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 permeabilityin vivoand the downregulation of TJ protein expressionin vitro. These findings indicate that the enhancement of BBB permeability and the reduction of TJ protein expression are due not to RABV infectionper sebut to virus-induced inflammatory chemokines/cytokines.IMPORTANCEPrevious 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 infectionper sebut 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.

Rabies, one of the oldest infectious diseases, still presents a public health threat in most parts of the world today. Its pathogen, rabies virus (RABV), can utilize its viral proteins, such as the nucleoprotein and phosphorylation protein, to subvert the host innate immune system. For a long time, the large (L) protein was believed to be essential for RABV transcription and replication, but its role in viral pathogenicity and immune evasion was not known. Recent studies have found that the conserved K-D-K-E tetrad motif in the L protein is related to the methyltransferase (MTase) activity in the viral mRNA process. In the present study, a series of RABV mutations in this motif was constructed with the recombinant CVS-B2c (rB2c) virus. Two of these mutants, rB2c-K1685A and rB2c-K1829A, were found to be stable and displayed an attenuated phenotype in both in vitro growth and in vivo pathogenicity in adult and suckling mice. Further studies demonstrated that these two mutants were more sensitive to the expression of the interferon-stimulated gene product IFIT2 than the parent virus. Taken together, our results suggest that K1685 and K1829 in the L protein play important roles in pathogenicity and immune evasion during RABV infection. IMPORTANCE Rabies continues to present a public health threat in most areas of the world, especially in the developing countries of Asia and Africa. The pathogenic mechanisms for rabies are not well understood. In the present study, it was found that the recombinant rabies viruses rB2c-K1685A and rB2c-K1829A, carrying mutations at the predicted MTase catalytic sites in the L protein, were highly attenuated both in vitro and in vivo . Further studies showed that these mutants were more sensitive to the expression of the interferon-stimulated gene product IFIT2 than the parent virus. These findings improve our understanding of rabies pathogenesis, which may help in developing potential therapeutics and an avirulent rabies vaccine.

Rabies is an ancient disease but remains endemic in most parts of the world and causes approximately 59,000 deaths annually. The mechanism through which the causative agent, rabies virus (RABV), evades the host immune response and infects the host central nervous system (CNS) has not been completely elucidated thus far. Our previous studies have shown that lab-attenuated, but not wild-type (wt), RABV activates the innate immune response in the mouse and dog models. In this present study, we demonstrate that lab-attenuated RABV causes abortive infection in astrocytes, the most abundant glial cells in the CNS. Furthermore, we found that lab-attenuated RABV produces more double-stranded RNA (dsRNA) than wt RABV, which is recognized by retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5). Activation of mitochondrial antiviral-signaling protein (MAVS), the common adaptor molecule for RIG-I and MDA5, results in the production of type I interferon (IFN) and the expression of hundreds of IFN-stimulated genes, which suppress RABV replication and spread in astrocytes. Notably, lab-attenuated RABV replicates in a manner identical to that of wt RABV in MAVS−/− astrocytes. It was also found that lab-attenuated, but not wt, RABV induces the expression of inflammatory cytokines via the MAVS- p38/NF-κB signaling pathway. These inflammatory cytokines increase the blood–brain barrier permeability and thus enable immune cells and antibodies infiltrate the CNS parenchyma, resulting in RABV control and elimination. In contrast, wt RABV restricts dsRNA production and thus evades innate recognition by RIG-I/MDA5 in astrocytes, which could be one of the mechanisms by which wt RABV evades the host immune response in resident CNS cells. Our findings suggest that astrocytes play a critical role in limiting the replication of lab-attenuated RABV in the CNS.

Rabies, as one of the most threatening zoonoses in the world, causes a fatal central nervous system (CNS) disease. So far, vaccination with rabies vaccines has been the most effective measure to prevent and control this disease. At present, inactivated rabies vaccines are widely used in humans and domestic animals. However, humoral immune responses induced by inactivated rabies vaccines are relatively low and multiple shots are required to achieve protective immunity. Supplementation with an adjuvant is a practical way to improve the immunogenicity of inactivated rabies vaccines. In this study, we found that monophosphoryl-lipid A (MPLA), a well-known TLR4 agonist, could significantly promote the maturation of bone marrow-derived dendritic cells (BMDC) through a TLR4-dependent pathway in vitro and the maturation of conventional DCs (cDCs) in vivo. We also found that MPLA, serving as an adjuvant for inactivated rabies vaccines, could significantly facilitate the generation of T follicular helper (Tfh) cells, germinal center (GC) B cells, and plasma cells (PCs), consequently enhancing the production of RABV-specific total-IgG, IgG2a, IgG2b, and the virus-neutralizing antibodies (VNAs). Furthermore, MPLA could increase the survival ratio of mice challenged with virulent RABV. In conclusion, our results demonstrate that MPLA serving as an adjuvant enhances the intensity of humoral immune responses by activating the cDC–Tfh–GC B axis. Our findings will contribute to the improvement of the efficiency of traditional rabies vaccines.

Untreated rabies virus (RABV) infection leads to death. Vaccine and postexposure treatment have been effective in preventing RABV infection. However, due to cost, rabies vaccination and treatment have not been widely used in developing countries. There are 55,000 human death caused by rabies annually. An efficacious and cost-effective rabies vaccine is needed. Parainfluenza virus 5 (PIV5) is thought to contribute to kennel cough, and kennel cough vaccines containing live PIV5 have been used in dogs for many years. In this work, a PIV5-vectored rabies vaccine was tested in mice. A recombinant PIV5 encoding RABV glycoprotein (G) (rPIV5-RV-G) was administered to mice via intranasal (i.n.), intramuscular (i.m.), and oral inoculation. The vaccinated mice were challenged with a 50% lethal challenge dose (LD ₅₀ ) of RABV challenge virus standard 24 (CVS-24) intracerebrally. A single dose of 10 ⁶ PFU of rPIV5-RV-G was sufficient for 100% protection when administered via the i.n. route. The mice vaccinated with a single dose of 10 ⁸ PFU of rPIV5-RV-G via the i.m. route showed very robust protection (90% to 100%). Intriguingly, the mice vaccinated orally with a single dose of 10 ⁸ PFU of rPIV5-RV-G showed a 50% survival rate, which is comparable to the 60% survival rate among mice inoculated with an attenuated rabies vaccine strain, recombinant LBNSE. This is first report of an orally effective rabies vaccine candidate in animals based on PIV5 as a vector. These results indicate that rPIV5-RV-G is an excellent candidate for a new generation of recombinant rabies vaccine for humans and animals and PIV5 is a potential vector for oral vaccines.