Systemic and brain macrophage infections in relation to the development of simian immunodeficiency virus encephalitis

The brains of individuals with lentiviral-associated encephalitis contain an abundance of infected and activated macrophages. It has been hypothesized that encephalitis develops when increased numbers of infected monocytes traffic into the central nervous system (CNS) during the end stages of immunosuppression. The relationships between the infection of brain and systemic macrophages and circulating monocytes and the development of lentiviral encephalitis are unknown. We longitudinally examined the extent of monocyte/macrophage infection in blood and lymph nodes of pigtailed macaques that did or did not develop simian immunodeficiency virus encephalitis (SIVE). Compared to levels in macaques that did not develop SIVE, more ex vivo virus production was detected from monocyte-derived macrophages and nonadherent peripheral blood mononuclear cells (PBMCs) from macaques that did develop SIVE. Prior to death, there was an increase in the number of circulating PBMCs following a rise in cerebrospinal fluid viral load in macaques that did develop SIVE but not in nonencephalitic macaques. At necropsy, macaques with SIVE had more infected macrophages in peripheral organs, with the exception of lymph nodes. T cells and NK cells with cytotoxic potential were more abundant in brains with encephalitis; however, T-cell and NK-cell infiltration in SIVE and human immunodeficiency virus encephalitis was more modest than that observed in classical acute herpes simplex virus encephalitis. These findings support the hypothesis that inherent differences in host systemic and CNS monocyte/macrophage viral production are associated with the development of encephalitis.     

High viral load in the cerebrospinal fluid and brain correlates with severity of simian immunodeficiency virus encephalitis

AIDS dementia and encephalitis are complications of AIDS occurring most frequently in patients who are immunosuppressed. The simian immunodeficiency virus (SIV) model used in this study was designed to reproducibly induce AIDS in macaques in order to examine the effects of a neurovirulent virus in this context. Pigtailed macaques (Macaca nemestrina) were coinoculated with an immunosuppressive virus (SIV/DeltaB670) and a neurovirulent molecularly cloned virus (SIV/17E-Fr), and more than 90% of the animals developed moderate to severe encephalitis within 6 months of inoculation. Viral load in plasma and cerebrospinal fluid (CSF) was examined longitudinally to onset of AIDS, and viral load was measured in brain tissue at necropsy to examine the relationship of systemic and central nervous system (CNS) viral replication to the development of encephalitis. In all animals, plasma viral load peaked at 10 to 14 days postinfection and remained high throughout infection with no correlation found between plasma viremia and SIV encephalitis. In contrast, persistent high levels of CSF viral RNA after the acute phase of infection correlated with the development of encephalitis. Although high levels of viral RNA were found in the CSF of all macaques (six of six) during the acute phase, this high level was maintained only in macaques developing SIV encephalitis (five of six). Furthermore, the level of both viral RNA and antigen in the brain correlated with the severity of the CNS lesions. The single animal in this group that did not have CNS lesions had no detectable viral RNA in any of the regions of the brain. The results substantiate the use of CSF viral load measurements in the postacute phase of SIV infection as a marker for encephalitis and CNS viral replication.     

Eastern equine encephalitis virus rapidly infects and disseminates in the brain and spinal cord of cynomolgus macaques following aerosol challenge

Eastern equine encephalitis virus (EEEV) is mosquito-borne virus that produces fatal encephalitis in humans. We recently conducted a first of its kind study to investigate EEEV clinical disease course following aerosol challenge in a cynomolgus macaque model utilizing the state-of-the-art telemetry to measure critical physiological parameters. Here, we report the results of a comprehensive pathology study of NHP tissues collected at euthanasia to gain insights into EEEV pathogenesis. Viral RNA and proteins as well as microscopic lesions were absent in the visceral organs. In contrast, viral RNA and proteins were readily detected throughout the brain including autonomic nervous system (ANS) control centers and spinal cord. However, despite presence of viral RNA and proteins, majority of the brain and spinal cord tissues exhibited minimal or no microscopic lesions. The virus tropism was restricted primarily to neurons, and virus particles (~61–68 nm) were present within axons of neurons and throughout the extracellular spaces. However, active virus replication was absent or minimal in majority of the brain and was limited to regions proximal to the olfactory tract. These data suggest that EEEV initially replicates in/near the olfactory bulb following aerosol challenge and is rapidly transported to distal regions of the brain by exploiting the neuronal axonal transport system to facilitate neuron-to-neuron spread. Once within the brain, the virus gains access to the ANS control centers likely leading to disruption and/or dysregulation of critical physiological parameters to produce severe disease. Moreover, the absence of microscopic lesions strongly suggests that the underlying mechanism of EEEV pathogenesis is due to neuronal dysfunction rather than neuronal death. This study is the first comprehensive investigation into EEEV pathology in a NHP model and will provide significant insights into the evaluation of countermeasure.     

Age-dependent resistance to lethal alphavirus encephalitis in mice: analysis of gene expression in the central nervous system and identification of a novel interferon-inducible protective gene,mouse ISG12

Several different mammalian neurotropic viruses produce an age-dependent encephalitis characterized by more severe disease in younger hosts. To elucidate potential factors that contribute to age-dependent resistance to lethal viral encephalitis, we compared central nervous system (CNS) gene expression in neonatal and weanling mice that were either mock infected or infected intracerebrally with a recombinant strain, dsTE12Q, of the prototype alphavirus Sindbis virus. In 1-day-old mice, infection with dsTE12Q resulted in rapidly fatal disease associated with high CNS viral titers and extensive CNS apoptosis, whereas in 4-week-old mice, dsTE12Q infection resulted in asymptomatic infection with lower CNS virus titers and undetectable CNS apoptosis. GeneChip expression comparisons of mock-infected neonatal and weanling mouse brains revealed developmental regulation of the mRNA expression of numerous genes, including some apoptosis regulatory genes, such as the proapoptotic molecules caspase-3 and TRAF4, which are downregulated during development, and the neuroprotective chemokine, fractalkine, which is upregulated during postnatal development. In parallel with increased neurovirulence and increased viral replication, Sindbis virus infection in 1-day-old mice resulted in both a greater number of host inflammatory genes with altered expression and greater changes in levels of host inflammatory gene expression than infection in 4-week-old mice. Only one inflammatory response gene, an expressed sequence tag similar to human ISG12, increased by a greater magnitude in infected 4-week-old mouse brains than in infected 1-day-old mouse brains. Furthermore, we found that enforced neuronal ISG12 expression results in a significant delay in Sindbis virus-induced death in neonatal mice. Together, our data identify genes that are developmentally regulated in the CNS and genes that are differentially regulated in the brains of different aged mice in response to Sindbis virus infection.     

Caspase 3-dependent cell death of neurons contributes to the pathogenesis of West Nile virus encephalitis

West Nile virus (WNV) is a neurotropic, arthropod-borne flavivirus that has become a significant global cause of viral encephalitis. To examine the mechanisms of WNV-induced neuronal death and the importance of apoptosis in pathogenesis, we evaluated the role of a key apoptotic regulator, caspase 3. WNV infection induced caspase 3 activation and apoptosis in the brains of wild-type mice. Notably, congenic caspase 3(-/-) mice were more resistant to lethal WNV infection, although there were no significant differences in the tissue viral burdens or the kinetics of viral spread. Instead, decreased neuronal death was observed in the cerebral cortices, brain stems, and cerebella of caspase 3(-/-) mice. Analogously, primary central nervous system (CNS)-derived neurons demonstrated caspase 3 activation and apoptosis after WNV infection, and treatment with caspase inhibitors or a genetic deficiency in caspase 3 significantly decreased virus-induced death. These studies establish that caspase 3-dependent apoptosis contributes to the pathogenesis of lethal WNV encephalitis and suggest possible novel therapeutic targets to restrict CNS injury.     

Role of microglial cells in selective replication of simian immunodeficiency virus genotypes in the brain

An accelerated, consistent macaque simian immunodeficiency virus (SIV) model in which over 90% of pigtailed macaques (Macaca nemestrina) coinoculated with SIV/17E-Fr and SIV/DeltaB670 developed encephalitis was used to determine whether central nervous system (CNS) lesions are associated with the replication of specific genotypes in the brain and, more specifically, in the microglia. Ten of 11 inoculated macaques had severe (n = 3), moderate (n = 5), or mild (n = 2) encephalitis at 3 months postinoculation. To compare actively replicating viral genotypes in the CNS and in microglia with those in the periphery, the V1 region of the SIV envelope gene was amplified and sequenced from RNA extracted from basal ganglia, from microglial cells isolated from the brain, and from peripheral blood mononuclear cells (PBMC) isolated from blood at the time of death. To distinguish between actively replicating with latent viral genotypes in the CNS, viral genotypes in RNA and DNA from basal ganglia were compared. Two macrophage-tropic, neurovirulent viruses, SIV/17E-Fr and SIV/DeltaB670 Cl-2, predominated in the brain RNA of macaques with encephalitis, comprising 95% of the genotypes detected. The same two viral genotypes were present at the same frequencies in microglial cell RNA, suggesting that microglia are pivotal in the selective replication of neurovirulent viruses. There was a significantly greater number of viral genotypes in DNA than there were in RNA in the brain (P = 0.004), including those of both the macrophage- and lymphocyte-tropic viral strains. Furthermore, significantly fewer viral genotypes were detected in brain RNA than in PBMC RNA at the time of death (P = 0.004) and the viral strain that predominated in the brain frequently was different from that which predominated in the PBMC of the same animal. These data suggest that many viral genotypes enter the brain, but only a limited subset of macrophage-tropic, neurovirulent viruses replicate terminally in the brains of macaques with encephalitis. They further suggest that the selection of macrophage-tropic, neurovirulent viruses occurs not at the level of the blood-brain barrier but at a stage after virus entry and that microglial cells may play an important role in that selection process.     

Influence of enriched environment on viral encephalitis outcomes: behavioral and neuropathological changes in albino Swiss mice

An enriched environment has previously been described as enhancing natural killer cell activity of recognizing and killing virally infected cells. However, the effects of environmental enrichment on behavioral changes in relation to virus clearance and the neuropathology of encephalitis have not been studied in detail. We tested the hypothesis that environmental enrichment leads to less CNS neuroinvasion and/or more rapid viral clearance in association with T cells without neuronal damage. Stereology-based estimates of activated microglia perineuronal nets and neurons in CA3 were correlated with behavioral changes in the Piry rhabdovirus model of encephalitis in the albino Swiss mouse. Two-month-old female mice maintained in impoverished (IE) or enriched environments (EE) for 3 months were behaviorally tested. After the tests, an equal volume of Piry virus (IEPy, EEPy)-infected or normal brain homogenates were nasally instilled. Eight days post-instillation (dpi), when behavioral changes became apparent, brains were fixed and processed to detect viral antigens, activated microglia, perineuronal nets, and T lymphocytes by immuno- or histochemical reactions. At 20 or 40 dpi, the remaining animals were behaviorally tested and processed for the same markers. In IEPy mice, burrowing activity decreased and recovered earlier (8-10 dpi) than open field (20-40 dpi) but remained unaltered in the EEPy group. EEPy mice presented higher T-cell infiltration, less CNS cell infection by the virus and/or faster virus clearance, less microgliosis, and less damage to the extracellular matrix than IEPy. In both EEPy and IEPy animals, CA3 neuronal number remained unaltered. The results suggest that an enriched environment promotes a more effective immune response to clear CNS virus and not at the cost of CNS damage.     

IL-22 Signaling Contributes to West Nile Encephalitis Pathogenesis

The Th17 cytokine, IL-22, regulates host immune responses to extracellular pathogens. Whether IL-22 plays a role in viral infection, however, is poorly understood. We report here that Il22(-/-) mice were more resistant to lethal West Nile virus (WNV) encephalitis, but had similar viral loads in the periphery compared to wild type (WT) mice. Viral loads, leukocyte infiltrates, proinflammatory cytokines and apoptotic cells in the central nervous system (CNS) of Il22(-/-) mice were also strikingly reduced. Further examination showed that Cxcr2, a chemokine receptor that plays a non-redundant role in mediating neutrophil migration, was significantly reduced in Il22(-/-) compared to WT leukocytes. Expression of Cxcr2 ligands, cxcl1 and cxcl5, was lower in Il22(-/-) brains than wild type mice. Correspondingly, neutrophil migration from the blood into the brain was attenuated following lethal WNV infection of Il22(-/-) mice. Our results suggest that IL-22 signaling exacerbates lethal WNV encephalitis likely by promoting WNV neuroinvasion.     

Bioluminescent Imaging and Histopathologic Characterization of WEEV Neuroinvasion in Outbred CD-1 Mice

Western equine encephalitis virus (WEEV; Alphavirus) is a mosquito-borne virus that can cause severe encephalitis in humans and equids. Previous studies have shown that intranasal infection of outbred CD-1 mice with the WEEV McMillan (McM) strain result in high mortality within 4 days of infection. Here in vivo and ex vivo bioluminescence (BLM) imaging was applied on mice intranasally infected with a recombinant McM virus expressing firefly luciferase (FLUC) to track viral neuroinvasion by FLUC detection and determine any correlation between BLM and viral titer. Immunological markers of disease (MCP-1 and IP-10) were measured and compared to wild type virus infection. Histopathology was guided by corresponding BLM images, and showed that neuroinvasion occurred primarily through cranial nerves, mainly in the olfactory tract. Olfactory bulb neurons were initially infected with subsequent spread of the infection into different regions of the brain. WEEV distribution was confirmed by immunohistochemistry as having marked neuronal infection but very few infected glial cells. Axons displayed infection patterns consistent with viral dissemination along the neuronal axis. The trigeminal nerve served as an additional route of neuroinvasion showing significant FLUC expression within the brainstem. The recombinant virus WEEV.McM.FLUC had attenuated replication kinetics and induced a weaker immunological response than WEEV.McM but produced comparable pathologies. Immunohistochemistry staining for FLUC and WEEV antigen showed that transgene expression was present in all areas of the CNS where virus was observed. BLM provides a quantifiable measure of alphaviral neural disease progression and a method for evaluating antiviral strategies.     

Development of Neurological Disease Is Associated with Increased Immune Activation in Simian Immunodeficiency Virus-Infected Macaques

Simian immunodeficiency virus (SIV) infection of macaques can result in central nervous system disorders, such as meningitis and encephalitis. We studied 10 animals inoculated with brain-derived virus from animals with SIV encephalitis. Over half of the macaques developed SIV-induced neurologic disease. Elevated levels of systemic immune activation were observed to correlate with viral RNA in the cerebral spinal fluid but not with plasma viral load, consistent with a role for SIV in the pathogenesis of neurologic disease.     

Anti-α4 Antibody Treatment Blocks Virus Traffic to the Brain and Gut Early,and Stabilizes CNS Injury Late in Infection

Four SIV-infected monkeys with high plasma virus and CNS injury were treated with an anti-α4 blocking antibody (natalizumab) once a week for three weeks beginning on 28 days post-infection (late). Infection in the brain and gut were quantified, and neuronal injury in the CNS was assessed by MR spectroscopy, and compared to controls with AIDS and SIV encephalitis. Treatment resulted in stabilization of ongoing neuronal injury (NAA/Cr by 1H MRS), and decreased numbers of monocytes/macrophages and productive infection (SIV p28⁺, RNA⁺) in brain and gut. Antibody treatment of six SIV infected monkeys at the time of infection (early) for 3 weeks blocked monocyte/macrophage traffic and infection in the CNS, and significantly decreased leukocyte traffic and infection in the gut. SIV – RNA and p28 was absent in the CNS and the gut. SIV DNA was undetectable in brains of five of six early treated macaques, but proviral DNA in guts of treated and control animals was equivalent. Early treated animals had low-to-no plasma LPS and sCD163. These results support the notion that monocyte/macrophage traffic late in infection drives neuronal injury and maintains CNS viral reservoirs and lesions. Leukocyte traffic early in infection seeds the CNS with virus and contributes to productive infection in the gut. Leukocyte traffic early contributes to gut pathology, bacterial translocation, and activation of innate immunity.     

Structural and nonstructural protein genome regions of eastern equine encephalitis virus are determinants of interferon sensitivity and murine virulence

Eastern equine encephalitis virus (EEEV) causes sporadic epidemics of human and equine disease in North America, but South American strains have seldom been associated with human neurologic disease or mortality, despite serological evidence of infection. In mice, most North American and South American strains of EEEV produce neurologic disease that resembles that associated with human and equine infections. We identified a South American strain that is unable to replicate efficiently in the brain or cause fatal disease in mice yet produces 10-fold higher viremia than virulent EEEV strains. The avirulent South American strain was also sensitive to human interferon (IFN)-alpha, -beta, and -gamma, like most South American strains, in contrast to North American strains that were highly resistant. To identify genes associated with IFN sensitivity and virulence, infectious cDNA clones of a virulent North American strain and the avirulent South American strain were constructed. Two reciprocal chimeric viruses containing swapped structural and nonstructural protein gene regions of the North American and South American strains were also constructed and found to replicate efficiently in vitro. Both chimeras produced fatal disease in mice, similar to that caused by the virulent North American strain. Both chimeric viruses also exhibited intermediate sensitivity to human IFN-alpha, -beta, and -gamma compared to that of the North American and South American strains. Virulence 50% lethal dose assays and serial sacrifice experiments further demonstrated that both structural and nonstructural proteins are important contributors to neurovirulence and viral tissue tropism. Together, the results of this study emphasize the complex and important influences of structural and nonstructural protein gene regions on EEEV virulence.     

MicroRNA targeting of neurotropic flavivirus: effective control of virus escape and reversion to neurovirulent phenotype

Neurotropic flaviviruses can efficiently replicate in the developing and mature central nervous systems (CNS) of mice causing lethal encephalitis. Insertion of a single copy of a target for brain-expressed microRNAs (miRNAs) in the 3' noncoding region (3'NCR) of the flavivirus genome (chimeric tick-borne encephalitis virus/dengue virus) abolished virus neurovirulence in the mature mouse CNS. However, in the developing CNS of highly permissive suckling mice, the miRNA-targeted viruses can revert to a neurovirulent phenotype by accumulating deletions or mutations within the miRNA target sequence. Virus escape from miRNA-mediated suppression in the developing CNS was markedly diminished by increasing the number of miRNA target sites and by extending the distance between these sites in the virus genome. Insertion of multiple miRNA targets into the 3'NCR altered virus neuroinvasiveness, decreased neurovirulence and neuroinflammatory responses, and prevented neurodegeneration without loss of immunogenicity. Although the onset of encephalitis was delayed, a small number of suckling mice still succumbed to lethal intracerebral infection with the miRNA-targeted viruses. Sequence analysis of brain isolates from moribund mice revealed that the viruses escaped from miRNA-mediated suppression exclusively through the deletion of miRNA targets and viral genome sequence located between the two miRNA targets separated by the greatest distance. These findings offer a general strategy to control the reversion of virus to a virulent phenotype: a simultaneous miRNA targeting of the viral genome at many different functionally important regions could prevent virus escape from miRNA-based attenuation, since a deletion of the targeted genomic sequences located between the inserted miRNA binding sites would be lethal for the virus.     

In vivo clearance of Japanese encephalitis virus by adoptively transferred virus specific cytotoxic T lymphocytes

Japanese encephalitis virus (JEV) is a positive stranded RNA virus that belongs to the flavivirus group. JEV infection damages the central nervous system (CNS) and is one of the main causative agents of acute encephalitis. H-2 restricted virus-specific cytotoxic T lymphocytes (CTL) have been generated specifically against JEV in our laboratory and these CTL have been shown to protect mice against lethal challenge with JEV. Virus replication was found to be inhibited in the brains of animals that were adoptively transferred with JEV specific CTL as revealed by immunohistological staining as well as viral plaque assays. We further show that virus specific CTL could be recovered from such protected mice as long as 45 days after adoptive transfer.     

The utilization of advance telemetry to investigate critical physiological parameters including electroencephalography in cynomolgus macaques following aerosol challenge with eastern equine encephalitis virus

Most alphaviruses are mosquito-borne and can cause severe disease in humans and domesticated animals. In North America, eastern equine encephalitis virus (EEEV) is an important human pathogen with case fatality rates of 30–90%. Currently, there are no therapeutics or vaccines to treat and/or prevent human infection. One critical impediment in countermeasure development is the lack of insight into clinically relevant parameters in a susceptible animal model. This study examined the disease course of EEEV in a cynomolgus macaque model utilizing advanced telemetry technology to continuously and simultaneously measure temperature, respiration, activity, heart rate, blood pressure, electrocardiogram (ECG), and electroencephalography (EEG) following an aerosol challenge at 7.0 log₁₀ PFU. Following challenge, all parameters were rapidly and substantially altered with peak alterations from baseline ranged as follows: temperature (+3.0–4.2°C), respiration rate (+56–128%), activity (-15-76% daytime and +5–22% nighttime), heart rate (+67–190%), systolic (+44–67%) and diastolic blood pressure (+45–80%). Cardiac abnormalities comprised of alterations in QRS and PR duration, QTc Bazett, T wave morphology, amplitude of the QRS complex, and sinoatrial arrest. An unexpected finding of the study was the first documented evidence of a critical cardiac event as an immediate cause of euthanasia in one NHP. All brain waves were rapidly (~12–24 hpi) and profoundly altered with increases of up to 6,800% and severe diffuse slowing of all waves with decreases of ~99%. Lastly, all NHPs exhibited disruption of the circadian rhythm, sleep, and food/fluid intake. Accordingly, all NHPs met the euthanasia criteria by ~106–140 hpi. This is the first of its kind study utilizing state of the art telemetry to investigate multiple clinical parameters relevant to human EEEV infection in a susceptible cynomolgus macaque model. The study provides critical insights into EEEV pathogenesis and the parameters identified will improve animal model development to facilitate rapid evaluation of vaccines and therapeutics.     

Protective Efficacy of Passive Immunization with Monoclonal Antibodies in Animal Models of H5N1 Highly Pathogenic Avian Influenza Virus Infection

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.     

Neuropathogenesis of chimeric simian human immunodeficiency virus infection in rhesus macaques

Comparative studies were performed to determine the neuropathogenesis of infection in macaques with simian human immunodeficiency virus (SHIV)89.6P and SHIV(KU). Both viruses utilize the CD4 receptor and CXCR4 co-receptor. However, in addition, SHIV89.6P uses the CCR5 co-receptor. Both agents are dual tropic for CD4+ T cells and blood-derived macrophages of rhesus macaques. Following inoculation into macaques, both caused rapid elimination of CD4+ T cells but they varied greatly in mechanisms of neuropathogenesis. Two animals infected with SHIV89.6P developed typical lentiviral encephalitis in which multinucleated giant cell formation, nodular accumulations of microglial cells, activated macrophages and astrocytes, and perivascular accumulations of mononuclear cells were present in the brain. Many of the macrophages in these lesions contained viral RNA. Three macaques infected with SHIV(KU) and killed on days 6, 11 and 18, respectively, developed a slowly progressive infection in the CNS but macrophages were not productively infected and there were no pathological changes in the brain. Two other animals infected with this virus and killed several months later showed minimal infection in the brain even though one of the two developed encephalitis of unknown etiology. The basic difference in the mechanisms of neuropathogenesis by the two viruses may be related to co-receptor usage. SHIV89.6P, in utilizing the CCR5 co-receptor, caused neuropathogenic effects that are similar to other neurovirulent primate lentiviruses.     

Herpes B virus replication and viral lesions in the liver of a cynomolgus macaque which died from severe disease with rapid onset

Herpes B virus (BV, Macacine alphaherpesvirus 1) infects macaques asymptomatically, with rare exceptions, but can cause fatal encephalitis in humans. Here, we report disseminated BV infection in a cynomolgus macaque that had died within 12 hour after the onset of unspecific symptoms. Multifocal lesions surrounded by viral antigen were detected in liver while other organs remained inconspicuous, indicating that the liver is a major target. Moreover, high copy numbers of viral DNA were found in feces, underlining the excrements are a potential source of transmission.     

Precise localization and dynamic distribution of Japanese encephalitis virus in the rain nuclei of infected mice

Japanese encephalitis virus (JEV) is a pathogen that causes severe vector-borne zoonotic diseases, thereby posing a serious threat to human health. Although JEV is potentially neurotropic, its pathogenesis and distribution in the host have not been fully elucidated. In this study, an infected mouse model was established using a highly virulent P3 strain of JEV. Immunohistochemistry and in situ hybridization, combined with anatomical imaging of the mouse brain, were used to dynamically localize the virus and construct three-dimensional (3D) images. Consequently, onset of mild clinical signs occurred in some mice at 3.5 d post JEV infection, while most mice displayed typical neurological signs at 6 d post-infection (dpi). Moreover, brain pathology revealed typical changes associated with non-suppurative encephalitis, which lasted up to 8 d. The earliest detection of viral antigen was achieved at 3 dpi in the thalamus and medulla oblongata. At 6 dpi, the positive viral antigen signals were mainly distributed in the cerebral cortex, olfactory area, basal ganglia, thalamus, and brainstem regions in mice. At 8 dpi, the antigen signals gradually decreased, and the localization of JEV tended to concentrate in the cerebrum and thalamus, while no viral antigen was detected in the brain at 21 dpi. In this model, the viral antigen was first expressed in the reticular thalamic nucleus (Rt), and the virus content is relatively stable. The expression of the viral antigen in the hippocampal CA2 region, the anterior olfactory nucleus, and the deep mesencephalic nucleus was high and persistent. The 3D images showed that viral signals were mostly concentrated in the parietal cortex, occipital lobe, and hippocampus, near the mid-sagittal plane. In the early stages of infection in mice, a large number of viral antigens were detected in denatured and necrotic neurons, suggesting that JEV directly causes neuronal damage. From the time of its entry, JEV is widely distributed in the central nervous system thereby causing extensive damage.