Estimation of inhibitory quotient using a comparative equilibrium dialysis assay for prediction of viral response to hepatitis C virus inhibitors

Summary. The relationship of inhibitory quotient (IQ) with the virologic response to specific inhibitors of human hepatitis C virus (HCV) and the best method to correct for serum protein binding in calculating IQ have not been addressed. A common method is to determine a fold shift by comparing the EC₅₀ values determined in cell culture in the absence and presence of human serum (fold shift in EC₅₀), but this method has a number of disadvantages. In the present study, the fold shifts in drug concentrations between 100% human plasma (HP) and cell culture medium (CCM) were directly measured using a modified comparative equilibrium dialysis (CED) assay for three HCV protease inhibitors (PIs) and for a novel HCV inhibitor GS‐9132. The fold shift values in drug concentration between the HP and CCM (CED ratio) were ～1 for SCH‐503034, VX‐950 and GS‐9132 and 13 for BILN‐2061. These values were ～3–10‐fold lower than the fold shift values calculated from the EC₅₀ assay for all inhibitors except BILN‐2061. Using the CED values, a consistent pharmacokinetic and pharmacodynamic relationship was observed for the four HCV inhibitors analysed. Specifically, an approximate 1 log₁₀ reduction in HCV RNA was achieved with an IQ close to 1, while 2–3 and greater log₁₀ reductions in HCV RNA were achieved with IQ values of 3–5 and greater, respectively. Thus, use of CED to define IQ provides a predictive and quantitative approach for the assessment of the in vivo potency of HCV PIs and GS‐9132. This method provides a framework for the evaluation of other classes of drugs that are bound by serum proteins but require the presence of serum for in vitro evaluation.     

Zero incidence of inhibitor development in previously treated Haemophilia A, HIV-negative patients upon exposure to a plasma-derived high-purity and double viral inactivated factor VIII concentrate

Thirty-six Haemophilia A, HIV-negative, previously treated patients were changed therapy to a high-purity and double-inactivated (solvent/detergent and dry-heating) previously unused factor VIII concentrate. The mean age of these patients was 27 years at the time of the change. Twenty-three patients were severe Haemophiliacs (FVIII:C < 0.02 IU mL⁻¹), seven moderate (FVIII:C between 0.02 and 0.05 IU mL⁻¹) and six mild (FVIII:C > 0.05 IU mL⁻¹). The mean follow-up with this single product was 16 months, with 82 accumulated exposure days and the mean consumption was 117 300 IU of FVIII corresponding to a mean of six batches per patient. No patient developed FVIII inhibitors (upper limit of the CI95: 7.98%), resulting in an incidence rate of 0/48 patient-years (upper limit of the CI95: 77/1000 patient-years). The change in therapy to this new factor VIII concentrate was not associated with the appearance of inhibitors.     

Inhibitors of the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B

More than 20 years after the identification of the hepatitis C virus (HCV) as a novel human pathogen, the only approved treatment remains a combination of pegylated interferon-α and ribavirin. This rather non-specific therapy is associated with severe side effects and by far not everyone benefits from treatment. Recently, progress has been made in the development of specifically targeted antiviral therapy for HCV (STAT-C). A major target for such direct acting antivirals (DAAs) is the HCV RNA-dependent RNA polymerase or non-structural protein 5B (NS5B), which is essential for viral replication. This review will examine the current state of development of inhibitors targeting the polymerase and issues such as the emergence of antiviral resistance during treatment, as well as strategies to address this problem.     

New drugs for hepatitis C virus

No new therapy has been approved for the treatment of chronic hepatitis C in the last 5 years in the USA since the approval of pegylated interferon (IFN)-α_2a and ribavirin. Multiple new drugs are currently in development and are expected to be approved for use in the USA and/or the EU by 2011–2013. Although the mechanism of action of pegylated IFN and ribavirin are not completely known, it is likely that they will continue to be used in combination regimens for a number of years. Direct antivirals are likely to be the first new drugs to be used in combination with pegylated IFN and ribavirin. Viral resistance will prove to be a significant barrier and require that consolidation therapy with at least 24 weeks of pegylated IFN and ribavirin be used to successfully prevent the selection or emergence of resistant variants. Numerous other compounds, such as ribavirin analogs, long-acting IFNs, hepatoprotectants and immunomodulators, are in development and may replace the drugs that are used currently. The combination of direct antivirals, such as protease and polymerase inhibitors, may rapidly follow in development, as has occurred in HIV drug therapy.     

Identification of Novel Antiviral Compounds Targeting Entry of Hantaviruses

Hemorrhagic fever viruses, among them orthohantaviruses, arenaviruses and filoviruses, are responsible for some of the most severe human diseases and represent a serious challenge for public health. The current limited therapeutic options and available vaccines make the development of novel efficacious antiviral agents an urgent need. Inhibiting viral attachment and entry is a promising strategy for the development of new treatments and to prevent all subsequent steps in virus infection. Here, we developed a fluorescence-based screening assay for the identification of new antivirals against hemorrhagic fever virus entry. We screened a phytochemical library containing 320 natural compounds using a validated VSV pseudotype platform bearing the glycoprotein of the virus of interest and encoding enhanced green fluorescent protein (EGFP). EGFP expression allows the quantitative detection of infection and the identification of compounds affecting viral entry. We identified several hits against four pseudoviruses for the orthohantaviruses Hantaan (HTNV) and Andes (ANDV), the filovirus Ebola (EBOV) and the arenavirus Lassa (LASV). Two selected inhibitors, emetine dihydrochloride and tetrandrine, were validated with infectious pathogenic HTNV in a BSL-3 laboratory. This study provides potential therapeutics against emerging virus infection, and highlights the importance of drug repurposing.     

白细胞介素17A对小鼠柯萨奇B3病毒性心肌炎心肌纤维化的影响

目的探讨白细胞介素17A（interleukin-17A,IL-17A）对小鼠病毒性心肌炎（viral myocarditis,VMC）心肌纤维化的影响。方法将100μL柯萨奇病毒B3（coxsackie-virus B3,CVB3）分别感染BALb/c小鼠野生型（wild-type,WT）和IL-17A基因敲除型（IL-17A-deficient,IL-17A-/-）建立小鼠心肌炎模型,造模后0 d、14 d、28 d、42 d分别取心肌组织作石蜡切片,行苏木素伊红及Masson染色观察病理改变,免疫组化（SP法）检测心肌基质金属蛋白酶（matrix metalloproteinases,MMP）-2、MMP-9及组织金属蛋白酶抑制剂-1（tissue inhibitors of metalloproteinase-1,TIMP-1）的表达。结果与WT小鼠比较,IL-17A-/-小鼠在感染CVB3后心肌炎症及纤维化程度明显减轻,生存率提高。并且MMP-2和MMP-9在IL-17A-/-小鼠心肌中的表达在14 d和28 d明显低于其在WT小鼠心肌中的表达,差异有统计学意义[14 d亚组：0.21±0.02 vs.0.32±0.04,0.22±0.02 vs.0.38±0.05,P均〈0.05;28 d亚组：0.14±0.02 vs.0.22±0.02,0.15±0.01 vs.0.26±0.03,P均〈0.05];而TIMP-1在IL-17A-/-小鼠心肌中的表达则在14 d显著高于其在WT小鼠心肌中的表达,差异有统计学意义（P〈0.05）。WT组小鼠心肌MMP-2/TIMP-1、MMP-9/TIMP-1的值先升高后降低,峰值出现在14 d左右。而IL-17A-/-组小鼠感染CVB3后14 d、28 d和42 d三个亚组心肌MMP-2/TIMP-1、MMP-9/TIMP-1的值较0 d亚组有所降低,差异有统计学意义（P〈0.05）,但以上各亚组之间比较则差异无统计学意义（P〉0.05）。结论 IL-17A基因敲除有助于维持MMP-2/TIMP-1和MMP-9/TIMP-1的动态平衡。IL-17A可能通过介导胶原合成/降解系统的失衡参与小鼠VMC心肌纤维化过程,提示阻断IL-17A的作用有助于减轻VMC心肌损伤后心肌纤维化的程度。     

Resistance against Integrase Strand Transfer Inhibitors and Relevance to HIV Persistence

Drug resistance prevents the successful treatment of HIV-positive individuals by decreasing viral sensitivity to a drug or a class of drugs. In addition to transmitted resistant viruses, treatment-naïve individuals can be confronted with the problem of drug resistance through de novo emergence of such variants. Resistant viruses have been reported for every antiretroviral drug tested so far, including the integrase strand transfer inhibitors raltegravir, elvitegravir and dolutegravir. However, de novo resistant variants against dolutegravir have been found in treatment-experienced but not in treatment-naïve individuals, a characteristic that is unique amongst antiretroviral drugs. We review here the issue of drug resistance against integrase strand transfer inhibitors as well as both pre-clinical and clinical studies that have led to the identification of the R263K mutation in integrase as a signature resistance substitution for dolutegravir. We also discuss how the topic of drug resistance against integrase strand transfer inhibitors may have relevance in regard to the nature of the HIV reservoir and possible HIV curative strategies.     

从病毒性心肌炎演变为扩张型心肌病:病毒的作用

病毒感染、宿主免疫反应、以及遗传和环境变化是决定病毒性心肌炎向扩张型心肌病演变的重要因素,其中病毒感染既是启动又是影响疾病发生发展的关键环节。近年研究发现,病毒不仅对心肌细胞有直接和间接损伤作用,而且还通过逃逸宿主先天免疫、诱导免疫因子分泌或表达异常等机制推动疾病进程。抗病毒治疗有益于病毒性心肌炎患者的恢复,在一定程度上抑制扩张型心肌病的发生。     

Viral Aggregation: The Knowns and Unknowns

Viral aggregation is a complex and pervasive phenomenon affecting many viral families. An increasing number of studies have indicated that it can modulate critical parameters surrounding viral infections, and yet its role in viral infectivity, pathogenesis, and evolution is just beginning to be appreciated. Aggregation likely promotes viral infection by increasing the cellular multiplicity of infection (MOI), which can help overcome stochastic failures of viral infection and genetic defects and subsequently modulate their fitness, virulence, and host responses. Conversely, aggregation can limit the dispersal of viral particles and hinder the early stages of establishing a successful infection. The cost–benefit of viral aggregation seems to vary not only depending on the viral species and aggregating factors but also on the spatiotemporal context of the viral life cycle. Here, we review the knowns of viral aggregation by focusing on studies with direct observations of viral aggregation and mechanistic studies of the aggregation process. Next, we chart the unknowns and discuss the biological implications of viral aggregation in their infection cycle. We conclude with a perspective on harnessing the therapeutic potential of this phenomenon and highlight several challenging questions that warrant further research for this field to advance.     

From the Cover: Ribonucleotide reductases reveal novel viral diversity and predict biological and ecological features of unknown marine viruses

Virioplankton play a crucial role in aquatic ecosystems as top-down regulators of bacterial populations and agents of horizontal gene transfer and nutrient cycling. However, the biology and ecology of virioplankton populations in the environment remain poorly understood. Ribonucleotide reductases (RNRs) are ancient enzymes that reduce ribonucleotides to deoxyribonucleotides and thus prime DNA synthesis. Composed of three classes according to O₂ reactivity, RNRs can be predictive of the physiological conditions surrounding DNA synthesis. RNRs are universal among cellular life, common within viral genomes and virioplankton shotgun metagenomes (viromes), and estimated to occur within >90% of the dsDNA virioplankton sampled in this study. RNRs occur across diverse viral groups, including all three morphological families of tailed phages, making these genes attractive for studies of viral diversity. Differing patterns in virioplankton diversity were clear from RNRs sampled across a broad oceanic transect. The most abundant RNRs belonged to novel lineages of podoviruses infecting α-proteobacteria, a bacterial class critical to oceanic carbon cycling. RNR class was predictive of phage morphology among cyanophages and RNR distribution frequencies among cyanophages were largely consistent with the predictions of the “kill the winner–cost of resistance” model. RNRs were also identified for the first time to our knowledge within ssDNA viromes. These data indicate that RNR polymorphism provides a means of connecting the biological and ecological features of virioplankton populations.Viruses are key players in biogeochemical cycling and energy flow and help shape the composition of aquatic microbial communities (1–3). Additionally, viruses influence microbial metabolism through horizontal gene transfer and expression of auxiliary metabolic genes during infection (4). Despite their impact, we understand little about the specific biological features and ecological strategies of viral populations within natural ecosystems. Constraining these second-order issues is critical to building better quantitative models of how viral processes affect ecosystems (5).Methodological limitations have hindered efforts to understand viral ecology. Viruses lack a universally conserved phylogenetic marker, akin to the 16S rRNA gene in cells, which can broadly assay viral distributions and diversity. Marker genes used as proxies of environmental viral diversity are typically limited to specific viral taxa. Furthermore, PCR-based approaches can fail to detect prominent and biologically important viral populations owing to the potential for low nucleotide similarity between homologous genes. Recent work examining the diversity of viral DNA polymerase A genes within virioplankton metagenomic (virome) sequence data revealed that low-efficiency DNA polymerases, undetected by PCR, were predominant within virioplankton (6). That work also highlighted the unique ability of DNA polA sequences to provide insights into the biological features of unknown phages within the virioplankton. In general, the ability to connect biological features with sequence diversity in marker genes—including those widely used in ecological studies, such as the 16S rRNA gene—can be tenuous (7).Ideally, a marker gene of viral diversity should (i) be widely distributed among diverse viral lineages and, therefore, evolutionarily ancient; (ii) be abundant within environmental viral assemblages; (iii) play an important role in viral biology; (iv) have a single evolutionary origin and not be replaceable through nonorthologous gene displacement; (v) be phylogenetically informative; and (vi) be well represented in reference databases. Ribonucleotide reductase (RNR) gene products fulfill these criteria. Nucleotide metabolism pathways, including biosynthesis, are among the most represented within the virioplankton (8, 9). RNRs are the only known enzymes capable of reducing ribonucleotides to deoxyribonucleotides (10), an essential step for DNA synthesis. As such, RNRs are key to nucleotide biosynthesis, under stringent evolutionary selection pressure, and among the most abundant annotated genes in marine virome libraries (11). Importantly, RNR genes are present in all three families of tailed phages in the order Caudovirales and have been identified in viruses infecting hosts within all three domains of life (10). RNRs are strongly tied to lytic marine phages (12), which significantly influence nutrient cycles within the global ocean (5). Therefore, RNRs easily fit the criteria of being functionally nonredundant, abundant, and widely distributed.In addition, RNRs are biologically informative and form three physiological classes according to reactivity with O₂. Class I RNRs are O₂-dependent. Class II RNRs are O₂-independent and rely upon adenosylcobalamin (vitamin B₁₂). Class III RNRs are sensitive to O₂. All three classes share a common catalytic center and use similar radical-based chemistry (13). Therefore, all three modern classes of RNR likely evolved from a single common ancestor (14). This study focused on the catalytic (alpha) subunit of the holoenzyme identified in virome libraries spanning a broad oceanic transect. Subsequently these data were used to examine the biological and ecological features of lytic phage populations within the Caudovirales. The outcomes of these analyses were interpreted within the context of known viral diversity and the “kill the winner–cost of resistance” (KTW–COR) model for viral–host interactions (15). Overall, these data show that RNR sequence diversity within the virioplankton connects broadly with phage morphological groups and can be predictive of the ecological strategies within the virioplankton.