Structural investigation of C6/36 and Vero cell cultures infected with a Brazilian Zika virus - Fig 6

<p><b>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.).</b> 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).</p

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

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

Zika Virus Outbreak in Rio de Janeiro, Brazil: Clinical Characterization, Epidemiological and Virological Aspects

<div><p>Background</p><p>In 2015, Brazil was faced with the cocirculation of three arboviruses of major public health importance. The emergence of Zika virus (ZIKV) presents new challenges to both clinicians and public health authorities. Overlapping clinical features between diseases caused by ZIKV, Dengue (DENV) and Chikungunya (CHIKV) and the lack of validated serological assays for ZIKV make accurate diagnosis difficult.</p><p>Methodology / Principal Findings</p><p>The outpatient service for acute febrile illnesses in Fiocruz initiated a syndromic clinical observational study in 2007 to capture unusual presentations of DENV infections. In January 2015, an increase of cases with exanthematic disease was observed. Trained physicians evaluated the patients using a detailed case report form that included clinical assessment and laboratory investigations. The laboratory diagnostic algorithm included assays for detection of ZIKV, CHIKV and DENV. 364 suspected cases of Zika virus disease were identified based on clinical criteria between January and July 2015. Of these, 262 (71.9%) were tested and 119 (45.4%) were confirmed by the detection of ZIKV RNA. All of the samples with sequence information available clustered within the Asian genotype.</p><p>Conclusions / Significance</p><p>This is the first report of a ZIKV outbreak in the state of Rio de Janeiro, based on a large number of suspected (n = 364) and laboratory confirmed cases (n = 119). We were able to demonstrate that ZIKV was circulating in Rio de Janeiro as early as January 2015. The peak of the outbreak was documented in May/June 2015. More than half of the patients reported headache, arthralgia, myalgia, non-purulent conjunctivitis, and lower back pain, consistent with the case definition of suspected ZIKV disease issued by the Pan American Health Organization (PAHO). However, fever, when present, was low-intensity and short-termed. In our opinion, pruritus, the second most common clinical sign presented by the confirmed cases, should be added to the PAHO case definition, while fever could be given less emphasis. The emergence of ZIKV as a new pathogen for Brazil in 2015 underscores the need for clinical vigilance and strong epidemiological and laboratory surveillance.</p></div

Detection of ZIKV RNA and viral load quantification from C6/36 and Vero cell supernatants at 24, 48 and 72 hr p.i. by quantitative real-time RT-PCR.

<p>Detection of ZIKV RNA and viral load quantification from C6/36 and Vero cell supernatants at 24, 48 and 72 hr p.i. by quantitative real-time RT-PCR.</p

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

<p>(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.</p

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

<p>(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.</p

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

<p>(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.</p

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

<p>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).</p

Baseline characteristics of confirmed and unconfirmed ZIKV disease patients.

<p>Baseline characteristics of confirmed and unconfirmed ZIKV disease patients.</p

Time series for number of cases confirmed (gray background) and not confirmed (white background) for ZIKV between January 1, 2015 and July 31, 2015 in Rio de Janeiro State.

<p>Time series for number of cases confirmed (gray background) and not confirmed (white background) for ZIKV between January 1, 2015 and July 31, 2015 in Rio de Janeiro State.</p