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Although vaccination is broadly used in North American swine breeding herds, managing swine influenza is challenging primarily due to the continuous evolution of influenza A virus (IAV) and the ability of the virus to transmit among vaccinated pigs. Studies that have simultaneously assessed the impact of vaccination on the emergence of IAV reassortment and genetic variation in pigs are limited. Here, we directly sequenced 28 bronchoalveolar lavage fluid (BALF) samples collected from vaccinated and unvaccinated pigs co-infected with H1N1 and H3N2 IAV strains, and characterized 202 individual viral plaques recovered from 13 BALF samples. We identified 54 reassortant viruses that were grouped in 17 single and 16 mixed genotypes. Notably, we found that prime-boost vaccinated pigs had less reassortant viruses than nonvaccinated pigs, likely due to a reduction in the number of days pigs were co-infected with both challenge viruses. However, direct sequencing from BALF samples revealed limited impact of vaccination on viral variant frequency, evolutionary rates, and nucleotide diversity in any IAV coding regions. Overall, our results highlight the value of IAV vaccination not only at limiting virus replication in pigs but also at protecting public health by restricting the generation of novel reassortants with zoonotic and/or pandemic potential.Swine influenza A viruses cause severe illness among pigs and financial losses on pig farms worldwide. These viruses can also infect humans and have caused deadly human pandemics in the past. Influenza A viruses are dangerous because viruses can be transferred between humans, birds and pigs. These co-infections can allow the viruses to swap genetic material. Viral genetic exchanges can result in new virus strains that are more dangerous or that can infect other types of animals more easily. Farmers vaccinate their pigs to control the swine influenza A virus. The vaccines are regularly updated to match circulating virus strains. But the virus evolves rapidly to escape vaccine-induced immunity, and infections are common even in vaccinated pigs. Learning about how vaccination affects the evolution of influenza A viruses in pigs could help scientists prevent outbreaks on pig farms and avoid spillover pandemics in humans. Li et al. show that influenza A viruses are less likely to swap genetic material in vaccinated and boosted pigs than in unvaccinated animals. In the experiments, Li et al. collected swine influenza A samples from the lungs of pigs that had received different vaccination protocols. Next, Li et al. used next-generation sequencing to identify new mutations in the virus or genetic swaps among different strains. In pigs infected with both the H1N1 and H3N2 strains of influenza, the two viruses began trading genes within a week. But less genetic mixing occurred in vaccinated and boosted pigs because they spent less time infected with both viruses than in unvaccinated pigs. The vaccination status of the pig did not have much effect on how many new mutations occurred in the viruses. The experiments show that vaccinating and boosting pigs against influenza A viruses may protect against genetic swapping among influenza viruses. If future studies on pig farms confirm the results, the information gleaned from the study could help scientists improve farm vaccine protocols to further reduce influenza risks to animals and people.

Broad-spectrum antiviral therapies hold promise as a first-line defense against emerging viruses by blunting illness severity and spread until vaccines and virus-specific antivirals are developed. The nucleobase favipiravir, often discussed as a broad-spectrum inhibitor, was not effective in recent clinical trials involving patients infected with Ebola virus or SARS-CoV-2. A drawback of favipiravir use is its rapid clearance before conversion to its active nucleoside-5′-triphosphate form. In this work, we report a synergistic reduction of flavivirus (dengue, Zika), orthomyxovirus (influenza A), and coronavirus (HCoV-OC43 and SARS-CoV-2) replication when the nucleobases favipiravir or T-1105 were combined with the antimetabolite 6-methylmercaptopurine riboside (6MMPr). The 6MMPr/T-1105 combination increased the C-U and G-A mutation frequency compared to treatment with T-1105 or 6MMPr alone. A further analysis revealed that the 6MMPr/T-1105 co-treatment reduced cellular purine nucleotide triphosphate synthesis and increased conversion of the antiviral nucleobase to its nucleoside-5′-monophosphate, -diphosphate, and -triphosphate forms. The 6MMPr co-treatment specifically increased production of the active antiviral form of the nucleobases (but not corresponding nucleosides) while also reducing levels of competing cellular NTPs to produce the synergistic effect. This in-depth work establishes a foundation for development of small molecules as possible co-treatments with nucleobases like favipiravir in response to emerging RNA virus infections.

Blastocyst complementation combined with gene editing is an emerging approach in the field of regenerative medicine that could potentially solve the worldwide problem of organ shortages for transplantation. In theory, blastocyst complementation can generate fully functional human organs or tissues, grown within genetically engineered livestock animals. Targeted deletion of a specific gene(s) using gene editing to cause deficiencies in organ development can open a niche for human stem cells to occupy, thus generating human tissues. Within this review, we will focus on the pancreas, liver, heart, kidney, lung, and skeletal muscle, as well as cells of the immune and nervous systems. Within each of these organ systems, we identify and discuss (i) the common causes of organ failure; (ii) the current state of regenerative therapies; and (iii) the candidate genes to knockout and enable specific exogenous organ development via the use of blastocyst complementation. We also highlight some of the current barriers limiting the success of blastocyst complementation.

The magnitude of SARS-CoV-2–specific T cell responses correlates inversely with human disease severity, suggesting T cell involvement in primary control. Whereas many COVID-19 vaccines focus on establishing humoral immunity to viral spike protein, vaccine-elicited T cell immunity may bolster durable protection or cross-reactivity with viral variants. To better enable mechanistic and vaccination studies in mice, we identified a dominant CD8 T cell SARS-CoV-2 nucleoprotein epitope. Infection of human ACE2 transgenic mice with SARS-CoV-2 elicited robust responses to H2-Db/N219-227, and 40% of HLA-A*02+ COVID-19 PBMC samples isolated from hospitalized patients responded to this peptide in culture. In mice, i.m. prime-boost nucleoprotein vaccination with heterologous vectors favored systemic CD8 T cell responses, whereas intranasal boosting favored respiratory immunity. In contrast, a single i.v. immunization with recombinant adenovirus established robust CD8 T cell memory both systemically and in the respiratory mucosa.

Influenza A viruses evolve rapidly to escape host immunity. In swine, this viral evolution has resulted in the emergence of multiple H1 and H3 influenza A virus (IAV) lineages in the United States (US) pig populations. The heterologous prime-boost vaccination strategy is a promising way to deal with diverse IAV infection in multiple animal models. However, whether or not this vaccination strategy is applicable to US swine to impart immunity against infection from North American strains of IAV is still unknown. We performed a vaccination-challenge study to evaluate the protective efficacy of using multivalent inactivated vaccine and/or a live attenuated IAV vaccine (LAIV) in pigs following multiple prime-boost vaccination protocols against a simultaneous H1N1 and H3N2 IAV infection. Our data show that pigs in the heterologous prime-boost vaccination group had more favorable outcomes consistent with a better response against virus challenge than non-vaccinated pigs. Additionally, delivering a multivalent heterologous inactivated vaccine boost to pigs following a single LAIV administration was also beneficial. We concluded the heterologous prime boost vaccination strategy may potentiate responses to suboptimal immunogens and holds the potential applicability to control IAV in the North American swine industry. However, more studies are needed to validate the application of this vaccination approach under field conditions.

Zika virus (ZIKV) exhibits a tropism for brain tumor cells and has been used as an oncolytic virus to target brain tumors in mice with modest effects on extending median survival. Recent studies have highlighted the potential for combining virotherapy and immunotherapy to target cancer. We postulated that ZIKV could be used as an adjuvant to enhance the long-term survival of mice with malignant glioblastoma and generate memory T-cells capable of providing long-term immunity against cancer remission. To test this hypothesis mice bearing malignant intracranial GL261 tumors were subcutaneously vaccinated with irradiated GL261 cells previously infected with the ZIKV. Mice also received intracranial injections of live ZIKV, irradiated ZIKV, or irradiated GL261 cells previously infected with ZIKV. Long-term survivors were rechallenged with a second intracranial tumor to examine their immune response and look for the establishment of protective memory T-cells. Mice with subcutaneous vaccination plus intracranial irradiated ZIKV or intracranial irradiated GL261 cells previously infected with ZIKV exhibited the greatest extensions to overall survival. Flow cytometry analysis of immune cells within the brains of long-term surviving mice after tumor rechallenge revealed an upregulation in the levels of T-cells, including CD4+ and tissue-resident memory CD4+ T-cells, in comparison to long-term survivors that were mock-rechallenged, and in comparison to naïve untreated mice challenged with intracranial gliomas. These results suggest that ZIKV can serve as an adjuvant to subcutaneous tumor vaccines that enhance long-term survival and generate protective tissue-resident memory CD4+ T-cells.

Understanding the immune responses against Porcine epidemic diarrhea virus (PEDV) is important to prevent infection and to design control strategies. We evaluated both systemic and mucosal immune responses to PEDV in pigs and assessed if prior exposure to virus protects against re-infection. Three-week-old pigs were infected with PEDV and immune response in blood, intestine, and mesenteric lymph node (MLN) was evaluated. At 30 dpi, virus exposed pigs were challenged with a field isolate of PEDV and immune response at 5 d post challenge was evaluated. We found that PEDV RNA persists in the intestine even after fecal shedding of the virus was stopped at 28 dpi and pigs previously exposed to PEDV are protected from virus shedding after re-infection. PEDV infection induced both humoral and cell mediated immune response with an increase in PEDV specific IgA and IgG antibodies in intestine and serum. Flow cytometry analysis showed a significantly higher frequency of B cells and lower frequency of T cells at 4 dpi. The frequency of CD4/CD8 double positive (DP) memory T cells was significantly increased in the MLN of challenged animals. These studies may provide further insights into understanding the mucosal immune response to PEDV and its role in protection against disease.

Influenza A virus in swine (IAV-S) is a prevalent respiratory pathogen in pigs that has deleterious consequences to animal and human health. Pigs represent an important reservoir for influenza and potential mixing vessel for novel gene reassortments. Despite the central role of pigs in recent influenza outbreaks, much remains unknown about the impact of swine immunity on IAV-S transmission, pathogenesis, and evolution. An incomplete understanding of interactions between the porcine immune system and IAV-S has hindered development of new diagnostic tools and vaccines. In order to address this gap in knowledge, we identified swine leukocyte antigen (SLA) restricted IAV-S peptides presented by porcine airway epithelial cells using an immunoproteomics approach. The majority of MHC-associated peptides belonged to matrix 1, nucleoprotein and nonstructural 1 proteins. Future investigation of the potential cross-reactive nature of these peptides is needed to confirm antigen recognition by cytotoxic T lymphocytes and their utility as vaccine candidates.

Background The effectiveness of biosecurity methods to mitigate the transmission of porcine epidemic diarrhea virus (PEDV) via farm personnel or contaminated fomites is poorly understood. This study was undertaken to evaluate the effectiveness of biosecurity procedures directed at minimizing transmission via personnel following different biosecurity protocols using a controlled experimental setting. Results PEDV RNA was detected from rectal swabs of experimentally infected (INF) and sentinel pigs by real-time reverse transcription polymerase chain reaction (rRT-PCR). Virus shedding in INF pigs peaked at 1 day post infection (dpi) and viral RNA levels remained elevated through 19 dpi. Sentinel pigs in the low biosecurity group (LB) became PEDV positive after the first movement of study personnel from the INF group. However, rectal swabs from pigs in the medium biosecurity (MB) and high biosecurity (HB) groups were negative during the 10 consecutive days of movements and remained negative through 24 days post movement (dpm) when the first trial was terminated. Viral RNA was detected at 1 dpm through 3 dpm from the personal protective equipment (PPE) of LB personnel. In addition, at 1 dpm, 2 hair/face swabs from MB personnel were positive; however, transmission of virus was not detected. All swabs of fomite from the HB study personnel were negative. Conclusions These results indicate that indirect PEDV transmission through contaminated PPE occurs rapidly (within 24 h) under modeled conditions. Biosecurity procedures such as changing PPE, washing exposed skin areas, or taking a shower are recommended for pig production systems and appear to be an effective option for lowering the risk of PEDV transmission between groups of pigs.

Haemophilus parasuis, the causative agent of Glasser's disease, is a pathogen that colonizes the upper respiratory tract (URT) of pigs, invades the bloodstream and causes polyserositis. Because antimicrobials are highly effective against H. parasuis, we hypothesized that they could have a detrimental effect on the establishment of an immune response if given at the time of URT colonization. In this study, we characterized clinical outcomes and antibody and IFN-γ responses to H. parasuis in pigs treated with enrofloxacin before or after low dose inoculation with a pathogenic H. parasuis strain. Pigs that were only inoculated with the agent (EXP group) and pigs that were treated with enrofloxacin and then inoculated (ABT/EXP group) developed signs of disease starting at 4days post inoculation (DPI), presented a significant increase in serum IgG and were protected against a subsequent homologous challenge. In contrast, pigs treated with antibiotic after inoculation (EXP/ABT group) neither showed signs of disease nor seroconverted (IgG) after low dose inoculation. EXP/ABT pigs as well as naive control pigs [enrofloxacin only (ABT) and challenge only (CHA)] were susceptible to challenge. Variable levels of antibodies in bronchioalveolar fluid and IFN-γ in peripheral blood mononuclear cells were observed after H. parasuis inoculation, but were not associated with protection. In summary, only pigs treated before low dose H. parasuis inoculation seroconverted and were protected against subsequent challenge. Results from this study can help determine timing of antimicrobial use and contribute to our current understanding of judicious antibiotic use.

Despite the high prevalence and devastating outcome, there remain a few options for treatment of ischemic stroke. Currently available treatments are limited by a short time window for treatment and marginal efficacy when used. We have tested a human umbilical cord blood-derived stem cell line that has been shown to result in a significant reduction in stroke infarct volume as well as improved functional recovery following stroke in the rat. In the present study we address the mechanism of action and compared the therapeutic efficacy of high- versus low-passage nonhematopoietic umbilical cord blood stem cells (nh-UCBSCs). Using the middle cerebral arterial occlusion (MCAo) model of stroke in Sprague–Dawley rats, we administered nh-UCBSC by intravenous (IV) injection 2 days following stroke induction. These human cells were injected into rats without any immune suppression, and no adverse reactions were detected. Both behavioral and histological analyses have shown that the administration of these cells reduces the infarct volume by 50% as well as improves the functional outcome of these rats following stroke for both high- and low-passaged nh-UCBSCs. Flow cytometry analysis of immune cells present in the brains of normal rats, rats with ischemic brain injury, and ischemic animals with nh-UCBSC treatment confirmed infiltration of macrophages and T cells consequent to ischemia and reduction to normal levels with nh-UCBSC treatment. Flow cytometry also revealed a restoration of normal levels of microglia in the brain following treatment. These data suggest that nh-UCBSCs may act by inhibiting immune cell migration into the brain from the periphery and possibly by inhibition of immune cell activation within the brain. nh-UCBSCs exhibit great potential for treatment of stroke, including the fact that they are associated with an increased therapeutic time window, no known ill-effects, and that they can be expanded to high numbers for, and stored for, treatment.

Wild-type mice control murine cytomegalovirus (MCMV) brain infection, but identical infection is lethal to animals deficient in interleukin (IL)-10. Here, we report that MCMV-infected IL-10 knockout (KO) mice displayed a marked increase in neutrophil infiltration into the infected, IL-10-deficient brain when compared to wild-type animals. Enhanced microglial cell activation, determined by MHC class II up-regulation, overexpression of CXCL2, and elevated P-selectin mRNA levels were observed. In vivo blocking of CXCL2 attenuated neutrophil infiltration and significantly improved the outcome of infection. Collectively, these data indicate that the absence of IL-10 results in pathologic neutrophil infiltration into MCMV-infected brains.

Interleukin (IL)-10 deficiency results in highly elevated levels of interferon (IFN)-gamma, as well as the IFN-gamma-inducible chemokines CXCL9 and CXCL10 within murine cytomegalovirus (MCMV)-infected brains. To test the hypothesis that these elevated chemokine levels would result in enhanced brain infiltration, we compared immune cell infiltration in response to MCMV brain infection between wild-type and IL-10 knockout (KO) mice. Longitudinal analysis following adoptive transfer of cells from beta-actin-luciferase transgenic wild-type mice showed maximal brain infiltration by peripheral immune cells occurred at 5 days post infection. Although the overall percentage of CD45(hi) cells infiltrating the brain was not altered by IL-10 deficiency, paradoxically, despite elevated chemokine levels, reduced T lymphocyte (CD8+) and natural killer (NK) (CD49b+) cell infiltration into the brain was observed in IL-10-deficient animals. This decreased lymphocyte infiltration was associated with elevated levels of the lymph node homing receptor L-selectin/CD62L on CD8+ T cells. Lymph node cells obtained from MCMV-infected mice deficient in IL-10 also displayed reduced migration towards CXCL10 when compared to wild-type animals. Taken together, these data show that despite elevated chemokine levels, absence of IL-10 results in reduced lymphocyte infiltration into MCMV-infected brains.

The authors have previously reported that adoptive transfer of splenocytes suppresses murine cytomegalovirus (MCMV) brain infection following intracerebroventricular injection of immunodeficient mice and that depletion of Thy 1.2+ T lymphocytes abolishes this suppressive effect. Here the authors report that splenocytes depleted of CD4+ T lymphocytes prior to adoptive transfer retained their ability to control viral expression in the brain. In sharp contrast, depletion of the CD8+ T-cell population prior to transfer abolished the suppressive effect, with sixfold greater expression in the brain than when undepleted splenocytes were used. The authors went on to examine the contributions of cytokine- and perforin-mediated mechanisms in controlling MCMV brain infection using splenocytes from major histocompatibility (MHC)-matched IFN-gamma -knockout (GKO), and perforin-knockout (PKO) mice. When used in adoptive transfer studies, splenocytes from GKO mice controlled viral expression; however, cells from PKO mice could not control reporter gene expression or viral DNA replication in brain tissues. The authors have previously reported that the levels of the T-cell chemoattractant CXCL10 are highly elevated in the brains of MCMV-infected mice. Here the authors found that the receptor for this ligand, CXCR3, was not essential in mediating the suppressive effects of adoptive transfer. These data indicate that peripheral CD8+ T cells control MCMV brain infection through a perforin-mediated mechanism and that neither IFN-gamma nor CXCR3 play a critical role in this neuroprotective response.

In recent years, West Nile virus (WNV) has emerged as a major cause of encephalitis in the United States. However, the neuropathogenesis of this flavivirus is poorly understood. In the present study, the authors used primary human brain cell cultures to investigate two neuropathogenic features: viral replication and induction of cytokines. Although neurons and astrocytes were found to support productive WNV infection, viral growth was poorly permissive in microglial cells. Compared to neuronal cultures that sustained viral growth for at least 2 weeks, replication peaked in astrocytes by 72 h post infection. In response to viral infection, astrocytes produced chemokines (CXCL10 and CCL5), but none of the cytokines (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, IL-6, interferon alpha or gamma) tested could be detected. Although microglial cells failed to support viral replication, WNV induced production of the proinflammatory cytokines IL-6 and TNF-alpha. Microglial cells also released robust amounts of the chemokines CXCL10 and CCL2, as well as lower levels of CCL5, in response to WNV infection. WNV-induced chemokine and cytokine production by microglia was coupled with activation of mitogen-activated protein kinase (MAPK) intracellular signaling pathways. Inhibition of p38 MAPK decreased chemokine production in response to WNV. Taken together, these findings suggest that microglial cell responses may influence the neuropathogenesis of WNV infection.

The ability of neural stem/progenitor cells (NSCs) to self-renew, migrate to damaged sites, and differentiate into neurons has renewed interest in using them in therapies for neurodegenerative disorders. Neurological diseases, including viral infections of the brain, are often accompanied by chronic inflammation, whose impact on NSC function remains unexplored. We have previously shown that chronic neuroinflammation, a hallmark of experimental herpes simplex encephalitis (HSE) in mice, is dominated by brain-infiltrating activated CD8 T-cells. In the present study, activated CD8 lymphocytes were found to suppress NSC proliferation profoundly. Luciferase positive (luc+) NSCs co-cultured with activated, MHC-matched, CD8+ lymphocytes (luc−) showed two- to five-fold lower luminescence than co-cultures with un-stimulated lymphocytes. On the other hand, similarly activated CD4+ lymphocytes did not suppress NSC growth. This differential lymphocyte effect on proliferation was confirmed by decreased BrdU uptake by NSC cultured with activated CD8 T-cells. Interestingly, neutralizing antibodies to interferon-gamma (IFN-γ) reversed the impact of CD8 lymphocytes on NSCs. Antibodies specific to the IFN-γ receptor-1 subunit complex abrogated the inhibitory effects of both CD8 lymphocytes and IFN-γ, indicating that the inhibitory effect of these cells was mediated by IFN-γ in a receptor-specific manner. In addition, activated CD8 lymphocytes decreased levels of nestin and Sox2 expression in NSCs while increasing GFAP expression, suggesting possible induction of an altered differentiation state. Furthermore, NSCs obtained from IFN-γ receptor-1 knock-out embryos were refractory to the inhibitory effects of activated CD8+ T lymphocytes on cell proliferation and Sox2 expression. Taken together, the studies presented here demonstrate a role for activated CD8 T-cells in regulating NSC function mediated through the production of IFN-γ. This cytokine may influence neuro-restorative processes and ultimately contribute to the long-term sequelae commonly seen following herpes encephalitis.

Neural stem cells (NSCs) respond to inflammatory cues induced during brain injury and are thought to be involved in recovery from brain damage. Little is known about NSC response during brain infections. The present study evaluated NSC proliferation during Herpes Simplex Virus-1 brain infection. Total numbers of nestin(+) NSCs increased significantly in infected brains at 6 days post infection (p.i.). However, by 15 days p.i. the nestin(+) population decreased significantly below levels observed in uninfected brains and remained depressed through 30 days p.i. This initial increase in NSC population occurred concurrently with increased brain cell proliferation, which peaked at 3 days p.i. On closer examination, we found that while actively proliferating Sox2(+) NSCs increased in number at 6 days p.i., proliferating DCX(+) neuroblasts contributed to the increased response at 3 days p.i. However, overall proliferation decreased steadily from 15 days p.i. to below control levels. To determine the mechanisms involved in altering NSC proliferation, neurotrophin and growth factor expression profiles were assessed. FGF-2 gene expression increased at 5 days p.i. and was robustly down-regulated at 15 days p.i. (>1000-fold), which was further confirmed by increased FGF-2 immunostaining around the lateral ventricles. Furthermore, supplementing infected animals with recombinant FGF-2, at 15 days p.i., significantly increased the number of proliferating brain cells. These findings demonstrate that the temporal changes in NSC proliferation are mediated through the regulation of FGF-2 and that the NSC niche may benefit from supplementation with FGF-2 during HSV-1 brain infection.

Background 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. Methodology/Principal Findings 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. Conclusions MCMV brain infection of newborn mice causes significant loss of NSCs, decreased proliferation of neuronal precursor cells, and marked loss of young neurons.

Long-term neurological sequela is common among herpes simplex encephalitis (HSE) survivors. Animal models for HSE are used to investigate mechanisms of acute disease, but little has been done to model chronic manifestations of HSE. The current study presents a detailed, systematic analysis of chronic neuropathology, including characterization of topography and sequential progression of degenerative lesions and inflammation. Subsequent to intranasal HSV-1 infection, inflammatory responses that were temporally and spatially distinct persisted in infected cortical and brain stem regions. Neutrophils were present exclusively within the olfactory bulb and brain stem regions during the acute phase of infection, while the chronic inflammation was marked by plasma cells, lymphocytes and activated microglia. The chronic lymphocytic infiltrate, cytokine production, and activated microglia were associated with the loss of cortical neuropile in the entorhinal cortex and hippocampus. Animals surviving the acute infection showed a spectrum of chronic lesions from decreased brain volume, neuronal loss, activated astrocytes, and glial scar formation to severe atrophy and cavitations of the cortex. These lesions were also associated with severe spatial memory deficits in surviving animals. Taken together, this model can be utilized to further investigate the mechanisms of neurological defects that follow in the wake of HSE.

Experimental murine herpes simplex virus (HSV)-1 brain infection stimulates microglial cell-driven proinflammatory chemokine production which precedes the presence of brain-infiltrating systemic immune cells. In the present study, we investigated the phenotypes and infiltration kinetics of leukocyte trafficking into HSV-infected murine brains. Using real-time bioluminescence imaging, the infiltration of luciferase-positive splenocytes, transferred via tail vein injection into the brains of HSV-infected animals, was followed over an 18-day time course. Flow cytometric analysis of brain-infiltrating leukocytes at 5, 8, 14, and 30 days postinfection (d.p.i.), was performed to assess their phenotype. A predominantly macrophage (CD45(high)CD11b(+)Ly6C(high)) and neutrophil (CD45(high)CD11b(+)Ly6G(+)) infiltration was seen early during infection, with elevated levels of TNF-alpha mRNA expression. By 14 d.p.i., the phenotypic profile shifted to a predominantly lymphocytic (CD45(high)CD3(+)) infiltrate. This lymphocyte infiltrate was detected until 30 d.p.i., when infectious virus could not be recovered, with CD8(+) and CD4(+) T cells present at a 3:1 ratio, respectively. This T lymphocyte infiltration paralleled increased IFN-gamma mRNA expression in the brain. Activation of resident microglia (CD45(int)CD11b(+)) was also detected until 30 d.p.i., as assessed by MHC class II expression. Activated microglial cells were further identified as the predominant source of IL-1beta. In addition, infected mice given primed immunocytes at 4 d.p.i. showed a significant increase in mortality. Taken together, these results demonstrate that intranasal infection results in early macrophage and neutrophil infiltration into the brain followed by prolonged microglial activation and T lymphocyte retention. Similar prolonged neuroimmune activation may contribute to the neuropathological sequelae observed in herpes encephalitis patients.

Cytomegalovirus (CMV) is the leading transmittable cause of congenital brain abnormalities in children and infection results in fatal ventriculoencephalitis in advanced acquired immunodeficiency syndrome (AIDS) patients. Pathology associated with CMV brain infection is seen predominantly in the periventricular region, an area known to harbor neural stem cells (NSCs). In the present study, using an adult model of murine CMV brain infection, the authors demonstrated that nestin-positive NSCs in the subventricular zone are susceptible to murine CMV infection. Furthermore, primary NSC cultures supported productive murine CMV replication with a 1000-fold increase in viral titers by 5 days post infection (d.p.i). Previous studies from the authors' laboratory demonstrated that CD8 lymphocytes were essential in protecting the brain against murine CMV infection. In the present study, the authors found that interferon (IFN)-gamma treatment increased the expression of major histocompatibility complex (MHC) class I on NSCs. Viral infection, on the other hand, inhibited this IFN-gamma-induced MHC up-regulation. In addition to increasing MHC class I expression, IFN-gamma (but not tumor necrosis factor [TNF]-alpha, interleukin [IL]-1 beta, or IL-10) also suppressed NSC proliferation in vitro. This decrease in proliferation was not accompanied by apoptosis or extracellular release of cellular lactate dehydrogenase (LDH), suggesting that the effects were not due to direct cytotoxicity. These studies demonstrate that NSCs are susceptible to murine CMV infection and inflammatory mediators, such as IFN-gamma, alter cellular characteristics which may have an impact on their reparative functions.

Murine models of tuberculous meningitis (TBM) have not reflected the severity of disease in humans. Based on reports that activated murine microglial cells, but not human microglial cells, express inducible nitric oxide synthase (iNOS), the objective of this study was to determine whether iNOS-knockout (iNOS -/- ) mice would provide such a model. iNOS -/- mice infected with M. tuberculosis developed serious clinical manifestations and granulomatous lesions containing tubercle bacilli throughout the meninges, all of which were absent in wild-type mice. This study underscores the importance of nitric oxide in defense against TBM and suggests that iNOS -/- mice are an appropriate model for human TBM.

Although production of reactive nitrogen and reactive oxygen species (RNS and ROS) is a component of innate defense against viral infection, their overproduction in the brain may also lead to deleterious consequences. To investigate potential immunopathologic roles of oxidative stress during herpes encephalitis, the authors examined the expression kinetics of inducible nitric oxide synthase (iNOS) as well as heme oxygenase-1 (HO-1), a marker of oxidative stress, and evaluated infection-induced oxidative brain damage. Results from these studies showed that both iNOS and HO-1 gene expression were highly elevated in the brain within 7 days post infection (d.p.i.) and remained elevated through 21 d.p.i. Real-time bioluminescence imaging of HO-1 promoter-luciferase transgenic mice confirmed HO-1 promoter activity in the brains of HSV-1-infected animals within 3 d.p.i., which peaked between 5 and 7 d.p.i. Immunohistochemical staining for both 3-nitrotyrosine and 8-hydroxydeoxyguanosine (8-OH-dG), as well as quantitative assessment of 8-isoprostane levels, demonstrated the presence of viral infection-induced oxidative brain damage. In addition, when brain leukocytes obtained from animals with experimental herpes encephalitis were sorted using fluorescence-activated cell sorting (FACS) and the individual cell populations analyzed, CD45(int)/CD11b(+) resident microglia were found to be the major cellular source of iNOS expression.

Interferons (IFNs) have been shown to inhibit influenza A virus (IAV) replication and play an essential role in controlling viral infection. Here we studied the kinetics and magnitude of induction of type I and type III IFN transcripts by primary porcine airway epithelial cells (pAECs) in response to swine and human origin IAV. We observed that swine influenza viruses (SIV) replicate more efficiently than the human pandemic influenza A/California/2009 (pH1N1 CA/09) in pAECs. Interestingly, we also found significant difference in kinetics of IFN-β, IFN-λ1 and IFN-λ3 gene expression by these viruses. While there was delay of up to 12 hours post infection (h p.i.) in induction of IFN genes in pAECs infected with swine IAV A/Sw/Illinois/2008 (H1N1 IL/08), human pH1N1 CA/09 rapidly induced IFN-β, IFN-λ1 and IFN-λ3 gene expression as early as 4 h p.i. However, the magnitude of IFN-β and IFN-λ3 induction at 24 h p.i. was not significantly different between the viral strains tested. Additionally, we found that swine H1N1 IL/08 was less sensitive to dsRNA induced antiviral response compared to human pH1N1 CA/09. Our data suggest that the human and swine IAVs differ in their ability to induce and respond to type I and type III interferons in swine cells. Swine origin IAV may have adapted to the pig host by subverting innate antiviral responses to viral infection.

Murine cytomegalovirus (MCMV) brain infection induces a transient increase in chemokine production, which precedes the infiltration of CD3(+) lymphocytes. In this study, we hypothesized that an absence of anti-inflammatory cytokines would result in sustained proinflammatory neuroimmune responses. Direct intracerebroventricular injection of MCMV into IL-10 knockout (KO) mice produced an unexpected result: while wild-type animals controlled MCMV, the infection was lethal in IL-10 KO animals. Identical infection of IL-4 KO animals did not produce lethal disease. To further characterize the role of IL-10, infected brain tissue from both wild-type and IL-10 KO animals was assessed for cytokine and chemokine levels, as well as viral gene expression. These data show vastly elevated levels of interferon (IFN)-gamma, and the IFN-gamma-inducible chemokines CXCL9 and CXCL10, as well as IL-6 in brain homogenates obtained from IL-10 KO animals. However, MCMV viral load, glycoprotein B mRNA levels, and titers of infectious virus were similar in both IL-10 KO and wild-type animals. Separation of cells isolated from murine brain tissue into distinct populations using FACS, along with subsequent quantitative RT real-time PCR, showed that brain-infiltrating CD45(hi)/CD11b(-) and CD45(hi)/CD11b(int) were the cellular source of IL-10 in the brain. Taken together, these data demonstrate that MCMV brain infection of IL-10-deficient mice causes lethal disease, which occurs in the presence of a dysregulated IFN-gamma-mediated neuroimmune response.

SUMMARYThe nature of microglia fascinated many prominent researchers in the 19th and early 20th centuries, and in a classic treatise in 1932, Pio del Rio-Hortega formulated a number of concepts regarding the function of these resident macrophages of the brain parenchyma that remain relevant to this day. However, a renaissance of interest in microglia occurred toward the end of the 20th century, fueled by the recognition of their role in neuropathogenesis of infectious agents, such as human immunodeficiency virus type 1, and by what appears to be their participation in other neurodegenerative and neuroinflammatory disorders. During the same period, insights into the physiological and pathological properties of microglia were gained from in vivo and in vitro studies of neurotropic viruses, bacteria, fungi, parasites, and prions, which are reviewed in this article. New concepts that have emerged from these studies include the importance of cytokines and chemokines produced by activated microglia in neurodegenerative and neuroprotective processes and the elegant but astonishingly complex interactions between microglia, astrocytes, lymphocytes, and neurons that underlie these processes. It is proposed that an enhanced understanding of microglia will yield improved therapies of central nervous system infections, since such therapies are, by and large, sorely needed.

Glial cells can respond to herpesvirus infections through the production of cytokines and chemokines. Although specific interactions between resident glia and lymphocytes that infiltrate the infected brain remain to be defined, the presence of T cell chemotactic signals in microglial cell supernatants following infection with cytomegalovirus or herpes simplex virus has led to the concept that chemokines initiate a cascade of neuroimmune responses that result in defense of the brain against herpesviruses. While chemokines may play a defensive role by attracting T cells into the brain, aberrant accumulation of lymphocytes may also induce brain damage. Host defense mechanisms must balance control of herpesvirus spread with associated undesirable immunopathologic effects. A growing body of evidence suggests that through complex networks of chemokines and cytokines produced in response to herpesvirus infection, glial cells orchestrate a cascade of events that result in successful defense of or damage to the brain.