Albert B. Sabin

Properties of ECHO types 22, 23 and 24 viruses

During our s tudies on summer d ia r rhea in 1956 (1), 7 viruses were isola ted from rec ta l swabs of infants wi th diarrhea, which could not be ident i f ied b y ant i sera p repa red wi th the known ECHO virus types . These 7 s t ra ins were considered as possible new ECHO viruses and 6 of t hem were s tudied. To exclude the poss ib i l i ty t h a t we were deal ing wi th mix tures of viruses, p laque pur i f ica t ion was carr ied out wi th al l 6 s trains. B y means of cross-neutra l iza t ion tes ts wi th an t i se ra p repared wi th the pur i f ied v i r u s s tocks i t could be shown t h a t these 6 s t ra ins fell into 3 new ECHO serotypes , des igna ted b y the Commit tee for Enterovi ruses of The Na t iona l F o u n d a t i o n for In fan t i l e Para lys is , Inc. , as ECHO 22, 23, and 24 vi rus (3 s t ra ins of ECHO 22, 1 s t ra in of ECHO 23, and 2 s t ra ins of ECHO 24 virus). I n th is communica t ion var ious proper t ies of these viruses as well as the serologic d i f ferent ia t ion from other viruses are described. Al though mos t d a t a refer to the selected p r o t o t y p e s t rains , reference to the o ther s t ra ins of ECHO 22 and 24 is made when the i r proper t ies dev ia t ed from those of the p r o t o t y p e strains.

Characteristics of poliomyelitis and other enteric viruses recovered in tissue culture from healthy American children.

Summary Rectal swabs from 1566 healthy American children who had no contact with clinically recognized cases of poliomyelitis were tested in monkey kidney tissue cultures. Among the 31 cytopathogenic agents which were recovered, 5 were poliomyelitis virus (three Type 2 and two Type 3), one was a group B Coxsackievirus, and 25 belonged to a hitherto unidentified group of enteric viruses. This new group of human enteric or HE viruses is antigenically unrelated to the so-called “orphan viruses” recovered in tissue culture by Melnick and by Steigman et al., from the stools of patients with suspected nonparalytic poliomyelitis. 22 of (these 25 new viruses belong to 3 distinct serologic types (8 were Type 1, 11 Type 2 and 3 Type 3). The HE 1, HE 3 and the 3 unclassified strains were neutralized by human gamma globulin while the HE 2 virus was not. The HE 1 and HE 2 strains produced neither paralysis nor any significant lesions after intraspinal injection in cynomolgus monkeys. The 5 poliomyelitis viruses were all intracerebrally aviru-lent spinal variants for cynomolgus monkeys. The Type 2 poliomyelitis viruses were pathogenic for mice by the spinal but not the intracerebral routes, while the Type 3 viruses were a virulent for mice by both routes. Among children under 10 years of age who came to clinics from families of lower income groups, poliomyelitis virus was recovered from about 1% and the new enteric viruses from about 6%.

Specificity of virus-induced resistance to transplantation of polyoma and SV 40 tumors in adult hamsters.

Summary Specificity of the transplantation resistance phenomenon was tested by using only 10 tissue cultured polyoma, SV 40, and F. Sa. No. 3 B hamster tumor cells in sufficiently large numbers of adult hamsters to yield definitive results. Pre-treatment with polyoma virus regularly produced resistance to transplantation of polyoma tumor cells but not of F. Sa. No. 3 B tumor cells; there was evidence of some resistance to SV 40 tumor cells in only 2 of 4 tests. Pre-treatment with SV 40 virus regularly produced resistance to SV 40 tumor cells, although in some tests of a lower order than in the polyoma virus vs polyoma tumor system, but not to F. Sa. No. 3 B cells; there was evidence of some resistance to polyoma tumor cells in only 2 of 4 tests. Pretreatment with adenoviruses, types 12 and 7A, herpesvirus and poliovirus type 1 produced no resistance against any of the 3 tumors tested. Reovirus, type 3, and vesicular stomatitis virus (Indiana strain) propagated in mouse embryo tissue culture, the same cells in which the polyoma virus was grown, produced no resistance against the polyoma tumor cells. The progeny of 10 polyoma hamster tumor cells which formed large tumors in occasional, polyoma-immune adult hamsters, nevertheless, were shown to possess the distinctive, cellular antigen. A sensitive test failed to reveal any cytotoxic antibody in the serum of polyoma-immune hamsters that resisted transplantation of polyoma tumor cells.

Characteristics and genetic potentialities of experimentally produced and naturally occurring variants of poliomyelitis virus.

Recently accumulated data are in accord with the hypothesis that the varying pathogenic and immunogenic activities of poliomyelitis viruses in different hosts and by different routes involving different cell types in the same host are determined by virus particles with distinct genetic characters. It follows from this that different variants can arise by mutation, and that the composition of any viral population would depend on the selective influences of the tissue and host employed for propagation. In order to make progress in this field, it is necessary to define clearly the characters exhibited by different poliomyelitis viruses and to study them in a quantitative manner. Characters having to do with virulence, immunogenic capacity, and cytopathogenic activity in. vilro readily lend themselves to such quantitative study. For poliomyelitis viruses, virulence is synonymous with paralytogenic activity resulting from damage to large numbers of lower motor neurons. The ultimate test for this character is inoculation in the gray matter of the spinal cord. It is already known that the characters that determine virulence for newborn mice, adult mice, monkeys, and chimpanzees are distinct. The position of hamsters and cotton rats has not as yet been clearly defined. Variants that are avirulent after intracerebral inoculation, but virulent after spinal inoculation lack the character that enables the virus to multiply sufficiently in certain areas of the brainstem to permit spread to the lower motor neurons. However, when very large quantities of intracerebrally avirulent variants are inoculated into the small space of a mouses brain, or even a monkeys brain, a sufficient number of virus particles may reach the lower motor neurons directly without having t3 pass through a barrier of insusceptible cells in the brainstem. Thus, virus populations can be characterized with respect to intracerebral and spinal virulence only by quantitative tests and by progeny tests on the virus recovered from the paralyzed animal. It has also been established that there are different grades of virulence for both intracerebral and spinal routes, depending on the capacity of the variant to multiply sufficiently in the initially invaded cells to permit extension to large enough numbers of others. Thus, the intracerebrally avirulent type 1 Brunhilde strain segregated by Enders, Weller, and Robbins in human tissue culture, the type 2 MEFI strain that we segregated by the terminal dilution technique in suckling mice, and the naturally occurring type 2 and type 3 strains recovered from the stools of healthy children by RamosAlvarez and myself2 all produced lesions of varying extent either only in the brainstem or in the brainstem and spinal cord of the intracerebrally inoculated monkeys that remained clinically well. On the other hand, the intracerebrally

The Complement Fixation Reaction in Toxoplasmic Infection.

Summary A frozen and thawed extract of toxoplasma-infected rabbit brain (but not similar extracts of certain other heavily parasitized tissues) yielded an antigen which fixed complement specifically in the presence of immune sera from experimentally infected rhesus monkeys but not rabbits, dogs, and cats. This complement fixing antibody appeared within 1 to 4 weeks after inoculation, but unlike the neutralizing antibody disappeared again in most monkeys, sometimes as early as 2 months. A certain number of human beings who were known or suspected to have experienced toxoplasmic infection also gave positive complement fixation reactions. However, twenty selected individuals whose sera had neutralizing antibodies against toxo-plasma gave negative complement fixation reactions, but in none of these was there evidence of recent or active infection. While a positive complement fixation reaction does not in all cases indicate active or even recent infection, it is believed that this test may have its greatest usefulness in the rapid diagnosis of active toxoplasmosis.

GENETIC, HORMONAL AND AGE FACTORS IN NATURAL RESISTANCE TO CERTAIN VIRUSES

Natural resistance is used here to designate a refractory state which does not depend on immunologic processes resulting from previous exposure of the host to a particular infectious agent or its component antigens. On this basis, there is evidence that the outcome of certain viral infections can be affected in different ways by the genetic constitution, age and hormonal status of the host. The relationship between the state of the host tissues and the special requirements of individual viruses is so close that the manner in which these factors operate can be illustrated best by an analysis of specific examples. The plant pathologists were the first to demonstrate by cross-breeding experiments that resistance of various plants to a variety of infectious agents was inborn and depended in some instances on a single pair of genes and in others on multiple pairs. They also showed that inborn resistance to one agent was generally independent of resistance to others. In some instances, susceptibility was inherited as a dominant and, in others, resistance was dominant. The studies of Webster’ with the viruses of louping ill and St. Louis encephalitis and those of Lynch and Hughes2 with the virus of yellow fever in mice provided the first experimental evidence that inherited host factors determined the outcome of mammalian viral infections. Here, too, it became apparent that one could not speak of “virus-susceptible” and “virus-resistant” mice, but only of mice which were susceptible or resistant to a specific virus or group of virses. The mice which were resistant to the viruses of louping ill, Russian Spring-Summe? and St. Louis encephalitis were not resistant to the viruses of vesicular stoma ti ti^,^ rabies’ and lymphocytic ch~riomeningitis.~ A most important contribution to this subject was Webster’s demonstration that the inherent resistance and susceptibility of mice to the virus of St. Louis encephalitis was correlated with the level of viral multiplication in the brain, not only in vivo6 but also in cultures containing minced mouse brain.6 Neither Lynch and Hughes nor Webster had strains of mice which were 100 per cent resistant, either originally or after crossing resistant parents. Accordingly, they had difficulty in obtaining satisfactory data for determining the mechanism of inheritance. I should now like to present some unpublished data of my own which throw additional light on the mechanism of inherited resistance to viral infections. I n 1944, I accidentally discovered that the albino mice which were inbred for many years a t The Rockefeller Institute a t Princeton, N. J. (henceforth called PRI mice) were uniformly resistant to the 17 D strain of yellow fever virus regardless of dosage administered. Early in 1950, I began to breed these mice* in Cincinnati and found that they were still 100 per

Unique Physico-Chemical Properties of Japanese B Encephalitis Virus Hemagglutinin.

Summary A specific hemagglutinin was recovered from mouse brains infected with the virus of Japanese B encephalitis, and it was found to possess properties different from all other viral hemagglutinins described thus far. Stable preparations of high potency were obtained after removal of an inactivating substance by differential centrifugation. The hemagglutinin combined with the receptors on the erythrocytes in a very narrow pH zone, the optimum being between 6.5 and 6.8. Marked enhancement of activity was obtained on dilution in certain buffers of optimum pH. No receptor destroying enzyme was found either in the infected mouse brains or in cultures of Vibrio cholerae, which destroyed the receptors for influenza virus. ZnSO4 in minute amounts inhibited the hemagglutinin of the Japanese B virus but not that of influenza virus. The inactive zinc-hemagglutinin complex was reactivated by dilution or removal of zinc with H2S. The inhibitor present in normal human serum was removed by shaking with chloroform, while the specific hemagglutination inhibiting antibody was unaffected, thus providing a simple test for diagnosis of infection with the Japanese B virus. Specific hemagglutinins, with properties different from that of the Japanese B virus, have also been found for the West Nile and Russian Spring-Summer encephalitis viruses.

THE SIGNIFICANCE OF VIRUSES RECOVERED FROM THE INTESTINAL TRACTS OF HEALTHY INFANTS AND CHILDREN

The viruses that may be recovered from the alimentary tract (throat and stools) of healthy infants and children vary with the method used for their detection, and they fall into two main categories: (1) those known to be the cause of specific diseases, e.g. poliomyelitis, herpangina (Coxsackie A), epidemic pleurodynia (Coxsackie B) , the pharyngeal-conjunctival syndrome (APC), herpes simplex, and presumably also those of infectious hepatitis, mumps and influenza B; (2) the large number of recently recognized viruses whose relationship to various disease syndromes is still a matter of current and future investigations. The newborn-mouse technique yields predominantly members of the Coxsackie group and also, less frequently, the herpex simplex virus. Rhesus or cynomolgus monkey-kidney tissue cultures most frequently yield the new group of many antigenically distinct “orphan”l or HE (human enteric)2 viruses recently established by committee action as the ECHO (enteric cytopathogenic human orphan) group of viruses3 and, less frequently, the poliomyelitis and certain other viruses. Certain members of the Coxsackie group (particularly A9 and group B) and of the new “APC”4 (adenoidal-pharyngealconjunctival) or R I group6 of viruses, as well as the herpes simplex, mumps, and influenza B viruses can also be detected by monkey-kidney cultures. Most of the APC viruses and certain types of Coxsackie virus, however, are more readily detected in cultures of HeLa cells, while many of the ECHO viruses multiply poorly in HeLa cells and may not be readily recovered in this type of culture medium. It is highly probable that the use of additional cell lines in tissue-culture media will reveal still more hitherto-unknown viruses. It is the classic tradition of man’s quest for knowledge about himself and his environment that we should want to learn as much as possible about the seemingly bewildering number of new viruses that appear to be part of the human heritage. I should like to begin by summarizing what my associate M. Ramos-Alvarez and I have learned during the past 2 years by testing rectal swabs from 3347 healthy children in cynomolgus monkey kidney tissue cultures. From 1566 healthy children aged 1 to 17 years tested in the region of Cincinnati, Ohio, during the months of July and August 1953, we recovered 31 strains of virus-5 were poliomyelitis, 1 a Coxsackie B4, and the remaining 25 constituted 5 new antigenic types of what we first called HE viruses, types 1 t o 5, and that will now be known as the ECHO viruses types 7 to 11. From 1771 healthy children aged 1 to 4 years tested in Mexico City and Veracruz, Mexico, during early June, 1954, we recovered 334 strains of virus, of which 73 were poliomyelitis viruses and 261 were other viruses requiring classification. It should be pointed out that in all 3 areas, the children came from the low socio-

Experimental studies on Phlebotomus (pappataci, sandfly) fever during World War II.

In conclusion it may be said that while the laboratory investigations on the virus or viruses of sandfly fever, which were encountered in the Mediterranean area and in the Middle East, gave us a good deal more information on the basic properties of the virus and, also, provided a supply for storage and future studies, the primary objectives of developing some method of propagating the virus outside of the human body and of a vaccine for the protection of exposed personnel, were not attained. It is, therefore, particularly fortunate that DDT was found to be so highly effective for the control ofPhlebotomus papatasi in dwellings22, and that the available mosquito repellents proved so effective in protecting those who may be exposed out of doors. Even if future efforts should not lead to the discovery of a vaccine, sandfly fever need no longer be the military problem that it had been in various operations in endemic areas in the past.

Human infection with Rift Valley fever virus and immunity twelve years after single attack.

Summary 1. Accidental human laboratory infection with a strain of Rift Valley Fever virus which had undergone at least about 300 intracerebral passages in mice, indicated that no modification in its pathogenicity for man resulted from this mode of passage. 2. The virus was recovered from the blood 10 hours after the first appearance of headache but titration revealed that the virus content was very low-only 666 LD50 per cc of serum. Four days after onset, no virus was detected in the blood, but neutralizing antibodies (index of 50) were already present and in the next 10 days increased to yield an index of at least 1,585,000. 3. Neutralizing antibodies for Rift Valley Fever virus (index of 3,160) were found 12 years after a single attack in the serum of an individual who had no further exposure to the virus during the intervening years.