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.

In Vitro and In Vivo Models to Study the Zoonotic Mosquito-Borne Usutu Virus

“…No strains from Europe 4 and Europe 5 lineages have been tested yet for their pathogenicity either in vitro or in vivo. Further, only one study [ 71 ] compared the replication kinetics, indicative of the virulence of four USUV strains (Africa 3 and Europe 1, 2, and 3 lineages), in chicken embryo primary cells and found marked replication of the Europe 3 strain compared to the others. However, in vivo studies using chicken embryos revealed similar pathogenicity between these strains, indicating the importance of combining different models from which to draw robust conclusions.…”

“…Similar to WNV, death due to USUV in domestic canaries was more likely attributed to a multi-systemic failure than to a pure neurologic disease, and the virus infected all major systems and a wide variety of cell types [ 110 ]. The myocardial cells strongly supported viral replication, as viral antigens were systematically detected by immunohistochemistry (IHC) in the experimentally infected chicken embryos and canaries [ 71 , 110 ].…”

“…In all these three models, USUV displayed a particular tropism for the eyes [ 71 , 110 , 111 ]. Visual impairment and ocular lesions have been described following infection of birds with other flaviviruses, such as WNV [ 112 , 113 ].…”

“…Microscopically, despite the detection of USUV antigens by IHC in many embryonic tissues, USUV replication was not associated with any significant lesions in the goose embryos, while multifocal necrosis and non-suppurative inflammation were observed only in the Chorioallantoic membrane (CAM) of the chicken embryo [ 71 ]. In canaries, these lesions affected several organs, including the brain, which, however, showed no IHC labeling.…”

“…Excretion of USUV via the immature feathers of birds during the early stages of infection was demonstrated [ 110 ]. Similarly, during embryonic development in chicken, the virus replicated in feather follicles [ 71 ]. These preliminary observations suggest that feathers can potentially play a role in the spread of the virus.…”