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  • 1.

    【2hr】Genome Stability of Pandemic Influenza A (H1N1) 2009 Based on Analysis of Hemagglutinin and Neuraminidase Genes

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    机译 基于血凝素和神经氨酸酶基因的分析，2009年甲型H1N1流感大流行的基因组稳定性
    摘要：Influenza A virus (H1N1), which arose in 2009, constituted the fourth pandemic after the cases of 1918, 1957, and 1968. This new variant was formed by a triple reassortment, with genomic segments from swine, avian, and human influenza origins. The objective of this study was to analyze sequences of hemagglutinin (n=2038) and neuraminidase (n=1273) genes, in order to assess the extent of diversity among circulating 2009-2010 strains, estimate if these genes evolved through positive, negative, or neutral selection models of evolution during the pandemic phase, and analyze the worldwide percentage of detection of important amino acid mutations that could enhance the viral performance, such as transmissibility or resistance to drugs. A continuous surveillance by public health authorities will be critical to monitor the appearance of new influenza variants, especially in animal reservoirs such as swine and birds, in order to prevent the potential animal-human transmission of viruses with pandemic potential.
  • 2.

    【2hr】Pilot Evaluation of RT-PCR/Electrospray Ionization Mass Spectrometry (PLEX-ID/Flu assay) on Influenza-Positive Specimens

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    机译 对流感阳性标本进行RT-PCR /电喷雾电离质谱（PLEX-ID / Flu分析）的初步评估
    摘要：The PLEX-ID/Flu assay has been recently developed to enable the detection and typing of influenza viruses based on the RT-PCR/electrospray ionization mass spectrometry technology.This novel assay was evaluated for typing performance on 201 positive influenza A or B nasopharyngeal swab specimens (NPS) detected by real-time RT-PCR during the 2010-2011 season. The PLEX-ID/Flu assay detected and characterized 91.3% and 95.3% of all influenza A and B samples, respectively. All non-typeable influenza A and B specimens by the assay showed low viral loads with threshold cycle values ≥ 33. Taken together, and although our results need to be confirmed by further prospective studies, the PLEX-ID/Flu assay detected positively and gave a typing result for 93% of all NPS detected positively by real-time RT-PCR, thus suggesting a potential role for influenza virus surveillance among other techniques.
  • 3.

    【2hr】The E89K Mutation in the Matrix Protein of the Measles Virus Affects In Vitro Cell Death and Virus Replication Efficiency in Human PBMC

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    机译 麻疹病毒基质蛋白中的E89K突变影响人PBMC的体外细胞死亡和病毒复制效率
    摘要：Matrix protein is known to have an important role in the process of virus assembly and virion release during measles virus replication. In the present in vitro study, a single mutation of E89K in the matrix protein was shown to affect cell death and virus replication efficiency in human PBMC. One strain with this mutation caused less cell death than the parental virus, and possessed high virus replication efficiency. Moreover, by Annexin V-FITC staining, polycaspase FLICA staining, and double labeling with poly-caspase FLICA and the Hoechst stain, the cell death seen was shown to be apoptosis.
  • 4.

    【2hr】Evaluation of Nucleocapsid and Phosphoprotein P Functionality as Critical Factors During the Early Phase of Paramyxoviral Infection

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    机译 副粘病毒感染早期阶段的关键因素评估核衣壳和磷蛋白P功能
    摘要：In the beginning of a paramyxovirus infection after cell entry viral survival depends on efficient primary (1°) transcription and on the stability of only one input nucleocapsid. Here we examined the influence of the viral polymerase co-factor phosphoprotein P on the very early phase of an infection, i.e. before progeny nucleocapsids are synthesized. We used a novel set-up with Sendai virus (SeV) mutants incapable of genome replication: SeV-ΔP with the entire P ORF deleted, SeV-PΔ2-77 with the deletion of aa 2-77. These mutants allow maintaining the state of the very beginning of an infection when statistically one viral genome is present in the cell. This single genome serves as template for transcription. During SeV-ΔP infections only early 1° transcription takes place at low levels. However, when the truncated P protein is expressed in SeV-PΔ2-77 infections, 1° transcription levels rise significantly up to an 8-fold increased amount of viral mRNA. This shows that the P protein is able to support transcription and thereby mediates the transition from early to late 1° transcription. Importantly, nucleocapsids of both mutants could be shown to remain stable and functional for at least 5 days – even without de novo P protein synthesis. These results describe a novel function of the P protein: enhancing viral gene expression even before genome replication has started. Thus, the since long postulated supportive function of the P protein is not related to stabilization of the nucleocapsid but rather enhances the processivity of the viral polymerase during late 1° and secondary (2°) transcription and genome replication.
  • 5.

    【2hr】Development and Field Testing of a Real-Time PCR Assay for Caprine Arthritis-Encephalitis-Virus (CAEV)

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    机译 山羊关节炎-脑炎病毒（CAEV）实时PCR分析的开发和现场测试
    摘要：Caprine arthritis/encephalitis (CAE) of goats and occasionally sheep are persistent virus infections caused by a lentivirus (CAEV). This viral infection results in arthritis in adult animals and encephalitis in kids. Prognosis for the encephalitic form is normally poor, with substantial economic loss for the farm. In this context an early/fast laboratory diagnosis for CAEV infection could be useful for effective prophylactic action. In this work we performed a quantitative real time PCR designed on the CAEV env gene to detect/quantify in goat/sheep samples, viral RNA or proviral DNA forms of CAEV. This procedure was validated in 15 sheep, experimentally infected with CAEV or with a highly correlated lentivirus (visna maedi, MVV); in addition, a total of 37 clinical goat specimens recruited in CAEV positive herds were analyzed and compared using serological analysis (Elisa and AGID). All samples infected with MVV resulted negative. In sheep experimentally infected with CAEV, proviral DNA was detectable 15 days post infection, whereas the serological methods revealed an indicative positivity after 40-60 days.This method showed a sensitivity of 102 env fragments/PCR) with a linear dynamic range of quantitation from 103 to 107 env fragments/PCR; the R2 correlation coefficient was 0.98. All subjects with a clinical diagnosis for Caprine Arthritis-Encephalitis (CAE) resulted CAEV DNA positive.
  • 6.

    【2hr】Suppression of Neointimal Hyperplasia Following Angioplasty-Induced Vascular Injury in Pigs Infected with Swinepox Virus

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    机译 猪瘟病毒感染猪血管成形术后血管损伤后新内膜增生的抑制
    摘要：Many patients suffering from angina pectoris are treated with percutaneous coronary intervention (PCI) and quickly develop angiographic renarrowing, or restenosis, at the site of PCI treatment. Restenosis is thought to arise from the combinatorial activation of thrombotic and inflammatory responses. The inflammatory response responsible for restenosis is also thought to involve the activation of a cascade of serine proteases and its subsequent regulation. Poxviruses are known to possess a variety of immunomodulatory strategies, some of which target serine proteases, cytokines, and chemokines. To this end we evaluated whether systemic species-specific swinepox virus (SPV) infection could induce sufficient host-immune modulation to promote an anti-inflammatory and anti-proliferative effect, thereby preventing restenosis. Two groups of domestic feeder pigs were used - the first group was experimentally infected with SPV (n= 11) and the second group served as an uninfected control (n= 5). A week after infection, the pigs were anesthetized and percutaneous transluminal coronary angioplasty (PTCA) was performed in the left anterior descending coronary artery using X-ray fluoroscopy to visualize the balloon and record angiograms. Three weeks post infection, the pigs were euthanized and balloon angioplasty injured arteries were harvested and examined. We observed a statistically significant reduction of restenosis in SPV-infected pigs (p = 0.05) compared to control pigs and conclude that systemic swinepox virus infection causes sufficient host immune suppression to significantly reduce restenosis in pigs after balloon angioplasty injury.
  • 7.

    【2hr】Development of a Flow Cytometry Live Cell Assay for the Screening of Inhibitors of Hepatitis C Virus (HCV) Replication

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    机译 流式细胞仪活细胞检测方法的发展，用于筛选丙型肝炎病毒（HCV）复制抑制剂
    摘要：In this study, we established a flow cytometry live cell-based assay that permits the screening of hepatitis C virus (HCV) inhibitors. Specifically, we created a stable cell line, which harbors a subgenomic replicon encoding an NS5A-YFP fusion protein. This system allows direct measurement of YFP fluorescence in live hepatoma cells in which the HCV replicon replicates. We demonstrated that this stable fluorescent system permits the rapid and sensitive quantification of HCV replication inhibition by direct-acting antiviral agents (DAA) including protease and NS5A inhibitors and host-targeting antiviral agents (HTA) including cyclophilin inhibitors. This flow cytometry-based live cell assay is well suited for multiple applications such as the evaluation of HCV replication as well as antiviral drug screening.
  • 8.

    【2hr】Suppl 1: Application of Molecular Diagnostic Techniques for Viral Testing

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    机译 增刊1：分子诊断技术在病毒检测中的应用
    • 作者：Fernando Cobo
    • 刊名：The Open Virology Journal
    • 2012年第期
    摘要：Nucleic acid amplification techniques are commonly used currently to diagnose viral diseases and manage patients with this kind of illnesses. These techniques have had a rapid but unconventional route of development during the last 30 years, with the discovery and introduction of several assays in clinical diagnosis. The increase in the number of commercially available methods has facilitated the use of this technology in the majority of laboratories worldwide. This technology has reduced the use of some other techniques such as viral culture based methods and serological assays in the clinical virology laboratory. Moreover, nucleic acid amplification techniques are now the methods of reference and also the most useful assays for the diagnosis in several diseases. The introduction of these techniques and their automation provides new opportunities for the clinical laboratory to affect patient care. The main objectives in performing nucleic acid tests in this field are to provide timely results useful for high-quality patient care at a reasonable cost, because rapid results are associated with improvements in patients care. The use of amplification techniques such as polymerase chain reaction, real-time polymerase chain reaction or nucleic acid sequence-based amplification for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range. This review is an up-to-date of the main nucleic acid techniques and their clinical applications, and special challenges and opportunities that these techniques currently provide for the clinical virology laboratory.
  • 9.

    【2hr】Suppl 1: Strategies for the Successful Implementation of Viral Laboratory Automation

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    机译 增刊1：成功实施病毒实验室自动化的策略
    摘要：It has been estimated that more than 70% of all medical activity is directly related to information providing analytical data. Substantial technological advances have taken place recently, which have allowed a previously unimagined number of analytical samples to be processed while offering high quality results. Concurrently, yet more new diagnostic determinations have been introduced - all of which has led to a significant increase in the prescription of analytical parameters. This increased workload has placed great pressure on the laboratory with respect to health costs. The present manager of the Clinical Laboratory (CL) has had to examine cost control as well as rationing - meaning that the CL’s focus has not been strictly metrological, as if it were purely a system producing results, but instead has had to concentrate on its efficiency and efficacy. By applying re-engineering criteria, an emphasis has had to be placed on improved organisation and operating practice within the CL, focussing on the current criteria of the Integrated Management Areas where the technical and human resources are brought together. This re-engineering has been based on the concepts of consolidating and integrating the analytical platforms, while differentiating the production areas (CORE Laboratory) from the information areas. With these present concepts in mind, automation and virological treatment, along with serology in general, follow the same criteria as the rest of the operating methodology in the Clinical Laboratory.
  • 10.

    【2hr】Suppl 1: Introduction of an Automated System for the Diagnosis and Quantification of Hepatitis B and Hepatitis C Viruses

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    机译 增刊1：介绍了一种自动系统，用于诊断和定量乙型肝炎和丙型肝炎病毒
    摘要：Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infections pose major public health problems because of their prevalence worldwide. Consequently, screening for these infections is an important part of routine laboratory activity. Serological and molecular markers are key elements in diagnosis, prognosis and treatment monitoring for HBV and HCV infections. Today, automated chemiluminescence immunoassay (CLIA) analyzers are widely used for virological diagnosis, particularly in high-volume clinical laboratories. Molecular biology techniques are routinely used to detect and quantify viral genomes as well as to analyze their sequence; in order to determine their genotype and detect resistance to antiviral drugs. Real-time PCR, which provides high sensitivity and a broad dynamic range, has gradually replaced other signal and target amplification technologies for the quantification and detection of nucleic acid. The next-generation DNA sequencing techniques are still restricted to research laboratories.The serological and molecular marker methods available for HBV and HCV are discussed in this article, along with their utility and limitations for use in Chronic Hepatitis B (CHB) diagnosis and monitoring.
  • 11.

    【2hr】Suppl 1: Improving Clinical Laboratory Efficiency: Introduction of Systems for the Diagnosis and Monitoring of HIV Infection

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    机译 增刊1：提高临床实验室效率：引入用于诊断和监测HIV感染的系统
    摘要：Since the first tests for identifying individuals with suspected human immunodeficiency virus (HIV) infection were introduced in the mid-1980s, diagnostic virology testing has greatly evolved. The technological advances, automating in the laboratories and the advances in molecular biology techniques have helped introduce invaluable laboratory methods for managing HIV patients. Tests for diagnosis, specially for screening HIV antibodies, are now fully automated; in the same way, tests for monitoring HIV viral load (HIV RNA copies/ml of plasma), which is used for monitoring infection and response to antiretroviral treatment, are also fully automated; however, resistance testing, tropism determination and minor variant detection, which are used to make decisions for changing antiretroviral treatment regimens in patients failing therapy, still remain highly laborious and time consuming. This chapter will review the main aspects relating to the automating of the methods available for laboratory diagnosis as well as for monitoring of the HIV infection and determination of resistance to antiretrovirals and viral tropism.
  • 12.

    【2hr】Suppl 1: Human Papillomavirus (HPV) Genotyping: Automation and Application in Routine Laboratory Testing

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    机译 增刊1：人乳头瘤病毒（HPV）基因分型：自动化及其在常规实验室检测中的应用
    摘要：A large number of assays designed for genotyping human papillomaviruses (HPV) have been developed in the last years. They perform within a wide range of analytical sensitivity and specificity values for the different viral types, and are used either for diagnosis, epidemiological studies, evaluation of vaccines and implementing and monitoring of vaccination programs. Methods for specific genotyping of HPV-16 and HPV-18 are also useful for the prevention of cervical cancer in screening programs. Some commercial tests are, in addition, fully or partially automated. Automation of HPV genotyping presents advantages such as the simplicity of the testing procedure for the operator, the ability to process a large number of samples in a short time, and the reduction of human errors from manual operations, allowing a better quality assurance and a reduction of cost. The present review collects information about the current HPV genotyping tests, with special attention to practical aspects influencing their use in clinical laboratories.
  • 13.

    【2hr】Suppl 1: Laboratory Detection of Respiratory Viruses by Automated Techniques

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    机译 补充1：通过自动化技术在实验室检测呼吸道病毒
    摘要：Advances in clinical virology for detecting respiratory viruses have been focused on nucleic acids amplification techniques, which have converted in the reference method for the diagnosis of acute respiratory infections of viral aetiology. Improvements of current commercial molecular assays to reduce hands-on-time rely on two strategies, a stepwise automation (semi-automation) and the complete automation of the whole procedure. Contributions to the former strategy have been the use of automated nucleic acids extractors, multiplex PCR, real-time PCR and/or DNA arrays for detection of amplicons. Commercial fully-automated molecular systems are now available for the detection of respiratory viruses. Some of them could convert in point-of-care methods substituting antigen tests for detection of respiratory syncytial virus and influenza A and B viruses. This article describes laboratory methods for detection of respiratory viruses. A cost-effective and rational diagnostic algorithm is proposed, considering technical aspects of the available assays, infrastructure possibilities of each laboratory and clinic-epidemiologic factors of the infection
  • 14.

    【2hr】Suppl 2: Loss of HPV16 E2 Protein Expression Without Disruption of the E2 ORF Correlates with Carcinogenic Progression

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    机译 补充2：HPV16 E2蛋白表达的丧失而不会破坏E2 ORF与致癌进展相关
    摘要：Integration of the viral DNA in the cellular genome has been suggested to be critical in carcinogenic progression of HPV-associated cervical neoplasia. This event can be accompanied by disruption of the open reading frame (ORF) encoding the E2 repressor, thus leading to transcriptional up-regulation of the E6 and E7 viral oncogenes. At this stage, it is unclear whether disruption of the E2 ORF is mandatory for carcinogenic progression. We measured E2 RNA and protein expression in clinical samples of various grades of HPV16-associated cervical neoplasia and compared it with the status of the viral genome. RNA extracted from paraffin embedded tissues was hybridized to specific probes and quantified by the NanoString technology. Protein expression was appreciated by immunohistochemistry and the status of viral DNA was determined by in situ hybridization, all performed on serial sections of the same samples. E2 protein was found highly expressed in CIN1, CIN2 lesions where the HPV DNA was highly replicative, while it was decreased in more advanced grade lesions where replication is decreased or lost (CIN3 and SCC). In contrast, E2 transcripts could be elevated even in conditions of no or low expression of the protein, as found in the Caski cell line. Our data demonstrate that integration of the viral DNA in the cellular genome does not always lead to disruption of the E2 ORF and drastic reduction of E2 transcripts, while in contrast, expression of the E2 protein is always drastically reduced.
  • 15.

    【2hr】Suppl 2: The HPV E2-Host Protein-Protein Interactions: A Complex Hijacking of the Cellular Network

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    机译 补充2：HPV E2-宿主蛋白-蛋白质相互作用：细胞网络的复杂劫持
    摘要：Over 100 genotypes of human papillomaviruses (HPVs) have been identified as being responsible for unapparent infections or for lesions ranging from benign skin or genital warts to cancer. The pathogenesis of HPV results from complex relationships between viral and host factors, driven in particular by the interplay between the host proteome and the early viral proteins. The E2 protein regulates the transcription, the replication as well as the mitotic segregation of the viral genome through the recruitment of host cell factors to the HPV regulatory region. It is thereby a pivotal factor for the productive viral life cycle and for viral persistence, a major risk factor for cancer development. In addition, the E2 proteins have been shown to engage numerous interactions through which they play important roles in modulating the host cell. Such E2 activities are probably contributing to create cell conditions appropriate for the successive stages of the viral life cycle, and some of these activities have been demonstrated only for the oncogenic high-risk HPV. The recent mapping of E2-host protein-protein interactions with 12 genotypes representative of HPV diversity has shed some light on the large complexity of the host cell hijacking and on its diversity according to viral genotypes. This article reviews the functions of E2 as they emerge from the E2/host proteome interplay, taking into account the large-scale comparative interactomic study.
  • 16.

    【2hr】Suppl 2: The Biology of Papillomavirus Latency

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    机译 补充2：乳头瘤病毒潜伏期生物学
    摘要：The presence of viral DNA in the absence of disease has suggested that papillomaviruses, like many other viruses, can exist as latent infections in the skin or other epithelial sites. In animal models, where detailed investigation has been carried out, papillomavirus DNA can be found at sites of previous infection following immune regression, with the site of latent infection being the epithelial basal layer. Such studies suggest that immune surveillance can restrict viral gene expression in the basal and parabasal layers without efficiently suppressing viral genome replication, most probably through the action of memory T-cells in the skin or dermis. Although gradual papillomavirus genome loss appears to occur over time at latent sites, immunosuppression can arrest this, and can lead to an elevation in viral genome copy number in experimental systems. In addition to immune-mediated latency, it appears that a similar situation can be achieved following infection at low virus titres and/or infection at epithelial sites where the virus life cycle is not properly supported. Such silent of asymptomatic infections do not necessarily involve the host immune system and may be controlled by different mechanisms. It appears that virus reactivation can be triggered by mechanical irritation, wounding or by UV irradiation which changes the local environment. Although the duration of papillomavirus latency in humans is not yet known, it is likely that some of the basic principles will resemble those elucidated in these model systems, and that persistence in the absence of disease may be the default outcome for at least some period of time following regression.
  • 17.

    【2hr】Suppl 2: Evidence and Impact of Human Papillomavirus Latency

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    机译 补充2：人类乳头瘤病毒潜伏期的证据和影响
    • 作者：Patti E Gravitt
    • 刊名：The Open Virology Journal
    • 2012年第期
    摘要：At present, there is no consensus in the scientific community regarding the ability for human papillomavirus (HPV) infections to establish latency. Based on animal studies, a model of papillomavirus latency has been proposed in which papillomaviruses can be retained in the basal epithelial stem cell pool as latent infections and periodically induced to reactivate when the stem cell divides and one daughter cell is committed to terminal differentiation and induction of the viral life cycle. Tissue resident memory T-cells are hypothesized to control these periodic reactivation episodes and thus limit their duration. In this paper, evidence from human studies consistent with this model of papillomavirus latency is reviewed. Given the strong circumstantial evidence supporting a natural history of HPV infection which includes a immunologically controlled latent state, the longer term implications of HPV latency on a highly infected and aging population may warrant a more serious evaluation.
  • 18.

    【2hr】Suppl 2: RNA Interference for the Treatment of Papillomavirus Disease

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    机译 补充2：RNA干扰素治疗乳头瘤病毒病
    摘要：Human Papillomavirus (HPV)-induced diseases are a significant burden on our healthcare system and current therapies are not curative. Vaccination provides significant prophylactic protection but effective therapeutic treatments will still be required. RNA interference (RNAi) has great promise in providing highly specific therapies for all HPV diseases yet this promise has not been realised. Here we review the research into RNAi therapy for HPV in vitro and in vivo and examine the various targets and outcomes. We discuss the idea of using RNAi with current treatments and address delivery of RNAi, the major issue holding back clinical adoption. Finally, we present our view of a potential path to the clinic.
  • 19.

    【2hr】Suppl 2: A Decade of Global mRNA and miRNA Profiling of HPV-Positive Cell Lines and Clinical Specimens

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    机译 补充2：HPV阳性细胞系和临床标本的全球mRNA和miRNA分析的十年
    摘要：For more than a decade, global gene expression profiling has been extensively used to elucidate the biology of human papillomaviruses (HPV) and their role in cervical- and head-and-neck cancers. Since 2008, the expression profiling of miRNAs has been reported in multiple HPV studies. Two major strategies have been employed in the gene and miRNA profiling studies: In the first approach, HPV positive tumors were compared to normal tissues or to HPV negative tumors. The second strategy relied on analysis of cell cultures transfected with single HPV oncogenes or with HPV genomes compared to untransfected cells considered as models for the development of premalignant and malignant transformations.In this review, we summarize what we have learned from a decade of global expression profiling studies. We performed comprehensive analysis of the overlap of the lists of differentially expressed genes and microRNAs, in both tissue samples and cell culture based studies. The review focuses mainly on HPV16, however reports from other HPV species are used as references. We discuss the low degree of consensus among different studies and the limitation of differential expression analysis as well as the fragmented miRNA-mRNA target correlation evidence. Furthermore, we propose an approach for future research to include more comprehensive miRNA-mRNA target correlation analysis and to apply systems biology/gene networks methodology.
  • 20.

    【2hr】Suppl 2: Human Papillomavirus Infections and Cancer Stem Cells of Tumors from the Uterine Cervix

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    机译 补充2：人乳头瘤病毒感染和子宫宫颈肿瘤干细胞
    摘要：Different rate of development of productive infections (as low grade cervical intraepithelial neoplasias), or high grade lesions and cervical malignant tumors associated with infections of the Transformation zone (TZ) by High-Risk Human Papillomavirus (HR-HPV), could suggest that different epithelial host target cells could exist. If there is more than one target cell, their differential infection by HR-HPV may play a central role in the development of cervical cancer. Recently, the concept that cancer might arise from a rare population of cells with stem cell-like properties has received support in several solid tumors, including cervical cancer (CC). According to the cancer stem cell (CSC) hypothesis, CC can now be considered a disease in which stem cells of the TZ are converted to cervical cancer stem cells by the interplay between HR-HPV viral oncogenes and cellular alterations that are thought to be finally responsible for tumor initiation and maintenance. Current studies of CSC could provide novel insights regarding tumor initiation and progression, their relation with viral proteins and interplay with the tumor micro-environment. This review will focus on the biology of cervical cancer stem cells, which might contribute to our understanding of the mechanisms responsible for cervical tumor development.
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