Construction and characterization of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus capsid proteins of Indian vaccine strain, O/IND/R2/75.

B. P. Sreenivasa,Ramesh Kumar,R. P. Tamilselvan

doi:10.14202/vetworld.2015.147-155

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.Materials and Methods:FMD viral RNA isolation, cDNA synthesis, and polymerase chain reaction were performed to synthesize expression cassettes (P1-2AB3BCwt and P1-2AB3BCm) followed by cloning in pShuttle-CMV vector. Chemically competent BJ5183-AD-1 cells were transformed with the recombinant pShuttle-CMV to produce recombinant adenoviral plasmids. HEK-293 cells were transfected with the recombinant adenoviral plasmids to generate recombinant adenoviruses (hAd5/P1-2AB3BCwt and hAd5/P1-2AB3BCm). 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).Results:The recombinant adenoviruses containing capsid protein genes of the FMDV O/IND/R2/75 were generated and amplified in HEK-293 cells. The titer of the recombinant adenoviruses was approximately 108, 109.5 and 1011 TCID50/ml in supernatant media, cell lysate and CsCl purified preparation, respectively. Expression of the FMDV capsid protein was detectable in sandwich ELISA and confirmed by immunofluorescence assay. Growth kinetics of the recombinant adenoviruses did not reveal a significant difference when compared with that of dAd5. A decrement of up to 10-fold at 4°C and 21-fold at 37°C was recorded in the virus titers during 60 h incubation period and found to be statistically significant (p<0.01).Conclusion:Recombinant adenoviruses expressing capsid proteins of the FMDV O/IND/R2/75 were constructed and produced in high titers. In vitro expression of the target proteins in the adenovirus vector system was detected by sandwich ELISA and immunofluorescence assay.

Highlights

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease of cloven-hoofed animals posing a great threat to the world economy [1]
The disease is caused by FMD virus (FMDV), a highly divergent small RNA virus of the genus Aphthovirus under the family Picornaviridae [2]
In colony polymerase chain reaction (PCR), out of 5 colonies screened, 3 each were found positive for P1-2AB3BCwt and P1-2AB3BCm

Summary

Introduction

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease of cloven-hoofed animals posing a great threat to the world economy [1]. The disease is caused by FMD virus (FMDV), a highly divergent small RNA virus of the genus Aphthovirus under the family Picornaviridae [2]. There are 7 serotypes of the virus namely O, A, C, Asia-1, SAT-1, SAT-2 and SAT-3. Infection with one serotype does not produce immunity to other serotypes. 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. The virus replicates rapidly, Copyright: The authors.

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