Molecular insights into respiratory syncytial virus: Genotyping and characterization in pediatric community-acquired lower respiratory tract infections

Replication cycle

The RSV replication cycle begins with the attachment of the G protein to heparan sulfate proteoglycans on host cells, followed by membrane fusion mediated by the F protein. The viral RNA genome is transcribed and replicated in cytoplasmic inclusion bodies, where the ribonucleoprotein complex orchestrates these processes. Newly formed virions assemble at the cell membrane and bud off to infect adjacent cells, often forming syncytia that contribute to tissue damage.^[13] Syncytium formation is a hallmark of RSV infection, facilitating cell-to-cell spread and immune evasion [Figure 2]. These features underscore RSV’s adaptability and persistence in host environments.

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Figure 2:

Photomicrographic detection of respiratory syncytial virus using indirect immunofluorescence technique. (fluorescein isothiocyanate 200x to 400x) (Source: National Institute of Allergy and Infectious Diseases)

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The replication process is tightly regulated, with each stage presenting potential targets for therapeutic intervention. For instance, inhibitors targeting the F protein’s conformational changes have shown promise in preclinical studies. Understanding RSV’s replication cycle is thus critical for developing antivirals and improving treatment outcomes [Figure 3].

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Figure 3:

Replication cycle of respiratory syncytial virus (RSV).

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Implications of genetic variability

The genetic evolution of RSV involves antigenic drift and recombination, particularly within the G protein gene, complicating the advancement of vaccinations and treatment approaches. Mutations in G protein’s mucin-like domain affect antigenicity and immune evasion. The F protein, although more conserved, exhibits variability in its prefusion and postfusion conformations, influencing viral infectivity and vaccine design.^[16] Genetic variety has pragmatic significance for RSV surveillance and vaccine development. For instance, ON1 Genotypes are linked to increased transmissibility and severity, necessitating genotype-specific interventions. By mapping genetic changes, researchers can anticipate shifts in RSV epidemiology and refine therapeutic strategies accordingly.

“Recent molecular analyses have revealed complex evolutionary patterns in RSV strains circulating globally. The G protein gene shows remarkable plasticity, with distinct duplication events characterizing major genotypes. The ON1 genotype of RSV-A, which emerged through a 72-nucleotide duplication in the C-terminal region of the G gene, has demonstrated enhanced transmissibility and virulence.^[17] Similarly, the BA genotype of RSV-B, characterized by a 60-nucleotide duplication, has shown increased fitness in population-level studies. Comprehensive genomic analyses have identified specific mutations in the F protein that affects neutralization sensitivity and vaccine efficacy.^[18]

Clinical studies correlating genotypes with disease severity have shown that certain ON1 variants are associated with increased hospitalization rates and oxygen requirements, particularly in infants under 6 months.^[19] The BA9 genotype of RSV-B has demonstrated enhanced replication efficiency in primary human airway epithelial cells, though its clinical implications remain under investigation.^[20] These findings have crucial implications for vaccine development and therapeutic strategies.”

Age and seasonal patterns

RSV infection rates and severity vary by age. Infants under 6 months face the greatest risk of severity disease, including pneumonia and bronchiolitis.^[24] RSV follows a seasonal pattern, peaking during winter in temperate regions and aligning with rainy seasons in tropical areas.^[25] The seasonality is influenced by climate factors such as temperature and humidity, which affect viral transmission rates.^[26]

Regional variations

The RSV-A: RSV-B ratio exhibits significant geographical variation. In North America and Europe, RSV-A predominates, while both subtypes co-circulate in Asia and Africa. These variations are influenced by local immunity, climate, and healthcare infrastructure.^[27] For example, RSV-B is more prevalent in sub-Saharan Africa, likely due to regional differences in immune pressure and viral evolution.^[28]

Regional studies have highlighted the importance of tailored public health interventions. In regions with high RSV-B prevalence, vaccination strategies may need to account for genotype-specific differences to achieve optimal protection. Surveillance data also inform resource allocation during seasonal peaks, minimizing the strain on healthcare systems.

RSV burden in India

Recent epidemiological studies from India have revealed a significant RSV disease burden among children. A comprehensive surveillance study across major Indian cities found that RSV accounts for 17–32% of hospitalizations due to acute lower respiratory infections in children under 5 years.^[29] The RSV-A subtype predominates in most Indian regions, with distinct seasonal patterns coinciding with monsoon months in different geographical areas. A multi-center study across six Indian states reported significantly higher hospitalization rates (45.3/1000) in infants below 6 months compared to older children.^[30] Urban areas showed higher detection rates, possibly due to better surveillance systems and healthcare access. Molecular surveillance data from 2020 to 2024 indicate the circulation of multiple genotypes, with ON1 being predominant among RSV-A strains and BA9 among RSV-B strains.^[31] The burden is particularly concerning in resource-limited settings where access to intensive care facilities is limited.”

Variability by host factors

Disease severity is influenced by host factors such as age, prematurity, and underlying conditions (e.g., preexisting heart defects or long-term lung conditions). Immunocompromised individuals and individuals with neuromuscular disorders are also at elevated risk.^[32] Genetic predispositions, including variations in innate immune genes, may also affect RSV susceptibility and outcomes.^[33]

Complications

Complications of severe RSV include apnea in neonates, secondary bacterial infections, and long-term sequelae such as recurrent wheezing and asthma in children. Studies suggest a strong correlation between severe RSV bronchiolitis and the subsequent development of chronic respiratory conditions.^[34] Severe cases often necessitate hospitalization, oxygen support, and, in extreme instances, mechanical ventilation. Early recognition and administration of complications are crucial for lowering illness and death rates.

RT-PCR

RT-PCR is the gold standard for RSV detection, offering high sensitivity and specificity. It targets conserved sections of the genome of RSV, including the F genes and N genes, and provides quantitative viral load data.^[35] Point-of-care RTPCR assays have improved the accessibility and speed of RSV diagnosis in clinical settings.

Sequencing and phylogenetics

Advancements in sequencing, including Sanger and NGS, have enhanced the characterization of RSV genotypes. Phylogenetic analysis aids in tracing RSV evolution and guiding vaccine design. NGS, despite its high cost, provides comprehensive insights into viral diversity.^[36] Phylogenetic trees constructed from G and F gene sequences reveal patterns of genotype dominance and interregional spread. Molecular tools have also facilitated real-time outbreak monitoring, enabling healthcare providers to implement timely interventions. These technologies underscore the importance of integrating diagnostics with epidemiological surveillance to mitigate RSV’s impact.

RSV genotyping is crucial to understanding its epidemiology, evolution, and clinical impact. Each molecular RSV genotyping method has pros and cons. This comparison compares the three most common genotyping methods, RTPCR, Sanger Sequencing, and NGS, based on their sensitivity, specificity, and clinical relevance^[37-39] [Table 2].

Challenges in vaccine development

RSV’s genetic diversity and continuous evolution present significant challenges for vaccine efficacy. Variability in the F and G proteins, principal targets for eliminating antibodies, complicates the formulation of broadly protective vaccines.^[40] In addition, incomplete and short-lived immunity following natural infection poses hurdles for vaccine-induced protection.

Recent advances

Promising vaccine candidates, including Pfizer’s ABRYSVO™ and GSK’s Arexvy, have shown efficacy in elderly individuals by focusing on the prefusion of F protein. These vaccines highlight the potential for age-specific formulations.^[41] Maternal vaccination strategies, aimed at transferring protective antibodies to neonates are also under investigation and have demonstrated encouraging outcomes in clinical trials [Figure 4].

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Figure 4:

Food and Drug Administration (FDA) approves respiratory syncytial virus vaccine in pregnancy to protect infants. The Washington post (Nirappil, 2023)

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“The RSV vaccine landscape has evolved significantly with recent approvals. GSK’s Arexvy (RSVPreF3) utilizes a stabilized prefusion F protein with AS01 adjuvant, demonstrating 83% efficacy against severe RSV disease in adults aged 60 and older.^[42] Pfizer’s ABRYSVO, a bivalent prefusion F protein-based vaccine, has shown efficacy in both maternal immunization (82% protection against severe RSV in infants through 6 months of age) and older adult populations.^[43] Moderna’s mRNA-1345, approved in late 2023, represents the first mRNA-based RSV vaccine, showing 84% efficacy against RSV-associated lower respiratory tract disease in adults 60 years and older.^[44]

Several promising vaccine candidates are in advanced clinical trials. These include novel live-attenuated vaccines for pediatric populations, showing favorable safety profiles and immunogenicity in phase 2 trials.^[45] Vector-based vaccines using adenovirus platforms have demonstrated robust immune responses in early-phase studies.^[46] In addition, combined RSV/influenza vaccines have shown promising results in phase 2 trials, potentially offering simplified vaccination schedules for older adults.^[47]

Recent advances in monoclonal antibodies have significantly expanded preventive options. Nirsevimab (Beyfortus), approved in 2023, provides single-dose protection through an infant’s first RSV season, showing 74.5% efficacy against medically attended RSV LRTIs.^[48] This represents a substantial improvement over palivizumab’s monthly dosing requirement. New monoclonal antibodies in development include those targeting multiple epitopes simultaneously and formulations optimized for cost-effectiveness in resource-limited settings.^[49]”

Surveillance and outbreak management

Global surveillance initiatives, such as those led by the World Health Organization, monitor RSV’s genetic diversity and seasonal trends, enabling targeted public health interventions.^[50] Early detection through molecular surveillance aids in outbreak management and resource allocation. Digital tools, such as real-time epidemic tracking and predictive modeling, enhance the timeliness and precision of responses to RSV outbreaks.

Tailored vaccination strategies

Population-specific vaccination strategies, informed by regional genotype prevalence, are essential for optimizing vaccine effectiveness. Molecular surveillance informs these strategies by identifying dominant strains and guiding vaccine updates.^[14] For instance, incorporating regionally prevalent genotypes into vaccine formulations could enhance their protective efficacy in high-burden areas.