A multiplex reverse transcription polymerase chain reaction method for the detection of foodborne viruses.

A multiplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the simultaneous detection of the human enteroviruses, hepatitis A virus (HAV) and Norwalk virus (NV). Poliovirus type 1 (PV1) was chosen as a model for the human enterovirus group. Three different sets of primers were used to produce three size-specific amplicons of 435 bp, 270 bp, and 192 bp for PV1, NV, and HAV, respectively. RT-PCR products were separated by agarose gel electrophoresis, and amplicon identity was confirmed by Southern transfer followed by DNA hybridization using nonradioactive, digoxigenin-labeled internal probes. When tested on mixed, purified virus suspensions, the multiplex method achieved detection limits of < or = 1 infectious unit (PV1 and HAV) or RT-PCR-amplifiable unit (NV) for all viruses. With further streamlining efforts such as single tube amplification and liquid hybridization, multiplex PCR offers advantages over cell culture methodology and monoplex PCR because it allows for rapid and cost-effective detection of several human enteric viruses in a single reaction tube.

Human immunodeficiency virus type 1 entry inhibitors PRO 542 and T-20 are potently synergistic in blocking virus-cell and cell-cell fusion.

Human immunodeficiency virus type 1 (HIV-1) entry proceeds via a cascade of events that afford promising targets for therapy. PRO 542 neutralizes HIV-1 by blocking its attachment to CD4 cells, and T-20 blocks gp41-mediated fusion. Both drugs have shown promise in phase 1/2 clinical trials. Here, the drugs were tested individually and in combination in preclinical models of HIV-1 infection, and inhibition data were analyzed for cooperativity by using the combination index method. Synergistic inhibition of virus-cell and cell-cell fusion was observed for phenotypically diverse viruses for a broad range of drug concentrations, often resulting in > or = 10-fold dose reductions in vitro. Additional mechanism-of-action studies probed the molecular basis of the synergies. The markedly enhanced activity observed for the PRO 542:T-20 combination indicates that the multistep nature of HIV-1 entry leaves the virus particularly vulnerable to combinations of entry inhibitors. These findings provide a strong rationale for evaluating combinations of these promising agents for therapy in vivo.