Identification and characterization of two human immunodeficiency virus type 1 unique recombinant forms from Yunnan,China

正Dear Editor,Recombination contributes greatly to the diversity of human immunodeficiency virus type 1(HIV-1).A large number of recombinant strains have been found in China,particularly in Yunnan,which is considered the HIV-1 epicenter of China.Surveillance of unique recombinant forms is helpful for prediction of new circulating recombinant forms.In this study,we identified two unique     

禽流感病毒A型和H5亚型RT-PCR检测试剂盒研究

目的　检测和鉴定A型、H5亚型禽流感病毒 (AIV) ,研发一种高效实用的检测手段。方法　根据Ming ShiuhLee报道的文献设计、合成引物 ,采用反转录和PCR一步法对A型、H5亚型禽流感病毒cDNA进行扩增和电泳鉴定 ,组装成禽流感病毒RT PCR试剂盒 ,对H1～ 15亚型AIV参考株、38份AIV国内分离株进行检测试验。结果　建立了A型、H5亚型禽流感病毒RT PCR检测方法 ,并在此基础上组装试剂盒 ,用A型试剂盒检测时 ,全部AIV毒株均为阳性 ,能检测 1 10 2 4血凝单位禽流感病毒 ;用H5亚型试剂盒检测时 ,仅有H5亚型AIV参考株和 19株H5亚型AIV分离株呈阳性 ,其余H1～H4、H6～H15参考株和H7、H9分离株以及 1株H5分株均为阴性 ,能检测1 6 4血凝单位禽流感病毒。 2种试剂盒对实验感染鸡病料检出率均为 10 0 %。结论　研制的AIVA型、H5亚型RT PCR试剂盒具有特异性强、敏感性高、稳定性和重复性好的特点。     

New prevalence estimate of Torque Teno virus(TTV) infection in healthy population and patients with chronic viral hepatitis in Jiujiang,China

<正>Dear Editor,Torque Teno virus(TTV)is a nonenveloped human DNA virus that was isolated from the serum of a patient with transfusion-transmitted hepatitis with unknown etiology in 1997(Nishizawa et al.,1997).TTV is the first human virus with a single-stranded circular DNA genome to be identified,and is recently classified as the Alphatorquevirus genus of the Anelloviridae family by the International Committee on Taxonomy of Viruses(ICTV)(King et al.,2011).TTV shows very high genetic     

Differentiation of velogenic, mesogenic and lentogenic strains of Newcastle disease virus by multiplex RT-PCR

A multiplex RT‐PCR technique has been developed for differentiation of velogenic, mesogenic and lentogenic pathotypes of Newcastle disease virus (NDV), using a set of three oligonucleotide primers designed from NDV genomic RNA (P1, P2 and P3). The primer pair P1 and P2 generated a RT‐PCR product of 204 bp, only with RNA from velogenic and mesogenic strains, whereas the P1 and P3 generated a 364 bp product only with RNA from mesogenic and lentogenic strains. Thirty four NDV strains, including some reference strains (known pathotypes), NDV field isolates and NDV vaccine strains, as well as other avian virus strains, were tested with multiplex RT‐PCR. All reference strains tested were differentiated in agreement with their intracerebral pathogenicity index (ICPI) values or with the pathotypes known in previous reports. The nucleotide sequence analysis of RT‐PCR products for four NDV strains was fully in agreement with the RT‐PCR characterisations of these strains. The RT‐PCR results of other avian RNA viruses further confirmed the reliability and specificity of this technique. However, the RT‐PCR failed to detect some other avian NDV, which may not originate from chicken. This multiplex RT‐PCR technique is simple and easy to perform. It could be applied not only to determine the origin of NDV, but also may be used diagnostically in molecular epidemiological analysis of ND and for prediction of pathotypes of NDV isolates.     

Relevance of the branch point adenosine, coordination loop, and 3' exon binding site for in vivo excision of the Sinorhizobium meliloti group II intron RmInt1

Excision of the bacterial group II intron RmInt1 has been demonstrated in vivo, resulting in the formation of both intron lariat and putative intron RNA circles. We show here that the bulged adenosine in domain VI of RmInt1 is required for splicing via the branching pathway, but branch site mutants produce small numbers of RNA molecules in which the first G residue of the intron is linked to the last C residue. Mutations in the coordination loop in domain I reduced splicing efficiency, but branched templates clearly predominated among splicing products. We also found that a single substitution at the EBS3 position (G329C), preventing EBS3-IBS3 pairing, resulted in the production of 50 to 100 times more RNA molecules in which the 5' and 3' extremities were joined. We provide evidence that these intron molecules may correspond to both, intron circles linked by a 2'-5' phosphodiester bond, and tandem, head-to-tail intron copies.     

一步多重PCR同时检测甲型肝炎和脊髓灰质炎病毒研究

建立的一步ＰＣＲ方法即反转录和ＰＣＲ在同一管中进行,同时检测甲型肝炎和脊髓灰质炎病毒病毒ＲＮＡ。实验中对不同的反转录温度以及一步多重ＰＣＲ的特异性和灵敏度进行了探讨。结果表明：４２℃、５０℃反转录时ｐｏｌｉｏ有非特异性条带出现,６０℃反转录特异性较好,而ＨＡＶ在三种不同的反转录温度下均得到牧场划性较好的条带;应用一步ＰＣＲ同时检测两种病毒与检测单一病毒的灵敏度基本一致,但在同等反应条件下后者的反应效率高于前者,特别是在检测ＨＡＶ时。     

A latest and promising approach for prediction of viral load in hepatitis B virus infected patients

INTRODUCTION:

Designing a rapid, reliable and sensitive assay for detection of hepatitis B virus (HBV) variants by real-time PCR is challenging at best. A recent approach for quantifying the viral load using a sensitive fluorescent principle was brushed in this study.

MATERIALS AND METHODS

: A total of 250 samples were collected from the outpatient unit, CLRD. Complete Human HBVDNA sequences (n = 944) were selected from the National Centre for Biotechnology Information (NCBI), primers and probes were designed and synthesized from the core, surface, and x region. Real-time based quantification was carried out using a standard kit and in-house generated standards and RT-PCR protocols.

RESULTS AND DISCUSSION:

The standard calibration curve was generated by using serial dilution 10² to 10⁸. The calibration curve was linear in a range from 10² to 10⁸ copies/ml, with an R² value of 0.999. Reproducibility as measured by dual testing of triplicates of serum samples was acceptable, with coefficients of variation at 6.5%, 7.5%, and 10.5%. Our results showed that amplification performance was good in the case of the x-region-based design (98%). Out of 100 negative samples screened by enzyme linked immunosorbent assay and the standard RT-PCR kit, one sample was detected as positive with the in-house developed RT-PCR assay, the positivity of the sample was confirmed by sequencing the amplified product, NCBI accession {"type":"entrez-nucleotide","attrs":{"text":"EU684022","term_id":"189176131","term_text":"EU684022"}}EU684022.

CONCLUSION:

This assay is reproducible showing limited inter- and intra-assay variability. We demonstrate that the results of our assay correlated well with the standard kit for the HBV viral load monitor.     

鸭甲肝病毒1型和3型间接ELISA方法的建立与应用

【目的】研究鸭甲肝病毒(Duck hepatitis A virus,DHAV)1型VP3和3型VP1串联基因在大肠杆菌中的表达,并以纯化的重组蛋白为抗原建立鸭病毒性肝炎抗体检测的间接ELISA方法。【方法】设计2对特异性引物,提取鸭甲肝病毒1型(DHAV-1)和3型(DHAV-3)的RNA,分别RT-PCR扩增得到714bp的DHAV-1VP3和720bp的DHAV-3 VP1基因,并克隆至p MD18-T载体中,然后依次将DHAV-1 VP3和DHAV-3 VP1定向插入p ET-32a(+)表达载体,获得重组表达载体p ET-1VP3-3VP1,进行IPTG(Isopropyl-beta-D-1-thiogalactopyranoside)诱导表达分析,以纯化的重组蛋白为抗原建立间接ELISA方法并应用于临床。【结果】经IPTG诱导表达,在大肠杆菌中可稳定、高效地表达DHAV-1VP3-3VP1蛋白。Western blot检测结果表明,表达的重组蛋白与DHAV-1和DHAV-3阳性血清均能发生特异性反应。确定间接ELISA方法的抗原最佳包被浓度为1.0μg/孔,血清最佳稀释度为1∶200,临界值为OD6 50值≥0.38,建立的间接ELISA方法具有较好的敏感性、特异性和重复性。该方法与中和试验分别检测DHAV-3阳性血清,两种方法的符合率各为96.3%和96.7%;初步临床应用结果表明该方法可用于雏鸭母源抗体和免疫后抗体的消长变化的检测。【结论】以大肠杆菌表达的DHAV-1VP3-3VP1重组蛋白建立的间接ELISA方法可用于DHAV-1和DHAV-3抗体的检测。     

乙型肝炎病毒DNA聚合酶反式调节蛋白HBVDNAPTP1的表达、纯化与初步鉴定

目的构建HBVDNAPTP1基因的原核表达载体,诱导其在大肠埃希菌中表达,并对融合蛋白进行纯化。方法利用逆转录-PCR获得乙型肝炎病毒(HBV)DNA聚合酶(Polymerase)反式调节人类新基因HBVD-NAPTP1,测序正确后插入至原核表达载体pET-32a(+)中,转化BL21(DE3)宿主菌进行诱导,并利用组氨酸亲和层析方法对融合蛋白进行纯化。结果 HBVDNAPTP1原核表达载体转化宿主菌后,经0.5 mmol/L IPTG、30℃诱导5 h获得了分子量约为31 kD的HBVDNAPTP1融合蛋白的优化表达,Western blotting证实融合蛋白的特异性。亲和层析纯化后得到较纯的HBVDNAPTP1融合蛋白,每升培养菌液中可获得2.24 mg的纯化蛋白。结论成功获得纯化的HBVDNAPTP1融合蛋白,为今后开展HBVDNAPTP1的生物学功能研究奠定了物质基础。     

Combined X-ray, NMR, and kinetic analyses reveal uncommon binding characteristics of the hepatitis C virus NS3-NS4A protease inhibitor BI 201335

Hepatitis C virus infection, a major cause of liver disease worldwide, is curable, but currently approved therapies have suboptimal efficacy. Supplementing these therapies with direct-acting antiviral agents has the potential to considerably improve treatment prospects for hepatitis C virus-infected patients. The critical role played by the viral NS3 protease makes it an attractive target, and despite its shallow, solvent-exposed active site, several potent NS3 protease inhibitors are currently in the clinic. BI 201335, which is progressing through Phase IIb trials, contains a unique C-terminal carboxylic acid that binds noncovalently to the active site and a bromo-quinoline substitution on its proline residue that provides significant potency. In this work we have used stopped flow kinetics, x-ray crystallography, and NMR to characterize these distinctive features. Key findings include: slow association and dissociation rates within a single-step binding mechanism; the critical involvement of water molecules in acid binding; and protein side chain rearrangements, a bromine-oxygen halogen bond, and profound pK(a) changes within the catalytic triad associated with binding of the bromo-quinoline moiety.     

Polymorphisms of glutathione S-transferases M1, T1, P1 and A1 genes in the Tunisian population: an intra and interethnic comparative approach

Genetic polymorphisms in glutathione S-transferases (GSTs) genes might influence the detoxification activities of the enzymes predisposing individuals to cancer risk. Owing to the presence of these genetic variants, inter-individual and ethnic differences in GSTs detoxification capacity have been observed in various populations. Therefore, the present study was performed to determine the prevalence GSTM1 0/0, GSTT1 0/0, GSTP1 Ile(105)Val, and GSTA1 A/B polymorphisms in 154 healthy individuals from South Tunisia, and to compare them with those observed in North and Centre Tunisian populations and other ethnic groups. GSTM1 and GSTT1 polymorphisms were analyzed by a Multiplex-PCR approach, whereas GSTP1 and GSTA1 polymorphisms were examined by PCR-RFLP. The frequencies of GSTM10/0 and GSTT1 0/0 genotypes were 53.9% and 27.9%, respectively. The genotype distribution of GSTP1 was 47.4% (Ile/Ile), 40.9% (Ile/Val), and 11.7% (Val/Val). For GSTA1, the genotype distribution was 24.7% (A/A), 53.9% (A/B), and 21.4% (B/B). The combined genotypes distribution of GSTM1, GSTT1, GSTP1 and GSTA1 polymorphisms showed that thirty one of the 36 possible genotypes were present in our population; eight of them have a frequency greater than 5%. To the best of our knowledge, this is the first report of GSTs polymorphisms in South Tunisian population. Our findings demonstrate the impact of ethnicity and reveal a characteristic pattern for Tunisian population. The molecular studies in these enzymes provide basis for further epidemiological investigations in the population where these functional polymorphisms alter therapeutic response and act as susceptibility markers for various clinical conditions.     

Characterization of the C3 receptor induced by herpes simplex virus type 1 infection of human epidermal, endothelial, and A431 cells

Herpes simplex virus type 1 (HSV-1) infection induces the appearance of viral analogues of human Fc IgG and C3 receptors on the surface of human cells. The virally induced C3 receptor(s) has been broadly defined as a C3b receptor, but its ligand binding characteristics have not been rigorously defined. In this study, human epidermal cells, A431 cells, and human umbilical vein endothelial cells infected with HSV-1 demonstrated rosetting with sheep erythrocytes (E) coated with IgG (E-IgG) or the complement components C3b (EAC3b) or iC3b (EAC3bi), but not with E-IgM, C4 (EAC14), C3d (EAC3d), or E alone. Rosetting was markedly enhanced by pretreatment of HSV-1-infected cells with neuraminidase. Unlike human C3 receptors, the HSV-1-induced C3 receptor was found to be trypsin resistant. To determine whether HSV-1 induced CR1-like receptors or CR3-like receptors, infected cells were pretreated with EDTA, which is known to inhibit native CR3 function. EDTA failed to prevent rosetting with EAC3bi. Furthermore, blocking studies using monoclonal antibodies against CR1 and CR3 revealed that the anti-CR1 antibody 5C11 consistently blocked EAC3b and EAC3bi rosetting with HSV-1-infected cells in a dose dependent manner, but monoclonal antibodies against CR3 did not. This study indicates that the HSV-1-induced C3 receptor is an analogue of CR1.     

SORL1 and SIRT1 mRNA expression and promoter methylation levels in aging and Alzheimer’s Disease

Alzheimer’s Disease (AD) is a neurodegenerative disorder and the most common cause of dementia among the elderly. Efforts have been made to understand the genetic and epigenetic mechanisms involved in the development of this disease. As SORL1 (sortilin-related receptor) and SIRT1 (sirtuin 1) genes have been linked to AD pathogenesis, we aimed to investigate their mRNA expression and promoter DNA methylation in post mortem brain tissues (entorhinal and auditory cortices and hippocampus) from healthy elderly subjects and AD patients. We also evaluated these levels in peripheral blood leukocytes from young, healthy elderly and AD patients, investigating whether there was an effect of age on these profiles. The comparative CT method by Real Time PCR and MALDI-TOF mass spectrometry were used to analyze gene expression and DNA methylation, respectively. SORL1 gene was differently expressed in the peripheral blood leukocytes and might act as a marker of aging in this tissue. Furthermore, we found that SORL1 promoter DNA methylation might act as one of the mechanisms responsible for the differences in expression observed between blood and brain for both healthy elderly and AD patients groups. The impact of these studied genes on AD pathogenesis remains to be better clarified.     

A quadruplex tetra-primer ARMS-PCR method for the simultaneous detection of TP53 Arg72Pro, IVS3 16bp Del/Ins and IVS6+62A>G, and NQO1 C609T polymorphisms

The apoptotic pathway has been shown to be crucial in the development of cancers in addition to a variety of neurodegenerative disorders. The tumor suppressor gene (TP53) encodes p53, the central protein in the apoptotic pathway. The NAD(P)H:quinone oxidoreductase 1, which is encoded by the NQO1 gene and, plays a direct role in apoptosis in addition to its recently discovered role as a regulator for p53. Three most commonly studied polymorphisms that were shown to affect the biochemical functions of p53 protein are the exon 4 Arg72pro, Intron 3 16bp Del/Ins, and Intron 6 A>G polymorphisms. The exon 6 C609T polymorphism was shown to significantly affect NQO1 enzymatic activity. The currently used methods for the separate detection of the four polymorphisms are either slow and laborious or extremely expensive. In this paper, a new highly optimized method for the simultaneous detection of the four polymorphisms is described. The proposed method utilizes 13 primers in a single PCR reaction to detect the four polymorphisms simultaneously based on the principle of tetra-primer ARMS-PCR (also known as PCR-CTPP). The proposed method offers extremely fast, economical, and simple detection. The proposed method was successfully applied to a sample of the Syrian population (n=144), where we found a unique distribution for TP53 polymorphisms that differed from the major ethnic groups. The proposed method is the first to simultaneously detect four polymorphisms including 3 SNPs in a single PCR reaction based on tetra-primer ARMS-PCR or PCR-CTPP, and can serve as an invaluable tool for the investigation of TP53 haplotypes and the combined effects of the TP53 and NQO1 genes with respect to apoptosis and susceptibility for various types of cancers and neurodegenerative disorders.