Rabies is a highly fatal zoonotic disease that is widely prevalent worldwide. Through large-scale immunization measures and other control strategies, rabies has been gradually brought under control. The World Health Organization (WHO) has called for the elimination of canine-mediated human rabies by 2030. Detection of the rabies virus is not only crucial for diagnosing cases but also an essential tool for measuring progress in rabies elimination. This article provides an overview and review of research on rabies virus detection, covering antigen detection techniques, antibody detection techniques, nucleic acid detection techniques, and other detection method.

Rabies virus (RABV) belonging to the Rhabdoviridae family and the Lyssavirus genus is the main pathogen of rabies and is a single stranded RNA virus with an envelope. The RABV genome encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA dependent RNA polymerase (RdRp). The N protein mainly plays an important role in the transcription and replication process of viruses, which is often used for diagnosis and virus identification; P protein can interact with some host protein partners, disrupt antiviral signaling pathways, and affect the pathogenicity of RABV; M protein plays an important role in regulating the balance between viral transcription and replication; as a target for inducing neutralizing antibodies, the trimeric structure analysis of G protein before fusion, the influence of fusion rings on successful trimerization and conformational stability, and the visualization of effective and widespread neutralizing antibody epitopes provide a basis for the development of broad-spectrum vaccines. The RdRp protein mainly catalyzes the transcription of viral RNA. In recent years, the analysis of the crystal structure of structural proteins, the discovery of related functional sites, and the interaction with small molecules have helped researchers better understand the pathogenicity of RABV, search for effective antiviral drug targets, and design broad-spectrum vaccines.

Objective:To compare the differences in the safety, efficacy and protective effects of rabies vaccine using the current pre-exposure prophylaxis schedule in China (0-7-21 or 28) and the newly recommended immunization program of WHO (0-7), aiming to provide data support for modifying the related content of Technical Guideline for Human Rabies Prevention and Control. Methods:The mice were randomly divided into five groups, namely 0-7-21 group (3-injection regimen), 0-7 group (2-injection regimen), 0-14 group, 0-21 group and control group, according to the current 3-injection regimen (0-7-21) in China and the 2-injection regimen (0-7) recommended by WHO. The survival status of the mice was observed. The mice were weighed every five days to compare the safety of different immunization procedures. Rabies virus neutralizing antibodies (RVNA) were detected 7, 14, 21, 28 and 35 d after the initial immunization. On day 35, the mice in each group were challenged with lethal dose of CVS-11 rabies virus to evaluate the protective effects of different pre-exposure immunization procedures.Results:There was no significant difference in weight gain of mice after vaccination. The positive rate of RVNA was 100% in all immunized groups from day 14, which could provide complete protection to mice. There was a significant difference in RVNA levels between 0-7-21 and 0-7 groups at 35 d( P<0.05), but there was no statistical difference at other time points ( P>0.05). RVNA level had a significant difference between 0-7 and 0-21 groups at 21 d and 35 d ( P<0.05). There was no statistical difference in RVNA level between other groups at each time point ( P>0.05). In the protective test, the survival rates of mice in all immune groups were 100%. Conclusions:The current 3-injection pre-exposure immunization procedure for rabies vaccine (0-7-21) and the newly recommended 2-injection immunization procedure (0-7) had similar efficacy and protective effects in animal tests. In view of the cost saving and better compliance of the 2-injection immunization procedure, it was recommended that relevant departments should conduct clinical trials as soon as possible to promote the implementation of this program.

Objective:To investigate the in vitro viability of rabies virus in tissues and body fluid samples. Methods:The viability of rabies virus in tissues and suspensions was analyzed by virus titer determination method, direct immunofluorescence, RT-PCR and laboratory techniques for virus isolation.Results:With the increase of temperature, the viability of rabies virus in brain tissues and suspensions decreased gradually. Rabies virus lost infectivity after 30 min at 56℃, but remained viable in tissues for 7 d at 37℃. The virus showed no viability after 1 h at pH9.6. The rabies virus in suspensions could be completely inactivated after the stimulation with ethanol at a final concentration above 30%, sodium hypochlorite above 500 mg/L or benzalkonium bromide above 100 mg/L for 3 min. It was found that 80% acetone had the strongest inactivation effect on rabies virus in tissues, and no virus could be isolated after soaking for 4 h.Conclusions:Rabies virus was not tolerant to high temperature and relatively stable in the environment with pH6.8-7.4. Common disinfectants could kill the virus. This study provided detailed data about the viability of rabies virus in vitro, which would be conducive to the prevention and control of rabies.

Objective:To analyze the in vitro inhibitory effects of resveratrol on rabies virus. Methods:The challenge virus standard (CVS)-11 strain of rabies virus and BHK-21 cells were used to establish the infection model. In vitro inhibitory effects of resveratrol on rabies virus were analyzed at different stages of infection by direct immunofluorescence and cell fluorescence focus unit assay. Results:Without affecting cell growth, resveratrol could block the adsorption of virus, interfere with the entry of virus into cells and inhibit virus proliferation in a concentration-dependent manner. The inhibition rate could reach up to about 95%. The results of co-incubation experiment showed that 40 μmol/L resveratrol could directly kill the virus.Conclusions:This study indicated that resveratrol inhibited the activity of rabies virus in a concentration-dependent manner.

Objective:To obtain the high-efficiency expression of the biological active rabies virus nucleoprotein in the prokaryotic expression system.Methods:This experiment uses codon optimization technology to re-encode the nucleoprotein gene of rabies virus CTN-1 strain, artificially synthesize the full-length gene and clone it into pET-43.1a prokaryotic expression vector, induced expression in BL21 (DE3) strain of Escherichia coli( E. coli), and used Western blot to detect its reactogenicity. Results:The results showed that after induction, SDS-PAGE electrophoresis analysis showed that an obvious expression band appeared at a molecular weight of 50×10 3, which was consistent with the expected protein band size. Among them, the E. coli concentration A600 is about 0.5, and the expression yield is the highest (about 32.3%) when induced at 37℃ for 5 h. Nucleoprotein expression product is mainly inclusion body when it is expressed in large quantities. After purification by Ni 2+ chelating chromatography, the purity of the target protein can reach over 95%. The purified product was identified by Western blot and positively reacted with the sera of mice immunized with rabies vaccine, indicating that the prokaryotic expression of the CTN-1 strain nucleoprotein has biological activity. Conclusions:This experiment successfully established a high-efficiency expression method for the nucleoprotein of the CTN-1 strain in the prokaryotic expression system, and obtained high-purity target protein, which provides a basis for further clinical diagnosis and preparation of new vaccines.

Objective:To understand the epidemiological characteristics of rabies in China from 2007 to 2018, and to provide reference information for formulating strategies to eliminate rabies.Methods:The incidence data were obtained from the national notifiable communicable disease reporting information system in China to analyze the spatial, population and time distribution of human rabies cases.Results:From 2007 to 2018, 17 848 cases of human rabies were reported nationwide. 57% of the cases were reported in 6 provinces, including Guangxi, Guangdong, Guizhou, Hunan, Henan and Sichuan. A total of 37 counties reported more than 50 cases. The number of reported cases decreased from 3 300 in 2007 to 422 in 2018, with an average annual decline rate of 7%. The number of districts and counties involved decreased from 984 in 2007 to 308 in 2018. The population aged 35-75 years accounted for 67%, followed by the population aged 0-15 years (17%). Farmers accounted for 70% of the total number of cases, followed by students (11%) and scattered children (6%).Conclusions:China′s human rabies epidemic has changed from a regional epidemic to a multi-regional spread.

Objective:To analyze epidemiological characteristics and explore control and prevention measures based on the rabies surveillance data in China from 2016 to 2018.Methods:The surveillance data of rabies in China from 2016-2018 were collected from the "Infectious Disease Surveillance Information Reporting Management System", eleven national surveillance sites in Hunan, Anhui, Guizhou, Jiangsu and Shandong provinces and National Bureau of Statistics. Descriptive epidemiological method was used to analyze the distributions of rabies outbreaks in China during this period, and univariate and multivariate analyses were conducted on the data using SPSS 25.0.Results:Rabies epidemics were mainly found in southern and central areas in China. Rabies mainly occurred in summer and autumn, and the majority of patients were farmers, students and children outside child care settings. The male to female ratio of the cases was 2.38∶1(1 114∶468). The cases were reported in all age groups and the incidence curve was bimodal, and more cases occurred in middle aged and old adults than in youths. In the case analysis of rabies from 2016 to 2018, the majority of the animals biting human were dogs, accounting for 91.46%(439/480). And about 71.88% (345/480) of the exposures were degree Ⅲ. The vaccination rate after exposure was only 8.13%(39/480). Age, degree of exposure, site of exposure, wound management, vaccination and injection of passive immunization product might influence the incubation period.Conclusions:The human rabies incidence in China declined consecutively from 2016 to 2018, and the affected areas decreased. No cases were reported in northwestern area. The rate of post exposure prophylaxis, vaccination and passive immunization product injection were all improved, but the level of standard wound management after exposure needs to be improved. Attention should be paid to epidemic surveillance in high-incidence provinces and neighboring provinces. It is necessary to strengthen professional treatment training in relevant medical institutions, strengthen immunization and management of dog to achieve the goal of "zero human rabies death by 2030".

Objective:To establish a rabies virus CVS-11 challenge model in BALB/c mice through intramuscular or intracerebral injection.Methods:The CVS-11 strain propagated in BSR cells with a titer of 2.7×10 7 FFU/ml was serially diluted 10 -1-10 -7 times to infect 4-week-old female mice through intramuscular or intracerebral injection. The morbidity and mortality of mice were observed after virus challenge. Moreover, brain tissues of all challenged mice were subjected to direct immunofluorescence assay (DFA) and reverse transcription polymerase chain reaction (RT-PCR) to analyze the cause of death. The median lethal doses (LD 50) in mice under different challenge methods were determined. Mouse challenge models were established to evaluate the immunoprotective effects of four domestically available rabies vaccines on mice after CVS-11 exposure. Results:BALB/c mice developed typical neurological symptoms and died 6-12 d after intracerebral challenge and the LD 50 was 18.3/0.1 ml. The mice intramuscularly challenged with CVS-11 showed clinical symptoms on 8-15 d and the LD 50 was 2.7×10 5/0.1 ml. DFA results showed that specific yellow-green fluorescence appeared in the brain tissue prints of all dead mice. RT-PCR results showed that all amplified products showed bright bands at about 250 bp. These results suggest that rabies virus infection was the cause of death in mice. The protective effect test results of four different rabies vaccines on the market without immunoglobulin application showed that the survival rate of mice after exposure to one of the vaccines was 50%, and the survival rates of mice immunized with the other three vaccines were all 30%. The above results indicated that the four rabies vaccines provided partial protection for mice exposed to CVS-11 without the use of rabies passive immunization preparations. Conclusions:This study established rabies virus CVS-11 challenge models in BALB/c mice under different challenge methods and provided a technical platform for related research on rabies and rabies vaccines.

Objective: To understand epidemiological characteristics of human rabies in China in 2017 and provide evidence for the development of strategy of human rabies control and prevention. Methods: The descriptive epidemiological analysis was conducted based on the epidemic data from Chinese Infectious Disease Surveillance Reporting System, sentinel surveillance system in 6 provinces (Hunan, Guangxi, Anhui, Guizhou, Jiangsu and Shandong) and National Bureau of Statistics in 2017. Results: A total of 516 human rabies cases, including 502 deaths, were reported by 27 provinces in 2017 with the morbidity rate and mortality rate of 0.037/100 000 and 0.036/100 000, respectively. The case number and death number decreased by 19.88% (128/644) and 15.20% (90/592) respectively compared with 2016. Rabies epidemics were mainly found in southern and central areas. The first 5 provinces reporting high case numbers were Hunan (71 cases), Henan (52 cases), Guangxi (41 cases), Anhui (39 cases) and Hubei (39 cases), their cases accounted for 46.90% (242/516) of the total reported cases in China. Rabies mainly occurred in summer and autumn, and the majority of patients were farmers, students and children outside child care settings. The male to female ratio of the cases was 2.46 ∶ 1 (367 ∶ 149). Cases was reported in all age groups, and more cases occurred in middle aged and old adults than in adolescents. Questionnaires survey was conducted for 186 cases, the results indicated that 94.89% (167/176) of exposures were caused by dog bites. The exposure degree was mainly category Ⅲ, accounting for 68.86% (115/167), and only 6.02% (10/166) of cases were immunized after exposure. The median of latent period of these cases was 72 days. Conclusions By 2017, the human rabies incidence in China had declined consecutively for ten years, more cases were reported in southern area than in northern area. The case number showed downward trends in provinces with high incidences and fluctuant increase in provinces with low incidence. Rabies cases mainly occurred in rural areas, and most cases were men and farmers. Low rate of post exposure prophylaxis, low rates of vaccination and passive immunization product injection were main causes for the onset of human rabies. It is necessary to strengthen the surveillance for human rabies, especially in rural areas, health education about treatment after rabies exposure and expend the coverage of canine immunization.