Adenovirus transformed monkey cell lines permissive to enteric adenovirus type 40
6
Citation
18
Reference
10
Related Paper
Keywords:
Mastadenovirus
HEK 293 cells
Adenoviruses are non-enveloped icosahedral viruses with trimeric fibre proteins protruding from their vertices. There are five known genera, from which only Mastadenoviruses have been widely studied. Apart from studying adenovirus as a biological model system and with a view to prevent or combat viral infection, there is a major interest in using adenovirus for vaccination, cancer therapy and gene therapy purposes. Adenoviruses from the Atadenovirus genus have been isolated from squamate reptile hosts, ruminants and birds and have a characteristic gene organization and capsid morphology. The carboxy-terminal virus-distal fibre head domains are likely responsible for primary receptor recognition. We determined the high-resolution crystal structure of the Snake Adenovirus 1 (SnAdV-1) fibre head using the multi-wavelength anomalous dispersion (MAD) method. Despite the absence of significant sequence homology, this Atadenovirus fibre head has the same beta-sandwich propeller topology as other adenovirus fibre heads. However, it is about half the size, mainly due to much shorter loops connecting the beta-strands. The detailed structure of the SnAdV-1 fibre head and other animal adenovirus fibre heads, together with the future identification of their natural receptors, may lead to the development of new strategies to target adenovirus vectors to cells of interest.
Mastadenovirus
Adenovirus genome
Adenovirus infection
Homology
Cite
Citations (17)
Currently, lentiviral vectors for research and gene therapy are produced from 293-T cells that are transiently transfected with plasmids encoding the vector and helper functions. However, transiently transfected vectors as well as the presence of SV40 virus large T-antigen (T-Ag) cause serious technical and safety considerations. We aimed to exploit single copy integration sites in the HEK293 genome supporting lentiviral vector production. We found that lentiviral vectors result in minimal infectious particle production from single copy integrants in HEK293. Moreover, once this cell line harbors single copy integrations of lentiviral vectors, its ability to transiently produce lentiviral vectors becomes strongly impaired. T-Ag has a dramatic effect on virus production. Low levels of constitutive T-Ag expression can overcome the production restriction imposed by integrated lentiviral vectors copies. Interestingly, T-Ag does not exert its role at the level of transcriptional activity of the vector; rather, it seems to impose an indirect effect on the cell thereby enabling lentiviral vector production. Altogether, our study highlights the restrictions for integrated lentiviral vectors that are relevant for the establishment of stable and safe producer cell lines. Gama-Norton and colleagues evaluate the feasibility of lentiviral vectors that are commonly used in transient production protocols to generate infectious viral particles on low copy integration into the HEK293 genome. They show that HEK293 cells do not support efficient lentiviral vector production when the vector is integrated at a low copy number in the cellular genome and that this impairment is partially dependent on the SV40 large T antigen.
HEK 293 cells
Cite
Citations (35)
Objective To explore an effective way for increasing amplification efficiency of adenoviral expression vector in vitro. Methods Approximately 70%-80% of confluent human embryonic kidney cells (HEK293) were co-cultured with recombinant adenoviral vector (Ad5CMVLacZ) for 36-48 h. X-gal histochemistry was performed to identify the transfection and expression of Ad5CMVLacZ. Results Under the normal condition, 70%-80% of confluent HEK 293 cells were logarithmically grown. Afler 36-48 h of co-culture with Ad5CMVLacZ, the shape of HEK 293 cells was gradually changed into spherical, and congregated into grape-like aggregation which is the typical cytopathic effect of Ad virus transfection of host cells. At this stage aboat 90% of transfected HEK 293 cells were staimed blue color by X-gal histochemistry. Conclusion The method is an effective way for amplification of adenoviral expression vector in vitro with paying more attention to ascertaining the time of transfectisn of HEK 293 cells with the viral vector and the time for harversting the transfected cells so as to obtain enough viral vector for gene therapy.
HEK 293 cells
Cytopathic effect
Cite
Citations (0)
Currently, exogenous gene expression system based on retroviral vector has been widely used as efficient gene expression system in both gene therapeutic research and RNA interference. In this study, we evaluated the efficiency of exogenous gene expression mediated by the retroviral vector in mammalian cells. First, we constructed EGFP (enhanced green fluorescent protein) vector using pcDNA3.1(+) and retroviral vector pQCXIN as backbone vector respectively. Then, we transfected or infected HEK293 cells and CHO-K1 cells with above vector or corresponding retroviral virus, and measured the relative fluorescence intensity (RFI) of EGFP. The results showed that the RFI of the retroviral virus-infected cells was two times higher than that of the plasmid-transfected cells. Further experiments revealed repeated virus infection enhanced the expression of EGFP markedly, with RFI increasing twice after four rounds of virus infection. Furthermore, the EGFP expression in HEK293 cells mediated by the retroviral vector was more stable than transfected with plasmid pcDNA3.1(+). Finally, we further validated the efficiency of exogenous gene expression system based on the retroviral vector by expressing recombinant human activated protein C (rhAPC) in HEK293 cells. We obtained HEK293 cell lines with rhAPC expression between 10 and 15 microg/(10(6) cells d). In conclusion, the exogenous gene expression system based on the retroviral vector is an alternative method for the generation of stable and high-expressing mammalian cell lines.
HEK 293 cells
Cite
Citations (1)
This study describes the prevalence of culture-positive adenovirus serotypes in culture-positive respiratory specimens sent to the Central Public Health Laboratory, Toronto, Ontario, Canada for the period September 2007-June 2008. Total nucleic acid was extracted from virus cultures using an automated extraction method followed by polymerase chain reaction and Sanger sequencing of the adenovirus hexon gene hypervariable region 7. 73% of specimens (n = 70) were from patients < or = 4 years of age. Of the 96 adenovirus isolates, the most common identified serotypes were serotype 3 (n = 44, 46%), serotype 2 (n = 25, 26%), serotype 1 (n = 17, 18%), and serotype 21 (n = 5, 5%). Adenovirus serotype 14 was not found in this study group. The leading serotype, Ad3, was identified throughout the duration of the study period. Molecular methods allow for the determination of circulating adenovirus serotypes and be used to document the spread of highly virulent adenoviral serotypes into a region.
Sanger sequencing
Hypervariable region
Mastadenovirus
Molecular Epidemiology
Cite
Citations (38)
Objective To determine if recombinant adenovirus can be amplified in HEK293T cells.Methods After HEK293T cells were infected with a preservation solution of adenovirus,its effect on cytopathy was observed.Infectivity of amplified adenovirus was assayed with 50% of tissue-culture infective dose.Infectivity of adenovirus was confirmed with infected myocardial cells and fibroblasts in vitro.Results HEK293T cells promoted generation of recombinant adenovirus with green fluorescent protein.The titer of adenoviruses could reach 5×106PFU/mL.Adenoviral particles could infect myocardial cells and fibroblasts in vitro.Conclusion Recombinant adenovirus with green fluorescent protein can be amplified in HEK293T cells.
HEK 293 cells
Infectivity
Adenovirus infection
Cite
Citations (0)
In the present paper, we examined the effect of the adenoviral vector dosage, the role of T cells, and the influence of the presence of replication-competent adenovirus (RCA) in adenoviral vector stocks, on the efficacy of adenoviral vector-directed transgene expression in the facial nucleus of immunocompetent Wistar and athymic nude rats. A small number of motor neurons and glial cells was transduced at low dosages of viral vector (1 × 106 pfu) and in the absence of RCA, and transgene-expressing cells persisted throughout the 3-week period of observation. Intraparenchymal infusion of 2 × 107 pfu of a recombinant adenoviral vector free of RCA was required for optimal transduction of facial motor neurons. In Wistar rats, a biphasic immune response occurred at higher dosages of the vector (5 × 106 and 2 × 107 pfu) that was characterized by early infiltration of macrophages and the occurrence of T cells during the second week after injection of the vector. The immune response was associated with the loss of transduced neural cells. In nude rats, administration of an adenoviral vector free of RCA resulted in a macrophage response comparable to that in the Wistar rat and long-term survival of transduced astroglial cells. However, transduced motor neurons degenerated according to a similar time course as observed in Wistar rats. Small amounts of RCA (2 × 105 pfu) injected with 2 × 107 pfu recombinant viral vector particles resulted in an accelerated T cell response and a rapid elimination of transduced cells within 1 week in Wistar rats, whereas in nude rats transgene expression continued during this period. Taken together, these observations suggest that at the high viral vector loads necessary to achieve optimal transduction of the facial nucleus, T cells play a role in the degeneration of adenoviral vector-transduced astroglial cells. The adverse effects on neurons appear to be due to the observed inflammatory response or to direct adenoviral vector toxicity. Adenoviral vectors have been shown to transduce neural cells in vitro and in vivo. The prevailing view is that adenoviral vector-directed gene transfer to the brain results in long-lasting transgene expression. We show that following administration of low dosages of viral vector (1 × 106 pfu) relatively small numbers of neurons expressed the transgene for at least 3 weeks. At moderate (5 × 106 and 2 × 107 pfu) and high dosages (1 × 108 pfu), transduction efficiency of neural cells improved significantly; however, this was accompanied by a biphasic immune response (comparable to that observed in lung and liver). In T cell-deficient nude rats, transduced astroglial cells persisted for at least 30 days but motor neurons degenerated as in Wistar rats, suggesting that T cells are involved in the elimination of transduced glial cells. Neuronal degeneration is apparently due to direct adverse effects of the viral vector or to the parenchymal inflammatory response.
Transduction (biophysics)
Cite
Citations (60)
Objective To construct mouse osteoinductive factor(OIF) gene recombinant retroviral vector,and investigate its expression in 293T cells. Methods OIF-3FLAG gene was amplified with PCR and cloned into retroviral vector(Puro-IRES-GFP) after sequencing,and recombinant retroviral vector pMSCV PIG-OIF-3FLAG was constructed and identified by sequencing.PMSCV PIG-OIF-3FLAG retroviral vector with helper vectors of VSAG and GAG-POL were co-transfected into 293T cells by lipofectamine2000.Forty-eight hours after transfection,the expression of green fluorescent protein(GFP) was examined by fluorescence microscopy.The supernatant of 293T cells,which were tansfected with pMSCV PIG-OIF-3FLAG retroviral vector and helper vectors of VSAG and GAG-POL,was used to reinfect 293T cells.After 48 h,293T cells stably expressing pMSCV PIG-OIF-3FLAG retroviral vector were screened by 3 μg/mL puromycin for 7 d,and the expression of OIF mRNA and protein of 293T cells was detected by Real-Time PCR and Western blotting,respectively. Results OIF gene retroviral vector pMSCV PIG-OIF-3FLAG was successfully constructed,and the cloning site and reading frame of objective gene were confirmed by enzyme digestion and sequencing.Then,293T cells were infected by retrovirus supernatant,and 293T cells with stable expression of mouse OIF were obtained by puromycin screening.Real-Time PCR and Western blotting revealed high expression of OIF mRNA and protein in 293T cells. Conclusion Mouse OIF gene retroviral vector has been successfully constructed,and 293T cells with stable expression of OIF gene are obtained.
HEK 293 cells
Puromycin
Cite
Citations (0)
HEK 293 cells
Cite
Citations (11)
During the period 1973-8 700 adenoviruses were isolated from the eyes of patients presenting at Moorfields Eye Hospital. Of these, 678 were serotyped by a neutralisation test. Twenty-one different serotypes were identified. Serotype 3, 7, and 10 accounted for 68% of the isolates, 4, 8, 15 (15/29), and 19 for 25%, and the other 14 serotypes for 7%. Community outbreaks of ocular infections by adenovirus 3, 4, 7, 10, and 15 (15/29) were observed. Outbreaks with adenovirus 3, 7, and 10 appeared to continue for 2 years or more, whereas outbreaks with 4 and 15 (15/29) were restricted to one year or less. Hospital outbreaks by adenovirus 8 and 19 were also recorded. During the same period 18 of these 21 adenovirus serotypes were isolated from the nonocular sites (mainly respiratory tract) in 7804 cases. There was a close association in the distribution of adenovirus 1, 2, 3, 5, and 6 in the ocular and nonocular sites. No such association was observed for adenovirus serotypes 4, 8, 10, 15 (15/29), and 19.
Mastadenovirus
Adenovirus infection
Cite
Citations (30)