequencies of RANTES-403 A, RANTES-28 G and RANTES-IN1.1 C alleles were higher in HIV infected patients than in healthy controls (-403: 28.2% vs 15.1%,P = 0.002; -28: 5.4% vs 2.8%, not significant; IN1.1:19.0% vs 11.0%, P = 0.038). In HIV/HCV coinfected patients, these RANTES alleles were less frequent than in patients with HIV infection alone (15.4% P = 0.002;1.7%; P = 0.048; 12.0%; not significant). Frequencies of these alleles were not significantly different between HIV/HCV positive patients, HCV positive patients and healthy controls.CONCLUSION: All three RANTES polymorphisms showed increased frequencies of the variant allele exclusively in patients with HIV monoinfection. The finding that the frequencies of these alleles remained unaltered in HIV/HCV coinfected patients suggests that HCV coinfection interferes with selection processes associated with these alleles in HIV infection.

No association between IRF3 polymorphism and susceptibility to hepatitis B virus infection in Chinese patients

AIM:To investigate the association between three tag single nucleotide polymorphisms (tagSNPs) in inter-feron regulatory factors (IRF3) and the genetic suscep-tibility to chronic hepatitis B virus (HBV) infection.METHODS:We performed a case-control study of 985 Chinese cases of chronic HBV infection and 294 self-limiting HBV-infected individuals as controls.Three tagSNPs in IRF3 (rs10415576,rs2304204,rs2304206) were genotyped with the Multiplex SNaPshot technique.The genotype and allele frequencies were calculatedand analyzed.RESULTS:The three SNPs showed no significant geno-type/allele associations with chronic HBV infection.Overall allele P values were:rs10415576,P=0.0908,odds ratio (OR) [95% confidence interval (CI)]=1.1798 (0.9740-1.4291);rs2304204,P=0.5959,OR (95% CI)=1.0597 (0.8552-1.3133);rs2304206,P=0.8372,OR (95% CI)=1.0250 (0.8097-1.2976).Overall genotype P values were:rs10415576,P=0.2106;rs2304204,P=0.8458;rs2304206,P=0.8315.There were no statisti-cally significant differences between patients with chron-ic HBV infection and controls.Haplotypes generated by these three SNPs were also not significantly different between the two groups.CONCLUSION:The three tagSNPs of IRF3 are not asso-ciated with HBV infection in the Han Chinese population.

A new polymorphism in the GRP78 is not associated with HBV invasion

AIM:To examine the association between -86 bp(T>A) in the glucose-regulated protein 78 gene(GRP78) and hepatitis B virus(HBV) invasion.METHODS:DNA was genotyped for the single-nucleotide polymorphism by polymerase chain reaction followed by sequencing in a sample of 382 unrelated HBV carriers and a total of 350 sex-and age-matched healthy controls.Serological markers for HBV infection were determined by enzyme-linked immunosorbent as-say kits or clinical chemistry testing.RESULTS:The distributions of allelotype and genotype in cases were not significantly different from those in controls.In addition,our fi ndings suggested that neither alanine aminotransferase/hepatitis B e antigen nor HBV-DNA were associated with the allele/genotype variation in HBV infected individuals.CONCLUSION:-86 bp T>A polymorphism in GRP78 gene is not related to the clinical risk and acute exacerbation of HBV invasion.

Association between MDM2-SNP309 and hepatocellular carcinoma in Taiwan Residents population

AIM:To investigate the risk association and compare the onset age of hepatocellular carcinoma(HCC) patients in Taiwan with different genotypes of MDM2- SNP309. METHODS:We analyzed MDM2-SNP309 genotypes from 58 patients with HCC and 138 cancer-free healthy controls consecutively.Genotyping of MDM2-SNP309 was conducted by restriction fragment length polymor- phism assay. RESULTS:The proportion of homozygous MDM2- SNP309 genotype(G/G)in cases and cancer-free healthy controls was similar(17.2%vs 16.7%).Multi-variate analysis showed that the risk of G/G genotypeof MDM2-SNP309 vs wild-type T/T genotype in patients with HCC was not significant(OR=1.265,95% CI=0.074-21.77)after adjustment for sex,hepatitis B or C virus infection,age,and cardiovascular disease/ diabetes.Nevertheless,there was a trend that GG genotype of MDM2-SNP309 might increase the risk in HCC patients infected with hepatitis virus(OR=2.568, 95%CI=0.054-121.69).Besides,the homozygous MDM2-SNP309 genotype did not exhibit a significantly earlier age of onset for HCC. CONCLUSION:Current data suggest that the asso- ciation between MDM2-SNP309 GG genotype and HCC is not significant,while the risk may be enhanced in patients infected by hepatitis virus in Taiwan.

Polymorphisms of interleukin-1R receptor antagonist genes in patients with chronic hepatitis B in Iran

AIM： To investigate the relationships between polymorphisms of interleukin-1R receptor antagonist genes and susceptibility to chronic hepatitis B in Iran population. METHODS： Genomic DNA was extracted from the peripheral blood of 80 patients with chronic hepatitis B （57 males, 23 females） aged 12-77 years （mean 36.1 ± 13.8 years） and 147 normal controls （96 males, 51 females） aged 6-75 years （mean 41 ± 18.7 years） who referred to a liver clinic of Tehran and then subjected to polymerase chain reaction （PCR） amplification. PCR products were resolved on a 3% agarose gel and stained with ethidium bromide. RESULTS： Only three of the five kinds of polymorphism （2/2, 2/4, and 4/4） were found in this study. The frequencies of 2/2, 2/4, and 4/4 were 12.5%, 17.5%, 70% respectively in chronic hepatitis B patients and 6.8%, 24.5%, and 68.7% respectively in controls. IL-1 R allele 2 was detected in 30% of chronic hepatitis B patients and in 31.3% of controls, while IL-1 R allele 4 was detected in 87.5% of chronic hepatitis B patients and in 93.2% of controls. The frequency of IL-1R alleles 2 and 4 was detected in 21.25% and 78.75% of the patients and 19.04% and 80.96% of the controls, respectively. CONCLUSION： Our results suggest that the carriage of IL-1R receptor antagonist alleles 2, 4, 6 may not play any role in the development of HBV infection. Large population-based studies are needed to investigate the role of IL-1 polymorphisms in the pathogenesis of developing chronic hepatitis B.

Genetic polymorphism of UL144 open reading frame of human cytomegalovirus DNA detected in colon samples from infants with Hirschsprung's disease

AIM： To explore the genetic diversities of UL144 open reading frame （ORF） of cytomegalovirus DNA detected in colon tissue from infants with Hirschsprung＇s disease （HD） by sequencing UL144 DNA in 23 aganglionic colon tissue and 4 urine samples from 25 HD infants. METHODS： Nest PCR was performed for amplification of the UL144 gene. The UL144 gene was analyzed with soffwares, such as DNAclub, BioEdit, PROSITE database, and DNAstar. RESULTS： The strains from HD patients were distributed among three genotypes of UL144： group 1A （64%）, group 2 （24%）, and group 3 （12%）. The UL144 genotypes between strains from HD and control group were compared by chi square test （x^2 = 1.870, P = 0.393）. Strains from the colon were sporadically distributed in UL144 genotypes. CONCLUSION： There are genetic diversities of UL144 ORF in colon tissue of infants with HD. However, cytomegalovirus UL144 genotypes are not associated with clinical manifestations of HD.

Infection frequency of Epstein-Barr virus in subgingival samples from patients with different periodontal status and its correlation with clinical parameters

Objective: To detect the infection frequencies of different genotypes of Epstein-Barr virus (EBV) in subgingival samples from chronic periodontitis (CP) patients, and to discuss the correlation between infection with EBV and clinical pa-rameters. Methods: Nested-PCR assay was used to detect EBV-1 and EBV-2 in subgingival samples from 65 CP patients, 65 gingivitis patients and 24 periodontally healthy individuals. The amplicons were further identified by restriction fragment length polymorphism analysis (RFLP) with endonucleases Afa I and Stu I. Clinical parameters mainly included bleeding on probing (BOP), probing depth (PD), attachment loss (AL) in six sites of the dentition. Results: In CP patients, gingivitis and periodontally healthy individuals, the infection frequencies were 47.7%, 24.6% and 16.7% for EBV-1, and 15.4%, 7.7% and 0% for EBV-2, respectively. In 2 out of the 65 CP patients co-infection of EBV-1 and EBV-2 was found. The positive rate of EBV-1 in chronic periodontitis patients was higher than that in gingivitis patients (P=0.01) and periodontally healthy individuals (P=0.01). But no significant difference was shown in EBV-1 frequency between gingivitis patients and healthy individuals (P>0.05) or in EBV-2 frequency among the three groups (P>0.05). In CP patients, higher mean BOP value was found in EBV-1 or EBV-2 positive patients than that in EBV negative ones (P<0.01), but with no statistical difference in the mean PD or AL value between EBV positive and negative patients (P>0.05). After initial periodontal treatment, 12 out of the 21 EBV-1 positive CP patients did not show detectable EBV-1 in subgingival samples. Conclusion: nPCR plus RFLP analysis is a sensitive, specific and stable method to detect EBV-1 and EBV-2 in subgingival samples. Subgingival infection with EBV-1 is closely associated with chronic perio-dontitis. Infection of EBV in subgingival samples was correlated with BOP.

IL28B polymorphisms associated with therapy response in Chilean chronic hepatitis C patients

AIM:To analyze the association of three IL28B single nucleotide polymorphisms with response to therapy in Chilean patients infected with hepatitis C virus CV.METHODS:We studied two groups of patients with chronic CV infection genotype 1,under standard combined treatment with pegylated interferon plus ribavirin.One group consisted of 50 patients with sustained virological response,whereas the second group consisted of 49 null responders.In order to analyze the IL28B single nucleotide polymorphisms rs12979860,rs12980275 and rs8099917,samples were used for polymerase chain reaction amplification,and the genotyping was performed by restriction fragment lengthpolymorphism.RESULTS:The IL28B rs12979860 CC,rs12980275 AA and rs8099917 TT genotypes were much more frequently found in patients with sustained virological response compared to null responders 38%,44% and 50% vs 2%,8.2% and 8.2%,respectively.These differences were highly significant in all three cases(P < 0.0001.CONCLUSION:The three IL28B polymorphisms studied are strongly associated with sustained virological response to therapy in Chilean patients with chronic CV genotype 1.

Analysis of Methylation-Sensitive Amplified Polymorphism and Prediction of Candidate Genes Infected by SCMV in Zea mays Genome

DNA methylation is an important component of the epigenetic network, and it plays important roles in gene expression regulation and epigenetic change response to various stresses. In this study, the authors assessed the methylation patterns stressed by SCMV （sugarcane mosaic virus） in maize by methylation-sensitive amplified polymorphism （MSAP）, and identified important candidate genes related to SCMV resistance through combining microarray analysis with CpG islands prediction. The results of MSAP indicated DNA methylation levels appeared dynamic changes inoculated for 0 d, 1 d, 4 d, 5 d and 10 d. 118 candidate genes were identified infected by SCMV, which may participate in DNA methylation modification. Among them, eight candidate genes were mapped on Scmvl and Scmv2 QTL regions, which are crucial for SCMV resistance. In conclusion, DNA methylation is closely related with maize resistance to SCMV and plays an important role in regulating gene expression responded to maize resistance.

计算机病毒与反病毒技术的新动向

本文较为全面地介绍了目前计算机病毒技术的发展新动向及反病毒技术的新发展,提出了未来反病毒技术中需要加强的功能。