Development of a Neutralization Assay for Influenza Virus Using an Endpoint Assessment Based on Quantitative Reverse-Transcription PCR

Teferedegne, Belete; Lewis, Andrew M.; Peden, Keith; Murata, Haruhiko

doi:10.1371/journal.pone.0056023

Cited by 18 publications

(24 citation statements)

References 33 publications

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“…Normally, the need for RNA/DNA purification from samples represents a significant constraint that can decrease throughput in qPCR-based assays. We recently developed a qPCR-based neutralization assay for influenza virus by making use of a commercial reagent that allows the generation of PCR-ready cell lysates with minimal effort, thus circumventing a previously rate-limiting technical obstacle [27]. In the present study, we have exploited the sensitivity afforded by qPCR to develop a rapid 96-well format microneutralization assay for RSV with an assessment of endpoint as early as 24 hours post-infection.…”

Section: Introductionmentioning

confidence: 99%

A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment

Varada

Teferedegne

Crim

et al. 2013

Virol J

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BackgroundFew studies have used quantitative polymerase chain reaction (qPCR) as an approach to measure virus neutralization assay endpoints. Its lack of use may not be surprising considering that sample nucleic acid extraction and purification can be expensive, labor-intensive, and rate-limiting.MethodsVirus/antibody mixtures were incubated for one hour at 37°C and then transferred to Vero cell monolayers in a 96-well plate format. At 24 (or 48) hours post-infection, we used a commercially available reagent to prepare cell lysates amenable to direct analysis by one-step SYBR Green quantitative reverse transcription PCR using primers specific for the RSV-N gene, thereby obviating the need for cumbersome RNA extraction and purification. The neutralization titer was defined as the reciprocal of the highest dilution needed to inhibit the PCR signal by 90% when compared with the mean value observed in virus control wells in the absence of neutralizing antibodies.ResultsWe have developed a qPCR-based neutralization assay for human respiratory syncytial virus. Due to the sensitivity of qPCR in detecting virus replication, endpoints may be assessed as early as 24 hours post-infection. In addition, the dynamic range of qPCR provides a basis for the assay to be relatively robust to perturbations in input virus dose (i.e., the assay is in compliance with the Percentage Law).ConclusionsThis qPCR-based neutralization assay is suitable for automated high-throughput applications. In addition, our experimental approach may be generalizable for the rapid development of neutralization assays for other virus families.

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Section: Introductionmentioning

confidence: 99%

A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment

Varada

Teferedegne

Crim

et al. 2013

Virol J

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“…Few studies have used PCR as an approach to measure virus neutralization assay endpoints (23)(24)(25). As far as we are aware, this is the first report of a real-time PCR-based neutralization assay being used in the diagnosis of a flavivirus infection.…”

Section: Discussionmentioning

confidence: 95%

Neutralization Assay for Zika and Dengue Viruses by Use of Real-Time-PCR-Based Endpoint Assessment

et al. 2017

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The global spread and infective complications of Zika virus (ZKV) and dengue virus (DENV) have made them flaviviruses of public health concern. Serological diagnosis can be challenging due to antibody cross-reactivity, particularly in secondary flavivirus infections or when there is a history of flavivirus vaccination. The virus neutralization assay is considered to be the most specific assay for measurement of anti-flavivirus antibodies. This study describes an assay where the neutralization endpoint is measured by real-time PCR, providing results within 72 h. It demonstrated 100% sensitivity (24/24 ZKV and 15/15 DENV) and 100% specificity (11/11 specimens) when testing well-characterized sera. In addition, the assay was able to determine the correct DENV serotype in 91.7% of cases. The high sensitivity and specificity of the real-time PCR neutralization assay makes it suitable to use as a confirmatory test for sera that are reactive in commercial IgM/IgG enzyme immunoassays. Results are objective and the PCR-based measurement of the neutralization endpoint lends itself to automation so that throughput may be increased in times of high demand.KEYWORDS dengue, flavivirus, serology, Zika Z ika virus (ZKV) and dengue virus (DENV) are members of the family Flaviviridae, genus Flavivirus, which also includes yellow fever, Japanese encephalitis, and West Nile viruses. There are at least 4 closely related but serologically distinct DENV, designated DENV serotypes 1 through 4 (1). There is only transient and weak crossprotection among the 4 DENV serotypes, so individuals living in an area where DENV is endemic can be infected up to 4 times (2).ZKV and DENV have spread across the tropics in Africa, Asia, the Pacific, and the Americas with their vector mosquitoes Aedes aegypti and A. albopictus (3, 4). Cocirculation of ZKV and DENV types 1 through 4 has been documented in both French Polynesia and Brazil (5, 6) and likely occurs in many other tropical countries. Incursions into temperate countries have also occurred, with autochthonous transmission observed in Europe (DENV in southeastern France, Croatia, and Madeira Island) and the United States (DENV and ZKV in Florida) (7-9).Both ZKV and DENV infection can produce a wide spectrum of illnesses with many asymptomatic or subclinical infections (10, 11). ZKV's potential to cause fetal central nervous system abnormalities, growth restriction, and death (12) has necessitated ZKV testing of potentially exposed pregnant women, among whom nonspecific serological activity may occur, and asymptomatic individuals with low pretest probabilities of positive ZKV serology.Direct detection of ZKV nucleic acids has limited utility in asymptomatic patients, as the periods of viremia and shedding in urine and saliva are relatively brief, occurring

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“…To measure VSV ebolavirus infectivity in a high-throughput methodology, we used the one-step RT-qPCR-based approach that we have applied to several viruses [29][30][31]. Briefly, after viral infection, a commercial lysis reagent was used to produce crude-cell lysates, which were used directly for RT-qPCR.…”

Section: Establishing An Rt-qpcr Assay To Detect Vsv Ebolavirus Infecmentioning

confidence: 99%

“…The neutralization titer was given by the reciprocal of the dilution that resulted in a 90% reduction in signal (NT90) compared with the samples with no antibody. We used NT90, since this value had yielded antibody titers that correlated with those obtained using other types of neutralization assays with other viruses [29,30]. The anti-EBOV antibody neutralized VSV.…”

Section: Evaluating Different Antibodies/sera With Different Ebolavirmentioning

confidence: 99%

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Development of a micro-neutralization assay for ebolaviruses using a replication-competent vesicular stomatitis hybrid virus and a quantitative PCR readout

Lee

Phy

Peden

et al. 2017

Vaccine

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Development of vaccines against highly pathogenic viruses that could also be used as agents of bioterrorism is both a public health issue and a national security priority. Methods that can quantify neutralizing antibodies will likely be crucial in demonstrating vaccine effectiveness, as most licensed viral vaccines are effective due to their capacity to elicit neutralizing antibodies. Assays to determine whether antibodies are neutralizing traditionally involve infectious virus, and the assay most commonly used is the plaque-reduction neutralization test (PRNT). However, when the virus is highly pathogenic, this assay must be done under the appropriate level of containment; for tier one select agents, such as Ebola virus (EBOV), it is performed under Biological Safety Level 4 (BSL-4) conditions. Developing high-throughput neutralization assays for these viruses that can be done in standard BSL-2 laboratories should facilitate vaccine development. Our approach is to use a replication-competent hybrid virus whose genome carries the envelope gene from the pathogenic virus on the genetic backbone of a non-pathogenic virus, such as vesicular stomatitis virus (VSV). We have generated hybrid VSVs carrying the envelope genes for several species of ebolavirus. The readout for infectivity is a one-step reverse transcriptase quantitative PCR (RT-qPCR), an approach that we have used for other viruses that allows robustness and adaptability to automation. Using this method, we have shown that neutralization can be assessed within 6-16h after infection. Importantly, the titers obtained in our assay with two characterized antibodies were in agreement with titers obtained in other assays. Finally, although in this paper we describe the VSV platform to quantify neutralizing antibodies to ebolaviruses, the platform should be directly applicable to any virus whose envelope is compatible with VSV biology.

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Development of a Neutralization Assay for Influenza Virus Using an Endpoint Assessment Based on Quantitative Reverse-Transcription PCR

Cited by 18 publications

References 33 publications

A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment

A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment

Neutralization Assay for Zika and Dengue Viruses by Use of Real-Time-PCR-Based Endpoint Assessment

Development of a micro-neutralization assay for ebolaviruses using a replication-competent vesicular stomatitis hybrid virus and a quantitative PCR readout

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