Visualizing real-time influenza virus infection, transmission and protection in ferrets

Abstract

Influenza transmission efficiency in ferrets is vital for risk-assessment studies. However, the inability to monitor viral infection and transmission dynamics in real time only provides a glimpse into transmissibility. Here we exploit a replication-competent influenza reporter virus to investigate dynamics of infection/transmission in ferrets. Bioluminescent imaging of ferrets infected with A/California/04/2009 H1N1 virus (CA/09) encoding NanoLuc (NLuc) luciferase provides the first real-time snapshot of influenza infection/transmission. Luminescence in the respiratory tract and in less well-characterized extra-pulmonary sites is observed, and imaging identifies infections in animals that would have otherwise been missed by traditional methods. Finally, the reporter virus significantly increases the speed and sensitivity of virological and serological assays. Thus, bioluminescent imaging of influenza infections rapidly determines intra-host dissemination, inter-host transmission and viral load, revealing infection dynamics and pandemic potential of the virus. These results have important implications for antiviral drug susceptibility, vaccine efficacy, transmissibility and pathogenicity studies.

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 TCID₅₀ 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 TCID₅₀ 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.

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 TCID₅₀ 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 TCID₅₀ assay using HAU as an end point (c), luminescent TCID₅₀ assay (d) or the nasal wash was directly assessed for luminescence (e). Lines represent individual animals.

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.

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.

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.