Additional file 1: of Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro virus and Pichinde virus

Absolute quantification of PTV and PICV RNA molecules using dd PCR. Viral cell culture supernatants spiked in water at specific PFU/ml were 10-fold serially diluted, extracted, and tested using the QX200 digital droplet system. Log10 transformation of PFU/ml of the spiked dilutions versus counts/Îźl determined by ddPCR are graphed. Linear regression analysis along with slope, R square value, and equation are shown. (PDF 112 kb

Neuropathogenesis of Zika Virus in a Highly Susceptible Immunocompetent Mouse Model after Antibody Blockade of Type I Interferon

<div><p>Animal models are needed to better understand the pathogenic mechanisms of Zika virus (ZIKV) and to evaluate candidate medical countermeasures. Adult mice infected with ZIKV develop a transient viremia, but do not demonstrate signs of morbidity or mortality. Mice deficient in type I or a combination of type I and type II interferon (IFN) responses are highly susceptible to ZIKV infection; however, the absence of a competent immune system limits their usefulness for studying medical countermeasures. Here we employ a murine model for ZIKV using wild-type C57BL/6 mice treated with an antibody to disrupt type I IFN signaling to study ZIKV pathogenesis. We observed 40% mortality in antibody treated mice exposed to ZIKV subcutaneously whereas mice exposed by intraperitoneal inoculation were highly susceptible incurring 100% mortality. Mice infected by both exposure routes experienced weight loss, high viremia, and severe neuropathologic changes. The most significant histopathological findings occurred in the central nervous system where lesions represent an acute to subacute encephalitis/encephalomyelitis that is characterized by neuronal death, astrogliosis, microgliosis, scattered necrotic cellular debris, and inflammatory cell infiltrates. This model of ZIKV pathogenesis will be valuable for evaluating medical countermeasures and the pathogenic mechanisms of ZIKV because it allows immune responses to be elicited in immunologically competent mice with IFN I blockade only induced at the time of infection.</p></div

Viral titers of ZIKV in Wild-type Mice Treated with an IFNAR1-Blocking MAb.

<p>Five week old wild-type mice were treated with an IFNAR1-blocking MAb or PBS by intraperitoneal (IP) injection and then exposed to 6 log₁₀ of ZIKV strain DAK AR D 41525 subcutaneously (SC) or IP. When mice succumbed or were euthanized, tissues were collected, weighed, homogenized, and analyzed by qRT-PCR. Data are shown as PFU equivalents (PFUe) per gram (g) after normalization to a standard curve. Symbols represent the individual mice, the line represents the geometric mean, and the error bars represent the 95% confidence interval.</p

Wild-type Mice Treated with an IFNAR1-Blocking MAb are Susceptible to ZIKV.

<p>Five week old wild-type mice were treated with an IFNAR1-blocking MAb or PBS by intraperitoneal (IP) injection and then exposed to 6 log₁₀ of ZIKV strain DAK AR D 41525 subcutaneously (SC) or IP. Mice were monitored for survival (A) and weight loss shown as percent change in baseline prior to infection (B). ZIKV RNA in serum was determined on day 4 post-infection (PI) (C) and when the mice were euthanized (D) by qRT-PCR. Data are shown as PFU equivalents (PFUe) per milliliter after normalization to a standard curve. Symbols represent the individual mice, the line represents the geometric mean, and the error bars represent the 95% confidence interval. The dotted line represents the assay limit of detection. Statistically significant differences are denoted by an asterisk (*p < 0.05; *p < 0.0001).</p

Histologic and ISH Findings in the spinal cord of ZIKV-Infected Wild-type Mice Treated with an IFNAR1-Blocking MAb and Uninfected Control Mice.

<p>(A-B) The spinal cord of a mouse that was exposed to ZIKV subcutaneously (SC) and euthanized on day 8 post-infection (PI). (A) Representative hematoxylin and eosin staining showed there is scattered necrotic cellular debris (representative areas indicated by circles), gliosis (representative cells indicated by black arrow), and perivascular cuffing (indicated by white arrow); scale bar represents 100 μm. (B) Representative ISH staining demonstrating that ZIKV RNA is detected throughout the same region; scale bar represents 200 μm. (C) ISH staining demonstrating massive ZIKV infection in the spinal cord of a mouse that was exposed to ZIKV intraperitoneally (IP) and was euthanized on day 7 PI; scale bar represents 100 μm. (D) Hematoxylin and eosin staining of spinal ganglion demonstrates a focal area of necrosis (indicated by an asterisk) with adjacent gliosis and few infiltrating neutrophils in a mouse exposed to ZIKV IP that succumbed on day 7 PI; scale bar represents 100 μm. (E) Representative hematoxylin and eosin staining in the spinal cord of an uninfected control mouse; scale bar represents 100 μm. Inset is a spinal ganglion from an uninfected control mouse; scale bar represents 100 μm. (F) Representative ISH staining demonstrating no ZIKV RNA is detected in the spinal cord of an uninfected control mouse; scale bar represents 200 μm. These findings are from one independent experiment where a total of 6 ZIKV-infected mouse spinal cords (3 uninfected controls) were analyzed by an unblinded, board-certified veterinary pathologist.</p

Histologic and ISH Findings in the Spleen of ZIKV-Infected Wild-type Mice Treated with an IFNAR1-Blocking MAb and Uninfected Control Mice.

<p>(A) Representative hematoxylin and eosin staining showed lymphocytolysis within the follicles (representative area indicated with an asterisk) in the spleen of a mouse exposed to ZIKV intraperitoneally (IP) that was euthanized on day 11 post-infection (PI); scale bar represents 100 μm. (B) Representative ISH staining demonstrating that multifocally, ZIKV RNA is detected in the lymphoid follicles in the spleen of a mouse exposed to ZIKV IP that was euthanized on day 11 PI; scale bar represents 200 μM (inset scale bar represents 50 μm). (C) Representative hematoxylin and eosin staining in the spleen of an uninfected control mouse; scale bar represents 100 μm. (D) Representative ISH staining demonstrating no ZIKV RNA is detected in the spleen of an uninfected control mouse; scale bar represents 100 μm. These findings are from one independent experiment where a total of 8 ZIKV-infected mouse spleens (3 uninfected controls) were analyzed by an unblinded, board-certified veterinary pathologist.</p

Iba1 and GFAP are Increased in the Brains of ZIKV-Infected Wild-type Mice Treated with an IFNAR1-Blocking MAb.

<p>(A-B) Brain sections from mice treated with IFNAR1-blocking MAb and infected with ZIKV or mock infected with PBS were imaged and analyzed by infrared imaging. Representative images showed increased Iba1 and GFAP staining in brain sections from ZIKV-infected mice compared to PBS-inoculated mice. (B) The average infrared intensity, expressed as fold increase relative to PBS, was significantly increased in ZIKV-infected mice compared to PBS-inoculated mice for both Iba1 (PBS: 0.9929 ± 0.1614, n = 5; ZIKV: 3.725 ± 0.2415, n = 10; *p < 0.0001, t-test) and GFAP (PBS: 1.000 ± 0.1022, n = 5; ZIKV: 2.429 ± 0.1252, n = 10; *p < 0.0001, t-test). (C) Representative images of Iba1 and GFAP immunofluorescent labeling of Iba1 (green) and GFAP (red) in brain sections of mock- or ZIKV-infected mice. Scale bar represents 50 μm.</p

Histologic and ISH Findings in the Hippocampus of ZIKV-Infected Wild-type Mice Treated with an IFNAR1-Blocking MAb and Uninfected Control Mice.

<p>(A) The hippocampus of a mouse exposed to ZIKV intraperitoneally (IP) that was euthanized on day 11 post-infection (PI). Representative hematoxylin and eosin staining showed neuropil vacuolation (representative area indicated with an asterisk), microgliosis (representative cells indicated by arrows), and necrotic cellular debris (representative area indicated with a circle); scale bar represents 100 μm. (B) The hippocampus of a mouse exposed to ZIKV IP that was euthanized on day 7 PI. The representative ISH staining demonstrates massive ZIKV infection of the brain; scale bar represents 500 μm (inset picture scale bar represents 100 μm). (C-D) The hippocampus of a mouse exposed to ZIKV IP that was euthanized on day 7 PI. (C) The representative hematoxylin and eosin staining demonstrates that pyramidal neurons are often necrotic (representative cells indicated by arrows) and the adjacent vacuolated neuropil (representative area indicated with an asterisk) contains necrotic scattered cellular debris (representative area indicated by a circle); scale bar represents 50 μm. (D) Representative ISH staining demonstrating that ZIKV RNA is detected in the same region; scale bar represents 200 μm. (E) Representative hematoxylin and eosin staining in the hippocampus of an uninfected control mouse; scale bar represents 100 μm. (F) Representative ISH staining demonstrating no ZIKV RNA is detected in the hippocampus of an uninfected control mouse; scale bar represents 200 μm. These findings are from one independent experiment where a total of 10 ZIKV-infected mouse brains (3 uninfected controls) were analyzed by an unblinded, board-certified veterinary pathologist.</p

Genomic Comparison of <em>Escherichia coli</em> O104:H4 Isolates from 2009 and 2011 Reveals Plasmid, and Prophage Heterogeneity, Including Shiga Toxin Encoding Phage stx2

<div><p>In May of 2011, an enteroaggregative <em>Escherichia coli</em> O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical <em>E. coli</em> O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C–3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL–2050 and 2009EL–2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the <em>mer/tet</em> antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of <em>Yersinia pestis</em>. In addition, multiphenotype analysis showed that 2009EL–2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing <em>E. coli</em> O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.</p> </div

Analysis of prophage content of O104:H4 strains.

<p><b>A)</b> Location of prophages in the genomes of the EAggEc strains analyzed in this study. Linear maps of the genomes and the location of prophages as boxes are shown. All genome sequences have the same starting position as described in methods. The prophage locations are drawn to scale. Phages are color-coded according to similarity; for example the red box indicates the stx2a phages. The exact genomic locations of the prophages in their respective genomes are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048228#pone-0048228-t006" target="_blank">Table 6</a>. <b>B)</b> Architecture of individual prophages. Phage proteins are colored according to their predicted functions. The <i>stx2ab</i> genes are boxed in red; the island of pyrimidine biosynthesis genes identified as a part of this prophage by Phage_Finder is indicated by the blue box. In all cases the <i>int</i> genes are positioned on the left, regardless of the orientation of the prophage within the chromosome.</p