Structural investigation of C6/36 and Vero cell cultures infected with a Brazilian Zika virus

Abstract

Zika virus (ZIKV) is a member of the flavivirus genus, and its genome is approximately 10.8 kilobases of positive-strand RNA enclosed in a capsid and surrounded by a membrane. Studies on the replication dynamics of ZIKV are scarce, which limits the development of antiviral agents and vaccines directed against ZIKV. In this study, Aedes albopictus mosquito lineage cells (C6/36 cells) and African green monkey kidney epithelial cells (Vero cells) were inoculated with a ZIKV sample isolated from a Brazilian patient, and the infection was characterized by immunofluorescence staining, phase contrast light microscopy, transmission electron microscopy and real-time RT-PCR. The infection was observed in both cell lineages, and ZIKV particles were observed inside lysosomes, the rough endoplasmic reticulum and viroplasm-like structures. The susceptibility of C6/36 and Vero cells to ZIKV infection was demonstrated. Moreover, this study showed that part of the replicative cycle may occur within viroplasm-like structures, which has not been previously demonstrated in other flaviviruses.

Fig 1. C6/36 mosquito cells infected with ZIKV analyzed by phase contrast light microscopy at different time points post infection.

(A) Uninfected cells with no monolayer changes; (B) 24 hr p.i.; (C) 48 hr p.i.; (D) 72 hr p.i. Cell individualization, rounding and detachment as well as cytolysis were observed at all time points and were more evident at 72 hr p.i.

Fig 2. Vero cell monolayer infected with ZIKV analyzed by phase contrast light microscopy at different time points post infection.

(A) Uninfected cells with no monolayer changes; (B) 24 hr p.i.; (C) 48 hr p.i.; (D) 72 hr p.i. Cell individualization, rounding and detachment as well as cytolysis were observed at all time points and were more evident at 72 hr p.i.

Fig 3. Immunolocalization of ZIKV E protein (4G2, green) [arrow] inside cytoplasm of C6/36 cells infected with ZIKV at different time points post infection.

(A) Uninfected cells; (B) 24 hr p.i.;(C/D) 48 hr p. i.; (E) 72 hr p.i. The nuclei (N) were counterstained with DAPI (blue). (F) The graph represents the mean ± standard deviation of the infection percentage (%). The highest number of infected cells was observed at 72 hr p.i.

Fig 4. Immunolocalization of ZIKV E protein (4G2, green) [arrow] in the cytoplasm of Vero cells infected with ZIKV at different time points post-infection.

(A) Uninfected cells; (B) 24 hr p.i; (C) 48 hr p.i.; (D) 72 hr p.i. The nuclei were counterstained with DAPI (blue). (E) The graph represents the mean ± standard deviation of the infection percentage (%). A higher number of infected cells was observed at 24 hr p.i.

Fig 5. Uninfected C6/36 cells (negative controls) at different time points in culture.

No cellular ultrastructural alterations were observed. (A–B) 24 hr of culture; (C) 48 hr of culture; (D) 72 hr of culture. Rough endoplasmic reticulum cisternae (rER), nucleus (N).

Fig 6

C6/36 cells infected with ZIKV analyzed by transmission electron microscopy (TEM) at different time points post-infection (A-B: 48 hr p.i., C-D: 72 hr p.i.). Several large viroplasm-like perinuclear compartments (V) (A-B) and ZIKV particles (*) measuring approximately 40–50 nm in diameter in the endoplasmic reticulum cisternae (RER) (A, B) and in lysosomes (L) (C-D) were observed. Nucleocapsids were observed inside the rER (D). Thickening of the nuclear membrane and rough endoplasmic reticulum cisternae (rER) (black head arrow) (C), numerous lysosomes (L) (C, D) and vesicular compartments associated with rER (arrow) (C) measuring approximately 100 nm in diameter were observed. Nucleus (N).

Fig 7

C6/36 cells infected with ZIKV at different time points post-infection (A-C: 48 hr p.i., D: 72 hr p.i.). The presence of several phagasomes (*) was observed. Marked areas of the image A (numbers 1 and 2). Virus particles (arrow) inside cytoplasmic vesicles. Nucleus (N), viroplasm-like perinuclear compartments (V).

Fig 8. Increasing magnifications of ZIKV-infected C6/36 cells at 48 hr p.i.

Several large viroplasm-like perinuclear compartments (V) containing ZIKV particles in the lumen (arrow) were observed (A, B, C); ZIKV particles were also observed in lysosomes (L). Rough endoplasmic reticulum cisternae (rER), mitochondria (*), microtubules (m). N = Nucleus.

Fig 9. Uninfected Vero cells (negative controls) at different culture time points.

No cellular ultrastructural alterations were observed. (A) 24 hr of culture; (B) 48 hr of cultures; (C): 72 hr of culture. Nucleus (N), mitochondria (M).

Fig 10

Vero cell monolayers infected with ZIKV analyzed by transmission electron microscopy at 24 hr (A-D) and 72 hr p.i. (E). Numerous phagosomes (*)(A-B), nucleocapsids (n) inside the rough endoplasmic reticulum cisternae rER (C). ZIKV particles (black arrow) were observed inside in cytoplasm vesicles (D) and in lumen of electron dense viroplasm-like structures (VL) (E). Nucleus (N), microtubules (m).