Comparison of the rescue efficiency of Sendai virus minigenome mediated by CMV and T7 promoter / 病毒学报

In this study, we constructed the plasmid of Sendai virus (SeV) BB1 strain minigenome with Gaussia luciferase (Gluc) as reporter and compared the rescue efficiency of SeV minigenome mediated by T7 promoter with that by CMV promoter. Firstly, the sequence was designed and synthesized which contained hammerhead ribozyme, sequence composed of the trailer, untranslated region of L gene, untranslated region of N gene, and the leader from SeV, and mutant hepatitis delta virus ribozyme sequence. Then, the synthesized sequence was inserted into pVAX1 containing CMV and T7 promoters and the general vector for SeV minigenome pVAX-miniSeV was obtained. Furthermore, pVAX-miniSeV-Gluc (+) and pVAX-miniSeV-Gluc(-) were obtained by inserting Gluc gene into pVAX-miniSeV. From the supernatant of BHK-21 cell transfected with pVAX-miniSeV-Gluc(+), high level of Gluc expression was detection indicating the normal transcription function of CMV promoter. pVAX-SeV-miniGluc(-) and plasmids expressing N,P and L protein of SeV were co-transfected into BST T7/5 cell which derived from BHK-21 and expressed T7 RNA polymerase stably. And high expression of Gluc was found, which indicated that SeV minigenome was efficiently rescued. However, we failed to repeat the result on BHK-21 cell, implying that T7 promoter and CMV promoter may have different effects on the rescue efficiency of SeV minigenome. Therefore, we further constructed SeV minigenome vectors pT7-miniSeV-Gluc (-) and pCMV-miniSeV-Gluc(-) with single promoter of T7 or CMV. Then, these vectors were transfected into BSR T7/ 5 cells respectively accompanied with the N, P, and L protein expression vectors. The result demonstrated that high expression of Gluc was found in the group of pT7-miniSeV-Gluc(-), which failed in the group of pCMV-miniSeV-Gluc(-). It indicated that T7 promoter significantly increased the rescue efficiency of SeV minigenome. We successfully constructed a SeV minigenome vector with secreted luciferase gene as report er and proved T7 promoter can enhance the rescue efficiency of SeV minigenome, which provides basis for construction of infectious clone containing SeV full-length genome.

Study on the differences of two mouse models of hepatitis B virus infection by transduction with rAAV8-1. 3HBV / 病毒学报

We recently developed a mouse model of hepatitis B virus (HBV) chronic infection by intravenous (i.v.) injection with rAAV8-1. 3HBV to C57BL/6 mice. To define the responses of different mouse strains after injection with rAAV8-1. 3HBV, we intravenously injected rAAV8-1. 3HBV at doses of 4 x10(9) (Viral genome,vg), 4 x 10(10) vg and 4 x 10(11) vg to C57BL/6 and BALB/c mice,respectively, and determined the levels of serum HBV antigen and antibody by ELISA,serum viral DNA by real-time PCR,and HBcAg expression in liver by immunohistochemical staining. For C57BL/6 mouse strain with injection of rAAV8-1. 3HBV at three doses, 100% of the mice carried HBV for more than 8 months. The levels of serum HBsAg and HBeAg, serum viral DNA and HBcAg-positive hepatocytes increased in a rAAV8-1. 3HBV dose-dependent manner. For C57BL/6 mice injected with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg,over 40% of hepatocytes expressed HBcAg and serum viral DNA reached over 10(5) IU/mL. No HBV antibody was detected in sera of C57BL/6 mice. For BALB/c mice with injection of rAAV8-1. 3HBV at three doses, serum HBeAg, serum viral DNA and HBcAg-positive hepatocytes persisted for more than 8 months, but serum HBsAg declined remarkably at 2 weeks after injection. The levels of serum HBeAg and HBcAg-positive hepatocytes in BALB/c mice increased in a rAAV8-1. 3HBV dose-dependent manner. Injection with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg resulted in over 50% of BALB/c mice hepatocytes expressing HBcAg. Serum anti-HBsAg were detected in BALB/c mice with rAAV8-1. 3HBV injection at the dose of 4 x10 (10) vg. In conclusion, both C57BL/6 and BALB/c strains can be developed to chronic HBV infection mouse models by i. v. injection with rAAV8-1. 3HBV at doses of 4 x10(9) - 4 x 10(11) vg and the levels of HBV replication increase in a rAAV8-1. 3HBV dose-dependent manner. In contrast to C57BL/6 strain, the BALB/c mice carry out humoral immunity to HBsAg, but fail to mediate HBV clearance.

Recombinant adeno-associated virus type 8 mediated dual-luciferase gene expression in mouse / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Recombinant adeno-associated virus type 8 (rAAV8) mediating transgene expression in mice was investigated using co-expressed report gene of secreted Gaussia princeps luciferase (Gluc) and non-secreted firefly luciferase(Fluc).</p><p><b>METHODS</b>rAAV8-Gluc/Fluc was prepared and infected HEK293 cells to test its performance in vitro. BALB/c mice were received rAAV8-Gluc/Fluc at different doses by intravenous injection (iv) or intramuscular injection (im). Then Glue activities in blood were measured,the whole-body images for Flue activities were performed and Flue activities of tissue lysate were also detected.</p><p><b>RESULTS</b>rAAV8-Gluc/Fluc was successfully prepared and could infected HEK293 cells. The Glue was mainly detected in the culture media while the Flue was mainly detected within cells. The blood Glue activities of mice received rAAV8-Gluc/Fluc by iv or im peaked at 10-20 d post injection and persisted for at least 120d. The blood Gluc activities increased at the rAAV8-Gluc/Fluc dose-dependent manner. For mice received rAAV8 by iv, Fluc mainly expressed in liver and minor Fluc expression was also found in cardiac muscle and skeletal muscle. For mice received rAAV8-Gluc/Fluc by im, Fluc mainly expressed in local skeletal muscle and secondly in liver.</p><p><b>CONCLUSION</b>rAAV8-Gluc/Fluc has been prepared successfully and its mediating transgene expression in mice has been investigated. This research will facilitate preclinical studies for rAAV8-mediated gene therapy.</p>

Effects of Lin28a and Lin28b on let-7 family activity / 病毒学报

In this report, we study the effects of over-expression of Lin28a and Lin28b on let-7 family activity in HeLaS3. Firstly, we constructed pAAV2neo-Lin28a and pAAV2neo-Lin28b to express Lin28a and Lin28b, respectively. Then, pAAV2neo-Lin28a and pAAV2neo-Lin28b were transfected into HeLaS3, selected with G418 and obtained cell lines, HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b, to express Lin28a and Lin28b stably. Thereafter, we constructed eight plasmid vectors for detection of let-7 family activity based on pAAV2neo-Gluc-(Fluc). These vectors were further packaged into recombinant adeno-associated viral vectors (rAAV) which were used as sensors, nominated as Asensors, to detect inhibition activity of miRNA at post-transcriptional level. Subsequently, with HeLaS3 as a control, we assayed expression levels of Lin28a and Lin28b by Western blot, detected expression levels of let-7 family by QRT-PCR, and tested let-7 family activity by Asensors in HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b. Results demonstrated that both HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b could express Lin28a and Lin28b effectively. Compared with HeLaS3, the expression level of let-7 family except let-7e declined in HeLaS3/pAAV2neo-Lin28a. But declining extent among members of let-7 family was different. The let-7 family activity also decreased while the decreasing extent varied among members. Furthermore, the activity level was not consistent with its expression level for the same member in let-7 family. Compared with HeLaS3, both expression level and activity level of let-7 family in HeLaS3/ pAAV2neo-Lin28b were decreased. However, the decreasing extent of let-7 family expression changes was larger than that of HeLaS3/pAAV2neo-Lin28a while the decreasing extent of activity changes was similar. In this study, we established a method to detect and compare post-transcriptional inhibition level mediated by miRNA complementary targets. We firstly clarified the effect of Lin28a and Lin28b on let-7 family activity profile and found that this effect was not the same as that at expression level of let-7 family, suggesting that it was more comprehensive to understand miRNA regulation roles to detect both miRNA expression and activity. This paves a way for further research on mechanism of regulation of let-7 family.

Deletion of IV a2 gene from adenoviral genome by lambda-Red recombinase system and packaging of the recombinant adenovirus / 病毒学报

This investigation is to delete the most of the coding sequence (1104 bp) of the IV a2 gene in an adenovirus genome by a lambda-Red recombinase system-mediated PCR-targeting approach and rescue a recombinant adenovirus with IV a2 deletion. First, the template pAK of PCR targeting, containing kanamycin cassette, was constructed. Then, a linear fragment for PCR targeting, which had 39 bp homologous arms at both of its terminus, was amplified by PCR from the pAK. The pFG140 and the linear fragment were electroporated into E. coli BW25113/pIJ790 sequentially and the recombinant pFG140-deltaIV a2 (1104) was established by homologous recombination between the linear fragment and the pFG140 with aid of X-Red recombinase. The precise deletion of 1 104 bp fragment from IV a2 was confirmed by restriction endonucleases digestion and DNA sequencing. ORF of IV a2 was amplified by PCR from pFG140 and then cloned into the pAAV2neo vector. The recombinant adenovirus Ad5delta IV a2 (1104) was rescued by co-transfection of pFG140-deltaIV a2 (1104) and pAAV2neo-IV a2 into HEK293 cells. It was shown by Western Blot that IV a2 could not be detected in the Ad5deltaIV a2 (1104)- infected HEK293 cells. This study established a PCR-targeting strategy for manipulating adenovirus genome directly by a lambda-Red recombinase system, and a recombinant adenovirus with IV a2 deletion was obtained.

Establishment of hepatitis B virus (HBV) chronic infection mouse model by in vivo transduction with a recombinant adeno-associated virus 8 carrying 1. 3 copies of HBV genome (rAAN8-1. 3HBV) / 病毒学报

In this report, we developed a HBV infection model in C57BL/6 mouse line by in vivo injection of a recombinant adeno-associated virus 8 vector carrying 1. 3 copies of HBV genome (ayw subtype) (rAAV8-1. 3HBV). We firstly prepared and purified the rAAV8-1. 3HBV and then injected it into three C57BL/6 mice with the dose of 2 x 10e11vg, respectively. HBsAg and HBeAg were assayed in sera collected at different time points post injection. Ten weeks post injection, the three mice were sacrificed and blood and liver tissue were taken for assay. Copies of HBV DNA were detected by real time PCR and the way of HBV DNA replication was identified by PCR. Subsequently, detection of HBV antigen by immunohistochemistry and pathology analysis of liver tissue of mice were performed. The results suggested that expression of HBsAg and HBeAg lasted for at least 10 weeks in mice sera. Among mice injected with rAAV8-1. 3HBV, HBsAg levels were showed an 'increasing-decreasing-increasing' pattern (the lowest level at the 4th week post injection), while HBeAg levels were kept high and relatively stable. HBV DNA copies were 4.2 x 10(3), 3.6 x 10(3), 2.5 x 10(3) copies/mL in sera and 8.0 x 10(6), 5.7 x 10(6), 2.6 x 10(6) copies/g in hepatic tissues of three mice, respectively. We found that the linear 1. 3HBV DNA in the rAAV8-1. 3HBV could self form into circular HBV genome and replicate in livers of HBV transfected mice. HBsAg and HBcAg were both positive in liver tissue of mice injected with rAAV8-1. 3HBV and no obvious pathological characters were found in liver of mice injected with rAAV8-1. 3HBV. In conclusion, we successfully developed a HBV chronic infection model in C57BL/6 mouse line by in vivo transduction with the recombinant virus rAAV8-1. 3HBV, in which HBV genes could be continuously expressed and replicated over 10 weeks, and paved a way for further characterization of the human chronic hepatitis B virus infection and evaluation of vaccine and anti-HBV agents.

Construction of a dual-luciferase co-expression vector and its characteristics in vitro and in vivo / 病毒学报

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.

The preparation of monoclonal antibodies against AAV2 and study on their characterizations / 病毒学报

7 strains of stable cell lines secreting monoclonal antibodies against AAV2 capsids were obtained by immunizing BALB/C mice with highly purified recombinant adeno-associated virus. Among them, the monoclonal antibodies B10 and G4 had neutralizing activity, and their subtypes were IgG1 and IgG2a, respectively. The binding characterizations of the two neutralizing antibodies were studied. Both B10 and G4 showed serotype specific binding activities to rAAV2 virus particles other than AAV1, AAV5, and AAV8, and the binding could not be blocked by heparin. After incubating with the two antibodies separately, rAAV2 viruses could still bind to sensitive cell line BHK-21, suggesting that the binding sites of the two antibodies to rAAV2 located at different positions on viral particle surface from the primary receptor binding sites of AAV2. Western blotting assay showed that B10 could bind to VP1, VP2 and VP3 of rAAV2. However, G4 bound none of them. The results suggested that B10 recognized a linear epitope of AAV2 capsid, whereas G4 probably recognized a conformational epitope on the surface of AAV2 virus particle. The two antibodies with different characteristics provided valuable tools for AAV2 virus particles detection and infection processes.

Comparison of TTR and CMV promoters in vivo and in vitro via a secreted luciferase reporter system / 病毒学报

GLuc (Gaussia luciferase) is a secreted luciferase with high sensitivity. In this study, we primarily compared expression character of PTTR with that of PCMV, relied on easy secretion, high sensitivity and simple and fast detection of GLuc. We firstly constructed two plasmids pAAV2-neo-TTR-GLuc and pAAV2-neo-CMV-GLuc. Then, 4 cell lines were transfected with the two plasmids in aid of Lipofectamine 2000, including Huh7 and HepG2, which are derived from liver cells, as well as HEK293 and HeLaS3 cells, which are non-liver cell lines. We monitored the expression of GLuc in the supernatant of these cell cultures at different time points post-transfection. Furthermore, we injected the two plasmids with different doses into BALB/c mice by the means of hydrodynamic delivery and monitored the GLuc expression in vivo with 2.5 microl tail tip blood since 2 h post-injection. The cell assay results suggested that the expression of GLuc driven by CMV promoter was significantly higher than that of GLuc driven by TTR promoter. And, the luciferase activity of GLuc driven by CMV promoter was 50-300 times higher than that of GLuc driven by TTR promoter in HEK293 and HeLaS3 cell lines, but less than 10 times higher than that of GLuc driven by TTR promoter in the HepG2 and Huh7 cell lines, indicating the relative liver-specificity of TTR promoter. In the animal assay, the higher luciferase activity was determined in CMV promoter group than in TTR promoter group at different doses of the two plasmids. But the expression patterns for the two promoters differed obviously. The expression of GLuc driven by CMV promoter reached the maximum 10 hours post-injection and declined rapidly; while the expression of GLuc driven by TTR promoter reached the maximum 48 hours after delivery, and declined very slowly. These results implied that PTTR could keep expression of driven gene in a long time although its expression intensity is lower than PCMV's. Thus, it is more suitable for maintaining longer expression of target genes in liver.