Effects of neutrophil extracellular traps during human respiratory syncytial virus infection in vitro

The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.5–16 μg/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.

Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation

Recoding viral genomes by introducing numerous synonymous nucleotide substitutions that create suboptimal codon pairs provides new live-attenuated vaccine candidates. Because recoding typically involves a large number of nucleotide substitutions, the risk of de-attenuation is presumed to be low. However, this has not been thoroughly studied. We previously generated human respiratory syncytial virus (RSV) in which the NS1, NS2, N, P, M and SH ORFs were codon-pair deoptimized (CPD) by 695 synonymous nucleotide changes (Min A virus). Min A exhibited a global reduction in transcription and protein synthesis, was restricted for replication in vitro and in vivo, and exhibited moderate temperature sensitivity. Here, we show that under selective pressure by serial passage at progressively increasing temperatures, Min A regained replication fitness and lost its temperature sensitivity. Whole-genome deep sequencing identified numerous missense mutations in several genes, in particular ones accumulating between codons 25 and 34 of the phosphoprotein (P), a polymerase cofactor and chaperone. When re-introduced into Min A, these P mutations restored viral transcription to wt level, resulting in increased protein expression and RNA replication. Molecular dynamic simulations suggested that these P mutations increased the flexibility of the N-terminal domain of P, which might facilitate its interaction with the nucleoprotein N, and increase the functional efficiency of the RSV transcription/replication complex. Finally, we evaluated the effect of the P mutations on Min A replication and immunogenicity in hamsters. Mutation P[F28V] paradoxically reduced Min A replication but not its immunogenicity. The further addition of one missense mutation each in M and L generated a version of Min A with increased genetic stability. Thus, this study provides further insight into the adaptability of large-scale recoded RNA viruses under selective pressure and identified an improved CPD RSV vaccine candidate.

Viral RNA N6-methyladenosine modification modulates both innate and adaptive immune responses of human respiratory syncytial virus

Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in humans. A well-known challenge in the development of a live attenuated RSV vaccine is that interferon (IFN)-mediated antiviral responses are strongly suppressed by RSV nonstructural proteins which, in turn, dampens the subsequent adaptive immune responses. Here, we discovered a novel strategy to enhance innate and adaptive immunity to RSV infection. Specifically, we found that recombinant RSVs deficient in viral RNA N6-methyladenosine (m6A) and RSV grown in m6A methyltransferase (METTL3)-knockdown cells induce higher expression of RIG-I, bind more efficiently to RIG-I, and enhance RIG-I ubiquitination and IRF3 phosphorylation compared to wild-type virion RNA, leading to enhanced type I IFN production. Importantly, these m6A-deficient RSV mutants also induce a stronger IFN response in vivo, are significantly attenuated, induce higher neutralizing antibody and T cell immune responses in mice and provide complete protection against RSV challenge in cotton rats. Collectively, our results demonstrate that inhibition of RSV RNA m6A methylation enhances innate immune responses which in turn promote adaptive immunity.

1199. Decreased Human Respiratory Syncytial Virus Activity during the COVID-19 Pandemic in Japan: An Ecological Time-Series Analysis, 2014 through 2020

Background Non-pharmaceutical interventions (NPIs), such as sanitary measures and travel restrictions, aimed at controlling the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), may affect the transmission dynamics of human respiratory syncytial virus (HRSV). We aimed to quantify the contribution of the sales of hand hygiene products and the number of international and domestic airline passenger arrivals on HRSV epidemic in Japan. Methods The monthly number of HRSV cases per sentinel site (HRSV activity) in 2020 was compared with the average of the corresponding period in the previous 6 years (from January 2014 to December 2020) using a monthly paired t-test. A generalized linear Poisson regression model was used to regress the time-series of the monthly HRSV activity against NPI indicators, including sale of hand hygiene products and the number of domestic and international airline passengers, while controlling for meteorological conditions (monthly average temperature and relative humidity) and seasonal variations between years (2014–2020). Results The average number of monthly HRSV case notifications in 2020 decreased by approximately 85% (P < 0.001) compared to those in the preceding 6 years (2014–2019) (Figure 1A). For every average ¥1 billion (approximately &9,000,000/£6,800,00) spent on hand hygiene products during the current month and 1 month before (lag 0-1 months) there was a 0.22% (P = 0.02) decrease in HRSV infections (Table 1). An increase of average 1,000 domestic and international airline passenger arrivals during the previous 1–2 months (lag 1–2 months) was associated with a 4.6×10−4% (P < 0.001) and 1.1×10−3% (P = 0.007) increase in the monthly number of HRSV infections, respectively. Figure 1. Monthly seasonal variations of number of HRSV activity, NPI indicators, and meteorological conditions during 2014-2020. (A) Monthly seasonal variations of number of HRSV cases per sentinel sites based on national HRSV surveillance data during 2014-2020. (B) Monthly seasonal variations of retail sales of hand hygiene products per ¥1 billion (unit: yen) during 2014-2020. (C) Monthly seasonal variations of number of domestic airline passengers per 1,000 population (unit: person) during 2014-2020. (D) Monthly seasonal variations of number of international airline passengers per 1,000 population (unit: person) during 2014-2020. (E) Monthly seasonal variations of average temperature (unit: ℃) throughout Japan during 2014-2020. (F) Monthly seasonal variations of relative humidity (unit: %) throughout Japan during 2014-2020. Table 1. Generalized linear Poisson regression model for the monthly number of human respiratory syncytial virus cases among prefectures in Japan. Conclusion This study suggests that there is an association between the decrease in the monthly number of HRSV cases and improved hygiene and sanitary measures and travel restrictions for COVID-19 in Japan, indicating that these public health interventions can contribute to the suppression of HRSV activity. These findings may help in public health policy and decision making. Disclosures All Authors: No reported disclosures

Comparison of 11 respiratory pathogens among hospitalized children before and during the COVID-19 epidemic in Shenzhen, China

Background The effect of SARS-CoV-2 on existing respiratory pathogens in circulation remains uncertain. This study aimed to assess the impact of SARS-CoV-2 on the prevalence of respiratory pathogens among hospitalized children. Methods This study enrolled hospitalized children with acute respiratory infections in Shenzhen Children’s Hospital from September to December 2019 (before the COVID-19 epidemic) and those from September to December 2020 (during the COVID-19 epidemic). Nasopharyngeal swabs were collected, and respiratory pathogens were detected using multiplex PCR. The absolute case number and detection rates of 11 pathogens were collected and analyzed. Results A total of 5696 children with respiratory tract infection received multiplex PCR examination for respiratory pathogens: 2298 from September to December 2019 and 3398 from September to December 2020. At least one pathogen was detected in 1850 (80.5%) patients in 2019, and in 2380 (70.0%) patients in 2020; the detection rate in 2020 was significantly lower than that in 2019.The Influenza A (InfA) detection rate was 5.6% in 2019, but 0% in 2020. The detection rates of Mycoplasma pneumoniae, Human adenovirus, and Human rhinovirus also decreased from 20% (460), 8.9% (206), and 41.8% (961) in 2019 to 1.0% (37), 2.1% (77), and 25.6% (873) in 2020, respectively. In contrast, the detection rates of Human respiratory syncytial virus, Human parainfluenza virus, and Human metapneumovirus increased from 6.6% (153), 9.9% (229), and 0.5% (12) in 2019 to 25.6% (873), 15.5% (530), and 7.2% (247) in 2020, respectively (p < 0.0001). Conclusions Successful containment of seasonal influenza as a result of COVID-19 control measures will ensure we are better equipped to deal with future outbreaks of both influenza and COVID-19.Caused by virus competition, the detection rates of Human respiratory syncytial virus, Human parainfluenza virus, and Human metapneumovirus increased in Shenzhen,that reminds us we need to take further monitoring and preventive measures in the next epidemic season.