RESUMO
Although the theoretical concern of genetic recombination has been raised related to the use of live attenuated flavivirus vaccines [Seligman, Gould, Lancet 2004;363:2073-5], it has little foundation [e.g., Monath TP, Kanesa-Thasan N, Guirakhoo F, Pugachev K, Almond J, Lang J, et al. Vaccine 2005;23:2956-8]. To investigate biological effects of recombination between a chimeric yellow fever (YF) 17D/Japanese encephalitis (JE) vaccine virus (ChimeriVax-JE) and a wild-type flavivirus Kunjin (KUN-cDNA), the prM-E envelope protein genes were swapped between the two viruses, resulting in new YF 17D/KUN(prM-E) and KUN/JE(prM-E) chimeras. The prM-E genes are easily exchangeable between flavivirues, and thus the exchange was expected to yield the most replication-competent chimeras, while other rationally designed recombinants would be more likely to be crippled or non-viable. The new chimeras proved highly attenuated in comparison with the KUN-cDNA parent, as judged by plaque size and growth kinetics in cell culture, low viremia in hamsters, and reduced neurovirulence/neuroinvasiveness in mice. These data provide strong experimental evidence that the potential of recombinants, should they ever emerge, to cause disease or spread (compete in nature with wild-type flaviviruses) would be indeed extremely low.
Assuntos
Flavivirus/genética , Flavivirus/imunologia , Engenharia Genética , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas contra o Vírus do Nilo Ocidental/genética , Vacinas contra o Vírus do Nilo Ocidental/imunologia , Animais , Sequência de Bases , Peso Corporal/imunologia , Linhagem Celular , Cricetinae , Feminino , Flavivirus/patogenicidade , Genoma Viral/genética , Humanos , Cinética , Camundongos , Vacinas Atenuadas/efeitos adversos , Virulência , Replicação Viral , Vacinas contra o Vírus do Nilo Ocidental/efeitos adversosRESUMO
St. Louis encephalitis (SLE) and West Nile (WN) flaviviruses are genetically closely related and cocirculate in the United States. Virus neutralization tests provide the most specific means for serodiagnosis of infections with these viruses. However, use of wild-type SLE and WN viral strains for laboratory testing is constrained by the biocontainment requirements. We constructed two highly attenuated yellow fever (YF) virus chimeras that contain the premembrane-envelope (prM-E) protein genes from the virulent MSI-7 (isolated in the United States) or the naturally attenuated CorAn9124 (Argentina) SLE strains. The YF/SLE (CorAn version) virus and the previously constructed YF/WN chimera were shown to specifically distinguish between confirmed human SLE and WN cases in a virus neutralization test using patient sera. These chimeras have the potential for use as diagnostic reagents and vaccines against SLE and WN.
Assuntos
Vírus da Encefalite de St. Louis/isolamento & purificação , Encefalite de St. Louis/prevenção & controle , Genes Virais/genética , Vacinas Virais/síntese química , Febre Amarela/prevenção & controle , Vírus da Febre Amarela/isolamento & purificação , Sequência de Aminoácidos , Animais , Argentina/epidemiologia , Culex/virologia , Vírus da Encefalite de St. Louis/genética , Vírus da Encefalite de St. Louis/imunologia , Encefalite de St. Louis/epidemiologia , Encefalite de St. Louis/transmissão , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Estados Unidos/epidemiologia , Vacinas Virais/uso terapêutico , Febre Amarela/epidemiologia , Febre Amarela/transmissão , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/imunologiaRESUMO
Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 x 10(-4) per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 x 10(-7) to 2.3 x 10(-7). Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Dengue/prevenção & controle , Vírus da Dengue/genética , Análise de Sequência de DNA , Inoculações Seriadas , Transcrição Gênica , Vacinas Sintéticas , Ensaio de Placa Viral , Vacinas Virais , Montagem de Vírus , Replicação Viral , Vírus da Febre Amarela/enzimologia , Vírus da Febre Amarela/genéticaRESUMO
Consensus sequencing of the genome of the ARILVAX live attenuated yellow fever (YF) 17D vaccine was performed directly on reconstituted virus from a vial of the vaccine secondary seed (without plaque-purification or cloning of cDNA). The genome of ARILVAX was identical in organization and size (10,862 nucleotides (nt)) to other published YF 17D sequences. A total of 12 nt heterogeneities were detected indicating that the vaccine is a heterogeneous population. Some of these indicated the presence of quasispecies with residues not reported previously for other sequenced YF 17D strains. A number of nts clearly differed from some YF vaccine strain sequences but coincided with the others, which could be due to the use of consensus sequencing approach in this study. Most (but not all) of the heterogeneities and nt differences were silent (i.e. did not result in an amino acid change). The differences are inconsequential to safety and effectiveness of ARILVAX. Other YF 17D vaccines are undoubtedly also heterogeneous and need to be re-examined using the consensus approach.