Viral infections of nonhuman primates.

Approximately 53,000 serologic tests and viral isolation studies were performed on 1,700 nonhuman primate specimens for evidence of past and/or current viral infection. Information, other than the requested test, generally was not provided with the specimen. This lack of information does not permit any attempt at interpretation of results. Requested testing included a large number of diverse viral agents in approximately 40 primate species. The resulting data are in keeping with those of previous studies and offer an insight into the needs of colony management, as well as some general information on the overall frequency of infection with the indicated viruses. Inasmuch as the results represent testing of single specimens, they are not to be construed as "diagnostic," and simply indicate past infection as represented by the presence of antibody in the test animal. Viral isolation results are listed, and the number of positive results versus the number of animals tested emphasizes the limitations of the procedure. Investigations such as these continue to assist in the maintenance of healthy nonhuman primate colonies. This information also supports continued use of nonhuman primates for research in human viral infections and may be helpful in terms of animal selection for use in xenotransplants.

Fatal disseminated cercopithecine herpesvirus 1 (herpes B infection in cynomolgus monkeys (Macaca fascicularis).

Two adult female cynomolgus monkeys (Macaca fascicularis) that had been housed together for 4 months died within 2 weeks of each other after brief illnesses. Monkey No. 1 presented with collapse, watery stool, and hypothermia and died overnight. Monkey No. 2 presented with dyspnea, nasal discharge, leukopenia, and hypoproteinemia and was euthanized after 2 days. Both animals had peritoneal effusions, massive necrosis of pharyngeal, esophageal, and gastric mucosa, and multifocal hepatic and pancreatic necrosis. Monkey No. 2 also had lingual ulcers and locally extensive necrosis of spleen, adrenal glands, and lymph nodes. Large numbers of eosinophilic intranuclear inclusion bodies were present in epithelial and syncytial cells adjoining the necrotic foci in Monkey No. 2 but were absent in Monkey No. 1. Monkey No. 1 seroconverted to cercopithecine herpesvirus 1 (CHV-1, commonly known as herpes B) in the month before death. CHV-1 was isolated from a sample of stomach from Monkey No. 2, and electron microscopy of liver from this animal demonstrated herpesvirus particles within hepatocytes. Both animals were seropositive for simian type D retrovirus, and the virus was cultured from the liver of Monkey No. 2. A diagnosis of disseminated CHV-1 infection was made, possibly occurring secondary to immunosuppression due to infection with simian type D retrovirus. Although a high percentage of cynomolgus monkeys are apparently infected with CHV-1, disseminated disease is rare. Because infection with CHV-1 in humans is associated with a high fatality rate, familiarity with the lesions of disseminated infection with this virus is important.

Detection of Ebola-Reston (Filoviridae) virus antibody by dot-immunobinding assay.

Thirty human and nonhuman primate sera tested at the Centers for Disease Control by enzyme-linked immunosorbent assay (ELISA), immunofluorescent antibody assay (IFA), and Western blotting were retested at the Virus Reference Laboratory, Inc. by the dot-immunobinding assay (DIA). The Ebola-Reston strain of virus received from the Centers for Disease Control was prepared into a suitable DIA antigen as described for other antigens. All six Western blotting-positive sera were also positive by DIA, as were the five ELISA-positive sera. Testing by IFA, the original test of choice, indicated an additional four seropositives, all negative by the other test systems. Of 288 randomly selected macaque sera, 19 were also found to be Ebola-Reston virus-positive by DIA.

Detection of antibody to immunodeficiency viruses by dot immunobinding assay.

The dot immunobinding assay (DIA), a modified enzyme immunoassay (EIA), has been demonstrated to be a highly sensitive and specific assay for the detection of antibody to a number of viruses. Different laboratory procedures are available for detecting antibody to the immunodeficiency viruses; however, these procedures require a certain amount of sophisticated equipment and trained personnel. Further, commercial kits for detecting antibody to human immunodeficiency virus, as now available, are not easy to use in the nonlaboratory setting. The DIA, as described herein, may be formatted to test up to 30 serum samples and is designed to be used in the absence of laboratory equipment. To determine the effectiveness of the DIA as a test kit for the detection of HIV and human T-cell leukemia virus type I (HTLV-I) antibodies, the kit was compared with commercial EIA and Western blot (WB; immunoblot) kits. Testing approximately 1,000 human serum samples for HIV antibody by DIA and EIA revealed a total agreement of 98.1%, a specificity of 99.0%, and a sensitivity of 95.9%. For 804 serum samples tested (200 were tested independently in two laboratories), eight results were discrepant: four DIA negatives which were EIA borderline positive and four DIA positives which were EIA negative. Testing the eight discrepant sera by immunofluorescence assay and WB resulted in their being either negative or indeterminate. The four DIA positives were indeterminate by WB. Close agreement was obtained when the remaining sera were compared by DIA, EIA, and WB. Of interest was finding that the DIA results compared favorably with those obtained by WB. Twenty-six suspect HTLV-I-positive serum samples tested by DIA also gave results comparable to those obtained by EIA and WB.

Detection and titration of measles virus antibody by hemagglutination inhibition and by dot immunobinding.

Measles continues to be a major disease of both human and nonhuman primates. The dot immunobinding assay, a modified enzyme immunoassay, permits the detection of measles virus antibody in the nonlaboratory setting with either serum or whole blood collected on filter paper.

Viral battery testing in nonhuman primate colony management.

Good colony management is associated with monitoring of animals for infectious agents. Of major current concern are B virus and simian AIDS (SAIDS) viruses. However, other viral agents frequently cause serious disease outbreaks which can be avoided if their presence is detected sufficiently early. The recent development of a rapid, sensitive and specific diagnostic test system, i.e., the dot immunobinding assay (DIA) permits the monitoring of a colony for many of the viruses that pose problems. By employing battery type testing using a panel of appropriate viral antigens, investigators are able to detect the increased presence of viral agents of concern and take necessary measures to prevent extension of the problem.

Primate viral diseases in perspective.

The recent occurrence of fatal Herpesvirus simiae (B virus) infection in human subjects has again focused the attention of primatologists on this virus. B virus, however, is only one of a number of viral diseases that plays a role in primate colony management. This report is to emphasize to the primatologist a number of viruses other than H. simiae, with high morbidity and mortality rates, of importance for health management of nonhuman primate animal colonies. This concept is supported by the recent occurrence in colonies of nonhuman primates of simian hemorrhagic fever virus, SA8, herpesvirus, respiratory syncytial virus, encephalomyocarditis virus, Ebola virus, and simian immunodeficiency viruses.

The collection of blood on filter paper and its use in the dot-immunobinding assay (DIA) for detection of antibody.

The ability to collect whole blood directly onto filter paper pre-cut to the size used in the dot-immunobinding assay (DIA) is a practical adjunct to this procedure. Its applicability for field studies is suggested.

Dot immunobinding assay compared with enzyme-linked immunosorbent assay for rapid and specific detection of retrovirus antibody induced by human or simian acquired immunodeficiency syndrome.

A rapid, specific, and sensitive modification of the dot immunobinding assay was compared with the standard enzyme immunoassay as a screening procedure for the detection of antibody in human or simian acquired immunodeficiency syndrome. Comparative testing with the available enzyme immunoassay procedures, either in commercial kit form or as provided by diagnostic laboratories, indicated excellent correlation. Ease of operation and cost are key features of the dot immunobinding assay procedure.

Serodiagnosis of rabies by dot immunobinding assay.

A dot immunobinding assay that uses inactivated antigen for the detection of rabies viral antibodies was compared with the rapid fluorescent focus inhibition test. Results of testing pre- and postvaccination sera from humans (n = 33) and canines (n = 22) were identical for both tests. Endpoint titers of positive sera also were approximately the same by both methods. When a mouse monoclonal antibody was used, the dot immunobinding assay antigen was shown to possess detectable rabies virus glycoprotein and core antigens.

A dot-immunobinding assay on nitrocellulose with psoralen inactivated Herpesvirus simiae (B virus).

An enzyme-immunoassay performed with Herpesvirus simiae (B virus) and H. simplex antigens inactivated with a psoralen derivative and long-wavelength ultraviolet light irradiation is described. Although B virus is a known human pathogen requiring extreme care in its handling, the use of inactivated antigens in the test allows its performance without biosafety containment. The test utilizes nitrocellulose sheets dotted with antigen for the assay of antibody against B virus in nonhuman primate sera. Antigen-antibody complexes are detected visually as red dots by the use of enzyme-conjugated antiglobulin second antibody and a substrate that produces an insoluble product. The test is more rapid, sensitive and specific than the serum neutralization test it is intended to replace. Of 150 macaque monkey sera tested, 83 were negative by the enzyme and neutralization tests, 56 were positive by both tests and 11 were positive by enzyme-assay but negative by neutralization. Positive sera reacted with both simian and human viral antigens in the enzyme assay but with greater intensity against B virus. Absorption with H. simplex removes reactivity with this virus without reducing the B virus response.

Rapid dot-immunobinding assay on nitrocellulose for viral antibodies.

A procedure is described for the routine laboratory diagnosis of viral serum antibodies. Antigens are dotted on nitrocellulose strips or sheets, and sera are applied on absorbent paper strips. Antigen-antibody complexes are detected with enzyme-conjugated antiglobulin and development of a colored, insoluble substrate product. The test allows processing of multiple sera in one 3- to 5-h operation and is equal to or more sensitive than serum neutralization, hemagglutination inhibition, and fluorescent antibody assays. Highly infectious viruses inactivated with a psoralen derivative and long-wavelength UV light irradiation can be used as antigens, allowing the study of human pathogens. Although the test detects cross-reacting, group-specific herpesvirus antigens, the intensity of the antibody reaction is greatest with type-specific antigens. Preliminary data suggest that the technique will be useful for the rapid typing of viruses from clinical specimens.

Dot immunobinding assay of viral antigen and antibodies.

A dot-immunobinding assay for the rapid and specific detection of viral antibody as well as the identification of a virus is described. Based on the use of nitrocellulose membranes for the adsorption of viral protein, this test may be used to detect the presence of virus antibody or identify an isolate within 4 to 6 hours. Use of goat anti-human IgG-alkaline phosphatase followed by a naphthol-AS-MX-phosphate: Fast Red substrate permits visual detection of a positive reaction. The use of psoralen inactivated herpes B virus permits the incorporation of this virus into the battery of test antigens.

Experimental Herpes neonatalis in SA 8-infected baboons (Papio cynocephalus).

13 newborn baboons were experimentally infected with a baboon-derived herpesvirus SA 8-strain. Intratracheal infection produced herpetic tracheobronchopneumonia, intravenous inoculation resulted in adrenal necrosis similar to that in human neonatal herpes infection.

Malignant lymphoma and Hodgkin's disease in baboons (Papio sp.).

Four cases of spontaneous malignant lymphoma and one of Hodgkin's lymphoma in baboons at the Southwest Foundation for Biomedical Research were studied and described. These cases were in animals of both sexes that varied in age from 6 to 25 years, and were in residence at the Foundation from 2 to 24 years, during which time there was no known exposure to carcinogenetic agents. Attempts to isolate an etiological viral agent or demonstrate viral particles in lymphoid tissue were unsuccessful.

Sensitivity of equine herpesviruses 1 and 3 in vitro to a new nucleoside analogue, 9-[[2-hydroxy-1-(hydroxymethyl) ethoxy] methyl] guanine.

The following members of the Herpetoviridae family were tested to determine their sensitivities to the new antiviral drug, BIOLF-62: equine herpesvirus types 1 and 3, human herpesvirus types 1 and 2, swine herpesvirus, bovine herpesvirus type 4, feline herpesvirus, canine herpesvirus, and herpes simiae virus. Equine herpesviruses 1 and 3, human herpesviruses 1 and 2, and herpes simiae virus were all sensitive to BIOLF-62 at concentrations of less than 0.55 micrograms/ml. Equine herpesvirus types 1 and 3 were particularly sensitive, viral median effective dose (ED50) concentrations of the drug being only 0.033 and 0.16 micrograms/ml, respectively. Such high antiviral potency and low cell toxicity indicate that BIOLF-62 might be useful in the treatment of infected animals.

Immunological tolerance induced by in utero injection.

Intrauterine injection of human whole blood into rabbit and rhesus monkey fetuses was found to result in long lasting unresponsiveness to human serum albumin. Intrauterine injection of viable allogeneic bone marrow cells into rabbit fetuses was without any apparent harmful effect and also resulted in permanent unresponsiveness demonstrated by donor red cell survival studies. The implication of these findings in respect of using this approach towards the correction of certain inherited diseases in man is discussed.

Infection of baboons ("Papio cynocephalus") with rotavirus (SA11).

A recent survey of nonhuman primate sera indicated that antibody to a rotavirus (SA11) was prevalent among a wide spectrum of animals. Both New and Old World species were found with antibody, many with surprisingly high titers (1:320). Whether or not infection per se was due to SA11 or an antigenically closely related agent could not be determined by that study; however, it is apparent from this study as well as from a survey of the literature that natural as well as experimental infection of nonhuman primates occurs. Recognizing that there is a need for an animal model for the study of viruses associated with human diarrhea, a preliminary investigation attempting to ascertain the susceptibility of the baboon (Papio cynocephalus) to a rotavirus was undertaken. Inoculation of four newborn baboons with SA11 resulted in diarrhea within 24 to 48 hours in all animals. One animal died 10 days postinoculation following severe diarrhea and dehydration. Virus was isolated as well as seen by electron microscopy in the stools of all four animals.