Objective In order to further understand the bioactivity of sphingosine kinase, high efficient replication defective recombinant adenovirus carrying the wild type SPK gene (SPK WT ) and SPK mutant gene (SPK DN ) were constructed respectively. Methods The SPK genes of interest were subcloned into a shuttle vector pshuttle cmv respectively. Pshuttle cmv SPK WT and Pshuttle cmv SPK DN were linearized by PmeI and transformed respectively into the competent cells of E. coli BJ AD 1 cells which contain adenoviral backbone plasmid pAdEasy 1. The linearized recombinant plasmids were transfected into adenovirus 293 packaging cells respectively,and then recombinant adenoviruses were harvested. We performed the PCR, Western blot and enzyme assays to identify the recombinant adenoviruses. Results The recombinant adenoviruses containing the interest gene and being able to infect ECV 304 cells were obtained. Overexpression of wild type SPK gene in ECV 304 cells increased the endogenous SPK activity, whereas overexpression of mutant SPK (SPK DN ) inhibited intracellular SPK activity. Conclusion Recombinant adenoviral vectors can mediate interesting gene expression in cells.

We wished to study the intracellular transport of adenoviruses. We constructed a novel recombinant adenovirus in which the structural protein IX was labeled with a mini-singlet oxygen generator (miniSOG). The miniSOG gene was synthesized by overlapping extension polymerase chain reaction (PCR), cloned to the pcDNA3 vector, and expressed in 293 cells. Activation of miniSOG generated sufficient numbers of singlet oxygen molecules to catalyze polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by transmission electron microscopy (TEM). To construct miniSOG-labelled recombinant adenoviruses, the miniSOG gene was subcloned downstream of the IX gene in a pShuttle plasmid. Adenoviral plasmid pAd5-IXSOG was generated by homologous recombination of the modified shuttle plasmid (pShuttle-IXSOG) with the backbone plasmid (pAdeasy-1) in the BJ5183 strain of Eschericia coli. Adenovirus HAdV-5-IXSOG was rescued by transfection of 293 cells with the linearized pAd5-IXSOG. After propagation, virions were purified using the CsC1 ultracentrifugation method. Finally, HAdV-5-IXSOG in 2.0 mL with a particle titer of 6 x 1011 vp/mL was obtained. Morphology of HAdV-5-IXSOG was verified by TEM. Fusion of IX with the miniSOG gene was confirmed by PCR. In conclusion, miniSOG-labeled recombinant adenoviruses were constructed, which could be valuable tools for virus tracking by TEM.
Adenoviruses, Human ; chemistry ; genetics ; metabolism ; Arabidopsis Proteins ; chemistry ; genetics ; metabolism ; Flavoproteins ; chemistry ; genetics ; metabolism ; Humans ; Phototropins ; chemistry ; genetics ; metabolism ; Singlet Oxygen ; chemistry ; Staining and Labeling ; Transfection

Human adenovirus type 41 (HAdV-41) is considered to be a "fastidious adenovirus". E1-deleted HAdV-41 cannot be rescued or amplified in 293 cells. To propagate recombinant HAdV-41 in 293 cells, the backbone plasmid pAdbone41 was reconstructed. That is, the E3 coding sequence of HAdV-41 was deleted and replaced with the HAdV-5 E4orf6 gene; and the E1A enhancer of HAdV-5 was inserted upstream of the E4 promoter of HAdV-41. Novel adenoviral plasmid pAd41E4EE-GFP was generated by homologous recombination of the shuttle plasmid pSh41-GFP with the modified backbone plasmid in the Escherichia coli BJ5183 strain. Adenovirus HAdV-41-E4EE-GFP was rescued by transfecting 293 cells with linearized pAd41E4EE-GFP. After seven rounds of propagation, viruses were purified by the CsCl ultracentrifugation method. HAdV-41-E4EE-GFP in 1.0 ml with a particle titer of 8 x 10(10) vp/mL was obtained which had a particle-to-infectious ratio of 50 : 1. The genome of HAdV-41-E4EE-GFP was confirmed by restriction analyses and polymerase chain reaction. These results showed that a novel HAdV-41 vector system was established in which recombinant HAdV-41 could be constructed and packaged in 293 cells.
Adenoviruses, Human ; genetics ; growth & development ; physiology ; Cell Line ; Genetic Vectors ; genetics ; physiology ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Plasmids ; genetics ; metabolism ; Recombination, Genetic ; Transfection ; Virus Assembly

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.

The baculovirus expression system was employed to prepare the virus-like particles (VLPs) of human parvovirus B19. The synthesized VP2 gene of B19 was inserted into the multi-cloning site (MCS) of pFastBac1 vector; the resulting plasmid was transferred to the Escherichia coli DH10Bac competent cells, which contain a baculovirus shuttle vector (Bacmid), to generate Bacmid-VP2 by site-specific transposition. Recombinant baculovirus carrying VP2 gene (rBac-VP2) was then rescued from Bacmid-VP2-transfected Sf9 cells. Indirect immunofluorescence and Western blotting were used to identify the VP2 protein in rBac-VP2-infected Sf9 cells, and the VLPs were observed under transmission electron microscope after being enriched by ultracentrifugation. The B19 VLPs were successfully produced in insect cells with baculovirus expression system, which will facilitate the development of diagnostic reagents to detect the antibody against B19 virus in human serum.
Animals ; Antibodies, Viral ; blood ; Baculoviridae ; genetics ; metabolism ; Capsid Proteins ; biosynthesis ; genetics ; Cell Line ; Cloning, Molecular ; Genetic Vectors ; genetics ; Parvovirus B19, Human ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Virion ; genetics ; metabolism

Objective:To recognize virus electron microscopic (EM) images automatically with deep learning techniques and to select an appropriate network for virus EM image classification.Methods:Multiple classic convolutional neural networks, such as AlexNet, VGG, ResNet, DenseNet, SqueezeNet, MobileNet, and ShuffleNet, were used to classify the seven classes of virus electron microscope images by increasing the network depth increasing the network depth and adjusting the learning rate, adjusting the learning rate, batch size and other parameters.Results:Overall, DenseNet169 achieved the best performance with 91.9% accuracy, 90.1% sensitivity and 98.6% specificity. In particular, the model performed best in parvovirus recognition. The precision, sensitivity, specificity, and F1 of papilloma virus, herpes virus, pox virus and rotavirus, were above 90%, even close to 100%, but the precision of adenoviruses and the sensitivity of polyomaviruses were not satisfied, leading to corresponding low F1 value. At the same time, Lightweight network ShuffleNet outperformed AlexNet and VGG deep networks with fewer parameters and fewer FLOPs and could achieve comparable result to ResNet with about 15 times fewer parameters and 90 times fewer FLOPs; Compared with DenseNet, Shufflenet sacrificed recognition performance in the acceptable range to achieve about 10 times fewer parameters and 80 times fewer FLOPs.Conclusions:Deep networks DenseNet169 could realize automatic recognition of virus electron microscopic images with the optimal performance and lightweight networks shuffleNet_v2_x0_5 could achieve suboptimal performcane with fewer parameters and FLOPs. In practical applications, tradeoffs can be made between deep networks and lightweight networks based on specific conditions.

Objective To develop an indicator cell line for detection of human cytomegalovirus (HCMV) based on inducible expression of green fluorescent protein (GFP) gene.Methods GFP reporter gene and the promoter of HCMV UL54 gene (UL54p) were amplified by PCR method;GFP gene was used to replace the luc2 coding sequence in pGL4.17 [luc2/Neo] vector,and then UL54p was inserted into the multiple cloning sites (MCS) to generate pGL4UL54p-GFP.pGL4UL54p-GFP plasmid was transfected into MRC-5T,a telomerase reverse transcriptase (TERT) gene-immortalized MRC-5 cell line,by mixing with lipofectamine 3000 reagent.G418-resistant cell colonies were isolated and subjected into screening by observing the inducible expression of GFP under fluorescence microscope after HCMV infection.The specificity to HCMV of the obtained cell line was tested through infection of human adenovirus type 5 or influenza A virus subtype H1N1.Results The recombinant plasmid pGL4UL54p-GFP was successfully constructed.Nine G418-resistant cell colonies were obtained,and two of them could express GFP after HCMV infection among which MRC-5TUG#7 gave brighter fluorescence.No GFP-positive cells were seen after challenging MRC-5TUG#7 with adenovirus or influenza virus,suggesting an acceptable specificity.Conclusion MRC-5-based indicator cell line for HCMV detection was successfully established,which may be used for HCMV isolation and titration.

Objective To study the role of protein Ⅸ(pⅨ) in the interaction between adenovirus and host cell dynamically,we attempt to construct a recombinant human adenovirus with the capsid protein pⅨ being labelled with Green Fluorescent Protein (GFP).Methods GFP gene was inserted into the downstream of pⅨ genein the shuttle plasmid to form pShuttle-IXGFP;and adenoviral plasmid pAd5-IXGFP was generated by homologous recombination of pShuttle-IXGFP with the backbone plasmid (pAdeasy-1) in E.coli BJ5183 strain.Recombinant adenovirus (Ad5-IXGFP) was rescued by transfecting 293 cells with the linearized pAd5-IXGFP.Results After 4 rounds of propagation in 293 cells,viruses were purified with CsCl ultracentrifugation method.Ad5-IXGFP in 1.0 ml with a particle titer of 6.9 × 1011 vp/ml was finally obtained.PCR was performed to confirm the fusion of Ⅸ with GFP gene in the genome of Ad5-IXGFP.Virions were incubated with suspended HEp-2 cells at 4 C for 5 h,and the attachment of Ad5-IXGFP on the cellular membrane could be observed under fluorescence microscope.Conclusion GFP-labelled recombinant adenovirus was successfully constructed,which could be a valuable tool for subcellular localization fo pⅨ after virus entry real-time single adenovirus tracking by confocal fluorescence microscopy.

Objective: To study the intracellular location and autophagosome production of rhinovirus 16 2B protein using miniSOG labeling technique. Methods: 2B was fused with miniSOG and flag tags to construct pcDNA3.1-2B-miniSOG-flag plasmid, which was used to transfect HEK293 cells, LC3 protein was detected by western blot. The transfected cells were fixed, stained with DAB through the photooxidation activity of miniSOG, and used to prepare ultrathin sections. Localization of 2B-miniSOG protein in cells and ultrastructural changes of cells were observed under electron microscope. Results: 2B-miniSOG protein glows green under a fluorescence microscopy. Green flourescence coold be observed in the cells expressing 2B-miniSOG protein.LC-II protein increased in the cells transfected with pcDNA3.1-2B-miniSOG-flag. Under electron microscopy it was observed that 2B-miniSOG protein was located in the mitochondria, and a large number of vesicular structures appeared in the cytoplasm. Both autophagosomes and autophagic lysosomes can be observed. Conclusions Non-structural protein 2B of HRV16 can induce autophagy.

Objective: To establish a method of correlative light and electron microscopy (CLEM) to study the intracellular location of adenovirus protein IX. Methods: MiniSOG (mini singlet oxygen generator) is a recently-invented genetically-encoded tag for CLEM. MiniSOG-fused adenovirus IX gene (IXSOG) was cloned by PCR, and inserted into pcDNA3 plasmid to form pTPL-IXSOG, which was used to transfect 293 cells. IXSOG expressing cells could be distinguished under fluorescence microscope due to the emission of green fluorescence of miniSOG. The transfected cells were fixed in 2.5% glutaraldehyde in situ, stained with diaminobenzidine(DAB) through the photooxidation activity of miniSOG, and used to prepare ultrathin sections. Intracellular location of IXSOG was studied by observing the sections under transmission electron microscope. Results: Eukaryotic expression plasmid carrying IXSOG fusion gene was constructed. IXSOG expressing cells were selected for DAB photooxidation and preparation of ultrathin sections. IXSOG fusion mainly formed punctate aggregations or inclusions in the nucleus. Conclusions The correlative light and electron microscopy method based on miniSOG was successfully established, and it could be used to study the intracellular localization of viral proteins.