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Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine.

Jimenez-Guardeño JM, Regla-Nava JA, Nieto-Torres JL, DeDiego ML, Castaño-Rodriguez C, Fernandez-Delgado R, Perlman S, Enjuanes L - PLoS Pathog. (2015)

Bottom Line: A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine.To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome.In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.

ABSTRACT
A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.

No MeSH data available.


Related in: MedlinePlus

Virulence and viral growth of SARS-CoV-nsp1* mutants in vivo.BALB/c mice were intranasally infected with 100,000 pfu of wt, and SARS-CoV-nsp1-ΔA, -ΔB, -ΔC and -ΔD viruses (5 mice per group). (A) Animals were monitored daily for weight loss and survival. (B) Viral titers in lungs were determined at 2 and 4 days post infection (3 mice per group and time point). Error bars represent the standard deviations from three independent mice in each case. (C) Lung tissue sections from mice infected with the different recombinant viruses were prepared and stained with hematoxylin and eosin at 2 and 4 dpi. Three independent mice per group were analyzed. Original magnification was 20x and representative images are shown.
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ppat.1005215.g009: Virulence and viral growth of SARS-CoV-nsp1* mutants in vivo.BALB/c mice were intranasally infected with 100,000 pfu of wt, and SARS-CoV-nsp1-ΔA, -ΔB, -ΔC and -ΔD viruses (5 mice per group). (A) Animals were monitored daily for weight loss and survival. (B) Viral titers in lungs were determined at 2 and 4 days post infection (3 mice per group and time point). Error bars represent the standard deviations from three independent mice in each case. (C) Lung tissue sections from mice infected with the different recombinant viruses were prepared and stained with hematoxylin and eosin at 2 and 4 dpi. Three independent mice per group were analyzed. Original magnification was 20x and representative images are shown.

Mentions: To analyze the effects of these deletions in vivo, mice were intranasally infected with mutants rSARS-CoV-nsp1-∆A, -∆B, -∆C and -∆D, and daily monitored for 10 days. SARS-CoV-nsp1-∆C and -∆D infected mice transiently lost a small amount of weight and all mice survived. In contrast, mice infected with SARS-CoV lost weight, and all died by day 5 (Fig 9A). Mice infected with rSARS-CoV-nsp1-∆A or rSARS-CoV-nsp1-∆B lost 20 and 15% of their initial weight by day 3, with survival reduced to 60 and 80%, respectively (Fig 9A). These data indicated that deletion of regions C and D within nsp1 protein led to attenuated mutants, whereas deletion of regions A and B were only partially attenuating. With the exception of rSARS-CoV-nsp1-∆A at day 2 p.i., virus titers were reduced compared to rSARS-CoV-infected mice (Fig 9B). There was not a strict correlation between virus titers and virulence, possibly because nsp1 is involved in the countering IFN production after infection.


Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine.

Jimenez-Guardeño JM, Regla-Nava JA, Nieto-Torres JL, DeDiego ML, Castaño-Rodriguez C, Fernandez-Delgado R, Perlman S, Enjuanes L - PLoS Pathog. (2015)

Virulence and viral growth of SARS-CoV-nsp1* mutants in vivo.BALB/c mice were intranasally infected with 100,000 pfu of wt, and SARS-CoV-nsp1-ΔA, -ΔB, -ΔC and -ΔD viruses (5 mice per group). (A) Animals were monitored daily for weight loss and survival. (B) Viral titers in lungs were determined at 2 and 4 days post infection (3 mice per group and time point). Error bars represent the standard deviations from three independent mice in each case. (C) Lung tissue sections from mice infected with the different recombinant viruses were prepared and stained with hematoxylin and eosin at 2 and 4 dpi. Three independent mice per group were analyzed. Original magnification was 20x and representative images are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4626112&req=5

ppat.1005215.g009: Virulence and viral growth of SARS-CoV-nsp1* mutants in vivo.BALB/c mice were intranasally infected with 100,000 pfu of wt, and SARS-CoV-nsp1-ΔA, -ΔB, -ΔC and -ΔD viruses (5 mice per group). (A) Animals were monitored daily for weight loss and survival. (B) Viral titers in lungs were determined at 2 and 4 days post infection (3 mice per group and time point). Error bars represent the standard deviations from three independent mice in each case. (C) Lung tissue sections from mice infected with the different recombinant viruses were prepared and stained with hematoxylin and eosin at 2 and 4 dpi. Three independent mice per group were analyzed. Original magnification was 20x and representative images are shown.
Mentions: To analyze the effects of these deletions in vivo, mice were intranasally infected with mutants rSARS-CoV-nsp1-∆A, -∆B, -∆C and -∆D, and daily monitored for 10 days. SARS-CoV-nsp1-∆C and -∆D infected mice transiently lost a small amount of weight and all mice survived. In contrast, mice infected with SARS-CoV lost weight, and all died by day 5 (Fig 9A). Mice infected with rSARS-CoV-nsp1-∆A or rSARS-CoV-nsp1-∆B lost 20 and 15% of their initial weight by day 3, with survival reduced to 60 and 80%, respectively (Fig 9A). These data indicated that deletion of regions C and D within nsp1 protein led to attenuated mutants, whereas deletion of regions A and B were only partially attenuating. With the exception of rSARS-CoV-nsp1-∆A at day 2 p.i., virus titers were reduced compared to rSARS-CoV-infected mice (Fig 9B). There was not a strict correlation between virus titers and virulence, possibly because nsp1 is involved in the countering IFN production after infection.

Bottom Line: A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine.To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome.In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.

ABSTRACT
A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.

No MeSH data available.


Related in: MedlinePlus