Fig 1.
Brightfield microscopy of tissues of Gallus gallus domesticus 72 hpi with yellow fever 17DD virus.
(A) Apoptotic bodies in skeletal muscular tissue; (B) detail of karyorrhexis in muscular bundles; (C) apoptotic bodies in tubular epithelium; (D) apoptotic bodies in muscular region of gizzard; (E) detail of apoptotic bodies in the muscular layer of the gizzard; (F) apoptotic bodies in fibroblastoid cells of perichondrium. Apoptotic nuclei are indicated by black arrows (→). Hematoxylin and Eosin stain.
Fig 2.
Immunofluorescence in skeletal muscle tissue of chicken embryos at 72 hpi with 17DD virus.
Polyclonal antibodies directed against the yellow fever virus were used to immunostain virus proteins in: (A) skeletal muscle cell bundles; (B) skeletal muscle cells showing perinuclear thickening and presenting an intense labeling in sarcoplasmic reticulum following the striations of the cytoskeleton–yellow arrows (→) show pyknosis and karyorrhexis close to infected cells; and (C) skeletal muscle cells evidenced by desmin antibody and showing the virus infection of the muscular bundles. (D) Detail of the infected muscular bundles, showing the perinuclear positivity together with striations. Yellow fever virus staining in green, nuclei stained with DAPI in blue and desmin in red.
Fig 3.
Heart muscular tissue of Gallus gallus domesticus at 72 hpi with yellow fever 17DD virus.
(A) Infected heart muscle cells; (B) desmin positive heart muscle cells showing perinuclear virus protein distribution and striated pattern compatible with sarcoplasmic virus protein distribution. Yellow fever viral antigen detection in green, nuclei stained with DAPI in blue and desmin in red.
Fig 4.
Nervous system of Gallus gallus domesticus at 72 hpi with yellow fever 17DD virus.
(A) Brain section presenting some infected neurons and glial cells; (B) spinal cord infected neurons; (C) one neuron of the brain showing perinuclear thickening and vesicles dispersed throughout the cytoplasm; (D) infected fibroblastoid cells along the meninges. Yellow fever virus protein detection in green and nuclei stained with DAPI in blue.
Fig 5.
Tubular epithelial cells in Gallus gallus domesticus at 72 hpi with yellow fever virus.
(A, B) Infected kidney tubular epithelium cells; (C) fluorescent vesicles inside tubular kidney cells, suggesting virus excretion to the lumen of the tubules–pyknotic nuclei are indicated by yellow arrows (→). Yellow fever virus detection in green and nuclei stained with DAPI in blue.
Fig 6.
Fibroblastoid cells in different sites of Gallus gallus domesticus at 72 hpi with 17DD virus.
(A) Parenchyma lung cells surrounding the parabronchi epithelium; (B) detail of parenchymal positive cells with desmin expression; (C) infected fibroblastoid cells along the perichondrium; (D) infected cell cluster in subepithelial connective tissue; (E) infected cells in the muscular layer of the gizzard; (F) infected cells in the muscular layer of the yolk stalk. Yellow fever virus proteins immunostained in green, nuclei stained with DAPI in blue and desmin in red.
Fig 7.
Intracellular aspects of muscle cells of Gallus gallus at 72 hpi with 17DD virus.
(A) Skeletal muscle cells detected by confocal microscopic analysis; (B) Airyscan super-resolution microscopy of the same field of view in a 0.16 μm optical slice showing a vesicular pattern of virus protein expression. Yellow fever virus proteins detected in green (A) or in white (B), nuclei stained with DAPI in blue (A).
Fig 8.
SR-SIM microscopy of chicken muscle cells at 72 hpi with 17DD virus.
The virus proteins follow the striations in a localization compatible with the sarcoplasmatic reticulum. In the center it is possible to see an agglomeration of viral proteins probably inside the endoplasmic reticulum.
Fig 9.
Detection of viral genomic RNA in YF 17DD-infected chicken embryos.
The amplicons generated by Nested-PCR were analyzed by 2% agarose gel electrophoresis. The lanes correspond to the following specimens: (1) and (2)—head; (3) and (4)—legs; (5) and (6)—wings; from (7) to (14)—trunks; (15) and (16)—vitelline membrane; (17) and (18)—chorioallantoic membrane; from (19) to (22)—negative control (water-inoculated animals). Even-numbered lanes indicate samples submitted to amplification of genomic RNA whereas odd-numbered lanes indicate samples submitted to amplification of the replicative intermediate RNA. The molecular length markers are indicated on the left of the figure. The black arrow indicates the 156bp amplicon obtained from the amplification of YF 17D RNA.