Respiratory Syncytial Virus Strains Research Articles

Crystal Structure and Immunogenicity of the DS-Cav1-Stabilized Fusion Glycoprotein From Respiratory Syncytial Virus Subtype B.

Background:Respiratory syncytial virus (RSV) subtypes, A and B, co-circulate in annual epidemics and alternate in dominance. We have shown that a subtype A RSV fusion (F) glycoprotein, stabilized in its prefusion conformation by DS-Cav1 mutations, is a promising RSV-vaccine immunogen, capable of boosting RSV-neutralizing titers in healthy adults. In both humans and vaccine-tested animals, neutralizing titers elicited by this subtype A DS-Cav1 immunogen were ~ 2- to 3-fold higher against the homologous subtype A virus than against the heterologous subtype B virus.Methods:To understand the molecular basis for this subtype difference, we introduced DS-Cav1 mutations into RSV strain B18537 F, determined the trimeric crystal structure, and carried out immunogenicity studies.Results:The B18537 DS-Cav1 F structure at 2-Å resolution afforded a precise delineation of prefusion F characteristics, including those of antigenic site Ø, a key trimer-apex site. Structural comparison with the subtype A prefusion F indicated 11% of surface residues to be different, with an alpha-carbon root-mean-square deviation (RMSD) of 1.2 Å; antigenic site Ø, however, differed in 23% of its surface residues and had an alpha-carbon RMSD of 2.2 Å. Immunization of vaccine-tested animals with DS-Cav1-stabilized B18537 F induced neutralizing responses ~100-fold higher than with postfusion B18537 F. Notably, elicited responses neutralized RSV subtypes A and B at similar levels and were directed towards both conserved equatorial and diverse apical regions.Conclusion:We propose that structural differences in apical and equatorial sites–coupled to differently focused immune responses–provide a molecular explanation for observed differences in elicited subtype A and B neutralizing responses.

Adenovectors encoding RSV-F protein induce durable and mucosal immunity in macaques after two intramuscular administrations

Respiratory Syncytial Virus (RSV) can cause severe respiratory disease, yet a licensed vaccine is not available. We determined the immunogenicity of two homologous and one heterologous intramuscular prime-boost vaccination regimens using replication-incompetent adenoviral vectors of human serotype 26 and 35 (Ad26 and Ad35), expressing a prototype antigen based on the wild-type fusion (F) protein of RSV strain A2 in adult, RSV-naive cynomolgus macaques. All regimens induced substantial, boostable antibody responses that recognized the F protein in pre- and postfusion conformation, neutralized multiple strains of RSV, and persisted for at least 80 weeks. Vaccination induced durable systemic RSV-F-specific T-cell responses characterized mainly by CD4+ T cells expressing Th1-type cytokines, as well as RSV-F-specific CD4+ and CD8+ T cells, IgG, and IgA in the respiratory tract. Intramuscular immunization with Ad26 and 35 vectors thus is a promising approach for the development of an optimized RSV vaccine expected to induce long-lasting humoral and cellular immune responses that distribute systemically and to mucosal sites.

Analysis of phylogenetic diversity and in vitro adherence characteristics of respiratory syncytial virus and Streptococcus pneumoniae clinical isolates obtained during pediatric respiratory co-infections.

Respiratory syncytial virus (RSV) and Streptococcus pneumoniae are frequently co-associated during acute respiratory infections, particularly amongst infants and young children. In this study, we aimed to identify strains of RSV and serotypes/sequence types of S. pneumoniae associated with co-infections within a cohort of paediatric patients, and to assess RSV-mediated adhesion of pneumococcal isolates. The RSV glycoprotein sequence was determined for 58 RSV-positive samples and molecular serotyping and MLST was used to analyse 26 pneumococcal isolates. We also compared 23 pneumococcal isolates for their adherence to RSV-infected or mock-infected airway epithelia cells using immunofluorescence microscopy and automated particle counting. The tight association between RSV and S. pneumoniae was also visualized using scanning electron microscopy. This study did not identify any statistically significant trend in the strains of RSV and S. pneumoniae associated with co-infections. Furthermore, almost all isolates (22 of 23) showed significantly increased adherence to RSV-infected cells. The level of adherence did not appear to correlate with pneumococcal strain or sequence type, and isolates obtained from RSV-infected patients displayed a similar level of adherence as those from RSV-negative patients. The absence of particular S. pneumoniae or RSV strains associated with co-infection, together with the near ubiquitous presence of RSV-mediated adhesion throughout the pneumococcal clinical isolates, may indicate that the mechanisms governing the association with RSV are of sufficient importance to be maintained across much of the species.

Evaluation of the role of respiratory syncytial virus surface glycoproteins F and G on viral stability and replication: implications for future vaccine design.

Respiratory syncytial virus (RSV) remains a leading cause of infant mortality worldwide and exhaustive international efforts are underway to develop a vaccine. However, vaccine development has been hindered by a legacy of vaccine-enhanced disease, poor viral immunogenicity in infants, and genetic and physical instabilities. Natural infection with RSV does not prime for enhanced disease encouraging development of live-attenuated RSV vaccines for infants; however, physical instabilities of RSV may limit vaccine development. The role of RSV strain-specific differences on viral physical stability remains unclear. We have previously demonstrated that the RSV fusion (F) surface glycoprotein is responsible for mediating significant differences in thermostability between strains A2 and A2-line19F. In this study, we performed a more comprehensive analysis to characterize the replication and physical stability of recombinant RSV A and B strains that differed only in viral attachment (G) and/or F surface glycoprotein expression. We observed significant differences in thermal stability, syncytia size, pre-fusion F incorporation and viral growth kinetics in vitro, but limited variations to pH and freeze-thaw inactivation among several tested strains. Consistent with earlier studies, A2-line19F showed significantly enhanced thermal stability over A2, but also restricted growth kinetics in both HEp2 and Vero cells. As expected, no significant differences in susceptibility to UV inactivation were observed. These studies provide the first analysis of the physical stability of multiple strains of RSV, establish a key virus strain associated with enhanced thermal stability compared to conventional lab strain A2, and further support the pivotal role RSV F plays in virus stability.

Attenuation of Human Respiratory Viruses by Synonymous Genome Recoding.

Using computer algorithms and commercial DNA synthesis, one or more ORFs of a microbial pathogen such as a virus can be recoded and deoptimized by several strategies that may involve the introduction of up to thousands of nucleotide (nt) changes without affecting amino acid (aa) coding. The synonymous recoding strategies that have been applied to RNA viruses include: deoptimization of codon or codon-pair usage, which may reduce protein expression among other effects; increased content of immunomodulatory CpG and UpA RNA, which increase immune responses and thereby restrict viral replication; and substitution of serine and leucine codons with synonymous codons for which single-nt substitutions can yield nonsense codons, thus limiting evolutionary potential. This can reduce pathogen fitness and create potential live-attenuated vaccines that may have improved properties. The combined approach of genome recoding, synthetic biology, and reverse genetics offers several advantages for the generation of attenuated RNA viruses. First, synonymous recoding involves many mutations, which should reduce the rate and magnitude of de-attenuation. Second, increasing the amount of recoding can provide increased attenuation. Third, because there are no changes at the aa level, all of the relevant epitopes should be expressed. Fourth, attenuation frequently does not compromise immunogenicity, suggesting that the recoded viruses have increased immunogenicity per infectious particle. Synonymous deoptimization approaches have been applied to two important human viral pathogens, namely respiratory syncytial virus (RSV) and influenza A virus (IAV). This manuscript will briefly review the use of these different methods of synonymous recoding to generate attenuated RSV and IAV strains. It also will review the characterization of these vaccine candidates in vitro and in animal models, and describe several surprising findings with respect to phenotypic and genetic instability of some of these candidates.

Respiratory syncytial virus strains differentially activate the inflammasome eliciting a pathogenic Th17 response

Abstract Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children worldwide with no licensed vaccine available. RSV-induced disease ranges from mild to severe, with severe disease correlated with elevated levels of pro-inflammatory cytokines, including the Th17-associated cytokine IL-17. To better understand the virus factors that influence the induction of a Th17 response, we compared the inflammatory responses elicited by different RSV strains. In contrast to the commonly used RSV A2 strain, RSV 2–20 infection resulted in increased levels of IL-6 and IL-1β, which were required to induce a pathogenic Th17 response. Th17 induction was associated with increased mucus production and airway hyperreactivity following RSV 2–20 infection indicating a strain-dependent increase in disease severity. A recombinant RSV A2 virus expressing the 2–20 fusion (F) protein induced IL-1β to similar levels as wild-type RSV 2–20 suggesting that the 2–20 F protein is necessary for increased pro-inflammatory cytokine production. Sequencing of the RSV A2 and 2–20 F protein genes revealed three point mutations in a region likely to interact with host cells. Reversion of these three amino acids in the RSV A2/2–20 F recombinant virus to the A2 sequence significantly reduced RSV-induced IL-6 and IL-1β protein production. Together our results demonstrate that the RSV F protein plays an essential role in the induction of pro-inflammatory cytokines which are critical for CD4 Th17 cell differentiation. Genetic variations in the RSV F protein between RSV strains play a crucial role in modulating RSV-induced disease severity in the animal model and may contribute to the differential RSV pathogenesis observed in humans.

Development and properties of neutralizing monoclonal antibodies for fusion protein of respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections in infants and the elderly. The absence of a wide range of therapeutic drugs and vaccines indicates to the high relevance of the development of new effective drugs for the prevention and treatment of RSV infections. to obtain highly active and specific monoclonal antibodies (MAbs) capable of detecting RSV in infected cells and neutralizing the infectious activity of the virus in vitro. RSV reference strains of group A 2 subgroups (A2 and Long) were propagated in HEp-2 and MA-104 cell lines, respectively. Mice were immunized with purified RSV A2 virus. MAbs were obtained using hybridoma technology. A panel of 6 MAbs reacting with RSV strains А2 and Long has been obtained. Four MAbs were IgG (IgG2a or IgG2b subtype), two MAbs were IgM. All MAbs reacted with RSV F-protein in immunochemical tests. The MAbs actively reacted with RSV in ELISA, in immufluorescence and peroxidase staining of infected cells, and in immunodot test. Five out of 6 MAbs neutralized of RSV in cell culture. Different properties of MAbs suggest that they target different antigenic sites of F-protein. Comparative analysis suggests that the obtained MAbs can be used for the development of diagnostic preparations, for RSV detection in clinical materials and confirmation of infection etiology by rapid culture method. High activity and specificity of MAbs indicate that they can serve as a basis for development vaccines and preventive medicines.

Therapeutic delivery of ALX‐0171 Nanobody reduces disease severity of human respiratory syncytial virus strain M37 after established RSV infection

Respiratory syncytial virus (RSV) is the most common reason for hospitalization of infants with viral pneumonia but lacks a therapy that is fully satisfactory and effective. ALX‐0171 is a Nanobody directed against RSV F protein that reduces RSV infection and disease severity. In order to determine the optimal dose of therapeutic delivery of ALX‐0171 after RSV infection, three doses of ALX‐0171 (0, 1.4, 2.8 and 11 mg) were delivered by nebulization to lambs at 3, 4, and 5 days after infection with a human strain of RSV (Memphis 37 strain) and assessed for efficacy at 2, 4 and 6 days post infection. Lambs not receiving ALX‐0171 had limited or lacked RSV gross and microscopic lesions, RSV viral antigen in lung and RSV titers at 2 days post RSV but these lesions and viral parameters increased progressively at days 4 and 6. There was a dose‐effect relationship yielding in vivo IC50 and lambs treated with ALX‐0171 lacked lesions 2 days post RSV infection and had reduced lesions and viral parameters at 4 and 6 days post RSV infection compared to lesions in lambs not receiving treatment. These studies determined that ALX‐0171 reduces RSV disease severity when administered after RSV inoculation. Clinically, infants commonly do not receive treatment for RSV until after clinical signs develop and work in this study suggests that ALX‐0171 has potential to reduce RSV disease severity in the face of established RSV infection at optimized doses.Support or Funding InformationThese studies were funded by Ablynx NVThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Persistence and continuous evolution of the human respiratory syncytial virus in northern Taiwan for two decades

The study aimed to characterize the molecular epidemiology, phylogenetic relationship, and population dynamics of the G protein gene in clinical respiratory syncytial virus (RSV) strains isolated from northern Taiwan. We analyzed a total of 160 and 116 G protein gene sequences of RSV-A and RSV-B representative strains, respectively, from 804 clinical viral stocks collected between July 2000 and June 2016. Population dynamic patterns of the RSV G protein gene were analyzed using Bayesian inference through the Markov chain Monte Carlo framework. A phylogenetic analysis revealed that RSV-A from Taiwan could be categorized into GA2, GA5, and GA7 lineages. GA2 of RSV-A could be further divided into NA1, NA2, NA4, and ON1 clades. These RSV-A lineages has been replaced over time, whereas RSV-B strains from Taiwan continually evolved from a single lineage with significant time-dependent waves. Four putative positive selection sites were observed in both RSV-A and RSV-B. The Bayesian skyline plot revealed that the local population dynamics of RSV were associated with lineage displacement events. Both circulating subtypes and population dynamics represented a unique local pattern. Our results affirm the necessity of continuing molecular surveillance of RSV to attain a more comprehensive understanding of epidemics.

High epidemic burden of RSV disease coinciding with genetic alterations causing amino acid substitutions in the RSV G-protein during the 2016/2017 season in The Netherlands

BackgroundWe found amino acid substitutions in the Gglycoprotein of respiratory syncytial virus (RSV) A during the 2016/2017 epidemic in The Netherlands. ObjectivesWe evaluated whether these alterations led to increased RSV incidence and disease burden. Study designWe sequenced the gene encoding the G-protein of prospectively collected clinical specimens from secondary care adult patients testing positive for RSV during the 2016/2017 and 2017/2018 epidemic RSV season. We evaluated associations between genetic, clinical and epidemiological data. ResultsWe included 49 RSV strains. In 2016/2017 28 strains were included, 20 community acquired RSV-A, 5 hospital acquired RSV-A and 3 community acquired RSV-B. In 2017/2018 21 strains were included, 8 community acquired RSV-A and 13 community acquired RSV-B. G-proteins of 10 out of the 20 community acquired 2016/2017 RSV-A strains shared a set of eight novel amino acid substitutions of which seven in mucin-like regions 1 and 2 and one in the heparin binding domain. This genetic variant was no longer detected among 2017/2018 RSV-A strains. Among patients carrying the novel RSV-A strain-type, 30% died. ConclusionsA set of eight amino acid substitutions was found in 50% of the 2016/2017 community acquired RSV-A G-proteins. This combination of substitutions was globally never observed before. The appearance of this new strain-type coincided with an increased RSV peak in The Netherlands and was associated with higher disease severity. The transient character of this epidemic strain-type suggests rapid clearance of this lineage in our study community.

Differences in Susceptibility of Human and Mouse Macrophage Cell Lines to Respiratory Syncytial Virus Infection

Objectives: Differences have been observed in the susceptibility of macrophage cell lines to respiratory syncytial virus (RSV) infection. In this study, we evaluated whether the type of macrophage cell line and RSV strain used have an influence on the infectivity and production of progeny virus. Methods: Both human and murine macrophage-like cell lines were infected with different RSV strains, both lab strains as well as clinical isolates. The infection was evaluated after 24 and 72 h by immunofluorescence staining and microscopic analysis, and the production of new virus particles was determined by plaque assay. Results: Susceptibility of macrophages to RSV was influenced by the RSV strain used but was mostly dependent on the macrophage cell line. Numbers of infected cells and virus production were generally very low or absent in murine cell lines. In human cell lines, clear infection was observed associated with production of new virus particles. Conclusion: Differences in susceptibility of macrophage cell lines to RSV infection are primarily related to the species of origin of the cell line but are also influenced by the RSV strain.

Complete Genome Sequences of 12 Human Respiratory Syncytial Virus (Human Orthopneumovirus) Strains Detected in Children with Repeated Subgroup B Infections in the Philippines.

Complete genome sequences were determined for 12 human respiratory syncytial virus strains collected from nasopharyngeal samples obtained from children with repeated subgroup B infections. Eight common amino acid polymorphisms in the G, F, and L proteins were identified between the viruses detected in initial and subsequent infections.

An overview of respiratory syncytial virus infections in Saudi Arabia.

Respiratory syncytial virus (RSV) is a major pathogen of acute respiratory tract infection (ARI) in different geographical regions including Saudi Arabia. Numerous hospital-based investigations have revealed the RSV prevalence between 0.2-54% in the paediatric population with ARI/ALRI from Saudi Arabia during 1991-2015. Maximum RSV infections occurred in children less than 1 year of age (51-97%) and male children (51-69%) were more commonly affected than females (31-49%). RSV infections are reported mostly during winter season suggesting seasonal distribution of the virus. Other respiratory viruses reported from this region are adenovirus, influenza, parainfluenza, human metapneumovirus and rhinovirus including many mixed infections. A few studies have reported the phylogenetic analysis of the circulating strains of RSV. These studies have revealed that circulating group A-RSV Saudi strains belonged to NA1 and ON1 genotypes and group B-RSV viruses clustered in the BA genotype. Molecular characterization of the Saudi strains was further carried out by mutational, selection pressure and glycosylation site analyses. We have compiled all the eighteen studies of RSV infection from Saudi Arabia in the form of this review and concluded that detailed comprehensive surveillance of RSV and other viruses in community and hospital settings is required. Information on the molecular characterization of currently circulating strains of RSV will contribute towards better understanding of the epidemiology and evolutionary dynamics of this viral pathogen. Moreover, the determination of the genetic composition of circulating RSV strains will be important during evaluation of initial vaccine trials.

Development of an allosteric inhibitor class blocking RNA elongation by the respiratory syncytial virus polymerase complex

Respiratory syncytial virus (RSV) represents a significant health threat to infants and to elderly or immunocompromised individuals. There are currently no vaccines available to prevent RSV infections, and disease management is largely limited to supportive care, making the identification and development of effective antiviral therapeutics against RSV a priority. To identify effective chemical scaffolds for managing RSV disease, we conducted a high-throughput anti-RSV screen of a 57,000-compound library. We identified a hit compound that specifically blocked activity of the RSV RNA-dependent RNA polymerase (RdRp) complex, initially with moderate low-micromolar potency. Mechanistic characterization in an in vitro RSV RdRp assay indicated that representatives of this compound class block elongation of RSV RNA products after initial extension by up to three nucleotides. Synthetic hit-to-lead exploration yielded an informative 3D quantitative structure-activity relationship (3D-QSAR) model and resulted in analogs with more than 20-fold improved potency and selectivity indices (SIs) of >1,000. However, first-generation leads exhibited limited water solubility and poor metabolic stability. A second optimization strategy informed by the 3D-QSAR model combined with in silico pharmacokinetics (PK) predictions yielded an advanced lead, AVG-233, that demonstrated nanomolar activity against both laboratory-adapted RSV strains and clinical RSV isolates. This anti-RSV activity extended to infection of established cell lines and primary human airway cells. PK profiling in mice revealed 34% oral bioavailability of AVG-233 and sustained high drug levels in the circulation after a single oral dose of 20 mg/kg. This promising first-in-class lead warrants further development as an anti-RSV drug.

Circulação do vírus respiratório sincicial humano em cinco Estados da Região Amazônica brasileira: primeira descrição do genótipo ON1 no Pará

OBJETIVO: Realizar a deteccao e caracterizacao das cepas de virus respiratorio sincicial humano (VRSH), nos casos de infeccao respiratoria aguda (IRA), circulantes nos estados brasileiros do Acre, Amapa, Amazonas, Para e Roraima, no ano de 2015. MATERIAIS E METODOS: Foram coletadas 1.082 amostras de aspirado de nasofaringe e swab combinado narina e garganta de pacientes de diferentes generos e faixas etarias com IRA e submetidas a qRT-PCR para VRSH. As amostras positivas foram inoculadas em cultura celular HEP-2 e as que apresentaram efeito citopatico foram submetidas a tres etapas: extracao do RNA viral, amplificacao do gene G pela RT-PCR e sequenciamento genetico. RESULTADOS: Das 1.082 amostras, 57 (5,3%) foram positivas para VRSH, sendo 38 (66,7%) de pacientes de 0 a 4 anos de idade. A circulacao predominou entre marco e julho, periodo de transicao climatica na Regiao. Apenas no Acre, Amazonas e Para foram detectadas amostras positivas, 15 (26,3%), 23 (40,4%) e 19 (33,3%), respectivamente. O efeito citopatico foi avaliado em 45 (78,9%) amostras, nas quais foram identificados os subgrupos: 40 (88,9%) do VRSH-B com genotipo BA e cinco (11,1%) do VRSH-A do genotipo ON1. CONCLUSAO: Este estudo revelou uma taxa de infeccao viral relevante em criancas de 0 a 4 anos de idade. A circulacao viral ocorreu no Acre, Amazonas e Para durante o periodo de mudanca climatica. Foi notoria a ocorrencia dos genotipos BA e ON1, sendo a primeira vez que se comprova a circulacao do ON1 no Para.

Characterization of circulating RSV strains among subjects in the OUTSMART-RSV surveillance program during the 2016-17 winter viral season in the United States.

BackgroundRespiratory syncytial virus (RSV) is an established cause of serious lower respiratory disease in infants, elderly and high-risk populations. The OUTSMART surveillance program aims to characterize patient populations and currently circulating RSV strains, and monitor temporal and geographic evolution of RSV F and G proteins in the U.S.MethodsThe OUTSMART 2016–17 study collected RSV-positive samples from 25 RSVAlert® laboratories from 4 U.S. regions and Puerto Rico during November 2016 through March 2017. Frequencies of A and B subtypes and genotypes were determined for several demographic and geographic variables. To gauge the representativeness of the OUTSMART patients, results were compared to discharge data from the NEDS and NIS databases.ResultsA total of 1,041 RSV-positive samples with associated demographic data were obtained and the RSV F gene and second variable region of the G gene were sequenced. The majority of samples (76.0%) came from children under 2 years old: <1 year (48.4%), 1–2 years (27.6%). The OUTSMART patient sample was similar to NEDS and NIS for age, gender, and geographic location. Both OUTSMART and national RSV cases peaked in January. Of OUTSMART samples, 45.3% were subtype A, 53.7% were subtype B and 1.0% were mixed A and B. The percentage of RSV B cases increased with increasing age. Hospitalization (length of hospital stay, LOS, >24 hrs) occurred in 29.0% of patients of which 52.0% had RSV B. Outpatients (LOS <24 hrs) were 64.4% of total of which 73.3% were diagnosed in the ER and discharged, while only 6% were diagnosed in other outpatient settings.ConclusionsThe OUTSMART 2016–17 study was representative of the U.S. RSV experience. Geographic and temporal information from the RSV surveillance program will be used to establish a molecular baseline of RSV F and G sequence variability and to help inform development of novel agents for RSV prophylaxis and treatment.

Etiology of viral respiratory infections in Northern Lao People's Democratic Republic.

In Lao People's Democratic Republic (PDR), acute respiratory infections overburden the health care system, but viral etiology, genetic diversity, and seasonality, especially in light of the introduction of influenza vaccination in the country, are poorly understood. From August 2010 to April 2011, 309 outpatients were recruited at the Luang Prabang Provincial Hospital covering highland Lao communities. Nasopharyngeal swabs were screened for the presence of 13 respiratory viruses. At least one virus was detected in 69.6% and dual/triple viral infections in 12.9%/1.9% of the patients. Influenza A and B viruses combined were the most frequently detected pathogens, followed by human adenovirus and respiratory syncytial virus (RSV). The other viruses were detected in less than 10% of the patients. Phylogenetic analyses on a representative set of RSV strains revealed that, while otherwise very rare, the RSV‐B CB1/THB genotype cocirculated with other common genotypes. A single wave of influenza virus and RSV activity was observed during the rainy season, providing further support to influenza vaccination before the onset of the rains. This study provides recommendations for influenza vaccination that still needs optimization and highlights the need for revised guidelines for treatment and prevention of respiratory infections in Lao PDR, as well as for increased surveillance efforts.

Improving ability of RSV microneutralization assay to detect G-specific and cross-reactive neutralizing antibodies through immortalized cell line selection

Respiratory syncytial virus (RSV) is a significant cause of bronchiolitis and pneumonia. Protection against RSV is associated with neutralizing antibodies against the fusion (F) and attachment (G) glycoproteins. Several RSV vaccine candidates are in development, but their immunogenicity is hard to compare due to the little-understood differences between multiple RSV neutralizing antibody assays used. Existing assays utilize primarily Vero or HEp-2 cells, but their ability to detect G-neutralizing antibodies or antibodies against specific RSV strains is unclear. In this work, we developed an RSV microneutralization assay (MNA) using unmodified RSV and immortalized cell line derived from human airway epithelial cells (A549). Performance of A549-, HEp-2- and Vero-based MNA was compared under the same assay conditions (fixed amount of virus and cells) with regards to detection of neutralizing antibodies against RSV A or B viruses, G-reactive neutralizing antibodies, and effect of complement. Our results indicate that A549 cells yield the highest MNA titers, particularly in the RSV A/A2 MNA, are least susceptible to complement-enhancing effect of neutralizing titer readout and are superior to Vero or HEp-2 MNA at recognizing G-reactive neutralizing antibodies when no complement is used. Vero cells, however, can be more consistent at recognizing neutralizing antibodies against multiple RSV strains. The choice of substrate cells thus affects the outcome of MNA, as some immortalized cells better support detection of broader range of neutralizing antibodies, while others facilitate detection of G-targeting neutralizing antibodies, a long-thought prerogative of primary airway epithelial cells.

Analysis of genetic variability of respiratory syncytial virus groups A and B in Kuwait.

Respiratory syncytial virus (RSV) is the most frequently identified viral agent in infants, children, and elderly people with acute respiratory tract infections (ARTIs). This study is the only one of its kind in Kuwait, and its purpose was to investigate the genetic variability of the G protein gene in RSV strains prevalent in Kuwait. Respiratory samples were collected from patients with ARTIs in various hospitals in Kuwait and subjected to reverse transcription PCR (RT-PCR) amplifying a fragment of the G gene of RSV. A total of 305 samples were collected between January and mid-December 2016, and 77 (25.2%) were positive for RSV. Group A viruses were predominant over group B viruses; the RSV-A group was detected in 52 (67.5%) of the positive samples, while the RSV-B group was detected in 25 (32.5%) of the positive samples. Phylogenetic analysis showed that all RSV-A strains grouped into eight clusters of identical sequences of untyped strains. Twelve RSV-B strains, on the other hand, belonged to the RSV-B/BA10 genotype, while the rest were untyped. These data suggest that new and untyped strains of RSV-A group likely predominated in Kuwait and that the BA10 genotype of the RSV-B group became the dominant genotype in the 2016 season.

Osteopontin plays a pivotal role in increasing severity of respiratory syncytial virus infection.

The molecular mechanisms underlying susceptibility to severe respiratory syncytial virus (RSV) infection remain poorly understood. Herein, we report on the role of osteopontin (OPN) in regulation of RSV infection in human epithelial cells and how interleukin-1 beta (IL-1β), a cytokine secreted soon after RSV infection, when persistently expressed can induce OPN expression leading to increased viral infection. We first compared OPN expression in two human epithelial cell lines: HEK-293 and HEp-2. In contrast to HEp-2, HEK-293 expresses low levels of pro-caspase-1 resulting in decreased IL-1β expression in response to RSV infection. We found a correlation between low IL-1β levels and a delay in induction of OPN expression in RSV-infected HEK-293 cells compared to HEp-2. This phenomenon could partially explain the high susceptibility of HEp-2 cells to RSV infection versus the moderate susceptibility of HEK-293 cells. Also, HEK-293 cells expressing low levels of pro-caspase-1 exhibit decreased IL-1β expression and delayed OPN expression in response to RSV infection. HEK-293 cells incubated with human rIL-1β showed a dose-dependent increase in OPN expression upon RSV infection. Also, incubation with rOPN increased RSV viral load. Moreover, HEp-2 cells or mice infected with a mucogenic RSV strain RSV-L19F showed elevated levels of OPN in contrast to mice infected with the laboratory RSV strain rA2. This correlated with elevated levels of OPN following infection with RSV-L19F compared to rA2. Together, these results demonstrate that increased OPN expression is regulated in part by IL-1β, and the interplay between IL-1β and OPN signaling may play a pivotal role in the spread of RSV infection.