Baculovirus expression system offers the advantage of expression of several large proteins simultaneously by a single recombinant virus. To date, expression of multiple large (>100 kDa) proteins has been hampered by the need to generate large constructs and repeat use of homologous sequence and promoter. The development of multi-loci baculovirus expression system overcomes these issues by enabling the recombination of large foreign sequences into different regions of the genome. In this paper, we have examined the co-expression of African horse sickness virus (AHSV) VP2 proteins from multiple serotypes in a single recombinant baculovirus. To this end, recombinant baculoviruses expressing multiple AHSV VP2 proteins were generated and it was found that up to six different AHSV serotypes (serotype 1, 3, 4, 5, 7 and 8) VP2 proteins (similar to 120 kDa) could be expressed simultaneously from different loci of baculovirus genome. The expression of VP2 of one serotype was not significantly hindered by the presence of other serotypes, although there were slight differences in expression level between different serotypes. The expression of VP2 of further serotypes from additional loci resulted in a lesser expression level of VP2 proteins. Based on these findings, three additional recombinant baculoviruses encompassing all nine AHSV serotypes were constructed (serotypes 1, 7, 8 or serotypes 2, 4, 5 or serotypes 3, 6, 9) and each of the triple recombinant viruses exhibited similar expression level of each VP2. This system allows for the expression of a number of large proteins that has the potential to be exploited for multivalent vaccines production. (C) 2013 Elsevier Inc. All rights reserved.