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

Aljofan, Mohamad; Porotto, Matteo; Moscona, Anne; Mungall, Bruce A.

doi:10.1016/j.jviromet.2008.01.016

Cited by 38 publications

(44 citation statements)

References 34 publications

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“…The N-terminally tagged peptide, however, inhibited viral replication by only 40% at the highest concentration (1 nM). In the multicycle replication assay for which results are shown here, cell monolayers were not covered with an overlay, and the extent of viral spread through the monolayer was measured by a chemiluminescent assay (2). Whereas the experiments for which results are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Viral Entry Inhibitors Targeted to the Membrane Site of Action

Porotto

Yokoyama

Palermo

et al. 2010

J Virol

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110

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The fusion of enveloped viruses with the host cell is driven by specialized fusion proteins to initiate infection. The "class I" fusion proteins harbor two regions, typically two heptad repeat (HR) domains, which are central to the complex conformational changes leading to fusion: the first heptad repeat (HRN) is adjacent to the fusion peptide, while the second (HRC) immediately precedes the transmembrane domain. Peptides derived from the HR regions can inhibit fusion, and one HR peptide, T20 (enfuvirtide), is in clinical use for HIV-1. For paramyxoviruses, the activities of two membrane proteins, the receptor-binding protein (hemagglutininneuraminidase [HN] or G) and the fusion protein (F), initiate viral entry. The binding of HN or G to its receptor on a target cell triggers the activation of F, which then inserts into the target cell and mediates the membrane fusion that initiates infection. We have shown that for paramyxoviruses, the inhibitory efficacy of HR peptides is inversely proportional to the rate of F activation. For HIV-1, the antiviral potency of an HRC-derived peptide can be dramatically increased by targeting it to the membrane microdomains where fusion occurs, via the addition of a cholesterol group. We report here that for three paramyxoviruses-human parainfluenza virus type 3 (HPIV3), a major cause of lower respiratory tract diseases in infants, and the emerging zoonotic viruses Hendra virus (HeV) and Nipah virus (NiV), which cause lethal central nervous system diseases-the addition of cholesterol to a paramyxovirus HRC-derived peptide increased antiviral potency by 2 log units. Our data suggest that this enhanced activity is indeed the result of the targeting of the peptide to the plasma membrane, where fusion occurs. The cholesterol-tagged peptides on the cell surface create a protective antiviral shield, target the F protein directly at its site of action, and expand the potential utility of inhibitory peptides for paramyxoviruses.Fusion of enveloped viruses with the host cell is a key step in viral infectivity, and interference with this process can lead to highly effective antivirals. Viral fusion is driven by specialized proteins that undergo an ordered series of conformational changes. These changes facilitate the initial, close apposition of the viral and host membranes, and they ultimately result in the formation of a fusion pore (reviewed in reference 12). The "class I" fusion proteins harbor two regions, typically two heptad repeat (HR) domains: the first one (HRN) adjacent to the fusion peptide and the second one (HRC) immediately preceding the transmembrane domain. Peptides derived from the HR regions can inhibit fusion, and one of them, T20 (enfuvirtide), is in clinical use for HIV-1 (19). Peptides derived from the HRN and HRC regions of paramyxovirus fusion (F) proteins can interact with fusion intermediates of F (3,20,22,37,46,49) and provide a promising antiviral strategy.The current model for class I-driven fusion postulates the existence of a so-called prehairpin intermedia...

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Section: Resultsmentioning

confidence: 99%

Viral Entry Inhibitors Targeted to the Membrane Site of Action

Porotto

Yokoyama

Palermo

et al. 2010

J Virol

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110

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“…The antiviral efficacy of ribavirin against henipaviruses has been shown in several in vitro studies; however, in vivo studies have had varied outcomes (1,2,8,29). While ribavirin treatment in hamsters was not protective against lethal HeV challenge (8), an open-label ribavirin treatment trial run during an NiV outbreak in Malaysia in 1998 reported a reduction in mortality by 36% (6).…”

Section: Discussionmentioning

confidence: 99%

“…Several in vitro studies have shown that ribavirin is effective against both HeV and NiV infection (1,2,29). An open-label ribavirin treatment trial was run during an outbreak of NiV in Malaysia in 1998 and reported to reduce mortality by 36% (6).…”

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confidence: 99%

A Novel Model of Lethal Hendra Virus Infection in African Green Monkeys and the Effectiveness of Ribavirin Treatment

Rockx¹,

Bossart

Feldmann³

et al. 2010

J Virol

115

121

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The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are emerging zoonotic paramyxoviruses that can cause severe and often lethal neurologic and/or respiratory disease in a wide variety of mammalian hosts, including humans. There are presently no licensed vaccines or treatment options approved for human or veterinarian use. Guinea pigs, hamsters, cats, and ferrets, have been evaluated as animal models of human HeV infection, but studies in nonhuman primates (NHP) have not been reported, and the development and approval of any vaccine or antiviral for human use will likely require efficacy studies in an NHP model. Here, we examined the pathogenesis of HeV in the African green monkey (AGM) following intratracheal inoculation. Exposure of AGMs to HeV produced a uniformly lethal infection, and the observed clinical signs and pathology were highly consistent with HeV-mediated disease seen in humans. Ribavirin has been used to treat patients infected with either HeV or NiV; however, its utility in improving outcome remains, at best, uncertain. We examined the antiviral effect of ribavirin in a cohort of nine AGMs before or after exposure to HeV. Ribavirin treatment delayed disease onset by 1 to 2 days, with no significant benefit for disease progression and outcome. Together our findings introduce a new disease model of acute HeV infection suitable for testing antiviral strategies and also demonstrate that, while ribavirin may have some antiviral activity against the henipaviruses, its use as an effective standalone therapy for HeV infection is questionable.

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“…The inoculum was then replaced with fresh EMEM and incubated for 24 h at 37°C. Viral protein immunoassays were performed as described previously (33). Detec-tion was performed with 1:1000 anti-HeV and anti-NiV antibody (rabbit polyclonal anti-N protein (34) provided by Brian Shiell), 1:2000 HRP-conjugated anti-rabbit antibody, and 10% chemiluminescent peroxidase substrate-3.…”

Section: Hendra Virus (Hev) and Nipah Virus (Niv) Experimentsmentioning

confidence: 99%

A Novel L-ficolin/Mannose-binding Lectin Chimeric Molecule with Enhanced Activity against Ebola Virus

Michelow

Dong

Mungall

et al. 2010

Journal of Biological Chemistry

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Ebola viruses constitute a newly emerging public threat because they cause rapidly fatal hemorrhagic fevers for which no treatment exists, and they can be manipulated as bioweapons. We targeted conserved N-glycosylated carbohydrate ligands on viral envelope surfaces using novel immune therapies. Mannose-binding lectin (MBL) and L-ficolin (L-FCN) were selected because they function as opsonins and activate complement. Given that MBL has a complex quaternary structure unsuitable for large scale cost-effective production, we sought to develop a less complex chimeric fusion protein with similar ligand recognition and enhanced effector functions. We tested recombinant human MBL and three L-FCN/MBL variants that contained the MBL carbohydrate recognition domain and varying lengths of the L-FCN collagenous domain. Non-reduced chimeric proteins formed predominantly nona-and dodecameric oligomers, whereas recombinant human MBL formed octadecameric and larger oligomers. Surface plasmon resonance revealed that L-FCN/MBL76 had the highest binding affinities for N-acetylglucosamine-bovine serum albumin and mannan. The same chimeric protein displayed superior complement C4 cleavage and binding to calreticulin (cC1qR), a putative receptor for MBL. L-FCN/MBL76 reduced infection by wild type Ebola virus Zaire significantly greater than the other molecules. Tapping mode atomic force microscopy revealed that L-FCN/ MBL76 was significantly less tall than the other molecules despite similar polypeptide lengths. We propose that alterations in the quaternary structure of L-FCN/MBL76 resulted in greater flexibility in the collagenous or neck region. Similarly, a more pliable molecule might enhance cooperativity between the carbohydrate recognition domains and their cognate ligands, complement activation, and calreticulin binding dynamics. L-FCN/ MBL chimeric proteins should be considered as potential novel therapeutics.

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

Cited by 38 publications

References 34 publications

Viral Entry Inhibitors Targeted to the Membrane Site of Action

Viral Entry Inhibitors Targeted to the Membrane Site of Action

A Novel Model of Lethal Hendra Virus Infection in African Green Monkeys and the Effectiveness of Ribavirin Treatment

A Novel L-ficolin/Mannose-binding Lectin Chimeric Molecule with Enhanced Activity against Ebola Virus

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