Clinical and diagnostic virology最新文献

Background: Flavivirus infection elicits an abundant immune response in the host which is directed against a number of the viral proteins. Resistance to flavivirus-induced disease can also be controlled via a non-immune mechanism involving the product of a naturally occurring murine gene, Flv.

Objectives: To review studies that have reported the mapping of epitopes on flavivirus proteins that elicit T- or B-cell immune responses in mice or humans and to discuss a possible mechanism for flavivirus-specific genetic resistance.

Study design: Purified viral proteins and synthetic peptides were used to map B-cell epitopes. Purified proteins, vaccinia-expressed viral protein fragments and synthetic peptides were used to map T-cell epitopes. Congenic-resistant, C3H/RV and congenic susceptible, C3H/He mice and cell cultures were used to study the mechanism of genetic resistance to flavivirus infection.

Results: T- and B-cell epitopes have been mapped to the E, NS1 and NS3 proteins of several flaviviruses. Immune responses to the C, PreM, NS2a, NS4a, and NS5 proteins have also been documented. Data suggest that the Flv gene product acts intracellularly to suppress the synthesis of viral genomic RNA.

Conclusions: Although flavivirus infection elicits an abundant immune response, this response is not always rapid enough to protect the host from developing encephalitis. During secondary infections both the humoral and cellular flavivirus-specific responses can confer protection. Dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) appear to be caused by an overly vigorous immune response. In genetically resistant animals reduced production of virus results in a slower spread of the infection, which in turn allows time for the immune response to develop and to clear the infection before disease symptoms appear.

Background: Chronic infection with hepatitis C virus (HCV) is associated with progressive liver damage, including the development of cirrhosis and hepatocellular carcinoma, and HCV is a leading cause of liver dysfunction worldwide. The current therapy for chronic HCV infection, interferon-α (IFN), is effective in a minority of HCV-infected patients. Several studies have demonstrated a correlation between therapeutic outcome and the amino acid sequence of a small region of the HCV non-structural 5A (NS5A) gene product. It has been suggested that this region, termed the interferon sensitivity-determining region (ISDR), may mediate IFN resistance by directly interacting with one or more cellular proteins associated with the IFN-mediated antiviral response.

Objectives: In an attempt to define the molecular mechanism by which the NS5A protein and the ISDR might contribute to HCV resistance to IFN, we examined whether NS5A could regulate the IFN-induced protein kinase, PKR, a primary mediator of the IFN-induced antiviral response.

Study design: Multiple approaches, including in vitro assays using recombinant proteins, the transfection of recombinant clones into cultured cells, and in vivo studies in yeast, were used to examine the interaction of NS5A with PKR, as well as the functional significance of the interaction. An ISDR deletion mutant was prepared to evaluate the importance of the ISDR in mediating the NS5A–PKR interaction and the requirement of this region for PKR inhibition.

Results: NS5A repressed PKR activity through a direct interaction with the protein kinase catalytic domain. Both PKR repression and interaction required the presence of the ISDR.

Conclusions: Inactivation of PKR may be one mechanism by which HCV avoids the antiviral effects of IFN. Thus, therapeutic strategies designed to block the NS5A–PKR interaction may increase the efficacy of IFN therapy in HCV-infected individuals.

Background: The development of antiviral drugs for hepatitis C virus (HCV) infection represents a substantial challenge. Similar to human immunodeficiency virus (HIV), HCV is highly prone to mutation. It is, therefore, expected that potential HCV therapeutics currently under development, such as protease inhibitors, will suffer from the same shortcomings of HIV therapeutic drugs; the emergence of drug resistant viral mutants. Ribozymes (Rz) are enzymatic RNA molecules that can be engineered to specifically target any given RNA molecule. A therapeutic Rz can be manufactured and administered as a drug, or a Rz gene can be delivered and expressed intracellularly by gene therapy. For HCV therapeutics, we favour the gene therapy approach as delivery and in vivo expression of Rz genes will result in a constant and continuous supply of multiple intracellular Rz, offering less opportunity for the development of drug-resistant viral variants.

Objectives: To utilise direct intravenous injection of hepatotropic viral vectors to transfer Rz genes directly into the hepatocytes of HCV-infected patients, resulting in degradation of the HCV positive strand RNA genome, the viral mRNAs, and even the negative strand RNA replication intermediate. We plan to circumvent the emergence of drug-resistant viral mutants by targeting multiple, highly conserved HCV RNA sequences simultaneously with multiple Rz genes expressed from a single vector.

Study design: Rzs targeting conserved regions of the HCV positive and negative RNAs were transcribed in vitro and used to cleave HCV target RNAs. The most effective Rzs identified were then incorporated into adeno associated viral (AAV) vectors and adenoviral (AV) vectors and tested for their ability to inhibit HCV core expression in a tissue culture model.

Results: Several Rzs targeting highly conserved HCV sequences effectively degraded positive and negative strands of HCV RNA in vitro. Furthermore, substantial inhibition of HCV gene expression was observed in tissue culture using viral vectors to deliver and express Rz genes.

Conclusions: Rz gene therapy has potential for the production of anti-viral drugs directed against HCV. Initial studies employing Rz gene therapy to produced anti-viral drugs against HCV have proved successful. Rz gene therapy may be a useful approach to overcome problems associated with anti-HCV drug design, such as the emergence of drug-resistant mutants.

Background: Pestiviruses are the veterinary viruses with genome homology to human hepatitis C virus (HCV). This group includes classical swine fever virus (CSFV), border disease virus of sheep (BDV) and bovine virus diarrhoea virus (BVDV). There are some similarities in the pathology of all three virus infections; in utero transmission to the foetus can cause early embryonic losses, severe congenital abnormalities and, particularly with BVDV, lifelong persistent infections. In situ hybridisation studies have demonstrated virus within reproductive tissues and the germinal centres of lymphoid tissue.

Objectives: To examine the immune response of cattle throughout their pregnancy following infection with bovine pestivirus (BVDV) during the first trimester (before 110 days).

Study design: In two experimental studies, heifers were infected with BVDV before 98 days gestation. Their antibody response was monitored during the remainder of the pregnancy. In another study, the antibody response of pregnant cattle was monitored following a natural infection of BVDV on a farm. Calves of the dams from all these three studies were examined, following birth, for persistent BVDV infection.

Results: It was observed that in dams carrying persistently infected foetuses, the immune response was markedly higher (13 811±1273 U ELISA antibody) than in those dams carrying uninfected foetuses (2542±588 U ELISA antibody). These results were used to establish an antibody threshold (10 000 U ELISA antibody) to predict the virus status of unborn calves during a farm outbreak of BVDV infection. The combined results of experimental and farm studies showed that in dams with low antibodies, 5/15 calves were infected whereas in dams with high antibodies, 17/19 calves were infected.

Conclusions: The predictive reliability of the assay appeared valuable but not secure. The ability of BVDV to infect the foetus with consequent maternal recognition, whilst remaining inaccessible to maternal immune exclusion, is a novel finding.

Background: The hepatitis G virus (HGV) or GB virus C (GBV-C) is a new member of the Flaviviridae family. The virus is transmitted by transfusion of blood, infusion of some blood products, and by parenteral exposure to blood during intravenous drug use (IVDU) and haemodialysis. Transmission from mother to infant and by sexual contact has also been documented. Although the virus has been found in patients with acute and chronic hepatitis, evidence of disease association has not been forthcoming. The majority of patients carry the virus in the absence of liver enzyme abnormalities.

Objectives: To review what is currently known about HGV/GBV-C in order to evaluate its similarity with other members of the Flaviviridae and the association of the virus with disease.

Results: The genomic organisation of the virus is typical for Flaviviridae, with long 5′ and 3′ untranslated regions (UTR). However, a clearly identifiable nucleocapsid encoding region is lacking. Polyprotein synthesis is mediated through an internal ribosome entry site (IRES) contained within the 5′ UTR. Phylogenetic tree analysis of sequences derived from this region has demonstrated the existence of at least three genotypes. Apart from serum, HGV-RNA has been detected in lymphocytes also, but the quasispecies present in the two compartments appear to be different. The envelope glycoprotein E2 lacks a hypervariable region and is potentially the target of a neutralising antibody response.

Conclusion: Molecular analysis of HGV reveals close similarity of the virus with HCV. However, an association of the virus with liver disease remains unresolved and no association of the virus with hepatocellular carcinoma has been reported.

Background: Vaccines against many flaviviruses, such as Japanese encephalitis virus (JEV), yellow fever virus (YFV) and tick-borne encephalitis virus (TBEV), have been successfully used for many years. Other diseases such as dengue fever (DF) and hepatitis C are still major public health problems as no licensed vaccines are in use.

Objectives: To review studies on the use of defective recombinant adenoviruses (Rads) as experimental flavivirus vaccines and comment on their use to prevent infections with other members of the Flaviviridae such as hepatitis C virus.

Study design: Recombinant adenoviruses, defective in their replication strategy, contain deletions in the E1 and E3 regions of the genome to increase the amount of foreign genetic material that can be inserted. The expression of foreign genes, inserted into these regions, can be driven by the adenovirus’s own promoter, or by an additional viral promoters.

Conclusions: Rads have been successfully used to raise protective immunity in experimental models of infection with several viruses. They can elicit both humoral and cell-mediated immunity and can be given parenterally or by oral administration. In addition, their hepatotropism makes them suitable for tackling diseases such as hepatitis C. Careful design of the vaccine vectors is advised to ensure their efficacy and safety, and as hepatitis C is a persistent infection, it may be advisable to design Rads containing genes encoding for non-structural proteins in preference to structural proteins.

Background: Hepatitis C virus (HCV) NS3 proteinase activity is required for the release of HCV nonstructural proteins and is thus a potential antiviral target. The enzyme requires a protein cofactor NS4A, located downstream of NS3 on the polyprotein, for activation and efficient processing.

Objectives: Comparison of the proteinase three-dimensional structure before and after NS4A binding should help to elucidate the mechanism of NS4A-dependent enzyme activation.

Study design: We determined the crystal structure of NS3 proteinase of HCV BK isolate (genotype 1b; residues 1–189) and also the crystal structure of this proteinase complexed with HCV BK-NS4A (residues 21–34).

Results: The core region (residues 30–178) of the enzyme without cofactor (NS3P) or with bound cofactor (NS3P/4A) is folded into a trypsin-like conformation and the substrate P1 specificity pocket is essentially unchanged. However, the D1-E1 β-loop shifts away from the cofactor binding site in NS3P/4A relative to NS3P, thereby accommodating NS4A. One result is that catalytic residues His-57 and Asp-81 move closer to Ser-139 and their sidechains adopt more ‘traditional’ (trypsin-like) orientation. The N-terminus (residues 1–30), while extended in NS3P, is folded into an α-helix and β-strand that cover the bound cofactor of NS3P/4A. A new substrate-binding surface is formed from both the refolded N-terminus and NS4A, potentially affecting substrate residues immediately downstream of the cleavage site.

Conclusions: Direct comparison of the crystal structures of NS3P and NS3P/4A shows that the binding of NS4A improves the anchoring and orientation of the enzyme's catalytic triad. This is consistent with the enhancement of NS3P's weak residual activity upon NS4A binding. There is also significant refolding of the enzyme's N-terminus which provides new interactions with P′-side substrate residues. The binding surface for P′-side substrate residues, including the P1 specificity pocket, changes little after NS4A binding. In summary, we observe a structural basis for improved substrate turnover and affinity that follows complexation of NS3P with its NS4A cofactor.

Background: In the 1970s Manfred Eigen and colleagues proposed a new model of molecular evolution to explain adaptability and rapid evolution of simple replicons, as those that probably populated the earth at the onset of life. This model of evolution placed emphasis on mutant generation, to the point of invalidating the concept of wild-type genomes as a defined sequence of nucleotides. In striking similarity with the proposals for such early replicons, present-day RNA viruses consist of complex distributions of nonidentical but closely related genomes termed quasispecies.

Objectives: To discuss indeterminations inherent to a quasispecies structure and to the analytical procedures to define it, biological implications of quasispecies, and the need to take into account this type of population structure, in order to design effective strategies to prevent and control diseases caused by highly variable viruses.

Results: Quasispecies have many biological implications, extending from viral pathogenesis to the emergence of new pathogens, rapid antigenic variation, and alterations in cell tropism, virulence, host range and viral gene expression.

Conclusions: Diseases caused by highly variable RNA viruses prove very difficult to control and vaccine development against such viruses are largely unsuccessful. It is important to understand quasispecies composition and dynamics, as quasispecies are an important step in the natural history of RNA viruses.