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Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications.

Spronken MI, Short KR, Herfst S, Bestebroer TM, Vaes VP, van der Hoeven B, Koster AJ, Kremers GJ, Scott DP, Gultyaev AP, Sorell EM, de Graaf M, Bárcena M, Rimmelzwaan GF, Fouchier RA - PLoS ONE (2015)

Bottom Line: Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1).The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo.Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses.

View Article: PubMed Central - PubMed

Affiliation: Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands.

ABSTRACT
Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses.

No MeSH data available.


Related in: MedlinePlus

In vivo assessment of selected reporter viruses.(A) Loss of bodyweight after intranasal inoculation of mice with PBS (grey square), WT (black circle), 2UP_PA_gLUC_sPR (yellow triangle), 2UP_PA_eGFP_sPR (green diamond), 2UP_PA_fRFP_sPR (red cross) and 2UP_PA_iRFP_dPR (purple square). Statistical significance was determined using two-way ANOVA with Bonferroni post-test and is depicted by two (p<0.01) or three (p<0.001) asterisks. Error bars indicate the standard error of the mean. Each group consisted of 6 mice for each time-point. Colours of the asterisk represent the different reporters (green for eGFP and red for fRFP virus). Lung (B) and nose (C) samples from 3 mice were homogenized and used for virus titrations at day 1, 3, 4 and 5. Black squares represent wild-type, orange squares gLUC, green squars eGFP, red squars fRFP and purple squares the iRFP virus. Statistical significance was determined using the Kruskal-Wallis and Dunn’s multiple comparison test and is depicted by one (p<0.05) or two (p<0.01) asterisks. Data are represented by x-y scatter plots showing individual virus titres, the bars represent the median and the dotted line the detection limit. (D) Lungs were imaged using the IVIS Spectrum. Colours represent the intensity of the signal with purple indicating the lowest and red the highest signal. (E) Confocal images of the entire lungs from mice inoculated with PBS, 2UP_PA_eGFP_sPR and 2UP_PA_fRFP_sPR virus. (F) Sections of lungs were stained for NP antigen. The red colour represents NP positive cells and arrows indicate examples of positive cells. (G) Lung homogenates and virus stocks were used to inoculated MDCK cells to determine the in vivo stability. The cells were stained for NP and within the influenza A positive population the percentage of fluorescent cells or luciferase activity (H) from the respective reporter was determined. Data are represented by x-y scatter plots showing individual virus titres, the bars represent the median and the dotted line the detection limit.
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pone.0133888.g004: In vivo assessment of selected reporter viruses.(A) Loss of bodyweight after intranasal inoculation of mice with PBS (grey square), WT (black circle), 2UP_PA_gLUC_sPR (yellow triangle), 2UP_PA_eGFP_sPR (green diamond), 2UP_PA_fRFP_sPR (red cross) and 2UP_PA_iRFP_dPR (purple square). Statistical significance was determined using two-way ANOVA with Bonferroni post-test and is depicted by two (p<0.01) or three (p<0.001) asterisks. Error bars indicate the standard error of the mean. Each group consisted of 6 mice for each time-point. Colours of the asterisk represent the different reporters (green for eGFP and red for fRFP virus). Lung (B) and nose (C) samples from 3 mice were homogenized and used for virus titrations at day 1, 3, 4 and 5. Black squares represent wild-type, orange squares gLUC, green squars eGFP, red squars fRFP and purple squares the iRFP virus. Statistical significance was determined using the Kruskal-Wallis and Dunn’s multiple comparison test and is depicted by one (p<0.05) or two (p<0.01) asterisks. Data are represented by x-y scatter plots showing individual virus titres, the bars represent the median and the dotted line the detection limit. (D) Lungs were imaged using the IVIS Spectrum. Colours represent the intensity of the signal with purple indicating the lowest and red the highest signal. (E) Confocal images of the entire lungs from mice inoculated with PBS, 2UP_PA_eGFP_sPR and 2UP_PA_fRFP_sPR virus. (F) Sections of lungs were stained for NP antigen. The red colour represents NP positive cells and arrows indicate examples of positive cells. (G) Lung homogenates and virus stocks were used to inoculated MDCK cells to determine the in vivo stability. The cells were stained for NP and within the influenza A positive population the percentage of fluorescent cells or luciferase activity (H) from the respective reporter was determined. Data are represented by x-y scatter plots showing individual virus titres, the bars represent the median and the dotted line the detection limit.

Mentions: To determine if the viruses produced in this study could be used in vivo, BALB/c mice were inoculated intranasally with 50 μl of 105 TCID50 of wild-type A/PR/8 virus, 2UP_PA_gLUC_sPR, 2UP_PA_eGFP_sPR, 2UP_PA_fRFP_sPR or 2UP_PA_iRFP_dPR virus. The mock group was inoculated with 50 μl of PBS. Bodyweight was monitored daily and if the loss in bodyweight exceeded 20% the mice were killed humanely. Mice (n = 6 per group) were imaged (n = 3) and virus titrations were performed (n = 3) at day 1, 3, 4 and 5 post-inoculation. All groups showed a reduction in bodyweight from day 2 onwards (Fig 4A). The reduction in bodyweight was statistically significant at day 2, 3 and 4 for the 2UP_PA_eGFP_sPR and 2UP_PA_fRFP_sPR group when compared to the mice inoculated with the wild-type virus. Mice inoculated with the 2UP_PA_gLUC_sPR, 2UP_PA_iRFP_dPR and wild-type virus showed ≥20% loss in bodyweight on day 4 whilst mice inoculated with the 2UP_PA_eGFP_sPR and 2UP_PA_fRFP_sPR virus reached this loss in bodyweight on day 5.


Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications.

Spronken MI, Short KR, Herfst S, Bestebroer TM, Vaes VP, van der Hoeven B, Koster AJ, Kremers GJ, Scott DP, Gultyaev AP, Sorell EM, de Graaf M, Bárcena M, Rimmelzwaan GF, Fouchier RA - PLoS ONE (2015)

In vivo assessment of selected reporter viruses.(A) Loss of bodyweight after intranasal inoculation of mice with PBS (grey square), WT (black circle), 2UP_PA_gLUC_sPR (yellow triangle), 2UP_PA_eGFP_sPR (green diamond), 2UP_PA_fRFP_sPR (red cross) and 2UP_PA_iRFP_dPR (purple square). Statistical significance was determined using two-way ANOVA with Bonferroni post-test and is depicted by two (p<0.01) or three (p<0.001) asterisks. Error bars indicate the standard error of the mean. Each group consisted of 6 mice for each time-point. Colours of the asterisk represent the different reporters (green for eGFP and red for fRFP virus). Lung (B) and nose (C) samples from 3 mice were homogenized and used for virus titrations at day 1, 3, 4 and 5. Black squares represent wild-type, orange squares gLUC, green squars eGFP, red squars fRFP and purple squares the iRFP virus. Statistical significance was determined using the Kruskal-Wallis and Dunn’s multiple comparison test and is depicted by one (p<0.05) or two (p<0.01) asterisks. Data are represented by x-y scatter plots showing individual virus titres, the bars represent the median and the dotted line the detection limit. (D) Lungs were imaged using the IVIS Spectrum. Colours represent the intensity of the signal with purple indicating the lowest and red the highest signal. (E) Confocal images of the entire lungs from mice inoculated with PBS, 2UP_PA_eGFP_sPR and 2UP_PA_fRFP_sPR virus. (F) Sections of lungs were stained for NP antigen. The red colour represents NP positive cells and arrows indicate examples of positive cells. (G) Lung homogenates and virus stocks were used to inoculated MDCK cells to determine the in vivo stability. The cells were stained for NP and within the influenza A positive population the percentage of fluorescent cells or luciferase activity (H) from the respective reporter was determined. Data are represented by x-y scatter plots showing individual virus titres, the bars represent the median and the dotted line the detection limit.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4524686&req=5

pone.0133888.g004: In vivo assessment of selected reporter viruses.(A) Loss of bodyweight after intranasal inoculation of mice with PBS (grey square), WT (black circle), 2UP_PA_gLUC_sPR (yellow triangle), 2UP_PA_eGFP_sPR (green diamond), 2UP_PA_fRFP_sPR (red cross) and 2UP_PA_iRFP_dPR (purple square). Statistical significance was determined using two-way ANOVA with Bonferroni post-test and is depicted by two (p<0.01) or three (p<0.001) asterisks. Error bars indicate the standard error of the mean. Each group consisted of 6 mice for each time-point. Colours of the asterisk represent the different reporters (green for eGFP and red for fRFP virus). Lung (B) and nose (C) samples from 3 mice were homogenized and used for virus titrations at day 1, 3, 4 and 5. Black squares represent wild-type, orange squares gLUC, green squars eGFP, red squars fRFP and purple squares the iRFP virus. Statistical significance was determined using the Kruskal-Wallis and Dunn’s multiple comparison test and is depicted by one (p<0.05) or two (p<0.01) asterisks. Data are represented by x-y scatter plots showing individual virus titres, the bars represent the median and the dotted line the detection limit. (D) Lungs were imaged using the IVIS Spectrum. Colours represent the intensity of the signal with purple indicating the lowest and red the highest signal. (E) Confocal images of the entire lungs from mice inoculated with PBS, 2UP_PA_eGFP_sPR and 2UP_PA_fRFP_sPR virus. (F) Sections of lungs were stained for NP antigen. The red colour represents NP positive cells and arrows indicate examples of positive cells. (G) Lung homogenates and virus stocks were used to inoculated MDCK cells to determine the in vivo stability. The cells were stained for NP and within the influenza A positive population the percentage of fluorescent cells or luciferase activity (H) from the respective reporter was determined. Data are represented by x-y scatter plots showing individual virus titres, the bars represent the median and the dotted line the detection limit.
Mentions: To determine if the viruses produced in this study could be used in vivo, BALB/c mice were inoculated intranasally with 50 μl of 105 TCID50 of wild-type A/PR/8 virus, 2UP_PA_gLUC_sPR, 2UP_PA_eGFP_sPR, 2UP_PA_fRFP_sPR or 2UP_PA_iRFP_dPR virus. The mock group was inoculated with 50 μl of PBS. Bodyweight was monitored daily and if the loss in bodyweight exceeded 20% the mice were killed humanely. Mice (n = 6 per group) were imaged (n = 3) and virus titrations were performed (n = 3) at day 1, 3, 4 and 5 post-inoculation. All groups showed a reduction in bodyweight from day 2 onwards (Fig 4A). The reduction in bodyweight was statistically significant at day 2, 3 and 4 for the 2UP_PA_eGFP_sPR and 2UP_PA_fRFP_sPR group when compared to the mice inoculated with the wild-type virus. Mice inoculated with the 2UP_PA_gLUC_sPR, 2UP_PA_iRFP_dPR and wild-type virus showed ≥20% loss in bodyweight on day 4 whilst mice inoculated with the 2UP_PA_eGFP_sPR and 2UP_PA_fRFP_sPR virus reached this loss in bodyweight on day 5.

Bottom Line: Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1).The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo.Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses.

View Article: PubMed Central - PubMed

Affiliation: Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands.

ABSTRACT
Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses.

No MeSH data available.


Related in: MedlinePlus