Early Diagnosis And Photothermal Therapy Of Rabies Post Infection

Rabies is a zoonotic disease caused by rabies virus(RABV).This disease poses a serious threat to the public health worldwide.To date,there is no effective method for rabies therapy.Although early diagnosis of rabies will lay the foundation for rabies clinical therapy,a sufficiently sensitive diagnostic method is still needed for early diagnosis of RABV infection.In recent studies,a new detection method based on CRISPR-Cas13 a combining with recombinase polymerase amplification(RPA)exhibited extremely high sensitivity(a single copy/μL)when detecting Zika virus,Dengue fever virus,and Influenza virus.In order to solve the problem of early diagnosis of rabies,CRISPR-Cas13 a nucleic acid detection was developed and was validated in early diagnosis of rat RABV infection model.Meanwhile,a luciferase-based in vivo bioimaging system was introduced to indicate the virus distribution in infected rats.As a highly sensitive nucleic acid detection method,the detection limit of CRISPR-Cas13 a to RABV genomic RNA and ss RNA was single copy/μL.Viral particles in the cerebrospinal fluid can be directly detected by CRISPR-Cas13 a by combining with HUDSON method.Secondly,the recombinant rabies virus r B2c-te Luc expressing luciferase was constructed through reverse genetic technology.The replication of r B2c-te Luc was not affected by inserting the gene te Luc.The recombinant virus was found to express te Luc in a dose-dependent manner.CRISPR-Cas13 a and in vivo bioimaging methods were used to detect the viral load in rats infected with recombinant virus r B2c-te Luc.It was found that CRISPR-Cas13 a could detect the virus in the cerebrospinal fluid as early as 3 days post infection(dpi),and this result was also verified by in vivo bioimaging technology.Since RABV is a typical neurotrophic virus,the blood-brain barrier(BBB)obstructs peripheral neutralizing antibodies and some antiviral drugs delivering into the central nervous system(CNS)to clear the RABV.Therefore,in addition to early diagnosis,the CNS drug delivery,drug targeting specificity,and antiviral effects are still challenging rabies clinical therapy.Photothermal therapy(PTT)based on near infrared spectroscopy(NIR)is a widely used cancer treatment strategy.Since the absorption of NIR by gold nanorods(Au NR)converts light energy into heat energy.Thus,Au NR is also widely used in in vivo photothermal therapy.In order to solve the challenge in rabies therapy,an RVG-Apt-Silica Au NR coupled with DNA aptamer and peptide from rabies virus G protein was constructed to develop the PTT strategy for rabies therapy.Nucleotide aptamers constitute of single-stranded DNA or single-stranded RNA(usually shorter than 100 nt)that can specifically bind target molecules with high affinity,including living cells,proteins,and compounds.The aptamer was conjugated on the surface of Au NR to target the RABV-G protein on the membrane of infected cells.Secondly,the RVG peptide was covalently modified on Au NR to improve the efficiency of CNS delivery.Through in vivo bioimaging and frozen section technology,it was indicated that Au NR could enter the CNS from peripheral blood post intravenous injection.In addition,the Au NR specifically targeted the RABV-G protein on the cell surface in the mouse brain.The viral particles in the supernatant was completely inactivated after 5 minutes of PTT in vitro.Inside the infected cells,the virus titer was reduced by about 100 times post PTT in vitro.In in vivo experiments,60% of mice survived after treated with PTT.In addition,RNA quantitative detection and immunohistochemistry tests were performed to further determine the viral load in the mouse brain.The results showed that the viral genomic RNA level and viral load were obviously reduced.These results showed that this PTT could effectively eliminate rabies virus post infection.This study developed a CRISPR-Cas13a-based detection method for the early diagnosis of RABV,laying the foundation for timely clinical therapy.At the same time,this study developed an aptamer-coupled RVG-Apt-Silica-Au NR for the photothermal therapy to effectively eliminate rabies virus in vivo and in vitro,which provides a useful exploration for post infection therapy of rabies.