Clinical Burden of Respiratory Syncytial Virus in Hospitalized Children Aged ≤5 Years (INSPIRE Study).

BACKGROUND: Respiratory syncytial virus (RSV) is a leading cause of hospitalizations in children (≤5 years of age); limited data compare burden by age. METHODS: This single-center retrospective study included children (≤5 years of age) hospitalized for >24 hours with reverse-transcription polymerase chain reaction (RT-PCR)-confirmed RSV infection (2015-2018). Hospital length of stay (LOS), intensive care unit (ICU) admissions, ICU LOS, supplemental oxygen, and medication use were assessed. Multivariate logistic regression analyses identified predictors of hospital LOS >5 days. RESULTS: Three hundred twelve patients had RSV infection (ages 0 to <6 months [35%], 6 to <12 months [15%], 1 to <2 years [25%], and 2-5 years [25%]); 16.3% had predefined comorbidities (excludes preterm infants). Median hospital LOS was 5.0 days and similar across age; 5.1% (16/312) were admitted to ICU (ICU LOS, 5.0 days), with those aged 0 to <6 months admitted most frequently (10/108 [9.3%]). Supplemental oxygen was administered in 57.7% of patients, with similar need across ages. Antibiotics were administered frequently during hospitalization (43.6%). Predictors of prolonged LOS included pneumonia (odds ratio [OR], 2.33), supplemental oxygen need (OR, 5.09), and preterm births (OR, 3.37). High viral load (RT-PCR RSV cycle threshold value <25) was associated with greater need for supplemental oxygen. CONCLUSIONS: RSV causes substantial burden in hospitalized children (≤5 years), particularly preterm infants and those aged <6 months.

Similar severity of influenza primary and re-infections in pre-school children requiring outpatient treatment due to febrile acute respiratory illness: prospective, multicentre surveillance study (2013-2015).

BACKGROUND: Influenza virus infections in immunologically naïve children (primary infection) may be more severe than in children with re-infections who are already immunologically primed. We compared frequency and severity of influenza virus primary and re-infections in pre-school children requiring outpatient treatment. METHODS: Influenza-unvaccinated children 1-5 years of age presenting at pediatric practices with febrile acute respiratory infection < 48 h after symptom onset were enrolled in a prospective, cross-sectional, multicenter surveillance study (2013-2015). Influenza types/subtypes were PCR-confirmed from oropharyngeal swabs. Influenza type/subtype-specific IgG antibodies serving as surrogate markers for immunological priming were determined using ELISA/hemagglutination inhibition assays. The acute influenza disease was defined as primary infection/re-infection by the absence/presence of influenza type-specific immunoglobulin G (IgG) and, in a second approach, by the absence/presence of subtype-specific IgG. Socio-demographic and clinical data were also recorded. RESULTS: Of 217 influenza infections, 178 were due to influenza A (87 [49%] primary infections, 91 [51%] re-infections) and 39 were due to influenza B (38 [97%] primary infections, one [3%] re-infection). Children with "influenza A primary infections" showed fever with respiratory symptoms for a shorter period than children with "influenza A re-infections" (median 3 vs. 4 days; age-adjusted p = 0.03); other disease characteristics were similar. If primary infections and re-infections were defined based on influenza A subtypes, 122 (87%) primary infections (78 "A(H3N2) primary infections", 44 "A(H1N1)pdm09 primary infections") and 18 (13%) re-infections could be classified (14 "A(H3N2) re-infections" and 4 "A(H1N1)pdm09 re-infections"). Per subtype, primary infections and re-infections were of similar disease severity. Children with re-infections defined on the subtype level usually had non-protective IgG titers against the subtype of their acute infection (16 of 18; 89%). Some patients infected by one of the influenza A subtypes showed protective IgG titers (≥ 1:40) against the other influenza A subtype (32/140; 23%). CONCLUSIONS: Pre-school children with acute influenza A primary infections and re-infections presented with similar frequency in pediatric practices. Contrary to expectation, severity of acute "influenza A primary infections" and "influenza A re-infections" were similar. Most "influenza A re-infections" defined on the type level turned out to be primary infections when defined based on the subtype. On the subtype level, re-infections were rare and of similar disease severity as primary infections of the same subtype. Subtype level re-infections were usually associated with low IgG levels for the specific subtype of the acute infection, suggesting only short-time humoral immunity induced by previous infection by this subtype. Overall, the results indicated recurring influenza virus infections in this age group and no or only limited heterosubtypic antibody-mediated cross-protection.

Respiratory viruses dynamics and interactions: ten years of surveillance in central Europe.

BACKGROUND: Lower respiratory tract infections are among the main causes of death. Although there are many respiratory viruses, diagnostic efforts are focused mainly on influenza. The Respiratory Viruses Network (RespVir) collects infection data, primarily from German university hospitals, for a high diversity of infections by respiratory pathogens. In this study, we computationally analysed a subset of the RespVir database, covering 217,150 samples tested for 17 different viral pathogens in the time span from 2010 to 2019. METHODS: We calculated the prevalence of 17 respiratory viruses, analysed their seasonality patterns using information-theoretic measures and agglomerative clustering, and analysed their propensity for dual infection using a new metric dubbed average coinfection exclusion score (ACES). RESULTS: After initial data pre-processing, we retained 206,814 samples, corresponding to 1,408,657 performed tests. We found that Influenza viruses were reported for almost the half of all infections and that they exhibited the highest degree of seasonality. Coinfections of viruses are frequent; the most prevalent coinfection was rhinovirus/bocavirus and most of the virus pairs had a positive ACES indicating a tendency to exclude each other regarding infection. CONCLUSIONS: The analysis of respiratory viruses dynamics in monoinfection and coinfection contributes to the prevention, diagnostic, treatment, and development of new therapeutics. Data obtained from multiplex testing is fundamental for this analysis and should be prioritized over single pathogen testing.

Performance and feasibility of universal PCR admission screening for SARS-CoV-2 in a German tertiary care hospital.

Anamnestic screening of symptoms and contact history is applied to identify coronavirus disease 2019 (COVID-19) patients on admission. However, asymptomatic and presymptomatic patients remain undetected although the viral load may be high. In this retrospective cohort study, all hospitalized patients who received polymerase chain reaction (PCR) admission testing from March 26th until May 24th, 2020 were included. Data on COVID-19-specific symptoms and contact history to COVID-19 cases were retrospectively extracted from patient files and from contact tracing notes. The compliance to the universal testing protocol was high with 90%. Out of 6940 tested patients, 27 new severe acute respiratory syndrome coronavirus-2 infections (0.4%) were detected. Seven of those COVID-19 cases (26% of all new cases) were asymptomatic and had no positive contact history, but were identified through a positive PCR test. The number needed to identify an asymptomatic patient was 425 in the first wave of the epidemic, 1218 in the low incidence phase. The specificity of the method was above 99.9%. Universal PCR testing was highly accepted by staff as demonstrated by high compliance. The costs to detect one asymptomatic case in future studies need to be traded off against the costs and damage caused by potential outbreaks of COVID-19.

Epidemiology of Human Parechovirus Type 3 Upsurge in 2 Hospitals, Freiburg, Germany, 2018.

In 2018, a cluster of pediatric human parechovirus (HPeV) infections in 2 neighboring German hospitals was detected. Viral protein 1 sequence analysis demonstrated co-circulation of different HPeV-3 sublineages and of HPeV-1 and -5 strains, thereby excluding a nosocomial outbreak. Our findings underline the need for HPeV diagnostics and sequence analysis for outbreak investigations.

Spread and clinical severity of respiratory syncytial virus A genotype ON1 in Germany, 2011-2017.

BACKGROUND: The Respiratory Syncytial Virus (RSV) A genotype ON1, which was first detected in Ontario (Canada) in 2010/11, appeared in Germany in 2011/12. Preliminary observations suggested a higher clinical severity in children infected with this new genotype. We investigated spread and disease severity of RSV-A ON1 in pediatric in- and outpatient settings. METHODS: During 2010/11 to 2016/17, clinical characteristics and respiratory samples from children with acute respiratory tract infections (RTI) were obtained from ongoing surveillance studies in 33 pediatric practices (PP), one pediatric hospital ward (PW) and 23 pediatric intensive care units (PICU) in Germany. RSV was detected in the respiratory samples by PCR; genotypes were identified by sequencing. Within each setting, clinical severity markers were compared between RSV-A ON1 and RSV-A non-ON1 genotypes. RESULTS: A total of 603 children with RSV-RTI were included (132 children in PP, 288 in PW, and 183 in PICU). Of these children, 341 (56.6%) were infected with RSV-A, 235 (39.0%) with RSV-B, and one child (0.2%) with both RSV-A and RSV-B; in 26 (4.3%) children, the subtype could not be identified. In the 341 RSV-A positive samples, genotype ON1 was detected in 247 (72.4%), NA1 in 92 (26.9%), and GA5 in 2 children (0.6%). RSV-A ON1, rarely observed in 2011/12, was the predominant RSV-A genotype in all settings by 2012/13 and remained predominant until 2016/17. Children in PP or PW infected with RSV-A ON1 did not show a more severe clinical course of disease compared with RSV-A non-ON1 infections. In the PICU group, hospital stay was one day longer (median 8 days, inter-quartile range (IQR) 7-12 vs. 7 days, IQR 5-9; p = 0.02) and duration of oxygen treatment two days longer (median 6 days, IQR 4-9 vs. 4 days, IQR 2-6; p = 0.03) for children infected with RSV-A ON1. CONCLUSIONS: In children, RSV-A ON1 largely replaced RSV-A non-ON1 genotypes within two seasons and remained the predominant RSV-A genotype in Germany during subsequent seasons. A higher clinical severity of RSV-A ON1 was observed within the group of children receiving PICU treatment, whereas in other settings clinical severity of RSV-A ON1 and non-ON1 genotypes was largely similar.

Detection of enterovirus D68 in patients hospitalised in three tertiary university hospitals in Germany, 2013 to 2014.

Enterovirus D68 (EV-D68) has been recognised as a worldwide emerging pathogen associated with severe respiratory symptoms since 2009. We here report EV-D68 detection in hospitalised patients with acute respiratory infection admitted to three tertiary hospitals in Germany between January 2013 and December 2014. From a total of 14,838 respiratory samples obtained during the study period, 246 (1.7%) tested enterovirus-positive and, among these, 39 (15.9%) were identified as EV-D68. Infection was observed in children and teenagers (0-19 years; n=31), the majority (n=22) being under five years-old, as well as in adults > 50 years of age (n=8). No significant difference in prevalence was observed between the 2013 and 2014 seasons. Phylogenetic analyses based on viral protein 1 (VP1) sequences showed co-circulation of different EV-D68 lineages in Germany. Sequence data encompassing the entire capsid region of the genome were analysed to gain information on amino acid changes possibly relevant for immunogenicity and revealed mutations in two recently described pleconaril binding sites.

Continued high incidence of children with severe influenza A(H1N1)pdm09 admitted to paediatric intensive care units in Germany during the first three post-pandemic influenza seasons, 2010/11-2012/13.

BACKGROUND: Previous influenza surveillance at paediatric intensive care units (PICUs) in Germany indicated increased incidence of PICU admissions for the pandemic influenza subtype A(H1N1)pdm09. We investigated incidence and clinical characteristics of influenza in children admitted to PICUs during the first three post-pandemic influenza seasons, using active screening. METHODS: We conducted a prospective surveillance study in 24 PICUs in Bavaria (Germany) from October 2010 to September 2013. Influenza cases among children between 1 month and 16 years of age admitted to these PICUs with acute respiratory infection were confirmed by PCR analysis of respiratory secretions. RESULTS: A total of 24/7/20 influenza-associated PICU admissions were recorded in the post-pandemic seasons 1/2/3; incidence estimates per 100,000 children were 1.72/0.76/1.80, respectively. Of all 51 patients, 80% had influenza A, including 65% with A(H1N1)pdm09. Influenza A(H1N1)pdm09 was almost absent in season 2 (incidence 0.11), but dominated PICU admissions in seasons 1 (incidence 1.35) and 3 (incidence 1.17). Clinical data was available for 47 influenza patients; median age was 4.8 years (IQR 1.6-11.0). The most frequent diagnoses were influenza-associated pneumonia (62%), bronchitis/bronchiolitis (32%), secondary bacterial pneumonia (26 %), and ARDS (21%). Thirty-six patients (77 %) had underlying medical conditions. Median duration of PICU stay was 3 days (IQR 1-11). Forty-seven per cent of patients received mechanical ventilation, and one patient (2%) extracorporeal membrane oxygenation; 19% were treated with oseltamivir. Five children (11%) had pulmonary sequelae. Five children (11%) died; all had underlying chronic conditions and were infected with A(H1N1)pdm09. In season 3, patients with A(H1N1)pdm09 were younger than in season 1 (p = 0.020), were diagnosed more often with bronchitis/bronchiolitis (p = 0.004), and were admitted to a PICU later after the onset of influenza symptoms (p = 0.041). CONCLUSIONS: Active screening showed a continued high incidence of A(H1N1)pdm09-associated PICU admissions in the post-pandemic seasons 1 and 3, and indicated possible underestimation of incidence in previous German studies. The age shift of severe A(H1N1)pdm09 towards younger children may be explained by increasing immunity in the older paediatric population. The high proportion of patients with underlying chronic conditions indicates the importance of consistent implementation of the current influenza vaccination recommendations for risk groups in Germany.

Molecular characterization of a respiratory syncytial virus outbreak in a hematology unit in Heidelberg, Germany.

In 2011 and 2012, a large outbreak of respiratory syncytial virus (RSV) infections affecting 57 laboratory-confirmed patients occurred in an adult hematology unit in Heidelberg, Germany. During the outbreak investigation, we performed molecular genotyping of RSV strains to differentiate between single versus multiple introductions of the virus into the unit. Furthermore, we assessed the time of viral shedding of consecutive samples from the patients in order to better understand the possible impact of prolonged shedding for outbreak control management. We used subtype-specific reverse transcription-PCR on nasopharyngeal and bronchoalveolar specimens for routine diagnostics and for measuring the viral shedding period. Samples of 47 RSV-infected patients involved in the outbreak were genotyped by sequence analysis and compared to samples from RSV-infected hospitalized children representing the timing of the annual RSV epidemic in the community. Molecular investigation of the virus strains from clinical samples revealed a unique cluster with identical nucleotide sequences of RSV type A (RSV A outbreak strain) for 41 patients, while 3 patients were infected with different RSV A (nonoutbreak) strains and three other patients with RSV type B. Outbreak strains were identified in samples from November 2011 until January 2012, while nonoutbreak strains were from samples coinciding with the community epidemic in February and March 2012. Median duration of viral shedding time was 24.5 days (range, 1 to 168 days) with no difference between outbreak and nonoutbreak strains (P = 0.45). Our investigation suggests a single introduction of the RSV A outbreak strain into the unit that spread among the immunocompromised patients. Prolonged viral shedding may have contributed to nosocomial transmission and should be taken into account in the infection control management of RSV outbreaks in settings with heavily immunosuppressed patients.

The innate antiviral factor APOBEC3G targets replication of measles, mumps and respiratory syncytial viruses.

The cytidine deaminase APOBEC3G (apolipoprotein B mRNA-editing enzyme-catalytic polypeptide 3G; A3G) exerts antiviral activity against retroviruses, hepatitis B virus, adeno-associated virus and transposable elements. We assessed whether the negative-strand RNA viruses measles, mumps and respiratory syncytial might be affected by A3G, and found that their infectivity was reduced by 1-2 logs (90-99 %) in A3G overexpressing Vero cells, and in T-cell lines expressing A3G at physiological levels. Viral RNA was co-precipitated with HA-tagged A3G and could be amplified by RT-PCR. Interestingly, A3G reduced viral transcription and protein expression in infected cells by 50-70 %, and caused an increased mutation frequency of 0.95 mutations per 1000 nt in comparison to the background level of 0.22/1000. The observed mutations were not specific for A3G [cytidine to uridine (C→U) or guanine to adenine (G→A) hypermutations], nor specific for ADAR (adenosine deaminase acting on RNA, A→G and U→C transitions, with preference for next neighbour-nucleotides U = A>C>G). In addition, A3G mutants with inactivated catalytic deaminase (H257R and E259Q) were inhibitory, indicating that the deaminase activity is not required for the observed antiviral activity. In combination, impaired transcription and increased mutation frequencies are sufficient to cause the observed reduction in viral infectivity and eliminate virus replication within a few passages in A3G-expressing cells.

Parainfluenza virus infections in patients with hematological malignancies or stem cell transplantation: Analysis of clinical characteristics, nosocomial transmission and viral shedding.

To assess morbidity and mortality of parainfluenza virus (PIV) infections in immunocompromised patients, we analysed PIV infections in a hematology and stem cell transplantation (SCT) unit over the course of three years. Isolated PIV strains were characterized by sequence analysis and nosocomial transmission was assessed including phylogenetic analysis of viral strains. 109 cases of PIV infection were identified, 75 in the setting of SCT. PIV type 3 (n = 68) was the most frequent subtype. PIV lower respiratory tract infection (LRTI) was observed in 47 patients (43%) with a mortality of 19%. Severe leukopenia, prior steroid therapy and presence of co-infections were significant risk factors for development of PIV-LRTI in multivariate analysis. Prolonged viral shedding was frequently observed with a median duration of 14 days and up to 79 days, especially in patients after allogeneic SCT and with LRTI. Nosocomial transmission occurred in 47 patients. Phylogenetic analysis of isolated PIV strains and combination with clinical data enabled the identification of seven separate clusters of nosocomial transmission. In conclusion, we observed significant morbidity and mortality of PIV infection in hematology and transplant patients. The clinical impact of co-infections, the possibility of long-term viral shedding and frequent nosocomial transmission should be taken into account when designing infection control strategies.

High prevalence of antibodies against polyomavirus WU, polyomavirus KI, and human bocavirus in German blood donors.

BACKGROUND: DNA of the polyomaviruses WU (WUPyV) and KI (KIPyV) and of human bocavirus (HBoV) has been detected with varying frequency in respiratory tract samples of children. However, only little is known about the humoral immune response against these viruses. Our aim was to establish virus-specific serological assays and to determine the prevalence of immunoglobulin G (IgG) against these three viruses in the general population. METHODS: The capsid proteins VP1 of WUPyV and KIPyV and VP2 of HBoV were cloned into baculovirus vectors and expressed in Sf9 insect cells. IgG antibodies against WUPyV VP1, KIPyV VP1, and HBoV VP2 were determined by immunofluorescence assays in 100 plasma samples of blood donors. RESULTS: The median age of the blood donors was 31 years (range 20 - 66 yrs), 52% were male. 89% of the samples were positive for WUPyV IgG (median age 31 yrs, 49.4% male), 67% were positive for KIPyV IgG (median age 32 yrs, 46.3% male), and 76% were positive for HBoV IgG (median age 32 yrs, 51.3% male). For WUPyV and HBoV, there were no significant differences of the seropositivity rates with respect to age groups or gender. For KIPyV, the seropositivity rate increased significantly from 59% in the age group 20 - 29 years to 100% in the age group > 50 years. CONCLUSIONS: High prevalences of antibodies against WUPyV, KIPyV, and HBoV were found in plasma samples of healthy adults. The results indicate that primary infection with these viruses occurs during childhood or youth. For KIPyV, the seropositivity appears to increase further during adulthood.

Subtype-specific Clinical Presentation, Medical Treatment and Family Impact of Influenza in Children 1-5 Years of Age Treated in Outpatient Practices in Germany During Three Postpandemic Years, 2013-2015.

BACKGROUND: Limited data on the influenza burden in pediatric outpatients are available, especially regarding direct comparison of the cocirculating (sub)types A(H1N1)pdm09, A(H3N2) and B. METHODS: Children 1-5 years of age, unvaccinated against influenza and presenting with febrile acute respiratory infections (ARIs), were enrolled in 33 pediatric practices in Germany from 2013 to 2015 (January-May). Influenza was confirmed by multiplex polymerase chain reaction from pharyngeal swabs and (sub)typed. RESULTS: In 805 children with ARI, influenza was the most frequently detected respiratory virus (n = 305; 37.9%). Of 217 influenza patients included, 122 (56.2%) were infected with A(H3N2), 56 (25.8%) with A(H1N1)pdm09 and 39 (18.0%) with B. Median age was 3.7 years [interquartile range (IQR), 2.1-4.8]; 11% had underlying conditions. Median fever duration was 4 days (IQR, 3-5), and the disease duration was 9 days (IQR, 7-12). Most frequent diagnoses were pharyngitis (26%), bronchitis (18%) and acute otitis media (10%). Children received mainly antipyretics (86%) and adrenergic nasal drops/spray (53%); 9% received antibiotics and 3% oseltamivir. Thirty-six percent required at least 1 additional practice visit; 1% was hospitalized. Median absences from childcare were 5 days (IQR, 3-7); parents lost 4 workdays (IQR, 2-6). Symptoms, severity and impact on the family were largely unrelated to (sub)type. However, patients with A(H1N1)pdm09 had fewer underlying conditions (P = 0.017), whereas patients with B more often had pharyngitis (P = 0.022), acute otitis media (P = 0.012) and stenosing laryngotracheitis (P = 0.007). CONCLUSIONS: Influenza was the most frequently detected viral pathogen in outpatient children with febrile, mostly uncomplicated ARI. In this setting, clinical manifestations and severity were similar across the (sub)types prevalent during the postpandemic seasons.

Long-Term Shedding of Influenza Virus, Parainfluenza Virus, Respiratory Syncytial Virus and Nosocomial Epidemiology in Patients with Hematological Disorders.

Respiratory viruses are a cause of upper respiratory tract infections (URTI), but can be associated with severe lower respiratory tract infections (LRTI) in immunocompromised patients. The objective of this study was to investigate the genetic variability of influenza virus, parainfluenza virus and respiratory syncytial virus (RSV) and the duration of viral shedding in hematological patients. Nasopharyngeal swabs from hematological patients were screened for influenza, parainfluenza and RSV on admission as well as on development of respiratory symptoms. Consecutive swabs were collected until viral clearance. Out of 672 tested patients, a total of 111 patients (17%) were infected with one of the investigated viral agents: 40 with influenza, 13 with parainfluenza and 64 with RSV; six patients had influenza/RSV or parainfluenza/RSV co-infections. The majority of infected patients (n = 75/111) underwent stem cell transplantation (42 autologous, 48 allogeneic, 15 autologous and allogeneic). LRTI was observed in 48 patients, of whom 15 patients developed severe LRTI, and 13 patients with respiratory tract infection died. Phylogenetic analysis revealed a variety of influenza A(H1N1)pdm09, A(H3N2), influenza B, parainfluenza 3 and RSV A, B viruses. RSV A was detected in 54 patients, RSV B in ten patients. The newly emerging RSV A genotype ON1 predominated in the study cohort and was found in 48 (75%) of 64 RSV-infected patients. Furthermore, two distinct clusters were detected for RSV A genotype ON1, identical RSV G gene sequences in these patients are consistent with nosocomial transmission. Long-term viral shedding for more than 30 days was significantly associated with prior allogeneic transplantation (p = 0.01) and was most pronounced in patients with RSV infection (n = 16) with a median duration of viral shedding for 80 days (range 35-334 days). Long-term shedding of respiratory viruses might be a catalyzer of nosocomial transmission and must be considered for efficient infection control in immunocompromised patients.

Novel respiratory syncytial virus (RSV) genotype ON1 predominates in Germany during winter season 2012-13.

Respiratory syncytial virus (RSV) is the leading cause of hospitalization especially in young children with respiratory tract infections (RTI). Patterns of circulating RSV genotypes can provide a better understanding of the molecular epidemiology of RSV infection. We retrospectively analyzed the genetic diversity of RSV infection in hospitalized children with acute RTI admitted to University Hospital Heidelberg/Germany between October 2012 and April 2013. Nasopharyngeal aspirates (NPA) were routinely obtained in 240 children younger than 2 years of age who presented with clinical symptoms of upper or lower RTI. We analyzed NPAs via PCR and sequence analysis of the second variable region of the RSV G gene coding for the attachment glycoprotein. We obtained medical records reviewing routine clinical data. RSV was detected in 134/240 children. In RSV-positive patients the most common diagnosis was bronchitis/bronchiolitis (75.4%). The mean duration of hospitalization was longer in RSV-positive compared to RSV-negative patients (3.5 vs. 5.1 days; p<0.01). RSV-A was detected in 82.1%, RSV-B in 17.9% of all samples. Phylogenetic analysis of 112 isolates revealed that the majority of RSV-A strains (65%) belonged to the novel ON1 genotype containing a 72-nucleotide duplication. However, genotype ON1 was not associated with a more severe course of illness when taking basic clinical/laboratory parameters into account. Molecular characterization of RSV confirms the co-circulation of multiple genotypes of subtype RSV-A and RSV-B. The duplication in the G gene of genotype ON1 might have an effect on the rapid spread of this emerging RSV strain.