Germacrone inhibits early stages of influenza virus infection

Highly pathogenic influenza viruses pose a serious public health threat to humans. Although vaccines are available, antivirals are needed to efficiently control disease progression and virus transmission due to the emergence of drug-resistant viral strains. In this study, germacrone, which is a major component of the essential oils extracted from Rhizoma Curcuma, was found to inhibit influenza virus replication. Germacrone showed antiviral activity against the H1N1 and H3N2 influenza A viruses and the influenza B virus in a dose-dependent manner. The viral protein expression, RNA synthesis and the production of infectious progeny viruses were decreased both in MDCK and A549 cells treated with germacrone. In a time-of-addition study, germacrone was found to exhibit an inhibitory effect on both the attachment/entry step and the early stages of the viral replication cycle. Germacrone also exhibited an effective protection of mice from lethal infection and reduced the virus titres in the lung. Furthermore, the combination of germacrone and oseltamivir exhibited an additive effect on the inhibition of influenza virus infection, both in vitro and in vivo. Our results suggest that germacrone may have the potential to be developed as a therapeutic agent alone or in combination with other agents for the treatment of influenza virus infection.     

Novel mode of action of ribavirin (Rebetol), a drug for the treatment of chronic hepatitis C: inducting the mutation of RNA viruses

Ribavirin (Rebetol) is an antiviral agent used in combination with interferon alpha-2b (IFN alpha-2b) for the treatment of chronic hepatitis C. Ribavirin has been reported to have a broad-spectrum antiviral activity mainly against RNA viruses. The effect of ribavirin was potentiated when administered in combination with IFN alpha-2b in an antiviral assay using bovine viral diarrhea virus as a surrogate for hepatitis C virus (HCV). Inhibition of host inosine monophosphate dehydrogenase and inhibition of RNA-dependent RNA polymerase (RdRp) of RNA viruses have been reported as the modes of action of ribavirin. Recently, ribavirin has been shown to induce mutation as template for newly generated RNA after uptake in RNA by RdRp of poliovirus, which is an RNA virus as in the case of HCV. It has also been shown that the infectivity of viruses is drastically reduced by the very slight increase in mutations induced by ribavirin. This effect as a mutagen on RNA viruses is a novel mode of ribavirin, and it is thought necessary to classify ribavirin into a new antiviral drug class.     

Evaluation of TV cell line viral susceptibility using conventional cell culture techniques

Despite the fact that a lot of methods have been developed for rapid virus detection, classic cell culture is still “the golden standard”. The range of viruses that can be isolated and cultured in cell line systems is often limited by the susceptibility of cells to support viral replication. Since the primary cell culture, the best cellular system available to support replication of a large number of viruses, is very expensive and diffcult to obtain, cell lines, which are easier to manipulate, are commonly used for virus growth and isolation. In two previous papers we described the TV cell line initiated by our team from a laryngeal tumor, which harbors human papillomavirus (HPV) gene sequences. In this paper we analyze its capacity to support virus replication. Depending on the virus, different cytopathic effects were produced. Comparison of viral effect observed on this cell line with the effect obtained on other cell lines has been performed. This cell line might be used in the clinical virology laboratory for virus isolation.     

Antiviral action of lysolecithin analogs against human pathogenic viruses

The virucidal effect, the inhibition of virus adsorption and penetration and the influence on later phases of replication of human pathogenic viruses were studied. The compounds showed a significant virucidal effect to enveloped viruses (measles virus, herpes virus type 1 and 2), whereas the compounds were ineffective against nonenveloped viruses (Coxsackie virus A9 and B1, attenuated poliovirus type 1, adenovirus type 1). Interactions with the viral envelope are supposed.     

SP600125 inhibits Orthopoxviruses replication in a JNK1/2 -independent manner: Implication as a potential antipoxviral

The pharmacological inhibitor SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone] has been largely employed as a c-JUN N-terminal kinase (JNK1/2) inhibitor. In this study, we evaluated whether pretreatment with SP600125 was able to prevent Orthopoxviruses Vaccinia virus (VACV), Cowpox virus (CPXV) and modified Vaccinia virus Ankara (MVA) replication. We found that incubation with SP600125 not only blocked virus-stimulated JNK phosphorylation, but also, significantly reduced virus production. We observed 1-3 log decline in viral yield depending on the cell line infected (A31, BSC-40 or BHK-21). The reduction in viral yield correlated with a dramatic impact on virus morphogenesis progress, intracellular mature viruses (IMV) were barely detected. Despite the fact that SP600125 can act as an efficient anti-orthopoxviral compound, we also provide evidence that this antiviral effect is not specifically exerted through JNK1/2 inhibition. This conclusion is supported by the fact that viral titers measured after infections of JNK1/2 knockout cells were not altered as compared to those of wild-type cells. In contrast, a decline in viral titers was verified when the infection of KO cells was carried out in the presence of the pharmacological inhibitor. SP600125 has been the focus of recent studies that have evaluated its action on diverse viral infections including DNA viruses. Our data support the notion that SP600125 can be regarded as a potential antipoxviral compound.     

病毒学研究与抗病毒治疗药物

人们在与病毒的长期斗争中取得了不少重要成就,一些病毒进入人体细胞的过程及其致病机制相继得到阐明。随着病毒学研究的进步,许多抗病毒治疗药物不断出现并被用于临床,人们治疗艾滋病、病毒性肝炎、流感和疱疹病毒感染等病毒性疾病也由此取得了明显进步。然而,面对众多病毒性疾病,人们现有的有效治疗措施仍然极为有限。加强病毒学研究以加深对病毒致病机制的认识是研发新型抗病毒治疗药物的关键。本文介绍目前对临床常见病毒的研究与治疗进展,强调病毒学研究的进步在促进抗病毒治疗药物开发中的重要作用。     

Effect of amantadine on rhabdovirus infection

Enveloped viruses enter host cells by fusion or viropexis. The latter mechanism is the prevalent entry pathway of rhabdoviruses into susceptible cells. Amantadine, a lysosomotropic agent, inhibits the multiplication of various groups of viruses. The effect of this drug was investigated on vesicular stomatitis virus and rabies fixed virus strain replication in fibroblasts. Amantadine was added to cells before, during and after infection to detect the phase of viral replication affected by the drug. Cells were inoculated with viruses at 4 degrees C and the incubation temperature was progressively raised to 37 degrees C in order to observe the effect of amantadine on attachment and early stages of viral replication. Experimental results indicated that the compound inhibited rhabdovirus infection in CER cells. Viral attachment and penetration did not appear to be affected by the drug, while later steps were inhibited, probably at the level of uncoating when the virus is released from the lysosomes into the intracytoplasmic compartment.     

Bovine lactoferrin prevents the entry and intercellular spread of herpes simplex virus type 1 in Green Monkey Kidney cells

Lactoferrin (Lf) is a multifunctional glycoprotein that plays an important role in immune regulation and defence mechanisms against bacteria, fungi and viruses. Bovine lactoferrin (bLf) has been recognized as a potent inhibitor of human herpetic viruses, such as cytomegalovirus and herpes simplex virus type 1 and 2. BLf has been found to prevent viral infection by binding to heparan sulphate containing proteoglycans that also act as cell receptors for herpetic viruses. In this study we further investigated the inhibiting activity of bLf against herpes simplex virus type 1 (HSV-1) in Green Monkey Kidney (GMK) cells and found that, in addition to the viral adsorption step, bLf also targets the HSV-1 entry process and cell-to-cell viral spread. Our study showed that the inhibition of HSV-1 infectivity by bLf is dependent on its interaction with specific structural viral proteins. Apart from the prevention of early phases of viral infection, cell-to-cell spread inhibition activity of HSV-1 by bLf confirmed that this protein is an outstanding candidate for the treatment of herpetic infections since it would offer the advantage to prevent also viral infections caused by cell-associated virus.     

HSV-1 viral oncolysis and molecular imaging with PET

Viral oncolysis, the destruction of cancer cells by replicating viruses, is a new modality of cancer therapy. This strategy involves use of viruses that are either genetically engineered to replicate preferentially in neoplastic cells, or use of viruses that display innate tropism for neoplastic cells. These viruses may also be modified to deliver transgenes to destroy cancer cells. While numerous viruses may be used for this form of cancer therapy, HSV-1 is an attractive vector for viral oncolysis due to several characteristics including its high infectivity, ease of genetic engineering, large transgene capacity, and the availability of an effective medical treatment for Herpes simplex virus infections. The HSV-1 viral genome has been manipulated to generate replication conditional viruses which target cancer cells. Although these viruses are programmed to replicate preferentially in cancer cells, there is some unintended replication in normal cells. Currently, biopsy is the gold standard for monitoring the therapeutic effects of viral oncolysis. However, a non-invasive test capable of serial monitoring of therapy during the treatment period is required for both preclinical and clinical studies. Positron emission tomography (PET) using HSV thymidine kinase as the PET reporter gene offers the desired qualities of a non-invasive test which can be easily repeated to determine the location and magnitude of viral replication and tumor lysis. We review viral oncolysis, focusing on HSV-1 viral oncolysis and therapeutic monitoring by PET.     

Oncolytic viruses as novel anticancer agents: turning one scourge against another

Although the use of viruses as oncolytic agents is an historic concept, the use of genetically modified viruses to selectively target tumour cells is relatively novel and recent. The ability of viruses to efficiently infect and lyse cells, combined with the potential augmentation of this effect by progeny viruses throughout the tumour provide justification for exploitation of these agents in cancer therapy. Before application to humans, though, issues related to tumour cell selectivity, lack of toxicity to normal tissues and the effect of the antiviral immune response, will have to be clarified. The more commonly used oncolytic viruses are based on mutant strains of herpes simplex virus, adenovirus and reovirus. The tumour selectivity of each of these strains is discussed, particularly the complementation of the viral defect by cellular pathways involved in tumourigenesis. The combination of oncolytic viruses with radiation, chemotherapy and gene therapy is also reviewed. Further study of the interaction of viral proteins with cellular pathways involved in cell cycle control will provide the rationale for viral mutants with increased selectivity for tumour cells.     

Application of bacteriophages as surrogates for mammalian viruses: a case for use in filter validation based on precedents and current practices in medical and environmental virology

Infectivity-based assays are the assays of choice for the detection of pathogenic mammalian viruses. While it is intuitively appropriate to conduct testing and validation studies with the known viral burden or a closely related mammalian species, logistic considerations often dictate otherwise. Consequently, bacteriophages have served as suitable surrogates for mammalian viruses in both medical and environmental virology applications. The wide range of bacteriophages available offers a powerful analytical tool amenable to several different applications: filter validation studies (where removal is based on size exclusion), investigations into virus contamination control issues, evaluation of barrier materials, etc. There is a considerable body of evidence to suggest and support the use of bacteriophages as surrogates for mammalian viruses. Use of appropriately sized bacteriophages provides an innocuous, efficacious and expeditious method for economical testing and validation of viral clearance capabilities of virus removal filters, thus facilitating performance of filter validation studies in biopharmaceuticals under product- and process-specific conditions in an overall effort towards ensuring the virological safety of biologicals. This paper discusses the limitations associated with mammalian virus assays and provides a rationale for the use of bacteriophages as surrogates for mammalian viruses. Data from published literature documenting applicability of bacteriophages in filter validation studies, especially when removal is based on size exclusion, is reviewed along with examples of studies from the fields of medical and environmental virology.     

肿瘤治疗的新突破:溶瘤病毒治疗

溶瘤病毒通过感染癌细胞,引起病毒性细胞病变并诱发宿主抗肿瘤免疫反应,从而选择性感染和杀死癌细胞.美国食品药品监督管理局(FDA)和欧洲药物管理局(EMA)最近批准了世界上第一个用于治疗恶性黑色素瘤的溶瘤病毒制剂,命名为Imlygic.Imlygic为表达人粒细胞-巨噬细胞集落刺激因子(GM-CSF)的单纯疱疹病毒I型(HSV-1)活病毒颗粒,经直接注射入肿瘤组织引起肿瘤细胞裂解和机体抗肿瘤免疫双重效应.除了HSV-1外,尚有许多不同类型的病毒已经进入临床试验,评估其生物安全性和抗肿瘤疗效.这些正在临床试验的溶瘤病毒包括腺病毒、痘病毒、麻疹病毒和呼肠孤病毒等.治疗的肿瘤包括卵巢癌、腹膜癌、肝癌、胶质瘤和黑色素瘤等.目前,溶瘤病毒治疗肿瘤的临床试验约有67项,一些临床试验已经取得了有希望的结果,从而进展到第三期临床试验.该文重点讨论目前正在临床试验中用于治疗各类癌症的溶瘤病毒,并讨论这些溶瘤病毒作为一类新的癌症治疗药物的机遇和挑战.     

Gamma-irradiation of serum for the inactivation of adventitious contaminants

Assurance of viral and mycoplasma safety in biopharmaceuticals is best achieved through elimination of animal-derived materials from the manufacturing process. Where this is not possible, the risk of introducing a contaminant through use of the material must be assessed and mitigated. Bovine-sourced serum represents an animal-derived material that is required for certain biopharmaceutical manufacturing processes. Gamma-irradiation of frozen bovine serum was investigated as an approach for mitigating the risk of introducing viral or mycoplasma contaminants. Mycoplasmas in frozen bovine serum were effectively inactivated by gamma-irradiation at 25-40 kGy. The larger viruses tested, respiratory enteric orphan (REO) and Cache Valley virus (CVV), were inactivated completely, while the smaller virus, simian virus type 40, was not inactivated. Gamma-irradiation of bovine-sourced serum is therefore useful for mitigating the risk of introduction of mycoplasmas and many of the viral contaminants found in biologics unprocessed bulk (e.g., CVV, REO virus, epizootic hemorrhagic disease virus). This mitigation strategy is not useful for the smaller viruses (e.g., polyomaviruses, parvoviruses, picornaviruses, caliciviruses).     

H1N1 influenza A virus neuraminidase modulates infectivity in mice

In the 2years since the onset of the H1N1 2009 pandemic virus (H1N1pdm09), sporadic cases of oseltamivir-resistant viruses have been reported. We investigated the impact of oseltamivir-resistant neuraminidase from H1N1 Brisbane-like (seasonal) and H1N1pdm09 viruses on viral pathogenicity in mice. Reassortant viruses with the neuraminidase from seasonal H1N1 virus were obtained by co-infection of a H1N1pdm09 virus and an oseltamivir-resistant H1N1 Brisbane-like virus. Oseltamivir-resistant H1N1pdm09 viruses were also isolated from patients. After biochemical characterization, the pathogenicity of these viruses was assessed in a murine model. We confirmed a higher infectivity, in mice, of the H1N1pdm09 virus compared to seasonal viruses. Surprisingly, the oseltamivir-resistant H1N1pdm09 virus was more infectious than its sensitive counterpart. Moreover, the association of H1N1pdm09 hemagglutinin and an oseltamivir-resistant neuraminidase improved the infectivity of reassortant viruses in mice, regardless of the NA origin: seasonal (Brisbane-like) or pandemic strain. This study highlights the need to closely monitor the emergence of oseltamivir-resistant viruses.     

Inhibition of influenza virus internalization by (−)-epigallocatechin-3-gallate

(−)-Epigallocatechin-3-gallate (EGCG), one of the major flavonoid components of green tea, is known to have a broad antiviral activity against several enveloped viruses, including the influenza virus. However, its mode of action and the mechanism that allows it to target influenza virus molecules have not been fully elucidated. Thus, this study investigated the molecular mechanism by which EGCG suppresses influenza virus infections. EGCG was found to block an early step in the influenza viral life cycle, but it did not affect viral adsorption to target cells or viral RNA replication. However, EGCG inhibited hemifusion events between virus particles and the cellular membrane by reducing the viral membrane integrity, thereby resulting in the loss of the cell penetration capacity of the influenza virus. EGCG also marginally suppressed the viral and nonviral neuraminidase (NA) activity in an enzyme-based assay system. In conclusion, it is suggested that the anti-influenza viral efficacy of EGCG is attributable to damage to the physical properties of the viral envelope and partial inhibition of the NA surface glycoprotein. These results may facilitate future investigations of the antiviral activity of EGCG against other enveloped viruses as well as influenza virus.     

Viral elements sense tumorigenic processes: approaching selective cancer therapy

Viruses can produce viral oncoproteins that drive multiple genetic alterations as the consequence of neoplastic transformation. Viral proteins encoded by onco-related viruses such as polyomavirus SV40 or Epstein-Barr virus are involved in cellular processes resulting in imbalance between proliferation and cell death, knowledge of which continues to be crucial for combating cancer. On the other hand, viruses also generate viral components that, from a cold viral protein, can become a tumor-selective killer by sensing cellular tumorigenic hallmarks. For instance, the avian virus derived apoptin protein has been proven to induce tumor-regression in various pre-clinical animal models without showing detectable side effects. In particular, apoptin-interacting protein partners such as components of the anaphase promoting complex were identified as potential anticancer drug targets. The adenovirus-derived protein E4orf4, another viral protein with tumor-specific apoptosis characteristics, has been proven to interact with the tumor-suppressor protein phosphatase 2A. This review aims to describe recent studies with representative viral elements that have contributed to our understanding of critical tumorigenic processes and have conferred an impact on the development of novel anti-cancer therapies.     

Arbidol: a broad-spectrum antiviral compound that blocks viral fusion

Arbidol (ARB; ethyl-6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-[(phenylthio)methyl]-indole-3-carboxylate hydrochloride monohydrate), is a Russian-made potent broad-spectrum antiviral with demonstrated activity against a number of enveloped and non-enveloped viruses. ARB is well known in Russia and China, although to a lesser extent in western countries. Unlike other broad-spectrum antivirals, ARB has an established molecular mechanism of action against influenza A and B viruses, which is different from that of available influenza antivirals, and a more recently established mechanism of inhibition of hepatitis C virus (HCV). For both viral infections the anti-viral mechanism involves ARB inhibition of virus-mediated fusion with target membrane and a resulting block of virus entry into target cells. However, ARB inhibition of fusion exploits different ARB modalities in case of influenza viruses or HCV. This review aims to summarize the available evidence of ARB effects against different groups of viruses, also, to compare various aspects of ARB anti-fusion mechanisms against influenza virus and HCV (with reference to different stringency of pH-dependence of these two viral fusogens) and to discuss further prospects for ARB and its improved derivatives of the parent compounds.     

Prospects and strategies for the discovery and development of small-molecule inhibitors of six-helix bundle formation in class 1 viral fusion proteins

Class I viral fusion proteins have an important role in the fusion of viral membranes with host cell membranes, a critical step in the viral life-cycle. These proteins all have similar structural features and form six-helix bundles in their fusogenic form, a general mechanism of action for virus-cell fusion. The successful discovery of peptide-based inhibitors of fusion proteins, in addition to the US Food and Drug Administration approval of one of these inhibitors as an anti-HIV-1 drug, confirmed that the inhibition of six-helix bundle formation is a viable strategy for identifying antiviral drugs. Because peptide-based drugs have several limitations, research has been undertaken to identify potent small-molecule inhibitors of six-helix bundle formation in a variety of viruses, including HIV-1, human respiratory syncytial virus and measles virus. Small-molecule inhibitors that disrupt six-helix bundle formation and prevent viral infection have been identified. This review will focus on the discovery of these small-molecule inhibitors.     

Herpes simplex virion entry into and intracellular transport within mammalian cells

Alphaherpesviruses, membrane-enveloped DNA viruses that are responsible for a host of human ailments, bind to, enter and are directly targeted to specific intracellular domains within their mammalian host cells. This review emphasizes recent work on the best studied of the alphaherpesviruses, Herpes simplex virus type 1 (HSV1). One area of focus is on recent work that has identified viral glycoproteins that are important in binding and internalization of the virus to the host cell. Complementary work on the receptors for those viral glycoproteins that reside on the host cell surface is also presented, with some discussion of how receptor variety might lead to the tissue tropism demonstrated by alphaherpes viruses. An additional area of focus in this review is how HSV uses the host cell transport systems to achieve intracellular targeting of the incoming virion toward the cell nucleus, and, after production of newly synthesized and assembled viral progeny, targeting them toward the plasma membrane for release.