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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.

A study of 2,510 contacts of 214 patients with human monkeypox was conducted in Zaire from 1980 to 1984. Among the contacts of 130 primary cases of human monkeypox, a further 22 co-primary and 62 secondary cases were detected, and an additional fourteen people who had no evidence of clinical disease had positive serological results. A majority of the clinical and subclinical cases of monkeypox occurred in children less than 10 years of age. Immunity in vaccinated persons now appears to be waning because 16 overt cases occurred in contacts who had been vaccinated. The overall attack rate for contacts without a vaccination scar (7.2%) differed significantly from the attack rate for those who had been vaccinated in the past (0.9%). The attack rate for household contacts was significantly higher than that for other contacts, among both unvaccinated (four times higher) and vaccinated (seven times higher) household contacts. Many unvaccinated contacts living in the same household as the index case under conditions of maximum exposure, however, escaped not only the disease but also infection.

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.

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.

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.

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.

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.

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.

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.

Two of the more heat-stable measles vaccines were field tested in Cameroon. Both maintained the minimum required infectivity titre and the ability to induce seroconversion after storage unreconstituted at 37 degrees C for 14 days. One of the vaccines, studied after reconstitution, maintained its ability to induce seroconversion after reconstitution and storage at 25 degrees C for 48 hours and at 37 degrees C for at least four hours. The increased heat stability of the studied vaccines will not eliminate the need for a well-monitored system of vaccine conservation and distribution but will ease the rigid cold-storage requirements of conventional measles vaccines.

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.

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.

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.

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.

Between September 1970 and May 1971 six cases of human infection with monkeypox virus were identified in three West African countries-Liberia, Sierra Leone, and Nigeria. Four of the cases were confirmed by viral isolation, and two were diagnosed on epidemiological and serological evidence. Poxvirus strains isolated from the four cases were indistinguishable from reference monkeypox strains (Copenhagen and Utrecht), and all were easily differentiated from variola and vaccinia viruses. The isolated strains produced small necrotic haemorrhagic pocks on CAM, grew well at 39.0 degrees C, formed large plaques in Vero cell cultures, showed markedly more virulence for chick embryos and mice than do variola strains, and produced large necrotic haemorrhagic local lesions with generalized illness and florid secondary exanthem when inoculated into rabbit skin.The finding of smallpox-like illness in humans resulting from infection with a poxvirus of lower animal origin serves to emphasize the importance of thorough epidemiological and laboratory evaluation of all suspect smallpox cases occurring in areas where smallpox has been or is about to be eradicated.

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.

SummaryStrain E-42, presently identified as a strain of C. tetanomorphum, is exacting in its nutritional requirements. Growth will occur in a basal salt-glucose-thioglycollate medium only following the addition of pantothenic and nicotinic acids, biotin, L-glutamic acid, and L-tryptophan. p-Aminobenzoic acid, choline, thiamine, leucine, tyrosine, and phenylalanine may exert slight stimulatory action on growth but are not essential for growth of this organism.