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

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 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

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

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

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