Expression of Interferon Gamma by a Recombinant Rabies Virus Strongly Attenuates the Pathogenicity of the Virus via Induction of Type I Interferon

Abstract

Previous animal model experiments have shown a correlation between interferon gamma (IFN-␥) expression and both survival from infection with attenuated rabies virus (RABV) and reduction of neurological sequelae. Therefore, we hypothesized that rapid production of murine IFN-␥ by the rabies virus itself would induce a more robust antiviral response than would occur naturally in mice. To test this hypothesis, we used reverse engineering to clone the mouse IFN-␥ gene into a pathogenic rabies virus backbone, SPBN, to produce the recombinant rabies virus designated SPBN␥. Morbidity and mortality were monitored in mice infected intranasally with SPBN␥ or SPBN(؊) control virus to determine the degree of attenuation caused by the expression of IFN-␥. Incorporation of IFN-␥ into the rabies virus genome highly attenuated the virus. SPBN␥ has a 50% lethal dose (LD 50) more than 100-fold greater than SPBN(؊). In vitro and in vivo mouse experiments show that SPBN␥ infection enhances the production of type I interferons. Furthermore, knockout mice lacking the ability to signal through the type I interferon receptor (IFNAR ؊/؊ ) cannot control the SPBN␥ infection and rapidly die. These data suggest that IFN-␥ production has antiviral effects in rabies, largely due to the induction of type I interferons. IMPORTANCE Survival from rabies is dependent upon the early control of virus replication and spread. Once the virus reaches the central nervous system (CNS), this becomes highly problematic. Studies of CNS immunity to RABV have shown that control of replication begins at the onset of T cell entry and IFN-␥ production in the CNS prior to the appearance of virus-neutralizing antibodies.Moreover, antibody-deficient mice are able to control but not clear attenuated RABV from the CNS. We find here that IFN-␥ triggers the early production of type I interferons with the expected antiviral effects. We also show that engineering a lethal rabies virus to express IFN-␥ directly in the infected tissue reduces rabies virus replication and spread, limiting its pathogenicity in normal and immunocompromised mice. Therefore, vector delivery of IFN-␥ to the brain may have the potential to treat individuals who would otherwise succumb to infection with rabies virus. R abies virus (RABV) is the type species of the Lyssavirus genus in the Rhabdoviridae family. Its small, negative-stranded RNA genome contains only five true genes (1, 2). Although relatively simple, this zoonotic virus has a devastating impact worldwide. The majority of human rabies deaths occur in children in the developing world, and it is estimated that at least 55,000 humans die of rabies each year in Africa and Asia alone (3).Although RABV infection historically has been viewed as a death sentence once the virus reaches the brain, there is a small but growing number of humans who have survived rabies even though the virus entered the brain (4, 5). Due to such cases and to research using animal models of RABV infection (6-8), many believe that the immune system may be cap...

T-bet Is Required for the Rapid Clearance of Attenuated Rabies Virus from Central Nervous System Tissue

“…However, the peak response was significantly lower in T-bet −/− mice compared to their congenic controls. The T-bet transcription factor has been established to be essential in the production of interferon-gamma (37) and we have recently found IFN-γ to be an important trigger of the elevated early production of type I interferons in attenuated RABV-infected mouse brain (23). Thus it seemed plausible that the lower IFN-β mRNA levels detected in the infected T-bet −/− cortex could be a consequence of lower IFN-γ expression but that does not appear to be the case.…”

“…On the other hand, the production of IFN-γ in CNS tissues has been found to strongly inhibit attenuated RABV replication independently of antibody, likely through the enhanced expression of innate antiviral agents (22). IFN-α expression was most closely related to IFN-γ-induced antiviral effects while the induction of IFN-β appeared to be more a consequence of viral replication (23). In the current study, we observed that peak virus replication, which occurred at day 8 post-infection, was lower in the brains of T-bet −/− by comparison with congenic C57BL/6 mice.…”

“…First, cells producing IFN-γ control virus replication, evidently through the enhanced induction of type 1 interferons and other innate antiviral mechanisms (22, 23). This explains why mice with T cells but no functional B cells can control attenuated RABV infection and survive for extended periods of time while mice lacking both T and B cells succumb (24).…”

Reverse genetics of rabies virus: new strategies to attenuate virus virulence for vaccine development

“…Besides the type I IFN, the type II IFN, IFN-γ, also has many important functions in both innate and adaptive immunity (Kotenko 2011). Previous studies have shown that control of RABV replication in CNS begins at the onset of T cell entry and IFN-γ production in the CNS prior to the production of VNAs (Barkhouse et al 2014). It was shown that SPBN expressing IFN-γ highly attenuated the pathogenicity of the virus predominantly via enhancing the production of type I IFNs, and the pathogenicity of the virus was reduced in both normal and immunocompromised mice (Barkhouse et al 2014).…”

“…Previous studies have shown that control of RABV replication in CNS begins at the onset of T cell entry and IFN-γ production in the CNS prior to the production of VNAs (Barkhouse et al 2014). It was shown that SPBN expressing IFN-γ highly attenuated the pathogenicity of the virus predominantly via enhancing the production of type I IFNs, and the pathogenicity of the virus was reduced in both normal and immunocompromised mice (Barkhouse et al 2014). Recent studies have confirmed the importance of IFN-γ during RABV clearance from the CNS and demonstrated the concept that I IFN-γ is located in the center of the signaling pathway in the brains of mice infected with attenuated CVS-B2c strain, compared to the central role of TNF-α in the network generated from mice infected with street DRV-Mexico (Chai et al 2014).…”