Persistent Humoral Immune Responses in the CNS Limit Recovery of Reactivated Murine Cytomegalovirus

Background: Experimental infection of the mouse brain with murine CMV (MCMV) elicits neuroimmune responses that terminate acute infection while simultaneously preventing extensive bystander damage. Previous studies have determined that CD8 + T lymphocytes are required to restrict acute, productive MCMV infection within the central nervous system (CNS). In this study, we investigated the contribution of humoral immune responses in control of MCMV brain infection. Methodology/Principal Findings: Utilizing our MCMV brain infection model, we investigated B-lymphocyte-lineage cells and assessed their role in controlling the recovery of reactivated virus from latently infected brain tissue. Brain infiltrating leukocytes were first phenotyped using markers indicative of B-lymphocytes and plasma cells. Results obtained during these studies showed a steady increase in the recruitment of B-lymphocyte-lineage cells into the brain throughout the timecourse of viral infection. Further, MCMV-specific antibody secreting cells (ASC) were detected within the infiltrating leukocyte population using an ELISPOT assay. Immunohistochemical studies of brain sections revealed co-localization of CD138 + cells with either IgG or IgM. Additional immunohistochemical staining for MCMV early antigen 1 (E1, m112–113), a reported marker of viral latency in neurons, confirmed its expression in the brain during latent infection. Finally, using B-cell deficient (Jh 2/2) mice we demonstrated that B-lymphocytes control recovery of reactivated virus from latently-infected brain tissue. A significantly higher rate of reactivated virus was recovered from the brains of Jh 2/2 mice when compared t

Murine Cytomegalovirus Infection of Neural Stem Cells Alters Neurogenesis in the Developing Brain

Congenital cytomegalovirus (CMV) brain infection causes serious neuro-developmental sequelae including: mental retardation, cerebral palsy, and sensorineural hearing loss. But, the mechanisms of injury and pathogenesis to the fetal brain are not completely understood. The present study addresses potential pathogenic mechanisms by which this virus injures the CNS using a neonatal mouse model that mirrors congenital brain infection. This investigation focused on, analysis of cell types infected with mouse cytomegalovirus (MCMV) and the pattern of injury to the developing brain.We used our MCMV infection model and a multi-color flow cytometry approach to quantify the effect of viral infection on the developing brain, identifying specific target cells and the consequent effect on neurogenesis. In this study, we show that neural stem cells (NSCs) and neuronal precursor cells are the principal target cells for MCMV in the developing brain. In addition, viral infection was demonstrated to cause a loss of NSCs expressing CD133 and nestin. We also showed that infection of neonates leads to subsequent abnormal brain development as indicated by loss of CD24(hi) cells that incorporated BrdU. This neonatal brain infection was also associated with altered expression of Oct4, a multipotency marker; as well as down regulation of the neurotrophins BDNF and NT3, which are essential to regulate the birth and differentiation of neurons during normal brain development. Finally, we report decreased expression of doublecortin, a marker to identify young neurons, following viral brain infection.MCMV brain infection of newborn mice causes significant loss of NSCs, decreased proliferation of neuronal precursor cells, and marked loss of young neurons

Plasma cell recruitment and retention in the brain following MCMV infection.

<p><b>A</b>. Single cell suspensions of infiltrating brain leukocytes from MCMV-infected mice were obtained and analyzed at the indicated time points. Contour plots depicting percentages of CD38⁺ and CD138⁺ double positive cells from the gated CD45hi population are shown. These cells also stained negative for CD3 and CD19. <b>B</b>. Data showing the mean (±SEM) absolute number of cells within the infiltrating CD45^(hi)CD3⁻CD19⁻ population pooled from 3 independent experiments. <b>C</b>. Immunofluorescent staining showing the distribution of CD138⁺ plasma cells in both the ventricles and brain parenchyma at 30 d p.i. (magnification 20×).</p

Entry and long-term persistence of B lymphocytes following MCMV brain infection.

<p>Single cell suspensions of brain tissue obtained from MCMV-infected mice (2–4 animals) per time point were banded on a 70% Percoll cushion. Brain leukocytes were collected and labeled with APC-conjugated Abs specific for CD45, Cy7-APC-labeled anti-CD11b, PE-CY7 anti-CD3, FITC anti-CD19 and analyzed using flow cytometry and FlowJo software. <b>A</b>. Representative FACS plots showing the percentages of CD45⁺CD19⁺ B lymphocytes within the infiltrating CD45^(hi)CD3⁻ population within infected brains at 7, 14, 21 and 30 d p.i.. <b>B</b>. FITC-labeled anti-CD19 Abs were used to determine the total number of CD19⁺ B lymphocytes within the infiltrating CD45^(hi)CD3⁻ population. Data shown are mean (±SEM) absolute number of infiltrating cells pooled from 3 independent experiments. <b>C</b>. mRNA levels for B-cell trophic factors were measured in total brain homogenates at the indicated time-points. <b>D</b>. Primed CD19⁺ B-cells from β-actin promoter-luciferase transgenic BALB/c mice were transferred via tail vein injection into MHC-matched, B-cell deficient (Jh^−/−) recipients 1 d prior to viral infection. Representative dorsal bioluminescence images of 2 saline-injected controls and 3 infected recipient animals are shown at the indicated time-points.</p

Reactivated infectious virus is recovered more efficiently from the brains of B-cell deficient animals.

<p><b>A</b>. Periventricular brain tissue obtained from latently-infected animals (>30 d p.i.) was cut into 1 mm pieces and placed onto primary murine glial cell cultures. At 10 d post-explant, these cultures were stained for MCMV-IE1 expression, indicative of viral reactivation, and processed for real-time PCR (MCMV IE1 and gB genes). <b>B</b>. Explant reactivation results in Wt versus Jh^−/− mice as determined using real-time PCR for IE1 and gB, as well as IE1 antigen staining. Data are shown as the number positive animals over total number examined (+/n). **p<0.01 Jh^−/− versus Wt animals.</p

Latently-infected neurons expresses MCMV early 1 (E1, m112–113), but not immediate early 1 (IE1) antigen.

<p>Coronal murine brain sections were prepared and stained for viral proteins at indicated time points. <b>A</b>. Detection of MCMV IE1 and <b>B</b>. E1antigens in wild-type mice during acute infection (5 d p.i.), lower magnifications (20×) demonstrate that MCMV is localized to cells surrounding the ventricles. <b>C–F</b>. Tissue slices from infected brains were stained with an antibody to MCMV E1 during chronic infection (30 d p.i.). Cells positive for E1 antigen in the cortex and hippocampus of wild-type (<b>C</b>), as well as B-cell-deficient (Jh^−/−) animals (<b>D, E and F</b>), are shown. Micrographs at higher magnification (40×) demonstrate the neuronal morphology of infected cells.</p

Correlation of ELISA method with three other automated serological tests for the detection of anti-SARS-CoV-2 antibodies.

Public health emergency of SARS-CoV-2 has facilitated diagnostic testing as a related medical countermeasure against COVID-19 outbreak. Numerous serologic antibody tests have become available through an expedited federal emergency use only process. This paper highlights the analytical characteristic of an ELISA based assay by AnshLabs and three random access immunoassay (RAIA) by DiaSorin, Roche, and Abbott that have been approved for emergency use authorization (EUA), at a tertiary academic center in a low disease-prevalence area. The AnshLabs gave higher estimates of sero-prevalence, over the three RAIA methods. For positive results, AnshLabs had 93.3% and 100% agreement with DiaSorin or Abbott and Roche respectively. For negative results, AnshLabs had 74.3% and 78.3% agreement with DiaSorin and Roche or Abbott respectively. All discrepant samples that were positive by AnshLabs and negative by RAIA tested positive by all-in-one step SARS-CoV-2 Total (COV2T) assay performed on the automated Siemens Advia Centaur XPT analyzer. None of these methods, however, are useful in early diagnosis of SARS-CoV-2