Multiple Respiratory Syncytial Virus (RSV) Strains Infecting HEp-2 and A549 Cells Reveal Cell Line-Dependent Differences in Resistance to RSV Infection.

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here, we reported a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with one of four strains of RSV representing both major subgroups as well as historic and more contemporary genotypes (RSV/A/Tracy [GA1], RSV/A/Ontario [ON], RSV/B/18537 [GB1], and RSV/B/Buenos Aires [BA]) via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response, including transcriptional changes and levels of secreted cytokines and growth factors. IMPORTANCE Infection with the respiratory syncytial virus (RSV) early in life is essentially guaranteed and can lead to severe disease. Most RSV studies have involved either of two historic RSV/A strains infecting one of two cell lines, HEp-2 or A549 cells. However, RSV contains ample variation within two evolving subgroups (A and B), and HEp-2 and A549 cell lines are genetically distinct. Here, we measured viral action and host response in both HEp-2 and A549 cells infected with four RSV strains from both subgroups and representing both historic and more contemporary strains. We discovered a subgroup-dependent difference in viral gene expression and found A549 cells were more potently antiviral and more sensitive, albeit subtly, to viral variation. Our findings revealed important differences between RSV subgroups and two widely used cell lines and provided baseline data for experiments with model systems better representative of natural RSV infection.

Viral Load of Severe Acute Respiratory Syndrome Coronavirus 2 in Adults During the First and Second Wave of Coronavirus Disease 2019 Pandemic in Houston, Texas: The Potential of the Superspreader.

BACKGROUND: During the coronavirus disease 2019 pandemic, a minority of index cases are associated with a majority of secondary cases suggesting that superspreaders could drive the pandemic. We identified a phenotype in individuals with extremely high viral load who could act as superspreaders. METHODS: Data were analyzed from individuals tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from 18 March through 15 August 2020. Outcomes were compared using contingency table and quantile regression to test the equality of medians between the pandemic waves and by viral load groups. RESULTS: Of the 11 564 samples tested, 1319 (11.4%) were positive for SARS-CoV-2. An increase in weekly median viral load occurred in the second wave of the SARS-CoV2 pandemic. This population was more likely to be women, outpatients, and symptomatic and to have an extremely high or high viral load. In patients with multiple reverse-transcription polymerase chain reaction-positive test results, the durations of viral shedding were comparable between individuals with asymptomatic/mild and mild/moderate illness severity. CONCLUSIONS: We detected a small group of individuals with extremely high SARS-CoV-2 viral loads and mild illness. We believe that these individuals' characteristics could be consistent with the superspreader phenomenon and that greater awareness of the social dynamics of these individuals is needed to understand the spread of SARS-CoV-2.

Comparison of Palivizumab-Like Antibody Binding to Different Conformations of the RSV F Protein in RSV-Infected Adult Hematopoietic Cell Transplant Recipients.

Background: Most respiratory syncytial virus (RSV) vaccine candidates include fusion (F) protein in different conformations. Antigenic site II found in the different F conformations is the target of palivizumab, the only US Food and Drug Administration approved monoclonal antibody (mAb). Serum palivizumab-like antibody (PLA) is a potential serologic correlate of immunity. Our objective was to determine if different conformations of F protein in a palivizumab competitive antibody (PCA) assay affect the PLA concentrations. Methods: Four PCA assays were standardized using mAbs. Each contained prefusion, postfusion, or intermediate F forms. PLA concentrations were measured in acute and convalescent sera from 22 RSV/A and 18 RSV/B-infected adult hematopoietic cell transplant (HCT) recipients. PLA concentrations were calculated using a 4-parameter logistic regression model and analyzed for statistical significance. Results: PCA assays revealed significantly greater PLA concentrations in convalescent sera; comparable increases in PLA concentration in RSV/A and RSV/B-infected HCT recipients; and significantly reduced PLA concentrations in HCT recipients who shed RSV ≥14 days. A significant positive correlation was observed between PCA assays and RSV neutralizing antibody titers. Conclusions: F protein conformation does not appear to have a measurable impact on PCA assays for measuring PLA induced by RSV/A or RSV/B infection.

Infection with novel respiratory syncytial virus genotype Ontario (ON1) in adult hematopoietic cell transplant recipients, Texas, 2011-2013.

BACKGROUND: Respiratory syncytial virus (RSV) can cause severe respiratory disease in adult hematopoietic cell transplant (HCT) recipients. RSV subgroups A and B have evolved into multiple genotypes. We report on a recently described RSV genotype (ON1) in a cohort of adult HCT recipients in Texas. METHODS: Twenty adult HCT recipients were enrolled as a part of an efficacy trial of ribavirin therapy. RSV identification and genotyping was performed using molecular techniques. RSV-specific neutralizing antibody (NAb) responses were measured. RESULTS: ON1 genotype was detected in 3 of 6 patients in the 2011-2012 season and in 8 of 14 patients in 2012-2013 season. Other genotypes detected were NA1 and BA. NAb levels were low at enrollment. Eight of 9 patients who cleared the RSV infection within 2 weeks mounted a ≥4-fold NAb response, compared with 2 of 8 who shed the virus for >2 weeks. The clinical course of those infected with ON1 was comparable to the course for individuals infected with other genotypes. CONCLUSION: This is the first report of RSV ON1 genotype in the United States, and ON1 genotype was dominant genotype in adult HCT recipients. Interestingly, faster viral clearance was associated with a ≥4-fold NAb response, likely indicating a reconstituted immune system.

Pediatric human nose organoids demonstrate greater susceptibility, epithelial responses, and cytotoxicity than adults during RSV infection.

Respiratory syncytial virus (RSV) is a common cause of respiratory infections, causing significant morbidity and mortality, especially in young children. Why RSV infection in children is more severe as compared to healthy adults is not fully understood. In the present study, we infect both pediatric and adult human nose organoid-air liquid interface (HNO-ALIs) cell lines with two contemporary RSV isolates and demonstrate how they differ in virus replication, induction of the epithelial cytokine response, cell injury, and remodeling. Pediatric HNO-ALIs were more susceptible to early RSV replication, elicited a greater overall cytokine response, demonstrated enhanced mucous production, and manifested greater cellular damage compared to their adult counterparts. Adult HNO-ALIs displayed enhanced mucus production and robust cytokine response that was well controlled by superior regulatory cytokine response and possibly resulted in lower cellular damage than in pediatric lines. Taken together, our data suggest substantial differences in how pediatric and adult upper respiratory tract epithelium responds to RSV infection. These differences in epithelial cellular response can lead to poor mucociliary clearance and predispose infants to a worse respiratory outcome of RSV infection.

Antibody responses of healthy adults to the p27 peptide of respiratory syncytial virus fusion protein.

The respiratory syncytial virus (RSV) fusion (F) protein undergoes two furin-cleavage events to become fusion competent, resulting in the release of a twenty-seven amino acid peptide (p27). Recent studies indicate that the p27 region of the F protein was an immunodominant antigen in young children. In this study, we evaluated the kinetics of the serum antibody response to the p27 peptide following natural RSV reinfection in adults. Nineteen healthy adults under sixty-five years of age were enrolled during the 2018-2019 RSV season in Houston, TX. Blood was collected at three study visits and RSV infection status was defined by changes in neutralizing antibody resulting in three groups: uninfected (n = 12), acutely infected (n = 4), and recently infected (n = 3). Serum IgG and IgA antibodies against RSV/A and RSV/B p27 peptides were measured by enzyme-linked immunosorbent assays, and serum p27-like antibodies were detected by a p27 competitive antibody assay. Anti-p27 antibodies were detected in all subjects at each study visit. The measured IgG and IgA anti-p27 antibody levels followed the same pattern as other RSV site-specific and neutralizing antibody responses described for this cohort previously: the uninfected group had stable responses for the duration of the study period, the acutely infected group had a significant increase following RSV infection, and the recently infected group had a decrease in anti-p27 antibody during the study period. These results indicate that antibodies to the p27 region of the F protein are generated following natural RSV reinfection and suggest that some of the F protein is potentially in a partially cleaved state on the surface of virions, expanding on the previous assumption that all of p27 is post-translationally released and not present on mature F. Additionally, antibody responses were significantly lower (1.4-1.5-fold) toward RSV/B than to RSV/A p27 at each study visit, despite being an RSV/B dominant outbreak. Understanding the mechanism for the differences in the magnitude of the RSV/A and RSV/B p27 antibody response may enhance our understanding of the intracellular processing of the F protein.

A prospective surveillance study on the kinetics of the humoral immune response to the respiratory syncytial virus fusion protein in adults in Houston, Texas.

Respiratory syncytial virus (RSV)-specific serum antibody has been correlated to protection of infection and reduction of severe disease, but reinfection is still frequent. In this study, we evaluated RSV-specific serum antibody activity following natural RSV re-infection to examine the longevity of the humoral immune response in adults. Nineteen healthy adult volunteers under sixty-five years of age were enrolled during the 2018-2019 RSV season in Houston, TX. Blood was collected at three study visits. The kinetics of RSV-neutralizing, RSV F site-specific competitive, and RSV-binding antibodies in serum samples were measured by microneutralization and enzyme-linked immunosorbent assays. Three distinct profiles of RSV-specific antibody kinetics were identified that were consistent with RSV infection status: uninfected, acutely infected, and recently infected. The uninfected group had stable antibody titers for the duration of the study period (185 days). The acutely infected group had lower antibody responses at the beginning of the study, supporting a correlate of infection, followed by a significant antibody response after infection that was maintained for at least 125 days. Unlike the acutely infected group, the recently infected group had a significant precipitous decrease in RSV antibody in only 60 days. This study is the first, to our knowledge, to describe this abrupt loss of RSV-specific antibody in detail. This rapid decline of antibody may present an obstacle for the development of vaccines with lasting protection against RSV, and perhaps other respiratory pathogens. Neutralizing antibody responses were greater to prototypic than contemporaneous RSV strains, regardless of infection status, indicating that original antigenic sin may impact the humoral immune response to new or emerging RSV strains.

Humoral and Mucosal Antibody Response to RSV Structural Proteins in RSV-Infected Adult Hematopoietic Cell Transplant (HCT) Recipients.

Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract infection in infants, the elderly, and immunocompromised patients. RSV antibodies play a role in preventing reinfection and in clearance of RSV, but data regarding the levels of viral protein-specific antibodies elicited and their contribution to patient recovery from RSV-induced disease are limited. We prospectively enrolled a cohort of RSV-infected adult hematopoietic cell transplant (HCT) recipients (n = 40). Serum and nasal-wash samples were obtained at enrollment (acute samples) and convalescence (convalescent samples). We measured (1) humoral IgG and mucosal IgA binding antibody levels to multiple RSV proteins (F, G, N, P, and M2-1) by Western blot (WB); (2) neutralizing antibody (Nt Ab) titers by microneutralization assay; and (3) palivizumab-like antibody (PLA) concentrations by an ELISA-based competitive binding assay developed in the lab. Finally, we tested for correlations between protein-specific antibody levels and duration of viral shedding (normal: cleared in <14 days and delayed: cleared ≥14 days), as well as RSV/A and RSV/B subtypes. Convalescent sera from HCT recipients had significantly higher levels of anti-RSV antibodies to all 5 RSV structural proteins assayed (G, F, N, P, M2-1), higher Nt Abs to both RSV subtypes, and higher serum PLAs than at enrollment. Significantly higher levels of mucosal antibodies to 3 RSV structural proteins (G, N, and M2-1) were observed in the convalescent nasal wash versus acute nasal wash. Normal viral clearance group had significantly higher levels of serum IgG antibodies to F, N, and P viral proteins, higher Nt Ab to both RSV subtypes, and higher PLA, as well as higher levels of mucosal IgA antibodies to G and M2-1 viral proteins, and higher Nt Ab to both RSV subtypes compared to delayed viral clearance group. Normal RSV clearance was associated with higher IgG serum antibody levels to F and P viral proteins, and PLAs in convalescent serum (p < 0.05). Finally, overall antibody levels in RSV/A- and/B-infected HCT recipients were not significantly different. In summary, specific humoral and mucosal RSV antibodies are associated with viral clearance in HCT recipients naturally infected with RSV. In contrast to the humoral response, the F surface glycoprotein was not a major target of mucosal immunity. Our findings have implications for antigen selection in the development of RSV vaccines.

Serum IgG anti-SARS-CoV-2 Binding Antibody Level Is Strongly Associated With IgA and Functional Antibody Levels in Adults Infected With SARS-CoV-2.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in December 2019 in Wuhan, China, and then rapidly spread causing an unprecedented pandemic. A robust serological assay is needed to evaluate vaccine candidates and better understand the epidemiology of coronavirus disease (COVID-19). Methods: We used the full-length spike (S) protein of SARS-CoV-2 for the development of qualitative and quantitative IgG and IgA anti-S enzyme linked immunosorbent assays (ELISA). A total of 320 sera used for assay development were comprised of pandemic sera from SARS-CoV-2 infected adults (n=51) and pre-pandemic sera (n=269) including sera from endemic human coronavirus infected adults. Reverse cumulative curves and diagnostic test statistics were evaluated to define the optimal serum dilution and OD cutoff value for IgG anti-S and IgA anti-S ELISAs. The IgG and IgA anti-S, and three functional antibodies (ACE-2 receptor blocking antibody, lentipseudovirus-S neutralizing antibody, and SARS-CoV-2 neutralizing antibody) were measured using additional SARS-CoV-2 PCR positive sera (n=76) and surveillance sera (n=25). Lastly, the IgG and IgA anti-S levels were compared in different demographic groups. Results: The optimal serum dilution for the qualitative IgG anti-S ELISA was at 1:1024 yielding a 99.6% specificity, 92.2% sensitivity, 92.9% positive predictive value (PPV), and 99.6% negative predictive value (NPV) at a SARS-CoV-2 seroprevalence of 5%. The optimal serum dilution for the qualitative IgA anti-S ELISA was at 1:128 yielding a 98.9% specificity, 76.5% sensitivity, 78.3% PPV, and 98.8% NPV at the same seroprevalence. Significant correlations were demonstrated between the IgG and IgA (r=0.833 for concentrations, r=0.840 for titers) as well as between IgG and three functional antibodies (r=0.811-0.924 for concentrations, r=0.795-0.917 for titers). The IgG and IgA anti-S levels were significantly higher in males than females (p<0.05), and in adults with moderate/severe symptoms than in adults with mild/moderate symptoms (p<0.001). Conclusion: We developed a highly specific and sensitive IgG anti-S ELISA assay to SARS-CoV-2 using full length S protein. The IgG anti-S antibody level was strongly associated with IgA and functional antibody levels in adults with SARS-CoV-2 infection. Gender and disease severity, rather than age, play an important role in antibody levels.

The Human Nose Organoid Respiratory Virus Model: an Ex Vivo Human Challenge Model To Study Respiratory Syncytial Virus (RSV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Pathogenesis and Evaluate Therapeutics.

There is an unmet need for preclinical models to understand the pathogenesis of human respiratory viruses and predict responsiveness to immunotherapies. Airway organoids can serve as an ex vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a noninvasive technique to generate human nose organoids (HNOs) as an alternative to biopsy-derived organoids. We made air-liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hypersecretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation), while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration-dependent manner. Thus, the HNO-ALI model can serve as an alternative to lung organoids to study respiratory viruses and test therapeutics. IMPORTANCE Preclinical models that recapitulate aspects of human airway disease are essential for the advancement of novel therapeutics and vaccines. Here, we report a versatile airway organoid model, the human nose organoid (HNO), that recapitulates the complex interactions between the host and virus. HNOs are obtained using noninvasive procedures and show divergent responses to SARS-CoV-2 and RSV infection. SARS-CoV-2 induces severe damage to cilia and the epithelium, no interferon-λ response, and minimal mucus secretion. In striking contrast, RSV induces hypersecretion of mucus and a profound interferon-λ response with ciliary damage. We also demonstrated the usefulness of our ex vivo HNO model of RSV infection to test the efficacy of palivizumab, an FDA-approved monoclonal antibody to prevent severe RSV disease in high-risk infants. Our study reports a breakthrough in both the development of a novel nose organoid model and in our understanding of the host cellular response to RSV and SARS-CoV-2 infection.

The human nose organoid respiratory virus model: an ex-vivo human challenge model to study RSV and SARS-CoV-2 pathogenesis and evaluate therapeutics.

There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.

Antigenic Site-Specific Competitive Antibody Responses to the Fusion Protein of Respiratory Syncytial Virus Were Associated With Viral Clearance in Hematopoietic Cell Transplantation Adults.

Background: Recent studies of human sera showed that the majority of the respiratory syncytial virus (RSV) neutralizing antibodies are directed against pre-fusion conformation of the fusion (F) protein of RSV and revealed the importance of pre-fusion antigenic site Ø specific antibodies. However, detailed analysis of multiple antigenic site-specific competitive antibody responses to RSV F protein and their contribution to virus clearance in humans are lacking. Methods: We prospectively enrolled a cohort of RSV infected hematopoietic cell transplantation (HCT) adults (n = 40). Serum samples were collected at enrollment (acute, n = 40) and 14 to 60 days post-enrollment (convalescent, n = 40). Antigenic site-specific F protein antibodies were measured against pre-fusion site Ø, post-fusion site I, and sites II and IV present in both the pre-fusion and post-fusion F protein conformations utilizing four different competitive antibody assays developed with biotinylated monoclonal antibodies (mAb) D25, 131-2A, palivizumab, and 101F, respectively. The lower limit of detection were 7.8 and 1.0 µg/mL for the competitive antibody assays that measured site Ø specific response, as well as sites I, II, and IV specific responses, respectively. Neutralizing antibody titers to RSV A and B subgroups was determined by microneutralization assays. Results: The overall findings in RSV infected HCT adults revealed: (1) a significant increase in antigenic site-specific competitive antibodies in convalescent sera except for site Ø competitive antibody (p < 0.01); (2) comparable concentrations in the acute and convalescent serum samples of antigenic site-specific competitive antibodies between RSV/A and RSV/B infected HCT adults (p > 0.05); (3) significantly increased concentrations of the antigenic site-specific competitive antibodies in HCT adults who had genomic RSV detected in the upper respiratory tract for <14 days compared to those for ≥14 days (p < 0.01); and (4) statistically significant correlation between the antigenic site-specific competitive antibody concentrations and neutralizing antibody titers against RSV/A and RSV/B (r ranged from 0.33 to 0.83 for acute sera, and 0.50-0.88 for convalescent sera; p < 0.05). Conclusions: In RSV infected HCT adults, antigenic site-specific antibody responses were induced against multiple antigenic sites found in both the pre-fusion and post-fusion F conformations, and were associated with a more rapid viral clearance and neutralizing antibody activity. However, the association is not necessarily the cause and the consequence.

Clinical characteristics and outcomes of respiratory syncytial virus infection in pregnant women.

OBJECTIVE: To describe the clinical presentation and laboratory diagnosis of pregnant women with respiratory syncytial virus (RSV) infection. METHODS: Pregnant women in their second and third trimester were enrolled during the course of routine prenatal care visits when they were asymptomatic within the preceding two weeks (healthy controls) or when they reported symptoms of acute respiratory illness (ARI) of ≤7 days of duration (cases). Clinical outcomes were assessed at enrollment and two weeks after. Re-enrollment was allowed. Nasal-pharyngeal secretions were evaluated for respiratory pathogens by real-time reverse transcription polymerase chain reaction (PCR). Sera were tested for RSV-specific antibody responses by Western Blot, microneutralization assay, and palivizumab competitive antibody assay. RESULTS: During the 2015-2016 respiratory virus season, 7 of 65 (11%) pregnant women with ARI at their initial enrollment and 8 of 77 (10%) pregnant women with ARI during the study period (initial or re-enrollment) had PCR-confirmed RSV infection. Four (50%) PCR-confirmed RSV ARI cases reported symptoms of a lower respiratory tract illness (LRTI), one was hospitalized. Combining PCR and serology data, the RSV attack rate at initial enrollment was 12% (8 of 65), and 13% (10 of 77) based on ARI episodes. Among healthy controls, 28 of 88 (32%) had a Western Blot profile suggestive of a recent RSV infection either in the prior and/or current season. CONCLUSION: RSV had an attack rate of 10-13% among ambulatory pregnant women receiving routine prenatal care during the respiratory virus season. The serology results of healthy controls suggest a potentially higher attack rate. Future studies should be aware of the combined diagnostic strength of PCR and serology to identify RSV infection. As maternal RSV vaccine candidates are evaluated to protect young infants, additional priority should be placed on outcomes of pregnant women.

The interdependencies of viral load, the innate immune response, and clinical outcome in children presenting to the emergency department with respiratory syncytial virus-associated bronchiolitis.

Respiratory syncytial virus (RSV) causes significant infant morbidity and mortality. For decades severe RSV-induced disease was thought to result from an uncontrolled host response to viral replication, but recent work suggests that a strong innate immune response early in infection is protective. To shed light on host-virus interactions and the viral determinants of disease, copy numbers of five RSV genes (NS1, NS2, N, G, F) were measured by quantitative real-time polymerase chain reaction (qPCR) in nasal wash samples from children with RSV-associated bronchiolitis. Correlations were sought with host cytokines/chemokines and biomarkers. Associations with disposition from the emergency department (hospitalized or sent home) and pulse oximetry O2 saturation levels were also sought. Additionally, RNase P copy number was measured and used to normalize nasal wash data. RSV gene copy numbers were found to significantly correlate with both cytokine/chemokine and biomarker levels; and RNase P-normalized viral gene copy numbers (NS1, NS2, N and G) were significantly higher in infants with less severe disease. Moreover, three of the normalized viral gene copy numbers (NS1, NS2, and N) correlated significantly with arterial O2 saturation levels. The data support a model where a higher viral load early in infection can promote a robust innate immune response that protects against progression into hypoxic RSV-induced lower respiratory tract illness.

Respiratory Syncytial Virus (RSV): Neutralizing Antibody, a Correlate of Immune Protection.

Assays that measure RSV-specific neutralizing antibody activity are very useful for evaluating vaccine candidates, performing seroprevalence studies, and detecting infection. Neutralizing antibody activity is normally measured by a plaque reduction neutralization assay or by a microneutralization assay with or without complement. These assays measure the functional capacity of serum (or other fluids) to neutralize virus infectivity in cells as compared to ELISA assays that only measure the binding capacity against an antigen. This chapter discusses important elements in standardization of the RSV-specific microneutralization assay for use in the laboratory.

Gene sequence variability of the three surface proteins of human respiratory syncytial virus (HRSV) in Texas.

Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004-2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community.

Lactate dehydrogenase and caspase activity in nasopharyngeal secretions are predictors of bronchiolitis severity.

BACKGROUND: Bronchiolitis is the leading cause of hospitalization in infants. Biomarkers of disease severity might help in clinical management. OBJECTIVE: To determine the clinical predictiveness of NW-LDH, NW-caspase 3/7, and NW-LDH/NW-caspase 3/7 ratio in bronchiolitis. METHODS: Previously healthy children less than 24 months of age with bronchiolitis were recruited from the Texas Children's emergency room and intensive care unit from October 2010 to April 2011. Demographic, clinical information, and NW samples were obtained at enrollment. NW samples were analyzed for respiratory viruses, caspase 3/7, and LDH. RESULTS: A viral pathogen was detected in 91·6% of 131 children, with the most common being respiratory syncytial virus and human rhinovirus. A single infection was found in 61·8% of subjects and co-infection in 29·8%. Children admitted to ICU had significantly higher NW-LDH than children sent home from the ER or admitted to the general floor (P = 0·02). Children infected with RSV had the highest NW-LDH concentration (P = 0·03) compared with other viral infections. NW-LDH and NW-caspase were significantly correlated (r = 0·77, P < 0·0001). The univariate models showed NW-LDH and NW-LDH/NW- caspase 3/7 ratio were directly associated with hospitalization. Mutivariate regression analyses suggested a complex interaction between the biomarkers, demographics, and disposition. CONCLUSIONS: NW-LDH, NW-caspase 3/7 and NW-LDH/NW-caspase 3/7 ratio and their interactions with demographic factors are predictive of bronchiolitis severity and can help distinguish children requiring ICU-level care from those admitted to the general floor, or discharged home from the emergency center.