Contribution of a single host genetic locus to mouse adenovirus type 1 infection and encephalitis

Abstract

Susceptibility to mouse adenovirus type 1 (MAV-1) is mouse strain dependent; susceptible mice die from hemorrhagic encephalomyelitis. The MAV-1 susceptibility quantitative trait locus Msq1 accounts for ~40% of the phenotypic (brain viral load) variance that occurs between resistant BALB/c and susceptible SJL mice after MAV-1 infection. Using an interval-specific congenic mouse strain (C.SJL-Msq1(SJL)), in which the SJL-derived allele Msq1(SJL) is present in a BALB/c background, we demonstrate that Msq1(SJL) controls the development of high brain viral titers in response to MAV-1 infection, yet does not account for the total extent of brain pathology or mortality in SJL mice. C.SJL-Msq1(SJL) mice had disruption of the blood-brain barrier and increased brain water content after MAV-1 infection, but these effects occurred later and were not as severe, respectively, as those noted in infected SJL mice. As expected, BALB/c mice showed minimal pathology in these assays. Infection of SJL- and C.SJL-Msq1(SJL)-derived primary mouse brain endothelial cells resulted in loss of barrier properties, whereas BALB/c-derived cells retained their barrier properties despite being equally capable of supporting MAV-1 infection. Finally, we provide evidence that organ pathology and inflammatory cell recruitment to the brain following MAV-1 infection were both influenced by Msq1. These results validate Msq1 as an important host factor in MAV-1 infection and refine the major role of the locus in development of MAV-1 encephalitis. They further suggest that additional host factors or gene interactions are involved in the mechanism of pathogenesis in MAV-1-infected SJL mice.

Importance: A successful viral infection requires both host and viral factors; identification of host components involved in viral replication and pathogenesis is important for development of therapeutic interventions. A genetic locus (Msq1) controlling mouse adenovirus type 1 (MAV-1) brain infection was previously identified. Genes in Msq1 belong to the same family of genes associated with susceptibility to other encephalitic viruses, HIV-1 and West Nile virus. We constructed an interval-specific congenic mouse strain to examine the contribution of Msq1 to MAV-1 susceptibility and brain morbidity. We compared infected resistant, susceptible, and congenic mice regarding known MAV-1 disease manifestations in the brain (survival, viral loads, blood-brain barrier disruption, edema, mouse brain endothelial cell barrier properties, pathology, and inflammatory cell recruitment) to determine the extent to which Msq1 influences MAV-1 infection outcome. Our results showed that Msq1 is a critical host genetic factor that controls many aspects of MAV-1 infection.

FIG 1

Congenic mice are susceptible to MAV-1 infection. (A) Mice of the indicated strains were infected with 10² PFU of MAV-1, and brains were harvested 8 dpi. Viral loads in brain homogenates were measured using capture ELISA. Each symbol represents the mean of three measurements per homogenate for an individual mouse; the number of mice is indicated below the axis. The means and standard deviations (SD) are indicated. Statistical significance was calculated by a two-tailed Mann-Whitney test. OD450, optical density at 450 nm. (B) SJL and C.SJL-Msq1^SJL (designated C.SJL-Msq1 here and in subsequent figures) mice were infected with either 10² PFU or 10⁴ PFU of virus. The numbers of mice in each group are indicated. For SJL mice infected at 10² PFU versus C.SJL-Msq1 mice infected at 10² PFU, P = 0.0001; for SJL mice infected at 10⁴ PFU versus C.SJL-Msq1 infected at 10⁴ PFU, P = 0.0022. Statistical significance for survival curves was calculated by the log-rank (Mantel-Cox) test.

FIG 2

Increased BBB (blood-brain barrier) permeability seen in SJL and C.SJL-Msq1^SJL mice. BALB/c, C.SJL-Msq1^BALB, SJL, and C.SJL-Msq1^SJL mice were either mock infected or infected with 10⁴ PFU of MAV-1, and brains were harvested 6 dpi. Sodium fluorescein levels in the brains and serum were measured in duplicate; the average amount of brain sodium fluorescein was normalized to average levels in serum for each mouse. After normalization, the amount of sodium fluorescein in brains of infected mice of the appropriate strain was determined and is represented as a ratio to the amount of dye in mock-infected brains. Each data point represents an individual mouse; numbers of mice in each group are indicated below the respective sample data. For SJL versus C.SJL-Msq1^BALB mice, P = 0.008; for C.SJL-Msq1^SJL versus C.SJL-Msq1^BALB mice, P = 0.034. Means and SDs are indicated. Statistical significance was calculated by one-way analysis of variance (ANOVA) (Kruskal-Wallis) with Dunn’s multiple comparison test.

FIG 3

Onset of BBB disruption is delayed in C.SJL-Msq1^SJL mice. Mice were either mock infected or MAV-1 infected at 10⁴ PFU and assayed for BBB disruption using Evans blue dye at 3, 4, and 5 dpi. (A) Viral loads of individual infected mouse brains were measured by capture ELISA. Each symbol represents the mean of three measurements per homogenate. (B) The amount of Evans blue dye in infected mouse brains is represented as a ratio to the amount of dye found in mock-infected brains for each individual strain. Each symbol represents the average of duplicate measurements for an individual mouse; numbers of mice in each group are indicated. Means and SDs are indicated. Statistical significance was calculated by a two-tailed Mann-Whitney test.

FIG 4

Edema in MAV-1-infected mouse strains. Mice of the indicated strains were either mock infected or MAV-1 infected (Inf) with 10⁴ PFU of MAV-1 and assayed 6 dpi. (A) Brain weights were determined prior to and after dehydration. Percent water content was calculated as described in Materials and Methods. (B and C) Ion contents of the brains represented in panel A were measured by flame photometry. Ion contents were then normalized to individual mouse brain weights. Each symbol represents a single mouse. Numbers of mice and means and SDs are indicated. Statistical significance was calculated by a two-tailed Mann-Whitney test.

FIG 5

SJL- and C.SJL-Msq1-derived pmBECs (primary mouse brain endothelial cells) lose BBB properties after MAV-1 infection. pmBECs from (A) BALB, (B) SJL, or (C) C.SJL-Msq1 mice were isolated, grown to confluence, and infected with MAV-1 at an MOI of 5. TEER (transendothelial electrical resistance) measured on the day of infection was normalized to 100%. After infection, measurements were taken at 24-h intervals in each sample well. Means and SDs at each time point are shown. Data for each graph were combined from the results of 3 independent experiments.

FIG 6

Growth curves of MAV-1 in SJL-, C.SJL-Msq1-, and BALB/c-derived pmBECs. pmBECs from SJL, C.SJL-Msq1, or BALB/c mice were isolated and infected at an MOI of 5. Cells were harvested at 0, 2, and 5 dpi and assayed for viral titers. Data are combined from the results of at least 2 experiments. Virus yields were determined by plaque assays performed in triplicate. Means and SDs are shown.

FIG 7

Increased pathology in brains of C.SJL-Msq1^SJL and SJL mice after MAV-1 infection. (A) Cerebellum sections from BALB/c, SJL, and C.SJL-Msq1 mice infected with 10⁴ PFU of virus at 6 dpi were stained with hematoxylin and eosin. Top panel, cerebellum from mock-infected BALB, SJL, and C.SJL-Msq1 mice. Bars, 100 µm. Middle panel, cerebellum from virus-infected BALB, SJL, and C.SJL-Msq1 mice. Bars, 50 µm. Bottom panel, high magnification of MAV-1-infected BALB, SJL, and C.SJL-Msq1 mice, showing vasculitis in the latter two images. Bars, 20 µm. (B) Histological changes were assigned scores and then totaled. Presence of vasculitis, microhemorrhages, perivascular edema, focal lesions, and meningeal lymphoid infiltrates each received a score of 1. Multifocal lesions received a score of 2. Each symbol represents the score of a single mouse. There were 5 mice per infected group, but 3 SJL mice died prior to analysis. Due to the small sample size, we did not determine whether differences between groups were statistically significant.

FIG 8

Increased inflammatory cell recruitment to brains of C.SJL-Msq1^SJL and SJL mice after MAV-1 infection. Mice from the indicated strains were infected with 10² PFU of MAV-1, and mice were euthanized 8 dpi. Cells isolated from brains from either mock-infected or MAV-1-infected mice were isolated and pooled and stained with relevant antibodies. The numbers of experiments for each group are indicated on the graphs. For the CD45^(hi) population, the numbers of cells were determined independently in at least 2 separate tubes per experiment.