Caroline K.Y. Fong

Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes.

A method of in situ cytohybridization is described for the detection of specific viral genomes in infected cell cultures or paraffin-embedded tissue sections without the use of radioisotopes. Biotin-labeled analogs of TTP are incorporated into viral DNA in vitro by nick translation and the resultant DNA probes hybridized to cytologic samples. Cells containing viral genetic material are then revealed by standard immunofluorescence, immunoperoxidase, or affinity cytochemical techniques that are based on the specific interaction between biotin and antibiotin IgG or avidin. Hybridization probes containing nucleotides that have an 11- or 16-atom spacer arm between the biotin molecule and the pyrimidine ring interact with these detector proteins more efficiently than probes containing biotin-nucleotides with a 4-atom spacer arm. The total procedure can be performed fairly rapidly (24 hr or less) and numerous samples can be processed simultaneously. Although the detection methods employed to date are not as sensitive as autoradiographic procedures with high specific activity probes, more sensitive protein detector complexes are currently being constructed. The speed, specificity, and resolving power of this technique should be of general utility in screening for the presence of infectious agents in cell or tissue samples. Here we report the visualization of parvovirus, polyomavirus, herpes simplex virus, adenovirus, and retrovirus genetic material in infected cell cultures and herpes simplex and adenovirus DNA in paraffin-embedded autopsy tissues.

Activity of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) against guinea pig cytomegalovirus infection in cultured cells and in guinea pigs

(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC, and two HPMPC-related nucleoside analogs, (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, HPMPA, and (2-phosphonylmethoxyethyl)guanine, PMEG, were evaluated for their antiviral activities against guinea pig cytomegalovirus (GPCMV) infection in guinea pig embryo (GPE) cells and human cytomegalovirus (HCMV) infection in human diploid fibroblast (MRC-5) cells. DHPG, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, was used for comparison. The antiviral activity of HPMPC against GPCMV infection in vivo and its toxicity to Hartley guinea pigs were also evaluated. The 50% antiviral effective doses (ED50) of HPMPC, HPMPA, PMEG and DHPG against GPCMV infection in GPE cells were 0.22, 1.4, 0.07 and 62 microM, respectively; and against HCMV infection in MRC-5 cells, the ED50s were 0.51, 0.72, 0.01 and 17.5 microM, respectively. Their cytotoxic doses (CyD50) in GPE replicating cells were 84, 35, 1.4 and 700 microM, respectively and in MRC-5 cells were approximately 114, 31, 0.86 and 750 microM, respectively. Based on their calculated therapeutic indexes, HPMPC was the most potent and selective of the four compounds tested. In vivo, during acute infection, the spleen indexes of all infected animals that were treated with 1.25 to 5.0 mg/kg/day of HPMPC for 5 days were significantly reduced as compared with sham-treated animals. Virus infectivity titers in blood and various tissues of infected animals treated with HPMPC, 2.5 or 1.25 mg/kg/day were not significantly lower than those of the infected, sham-treated animals; with 5 mg/kg/day, infectivity titers in the blood, spleen, and salivary gland were significantly lower in HPMPC-treated than in sham-treated animals. However, HPMPC was toxic to guinea pigs especially at doses of 5 to 10 mg/kg/day. These data showed that HPMPC was highly active and selective in cultured guinea pig cells and human fibroblast cells against CMV infection but did not effectively inhibit GPCMV infection in guinea pigs at minimum toxic concentrations.

Disseminated adenovirus infection in an immunocompromised host: Pitfalls in diagnosis

In this report, a bone marrow transplant recipient with rapidly fatal gastroenteritis is presented. The presence of intranuclear inclusions on postmortem light microscopic examination of liver, lung, and small bowel tissue was considered diagnostic of cytomegalovirus infection. However, electron microscopic examination of liver tissue demonstrated adenovirus infection. This was confirmed by isolation of an adenovirus type 2 with unusual laboratory features from liver, lung, colon contents, serum, esophageal swab, and oral ulcerations. Results of a complement fixation test for antibodies to adenovirus performed on postmortem serum samples were negative, and a titer of 1:4 was noted for antibody against cytomegalovirus. This case illustrates the diagnostic pitfalls that may be encountered in establishing a specific viral diagnosis in severely ill patients.

Antiviral effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine against cytomegalovirus infection in a guinea pig model.

The antiviral activity of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) against guinea pig cytomegalovirus (GPCMV) was evaluated in guinea pig cell cultures and in Hartley guinea pigs. The 50% effective dose of DHPG against GPCMV replication in cell cultures was 71 microM. Ultrastructural studies revealed that DHPG inhibited the formation of viral cores and the production of nucleocapsids, enveloped virions and dense bodies, but the drug did not prevent the formation of virus induced intranuclear tubular structures. In vivo, guinea pigs inoculated intraperitoneally with GPCMV were treated with DHPG, 25 mg/kg subcutaneously, twice daily. Treatment was initiated 24 h after infection and continued for 7 days. During the acute infection, the average body weights of DHPG-treated, virus infected guinea pigs were approximately 14% lower than the sham-treated counterparts on day 10, 11 and 13 post-virus inoculation. Virus infectivity titers were higher in the lungs of DHPG-treated guinea pigs on day 10 than the sham-treated ones. Although there was no significant difference on histopathologic lesions in the spleen, liver and lungs of the drug-treated and the sham-treated guinea pigs, DHPG treated animals appeared to have fewer virus-induced lesions or inclusions in the kidneys and salivary glands than the sham-treated ones. In addition, virus infectivity titers in the salivary gland of DHPG treated guinea pigs were consistently lower than the sham-treated animals.

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

SummarySalivary 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.

Ultrastructural studies of the envelopment and release of guinea pig herpes-like virus in cultured cells.

Abstract The process of envelopment and release of guinea pig herpes-like virus was examined in both infected guinea pig kidney and thymus tissue culture cells by electron microscopy. The majority of the nucleocapsids were enveloped by budding into nuclear vacuoles; some were enveloped by budding from the inner nuclear membrane. Budding into cytoplasmic vacuoles was also seen. Many enveloped virus particles inside the nuclear vacuoles were pear shaped with a tail-like structure. Approximately 23% of pear-shaped virus particles were seen in the infected thymus fibroblastic cells, but only 6% were found in the infected epithelial cells. The envelopes of all nuclear enveloped virus particles appeared as smooth membranes, while the majority of particles exhibiting fuzzy and thick dense envelopes were seen in the cytoplasm or extracellular space. The average diameter of the cytoplasmic or extracellular enveloped virus particles was approximately 167 nm, and the average diameter of the nuclear enveloped virus particles was about 146 nm. Data also showed that mature nuclear virus particles were first released into perinuclear cisterna and then traveled through cytoplasmic channels to the extracellular space.

Productive and abortive infections of simian and nonsimian cells with a simian adenovirus SV15: I. Microscopic observations

Abstract The uptake and development of simian adenovirus SV15 in rhesus monkey and hamster kidney cells was followed by light and electron microscopy. Uptake of virus particles was noted in both cell systems after virus inoculation. Sequential nuclear changes were observed only in SV15-infected monkey cells. These changes included the early appearance of eosinophilic inclusions, followed by the formation of basophilic inclusions, then nuclear vacuolation, and finally nuclear distortion. Progeny virus particles were seen in the infected nuclei of monkey cells as early as 14 hours postinfection. In hamster cells, on the other hand, an abortive growth cycle developed after infection by this virus. Only eosinophilic inclusions were observed in the infected hamster cells, Progeny virus particles could not be found even at 40 hours postinoculation, at which time complete cellular degeneration occurred. Formation of eosinophilic inclusions in hamster cells appeared to be induced by the infectious virus, but not by the noninfectious virus preparations.

Oncornavirus of guinea pigs: I. Morphology and distribution in normal and leukemic guinea pig cells

Abstract Two morphologically distinct types of oncornavirus particles were observed in guinea pig cells. In tissues of leukemic guinea pigs, intracisternal A-type particles 90–100-nm diameter, predominated. Extracellular particles with dense core, approximately 90–110 nm in diameter, were seen only occasionally at the intercellular space of the tissues, but were predominant in plasma and sera of the same animals. Placental and fetal tissues obtained from normal guinea pigs showed only intracisternal A-type particles. Cultured guinea pig cells when treated with BrdU revealed many intracytoplasmic A-type particles 90–100-nm diameter. Budding of these A-type particles at the cell membrane to form extracellular enveloped A-type particles was observed. Extracellular virus particles with dense cores, 110–120 nm in diameter, similar to those seen in tissues and plasma of leukemic guinea pigs, were abundant in the BrdU-treated cultures. The morphology and distribution of the intracellular and extracellular virus particles in tissues and tissue cultures derived from leukemic and normal guinea pigs are compared, and the relationships between these virus particles are discussed.

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

SummaryIntranuclear 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.

Productive and abortive infections of simian and nonsimian cells with a simian adenovirus SV15: II. Viral biosynthesis and effects of chemical inhibitors

Abstract The viral biosynthesis of a simian adenovirus, SV15, was studied in both simian and nonsimian cells. In rhesus monkey kidney cells, viral complement-fixing (CF) antigen and infectious progeny virus were first detected 12 hours after virus inoculation. Synthesis of viral DNA was initiated at about 5–6 hours before the first appearance of complete virus; viral protein was first obtained 2–3 hours before the maturation of virus. In primary hamster kidney cells, SV15 induced an abortive infection. DNA synthesis was stimulated in SV15-infected hamster kidney cells as determined by the increased uptake of thymidine- 3 H, but viral antigen could not be detected by CF test, immunofluorescence or agar gel double-diffusion methods. At no time could progeny infectious virus be detected by infectivity titrations. The use of polyoma virus or SV40 as a “helper” virus did not induce the production of SV15 infectious virus in hamster kidney cells. Fluorouracil and cytosine arabinoside were unable to inhibit cytopathic effect as well as the formation of eosinophilic inclusions in SV15-infected RhMK or HamK cells; these inhibitors did prevent the development of basophilic inclusions in infected RhMK cells. Actinomycin D and streptovitacin A inhibited cellular changes and eosinophilic inclusions induced by SV15 in both cell systems tested. In monkey cells, fluorouracil at concentrations of 12–50 μg/ml inhibited 90% or more of the infectious virus, whereas only a 50% reduction of complement-fixing antigen occurred at concentrations of 25–50 μg/ml. To obtain a greater than 90% reduction of CF antigen production, 100 μg/ml of FU was required. Cytosine arabinoside (10 μg/ml), actinomycin D (1 μg/ml), and streptovitacin A (3 μg/ml) completely inhibited viral antigen as well as infectious virus production.