A rapid method for infectivity titration of Andes hantavirus using flow cytometry

Barriga, Gonzalo P.; Martínez-Valdebenito, Constanza; Galeno, H.; Ferrés, Marcela; Lozach, Pierre‐Yves; Tischler, Nicole D.

doi:10.1016/j.jviromet.2013.06.022

Cited by 17 publications

(18 citation statements)

References 28 publications

(29 reference statements)

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“…The NP production of hantaviruses is used to quantify the amount of infectious particles and reflect the viral replication level (Koma et al, 2012; Barriga et al, 2013; Guo et al, 2015; Cheng et al, 2016; Ma et al, 2016). Compared with conventional methods, such as ELISA, the ICW assay promptly detected HTNV NP expression, and the results were characterized by high accuracy and quality.…”

Section: Discussionmentioning

confidence: 99%

“…Nevertheless, one significant characteristic of hantaviruses is that their replication in mammalian cell culture tends to be slow and non-lytic (McCaughey et al, 1999). Several traditional methods have been developed to detect hantavirus replication, such as the improved plaque formation test (McCaughey et al, 1999), enzyme-labeled immunosorbent assay (ELISA; Cheng et al, 2014), quantitative real-time RT-PCR (qRT-PCR; Machado et al, 2013), immunofluorescence assay (IFA; Xu et al, 2002; Jin et al, 2012) and flow cytometry (FCM; Barriga et al, 2013). The improved plaque formation test is dependent on the low pH-induced cytopathic effects of hantavirus but is time-consuming and has low reproducibility.…”

Section: Introductionmentioning

confidence: 99%

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In-Cell Western Assays to Evaluate Hantaan Virus Replication as a Novel Approach to Screen Antiviral Molecules and Detect Neutralizing Antibody Titers

Chen

et al. 2017

Front. Cell. Infect. Microbiol.

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Hantaviruses encompass rodent-borne zoonotic pathogens that cause severe hemorrhagic fever disease with high mortality rates in humans. Detection of infectious virus titer lays a solid foundation for virology and immunology researches. Canonical methods to assess viral titers rely on visible cytopathic effects (CPE), but Hantaan virus (HTNV, the prototype hantavirus) maintains a relatively sluggish life cycle and does not produce CPE in cell culture. Here, an in-cell Western (ICW) assay was utilized to rapidly measure the expression of viral proteins in infected cells and to establish a novel approach to detect viral titers. Compared with classical approaches, the ICW assay is accurate and time- and cost-effective. Furthermore, the ICW assay provided a high-throughput platform to screen and identify antiviral molecules. Potential antiviral roles of several DExD/H box helicase family members were investigated using the ICW assay, and the results indicated that DDX21 and DDX60 reinforced IFN responses and exerted anti-hantaviral effects, whereas DDX50 probably promoted HTNV replication. Additionally, the ICW assay was also applied to assess NAb titers in patients and vaccine recipients. Patients with prompt production of NAbs tended to have favorable disease outcomes. Modest NAb titers were found in vaccinees, indicating that current vaccines still require improvements as they cannot prime host humoral immunity with high efficiency. Taken together, our results indicate that the use of the ICW assay to evaluate non-CPE Hantaan virus titer demonstrates a significant improvement over current infectivity approaches and a novel technique to screen antiviral molecules and detect NAb efficacies.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

In-Cell Western Assays to Evaluate Hantaan Virus Replication as a Novel Approach to Screen Antiviral Molecules and Detect Neutralizing Antibody Titers

Chen

et al. 2017

Front. Cell. Infect. Microbiol.

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“…ANDV isolate CHI-7913 (kindly provided by Héctor Galeno, Instituto de Salud Pública, Chile) was propagated in Vero E6 cells (ATTC) as described before [57]. All work involving the infectious virus was performed under biosafety level 3 conditions (Centro de Investigaciones Médicas, Pontificia Universidad Católica de Chile, Chile).…”

Section: Virus and Cellsmentioning

confidence: 99%

Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc

et al. 2016

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Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses.

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“…In this cellular context, a pH of 5.9 was found to activate fusion of Andes virus (ANDV), whilst a pH of 6.3 was reported for Hantaan virus (Cifuentes-Muñoz et al, 2011;Ogino et al, 2004), most probably influencing the site of endosomal escape. A role for endosomal pH in ANDV cell entry has been confirmed by the use of the weak base ammonium chloride, which inhibits ANDV entry by raising the pH of endosomes (Barriga et al, 2013). For the bunyavirus Rift Valley fever virus, it has been shown that low pH is enough to trigger Gc multimerization changes which seem to be mediated by protonation of specific histidines (de Boer et al, 2012).…”

mentioning

confidence: 99%

Acidification triggers Andes hantavirus membrane fusion and rearrangement of Gc into a stable post-fusion homotrimer

Acuña

Bignon

Mancini

et al. 2015

Journal of General Virology

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The hantavirus membrane fusion process is mediated by the Gc envelope glycoprotein from within endosomes. However, little is known about the specific mechanism that triggers Gc fusion activation, and its pre-and post-fusion conformations. We established cell-free in vitro systems to characterize hantavirus fusion activation. Low pH was sufficient to trigger the interaction of virus-like particles with liposomes. This interaction was dependent on a pre-fusion glycoprotein arrangement. Further, low pH induced Gc multimerization changes leading to non-reversible Gc homotrimers. These trimers were resistant to detergent, heat and protease digestion, suggesting characteristics of a stable post-fusion structure. No acid-dependent oligomerization rearrangement was detected for the trypsin-sensitive Gn envelope glycoprotein. Finally, acidification induced fusion of glycoprotein-expressing effector cells with non-susceptible CHO cells. Together, the data provide novel information on the Gc fusion trigger and its non-reversible activation involving lipid interaction, multimerization changes and membrane fusion which ultimately allow hantavirus entry into cells.

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A rapid method for infectivity titration of Andes hantavirus using flow cytometry

Cited by 17 publications

References 28 publications

In-Cell Western Assays to Evaluate Hantaan Virus Replication as a Novel Approach to Screen Antiviral Molecules and Detect Neutralizing Antibody Titers

In-Cell Western Assays to Evaluate Hantaan Virus Replication as a Novel Approach to Screen Antiviral Molecules and Detect Neutralizing Antibody Titers

Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc

Acidification triggers Andes hantavirus membrane fusion and rearrangement of Gc into a stable post-fusion homotrimer

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