Plaque Assay for Ebola Virus

Moe, James B.; Lambert, Rhonda D.; Lupton, H W

doi:10.1128/jcm.13.4.791-793.1981

Cited by 78 publications

(42 citation statements)

References 7 publications

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“…All cell cultures were tested for viral antigen by immunofluorescent staining with fluorescein isothiocyanate-conjugated convalescentphase serum from an experimentally infected monkey; those with visible cytopathic effect were harvested, fixed, and stained, and the remaining cultures were similarly immunostained at the end of a 2-week cultivation. Virus infectivity was assayed by counting plaques on monolayers of MA104 cells in multiwell plastic plates according to a standard method (10).…”

Section: Methodsmentioning

confidence: 99%

Enzyme immunosorbent assay for Ebola virus antigens in tissues of infected primates

et al. 1992

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Section: Methodsmentioning

confidence: 99%

Enzyme immunosorbent assay for Ebola virus antigens in tissues of infected primates

et al. 1992

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“…Supernatants were then layered on TNE buffer (20 mM Tris [pH 7.5], 0.1 M NaCl, 0.1 mM EDTA) containing 20% sucrose and spun at 28,000 rpm at 4uC for 2 h by using an SW28 rotor with a Beckman Optima L-70K ultracentrifuge. The virion pellet was resuspended in RPMI 1640 and titers were determined by plaque assay as previously described [25]. As a control, supernatant from mock-infected Vero E6 cells was purified and quantified by the same procedures.…”

Section: Cells and Virusesmentioning

confidence: 99%

Ebola Virion Attachment and Entry into Human Macrophages Profoundly Effects Early Cellular Gene Expression

et al. 2011

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Zaire ebolavirus (ZEBOV) infections are associated with high lethality in primates. ZEBOV primarily targets mononuclear phagocytes, which are activated upon infection and secrete mediators believed to trigger initial stages of pathogenesis. The characterization of the responses of target cells to ZEBOV infection may therefore not only further understanding of pathogenesis but also suggest possible points of therapeutic intervention. Gene expression profiles of primary human macrophages exposed to ZEBOV were determined using DNA microarrays and quantitative PCR to gain insight into the cellular response immediately after cell entry. Significant changes in mRNA concentrations encoding for 88 cellular proteins were observed. Most of these proteins have not yet been implicated in ZEBOV infection. Some, however, are inflammatory mediators known to be elevated during the acute phase of disease in the blood of ZEBOV-infected humans. Interestingly, the cellular response occurred within the first hour of Ebola virion exposure, i.e. prior to virus gene expression. This observation supports the hypothesis that virion binding or entry mediated by the spike glycoprotein (GP1,2) is the primary stimulus for an initial response. Indeed, ZEBOV virions, LPS, and virus-like particles consisting of only the ZEBOV matrix protein VP40 and GP1,2 (VLPVP40-GP) triggered comparable responses in macrophages, including pro-inflammatory and pro-apoptotic signals. In contrast, VLPVP40 (particles lacking GP1,2) caused an aberrant response. This suggests that GP1,2 binding to macrophages plays an important role in the immediate cellular response.

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“…Zaire EBOV strain Mayinga or MARV strain Musoke was used for infection of cells in tissue culture (33,34). The filoviruses were propagated in Vero or Vero E6 cells and titrated by a 50% tissue culture infective dose (TCID 50 ) assay or standard plaque assay (25). All experiments with filovirusinfected cells or mice were performed in the BSL-4 facilities of the Philipps-University Marburg or the United States Army Medical Research Institute of Infectious Diseases (USAMRIID).…”

Section: Cells and Virusesmentioning

confidence: 99%

“…Blood samples (n ϭ 6 mice per group) were obtained on 3 and 6 dpi under anesthesia by cardiac puncture. Viral titers in the serum were determined by traditional plaque assay (25), and IFN-␣ levels were determined using a commercially available enzyme-linked immunosorbent assay per the manufacturer's directions under BSL-4 conditions (PBL Laboratories, Piscataway, NJ).…”

Section: Cells and Virusesmentioning

confidence: 99%

VP35 Knockdown Inhibits Ebola Virus Amplification and Protects against Lethal Infection in Mice

Enterlein

Warfield

Swenson

et al. 2006

Antimicrob Agents Chemother

115

108

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Phosphorodiamidate morpholino oligomers (PMO) are a class of uncharged single-stranded DNA analogs modified such that each subunit includes a phosphorodiamidate linkage and morpholine ring. PMO antisense agents have been reported to effectively interfere with the replication of several positive-strand RNA viruses in cell culture. The filoviruses, Marburg virus and Ebola virus (EBOV), are negative-strand RNA viruses that cause up to 90% lethality in human outbreaks. There is currently no commercially available vaccine or efficacious therapeutic for any filovirus. In this study, PMO conjugated to arginine-rich cell-penetrating peptide (P-PMO) and nonconjugated PMO were assayed for the ability to inhibit EBOV infection in cell culture and in a mouse model of lethal EBOV infection. A 22-mer P-PMO designed to base pair with the translation start site region of EBOV VP35 positive-sense RNA generated sequence-specific and time-and dose-dependent inhibition of EBOV amplification in cell culture. The same oligomer provided complete protection to mice when administered before or after an otherwise lethal infection of EBOV. A corresponding nonconjugated PMO, as well as nonconjugated truncated versions of 16 and 19 base residues, provided length-dependent protection to mice when administered prophylactically. Together, these data suggest that antisense PMO and P-PMO have the potential to control EBOV infection and are promising therapeutic candidates.The Filoviridae family consists of only two genera, Ebola virus (EBOV) and Marburg virus (MARV), and belongs to the order Mononegavirales. Filoviruses possess a nonsegmented, single-stranded RNA genome of negative polarity that contains seven genes. The nontranscribed genomic ends, termed "leader" (3Ј end) and "trailer" (5Ј end), harbor cis-acting signals important for viral transcription, replication, and encapsidation. Transcription and replication of the viral genome requires viral proteins (VP) VP30 and VP35, the nucleoprotein (NP), and viral polymerase L (27, 28). Along with its role in viral RNA synthesis, VP35 has also been shown to act as a type I interferon antagonist (2). As the only viral surface protein, the glycoprotein (GP) is involved in binding and entry of the virion into host cells. The matrix protein VP40 functions in virus assembly and budding, and it has been suggested that the minor matrix protein VP24 also plays a role in these processes (7,14).Most MARV and EBOV species cause a severe hemorrhagic fever associated with fatality rates up to 90% in humans and nonhuman primates (8, 32). Due to the high mortality rates and the lack of any approved effective human therapeutic or vaccine (5), filoviruses have been classified as biological safety level 4 (BSL-4) agents. Many approaches have been employed in attempts to develop effective therapies for EBOV, including the administration of nucleoside analogues (i.e., ribavirin), immune globulins, type I interferons and other cytokines, anticoagulants, and therapeutic vaccines (10,13,19,20,24,37,39,40). To date, th...

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Plaque Assay for Ebola Virus

Abstract: A plaque assay for Ebola virus is reported. The procedure has real potential for future research, although it is less sensitive than indirect fluorescent-antibody and mouse inoculation tests.

Cited by 78 publications

References 7 publications

Enzyme immunosorbent assay for Ebola virus antigens in tissues of infected primates

Enzyme immunosorbent assay for Ebola virus antigens in tissues of infected primates

Ebola Virion Attachment and Entry into Human Macrophages Profoundly Effects Early Cellular Gene Expression

VP35 Knockdown Inhibits Ebola Virus Amplification and Protects against Lethal Infection in Mice

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