Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

ABSTRACT The M2 gene of respiratory syncytial virus (RSV) encodes two putative proteins: M2-1 and M2-2; both are believed to be involved in the RNA transcription or replication process. To understand the function of the M2-2 protein in virus replication, we deleted the majority of the M2-2 open reading frame from an infectious cDNA clone derived from the human RSV A2 strain. Transfection of HEp-2 cells with the cDNA clone containing the M2-2 deletion, together with plasmids that encoded the RSV N, P, and L proteins, produced a recombinant RSV that lacked the M2-2 protein (rA2ΔM2-2). Recombinant virus rA2ΔM2-2 was recovered and characterized. The levels of viral mRNA expression for 10 RSV genes examined were unchanged in cells infected with rA2ΔM2-2, except that a shorter M2 mRNA was detected. However, the ratio of viral genomic or antigenomic RNA to mRNA was reduced in rA2ΔM2-2-infected cells. By use of an antibody directed against the bacterially expressed M2-2 protein, the putative M2-2 protein was detected in cells infected with wild-type RSV but not in cells infected with rA2ΔM2-2. rA2ΔM2-2 displayed a small-plaque morphology and grew much more slowly than wild-type RSV in HEp-2 cells. In infected Vero cells, rA2ΔM2-2 exhibited very large syncytium formation compared to that of wild-type recombinant RSV. rA2ΔM2-2 appeared to be a host range mutant, since it replicated poorly in HEp-2, HeLa, and MRC5 cells but replicated efficiently in Vero and LLC-MK2 cells. Replication of rA2ΔM2-2 in the upper and lower respiratory tracts of mice and cotton rats was highly restricted. Despite its attenuated replication in rodents, rA2ΔM2-2 was able to provide protection against challenge with wild-type RSV A2. The genotype and phenotype of the M2-2 deletion mutant were stably maintained after extensive in vitro passages. The attenuated phenotype of rA2ΔM2-2 suggested that rA2ΔM2-2 may be a potential candidate for use as a live attenuated vaccine.

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Considerations for a Respiratory Syncytial Virus Vaccine Targeting an Elderly Population

Vaccines ◽

10.3390/vaccines9060624 ◽

2021 ◽

Vol 9 (6) ◽

pp. 624

Author(s):

Laura M. Stephens ◽

Steven M. Varga

Keyword(s):

Respiratory Syncytial Virus ◽

Disease Burden ◽

Elderly Population ◽

Vaccine Development ◽

The Elderly ◽

The United States ◽

Susceptible Population ◽

Age Related ◽

Syncytial Virus ◽

Tract Infections

Respiratory syncytial virus (RSV) is most commonly associated with acute lower respiratory tract infections in infants and children. However, RSV also causes a high disease burden in the elderly that is often under recognized. Adults >65 years of age account for an estimated 80,000 RSV-associated hospitalizations and 14,000 deaths in the United States annually. RSV infection in aged individuals can result in more severe disease symptoms including pneumonia and bronchiolitis. Given the large disease burden caused by RSV in the aged, this population remains an important target for vaccine development. Aging results in lowered immune responsiveness characterized by impairments in both innate and adaptive immunity. This immune senescence poses a challenge when developing a vaccine targeting elderly individuals. An RSV vaccine tailored towards an elderly population will need to maximize the immune response elicited in order to overcome age-related defects in the immune system. In this article, we review the hurdles that must be overcome to successfully develop an RSV vaccine for use in the elderly, and discuss the vaccine candidates currently being tested in this highly susceptible population.

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The varicella–zoster virus–mediated delayed host shutoff: open reading frame 17 has no major function, whereas immediate–early 63 protein represses heterologous gene expression

Microbes and Infection ◽

10.1016/j.micinf.2005.05.010 ◽

2005 ◽

Vol 7 (15) ◽

pp. 1519-1529 ◽

Cited By ~ 12

Author(s):

Nathalie Desloges ◽

Markus Rahaus ◽

Manfred H. Wolff

Keyword(s):

Gene Expression ◽

Varicella Zoster Virus ◽

Heterologous Gene Expression ◽

Open Reading Frame ◽

Heterologous Gene ◽

Immediate Early ◽

Reading Frame ◽

Varicella Zoster ◽

Major Function ◽

Host Shutoff

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Gene Expression of Nucleic Acid-Sensing Pattern Recognition Receptors in Children Hospitalized for Respiratory Syncytial Virus-Associated Acute Bronchiolitis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00445-08 ◽

2009 ◽

Vol 16 (6) ◽

pp. 816-823 ◽

Cited By ~ 38

Author(s):

Carolina Scagnolari ◽

Fabio Midulla ◽

Alessandra Pierangeli ◽

Corrado Moretti ◽

Enea Bonci ◽

...

Keyword(s):

Gene Expression ◽

Pattern Recognition ◽

Viral Load ◽

Respiratory Syncytial Virus ◽

Pattern Recognition Receptors ◽

Mrna Levels ◽

Acute Bronchiolitis ◽

Expression Levels ◽

Rsv Infection ◽

Syncytial Virus

ABSTRACT Given the critical role of pattern recognition receptors (PRRs) in acid nucleic recognition in the initiation of innate immunity and the orchestration of adaptive immunity, the aim of this study was to determine whether any heterogeneity of PRR expression in the airway tracts of infants with respiratory syncytial virus (RSV) infection might explain the broad clinical spectrum of RSV-associated bronchiolitis in infants. For this purpose, the levels of melanoma differentiation-associated protein-5 (MDA-5), retinoic acid inducible gene-1 (RIG-1), and Toll-like receptor 3 (TLR-3), TLR-7, TLR-8, and TLR-9 mRNAs were evaluated, using TaqMan quantitative reverse transcription-PCR, in cells from nasopharyngeal washes collected from 157 infants suffering from acute bronchiolitis whether or not they were associated with respiratory viruses. High interindividual variability was observed in both virus-positive and -negative infants; however, the relative gene expression levels of MDA-5, RIG-1, TLR-7, and TLR-8 were significantly higher in the virus-infected group, whereas the expression levels of TLR-3 and TLR-9 were not significantly different. The differences in the gene expression of MDA-5, RIG-1, TLR-7, and TLR-8 were more evident in infants with RSV infection than in those with bocavirus or rhinovirus infection. In RSV-infected infants, PRR-mRNA levels also were analyzed in relation to interferon protein levels, viral load, clinical severity, days of hospitalization, age, and body weight. A significant positive correlation was observed only between RSV viral load and RIG-1 mRNA levels. These findings provide the first direct evidence that, in infants with respiratory virus-associated bronchiolitis, especially RSV, there are substantial changes in PRR gene expression; this likely is an important determinant of the clinical outcome of bronchiolitis.

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Respiratory syncytial virus increases IL-8 gene expression and protein release in A549 cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1995.269.6.l865 ◽

1995 ◽

Vol 269 (6) ◽

pp. L865-L872 ◽

Cited By ~ 13

Author(s):

M. A. Fiedler ◽

K. Wernke-Dollries ◽

J. M. Stark

Keyword(s):

Gene Expression ◽

Respiratory Syncytial Virus ◽

Viral Replication ◽

Mrna Expression ◽

A549 Cells ◽

Protein Release ◽

Tnf Alpha ◽

Epithelial Cell Line ◽

Airway Epithelial ◽

Syncytial Virus

The mechanism of respiratory syncytial virus (RSV)-induced inflammation in the airways of infants and children is not fully understood. We hypothesized that RSV directly induces interleukin (IL)-8 gene expression in airway epithelial cells, independent of IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) production. Exposure of A549 cells (an airway epithelial cell line) to RSV resulted in increased IL-8 mRNA expression and IL-8 protein release from the cells as early as 2 h after treatment. Neither IL-1 beta nor TNF-alpha (mRNA or protein) were detected. Viral replication was not necessary for the effects of RSV on IL-8 mRNA expression and protein release early in the infectious process. However, sustained levels of increased IL-8 production required RSV replication. A dose-response relationship was observed between the multiplicity of infection and IL-8 production with both active and nonreplicative RSV at the 2-h time point. Both active RSV and nonreplicative RSV increased the transcriptional activity of the 1.6-kb 5' flanking region of the IL-8 gene. Neither active RSV nor nonreplicative RSV increased the stability of the IL-8 mRNA in A549 cells. We conclude that RSV increases IL-8 gene expression in A549 cells in a biphasic pattern independent of viral replication early (2 h) but dependent on viral replication late (24 h).

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Differential Gene Expression in Young Vs Aged Mice Infected with Respiratory Syncytial Virus

Journal of Allergy and Clinical Immunology ◽

10.1016/j.jaci.2012.12.705 ◽

2013 ◽

Vol 131 (2) ◽

pp. AB8

Author(s):

Terianne M. Wong ◽

Sandhya Boyapalle ◽

Siddarth Kamath ◽

Huy Nguyen ◽

Subhra Mohapatra ◽

...

Keyword(s):

Gene Expression ◽

Respiratory Syncytial Virus ◽

Differential Gene Expression ◽

Aged Mice ◽

Differential Gene ◽

Syncytial Virus

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The Immediate-Early Gene Product Encoded by Open Reading Frame 57 of Herpesvirus Saimiri Modulates Gene Expression at a Posttranscriptional Level

Journal of Virology ◽

10.1128/jvi.72.1.857-861.1998 ◽

1998 ◽

Vol 72 (1) ◽

pp. 857-861 ◽

Cited By ~ 48

Author(s):

Adrian Whitehouse ◽

Matthew Cooper ◽

David M. Meredith

Keyword(s):

Gene Expression ◽

Gene Product ◽

Target Gene ◽

Immediate Early Gene ◽

Open Reading Frame ◽

Early Gene ◽

Immediate Early ◽

Herpesvirus Saimiri ◽

Amino Acid Homology ◽

Reading Frame

ABSTRACT The herpesvirus saimiri (HVS) immediate-early gene product encoded by open reading frame (ORF) 57 shares limited amino acid homology with HSV-1 ICP27 and Epstein-Barr virus BMLF1, both regulatory proteins. The ORF 57 gene has been proposed to be spliced based on the genome sequence, and here we confirm the intron-exon structure of the gene. We also demonstrate that a cDNA construct of the ORF 57 gene product represses the transactivating capability of the ORF 50a gene product (which is produced from a spliced transcript), but activates that of ORF 50b (an unspliced transcript). Further analyses with cotransfection experiments show that ORF 57 can either activate or repress expression from a range of both early and late HVS promoters, depending on the target gene. These results indicate that repression of gene expression mediated by the ORF 57 gene product is dependent on the presence of an intron within the target gene encoding region. Furthermore, Northern blot analysis demonstrates that the levels of mRNA transcribed from genes not containing an intron are not significantly affected in the presence of the ORF 57 gene product. This suggests that it regulates gene expression through a posttranscriptional mechanism.

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Local Evolutionary Patterns of Human Respiratory Syncytial Virus Derived from Whole-Genome Sequencing

Journal of Virology ◽

10.1128/jvi.03391-14 ◽

2015 ◽

Vol 89 (7) ◽

pp. 3444-3454 ◽

Cited By ~ 41

Author(s):

Charles N. Agoti ◽

James R. Otieno ◽

Patrick K. Munywoki ◽

Alexander G. Mwihuri ◽

Patricia A. Cane ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Large Scale ◽

Vaccine Development ◽

Human Respiratory Syncytial Virus ◽

Genomic Variation ◽

Genomic Sequences ◽

The Novel ◽

Sequencing Method ◽

The World ◽

Syncytial Virus

ABSTRACTHuman respiratory syncytial virus (RSV) is associated with severe childhood respiratory infections. A clear description of local RSV molecular epidemiology, evolution, and transmission requires detailed sequence data and can inform new strategies for virus control and vaccine development. We have generated 27 complete or nearly complete genomes of RSV from hospitalized children attending a rural coastal district hospital in Kilifi, Kenya, over a 10-year period using a novel full-genome deep-sequencing process. Phylogenetic analysis of the new genomes demonstrated the existence and cocirculation of multiple genotypes in both RSV A and B groups in Kilifi. Comparison of local versus global strains demonstrated that most RSV A variants observed locally in Kilifi were also seen in other parts of the world, while the Kilifi RSV B genomes encoded a high degree of variation that was not observed in other parts of the world. The nucleotide substitution rates for the individual open reading frames (ORFs) were highest in the regions encoding the attachment (G) glycoprotein and the NS2 protein. The analysis of RSV full genomes, compared to subgenomic regions, provided more precise estimates of the RSV sequence changes and revealed important patterns of RSV genomic variation and global movement. The novel sequencing method and the new RSV genomic sequences reported here expand our knowledge base for large-scale RSV epidemiological and transmission studies.IMPORTANCEThe new RSV genomic sequences and the novel sequencing method reported here provide important data for understanding RSV transmission and vaccine development. Given the complex interplay between RSV A and RSV B infections, the existence of local RSV B evolution is an important factor in vaccine deployment.

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Differential Histopathology and Chemokine Gene Expression in Lung Tissues following Respiratory Syncytial Virus (RSV) Challenge of Formalin-Inactivated RSV- or BBG2Na-Immunized Mice

Journal of Virology ◽

10.1128/jvi.75.24.12421-12430.2001 ◽

2001 ◽

Vol 75 (24) ◽

pp. 12421-12430 ◽

Cited By ~ 33

Author(s):

Ultan F. Power ◽

Thierry Huss ◽

Vincent Michaud ◽

Hélène Plotnicky-Gilquin ◽

Jean-Yves Bonnefoy ◽

...

Keyword(s):

Gene Expression ◽

Respiratory Syncytial Virus ◽

Mouse Model ◽

Subunit Vaccine ◽

Late Time ◽

Chemokine Gene ◽

Time Points ◽

Rsv Infection ◽

Syncytial Virus ◽

Chemokine Gene Expression

ABSTRACT A BALB/c mouse model of enhanced pulmonary pathology following vaccination with formalin-inactivated alum-adsorbed respiratory syncytial virus (FI-RSV) and live RSV challenge was used to determine the type and kinetics of histopathologic lesions induced and chemokine gene expression profiles in lung tissues. These data were compared and contrasted with data generated following primary and/or secondary RSV infection or RSV challenge following vaccination with a promising subunit vaccine, BBG2Na. Severe peribronchiolitis and perivascularitis coupled with alveolitis and interstitial inflammation were the hallmarks of lesions in the lungs of FI-RSV-primed mice, with peak histopathology evident on days 5 and 9. In contrast, primary RSV infection resulted in no discernible lesions, while challenge of RSV-primed mice resulted in rare but mild peribronchiolitis and perivascularitis, with no evidence of alveolitis or interstitial inflammation. Importantly, mice vaccinated with a broad dose range (20 to 0.02 μg) of a clinical formulation of BBG2Na in aluminium phosphate demonstrated histopathology similar to that observed in secondary RSV infection. At the molecular level, FI-RSV priming was characterized by a rapid and strong up-regulation of eotaxin and monocyte chemotactic protein 3 (MCP-3) relative gene expression (potent lymphocyte and eosinophil chemoattractants) that was sustained through late time points, early but intermittent up-regulation of GRO/melanoma growth stimulatory activity gene and inducible protein 10 gene expression, while macrophage inflammatory protein 2 (MIP-2) and especially MCP-1 were up-regulated only at late time points. By comparison, primary RSV infection or BBG2Na priming resulted in considerably lower eotaxin and MCP-3 gene expression increases postchallenge, while expression of lymphocyte or monocyte chemoattractant chemokine genes (MIP-1β, MCP-1, and MIP-2) were of higher magnitude and kinetics at early, but not late, time points. Our combined histopathologic and chemokine gene expression data provide a basis for differentiating between aberrant FI-RSV-induced immune responses and normal responses associated with RSV infection in the mouse model. Consequently, our data suggest that BBG2Na may constitute a safe RSV subunit vaccine for use in seronegative infants.

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