ULTRAFILTRATION OF THE VIRUS OF POLIOMYELITIS

From the ultrafiltration analysis the size of the virus of human poliomyelitis has been estimated to be somewhere between 12 and 17 mµ. Technical difficulties encountered and the low concentration of the virus make it seem possible that the virus is even smaller.     

SURVIVAL OF THE VIRUS OF POLIOMYELITIS FOR EIGHT YEARS IN GLYCEROL

1. An instance of successful inoculation of poliomyelitis virus after preservation for 8 years in 50 per cent glycerol is reported. 2. The virulence of the material injected remained essentially unchanged during this period. 3. The fact that poliomyelitis virus will survive in glycerol for so great a period may be taken as further indication of the improbability of streptococci as the inciting organisms. 4. Poliomyelitis virus would seem to vary in its resistance to glycerolation. 5. The remarkable persistence of active virus outside of the body may have a bearing on the epidemiology of poliomyelitis.     

REVIVED ACTIVITY OF THE VIRUS OF POLIOMYELITIS

A strain of the virus of poliomyelitis has been described which has passed through several stages of virulence as tested upon monkeys. The first stage consisted of the adaptation of the original human virus to the monkey. In this process high virulence was readily achieved. The adapted, virulent strain of virus was passed regularly through monkeys and maintained its activity for about 3 years, when diminution became apparent. The loss of power of the virus was such that it may be said to have returned approximately to the level of the original human virus. This change constituted the second stage. The third stage is represented by recovery of the high virulence. This revival occurred, it seems, during the sojourn of the virus in glycerol and required several years for its consummation. It was first noticed nearly 6 years after the low level of the second stage became established. The potent virus of the third stage has been found to remain active over a period of at least 4 years while preserved in glycerol. What constitutes at least a superficial resemblance between the wave-like rises and falls of the incidence of epidemic poliomyelitis and the phenomena of increase and decrease in virulence of the specimen of virus has been alluded to. The two processes differ, however, essentially in respect to the time factor, since the fluctuations of the epidemic wave occupy small and those of the virulence occupy large increments of time.     

EXPERIMENTS WITH THE VIRUS OF POLIOMYELITIS

1. Efforts to adapt the virus of poliomyelitis to the rabbit organism and to produce poliomyelitis in rabbits by testicular injection and by brain injection after testicular passage produced no evidence that the virus could be adapted in this manner. Suggestive symptoms produced in very young rabbits were duplicated in non-specifically treated and in uninoculated controls. The admixture of a vaccine virus, adapted to the rabbit organism, with the poliomyelitis virus in similar injections and passages did not aid the adaptation. The virus of poliomyelitis did not survive 24 hours in the rabbit testicle—whether alone or mixed with vaccine virus. Repeated intraperitoneal and intradermal injection of poliomyelitis virus and of poliomyelitis and vaccinia virus mixtures produced no disease in rabbits. Massive doses of concentrated virus by stomach tube in conjunction with meningeal irritation produced no symptoms in rabbits. 2. No neutralizing substances against poliomyelitis virus could be produced in rabbits by the repeated intraperitoneal and intradermal injection of poliomyelitis virus or of poliomyelitis-vaccinia virus mixtures. 3. Although attempts to infect monkeys by intrastomachic injections, after bile irritation of the mucosa, were entirely negative, evidence was obtained that repeated intrastomachic injection after bile irritation may produce an appreciable degree of immunity. 4. No evidence could be obtained that the cellular elements of the blood contain the virus in any greater proportion than the whole blood. 5. One attempt to immunize by neutral virus-serum mixtures was entirely negative.     

PERSISTENCE OF THE VIRUS OF POLIOMYELITIS IN THE NASOPHARYNX

The virus of poliomyelitis occurs in the nasopharynx of man and monkeys. In man it has been detected by the inoculation test in washings from acute cases, rarely in similar washings from healthy contacts, in the nasopharyngeal tissues obtained from fatal cases in the 1st week of infection, but rarely, if ever, from nasopharyngeal tissues removed surgically at later periods in the course of the disease. In monkeys, also, the virus has been detected in the secretions from acute experimental infections, in the nasopharyngeal tissues derived from early cases, and rarely from cases several weeks or months after recovery from the acute symptoms. The inoculation of tonsils and adenoids obtained from cases of undoubted poliomyelitis either yielded definite results in the form of typical paralysis and histological lesions in the central nervous organs of the monkeys injected, or no symptoms or lesions which could be confounded with poliomyelitis. The indefinite symptoms and atypical lesions described in a certain class of inoculated animals by Kling, Pettersson, and Wernstedt were not encountered in our experiments. The deduction from the experiments reported is to the effect that the virus is regularly present in the nasopharynx in cases of poliomyelitis in the first days of illness, and especially in fatal cases; that it diminishes relatively quickly as the disease progresses, except in rare instances; and that it is unusual for a carrier state to be developed. Hence the period of greatest infectivity of patients would appear to be early in the disease, which is probably the time at which communication of the virus from person to person takes place. Available evidence proves that healthy carriers of the virus occur. We do not, however, possess data which indicate the frequency with which carriage arises. The fact that even after a severe and wide epidemic, such as occurred in the United States in 1916, the disease may virtually disappear within 2 or 3 years, points to the probability that enduring carriers of the active virus, whether healthy or chronic, are of exceptional occurrence.     

ISOLATION OF POLIOMYELITIS VIRUS FROM THE NASOPHARYNX

A single example of mild illness diagnosed as suspected abortive poliomyelitis is described in which the virus of poliomyelitis was recovered from the nasopharynx by three different methods. Failure to recover virus from a total of twenty-six cases diagnosed as suspected or abortive poliomyelitis and fourteen contacts is also reported. The original material from the nasopharynx of the positive case proved unusually infective for the monkey, apparently even more so than are the majority of suspensions of spinal cords from fatal human cases of poliomyelitis. An explanation of this fact is not clear. The method of isolating human virus from the throat, by means of preserving the sediment of washings from this site in glycerine, has been shown to be efficient in one case for a period of 101 days.     

AN IMMUNIZING STRAIN OF THE VIRUS OF POLIOMYELITIS

In this communication we have described a strain of the virus of poliomyelitis derived from a rapidly fatal human case, which exhibits mild degrees of infective power and marked degrees of protective effect for the monkey. While Macacus rhesus displays perceptible differences in suceptibility to the attenuated virus, nearly all individuals respond to the inoculation and none succumbs to the infection induced. As compared with previously described examples of experimental poliomyelitis, the modified disease described in this paper is distinguished by its relatively benign nature and its tendency to end in recovery rather than, as with the earlier observed instances, in death.     

THE EFFECT OF CATAPHORESIS ON POLIOMYELITIS VIRUS

1. Under ordinary conditions of hydrogen ion concentration the virus of poliomyelitis, as such, or associated with particles in fine suspension, migrates in an electrical field to the anode. It follows that the virus bears an electronegative charge. 2. By means of cataphoresis, the virus can be recovered from a non-infective mixture of virus and specific immune serum. 3. By the same means it is possible to reveal the presence of virus in the central nervous system of a monkey which has recovered from the active stage of experimental poliomyelitis.     

NEUTRALIZATION OF THE VIRUS OF POLIOMYELITIS BY NASAL WASHINGS

1. The results of 56 experiments have shown that washings of the nasal and pharyngeal mucosas possess definite power to inactivate or neutralize the active virus of poliomyelitis. 2. This power is not absolutely fixed, but is subject to fluctuation in a given person. Apparently inflammatory conditions of the upper air passages tend to remove or diminish the power of neutralization. But irregularities have been noted, even in the absence of these conditions. 3. Too few tests have been made thus far to ascertain whether adults and children differ with respect to the existence of this neutralizing property in the nasal secretions. While the inactivating property was absent from the secretions of one child during the first days of poliomyelitis, it was present in another to whom immune serum was administered, and in still another on the 15th day of illness when convalescence was established. 4. The neutralizing substance is water-soluble and appears not to be inorganic; it appears to be more or less thermolabile, and its action does not depend upon the presence of mucin as such. 5. It is suggested that the production of healthy carriers through contamination with the virus of poliomyelitis may be determined by the presence or absence of this inactivating or neutralizing property in the secretions. Whether this effect operates to prevent actual invasion of the virus and production of infection can only be conjected. Probably accessory and not the essential element on which defense against infection rests. It is more probable that other factors exist which help to determine the issue of the delicate adjustment between contamination and infection.     

THE RELATION TO THE BLOOD OF THE VIRUS OF EPIDEMIC POLIOMYELITIS

Specimens of human blood taken during the paralytic stage of poliomyelitis and post mortem have proved not to be capable of infecting Macacus monkeys. Specimens of monkey blood taken at various stages of experimental poliomyelitis have not proved as a rule to be capable of infecting monkeys. In a single instance, among ten tests, infection was secured with a specimen of blood removed at the beginning of the paralysis on the seventh day following an intracerebral inoculation. When suspensions of the spinal cord from a paralyzed monkey have been injected into the brain or simultaneously into the brain and spinal canal, the blood removed from one to forty-eight hours later failed to cause paralysis after intracerebral injection. When large volumes of active filtrate are injected into the circulation the blood remains infective for seventy-two hours at least, but may be no longer infective after ten days when the paralytic symptoms first appear. When, however, the filtrate is injected in smaller amount or when a filtrate of a less active virus is employed in large quantity, the blood either fails to convey infection or conveys it irregularly. It is only when overwhelming quantities of an active virus are injected into the blood that paralysis results. The injection of moderate doses is not followed by paralysis, although the virus may still be detected in a blood sample twenty-four hours after the injection. The existence of a mechanism capable of excluding the virus within the blood from the central nervous organs is therefore inferred. Infection is accomplished far less readily through the circulation than by means of the more direct lymphatic and nervous channels of communication with the central nervous system. Several series of feeding experiments conducted with the biting stable fly (Stomoxys calcitrans) resulted negatively.     

LOCALIZATION OF THE VIRUS AND PATHOGENESIS OF EPIDEMIC POLIOMYELITIS

The virus of poliomyelitis is capable of penetrating the retina without producing apparent injury, to reach the central nervous organs. The virus injected into the blood is deposited promptly in the spleen and bone marrow, but not in the kidneys, spinal cord, or brain. Notwithstanding the affinity which the nervous tissues possess for the virus, it is not removed from the blood by the spinal cord and brain until the choroid plexus and blood vessels have suffered injury. The intervertebral ganglia remove the virus from the blood earlier than do the spinal cord and brain. An aseptic inflammation produced by an intraspinous injection of horse serum facilitates and insures the passage of the virus to the central nervous organs, and the production of paralysis. The unaided virus, even when present in large amounts, passes inconstantly from the blood to the substance of the spinal cord and brain. When the virus within the blood fails to gain access to the central nervous organs, and to set up paralysis, it is destroyed by the body, in course of which destruction it undergoes, as a result of the action of the spleen and, perhaps, other organs, diminution of virulence. The histological lesions that follow the intravenous injections of the virus in some but not in all cases differ from those which result from intraneural modes of infection. In escaping from the blood into the spinal cord and brain, the virus causes a lymphatic invasion of the choroid plexus and widespread perivascular infiltration, and from the latter cellular invasions enter the nervous tissues. A similar lymphoid infiltration of the choroid plexus may arise also from an intracerebral injection of the virus. The histological lesions present in the central nervous organs in human cases of poliomyelitis correspond to those that arise from the intraneural method of infection in the monkey. The virus in transit from the blood through the cerebrospinal fluid to the substance of the spinal cord and brain is capable of being neutralized by intraspinous injection of immune serum, whereby the production of paralysis is averted. Carmin in a sterile and finely divided state introduced into the meninges and ventricles sets up an aseptic inflammation, but is quickly taken up by cells, including ependymal cells. When an aseptic inflammation has been previously established by means of horse serum, or when the nervous tissues are already injured by the poliomyelitic virus, the pigment appears to enter the ependymal cells more freely. The experiments described support the view that infection in epidemic poliomyelitis in man is local and neural, and by way of the lymphatics, and not general and by way of the blood. Hence they uphold the belief that the infection atrium is the upper respiratory mucous membrane.     

EXPERIMENTS ON INSECT TRANSMISSION OF THE VIRUS OF POLIOMYELITIS

The domestic fly (Musca domestica) can carry the virus of poliomyelitis in an active state for several days upon the surface of the body and for several hours within the gastro-intestinal tract. Mosquitos (Culex pipiens, Culex sollicitans, and Culex cantator), in our experiments, have not taken up and maintained in a living state the virus from the spinal cord of monkeys. Lice (Pediculus capitis and Pediculus vestimenti) have not taken the virus out of the blood of monkeys or maintained it in a living state. The bedbug (Cimex lectularius) has taken the virus with the blood from infected monkeys and maintained it in a living state within the body for a period of seven days.     

EXPERIMENTS ON THE PURIFICATION AND CONCENTRATION OF THE VIRUS OF POLIOMYELITIS

Methods employed by Willstätter and his coworkers in the isolation and purification of enzymes have been applied to the virus of poliomyelitis. Rhoads (6) showed that alumina gel C mixed with poliomyelitis virus in certain proportions at an acid pH resulted in the adsorption and inactivation of the virus. The experiments in this communication confirm Rhoads'' observation, and show further that the adsorption as well as the inactivation are reversible; i.e., by changing the pH to the alkaline side with M/15 Na₂HPO₄ it is possible to free the virus in a state in which it is again capable of producing typical poliomyelitis. These experiments show also that by this process the virus undergoes considerable purification by diminution in the concentration of non-virus-containing substances. Washing the alumina gel C-virus complex with M/5 NaCl-M/15 KH₂PO₄ apparently dissociates no virus but is capable of freeing a certain amount of extractible organic substances. Furthermore, it is possible to increase the degree of purity and concentration of the virus by distillation in vacuo, and subsequent repeated adsorptions and elutions. By such partial purification and concentration, a virus solution with a minimal effective dose (as regards the production of typical poliomyelitis) of 0.0003 cc. was obtained. This solution had 0.04 mg. N per cc., and gave negative biuret, xanthoproteic, and ninhydrin reactions. These methods offer an opportunity for the preparation of a quantity of sufficiently purified and concentrated poliomyelitis virus to warrant the beginning of a study of its chemical nature; they also offer a means of anchoring the virus to an insoluble, and centrifugable substance (alumina gel C), which promises to facilitate various immunological studies, that might otherwise have been impossible.     

STUDIES OF THE DISTRIBUTION OF POLIOMYELITIS VIRUS : V. THE VIRUS IN FAMILIAL ASSOCIATES OF CASES

The occurrence and duration of the carrier state in familial associates of recognized cases of poliomyelitis was studied by the examination for virus of stool specimens collected from the members of four families at regular intervals for a period of over 2 months. The results indicate that: (1) virus may persist in their stools continuously for 4 to 5 weeks; (2) virus may be encountered intermittently in the stools; (3) in some instances virus may be present for brief periods only; (4) children are more likely to maintain virus than are adults in the same family; (5) infection of a family takes place rapidly, suggesting again simultaneous infection from a common source.     

CARRIAGE OF THE VIRUS OF POLIOMYELITIS,WITH SUBSEQUENT DEVELOPMENT OF THE INFECTION

A family group containing four children of whom all showed in varying degree symptoms of poliomyelitis is described. The source of infection and periods of incubation have been followed. Two of the children were proven by inoculation tests to carry the virus of poliomyelitis in the nasopharynx. Of these, one was detected to be a carrier after recovering from a non-paralytic attack of the disease, and the other was discovered to be a carrier about 5 days before the initial symptoms, attended later by paralysis, appeared. The original case from which the three others took origin was fatal; the youngest child, after quite a severe onset, was treated with immune serum, and made a prompt and almost perfect recovery. The nasopharyngeal secretions of two of the cases, taken 1 month after the attack, proved incapable of neutralizing an active poliomyelitic virus. The proposition is presented that every case of poliomyelitis develops from a carrier of the microbic cause, or virus, of poliomyelitis.     

SURVIVAL AND MULTIPLICATION OF THE VIRUS OF POLIOMYELITIS IN VITRO

The study here reported concerns attempts at bacteriological cultivations with fragments of brain or cord, or with Berkefeld V filtrates of the nervous tissues, from seven monkeys successfully inoculated with poliomyelitic virus. With these materials, 315 tubes were inoculated, of which thirty-six showed minute bodies resembling the globoid bodies described by Flexner and Noguchi. However, a study of subplants from these minute, morphological particles did not convince us that we had in hand actual cultures of the globoid bodies, or indeed of any living microorganism. Nevertheless, when washed sediments from subplants of one of the strains, representing the seventh, eighth, ninth, and tenth transfers, were inoculated into monkeys, the clinical signs and pathological effects characteristic of experimental poliomyelitis could be induced. The virulence of the "cultures" could not be ascribed to carrying over the original material into these remote subplants since the seventh transfer represented a dilution of the original cultivated material to about 1.5 x 10^–12, and the tenth, to about 1.3 x 10^–18 if one assume, as the transfer technic justifies, a thorough mixing of the contents of each tube. On the contrary, it appears as if the poliomyelitic virus had multiplied in vitro, and had increased as a consequence of being in a medium of a modified living tissue-cell culture. For in practically all specimens we observed many well-preserved kidney tissue cells and leucocytes, the latter probably derived from human ascitic fluid, a component of the Smith-Noguchi medium. In this connection, it should be mentioned that the several lots of ascitic fluid used in the cultivation tests were recently obtained from patients and employed from a week to a month after their collection. There remains for consideration the problem of the selective pathogenicity of the "cultures:" only the material of those tubes of the ninth and tenth transfers which showed the "globoid bodies" proved pathogenic; those respective tubes of the same transfers which were free from the minute bodies but apparently identical in all other respects, were avirulent. It may be that the virus was adsorbed to the particular bodies which we have found in the "cultures" and which resemble closely the globoid bodies of Flexner and Noguchi. Further elaboration of this study would be necessary, however, before such an inference could be regarded as a definite hypothesis.     

THE ULTRACENTRIFUGE AS AN AID IN THE DETECTION OF POLIOMYELITIS VIRUS

1. By means of differential ultracentrifugation, a purified and concentrated macromolecular fraction has been regularly obtained from infected human, monkey, and chimpanzee stools. This fraction was isolated from sixteen stools in which virus was thought to be present, and inoculated intracerebrally into sixteen monkeys, of which fifteen developed poliomyelitis. 2. Eleven stool specimens in which virus was suspected, when tested separately in eleven monkeys by the intra-abdominal-intranasal method, produced poliomyelitis in two of these animals. When the same specimens were tested separately by the ultracentrifugal-intracerebral method, poliomyelitis developed in ten monkeys out of eleven inoculated. 3. With the intra-abdominal-intranasal method, it has been customary to inoculate the virus present in 1.6 gm. of stool. With the ultracentrifugal-intracerebral method, the virus present in as much as 30 gm. of stool has been inoculated. 4. From one titration experiment it would appear that the ultracentrifugal-intracerebral method is at least 100 times more sensitive than the intra-abdominal-intranasal method.     

PENETRATION OF THE VIRUS OF POLIOMYELITIS FROM THE BLOOD INTO THE CEREBROSPINAL FLUID

The virus of poliomyelitis introduced into the blood may pass indirectly by way of the cerebrospinal fluid to the interstices of the central nervous organs. To reach the cerebrospinal fluid the virus must first penetrate the barrier of the choroid plexus, which operation requires time. By the inoculation test, no virus was detected in the fluid at the expiration of 48 hours, only small amounts at the expiration of 72 hours, while at the expiration of 96 hours the virus had passed more freely. The virus was still detectable in the fluid at the onset of paralysis 19 days after the intravenous injection. Pathological conditions of the leptomeninges and the cerebrospinal fluid play an important part in the pathogenesis of epidemic poliomyelitis.