Molecular Determinants of Antiviral Potency of Paramyxovirus Entry Inhibitors

Porotto, Matteo; Carta, Patrizia; Deng, Yiqun; Kellogg, Glen E.; Whitt, Michael A.; Lu, Min; Mungall, Bruce A.; Moscona, Anne

doi:10.1128/jvi.01181-07

Cited by 70 publications

(97 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…5a). The level of inhibition (defined as the concentration of antiviral drug resulting in 50% inhibition, IC 50 ) observed with this assay was comparable to those previously reported in this laboratory (208 nM HPIV3-wt PN-IC 50 (Porotto et al, 2007), 2 µM Ribavirin (Wright et al, 2005)) (Figs. 5a and b).…”

Section: Assay Validationsupporting

confidence: 75%

“…5a and b). Non-linear regression analysis (GraphPad Prism) indicated the concentration of each positive control resulting in 50% inhibition of NiV infection (IC 50 ) was comparable in both fluorescent and chemiluminescent assays and with that previously reported (Porotto et al, 2007).…”

Section: Assay Validationmentioning

confidence: 93%

“…Based on previous studies in this laboratory (Porotto et al, 2007;Wright et al, 2005), assay performance was assessed using two positive controls: (Ribavirin (1-100 uM) and a fusion inhibitory peptide derived from the fusion protein of human parainfluenza virus-3 (HPIV-3; Wt, 10-1000 nM) in addition to the corresponding negative control scrambled peptide (Sc, 10-1000nM). These previous studies utilised a CPE based titration readout (Ribavirin, (Wright et al, 2005)) or fluorescent immunoassay format with inhibition endpoints assessed by a reduction in the number of detectable syncytia, as well as a pseudotyped entry assay with inhibition assessed by reduction in entry or multicycle replication of HeV pseudotyped viruses (HPIV-3, (Porotto et al, 2007). When infecting with 10,000 TCID 50 /ml NiV (m.o.i =0.25), the effect of the controls was clearly and repeatably detected using the chemiluminescent-HRP immunodetection assay (Fig.…”

Section: Assay Validationmentioning

confidence: 99%

“…To evaluate the effect of plate storage at 4°C on assay sensitivity and specificity, a number of methanol fixed, air dried plates were sealed and stored at 4°C (for 7 days) prior to immunolabeling. To evaluate the reproducibility of virus inhibition, the broad spectrum antiviral Ribavirin and an experimental inhibitory peptide derived from the F protein of human parainfluenza virus type 3 (HPIV-3) (Porotto et al, 2007;Porotto et al, 2006) were used as positive controls for NiV and HeV inhibition, with the associated scrambled peptide acting as a negative control.…”

Section: Hendra and Nipah Viral Infection Of Cellsmentioning

confidence: 99%

“…Recently, a multicycle pseudotyped virus replication assay was described that detects Hendra virus antibodies, as well as other antiviral compounds that target the viral entry steps, and is amenable to High Throughput Screening (HTS) (Porotto et al, 2007). This assay can be carried out entirely in BSL2 conditions, however it targets only entry inhibitors and requires validation using live viruses.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Development and validation of a chemiluminescent immunodetection assay amenable to high throughput screening of antiviral drugs for Nipah and Hendra virus

Aljofan

Porotto

Moscona

et al. 2008

Journal of Virological Methods

View full text Add to dashboard Cite

There are currently no antiviral drugs approved for the highly lethal Biosafety Level Four pathogens Nipah and Hendra virus. A number of researchers are developing surrogate assays amenable to Biosafety Level Two biocontainment but ultimately, the development of a High Throughput Screening method for directly quantifying these viruses in a Biosafety Level Four environment will be critical for final evaluation of antiviral drugs identified in surrogate assays, in addition to reducing the time required for effective antiviral drug development. By adapting an existing immunoplaque assay and using enzyme linked immunodetection in a microtitre plate format, the current experiments describe a simple two step assay protocol involving an overnight virus inoculation of Vero cell monolayers (with or without antiviral drug treatment) at Biosafety Level Four, followed by cell fixation and virus inactivation enabling removal of plates from the Biosafety Level Four laboratory and a subsequent immunodetection assay using a chemiluminescent Horse Radish Peroxidase substrate to be performed at Biosafety Level Two. The analytical sensitivity (limit of detection) of this assay is 100 Tissue Culture Infectious Dose 50 /ml of either Nipah or Hendra virus. In addition this assay enables linear quantitation of virus over three orders of magnitude and is unaffected by Dimethyl Sulfoxide concentrations of 1% or less. Intra-assay coefficients of variation are acceptable (less than 20%) when detecting a minimum of 1,000 Tissue Culture Infectious Dose 50 /ml of either virus although inter-assay variation is considerably greater. By an assessment of efficacies of the broad spectrum antiviral Ribavirin and an experimental fusion inhibitory peptide, this assay reveals a good correlation with previously published fluorescent immunodetection assays. The current experiments describe for the first time, a High Throughput Screening method amenable for direct assessment of live henipavirus antiviral drug activity.

show abstract

Section: Assay Validationsupporting

confidence: 75%

Section: Assay Validationmentioning

confidence: 93%

Section: Assay Validationmentioning

confidence: 99%

Section: Hendra and Nipah Viral Infection Of Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Development and validation of a chemiluminescent immunodetection assay amenable to high throughput screening of antiviral drugs for Nipah and Hendra virus

Aljofan

Porotto

Moscona

et al. 2008

Journal of Virological Methods

View full text Add to dashboard Cite

show abstract

Henipavirus Membrane Fusion and Viral Entry

Aguilar

Iorio

2012

Current Topics in Microbiology and Immunology

View full text Add to dashboard Cite

Single-Vector, Single-Injection Recombinant Vesicular Stomatitis Virus Vaccines Against High-Containment Viruses

2016

View full text Add to dashboard Cite

There are many avenues for making an effective vaccine against viruses. Depending on the virus these can include one of the following: inactivation of whole virions; attenuation of viruses; recombinant viral proteins; non-replication-competent virus particles; or surrogate virus vector systems such as vesicular stomatitis virus (VSV). VSV is a prototypic enveloped animal virus that has been used for over four decades to study virus replication, entry, and assembly due to its ability to replicate to high titers in a wide variety of mammalian and insect cells. The use of reverse genetics to recover infectious and single-cycle replicating VSV from plasmid DNA transfected in cell culture began a revolution in the study of recombinant VSV (rVSV). This platform can be manipulated to study the viral genetic sequences and proteins important in the virus life cycle. Additionally, foreign genes can be inserted between naturally occurring or generated start/stop signals and polyadenylation sites within the VSV genome. VSV has a tolerance for foreign gene expression which has led to numerous rVSVs reported in the literature. Of particular interest are the very effective single-dose rVSV vaccine vectors against high-containment viruses such as filoviruses, henipaviruses, and arenaviruses. Herein we describe the methods for selecting foreign antigenic genes, selecting the location within the VSV genome for insertion, generation of rVSV using reverse genetics, and proper vaccine study designs.

show abstract

Molecular Determinants of Antiviral Potency of Paramyxovirus Entry Inhibitors

Cited by 70 publications

References 51 publications

Development and validation of a chemiluminescent immunodetection assay amenable to high throughput screening of antiviral drugs for Nipah and Hendra virus

Development and validation of a chemiluminescent immunodetection assay amenable to high throughput screening of antiviral drugs for Nipah and Hendra virus

Henipavirus Membrane Fusion and Viral Entry

Single-Vector, Single-Injection Recombinant Vesicular Stomatitis Virus Vaccines Against High-Containment Viruses

Contact Info

Product

Resources

About