Evaluation of arthropod-borne virus recombination potential and phenotype: In vitro and in vivo analysis of flavivirus and alphavirus model systems.

摘要

Recombination is a mechanism whereby positive sense single stranded RNA viruses exchange segments of genetic information. Recent phylogenetic analyses of naturally occurring recombinant flaviviruses have raised concerns regarding the potential for the emergence of virulent recombinants either post-vaccination or following co-infection with two distinct wild-type viruses. In order to characterize the conditions and sequences that favor RNA arthropod-borne virus recombination I designed, constructed, and evaluated various alphavirus (chikungunya virus (CHIKV) and Sindbis virus (SINV)) and flavivirus (yellow fever virus (YFV) 17D) recombinant crosses. Standard molecular cloning methodologies were used to generate CHIKV, SINV, and YFV 17D replicon/defective helper systems possessing trans-complementary sequences necessary and sufficient for the generation of full length viable recombinant viruses following transfection, replication, and purification in cell culture.;Alphavirus recombinant crosses were utilized to both establish standardized conditions for the reproducible generation and cell culture purification of full length recombinant viruses, and to elucidate general characteristics about the nature of RNA arbovirus recombination. The efficiency of CHIKV recombination was related to the functional constraints on the sequence participating in the event with inter-genic recombination being ∼100 fold more efficient (minimum co-infection: ∼103) than intra-genic (minimum co-infection: ∼105). Contrary to previous reports full length SINV recombinants were readily and reproducibly purified in cell culture to a minimum co-infection of ∼104. Furthermore, heterotypic recombination was observed to reproducibly result in the generation of viable chimeric CHIKV/SINV and SINV/CHIKV genomes capable of replication, transcription, translation, assembly, and release.;Flavivirus recombination was evaluated via co-transfection of YFV 17D replicon genomes, containing complementary in-frame deletions of the envelope protein coding sequence. However, full length recombinant YFV 17D virus was not detected under any of the experimental conditions examined, despite achieving estimated YFV replicon co-infection levels of ∼2.4x106 in BHK-21 (vertebrate) cells and ∼1.05x105 in C710 (arthropod) cells. Additionally, to address concerns that a flavivirus vaccine/wild-type recombinant virus might have a high mosquito infectivity and/or vertebrate virulent phenotype, the YFV backbone vaccine sequences of YFV 17D and ChimeriVax Dengue 4 were wholly replaced with the corresponding YFV wild-type sequences. The phenotype of the resulting recombinant chimeras were then evaluated in Aedes aegypti mosquitoes and Cynomolgus macaques. Although these chimeras were observed to efficiently replicate in vitro they were significantly attenuated with respect to mosquito infectivity and pathogenicity in non-human primates. Therefore it is concluded that even in the unlikely event of recombination between wild-type and vaccine flaviviruses virulent vaccine associated transmission cycles would not be established.