Multi-spectral fluorescent reporter influenza viruses (Color-flu) as powerful tools for in vivo studies

Abstract

Seasonal influenza A viruses cause annual epidemics of respiratory disease; highly pathogenic avian H5N1 and the recently emerged H7N9 viruses cause severe infections in humans, often with fatal outcomes. Although numerous studies have addressed the pathogenicity of influenza viruses, influenza pathogenesis remains incompletely understood. Here we generate influenza viruses expressing fluorescent proteins of different colours ('Color-flu' viruses) to facilitate the study of viral infection in in vivo models. On adaptation to mice, stable expression of the fluorescent proteins in infected animals allows their detection by different types of microscopy and by flow cytometry. We use this system to analyse the progression of viral spread in mouse lungs, for live imaging of virus-infected cells, and for differential gene expression studies in virus antigen-positive and virus antigen-negative live cells in the lungs of Color-flu-infected mice. Collectively, Color-flu viruses are powerful tools to analyse virus infections at the cellular level in vivo to better understand influenza pathogenesis.

ARTICLE

Received 23 Aug 2014 | Accepted 10 Feb 2015 | Published 25 Mar 2015

Satoshi Fukuyama1,2,*, Hiroaki Katsura2,*, Dongming Zhao1,2,*, Makoto Ozawa3,4,*, Tomomi Ando1,2, Jason E. Shoemaker1,2, Izumi Ishikawa1,2, Shinya Yamada2, Gabriele Neumann5, Shinji Watanabe1,6, Hiroaki Kitano1,7,8,9 & Yoshihiro Kawaoka1,2,5,10

Seasonal inuenza A viruses cause annual epidemics of respiratory disease; highly pathogenic avian H5N1 and the recently emerged H7N9 viruses cause severe infections in humans, often with fatal outcomes. Although numerous studies have addressed the pathogenicity of inuenza viruses, inuenza pathogenesis remains incompletely understood. Here we generate inuenza viruses expressing uorescent proteins of different colours (Color-u viruses) to facilitate the study of viral infection in in vivo models. On adaptation to mice, stable expression of the uorescent proteins in infected animals allows their detection by different types of microscopy and by ow cytometry. We use this system to analyse the progression of viral spread in mouse lungs, for live imaging of virus-infected cells, and for differential gene expression studies in virus antigen-positive and virus antigen-negative live cells in the lungs of Color-u-infected mice. Collectively, Color-u viruses are powerful tools to analyse virus infections at the cellular level in vivo to better understand inuenza pathogenesis.

1 Exploratory Research for Advanced Technology Infection-Induced Host Responses Project, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan. 2 Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan. 3 Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima 890-0065, Japan. 4 Transboundary Animal Distance Center, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima 890-0065, Japan. 5 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53711, USA. 6 Laboratory of Veterinary Microbiology, Department of Veterinary Sciences, University of Miyazaki, Miyazaki 889-2192, Japan. 7 The Systems Biology Institute, Minato-ku, Tokyo 108-0071, Japan. 8 Sony Computer Science Laboratories, Shinagawa-ku, Tokyo 141-0022, Japan. 9 Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa 904-0495, Japan. 10 Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.K. (email: mailto:[email protected]

Web End [email protected] ).

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DOI: 10.1038/ncomms7600 OPEN

Multi-spectral uorescent reporter inuenza viruses (Color-u) as powerful tools for in vivo studies

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7600

Inuenza A virus is a respiratory pathogen that causes annual epidemics and sporadic pandemics1. Moreover, highly pathogenic avian H5N1 and the recently emerged H7N9

inuenza viruses have caused an appreciable number of human infections with high mortality rates2,3. Inuenza viruses infect respiratory epithelial cells and alveolar macrophages in mammalian hosts4. The host immune system recognizes the RNA genome of inuenza viruses via cytosolic sensors5,6, which trigger innate immune responses that lead to the production of type I interferons (IFNs) and proinammatory cytokines and chemokines7. Type I IFNs upregulate the production of antiviral proteins including myxovirus resistance (Mx), oligoadenylate synthetase (OAS) and interferon-stimulated gene 15 (ISG15)8. Dysregulation of the innate immune responses to inuenza virus infection causes lung pathology mediated by inltrating immune cells, including macrophages and neutrophils9,10. Although several studies have addressed host responses to inuenza virus infections11, the mechanisms of inuenza virus-induced pathology are still not fully understood.

To analyse the immune responses to inuenza virus infection in vivo, viruses have been generated that expressed a uorescent reporter protein12,13. However, these viruses were signicantly attenuated12,13 and may not accurately reect natural infections. Manicassamy et al.14 generated a GFP-expressing inuenza virus, which they used to assess the route of antigen presentation on inuenza virus infection15. However, the GFP gene was not stably maintained during replication in mouse lung or cultured cells, even though they isolated the GFP-expressing virus by repeated plaque purications14.

Here we generated appreciably improved versions of uorescent inuenza viruses each of which stably expresses one of four different uorescent proteins that can be monitored simultaneously. Plaque purication is not required for our strains to maintain the uorescent expression. Using these viruses, we performed several studies to demonstrate the versatility of this novel tool set.

ResultsGeneration of Color-u viruses. To generate a uorescent inuenza virus expressing a reporter protein fused to the NS1 open reading frame, we chose Venus, a GFP variant with eight mutations including F46L, which improves chromophore formation and increases brightness compared with GFP16. As expected based on previous ndings of attenuation for inuenza viruses expressing reporter proteins12,13, the mouse pathogenicity of A/Puerto Rico/8/34 (PR8; H1N1) virus expressing Venus (WT-Venus-PR8) was substantially lower than that of wild-type PR8 (WT-PR8); the dose required to kill 50% of infected mice (MLD50) was more than 104.3 plaque-forming units (PFU) for

WT-Venus-PR8 compared with 102.5 PFU for WT-PR8 (Fig. 1). We therefore serially passaged WT-Venus-PR8 in C57BL/6 (B6) mice. After six consecutive passages, we identied a variant (designated mouse-adapted (MA)-Venus-PR8; possessing a T-to-A mutation at position 380 of the hemagglutinin protein, and an E-to-D mutation at position 712 of the polymerase subunit PB2) with appreciably higher pathogenicity (MLD50 103.3 PFU)

compared with WT-Venus-PR8, although it was still slightly less pathogenic than the original PR8 virus (Fig. 1). To assess the replicative ability of MA-Venus-PR8 in mouse lungs, we intranasally infected B6 mice with 104 PFU of MA-Venus-PR8 or WT-PR8. At all time points tested, the lung virus titres were similar for MA-Venus-PR8- and WT-PR8-infected mice (Table 1). To test the stability of Venus expression, we performed plaque assays using lung homogenate from infected mice and found that only one of 150 plaques on each of days 3, 5 and 7 post-infection (p.i.) was Venus-negative, attesting to the

high genetic stability of Venus expression in this recombinant virus. In contrast, only 70% of NS1-GFP virus expressed the reporter protein14. The robust virulence and genetic stability of MA-Venus-PR8 indicate that this virus represents a highly attractive reporter system to visualize inuenza virus-infected cells in vivo.

To increase the versatility of uorescent inuenza viruses as imaging tools, we next generated additional MA-PR8 variants expressing different spectral GFP mutants, namely, eCFP (ex. 434 nm, em. 477 nm) and eGFP (ex. 489 nm, em. 508 nm)17. We also generated an mCherry variant (ex. 587 nm, em. 610 nm), which emits uorescence at a longer wavelength than Venus (ex. 515 nm, em. 528 nm)16,18. These inuenza viruses encoding the multi-spectral uorescent reporter proteins were collectively named Color-u. To determine the pathogenicity of Color-u viruses, we compared the virus titres in mouse lung tissues and the MLD50 values of MA-eCFP, eGFP and mCherry-PR8 with those of MA-Venus-PR8 and MA-PR8. All of virus strains showed comparatively high replication in the lungs and the MLD50 values were similar among the Color-u viruses (Supplementary Fig. 1 and Table 1). We also tested the stability of the uorescent expression of the Color-u viruses in vivo and in vitro by plaque assay. When we collected virus from the lungs of mice on day 7 p.i., the percentages of uorescent-positive plaques were 98.0% (MA-eCFP-PR8), 100.0% (MA-eGFP-PR8) and 96.4% (MA-mCherry-PR8). We also measured the percentages of uorescent-positive plaque in the sample from the culture medium of MDCK cells after 72 h p.i. and found them to be 100.0% (MA-eCFP-PR8), 99.2% (MA-eGFP-PR8) and98.2% (MA-mCherry-PR8). In addition, we examined the stability of an NS1-uorescent protein chimera in virus-infected cells by infecting MDCK cells with MA-Venus-PR8 virus and detecting NS1-Venus chimeric protein by using anti-GFP and anti-NS1 antibodies (Supplementary Fig. 2). We found that the NS1-Venus chimeric protein was not degraded until the time point we examined (that is, 12 h p.i.), indicating that the uorescent signal is mainly emitted from the NS1-uorescent protein chimera and not from degradation products in cells infected with Color-u viruses. These ndings indicate that the pathogenicity and stability of the Color-u viruses were not affected by the different uorescent reporter genes. To assess the expression of Color-u viruses in mouse lungs, we collected lungs from B6 mice infected with each of the Color-u viruses and processed them for visualization as described in the Methods section. All four colours were clearly visible in whole transparent lung tissue when analysed with a uorescent stereomicroscope (Fig. 2a). Fluorescent signals were mainly seen in the bronchial epithelial layer at day 3 p.i. At day 5 p.i., uorescent signals extended to the peripheral alveolar regions. These data indicate that all four Color-u viruses are useful for analysing the distribution of inuenza virus-infected cells in mouse lungs.

Next, we employed the Nuance spectral imaging system to test whether the uorescent signals of all four Color-u viruses could be detected simultaneously. Lung tissues were collected from B6 mice intranasally inoculated with a mixture of the four strains(2.5 104 PFU each in a total volume of 50 ml). Analysis of lung

sections obtained at days 2 and 5 p.i. showed that the uorescent signals of all four Color-u viruses were distinguishable from each other (Fig. 2b). At day 2 p.i., we found clusters of the same uorescent colour in bronchial epithelial cells, suggesting local spread of the individual viruses. At this time point, a limited number of alveolar cells were infected. At day 5 p.i., we detected a cluster of alveolar cells expressing a single uorescent protein, indicative of the initiation of infection with a single virus and its local spread (Fig. 2b). Interestingly, we also detected epithelial cells simultaneously expressing two or three uorescent proteins,

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Weight change Survival

106 PFU 105 PFU 103 PFU

104 PFU 102 PFU 101 PFU

60 0 2 4 6 8 10 12 14

WT-PR8

WT-Venus-PR8

MA-Venus-PR8

125

100

75

50

125

100

75

50

125

100

75

50

Weight change

120

100

80

MLD50:

25 102.5 PFU

MLD50:

25 >104.3 PFU

120

100

80

Weight change Weight change

120

100

80

Percent survival Percent survival Percent survival

60 0 2 4 6 8 10 12 14

60 0 2 4 6 8 10 12 14

0 0 2 4 6 8 10 12 14

0 0 2 4 6 8 10 12 14

MLD50:

25 103.3 PFU

Days post-infection Days post-infection

0 0 2 4 6 8 10 12 14

Figure 1 | Characteristics of mouse-adapted Venus-PR8 in mice. Four B6 mice per group were intranasally inoculated with WT-PR8, WT-Venus-PR8 or MA-Venus-PR8. Body weight and survival of mice were monitored for 14 days.

Table 1 | Replication and virulence of Color-u in mice*.

Virus Mean virus titer (log10 PFU/gs.d.) in the mouse lung on the indicated day p.i.

MLD50

(PFU)

Day 3 p.i. Day 5 p.i. Day 7 p.i.

MA-eCFP-PR8 8.10.2w 8.00.1 6.30.1 103.0

MA-eGFP-PR8 8.60.1 8.30.1 6.30.1 103.5 MA-Venus-PR8 8.60.2 8.40.1 6.50.3 103.3 MA-mCherry-PR8 7.70.3w 7.50.7 6.10.4 102.7 WT-Venus-PR8 5.60.3w 5.30.3w 5.20.2w 4104.3

WT-PR8 8.80.1 8.20.5 6.90.2 102.5 MA-PR8 8.90.1 9.00.0 7.90.2w 102.3

*B6 mice were inoculated intranasally with 104 PFU of each virus in a 50 ml volume. Three mice from each group were killed on days 3, 5 and 7 p.i., and virus titres in the lungs were determined in MDCK cells.

wStatistical signicance was calculated by using the Students t-test; the P value was o0.01 compared with the titres in the lungs of mice infected with WT-PR8 virus.

albeit at a low frequency, suggesting co-infection of these cells (Fig. 2c). To quantitatively analyse the co-infection in the bronchial epithelium, we utilized the inForm multispectral imaging software for automated user-trained tissue and cell segmentation, together with Nuance. We found that B20% of epithelial cells were infected with multiple strains of Color-u viruses in bronchus isolated from mice on day 2 p.i. (Supplementary Fig. 3). The ability to visualize cells co-infected with different inuenza viruses in vivo is a major advance in technology and will allow us to provide novel insights into inuenza co-infection and reassortment processes.

The innate host response to Color-u viruses. We next tested the utility of Color-u viruses for the analysis of host responses to infection. As macrophages are involved in innate immunity and acute inammation in inuenza virus-infected lungs, we examined lung sections stained with an antibody to macrophages (PE-Mac3) by using confocal microscopy. Macrophages inltrated regions containing Venus-positive bronchial epithelial cells at day 2 p.i. of mice with MA-Venus-PR8 (Fig. 3a); by contrast, only a few Mac3-positive cells were detected in the alveoli of lungs from mock-infected animals. On the basis of this nding, we next employed live imaging to further study the interaction between inuenza virus-infected epithelial cells and macrophages in mouse lungs. In the lung tissue of naive B6 mice, CD11b alveolar macrophages were detected by use of a

two-photon laser microscope. Most of these macrophages did not migrate (that is, showed little movement) during the observation period (49 min; Supplementary Movie 1). In mice infected with MA-eGFP-PR8 virus, many CD11b macro

phages appeared to be attached to eGFP-positive epithelial cells (Supplementary Movie 2); moreover, some of these eGFP-positive epithelial cells exhibited blebbing similar to apoptotic cells (Fig. 3b). Interestingly, a number of CD11b macro

phages quickly moved around the eGFP-positive epithelial cells, suggesting possible macrophage responses to inammatory signals such as IFNs or chemokines. We also analysed the kinetics of the lung macrophages by tracking individual cells (Fig. 3b) and found that there were no obvious differences between the kinetics of macrophages in the naive lung and those in the infected lung. Our system can thus be used to

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Day 3 p.i. Day 5 p.i.

AF eCFP eGFP

Venus mCherry

mCherry -PR8

Venus

-PR8

eGFP

-PR8

eCFP

-PR8

Merge Fluorescence Merge Fluorescence

Day 2 p.i. Day 5 p.i.

merge

Figure 2 | Distribution of Color-u viruses in lungs. (a) Lung tissues were harvested from B6 mice at days 3 and 5 p.i. with Color-u viruses (105 PFU of MA-eCFP, eGFP, Venus and mCherry-PR8). Whole-mount images of transparent lung tissues were obtained by using a uorescent stereomicroscope. Scale bar, 5 mm. (b,c) B6 mice were intranasally inoculated with a mixture of MA-eCFP, eGFP, Venus and mCherry-PR8(2.5 104 PFU per strain). (b) The sections of lungs at days 2 and 5 p.i.

were analysed by using an inverted uorescence microscope with a Nuance FX multispectral imaging system with inForm software. Scale bar, 100 mm.

(c) Enlarged images of the indicated area in (b) were unmixed and separated into autouorescence (AF), eCFP, eGFP, Venus and mCherry uorescence. Arrows in the merged image indicate cells infected with different colour variants of Color-u viruses.

monitor the in vivo interactions between virus-infected and immune cells.

A number of studies have assessed the transcriptomics and proteomics proles of inuenza virus-infected mice19,20. As these studies used whole lung samples, the results are the sum of virus-infected and uninfected cells, leading to the dilution of host responses and not allowing one to distinguish the proles of infected cells from those of uninfected, bystander cells. As a rst step to overcome this shortcoming, we sorted macrophages (known to be infected by inuenza viruses (Fig. 3c)) from the lungs of mice infected with MA-Venus-PR8 on the basis of their uorescent protein expression and performed microarray analysis. Macrophages isolated from the lungs of mice inoculated with PBS (naive macrophages) served as controls. In uorescent-positive macrophages, 6,199 transcripts were differently expressed relative to naive macrophages. By contrast, in uorescent-negative macrophages obtained from infected mice, only 4,252 transcripts were differentially expressed relative to the

naive macrophages. This difference likely reects differences in gene transcription induced by active inuenza virus infection. However, it should be noted that the uorescent-negative cell populations obtained from infected animals may have included infected cells in which the uorescent signal had not yet been detected as would be expected at an early stage of virus infection. In fact, confocal microscopy revealed that it took 9 h to detect uorescent protein expression in the majority of MDCK cells (Supplementary Fig. 4). Hierarchical clustering of differentially expressed transcripts, followed by functional enrichment analysis of each cluster, indicated that both uorescent-positive and uorescent-negative macrophages obtained from infected animals exhibit activation of pathways associated with the immune response, cytokine production and inammation (Fig. 3d, green cluster). The upregulation of these pathways in the uorescent-negative cells may have resulted from cell activation by IFN and cytokines released from infected cells, and/or from cells that were at an early stage of virus infection (as discussed earlier). Yet, a subset of enriched annotations, for example, type I IFN-mediated signalling (Fig. 3d, light blue cluster), included transcripts that were more highly expressed in uorescent-positive macrophages. In addition, we observed that type I IFN genes were among the most upregulated transcripts in the uorescent-positive macrophages (Fig. 3e). Taken together, this enhanced type I IFN activity is consistent with the suggestion that the uorescent-positive cells had been infected, whereas the uorescent-negative cells included both uninfected (but potentially stimulated) cells and cells at early stages of inuenza virus infection. Indeed, it took at least 5 h to detect uorescent protein expression after infection with Color-u viruses, although all of the uorescent proteins (that is, eCFP, eGFP, Venus, and mCherry) were detectable in the majority of cells by 9 h p.i. (Supplementary Fig. 4). These ndings open new avenues in infectious disease research to compare gene expression (or other types of expression) patterns of reporter protein-positive cells with those of reporter protein-negative cells (but potentially stimulated by released cytokines and/or are at an early stage of infection).

Avian inuenza A (H5N1) virus expressing Venus protein. Finally, we tested whether the concept of mouse-adapted uorescent inuenza viruses could be applied to other inuenza virus strains, such as highly pathogenic avian inuenza A (H5N1) (HPAI) viruses, which are a research priority due to the threat they pose to humans. We generated an MA-Venus-HPAI virus based on A/Vietnam/1203/2004 (VN1203; H5N1), employing the same strategy used to create MA-Venus-PR8; we used the PR8 NS gene to express NS1-Venus chimeric protein because the NS gene did not contribute to the pathogenicity of VN1203 strain in mice21. The pathogenicity of MA-Venus-HPAI virus for B6 mice was comparable to that of VN1203, with MLD50 values for both viruses being less than 5 PFU (Fig. 4a and ref. 22). MA-Venus-HPAI virus also shared with other HPAI viruses the ability to spread systemically and replicate in various organs including the spleen, kidney and brain (Fig. 4b and ref. 22). Moreover, taking advantage of the strong uorescent signal emitted by MA-Venus-HPAI virus- and MA-Venus-PR8-infected cells, we successfully constructed a three-dimensional image of an HPAI virus and PR8-infected bronchus as well as alveolar areas inside the lung tissues by using two-photon laser microscopy (Fig. 4c, Supplementary Movies 36)). This type of three-dimensional imaging analysis improves our understanding of the spatial distribution of inuenza virus-infected bronchi. When we compared the distribution of virus-infected cells between HPAI virus- and PR8-infected lungs, we found that HPAI virus spreads from the bronchial epithelium to alveolar sites more quickly than did PR8 (Fig. 4c,d). We also found, by using ow cytometric

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Mock (PBS)

Day 2 p.i.

Mock (PBS) Day 3 p.i.

CD11b (F4/80-positive fraction)

0.07% 7.48%

Venus

40 m 40 m

Time 0:15:02.527

Time 0:24:17.467

1

2

3 4

Immune response (10) Inflammatory response (10)

Defense response (10) Response to virus (10) Type 1 Interferon-mediated signalling (10)

Response to Type 1 interferon (10)

CD Molecules (3.6) Response to wounding (10)

Extracellular matrix organization (10)

Cell adhesion (10) Cell-cell junction (3) Immune response (10) Inflammatory response (10)

Defense response (10) Response to virus (10) Cytokine production (10) Leukocyte activation (10)

DAG/PE domain (10) Lipid metabolic process (3.1)

Cell migration (10)

4 2 0 2

Venus(+) versus naive

4

Gene symbol

Venus(+) versus naive

Venus() versus naive

Ifnb1 Ifna5 Ifna12

Ifna2 Ifnab Ifna4 Ifna13

Ifna1 Ifna14

Ifna7 Ifna11 Ifna12

Ifna2

Log2 fold change

4 0 +4

NS

NS NS NS NS

Venus() versus naive

Figure 3 | Analysis of lung macrophages. (a) Analysis of macrophage inltration. Fixed lung sections of PBS-inoculated mice (mock) or mice infected with 105 PFU of MA-Venus-PR8 at day 2 p.i. were incubated with an PE-anti-Mac3 antibody (red) and Hoechst dye (blue). The Venus uorescent signal is shown in green. Scale bar, 200 mm. (b) Kinetics of the interactions between virus-infected cells and lung macrophages. Images of eGFP-positive cells (green) and CD11b macrophages (red) in lung tissue from naive B6 mice (upper left panel) and B6 mice on day 3 p.i. with 105 PFU of MA-eGFP-PR8

(upper right panel) were obtained by using a two-photon microscope. The tracks of individual macrophages are indicated as coloured lines. Sequential images in the lower panel (14) show an enlarged view of the box in the upper right panel. Arrowheads indicate the blebbing of eGFP-positive cells. Scale bar, 40 mm. (c) Infection of macrophages by inuenza viruses. Single-cell suspensions were obtained from lungs of PBS-inoculated (mock) mice or mice infected with 105 PFU of MA-Venus-PR8 at day 3 p.i., The FACS analysis shows Venus expression from cells gated on F4/80 and CD45 expression levels.

(d,e) Gene expression analysis. Total RNA was isolated from sorted macrophages of PBS-inoculated (naive) mice, and from sorted Venus-positive (Venus( )) and Venus-negative (Venus( )) macrophages of mice inoculated with 105 PFU of MA-Venus-PR8 at day 3 p.i. (9 mice per treatment), and

microarray analysis was performed. (d) Differentially expressed (DE) transcripts were identied by comparing gene expression levels in naive macrophages with those in Venus( ) and Venus( ) macrophages from infected mice. A heat map of the clustered transcripts for each condition is displayed, with

enriched annotations and the enrichment scores for each cluster. A shift of the blue line in the heat map to the left or right indicates that the DE transcript is more highly expressed in Venus( ) or Venus( ) macrophages, respectively. (e) Heat map comparing expression levels of type I interferons (IFNs)

between Venus( ) and Venus( ) macrophages. NS denotes not statistically signicant between Venus( ) cells from infected animals and naive

macrophages from uninfected animals.

analysis, that CD45-negative, non-hematopoietic cells and F4/80-positive macrophages more frequently expressed Venus in the lungs of mice infected with MA-Venus-HPAI virus than in the lungs of animals inoculated with MA-Venus-PR8 (Fig. 4e,f), supporting ndings that H5N1 HPAI viruses induce more severe inammatory responses in the lung than does PR8. Taken together, these ndings demonstrate the utility

of Color-u viruses for comparative studies of inuenza

pathogenesis.

DiscussionIn this study, we established Color-u viruses to study inuenza virus infections at the cellular level. Color-u viruses combine several improvements over existing systems, including robust

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100 PFU 101 PFU 104 PFU

103 PFU

102 PFU

Day 1 p.i. Day 2 p.i.

Percent survival

125 100

75 50 25

0 0 2 4 6 8 10 12 14Days post-infection

1 )

Viral titer

(log 10PFU g

10

8 6 4 2 0

MLD50:

3.2 PFU

MA-Venus

-PR8

MA-Venus

-HPAI

Lung Spleen Kidney Brain

Alveolar

Bronchus MA-Venus-PR8

MA-Venus-HPAI

MA-Venus-PR8 MA-Venus-HPAI

P=0.04

*

6107

4107

2107

0

2.0107

1.5107

1.0107

5.0106

0

3 )

3 )

Volume (m

Volume (m

P=0.008

**

1 2 1 2

Venus-positive CD45neg cell

0 1 2 3 4

Days post-infection Days post-infection

MA-Venus-HPAI MA-Venus-PR8

30

20

10

CD45neg cell Macrophage

Venus+

(% Of CD45neg cells)

MA-Venus-HPAI

MA-Venus-PR8

PBS

CD45

17.6%

**

*

31.6%

10.3%

2.2%

Days post-infection

Days post-infection

9.6%

Venus-positive macrophage

F4/80

40 MA-Venus-HPAI

MA-Venus-PR8

30

** *

0

Venus+

(% Of macrophage)

20

10

Venus

0 1 2 3 4

Figure 4 | Characterization of MA-Venus-HPAI virus. (a) Four B6 mice per group were intranasally inoculated with MA-Venus-HPAI virus. Mouse body weight and survival were monitored for 14 days. (b) Lungs, spleens, kidneys and brains were harvested from B6 mice at day 3 p.i. with 105 PFU of MA-Venus-HPAI virus. Virus titres of tissue homogenates were determined by use of plaque assays in MDCK cells. Each data point represents the means.d. (n 3). (c,d) Lung tissues were harvested from B6 mice at day 1 and 2 p.i. with 105 PFU of MA-Venus-HPAI and PR8. Images of transparent

lung tissues with Venus-positive cells in the bronchus (red) and alveolar area (green) were obtained by using a two-photon microscope. Each data point represents the means.d. (n 3). Statistical signicance was calculated by using the Students t-test. (d) The distribution of Venus-positive cells was

evaluated via volume analysis of the Venus-positive bronchus and alveolar area by using 3D images of the transparent lung tissues. (e,f) Cells were collected from lungs of B6 mice at days 1, 2, 3 and 4 p.i. with 105 PFU of MA-Venus-PR8 or MA-Venus-HPAI virus, and stained for CD45, CD11b and F4/80. Venus expression in CD45-negative cells, and the Venus versus F4/80 staining prole gated on CD45-positive cells were analysed by ow cytometry. (e) A representative dot plot from day 2 p.i. is shown with the percentage of Venus-positive cells.

viral replication, virulence, stable uorescent protein expression and a set of four different colours that can be visualized simultaneously. We also demonstrated that Color-u viruses are applicable to a different inuenza virus strain. These improvements allowed global transcriptomics analyses of infected and bystander cells and, for the rst time, live-imaging of inuenza virus-infected cells in the mouse lung.

Previous versions of uorescent inuenza viruses12,13 including our original construct (that is, WT-Venus-PR8) were appreciably attenuated in mice. These attenuated uorescent viruses may still be useful for identifying initial target cells. However, the immune responses elicited by these highly attenuated, non-lethal viruses most likely differ considerably from those of the mouse-lethal parent virus, making their use for pathogenesis studies problematic. Here, we solved this problem by passaging viruses

in mice. This strategy proved to be successful for two different inuenza virus strains, suggesting its broad applicability. A second drawback of previously tested uorescent inuenza viruses is the genetic instability of the added reporter protein14. We, however, found that 495% of virus plaques examined from mouse lung samples on day 7 p.i. expressed the reporter protein. We are currently studying the mechanism by which our mouse-adapted viruses stably express uorescent proteins.

At present, Color-u viruses cannot be monitored in live animals non-invasively because uorescent reporter proteins must be within a biological optical window (650900 nm) to be detected for imaging of tissues in live animals using uorescent probes23,24, and none of the uorescent reporter proteins including mCherry, which has the longest emission among the reporter proteins of Color-u, is inside this biological optical

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window. Several groups generated a luciferase reporter-expressing inuenza virus that can be used to monitor virus replication in live animals2527; however, this system needs systemic inoculation of substrate into the animals at every observation point. In addition, the resolution of their imaging system (based on the IVIS system) is not adequate for the analysis of cellular immune mechanisms in vivo, which we are able to achieve with our system.

Novel technologies for imaging analysis28 have enabled us to develop a set of four different inuenza colour variants that can be distinguished from one another by using Nuance, hence allowing their simultaneous detection. In fact, our pilot study identied lung epithelial cells expressing two or three different uorescent proteins (Fig. 2c, Supplementary Fig. 3). To our knowledge, this is the rst visualization of mouse lung cells infected with more than one inuenza virus strain. In future studies, these colour variants could be used to address longstanding questions in inuenza virus research, such as the frequency of viral co-infections in vivo, which may be critical to better understand inuenza virus reassortment and, hence, the generation of novel inuenza viruses such as the pandemic viruses of 1957 (refs 29,30), 1968 (refs 29,30) and 2009 (refs 31,32).

By employing our novel tool sets, we were able to detect inuenza virus-infected cells in whole-lung tissues of mice, allowing us to observe the location and distribution of inuenza viruses in the lung. Moreover, we were able to observe interactions of virus-infected epithelial cells with immune cells. Such studies will allow us to directly monitor inuenza disease progression from acute bronchitis to severe viral pneumonia, which causes considerable morbidity and mortality in highly pathogenic inuenza virus infections33,34.

In conclusion, Color-u viruses in combination with advanced imaging technologies are a powerful and versatile tool to elucidate the mechanisms of inuenza virus pathogenicity at the cellular level in animals.

Methods

Generation of Color-u. The NS segments of PR8 fused with different uorescent reporter genes including eCFP, eGFP, Venus and mCherry were constructed by overlapping fusion PCR as described previously14. In brief, the open reading frame (ORF) of the NS1 gene without the stop codon was fused with the N terminus of uorescent reporter genes via a sequence encoding the amino-acid linker GSGG. The uorescent reporter ORFs were followed by a sequence encoding the GSG linker, a foot-and-mouth virus protease 2A autoproteolytic site with 57 nucleotides from porcine teschovirus-1 (ref. 14), and by the ORF of nuclear export protein (Supplementary Fig. 5). In addition, silent mutations were introduced into the endogenous splice acceptor site of the NS1 gene to abrogate splicing35. The constructed NS segments (designated eCFP-NS, eGFP-NS, Venus-NS and mCherry-NS) were subsequently cloned into a pPolI vector for reverse genetics as described previously36. The plasmid encoding the Venus reporter protein was a kind gift from Dr A. Miyawaki (Laboratory for Cell Function Dynamics, RIKEN Brain Science Institute, Wako, Japan)16. WT-Venus-PR8 was generated by using the reverse genetics system as described previously36. As WT-Venus-PR8 pathogenicity and Venus expression levels were appreciably attenuated in mice, we serially passaged WT-Venus-PR8 in mice. After six passages, we obtained a variant (MA-Venus-PR8) with increased pathogenicity and strong Venus expression.

A stock of MA-Venus-PR8 was generated in MDCK cells. As serial passage in animals typically results in virus populations composed of genetic variants, we recreated MA-Venus-PR8 by using reverse genetics. Likewise, MA-eCFP-PR8, -eGFP-PR8 and -mCherry-PR8 were generated with the same genetic backbone as MA-Venus-PR8. To generate a Venus-HPAI virus by reverse genetics, the NS segment of A/Vietnam/1203/2004 (H5N1; VN1203) was replaced with Venus-NS of PR8, and the virus was adapted to mice as described for MA-Venus-PR8.

A stock of MA-Venus-HPAI virus was made in MDCK cells. The set of these inuenza viruses carrying various uorescent proteins was collectively termed Color-u.

Mouse experiments. Female, 6-week-old C57BL/6 (B6) mice, which were purchased from Japan SLC, Inc. (Shizuoka, Japan), were intranasally inoculated with Color-u viruses, at the dosages indicated in the gure panels, in 50 ml of PBS under sevourane anaesthesia, and body weights and survival were monitored for 14 days. Lungs were harvested from PBS-inoculated or Color-u virus-infected

mice for virus titration, ow cytometric analysis and histological experiments at the times indicated in the gure panels. All animal experiments were performed in accordance with the regulations of the University of Tokyo Committee for Animal Care and Use and were approved by the Animal Experiment Committee of the Institute of Medical Science of the University of Tokyo.

Histology and cytology. Lungs were xed in 4% paraformaldehyde (PFA) phosphate buffer solution. Fixed tissues were embedded in OCT compound (Sakura Finetek, Tokyo, Japan), frozen by liquid N2 and stored at 80 C. Cryostat

6-mm sections were treated for 30 min with PBS containing 1% BSA (PBS-BSA) to block nonspecic binding, and then incubated with phycoerythrin (PE)-Mac3 (M3/84, BD Biosciences, San Jose, CA). To examine the cytology of the MDCK cells, we infected them with Color-u virus and then xed them in 4% PFA phosphate buffer solution. Nuclei were stained with Hoechst33342 (Invitrogen, Carlsbad, CA). Sections and cells were visualized by using a confocal microscope (Nikon A1Rsi, Nikon, Tokyo, Japan), controlled by NIS-Elements software. For quantitative multi-colour imaging analysis, the slides were visualized by use ofan inverted uorescence microscope (Nikon Eclipse TS100) with a Nuance FX multispectral imaging system with inForm software (PerkinElmer, Waltham, MA).

Whole-mount imaging of lung tissue. Mice were killed and intracardially per-fused with PBS to remove blood cells from the lung. The lungs were isolated after intratracheal perfusion with 4% PFA phosphate buffer solution. The lung tissues were cleared with SCALEVIEW-A2 solution (Olympus, Tokyo, Japan) according to the manufacturers instructions. Images were acquired by using a stereo uorescence microscope (M205FA, Leica Microsystems, Wetzlar, Germany) equipped with a digital camera (DFC365FX, Leica Microsystems).

Two-photon laser microscopy. A total of 105 PFU of MA-eGFP-PR8 was intranasally inoculated into B6 mice. To label lung macrophages, 50 ml of PECD11b (M1/70, BioLegend, San Diego, CA) was injected intravenously to the mice at day 3 p.i. Thirty minutes after the antibody injection, the lungs of the mice were harvested. The kinetics of eGFP- and PE-positive cells in the lungs was imaged with a two-photon laser microscope (LSM 710 NLO, Carl Zeiss, Oberkochen, Germany). During the analysis, the lungs were maintained in complete medium (RPMI 1640 with 10% fetal calf serum) in a humid chamber (37 C, 5% CO2). The data were processed with LSM software Zen 2009 (Carl Zeiss). For three-dimensional imaging of HPAI virus-infected lung tissues, B6 mice were intranasally inoculated with 105 PFU of MA-Venus-HPAI virus and MA-Venus-PR8. The lung tissues were collected from the mice at day 1 and 2 p.i., and treated with SCALEVIEW-A2 solution (Olympus) to make tissues transparent as described above. Three-dimensional images of lung tissues were obtained from a two-photon laser microscope (LSM 710 NLO).

Flow cytometric analysis and cell sorting. To obtain single-cell suspensions, lungs were dissociated with Collagenase D (Roche Diagnostics, Mannheim, Germany; nal concentration: 2 mg ml 1) and DNase I (Worthington Biochemical,

Lakewood, NJ; nal concentration: 40 U ml 1) for 30 min at 37 C by grinding the tissue through nylon lters (BD Biosciences). Red blood cells (RBCs) were lysed by treatment with RBC lysing buffer (Sigma Aldrich, St Louis, MO). To block nonspecic binding of antibodies, cells were incubated with puried anti-mouse CD16/32 (Fc Block, BD Biosciences, San Diego, CA). Cells were stained with appropriate combinations of uorescent antibodies to analyse the population of each immune cell subset. The following antibodies were used: anti-CD45 (30-F11: eBioscience, San Diego, CA), anti-CD11b (M1/70: BioLegend), anti-F4/80 (BM8: eBioscience) and anti-CD11c (HL3: BD Biosciences). All samples were also incubated with 7-aminoactinomycin D (Via-Probe, BD Biosciences) for deadcell exclusion. Data from labelled cells were acquired on a FACSAria II (BD Biosciences) and analysed with FlowJo software version 9.3.1 (Tree Star, San Carlos, CA). To isolate Venus-positive and -negative macrophages from lungs, stained cells were sorted using a FACSAria II (BD Biosciences).

Microarray analysis. Total RNA of sorted macrophages was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA) and precipitated with isopropanol. RNA amplication was performed using the Arcturus Riboamp Plus RNA Amplication Kit (Life technologies) in accordance with the manufacturers instructions. RNA was labelled by using the Agilent Low Input Quick Amp Labelling kit, one colour (Agilent Technologies, Santa Clara, CA) and hybridized to the SurePrint G3 Mouse GE 8X60K microarray (Agilent Technologies). Arrays were scanned with a DNA Microarray Scanner with SureScan High-Resolution Technology, (G2565CA; Agilent Technologies), and data were acquired using Agilent Feature Extraction software ver. 10.7.3.1. (Agilent Technologies). Probe annotations were provided by Agilent Technologies (AMADID 028005). Probe intensities were background-corrected and normalized using the normal-exponential and quantile methods, respectively. The log2 of the intensities were then t to a linear model that compared the groups of interest37. All reported

P values were adjusted for multiple hypothesis comparisons using the Benjamini Hochberg method. Transcripts were considered differentially expressed if there was

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at least a twofold change in the mean probe intensity between contrasts with an adjusted Po0.01. Hierarchical clustering was performed in R. The resultant gene clusters were then analysed with ToppCluster38 to identify gene annotations that were enriched in each cluster. The reported scores are the log10 of the

BenjaminiHochberg adjusted P value.

Western blot analysis. Whole-cell lysates of MDCK cells were electrophoresed through sodium dodecylsulfate polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA) and transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was then blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) and incubated with polyclonal rabbit anti-GFP (MBL, Nagoya, Japan), mouse anti-NS1 (188/5, a gift from Prof. H. Kida, Hokkaido Univ., Sapporo, Japan), rabbit anti-WSN (R309, prepared in our laboratory) or mouse anti-b-actin (A2228; Sigma-Aldrich), followed by HRP-conjugated anti-mouse or anti-rabbit IgG antibody (GE Healthcare, Waukesha, WI), respectively. After the membrane was washed with PBS-Tween, specic proteins were detected by using the ECL Plus Western Blotting Detection System (GE Healthcare). The specic protein bands were visualized by use of the VersaDoc Imaging System (Bio-Rad Laboratories).

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Acknowledgements

We thank Susan Watson for editing the manuscript. We also thank to Dr A. Miyawaki (Cell Function and Dynamics, Brain Science Institute, RIKEN, Saitama, Japan) for Venus-plasmid. This work was supported by the Japan Initiative for Global Research Network on Infectious Diseases from the Ministry of Education, Culture, Sports, Science and Technology, Japan; by grants-in-aid from the Ministry of Health, Labour and Welfare, Japan; by ERATO and Strategic Basic Research Programs of Japan Science and Technology Agency, and by an NIAID-funded Center for Research on Inuenza Pathogenesis (CRIP, HHSN266200700010C) to Y.K. H. Katsura is supported by JSPS Research Fellowships for young scientists.

Author contributions

S.F., H. Katsura, D.Z., M.O., H. Kitano, G.N., S.W. and Y.K. designed the experiments and wrote the paper. S.F., H. Katsura, D.Z., M.O., T.A., I.I. and S.Y. performed experiments. S.F., H. Katsura, D.Z., M.O., T.A., I.I., J.E.S., S.W. and Y.K. analysed the data.

Additional information

Accession codes: Microarray data have been deposited in the GEO data repository under the accession code GSE64473.

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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How to cite this article: Fukuyama, S. et al. Multi-spectral uorescent reporter inuenza viruses as powerful tools for in vivo studies. Nat. Commun. 6:6600 doi: 10.1038/ncomms7600 (2015).

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