L. V. Meléndez

Herpesvirus Type 2 Isolated from Cervical Tumor Cells Grown in Tissue Culture

A herpesvirus has been isolated from spontaneously degenerating cultures of cervical tumor cells grown in vitro. The virus was identified as a type 2 herpesvirus on the basis of biologic properties, including plaque morphology and microtubule formation in infected HEp-2 cells, and of immunologic specificity as determined by neutralization. Herpesvirus antigens and virus particles were not seen in duplicate cultures of viable cervical tumor cells.

Acute Lymphocytic Leukemia in Owl Monkeys Inoculated with Herpesvirus saimiri

Our study demonstrates for the first time that Herpesvirus saimiri can induce acute lymphocytic leukemia in owl monkeys (Aotus trivirgatus) and that malignant lymphoma can be induced in this species of nonhuman primates by the inoculation of the virus by various routes (intravenous, sub-cutaneous, and intradermal).

Spontaneous Herpes-T Infection in the Owl Monkey (Aotus trivirgatus):

The pathological features of spontaneous herpes-T infection in 6 owl monkeys (Aotus trivirgatus) were characterized by the presence of intranuclear inclusion bodies in epithelial and mesenchymal cells associated with necrosis of the skin, oral mucous membranes, esophagus, small intestine, cecum, colon, nasal mucous membranes, larynx, trachea, lung, liver, spleen, lymph nodes, adrenal gland, and bone marrow. The inclusion bodies and the lesions are similar to herpes virus infections of other animal species. A virus was isolated from 5 monkeys, which on the basis of serum neutralization tests in vitro was identified as herpes-T.

Isolation of Herpes-T virus from a spontaneous disease in squirrel monkeys (Saimiri sciureus)

Herpes-T virus was isolated from 2 of 4 clinically ill squirrel monkeys. The clinical manifestations of the disease in the monkeys was characterized by oral and labial lesions. From one of two animals sacrificed for histopathological examination, Herpes-T virus was isolated from the tongue and salivary gland. Herpes-T was isolated from the oral swab of one of the two live monkeys on the day of arrival in our laboratory and again on the 4th day. The anal swabs collected from both these monkeys failed to yield virus. The sera of these monkeys collected on the day of arrival showed a neutralization index (NI) of 1.0 and 1.5 against Herpes-T virus. The convalescent sera, collected two weeks later, showed a NI of 3.5 and 4.0 respectively for the same virus. Clear plaques ranging in size from 2 to 3 mm were produced in chick fibroblast and rabbit kidney primary monolayers. This is the first report of the isolation of Herpes-T virus from naturally infected squirrel monkeys. This data further supports that the squirrel monkey is a natural host of Herpes-T virus.

Herpesvirus saimiri. III. Plaque Formation Under Multi Agar, Methyl Cellulose and Starch Overlays

Summary Herpesvirus saimiri produced plaques in cultures of fetal squirrel monkey heart, lung, intestine, and kidney and in owl monkey kidney and marmoset kidney cell cultures. The plaque populations in all cell types were heterogeneous in respect to size. Harvests of single plaques produced heterogeneous plaque populations on serial plating. Plaques were most numerous under methyl cellulose. Under agar, plaques were less numerous but were larger. Protamine sulfate and, to a limited degree, DEAE-dextran stimulated plaque production under agar while arginine had no effect. Plaques also formed under starch but were difficult to visualize. The number of plaques was directly proportional to the virus dilution, was also proportional to the volume of the inoculum, and was reduced by specific antiserum.

Herpes virus aotus: a latent herpesvirus from owl monkeys (Aotus trivirgatus) isolation and characterization.

Summary An uninoculated batch of owl monkey kidney cell cultures yielded a viral agent 23 days after the cell culture was initiated. This agent possessed the physical, chemical, cytopathic, histological, and ultrastructural properties of a herpesvirus. Random testing of owl monkey sera showed the presence of high titered neutralizing antibodies against this new agent. Herpesvirus simplex, Herpesvirus T., Herpesvirus B, Herpesvirus suis, Herpesvirus saimiri, infectious bovine rhinotraecheitis, and Herpesvirus saguinus, OMKI 372, and OMKI 68-69 antisera failed to neutralize the infectivity of this new agent. In cell cultures the virus grew best in cells of owl monkey origin. It also grew (poorly) in cebus monkey kidney cell cultures, some batches of squirrel monkey kidney cells, in Vero, BSC-1, human embryonic lung, whole human embryo and human embryo skin and muscle cells. It failed to form plaques under an agar overlay and does not form pocks on chorioallantoic membranes of embryonated eggs. Based on these findings, the name Herpesvirus aotus has been suggested. Owl monkey kidney cell cultures and owl monkey sera, when used for any work with other members of the herpesvirus group, ought to be employed with caution. The case reported here represents the first herpesvirus isolated from an owl monkey. Even though the pathogenic spectrum of this virus has not been established, being a herpesvirus, it should be regarded as potentially dangerous. Investigators who use owl monkeys should be aware of this new herpesvirus.

Herpesvirus Saimiri: In Vitro Sensitivity to Virus-Induced Interferon and to Polyriboinosinic Acid: Polyribocytidylic Acid

Summary The sensitivity of H. saimiri to virus-induced interferon and to the synthetic polynucleotide Poly I:C was determined in OMK cell cultures. These studies indicated that H. saimiri is relatively insensitive to the interferon activity in cultures pretreated for 6 hr with NDV-induced interferon. The degree of protection was improved when the cells were kept in interferon medium after the challenge with the virus. A direct pretreatment of the cells with low doses of Poly I:C induced a good protection which was greatly increased by a simultaneous addition of DEAE-D in the medium. The action of the Poly I:C/DEAE-D complex was considerably more effective when the treatment was initiated 1 hr after the inoculation of the virus. These findings suggest that the use of synthetic polynucleotides might prove to be useful for the inhibition of the malignancy produced by H. saimiri in susceptible nonhuman primates.

Herpes T virus variants. Isolation and characterization.

Herpes T virus obtained from squirrel monkeys appears to be composed of a mixed population of large and small plaques. By clonal selection it was possible to obtain a large plaque and a small plaque strain. The latter which formed a minor component in the parent population differed from its companion large plaque type in all in-vitro markers studied: plaque size, CPE development, infectivity to host cell cultures both in degree and in titer, polykaryocyte development, rate of adsorption and thermostability characteristics. LPV measured 3 mm and SPV 1 mm as seen in rabbit kidney cell cultures 6 days post inoculation. This size difference was also seen in simian, mammalian and avian cell cultures. The CPE caused by LPV differed from SPV both in appearance and in the time of development. LPV had a greater capacity to form polykaryocytes in almost all cell cultures examined, while SPV was deficient in this regard and failed to form polykaryocytes in rabbit kidney, mouse embryo kidney, cat kidney and chick fibroblast cell cultures. LPV was more thermostable than SPV — at 56° C SPV lost 100% infectivity within 10 minutes, while LPV lost 63%. When the adsorption rates of the two clones were compared, LPV adsorbed at a faster rate with 60% being adsorbed within 15 minutes, in contrast to 31% with SPV. Plaque morphology, development of CPE, in-vitro virulence to cell cultures, formation of polykaryocytes, rate of adsorption and thermostability were useful markers for the characterization of Herpes T virus variants.

Incidence of neutralizing antibodies to Herpes B virus in the Taiwan monkey (Macaca cyclopis)

A group of 46 recently captured Taiwan monkeys (Macaca cyclopis) was tested for neutralizing antibody for Herpes B virus. Results indicated that 11 (24%) were positive, 25 (54%) were negative, and 10 (22%) were suspect. This is thought to reflect the incidence in nature. Subsequent samples from the same animals, acquired 7 months postarrival, indicated 10 (22%) positive, 27 (59%) negative, and 9 (20%) suspects. A number of animals (18) converted from one category to another, assumedly from loss or acquisition of immunity during the period. A suggestion of geographic difference in incidence of titers was detected on the first sample. Twenty-eight human caretakers and veterinarians were tested with no positives, 25 negatives and 3 suspect.

Herpes T virus plaque assay studies

A plaque assay system for the study of Herpes T virus in various host cells was examined. Squirrel monkey kidney cells were found to be the most sensitive for plaque assay. The difficulty of obtaining kidney cultures without indigenous viral agents hampered the utilization of this tissue for routine purposes and favored the use of rabbit kidney cells. The optimum temperature for viral adsorption and plaque development was found to be 37°C. There was total absence of plaque development at 25° C. Addition of agar to the infected cultures without washing the monolayers prevented diffusion of virus through the agar, indicating the need for washing cell cultures when used for viral cloning procedures. Plaque yield was influenced by the time of adsorption. Maximum plaque development resulted when the adsorption time was extended to 3 hours. The influence of the inoculum size on plaque titer was paradoxical: the smaller the inoculum size (0.05 ml) the higher the plaque yield. The plating efficiency of Herpes T virus was observed by a linear relationship between virus concentration and plaque numbers. The reproducibility of replicate plaque assay and the ease with which Herpes T plaques developed in most cell cultures studied made this a reliable technique for quantitation purposes.