Genetic variability of adenoviruses. N Engl J Med. B Histological analysis of tumors after 17 days of treatment from sacrificed mice. Functional study of the novel multidrug resistance gene HA and its comparison to multidrug resistance gene 1. Representative image of tumors at the end of the treatment are also shown. Graham F, Prevec L.
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Sreenivasa, e-mail: moc. Copyright : The authors. This article has been cited by other articles in PMC. Abstract Aim: Generation of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus FMDV capsid protein genes along with full-length 2B, 3B and 3Cpro and its characterization.
Expression of the target proteins was analyzed by sandwich ELISA and indirect immunofluorescence assay. The recombinant adenoviruses were purified and concentrated by CsCl density gradient ultracentrifugation.
Growth kinetics and thermostability of the recombinant adenoviruses were compared with that of non-recombinant replication-defective adenovirus dAd5. The titer of the recombinant adenoviruses was approximately , Growth kinetics of the recombinant adenoviruses did not reveal a significant difference when compared with that of dAd5.
In vitro expression of the target proteins in the adenovirus vector system was detected by sandwich ELISA and immunofluorescence assay. Infection with one serotype does not produce immunity to other serotypes. Among domesticated animals, cattle, buffalo, swine, sheep and goat are susceptible to the disease.
African wild buffaloes maintain SAT serotypes of the virus in oropharyngeal region and act as carriers to cloven-hoofed wildlife [ 3 ]. The disease is transmitted via contaminated air and fomites or direct contact with infected animal. After infection, the virus replicates rapidly, and viremia occurs within a day. The virus transmission occurs at 0. Generally, FMD is characterized by formation of vesicles on the feet, buccal mucosa and mammary glands, and drooling of saliva in the form of string.
After recovery, the affected animals retain the virus in their oro-pharynx and may act as a carrier for the disease transmission to the susceptible animals. The ORF is translated to single polyprotein, which is subsequently processed by virus-encoded proteases e.
The disease is endemic and prevalent in many countries in Africa, the Middle East, Asia and South America, and occurs in the form of outbreaks. In India, the disease is caused by the serotypes O, A, and Asia-1 among which the serotype O is the most prevalent Currently, the disease is controlled by vaccination in developing countries while developed countries follow stamping out policy [ 8 ].
Inactivated whole virus vaccine has been widely used to control and prevent the disease. However, the preparation of the inactivated virus vaccine requires costly biocontainment facilities, large quantities of live virus and cold chain maintenance, and is associated with risk of escape of the live virus from the vaccine manufacturing unit to the environment and from vaccine due to incomplete activation, and is also not suitable for differentiating infected from vaccinated animals DIVA strategy [ 1 ].
Various approaches have been tried to develop alternatives to the inactivated whole virus vaccine. VLPs based vaccine has received a lot of attention and considered one of the most appealing approach for contemporary vaccine design [ 9 ] due to their immunogenic properties and high safety profile for vaccine delivery platforms [ 10 ].
VLPs have been generated by use of various expression systems like vaccinia virus [ 11 ], adenovirus [ 12 ], Escherichia coli [ 13 ] and baculovirus [ 14 ].
These are structurally similar to whole virus particles but noninfectious and safe, induce efficient humoral and cellular immune response [ 17 , 18 ] and also suitable for DIVA strategy. Recombinant adenoviruses have become vectors of choice for target gene delivery, expression of foreign antigens, and have been used in gene therapy, vaccination and cancer therapy [ 19 , 20 ].
The adenoviruses are considered as powerful vectors because of their ability to recombine in culture, high production titers, relatively high capacity for transgene insertion, efficient transduction of both quiescent and actively dividing cells, and for inducing humoral and cellular immune responses [ 21 - 23 ].
The hAd5 carrying FMDV capsid protein antigen PA along with 3Cpro have been tested in mice [ 17 , 25 ], guinea pigs [ 26 ], swine [ 17 , 26 ] and cattle [ 27 ] to protect them from homologous challenge virus. The hAd5 containing full length 2B has been reported to induce a rapid and increased FMDV-specific humoral and cellular immune responses as compared to the original vector [ 28 , 29 ]. Synthesis of 3BCm was carried out in two steps.
In the first step, two segments of the 3BCm 3BC1 and 3C2 were synthesized using the mutagenic primers enlisted in Table Both the expression cassettes were similar except glycine G at position 38 and phenylalanine F at position 48 of 3Cwt were replaced with serine S in 3Cm.
A protocol for rapid generation of recombinant adenoviruses using the AdEasy system
A protocol for rapid generation of recombinant adenoviruses using the AdEasy system.
Sreenivasa, e-mail: moc. Copyright : The authors. This article has been cited by other articles in PMC. Abstract Aim: Generation of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus FMDV capsid protein genes along with full-length 2B, 3B and 3Cpro and its characterization. Expression of the target proteins was analyzed by sandwich ELISA and indirect immunofluorescence assay.
ADEASY PROTOCOL PDF
All solutions, reagents and equipment coming into contact with cells must be sterile, and proper sterile and antiseptic techniques should be used accordingly. Biohazard wastes containing adenoviruses should be disinfected with chlorine bleach. Sambrook, E. Fritsch or other protocols in this series for full details on cloning techniques and protocols. If the GOI and the shuttle vector do not have correctly positioned restriction sites, it may be necessary to blunt-end one or both restriction sites with T4 DNA polymerase. In some cases, it may be more convenient to introduce new restriction sites at one or both ends by linker ligation or by PCR amplification. Introduction of restriction sites by PCR is quick and efficient, but the sequence of the amplified segments must be verified by DNA sequencing.