Correction: Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm

<p>Correction: Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm</p

Generation of WSN PA-GFP influenza virus.

<p>Schematic of the reverse genetics pHW2000 plasmid created by inserting the coding region of full length GFP into the C-terminal domain of the WSN PA open reading frame (A). This insertion included a duplication of 162 nucleotides of WSN PA prior to the 5′ non-coding region (NCR) depicted in the black rectangle. Western blot of purified recombinant WT WSN or WSN PA-GFP virus grown in embryonated eggs (B). The GFP signal corresponds to the appropriate size of a PA-GFP fusion protein. Viral NP protein was used as a control to ensure loading of equivalent amounts of virus. Immunofluorescence of MDCK cells infected with WSN PA-GFP (MOI = 3) for 16 hpi with anti-influenza NP antibody (C). Images to the right are enlarged regions identified by the dashed square. White arrows show areas of co-localization. All scale bars are 5 µm.</p

Proposed model for vRNA assembly.

<p>The data presented in this paper supports a model of vRNA assembly whereby 1) vRNA segments export from the nucleus as complexes containing multiple vRNA segments, 2) cytoplasmic foci containing vRNA segments transport through the cytoplasm and 3) these cytoplasmic foci fuse together in the cytoplasm prior to budding. Each vRNA segment is shown as a loop where the ends are associated with a heterotrimeric polymerase complex identified as 3 circles. The PA protein of the polymerase complex is shown in green since this was the protein used as a surrogate for vRNA transport. The cellular Rab11a-containing endosomes that colocalize with vRNA-containing cytoplasmic foci are indicated.</p

Colocalization of WSN PA-GFP and influenza vRNA segments.

<p>Fluorescent <i>in situ</i> hybridization (FISH) for viral RNA segments 1 (PB2) and 4 (HA) on MDCK (A) or A549 cells (B) cells 16 hpi with WSN PA-GFP (MOI = 0.5). Images to the right are enlarged regions identified by the dashed box. White arrowheads show colocalization of PA-GFP and vRNA, and open arrowheads indicate GFP foci not colocalized with viral RNA signal. DAPI marks the cellular nucleus. Scale bars are 5 µm in all panels of part A. In part B scale bars are 10 µm in the whole cell images and 2 µm in length in the enlarged region.</p

Colocalization coefficients between influenza vRNA segments.

<p>Graphical representation of the Pearson correlation coefficient values between each vRNA segment and all other vRNA segments from exclusively cytoplasmic staining. Dual staining of infected cells with FISH probes against the same target served as a positive control and is indicated in red. Each spot represents a single analyzed infected cell. Gray shading highlights the region of low colocalization based on the Pearson correlation coefficient.</p

Enhanced inflammation in New Zealand white rabbits when MERS-CoV reinfection occurs in the absence of neutralizing antibody

<div><p>The Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that was first detected in humans in 2012 as a cause of severe acute respiratory disease. As of July 28, 2017, there have been 2,040 confirmed cases with 712 reported deaths. While many infections have been fatal, there have also been a large number of mild or asymptomatic cases discovered through monitoring and contact tracing. New Zealand white rabbits are a possible model for asymptomatic infection with MERS-CoV. In order to discover more about non-lethal infections and to learn whether a single infection with MERS-CoV would protect against reinfection, we inoculated rabbits with MERS-CoV and monitored the antibody and inflammatory response. Following intranasal infection, rabbits developed a transient dose-dependent pulmonary infection with moderately high levels of viral RNA, viral antigen, and perivascular inflammation in multiple lung lobes that was not associated with clinical signs. The rabbits developed antibodies against viral proteins that lacked neutralizing activity and the animals were not protected from reinfection. In fact, reinfection resulted in enhanced pulmonary inflammation, without an associated increase in viral RNA titers. Interestingly, passive transfer of serum from previously infected rabbits to naïve rabbits was associated with enhanced inflammation upon infection. We further found this inflammation was accompanied by increased recruitment of complement proteins compared to primary infection. However, reinfection elicited neutralizing antibodies that protected rabbits from subsequent viral challenge. Our data from the rabbit model suggests that people exposed to MERS-CoV who fail to develop a neutralizing antibody response, or persons whose neutralizing antibody titers have waned, may be at risk for severe lung disease on re-exposure to MERS-CoV.</p></div

Viral RNA composition and spatial location of cytoplasmic foci.

<p>3D rendering of an MDCK cell infected with WT WSN (MOI = 3) for 8 hpi were stained with probe B from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003971#ppat.1003971.s010" target="_blank">Table S1</a>: WSN PB2 Quasar 570 (red spots), PB1 Alexa-Fluor 488 (green spots), PA Cal Fluor Red 590 (orange spots), and NP Quasar 670 (yellow spots) (A). The DAPI-stained nucleus is labeled in blue. An enlarged region of the 3D image was tilted 90° in the z-direction to provide an axial-view of the cytoplasmic foci and nucleus. Scale bars are 4 µm in the whole cell image and 2 µm in the rotated images. The total number of foci containing 1, 2, 3, or 4 vRNA segments was quantified within a given cell (B). Each bar represents the average from 3 independently analyzed cells with standard error indicated. The distance from the nucleus for each focus from four independent cells was calculated (C). The proportion of foci containing 4,3,2,or 1 distinct vRNA segment with a given range from the nucleus is represented graphically as a scatter plot. Each spot is an average from 4 independent cells and the standard error is indicated.</p

Visualization of four distinct influenza viral gene segments within a single cell.

<p>MDCK cells were infected with wild type (WT) WSN at a multiplicity of infection (MOI) of 3 for 8 hours post infection (hpi) and then imaged with FISH probes directed against four distinct vRNA species. The first row displays a cell probed for influenza vRNA segments PB2, PB1, PA, and NP. Row 3 contains images of a cell probed for the other 4 influenza vRNA segments: HA, NA, M and NS. The second and fourth row of images show an enlargement of the area defined by the dashed boxes in the first and third rows. Solid white arrowheads identify a cytoplasmic focus with all four distinct vRNA segments, turquoise arrowheads indicate a focus with only three vRNA species, and open arrowheads show a focus with only two vRNA species. All scale bars are 10 µm. DAPI marks the cellular nucleus.</p

Fusion event of an WSN PA-GFP focus in the cytoplasm of MDCK cells.

<p>Maximum intensity projection from inverted selective-plane illumination microscopy (iSPIM) movie of MDCK cells infected with WSN PA-GFP (MOI = 5) at 16 hpi, 36 seconds (s) after onset of tracking (A). The red-boxed region is highlighted in (B and C), and the red star indicates the region where colocalization of two GFP foci (marked in red and blue arrows) occurred. A single WSN PA-GFP focus trajectory; each red dot is a spot tracked in time, the yellow line is the overall trace, and white arrows indicate the direction of the PA-GFP movement (B). The total track duration is 46 s. Part C contains still frames of the red-boxed region in part A from 26–44 s, and illustrates colocalization and subsequent fusion of two foci. The red arrow indicates the PA-GFP focus that is being tracked and the blue arrow indicates the PA-GFP focus with which it fuses. All scale bars are 2 µm. An intensity versus depth plot of the colocalized foci, indicated by the red star in part A, demonstrates a single peak indicating that the fusion occurs in a single diffraction-limited spot (D). An intensity vs. time plot of the fused focus compared to an adjacent stationary focus, identified by the white circle in part C, during the same time frame (E). The asterisk marks the time corresponding to the colocalization event.</p

CD3+ cells in the lungs following primary infection and reinfection.

<p>DAB images from primary infection (A) and reinfection (B). Immunofluorescence (IF) image of CD3 (green) and virus antigen (red) within the same perivascular region following reinfection (C). DAB images from day 3 post-infection at 10x, bar equivalent to 100μm. IF images at 40x, bar equivalent to 20μm.</p