Respiratory Syncytial Virus (RSV) is a common, contagious virus that causes infections of the respiratory tract. It is a negative-sense, single-stranded RNA virus that belongs to the genus Orthopneumovirus, subfamily Pneumovirinae, and family Paramyxvoviridae. It consists of two major antigenic subgroups, RSV/A and RSV/B viruses, which have multiple genotypes with high diversity in the attachment (G) glycoprotein. RSV is the single most common cause of respiratory hospitalization in infants, and can also cause severe illness in older adults, people with heart and lung disease, or anyone with a weak immune system.
The diagnosis of RSV infection can be done by detecting viral antigens, nucleic acids, or antibodies in clinical specimens. Antigen detection methods include immunofluorescence assay (IFA), enzyme immunoassay (EIA) and rapid antigen tests. Nucleic acid detection methods include reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and multiplex PCR. Antibody detection methods include enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody assay (IFA) and neutralization assay.
The serology of RSV is important for understanding the epidemiology, immunity, and vaccine efficacy of RSV infection. The most widely used serological marker of RSV infection is the neutralizing antibody, which reflects the functional capacity of serum to inhibit viral infectivity in cells. Neutralizing antibody activity is correlated with protection against RSV disease in animal models and humans.
Microneutralization Assay for RSV
RSV-specific neutralizing antibody activity is a correlate of immune protection and a useful marker for evaluating vaccine candidates, performing seroprevalence studies, and detecting infection. Microneutralization assay is a method that measures the functional capacity of serum (or other fluids) to neutralize virus infectivity in cells. It involves incubating serial dilutions of serum with a known amount of RSV in cell culture plates, followed by measuring the viral replication after a certain period. Viral replication can be measured by different methods, such as plaque counting, immunostaining, or quantitative PCR. Microneutralization assay is more sensitive and specific than ELISA for detecting RSV antibodies and can distinguish between RSV subgroups and genotypes. However, it is also more labor-intensive and time-consuming than ELISA and requires standardized protocols and reagents.
To ensure the reliability and reproducibility of the microneutralization assay for RSV, it is essential to validate the assay using standardized protocols and quality control measures. Some of the aspects that need to be validated are:
- The source and quality of RSV strains used for the assay, which should represent the diversity and prevalence of RSV subgroups and genotypes in the population.
- The cell line and culture conditions used for the assay, should support optimal viral growth and infection without cytotoxicity or interference.
- The serum dilution range and incubation time used for the assay, which should cover the expected range of neutralizing antibody titers and allow sufficient time for viral replication.
- The method of viral replication measurement used for the assay, which should be accurate, precise, and consistent across different operators and batches.
- The criteria for defining positive and negative controls, cut-off values and neutralization titers used for the assay should be based on statistical analysis and biological relevance.
A recent study (Bonifazi et al., 2023) found that a micro-neutralization assay is a reliable and standardized method for assessing the neutralizing activity of serum samples against respiratory syncytial virus (RSV). The method is based on a recombinant RSV expressing a reporter gene, which enables fast and accurate measurement of virus infection in cell culture. This paper describes the development and optimization of the micro-neutralization assay using clinical samples from RSV-infected patients and vaccinated volunteers and demonstrates that the micro-neutralization assay has several benefits over the PRNT, such as higher sensitivity, specificity, reproducibility, and throughput. It also shows that the micro-neutralization assay can differentiate between RSV subtypes A and B, and can measure neutralizing antibodies in various sample types, such as serum, plasma, and nasal washes. Emphasis is placed on the significance and applications of the micro-neutralization assay for RSV research and vaccine development and on the possible use of micro-neutralization assay to assess the immunogenicity and efficacy of RSV vaccines, as well as to monitor the circulation and diversity of RSV strains in different populations.
Other studies have attempted to validate the microneutralization assay for RSV using different protocols and parameters. For example:
- Zielinska et al. (2005) developed an improved microneutralization assay using an image analyzer for automated plaque counting, which reduced the labor and variability of manual counting. They validated their assay using a reference serum and two control sera from different age groups.
- Piedra et al. (2016) described a standard protocol for performing the microneutralization assay using complement-mediated enhancement of neutralization, which increased the sensitivity and specificity of the assay. They validated their assay using sera from infants and adults vaccinated with different RSV vaccine candidates.
- Varada et al. (2013) proposed a quantitative PCR-based microneutralization assay, which measured the viral RNA levels instead of the viral protein levels. They validated their assay using a pooled human immunoglobulin reference standard and sera from children with RSV infection.
These studies demonstrate the feasibility and utility of validating the microneutralization assay for RSV using different approaches and criteria. However, there is still a need for a more comprehensive and standardized validation of the assay across different laboratories and settings, especially for the evaluation of RSV vaccines and immunotherapies.
Conclusion
The microneutralization assay is a valuable tool for measuring the neutralizing antibody activity of serum against RSV. It can provide information on the epidemiology, immunity, and vaccine efficacy of RSV infection. However, the assay requires careful validation and quality control to ensure its reliability and reproducibility. Further research and collaboration are needed to establish a universal and robust microneutralization assay for RSV that can be applied to various clinical and research purposes.
References
Bonifazi, C., Trombetta, C. M., Barneschi, I., Latanza, S., Leopoldi, S., Benincasa, L., Leonardi, M., Semplici, C., Piu, P., Marchi, S., Montomoli, E., & Manenti, A. (2023). Establishment and validation of a high-throughput micro-neutralization assay for respiratory syncytial virus (subtypes A and B). Journal of Medical Virology, 95(7), e28923. https://doi.org/10.1002/jmv.
Zielinska, E., Liu, D., Wu, HY. et al. Development of an improved microneutralization assay for respiratory syncytial virus by automated plaque counting using imaging analysis. Virol J 2, 84 (2005). https://doi.org/10.1186/1743-
Piedra PA, Hause AM, Aideyan L. Respiratory Syncytial Virus (RSV): Neutralizing Antibody, a Correlate of Immune Protection. Methods Mol Biol. 2016;1442:77-91. doi: 10.1007/978-1-4939-3687-8_7. PMID: 27464689.
Varada, J.C., Teferedegne, B., Crim, R.L. et al. A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment. Virol J 10, 195 (2013). https://doi.org/10.1186/1743-
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