Secreted Virus–Encoded Proteins Reflect Murine Cytomegalovirus Productivity in Organs

Brune, Wolfram; Hasan, Milena; Krych, M.; Bubić, Ivan; Jonjić, Stipan; Koszinowski, Ulrich H.

doi:10.1086/323993

Cited by 16 publications

(10 citation statements)

References 16 publications

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“…Our data show for the first time that a herpesvirus infection can be launched directly from bacteria carrying the infectious virus genome, omitting the need for technical preparatory work to isolate BAC DNA. We have also demonstrated that additional genes introduced into the virus are effectively expressed (3). Thus, the use of BAC-derived viruses has a potential as vaccine and vaccine vector, and this is also the case when the viral genome is launched from bacteria.…”

Section: Discussionmentioning

confidence: 85%

“…Plasmid pRep4-HBs was generated by inserting the gene of the hepatitis B virus surface antigen (HBsAg) into pRep4 (Invitrogen) as described (3). To obtain MCMV-HBs, the HBs gene bracketed by a Rous sarcoma virus promoter and a simian virus 40 polyadenylation signal was excised from pRep4-HBs and inserted into the ie2 gene locus of the cloned full-length MCMV BAC, pSM3fr (37), as previously described (3).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Vaccination of Mice with Bacteria Carrying a Cloned Herpesvirus Genome Reconstituted In Vivo

Čičin-Šain¹,

Brune

Bubić

et al. 2003

J Virol

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Bacterial delivery systems are gaining increasing interest as potential vaccination vectors to deliver either proteins or nucleic acids for gene expression in the recipient. Bacterial delivery systems for gene expression in vivo usually contain small multicopy plasmids. We have shown before that bacteria containing a herpesvirus bacterial artificial chromosome (BAC) can reconstitute the virus replication cycle after cocultivation with fibroblasts in vitro. In this study we addressed the question of whether bacteria containing a single plasmid with a complete viral genome can also reconstitute the viral replication process in vivo. We used a natural mouse pathogen, the murine cytomegalovirus (MCMV), whose genome has previously been cloned as a BAC in Escherichia coli. In this study, we tested a new application for BAC-cloned herpesvirus genomes. We show that the MCMV BAC can be stably maintained in certain strains of Salmonella enterica serovar Typhimurium as well and that both serovar Typhimurium and E. coli harboring the single-copy MCMV BAC can reconstitute a virus infection upon injection into mice. By this procedure, a productive virus infection is regenerated only in immunocompromised mice. Virus reconstitution in vivo causes elevated titers of specific anti-MCMV antibodies, protection against lethal MCMV challenge, and strong expression of additional genes introduced into the viral genome. Thus, the reconstitution of infectious virus from live attenuated bacteria presents a novel concept for multivalent virus vaccines launched from bacterial vectors.Live, attenuated bacteria have a potential to serve as vaccine vectors for the oral delivery of foreign proteins (29) or DNA (11,13,23). Oral vaccination procedures do not require educated personnel, and lyophilized bacterial preparations can be kept and distributed at room temperature, without the costly and difficult necessity of keeping the preparations cold. Moreover, oral vaccination can be performed simultaneously on large numbers of subjects, which should make it an efficient strategy for livestock vaccination.Live antiviral vaccines are generally considered more efficient than subunit or inactivated vaccines because they are more likely to induce a broad range of immune responses to the expressed gene products and provide a better protection. Furthermore, since these vaccines replicate in the recipient, the immunity they confer should be long lasting (39). Therefore, a bacterial vaccination vector delivering an attenuated, yet infectious virus may present the basis for efficient vaccines that are easy to store, distribute, and administer.Herpesviruses are important pathogens for humans and livestock. We have previously shown that the large genomes of herpesviruses (up to 230 kb) can be cloned as bacterial artificial chromosomes (BACs) into Escherichia coli (1, 2, 24). Others have demonstrated that BAC DNA encoding the genome of a herpesvirus can induce specific protective immunity (35,36). However, in these experiments, the BAC DNA was isolated from bac...

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Section: Discussionmentioning

confidence: 85%

Section: Methodsmentioning

confidence: 99%

Vaccination of Mice with Bacteria Carrying a Cloned Herpesvirus Genome Reconstituted In Vivo

Čičin-Šain¹,

Brune

Bubić

et al. 2003

J Virol

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“…(A) Virus yield (log) per gram of homogenized liver and brain determined by plaque assay and results expressed as infectious units (PFU)/g of tissue as described previously (reference 21). (B) Real-time PCR performed with MCMV IE-1 primers using DNA extracted from the livers or brains of infected animals as described in Results and Materials and methods, expressed as IE-1 copy number per 200 μl of 10% homogenate as a function of time after infection (reference 77). (C–F) IHC detection of virus-infected cells using anti-pp89 (IE-1) in (C) the frontal cortex from a PN day 12 mouse, (D) in neurons of hippocampus from a PN day 11–infected mouse, (E) granular neurons of cerebellum, and (F) Purkinje neurons of cerebellum from PN day 11 mice.…”

Section: Resultsmentioning

confidence: 99%

Altered development of the brain after focal herpesvirus infection of the central nervous system

Koontz¹,

Bralić

Tomac

et al. 2008

The Journal of Experimental Medicine

164

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Human cytomegalovirus infection of the developing central nervous system (CNS) is a major cause of neurological damage in newborn infants and children. To investigate the pathogenesis of this human infection, we developed a mouse model of infection in the developing CNS. Intraperitoneal inoculation of newborn animals with murine cytomegalovirus resulted in virus replication in the liver followed by virus spread to the brain. Virus infection of the CNS was associated with the induction of inflammatory responses, including the induction of a large number of interferon-stimulated genes and histological evidence of focal encephalitis with recruitment of mononuclear cells to foci containing virus-infected cells. The morphogenesis of the cerebellum was delayed in infected animals. The defects in cerebellar development in infected animals were generalized and, although correlated temporally with virus replication and CNS inflammation, spatially unrelated to foci of virus-infected cells. Specific defects included decreased granular neuron proliferation and migration, expression of differentiation markers, and activation of neurotrophin receptors. These findings suggested that in the developing CNS, focal virus infection and induction of inflammatory responses in resident and infiltrating mononuclear cells resulted in delayed cerebellar morphogenesis.

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“…For dexa experiments, organs were collected, weighed and homogenized. A 10% homogenate in media was utilized for standard plaque assays [134]. For pred experiments, organs were collected and DNA was isolated using Trizol according to the manufacturer's instructions (Roche Applied Science).…”

Section: Methodsmentioning

confidence: 99%

Glucocortiocoid Treatment of MCMV Infected Newborn Mice Attenuates CNS Inflammation and Limits Deficits in Cerebellar Development

et al. 2013

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Infection of the developing fetus with human cytomegalovirus (HCMV) is a major cause of central nervous system disease in infants and children; however, mechanism(s) of disease associated with this intrauterine infection remain poorly understood. Utilizing a mouse model of HCMV infection of the developing CNS, we have shown that peripheral inoculation of newborn mice with murine CMV (MCMV) results in CNS infection and developmental abnormalities that recapitulate key features of the human infection. In this model, animals exhibit decreased granule neuron precursor cell (GNPC) proliferation and altered morphogenesis of the cerebellar cortex. Deficits in cerebellar cortical development are symmetric and global even though infection of the CNS results in a non-necrotizing encephalitis characterized by widely scattered foci of virus-infected cells with mononuclear cell infiltrates. These findings suggested that inflammation induced by MCMV infection could underlie deficits in CNS development. We investigated the contribution of host inflammatory responses to abnormal cerebellar development by modulating inflammatory responses in infected mice with glucocorticoids. Treatment of infected animals with glucocorticoids decreased activation of CNS mononuclear cells and expression of inflammatory cytokines (TNF-α, IFN-β and IFNγ) in the CNS while minimally impacting CNS virus replication. Glucocorticoid treatment also limited morphogenic abnormalities and normalized the expression of developmentally regulated genes within the cerebellum. Importantly, GNPC proliferation deficits were normalized in MCMV infected mice following glucocorticoid treatment. Our findings argue that host inflammatory responses to MCMV infection contribute to deficits in CNS development in MCMV infected mice and suggest that similar mechanisms of disease could be responsible for the abnormal CNS development in human infants infected in-utero with HCMV.

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Secreted Virus–Encoded Proteins Reflect Murine Cytomegalovirus Productivity in Organs

Cited by 16 publications

References 16 publications

Vaccination of Mice with Bacteria Carrying a Cloned Herpesvirus Genome Reconstituted In Vivo

Vaccination of Mice with Bacteria Carrying a Cloned Herpesvirus Genome Reconstituted In Vivo

Altered development of the brain after focal herpesvirus infection of the central nervous system

Glucocortiocoid Treatment of MCMV Infected Newborn Mice Attenuates CNS Inflammation and Limits Deficits in Cerebellar Development

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