Complete Genome Sequence of Cell Culture-Attenuated Guinea Pig Cytomegalovirus Cloned as an Infectious Bacterial Artificial Chromosome

Yang, Dongmei; Alam, Zohaib; Cui, Xiaohong; Chen, Michael I.; Sherrod, Carly J.; McVoy, Michael A.; Schleiss, Mark R.; Dittmer, Dirk P.

doi:10.1128/genomea.00928-14

Cited by 6 publications

(15 citation statements)

References 14 publications

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“…Although the gp145 mutant was able to replicate sufficiently to generate virus for vaccination, for ideal production strategies, it may be necessary for stocks to be propagated on complementing cells, particularly for an HCMV vaccine deleted of all PKR antagonists. Additionally, the fact that sterilizing immunity protecting against congenital GPCMV transmission was not conferred by the ⌬145 vaccine may relate to the inability of the virus to form a functional PC, based on the known mutation in the gp129 gene in the parent N13R10 virus (25). Generation of a gp145 deletion against the background of a gp129-repaired BAC will help clarify the potential for the development and optimal design of HCMV vaccines based on knockout of PKR-inhibitory functions.…”

Section: Discussionmentioning

confidence: 99%

“…The genome structure of vJZ848 was confirmed by restriction endonuclease digestion, PCR, and targeted sequencing. Previous GFP-tagged GPCMVs contain 4-bp or 1.6-kb deletions that impact the gp129/gp133 locus and that confer an in vivo attenuation phenotype (25,42). Virus vJZ848 retained the 1.6-kb locus, demonstrated no loss of the 1.6-kb locus after over 10 passages in GPL cells (as assessed by PCR), and maintained the wild-type sequence, as demonstrated by sequence analysis of PCR-amplified DNA from multiple serial passages for the gp129, gp131, and gp133 ORFs encoding components of the GPCMV pentameric complex (43, 44) (data not shown).…”

Section: Methodsmentioning

confidence: 99%

“…BAC construct N13R10 contains the complete GPCMV strain 22122 genome, while the BAC N2 contains a 17.9-kb deletion (39). BAC N2 with a targeted deletion of gp145 (N2-⌬145) and N13R10 with a targeted deletion of gp145 (N13R10-⌬145 or ⌬145) were constructed using a linear recombination approach to result in the substitution of a kanamycin resistance cassette (Kn) for nucleotides 224206 to 226104 (GenBank accession number KM384022 [25]) comprising the gp145 open reading frame (Fig. 1A).…”

Section: Methodsmentioning

confidence: 99%

“…The live attenuated vaccine approach has also been modeled in the guinea pig, in which GPCMV vaccines generated using bacterial artificial chromosome (BAC)-based mutagenesis strategies have been evaluated (23,24). One limitation of these studies is that the BAC containing the GPCMV genome was found on sequence analysis to contain a 4-bp deletion that frameshifts the gp129 open reading frame (ORF) (25), hence rendering viruses reconstituted from the BAC unable to assemble the GPCMV homolog of the HCMV pentameric complex (PC), composed of GPCMV proteins gH, gL, gp129, gp131, and gp133. In this respect, live GPCMV vaccines derived from the N13R10 BAC have an attenuated parental genetic background similar to that of the Towne vaccine.…”

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confidence: 99%

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Vaccination with a Live Attenuated Cytomegalovirus Devoid of a Protein Kinase R Inhibitory Gene Results in Reduced Maternal Viremia and Improved Pregnancy Outcome in a Guinea Pig Congenital Infection Model

Schleiss

Bierle

Swanson

et al. 2015

J Virol

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Development of a vaccine to prevent congenital cytomegalovirus infection is a major public health priority. Live vaccines attenuated through mutations targeting viral mechanisms responsible for evasion of host defense may be both safe and efficacious. Safety and vaccine efficacy were evaluated using a guinea pig cytomegalovirus (GPCMV) model. Recombinant GPCMV with a targeted deletion of gp145 (designated ⌬145), a viral protein kinase R (PKR) inhibitor, was generated. Attenuation was evaluated following inoculation of 10 7 PFU of ⌬145 or parental virus into guinea pigs immunosuppressed with cyclophosphamide. Efficacy was evaluated by immunizing GPCMV-naive guinea pigs twice with either 10 5 or 10 6 PFU of ⌬145, establishing pregnancy, and challenging the guinea pigs with salivary gland-adapted GPCMV. The immune response, maternal viral load, pup mortality, and congenital infection rates in the vaccine and control groups were compared. ⌬145 was substantially attenuated for replication in immunocompromised guinea pigs. Vaccination with ⌬145 induced enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody levels comparable to those achieved in natural infection. In the higher-and lower-dose vaccine groups, pup mortality was reduced to 1/24 (4%) and 4/29 (14%) pups, respectively, whereas it was 26/31 (81%) in unvaccinated control pups (P < 0.0001 for both groups versus the control group). Congenital infection occurred in 20/31 (65%) control pups but only 8/24 (33%) pups in the group vaccinated with 10 6 PFU (P < 0.05). Significant reductions in the magnitude of maternal DNAemia and pup viral load were noted in the vaccine groups compared to those in the controls. Deletion of a GPCMV genome-encoded PKR inhibitor results in a highly attenuated virus that is immunogenic and protective as a vaccine against transplacental infection. Infection with human cytomegalovirus (HCMV) causes considerable morbidity and occasional mortality in immunocompromised solid organ transplant and hematopoietic stem cell transplant patients, HIV-infected individuals, and newborns that acquire infection in utero (1, 2). Due to the lifelong morbidity associated with congenital CMV infection, a preconception vaccine capable of preventing virus transmission to the fetus would provide a highly cost-effective public health advance (3). Unfortunately, the lack of clear immunological correlates of protective immunity has hampered development of an HCMV vaccine. In spite of this uncertainty, there is evidence that virus-neutralizing antibody responses targeting viral envelope glycoproteins, as well as cellular immune responses (CD4 ϩ and CD8 ϩ ) targeting multiple structural and regulatory proteins, play important roles in protection against acquisition and reactivation of infection (4-7).

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Vaccination with a Live Attenuated Cytomegalovirus Devoid of a Protein Kinase R Inhibitory Gene Results in Reduced Maternal Viremia and Improved Pregnancy Outcome in a Guinea Pig Congenital Infection Model

Schleiss

Bierle

Swanson

et al. 2015

J Virol

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“…Based on these comparisons to SG virus, adult guinea pigs tolerate a 5,880-fold-higher dose of N13R10 without significant illness or mortality, while a 140-foldhigher dose of N13R10 during pregnancy produces lower rates of fetal transmission and pup loss (11,12). The molecular basis for the attenuation of N13R10 is uncertain, but DNA sequence comparisons of the SG virus and N13R10 genomes identified only two differences resulting in alteration of annotated protein coding sequences: (i) a 4-bp deletion that disrupts overlapping genes GP129 and GP130 and (ii) a missense mutation in GP130 3= of the 4-bp deletion/frameshift (14,15).…”

mentioning

confidence: 99%

Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired during Fibroblast Passage Restores Pathogenesis in Immune-Suppressed Guinea Pigs and in the Context of Congenital Infection

McVoy

Wang

Dittmer

et al. 2016

J Virol

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Guinea pig cytomegalovirus (GPCMV) provides a valuable model for congenital cytomegalovirus transmission. Salivary gland (SG)-passaged stocks of GPCMV are pathogenic, while tissue culture (TC) passage in fibroblasts results in attenuation. Nonpathogenic TC-derived virus N13R10 (cloned as a bacterial artificial chromosome [BAC]) has a 4-bp deletion that disruptsGP129, which encodes a subunit of the GPCMV pentameric complex (PC) believed to govern viral entry into select cell types, and GP130, an overlapping open reading frame (ORF) of unknown function. To determine if this deletion contributes to attenuation of N13R10, markerless gene transfer in Escherichia coli was used to construct virus r129, a variant of N13R10 in which the 4-bp deletion is repaired. Virions from r129 were found to contain GP129 as well as two other PC subunit proteins, GP131 and GP133, whereas these three PC subunits were absent from N13R10 virions. Replication of r129 in fibroblasts appeared unaltered compared to that of N13R10. However, following experimental challenge of immunocompromised guinea pigs, r129 induced significant weight loss, longer duration of viremia, and dramatically higher (up to 1.5 ؋ 10 6 -fold) viral loads in blood and end organs compared to N13R10. In pregnant guinea pigs, challenge with doses of r129 virus of >5 ؋ 10 6 PFU resulted in levels of maternal viremia, congenital transmission, pup viral loads, intrauterine growth restriction, and pup mortality comparable to that induced by pathogenic SG virus, although higher doses of r129 were required. These results suggest that the GP129-GP130 mutation is a significant contributor to attenuation of N13R10, likely by abrogating expression of a functional PC. IMPORTANCETissue culture adaptation of cytomegaloviruses rapidly selects for mutations, deletions, and rearrangements in the genome, particularly for viruses passaged in fibroblast cells. Some of these mutations are focused in the region of the genome encoding components of the pentameric complex (PC), in particular homologs of human cytomegalovirus (HCMV) proteins UL128, UL130, and UL131A. These mutations can attenuate the course of infection when the virus is reintroduced into animals for vaccine and pathogenesis studies. This study demonstrates that a deletion that arose during the process of tissue culture passage can be repaired, with subsequent restoration of pathogenicity, using BAC-based mutagenesis. Restoration of pathogenicity by repair of a frameshift mutation in GPCMV gene GP129 using this approach provides a valuable genetic platform for future studies using the guinea pig model of congenital CMV infection. Infection with human cytomegalovirus (HCMV) is a leading cause of disability in newborns, and development of an effective vaccine is a major public health priority (1, 2). Preclinical modeling of vaccines against congenital infection must rely on the study of species-specific CMVs, since HCMV will not infect nonhuman cells (3, 4). The study of guinea pig cytomegalovirus (GPCMV) is particularl...

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Cytomegalovirus UL128 homolog mutants that form a pentameric complex produce virus with impaired epithelial and trophoblast cell tropism and altered pathogenicity in the guinea pig

2017

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Guinea pig cytomegalovirus (GPCMV) encodes a homolog pentameric complex (PC) for specific cell tropism and congenital infection. In human cytomegalovirus, the PC is an important antibody neutralizing target and GPCMV studies will aid in the development of intervention strategies. Deletion mutants of the C-terminal domains of unique PC proteins (UL128, UL130 and UL131 homologs) were unable to form a PC in separate transient expression assays. Minor modifications to the UL128 homolog (GP129) C-terminal domain enabled PC formation but viruses encoding these mutants had altered tropism to renal and placental trophoblast cells. Mutation of the presumptive CC chemokine motif encoded by GP129 was investigated by alanine substitution of the CC motif (codons 26–27) and cysteines (codons 47 and 62). GP129 chemokine mutants formed PC but GP129 chemokine mutant viruses had reduced epitropism. A GP129 chemokine mutant virus pathogenicity study demonstrated reduced viral load to target organs but highly extended viremia.

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Complete Genome Sequence of Cell Culture-Attenuated Guinea Pig Cytomegalovirus Cloned as an Infectious Bacterial Artificial Chromosome

Cited by 6 publications

References 14 publications

Vaccination with a Live Attenuated Cytomegalovirus Devoid of a Protein Kinase R Inhibitory Gene Results in Reduced Maternal Viremia and Improved Pregnancy Outcome in a Guinea Pig Congenital Infection Model

Vaccination with a Live Attenuated Cytomegalovirus Devoid of a Protein Kinase R Inhibitory Gene Results in Reduced Maternal Viremia and Improved Pregnancy Outcome in a Guinea Pig Congenital Infection Model

Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired during Fibroblast Passage Restores Pathogenesis in Immune-Suppressed Guinea Pigs and in the Context of Congenital Infection

Cytomegalovirus UL128 homolog mutants that form a pentameric complex produce virus with impaired epithelial and trophoblast cell tropism and altered pathogenicity in the guinea pig

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