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An antigenically atypical strain of variola virus.

The Kuwait-5-67 strain of variola virus, although indistinguishable from variola virus in its other properties, behaved atypically in immuno-diffusion tests with antivaccinia serum. The precipitation line pattern produced was similar to that of cowpox virus but lacked one of the main precipitation bands given by all other strains of variola, alastrim and vaccinia viruses tested. Trypsin treatment of cowpox virus preparations revealed the missing precipitation line but did not do so with the Kuwait-5-67 strain. The antivaccinia serum absorbed by Kuwait-5-67 strain lost its ability to interact with this strain, but still gave precipitation lines with variola, vaccinia and cowpox viruses.

Antigens, Viral

Effect of temperature and relative humidity on variola virus in crusts.

The viability of variola virus in crusts under different conditions of temperature and relative humidity was studied for 16 weeks. At the ambient temperature of 25.8-26.4 degrees C and 85-90% relative humidity, the virus survived only 8 weeks but at lower temperatures and relative humidities the survival time was considerably prolonged.

Humans

Variola virus strains of 1960--1975: the range of intraspecies variability and relationship between properties and geographic origin.

A comparative study of 61 variola virus strains recovered from patients in various geographic regions of the world revealed significant intraspecies variability in some biological properties, established the range of this variability and demonstrated the existence of strains with atypical properties. There was a certain correlation between some laboratory markers of variola virus and the degree of its pathogenicity for man. The prevalence of more virulent and thermoresistant strains in the Hindustan subcontinent and the peculiarity and heterogeneity of the strains circulating in Africa were demonstrated. Alastrim virus was detected among the African isolates.

Africa

Effect of disinfectants on variola virus in cell culture.

Twenty kinds of disinfectants were examined for ability to inactivate variola virus. Cytopathic effect and plaque formation on monolayer cultures of an established monkey kidney cell line were used as indicators of virus inactivation. A micromethod using microplate cultures, and not requiring a CO2 incubator, was adopted. The procedures were straightforward, showing good reproducibility. Among the compounds tested, several were found to be superior because of the minimum concentrations required for complete inactivation of virus. The purified viruses were shown to be more sensitive to the compounds than were the crude samples. The virus inactivation kinetics curves were determined by plaque counting. The usefulness of this method for quantitative analysis of disinfecting effect is suggested.

Cells, Cultured

High resolution polyacrylamide gradient gel electrophoresis.

A method was developed for high resolution electrophoresis of proteins in linear gradient (3 to 30%) polyacrylamide gel rods in a neutral phosphate buffer containing 0.1% sodium dodecyl sulfate. Well-defined protein zones were observed and improved resolution was attained especially for low molecular weight proteins in preparations containing a variety of polypeptides, e.g. viruses that are often separated by continuous gel methods. Electropherograms of continuous (8%) and gradient (3 to 30%) gels were made of purified vesicular stomatitis virus, variola virus, Rickettsia rickettsii, and alpha and beta chains of hemoglobin in order to demonstrate the resolution of the gradient system.

Acrylamides

[Abolition of compulsory vaccination against smallpox. Increased danger to humans from animal smallpox? (author's transl)].

After the world-wide eradication of human smallpox, compulsory vaccination against smallpox will no longer be applicable in future in most countries. Although the human smallpox virus (variola virus) appears to have no animal reservoir, humanity is increasingly endangered in future by animal smallpox viruses pathogenic in man against which he has been so far protected by the general smallpox vaccination. This is a virus type of the Genus Orthopox virus. The risk from animal pox viruses, which are not related to the Orthopox virus, remains unchanged. Of the animal Orthopox viruses the monkey pox viruses deserve special attention; possibly rodents also play a decisive role in transmission of smallpox.

Animals

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.

Antibodies, Viral

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.

Complement Fixation Tests

[Determination of autohemolysin formation using the plaque test. Demonstration of autoimmune phenomena following vaccinations and infections].

A simple and sensitive modification of the plaque assay for the evaluation of autohaemolysin-producing cells (APC) in the peripheral blood is described: Very small chambers, (volume less than or equal to 0.03 cc) made out of glass slides (75 X 25 mm), are filled with a diluted (1:7) blood cell suspension. After incubation for 26 hours the haemolytic plaques produced by APC in the thin monolayer of blood cells can be counted under the microcope at a magnification up to 320-fold. The number of APC in guinea pigs immunized with cholera vaccine and in monkeys infected with variola-virus was evaluated by means of this method. Cholera vaccination significantly raised the APC up to 7 days after immunization. In monkeys these cells increased markedly between 4 and 7 days after infection with variola-virus. The increase was less pronounced in animals partially protected by prior vaccination with attenuated vaccinia-virus (MVA-strain) and showing a mild course of variola. Autoimmune mechanisms may exist in apparently normal organisms. They may rise after tissue destruction or cell alteration by infectious or other exogenous agents. The method described might be useful to determine the degree of reactogenicity of bacterial and viral vaccines.

Animals

[Results of examining wild monkeys for the presence of smallpox antibodies and smallpox group viruses].

The results of examinations of sera, blood and organs of different species of monkeys from some Asian and African countries for the presence of antibody to smallpox and viruses of the smallpox group. Significant titers of smallpox antibodies (antihemagglutinins virus-neutralizing and, in some cases, precipitating antibody) were found in a considerable number of monkeys shot near foci with human cases (Equatorial province of Zair Republic). In the same monkeys kidney tissues yielded 3 isolates of smallpox virus group two of which were indistinguishable in the laboratory tests from variola virus. On the basis of these data it is concluded that smallpox viruses circulate among wildlife monkeys in some areas of Equatorial Africa. Further studies along these lines are necessary.

Africa

Evaluation of smallpox vaccination policy.

During 1978 and 1979 the Global Commission for the Certification of Smallpox Eradication will proceed with verification of smallpox eradication in 31 countries. If current surveillance activities do not discover any further cases before the end of 1979, the world will be declared smallpox free. However, the recent occurrence of two laboratory associated smallpox cases in Birmingham, England, revealed that the stocks of variola virus held in at least 12 laboratories in the world pose a real danger to the achievement of smallpox eradication and efforts are being made to reduce the number of laboratories retaining the virus to not more than 4-all WHO collaborating centres-by 1980. Scientific data indicate the unlikelihood of smallpox recurring once it has been eradicated from the human population, although further confirmatory studies are continuing. If the Global Commission verifies the eradication of smallpox, continuation of smallpox vaccination will be unjustifiable in view of its severe complications, however infrequent, and the expected global savings that would result from the termination of vaccination programmes.

Animals

Monkeypox and whitepox viruses in West and Central Africa.

Prospects for the eradication of smallpox are now highly encouraging. With the cessation of man-to-man transmission, the question of possible animal reservoirs of smallpox becomes increasingly important. During the period 1970-1975, 20 cases of a smallpox-like disease were detected in smallpox-free areas of tropical rain forest in West and Central Africa. Epidemiological and virological investigations revealed that the disease was caused by an animal poxvirus termed monkeypox virus, a member of the orthopox virus group. The disease spread with difficulty even among susceptible close contacts and does not appear to be sufficiently transmissible to permit continuing infection to become established in man. During the investigations, four orthopox viruses termed whitepox viruses were isolated from rodents and monkeys. The isolates were not distinguishable from variola virus with currently available laboratory techniques, but there is no evidence so far that viruses of this group have infected man. Although there is now substantial and accumulating evidence that there is no animal reservoir for smallpox, continued surveillance and studies in West and Central Africa are warranted.

Africa, Central

An Evolutionary Framework Exploiting Virologs and Their Host Origins to Inform Poxvirus Protein Functions.

Poxviruses represent evolutionary successful infectious agents. As a family, poxviruses can infect a wide variety of species including humans, fish, and insects. While many other viruses are species-specific, an individual poxvirus species is often capable of infecting diverse hosts and cell types. For example, the prototypical poxvirus, vaccinia, is well known to infect numerous human cell types but can also infect cells from divergent hosts like frog neurons. Notably, poxvirus infections result in both detrimental human and animal diseases. The most infamous disease linked to a poxvirus is smallpox caused by variola virus. Poxviruses are large double-stranded DNA viruses, which uniquely replicate in the cytoplasm of cells. The model poxvirus genome encodes ~200 nonoverlapping protein-coding open reading frames (ORFs). Poxvirus gene products impact various biological processes like the production of virus particles, the host range of infectivity, and disease pathogenesis. In addition, poxviruses and their gene products have biomedical application with several species commonly engineered for use as vaccines and oncolytic virotherapy. Nevertheless, we still have an incomplete understanding of the functions associated with many poxvirus genes. In this chapter, we outline evolutionary insights that can complement ongoing studies of poxvirus gene functions and biology, which may serve to elucidate new molecular activities linked to this biomedically relevant class of viruses.

Animals

Single radial immunodiffusion test for detecting antibodies against surface antigens of intracellular and extracellular vaccinia virus.

Antibodies to surface antigens of intracellular naked vaccinia virus (INV) and in limited studies extracellular enveloped virus (EEV) were determined by single radial immunodiffusion tests (SRDT) with immobilized virions in agarose gels. Antibodies to INV were demonstrable in rabbit hyperimmune sera (one to four visible zones), smallpox convalescent sera and sera from re-vaccinated individuals. A difference in specificity of antibodies reacting with INV and EEV was detectable by SRDT. The SRDT provides a simple, reproducible and specific test for detection of antibodies against vaccinia or variola virus, but the technique requires large quantities of virions.

Animals

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.

Antigens, Viral