Usutu Virus Infection of Embryonated Chicken Eggs and a Chicken Embryo-Derived Primary Cell Line

Abstract

Usutu virus (USUV) is a mosquito-borne flavivirus, closely related to the West Nile virus (WNV). Similar to WNV, USUV may cause infections in humans, with occasional, but sometimes severe, neurological complications. Further, USUV can be highly pathogenic in wild and captive birds and its circulation in Europe has given rise to substantial avian death. Adequate study models of this virus are still lacking but are critically needed to understand its pathogenesis and virulence spectrum. The chicken embryo is a low-cost, easy-to-manipulate and ethically acceptable model that closely reflects mammalian fetal development and allows immune response investigations, drug screening, and high-throughput virus production for vaccine development. While former studies suggested that this model was refractory to USUV infection, we unexpectedly found that high doses of four phylogenetically distinct USUV strains caused embryonic lethality. By employing immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction, we demonstrated that USUV was widely distributed in embryonic tissues, including the brain, retina, and feather follicles. We then successfully developed a primary cell line from the chorioallantoic membrane that was permissive to the virus without the need for viral adaptation. We believe the future use of these models would foster a significant understanding of USUV-induced neuropathogenesis and immune response and allow the future development of drugs and vaccines against USUV.

Keywords: Usutu virus; chicken embryo; chorioallantoic membrane; flavivirus; model; primary culture; replication.

Figure 1

Kaplan–Meier survival curves for chicken embryos inoculated with three different doses of USU-BE-Seraing/2017 strain using the allantoic route.

Figure 2

Chicken embryos after infection with USU-BE-Seraing/2017 strain using the allantoic route. (A) The infected chicken embryos showed cutaneous hemorrhage compared with the non-infected controls. (B) Unlike the non-infected embryo, the infected embryos (in the middle and on the right of the picture) died and showed cutaneous hemorrhage and pallor in the liver.

Figure 3

Chorioallantoic membrane from chicken embryos inoculated with the USU-BE-Seraing/2017 strain via the allantoic route. (A) Negative control two days after mock inoculation; (B) diffuse necrosis in the chorionic layer indicated by cell vacuolization (arrows) and massive nuclear fragmentation (stars) at two days post-infection (dpi); (C) massive infiltration of lymphocytes and heterophils in the stroma on day 5 post-infection; (D) Severe degeneration with vacuolization (arrows) and necrosis (stars) of cells in both epithelial layers (5 dpi). Abbreviations: ae, allantoic epithelium; ce, chorionic epithelium; st, stroma. Hematoxylin and eosin stain. Scale bars = 50 µm.

Figure 4

Immunohistochemical staining of Usutu virus antigens and chicken embryos. (A) Chorioallantoic membrane (CAM) on day 3 post-infection (pi); (B) skeletal muscle on day 3 pi; (C) heart on day 5 pi; (D) retina on day 3 of negative control; (E) retina on day 3 pi, degeneration of the neuronal layer with focal loss of the pigmented epithelium; (F) epidermis and feather follicle pulp on day 5 pi; (G) intestine, on day 5 pi; (H) brain on day 5 pi, UR-10-Tm strain; (I) pituitary gland on day 6 pi, USU-BE-Grivegnee/2017 strain. Mayer hematoxylin counterstain. Scale bars = 50 µm.

Figure 5

Viral RNA loads in the allantoic fluids from embryonated chicken eggs infected with USU-BE-Seraing/2017 strain at a dose of 10⁵ 50% tissue culture infective dose (TCID₅₀). Data are representative of five samples per day (error bars represent the standard deviations). n = 5 per day of infection; “*” indicates a p-value < 0.05.

Figure 6

Comparison of the body weights on day 5 of the experiment between control and infected chicken embryos with the USU-BE-Seraing/2017 strain using the allantoic route. Bars indicate means ± standard deviation; n = 5 per condition; “*” indicates a p-value < 0.05.

Figure 7

Usutu virus RNA loads detected by RT-qPCR in the brain, heart, liver, and chorioallantoic membrane (CAM) samples of chicken embryos inoculated with USU-BE-Seraing/2017 strain (10⁵ TCID₅₀) via the allantoic route. The data show the mean log 10 viral RNA copies/mL ± standard deviation. n = 5 per tissue per day of infection.

Figure 8

Kaplan–Meier survival curves for chicken embryos inoculated with three different doses of (A) Vienna 2001, (B) UR-10-Tm, and (C) USU-BE-Grivegnee/2017 Usutu virus strains using the allantoic route.

Figure 9

Viral RNA loads in the supernatants of primary cultures of chorioallantoic membrane (CAM) cells infected with different USUV strains (a) USU-BE-Seraing/2017 (b) Vienna 2001, (c) UR-10-Tm, and (d) USU-BE-Grivegnee/2017, as determined by RT-qPCR. CAM cells were infected with USUV at MOIs of 0.1, 0.01, and 0.001. Data are representative of three wells per day for each MOI, each performed in duplicate (error bars represent standard deviations).

Figure 9

Viral RNA loads in the supernatants of primary cultures of chorioallantoic membrane (CAM) cells infected with different USUV strains (a) USU-BE-Seraing/2017 (b) Vienna 2001, (c) UR-10-Tm, and (d) USU-BE-Grivegnee/2017, as determined by RT-qPCR. CAM cells were infected with USUV at MOIs of 0.1, 0.01, and 0.001. Data are representative of three wells per day for each MOI, each performed in duplicate (error bars represent standard deviations).

Figure 10

Immunohistochemical staining of USUV antigens performed on chicken chorioallantoic membrane cells. (A) Mock-inoculated cells; (B) USUV-infected cells. Mayer hematoxylin counterstain. Scale bars = 50 µm.