Zhen F. Fu

Attenuated Rabies Virus Activates, while Pathogenic Rabies Virus Evades, the Host Innate Immune Responses in the Central Nervous System

ABSTRACT Rabies virus (RV) induces encephalomyelitis in humans and animals. However, the pathogenic mechanism of rabies is not fully understood. To investigate the host responses to RV infection, we examined and compared the pathology, particularly the inflammatory responses, and the gene expression profiles in the brains of mice infected with wild-type (wt) virus silver-haired bat RV (SHBRV) or laboratory-adapted virus B2C, using a mouse genomic array (Affymetrix). Extensive inflammatory responses were observed in animals infected with the attenuated RV, but little or no inflammatory responses were found in mice infected with wt RV. Furthermore, attenuated RV induced the expression of the genes involved in the innate immune and antiviral responses, especially those related to the alpha/beta interferon (IFN-α/β) signaling pathways and inflammatory chemokines. For the IFN-α/β signaling pathways, many of the interferon regulatory genes, such as the signal transduction activation transducers and interferon regulatory factors, as well as the effector genes, for example, 2′-5′-oligoadenylate synthetase and myxovirus proteins, are highly induced in mice infected with attenuated RV. However, many of these genes were not up-regulated in mice infected with wt SHBRV. The data obtained by microarray analysis were confirmed by real-time PCR. Together, these data suggest that attenuated RV activates, while pathogenic RV evades, the host innate immune and antiviral responses.

Hantavirus Infections in Humans and Animals, China

Hemorrhagic fever with renal syndrome is a serious public health problem in China.

Silver-haired bat rabies virus variant does not induce apoptosis in the brain of experimentally infected mice.

To examine whether induction of apoptosis plays a role in the pathogenesis of street rabies, we compared the distribution of viral antigens, histopathology, and the induction of apoptosis in the brain of mice infected with a street rabies virus (silver-haired bat rabies virus, SHBRV) and with a mouse-adapted laboratory rabies virus strain (challenge virus standard, CVS-24). Inflammation was identified in the meninges, but not in the parenchyma of the brain of mice infected with either CVS-24 or SHBRV. Necrosis was present in numerous cortical, hippocampal, and Purkinje neurons in CVS-24-infected mice, but only minimal necrosis was identified in mice infected with SHBRV. Likewise, extensive terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) staining was observed in the brain of mice infected with CVS-24 but little or none in the brain of mice infected with SHBRV. Rabies virus antigens were distributed similarly in the CNS infected with either virus. However, the expression of the glycoprotein (G) is more widespread and the staining of G is generally stronger in CVS- than SHBRV-infected mice, whereas the expression of rabies virus nucleoprotein (N) is similar in mice infected with either CVS or SHBRV. The positive TUNEL staining thus correlates with the high level of G expression in CVS-infected mouse brain. Northern blot hybridization revealed that the ratio between the N and G transcripts is similar in brains infected with either virus, indicating that the reduced expression of G protein is not caused by reduced transcription in SHBRV-infected animals. Taken together, these observations suggest that apoptosis is not an essential pathogenic mechanism for the outcome of a street rabies virus infection and that other pathologic processes may contribute to the profound neuronal dysfunction characteristic of street rabies.

Neuronal dysfunction and death in rabies virus infection

Because morphologic changes in natural rabies are usually relatively mild, it is thought that the severe clinical disease with a fatal outcome must be due to neuronal dysfunction of rabies virus-infected neurons. The precise bases of this functional impairment are unknown, and current knowledge on electrophysiological alterations, effects on ion channels and neurotransmission, and neurotoxicity are reviewed. Rabies virus may induce neuronal death, possibly through apoptotic mechanisms. Neuronal apoptosis has been observed in vitro and also in vivo under particular experimental conditions. The relevance of neuronal apoptosis in these situations to natural rabies has not yet been fully elucidated.

Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination

ABSTRACT Our previous studies indicated that recruitment and/or activation of dendritic cells (DCs) is important in enhancing the protective immune responses against rabies virus (RABV) (L. Zhao, H. Toriumi, H. Wang, Y. Kuang, X. Guo, K. Morimoto, and Z. F. Fu, J. Virol. 84:9642-9648). To address the importance of DC activation for RABV vaccine efficacy, the genes for several DC recruitment and/or activation molecules, e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-derived chemokine (MDC), and macrophage inflammatory protein 1α (MIP-1α), were individually cloned into RABV. The ability of these recombinant viruses to activate DCs was determined in vitro and in vivo. Infection of mouse bone marrow-derived DCs with each of the recombinant viruses resulted in DC activation, as shown by increased surface expression of CD11c and CD86 as well as an increased level of alpha interferon (IFN-α) production compared to levels observed after infection with the parent virus. Intramuscular infection of mice with each of the viruses recruited and/or activated more DCs and B cells in the periphery than infection with the parent virus, leading to the production of higher levels of virus-neutralizing antibodies. Furthermore, a single immunization with recombinant RABV expressing GM-CSF or MDC protected significantly more mice against intracerebral challenge with virulent RABV than did immunization with the parental virus. Yet, these viruses did not show more virulence than the parent virus, since direct intracerebral inoculation with each virus at up to 1 × 107 fluorescent focus units each did not induce any overt clinic symptom, such as abnormal behavior, or any neurological signs. Together, these data indicate that recombinant RABVs expressing these molecules activate/recruit DCs and enhance protective immune responses.

Role of chemokines in the enhancement of BBB permeability and inflammatory infiltration after rabies virus infection

Induction of innate immunity, particularly through the induction of interferon and chemokines, by rabies virus (RABV) infection has been reported to be inversely correlated with pathogenicity. To further investigate the association between the expression of chemokines and RABV infection, laboratory-attenuated RABV (B2C) and wild-type (wt) RABV (DRV) were administered to Balb/c mice intramuscularly. Chemokine expression, inflammatory cell infiltration, and blood-brain barrier (BBB) permeability were evaluated at various time points after infection. At day 3 post-infection (p.i.) there was very little inflammation in the central nervous system (CNS) and BBB permeability did not change in mice infected with either virus when compared with mock-infected mice. At 6 day p.i., infection with B2C induced the expression of inflammatory chemokines and infiltration of inflammatory cells into the CNS, while these changes were minimal in DRV-infected mice. Furthermore, infection with B2C significantly enhanced BBB permeability comparing to infection with DRV. Among the upregulated chemokines, the expression of IP-10 was best correlated with infiltration of inflammatory cells into the CNS and enhancement of BBB permeability. These data indicate that laboratory-attenuated RABV induces expression of chemokines and infiltration of inflammatory cells into the CNS. Upregulation of chemokines by B2C may have triggered the change in BBB permeability, which helps infiltration of inflammatory cells into the CNS, and thus attenuation of RABV.

Enhancement of Blood-brain Barrier Permeability and Reduction of Tight Junction Protein Expression Are Modulated by Chemokines/Cytokines induced by Rabies Virus Infection

ABSTRACT Infection with laboratory-attenuated rabies virus (RABV) enhances blood-brain barrier (BBB) permeability, which has been demonstrated to be an important factor for host survival, since it allows immune effectors to enter the central nervous system (CNS) and clear RABV. To probe the mechanism by which RABV infection enhances BBB permeability, the expression of tight junction (TJ) proteins in the CNS was investigated following intracranial inoculation with laboratory-attenuated or wild-type (wt) RABV. BBB permeability was significantly enhanced in mice infected with laboratory-attenuated, but not wt, RABV. The expression levels of TJ proteins (claudin-5, occludin, and zonula occludens-1) were decreased in mice infected with laboratory-attenuated, but not wt, RABV, suggesting that enhancement of BBB permeability is associated with the reduction of TJ protein expression in RABV infection. RABV neither infects the brain microvascular endothelial cells (BMECs) nor modulates the expression of TJ proteins in BMECs. However, brain extracts prepared from mice infected with laboratory-attenuated, but not wt, RABV reduced TJ protein expression in BMECs. It was found that brain extracts from mice infected with laboratory-attenuated RABV contained significantly higher levels of inflammatory chemokines/cytokines than those from mice infected with wt RABV. Pathway analysis indicates that gamma interferon (IFN-γ) is located in the center of the cytokine network in the RABV-infected mouse brain, and neutralization of IFN-γ reduced both the disruption of BBB permeability in vivo and the downregulation of TJ protein expression in vitro. These findings indicate that the enhancement of BBB permeability and the reduction of TJ protein expression are due not to RABV infection per se but to virus-induced inflammatory chemokines/cytokines. IMPORTANCE Previous studies have shown that infection with only laboratory-attenuated, not wild-type, rabies virus (RABV) enhances blood-brain barrier (BBB) permeability, allowing immune effectors to enter the central nervous system (CNS) and clear RABV from the CNS. This study investigated the mechanism by which RABV infection enhances BBB permeability. It was found that RABV infection enhances BBB permeability by downregulation of tight junction (TJ) protein expression in the brain microvasculature. It was further found that it is not RABV infection per se but the chemokines/cytokines induced by RABV infection that downregulate the expression of TJ proteins and enhance BBB permeability. Blocking some of these cytokines, such as IFN-γ, ameliorated both the disruption of BBB permeability and the downregulation of TJ protein expression. These studies may provide a foundation for developing therapeutics for clinical rabies, such as medication that could be used to enhance BBB permeability.

The Roles of Chemokines in Rabies Virus Infection: Overexpression May Not Always Be Beneficial

ABSTRACT It was found previously that induction of innate immunity, particularly chemokines, is an important mechanism of rabies virus (RABV) attenuation. To evaluate the effect of overexpression of chemokines on RABV infection, chemokines macrophage inflammatory protein 1α (MIP-1α), RANTES, and IP-10 were individually cloned into the genome of attenuated RABV strain HEP-Flury. These recombinant RABVs were characterized in vitro for growth properties and expression of chemokines. It was found that all the recombinant viruses grew as well as the parent virus, and each of the viruses expressed the intended chemokine in a dose-dependent manner. When these viruses were evaluated for pathogenicity in the mouse model, it was found that overexpression of MIP-1α further decreased RABV pathogenicity by inducing a transient innate immune response. In contrast, overexpression of RANTES or IP-10 increased RABV pathogenicity by causing neurological diseases, which is due to persistent and high-level expression of chemokines, excessive infiltration and accumulation of inflammatory cells in the central nervous system, and severe enhancement of blood-brain barrier permeability. These studies indicate that overexpression of chemokines, although important in controlling virus infection, may not always be beneficial to the host.

Degeneration of Neuronal Processes after Infection with Pathogenic, but Not Attenuated, Rabies Viruses

ABSTRACT The structural alterations of neuronal processes in mice were investigated after the mice were infected with rabies virus (RV). Silver staining of infected brain sections showed severe destruction and disorganization of neuronal processes in mice infected with pathogenic RV but not with attenuated RV. However, neuronal bodies showed very little pathological changes. Electron microscopy revealed the disappearance of intracellular organelles, as well as the disappearance of synaptic structures and vesicles. Infection of primary neurons with pathogenic, but not attenuated, RV resulted in the destruction of neuronal processes and disappearance of microtubule-associated protein 2 and neurofilament immunoreactivity, which suggests that pathogenic RV causes degeneration of neuronal processes possibly by interrupting cytoskeletal integrity.

Viral Infection of the Central Nervous System and Neuroinflammation Precede Blood-Brain Barrier Disruption during Japanese Encephalitis Virus Infection

ABSTRACT Japanese encephalitis is an acute zoonotic, mosquito-borne disease caused by Japanese encephalitis virus (JEV). Japanese encephalitis is characterized by extensive inflammation in the central nervous system (CNS) and disruption of the blood-brain barrier (BBB). However, the pathogenic mechanisms contributing to the BBB disruption are not known. Here, using a mouse model of intravenous JEV infection, we show that virus titers increased exponentially in the brain from 2 to 5 days postinfection. This was accompanied by an early, dramatic increase in the level of inflammatory cytokines and chemokines in the brain. Enhancement of BBB permeability, however, was not observed until day 4, suggesting that viral entry and the onset of inflammation in the CNS occurred prior to BBB damage. In vitro studies revealed that direct infection with JEV could not induce changes in the permeability of brain microvascular endothelial cell monolayers. However, brain extracts derived from symptomatic JEV-infected mice, but not from mock-infected mice, induced significant permeability of the endothelial monolayer. Consistent with a role for inflammatory mediators in BBB disruption, the administration of gamma interferon-neutralizing antibody ameliorated the enhancement of BBB permeability in JEV-infected mice. Taken together, our data suggest that JEV enters the CNS, propagates in neurons, and induces the production of inflammatory cytokines and chemokines, which result in the disruption of the BBB. IMPORTANCE Japanese encephalitis (JE) is the leading cause of viral encephalitis in Asia, resulting in 70,000 cases each year, in which approximately 20 to 30% of cases are fatal, and a high proportion of patients survive with serious neurological and psychiatric sequelae. Pathologically, JEV infection causes an acute encephalopathy accompanied by BBB dysfunction; however, the mechanism is not clear. Thus, understanding the mechanisms of BBB disruption in JEV infection is important. Our data demonstrate that JEV gains entry into the CNS prior to BBB disruption. Furthermore, it is not JEV infection per se, but the inflammatory cytokines/chemokines induced by JEV infection that inhibit the expression of TJ proteins and ultimately result in the enhancement of BBB permeability. Neutralization of gamma interferon (IFN-γ) ameliorated the enhancement of BBB permeability in JEV-infected mice, suggesting that IFN-γ could be a potential therapeutic target. This study would lead to identification of potential therapeutic avenues for the treatment of JEV infection.