RESUMO
An increasing number of studies are showing that autophagy plays a vital role in viral replication and escape. Rabies virus (RABV), a typical neurotropic virus, has been proven to induce autophagy in neurons. However, there are no reports indicating that RABV can cause autophagy in other cells of the central nervous system. Thus, we aimed to explore the relationship between autophagy and RABV infection in BV2 cells in this study. Results of viral growth curves showed that the titers of microglial BV2 cells infected with RABV peaked at 12 hours post-infection (hpi) and then decreased continuously over time. However, it was found that the viral genome RNA and structural proteins can express normally in BV2 cells. In addition, Western blotting indicated that RABV infection increased LC3-II and p62 expression in BV2 cells. LC3 punctate increased with RABV infection in BV2 cells after the transfection of fluorescent protein-tagged LC3 plasmids. Moreover, autophagy cargo protein further accumulated with RABV infection in Bafilomycin A1-treated cells. Subsequently, RABV infection inhibited the fusion of autophagosomes with lysosomes by using a tandem fluorescent marker. Furthermore, a higher multiplicity of infection induced stronger autophagy. Thus, RABV can induce autophagy in BV2 cells, and the autophagy is positively associated with the viral load.
RESUMO
Enhancement of blood-brain barrier (BBB) permeability is necessary for clearing virus in the central nervous system (CNS). It has been reported that only laboratory-attenuated rabies virus (RABV) induces inflammatory response to lead BBB transient breakdown rather than wild-type (wt) strains. As a component of ribonucleoprotein (RNP), phosphoprotein (P) of RABV plays a key role in viral replication and pathogenicity. To our knowledge, the function of RABV P gene during RABV invasion was unclear so far. In order to determine the role of RABV P gene during RABV infection, we evaluated the BBB permeability in vivo after infection with wt RABV strain (GD-SH-01), a lab-attenuated RABV strain (HEP-Flury), and a chimeric RABV strain (rHEP-SH-P) whose P gene cloned from GD-SH-01 was expressed in the genomic backbone of HEP-Flury. We found that rHEP-SH-P caused less enhancement of BBB permeability and was less pathogenic to adult mice than GD-SH-01 and HEP-Flury. In an effort to investigate the mechanism, we found that the replication of rHEP-SH-P has been limited due to the suppressed P protein expression and induced less response to maintain BBB integrity. Our data indicated that the P gene of wt RABV was a potential determinant in hampering viral replication in vivo, which kept BBB integrity. These findings provided an important foundation for understanding the viral invasion and development of novel vaccine.
RESUMO
Rabies, which is caused by the rabies virus (RABV), is an ancient zoonosis that has a high mortality rate. Previous studies have indicated that recombinant RABV expressing canine interleukin-6 (rHEP-CaIL6), induced more virus-neutralizing antibodies than parental RABV in mice following intramuscular immunization. To investigate the immune response induced in the CNS by rHEP-CaIL6 after intranasal or intracranial administration in mice, the permeability of the blood-brain barrier (BBB), the infiltration of CD3 T cells, and innate immune response-related effector molecules in the CNS were examined. It was observed that infection of rHEP-CaIL6 led to enhanced BBB permeability following intranasal infection. More CD3 T cells infiltrated into the central nervous system (CNS) in mice infected with rHEP-CaIL6 than in those infected with the HEP-Flury strain. Furthermore, rHEP-CaIL6 induced an increased expression of innate immune response-related effector molecules, compared with the parental HEP-Flury strain, within the CNS. Taken together, these findings suggest that rHEP-CaIL6 induced stronger immune responses in mice brains, which is more beneficial for virus clearance. These results may also partly illustrate the role of IL6 in RABV infection.