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Respiratory syncytial virus (RSV) is a pleomorphic enveloped virus that buds as both spherical and filamentous particles. The determinants of RSV particle morphology and the roles of these forms in transmission and pathogenicity are not clearly defined, owing to a complex interplay of viral proteins and host factors that remains poorly understood. To further characterize RSV morphology, we developed a sucrose gradient velocity sedimentation method to separate spherical and filamentous virions. Fluorescence microscopy and electron microscopy (EM) confirmed two distinct peaks containing predominantly spherical or filamentous particles, respectively. Notably, EM images revealed a distinctive “honeycomb” pattern on the RSV envelope, suggesting an ordered lattice of glycoproteins on the virion surface. Biochemical analyses of viral protein and lipid content showed that filamentous particles contained higher levels of uncleaved fusion protein F0 and exhibited distinct phospholipid profiles compared to spherical particles. Both forms were enriched in cholesterol and phospholipids characteristic of lipid rafts, consistent with the idea that RSV buds from lipid raft microdomains. This enrichment in raft lipids is linked to cell-to-cell fusion (syncytium) formation and virion assembly. Quantitative real-time PCR analysis indicated a high particle-to-PFU ratio (~4:1), meaning only about one in four RSV virions was infectious. Spherical particles contained on average ~3 genomic RNA copies per virion, whereas filamentous particles contained ~2 copies. These data reveal several structural and compositional differences between RSV particle morphologies that may influence viral pathogenesis, and they provide a foundation for new antiviral approaches targeting virion assembly and morphology.

Respiratory syncytial virus (RSV) is a major human respiratory pathogen, particularly affecting infants, the elderly, and immunocompromised individuals. RSV exists in both spherical and filamentous forms, with the filamentous morphology associated with enhanced infectivity and cell-to-cell spread. Here, we demonstrate that RhoA, a small GTPase involved in cytoskeletal regulation, is essential for filamentous RSV morphogenesis through its role in organizing lipid raft microdomains. Rhosin, a selective RhoA inhibitor developed through structure-guided screening, disrupts GEF–RhoA interactions to block RhoA activation. The pharmacological inhibition of RhoA with Rhosin significantly reduced filamentous virion formation, disrupted RSV fusion (F) protein colocalization with lipid rafts, and diminished cell-to-cell fusion, without affecting overall viral replication. Scanning electron microscopy revealed that Rhosin-treated infected HEp-2 cells exhibited fewer and shorter filamentous projections compared to the extensive filament formation seen in untreated cells. β-galactosidase-based fusion assays confirmed that reduced filamentation corresponded with decreased cell-to-cell fusion. The biophysical separation of RSV spherical and filamentous particles by sucrose gradient velocity sedimentation, coupled with fluorescence and transmission electron microscopy, showed that Rhosin treatment shifted virion morphology toward spherical forms. This suggests that RhoA activity is critical for filamentous virion assembly, which may enhance viral spread. Immunofluorescence microscopy using lipid raft-selective dyes (DiIC16) and fusion protein-specific antibodies revealed the strong co-localization of RSV proteins with lipid rafts. Importantly, the pharmacological inhibition of RhoA with Rhosin disrupted F protein partitioning into raft domains, underscoring the requirement for intact lipid rafts in assembly. These findings highlight a novel role for host RhoA signaling in regulating viral assembly through raft microdomain organization, offering a potential target for host-directed antiviral intervention aimed at altering RSV structural phenotypes and limiting pathogenesis.

Background Estimating population prevalence and incidence of prior SARS-CoV-2 infection is essential to formulate public health recommendations concerning the COVID-19 pandemic. However, interpreting estimates based on sero-surveillance requires an understanding of the duration of elevated antibodies following SARS-CoV-2 infection, especially in the large number of people with pauci-symptomatic or asymptomatic disease. Methods We examined > 30,000 serology assays for SARS-CoV-2 specific IgG and IgM assays acquired longitudinally in 11,468 adults between April and November 2020 in the COVID-19 Community Research Partnership. Results Among participants with serologic evidence for infection but few or no symptoms or clinical disease, roughly 50% sero-reverted in 30 days of their initial positive test. Sero-reversion occurred more quickly for IgM than IgG and for antibodies targeting nucleocapsid protein compared with spike proteins, but was not associated with age, sex, race/ethnicity, or healthcare worker status. Conclusions The short duration of antibody response suggests that the true population prevalence of prior SARS-CoV-2 infection may be significantly higher than presumed based on earlier sero-surveillance studies. The impact of the large number of minimally symptomatic COVID-19 cases with only a brief antibody response on population immunity remains to be determined.

Modified vaccinia Ankara (MVA) was evaluated as an alternative to Dryvax ® in vaccinia-naïve and vaccinia-immune adult volunteers. Subjects received intramuscular MVA or placebo followed by Dryvax ® challenge at 3 months. Two or more doses of MVA prior to Dryvax ® reduced severity of lesion formation, decreased magnitude and duration of viral shedding, and augmented post-Dryvax ® vaccinia-specific CD8+ T cell responses and extracellular enveloped virus protein-specific antibody responses. MVA vaccination is safe and immunogenic and improves the safety and immunogenicity of subsequent Dryvax ® vaccination supporting the potential for using MVA as a vaccine in the general population to improve immunity to orthopoxviruses.

Despite the declaration of smallpox eradication in 1980, the existence of variola stockpiles and the threat of bioterrorism demand that immunity to smallpox through vaccination be maintained. Although the currently available vaccine was used for the most successful medical intervention ever accomplished, it also is associated with side effects that are difficult to accept in a vaccine for a disease that has not been present for >25 years. Herein, we review alternative approaches to maintaining immunity to smallpox through vaccination with attenuated poxviruses, and we suggest modified vaccinia Ankara (MVA) as a leading candidate for an alternative smallpox vaccine.

Background. Dolutegravir (DTG; S/GSK1349572), a human immunodeficiency virus type 1 (HIV-1) integrase inhibitor, has limited cross-resistance to raltegravir (RAL) and elvitegravir in vitro. This phase IIb study assessed the activity of DTG in HIV-1—infected subjects with genotypic evidence of RAL resistance. Methods. Subjects received DTG 50 mg once daily (cohort I) or 50 mg twice daily (cohort II) while continuing a failing regimen (without RAL) through day 10, after which the background regimen was optimized, when feasible, for cohort I, and at least 1 fully active drug was mandated for cohort II. The primary efficacy end point was the proportion of subjects on day 11 in whom the plasma HIV-1 RNA load decreased by ≥0.7 log10 copies/mL from baseline or was <400 copies/mL. Results. A rapid antiviral response was observed. More subjects achieved the primary end point in cohort II (23 of 24 [96%]), compared with cohort I (21 of 27 [78%]) at day 11. At week 24, 41% and 75% of subjects had an HIV-1 RNA load of <50 copies/mL in cohorts I and II, respectively. Further integrase genotypic evolution was uncommon. Dolutegravir had a good, similar safety profile with each dosing regimen. Conclusion. Dolutegravir 50 mg twice daily with an optimized background provided greater and more durable benefit than the once-daily regimen. These data are the first clinical demonstration of the activity of any integrase inhibitor in subjects with HIV-1 resistant to RAL.

The COVID-19 Community Research Partnership is a population-based longitudinal syndromic and sero-surveillance study. The study includes over 17,000 participants from six healthcare systems in North Carolina who submitted over 49,000 serology results. The purpose of this study is to use these serology data to estimate the cumulative proportion of the North Carolina population that has either been infected with SARS-CoV-2 or developed a measurable humoral response to vaccination. Adult community residents were invited to participate in the study between April 2020 and February 2021. Demographic information was collected and daily symptom screen was completed using a secure, HIPAA-compliant, online portal. A portion of participants were mailed kits containing a lateral flow assay to be used in-home to test for presence of anti-SARS-CoV-2 IgM or IgG antibodies. The cumulative proportion of participants who tested positive at least once during the study was estimated. A standard Cox proportional hazards model was constructed to illustrate the probability of seroconversion over time up to December 20, 2020 (before vaccines available). A separate analysis was performed to describe the influence of vaccines through February 15, 2021. 17,688 participants contributed at least one serology result. 68.7% of the population were female, and 72.2% were between 18 and 59 years of age. The average number of serology results submitted per participant was 3.0 (±1.9). By December 20, 2020, the overall probability of seropositivity in the CCRP population was 32.6%. By February 15, 2021 the probability among healthcare workers and non-healthcare workers was 83% and 49%, respectively. An inflection upward in the probability of seropositivity was demonstrated around the end of December, suggesting an influence of vaccinations, especially for healthcare workers. Among healthcare workers, those in the oldest age category (60+ years) were 38% less likely to have seroconverted by February 15, 2021. Results of this study suggest more North Carolina residents may have been infected with SARS-CoV-2 than the number of documented cases as determined by positive RNA or antigen tests. The influence of vaccinations on seropositivity among North Carolina residents is also demonstrated. Additional research is needed to fully characterize the impact of seropositivity on immunity and the ultimate course of the pandemic.

Introduction The COVID-19 Community Research Partnership is a population-based longitudinal syndromic and sero-surveillance study. The study includes over 17,000 participants from six healthcare systems in North Carolina who submitted over 49,000 serology results. The purpose of this study is to use these serology data to estimate the cumulative proportion of the North Carolina population that has either been infected with SARS-CoV-2 or developed a measurable humoral response to vaccination. Methods Adult community residents were invited to participate in the study between April 2020 and February 2021. Demographic information was collected and daily symptom screen was completed using a secure, HIPAA-compliant, online portal. A portion of participants were mailed kits containing a lateral flow assay to be used in-home to test for presence of anti-SARS-CoV-2 IgM or IgG antibodies. The cumulative proportion of participants who tested positive at least once during the study was estimated. A standard Cox proportional hazards model was constructed to illustrate the probability of seroconversion over time up to December 20, 2020 (before vaccines available). A separate analysis was performed to describe the influence of vaccines through February 15, 2021. Results 17,688 participants contributed at least one serology result. 68.7% of the population were female, and 72.2% were between 18 and 59 years of age. The average number of serology results submitted per participant was 3.0 (±1.9). By December 20, 2020, the overall probability of seropositivity in the CCRP population was 32.6%. By February 15, 2021 the probability among healthcare workers and non-healthcare workers was 83% and 49%, respectively. An inflection upward in the probability of seropositivity was demonstrated around the end of December, suggesting an influence of vaccinations, especially for healthcare workers. Among healthcare workers, those in the oldest age category (60+ years) were 38% less likely to have seroconverted by February 15, 2021. Conclusions Results of this study suggest more North Carolina residents may have been infected with SARS-CoV-2 than the number of documented cases as determined by positive RNA or antigen tests. The influence of vaccinations on seropositivity among North Carolina residents is also demonstrated. Additional research is needed to fully characterize the impact of seropositivity on immunity and the ultimate course of the pandemic.

Modified vaccinia Ankara (MVA) was evaluated as an alternative to Dryvax super([registered]) in vaccinia-naive and vaccinia-immune adult volunteers. Subjects received intramuscular MVA or placebo followed by Dryvax super([registered]) challenge at 3 months. Two or more doses of MVA prior to Dryvax super([registered]) reduced severity of lesion formation, decreased magnitude and duration of viral shedding, and augmented post-Dryvax super([registered]) vaccinia-specific CD8+ T cell responses and extracellular enveloped virus protein-specific antibody responses. MVA vaccination is safe and immunogenic and improves the safety and immunogenicity of subsequent Dryvax super([registered]) vaccination supporting the potential for using MVA as a vaccine in the general population to improve immunity to orthopoxviruses.

Modified vaccinia Ankara (MVA) was evaluated as an alternative to Dryvax® in vaccinia-naïve and immune adult volunteers. Subjects received intramuscular MVA or placebo followed by Dryvax® challenge at 3 months. Two or more doses of MVA prior to Dryvax® reduced severity of lesion formation, decreased magnitude and duration of viral shedding, and augmented post-Dryvax® vaccinia-specific CD8+ T cell responses and extracellular enveloped virus protein-specific antibody responses. MVA vaccination is safe and immunogenic and improves the safety and immunogenicity of subsequent Dryvax® vaccination supporting the potential for using MVA as a vaccine in the general population to improve immunity to orthopoxviruses.