Bardoxolone methyl inhibits the infection of rabies virus via Nrf2 pathway activation in vitro.

BACKGROUND: Rabies is a widespread, fatal, infectious disease. Several antivirals against rabies virus (RABV) infection have been reported, but no approved, RABV-specific antiviral drugs that inhibit RABV infection in the clinic after symptom onset are available. Therefore, more effective drugs to reduce rabies fatalities are urgently needed. Bardoxolone methyl (CDDO-Me), an FDA-approved compound that has long been known as an antioxidant inflammatory modulator and one of the most potent nuclear factor erythroid-derived 2-like 2 (Nrf2) activators, protects myelin, axons, and CNS neurons by Nrf2 activation. Therefore, we investigated the potency of its anti-RABV activity in vitro. METHODS: The mouse neuroblastoma cell line Neuro2a (N2a) and three RABV strains of different virulence were used; the cytotoxicity and anti-RABV activity of CDDO-Me in N2a cells were evaluated by CCK-8 assay and direct fluorescent antibody (DFA) assay. Pathway activation in N2a cells infected with the RABV strains SC16, CVS-11 or CTN upon CDDO-Me treatment was evaluated by western blotting (WB) and DFA assay. RESULTS: CDDO-Me significantly inhibited infection of the three RABV strains of differing virulence (SC16, CVS-11 and CTN) in N2a cells. We also examined whether CDDO-Me activates the Nrf2-associated pathway upon infection with RABV strains of differing virulence. Nrf2, phosphorylated sequestosome (SQSTM1), SQSTM1, hemoglobin oxygenase (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1) expression in N2a cells increased to varying degrees with CDDO-Me treatment, accompanied by Kelch-like ECH-associated protein 1 (Keap1) dissociation, upon infection with SC16, CVS-11 or CTN. The activation of SQSTM1 phosphorylation was significantly associated with the degradation of Keap-1 in CDDO-Me-treated N2a cells upon RABV infection. Furthermore, N2a cells pretreated with the Nrf2-specific inhibitor ATRA showed a significant decrease in HO-1 and NQO1 expression and a decrease in the anti-RABV efficacy of CDDO-Me. These inhibitory effects were observed upon infection with three RABV strains of differing virulence. CONCLUSION: CDDO-Me inhibited RABV infection via Nrf2 activation, promoting a cytoprotective defense response in N2a cells. Our study provides a therapeutic strategy for RABV inhibition and neuroprotection during viral infection.

BCX4430 inhibits the replication of rabies virus by suppressing mTOR-dependent autophagy invitro.

Rabies is a fatal neurological infectious disease caused by rabies virus (RABV). However, no effective anti-RABV drugs for treatment during the symptomatic phase are available. The novel adenosine nucleoside analog galidesivir (BCX4430) has broad-spectrum activity against a wide variety of highly pathogenic RNA viruses. In this study, we observed no apparent cytotoxicity of BCX4430 at the highest concentration of 250 µΜ, and which was displayed stronger antiviral activity against different virulent RABV in N2a or BHK-21 cells until 72 hpi. Meanwhile, BCX4430 showed greater anti-RABV activity than T-705 and anti-RABV activity similar to that of ribavirin in N2a cells. Furthermore, BCX4430 dose- and time-dependently inhibited RABV replication via mTOR-dependent autophagy inhibition in N2a cells with increased phospho-mTOR and phospho-SQSTM1 and decreased LC3-II levels. Taken together, these findings suggest that BCX4430 has potent anti-RABV activity in vitro and might provide a basis for the development of novel drug therapies against RABV.

Enhancement of Virus Replication in An Influenza A Virus NS1-Expresssing 293 Cell Line.

The nonstructural protein 1 (NS1) of influenza A virus, which is absent from the viral particle, but highly expressed in infected cells, strongly antagonizes the interferon (IFN)-mediated antiviral response. We engineered an NS1-expressing 293 (293-NS1) cell line with no response to IFN stimulation. Compared with the parental 293 cells, the IFN-nonresponsive 293-NS1 cells improved the growth capacity of various viruses, but the introduction of NS1 barely enhanced the propagation of Tahyna virus, a negative-strand RNA virus. In particular, fastidious enteric adenovirus that replicates poorly in 293 cells may grow more efficiently in 293-NS1 cells; thus, IFN-nonresponsive 293-NS1 cells might be of great value in diagnostic laboratories for the cultivation and isolation of human enteric adenoviruses.

[Molecular characteristics of the genome of G I of Japanese encephalitis virus isolated from the specimen collected from viral encephalitis case for the first time].

OBJECTIVE: To investigate the molecular basis of pathogenicity of Japanese encephalitis virus (JEV) by sequencing of complete nucleotide sequence and analyze the characteristics of full-length genome of genotype I Japanese encephalitis virus strains (GZ56) which was isolated from the first cerebrospinal fluid (CSF) of Japanese encephalitis patients. METHODS: The complete nucleotide sequence was obtained by RT-PCR and sequencing was performed directly. Bioinformatics was used to analyze the nucleic acid data, deduced amino acid sequence and phylogenetic trees. RESULTS: The result of sequence analysis showed that the genome of GZ56 strains had 10 965 nucleotides, which coded for a 3432-amino acid polyprotein. Phyolngenetic analysis based on full-length genome showed that GZ56 strains and M-28 strains which were the first isolated from mosquitoes in Yunnan in 1977 were in the same evolutionary branch. GZ56 strains belongs to genotype I of Japanese encephalitis virus, the homology of genome ranged from 96.2% to 98.6% in nucleotide and from 98.2% to 99.7% in amino acid sequences respectively when compared with selected genotype I of JEV strains in GenBank. There were 11 amino acid divergences in E protein when compared with the JEV inactivated P3 strain but they are not the key virulence sites. However, there were 14 amino acid divergences in E protein when compared with the JEV live attenuated vaccine SA14-14-2 strain and 8 amino acid divergences were the key virulence sites. CONCLUSION: This study indicated that the full length of genome GZ56 strains had no ignificant change. It can be hypothesized from genomic level that the currently available JEV vaccines(inactivated and live attenuated) can protect against GZ56 strains infection, meanwhile, the JEV live attenuated vaccine (SA14-14-2) formulation conferred higher levels of protection.

[Preparation and identification of a recombinant hepatitis C virus (HCV) based on sindbis virus vector].

OBJECTIVE: To construct the recombinant virus-like particles containing HCV envelope glycoprotein E1E2 based on sindbis virus vector. METHODS: The gene encoding HCV envelope glycoprotein E1E2 was cloned into sindbis virus vector to construct recombinant plasmids pBR-XJE1E2 and pVA-XJE1E2, and transfect them into BHK-21 cells to obtain recombinant virus-like particles. The expression of E1 and E2 protein were verified by Western Blot and indirect immunofluorescent assay (IFA). RESULTS: The results of restriction enzyme digestion, PCR and sequencing analysis showed that the recombinant plasmids were constructed successfully. And the results of RT-PCR, Western blotting and IFA detection showed that the transfect cells could package HCV-like particles of expressing structural proteins E1E2. CONCLUSION: The recombinant expression plasmids pBR-XJE1E2 and pVA-XJE1E2 based on sindbis virus vector could package HCV-like particles in eukaryotic cell, which provides a foundation for further study of its in vivo animal immune response.

Effects of the nsP2-726 Pro mutation on infectivity and pathogenesis of Sindbis virus derived from a full-length infectious cDNA clone.

The point mutations at residue 726 Pro in the nonstructural gene 2 (nsP2-726P) could make Sindbis virus (SINV) replicons lacking the structural protein-coding region less cytopathic and capable of persisting in some vertebrate cell lines. However, the effects of nsP2-726P mutations on characteristics of SINV in the context of genomic-RNA are poorly understood. To investigate the effects of point mutations at nsP2-726P on the infectivity and the pathogenesis of SINV, based on the infectious clone (pBR-XJ160) of a Sindbis-like XJ-160 virus, we constructed mutants BR-726L, BR-726S, BR-726V and BR-726A containing point mutations Pro-to-Leu, Pro-to-Ser, Pro-to-Val and Pro-to-Ala. The BR-726V virus and BR-726A virus exhibited similar growth characteristics to the wild-type BR-XJ160 in cultured cells, including cytopathic effects (CPE), plaque morphology and growth kinetics. For the Leu substitution, no CPE or plaques were seen after six passages through BHK-21 cells, although expression of XJ-160 virus-specific protein was detectable by indirect immunofluorescence assay (IFA). The Ser substitutions gave an intermediate phenotype. The mutant viruses exhibited different levels of neurovirulence in 3-day-old suckling mice, which did not match their propagation in cultured cells or in the mouse brain. Compared with BR-XJ160, BR-726A with the Ala substitution showed highly increased neurovirulence, while BR-726V with the Val substitution exhibited an attenuated phenotype. In contrast, BR-726S, with reduced growth capacity in cultured cells and mouse brain, showed intermediate neurovirulence. BR-726L virus produced no lethality or morbidity in suckling mice. Thus, the nsP2-726 Pro residue regulates virus-host cell interactions directly and is also important in viral pathogenesis in suckling mice.

Substitutions of 169Lys and 173Thr in nonstructural protein 1 influence the infectivity and pathogenicity of XJ-160 virus.

An infectious clone (pBR-XJ160) was constructed using the full-length cDNA of the Sindbis-like XJ-160 virus. Two nucleotide mutations, causing amino acid changes at residue 169 from Lys to Arg and at residue 173 from Thr to Ile in the nonstructural protein (nsP) 1 coding region, strongly influenced the infectivity of in vitro-synthesized RNA. We used site-directed mutagenesis to obtain clones encoding a change to Arg at residue 169 of nsP1 (pBR-169), a change to Ile at residue 173 (pBR-173), or both changes (pBR-6973). Infectivity of RNA from pBR-169 was abolished, but viral forms BR-173 and BR-6973 were obtained from pBR-173 and pBR-6973, respectively. Further, BR-173 exhibited higher propagation than BR-XJ160 in cell culture and higher neurovirulence in a suckling mouse model. BR-6973 possessed an intermediate phenotype. BR-173 and BR-6973 showed increased sensitivity to 3-deazaadenosine (3-DZA), which inhibits S-adenosylhomocysteine hydrolase. Thus, mutagenesis at residue 169 in the nsP1 region of XJ-160 is lethal, but mutation at residue 173 from Thr to Ile enhances viral infectivity and neurovirulence and suppresses the lethal effect of the mutation at residue 169. These mutations might be associated with the RNA methyltransferase (MTase) activity of nsP1.