Respiratory syncytial virus seasonality, transmission zones, and implications for seasonal prevention strategy in China: a systematic analysis.

BACKGROUND: Respiratory syncytial virus (RSV) represents a substantial global health challenge, with a disproportionately high disease burden in low-income and middle-income countries. RSV exhibits seasonality in most areas globally, and a comprehensive understanding of within-country variations in RSV seasonality could help to define the timing of RSV immunisation programmes. This study focused on China, and aimed to describe the geographical distribution of RSV seasonality, identify distinct RSV transmission zones, and evaluate the potential suitability of a seasonal RSV prevention strategy. METHODS: We did a systematic analysis of RSV seasonality in China, with use of data on RSV activity extracted from a systematic review of studies published on Embase, MEDLINE, Web of Science, China National Knowledge Infrastructure, Wanfang Data, Chongqing VIP Information, and SinoMed, from database inception until May 5, 2023. We included studies of any design in China reporting at least 25 RSV cases, which aggregated RSV case number by calendar month or week at the province level, and with data covering at least 12 consecutive months before the year 2020 (prior to the COVID-19 pandemic). Studies that used only serology for RSV testing were excluded. We also included weekly data on RSV activity from open-access online databases of the Taiwan National Infection Disease Statistics System and Hong Kong Centre for Health Protection, applying the same eligiblity requirements. Across all datasets, we excluded data on RSV activity from Jan 1, 2020, onwards. We estimated RSV seasonal epidemic onset and duration using the annual average percentage (AAP) approach, and summarised seasonality at the provincial level. We used Pearson's partial correlation analysis to assess the correlation between RSV season duration and the latitude and longitude of the individual provinces. To define transmission zones, we used two independent approaches, an infant-passive-immunisation-driven approach (the moving interval approach, 6-month interval) and a data-driven approach (k-means), to identify groups of provinces with similar RSV seasonality. The systematic review was registered on PROSPERO, CRD42022376993. FINDINGS: A total of 157 studies were included along with the two online datasets, reporting data on 194 596 RSV cases over 442 study-years (covering the period from Jan 1, 1993 to Dec 31, 2019), from 52 sites in 23 provinces. Among 21 provinces with sufficient data (≥100 reported cases), the median duration of RSV seasonal epidemics was 4·6 months (IQR 4·1-5·4), with a significant latitudinal gradient (r=-0·69, p<0·0007), in that provinces on or near the Tropic of Cancer had the longest epidemic duration. We found no correlation between longitude and epidemic duration (r=-0·15, p=0·53). 15 (71%) of 21 provinces had RSV epidemics from November to March. 13 (62%) of 21 provinces had clear RSV seasonality (epidemic duration ≤5 months). The moving interval approach categorised the 21 provinces into four RSV transmission zones. The first zone, consisting of five provinces (Fujian, Guangdong, Hong Kong, Taiwan, and Yunnan), was assessed as unsuitable for seasonal RSV immunisation strategies; the other three zones were considered suitable for seasonal RSV immunisation strategies with the optimal start month varying between September (Hebei), October (Anhui, Chongqing, Henan, Hubei, Jiangsu, Shaanxi, Shandong, Shanghai, Sichuan, and Xinjiang), and November (Beijing, Gansu, Guizhou, Hunan, and Zhejiang). The k-means approach identified two RSV transmission zones, primarily differentiated by whether the province was on or near the Tropic of Cancer (Fujian, Guangdong, Hong Kong, Taiwan, Yunan, and Hunan) or not (the remaining 15 provinces). INTERPRETATION: Although substantial variations in RSV seasonality were observed across provinces of China, our study identified distinct transmission zones with shared RSV circulating patterns. These findings could have important implications for decision making on RSV passive immunisation strategy. Furthermore, the methodological framework in this study for defining RSV seasons and identifying RSV transmission zones is potentially applicable to other countries or regions. FUNDING: Nanjing Medical University. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Continuous Invasion by Respiratory Viruses Observed in Rural Households During a Respiratory Syncytial Virus Seasonal Outbreak in Coastal Kenya.

Background: Households are high-intensity close-contact environments favorable for transmission of respiratory viruses, yet little is known for low-income settings. Methods: Active surveillance was completed on 47 households in rural coastal Kenya over 6 months during a respiratory syncytial virus (RSV) season. Nasopharyngeal swabs (NPSs) were taken from 483 household members twice weekly irrespective of symptoms. Using molecular diagnostics, NPSs from 6 households were screened for 15 respiratory viruses and the remainder of households only for the most frequent viruses observed: rhinovirus (RV), human coronavirus (HCoV; comprising strains 229E, OC43, and NL63), adenovirus (AdV), and RSV (A and B). Results: Of 16928 NPSs tested for the common viruses, 4259 (25.2%) were positive for ≥1 target; 596 (13.8%) had coinfections. Detection frequencies were 10.5% RV (1780), 7.5% HCoV (1274), 7.3% AdV (1232), and 3.2% RSV (537). On average, each household and individual had 6 and 3 different viruses detected over the study period, respectively. Rhinovirus and HCoV were detected in all the 47 households while AdV and RSV were detected in 45 (95.7%) and 40 (85.1%) households, respectively. The individual risk of infection over the 6-month period was 93.4%, 80.1%, 71.6%, 61.5%, and 37.1% for any virus, RV, HCoV, AdV, and RSV, respectively. NPSs collected during symptomatic days and from younger age groups had higher prevalence of virus detection relative to respective counterparts. RSV was underrepresented in households relative to hospital admission data. Conclusions: In this household setting, respiratory virus infections and associated illness are ubiquitous. Future studies should address the health and economic implications of these observations.

Sixteen years of evolution of human respiratory syncytial virus subgroup A in Buenos Aires, Argentina: GA2 the prevalent genotype through the years.

Human respiratory syncytial virus (HRSV) is the main viral cause of acute lower respiratory tract infections (LRTI) in children worldwide. In recent years, several preclinical trials with vaccine candidates have been reported. It is in this sense that molecular epidemiological studies become important. Understanding viral dispersion patterns before and after the implementation of a vaccine can provide insight into the effectiveness of the control strategies. In this work we analyzed the molecular epidemiology of HRSV-A over a period of sixteen years (1999-2014) in Buenos Aires. By bioinformatic tools we analyzed 169 sequences of the G glycoprotein gene from hospitalized pediatric patients with LRTI. We found that GA2 was the most prevalent genotype (73.35%). GA5 genotype co-circulated in our region until 2009 when it was no longer detected, except in 2011. The recently globally emerging ON1 lineage with a 72-nt duplication increased its frequency to become the only lineage detected in Buenos Aires in 2014. By discrete phylogeographic analysis of global ON1 strains we could determine that Panama could be the location of the MRCA dated June 20, 2010; and this lineage could be introduced in Argentina from Spain in April 2011. This analysis also showed temporary and geographical clustering of ON1 strains observed as phylogenetic clades with strains exclusively associated from a single country, nevertheless among our 44 ON1 strains from three outbreaks (2012-2014) we could also detect posterior reintroductions and circulation from United States, Cuba, South Korea, and Spain. The continuous phylogeographic analysis of one sublineage of Argentine ON1 strains allowed us to establish that there could be a local clustering of some strains even in neighborhoods. This work shows the potential of this type of bioinformatic tools in the context of a future vaccine surveillance network to trace the spread of new genetic lineages in human populations.

Human respiratory syncytial virus and metapneumovirus in patients with acute respiratory infection in Colombia, 2000 - 2011 / Virus sincitial respiratorio y metapneumovirus humano en pacientes con infección respiratoria aguda en Colombia, 2000 - 2011

OBJECTIVE: To describe the epidemiology of respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) in Colombia from 2000 - 2011, including seasonal trends. METHODS: Nasopharyngeal aspirates and/or throat swabs from 14 870 patients with acute respiratory infections (ARI) were studied. Two subgroups were analyzed using molecular biology techniques. The first consisted of 264 RSV indirect fluorescence assay (IFA)-positive samples, the second of 264 RSV IFA-negative samples. RSV and hMPV were detected using reverse transcription polymerase chain reaction (RT-PCR). RESULTS: 2 799 samples (18.8%) contained a respiratory virus. RSV was detected by IFA in 1 333 samples (8.9%). RSV was detected by RT-PCR in 192 samples from the RSV IFA-positive subgroup and in 25 samples from the RSV IFA-negative subgroup. hMPV was detected in eight samples from the RSV IFA-positive subgroup and in 11 samples from the RSV IFA-negative subgroup. Among the RSV infections, subtype A was dominant in two-year intervals, subtype B was dominant in one-year intervals. 85.3% of RSV and 74% of hMPV infections occurred in children younger than 5 years old. RSV and hMPV infections were associated with rainy seasons. Co-infection with RSV A and RSV B was detected in two patients. Five cases of co-infection with RSV and hMPV were detected. CONCLUSIONS: This report is the first to examine the epidemiology of ARIs in Colombia, with an emphasis on RSV and hMPV. The samples studied here were obtained over a 12-year period and represent all age groups and both genders.

OBJETIVO: Describir la epidemiología y tendencias estacionales del virus sincitial respiratorio (VSR) y metapneumovirus humano (MPVh) en Colombia durante el período 2000 - 2011. MÉTODOS: Se recolectaron aspirados nasofaríngeos o hisopados faríngeos de 14 870 pacientes con infección respiratoria aguda. Se analizaron dos subgrupos por técnicas de biología molecular. El primero estuvo compuesto por 264 muestras positivas para el VSR por inmunofluorescencia indirecta (IFI), y el segundo estuvo compuesto por 264 muestras negativas para el VSR por IFI. La técnica utilizada para la detección del VSR y el MPVh en ambos subgrupos fue la transcripción inversa asociada a la reacción en cadena de la polimerasa (RT-PCR). RESULTADOS: 2 799 muestras (18,8%) contenían algún virus respiratorio. Se detectó VSR por IFI en 1 333 muestras (8,9%), e igualmente fue detectado por RT-PCR en 192 muestras en el subgrupo de muestras positivas para el VSR por IFI y en 25 muestras en el subgrupo de muestras negativas para el VSR por IFI. Se detectó MPVh en 8 muestras en el subgrupo de muestras positivas para el VSR por IFI, y en 11 muestras en el subgrupo de muestras negativas para el VSR por IFI. De las infecciones causadas por el VSR, el subtipo A fue dominante en períodos bianuales; en contraste, el subtipo B fue dominante en períodos anuales. El 85,3% de las infecciones por el VSR y 74% de las infecciones por el MPVh ocurrieron en niños menores de 5 años de edad. Las infecciones causadas por el VSR y el MPVh se asociaron con las estaciones de lluvia. Se encontró coinfección con VSR A y VSR B en 2 pacientes, y coinfección con VSR y MPVh en 5 pacientes. CONCLUSIONES: Esta investigación es la primera en estudiar la epidemiología de las infecciones respiratorias agudas en Colombia, con énfasis en las causadas por el VSR y el MPVh. Las muestras estudiadas fueron recolectadas durante un período de 12 años y representan todos los grupos etarios de ambos sexos.

Rapid spread and diversification of respiratory syncytial virus genotype ON1, Kenya.

Respiratory syncytial virus genotype ON1, which is characterized by a 72-nt duplication in the attachment protein gene, has been detected in >10 countries since first identified in Ontario, Canada, in 2010. We describe 2 waves of genotype ON1 infections among children admitted to a rural hospital in Kenya during 2012. Phylogenetic analysis of attachment protein gene sequences showed multiple introductions of genotype ON1; variants distinct from the original Canadian viruses predominated in both infection waves. The genotype ON1 dominated over the other group A genotypes during the second wave, and some first wave ON1 variants reappeared in the second wave. An analysis of global genotype ON1 sequences determined that this genotype has become considerably diversified and has acquired signature coding mutations within immunogenic regions, and its most recent common ancestor dates to ≈2008-2009. Surveillance of genotype ON1 contributes to an understanding of the mechanisms of rapid emergence of respiratory viruses.

Nosocomial transmission of respiratory syncytial virus in an outpatient cancer center.

Respiratory syncytial virus (RSV) outbreaks in inpatient settings are associated with poor outcomes in cancer patients. The use of molecular epidemiology to document RSV transmission in the outpatient setting has not been well described. We performed a retrospective cohort study of 2 nosocomial outbreaks of RSV at the Seattle Cancer Care Alliance. Subjects included patients seen at the Seattle Cancer Care Alliance with RSV detected in 2 outbreaks in 2007-2008 and 2012 and all employees with respiratory viruses detected in the 2007-2008 outbreak. A subset of samples was sequenced using semi-nested PCR targeting the RSV attachment glycoprotein coding region. Fifty-one cases of RSV were identified in 2007-2008. Clustering of identical viral strains was detected in 10 of 15 patients (67%) with RSV sequenced from 2007 to 2008. As part of a multimodal infection control strategy implemented as a response to the outbreak, symptomatic employees had nasal washes collected. Of 254 employee samples, 91 (34%) tested positive for a respiratory virus, including 14 with RSV. In another RSV outbreak in 2012, 24 cases of RSV were identified; 9 of 10 patients (90%) had the same viral strain, and 1 (10%) had another viral strain. We document spread of clonal strains within an outpatient cancer care setting. Infection control interventions should be implemented in outpatient, as well as inpatient, settings to reduce person-to-person transmission and limit progression of RSV outbreaks.

Infection prevention and control measures for acute respiratory infections in healthcare settings: an update.

Viruses account for the majority of the acute respiratory tract infections (ARIs) globally with a mortality exceeding 4 million deaths per year. The most commonly encountered viruses, in order of frequency, include influenza, respiratory syncytial virus, parainfluenza and adenovirus. Current evidence suggests that the major mode of transmission of ARls is through large droplets, but transmission through contact (including hand contamination with subsequent self-inoculation) and infectious respiratory aerosols of various sizes and at short range (coined as "opportunistic" airborne transmission) may also occur for some pathogens. Opportunistic airborne transmission may occur when conducting highrisk aerosol generating procedures and airborne precautions will be required in this setting. General infection control measures effective for all respiratory viral infections are reviewed and followed by discussion on some of the common viruses, including severe acute respiratory syndrome (SARS) coronavirus and the recently discovered novel coronavirus.

Mucosal delivery of human papillomavirus pseudovirus-encapsidated plasmids improves the potency of DNA vaccination.

Mucosal immunization may be important for protection against pathogens whose transmission and pathogenesis target the mucosal tissue. The capsid proteins of human papillomavirus (HPV) confer tropism for the basal epithelium and can encapsidate DNA during self-assembly to form pseudovirions (PsVs). Therefore, we produced mucosal vaccine vectors by HPV PsV encapsidation of DNA plasmids expressing an experimental antigen derived from the M and M2 proteins of respiratory syncytial virus (RSV). Intravaginal (IVag) delivery elicited local and systemic M-M2-specific CD8+ T-cell and antibody responses in mice that were comparable to an approximately 10,000-fold higher dose of naked DNA. A single HPV PsV IVag immunization primed for M-M2-specific-IgA in nasal and vaginal secretions. Based on light emission and immunofluorescent microscopy, immunization with HPV PsV-encapsidated luciferase- and red fluorescent protein (RFP)-expressing plasmids resulted in transient antigen expression (<5 days), which was restricted to the vaginal epithelium. HPV PsV encapsidation of plasmid DNA is a novel strategy for mucosal immunization that could provide new vaccine options for selected mucosal pathogens.

Nosocomial transmission of respiratory syncytial virus in neonatal intensive care and intermediate care units: a prospective epidemiologic study.

To test the hypothesis that a considerable number of preterm infants acquire respiratory syncytial virus (RSV) within the hospital during the postnatal stay, a prospective epidemiologic survey was performed. Nasopharyngeal swabs were taken twice weekly for a period of 8 weeks from preterm infants, medical/nursing staff, and parents during the peak of RSV season 2007/2008 and tested for RSV by polymerase chain reaction. Of 1002 samples, only 4 tested positive (2 from a patient, 2 from staff). Sequence analyses of the G protein demonstrated that nosocomial transmission did not occur between these individuals.

Healthcare-associated atypical pneumonia.

Atypical pneumonia was first described in 1938, and over time, Mycoplasma, Legionella, and Chlamydophila were the agents commonly linked with community-associated atypical pneumonia. However, as technology has improved, so has our understanding of this clinical entity. It is now known that there are many agents linked with atypical pneumonia in the community, and many of these agents are also major causes of healthcare-associated pneumonia. This article discusses the history, epidemiology, and pathogenesis of infection; control of infection; clinical findings; diagnosis; and, where applicable, treatment of the agents of healthcare-associated atypical pneumonia. Bacterial agents include Legionella species, Mycoplasma pneumoniae, Chlamydophila species, and Coxiella burnetii. Although there are over 100 viruses that can cause respiratory tract infections, only a fraction of those have been defined in the context of healthcare-associated atypical pneumonia: adenovirus and human bocavirus (HBoV); rhinovirus; human coronaviruses (HCoV), including HCoV 229E, HCoV OC43, HCoV NL63, HCoV HKU1; members of the paramyxoviridae (parainfluenza viruses, human metapneumovirus, and respiratory syncytial virus); hantavirus; influenza; and severe acute respiratory syndrome (SARS) Co-V. Our knowledge about healthcare-associated atypical pneumonia will continue to evolve as newer pathogens are identified and as newer diagnostic modalities such as multiplex polymerase chain reaction are introduced.

Molecular characterization of strains of respiratory syncytial virus identified in a hematopoietic stem cell transplant outpatient unit over 2 years: community or nosocomial infection?

Respiratory syncytial virus (RSV) is recognized as the leading cause of nosocomial respiratory infection among hematopoietic stem cell transplant (HSCT) recipients, causing considerable morbidity and mortality. RSV is easily transmitted by contact with contaminated surfaces, and in HSCT units, more than 50% of RSV infections have been characterized as of nosocomial origin. From April 2001 to October 2002, RSV was identified by direct immunofluorescent assay in 42 symptomatic HSCT recipients. Seven RSV strains from 2001 and 12 RSV strains from 2002 were sequenced. RNA extraction, cDNA synthesis, and seminested polymerase chain reaction (PCR) with primers complementary to RSV genes G and F were performed. PCR products were analyzed by nucleotide sequencing of the C-terminal region of gene G for typing (in group A or B). Of the 7 strains analyzed in 2001, only 2 belonged to group B; the other 5 belonged to group A. Of these 7 strains, 3 were identical and were from recipients receiving outpatient care. In 2002, of the 12 strains analyzed, 3 belonged to group A and the other 9 belonged to group B. Of these 9 strains, 7 were genetically identical and were also from recipients receiving outpatient care. Therefore, multiple strains of RSV cocirculated in the hematopoietic stem cell transplant units (ward and outpatient units) between 2001 and 2002. Nosocomial transmission was more likely to occur at the HSCT outpatient unit than in the HSCT ward. Infection control practices should also be implemented in the outpatient setting.

Strategies for prevention of RSV nosocomial infection.

OBJECTIVE: To review the literature on respiratory syncytial virus (RSV) as a cause of nosocomial infections (NI) on neonatal intensive care units (NICUs) and pediatric wards, and the effectiveness of various containment strategies. STUDY DESIGN: We conducted a literature review to define characteristics of RSV NI, and to evaluate the relative effectiveness of various infection containment programs, including the use of palivizumab on the reported incidence of RSV NI on NICUs and pediatric wards. RESULT: Highly variable rates of RSV NI have not significantly changed since RSV was first identified. The evaluation of the effectiveness of containment strategies has relied on before/after study designs. Focus on rapid patient diagnosis, compliance of acceptable handwashing techniques and cohorting of patients and staff appears to form the backbone of most prevention and containment programs. When these or other measures have failed, the administration of palivizumab has been useful in halting the spread of RSV NI in children. CONCLUSION: RSV NI continues to be prevalent in the NICU despite adoption of infection control programs. Preventive measures should be employed to lower the risk of RSV NI and, if identified, appropriate containment strategies should be rapidly implemented.

Preventing respiratory syncytial virus in homebound premature infants.

This article explores the home health nurse's role in preventing respiratory syncytial virus (RSV) among premature infants. Thousands of children infected with RSV require hospitalization each year. Consistent contact with the infant alerts the nurse to subtle signs and symptoms of RSV infection, which may include nasal congestion, cough, low-grade fever, and malaise. By developing patient and caregiver trust, the home health nurse can implement an RSV prevention plan, leading to a decrease in hospitalization episodes of premature infants with RSV. Identification of patient risk factors contributing to RSV together with caregiver education is addressed in this article.

Viral infection of the lungs through the eye.

Respiratory syncytial virus (RSV) is the foremost respiratory pathogen in newborns and claims millions of lives annually. However, there has been no methodical study of the pathway(s) of entry of RSV or its interaction with nonrespiratory tissues. We and others have recently established a significant association between allergic conjunctivitis and the presence of RSV in the eye. Here we adopt a BALB/c mouse model and demonstrate that when instilled in the live murine eye, RSV not only replicated robustly in the eye but also migrated to the lung and produced a respiratory disease that is indistinguishable from the standard, nasally acquired RSV disease. Ocularly applied synthetic anti-RSV small interfering RNA prevented infection of the eye as well as the lung. RSV infection of the eye activated a plethora of ocular cytokines and chemokines with profound relevance to inflammation of the eye. Anticytokine treatments in the eye reduced ocular inflammation but had no effect on viral growth in both eye and lung, demonstrating a role of the cytokine response in ocular pathology. These results establish the eye as a major gateway of respiratory infection and a respiratory virus as a bona fide eye pathogen, thus offering novel intervention and treatment options.

Temperature, humidity, and ultraviolet B radiation predict community respiratory syncytial virus activity.

To obtain knowledge of how meteorological conditions affect community epidemics of respiratory syncytial virus (RSV) infection, RSV activity was recorded year-round in 9 cities that differ markedly in geographic location and climate. Local weather conditions were correlated with weekly or monthly RSV cases. Similar reports from other areas varying in climate were also reviewed. Results demonstrated that for all sites combined, weekly RSV activity was related to temperature in a bimodal fashion, with peaks of activity at temperatures more than 24-30 degrees C and at 2-6 degrees C. RSV activity also was greatest at 45-65% relative humidity. RSV activity was inversely related to ultraviolet B (UVB) radiance at 3 sites where this could be analyzed; the fourth site had minimal amounts of annual UVB radiance. At sites with persistently warm temperatures and high humidity, RSV activity was continuous throughout the year, peaking in summer and early autumn. In temperate climates, RSV activity was maximal during winter, correlating with lower temperatures. In areas where temperatures remained cold throughout the year, RSV activity again became nearly continuous. These data led us to conclude that community activity of RSV is substantial when both ambient temperatures and absolute humidity are very high, perhaps reflecting greater stability of RSV in aerosols; transmission of RSV in cooler climates is inversely related to temperature, possibly as a result of increased stability of the virus in secretions in the colder environment; and UVB radiation may inactivate the virus in the environment or influence susceptibility to RSV by altering host resistance.

trans-Complementation allows recovery of human respiratory syncytial viruses that are infectious but deficient in cell-to-cell transmission.

Human respiratory syncytial virus (HRSV) expresses three transmembrane glycoproteins: small hydrophobic protein SH, attachment protein G, and fusion protein F. The genes encoding SH and G can be deleted from the HRSV genome and infectious virus recovered. In contrast, HRSVs lacking the F gene or a functional replacement thereof have not been reported. To generate a system with which to study the roles of the viral transmembrane glycoproteins, including F, in the HRSV life cycle, we generated a cell line expressing a heterologous viral glycoprotein for complementation of glycoprotein function in trans. We previously demonstrated that the baculovirus GP64 protein or a chimeric form of GP64 carrying the 12 C-terminal amino acids of the HRSV F protein (GP(64/F)) can efficiently mediate HRSV infectivity and improve its stability, when expressed from an engineered HRSV genome. Here, we report the development of a stably transfected Vero cell line (Vbac) constitutively expressing the GP(64/F) protein. From the Vbac cell line, viruses that lacked the SH and F open reading frames (ORFs) or the SH, G, and F ORFs could be recovered from cDNAs. These viruses, designated RSDeltaSH,F and RSDeltaSH,G,F, respectively, had place-keeper ORFs inserted in place of the deleted ORFs to maintain authentic transcription levels. In the Vbac cell line, RSDeltaSH,f and RSDeltaSH,G,F could be amplified to near wild-type-level titers, and the resulting viruses were infectious to Vero and HEp-2 cells. After entry into Vero or HEp-2 cells, however, neither virus RSDeltaSH,F nor virus RSDeltaSH,G,F was able to spread in the infected cultures. Growth analyses showed that infectious virions were not produced in Vero or HEp-2 cells infected with RSDeltaSH,F and RSDeltaSH,G,F. Combined, these data provide direct evidence that HRSV F is an essential viral protein required for cell-to-cell transmission and demonstrate that complementation with the GP64 protein in trans constitutes a powerful tool for the study of the role of individual HRSV transmembrane glycoproteins in virus assembly, morphogenesis, and pathogenesis. In addition, the ability to generate infectious but nonspreading viruses may provide an alternative approach for the development of safe and stable HRSV vaccine candidates.