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.

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.

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 prepare immortalized MRC-5 cells by transduction of human telomerase reverse transcriptase gene (hTERT),which can be used for the routine propagation of human cytomegalovirus (HCMV).Methods The recombinant plasmid pLVX-EFla-hTERT was constructed by cloning hTERT gene into the vector pLVX-EFla-Tet3G,and hTERT gene was amplified from another plasmid pLVX-EFla-Tet3G.Lentvirus particles LVX-EF1a-hTERT were prepared by co-transfected 293T cells with pLVX-EF1 a-hTERT and Lenti-X HTX Packaging Mix.After that,Lentvirus particles were used to infect MRC-5 cells,then both the propagation and expression of hTERT in the passaged MRC-5T were examined.Results We obtained Lentvirus particles LVX-EF1a-hTERT and immortalized MRC-5 cells.Conclusion Method of human telomerase reverse transcriptase-immortalized MRC-5 cells will contribute to the construction of immortalized primary cells.

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 provide a scientific basis for rabies control and prevention,the epidemic distribution data of 1996-2007 and 2008-2014 were analyzed contrastively.Methods The epidemic surveillance data were collected,the rising (1996-2007) and declining (2008-2014) of the epidemics were divided according to human rabies case number reported in China.The epidemic reporting,region distribution,geographical distribution,population and seasonal distribution were analyzed contrastively.Results From 1996 to 2014,29 656 human rabies cases were reported in China,average 1 561 cases each year,with a morbidity of 0.1198 per hundred thousand.From 1996 to 2007,total 17 459 cases were reported,20 to 24 provinces reporting rabies annually;2008-2014,total 12 197 cases were reported,23 to 28 provinces reporting rabies annually.Compared to the rising period of the epidemic,during the declining period,the regions surrounding the previous high-epidemic provinces and 14 north provinces had a rising incidence;the constitute of male case rose from 68.71％ to 70.54％ (P =0.001);the cases of 45 to 74 years old were 52.60％ instead of 42.56％ (P =0.000);the farmer cases increased to 69.76％ compared to 62.42％ (P =0.000);the cases from winter and spring rose to 42.34％ from 38.27％ (P =0.004).Conclusion It' s very important to strengthen dog rabies prevention and control in China,strengthen rabies knowledge education among the elderly population in rural areas,and never lose attention to rabies prevention and control in winter and spring.

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.

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