Small hydrophobic (SH) proteins of Pneumoviridae and Paramyxoviridae: small but mighty.

Small hydrophobic (SH) proteins are a class of viral accessory proteins expressed by many members of the negative-stranded RNA viral families Paramyxoviridae and Pneumoviridae. Identified SH proteins are type I or II transmembrane (TM) proteins with a single-pass TM domain. Little is known about the functions of SH proteins; however, several possess viroporin activity, enhancing membrane permeability of infected cells or those expressing SH protein. Moreover, several SH proteins inhibit apoptosis and immune signaling pathways within infected cells, including TNF and interferon signaling, or activate inflammasomes. SH proteins are generally nonessential for viral replication in vitro, but loss of SH is often associated with reduced replication in vivo, suggesting a role in enhancing viral replication or evading host immunity. Analogous proteins are expressed by a variety of pathogens of public health importance; thus, understanding the functional importance and mechanisms of SH proteins provides insight into the pathogenesis and replication of negative-sense RNA viruses.

Structural basis of paramyxo- and pneumovirus polymerase inhibition by non-nucleoside small-molecule antivirals.

Small-molecule antivirals can be used as chemical probes to stabilize transitory conformational stages of viral target proteins, facilitating structural analyses. Here, we evaluate allosteric pneumo- and paramyxovirus polymerase inhibitors that have the potential to serve as chemical probes and aid the structural characterization of short-lived intermediate conformations of the polymerase complex. Of multiple inhibitor classes evaluated, we discuss in-depth distinct scaffolds that were selected based on well-understood structure-activity relationships, insight into resistance profiles, biochemical characterization of the mechanism of action, and photoaffinity-based target mapping. Each class is thought to block structural rearrangements of polymerase domains albeit target sites and docking poses are distinct. This review highlights validated druggable targets in the paramyxo- and pneumovirus polymerase proteins and discusses discrete structural stages of the polymerase complexes required for bioactivity.

Genomic analysis of canine pneumoviruses and canine respiratory coronavirus from New Zealand.

AIMS: To isolate canine respiratory coronavirus (CRCoV) and canine pneumovirus (CnPnV) in cell culture and to compare partial genomic sequences of CRCoV and CnPnV from New Zealand with those from other countries. METHODS: Oropharyngeal swab samples from dogs affected by canine infectious respiratory disease syndrome that were positive for CnPnV (n = 15) or CRCoV (n = 1) by virus-specific reverse transcriptase quantitative PCR (RT-qPCR) in a previous study comprised the starting material. Virus isolation was performed in HRT-18 cells for CRCoV and RAW 264.7 and Vero cells for CnPnV. The entire sequence of CnPnV G protein (1,266 nucleotides) and most (8,063/9,707 nucleotides) of the 3' region of CRCoV that codes for 10 structural and accessory proteins were amplified and sequenced. The sequences were analysed and compared with other sequences available in GenBank using standard molecular tools including phylogenetic analysis. RESULTS: Virus isolation was unsuccessful for both CRCoV and CnPnV. Pneumovirus G protein was amplified from 3/15 (20%) samples that were positive for CnPnV RNA by RT-qPCR. Two of these (NZ-048 and NZ-049) were 100% identical to each other, and 90.9% identical to the third one (NZ-007). Based on phylogenetic analysis of the G protein gene, CnPnV NZ-048 and NZ-049 clustered with sequences from the USA, Thailand and Italy in group A, and CnPnV NZ-007 clustered with sequences from the USA in group B. The characteristics of the predicted genes (length, position) and their putative protein products (size, predicted structure, presence of N- and O-glycosylation sites) of the New Zealand CRCoV sequence were consistent with those reported previously, except for the region located between open reading frame (ORF)3 (coding for S protein) and ORF6 (coding for E protein). The New Zealand virus was predicted to encode 5.9 kDa, 27 kDa and 12.7 kDa proteins, which differed from the putative coding capacity of this region reported for CRCoV from other countries. CONCLUSIONS: This report represents the first characterisation of partial genomic sequences of CRCoV and CnPnV from New Zealand. Our results suggest that the population of CnPnV circulating in New Zealand is not homogeneous, and that the viruses from two clades described overseas are also present here. Limited conclusions can be made based on only one CRCoV sequence, but the putative differences in the coding capacity of New Zealand CRCoV support the previously reported variability of this region. The reasons for such variability and its biological implications need to be further elucidated.

Antibody recognition of the Pneumovirus fusion protein trimer interface.

Human metapneumovirus (hMPV) is a leading cause of viral respiratory infection in children, and can cause severe lower respiratory tract infection in infants, the elderly, and immunocompromised patients. However, there remain no licensed vaccines or specific treatments for hMPV infection. Although the hMPV fusion (F) protein is the sole target of neutralizing antibodies, the immunological properties of hMPV F remain poorly understood. To further define the humoral immune response to the hMPV F protein, we isolated two new human monoclonal antibodies (mAbs), MPV458 and MPV465. Both mAbs are neutralizing in vitro and were determined to target a unique antigenic site using competitive biolayer interferometry. We determined both MPV458 and MPV465 have higher affinity for monomeric hMPV F than trimeric hMPV F. MPV458 was co-crystallized with hMPV F, and the mAb primarily interacts with an alpha helix on the F2 region of the hMPV F protein. Surprisingly, the major epitope for MPV458 lies within the trimeric interface of the hMPV F protein, suggesting significant breathing of the hMPV F protein must occur for host immune recognition of the novel epitope. In addition, significant glycan interactions were observed with a somatically mutated light chain framework residue. The data presented identifies a novel epitope on the hMPV F protein for epitope-based vaccine design, and illustrates a new mechanism for human antibody neutralization of viral glycoproteins.

Pneumoviral Phosphoprotein, a Multidomain Adaptor-Like Protein of Apparent Low Structural Complexity and High Conformational Versatility.

Mononegavirales phosphoproteins (P) are essential co-factors of the viral polymerase by serving as a linchpin between the catalytic subunit and the ribonucleoprotein template. They have highly diverged, but their overall architecture is conserved. They are multidomain proteins, which all possess an oligomerization domain that separates N- and C-terminal domains. Large intrinsically disordered regions constitute their hallmark. Here, we exemplify their structural features and interaction potential, based on the Pneumoviridae P proteins. These P proteins are rather small, and their oligomerization domain is the only part with a defined 3D structure, owing to a quaternary arrangement. All other parts are either flexible or form short-lived secondary structure elements that transiently associate with the rest of the protein. Pneumoviridae P proteins interact with several viral and cellular proteins that are essential for viral transcription and replication. The combination of intrinsic disorder and tetrameric organization enables them to structurally adapt to different partners and to act as adaptor-like platforms to bring the latter close in space. Transient structures are stabilized in complex with protein partners. This class of proteins gives an insight into the structural versatility of non-globular intrinsically disordered protein domains.

Phylogenetic evidence of a novel lineage of canine pneumovirus and a naturally recombinant strain isolated from dogs with respiratory illness in Thailand.

BACKGROUND: Canine pneumovirus (CPV) is a pathogen that causes respiratory disease in dogs, and recent outbreaks in shelters in America and Europe have been reported. However, based on published data and documents, the identification of CPV and its variant in clinically symptomatic individual dogs in Thailand through Asia is limited. Therefore, the aims of this study were to determine the emergence of CPV and to consequently establish the genetic characterization and phylogenetic analysis of the CPV strains from 209 dogs showing respiratory distress in Thailand. RESULTS: This study identified and described the full-length CPV genome from three strains, designated herein as CPV_CP13 TH/2015, CPV_CP82 TH/2016 and CPV_SR1 TH/2016, that were isolated from six dogs out of 209 dogs (2.9%) with respiratory illness in Thailand. Phylogenetic analysis suggested that these three Thai CPV strains (CPV TH strains) belong to the CPV subgroup A and form a novel lineage; proposed as the Asian prototype. Specific mutations in the deduced amino acids of these CPV TH strains were found in the G/glycoprotein sequence, suggesting potential substitution sites for subtype classification. Results of intragenic recombination analysis revealed that CPV_CP82 TH/2016 is a recombinant strain, where the recombination event occurred in the L gene with the Italian prototype CPV Bari/100-12 as the putative major parent. Selective pressure analysis demonstrated that the majority of the nucleotides in the G/glycoprotein were under purifying selection with evidence of positive selection sites. CONCLUSIONS: This collective information on the CPV TH strains is the first evidence of CPV emergence with genetic characterization in Thailand and as first report in Asia, where homologous recombination acts as a potential force driving the genetic diversity and shaping the evolution of canine pneumovirus.

First demonstration of the circulation of a pneumovirus in French pigs by detection of anti-swine orthopneumovirus nucleoprotein antibodies.

The presence of pneumoviruses in pigs is poorly documented. In this study, we used the published sequence of the nucleoprotein (N) of the recently identified Swine Orthopneumovirus (SOV) to express and purify SOV N as a recombinant protein in Escherichia coli. This protein was purified as nanorings and used to set up an enzyme-linked immunosorbent assay, which was used to analyse the presence of anti-pneumovirus N antibodies in swine sera. Sera collected from different pig farms in the West of France and from specific pathogen free piglets before colostrum uptake showed indirectly that a pneumovirus is circulating in pig populations with some variations between animals. Piglets before colostrum uptake were sero-negative for anti-pneumovirus antibodies while most of the other pigs showed positivity. Interestingly, in two farms presenting respiratory clinical signs and negative or under control for some common respiratory pathogens, pigs were detected positive for anti-pneumovirus antibodies. Globally, anti-pneumovirus N antibody concentrations were variable between and within farms. Further studies will aim to isolate the circulating virus and determine its potential pathogenicity. SOV could potentially become a new member of the porcine respiratory complex, important on its own or in association with other viral and bacterial micro-organisms.

B cells are not essential for Lactobacillus-mediated protection against lethal pneumovirus infection.

We have shown previously that priming of respiratory mucosa with live Lactobacillus species promotes robust and prolonged survival from an otherwise lethal infection with pneumonia virus of mice, a property known as heterologous immunity. Lactobacillus priming results in a moderate reduction in virus recovery and a dramatic reduction in virus-induced proinflammatory cytokine production; the precise mechanisms underlying these findings remain to be elucidated. Because B cells have been shown to promote heterologous immunity against respiratory virus pathogens under similar conditions, in this study we explore the role of B cells in Lactobacillus-mediated protection against acute pneumovirus infection. We found that Lactobacillus-primed mice feature elevated levels of airway Igs IgG, IgA, and IgM and lung tissues with dense, B cell (B220(+))-enriched peribronchial and perivascular infiltrates with germinal centers consistent with descriptions of BALT. No B cells were detected in lung tissue of Lactobacillus-primed B cell deficient µMT mice or Jh mice, and Lactobacillus-primed µMT mice had no characteristic infiltrates or airway Igs. Nonetheless, we observed diminished virus recovery and profound suppression of virus-induced proinflammatory cytokines CCL2, IFN-γ, and CXCL10 in both wild-type and Lactobacillus-primed µMT mice. Furthermore, Lactobacillus plantarum-primed, B cell-deficient µMT and Jh mice were fully protected from an otherwise lethal pneumonia virus of mice infection, as were their respective wild-types. We conclude that B cells are dispensable for Lactobacillus-mediated heterologous immunity and were not crucial for promoting survival in response to an otherwise lethal pneumovirus infection.

Production and differentiation of myeloid cells driven by proinflammatory cytokines in response to acute pneumovirus infection in mice.

Respiratory virus infections are often pathogenic, driving severe inflammatory responses. Most research has focused on localized effects of virus infection and inflammation. However, infection can induce broad-reaching, systemic changes that are only beginning to be characterized. In this study, we assessed the impact of acute pneumovirus infection in C57BL/6 mice on bone marrow hematopoiesis. We hypothesized that inflammatory cytokine production in the lung upregulates myeloid cell production in response to infection. We demonstrate a dramatic increase in the percentages of circulating myeloid cells, which is associated with pronounced elevations in inflammatory cytokines in serum (IFN-γ, IL-6, CCL2), bone (TNF-α), and lung tissue (TNF-α, IFN-γ, IL-6, CCL2, CCL3, G-CSF, osteopontin). Increased hematopoietic stem/progenitor cell percentages (Lineage(-)Sca-I(+)c-kit(+)) were also detected in the bone marrow. This increase was accompanied by an increase in the proportions of committed myeloid progenitors, as determined by colony-forming unit assays. However, no functional changes in hematopoietic stem cells occurred, as assessed by competitive bone marrow reconstitution. Systemic administration of neutralizing Abs to either TNF-α or IFN-γ blocked expansion of myeloid progenitors in the bone marrow and also limited virus clearance from the lung. These findings suggest that acute inflammatory cytokines drive production and differentiation of myeloid cells in the bone marrow by inducing differentiation of committed myeloid progenitors. Our findings provide insight into the mechanisms via which innate immune responses regulate myeloid cell progenitor numbers in response to acute respiratory virus infection.

New and emerging pathogens in canine infectious respiratory disease.

Canine infectious respiratory disease is a common, worldwide disease syndrome of multifactorial etiology. This review presents a summary of 6 viruses (canine respiratory coronavirus, canine pneumovirus, canine influenza virus, pantropic canine coronavirus, canine bocavirus, and canine hepacivirus) and 2 bacteria (Streptococcus zooepidemicus and Mycoplasma cynos) that have been associated with respiratory disease in dogs. For some pathogens a causal role is clear, whereas for others, ongoing research aims to uncover their pathogenesis and contribution to this complex syndrome. Etiology, clinical disease, pathogenesis, and epidemiology are described for each pathogen, with an emphasis on recent discoveries or novel findings.

Structures of the Foamy virus fusion protein reveal an unexpected link with the F protein of paramyxo- and pneumoviruses.

Foamy viruses (FVs) constitute a subfamily of retroviruses. Their envelope (Env) glycoprotein drives the merger of viral and cellular membranes during entry into cells. The only available structures of retroviral Envs are those from human and simian immunodeficiency viruses from the subfamily of orthoretroviruses, which are only distantly related to the FVs. We report the cryo-electron microscopy structures of the FV Env ectodomain in the pre- and post-fusion states, which unexpectedly demonstrate structural similarity with the fusion protein (F) of paramyxo- and pneumoviruses, implying an evolutionary link between the viral fusogens. We describe the structural features that are unique to the FV Env and propose a mechanistic model for its conformational change, highlighting how the interplay of its structural elements could drive membrane fusion and viral entry. The structural knowledge on the FV Env now provides a framework for functional investigations, which can benefit the design of FV Env variants with improved features for use as gene therapy vectors.

Detection of canine pneumovirus in dogs with canine infectious respiratory disease.

Canine pneumovirus (CnPnV) was recently identified during a retrospective survey of kenneled dogs in the United States. In this study, archived samples from pet and kenneled dogs in the United Kingdom were screened for CnPnV to explore the relationship between exposure to CnPnV and the development of canine infectious respiratory disease (CIRD). Within the pet dog population, CnPnV-seropositive dogs were detected throughout the United Kingdom and Republic of Ireland, with an overall estimated seroprevalence of 50% (n = 314/625 dogs). In the kennel population, there was a significant increase in seroprevalence, from 26% (n = 56/215 dogs) on the day of entry to 93.5% (n = 201/215 dogs) after 21 days (P <0001). Dogs that were seronegative on entry but seroconverted while in the kennel were 4 times more likely to develop severe respiratory disease than those that did not seroconvert (P < 0.001), and dogs with preexisting antibodies to CnPnV on the day of entry were significantly less likely to develop respiratory disease than immunologically naive dogs (P < 0.001). CnPnV was detected in the tracheal tissues of 29/205 kenneled dogs. Detection was most frequent in dogs with mild to moderate respiratory signs and histopathological changes and in dogs housed for 8 to 14 days, which coincided with a significant increase in the risk of developing respiratory disease compared to the risk of those housed 1 to 7 days (P < 0.001). These findings demonstrate that CnPnV is present in the United Kingdom dog population; there is a strong association between exposure to CnPnV and CIRD in the kennel studied and a potential benefit in vaccinating against CnPnV as part of a wider disease prevention strategy.

[Etiological structure of acute respiratory viral infections morbidity in Novosibirsk and Novosibirsk region in epidemic season 2011-2012].

INTRODUCTION: ARI occupying the first place in the structure of total human morbidity. The aim of the study was to investigate the species diversity of the viruses causing AR among residents of the Novosibirsk region during epidemic season (October to April). MATERIALS AND METHODS: 164 nasopharyngeal swabs were collected and analyzed. Viral RNA/DNA, cDNA synthesis and PCR were carried out employing "RIBO-prep" "eReverta-L", "AmpliSens Influenza virus A/B-FL" and "AmpliSens ARI-screen-FL" kits (CRI of Epidemiology). RESULTS: Etiological agent of the disease was found in 69(43%) samples. Monoinfection was found in 58 (35%). In 14 (9%) samples were detected serogroup I coronaviruses, in 13 (8%) rhinoviruses, in 7 (4%) respiratory syncytial virus, in 6 (4%) parainfluenza virus type 1, in 5 (3%) parainfluenza virus type 3. Adenoviruses and bocavirus were identified in 3 (2%) samples. Parainfluenza virus type 2 and 4, metapneumovirus, serogroup Il coronaviruses (HKU1 and OC43) were presented in 2 (1%) samples. In 11 (7%) samples was found mixed infection. CONCLUSION: The majority of common colds were caused by serogroup I coronaviruses (NL63 and 229E), rhinoviruses and mixed infections. The peak of species variability of viruses caused acute respiratory infections was determined in age group of children 2-4 years old. In older age groups the species variability of analyzed viruses was decreased, rhinovirus infection becomes prevalent.

RSV infection in children hospitalised with severe lower respiratory tract infection in a low-middle-income setting: A cross-sectional observational study.

INTRODUCTION: Low- and middle-income countries carry the largest burden of Respiratory syncytial virus (RSV) disease, with most deaths occurring in these settings. This study aimed to investigate the burden of RSV disease in South African children hospitalised with lower respiratory tract infection (LRTI), with specific reference to incidence, risk factors, and co-infections. METHODS: A database from a previous prospective study containing demographic, laboratory and clinical data on children hospitalised with LRTIs in Cape Town, South Africa, was used. A nasopharyngeal swab (NP) and induced sputum (IS) were tested for RSV PCR. Descriptive statistics were used to characterise the study population, and a multivariable analysis of risk factors and co-infections was done. RESULTS: RSV was detected in 142 (30.9%; 95% CI 26.7-35.3) of the included 460 study children with LRTI. The median age of RSV-positive children was 4.6 (IQR 2.4-9.7) months compared to RSV-negative children of 10.5 (IQR 4.4-21.3) months, P = <0.001. Most cases occurred in autumn and winter with 126 (89%) cases over this period. IS demonstrated greater sensitivity for RSV diagnosis with 135 cases (95.1%) detected on IS and 57 cases (40.1%) identified on NP; P<0.001. The median length of hospital stay was 3.3 (SD 4.2) days in the RSV positive group and 2.7 (SD 3.3) days in the RSV negative group; P<0.001. The median number of detected viral pathogens was 1 (IQR 0-2) in RSV-positive children (when RSV was excluded from the count) compared to 2 (IQR 2-3) in RSV negative children; P<0.001. The presence of RSV was independently associated with a reduction in the frequency of most viruses tested for on PCR. CONCLUSIONS: RSV is common in children hospitalised with LRTI and mainly affects younger children. There is an urgent need to find an effective vaccine to prevent RSV pneumonia in children worldwide, especially in LMICs that carry the greatest burden of disease.

The HRA2pl fusion peptide exerts in vitro antiviral activity against human respiratory paramyxoviruses and pneumoviruses.

Acute respiratory infections are a group of diseases caused by viruses, bacteria, and parasites that mainly affect children until the age of 5 and immunocompromised senior adults. In Mexico, these infections are the main cause of morbidity in children, with more than 26 million cases of respiratory infections reported by the Secretariat of Health, in 2019. The human respiratory syncytial virus (hRSV), the human metapneumovirus (hMPV), and the human parainfluenza-2 (hPIV-2) are responsible for many respiratory infections. Currently, palivizumab, a monoclonal antibody against the fusion protein F, is the treatment of choice against hRSV infections. This protein is being studied for the design of antiviral peptides that act by inhibiting the fusion of the virus and the host cell. Therefore, we examined the antiviral activity of the HRA2pl peptide, which competes the heptad repeat A domain of the F protein of hMPV. The recombinant peptide was obtained using a viral transient expression system. The effect of the fusion peptide was evaluated with an in vitro entry assay. Moreover, the effectiveness of HRA2pl was examined in viral isolates from clinical samples obtained from patients with infections caused by hRSV, hMPV, or hPIV-2, by evaluating the viral titer and the syncytium size. The HRA2pl peptide affected the viruses' capacity of entry, resulting in a 4-log decrease in the viral titer compared to the untreated viral strains. Additionally, a 50% reduction in the size of the syncytium was found. These results demonstrate the antiviral potential of HRA2pl in clinical samples, paving the way toward clinical trials.

First Detection and Genetic Characterization of Swine Orthopneumovirus from Domestic Pig Farms in the Republic of Korea.

Novel swine orthopneumovirus (SOV) infections have been identified in pigs in the USA and some European countries but not in Asian countries, including South Korea, to date. The current study reports the first SOV infections in four domestic pig farms located in four provinces across South Korea. The detection rate of SOV in oral fluid samples using qRT-PCR was 4.4% (14/389), indicating the presence of the virus in pigs at commercial farms in Korea. Two complete genome sequences and one glycoprotein (G) gene sequence were obtained from SOV-positive samples. The complete genome analysis of KSOV-2201 and KSOV-2202 strains showed 98.2 and 95.4% homologies with a previously reported SOV, and the phylogenetic tree exhibited a high correlation with a previously reported SOV strain from the US and a canine pneumovirus (CPnV) strain from China. Based on the genetic analysis of the viral G gene, the murine pneumonia virus (MPV)-like orthopneumoviruses (MLOVs) were divided into two genogroups (G1 and G2). Seventeen CPnVs and two feline pneumoviruses were grouped into G1, while the Korean SOV strains identified in this study were grouped into G2 along with one SOV and two CPnVs. These results will contribute to expanding our understanding of the geographical distribution and genetic characteristics of the novel SOV in the global pig population.

Emergence of swine influenza A virus, porcine respirovirus 1 and swine orthopneumovirus in porcine respiratory disease in Germany.

Respiratory disease is a significant economic issue in pig farming, with a complex aetiology that includes swine influenza A viruses (swIAV), which are common in European domestic pig populations. The most recent human influenza pandemic in 2009 showed swIAV's zoonotic potential. Monitoring pathogens and disease control are critical from a preventive standpoint, and are based on quick, sensitive, and specific diagnostic assays capable of detecting and distinguishing currently circulating swIAV in clinical samples. For passive surveillance, a set of multiplex quantitative reverse transcription real-time PCRs (mRT-qPCR) and MinION-directed sequencing was updated and deployed. Several lineages and genotypes of swIAV were shown to be dynamically developing, including novel reassortants between human pandemic H1N1 and the avian-derived H1 lineage of swIAV. Despite this, nearly 70% (842/1216) of individual samples from pigs with respiratory symptoms were swIAV-negative, hinting to different aetiologies. The complex and synergistic interactions of swIAV infections with other viral and bacterial infectious agents contribute to the aggravation of pig respiratory diseases. Using a newly developed mRT-qPCR for the combined detection of swIAV and the recently described porcine respirovirus 1 (PRV1) and swine orthopneumovirus (SOV) widespread co-circulation of PRV1 (19.6%, 238/1216 samples) and SOV (14.2%, 173/1216 samples) was evident. Because of the high incidence of PRV1 and SOV infections in pigs with respiratory disease, these viruses may emerge as new allies in the porcine respiratory disease syndrome.

Granzyme A- and B-cluster deficiency delays acute lung injury in pneumovirus-infected mice.

Lower respiratory tract infection by the human pneumovirus respiratory syncytial virus is a frequent cause of acute lung injury in children. Severe pneumovirus disease in humans is associated with activation of the granzyme pathway by effector lymphocytes, which may promote pathology by exaggerating proapoptotic caspase activity and proinflammatory activity. The main goal of this study was to determine whether granzymes contribute to the development of acute lung injury in pneumovirus-infected mice. Granzyme-expressing mice and granzyme A- and B-cluster single- and double-knockout mice were inoculated with the rodent pneumovirus pneumonia virus of mice strain J3666, and were studied for markers of lung inflammation and injury. Expression of granzyme A and B is detected in effector lymphocytes in mouse lungs in response to pneumovirus infection. Mice deficient for granzyme A and the granzyme B cluster have unchanged virus titers in the lungs but show a significantly delayed clinical response to fatal pneumovirus infection, a feature that is associated with delayed neutrophil recruitment, diminished activation of caspase-3, and reduced lung permeability. We conclude that granzyme A- and B-cluster deficiency delays the acute progression of pneumovirus disease by reducing alveolar injury.

Cytokine expression in rhinovirus- vs. respiratory syncytial virus-induced first wheezing episode and its relation to clinical course.

Rhinovirus (RV) and respiratory syncytial virus (RSV) are common causes of bronchiolitis. Unlike an RSV etiology, an RV etiology is associated with a markedly increased risk of asthma. We investigated the cytokine profiles of RV- and RSV-induced first wheezing episode and their correlation with prognosis. We recruited 52 sole RV- and 11 sole RSV-affected children with a severe first wheezing episode. Peripheral blood mononuclear cells (PBMCs) were isolated during acute illness and 2 weeks later and stimulated in vitro with anti-CD3/anti-CD28. Culture medium samples were analyzed for 56 different cytokines by multiplex ELISA. Recurrences were prospectively followed for 4 years. In adjusted analyses, the cytokine response from PBMCs in the RV group was characterized by decreased expression of interleukin 1 receptor antagonist (IL-1RA), interleukin 1 beta (IL-1ß), and monocyte chemoattractant protein-1 (MCP-1) and increased expression of eosinophil chemotactic protein 2 (eotaxin-2), thymus- and activation-regulated chemokine (TARC), and epithelial-derived neutrophil-activating peptide 78 (ENA-78) in the acute phase and increased expression of fractalkine in the convalescent phase compared to those in the RSV group. An analysis of the change in cytokine expression between study points revealed an increased expression of fractalkine and IL-1ß and decreased expression of I-309 (CCL1) and TARC in the RV group compared to those in the RSV group.. Considering hospitalization time, a significant non-adjusted group × cytokine interaction was observed in the levels of interferon gamma (IFN-γ), macrophage-derived chemokine (MDC), IL-1RA, and vascular endothelial growth factor (VEGF), indicating that a higher expression of cytokine was associated with shorter hospitalization time in the RSV group but not in the RV group. A significant interaction was also found in interleukin 6 (IL-6), but the cytokine response was not associated with hospitalization time in the RSV or RV group. In the RV group, increased expression of I-309 (CCL1) and TARC was associated with fewer relapses within 2 months, and decreased expression of interleukin 13 (IL-13) and increased expression of I-309 (CCL1) were associated with less relapses within 12 months. Differences in cytokine response from PBMCs were observed between RV- and RSV-induced first severe wheezing episode. Our findings also reveal new biomarkers for short- and medium-term prognosis in first-time wheezing children infected with RV or RSV.

Elicitation of pneumovirus-specific B cell responses by a prefusion-stabilized respiratory syncytial virus F subunit vaccine.

Respiratory syncytial virus (RSV) is a substantial cause of morbidity and mortality globally. A candidate RSV prefusion (pre-F)-stabilized subunit vaccine, DS-Cav1, has previously been shown to elicit potent and durable neutralizing activity in a phase 1 clinical trial in healthy adults. Here, we used fluorescently labeled probes and flow cytometry to evaluate the antigen specificity and phenotype of RSV F-specific B cells longitudinally after DS-Cav1 immunization. Peripheral blood mononuclear cells (PBMCs) collected at time points before the first immunization through the end of the trial at 44 weeks were assessed by flow cytometry. Our data demonstrate a rapid increase in the frequency of pre-F-specific IgG+ and IgA+ B cells after the first immunization and a modest increase after a second immunization at week 12. Nearly all F-specific B cells down-regulated CD21 and up-regulated the proliferation marker CD71 after the first immunization, with less pronounced activation after the second immunization. Memory B cells (CD27+CD21+) specific for pre-F remained elevated above baseline at 44 weeks after vaccination. DS-Cav1 vaccination also activated human metapneumovirus (HMPV) cross-reactive B cells capable of binding prefusion-stabilized HMPV F protein and increased HMPV F-binding antibodies and neutralizing activity for HMPV in some participants. In summary, vaccination with RSV pre-F resulted in the expansion and activation of RSV and HMPV F-specific B cells that were maintained above baseline for at least 10 months and could contribute to long-term pneumovirus immunity.