Development of an RNA Strand-Specific Hybridization Assay To Differentiate Replicating versus Nonreplicating Influenza A Viruses.

Replication of influenza A virus (IAV) from negative-sense viral RNA (vRNA) requires the generation of positive-sense RNA (+RNA). Most molecular assays, such as conventional real-time reverse transcriptase PCR (rRT-PCR), detect total RNA in a sample without differentiating vRNA from +RNA. These assays are not designed to distinguish IAV infection versus exposure of an individual to an environment enriched with IAVs but wherein no viral replication occurs. We therefore developed a strand-specific hybridization (SSH) assay that differentiates between vRNA and +RNA and quantifies relative levels of each RNA species. The SSH assay exhibited a linearity of 7 logs with a lower limit of detection of 6.0 × 102 copies of molecules per reaction. No signal was detected in samples with a high load of nontarget template or influenza B virus, demonstrating assay specificity. IAV +RNA was detected 2 to 4 h postinoculation of MDCK cells, whereas synthesis of cold-adapted IAV +RNA was significantly impaired at 37°C. The SSH assay was then used to test IAV rRT-PCR positive nasopharyngeal specimens collected from individuals exposed to IAV at swine exhibitions (n = 7) or while working at live bird markets (n = 2). The SSH assay was able to differentiate vRNA and +RNA in samples collected from infected, symptomatic individuals versus individuals who were exposed to IAV in the environment but had no active viral replication. Data generated with this technique, especially when coupled with clinical data and assessment of seroconversion, will facilitate differentiation of actual IAV infection with replicating virus versus individuals exposed to high levels of environmental contamination but without virus infection.

Translational control by negative-strand RNA viruses: methods for the study of a crucial virus/host interaction.

Protein synthesis is a vital step in the successful replication of negative-strand RNA viruses. Protein synthesis is also a critical step in the development of a successful antiviral response from the host. This makes understanding the interplay between host and viral translation an important aspect of defining the virus/host interaction. For the negative-strand RNA viruses there are disparate mechanism of how viruses interact with the host protein synthesis apparatus, ranging from the complete takeover of all protein synthesis to the subtle insertion of viral mRNAs into an otherwise unchanged protein synthesis pattern. In this article, we discuss different ways to investigate protein synthesis in virus-infected cells, ranging from the use of metabolic labeling for the study of general translation changes to using fluorescence-coupled labeling techniques that allow the pinpointing of any subcellular localization of protein synthesis during virus replication. We also discuss methods for analyzing the translation initiation factors that are frequently modified in virus-infected cells.

A vesiculovirus showing a steepened transcription gradient and dominant trans-repression of virus transcription.

Vesicular stomatitis virus (VSV) is a prototype nonsegmented, negative-sense virus used to examine viral functions of a broad family of viruses, including human pathogens. Here we demonstrate that S(2) VSV, an isolate with a small plaque phenotype compared to other Indiana strain viruses, has a transcription defect resulting in an altered pattern and rapid decline of transcription. The S(2) VSV transcription gradient is dominant over the wild-type transcription in a coinfection. This is the first characterization of an altered gradient of transcription not dependent on RNA template sequence or host response and may provide insight into new approaches to viral attenuation.

Identification of a pyridopyrimidinone inhibitor of orthopoxviruses from a diversity-oriented synthesis library.

Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential biothreat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drug-resistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals.

Detection of oseltamivir-resistant zoonotic and animal influenza A viruses using the rapid influenza antiviral resistance test.

Mutations in the influenza virus neuraminidase (NA) that cause reduced susceptibility to the NA inhibitor (NAI) oseltamivir may occur naturally or following antiviral treatment. Currently, detection uses either a traditional NA inhibition assay or gene sequencing to identify known markers associated with reduced inhibition by oseltamivir. Both methods are laborious and require trained personnel. The influenza antiviral resistance test (iART), a prototype system developed by Becton, Dickinson and Company for research use only, offers a rapid and simple method to identify such viruses. This study investigated application of iART to influenza A viruses isolated from non-human hosts with a variety of NA subtypes (N1-N9).

Identification of a broad-spectrum inhibitor of viral RNA synthesis: validation of a prototype virus-based approach.

There are no approved therapeutics for the most deadly nonsegmented negative-strand (NNS) RNA viruses, including Ebola (EBOV). To identify chemical scaffolds for the development of broad-spectrum antivirals, we undertook a prototype-based lead identification screen. Using the prototype NNS virus, vesicular stomatitis virus (VSV), multiple inhibitory compounds were identified. Three compounds were investigated for broad-spectrum activity and inhibited EBOV infection. The most potent, CMLDBU3402, was selected for further study. CMLDBU3402 did not show significant activity against segmented negative-strand RNA viruses, suggesting proscribed broad-spectrum activity. Mechanistic analysis indicated that CMLDBU3402 blocked VSV viral RNA synthesis and inhibited EBOV RNA transcription, demonstrating a consistent mechanism of action against genetically distinct viruses. The identification of this chemical backbone as a broad-spectrum inhibitor of viral RNA synthesis offers significant potential for the development of new therapies for highly pathogenic viruses.