Guinea Pig Cytomegalovirus Genome Research Articles

Intravaginal cytomegalovirus (CMV) challenge elicits maternal viremia and results in congenital transmission in a guinea pig model

BackgroundThe objective of this study was to compare intravaginal (ivg) and subcutaneous (sc) administration of the guinea pig cytomegalovirus (GPCMV) in pregnant and non-pregnant guinea pigs. These studies tested the hypotheses that ivg infection would elicit immune responses, produce maternal viremia, and lead to vertical transmission, with an efficiency similar to the traditionally employed sc route.ResultsFour groups of age- and size-matched guinea pigs were studied. Two groups were pregnant, and two groups were not pregnant. Animals received 5x105 plaque-forming units (PFU) of a GPCMV reconstituted from an infectious bacterial artificial chromosome (BAC) construct containing the full-length GPCMV genome. Seroconversion was compared by IgG ELISA, and viremia (DNAemia) was monitored by PCR. In both pregnant and non-pregnant animals, sc inoculation resulted in significantly higher serum ELISA titers than ivg inoculation at 8 and 12 weeks post-infection. Patterns of viremia (DNAemia) were similar in animals inoculated by either sc or ivg route. However, in pregnant guinea pigs, animals inoculated by both routes experienced an earlier onset of DNAemia than did non-pregnant animals. Neither the percentage of dead pups nor the percentage of GPCMV positive placentas differed by inoculation route.ConclusionsIn the guinea pig model of congenital CMV infection, the ivg route is as efficient at causing congenital infection as the conventional but non-physiologic sc route. This finding could facilitate future experimental evaluation of vaccines and antiviral interventions in this highly relevant animal model.

Characterization of the guinea pig cytomegalovirus genome locus that encodes homologs of human cytomegalovirus major immediate-early genes, UL128, and UL130

We reported previously that the guinea pig cytomegalovirus (CMV) stock purchased from the American Type Culture Collection contained two types of strains, one containing and the other lacking a 1.6 kb locus, and that the 1.6 kb locus was required for efficient viral growth in animals but not in cell culture. In this study, we characterized the genetic contents of the locus, and found that i) the 1.6 kb locus encodes homologs of human CMV UL128 and UL130, GP129 and GP131, respectively, ii) these genes are expressed with late gene kinetics, iii) GP131 protein (pGP131) localized to cell surface only in the presence of glycoproteins H and L, and iv) pGP131 is a virion component. Therefore, it is plausible that pGP131 forms a complex with glycoproteins H and L and becomes a virion component as does UL130 protein (pUL130). Since pUL130 is one of the glycoproteins essential for infection of endothelial and epithelial cells in human and primates, functional and immunological analyses of this GPCMV homolog of pUL130 may help to illuminate the in vivo role of pUL130.

A live guinea pig cytomegalovirus vaccine deleted of three putative immune evasion genes is highly attenuated but remains immunogenic in a vaccine/challenge model of congenital cytomegalovirus infection

Live attenuated vaccines for prevention of congenital cytomegalovirus infections encode numerous immune evasion genes. Their removal could potentially improve vaccine safety and efficacy. To test this hypothesis, three genes encoding MHC class I homologs (presumed NK evasins) were deleted from the guinea pig cytomegalovirus genome and the resulting virus, 3DX, was evaluated as a live attenuated vaccine in the guinea pig congenital infection model. 3DX was attenuated in vivo but not in vitro. Vaccination with 3DX produced elevated cytokine levels and higher antibody titers than wild type (WT) virus while avidity and neutralizing titers were similar. Protection, assessed by maternal viral loads and pup mortality following pathogenic viral challenge during pregnancy, was comparable between 3DX and WT and significant compared to naïve animals. These results suggest that the safety and perhaps efficacy of live attenuated human cytomegalovirus vaccines could be enhanced by deletion of viral immunomodulatory genes.

Identification of a 1.6 kb genome locus of guinea pig cytomegalovirus required for efficient viral growth in animals but not in cell culture

Guinea pig cytomegalovirus (GPCMV) provides a useful model for studies of congenital CMV infection. During characterization of the GPCMV genome sequence, we identified two types of strains in a virus stock purchased from ATCC. One of them, GPCMV/del, lacks a 1.6 kb locus that positionally corresponds to murine CMV (MCMV) M129–M133. Growth of GPCMV/del in cell culture was marginally better than that of the other strain, GPCMV/full, which harbors the 1.6 kb locus. However, in animals infected intraperitoneally with virus stocks containing both strains, GPCMV/full disseminated more efficiently than GPCMV/del, including 200-fold greater viral load in salivary glands. Viral DNA, transcripts of the immediate-early 2 gene homolog, and viral antigens were more abundant in animals infected with GPCMV/full than in those infected with GPCMV/del. Although the observed phenomena have some similarity with the growth properties of MCMV strains defective in mck-1/mck-2(M129/131) and those defective in sgg(M132), no M129–M132 homologs were found in the 1.6 kb locus. Since one of the ORFs in the locus has a weak sequence similarity with HCMV UL130, which relates to cell tropism, further studies will be required to learn the mechanism for efficient GPCMV growth in animal.

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

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 232 kb 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.

Expression of the human cytomegalovirus UL97 gene in a chimeric guinea pig cytomegalovirus (GPCMV) results in viable virus with increased susceptibility to ganciclovir and maribavir

In lieu of a licensed vaccine, antivirals are being considered as an intervention to prevent congenital human cytomegalovirus (HCMV) infection. Ideally, antiviral therapies should undergo pre-clinical evaluation in an animal model prior to human use. Guinea pig cytomegalovirus (GPCMV) is the only small animal model for congenital CMV. However, GPCMV is not susceptible to the most commonly used HCMV antiviral, ganciclovir (GCV), rendering in vivo study of this agent problematic in the guinea pig model. Human cytomegalovirus (HCMV) susceptibility to GCV is linked to the UL97 gene. We hypothesized that GPCMV susceptibility to GCV could be improved by inserting the HCMV (Towne) UL97 gene into the GPCMV genome in place of the homolog, GP97. A chimeric GPCMV (GPCMV:: UL97) expressed UL97 protein, and replicated efficiently in cell culture, with kinetics similar to wild-type GPCMV. In contrast, deletion of GP97 resulted in a virus (GPCMVd GP97) that grew poorly in culture. GPCMV:: UL97 had substantially improved susceptibility to the inhibitory effects of GCV in comparison to wild-type GPCMV. Additionally, GPCMV:: UL97 exhibited improved susceptibility to another antiviral undergoing clinical trials, maribavir (MBV; benzimidazole riboside 1263W94), which also acts through UL97.

Analysis of the nucleotide sequence of the guinea pig cytomegalovirus (GPCMV) genome

In this report we describe the genomic sequence of guinea pig cytomegalovirus (GPCMV) assembled from a tissue culture-derived bacterial artificial chromosome clone, plasmid clones of viral restriction fragments, and direct PCR sequencing of viral DNA. The GPCMV genome is 232,678 bp, excluding the terminal repeats, and has a GC content of 55%. A total of 105 open reading frames (ORFs) of > 100 amino acids with sequence and/or positional homology to other CMV ORFs were annotated. Positional and sequence homologs of human cytomegalovirus open reading frames UL23 through UL122 were identified. Homology with other cytomegaloviruses was most prominent in the central ~60% of the genome, with divergence of sequence and lack of conserved homologs at the respective genomic termini. Of interest, the GPCMV genome was found in many cases to bear stronger phylogenetic similarity to primate CMVs than to rodent CMVs. The sequence of GPCMV should facilitate vaccine and pathogenesis studies in this model of congenital CMV infection.

Animal models of congenital cytomegalovirus infection: an overview of progress in the characterization of guinea pig cytomegalovirus (GPCMV).

The strict species-specificity of cytomegalovirus (CMV) precludes preclinical evaluation of human CMV (HCMV) vaccines in animal models and necessitates the study of nonhuman CMVs. Among the CMVs of small mammals, the guinea pig cytomegalovirus (GPCMV) has unique advantages, due to its ability to cross the placenta, causing infection in utero. OBJECTIVE AND STUDY DESIGNS: Progress in GPCMV studies has been hampered by a lack of detailed molecular characterization of the viral genome. Therefore, recent efforts have been undertaken to characterize the GPCMV genome, and apply this information to in vivo subunit vaccine studies. Progress in the sequencing of the GPCMV genome has revealed the presence of both highly conserved as well as novel open reading frames (ORFs). Cloning of GPCMV vaccine candidates, such as the glycoprotein B (gB) and UL83 proteins, has facilitated subunit vaccine evaluation. Protein vaccines and DNA vaccines have shown evidence of protection in pregnancy/challenge experiments. In addition, the GPCMV genome has proved amenable to cloning as a bacterial artificial chromosome (BAC) in Escherichia coli, and BAC-derived recombinants retain the ability to replicate in vivo. Progress has been made in molecular characterization of GPCMV. Insights from these studies should prove germane to the understanding of the correlates of protective immunity for the fetus in vaccine studies, and should assist in prioritization of vaccine strategies in HCMV vaccine trials.

Molecular Cloning of the Guinea Pig Cytomegalovirus (GPCMV) Genome as an Infectious Bacterial Artificial Chromosome (BAC) in Escherichia coli

Since cytomegalovirus (CMV) infection is highly species-specific, it is necessary to study animal cytomegaloviruses to assess viral factors which contribute to pathogenesis. The generation of recombinant viruses carrying reporter genes would provide useful tools for studying the genetics of CMV pathogenicity in vivo. We evaluated whether the guinea pig cytomegalovirus (GPCMV) was amenable to such manipulation. Metabolic selection using the guanosylphosphoribosityl transferase (gpt) gene facilitated recovery of a recombinant virus, vAM403, containing a gpt/green fluorescent protein (eGFP) cassette introduced into the HindIII “N” region of the viral genome. This virus had replication kinetics identical to wild-type virus. We next attempted to clone the GPCMV genome as a bacterial artificial chromosome (BAC). A BAC plasmid containing a gpt/eGFP cassette and the chloramphenicol resistance marker was introduced into HindIII “N” to generate another GPCMV recombinant, vAMBGPCMV. Circular viral DNA isolated from vAMBGPCMV-infected cells was used to transform Escherichia coli. Restriction profiles revealed that the GPCMV genome had been cloned as a BAC plasmid, and transfection of BAC plasmid DNA confirmed that the BAC clone was infectious. A novel strategy based on a unique PmeI site was devised to quickly modify the BAC GPCMV plasmid. Recombinants retained the capability to replicate and express reporter genes in guinea pigs, suggesting that these viruses will be useful for in vivo pathogenesis studies.

Molecular characterization of the guinea-pig cytomegalovirus glycoprotein L gene.

Although the guinea pig cytomegalovirus (GPCMV) model is well suited to the study of vaccines for prevention of congenital CMV infection, there has been limited molecular characterization of GPCMV glycoproteins. Since the in vivo co-expression of the human cytomegalovirus (HCMV) glycoprotein H (gH, gpUL75) with glycoprotein L (gL, gpUL115) may have relevance to CMV vaccine studies, these experiments were undertaken to test whether the GPCMV encodes a gL homolog. Sequencing of the EcoR I "G" fragment of the GPCMV genome identified an open reading frame (ORF) of 774 nucleotides capable of encoding a protein of 258 amino acids. Computer matrix analyses demonstrated identity between this ORF and the gL coding sequences of other betaherpesviruses. Sequence analysis also identified an ORF with identity to the HCMV uracil DNA glycosylase (UDG, UL114 gene). The GPCMV gL ORF encodes 6 cysteine residues, contains 3 potential N-linked glycosylation sites, and has a predicted Mr of 29.7 kDa. Northern blot studies identified an abundant 2.7 kb "early" transcript from infected cells, the putative gL message. In vitro translation of gL mRNA in reticulocyte lysate resulted in synthesis of 30 kDa polypeptide. A polyclonal antiserum was raised against a gL/glutathione-S-transferase fusion protein generated in E. coli using the pGEX expression system. This antibody identified a 40-kDa virion-associated protein, the putative GPCMV gL, in immunoblot assays.

Sequence and transcriptional analysis of the guinea pig cytomegalovirus UL97 homolog.

Guinea pig cytomegalovirus (GPCMV) displays a similar pathogenesis to human cytomegalovirus (HCMV), and the guinea pig has been used as a model system for testing anti-CMV therapies. However, not all agents active against HCMV share antiviral activity against GPCMV. For example, GPCMV appears resistant to the nucleoside analog, ganciclovir. The molecular basis for this discrepancy in antiviral susceptibility is unknown because to date there has been little analysis of the GPCMV genome. For HCMV, the antiviral effect of ganciclovir depends upon phosphorylation of the drug to its active form. This effect is mediated by the viral UL97 gene product. In order to begin to explore the molecular basis of the resistance of GPCMV to ganciclovir, experiments were undertaken to test whether the GPCMV genome encoded a homolog of the HCMV UL97 gene. Based on the prediction of co-linearity of UL97 homologs within the respective viral genomes, the EcoR I S and F fragments of the GPCMV genome were cloned and partially sequenced. A 1815 base pair open reading frame (ORF) capable of encoding a 604 amino acid (aa) protein was identified spanning portions of the EcoR I S and adjacent EcoR I F genome fragments. Computer-assisted matrix analyses revealed identity between this ORF and the HCMV UL97 gene. ORFs upstream of the GPCMV UL97 gene were identified which shared homology with the HCMV UL95 and 96 genes. Northern blot analyses identified a UL97-specific mRNA of 3.9 kb which was expressed at "early" times post-infection. RNA transcripts of 6.0 and 4.6 kb were identified which corresponded to the UL95 and UL96 homolog coding sequences, respectively. Comparison of the GPCMV UL97 sequence to that of other herpesvirus homologs as well as that of ganciclovir-resistant clinical isolates of HCMV identified nonconservative aa substitutions in two domains involved in catalysis and substrate recognition.

Sequence and transcriptional analysis of the guinea-pig cytomegalovirus DNA polymerase gene.

Although the guinea-pig cytomegalovirus (GPCMV) displays a similar pathogenesis to human cytomegalovirus (HCMV), there have unfortunately been few molecular analyses of the GPCMV genome to date. The guinea-pig has proved useful for the testing of drugs active against CMV infection, and insights derived from characterization of the specific virally encoded molecular targets of antiviral therapies would allow this model system to be more fully developed. Because the DNA polymerase serves as an important target for nucleoside antiviral agents active against herpesviruses, experiments were undertaken to identify, clone and sequence the GPCMV DNA polymerase gene (pol). A 3285 bp ORF capable of encoding a 1094 amino acid protein was identified spanning portions of the HindIII Q and P fragments of the genome. This ORF contained extensive homology to other herpesvirus DNA pol genes. Northern blot analyses identified two 3' coterminal pol-specific mRNAs of 3.9 and 1.9 kb at early times post-infection. Primer extension and nuclease protection analyses mapped the 5' end of the 3.9 kb transcript to a site 275 bases upstream of the pol initiation codon. Comparison of the GPCMV pol-encoded sequence to those of other herpesvirus polymerases identified non-conservative amino acid substitutions in a domain involved in substrate recognition.

Guinea pig cytomegalovirus immediate-early transcription

Guinea pig cytomegalovirus (GPCMV) immediate-early (IE) gene expression was analyzed. GPCMV IE RNA was defined as RNA obtained from GPCMV-infected guinea pig cells treated with cycloheximide for 1 h before infection and for 4 h postinfection. Mapping studies showed that GPCMV IE genes are located at several distinct sites on the GPCMV genome. A total of 17 GPCMV IE transcripts were identified, and 9 IE transcripts coded for by three specific regions of the genome (regions I, II, and III) were characterized in detail. A series of recombinant DNA clones were generated to identify the nine IE transcripts. Three of the IE transcripts from region I and three from region III were transcribed in the same direction from overlapping sequences. The 2.0-kilobase (kb) transcript encoded by the EcoRI E DNA fragment (region II) was the most abundant IE GPCMV transcript. The cloned GPCMV DNA subfragment that was used to identify the region II EcoRI E 2.0-kb transcript did not hybridize to GPCMV early or late RNA, indicating that this transcript is expressed only under IE conditions. Expression of RNAs from the IE genes was also measured during a natural GPCMV infection in the absence of cycloheximide. During the natural infection, the transcripts previously identified under IE cycloheximide block conditions were expressed, and the region II EcoRI E 2.0-kb transcript was the most abundant transcript at 1 h postinfection. In addition, a rise and fall in RNA levels was observed during the natural infection, demonstrating the transient nature of expression of these transcripts. We conclude that GPCMV IE gene expression is complex, involving a reasonably large number of genes, and demonstrates some similarities with IE transcription by other CMVs.

Characterization of the guinea pig cytomegalovirus genome by molecular cloning and physical mapping.

Fragments of guinea pig cytomegalovirus (GPCMV) DNA produced by HindIII or EcoRI restriction endonuclease digestion were cloned into vectors pBR322 and pACYC184, and recombinant fragments representing ca. 97% of the genome were constructed. Hybridization of 32P-labeled cloned and gel-purified HindIII, EcoRI, and XbaI fragments to Southern blots of HindIII-, EcoRI-, and XbaI-cleaved GPCMV DNA verified the viral origin of cloned fragments and allowed construction of HindIII, EcoRI, and XbaI restriction maps. On the basis of the cloning and mapping experiments, the size of GPCMV DNA was calculated to include 239 kilobase pairs, corresponding to a molecular weight of 158 X 10(6). No cross-hybridization between any internal fragments was seen. We conclude that the GPCMV genome consists of a long unique sequence with terminal repeat sequences but without internal repeat regions. In addition, GPCMV DNA molecules exist in two forms. In the predominant form, the molecules demonstrate sequence homology between the terminal fragments; in the minor population, one terminal fragment is smaller by 0.7 X 10(6) daltons and is not homologous with the fragment at the other end of the physical map. The structural organization of GPCMV DNA is unique for a herpesvirus DNA, similar in its simplicity to the structure reported for murine cytomegalovirus DNA and quite dissimilar from that of human cytomegalovirus DNA.