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Immunostimulatory Defective Viral Genomes from Respiratory Syncytial Virus Promote a Strong Innate Antiviral Response during Infection in Mice and Humans.

Sun Y, Jain D, Koziol-White CJ, Genoyer E, Gilbert M, Tapia K, Panettieri RA, Hodinka RL, López CB - PLoS Pathog. (2015)

Bottom Line: RSV iDVGs were detected in respiratory secretions of hospitalized patients, and their amount positively correlated with the level of expression of antiviral genes in the samples.Infection of explanted human lung tissue from different donors revealed that most humans can respond to RSV iDVGs and that the rate of accumulation of iDVGs during infection directly correlates with the quality of the antiviral response.Taken together, our data establish iDVGs as primary triggers of robust antiviral responses to RSV and provide the first evidence for an important biological role for naturally occurring iDVGs during a paramyxovirus infection in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Human respiratory syncytial virus (RSV) is a major cause of severe respiratory illness in children and susceptible adults. RSV blocks the development of the innate antiviral immune response and can grow to high titers in the respiratory tract. Here we demonstrate that immunostimulatory defective viral genomes (iDVGs) that are naturally generated during RSV replication are strong inducers of the innate antiviral response to RSV in mice and humans. In mice, RSV iDVGs stimulated the expression of antiviral genes, restricted viral replication, and prevented weight loss and lung inflammation. In human cells, the antiviral response to RSV iDVGs was dominated by the expression of IFN-λ1 over IFN-β and was driven by rapid intranuclear accumulation of the transcription factor IRF1. RSV iDVGs were detected in respiratory secretions of hospitalized patients, and their amount positively correlated with the level of expression of antiviral genes in the samples. Infection of explanted human lung tissue from different donors revealed that most humans can respond to RSV iDVGs and that the rate of accumulation of iDVGs during infection directly correlates with the quality of the antiviral response. Taken together, our data establish iDVGs as primary triggers of robust antiviral responses to RSV and provide the first evidence for an important biological role for naturally occurring iDVGs during a paramyxovirus infection in humans.

No MeSH data available.


Related in: MedlinePlus

iDVGs associate with high expression of antiviral genes in respiratory secretions from patients infected with RSV.(A) Representative PCR results for gRSV and DVGs in human nasopharyngeal control samples infected with adenovirus (A1-A5) and samples infected with RSV (R1-R4). (B) Gene expression determined by RT-qPCR shown as copy number relative to house keeping genes (*p<0.05, **p<0.01, by two-tailed Mann Whitney test). (C) Samples were scored based on the intensity of the DVG amplicons (1–4, absent to highest intensity) and correlated with the level of expression of antiviral genes. (r = correlation coefficient, p<0.0001 for slope deviation from 0).
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ppat.1005122.g005: iDVGs associate with high expression of antiviral genes in respiratory secretions from patients infected with RSV.(A) Representative PCR results for gRSV and DVGs in human nasopharyngeal control samples infected with adenovirus (A1-A5) and samples infected with RSV (R1-R4). (B) Gene expression determined by RT-qPCR shown as copy number relative to house keeping genes (*p<0.05, **p<0.01, by two-tailed Mann Whitney test). (C) Samples were scored based on the intensity of the DVG amplicons (1–4, absent to highest intensity) and correlated with the level of expression of antiviral genes. (r = correlation coefficient, p<0.0001 for slope deviation from 0).

Mentions: To determine if iDVGs are present in humans naturally infected with RSV and if they correlate with innate antiviral activity in respiratory samples of infected patients, we analyzed nasopharyngeal aspirates from pediatric patients with confirmed RSV infection. As controls for the specificity of genomic and DVG detection assays, we analyzed samples from patients infected with adenovirus (AdV). Only samples with comparable amounts of virus, as determined by their RT-qPCR cycle threshold (Ct) for genomic RSV (gRSV), and with sufficient amount of total cellular RNA for the full analysis were considered for the study (n = 41). While no gRSV or RSV DVGs were detected in samples from AdV-infected patients, DVGs were detected in 48.8% of the RSV positive samples (20/41) (Fig 5A, additional samples and quantification in S8 Fig). Remarkably, among the RSV positive samples, those with detectable levels of DVGs showed significantly higher expression of a number of antiviral genes including IFNA4, IFIT1, and RSAD2 (also known as viperin) (Fig 5B). Expression of these genes was positively correlated with the amount of DVGs detected as scored based on the intensity of the amplicon band in the PCR (Fig 5C). Notably, expression of IFNL1 and IFNB1 was not detectable in most of the patients (S9 Fig), likely because these primary genes are not longer expressed at high levels at the moment of sampling (when patients are very sick and go to the hospital) and only secondary ISGs can be measured. Of note, only patients admitted to the hospital and with equivalent levels of RSV genome were analyzed in this study. This study design reduced potential false negative results from samples that either contained very low levels of virus or viral RNA was degraded. This study did not consider the timing of infection during sampling, co-morbidities, previous or current treatment, or infection outcome. Studies with the appropriate patient populations need to be designed to evaluate these parameters. However, our data demonstrate that iDVGs are naturally generated during infections with RSV and indicate that iDVG accumulation correlates positively with the expression of genes with antiviral activity in patients.


Immunostimulatory Defective Viral Genomes from Respiratory Syncytial Virus Promote a Strong Innate Antiviral Response during Infection in Mice and Humans.

Sun Y, Jain D, Koziol-White CJ, Genoyer E, Gilbert M, Tapia K, Panettieri RA, Hodinka RL, López CB - PLoS Pathog. (2015)

iDVGs associate with high expression of antiviral genes in respiratory secretions from patients infected with RSV.(A) Representative PCR results for gRSV and DVGs in human nasopharyngeal control samples infected with adenovirus (A1-A5) and samples infected with RSV (R1-R4). (B) Gene expression determined by RT-qPCR shown as copy number relative to house keeping genes (*p<0.05, **p<0.01, by two-tailed Mann Whitney test). (C) Samples were scored based on the intensity of the DVG amplicons (1–4, absent to highest intensity) and correlated with the level of expression of antiviral genes. (r = correlation coefficient, p<0.0001 for slope deviation from 0).
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Related In: Results  -  Collection

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ppat.1005122.g005: iDVGs associate with high expression of antiviral genes in respiratory secretions from patients infected with RSV.(A) Representative PCR results for gRSV and DVGs in human nasopharyngeal control samples infected with adenovirus (A1-A5) and samples infected with RSV (R1-R4). (B) Gene expression determined by RT-qPCR shown as copy number relative to house keeping genes (*p<0.05, **p<0.01, by two-tailed Mann Whitney test). (C) Samples were scored based on the intensity of the DVG amplicons (1–4, absent to highest intensity) and correlated with the level of expression of antiviral genes. (r = correlation coefficient, p<0.0001 for slope deviation from 0).
Mentions: To determine if iDVGs are present in humans naturally infected with RSV and if they correlate with innate antiviral activity in respiratory samples of infected patients, we analyzed nasopharyngeal aspirates from pediatric patients with confirmed RSV infection. As controls for the specificity of genomic and DVG detection assays, we analyzed samples from patients infected with adenovirus (AdV). Only samples with comparable amounts of virus, as determined by their RT-qPCR cycle threshold (Ct) for genomic RSV (gRSV), and with sufficient amount of total cellular RNA for the full analysis were considered for the study (n = 41). While no gRSV or RSV DVGs were detected in samples from AdV-infected patients, DVGs were detected in 48.8% of the RSV positive samples (20/41) (Fig 5A, additional samples and quantification in S8 Fig). Remarkably, among the RSV positive samples, those with detectable levels of DVGs showed significantly higher expression of a number of antiviral genes including IFNA4, IFIT1, and RSAD2 (also known as viperin) (Fig 5B). Expression of these genes was positively correlated with the amount of DVGs detected as scored based on the intensity of the amplicon band in the PCR (Fig 5C). Notably, expression of IFNL1 and IFNB1 was not detectable in most of the patients (S9 Fig), likely because these primary genes are not longer expressed at high levels at the moment of sampling (when patients are very sick and go to the hospital) and only secondary ISGs can be measured. Of note, only patients admitted to the hospital and with equivalent levels of RSV genome were analyzed in this study. This study design reduced potential false negative results from samples that either contained very low levels of virus or viral RNA was degraded. This study did not consider the timing of infection during sampling, co-morbidities, previous or current treatment, or infection outcome. Studies with the appropriate patient populations need to be designed to evaluate these parameters. However, our data demonstrate that iDVGs are naturally generated during infections with RSV and indicate that iDVG accumulation correlates positively with the expression of genes with antiviral activity in patients.

Bottom Line: RSV iDVGs were detected in respiratory secretions of hospitalized patients, and their amount positively correlated with the level of expression of antiviral genes in the samples.Infection of explanted human lung tissue from different donors revealed that most humans can respond to RSV iDVGs and that the rate of accumulation of iDVGs during infection directly correlates with the quality of the antiviral response.Taken together, our data establish iDVGs as primary triggers of robust antiviral responses to RSV and provide the first evidence for an important biological role for naturally occurring iDVGs during a paramyxovirus infection in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Human respiratory syncytial virus (RSV) is a major cause of severe respiratory illness in children and susceptible adults. RSV blocks the development of the innate antiviral immune response and can grow to high titers in the respiratory tract. Here we demonstrate that immunostimulatory defective viral genomes (iDVGs) that are naturally generated during RSV replication are strong inducers of the innate antiviral response to RSV in mice and humans. In mice, RSV iDVGs stimulated the expression of antiviral genes, restricted viral replication, and prevented weight loss and lung inflammation. In human cells, the antiviral response to RSV iDVGs was dominated by the expression of IFN-λ1 over IFN-β and was driven by rapid intranuclear accumulation of the transcription factor IRF1. RSV iDVGs were detected in respiratory secretions of hospitalized patients, and their amount positively correlated with the level of expression of antiviral genes in the samples. Infection of explanted human lung tissue from different donors revealed that most humans can respond to RSV iDVGs and that the rate of accumulation of iDVGs during infection directly correlates with the quality of the antiviral response. Taken together, our data establish iDVGs as primary triggers of robust antiviral responses to RSV and provide the first evidence for an important biological role for naturally occurring iDVGs during a paramyxovirus infection in humans.

No MeSH data available.


Related in: MedlinePlus