Rabies virus viability in vitro / 中华微生物学和免疫学杂志

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.

Comparison of immunization strategies for human rabies pre-exposure prophylaxis in laboratory animals / 中华微生物学和免疫学杂志

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.

In vitro inhibitory effects of resveratrol on rabies virus / 中华微生物学和免疫学杂志

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.

Codon optimization improves the expression of nucleoprotein of rabies virus CTN-1 strain in E. coli / 中华微生物学和免疫学杂志

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.

Establishment and preliminary application of rabies virus CVS-11 challenge model in mice / 中华微生物学和免疫学杂志

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.

Epidemiological characteristics of human rabies in China, 2017 / 中华流行病学杂志

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.

Expert consensus on rabies exposure prophylaxis / 中华预防医学杂志

Rabies is a zoonotic infectious disease caused by lyssavirus and characterized by central nervous system symptoms. The fatality rate of rabies is almost 100%. About 59 000 cases die of rabies worldwide every year, mainly in Asia and Africa. China is an epidemic country of rabies. Grade II and III exposures are the main types of rabies exposures in China. Standardized post-exposure prophylaxis (PEP) can prevent rabies almost 100%. Human Rabies Vaccine Technical Working Group, National Immunization Advisory Committee and invited experts reached an expert consensus on PEP by referring to the World Health Organization′s position paper on rabies vaccine in 2018 and related research progress in recent.

Differences on geographic distribution of rabies virus lineages in China / 中华流行病学杂志

Objective To study the lineages of rabies virus and the epidemic characteristics in different provincial populations of China,to provide information for the development of control and prevention measures in each respective provinces.Methods Full length N and G genes and full-genome of epidemic strains of rabies virus collected in China were downloaded from GenBank and combined with newly sequenced strains by our lab.Each strain was classified under six lineages of China rabies by constructing phylogenetic trees based on the N or G sequences.Numbers of strains and lineages in each province were counted and compared.Results Six lineages (China Ⅰ-Ⅵ) were prevalent in China,with 4 found in Yunnan and Hunan.In 6 provinces,including Henan and Fujian,3 lineages were found.In 8 provinces,including Shanghai and Jiangxi,2 lineages were found Only 1 lineage,were found in Beijing,Tianjin and other 12 provinces.the China Ⅰ,was the dominant one in 25 provinces.In recent years,China Ⅲ had been found in wild animals and spread over livestock in Inner Mongolia and Xinjiang areas.Qinghai and Tibet had been influenced by China Ⅳ,which also been found in wild animals of Inner Mongolia and Heilongjiang.Conclusion There had been obvious differences in lineages and strain numbers of rabies virus identified in different provinces in China.

Epidemiology of human rabies in China, 2016 / 中华流行病学杂志

Objective To understand the epidemiological characteristics of human rabies in China in 2016 and provide evidence for the control and prevention of human rabies.Methods The incidence data of human rabies in China in 2016 were collected from national infectious disease reporting information management system.The surveillance data were collected from provinces of Shandong,Guizhou,Anhui,Hunan,Jiangsu and Guangxi Zhuang Autonomous Region.Excel 2013 software was used to process and summarize the data,the epidemiological characteristics of human rabies in China in 2016 were described by using indicators such as morbidity,mortality and constituent ratio.Results A total of 644 human rabies cases were reported in 28 provinces in China in 2016,a decrease of 19.60％ (157/801) compared with 2015.The provinces reporting high incidences of human rabies were Henan,Hunan,Guangxi and Guizhou,accounting for 39.44％ (254/644) of the total cases.One case was reported in Qinghai province and Xinjiang Uygur Autonomous Region respectively.The male to female ratio of the cases was 2.14 ∶ 1 (439/205),and the majority of the patients were farmers (444/644).Surveillance points in 6 provinces reported 1 281 340 persons seeking post-exposure treatment,of whom 1 018 367 were treated for dog bite or scratch.A total of 764 234 persons completed the vaccination series,accounting for 63.90％ (764 234/1 195 956) of the persons with grade Ⅱ and Ⅲ exposures,and 28.89％ (165 677/573 571) of the persons with grade Ⅲ exposure were treated with passive immunization product.The average density of dogs in each surveillance area was 7.03/100 persons,the average canine immunization rate was 37.64％.Conclusion The incidence of human rabies has remained decline in China in 2016,the number of the affected provinces has increased and that has the tendency of spreading to low-risk regions.The cases mainly occurred in men and farmers,and caused by dog bite or scratch.It is necessary to strengthen the health education about rabies prevention and control in rural areas and expand the coverage of canine immunization to prevent and control human rabies.

Differences on geographic distribution of rabies virus lineages in China / 中华流行病学杂志

Objective To study the lineages of rabies virus and the epidemic characteristics in different provincial populations of China,to provide information for the development of control and prevention measures in each respective provinces.Methods Full length N and G genes and full-genome of epidemic strains of rabies virus collected in China were downloaded from GenBank and combined with newly sequenced strains by our lab.Each strain was classified under six lineages of China rabies by constructing phylogenetic trees based on the N or G sequences.Numbers of strains and lineages in each province were counted and compared.Results Six lineages (China Ⅰ-Ⅵ) were prevalent in China,with 4 found in Yunnan and Hunan.In 6 provinces,including Henan and Fujian,3 lineages were found.In 8 provinces,including Shanghai and Jiangxi,2 lineages were found Only 1 lineage,were found in Beijing,Tianjin and other 12 provinces.the China Ⅰ,was the dominant one in 25 provinces.In recent years,China Ⅲ had been found in wild animals and spread over livestock in Inner Mongolia and Xinjiang areas.Qinghai and Tibet had been influenced by China Ⅳ,which also been found in wild animals of Inner Mongolia and Heilongjiang.Conclusion There had been obvious differences in lineages and strain numbers of rabies virus identified in different provinces in China.

Epidemiology of human rabies in China, 2016 / 中华流行病学杂志

Objective To understand the epidemiological characteristics of human rabies in China in 2016 and provide evidence for the control and prevention of human rabies.Methods The incidence data of human rabies in China in 2016 were collected from national infectious disease reporting information management system.The surveillance data were collected from provinces of Shandong,Guizhou,Anhui,Hunan,Jiangsu and Guangxi Zhuang Autonomous Region.Excel 2013 software was used to process and summarize the data,the epidemiological characteristics of human rabies in China in 2016 were described by using indicators such as morbidity,mortality and constituent ratio.Results A total of 644 human rabies cases were reported in 28 provinces in China in 2016,a decrease of 19.60％ (157/801) compared with 2015.The provinces reporting high incidences of human rabies were Henan,Hunan,Guangxi and Guizhou,accounting for 39.44％ (254/644) of the total cases.One case was reported in Qinghai province and Xinjiang Uygur Autonomous Region respectively.The male to female ratio of the cases was 2.14 ∶ 1 (439/205),and the majority of the patients were farmers (444/644).Surveillance points in 6 provinces reported 1 281 340 persons seeking post-exposure treatment,of whom 1 018 367 were treated for dog bite or scratch.A total of 764 234 persons completed the vaccination series,accounting for 63.90％ (764 234/1 195 956) of the persons with grade Ⅱ and Ⅲ exposures,and 28.89％ (165 677/573 571) of the persons with grade Ⅲ exposure were treated with passive immunization product.The average density of dogs in each surveillance area was 7.03/100 persons,the average canine immunization rate was 37.64％.Conclusion The incidence of human rabies has remained decline in China in 2016,the number of the affected provinces has increased and that has the tendency of spreading to low-risk regions.The cases mainly occurred in men and farmers,and caused by dog bite or scratch.It is necessary to strengthen the health education about rabies prevention and control in rural areas and expand the coverage of canine immunization to prevent and control human rabies.

Epidemiological characteristics of rabies and its prevention and control in China / 中华实验和临床病毒学杂志

There were three rabies epidemic stages from 1950 to 2016 in China. This article describes the epidemiological characteristics, summarizes the experience and lessons of prevention and control of each stage. The prevalence of rabies in China was closely related to the social factors, the level of economic development, national policies and technology. Therefore, to eliminate rabies in humans in China, joint efforts including national policies and various norms of support are needed. Also, animal immunization and human disposal after exposure are important for the protection of human life and safety.

Experimental study on the inhibitory effect of soap solution on rabies viruses / 中华实验和临床病毒学杂志

Objective@#To investigate the inactivating effect of soap solution on rabies virus and to explore the concentration of soap solutions which could be effective in post-exposure prophylaxis (PEP) of rabies virus infection.@*Methods@#The BSR and N2a cells were respectively infected by the mixture of different concentrations of soap solution and rabies virus CVS-11. Based on the direct immunofluorescent method (DFA) and reverse transcription PCR (RT-PCR), the inactivating effects of soap solutions on rabies viruse in BSR and N2a cells were detected, respectively.@*Results@#This experiment established the BSR or N2a cells model in 24 well cell culture plates, and we found that the upper limit of soap solution concentration which BSR or N2a cells could tolerate was 1%. The inhibitory effect test of different soap solution on rabies virus showed that the 0.5% concentration of soap solution could completely inhibit the survival of CVS-11 strain in both the BSR and N2a cells, but the 0.1% concentration of soap solution could not inhibit the rabies viruses completely.@*Conclusions@#The 0.5%-1% concentration of soap solutions could inhibit the survival of CVS-11 strain in three minutes in vivo experiment.

Establishment and application of a noval CVS-11 pseudovirus-based assay for detection of neutrali-zing antibody against rabies virus / 中华微生物学和免疫学杂志

Objective To establish a CVS-11 pseudovirus particles ( pp)-based assay for detec-tion of neutralizing antibody against rabies virus. Methods An improved rapid fluorescence focus inhibition test ( RFFIT) for detection of neutralizing antibody against rabies virus ( RVNA) was established based on the CVS-11 pseudovirus expressing a luciferase reporter gene. Forty-six human serum samples were analyzed with the improved RFFIT and the results were compared with those by using standard RFFIT. Moreover, the improved RFFIT was used to detect the titers of RVNA in 91 serum samples collected from pet dogs and pet-breeders in Beijing. Results The coincidence rate of the improved RFFIT and the standard RFFIT was 100% regarding to the analysis of 46 human serum samples and 5 negative reference serum samples. Moreo-ver, the RVNA titers of all serum samples obtained with CVS-11 pseudovirus-based assay showed a signifi-cant high correlation with those obtained with standard RFFIT (n=46, r=0. 94, P<0. 000 1). All of the 91 serum samples collected from pet dogs and pet-breeders in Beijing were positive for RVNA as indicated by the improved RFFIT with a mean titer of 33. 01 IU/ml. Conclusion We established an improved RFFIT based on the CVS-11 pp expressing luciferase reporter gene, which might be used as a reliable alternative RFFIT for measuring RVNA titer. Analysis of the 91 serum samples collected in Beijing with the improved RFFIT showed that all samples were positive for RVNA.

Development and Application of An Assay for High-throughput Antiviral Compounds Screening against Alphaviruses / 病毒学报

To establish a cell-based rapid luciferase suppression assay for high-throughput screening (HTS) anti-alphaviruses compounds screening, which could cause viral encephalitis, raise the social issues associated directly with public health and huge economic burden to the society. The Gaussia luciferase assay system was used for HTS model for identifying inhibitors of labeled virus XJ160-GLUC. The decreased 50% GLUC activity inhibition ratio was deemed to be the screening positive index. The reaction system in this model was optimized, and the reliability of the model was evaluated. For HTS model's optimization, cells were infected with XJ160-GLUC at an MOI of 0.025 PFU/cell. The supernatant treated with compounds 48h were collected for GLUC expression detection. In the model, Z' factor was up to 0.71, demonstrating that HTS assay for identifying inhibitors that target all aspects of the viral life cycle of XJ160-GLUC was stable and reliable. After screening 8080 compounds (five-in-one), 341 positive samples were selected, and the positive rate was 4.2% with a cutoff at 50% inhibition. Then 1705 compounds were screened subsequently and the positive rate was 1.1% with obtaining 19 positive compounds. These results will lay the foundation for finding the anti-alphaviruses' drug targets.

Development of EGFP/GLUC-tagged Sindbis-like virus XJ-160.

Based on an infectious clone of Sindbis-like virus XJ-160, recombinant vectors containing a reporter gene (enhanced green fluorescence protein [EGFP] or Gaussia luciferase [GLUC]) were constructed by placing the reporter gene cassette containing the subgenomic promoter behind the 3' terminus of the viral structural protein gene. EGFP/GLUC-tagged Sindbis-like viruses were rescued in BHK-21 cells transfected with transcripts produced from the recombinant vectors. EGFP expression and strong luciferase activity were detected in BHK-21 cells infected with repeated passages of the EGFP/GLUC-tagged viruses, revealing the genetic stability of the chimeric viruses. The EGFP/GLUC-tagged Sindbis viruses reported will contribute to the assessment of viral replication and proliferation, tracking and elucidating Alphavirus-host interactions, and screening for antiviral compounds.

Phenotypic and molecular characteristics of plaque-purified MX10 virus, an Oriental-Australian genotype of Sindbis virus from Yunnan, China.

A previous investigation showed that MX10 virus, recently isolated in China, belongs to the Oriental-Australian (O/A) genotype of Sindbis virus (SINV) (Wang Jinglin, 2011, ATMH). Similar to the MRE16 isolate, the prototype O/A genotype of SINV, two derivate viruses with obviously different plaque morphologies were derived from MX10 virus, which were accordingly denoted as MX10-LP and MX10-SP. MX10-LP virus exhibited higher neurovirulence in neonatal mice than MX10-SP virus. Analysis of the complete genome revealed seven nucleotide differences between MX10-LP and MX10-SP. Compared with MRE16 virus, MRE16SP virus has a deletion of 30 aa in the E2 gene (200-229), which has been shown to be the molecular basis for the different plaque morphology. However, the MX10-SP virus did not have the 30-amino-acid deletion in the E2 gene. These results demonstrate that the molecular basis for the different plaque morphology of MX10 virus, the first strain of the O/A genotype of SINV isolated from China, is different from that of the prototype MRE16 virus.

How does cellular heparan sulfate function in viral pathogenicity? / 生物医学与环境科学（英文）

Heparan sulfate (HS) is ubiquitously expressed on the surfaces and in the extracellular matrix of virtually all cell types, making it an ideal receptor for viral infection. Compared with wild-type viruses, cell culture-adapted laboratory strains exhibit more efficient binding to cellular HS receptors. HS-binding viruses are typically cleared faster from the circulation and cause lower viremia than their non-HS-binding counterparts, suggesting that the HS-binding phenotype is a tissue culture adaptation that lowers virus fitness in vivo. However, when inoculated intracranially, efficient cell attachment through HS binding can contribute to viral neurovirulence. The primary aim of this review is to discuss the roles of HS binding in viral pathogenicity, including peripheral virulence and neurovirulence. Understanding how heparan sulfate functions during virus infection in vivo may prove critical for elucidating the molecular mechanism of viral pathogenesis, and may contribute to the development of therapeutics targeting HS.

Role of the E gene in the infectivity of SINV and SINL in infected cells / 中华微生物学和免疫学杂志

Objective To elucidate the molecular basis on the differences of infectivity in infected cells between Sindbis virus(SINV:YN87448 virus)and Sindbis like virus(SINLV:XJ-160 virus).Methods Compare the E(glycoprotein)gene sequence and secondary structure of YN87448 virus and XJ-160 virus by bioinformatics analysis.Analyze the contribution of E gene to the biological differences between SINV and SINLV by constructing recombinant virus.Results By bioinformatics analysis,YN87448 virus and XJ-160 virus have the same genomic structure,which has 11 717 nt and 11 626 nt respectively.There are 82 amino acid differences between E gene of these two viruses,and showed scattered distribution.The main peak is basically the same for the hydrophobic of the E gene protein,but in some region existing small differences.The recombinant virus which exchanged the E gene of XJ-160 virus with YN87448 virus totally showed the biological character of YN87448 virus,either in the showing time of CPE,plaque forming time and plaque diameter,or in expression of functional proteins.Conclusion E gene plays a major role in the differences of infectivity in infected cells between SINV and SINLV,this result provide the molecular biological evidences for elucidating the biological differences between SINV and SINLV.