Cellular localization of cytomegalovirus nucleic acids in guinea pig salivary glands by in situ hybridization

In situ hybridization with guinea pig cytomegalovirus (CMV) DNA probes was used to detect guinea pig CMV nucleic acid sequences in salivary glands during the course of infection. Optimum conditions for detection of guinea pig CMV gene sequences at the cellular level were determined. The technique of nucleic acid hybridization was compared to antigen detection and histologic examination for cellular localization of guinea pig CMV during acute and chronic infection. Tissue sections fixed for 1 h in 4% paraformaldehyde demonstrated darker specific staining and contained significantly larger numbers of cells positive for guinea pig CMV nucleic acids and antigens than sections fixed longer and in other fixatives. The method of in situ hybridization detected more guinea pig CMV infected cells than did routine histological evaluation. Histologically normal salivary gland duct cells as well as cells bearing typical inclusions were found to contain guinea pig CMV nucleic acids. Guinea pig CMV nucleic acids were also detected although less frequently in cells outside of the ducts. These results suggest that in situ hybridization allows for the detection of histologically inapparent guinea pig CMV infections at the cellular level.     

Comparison of guinea pig cytomegalovirus and guinea pig herpes-like virus: growth characteristics and antigentic relationship.

The growth characteristics of guinea pig cytomegalovirus (GPCMV) and guinea pig herpes-like virus (GPHLV) in cell cultures were compared. Guinea pig fibroblast cells were highly susceptible to infection with both viruses, whereas guinea pig kidney cells were sensitive only to GPHLV. No cytopathic effect was observed in the latter cell system after infection with GPCMV,nor was there an increase in virus titer, although the cirus persisted in the kidney cells for 2 to 3 weeks postinfection. Electron microscope studies showed nonvirion tubular structures in GPCMV -infected fibroblast cells, but not in GPHLV- infected cells. Large packages of enveloped nuclear virus particles were commonly seen in GPHLV -infected cells, especially kidney epithelial cells, but none were found in the GPCMV -infected fibroblasts. Complete enveloped extracellular virus particles were present in both virus-cell systems. Both viruses showed narrow host spectra and replicated well only in guinea pig cells although GPHLV multiplied to some degree in rabbit cells. No antigenic relationship could be demonstrated between the two viruses using antisera specific for each virus that was produced in rabbits and guinea pigs. Rabbits produced high neutralizing antibody titers to GPHLV, whereas guinea pigs were the animals of choice for GPCMV antiserum production.     

Ultrastructural localization of viral antigen in nuclear inclusions of cytomegalovirus infected guinea pig cells

Summary Intranuclear localization of viral antigens in guinea pig cytomegalovirus (GPCMV) infected guinea pig embryo (GPE) cells was investigated by cross-reactive indirect immunoperoxidase and immunoferritin techniques utilizing guinea pig antisera to GPCMV. Following primary fixation with 4 percent paraformal-dehyde, a brief treatment of infected cells with 0.25 percent trypsin was found to enhance penetration of antibodies and the conjugates. Ferritin or horseradish peroxidase conjugated goat anti-rabbit IgG was used as a secondary antibody that cross reacted with guinea pig immunoglobulins in order to reduce non-specific immunochemical reactions. Using light microscopy following immunoperoxidase staining, GPCMV antigens in an intranuclear location were not discernable when the infected cells were stained without pretreatment with trypsin, however intranuclear GPCMV antigens could be visualized after the fixed cells were treated with trypsin for 2–4 minutes prior to addition of the antiserum. Electron microscopic examination following indirect immunoferritin staining revealed viral antigens localized on viral capsids and on scattered electrondense amorphous matrices but not on the surrounding tubular structures or fibrils. The possibility that tubular structures may be a host cell product produced in response to GPCMV infection is discussed.With 3 Figures     

Histopathologic and ultrastructural studies of disseminated cytomegalovirus infection in strain 2 guinea pigs

Inbred strain 2 guinea pigs developed severe disseminated disease during acute experimental guinea pig cytomegalovirus (GPCMV) infection. A high mortality rate (100%) resulted, with most animals dying between 10 and 14 days after high dose (7.5 X 10(5) TCID50) virus inoculation. Infectious virus was recovered from many tissues, including spleen, lungs, liver, pancreas, heart, adrenals, kidneys, and salivary glands. The rate of GPCMV isolation from these tissues ranged from 50 to 100%. Gross lesions were observed in the spleen, liver, and lungs. On histologic examination, lesions were also seen in many other organs, including heart, pancreas, kidneys, adrenals, brain, intestines, and salivary glands. Intranuclear viral inclusions were present in many cell types of various organs. Under electron microscopic examination, cells with viral inclusions were easily found in the spleen, and liver, but less readily in the lungs, kidneys, salivary glands, and other organs. Most of the intranuclear inclusions consisted of electron-dense fibrils (10 nm diameter), viral nucleocapsids (100 nm), and tubular structures (60 nm diameter). Dense bodies and enveloped dense virions containing single or multiple capsids were present in the cytoplasm of many infected cells. The morphologic developments of GPCMV in these visceral tissues of strain 2 guinea pigs resembled those seen in GPCMV-infected cultured guinea pig cells but differed from those observed in the infected salivary gland duct cells. Strain 2 guinea pigs are a useful animal model for studying disseminated infection in CMV-associated human diseases.     

Immunogenicity evaluation of DNA vaccines that target guinea pig cytomegalovirus proteins glycoprotein B and UL83

Vaccines are needed for control of congenital human cytomegalovirus (HCMV) infection. Although the species-specificity of cytomegaloviruses precludes preclinical evaluation of HCMV vaccines in animal models, the guinea pig cytomegalovirus (GPCMV), which causes disease in utero, is a relevant model for the study of vaccines against congenital infection. We investigated whether DNA vaccines that target two GPCMV proteins, glycoprotein B (gB) and UL83 (pp65), are capable of eliciting immune responses in vivo. After cloning each gene into an expression vector, DNA was delivered by intramuscular inoculation and by pneumatic epidermal delivery. In Swiss-Webster mice, anti-gB titers were significantly higher after epidermal delivery. After epidermal inoculation in guinea pigs, all gB-immunized animals (n = 6) had antibody responses comparable to those induced by natural infection. Viral neutralization titers ranged from 1:64 to greater than 1:128. A GPCMV UL83 DNA vaccine also elicited an antibody response in all immunized guinea pigs (n = 6) after epidermal administration. Immunoprecipitation and Western blot assays confirmed that immune sera were immunoreactive with virion-associated UL83 and gB proteins. We conclude that DNA vaccines against GPCMV structural proteins are immunogenic, and warrant further investigation in the guinea pig model of congenital CMV infection.     

Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA.

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxigenin-labeled probes, and the spatial morphological resolution and localization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes constructed in our laboratory that were immunoenzymatically visualized employing anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal from the hybrid formation was detected, analyzed, and measured with a high performance, low light level imaging luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell, corresponding to the presence of hybridized CMV DNA, could be found in infected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive. The chemiluminescent in situ hybridization assay developed in this work can be a useful tool for a sensitive and specific diagnosis of viral infection and can be easily adapted to detect and study any specific gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.     

Identification and characterization of the guinea-pig cytomegalovirus glycoprotein H gene

Summary Subunit vaccines which target viral envelope glycoproteins offer promise for the prevention of congenital cytomegalovirus (CMV) infection. The guinea pig model of CMV infection is uniquely well suited to testing vaccines for prevention of congenital infection, since, in contrast to other animal cytomegaloviruses, the guinea pig CMV (GPCMV) crosses the placenta, producing intrauterine infection. Antibody to the CMV glycoproteins B (gB) and H (gH) appears to be important in conferring protective immunity. Unfortunately, little is known about specific GPCMV envelope glycoproteins. Sequencing of GPCMV genome fragments was therefore undertaken to test whether GPCMV encodes a gH homologue. Partial sequencing of theHind III A fragment of the GPCMV genome revealed an open reading frame of 2 169 nucleotides capable of encoding a protein of 723 amino acids. Computer matrix analyses demonstrated identity between this ORF and the gH coding sequences of other herpesviruses. The GPCMV gH ORF encodes 12 highly conserved cysteine residues, contains 9 potential N-linked glycosylation sites, and has a predicted M_r of 81.6 kDa. Northern blot hybridizations with gH-specific probes identified an abundant 5.1 kb mRNA with expression kinetics of an early gene. A polyclonal antiserum raised against a synthetic peptide derived from the deduced amino acid sequence of the gH ORF identified a virion-associated protein with an approximate M_r of 85-kDa, the putative GPCMV gH, in immunoblot assays.The nucleotide sequences reported in this paper have been submitted to the GenBank database and assigned the accession number U49361.     

Cloning and expression of the guinea pig cytomegalovirus glycoprotein B (gB) in a recombinant baculovirus: utility for vaccine studies for the prevention of experimental infection

The guinea pig cytomegalovirus (GPCMV) is unique among the cytomegaloviruses of small mammals, insofar as during pregnancy it crosses the placenta, causing infection of the fetus. Although the guinea pig model is well suited to vaccine studies, the lack of cloned, recombinant forms of immunogenic GPCMV proteins, such as envelope glycoproteins, has hindered experimental evaluations of subunit immunization for prevention of fetal disease. Since the glycoprotein B (gB) is a major target of neutralizing antibody responses, the GPCMV gB was cloned and expressed in a recombinant baculovirus. A recombinant was generated which expressed gB, truncated at codon 692, upstream of the putative transmembrane domain. Processing and expression of the recombinant protein, designated Bac-gB, was assessed, and the protein was characterized immunologically. Anti-gB antibodies were immunoreactive with Bac-gB by enzyme linked immunosorbent assay (ELISA) and immunoblot assay. Immunoprecipitation with polyclonal anti-GPCMV antisera identified protein species of 120, 80 and 30 kDa by reducing SDS-PAGE, suggesting that authentic cleavage and processing of Bac-gB occurred in insect cells. Sera from guinea pigs immunized with lectin-column purified native glycoproteins had high ELISA titers to Bac-gB. Recombinant GPCMV gB expressed in insect cells should prove useful in defining correlates of protective immunity in the GPCMV congenital infection model.     

Identification of essential and non-essential genes of the guinea pig cytomegalovirus (GPCMV) genome via transposome mutagenesis of an infectious BAC clone

We report application of a transposition methodology that allows the easy characterization and mutation of genes encoded on an infectious bacterial artificial chromosome (BAC) clone. We characterized mutants generated by transposome (Tn) mutagenesis of a BAC clone of guinea pig cytomegalovirus (GPCMV). A pool of Tn mutant GPCMV BACs were screened initially by restriction profile analysis to verify they were full-length, and subsequently GPCMV BAC DNA from individual mutants was transfected onto guinea pig lung fibroblast cells in order to generate virus. Tn GPCMV BAC mutants were classed as either essential or non-essential gene insertions, depending upon their ability to regenerate viable, replication-competent virus. Representative mutants were more fully characterized. Analysis by sequencing the Tn insertion site on the mutated BACs, and by regeneration of virus using transfection of guinea pig fibroblasts (GPL), demonstrated that a recombinant with a Tn insertion in the UL35 homolog gene (GP35) was a non-essential gene for viral replication in tissue culture. A mutant with an insertion in the UL46 homolog (GP46) was nonviable, a phenotype which could be rescued by homologous recombination of BAC DNA with wild-type UL46 sequences, suggesting an essential role of this putative capsid gene in virus replication.     

Comparison of guinea pig cytomegalovirus and guinea pig herpes-like virus: pathogenesis and persistence in experimentally infected animals.

The pathogenesis of guinea pig cytomegalovirus (GPCMV) and guinea pig herpes-like virus (GPHLV) in guinea pigs was compared. Animals were inoculated with the two viruses by different routes and sacrificed after varying periods of time. GPCMV was consistently isolated from salivary gland 2 weeks postinoculation and thereafter following intraperitoneal or subcutaneous incoulaton. Virus was less frequently found in other tissues including blood, spleen, and kidney. Intranuclear inclusions were seen in tissue sections of salivary gland after inoculation with GPCMV- infected tissue suspension, but were only rarely found after inoculation with tissue culture virus. In GPHLV-infected guinea pigs, consistent latent infection of leukocytes and other tissues was detected by cocultivation techniques. Intranuclear inclusions were not found in the spleen, salivary gland, or other infected tissues after GPHLV infection with either tissue culture virus or infected tissue suspension. Guinea pigs inoculated with GPCMV produced high titers of specific neutralizing antibody to the homologous virus; those inoculated with GPHLV developed long-term viremia accompanied by minimal neutralizing antibody levels to the virus.     

Quantitative-competitive PCR monitoring of viral load following experimental guinea pig cytomegalovirus infection

Human cytomegalovirus (HCMV) is the most common cause of congenital viral infection in the developed world, and can lead to significant morbidity. Animal models of HCMV infection are required for study of pathogenesis, because of the strict species-specificity of cytomegalovirus (CMV). Among the small animal CMV models, the guinea pig CMV (GPCMV) has unique advantages, in particular its propensity to cross the placenta, causing disease in utero. In order to develop quantitative endpoints for vaccine and antiviral therapeutic studies in the GPCMV model, a quantitative-competitive PCR (qcPCR) assay was developed, based on the GPCMV homolog of the HCMV UL83 gene, GP83. Optimal amplification of GPCMV DNA was observed using primers spanning a 248 base pair (bp) region of this gene. A 91 bp deletion of this cloned fragment was generated for use as an internal standard (IS) for PCR amplification. Standard curves based upon the fluorescent intensity of full-length external target to IS were compared with signal intensity of DNA extracted from blood and organs of experimentally infected guinea pigs in order to quantify viral load. Viral load in newborn guinea pigs infected transplacentally was determined and compared with that of pups infected with GPCMV as neonates. Viral loads were highest in pups infected as neonates. The most consistent isolation and highest quantities of viral DNA were observed in liver and spleen, although viral genome could be readily identified in brain, lung, and salivary gland. Viral load determination should be useful for monitoring outcomes following vaccine studies, as well as responses to experimental antiviral agents.     

Guinea pig cytomegalovirus: Transplacental transmission

Summary A well characterized strain of guinea pig cytomegalovirus (GPCMV) was used to infect pregnant guinea pigs during various periods of pregnancy. Transplacental transmission of virus with invasion of the fetus was observed, even in some mothers with preinoculation evidence of GPCMV antibody. Fetal infection occurred during the middle third of pregnancy and GPCMV was isolated from many fetal tissues although histologic evidence of infection was not noted. During the last third, abortion of the pregnancy occurred in some animals.This report demonstrates that GPCMV may invade the fetus producing a sublethal, possibly mild infection which may be very similar to the usual type of CMV infection observed in the human newborn.     

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.     

Comparison of vaccine strategies against congenital CMV infection in the guinea pig model.

Vaccines are urgently needed to protect newborns against the devastating sequelae of congenital cytomegalovirus infection. Evaluation of candidate vaccines in the guinea pig model of congenital infection can shed light on potentially useful strategies for humans, since guinea pig CMV (GPCMV) is transmitted to the fetus transplacentally, causing infection and disease in utero. A number of vaccine strategies have been evaluated in this model, including DNA vaccines, live attenuated vaccines, and recombinant glycoprotein vaccines. Induction of virus-neutralizing antibody appears to play a key role in protection of the fetus. Recently, a vectored vaccine based on the GPCMV homolog of the UL83 (pp65) protein has also been shown to be effective when used as a preconceptual vaccine in this model, suggesting that strategies designed to elicit T-cell responses may be of value in protection of the fetus.     

Ultrastructural development and persistence of guinea pig cytomegalovirus in duct cells of guinea pig submaxillary gland

Summary Salivary glands from Hartley guinea pigs were experimentally infected with guinea pig cytomegalovirus (GPCMV) and examined by light and electron microscopy at different time intervals. Characteristic intranuclear and intracytoplasmic viral inclusions were observed in duct cells of infected animals. Viral inclusion counts and infectivity titers in the salivary gland reached maximum levels by 3 to 4 weeks after infection; infectivity persisted, though at reduced levels, for at least 30 weeks. Electron microscopic examination of viral inclusions revealed several developmental events including nucleocapsid assembly, envelopment of nucleocapsids at the inner nuclear membrane and their enclosure by a thin vacuolar membrane. While contained within cytoplasmic vacuoles, enveloped virions acquired surface spikes. Cytoplasmic vacuoles containing virions subsequently coalesced and discharged mature virions at the cell surface into the lumen of the salivary gland duct. The data indicate that the ultrastructural development of GPCMV in the guinea pig salivary gland shows many similarities to that of human cytomegalovirus in humans. The salivary gland may provide a primary locus for virus shedding and horizontal transmission of cytomegalovirus.With 7 Figures     

An immunocytochemical method for the detection of fluorochrome-labelled DNA probes hybridized in situ with cellular RNA

Various detection systems for in situ hybridization of nucleic acids are currently used. We report here an immunocytochemical detection system which is based on the detection of FITC-labelled DNA/mRNA hybrids and takes advantage of FITC molecules attached covalently to the DNA probes prior to hybridization. In situ hybridization on cytocentrifuge spots is followed by the application of an anti-fluorescein antibody thus permitting detection of mRNA/DNA-FITC hybrids. The anti-FITC antibody reaction is demonstrated by an indirect immunocytochemical peroxidase-staining method. The T lymphoblast cell line Jurkat and the cDNA for the TCR-beta chain were chosen to establish the technique.     

Detection of Epstein-Barr virus by in situ hybridization. Progress toward development of a nonisotopic diagnostic test.

This work presents some initial quantitation of an in situ hybridization method for detection of Epstein-Barr (EB) virus nucleic acids. The purpose is to develop evaluative criteria for diagnosis of viral presence in clinical tissue specimens. In this work simultaneous denaturation of probe and target DNA and an alkaline phosphatase conjugate to detect biotinated probe were used as described by Unger et al. For evaluation of the hybridization, a variety of cell lines, both productively and latently infected, that were hybridized in situ using nick translated 32P-labeled viral probe sequences and counted by scintillation after the method of Lawrence and Singer were used. Producer cells (B95-8) showed intense foci of staining in approximately 5% of cells, with most of the other cells showing varying staining intensity. Raji cells showed varying amounts of signal from cell to cell. Namalwa cells exhibited one spot in most cells that was decreased after cells were treated with Actinomycin D (dactinomycin, Merck Sharp & Dohme, West Point, PA). Signal was identified in only a third of these same cells after sectioning. EB virus-negative Ramos cells showed no signal. The nuclear punctate nature of the signal generated is diagnostic of infected cells, and may be a useful test for cultured cells or pathologic specimens.