FIGURE SUMMARY
Title

Influenza a virus infection in zebrafish recapitulates mammalian infection and sensitivity to anti-influenza drug treatment

Authors
Gabor, K.A., Goody, M.F., Mowel, W.K., Breitbach, M.E., Gratacap, R.L., Witten, P.E., Kim, C.H.
Source
Full text @ Dis. Model. Mech.

Zebrafish embryos are susceptible to IAV infection, and IAV replicates in zebrafish hosts. PBS- or IAV-injected zebrafish embryos were monitored for 5 days post infection for quantification of cumulative percent survival (A) and viral burden (B). The results represent three separate experiments. (A) Cumulative percentage survival of zebrafish infected at 48 hpf with IAV APR8 or X-31 (n=50 fish per treatment). Significantly fewer zebrafish survived in either IAV-infected group compared with controls (***P<0.001). (B) Quantification of viral burden in zebrafish infected with APR8 or X-31 (n=20 fish per treatment). Viral burden increases 10- to 20-fold in APR8- or X-31-infected zebrafish. In APR8-infected embryos, viral burden increased steadily over time through to 96 hpi. In X-31-infected zebrafish, viral burden peaked around 48 hpi. These data demonstrate IAV replicates within zebrafish and suggest that death in IAV-infected zebrafish is likely to be caused by infection with the virus.

PHENOTYPE:
Fish:
Condition:
Observed In:
Stage Range: Long-pec to Days 7-13

IAV infection causes a disease phenotype in zebrafish. Brightfield microscopy of live zebrafish at 24, 48 and 72 hpi, side oriented, anterior left, dorsal top, magnification ×25. The data presented represent three individual experiments, a mean of n=7 fish per treatment. (A,C,E) PBS-injected controls. Development proceeded normally in PBS-injected controls at 24 hpi (A), 48 hpi (C) and 72 hpi (E). (B,D,F) APR8-infected zebrafish. Embryos infected with APR8 or X-31 (data not shown) displayed symptoms of infection. IAV-infected zebrafish had pericardial edema (black arrowheads in B,D,F) and yolk sac edema (white arrowheads in B,D,F). IAV-infected zebrafish also had craniofacial abnormalities (red arrowhead in D) and arched backs (red arrowhead in F). The most striking phenotype of IAV-infected zebrafish was generalized edema that worsened over time.

Histopathology of zebrafish infected with IAV. Parasagittal semi-thin sections from PBS-, APR8- or X-31-injected zebrafish at 48 hpi, stained with Toluidine Blue (n=3 per group, 350 sections per fish). Anterior to the left, dorsal top. PBS control (A-D), APR8-infected (E-H) and X-31-infected (I,J) zebrafish. (B) Head kidney (hk), liver (lv) and yolk sac (ys) of a control zebrafish. No degeneration of cells was observed. (C) Gill and pharyngeal arches adjoined to the occipital region of a control zebrafish. All tissues are without pathological anomalies: semicircular canal of the inner ear (scc) with macula (mc), cartilage of fifth pharyngeal arch (pa) and anterior located gill filaments (gf). (D) The same control fish with healthy heart in front of the yolk sac, atrium (at), ventricle (vt), conus ateriosus (ca) and pericardial volume (pc). (F) The liver of APR8-infected zebrafish. The white arrowheads indicate the presence of degenerative liver tissue, indicated by the necrotic disintegration of nuclei. (G) Gill chamber of APR8-infected zebrafish. The white arrowheads indicate necrotic gill filaments that contain many vacuolated cells. (H) Heart region of APR8-infected zebrafish. The white arrowhead indicates the fluid-filled pericardium. The histological analysis confirms the edema seen in IAV-infected zebrafish (Fig. 3B,D,F). (J) The head kidney of X-31-infected zebrafish shows clear signs of necrotic tissue degeneration. The white arrowheads indicate the presence of degenerating cells in the hematopoietic part of the head kidney.

NS1-GFP shows infection of the zebrafish cardiovascular system and swimbladder. (A–D) Single focal planes of PBS- or NS1-GFP-injected, fixed casper mutant zebrafish at 48 hpi, side mounted, anterior left, dorsal top, ×4 magnification. (A) Fluorescence micrograph of a PBS-injected control showing background autofluorescence. The data presented represent three individual experiments, a mean of n=6 fish per treatment. (B) Panel of PBS control showing merged fluorescence and brightfield micrographs. (C) Fluorescence micrograph of NS1-GFP-infected zebrafish. Punctate fluorescence confirms viral replication. Inset is a computer magnification of the pericardial region. White arrowhead points to fluorescence in the heart. (D) Merge of NS1-GFP-infected embryo fluorescence and brightfield micrographs. (E,F) 3D reconstruction of NS1-GFP-infected, live zebrafish, 24 hpi, side mounted, anterior left, dorsal top, magnification ×10. Punctate fluorescence was observed in major blood vessels. Inset is a computer zoom of two infected blood vessels. White arrowhead denotes fluorescence in an intersomitic blood vessel. (G–J) 3D reconstruction of casper mutant zebrafish after injection of either PBS or NS1-GFP into the swim bladder at 5 dpf, fixed at 24 hpi, side mounted, anterior left, dorsal top. (G) Fluorescence micrograph of zebrafish with PBS injected into the swim bladder, the image shows background autofluorescence. The data represent two individual experiments, a mean of n=5 fish per treatment. (H) Merged panel of PBS-injected control fluorescence and brightfield micrographs. (I) Fluorescence micrograph of NS1-GFP swim bladder infection. Green punctate fluorescence is likely to show infection of epithelial cells around the swim bladder. (J) Fluorescence and brightfield micrograph merge for NS1-GFP swim bladder infection. Infection with the NS1-GFP reporter virus in transparent zebrafish larva allows for visualization of viral replication, spread, resolution of infection and cell tropism in vivo.

Antiviral treatment with Zanamivir confers protection after infection in zebrafish. Zanamivir (16.7 ng/ml or 33.3 ng/ml) was administered at 3 hpi, and Zanamivir-containing media was changed twice daily. (A–C1) Single focal planes of NS1-GFP-injected, live zebrafish at 48 hpi, side mounted, anterior left, dorsal top, ×4 magnification. Lettered panels are brightfield images, and numbered panels are fluorescence micrographs. (A,A1) NS1-GFP-infected zebrafish. Note fluorescence in the yolk region, as well as punctate fluorescence throughout the body. (B,B1) NS1-GFP-infected zebrafish treated with 16.7 ng/ml Zanamivir. (C,C1) NS1-GFP-infected zebrafish treated with 33.3 ng/ml Zanamivir. Note that treatment with Zanamivir at either dose drastically reduced the punctate fluorescence in the body of the zebrafish, whereas fluorescence in the yolk region remained despite antiviral treatment. The data presented represent three individual experiments, a mean of n=20 fish per treatment. (D) Quantification of healthy, morbid and dead zebrafish at 5 days post infection with NS1-GFP. These data are compiled from three independent experiments, and the number of zebrafish per treatment group is labeled on the graph. e, embryos. Fisher’s exact test two-tailed P-values (with Bonferroni correction) were used to determine whether the proportion of dead versus alive (healthy and morbid) zebrafish significantly differed between treatments. Addition of either dose of Zanamivir to PBS-injected controls did not significantly affect the proportion of zebrafish that died. Infection with NS1-GFP significantly increased the proportion of dead zebrafish compared with the PBS-injected group (P=0.0018). Addition of either dose of Zanamivir significantly decreased the proportion of NS1-GFP-infected zebrafish that died compared with the infected, untreated group (P=0.0012 and P=0.0246 for 16.7 ng/ml and 33.3 ng/ml Zanamivir, respectively). *P<0.05, **P<0.01.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Dis. Model. Mech.