Human poxvirus disease after smallpox eradication.

A 5-year-old boy living in a small camp in the rural Ivory Coast had a disease resembling smallpox. This occurred 4 years after smallpox had been eradicated from the Ivory Coast and 1.5 years after the last case of smallpox was detected in West and Central Africa. Clinical, serological, and epidemiological evidence indicated this disease was probably monkeypox, a poxvirus of the variola/vaccina subgroup. A serologic survey of poxvirus antibodies in the wild animal population detected neutralizing antibodies in rodents, larger mammals, primates, and birds. The laboratory and ecological characteristics of poxviruses require further elucidation, especially those which have been found in animals near human monkeypox cases.

Evaluation of virological laboratory methods for smallpox diagnosis.

Between July 1966 and May 1972 the Vesicular Disease Laboratory, Center for Disease Control, Atlanta, Ga., USA, tested specimens from 849 suspected smallpox cases by at least 2 methods, electron microscopy and chick embryo chorioallantoic membrane (CAM) cultures. A smaller number of specimens was tested by each of 4 methods: electron microscopy, CAM culture, agar gel precipitation, and tissue culture. For specimens handled in the field the CAM culture method was less sensitive than electron microscopy because the adverse conditions often inactivated the virus. CAM cultures were valuable for identifying members of the poxvirus subgroups, however, particularly when supplemented by tissue culture. The agar gel precipitation test was the least sensitive but was of value in confirming the results of electron microscopy. The latter was highly effective for the diagnosis of varicella, but dependably identified only about half of the vaccinia infections; for vaccinia, the CAM technique was essential. The occurrence of human monkeypox cases in West Africa emphasized that the usual smallpox diagnostic methods were inadequate. More sophisticated tests, such as the rabbit dermal sensitivity test, are necessary for accurate diagnosis of these cases as monkeypox.

Naturally acquired measles immunity in Nepal and Sri Lanka.

Serological surveys of naturally acquired measles antibodies in children 6--72 months of age were done in Nepal and Sri Lanka. The prevalences of naturally acquired measles immunity are compared by age groups with serological studies done in other countries. Suggestions are made regarding age ranges for measles vaccination programs.

Can variola-like viruses be derived from monkeypox virus? An investigation based on DNA mapping.

The results are presented of a special study to determine whether variola-like "whitepox" viruses could arise as white pock variants of monkeypox virus after one or a few mutations. DNA mapping by cross-hybridization of restriction endonuclease DNA fragments was carried out on 18 orthopoxviruses relevant to this study, including variola and monkeypox viruses and white (non-haemorrhagic) pock producers recovered from chorioallantoic membranes infected with red (haemorrhagic) pock-producing monkeypox viruses. The distinctiveness of the DNA maps of true variola and monkeypox viruses indicated that spontaneous production of "whitepox" from monkeypox virus was genetically impossible. These and other observations led to the conclusion that the "whitepox" viruses recovered from monkeypox virus stocks had an exogenous origin.

Serological relatedness of monkeypox, variola, and vaccinia viruses.

Closely related human and monkey orthopoxviruses were differentiated by serologic techniques. Antiviral sera were tested by immunodiffusion for reactivity against six different viral antigens prepared from either infected cell cultures or infected chorioallantoic membranes (CAMs) of embryonated eggs. Portions of each antiserum were separately absorbed with heterologous antigens from infected CAMs to remove common reactivity. The absorbed sera formed immunodiffusion precipitates with both types of antigen preparation and revealed specific-character differences that made it possible to classify the viruses as variola, vaccinia, or monkeypox. Cross-complement fixation tests were also used to examine the immunologic reactivities of antisera to detergent-treated, purified preparations of three orthopoxviruses. Only common reactivities were detected by this method, however, and differentiating reactivities were not observed.

The virion and soluble antigen proteins of variola, monkeypox, and vaccinia viruses.

The structural proteins in purified preparations of variola, monkeypox, and vaccinia viruses were separated and compared by using a high resolution SDS-polyacrylamide gel electrophoresis system. About 30 proteins were resolved for each virus by autoradiography of longitudinally-sliced gel rods. Although the autoradioelectropherograms of each virus were similar, it was possible to differentiate them by their unique protein pattern in the 30,000 to 40,000 molecular weight region of the gels. A single virion glycoprotein (mol. wt. = 38 X 10(3)) and a virion phosphoprotein (mol. wt. = 12 X 10(3)) were associated with each of the virus preparations. Cross-absorbed monospecific immune sera against variola, monkeypox, and vaccinia virus-infected cells were used in immunodiffusion tests to precipitate radiolabeled, homologous, soluble antigen proteins. The predominant antigen protein associated with each immunospecific precipitate had a molecular weight of approximately 73,000.

Poxvirus in West African nonhuman primates: serological survey results.

Ten species of nonhuman primates in West African habitat were analysed for variolavaccinia subgroup haemagglutination-inhibition (HI) and neutralization antibodies. The animals were taken in 27 different sampling zones in parts of the Ivory Coast, Mali, and Upper Volta. Of the 195 tested, 15 (8%) had elevated HI antibodies after nonspecific reactions were reduced with potassium periodate pretreatment. Positive neutralization antibodies were found in 21% (44 of 206). Antibodies were detected in serum from monkeys living near two areas where monkeypox cases in humans had occurred. Four samples were tested for monkeypox specific antibodies using an indirect immunofluorescent test; 3 were positive. Despite the prevalence of poxvirus antibodies in monkeys (and other animals) in West Africa, smallpox eradication has been maintained in the area since 1970; thus, animal reservoirs of poxvirus appear to pose no threat to the worldwide smallpox eradication programme.

A study of the specificities of sequential antisera to variola and monkeypox viruses by radioimmunoassay.

The specificities of antisera during development of the humoral antibody response to poxvirus antigens were examined in monkeys injected with chimp-9 whitepox virus or monkeypox virus. Sera were obtained from 3 African green (vervet) monkeys inoculated with chimp-9 whitepox virus, 1 rhesus monkey inoculated with monkeypox virus, and 2 rhesus monkeys inoculated with soluble monkeypox viral antigen. The sequentially obtained sera from each animal were adsorbed with uninfected chicken chorioallantoic membranes (CAM) or vaccinia virus-infected CAM. The adsorbed sera were tested by radioimmunoassay to determine the specificity of the residual antibodies to vaccinia, variola, and monkeypox viruses. The adsorbed sera at different stages of the immune response showed increasing specificity with time after inoculation. Generally, antibodies in sera collected earlier than 21-27 days after immunization could not be identified after adsorption, but late sera could be identified unequivocally.

Characterization of antibodies to orthopoxviruses in human sera by radioimmunoassay.

Serological surveillance of suspected orthopoxvirus infections in man is important for confirming the success of the worldwide smallpox eradication programme. An adsorption radioimmunoassay (RIA) was used to differentiate sera from patients who were naturally infected with human monkeypox or variola virus, and individuals who were immunized with vaccinia virus. The antisera were adsorbed with uninfected chicken chorioallantoic membrane (CAM) and vaccinia-infected CAM before reacting in RIA with vaccinia, monkeypox, and variola antigens. Each serum group showed characteristic patterns of residual antibody activity which made it possible to identify antibody specificities.When 45 human sera were tested by this method, 71% were identified as having vaccinia, variola, or monkeypox adsorption characteristics, while the remaining 29% could not be identified. Of the identified sera, 9 were characteristic of vaccinia, 8 of variola, and 15 of monkeypox. Six of the 15 monkeypox sera were virologically confirmed monkeypox infections, 6 were suspected monkeypox infections but were not virologically confirmed, and 3 were of unknown aetiology.The adsorption RIA provides a method of identifying serologically the poxvirus responsible for infection long after the acute phase of illness.

Four generations of probable person-to-person transmission of human monkeypox.

This paper examines an outbreak of five cases of human monkeypox which occurred in children belonging to two families living in the West Kasai region of Zaire during May-July 1983. Epidemiologic investigations suggest that the first case was infected from an animal source, possibly a monkey, and that each of the other four cases was infected from a previous human case. Three of these cases of presumed person-to-person transmission occurred in close household contacts. The other case infection occurred either by casual contact within the hospital compound, or possibly because of infection due to use of the same syringe for injections. Human monkeypox is the most important orthopoxvirus infection in the post-smallpox eradication period. The disease is a zoonosis and person-to-person transmission is rather difficult. Thus, this episode is a rare event and special analysis of the circumstances is discussed. However, it supports the necessity to carry out surveillance and research on this disease as recently reported by Arita et al.

Human tanapox in Zaire: clinical and epidemiological observations on cases confirmed by laboratory studies.

Human tanapox, a mild disease characterized by a short febrile illness associated with one or more skin lesions, is important because of its possible confusion with smallpox. The article describes clinical and epidemiological features of 264 laboratory-confirmed tanapox cases observed in a geographically limited area in northern Zaire over the period 1979-83.

Microtiter determination of measles hemagglutination inhibition antibody with filter papers.

Epidemiological studies of measles and measles immunization frequently require determination of measles antibody status. In developing countries, where venipuncture is frequently unacceptable and where refrigerated storage of serum specimens is often unavailable, microtiter techniques not requiring refrigeration are required. We developed a filter paper technique that measures measles hemagglutination inhibition antibody and meets these criteria. Comparison of separately collected venous blood and peripheral blood collected on filter paper demonstrated 97% agreement in terms of presence or absence of antibody. In 30 of 32 measles specimens, 94% of titers were the same or varied by less than 2 twofold dilutions.

Further characterization of Raccoonpox virus.

A poxvirus isolated from the respiratory tract of raccoons in a forest and swamp area near Aberdeen, Maryland, was characterized by biological, serological, and biophysical methods. The virus was shown to be related to the vaccinia-variola subgroup by serological and biophysical methods, but measurably different from the other viruses of this group by biological methods. It causes flaccid paralysis in 1-day-old suckling mice and does not grow well on CAM after two or three passages.

Isolation of poxvirus from an African Rodent.

A poxvirus was isolated from a wild gerbil (Tatera kempii) caught in northern Dahomey, Africa at the time of an epidemic of human smallpox. Electron microscopic appearance and serologic reactions placed it in the vaccinia subgroup of poxviruses. The isolate differed from ectromelia, rabbitpox, vaccinia, monkeypox, and cowpox viruses in pock morphology on chorioallantoic membrane, ceiling temperature, relative innocuity for mice, and cytopathic effect in tissue culture. Like variola minor virus, it had a ceiling temperature of 38 C, produced small hypertrophic foci in tissue culture, and failed to grow in rabbit skin. Inoculated into a rhesus monkey, it caused fever but no skin eruption and produced seroconversion and protection from subsequent challenge with monkeypox virus. The growing list of animal viruses that differ only slightly from smallpox virus suggests the hypothesis that long-term survival of variola virus may be based on inapparent infection in animals as well as virulent spread among humans.

Detection by radioimmunoassay of antibodies in human smallpox patients and vaccinees.

A radioimmunoassay procedure was developed for determining smallpox and vaccinia antibodies in human sera. The test detected and measured both primary and secondary immune responses in persons infected with variola virus or vaccinia virus. The antibody titers obtained by complement fixation, hemagglutination inhibition, plaque reduction neutralization, and radioimmunoassay methods were compared. In sequential serum specimens, the radioimmunoassay test indicated fourfold or greater increases in all of the smallpox patients and in six of eight vaccinated persons. Both the complement fixation and the hemagglutination inhibition tests were less effective. In persons who had been vaccinated, radioimmunoassay and plaque reduction neutralization tests appeared to measure the same immune response. However, in smallpox patients the immune response was readily detected by radioimmunoassay, whereas an immune response was not detected by the plaque reduction neutralization test when vaccinia virus was the antigen in the test system. Radioimmunoassay is an operationally simple procedure which provides objective and quantitative end-point titers in serological determinations.

Preparation and evaluation of a noninfectious monkey pox virus vaccine.

Monkey pox virus was mechanically disrupted by low temperature and high pressure into soluble and insoluble fractions. Soluble fractions elicited virus-neutralizing antibodies (1:20 to 1:160) in rabbits, whereas the insoluble (in saline) fractions did not (less than 1:5). No infectious virus was detected after the disruption procedure. Rhesus monkeys immunized with the soluble fraction elicited virus-neutralizing (1:1,200), complement-fixing (1:16), and hemagglutinating-inhibiting (1:80 to 1:160) antibody titers and were completely protected against monkey pox virus-induced disease. This model of monkey pox virus subunit vaccine preparation may prove to be useful in developing an efficacious noninfectious vaccinia vaccine for use in high-risk individuals.

Differentiation of variola, monkeypox, and vaccinia antisera by radioimmunoassay.

Poxvirus antisera adsorbed with "homologous" and "heterologous" poxvirus-infected chorioallantoic membranes (CAM) were differentiated by solid-phase radioimmunoassay (RIA). Mixtures of the antiserum dilutions and infected CAM were added directly (without centrifugation) to poxvirus-infected CAM antigens affixed to wells of microtitration plates. The affixed antigens combined with unadsorbed antibodies, and the cross-reactive antigen-antibody complexes were removed by washing. The results showed that adsorption of an antiserum with variola-, vaccinia-, or uninfected-CAM antigen and subsequent reaction of each in RIA with monkeypox- and uninfected-CAM antigens allowed the identification of antivariola, antivaccinia, or antimonkeypox sera.

Immunofluorescence staining for detection of variola virus.

In September 1975 Bangladesh was the only country in the world with endemic variola major, and the eradication of the disease was imminent. A rapid and accurate laboratory diagnostic method was required to supplement immunodiffusion in agar gel and culture on chorioallantoic membrane of embryonated egg available at the Institute of Public Health in Dacca, Bangladesh. To determine its effectiveness, a new, improved immunofluorescence (IF) staining technique was introduced. Laboratory specimens (scabs or vesicular or pustular impressions) were collected from patients who had, or were suspected of having, smallpox. Seventy-eight of 144 specimens collected were found to be IF positive for smallpox. As the number of laboratory-positive cases far exceeded the number of clinically diagnosed smallpox cases, IF-positive cases were reinvestigated and subsamples of the IF-positive specimens were tested at a World Health Organization poxvirus reference laboratory at the Centers for Disease Control in Atlanta, Ga. The results indicated 100% sensitivity for the IF technique (no false-negative results) in diagnosing variola major but also showed a high rate of false-positive results. Consequently, IF could not be recommended as a routine screening test for smallpox.

Occupationally-acquired smallpox in an IgM-deficient health worker.

A 21-year-old Indian who worked as a vaccinator in the smallpox programme developed an atypical case of variola major despite several previous, successful vaccinations. The clinical course of the disease was unusual, as there were two distinctly separate "crops" of smallpox. The disease began less than one month after a successful vaccination, which therefore gave the briefest period of protection recorded for this reliable immunization. The patient was found to have a virtually complete IgM deficiency with normal levels of circulating antibody against orthopox virus.