A novel dual luciferase expression vector was designed and its expression characteristics were studied in vitro and in vivo. Firstly, the Gluc and Fluc genes were connected via the TaV 2A sequence by overlaping PCR, and inserted into the expression vector pAAV2neoCAG, obtaining the recombinant plasmid pAAV2neoCAG-Gluc-2A-Fluc. Then pAAV2neoCAG-Gluc-2A-Fluc was transfected into BHK21 cells and the activity of Gluc and Fluc in the supernatant and cell lysates were assayed respectively. Results showed that both Gluc and Fluc were expressed successfully. The Gluc was mainly detected in the culture media while the Fluc was mostly within cells. The activity of Gluc in the supernatant increased gradually with time while the Fluc activity in cells almost kept stable. To investigate the expression of pAAV2neoCAG-Gluc-2A-Fluc in vivo, the plasmid was hydrodynamically injected into BALB/c mice through tail vein. The Gluc activity was assayed in a small volume of blood taken by tail vein at different time points. Results showed that Gluc was expressed stably at least 7 days. Live bioluminescence imaging technology was used to compare the expression characteristics of Gluc and Fluc. Whole body imaging was seen when coelenterazine, a specific substrate for Gluc, was injected, and the imaging signals decreased rapidly within 10 minutes. Liver imaging was showed when Flue specific substrate named D-Luciferin was injected, and the imaging remained stable at least for half an hour. The dual luciferase expression vector pAAV2neoCAG-Gluc-2A-Fluc combines the advantages of the secreted report gene Gluc and the non-secreted report gene Fluc, and will provide a new tool for cell labeling and tracing.
Animals ; Base Sequence ; Cricetinae ; Crustacea ; enzymology ; Fireflies ; enzymology ; Gene Expression ; Genes, Reporter ; Genetic Vectors ; genetics ; metabolism ; Luciferases ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data

A combinatorial human Fab library to the rabies virus was constructed using antibody genes derived from the blood of vaccinated donors. The library were panned and selected on purified rabies virus particles of aG or CTN strain with phage display. Eleven unique human Fab antibodies specific for the rabies virus glycoprotein were obtained by ELISA, IFA and DNA sequences analysis of these antibodies. Among these Fab antibodies, five human Fab antibodies were converted to full-length human IgG antibodies with recombinant baculovirus system. The five full-length human IgG antibodies were tested in vitro for rabies virus neutralization, resulting in all specificities to neutralize the virus. The obtained human anti-rabies antibodies lay the basis for the production of cocktail of anti-rabies monoclonal antibody with chinese intellectual property.
Amino Acid Sequence ; Animals ; Antibodies, Neutralizing ; immunology ; Antibodies, Viral ; immunology ; Cell Line ; Cricetinae ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Immunoglobulin G ; genetics ; immunology ; Molecular Sequence Data ; Neutralization Tests ; Rabies ; immunology ; virology ; Rabies virus ; genetics ; immunology

The biological and genetic characteristics of a highly neurovirulent JE virus strain SA4 were studied. Mice were inoculated intracerebrally with strain SA4 and SA14, and observed for 14 days, respectively. On different days, mice brains were harvested for titrations of the virus content in the brains. Full-length genome of SA4 was sequenced and compared with SA14 as well as other JE virus strains in the world. The results indicated that the mice inoculated by SA4 induced sickness and death more rapidly (24 hours faster) than those induced by the SA14. The virus titers in the brains of mice infected with SA4 were 0.5-1.0 lg PFU/mL higher than that infected with SA14. The sequence comparison indicated that the nucleotide and amino acid homology between SA4 and the other 21 JE strains were 84.6%-99.0% and 95.2%-99.7% respectively. Comparison with strain SA14 revealed that there were 17 amino acid differences between the two strains, of which 5 were in the E protein region. The results demonstrate that strain SA4 is a highly neurovirulent strain. The substitutions of the 17 amino acids in the SA4 strain can be the molecular basis for the biological characteristics of high neurovirulence.
Animals ; Brain ; virology ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; pathogenicity ; Encephalitis, Japanese ; mortality ; virology ; Genotype ; Humans ; Mice ; Sequence Analysis ; Viral Envelope Proteins ; genetics ; Virulence

Dendritic Cells ; cytology ; immunology ; virology ; HIV ; physiology ; HIV Infections ; immunology ; Humans ; Immunity, Mucosal ; immunology ; Mucous Membrane ; cytology ; immunology ; Virus Attachment ; Virus Internalization

Porcine sapoviruses (SaVs), which belong to the family Caliciviridae, have been considered potential zoonotic agents for human infection, and several cases have been reported in Asian countries. In this study, a total of 200 porcine fecal samples collected from Lulong county of China were tested. Among 200 samples, porcine sapoviruses were detected by RT-PCR in 17 samples (8.5%) showing their circulation in China. 14 out of 17 positive sapovirus strains were genetically related to the genogroup III (GIII) and were further divided into three different clusters or genotypes according to the phylogenetic analysis. In addition, the remaining three sapovirus strains belonged to GVII (one strain) and a potential novel genogroup (two strains) according to the phylogenetic analysis and the nucleotide identity and amino acid identity. These data suggested the genetic diversity of porcine sapoviruses in China.
Animals ; China ; Genetic Variation ; Genotype ; Phylogeny ; Sapovirus ; classification ; genetics ; Sequence Homology, Nucleic Acid ; Swine ; virology

Four dsRNA bands were extracted from Pleurotus ostreatus TD300 by the dsRNA isolation technique with sizes of 8.2 kb, 2.5 kb, 2.1 kb, and 1.1 kb, respectively. Four virus-eliminated methods, i. e. hyphal tips cut (HTC), protoplast regeneration (PR), single spore hybridization (SSH), and frozen and lyophilized (FL), were applied to prepare virus-eliminated strains, and one virus-eliminated strain was selected for each virus-elimination method. The virus-eliminated strains were named as HTC8, PR15, FL01, and SSH11, respectively. There were low concentration of 8.2 kb dsRNA remained in HTC8, as well as low concentration of 8.2 kb and 2.5 kb dsRNA remained in FL01. However, no dsRNA remained in PR15 and SSH11. The hyphal growth rate and laccase activity of the virus-eliminated strains increased, especially HTC8 and PR15, whose hyphal growth rate was higher by 22.73% and 18.18%, and laccase activities higher by 145.83% and 134.38% than that of the original strain, respectively. The conclusion is that hyphal tips cut and protoplast regeneration are suitable to prepare virus-eliminated strains of edible fungi.
Food Microbiology ; Freeze Drying ; Hybridization, Genetic ; Hyphae ; virology ; Pleurotus ; cytology ; genetics ; growth & development ; virology ; Protoplasts ; virology ; RNA, Double-Stranded ; analysis ; isolation & purification ; RNA, Fungal ; analysis ; isolation & purification ; Spores, Fungal ; genetics ; virology ; Viruses ; isolation & purification

Based on the NSP4 sequence of bovine rotavirus (BRV), the shRNA was designed and synthesized, and a shRNA recombinant lenti-virus vector RNAi-H1-89 was constructed. The recombinant RNAi-H1-89 Lenti-virus was packaged by transfecting the 293T cell with the recombinant vector RNAi-H1-89 and two helper plasmids using lipofectamine, and then used to infect MA104 cells. The MA104 cells were further infected with BRV strain G6 24h post-infection, with the LacZ shRNA recombinant lenti-virus as control. Thirty-six hours later, the CPE of the infected cells was observed under microscope, shRNA of NSP4 gene inhibited CPE in MA104 cell; the shRNA against NSP4 gene also inhibited NSP4 gene expression by RT-PCR, The virus titer in the cell culture supernatant was significant lower compared with the control group. The above results showed that RNAi-H1-89 against NSP4 gene could specifically silence NSP4 gene expression, and inhibit the proliferation of BRV.
Animals ; Base Sequence ; Cattle ; Cell Line ; DNA, Recombinant ; genetics ; Glycoproteins ; deficiency ; genetics ; Molecular Sequence Data ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; Rotavirus ; genetics ; physiology ; Toxins, Biological ; genetics ; Viral Load ; genetics ; Viral Nonstructural Proteins ; deficiency ; genetics ; Virus Replication ; genetics

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, which is a widespread problem for beef and dairy herds, and has given rise to a significant loss in the livestock industry all over the world. The BVDV strain JZ05-1 isolated from cattle in Jilin, China generated cytopathic effect (CPE) in MDBK cells. Eight overlapped gene fragments were amplified by RT-PCR and sequenced, the complete genom sequence of BVDV strain JZ05-1 was assembled. According to the results, the JZ05-1 genome was composed of 12285 nucleotides in length (GenBank accession No. GQ888686), which could be divided into three regions: a 387 nt 5'-untranslated region (UTR), a 11694 nt single large open reading frame encoding a polyprotein, and a 204 nt 3'-UTR. The 5'-UTR and genome sequences were analyzed by sequence alignment and construction of phylogenetic trees. The strain JZ05-1 was classified as BVDV type 2a. The BVDV-2 strain JZ05-1 genome showed high similarity to the p11Q isolated in Canada and the XJ-04 isolated in China, with 90% and 91% identity in nucleotide sequence, respectively. Compared with the similarity within the BVDV-2 genotype (96%), the JZ05-1 had low sequence similarity to other BVDV-2 strains.
5' Untranslated Regions ; genetics ; Animals ; Cattle ; China ; Diarrhea Virus 2, Bovine Viral ; classification ; genetics ; isolation & purification ; Genome, Viral ; genetics ; Genotype ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

Four hundreds and ninety sheep sera from seven breeds raised at eight counties and one city of Aksu region in Xinjiang were tested by ELISA for the presence of antibodies against hepatitis E virus (HEV), and the positive rate differences were compared based on administrative areas, breeds and age by Chi-square test. The result showed that the general positive rate was 28.98% (142/490), the positive rate were 35.44% (28/79), 29.67% (27/91), 20% (4/20), 40% (12/30), 32.5% (26/80), 38% (19/50), 22.5% (9/40), 8% (4/50) and 26% (13/50) respectively in eight counties and one city, there was a significant difference between Xayar and other administrative areas (P<0.01); there was also a significant difference among age ranges (P<0.01), being 38.75% (31/80) over 2 years old, 15.45% (17/110) below 1 year old; The seroprevalence was still related to breeds, i. e. there was a significant difference between Mongolia sheep and other breeds (P<0.01). From these data, it is confirmed that there is a possibility of previous and potential infection of sheep HEV in Aksu region of Xinjiang Autonomous.
Animals ; Antibodies, Viral ; blood ; China ; epidemiology ; Hepatitis E ; epidemiology ; transmission ; veterinary ; virology ; Hepatitis E virus ; physiology ; Seroepidemiologic Studies ; Sheep ; blood ; virology ; Sheep Diseases ; epidemiology ; transmission ; virology

The purpose of this study is to elucidate the molecular mechanism of one-way serological reaction between XJ-160 virus and SINV by recombinant viruses which exchanged the glycoprotein genes individually or simultaneously. Three recombinant viruses were obtained based on the whole-length infectious cDNA clone of XJ-160 virus. The infectivity and pathogenesis to BHK-21 cells and animals were studied and the gene which controlled this one-way serological reaction phenomenon was searched by MCPENT. The results showed that the E2 glycoprotein was the main factor which influenced the growth rate, plaque morphology and pathogenicity of BHK-21 cells and suckling mice. The results of MCPENT showed that the E2 glycoprotein of SINV played a major role in this one-way serological reaction phenomenon. Our study identified the SINE2 gene was the determined gene for one way serological reaction between XJ-160 virus and SINV, and this research laid the foundation for further analysis of the genomic structure and function of SINV.
Alphavirus ; genetics ; immunology ; physiology ; Amino Acid Sequence ; Animals ; Cell Line ; DNA, Recombinant ; genetics ; Female ; Genetic Engineering ; Glycoproteins ; chemistry ; metabolism ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Neutralization Tests ; Sindbis Virus ; immunology ; Viral Load ; Viral Proteins ; chemistry ; metabolism