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

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.

Antimicrobial Resistance: Its Surveillance, Impact, and Alternative Management Strategies in Dairy Animals

“…Virus-like particles, being structurally similar to virus, but being non-infectious, safe, and having good immunogenic properties are being explored as an alternate to whole virus vaccines. Recently Kumar et al ( 96 ) constructed and characterized recombinant adenovirus expressing capsid proteins of Indian FMD virus strain O/IND/R2/75 to a high titer. Later, Eri silkworm larvae were explored for production of VLPs of FMD O/IND/R2/75 using recombinant baculovirus encoding polyprotein of FMD virus ( 97 ).…”

Deciphering Molecular Dynamics of Foot and Mouth Disease Virus (FMDV): A Looming Threat to Pakistan’s Dairy Industry

“…The global proliferation of trade and commerce, people's movement, and the lack of transboundary animal movement control is crucial for understanding the molecular epidemiology of FMDV [29]. South America FMD free [35] The FMDV genome encodes a polyprotein precursor that is firstly cleaved into three structural proteins, viz, VP1, VP3 and VP0, and later, during capsid assembly, it is divided into VP2 and VP4 along with 10 non-structural proteins (NSPs) [36]. The three structural proteins (VP1, VP2, and VP3) are surfaced exposed, while VP4 is highly conserved between serotypes [37].…”