G. D. Hsiung

Ultrastructural development of guinea pig cytomegalovirus in cultured guinea pig embryo cells.

The ultrastructural development of guinea pig cytomegalovirus (GPCMV) in guinea pig embryo cells was studied using electron microscopy. Tubular structures were found in nuclei of virus infected cells, followed by the appearance of intranuclear inclusions containing virus nucleocapsids. While some nucleocapsids were enveloped at the inner nuclear membrane, others were released into the cytoplasm where they were associated with, or within, dense matrix which was subsequently enveloped by cytoplasmic membranes to form enveloped dense virions. Dense bodies without virus capsids were formed in the cytoplasm and enveloped in a similar manner. An involvement of the nuclear pores in the release of unenveloped virus capsids from the nucleus to the cytoplasm was postulated. Evidence that the enveloped dense virions and dense bodies shared common envelope antigen(s) was obtained by immunoelectron microscopy. The similarities and differences in the ultrastructural development of GPCMV and other cytomegaloviruses are discussed.

Prophylactic and Therapeutic Treatment with Acyclovir of Genital Herpes in the Guinea Pig

Abstract The antiviral drug, acyclovir, was tested on experimentally infected guinea pigs with either of two herpes simplex virus type 1 (HSV-1) isolates following intravaginal inoculation. The drug was continuously infused subcutaneously utilizing an osmotic pump. Infusion was begun either prior to virus inoculation (prophylactic) or after virus inoculation at the time of first appearance of lesions (therapeutic). Prophylactic treatment markedly reduced the severity of the genital lesions, the appearance of acute neurologic sequellae, and the virus excretion in the genital tract of guinea pigs infected with either of the two strains tested. Therapeutic acyclovir treatment, however, did not decrease the incidence of acute neurologic sequellae with one of the two HSV-1 strains tested, nor did it reduce the severity of the genital lesions of either strain. These neurologic sequellae may be due to insufficient levels of ACV in the central nervous system as the concentration of ACV in the dorsal root ganglia was found to exceed that of the plasma, but only trace amounts of acyclovir were present in the brain and spinal cord. Continuous perfusion of ACV gave far higher tissue levels than intermittent injections. These findings suggest that prophylactic ACV is far more effective than therapeutic treatment for genital herpes in the guinea pig model.

Effect of heparin on cytomegalovirus replication.

Summary The presence of heparin in cell culture medium was found to reduce the infectivity titers of cytomegaloviruses isolated from human, monkey, guinea pig and mouse. This inhibition was shown to increase as the heparin concentration was increased. When heparin was added to cultures after virus adsorption had taken place, the effect on virus infectivity was negligible. Other anticoagulants studied included Alsevers solution, sodium citrate and EDTA, none of which showed any inhibitory effect on cytomegalovirus replication in cell culture.

Growth Characteristics of Bovine Herpesvirus 1 (Infectious Bovine Rhinotracheitis) in Human Diploid Cell Strain WI-38

Summary IBR virus was found to replicate in WI-38 cells. At a high input multiplicity the virus yield was comparable to that obtained in bovine cells, but comparable degree of CPE took longer to achieve. At a low input multiplicity of IBR virus, such as may be encountered in virus contaminated bovine serum, virus yield was only about 1% of that in bovine cells, with 50% of the cells showing CPE, followed by cell regrowth. Infectious virus was not recoverable from the regrown cells by 5 weeks after initial infection, and these regrown cells were susceptible to reinfection with IBR virus. Aging of WI-38 cells in cultures for as little as 1 week reduced IBR virus yield to 90% less than the yield from the same lot of cells inoculated 7 days earlier. This investigation was supported by NIH Contract No. FDA 233-74-1035 from the Food and Drug Administration, and USPHS Research Grant No. AI-08648-07 from the Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. the excellent technical assistance of Susan Rake is appreciated.

Malignant Transformation of Hamster Cells following Infection with a Bovine Herpesvirus (Infectious Bovine Rhinotracheitis Virus)

Summary Hamster embryo cells, following infection with IBR virus, showed malignant transformation. Hamsters of all ages, inbred or random bred, inoculated with two of the transformed cell lines developed solid tumors. Preliminary characterization of the tumors induced by one of the cell lines has indicated undifferentiated sarcomas. Viral specific antigen was detected in about 5 % of the transformed cells and 10% of primary tumor cells in culture. Viral specific antibody was detected in the serum of tumor-bearing hamsters by the indirect immunofluorescent method, but no neutralizing antibodies were found. Infectious virus has not been recovered from either the transformed or tumor cells by cocultivation with bovine embryonic kidney cells. This investigation was supported by NIH Contract No. DBS-72-2105 from the Food and Drug Administration and USPHS Grant No. AI 08648-05 from the Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. The authors are grateful to Dr. T. W. Reid, Yale University School of Medicine for the reverse tran-scriptase assay. The excellent technical assistance of Edilea Ellinger and Andrea Tomanik is appreciated. The HSV-1 transformed hamster cells were obtained from Dr. William Summers of Yale University who had originally obtained them as line 14-012-8-1 from Dr. Fred Rapp of Pennsylvania State University.

Dual infection with endogenous herpes- and C-type viruses in cultured cells derived from normal and leukemic guinea pigs.

Summary Dual infection with endogenous herpes- and C-type viruses in cultured cells derived from normal guinea pigs as well as in single guinea pig cells, was observed. The guinea pig C-type virus was activated by maintenance of the cultured cells in BrdU containing medium and was detected in all guinea pigs tested, regardless of age, strain or source of animals. Manifestation of the herpesvirus required prolonged cultivation of the cells or co-cultivation with susceptible monolayer cultures. The herpesvirus isolation was age and strain dependent; it occurred most frequently in older inbred animals. Note added in proof: Guinea pig No. AD 185 showed GPHLV infection when tested at 11 mo. Furthermore, both GPHLV and C-type virus were observed in cultured spleen and/or kidney cells derived from leukemic guinea pigs.

Characterization of conditions for the activation of endogenous guinea pig retrovirus in cultured cells by 5-bromo-2′-deoxyuridine

Human endogenous retroviral sequences recently have been shown to be associated with breast cancer and some leukemias. These retroviral sequences have similarities to an endogenous retrovirus expressed in guinea pigs. The conditions for activation of this guinea pig retrovirus (GPRV) in cultured guinea pig embryo (GPE) cells using 5-bromo-2′-deoxyuridine (BrdU) was investigated. These studies employed the reverse transcriptase activity (RT) assay and electron microscopy (EM), in conjunction with Northern blot analysis that utilized a 2.6 kb GPRV-specific cDNA probe. Contrary to published studies, dexamethasone at concentrations ranging from 10−8 to 10−5 M appeared to play a minimal role in enhancing the production of GPRV. Following a 6 hr incubation with BrdU, GPRV mRNA was present in cultured GPE cells. Extracellular virion release was also observed by EM 12 hr later, although RT activity was not detected. All three methods detected viral expression at 48 hr after the addition of the drug. Additionally, after 6 hr exposure to BrdU, detectable RT and mRNA levels were maintained through 44 days after the removal of BrdU in a stationary culture condition and through 31 days in cultures that were subcultured weekly in media not containing BrdU. Low levels of extracellular viruses were detected in these cultures by electron microscopy through 49 days. Therefore, after only a 6 hr exposure to BrdU was extracellular GPRV detected 12 hr after drug removal and virus production continued for up to 49 days. This study provides information about an animal endogenous retroviral system that may be used as a model for the study of human endogenous retroviruses.

Neonatal herpes simplex virus infection in guinea pigs.

Abstract The mode of neonatal herpes simplex virus types 1 and 2 infection in guinea pigs was explored. There was no evidence of transplacental transmission of either virus type from mother to fetus following genital infection of the former. Infectious virus was not recoverable from placenta, cord blood, or amniotic fluid following either genital or intracardiac inoculation of HSV-2 into pregnant guinea pigs. However, a low rate of neonatal transmission of HSV-2 from mother to newborn via an infected birth canal during delivery was demonstrated.

In Vitro Transformation of Hamster Embryo Cells by a Guinea Pig Herpes-Like Virus

Summary Hamster embryo cells infected with GPHLV showed morphological cellular transformation. Virus strains isolated from leukemic guinea pigs showed somewhat higher transforming capacity than the isolates from “normal” Hartley guinea pigs. Virus specific antigen was detected in the cytoplasm of 7-20% of the transformed hamster cells. Infectious virus could be recovered from the transformed cells by co-cultivation with guinea pig kidney cells. Quantitative analysis showed that in these cell lines 12-47/100,000 transformed hamster cells yielded infectious virus. The authors are grateful to Dr. R. H. Green for his valuable suggestions during the preparation of this manuscript. The excellent technical assistance of JoEllyn Bradley and Judy Wnek is appreciated.

Persistence and expression of herpes virus in guinea pig B and T spleen cells.

Summary The role of spleen lymphocytes in acute and latent guinea pig herpes-like virus (GPHLV) infection and the in vitro susceptibility of the lymphocytes to GPHLV were explored. Macrophage-, B-cell-, and T-cell-enriched populations obtained from infected guinea pigs were examined for infectious GPHLV. During acute infection, virus was first detected in the macrophage and B-cell fractions, whereas, infectious virus was only evident in the T-cell fraction 5 days or more after inoculation. During latent infection, infectivity titers in the B fractions were consistently higher than in the T fractions. In both the B and the T lymphocytes derived from latently infected guinea pigs, virus was expressed only after in vitro cultivation or cocultivation with susceptible cells. Lymphocytes infected in vitro did not support GPHLV replication, although latent infection of lymphocytes with GPHLV was readily accomplished in vivo. We thank Christine Cote for excellent technical assistance. This research was partially supported by a Research Training Grant 5 T32 AI 07018 from the Institute of Allergy and Infectious Diseases, National Institutes of Health, and by the Medical Research Service of the Veterans Administration.