The effect of storage at different temperatures on the stability of Hepatitis C virus RNA in plasma samples.

One important issue related to Hepatitis C virus (HCV) RNA nucleic acid amplification testing (NAT) is the storage conditions of plasma samples in order to obtain reliable results. Many authors have reported that the storage conditions could affect the RNA stability and, hence, HCV RNA detection. We have studied HCV RNA stability in plasma samples after storage at different temperatures (-70, -20, 5 and 25 degrees C). Samples containing different HCV titres were stored and analysed by qualitative or quantitative NAT techniques at defined time points. At -20 degrees C, samples containing high HCV RNA titres were followed-up during approximately 2.6-2.7 years, samples with intermediate concentrations during approximately 1 year and samples with 100 International Units/millilitre (IU/ml) during 2.5 years. Independently of the HCV RNA concentration, the results show absence of decay in HCV RNA detectability. Samples stored at 25 degrees C maintain their HCV RNA titre during 14 days and samples at 5 degrees C were stable for at least 3 months.     

cis-acting RNA elements required for replication of bovine viral diarrhea virus-hepatitis C virus 5' nontranslated region chimeras.

Pestiviruses, such as bovine viral diarrhea virus (BVDV), share many similarities with hepatitis C virus (HCV) yet are more amenable to virologic and genetic analysis. For both BVDV and HCV, translation is initiated via an internal ribosome entry site (IRES). Besides IRES function, the viral 5' nontranslated regions (NTRs) may also contain cis-acting RNA elements important for viral replication. A series of chimeric RNAs were used to examine the function of the BVDV 5' NTR. Our results show that: (1) the HCV and the encephalomyocarditis virus (EMCV) IRES element can functionally replace that of BVDV; (2) two 5' terminal hairpins in BVDV genomic RNA are important for efficient replication; (3) replacement of the entire BVDV 5' NTR with those of HCV or EMCV leads to severely impaired replication; (4) such replacement chimeras are unstable and efficiently replicating pseudorevertants arise; (5) pseudorevertant mutations involve deletion of 5' sequences and/or acquisition of novel 5' sequences such that the 5' terminal 3-4 bases of BVDV genome RNA are restored. Besides providing new insight into functional elements in the BVDV 5' NTR, these chimeras may prove useful as pestivirus vaccines and for screening and evaluation of anti-HCV IRES antivirals.     

Establishment of a subgenomic replicon for bovine viral diarrhea virus in Huh-7 cells and modulation of interferon-regulated factor 3-mediated antiviral response

We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease N(pro), and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2'-beta-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease N(pro) of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system.     

Standardization: a Progress Report

The introduction of nucleic acid amplification technology (NAT) assays for the detection of viral contamination of blood and blood products requires the availability of well-characterized reference reagents. Working reagents for hepatitis C virus RNA, hepatitis B virus DNA, HIV-1 RNA and human parvovirus B19 DNA have been established at NIBSC and at many other laboratories (both official medicinal control laboratories and commercial laboratories). However, as these reagents have been characterised independently, it is difficult to compare results from assays using different working reagents. Recently, a WHO International Standard was established for HCV RNA NAT assays. This standard has been calibrated in International Units (IU) and provides a common standard against which all working reagents can be calibrated. Collaborative studies to characterise two further candidate International Standards for HBV DNA and HIV-1 RNA NAT assays have been completed.     

Mutational analysis of bovine viral diarrhea virus RNA-dependent RNA polymerase

Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5BDeltaCT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (DeltaCT179 and DeltaCT218) that lacked RdRp activities.     

Improvement of virus safety of an antihemophilc factor IX by virus filtration process

Viral safety is an important prerequisite for clinical preparations of plasma-derived pharmaceuticals. One potential way to increase the safety of therapeutic biological products is the use of a virus-retentive filter. In order to increase the viral safety of human antihemophilic factor IX, particularly in regard to non-enveloped viruses, virus removal process using a polyvinylidene fluoride membrane filter (Viresolve NFP) has been optimized. The most critical factor affecting the filtration efficiency was operating pH and the optimum pH was 6 or 7. Flow rate increased with increasing operating pressure and temperature. Recovery yield in the optimized productionscale process was 96%. No substantial changes were observed in the physical and biochemical characteristics of the filtered factor IX in comparison with those before filtration. A 47-mm disk membrane filter was used to simulate the process performance of the production-scale cartridges and to test if it could remove several experimental model viruses for human pathogenic viruses, including human hepatitis A virus (HAV), porcine parvovirus (PPV), murine encephalomyocarditis virus (EMCV), human immunodeficiency virus type 1 (HIV), bovine viral diarrhea virus (BVDV), and bovine herpes virus (BHV). Nonenveloped viruses (HAV, PPV, and EMCV) as well as enveloped viruses (HIV, BVDV, and BHV) were completely removed during filtration. The log reduction factors achieved were (i)v.12 for HAV, (i)t.28 for PPV, (i)u.33 for EMCV, (i)u.51 for HIV, (i)u.17 for BVDV, and (i)u.75 for BHV. These results indicate that the virus filtration process successfully improved the viral safety of factor IX.     

The polymerase chain reaction and hepatitis C virus diagnosis

Abstract: In the absence of tissue culture, electron microscopy or assays for viral antigen, the direct detection of hepatitis C virus (HCV) is by necessity dependent upon nucleic acid hybridisation methods. Of the available methods, amplification of HCV cDNA by polymerase chain reaction (PCR) commends itself by virtue of its extreme sensitivity and its consequent ability to detect the very low levels of HCV-RNA that are present in many clinical samples. In this review the development and evolution of PCR techniques for HCV detection are described and a number of clinical applications are considered in detail. The application include diagnosis of acute infection during the seronegative window period prior to the appearance of HCV antibodies, and diagnosis of HCV infection in the immunosuppressed. PCR also enables identification of chronic viraemic carrier state and it permits accurate monitoring of the antiviral effects of drugs such as interferon. Confirmation of the specificity HCV antibody assays and detection of HCV contamination of blood donations and blood products are other important areas in which PCR techniques have proved invaluable. In addition, PCR-based techniques underlie an increasing number of molecular epidemiological and genotyping studies and they are providing insights into the details of HCV cellular tropism and replication. A number of logistic problems and operational difficulties are also discussed. Despite these limitations it is concluded that PCR will continue to make significant contributions to both clinical practice and to our understanding of the basic biology of HCV infection.     

Mechanism of de novo initiation by the hepatitis C virus RNA-dependent RNA polymerase: role of divalent metals

We functionally analyzed the role of metal ions in RNA-dependent RNA synthesis by three recombinant RNA-dependent RNA polymerases (RdRps) from GB virus-B (GBV), bovine viral diarrhea virus (BVDV), and hepatitis C virus (HCV), with emphasis on the HCV RdRp. Using templates capable of both de novo initiation and primer extension and RdRps purified in the absence of metal, we found that only reactions with exogenously provided Mg(2+) and Mn(2+) gave rise to significant amounts of synthesis. Mg(2+) and Mn(2+) affected the mode of RNA synthesis by the three RdRps. Both metals supported primer-dependent and de novo-initiated RNA by the GBV RdRp, while Mn(2+) significantly increased the amount of de novo-initiated products by the HCV and BVDV RdRps. For the HCV RdRp, Mn(2+) reduced the K(m) for the initiation nucleotide, a GTP, from 103 to 3 micro M. However, it increased de novo initiation even at GTP concentrations that are comparable to physiological levels. We hypothesize that a change in RdRp structure occurs upon GTP binding to prevent primer extension. Analysis of deleted proteins revealed that the C terminus of the HCV RdRp plays a role in Mn(2+)-induced de novo initiation and can contribute to the suppression of primer extension. Spectroscopy examining the intrinsic fluorescence of tyrosine and tryptophan residues in the HCV RdRp produced results consistent with the protein undergoing a conformational change in the presence of metal. These results document the fact that metal can affect de novo initiation or primer extension by flaviviral RdRps.     

Secondary structure of the 5'' nontranslated regions of hepatitis C virus and pestivirus genomic RNAs.

The RNA genomes of human hepatitis C virus (HCV) and the animal pestiviruses responsible for bovine viral diarrhea (BVDV) and hog cholera (HChV) have relatively lengthy 5' nontranslated regions (5'NTRs) sharing short segments of conserved primary nucleotide sequence. The functions of these 5'NTRs are poorly understood. By comparative sequence analysis and thermodynamic modeling of the 5'NTRs of multiple BVDV and HChV strains, we developed models of the secondary structures of these RNAs. These pestiviral 5'NTRs are highly conserved structurally, despite substantial differences in their primary nucleotide sequences. The assignment of similar structures to conserved segments of primary nucleotide sequence present in the 5'NTR of HCV resulted in a model of the secondary structure of the HCV 5'NTR which was refined by determining sites at which synthetic HCV RNA was cleaved by double- and single-strand specific RNases. These studies indicate the existence of a large conserved stem-loop structure within the 3' 200 bases of the 5'NTRs of both HCV and pestiviruses which corresponds to the ribosomal landing pad (internal ribosomal entry site) of HCV. This structure shows little relatedness to the ribosomal landing pad of hepatitis A virus, suggesting that these functionally similar structures may have evolved independently.     

Scavenger receptor BI and BII expression levels modulate hepatitis C virus infectivity

Hepatitis C virus (HCV) enters cells via a pH- and clathrin-dependent endocytic pathway. Scavenger receptor BI (SR-BI) and CD81 are important entry factors for HCV internalization into target cells. The SR-BI gene gives rise to at least two mRNA splice variants, SR-BI and SR-BII, which differ in their C termini. SR-BI internalization remains poorly understood, but SR-BII is reported to endocytose via a clathrin-dependent pathway, making it an attractive target for HCV internalization. We demonstrate that HCV soluble E2 can interact with human SR-BI and SR-BII. Increased expression of SR-BI and SR-BII in the Huh-7.5 hepatoma cell line enhanced HCV strain J6/JFH and JFH infectivity, suggesting that endogenous levels of these receptors limit infection. Elevated expression of SR-BI, but not SR-BII, increased the rate of J6/JFH infection, which may reflect altered intracellular trafficking of the splice variants. In human plasma, HCV particles have been reported to be complexed with lipoproteins, suggesting an indirect interaction of the virus with SR-BI and other lipoprotein receptors. Plasma from J6/JFH-infected uPA-SCID mice transplanted with human hepatocytes demonstrates an increased infectivity for SR-BI/II-overexpressing Huh-7.5 cells. Plasma-derived J6/JFH infectivity was inhibited by an anti-E2 monoclonal antibody, suggesting that plasma virus interaction with SR-BI was glycoprotein dependent. Finally, anti-SR-BI antibodies inhibited the infectivity of cell culture- and plasma-derived J6/JFH, suggesting a critical role for SR-BI/II in HCV infection.     

A sensitive and efficient detection method for bovine viral diarrhea virus (BVDV) in single preimplantation bovine embryos

The objective was to develop a method to accurately and efficiently detect minute amounts of bovine viral diarrhea virus (BVDV) associated with a single embryo. There are two major challenges for BVDV detection in a single embryo: the test sensitivity and the efficiency of viral molecule recovery. These become even more critical when attempts are made to detect BVDV infections that occurred naturally, not through artificial exposure of the embryos to high affinity BVDV strains. We have developed a one-step sample preparation method that has increased the viral molecule recovery rate compared to the standard RNA isolation procedure by 7-100-fold. Instead of using the traditional virus exposure approach, we generated BVDV positive embryos via somatic cell nuclear transfer (SCNT) technology using BVDV positive donor cells. By combining the highly efficient sample preparation procedure with a sensitive one-step, real-time PCR system, we have developed a sensitive test that allows detection of as low as two copies of BVDV in a single embryo. This method will allow systematic risk assessment for BVDV transmission during in vitro embryo production via IVF or SCNT procedures.     

The NS5A protein of bovine viral diarrhea virus contains an essential zinc-binding site similar to that of the hepatitis C virus NS5A protein

The recent demonstration that the NS5A protein of hepatitis C virus (HCV) contains an unconventional zinc-binding site with the format Cx(17)CxCx(20)C and the presence of a similar sequence element in the NS5A proteins of members of the Pestivirus genus has led to the hypothesis that the NS5A protein of the pestivirus bovine viral diarrhea virus (BVDV) is a zinc-binding protein. A method for the expression and partial purification of BVDV NS5A was developed, and the partially purified protein was analyzed for zinc content by atomic absorption spectroscopy. BVDV NS5A was found to coordinate a single zinc atom per protein molecule. Mutation of any of the four cysteines of the predicted zinc-binding motif eliminated zinc coordination. Furthermore, analysis of mutations at these cysteine residues in the context of a BVDV replicon system indicated that these residues were absolutely essential for RNA replication. The recently determined crystal structure of the N-terminal zinc-binding domain of the HCV NS5A protein, combined with secondary structure predictions of the region surrounding the mapped BVDV zinc-binding region, indicates that the BVDV zinc-binding motif fits the general template Cx(22)CxCx(24)C and likely comprises a three-stranded antiparallel beta-sheet fold. These data highlight the similarities between the Hepacivirus and Pestivirus NS5A proteins and suggest that both proteins perform a not-yet-defined function in RNA replication that requires coordination of a single zinc atom.     

Assessment of Needs for Plasma for Fractionation in Europe

In the early 1990s, a series of outbreaks of hepatitis C (HCV) infections clustering among recipients of certain lots of plasma-derived medicinal products (PDMP) alarmed regulatory authorities, manufacturers and the public alike. Also, a few episodes of Hepatitis A (HAV) infections occurred in haemophiliacs receiving solvent-detergent-treated factor VIII concentrates. Thus, several measures were brought into effect to reestablish the safety of the incriminated products and to further increase the margin of safety of PDMP in general. Therefore, intramuscular immunoglobulins had to be free of HCV RNA as shown by nucleic acid amplification technology (NAT) in the final products. Furthermore, the manufacturing process of PDMP had to be validated for both viral inactivation and elimination. Finally, HCV-NAT was to be standardised and implemented as a validated test of plasma pool samples.In 1994, a joint meeting of EPFA, EAPPI and Regulatory Authorities was held in Brussels to outline the state of the art and to delineate the actions to be taken. Five years later, in 1999, the incidence rates of HIV, HBV and HCV in unpaid blood donors have been minimized, especially in European countries. With probabilities for window period donations as low as 0.6 in 1 million for both HIV and HCV and 2.1 in 1 million for HBV in Switzerland, labile blood products have reached extreme, but not absolute safety. The introduction of HCV-NAT roughly doubles this safety resulting in a 1 in 3 million probability of a window donation.Concomittantly, extensive viral validation studies document effective inactivation and removal of viruses in PDMP. The demonstrated margins of safety, expressed as logarithmical reduction factors (LRF), range from 4 to over 20 log(10), depending on product, virus, and inactivation procedure used. Further progress to even safer PDMP shall be acomplished by consolidating the GMP processes, abandoning of obsolete requirements and harmonising national regulations within Europe. Before introducing new measures for additional agents such as HAV or Parvovirus B 19, gains and risks and even potential new threats have to be carefully assessed. Alternative efforts for the safeguard of patients, e.g. vaccination for HAV, need to be balanced against the risks of changing established and validated manufacturing procedures of PDMP with long-lasting safety records.