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 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.

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.
Alphavirus ; drug effects ; genetics ; metabolism ; Animals ; Antiviral Agents ; pharmacology ; Drug Evaluation, Preclinical ; methods ; Genes, Reporter ; High-Throughput Screening Assays ; methods ; Luciferases ; genetics ; metabolism

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.

This paper studied the diagnosis and embryology of 20 cases lacking one pulmonary artery out of 2040 patients operated for tetralogy of Fallot. History of severe cyanosis syncope and hemoptysis was usually present in the majority of these patients. The important diagnosis characteristic was asymmetry of plumonary vascularity on X-ray film and the decrease of pulmonary cascular markings on the side without plumonary artery. A right ventricular cardiogram can confirm the absence of one pulmonary artery or the blind-end like change.

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 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.

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.
Heparitin Sulfate ; physiology ; Humans ; Receptors, Virus ; physiology ; Virulence ; Viruses ; pathogenicity

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.