Native replication of CA/09-PA NLuc virus in vitro

To assess the utility of the NLuc reporter virus in ferrets, a replication-competent NanoLuc A/California/04/2009 (CA/09-PA NLuc) virus was generated by inserting NanoLuc into the polymerase gene as described16. We successfully rescued the CA/09-PA NLuc virus by transfecting 293T cells using an eight-plasmid reverse genetics system and propagating the resultant virus in embryonated chicken eggs19. Virus propagation through three passages in eggs did not result in attenuation of viral luminescence, suggesting stable maintenance of the reporter construct, and the replication kinetics of the reporter virus are indistinguishable from the parental strain in vitro (Fig. 1a).

Figure 1: Validation of CA/09-PA NLuc virus against parental rgCA/09 virus. (a) Replication kinetics of the CA/09-PA NLuc virus were compared with parental rgCA/09 virus by TCID 50 analysis on MDCK cells. Samples were run in triplicate, Error bars=s.d. (b) Eight-week-old male ferrets were lightly anaesthetised and intranasally inoculated with 105 TCID 50 rgCA/09 virus. Animals were administered 200 μl substrate in 1 ml PBS via the cephalic vein and images taken over a 4-min exposure. No luminescence is seen since the virus does not contain the swapped PA NLuc gene. (c) Comparison of time to maximal titre in MDCK cells between luminescent and traditional hemagglutination (HAU) end points in ferret nasal wash from triplicate samples. Full size image

Imaging real-time dynamics and viral replication patterns

To test whether CA/09-PA NLuc measures viral infection dynamics in real time and is suitable for transmission studies, donor ferrets (n=5) were inoculated with parental CA/09 virus (Fig. 1b) or CA/09-PA NLuc virus and serially imaged throughout infection (Fig. 2a). Nasal washes were collected from 2 to 14 days post infection (d.p.i.) and viral titres, as measured by traditional TCID 50 analysis, demonstrated that all the donor parental- and NLuc-inoculated animals became infected with titres peaking at ~106 TCID 50 (Fig. 2b,c). Titres were comparable between CA/09 and CA/09-PA NLuc viruses (Fig. 2b,c, Table 1), demonstrating that the reporter virus replicates similarly to the parental, and titres mirror previously reported studies using pdmH1N1 viruses in ferrets20,21,22,23. A TCID 50 assay with a bioluminescent endpoint was also developed (Fig. 2d). The bioluminescent TCID 50 (RLU) assay yielded titres nearly identical to the classical TCID 50 (HAU) assay, but dramatically accelerated determination of viral titre requiring only 18 h compared with TCID 50 (HAU) measurements that require 72 h (Fig. 1c, Table 1). Finally, we directly measured NLuc activity in the nasal washes (Fig. 2e). Luminescence in nasal wash was significantly correlated to viral titres determined by both TCID 50 assays and thus permits instantaneous measurement of changes in viral load (Fig. 2e). Significant correlation between nasal wash luminescence and viral titre was confirmed using Pearson’s correlation coefficient (Table 1).

Figure 2: CA/09-PA NLuc reporter virus has similar dynamics of transmission as rgCA/09 parental virus. (a) Ferrets (n=5) were directly inoculated with CA/09-PA NLuc reporter virus. Upper respiratory and lung bioluminescence was imaged every 48 h.p.i. through 14 d.p.i. Imaging ended when viral clearance was obtained as monitored by both luminescence and viral titre determination. Arrows indicate areas of interest where luminescence was detected. (b) Nasal washes from donor ferrets directly inoculated with parental rgCA/09 (black lines, n=5) virus, direct contacts (blue lines, n=3) and respiratory contact ferrets (red lines, n=2) were titrated by TCID 50 assay. (c–e) Nasal washes from ferrets directly inoculated with rgCA/09-PA NLuc virus (black lines) or direct (blue lines) and respiratory contact (red lines) animals were titrated by TCID 50 assay using HAU as an end point (c), luminescent TCID 50 assay (d) or the nasal wash was directly assessed for luminescence (e). Lines represent individual animals. Full size image

Table 1 Correlation between titres obtained by different methodologies and comparing rgCA/09-PA NLuc with rgCA/09 parental virus. Full size table

Imaging also revealed heterogeneous tissue distribution in influenza virus-infected ferrets (Fig. 2a). Bioluminescence was detected in the URT and LRT of all directly inoculated (donor) animals, indicating infection in the nasal turbinates and lungs respectively. All animals showed infection in the URT by 2 d.p.i., waning by 4–6 d.p.i. and resolving to below the level of detection by 8 d.p.i. Robust bioluminescence was detected in both the URT and LRT for two of the animals (donors 1 and 3). Multifocal bioluminescence detected in the LRT 2 and 4 d.p.i. is consistent with infection in multiple lobes of the lung and the trachea. Low-level infection was detected in the lungs of donor 2 that was completely resolved by 4 d.p.i. In contrast, there was significantly less lung infection evident in donors 4 and 5. Imaging of resected animals confirmed the ability to accurately localize URT and LRT infections as well as distinguish infections in the left and right lobes of the lung (Fig. 3a).

Figure 3: Correlation of viral titre and bioluminescence in CA/09-PA NLuc infected ferrets. (a) Ferrets (n=6) infected with CA/09-PA NLuc were imaged whole and resected. (b) Viral titres from nasal tissue were significantly correlated with nasal flux. (c) Viral load was measured in each lung lobe (UL, upper left; LL, lower left; UR, upper right; MR; middle right; LR, lower right) above the limit of detection (dashed line). Increased flux was associated with higher viral titres above background (dashed line) when data were considered for the right or left lobes (d) and whole lung (e). (f) Comparison of bioluminescence between whole and resected animals showed that overlying tissue can reduce flux from the lungs up to 4.5-fold. Bars represent individual animals. Full size image

Bioluminescent detection correlates with titres

Luminescent flux (radiance defined as photons s−1 cm−2 sr−1) has been directly correlated with viral titres in mice infected with influenza, Dengue and Sendai reporter viruses11,13,14,24,25. However, light attenuation by diffusion and scattering in tissue can complicate quantification of in vivo bioluminescent imaging and precise localization of the bioluminescent tissue, especially in larger animals. Bioluminescence from firefly luciferase can be imaged as deep as several centimeters within an animal; however, light from shallow tissues can be detected to a greater extent than that from deeper areas where attenuation by overlying tissues can be up to 10-fold26,27,28. NLuc, however, possesses 150-fold greater specific activity (that is, light output) than both Renilla and firefly luciferases and may therefore have increased sensitivity and minimized effects due to attenuation15. To test this, we correlated bioluminescent flux before and after resection with tissue titres to establish the extent to which overlying tissues attenuate NLuc bioluminescence. Briefly, ferrets (n=5) infected with CA/09-PA NLuc virus were imaged, humanely euthanized, breast plates removed and then bioluminescence was immediately measured for a second time (Fig. 3). Infected tissues were recovered and viral titres were determined by TCID 50 . Viral titres from nasal tissue were significantly correlated with flux emitted from this relatively shallow site of replication (Spearman’s ρ=0.9910, P=0.008) (Fig. 3b). In the lung tissue, increased flux was associated with higher viral titres when data were considered for the whole lung (Fig. 3c,d). Similar results were obtained when analysis was performed on the right or left lobes of the lung, further establishing the linkage between flux and viral titer and allowing more discrete localization of sites of replication. All animals with virus in the lung (as measured by TCID 50 ) also displayed flux when resected. Quantitative analyses indicated that a viral load as low as 102 TCID 50 per gram tissue was sufficient for imaging in intact animals (Fig. 3a,c). Comparison of bioluminescence between whole and resected animals showed that overlying tissue can reduce flux from the lungs up to 4.5-fold (Fig. 3e, ferret 1) and that at least twofold more flux is required to detect bioluminescence in an intact compared with resected animal (Fig. 3f, ferrets 2 and 3). Thus, flux is a highly sensitive measure of viral titre and attenuation of bioluminescence by overlying tissue has a minor impact on the measurement of lung viral loads, although care must be taken with direct comparisons between different replication sites.

Monitoring real-time influenza virus transmission in ferrets

Having established bioluminescent imaging as a noninvasive and quantitative measure of viral replication, we exploited this system to examine real-time dynamics during influenza virus transmission. Since CA/09 virus is known to transmit by both direct and respiratory contact21,22,23, naive ferrets were placed in the same cages as the inoculated donor ferrets (direct contact, DC) or in cages separated by 4–6 inches from the inoculated group (respiratory contact, RC) at 1 d.p.i. Imaging and viral titres were performed as for the donor ferrets. Transmission occurred in 100% of the DC animals (n=3). Bioluminescent flux and viral load peaked at 4–6 d.p.i. with maximum titres reaching ~105 TCID 50 ml−1 in nasal wash (Figs 2b–d and 4). Infections were subsequently detected in all of the RC ferrets (n=2), although viral peaks were delayed occurring 8–12 dpi (Figs 2 and 4). Similar to donor animals, luminescence was readily detected directly in the nasal wash of all contact animals supporting rapid analysis of viral shedding (Fig. 2e).

Figure 4: Variable patterns of infection in direct and respiratory contact animals. Naive ferrets were placed in direct contact (n=3) or respiratory contact (n=2) with inoculated donor animals 1 d.p.i. Upper respiratory and lung bioluminescence was imaged every 48 h.p.i. through 14 d.p.i. Imaging ended when viral clearance was obtained as monitored by both luminescence and viral titre determination. Arrows indicate areas of interest where luminescence was detected. Full size image

Intriguingly, the spatial resolution of bioluminescent imaging allowed identification of distinct patterns of infection. All of the donor animals displayed at least some degree of replication in both the URT and LRT (Fig. 2a). DC animals displayed highly localized infections in the URT alone (direct contact 1), LRT alone (direct contact 2) and both URT/LRT patterns (direct contact 3, Fig. 4). Nasal wash titres from the DC2 ferret were negative at all time points tested (Fig. 2), although this animal displayed obvious lung bioluminescence. In the absence of bioluminescent imaging, this animal would have been considered uninfected until seroconversion was assessed. In contrast to DC, diversity of tissue tropism was not detected in the RC ferrets where replication occurred in both the URT and LRT (Fig. 4). In spite of the distinct patterns of infection between cohorts, there were no differences in clinical signs including temperature and weight loss (Supplementary Fig. 1) suggesting that the relatively mild CA/09 replication in the lungs may not be associated with more severe clinical disease.

Luminescence was also detected at unexpected sites outside of the respiratory tract, including bioluminescent foci suggesting replication in areas we hypothesize, are the kidney (Fig. 2, donor 3, arrow), the ears (Fig. 2, donor 3, arrow) and the eye or conjunctiva (Fig. 4, direct contact 1, arrow). Although previous studies have shown replication of CA/09 and other influenza viruses in these extra-pulmonary sites29,30,31,32, we were unable to euthanize animals to confirm viral replication in these unconventional tissues due to the longitudinal nature of the study. Importantly, results from unbiased whole-body imaging can be used to better understand influenza virus tropism.

Visualizing immunological protections upon re-challenge

Ferrets are also important models for establishing the efficacy of antiviral therapies and vaccines7,8. To demonstrate the utility of the NLuc reporter virus for therapeutic studies, naive ferrets or those previously infected with CA/09-PA NLuc were challenged with CA/09-PA NLuc or CA/09 viruses at 28 days post primary infection. Images (Fig. 5a) and nasal washes were collected at 1 and 3 days post challenge (d.p.c.) and nasal turbinates, trachea and lungs collected at 3 d.p.c. Viral titres in naive ferrets inoculated with either the parental rgCA/09 or CA/09-PA NLuc viruses were nearly identical in nasal washes 1 and 3 d.p.c. as well as in the nasal epithelium, trachea and lungs at 3 d.p.c. (Table 2). Although all of the previously infected animals had neutralizing antibodies (Table 2) and were protected from challenge, the ferret that had a primary infection in the lung (Fig. 4, direct contact 2) had measurable virus in the nasal wash, trachea and lungs suggesting that the site of primary infection may impact protection from re-infection (Fig. 5a, direct contact 2). Though this has been shown in infected mice24,33, further studies are needed to evaluate this in influenza virus-infected ferrets.

Figure 5: Patterns of infection during primary infection impact the outcome during re-challenge. (a) Naive or previously infected animals (n=4 per group) exhibiting different patterns of infection were re-challenged with either rgCA/09 or CA/09-PA NLuc virus and images collected at 1 and 3 d.p.c. (b–d) Microneutralization titres were conducted by luminescent (b,c) and traditional (d) methods on duplicate samples from each animal run in triplicate. Error bars=s.d. Full size image

Table 2 Serology, nasal wash and tissue titres in virus re-challenged ferrets. Full size table

Finally, we asked whether the NLuc reporter virus was a more sensitive means to measure neutralizing antibody responses by microneutralization (MN) assay. To test this, sera collected from animals used for the re-challenge studies were assessed for neutralizing antibody titres by classical or luminescent-based MN utilizing the CA/09-PA NLuc virus (Fig. 5b–d). The luminescent-based MN assay showed increased sensitivity with neutralization titres two- to four-times higher using the infectious luminescent assay versus traditional enzyme-linked immunosorbent assay (Fig. 5b–d and Table 2). Interestingly, this increase in sensitivity may represent titres lower than previous methodology was able to detect as well as possible infections by defective particles or semi-infectious virions, which do not productively replicate in the cell.