RESUMO
In virus clearance study (VCS) design, the amount of virus loaded onto the virus filters (VF) must be carefully controlled. A large amount of virus is required to demonstrate sufficient virus removal capability; however, too high a viral load causes virus breakthrough and reduces log reduction values. We have seen marked variation in the virus removal performance for VFs even with identical VCS design. Understanding how identical virus infectivity, materials and operating conditions can yield such different results is key to optimizing VCS design. The present study developed a particle number-based method for VCS and investigated the effects on VF performance of discrepancies between apparent virus amount and total particle number of minute virus of mice. Co-spiking of empty and genome-containing particles resulted in a decrease in the virus removal performance proportional to the co-spike ratio. This suggests that empty particles are captured in the same way as genome-containing particles, competing for retention capacity. In addition, between virus titration methods with about 2.0 Log10 difference in particle-to-infectivity ratios, there was a 20-fold decrease in virus retention capacity limiting the throughput that maintains the required LRV (e.g., 4.0), calculated using infectivity titers. These findings suggest that ignoring virus particle number in VCS design can cause virus overloading and accelerate filter breakthrough. This article asserts the importance of focusing on virus particle number and discusses optimization of VCS design that is unaffected by virological characteristics of evaluation systems and adequately reflect the VF retention capacity.
Assuntos
Filtração , Vírus , Animais , Filtração/métodos , Cinética , Camundongos , VírionRESUMO
A rapid and sensitive screening assay has been established for in vitro evaluation of antiviral compounds against bovine viral diarrhoea virus (BVDV), which is widely used as a surrogate for hepatitis C virus (HCV). The procedure is based on photospectrometrical assessment for the viability of virus-infected cells via extracellular leakage of lactic dehydrogenase (LDH). The level of LDH in culture supernatants of BVDV-infected Madin-Darby bovine kidney (MDBK) cells was significantly higher than those of mock-infected MDBK cells. Under optimized assay conditions, the LDH level was found to correlate well with the degree of viral replication. When the 50% effective concentrations (EC50s) of ribavirin, cyclosporine A and human interferon-alpha for BVDV replication were determined by the established LDH method and compared with those obtained by a conventional tetrazolium colorimetric (MTT) method, there was a complete correlation in EC50s between the two methods. Furthermore, a much higher ratio of background activity (noise) to sample activity (signal) could be achieved with the LDH method than with the MTT method, indicating that the present LDH assay permits a sensitive, rapid and reliable screening of compounds for their anti-BVDV activity and may be useful for the discovery of novel anti-HCV agents.
Assuntos
Antivirais/farmacologia , Vírus da Diarreia Viral Bovina/efeitos dos fármacos , Interferon Tipo I/farmacologia , L-Lactato Desidrogenase/análise , Replicação Viral/fisiologia , Animais , Bovinos , Células Cultivadas , Colorimetria , Vírus da Diarreia Viral Bovina/fisiologia , Humanos , Proteínas Recombinantes , Ribavirina/farmacologia , Sais de Tetrazólio , Tiazóis , Replicação Viral/efeitos dos fármacosRESUMO
Bovine viral diarrhea virus (BVDV) is a well-characterized member of the Flaviviridae family. BVDV may be a surrogate model for human hepatitis C virus (HCV), since HCV does not replicate efficiently in cell cultures and animals. Mizoribine, a nucleoside analog clinically used as an immunosuppressant, was found to be active against the replication of BVDV in cell culture. We further investigated the combination of mizoribine and interferon (IFN)-alpha for antiviral activity and found that the combination synergistically inhibited BVDV replication in bovine kidney cells, as monitored by the inhibition of virus-induced cytopathicity. The combination of ribavirin and IFN-alpha was also synergistic in inhibiting BVDV replication. Treatment of infected cells with a combination of mizoribine and IFN-alpha at the concentrations, at which the respective compounds proved to be inactive, markedly reduced viral infectivity in culture supernatants. These results indicate that mizoribine in combination with IFN-alpha may have potential for the treatment of HCV infection.