Application of the pseudo-plaque assay for detection and titration of Crimean-Congo hemorrhagic fever virus

Berber, Engin; Canakoglu, Nurettin; Yörük, Mustafa Deniz; Tonbak, Şükrü; Aktaş, Münir; Ertek, Mustafa; Bolat, Yusuf; Kalkan, Ahmet; Özdarendeli, Aykut

doi:10.1016/j.jviromet.2012.07.025

Cited by 13 publications

(10 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The SARS-CoV-2 virus lysate was then harvested at 48 h post-infection and the supernatants were collected, clarified, and stored at -80°C. To determine the titer of the passage 2 virus a focus forming assay (FFA) was performed as described previously [ 22 ]. Briefly, Vero E6 cells were seeded on 96 well-plates and incubated at 37°C with 5% CO 2 for overnight.…”

Section: Methodsmentioning

confidence: 99%

“…The antibody-labeled cells were detected and analyzed by immunofluorescence microscopy (Leica, UK). The fluorescent foci in each well were counted, and the virus titers were calculated and expressed as fluorescent focus units (FFU) per ml as described previously [ 22 ]. To obtain the virus passage 3 virus stock, the Vero E6 cells were infected with the virus passage 2 virus at an MOI of 0.01, and the viral lysate were was harvested at 48 h post-infection and the supernatants were collected.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Isolation and characterization of severe acute respiratory syndrome coronavirus 2 in Turkey

et al. 2020

Self Cite

View full text Add to dashboard Cite

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated with severe respiratory illness emerged in Wuhan, China, in late 2019. The virus has been able to spread promptly across all continents in the world. The current pandemic has posed a great threat to public health concern and safety. Currently, there are no specific treatments or licensed vaccines available for COVID-19. We isolated SARS-CoV-2 from the nasopharyngeal sample of a patient in Turkey with confirmed COVID-19. We determined that the Vero E6 and MA-104 cell lines are suitable for supporting SARS-CoV-2 that supports viral replication, development of cytopathic effect (CPE) and subsequent cell death. Phylogenetic analyses of the whole genome sequences showed that the hCoV-19/Turkey/ERAGEM-001/2020 strain clustered with the strains primarily from Australia, Canada, England, Iran and Kuwait and that the cases in the nearby clusters were reported to have travel history to Iran and to share the common unique nucleotide substitutions.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of severe acute respiratory syndrome coronavirus 2 in Turkey

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…The virus was released from the cells by three freeze and thaw cycles. Titres of the virus stocks were determined by a pseudo-plaque assay (PPA) as described previously [ 14 ]. Polyclonal rabbit and mouse anti-CCHF virus sera were generated in rabbits and mice as described previously [ 15 ].…”

Section: Methodsmentioning

confidence: 99%

Immunization of Knock-Out α/β Interferon Receptor Mice against High Lethal Dose of Crimean-Congo Hemorrhagic Fever Virus with a Cell Culture Based Vaccine

et al. 2015

Self Cite

View full text Add to dashboard Cite

Crimean-Congo hemorrhagic fever (CCHF) is an acute tick-borne zoonotic disease. The disease has been reported in many countries of Africa, Asia, the Middle East, and in Eurasia. During the past decade, new foci of CCHF have emerged in the Balkan Peninsula, southwest Russia, the Middle East, western China, India, Africa, and Turkey. CCHF virus produces severe hemorrhagic manifestations in humans with fatality rates up to 30%. Vaccine development efforts have been significantly hampered by a lack of animal models and therefore, no protective vaccine has been achieved. Lately, IFN α/β receptor deficient (IFNAR−/−) mice have been established as a novel small animal model of CCHF virus infection. In the present study, we found that IFNAR−/− mice highly susceptible to CCHF virus Turkey-Kelkit06 strain. Immunization with the cell culture based vaccine elicited a significant level of protection against high dose challenge (1,000 PPFU) with a homologous CCHF virus in IFNAR−/− mice.

show abstract

“…Supernatants were collected on days 4 and 5 post-infection (PI), cleared of cell debris by centrifugation at 500 g for 20 min at 4°C, and aliquots were stored at -80°C. The titer of the viral stock was quantified at 4.3×10 5 ml -1 using a pseudo-plaque assay [34]. All handling of virus was conducted in a biosafety level 3 laboratory (BSL-3).…”

Section: Methodsmentioning

confidence: 99%

Pseudo-plaque reduction neutralization test (PPRNT) for the measurement of neutralizing antibodies to Crimean-Congo hemorrhagic fever virus

Canakoglu

Berber

Ertek³

et al. 2013

Virol J

Self Cite

View full text Add to dashboard Cite

BackgroundCrimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus family Bunyaviridae, which are enveloped viruses containing tripartite, negative polarity, single-stranded RNA. CCHF is characterized by high case mortality, occurring in Asia, Africa, the Middle East and Europe. Currently, there are no specific treatments or licensed vaccines available for CCHFV. Recently, two research groups have found adult mice with defective interferon responses allowed to lethal CCHFV infection. These mouse models could provide invaluable information for further studies. Efforts to develop a vaccine against CCHFV are being made. To determine the efficacy of vaccine candidates it is important to conduct serological studies that can accurately measure levels of protective antibodies. In the present study, a pseudo-plaque reduction neutralization test (PPRNT) based on enzyme-catalyzed color development of infected cells probed with anti-CCHFV antibodies was used to measure neutralization antibody of CCHFV.MethodsSixty-nine human serum samples (20 acute and 49 convalescent) were tested. The presence of CCHFV antibodies was determined and confirmed by a commercial ELISA kit. CCHFV RNA was determined by RT-PCR. All the samples were analyzed by PPRNT and fluorescent focus reduction neutralization test (FFRNT) to measure of CCHFV-neutralizing antibodies.ResultsPseudo-plaque reduction neutralization test showed a high sensitivity (98%), specificity (100%) and agreement (96,6%) in qualitative comparison with those of the FFRNT. There was a high correlation between the titers obtained in PPRNT and FFRNT (R2 = 0.92). The inter- and intra-assay variation of PPRNT revealed good reproducibility and positive cut-off of PPRNT was defined as 1:4 by the geometric mean titers for the individual samples distributed.ConclusionThe pseudo-plaque reduction neutralization test described in this study is a fast, reproducible and sensitive method for the measurement of CCHF neutralizing antibodies. This novel assay could serve as useful tools for CCHF research in epidemiology, vaccine development and other studies of immunity. It also provides an alternative to PRNT when viruses with no or poor CPE in cell culture.

show abstract

Application of the pseudo-plaque assay for detection and titration of Crimean-Congo hemorrhagic fever virus

Cited by 13 publications

References 22 publications

Isolation and characterization of severe acute respiratory syndrome coronavirus 2 in Turkey

Isolation and characterization of severe acute respiratory syndrome coronavirus 2 in Turkey

Immunization of Knock-Out α/β Interferon Receptor Mice against High Lethal Dose of Crimean-Congo Hemorrhagic Fever Virus with a Cell Culture Based Vaccine

Pseudo-plaque reduction neutralization test (PPRNT) for the measurement of neutralizing antibodies to Crimean-Congo hemorrhagic fever virus

Contact Info

Product

Resources

About