Connective Tissue Growth Factor: Regulation, Diseases, and Drug Discovery.

In drug discovery, selecting targeted molecules is crucial as the target could directly affect drug efficacy and the treatment outcomes. As a member of the CCN family, CTGF (also known as CCN2) is an essential regulator in the progression of various diseases, including fibrosis, cancer, neurological disorders, and eye diseases. Understanding the regulatory mechanisms of CTGF in different diseases may contribute to the discovery of novel drug candidates. Summarizing the CTGF-targeting and -inhibitory drugs is also beneficial for the analysis of the efficacy, applications, and limitations of these drugs in different disease models. Therefore, we reviewed the CTGF structure, the regulatory mechanisms in various diseases, and drug development in order to provide more references for future drug discovery.

Corrigendum: Isolation, identification, recombination analysis and pathogenicity experiment of a PRRSV recombinant strain in Sichuan Province, China.

[This corrects the article DOI: 10.3389/fmicb.2024.1362471.].

Pan-genome analysis of Streptococcus suis serotype 2 highlights genes associated with virulence and antibiotic resistance.

Streptococcus suis serotype 2 (SS2) is a Gram-positive bacterium. It is a common and significant pathogen in pigs and a common cause of zoonotic meningitis in humans. It can lead to sepsis, endocarditis, arthritis, and pneumonia. If not diagnosed and treated promptly, it has a high mortality rate. The pan-genome of SS2 is open, and with an increasing number of genes, the core genome and accessory genome may exhibit more pronounced differences. Due to the diversity of SS2, the genes related to its virulence and resistance are still unclear. In this study, a strain of SS2 was isolated from a pig farm in Sichuan Province, China, and subjected to whole-genome sequencing and characterization. Subsequently, we conducted a Pan-Genome-Wide Association Study (Pan-GWAS) on 230 strains of SS2. Our analysis indicates that the core genome is composed of 1,458 genes related to the basic life processes of the bacterium. The accessory genome, consisting of 4,337 genes, is highly variable and a major contributor to the genetic diversity of SS2. Furthermore, we identified important virulence and resistance genes in SS2 through pan-GWAS. The virulence genes of SS2 are mainly associated with bacterial adhesion. In addition, resistance genes in the core genome may confer natural resistance of SS2 to fluoroquinolone and glycopeptide antibiotics. This study lays the foundation for further research on the virulence and resistance of SS2, providing potential new drug and vaccine targets against SS2.

Isolation, identification, recombination analysis and pathogenicity experiment of a PRRSV recombinant strain in Sichuan Province, China.

Since 2013, the porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2), lineage 1.8 (NADC30-like PRRSV) has emerged and become widely prevalent in China. The NADC30-like PRRSV poses significant challenges for disease control, primarily because of its propensity for frequent mutations and recombinations. We successfully isolated and identified a NADC30-like strain, designated SCCD22, in Chengdu, Sichuan Province, China. We meticulously examined the genetic recombination properties and evaluated its pathogenicity in 28-day-old piglets. SCCD22 showed 93.02% nucleotide homology with the NADC30 PRRSV strain, and its non-structural protein 2 coding region showed the same 131 amino acid deletion pattern as that seen in NADC30. Furthermore, we identified two recombination events in SCCD22: one in the NSP2 region (1,028-3,290 nt), where it was highly similar to the JXA1-like strain GZ106; and another in the NSP10 ~ 12 region (9,985-12,279 nt), closely resembling the NADC30-like strain CY2-1604. Piglets infected with SCCD22 exhibited clinical symptoms such as elevated body temperature, prolonged fever, reduced appetite, and roughened fur. Postmortem examinations underscored the typical lung pathology associated with PRRSV, indicating that the lungs were the primary affected organs. Furthermore, extended viral shedding accompanied by progressive viremia was observed in the serum and nasal excretions of infected piglets. In summary, this study reports a domestic PRRSV recombination strain in the Sichuan Province that can provide critical insights into preventing and controlling PRRSV in this region.

Epidemiological investigation and pathogenicity of porcine reproductive and respiratory syndrome virus in Sichuan, China.

Porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) lineage 8 was first detected in mainland China in 2006 and has since rapidly spread to become the primary epidemic strain in the country. In this study, samples such as lung tissue, hilar lymph nodes, abortion fetuses, and blood were collected from large-scale pig farms across 11 prefecture-level cities in Sichuan province between 2019 and 2020 for antigen detection and PRRS virus isolation. The antigen detection results indicated that the positive rate of HP-PRRSV (JXA1-Like strain) was 44.74% (51/114), NADC30-Like PRRSV was 17.54% (20/114), and classical PRRSV (VR2332-Like strain) was 37.72% (43/114). The predominant strain was HP-PRRSV. Positive samples were further inoculated into Marc-145 cells for virus isolation and identification, leading to the isolation of a new JXA1-Like PRRSV strain named SCSN2020. The strain was characterized by RT-qPCR, indirect immunofluorescence assay (IFA), plaque purification, electron microscopy, and whole genome sequencing. The total length of the viral genome was determined to be approximately 15,374 bp. A comparison of the SCSN2020 genome with VR2332 revealed that both strains had the same discontinuous 30-amino acid deletion on the Nsp2 gene. ORF5 genotyping classified the SCSN2020 strain as sublineage 8.7, with a whole genome sequence identity of 99.34% with JXA1. Furthermore, we evaluated the pathogenicity of the SCSN2020 strain in 28-day-old piglets and observed persistent fever from day 4 to day 10, weight loss started on day 7, dyspnea and severe lung lesions began started on day 14. The results of this study highlight the current PRRSV epidemic situation in Sichuan province and provide a scientific reference for subsequent prevention and control measures.

Molecular cloning, prokaryotic expression and its application potential evaluation of interferon (IFN)-ω of forest musk deer.

Forest musk deer (Moschus berezovskii) are currently a threatened species under conservation, and the development of captive populations is restricted by health problems. To evaluate the application potential of interferon (IFN)-ω in the prevention and control of forest musk deer disease, 5 forest musk deer IFN-ω (fmdIFNω) gene sequences were successfully obtained by homologous cloning method for the first time. FmdIFNω5 was selected and recombinant fmdIFNω protein (rIFNω) was successfully expressed by pGEX-6P-1 plasmid and E. coli expression system. The obtained protein was used to stimulate forest musk deer lung fibroblasts cells FMD-C1 to determine its regulatory effect on interferon-stimulated genes (ISGs). In addition, an indirect ELISA method based on anti-rIFNω serum was established to detect endogenous IFN-ω levels in 8 forest musk deer. The results showed that there were 18 amino acid differences among the 5 fmdIFNω subtypes, all of which had the basic structure to exert the activity of type I IFN and were close to Cervus elaphus IFN-ω in the phylogenetic tree. The protein expressed was 48 kDa, and the transcription levels of all ISGs were increased in FMD-C1 cells stimulated by rIFNω, and the amount of transcription accumulation was time-dependent. Meanwhile, Anti-rIFNω serum of mice could react with both rIFNω and forest musk deer serum, and the OD450nm value of forest musk deer serum with the most obvious symptoms was the highest, suggesting that the level of natural IFN-ω in different forest musk deer could be monitored by the rIFNω-based ELISA method. These results indicate that fmdIFNω has the potential as an antiviral drug and an early indication of innate immunity, which is of great significance for the prevention and control of forest musk deer diseases.

Pan-genome wide association study of Glaesserella parasuis highlights genes associated with virulence and biofilm formation.

Glaesserella parasuis is a gram-negative bacterium that causes fibrotic polyserositis and arthritis in pig, significantly affecting the pig industry. The pan-genome of G. parasuis is open. As the number of genes increases, the core and accessory genomes may show more pronounced differences. The genes associated with virulence and biofilm formation are also still unclear due to the diversity of G. parasuis. Therefore, we have applied a pan-genome-wide association study (Pan-GWAS) to 121 strains G. parasuis. Our analysis revealed that the core genome consists of 1,133 genes associated with the cytoskeleton, virulence, and basic biological processes. The accessory genome is highly variable and is a major cause of genetic diversity in G. parasuis. Furthermore, two biologically important traits (virulence, biofilm formation) of G. parasuis were studied via pan-GWAS to search for genes associated with the traits. A total of 142 genes were associated with strong virulence traits. By affecting metabolic pathways and capturing the host nutrients, these genes are involved in signal pathways and virulence factors, which are beneficial for bacterial survival and biofilm formation. This research lays the foundation for further studies on virulence and biofilm formation and provides potential new drug and vaccine targets against G. parasuis.

Characteristics of a novel temperate bacteriophage against Staphylococcus arlettae (vB_SarS_BM31) / Características de un nuevo bacteriófago templado contra Staphylococcus arlettae (vB_SarS_BM31)

Background: Staphylococcus arlettae is a rarely reported coagulase-negative staphylococcus (CoNS) isolated from infected humans and livestock. Observing phage-bacteria interaction could improve the understanding of bacterial pathogenetic mechanisms, providing foundational evidence for phage therapy or phage detection. Herein, we aimed to characterise and annotate a novel bacteriophage, vB_SarS_BM31 (BM31), specific to S. arlettae. This bacteriophage was isolated from a milk sample associated with bovine mastitis and collected in the Sichuan Province, China. Results: The BM31 genome comprised a linear double-stranded DNA of 42,271 base pair in length with a G + C content of 34.59%. A total of 65 open reading frames (ORFs) were assembled from phage DNA, of which 29 were functionally annotated. These functional genes were divided into four modules: the structural, DNA packing and replication, lysis, and lysogeny modules. Holin (ORF25), lysin (ORF26), and integrase (ORF28) were located closely in the entire BM31 genome and were important for lyse or lysogeny cycle of BM31. The phage was identified as a temperate phage according to whole genome analysis and life cycle assay, with basic biological characteristics such as small burst size, short latency period, and narrow host range, consistent with the characteristics of the family Siphoviridae, subcluster B14 of the Staphylococcus bacteriophage. Conclusions: The present isolation and characterisation of BM31 contributes to the Staphylococcus bacteriophage database and provides a theoretical foundation for its potential applications. To the best of our knowledge, BM31 is the only shared and completely reported phage against S. arlettae in the entire public database.(AU)

Development of a Real-Time TaqMan RT-PCR Assay for the Detection of NADC34-like Porcine Reproductive and Respiratory Syndrome Virus.

NADC34-like porcine reproductive and respiratory syndrome virus first appeared in 2017 in a herd of pigs in Liaoning Province, China. The virus was subsequently found in other provinces. Given the potential for this virus to cause an epidemic, rapid, sensitive, and specific detection of NADC34-like PRRSV is required. The virus' ORF5 gene was artificially synthesized based on a Chinese reference strain, and specific primers/probes for the ORF5 gene were designed. Then, the amplified target fragment was cloned into the pMD19-T vector, and a series of diluted recombinant plasmids were used to generate a standard curve. An optimized real-time TaqMan RT-PCR method was established. The method was highly specific for NADC34-like PRRSV, without cross-reactions with other non-targeted pig viruses. The detection limit of this assay was 101 copies/µL. The method had an efficiency of 98.8%, a squared regression value (R2) of 0.999, and showed a linear range of 103-108 copies/µL of DNA per reaction. This method was shown to be analytically specific and sensitive with a low intra- and inter-assay coefficient of variation (<1.40%). A total of 321 clinical samples were tested using the established method, and four were shown to be positive (1.24%). This study confirmed the existence of NADC34-like PRRSV and HP-PRRSV co-infection in Sichuan and provided a promising alternative tool for the rapid detection of NADC34-like PRRSV.

RVG Peptide-Functionalized Favipiravir Nanoparticle Delivery System Facilitates Antiviral Therapy of Neurotropic Virus Infection in a Mouse Model.

Neurotropic viruses severely damage the central nervous system (CNS) and human health. Common neurotropic viruses include rabies virus (RABV), Zika virus, and poliovirus. When treating neurotropic virus infection, obstruction of the blood-brain barrier (BBB) reduces the efficiency of drug delivery to the CNS. An efficient intracerebral delivery system can significantly increase intracerebral delivery efficiency and facilitate antiviral therapy. In this study, a rabies virus glycopeptide (RVG) functionalized mesoporous silica nanoparticle (MSN) packaging favipiravir (T-705) was developed to generate T-705@MSN-RVG. It was further evaluated for drug delivery and antiviral treatment in a VSV-infected mouse model. The RVG, a polypeptide consisting of 29 amino acids, was conjugated on the nanoparticle to enhance CNS delivery. The T-705@MSN-RVG caused a significant decrease in virus titers and virus proliferation without inducing substantial cell damage in vitro. By releasing T-705, the nanoparticle promoted viral inhibition in the brain during infection. At 21 days post-infection (dpi), a significantly enhanced survival ratio (77%) was observed in the group inoculated with nanoparticle compared with the non-treated group (23%). The viral RNA levels were also decreased in the therapy group at 4 and 6 dpi compared with that of the control group. The T-705@MSN-RVG could be considered a promising system for CNS delivery for treating neurotropic virus infection.

Characteristics of a novel temperate bacteriophage against Staphylococcus arlettae (vB_SarS_BM31).

BACKGROUND: Staphylococcus arlettae is a rarely reported coagulase-negative staphylococcus (CoNS) isolated from infected humans and livestock. Observing phage-bacteria interaction could improve the understanding of bacterial pathogenetic mechanisms, providing foundational evidence for phage therapy or phage detection. Herein, we aimed to characterise and annotate a novel bacteriophage, vB_SarS_BM31 (BM31), specific to S. arlettae. This bacteriophage was isolated from a milk sample associated with bovine mastitis and collected in the Sichuan Province, China. RESULTS: The BM31 genome comprised a linear double-stranded DNA of 42,271 base pair in length with a G + C content of 34.59%. A total of 65 open reading frames (ORFs) were assembled from phage DNA, of which 29 were functionally annotated. These functional genes were divided into four modules: the structural, DNA packing and replication, lysis, and lysogeny modules. Holin (ORF25), lysin (ORF26), and integrase (ORF28) were located closely in the entire BM31 genome and were important for lyse or lysogeny cycle of BM31. The phage was identified as a temperate phage according to whole genome analysis and life cycle assay, with basic biological characteristics such as small burst size, short latency period, and narrow host range, consistent with the characteristics of the family Siphoviridae, subcluster B14 of the Staphylococcus bacteriophage. CONCLUSIONS: The present isolation and characterisation of BM31 contributes to the Staphylococcus bacteriophage database and provides a theoretical foundation for its potential applications. To the best of our knowledge, BM31 is the only shared and completely reported phage against S. arlettae in the entire public database.

The pathogenicity comparison of Lagovirus europaeus GI.1 and GI.2 strains in China by using relative quantitative assay.

Lagovirus europaeus GI.1 belongs to Lagovirus in the Caliciviridae family. GI.1 causes an acute, septic, and highly lethal disease in rabbits. Lagovirus europaeus GI.2, a new variant of GI.1, has caused explosive mortality in rabbits of all ages in Sichuan Province, China. To explore the differences in pathogenicity of rabbits infected with GI.1/GI.2, we investigated the virulence and disease progression of a naturally occurring GI.1/GI.2 in 4-week-old, 13-week-old, and 25-week-old New Zealand White laboratory rabbits after GI.1/GI.2 infection. Objective measures of disease progression were recorded using continuous body-temperature monitoring. We observed the kittens were infected with GI.2 during the most urgent course of the disease, and GI.1 was not lethal to kittens. We found that the target organ of both GI.1 and GI.2 was the liver, but the disease course of the two viruses was differed. Our study enriches the research on the pathogenicity of GI.1 and GI.2 under the same conditions.

MicroRNA let-7f-5p regulates PI3K/AKT/COX2 signaling pathway in bacteria-induced pulmonary fibrosis via targeting of PIK3CA in forest musk deer.

Background: Recent studies have characterized that microRNA (miRNA) is a suitable candidate for the study of bleomycin/LPS-induced pulmonary fibrosis, but the knowledge on miRNA in bacteria-induced pulmonary fibrosis (BIPF) is limited. Forest musk deer (Moschus berezovskii, FMD) is an important endangered species that has been seriously affected by BIPF. We sought to determine whether miRNA exist that modulates the pathogenesis of BIPF in FMD. Methods: High-throughput sequencing and RT-qPCR were used to determine the differentially expressed miRNAs (DEmiRNAs) in the blood of BIPF FMD. The DEmiRNAs were further detected in the blood and lung of BIPF model rat by RT-qPCR, and the targeting relationship between candidate miRNA and its potential target gene was verified by dual-luciferase reporter activity assay. Furthermore, the function of the candidate miRNA was verified in the FMD lung fibroblast cells (FMD-C1). Results: Here we found that five dead FMD were suffered from BIPF, and six circulating miRNAs (miR-30g, let-7f-5p, miR-27-3p, miR-25-3p, miR-9-5p and miR-652) were differentially expressed in the blood of the BIPF FMD. Of these, let-7f-5p showed reproducibly lower level in the blood and lung of the BIPF model rat, and the expression levels of PI3K/AKT/COX2 signaling pathway genes (PIK3CA, PDK1, Akt1, IKBKA, NF-κB1 and COX2) were increased in the lung of BIPF model rats, suggesting that there is a potential correlation between BIPF and the PI3K/AKT/COX2 signaling pathway. Notably, using bioinformatic prediction and experimental verification, we demonstrated that let-7f-5p is conserved across mammals, and the seed sequence of let-7f-5p displays perfect complementarity with the 3' UTR of PIK3CA gene and the expression of the PIK3CA gene was regulated by let-7f-5p. In order to determine the regulatory relationship between let-7f-5p and the PI3K/AKT/COX2 signaling pathway in FMD, we successfully cultured FMD-C1, and found that let-7f-5p could act as a negative regulator for the PI3K/Akt/COX2 signaling pathway in FMD-C1. Collectively, this study not only provided a study strategy for non-invasive research in pulmonary disease in rare animals, but also laid a foundation for further research in BIPF.

Application of Nanopore Sequencing in the Detection of Foodborne Microorganisms.

Foodborne pathogens have become the subject of intense interest because of their high incidence and mortality worldwide. In the past few decades, people have developed many methods to solve this challenge. At present, methods such as traditional microbial culture methods, nucleic acid or protein-based pathogen detection methods, and whole-genome analysis are widely used in the detection of pathogenic microorganisms in food. However, these methods are limited by time-consuming, cumbersome operations or high costs. The development of nanopore sequencing technology offers the possibility to address these shortcomings. Nanopore sequencing, a third-generation technology, has the advantages of simple operation, high sensitivity, real-time sequencing, and low turnaround time. It can be widely used in the rapid detection and serotyping of foodborne pathogens. This review article discusses foodborne diseases, the principle of nanopore sequencing technology, the application of nanopore sequencing technology in foodborne pathogens detection, as well as its development prospects.

The Antiviral Effect of Isatis Root Polysaccharide against NADC30-like PRRSV by Transcriptome and Proteome Analysis.

(1) Background: In recent years, the porcine reproductive and respiratory syndrome virus (PRRSV) has become a virulent pathogen that has caused devastating diseases and economic losses worldwide in the swine industry. IRPS has attracted extensive attention in the field of virology. However, it is not clear that IRPS has an antiviral effect on PRRSV at gene and protein levels. (2) Methods: We used transcriptomic and proteomic analysis to investigate the antiviral effect of IRPS against PRRSV. Additionally, a microbiome was used to explore the effects of IRPS on gut microbes. (3) Results: IRPS significantly extenuated the pulmonary pathological lesions and inflammatory response. We used transcriptomic and proteomic analysis to investigate the antiviral effect of IRPS against PRRSV. In the porcine model, 1669 differentially expressed genes (DEGs) and 370 differentially expressed proteins (DEPs) were identified. Analysis of the DEG/DEP-related pathways indicated immune-system and infectious-disease (viral) pathways, such as the NOD-like receptor (NLR) signaling pathway, toll-like receptor (TLR) signaling pathway, and Influenza A-associated signaling pathways. It is noteworthy that IRPS can inhibit NLR-dependent gene expression, then reduce the inflammatory damage. IRPS could exert beneficial effects on the host by regulating the structure of intestinal flora. (4) Conclusions: The antiviral effect of IRPS on PRRSV can be directly achieved by omics techniques. Specifically, the antiviral mechanism of IPRS can be better elucidated by screening target genes and proteins using transcriptome and proteome sequencing, and then performing enrichment and classification according to DEGs and DEPs.

Isolation of Klebsiella pneumoniae Phage vB_KpnS_MK54 and Pathological Assessment of Endolysin in the Treatment of Pneumonia Mice Model.

With the improper use of antibiotics, an increasing number of multidrug-resistant bacteria have been reported worldwide, posing challenges for disease treatment. Klebsiella pneumoniae is an important zoonotic pathogen that colonises the respiratory tract. Endolysin therapy has emerged with the development of phages. In this study, a lytic phage vB_KpnS_MK54 was isolated from the drinking water of a forest musk deer (FMD) farm in Sichuan Province. It was the first reported phage obtained from FMD. The primary biological characteristics were determined, and whole-genome sequencing analysis was performed. The phage which belongs to the family Siphoviridae is highly specific for lytic host bacteria and is moderately adaptable to different environments. Whole-genome sequencing results showed that the phage genome size was 46,218 bp. There were 80 coding DNA sequences (CDSs) in total, 32 of which had known functions. The last CDS is the phage endolysin LysG24. A new peptide-modified endolysin (LysCA) was constituted by connecting the cecropin A peptide residues with LysG24 to investigate the antibacterial activities of both LysG24 and LysCA. The results showed that the lytic profile of LysG24 and LysCA was wider than that of phage MK54. For in vitro tests, both endolysins destroyed 99% of the host bacteria within 6 h. The lysing ability and environmental adaptability of LysCA were significantly stronger than those of LysG24. For in vivo tests, LysG24 and LysCA exhibited therapeutic effects in a mouse model of pneumonia wherewith the mice were infected with K. pneumoniae (LPKP), wherein both LysG24 and LysCA can effectively reduce the pulmonary inflammatory response. The LPKP bacterial load in the treatment group was significantly lower than that in the bacterial group, among which LysCA displayed a more obvious therapeutic effect. Furthermore, the safety test showed that the endolysins had no toxic effects on mice. In general, both LysG24 and LysCA showed excellent antibacterial activity in vivo and in vitro, with high safety and strong adaptability to the environment, manifesting their latent potential as new antimicrobial agents.

Design of a multi-epitope vaccine against Haemophilus parasuis based on pan-genome and immunoinformatics approaches.

Background: Glässer's disease, caused by Haemophilus parasuis (HPS), is responsible for economic losses in the pig industry worldwide. However, the existing commercial vaccines offer poor protection and there are significant barriers to the development of effective vaccines. Methods: In the current study, we aimed to identify potential vaccine candidates and design a multi-epitope vaccine against HPS by performing pan-genomic analysis of 121 strains and using a reverse vaccinology approach. Results: The designed vaccine constructs consist of predicted epitopes of B and T cells derived from the outer membrane proteins of the HPS core genome. The vaccine was found to be highly immunogenic, non-toxic, and non-allergenic as well as have stable physicochemical properties. It has a high binding affinity to Toll-like receptor 2. In addition, in silico immune simulation results showed that the vaccine elicited an effective immune response. Moreover, the mouse polyclonal antibody obtained by immunizing the vaccine protein can be combined with different serotypes and non-typable Haemophilus parasuis in vitro. Conclusion: The overall results of the study suggest that the designed multi-epitope vaccine is a promising candidate for pan-prophylaxis against different strains of HPS.

Functionalized Nanoparticles in Prevention and Targeted Therapy of Viral Diseases With Neurotropism Properties, Special Insight on COVID-19.

Neurotropic viruses have neural-invasive and neurovirulent properties to damage the central nervous system (CNS), leading to humans' fatal symptoms. Neurotropic viruses comprise a lot of viruses, such as Zika virus (ZIKV), herpes simplex virus (HSV), rabies virus (RABV), and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Effective therapy is needed to prevent infection by these viruses in vivo and in vitro. However, the blood-brain barrier (BBB) usually prevents macromolecules from entering the CNS, which challenges the usage of the traditional probes, antiviral drugs, or neutralizing antibodies in the CNS. Functionalized nanoparticles (NPs) have been increasingly reported in the targeted therapy of neurotropic viruses due to their sensitivity and targeting characteristics. Therefore, the present review outlines efficient functionalized NPs to further understand the recent trends, challenges, and prospects of these materials.

Preexposure and Postexposure Prophylaxis of Rabies With Adeno-Associated Virus Expressing Virus-Neutralizing Antibody in Rodent Models.

Rabies, a fatal disease in humans and other mammals, is caused by the rabies virus (RABV), and it poses a public health threat in many parts of the world. Once symptoms of rabies appear, the mortality is near 100%. There is currently no effective treatment for rabies. In our study, two human-derived RABV-neutralizing antibodies (RVNA), CR57 and CR4098, were cloned into adeno-associated virus (AAV) vectors, and recombinant AAVs expressing RVNA were evaluated for postexposure prophylaxis after intrathecal injection into RABV-infected rats. At 4days post-infection with a lethal dose of RABV, 60% of the rats that received an intrathecal injection of AAV-CR57 survived, while 100% of the rats inoculated with AAV-enhanced green fluorescent protein (EGFP) succumbed to rabies. Overall, these results demonstrate that AAV-encoding RVNA can be utilized as a potential human rabies postexposure prophylaxis.

Early diagnosis of rabies virus infection by RPA-CRISPR techniques in a rat model.

Rabies, which is caused by rabies virus (RABV), poses an ever-present threat to public health in most countries of the world. Once clinical signs appear, the mortality of rabies approaches 100%. To date, no effective method for early rabies diagnosis has been developed. In this study, an RPA-CRISPR nucleic-acid-based assay was developed for early rabies diagnosis by detecting viral RNA shedding in the cerebrospinal fluid (CSF) of rats. This method can detect a single copy of RABV genomic RNA in 1 µL of liquid. RABV genomic RNA released from viral particles in the CSF could be detected via RPA-CRISPR as early as 3 days postinfection in a rat model. This study provides an RPA-CRISPR technique for early detection of RABV with potential application in the clinical diagnosis of human rabies.