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. 2017 Jan 31;11(1):e0005346.
doi: 10.1371/journal.pntd.0005346. eCollection 2017 Jan.

Modeling Powassan virus infection in Peromyscus leucopus, a natural host

Luwanika Mlera  1 , Kimberly Meade-White  2 , Greg Saturday  2 , Dana Scott  2 , Marshall E Bloom  1
Affiliations

Modeling Powassan virus infection in Peromyscus leucopus, a natural host

Luwanika Mlera et al. PLoS Negl Trop Dis. .

Abstract

The tick-borne flavivirus, Powassan virus (POWV) causes life-threatening encephalitis in humans in North America and Europe. POWV is transmitted by ixodid tick vectors that feed on small to medium-sized mammals, such as Peromyscus leucopus mice, which may serve as either reservoir, bridge or amplification hosts. Intraperitoneal and intracranial inoculation of 4-week old Peromyscus leucopus mice with 103 PFU of POWV did not result in overt clinical signs of disease. However, following intracranial inoculation, infected mice seroconverted to POWV and histopathological examinations revealed that the mice uniformly developed mild lymphocytic perivascular cuffing and microgliosis in the brain and spinal cord from 5 to 15 days post infection (dpi), suggesting an early inflammatory response. In contrast, intracranial inoculation of 4-week old C57BL/6 and BALB/c mice was lethal by 5 dpi. Intraperitoneal inoculation was lethal in BALB/c mice, but 40% (2/5) of C57BL/6 mice survived. We concluded that Peromyscus leucopus mice infected i.c. with a lethal dose of POWV support a limited infection, restricted to the central nervous system and mount an antibody response to the virus. However, they fail to develop clinical signs of disease and are able to control the infection. These results suggest the involvement of restriction factors, and the mechanism by which Peromyscus leucopus mice restrict POWV infection remains under study.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Outcomes of i.c. inoculation of POWV.
(A) Survival curve of P. leucopus, BALB/c and C57BL/6 mice after i.c. inoculation. (B) Terminal POWV (+)RNA copy numbers in brain homogenates determined by qPCR. 28 dpi copy numbers are presented for P. leucopus because these mice survived to the experimental end-point, set at 28 days. **** p<0.0001 (t-test).
Fig 2
Fig 2. POWV replication kinetics in P. leucopus following i.c. inoculation.
(A) POWV titers from brain homogenates harvested from i.c. inoculated P. leucopus determined by immunofocus assay. (B) POWV (+)RNA copy numbers in brain after i.c. inoculation. (C) POWV (+)RNA copy numbers in blood after i.c. inoculation. * p<0.05; ns: not significant (t-test).
Fig 3
Fig 3. Analysis of i.p. inoculation of POWV in BALB/c and C57BL/6 mice.
(A) Survival curve for i.p. inoculated BALB/c, C57BL/6 and P. leucopus mice. (B) Changes in weights of BALB/c mice after i.p. inoculation of POWV. (C) Changes in weights of BALB/c mice after ip inoculation of POWV. (D) POWV (+)RNA copy numbers in brains of i.p.-inoculated BALB/c and C57BL/6 mice. The red dot for BALB/c mice denotes RNA copy numbers from the mouse which succumbed first. The red square for C57BL/6 mice denotes the mouse that succumbed at 8 dpi, the black squares represent the mice that succumbed at 11 dpi and the survivors are denoted by the purple squares. ns: not significant (t-test).
Fig 4
Fig 4. Hematoxylin and eosin-stained sections of brain and spinal cords obtained from i.c.-inoculated mice.
The terminal brain sections of C57BL/6 (5 dpi) and BALB/c (4 dpi) mice show moderate lymphocytic perivascular cuffing and microgliosis, but these lesions were mild in P. leucopus brains (image obtained from mouse sacrificed at 15 dpi). Peromyscus leucopus spinal cords showed minimal lymphocytic perivascular cuffing and microgliosis, but BALB/c and C57BL/6 spinal cords showed moderate lesions. Normal meninges were observed for Peromyscus leucopus, but the meninges of BALB/c and C57BL/6 mice had moderate lymphocytic infiltrates.
Fig 5
Fig 5. Distribution of POWV RNA in the CNS of i.c. inoculated determined by in situ hybridization.
(A) POWV (+)RNA (stained brown) in the brain and spinal cord of i.c. inoculate P. leucopus mice. (i) Negative control showing no POWV (+)RNA. (ii) Diffuse detection of (+)RNA in the olfactory bulb (magnification x20). (iii) Close up image of the olfactory bulb (magnification x200). (iv) Multifocal detection of POWV (+)RNA in the paraventricular region of the brain. (v) Multifocal detection of POWV (+) RNA in the cerebellum. (vi) Multifocal detection of POWV (+)RNA in the spinal cord. (B) Detection of POWV negative RNA strand ((-)RNA) in the brains of Peromyscus leucopus, BALB/c and C57BL/6 mouse brains after ic inoculation. We used probes targeting the (-)RNA of POWV, which is only present during virus replication. (i) No viral RNA was detected in the mock-inoculated P. leucopus control. (ii) POWV (-)RNA was strongly and diffusely detected in the olfactory lobe of i.c. inoculated P. leucopus. (iii) Detection of diffuse POWV (-)RNA in the brain of an i.c. inoculated C57BL/6 mouse. (iv) Detection of diffuse POWV (-)RNA in the brain of an i.c. inoculated BALB/c mouse.
Fig 6
Fig 6. Detection of POWV (+)RNA in the brain and spinal cord by in situ hybridization after i.c. inoculation.
Diffuse positivity was observed in the brains of both BALB/c and C57BL/6 mice at 4 or 5 dpi. Positivity in the spinal cord was diffuse in BALB/c mice, but multi-focal in C57BL/6 mice. No viral RNA could be visualized in the brain and spinal cord of P. leucopus mice at 28 dpi.
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The study was supported by the Division of Intramural Research of the NIAID/NIH. The funders had no role in in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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