HCV-Mediated Apoptosis of Hepatocytes in Culture and Viral Pathogenesis.

Chronic Hepatitis C Virus (HCV) infection is associated with progressive liver injury and subsequent development of fibrosis and cirrhosis. The death of hepatocytes results in the release of cytokines that induce inflammatory and fibrotic responses. The mechanism of liver damage is still under investigation but both apoptosis and immune-mediated processes may play roles.

The Food and Drug Administration Office of Women's Health: Impact of Science on Regulatory Policy: An Update.

The U.S. Food and Drug Administration Office of Women's Health (FDA OWH) has supported women's health research for ∼20 years, funding more than 300 studies on women's health issues, including research on diseases/conditions that disproportionately affect women in addition to the evaluation of sex differences in the performance of and response to medical products.

A New Signaling Pathway for HCV Inhibition by Estrogen: GPR30 Activation Leads to Cleavage of Occludin by MMP-9.

Poor outcome in response to hepatitis C virus, including higher viral load, hepatocellular carcinoma and cirrhosis, is more associated with men and postmenopausal women than with premenopausal women and women receiving hormone replacement therapy, suggesting that β-estradiol plays an innate role in preventing viral infection and liver disease. Consequently, most research in the field has concluded that estrogen affects HCV replication through viral interactions with estrogen receptor-α. Previously, estrogen-like antagonists, including Tamoxifen, were shown to reduce HCV RNA production and prevent viral entry, although the authors did not identify host factors involved.

Rapid detection of hepatitis B virus in blood plasma by a specific and sensitive loop-mediated isothermal amplification assay.

Background: Hepatitis B virus (HBV) is an important blood-borne pathogen that causes hepatic inflammation and can lead to liver cirrhosis and hepatocellular carcinoma. Conventional methods of HBV detection are time consuming and require highly trained personnel and elaborate equipment. This report describes the development of a rapid, simple, specific, and sensitive loop-mediated isothermal amplification assay (LAMP) for detection of HBV genotypes A, B, C, D, E, and F in blood samples.

Evolution of cell culture systems for HCV.

Many challenges exist for the study of HCV in the laboratory. Therapy using interferon (IFN) is expensive, not well tolerated and ineffective for many patients. HCV research has been hampered by the lack of a robust tissue culture system, but recent advances have made virus growth in culture possible.

Immunogenicity and protection efficacy of monomeric and trimeric recombinant SARS coronavirus spike protein subunit vaccine candidates.

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease, and an effective vaccine is not available. In this study, we compared the immunogenicity and protection efficacy of recombinant proteins corresponding to different domains of the SARS-coronavirus spike protein. Trimeric recombinant proteins were created by fusing the foldon domain derived from T4 bacteriophage to the carboxy-termini of individual domains of the spike protein.

Altering SARS coronavirus frameshift efficiency affects genomic and subgenomic RNA production.

In previous studies, differences in the amount of genomic and subgenomic RNA produced by coronaviruses with mutations in the programmed ribosomal frameshift signal of ORF1a/b were observed. It was not clear if these differences were due to changes in genomic sequence, the protein sequence or the frequency of frameshifting. Here, viruses with synonymous codon changes are shown to produce different ratios of genomic and subgenomic RNA.

RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus.

Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem-loop suggested an important hitherto unknown function.

Biochemical characterization of a recombinant SARS coronavirus nsp12 RNA-dependent RNA polymerase capable of copying viral RNA templates.

The severe acute respiratory syndrome coronavirus (SARS-CoV) RNA genome is replicated by a virus-encoded RNA replicase, the key component of which is the nonstructural protein 12 (nsp12). In this report, we describe the biochemical properties of a full-length recombinant SARS-CoV nsp12 RNA-dependent RNA polymerase (RdRp) capable of copying viral RNA templates. The purified SARS-CoV nsp12 showed both primer-dependent and primer-independent RNA synthesis activities using homopolymeric RNA templates.

Interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses SARS coronavirus replication.

The programmed -1 ribosomal frameshifting (-1 PRF) utilized by eukaryotic RNA viruses plays a crucial role for the controlled, limited synthesis of viral RNA replicase polyproteins required for genome replication. The viral RNA replicase polyproteins of severe acute respiratory syndrome coronavirus (SARS-CoV) are encoded by the two overlapping open reading frames 1a and 1b, which are connected by a -1 PRF signal. We evaluated the antiviral effects of antisense peptide nucleic acids (PNAs) targeting a highly conserved RNA sequence on the - PRF signal.

Persistent growth of a human plasma-derived hepatitis C virus genotype 1b isolate in cell culture.

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNA(I) (VA RNA(I)), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b.

Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.

In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed -1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before.

Characterization of aurintricarboxylic acid as a potent hepatitis C virus replicase inhibitor.

Background: Hepatitis C virus (HCV) NS5B is an essential component of the viral replication machinery and an important target for antiviral intervention. Aurintricarboxylic acid (ATA), a broad-spectrum antiviral agent, was evaluated and characterized for its anti-NS5B activity in vitro and in HCV replicon cells.

Methods: Recombinant NS5B, HCV replicase and Huh-7 cells harbouring the subgenomic HCV replicon of genotype 1b were employed for biochemical and mechanistic investigations.

Innate immunity and hepatitis C virus: eluding the host cell defense.

Interferon-alpha (IFN-alpha) mono-therapy is largely ineffective for most of the hepatitis C virus (HCV)-infected patients that receive it. The addition of ribavirin to IFN therapy has increased the response rate dramatically. While many factors are implicated in determining the success rate for IFN therapy, viral genotype seems to play a crucial role.

Overcoming hurdles in hepatitis C virus research: efficient production of infectious virus in cell culture.

Hepatitis C virus is a flavivirus that infects nearly 2% of the world population. There is no vaccine available and current therapy with interferon and ribavirin is expensive, not well tolerated and effective in only 60% of patients. HCV research has been hampered by the lack of a robust tissue culture system, but recent advances have made virus growth in culture possible.

Severe acute respiratory syndrome coronavirus infection in vaccinated ferrets.

Background: Development of vaccines to prevent severe acute respiratory syndrome (SARS) is limited by the lack of well-characterized animal models. Previous vaccine reports have noted robust neutralizing antibody and inflammatory responses in ferrets, resulting in enhanced hepatitis.

Methods: We evaluated the humoral immune response and pathological end points in ferrets challenged with the Urbani strain of SARS-associated coronavirus (SARS-CoV) after having received formalin-inactivated whole-virus vaccine or mock vaccine.

Evaluation of inactivation methods for severe acute respiratory syndrome coronavirus in noncellular blood products.

Background: Severe acute respiratory syndrome coronavirus (SARS-CoV) has been detected in the blood of infected individuals, which may have the potential to contaminate donated blood and plasma-derived products in the event of a future outbreak. Effective methods for inactivating the SARS-CoV in protein solutions are described in this report.

Study Design And Methods: Heat, ultraviolet (UV) irradiation, octanoic acid, and solvent/detergent (S/D) methods were tested individually for their ability to inactivate SARS-CoV in protein solutions appropriately mimicking blood-derived products.

Obstacles and advances in SARS vaccine development.

The emergence of the severe acute respiratory syndrome (SARS) that resulted in a pandemic in 2003 spurred a flurry of interest in the development of vaccines to prevent and treat the potentially deadly viral infection. Researchers around the world pooled their scientific resources and shared early data in an unprecedented manner in light of the impending public health crisis. There are still large gaps in knowledge about the pathogenesis of this virus.

New antiviral pathway that mediates hepatitis C virus replicon interferon sensitivity through ADAR1.

While many clinical hepatitis C virus (HCV) infections are resistant to alpha interferon (IFN-alpha) therapy, subgenomic in vitro self-replicating HCV RNAs (HCV replicons) are characterized by marked IFN-alpha sensitivity. IFN-alpha treatment of replicon-containing cells results in a rapid loss of viral RNA via translation inhibition through double-stranded RNA-activated protein kinase (PKR) and also through a new pathway involving RNA editing by an adenosine deaminase that acts on double-stranded RNA (ADAR1). More than 200 genes are induced by IFN-alpha, and yet only a few are attributed with an antiviral role.

Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV.

Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by a novel coronavirus termed SARS-CoV. Due to the severity of this disease, the World Health Organization (WHO) recommends that manipulation of active viral cultures of SARS-CoV be performed in containment laboratories at biosafety level 3 (BSL3). The virus was inactivated by ultraviolet light (UV) at 254 nm, heat treatment of 65 degrees C or greater, alkaline (pH > 12) or acidic (pH < 3) conditions, formalin and glutaraldehyde treatments.

Protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis C virus envelope protein E2 through the eukaryotic initiation factor 2alpha kinase PERK.

The hepatitis C virus envelope protein, E2, is an endoplasmic reticulum (ER)-bound protein that contains a region of sequence homology with the double-stranded RNA-activated protein kinase PKR and its substrate, the eukaryotic translation initiation factor 2 (eIF2). We previously reported that E2 modulates global translation through inhibition of the interferon-induced antiviral protein PKR through its PKR-eIF2alpha phosphorylation site homology domain (PePHD). Here we show that the PKR-like ER-resident kinase (PERK) binds to and is also inhibited by E2.

Hepatitis C virus NS5A colocalizes with the core protein on lipid droplets and interacts with apolipoproteins.

The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) has been shown to interact with a variety of cellular proteins and implicated in the regulation of cell growth, interferon resistance, and other cellular signaling pathways, but the role of NS5A in HCV pathogenesis has not been firmly established. To further characterize this multifunctional protein, we instigated the studies of the subcellular localization of NS5A in a hepatoma cell line. NS5A was localized to the perinuclear membrane structures, including the endoplasmic reticulum (ER) and the Golgi apparatus, by immunofluorescence staining and confocal microscopy.

Detection of a novel unglycosylated form of hepatitis C virus E2 envelope protein that is located in the cytosol and interacts with PKR.

The hepatitis C virus (HCV) envelope protein E2 has been shown to accumulate in the lumen of the endoplasmic reticulum (ER) as a properly folded glycoprotein as well as large aggregates of misfolded proteins. In the present study, we have identified an additional unglycosylated species, with an apparent molecular mass of 38 kDa (E2-p38). In contrast to the glycosylated E2, E2-p38 is significantly less stable and is degraded through the proteasome pathway.