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Defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity.

Tapia K, Kim WK, Sun Y, Mercado-López X, Dunay E, Wise M, Adu M, López CB - PLoS Pathog. (2013)

Bottom Line: Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent of IFN signaling.Remarkably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing DVGs, while cells within the lung that contain standard viral genomes alone do not express this cytokine.Together, our data indicate that DVGs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection.

View Article: PubMed Central - PubMed

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

ABSTRACT
The innate immune response to viruses is initiated when specialized cellular sensors recognize viral danger signals. Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent of IFN signaling. We demonstrate that these defective viral genomes (DVGs) are generated naturally during respiratory infections in vivo even in mice lacking the type I IFN receptor, and their appearance coincides with the production of cytokines during infections with Sendai virus (SeV) or influenza virus. Remarkably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing DVGs, while cells within the lung that contain standard viral genomes alone do not express this cytokine. Together, our data indicate that DVGs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection.

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SeV DVGs reduce virulence in vivo.(A–C) Mice were infected with 105 TCID50/mouse of SeV Cantell HD (HD) or SeV Cantell LD (LD). (A) Weight loss (***p<0.001, ****p<0.0001; Two-way ANOVA with Bonferroni's post hoc test), (B) Virus titers in the lung, (n = 11 for day 1, n = 6 for day 3), and (C) expression of Ifnb mRNA by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11 (***p<0.001, Unpaired, two tailed, t student test). (D–F) Mice were infected with 104 TCID50/mouse SeV Cantell LD alone, in the presence of 5,000 HA Units/mouse purified defective particles (DPs) or in the presence of UV-inactivated DPs (UVDP). Mice received DPs or UVDPs immediately following virus inoculation. (D) Weight loss, (†, mice sacrificed due to severe weight loss; ***p<0.001, ****p<0.0001; Two-way ANOVA with Bonferroni's post hoc test), (E) lung lesion score at day 7 post-infection (n = 3)(*p<0.05, Mann-Whitney test). (F) Photos of the lung of a representative mouse at day 7 post-infection. Arrowhead indicates areas of lesions. (G) Lungs from mice infected with SeV Cantell LD alone, or in the presence of DPs or UVDPs were analyzed by flow cytometry for the expression of the SeV NP protein.
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ppat-1003703-g004: SeV DVGs reduce virulence in vivo.(A–C) Mice were infected with 105 TCID50/mouse of SeV Cantell HD (HD) or SeV Cantell LD (LD). (A) Weight loss (***p<0.001, ****p<0.0001; Two-way ANOVA with Bonferroni's post hoc test), (B) Virus titers in the lung, (n = 11 for day 1, n = 6 for day 3), and (C) expression of Ifnb mRNA by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11 (***p<0.001, Unpaired, two tailed, t student test). (D–F) Mice were infected with 104 TCID50/mouse SeV Cantell LD alone, in the presence of 5,000 HA Units/mouse purified defective particles (DPs) or in the presence of UV-inactivated DPs (UVDP). Mice received DPs or UVDPs immediately following virus inoculation. (D) Weight loss, (†, mice sacrificed due to severe weight loss; ***p<0.001, ****p<0.0001; Two-way ANOVA with Bonferroni's post hoc test), (E) lung lesion score at day 7 post-infection (n = 3)(*p<0.05, Mann-Whitney test). (F) Photos of the lung of a representative mouse at day 7 post-infection. Arrowhead indicates areas of lesions. (G) Lungs from mice infected with SeV Cantell LD alone, or in the presence of DPs or UVDPs were analyzed by flow cytometry for the expression of the SeV NP protein.

Mentions: To evaluate the impact of DVGs during SeV infection in vivo, we infected mice with SeV Cantell HD or LD. Mice infected with SeV Cantell HD showed diminished morbidity than mice infected with the same infectious dose of SeV Cantell LD (Fig. 4A) despite equivalent levels of virus in the lungs at early times post-infection (Fig. 4B), agreeing with reports of reduced virulence in virus stocks with a high content of DVGs [30], [31], [32], [33], [34], [35]. Reduced virulence of SeV Cantell HD was associated with a stronger stimulation of the host antiviral response as shown by the expression of Ifnb mRNA (Fig. 4C). To conclusively demonstrate the role of DVGs in diminishing virulence in vivo, we co-infected mice with SeV Cantell LD and purified viral particles containing DVGs (defective particles; DPs). Confirming their critical role, DVGs reduced the pathogenicity of SeV Cantell LD in mice, while UV-inactivated DP particles did not provide significant protection (Figs. 4D–F). Interestingly, infection in the presence of DPs resulted in reduced expression of SeV NP protein in the lung at day 7 post-infection, suggesting that in this system, DPs reduce virulence by interfering with virus replication.


Defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity.

Tapia K, Kim WK, Sun Y, Mercado-López X, Dunay E, Wise M, Adu M, López CB - PLoS Pathog. (2013)

SeV DVGs reduce virulence in vivo.(A–C) Mice were infected with 105 TCID50/mouse of SeV Cantell HD (HD) or SeV Cantell LD (LD). (A) Weight loss (***p<0.001, ****p<0.0001; Two-way ANOVA with Bonferroni's post hoc test), (B) Virus titers in the lung, (n = 11 for day 1, n = 6 for day 3), and (C) expression of Ifnb mRNA by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11 (***p<0.001, Unpaired, two tailed, t student test). (D–F) Mice were infected with 104 TCID50/mouse SeV Cantell LD alone, in the presence of 5,000 HA Units/mouse purified defective particles (DPs) or in the presence of UV-inactivated DPs (UVDP). Mice received DPs or UVDPs immediately following virus inoculation. (D) Weight loss, (†, mice sacrificed due to severe weight loss; ***p<0.001, ****p<0.0001; Two-way ANOVA with Bonferroni's post hoc test), (E) lung lesion score at day 7 post-infection (n = 3)(*p<0.05, Mann-Whitney test). (F) Photos of the lung of a representative mouse at day 7 post-infection. Arrowhead indicates areas of lesions. (G) Lungs from mice infected with SeV Cantell LD alone, or in the presence of DPs or UVDPs were analyzed by flow cytometry for the expression of the SeV NP protein.
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Related In: Results  -  Collection

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ppat-1003703-g004: SeV DVGs reduce virulence in vivo.(A–C) Mice were infected with 105 TCID50/mouse of SeV Cantell HD (HD) or SeV Cantell LD (LD). (A) Weight loss (***p<0.001, ****p<0.0001; Two-way ANOVA with Bonferroni's post hoc test), (B) Virus titers in the lung, (n = 11 for day 1, n = 6 for day 3), and (C) expression of Ifnb mRNA by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11 (***p<0.001, Unpaired, two tailed, t student test). (D–F) Mice were infected with 104 TCID50/mouse SeV Cantell LD alone, in the presence of 5,000 HA Units/mouse purified defective particles (DPs) or in the presence of UV-inactivated DPs (UVDP). Mice received DPs or UVDPs immediately following virus inoculation. (D) Weight loss, (†, mice sacrificed due to severe weight loss; ***p<0.001, ****p<0.0001; Two-way ANOVA with Bonferroni's post hoc test), (E) lung lesion score at day 7 post-infection (n = 3)(*p<0.05, Mann-Whitney test). (F) Photos of the lung of a representative mouse at day 7 post-infection. Arrowhead indicates areas of lesions. (G) Lungs from mice infected with SeV Cantell LD alone, or in the presence of DPs or UVDPs were analyzed by flow cytometry for the expression of the SeV NP protein.
Mentions: To evaluate the impact of DVGs during SeV infection in vivo, we infected mice with SeV Cantell HD or LD. Mice infected with SeV Cantell HD showed diminished morbidity than mice infected with the same infectious dose of SeV Cantell LD (Fig. 4A) despite equivalent levels of virus in the lungs at early times post-infection (Fig. 4B), agreeing with reports of reduced virulence in virus stocks with a high content of DVGs [30], [31], [32], [33], [34], [35]. Reduced virulence of SeV Cantell HD was associated with a stronger stimulation of the host antiviral response as shown by the expression of Ifnb mRNA (Fig. 4C). To conclusively demonstrate the role of DVGs in diminishing virulence in vivo, we co-infected mice with SeV Cantell LD and purified viral particles containing DVGs (defective particles; DPs). Confirming their critical role, DVGs reduced the pathogenicity of SeV Cantell LD in mice, while UV-inactivated DP particles did not provide significant protection (Figs. 4D–F). Interestingly, infection in the presence of DPs resulted in reduced expression of SeV NP protein in the lung at day 7 post-infection, suggesting that in this system, DPs reduce virulence by interfering with virus replication.

Bottom Line: Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent of IFN signaling.Remarkably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing DVGs, while cells within the lung that contain standard viral genomes alone do not express this cytokine.Together, our data indicate that DVGs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection.

View Article: PubMed Central - PubMed

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

ABSTRACT
The innate immune response to viruses is initiated when specialized cellular sensors recognize viral danger signals. Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent of IFN signaling. We demonstrate that these defective viral genomes (DVGs) are generated naturally during respiratory infections in vivo even in mice lacking the type I IFN receptor, and their appearance coincides with the production of cytokines during infections with Sendai virus (SeV) or influenza virus. Remarkably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing DVGs, while cells within the lung that contain standard viral genomes alone do not express this cytokine. Together, our data indicate that DVGs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection.

Show MeSH
Related in: MedlinePlus