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The results of the 3-year controlled trials of a new method of nonspecific urgent prophylaxis of influenza and acute respiratory diseases (ADR) by immunization of healthy adults with standard live enterovirus oral vaccines, introduced in 2-3 administrations at intervals of 7-10 days, at the initial stages of autumn and winter epidemics are presented. Observations, carried out in three republics, covered more than 150,000 persons immunized with enterovirus interferonogenic vaccines. A considerable decrease in morbidity rate among the vaccinees was achieved (on the average, by 3.2 times) in comparison to that among nonimmunized subjects. The method of nonspecific prophylaxis with live enterovirus interferonogenic vaccines is recommended during outbreaks of diseases induced simultaneously by several causative agents of influenza and ARD, as well as by pathogenic enterovirus strains.

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Until recently, it has been generally assumed that all human viruses are causative agents of diseases and should be regarded as harmful pathogenic agents that require control measures. In the early 1950s we began to doubt this view. In the course of experiments on virus isolations from feces of normal children, as well as in studies of isolates from animals and from tissue cultures, data accrued which suggested that some conditionally pathogenic and some completely nonpathogenic strains of enteroviruses may provide some benefit to their host by inhibition of pathogenic viruses and by activating nonspecific protective functions of the organism. The novel concept of beneficial viruses was proposed which suggested that the process of co-evolution of the host organism and its associated viral flora led to a specific interaction between them that was beneficial for both. This concept provides a potential approach to the nonspecific prevention of viral diseases by means of the interference between beneficial enteroviruses and pathogenic viruses belonging to different classes.

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In 1975 in Bulgaria a severe epidemic of central nervous system (CNS) disease occurred. Clinically, histopathologically, and epidemiologically the cases resembled poliomyelitis, aseptic meningitis, meningoencephalitis, and, in some cases, encephalomyocarditis. About 21% of the 700 reported cases developed paralysis, 44 with fatal outcome [ref. 1]. In 65 cases, 92 strains of enterovirus of the same serologic type were isolated: 38 strains from the CNS, 10 from mesenteric lymph nodes and tonsils, and 44 from feces [ref. 1,2]. A typical representative strain, No. 258, isolated from the spinal cord of a 3-month-old baby who died on the 5th day of disease with signs of focal polioencephalitis, was selected for intensive study. Cross-neutralization tests established the antigenic identity of the Bulgarian 258 strain (Stanchev) with American strains of enterovirus type 71 (BrCr and JaFr strains) and Swedish strains of the same type (Nos. 52343, 52500, and 6041). From its biological [ref. 1,2], physicochemical [ref. 3], and antigenic properties, the etiological agent of this large epidemic can be classified as a member of enterovirus type 71, one of the most highly pathogenic of the recently recognized enteroviruses.

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Virological and serological studies of an epidemic disease in Bulgaria, 1975, were carried out. Epidemiologically, clinically and pathomorphologically, the disease simulated almost all known forms of poliomyelitis, acute stem encephalitis, encephalomyocarditis and aseptic meningitis. The studies completely rules out the participation of polioviruses and provided comprehensive evidence for the etiological role of a peculiar enterovirus subsequently identified as enterovirus (EV) type 71 known in the literature since 1974. Altogether, in 1975 and 1976 from 65 cases of poliomyelitis-like disease (PLD) 92 strains of EV71 were isolated, including 37 strains from the brain and medulla, 1 from the cerebrospinal fluid, 10 from mesenterial lymph nodes and tonsils and 44 from faeces. In addition, in 282 convalescent cases of the disease, diagnostic seroconversion or high titers of antibody to this virus were demonstrated. The most successful virus isolation was achieved by inoculation of green monkey kidney cell cultures and newborn white mice. Bulgarian strains of enterovirus 71 regularly caused paralysis in monkeys and morphological poliomyelitis-like lesions in their CNS, and paralysis and myositis with Zenker necrosis in newborn white mice, cotton rats, Syrian hamsters, and 3-week-old cotton rats. The diseased rodents had much more virus in their mucles than in brains.

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In the present report some general physicochemical properties and RNA characteristics of strain 258 of enterovirus (EV) 71, isolated in 1975 from cases of poliomyelitis-like disease in Bulgaria (1), are described. It is concluded, that the features of the virus are consistent with the previous identification, based on biological studies, of this virus strain as a member of enterovirus subgroup of picornaviridae family.

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In May-September, 1975, an outbreak of epidemic disease clinically and pathomorphologically simulating nearly all known forms of poliomyelitis occurred predominantly among young infants in Bulgaria. Most cases presented benign aseptic meningitis, sometimes with a short period of general cerebra- symptoms. Paralytic forms, such as bulbar polioencephalitis, anterior poliomyelitis, isolated pareses of the facial nerve, occasional cases of encephalomyocarditis, etc., were observed in about 21% of all cases. Over one-fourth of the paralytic cases with bulbar symptoms ended fatally. In March, 1976 another sporadic fatal case of this disease was examined. No new cases occurred in 1977. Histopathological examinations in all fatal cases regularly revealed lesions in the grey matter of the medulla and spinal cord typical of acute anterior poliomyelitis and bulbar polioencephalitis, with some peculiar features of localization and depth of the involvement of the brain stem. The similarity to poliomyelitis and precariously rapid increase in the incidence led to the decision to urgently vaccinate the entire human population with Sabin's live poliovirus vaccine simultaneously in the whole country in order to produce interference with the circulating agent. This aim appeared to have been achieved partially because soon the number of new cases of the disease began to decrease; however, no sharp and complete break in the curve of the incidence occurred. By the time of mass vaccination, the results of virological examinations started before were not yet available. Later, comprehensive complete evidence was obtained that over 25 fatal and many other typical cases of the disease were associated with an enterovirus proved to be antigenically related to enterovirus 71. The diseases in Bulgaria, 1975, differ considerably in the frequency of paralytic forms and in severity from the epidemics caused by enterovirus 71 in Sweden, 1973, Australia, 1972--1973, USA, 1969--1972, and Japan, 1972--1973.

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It is shown that in using the method of multiple organ cultivation, the tissues of the CNS in the human and animal embryo preserve their organotypical characteristics. The use of a cartilage plate increases the viability of the tissues and the duration of its maintenance in cultures. During the process of explantation the cultivated tissue undergoes some morphological modifications. The data obtained are discussed in the light of a possible use of the CNS cultures for experimental purposes.

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Virological examinations of 72 specimens obtained during an outbreak of a poliomyelitis-like disease in Bulgaria, 1975, yielded 23 strains of enterovirus type 71 (E71) and 5 strains of enteroviruses of other types. The viruses were isolated in green monkey kidney cell cultures, in newborn cotton rats, newborn white mice and in monkeys. Most of E71 strains (16 out of 23) were isolated from the autopsy materials (brains and spinal cords) from 9 children dying of poliomyelitis, meningoencephalitis, and encephalitis. The remaining E71 strains and all the strains of other enterovirus serotypes, with the exception of ECHO7, were isolated from stool specimens of children with aseptic meningitis and their contacts. The antigenic similarity of the group of Bulgarian strains and their relationship with the prototype BrCr strain of E71 was established. The conclusion is that E71 was the causative agent of an outbreak of a poliomyelitis-like disease in Bulgaria.

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Multiplication of virulent and vaccine strains of poliovirus type I, II and III in laboratory animals of different species was studied comparatively. The main criterion of virus reproduction was the production of the photoresistant virus progeny after inoculation of the animals with proflavin-photosensitized virus strains. On the whole, virulent poliovirus strains were characterized by replication in a wide range of hosts (monkeys, cotton rats, white mice, guinea pigs, rabbits, chickens, chick embryos), a low infective dose, production of the photoresistant progeny to a high titre, clinically overt disease in some animal species. The vaccine strains multiplied in a norrower range of hosts, had a high infective dose, a low titre of virus progeny, and caused no clinical symptoms of infection. These differences may serve as a marker for differentiation between virulent and attenuated strains in vivo. Administration of guanidine before inoculation of newborn cotton rats completely prevented or delayed by several days the production of photoresistant virus progeny. This fact confirms the stability of the proflavin-poliovirus complex under conditions ruling out virus replication.

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A morphologic study of 57 cancers of the human gastrointestinal tract maintained in short-term organ culture was carried out, and the response of part of these tumors to infection with poliovirus was studied. The tumors, grown in vitro, mainly retained their original characteristics and histologic types. A certain correlation between the histologic type and its viability in short-term organ culture was revealed: the more differentiated forms were maintained better than the poorly differentiated ones. A comparative study of the polio-virus-infected and the control cultures indicated that wild poliovirus showed the most marked oncolytic activity in short-term organ culture, whereas attenuated poliovirus III produced a less marked, although still distinct, destructive effect.

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A cytopathic filterable agent designated EMC-70 was isolated in 1970 from Macaca rhesus monkeys with encephalomyocarditis. Its biologic and physico-chemical properties were studied. A number of primary and continuous cell cultures were found to be susceptible to the virus, and it was pathogenic for monkeys and small laboratory animals but did not multiply in chick embryos. The virus was stable to chlorofrom, thermolabile, stable to acids, and agglutinated group O erythrocytes of man, sheep, guinea pig. Electron microscopic examinations of infected cells revealed crystalline packings of subunits about 50 nm in diameter typical of picornaviruses. Serological studies demonstrated close antigenic relationship of the isolate with the virus belonging to encephalomyocarditis group.

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The capacity of vaccine and virulent strains of poliomyelitis virus to multiply in laboratory animals of different species was studied. Virus reproduction was judged by formation of photoresistant virus progeny in response to inoculation of the animals with photosensitized virus. Multiplication of virulent poliomyelitis virus strains observed in the majority of animal species examined (monkeys, newborn and adult cotton rats, newborn and adult white mice, chickens, chick embryos) resulted in active formation of photoresistant virus population and in some cases was accompanied by clinical symptoms of the disease. Multiplication of vaccine strains was observed in a smaller number of animal species and was limited, as a rule. Among non-primate animals, newborn cotton rats were most susceptible to poliovirus infection. Newborn guinea pigs were the only species of laboratory animals in which no multiplication of any of the six strains under study could be detected.

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A new method for intratypic antigenic differentiation of poliovirus strains has been developed. The method is based on the use of cross-absorbed sera in neutralization tests where virus is titrated by the cytopathic effect. Qualitative differences between the antigenic structure of vaccine and "wild" strains as well as between some "wild" strains of the same serologic type have been found. The method may be used in practical laboratories.

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The paper presents the results of 3-year observations of the status of herd immunity to poliomyelitis in 7 towns of the Moscow region. In individual years antibody for poliomyelitis virus of 3 types was found in 63-74% of the examined children and from 2.7 to 7.5% of children were triple-negative. Insufficient levels of herd immunity to poliovirus types I and III were found. Some factors are analysed which could have some influence on the decline of herd immunity.

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