RESUMO
Widespread use of a live-attenuated influenza vaccine (LAIV) in the United States (licensed as FluMist) raises the possibility that vaccine viruses will contribute gene segments to the type A influenza virus gene pool. Progeny viruses possessing new genotypes might arise from genetic reassortment between circulating wild-type (wt) and vaccine strains, but it will be difficult to predict whether they will be viable or exhibit novel properties. To begin addressing these uncertainties, reverse-genetics was used to generate 34 reassortant viruses derived from wt influenza virus A/Sydney/5/97 and the corresponding live vaccine strain. The reassortants contained different combinations of vaccine and wt PB2, PB1, PA, NP, M, and NS gene segments whereas all strains encoded wt HA and NA glycoproteins. The phenotypes of the reassortant strains were compared to wt and vaccine viruses by evaluating temperature-sensitive (ts) plaque formation and replication attenuation (att) in ferrets following intranasal inoculation. The results demonstrated that the vaccine virus PB1, PB2, and NP gene segments were dominant when introduced into the wt A/Sydney/5/97 genetic background, producing recombinant viruses that expressed the ts and att phenotypes. A dominant attenuated phenotype also was evident when reassortant strains contained the vaccine M or PA gene segments, even though these polypeptides are not temperature-sensitive. Although the vaccine M and NS gene segments typically are not associated with temperature sensitivity, a number of reassortants containing these vaccine gene segments did exhibit a more restricted ts phenotype. Overall, no reassortant strains were more virulent than wt, and in fact, 33 of the 34 recombinant viruses replicated less efficiently in infected ferrets. These results suggest that genetic reassortment between wt and vaccine strains is unlikely to produce viruses having novel properties that differ substantially from either progenitor, and that the likely outcome of reassortment will be attenuated viruses.
Assuntos
Genes Virais , Vírus da Influenza A/genética , Vacinas contra Influenza/biossíntese , Vírus Reordenados/genética , Recombinação Genética , Vacinas Atenuadas/genética , Proteínas Virais/genética , Animais , Furões , Engenharia Genética , Genótipo , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Fenótipo , RNA Polimerase Dependente de RNA/metabolismo , Vírus Reordenados/fisiologia , Temperatura , Células Tumorais Cultivadas , Vacinas Atenuadas/química , Vacinas Atenuadas/imunologia , Proteínas do Core Viral/química , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Ensaio de Placa Viral , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Measles virus V protein is a Cys-rich polypeptide that is dispensable for virus propagation in continuous cell lines, but necessary for efficient viral replication in animals. Those functions modulating virus propagation in vivo are not understood completely, although V protein is known to interfere with the host interferon response and control of viral gene expression. The ability to modulate gene expression was investigated further with a minireplicon transient expression system in which V protein was found to repress reporter activity. Two regions of the polypeptide contributed to this repressive effect including the carboxy-terminus and a region conserved in morbillivirus V proteins located between amino acids 110-131, whereas domains known to mediate the interaction between V and the nucleocapsid (N) protein were not essential. Accumulation of encapsidated minigenome in transfected cells was inhibited by V protein suggesting that it acted as a repressor of genome replication thereby limiting availability of template for reporter gene mRNA transcription.
Assuntos
Cloranfenicol O-Acetiltransferase/análise , Regulação Viral da Expressão Gênica , Vírus do Sarampo/fisiologia , Fosfoproteínas/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Fusão Gênica Artificial , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Sequência Conservada , Análise Mutacional de DNA , Genes Reporter , Genoma Viral/genética , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Fosfoproteínas/genética , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , RNA Viral/análise , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas Virais/genética , Replicação ViralRESUMO
An intranasally delivered, live attenuated, temperature sensitive (ts) respiratory syncytial virus vaccine candidate, rA2cp248/404/1,030DeltaSH, exhibits a low level of genetic instability in clinical studies, in contrast to the relatively high stability of two similar candidates, cpts248/404 and rA2cp248/404DeltaSH. The latter strains, containing two ts mutations (248ts and 404ts), are partially growth restricted at 37 degrees C, whereas, rA2cp248/404/1,030DeltaSH contains an additional ts mutation (1,030ts) that increases attenuation and partially restricts virus growth at 35 degrees C. Since the maximum human airway temperature is 35.5 degrees C, we investigated whether growth restriction at 35 degrees C contributes to genetic instability of rA2cp248/404/1,030DeltaSH in vitro. We conducted in vitro passage studies with the three strains at 32 degrees C (a fully permissive growth temperature) and 35 degrees C (restrictive for only rA2cp248/404/1,030DeltaSH). Instability of the ts phenotype was observed only in rA2cp248/404/1,030DeltaSH passaged at 35 degrees C, and corresponded with reversion at the 248ts or 1,030ts mutation sites, as observed in clinical studies. This study indicates that ts mutations that partially restrict replication at physiologic temperatures may contribute to genetic instability of viruses in vivo. In vitro passage studies performed at appropriate temperatures can be used to assess genetic stability and to prioritize ts vaccine candidates for clinical evaluation.
Assuntos
Instabilidade Genômica , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/genética , Vacinação , Vacinas Virais/genética , Administração Intranasal , Animais , Chlorocebus aethiops , Humanos , Mutação , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Inoculações Seriadas , Temperatura , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Células Vero , Vacinas Virais/administração & dosagemRESUMO
A canine distemper virus (CDV) genomic cDNA clone and expression plasmids required to establish a CDV rescue system were generated from a laboratory-adapted strain of the Onderstepoort vaccine virus. In addition, a CDV minireplicon was prepared and used in transient expression studies performed to identify optimal virus rescue conditions. Results from the transient expression experiments indicated that minireplicon-encoded reporter gene activity was increased when transfected cell cultures were maintained at 32 rather than 37 degrees C, and when the cellular stress response was induced by heat shock. Applying these findings to rescue of recombinant CDV (rCDV) resulted in efficient recovery of virus after transfected HEp2 or A549 cells were co-cultured with Vero cell monolayers. Nucleotide sequence determination and analysis of restriction site polymorphisms confirmed that rescued virus was rCDV. A rCDV strain also was engineered that contained the luciferase gene inserted between the P and M genes; this virus directed high levels of luciferase expression in infected cells.
