Performance validation of hepatitis B virus DNA quantification by AUTRAX automatic nucleic acid extraction workstation / 国际检验医学杂志

Objective To verify the performance of a novel HBV DNA assay based on AUTRAX automatic nucleic acid extrac-tion workstation .Methods According to the evaluation protocols of Clinical and Laboratory Standards Institute (CLSI) ,the per-formance of a novel HBV DNA assay was assessed in the aspects of accuracy ,precision ,lower limit detection ,linearity ,interference rejection and pollution prevention .Results The accuracy rate of this assay for laboratory evaluation samples of Shanghai Clinical Laboratory Center was 100% during 2015 to 2016 ,with each deviation between observed and expected values within ± 0 .18 lg IU/mL .The intra-assay and inter -assay CV of two levels (106 ,104 ) samples were both less than 5% .Detectable rates of lower limit of detection (LOD) and lower limit of quantitation (LOQ) were 5/5 at 20IU/mL and 40 IU/mL ,respectively .And intra-assay CV of LOQ was 3 .18% ,less than 5% .Linearity assessment exhibited an excellent dynamic range of linear quantification from 40 to 108 IU/mL(Y=1 .0182X-0 .3182 ,R2 =0 .978) .According to product manual ,conjugated bilirubin ,hemoglobin and triglyceride as interfering substance were made at concentration of 600 μmol/L ,7 g/L and 4 .5 mmol/L ,separately ,which had no interference for two levels (106 ,104 ) samples .Each deviation value between interference group and control group was within ± 0 .45 lg IU/mL .No pollution phenomenon was found .Conclusion The novel HBV DNA quantification by AUTRAX automatic nucleic acid extraction workstation has excellent performance in aspects of accuracy ,precision ,lower limit detection ,linearity ,interference rejection and pollution prevention ,which can be used for the detection of clinical specimens .

Evaluation of a novel fully automated real-time PCR assay for hepatitis B virus DNA quantification / 中华检验医学杂志

Objective To evaluate the analytical performance of a novel HBV DNA assay based on automated DNA extraction and real-time fluorescence quantitative PCR .Methods Analytic verification studies.Accuracy and lower limit of detection were assessed by determining a panel of HBV standard plasma of WHO.HBV standard plasma (genotype A, B, C and D) at 6 different concentrations were measured 18 times to evaluate precision and reproducibility .Pseudo-viral particles at high HBV DNA concentration were serially diluted to assess linear range .One hundred and forty-four clinical specimens were quantified for HBV DNA so as to evaluate the correlation between the new test and COBAS ? system. Results Quantification of HBV standard plasma showed acceptable accuracy , with each deviation between observed and expected values within ±0.35 lg IU/ml (-0.17-0.32 lg IU/ml).Intra-assay coefficients of variation ( CV) for genotype A , B, C and D were 3.87% -6.32%, 0.45% -14.68%, 0.16% -8.36% and 0.64%-13.01%respectively, and the inter-assay CV were 5.67%-9.69%, 1.28%-15.68%, 0.36%-9.05%and 1.69%-13.65%, separately.Linearity assessment exhibited an excellent dynamic range of linear quantification from 20 to 1.0 ×1010 IU/ml ( r =0.998, P <0.001 ) .And the satisfactory results obtained at 3 levels of HBV DNA concentration (10, 20, 50 IU/ml, respectively) confirmed the claimed lower limit of detection with 5/5 detectable rate at 20 IU/ml.Furthermore, good correspondence was observed between the new HBV DNA assay and the COBAS ? system with 100% ( 144/144 ) qualitative coincidence and significant correlation based on 104 positive data ( r=0.984, P<0.000 1).Conclusions The novel fully-automated real-time PCR assay displayed good analytical and clinical performance for highly sensitive detection of HBV DNA.It was well suited for monitoring antiviral responses as well as drug resistance according to current clinical practice guidelines for the management of chronic HBV infection .

Detection of DNA virus in respiratory samples of children by multiplex PCR combined with RDB / 中国医师杂志

Objective To develop multiplex polymerase chain reaction (PCR) combined with reversedotblothy bridization (RDB) method for detection of DNA virus in respiratory samples,and provide a surveillance and rapid diagnosis tool of acute viral respiratory infection.Methods We designed multiple PCR primers and the probes referenced to virus nucleic acid sequences in the National Center for Biotechnology Information (NCBI) database,and fixed specific oligonucleotide probes on the nylon membrane.After multiple PCR amplification of virus DNA of human bocavirus (hBOV),karolinska Institutet (KI),adenovirus (AdV),Washington University polyomaviros (WUPyV),and human parvovirus B19 (HPVBI9),the denaturalized amplification products were hybridized with various specific probes,followed by visualization and analysis of the results.The sensitivity and specificity were tested.At the same time,108 cases of clinical specimens of multiple PCR products were analyzed by reverse spot hybridization detection,and compared to the results of culture method.Results The specific probes of multiple PCR-RDB only hybridized with corresponding amplification products without cross-hybridization reaction with other pathogen.The sensitivity of RDB hybridization was 1 colony-forming units (CFU).The positive rate of 34.26％ (37 cases out of 108 cases) with PCR-RDB method was significandy higher than that 27.78％ (30 cases out of 108 cases) with common test method.Conclusions The multiplex PCR combined with RDB might become a rapid and simple method to detect the DNA virus in respiratory samples,which might be a promising tool for clinical application.

Changes of programmed death receptor-1 in patients with different baseline hepatitis B virus DNA levels after treatment with adefovir dipivoxil / 中华传染病杂志

Objective To explore the changes of programmed death receptor-1 (PD-1) in chronic hepatitis B (CHB) patients with different baseline of hepatitis B virus (HBV) DNA levels after treatment with adefovir dipivoxil (ADV).Methods One hundred CHB patients with positive hepatitis B e antigen (HBeAg),1 × 104 copy/mL≤HBV DNA≤1 × 107 copy/mL,and positive human leukocyte antigen-A2 were divided into two groups according to the baseline HBV DNA level:47 cases in low virus load group whose HBV DNA level was ≤1 × 105 copy/mL; 53 cases in high virus load group whose HBV DNA level was＞1 × 105 copy/mL.Both groups were treated with ADV 10 mg/d.Serum HBV DNA,HBeAg seroconversion rate,alanine aminotransferase (ALT) and total bilirubin (TBil) levels of both groups before treatment and 12 months after treatment were compared.Flow cytometry was used to test peripheral blood HBV-specific cytotoxic T lymphocyte (CTL) surface PD-1 and peripheral blood HBV-specific CTL level.Categorical data were tested by x2 test; quantitative data was compared with t-test.Results Peripheral blood HBV-specific CTL surface PD-1 of CHB patients in low virus load group was 20.17 ％±1.69％,which was lower than that in high virus load group (41.38％±2.30％,t =53.02,P＜0.01) ; peripheral blood HBV specific CTL levels in two groups were 0.37％±0.02％ and 0.17％± 0.02％,respectively (t=50.47,P＜0.01) ; ALT and TBil levels in low virus load group were both lower than those of high virus load group (t=13.07,P＜0.01; t=5.06,P＜0.01).Twelve months after treatment,HBV DNA of 25 cases (53.2％) in low virus load group and 10 cases (18.9％) in high virus load group were lower than the detectable level (HBV DNA＜500 copy/mL,x2 =12.89,P＜0.01);HBeAg seroconversion was achieved in 15 cases(31.9％) and 1 case (1.9％),respectively (x2 =16.72,P＜0.01) ; peripheral blood HBV-specific CTL surface PD-1 expression levels were 9.00 ％ ±1.38 ％ and 29.40 ％ ± 3.76 ％,respectively (t =36.80,P＜ 0.01) ; peripheral blood HBV-specific CTL levels were 0.65％±0.10％ and0.48％±0.07％,respectively (t=9.61,P＜0.01).Conclusions After treatment with ADV,along with the decrease of HBV DNA load,HBV-specific CTL surface PD-1 expression decreases,while HBV-specific CTL level increases.The changes in low virus load group are much more remarkable.

Comparative Viral Metagenomics of Environmental Samples from Korea

The introduction of metagenomics into the field of virology has facilitated the exploration of viral communities in various natural habitats. Understanding the viral ecology of a variety of sample types throughout the biosphere is important per se, but it also has potential applications in clinical and diagnostic virology. However, the procedures used by viral metagenomics may produce technical errors, such as amplification bias, while public viral databases are very limited, which may hamper the determination of the viral diversity in samples. This review considers the current state of viral metagenomics, based on examples from Korean viral metagenomic studies-i.e., rice paddy soil, fermented foods, human gut, seawater, and the near-surface atmosphere. Viral metagenomics has become widespread due to various methodological developments, and much attention has been focused on studies that consider the intrinsic role of viruses that interact with their hosts.

Sensing DNA Viruses and Bacteria by Intracellular DNA Sensors

The innate immune system confers first-line defense against various pathogens including bacteria and viruses. Early detection of invading pathogens by the host depends on a limited number of specific pattern recognition receptors (PRRs) that detect pathogen associated molecular patterns (PAMPs) and activate signal transduction cascades that lead to activation of defense mechanisms. Among those sensors, RIG-I-like receptors (RLRs) play crucial roles in the detection of viruses by recognizing intracellular viral patterns such as viral RNAs to induce type-I interferon production. The discovery of intracellular RNA sensing mechanism by RIG-I prompted the investigations to find out intracellular DNA sensors. Recently, several proteins including DAI, AIM2, IFI16, and cGAS have been suggested as DNA sensing molecules to detect DNA viruses and bacteria, suggesting there are multiple receptors for microbial DNA. In this review, we discuss the current our understanding of sensing microbial DNA and subsequent induction of immune responses.

Infecção pelo papilomavírus humano: etiopatogenia, biologia molecular e manifestações clínicas / Human papillomavirus infection: etiopathogenesis, molecular biology and clinical manifestations

O papilomavírus humano (HPV) é um vírus DNA que apresenta tropismo por células epiteliais, causando infecções na pele e nas mucosas. A replicação do HPV ocorre no núcleo das células escamosas e o seu ciclo de vida é diretamente relacionado ao programa de diferenciação da célula hospedeira. Até o momento, foram completamente caracterizados cerca de 100 tipos diferentes de HPVs e há um grande número adicional de tipos ainda não sequenciados. Além de ser o responsável por lesões benignas de pele e mucosas, o HPV também está envolvido no desenvolvimento de diversos tumores cutaneomucosos: doença de Bowen, cânceres de pele não melanoma e carcinomas genitais. Esta revisão aborda as características do HPV, quadros cutâneos e mucosos benignos e malignos causados por ele e os principais métodos empregados em sua detecção e tipagem.

Human papillomavirus (HPV) is a DNA virus that presents tropism for epithelial cells, causing infections of the skin and mucous membranes. Replication of HPV occurs in the nuclei of squamous cells and its life cycle is directly related to the differentiation program of the host cell. To date, nearly 100 different types of HPV have been characterized and there is a large number of other types that have not been sequenced yet. Besides being responsible for benign lesions of the skin and mucous membranes, HPV is also involved in the development of various mucocutaneous tumors: Bowen's disease, non-melanoma skin cancers and genital carcinomas. This review discusses the characteristics of HPV, malignant and benign mucous and skin manifestations caused by HPV, besides the main methods of detection and typing of the virus.

Papilomavírus humano e neoplasia cervical: [revisão] / Human papillomavirus and cervical neoplasia: [revision]

Human papillomavirus (HPV) has been established as an important etiological factor for the development of cervical cancer. This DNA virus primarily infects the epithelium and can induce benign and malignant lesions of the mucous membranes and skin. Some HPVs are considered high risk due to their role in malignant progression of cervical tumors. Genital HPV infections are common and usually transient among young sexually active women. Only a small fraction of infected women develop cervical cancer, implying the involvement of environmental and genetic cofactors in cervical carcinogenesis. Classification, virology, pathology, natural history, epidemiological features of genital HPV infection, and future prospects for cervical cancer prevention with HPV vaccines will be reviewed here.

O papilomavírus humano (HPV) é um fator etiológico bem estabelecido para o câncer cervical. Esse vírus de DNA infecta primariamente o epitélio e pode induzir lesões benignas ou malignas na pele e na mucosa. Alguns HPVs são considerados de alto risco, responsáveis pela progressão das lesões precursoras até câncer cervical. A infecção genital pelo HPV é comum em mulheres jovens e geralmente é transitória. Uma pequena proporção de mulheres infectadas desenvolve câncer cervical, implicando o envolvimento de fatores ambientais e fatores genéticos na carcinogênese. Essa revisão aborda a estrutura viral, classificação e patologia do HPV, história natural e fatores de risco para neoplasia cervical e perspectivas futuras com a vacina anti-HPV.

Virion Proteomics of Large DNA Viruses / 中国病毒学·英文版

Large DNA viruses normally have complex structures with many of protein components derived from both viral and host origins. The development in proteomics, especially mass spectrometry identification techniques provide powerful tools for analyzing large viruses. In this review, we have summarized the recent achievements on proteomic studies of large DNA viruses, such as herpesvirus, poxvirus, nimavirus and baculoviruse. The proteomics of baculovirus occlusion-derived virions (ODV) were emphasized. Different mass spectrometry techniques used on ,carious baculoviruses were introduced, and the identified structurally associated proteins of baculoviruses are summarized.

Ascovirus and its Evolution / 中国病毒学

Ascoviruses, iridoviruses, asfarviruses and poxviruses are all cytoplasmic DNA viruses. The evolutionary origins of cytoplasmic DNA viruses have never been fully addressed. Morphological, genetic and molecular data were used to test if all four cytoplasmic virus families (Ascoviridae, Iridoviridae, Asfarviridae, and Poxvirirdae) evolved from nuclear replicating baculoviruses and how the four virus groups are related. Molecular phylogenetic analyses using DNA polymerase predicted that cytoplasmic DNA viruses might have evolved from nuclear replicating baculoviruses, and that poxviruses and asfarviruses share a common ancestor with iridoviruses. These three cytoplasmic viruses again shared a common ancestor with ascoviruses. Morphological and genetic data predicted the same evolutionary trend as molecular data predicted. A genome sequence comparison showed that ascoviruses have more baculovirus protein homologues than do iridoviruses, which suggested that ascoviruses have evolved from baculoviruses and iridoviruses evolved from ascoviruses. Poxviruses showed genetic and morphological similarity to other cytoplamic viruses, such as ascoviruses, suggesting it has undergone reticulate evolution via hybridization, recombination and lateral gene transfer with other viruses. Within the ascovirus family, we tested if molecular phylogenetic analyses agree with biological inference; that is, ascovirus had an evolutionary trend of increasing genome size, expanding host range and widening tissue tropism for these viruses. Both molecular and biological data predicted this evolutionary trend. The phylogenetic relationship among the four species of ascovirus was predicted to be that TnAV-2 and HvAV-3 shared a common ancestor with SfAV-1 and the three virus species again shared a common ancestor with DpAV-4.

Baculoviruses as Vectors in Mammalian Cells / 中国病毒学

The Baculoviridae are a large family of enveloped DNA viruses exclusively pathogenic to arthropods. Baculoviruses have been extensively used in insect cell-based recombinant protein expression system and as biological pesticides. They have been deomostrated to be safe to mammals, birds and fish. Recently, baculoviruses has been shown to transduce different mammalian cells in spite of the fact that they cannot replicate in mammalian cells (11, 73, 76). This has resulted in the development of baculoviruses as mammalian expression systems and even as vestors for gene therapy.

Detection of transfusion transmitted virus infection and genotypes in pregnant women / 中华妇产科杂志

Objective To clarify the frequency, the routes of transmission from mother to infant, the correlation factors and distribution of genotypes of transfusion transmitted virus (TTV) infection. Methods Nested polymerase chain reaction (n PCR) was performed to detect TTV DNA and genotypes in serums and breast milks from 160 cases of pregnant women. Results TTV DNA in serum and breast milk was detected in 64 (40 0%) and 60 (37 5%) of 160 cases of pregnant women respectively. The positive rates of TTV DNA from HBV markers (+) and normal groups were 50 0%, 43 1% and 13 6%, 22 7% respectively in serums and breast milk. There were significant difference between the two groups ( P

Investigation of HBV-DNA in peripheral blood mononuclear cells and hepatocytes with molecular hybridization / 第三军医大学学报

The existence of HBV-DNA in the mononuclear cells of peripheral blood and in the serum of 61 patients with HBV infection was determined with southern blot and dot blot hybridization,and that in the liver tissue of 31 patients out of the 61 with southern blot and in situ hybridization.The positive rate of HBV-DNA in the serum,mononuclear cells and hepatocytrs was 26.2% (16/61),24.6% (15/61) and 44.8% (13/31) respectively.There was no concordance of the existence of HBV-DNA in the serum,peripheral mono-nuclear cells and hepatocytes in an individual.For example,HBV-DNA was absent in the serum but present in mononuclear cells and hepatocytes in 11 cases.In fact,peripheral mononuclsar cells can serve as the reservoir for HBV to replicate.It cannot be denied that HBV can replicate in an individual even though HBV-DNA is negative in the serum.