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Cloning the complete guinea pig cytomegalovirus genome as an infectious bacterial artificial chromosome with excisable origin of replication.

ABSTRACT: Congenital human cytomegalovirus infections are the major infectious cause of birth defects in the United States. How this virus crosses the placenta and causes fetal disease is poorly understood. Guinea pig cytomegalovirus (GPCMV) is a related virus that provides an important model for studying cytomegaloviral congenital transmission and pathogenesis. In order to facilitate genetic analysis of GPCMV, the 232kb GPCMV genome was cloned as an infectious bacterial artificial chromosome (BAC). The BAC vector sequences were flanked by LoxP sites to allow efficient excision using Cre recombinase. All initial clones contained spontaneous deletions of viral sequences and reconstituted mutant viruses with impaired growth kinetics in vitro. The deletions in one BAC were repaired using Escherichia coli genetics. The resulting repaired BAC reconstituted a virus with in vitro replication kinetics identical to the wild type parental virus; moreover, its genome was indistinguishable from that of the wild type parental virus by restriction pattern analysis using multiple restriction enzymes. These results suggest that the repaired BAC is an authentic representation of the complete GPCMV genome. It should provide a valuable tool for evaluating the impact of genetic modifications on the safety and efficacy of live attenuated vaccines and for identifying genes important for congenital transmission and fetal disease.

SUBMITTER: Cui X

PROVIDER: S-EPMC2831993 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Cloning the complete guinea pig cytomegalovirus genome as an infectious bacterial artificial chromosome with excisable origin of replication.

Cui Xiaohong X McGregor Alistair A Schleiss Mark R MR McVoy Michael A MA

Journal of virological methods 20080324 2

Congenital human cytomegalovirus infections are the major infectious cause of birth defects in the United States. How this virus crosses the placenta and causes fetal disease is poorly understood. Guinea pig cytomegalovirus (GPCMV) is a related virus that provides an important model for studying cytomegaloviral congenital transmission and pathogenesis. In order to facilitate genetic analysis of GPCMV, the 232kb GPCMV genome was cloned as an infectious bacterial artificial chromosome (BAC). The B ...[more]

PMID: 18359520

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Project description:BackgroundCytomegalovirus (CMV) is a leading infectious cause of neurologic deficits, both in the settings of congenital and perinatal infection, but few animal models exist to study neurodevelopmental outcomes. This study examined the impact of neonatal guinea pig CMV (GPCMV) infection on spatial learning and memory in a Morris water maze (MWM) model.MethodsNewborn pups were challenged intraperitoneally (i.p.) with a pathogenic red fluorescent protein-tagged GPCMV, or sham inoculated. On days 15-19 post infection (p.i.), pups were tested in the MWM. Viral loads were measured in blood and tissue by quantitative PCR (qPCR), and brain samples collected at necropsy were examined by histology and immunohistochemistry.ResultsViremia (DNAemia) was detected at day 3 p.i. in 7/8 challenged animals. End-organ dissemination was observed, by qPCR, in the lung, liver, and spleen. CD4-positive (CD4+) and CD8-positive (CD8+) T cell infiltrates were present in brains of challenged animals, particularly in periventricular and hippocampal regions. Reactive gliosis and microglial nodules were observed. Statistically significant spatial learning and memory deficits were observed by MWM, particularly for total maze distance traveled (p < 0.0001).ConclusionNeonatal GPCMV infection in guinea pigs results in cognitive defects demonstrable by the MWM. This neonatal guinea pig challenge model can be exploited for studying antiviral interventions.ImpactCMV impairs neonatal neurocognition and memory in the setting of postnatal infection. The MWM can be used to examine memory and learning in a guinea pig model of neonatal CMV infection. CD4+ and CD8+ T cells infiltrate the brain following neonatal CMV challenge. This article demonstrates that the MWM can be used to evaluate memory and learning after neonatal GPCMV challenge. The guinea pig can be used to examine central nervous system pathology caused by neonatal CMV infection and this attribute may facilitate the study of vaccines and antivirals.

Dataset Information

Cloning the complete guinea pig cytomegalovirus genome as an infectious bacterial artificial chromosome with excisable origin of replication.

Publications

Cloning the complete guinea pig cytomegalovirus genome as an infectious bacterial artificial chromosome with excisable origin of replication.

Similar Datasets

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