Howard A. Howe

Portals of Entry of Poliomyelitis Virus in the Chimpanzee.

Summary Typical poliomyelitis has been produced in 4 chimpanzees by the intranasal, intragastric, and oral inoculation of untreated human stools. In 2 instances the olfactory portal was ruled out by section of both olfactory tracts.

Poliomyelitis Virus in the Human Oro-pharynx.

Summary Throat swabs were taken from 14 cases of human poliomyelitis during the first week of the disease. Seven of these (50%) were found to contain active virus.

Experimental Poliomyelitis Without Paralysis.

Preliminary studies on a series of 13 Rhesus monkeys (Macaco mulatta) indicate that by section of the olfactory tracts it is possible to confine the activity of poliomyelitis virus after intranasal inoculation to the olfactory bulbs. Previous experiments (Brodie and Elvidge, 1 Schultz and Gebhardt 2 ) have shown that monkeys in which both olfactory tracts and bulbs were destroyed did not develop paralysis after introduction of the virus into the nose. These findings, however, give no information regarding the ultimate fate of the virus after such a method of inoculation. The question remains as to whether the virus dies out immediately or may survive in the body—whether it can enter into any reactions with non-nervous tissue, or is capable of conferring any immunity upon its host although the typical picture of clinical poliomyelitis does not appear. Eight animals were prepared with very careful sections of both olfactory tracts. This was done by an approach through the medial wall of the orbit, well proximal to the olfactory bulbs. The olfactory tracts were delivered through a small slit in the dura and were cut, in most cases without blood loss or damage to the underlying frontal cortex. Most important of all, there was no disturbance to the nervous tissue or blood vessels of the olfactory bulbs, so that the fibrils of the olfactory nerves presumably maintained their normal relations within the nasal mucous membrane. The operated animals and normal controls were inoculated intranasally on 2 successive days with one cc. of a 10-20% suspension of monkey cord containing MV virus. The controls showed a febrile reaction in 3-7 days with paralysis in 7-12 days from the date of inoculation. On the other hand the operated animals remained unparalyzed, although 6 of them developed a marked fever at the same time as the controls.

Neuronal Pathways as Determining Factors in Dissemination of Poliomyelitis in the Central Nervous System.

There has been hitherto no clear-cut evidence regarding the mode of progression of the virus of poliomyelitis from the portal of entry into and through the central nervous system (CNS) of man. In the experimental animal more information is available and there is now a general belief, supported by considerable evidence, that the principal rôle in the dissemination of the virus is played by neuronal pathways rather than by humoral ones, and that within the CNS there occurs a progression of the virus from the point of entry to certain susceptible regions, especially the motor centers in the hind-brain and spinal cord, where the most serious effects of the virus-host reaction become apparent. Little is known as yet concerning the determining factors in the transmission and localization of the virus throughout the CNS, although studies of some of the neuronal pathways involved have been made by Fairbrother and Hurst, 1 and by others. In this report, additional evidence bearing on these problems will be presented. The material examined up to the present time consists of some 50 brains of Rhesus monkeys in preparalytic and paralytic stages of poliomyelitis, induced by introduction of the MV virus intra-nasally, intracerebrally, intraocularly and intraneurally. In a few-cases the Wallingford strain (Trask and Paul 2 ), inoculated intracerebrally and by skin rub, was used. The brains were prepared under optimal conditions for histological study chiefly by the gallocyanin method of Einarson, 3 which satisfactorily demonstrates nerve cells, neuroglia, and inflammatory cells. In some cases various experimental procedures, such as section of the olfactory tracts, the corpus callosum, the bulbar pyramids, or the spinal cord, were carried out in order to modify if possible the mode of dissemination of the virus.

A note on the penetration of poliomyelitis virus from the gastrointestinal tract in the chimpanzee

Summary Evidence is presented that in the chimpanzee poliomyelitis virus may reach the central nervous system from the gut via the abdominal sympathetic nerves.

Modification of the Site of Paralysis in Experimental Poliomyelitis.

Römer 1 noted that monkeys inoculated intraeranially with poliomyelitis virus frequently showed paralysis on the opposite side of the body. This suggested the participation of some decussating pathway in the propagation of the virus, but threw no light on the actual mechanisms which might be thus involved. A demonstration that virus may be routed along known fiber tracts in the central nervous system would go far toward explaining the fact that although the infective agent may enter through the olfactory tract, it usually traverses the entire brain stem without causing serious damage above the cord. Various cytologically distinct areas of the cerebral cortex provide the ideal conditions for such an experiment since their fiber connections with lower centers are sufficiently well known to provide a ready interpretation of the results obtained. Accordingly 3 representative cortical areas were selected as sites for the introduction of the virus. The first, the so-called motor cortex, known cytologically as area 4 of Brodmann, was chosen because its known lower connections both ascending and descending are predominantly crossed. Next, the visual cortex, area 17 of Brodmann, presented the picture of an area with uncrossed connections. Its relations are with the lateral geniculate body of the same side, a region receiving the optic tracts, but possessing no direct connections with the lower brain stem or cord. The third region selected was the pre-motor cortex, or area 6 of Brodmann. The course of the fibers connecting this field with lower centers is not so well known as in the case of the other cortical areas. It is certain that its descending fibers run outside the pyramidal tract, proceeding as far as the midbrain and pons. 2 Stimulation of area 6 with the cortico-spinal tract eliminated 3 indicates that it is primarily concerned with movements of both arms, although there is a preponderance of crossed representation.

An Effective Method of Intraneural Inoculation of Poliomyelitis Virus.

The uncertainty of results obtained by most investigators with intraneural injections of poliomyelitis virus has limited the use of this method of inoculation for inducing experimental poliomyelitis. Neveretheless, if the virus of poliomyelitis is truly neurotropic, one would expect that intraneural inoculation would be as effective as intracerebral inoculation, and simpler, if the virus could actually be made to come into contact with numerous nerve fibers, rather than being forced along and between connective tissue sheaths within the peripheral nerve. This of course is strongly suggested by the work of Fairbrother and Hurst, 1 who showed that trauma during intraneural injection facilitates “takes” by this method of inoculation. Going one step farther, and with the knowledge that during the first few days after nerve section the nerve cells with axons cut are more susceptible to the virus than normal cells, 2 it was decided to determine whether simple section of a peripheral nerve and immersion of the central stump in virus suspension for a few minutes was sufficient to produce poliomyelitis. This method, which involves no mechanical injection pressure, and which places the virus in contact with the protoplasm of every axon in the nerve, was found to be highly successful in producing poliomyelitis. When a large nerve, such as the sciatic nerve, was used, this method of inoculation was invariably successful with two strains of known potency, the MV and Wfd 3 strains. In 9 Rhesus monkeys the sciatic nerve was sectioned with sharp scissors peripheral to the sciatic notch or at the mid-thigh level, and the central cut end then soaked in as little as 0.1 cc of 20% virus suspension for several minutes. Poliomyelitis resulted after an incubation period of 4-6 days.

Production of Experimental Poliomyelitis from Untreated Stools.

It is now a well established fact that poliomyelitis virus may be present in considerable quantities in the stools of patients suffering from either paralytic or abortive attacks of the disease. These findings have been summarized by Trask, Vignec and Paul 1 who recently described a method for treating human stools in order to render them suitable for intraperitoneal inoculation into monkeys. Such procedures unfortunately involve the use of bactericidal substances of which the effect upon poliomyelitis virus is unknown. It is thus desirable to obtain a method of utilizing the material from stools without the intervention of measures which may cause attenuation or other change in virus. Such a method has been found in the simple intranasal inoculation of monkeys with an untreated stool suspension. On September 15, 1938, a stool specimen was obtained from a 3–year-old quadriplegic child through the courtesy of Dr. Harold Hobart of the Childrens Hospital, Washington, D. C. The stool was collected on the third day after the onset of paralysis. At the time the child was still febrile. The specimen was immediately made up into a thick suspension with distilled water and the supernatant fluid was placed in the ice box. Two Rhesus monkeys were inoculated intranasally with this fluid on 3 successive days, each animal receiving a total of 3 cc per nostril. Following the introduction of the material the nasal passages were rubbed gently with a pipe cleaner. On the fourteenth and eighteenth days respectively the animals developed typical but not extensive paralyses. They were killed and portions of the cord were removed for histological study and rein-oculation. Typical poliomyelitic lesions were found in the grey matter of each spinal cord. Cultures of a suspension of each cord on blood agar and in blood broth were negative except for a B. subtilis contamination in one sample.

The Pia-Arachnoid as a Barrier in Experimental Poliomyelitis.∗

The occasional demonstration of poliomyelitis virus in the cerebrospinal fluid of experimental animals has given rise to a series of experiments and speculations regarding the rôle of this body liquid in the dissemination of the disease through the central nervous system. Such investigations as those of Clark and Amoss 1 and Hurst 2 who produced experimental poliomyelitis regularly by intra-cisternal and intrathecal inoculation have led to the assumption recently made articulate by Schaeffer and Muckenfuss 3 that intracerebral inocula may be effective at sites far distant from the point of introduction. In previous observations the permeability of the ependyma and the possibility of injury to the pia-arachnoid are factors which have not been properly controlled. The following experiments indicate that under ordinary conditions the leptomeninges constitute an effective barrier—so effective that large amounts of active virus may be present in the cerebrospinal fluid without any clinical indication of poliomyelitis. These experiments fall into 3 groups: (a) virus dripped over an exposed cortical surface with intact pia; (b) virus introduced into the lumbar cistern with intact pia; (c) virus introduced at either of the above sites after deliberate pial injury. Fifteen animals were used in the first group. The cerebral cortex was exposed by opening an oval dural flap 3 cm by 2 cm which was centered over the paracentral lobule with the long axis in a rostro-caudal direction. Great care was taken to avoid injury to the pia, although the arachnoid was undoubtedly torn in places. One-quarter to three-quarters cc of 20% MV virus in salt solution was then dripped over the exposed cortical surface, the edges of the dural flap being elevated to insure as wide dissemination as possible. The dura was then carefully sutured and the incision closed.

Production of Immunity to Poliomyelitis Virus in Motor Cells of the Monkey's Spinal Cord.∗

The following experiments constitute a demonstration that, as in the case of the olfactory bulbs already reported, 1 certain motor cells in the spinal cord of the rhesus monkey may be rendered resistant to the effects of invasion by poliomyelitis virus by means of procedures which produce alterations in their normal metabolism. Eighteen animals were subjected to section of the sciatic nerve in the sciatic notch at intervals varying between 3 and 57 days previous to the induction of complete leg paralysis by intranasal inoculation of poliomyelitis virus. A series of 6 uninoculated controls were operated upon in a similar fashion and sacrificed at intervals between 6 and 30 days. These animals may be grouped under two headings. The first includes those monkeys in which the nerve section either produced no effect upon the susceptibility of the cells involved, or may have slightly enhanced it. This situation was found in 3 cases where nerve section had preceded leg paralysis by 3-4 days. In this group the cells of the operated and control sides were completely destroyed and it appeared that the destruction of the former had preceded that of the control cells. Exactly the opposite effects were observed in the second group of 15 animals in which nerve section had been carried out 6-57 days previous to leg paralysis. In these cases the entire anterior horn of the control side was destroyed wrhile on the operated side the vast majority of the cells giving rise to the sciatic nerve were spared. In the cases of longest duration of nerve section (13-57 days) the differences between the two sides were most striking (Figs. 1 and 2). As might be expected from the analysis of the cases in the first group, showing the opposite effect, the sparing was less extensive in those animals where nerve section was of shorter duration (6-8 days).