John G. Kidd

A Virus Causing Oral Papillomatosis in Rabbits

Summary Oral papillomatosis is common in domestic rabbits bred in the New York area. The disease is due to an hitherto undescribed virus which is evidently pathogenic for the oral mucous membrane only.

Tissue Affinity of Shope Papilloma Virus

Summary It is plain that the Shope papilloma virus is remarkably specific in its action, affecting only the epidermis of rabbits and hares and failing to influence embryonic epidermis or other kinds of epithelium, even when this is keratinizing as the result of avitaminosis A, or proliferating in consequence of Scharlach R stimulation.

A Complement-binding Antigen in Extracts of the Brown-Pearce Carcinoma of Rabbits

Summary and Comment A substance is present in extracts of the Brown-Pearce tumor which binds complement specifically when mixed with the sera of rabbits bearing the growth, or in which it has retrogressed. The substance is readily distinguishable from the complement-binding antigen which can be extracted from the virus-induced papillomas of rabbits, though it resembles the latter in its general traits. Further work is under way to determine the significance of the findings.

Antigenic Individuality of Certain Papilloma Viruses

Papillomas of the skin and mucous membranes are frequent in man and many of the lower animals. That certain of these growths are caused by filterable viruses has been demonstrated, notably verruca vulgaris in man, 1 oral papillomatosis of dogs, 2 cattle warts 3 and the papillomatosis of American cottontail rabbits. 4 All these growths are readily induced in susceptible hosts by inoculation with the respective viruses. In general, animals of other species than that furnishing the virus prove resistant on inoculation, though some problematic transfers have been reported. The virus of the Shope papilloma of cottontails is undoubtedly effective in other kinds of rabbits, 5 but guinea pigs, dogs, cats, rats and mice have proved insusceptible. Despite the limitations thus illustrated in the pathogenic range of the viruses, the strikingly similar characters of the growth produced by those deriving from various animals suggest that they may be related, like the viruses causing the pox diseases and the swine and human influenza viruses. We have tested this possibility immunologically, seeking for evidence of cross neutralization between the virus of rabbit papillomatosis and the sera of dogs, rabbits, cattle, and men bearing virus-induced papillomas or recently recovered from them. It is known that these growths induce neutralizing antibodies for the homologous agents with fair regularity. It was impracticable for us to test most of the sera against the homologous agents; and hence we have assumed that they were actively neutralizing. Serum was procured from a cottontail rabbit trapped in Texas and carrying many cutaneous papillomas, as also from 2 cows of the same Texas neighborhood,—one a yearling heifer bearing characteristic papillomas of the kind due to virus, the other a cow about 2 years old in which similar growths had retrogressed about 4 months previously.

Effect of the Papilloma Virus (Shope) Upon the Tar Warts of Rabbits

When the papilloma virus is injected into the blood stream of rabbits previously tarred on the ears for 1 1/2 to 3 months, growths rapidly arise on the tarred skin, often in great numbers. Some are squamous cell carcinomas, 1 usually multiple, and frequently metas-tasing.∗ We have studied the phenomenon in more than 70 rabbits, with 90 tarred controls. In none of the latter has a cancer developed. Many of the growths that follow upon injection of the virus appear where no localized proliferation was previously visible, but others derive from pre-existing tar warts, which start growing rapidly, alter in aspect, and not infrequently manifest malignancy. After tarring is stopped, most ordinary tar warts disappear and the others become indolent, whereas the virus-stimulated warts keep on enlarging. Some are now carcinomatous but may undergo conversion into characteristic virus papillomas, as a scrutiny of several hundred specimens has shown; and others have become hybrids, neither ordinary tar tumors nor ordinary virus tumors but peculiar papillomas of malignant appearance, which change to squamous cell carcinomas almost at once. Not a few of the warts, however, though greatly stimulated by the virus (as proven by weekly records of their size), retain the morphology of the tar tumors. To learn more, opportunity was provided for the virus to infect tar warts in vitro. The warty tissue was punched from the ears, a slice taken for section, and equal portions of the hashed remainder were steeped for some minutes in Tyrode, and in a Berkefeld filtrate of Tyrode containing potent virus, respectively; after which they were implanted at corresponding situations in the leg muscles and subcutaneous tissue of the hosts. Four or 5 wart materials from each rabbit were so treated, as was also some of the tarred skin devoid of warting. Tarring was kept up during a few later days because of its presumptive general influence to favor proliferation. The warts utilized proved to be the familiar tar papillomas and car-cinoids, 2 so called because, though having the histology of carcinomas, and often ulcerating and growing through the ear, they ultimately disappear or revert to the benign, papillomatous form, even though tarring is continued. One implanted animal died 20 days later and 9 were killed after 38 to 64 days. None of the 44 wart materials steeped in Tyrode had given rise to a growth, nor had the skin specimens done so. Tiny cysts lined with ordinary, stratified squamous epithelium resulted from 5 of the former and from 4 of the latter, the others undergoing resorption. Ferrero 3 has reported similar findings. Very different was the outcome with the tissues steeped in virus. Then the bits of skin regularly gave rise to nodules of papillomatous tissue such as form on direct implantation of the Shope papilloma. 2 Similar nodules resulted from the implantation of 15 of the 24 tar papillomas, 4 yielded nothing, and 3 gave rise to small cysts like those just described. From one of the remaining 2 a malignant papilloma arose, markedly different from the original wart, and from the other a multicentric growth retaining the distinctive morphology of the latter. Nodules of Shope papillomatosis resulted from 8 of the 20 virus-steeped carcinoids, while 9 yielded nothing. In the remaining 3 instances, growths of carcinomatous aspect developed. The morphological characteristics of the original carcinoid had been retained in 2 of these cases, and the growths, though invasive, were small. The third was large, anaplastic and notably aggressive, to all appearances a malignant cancer, with but slight resemblance to the original carcinoid and none to those Shope papillomas that invade the muscle in which they are implanted. 4 The warts from which the implantation growths of cancerous aspect derived had been large at biopsy in 4 of the 5 cases, and much of their tissue had been left in situ. After tarring was stopped one of them continued to enlarge slowly, but became a benign papilloma histologically, while the other 3 disappeared. The biggest to disappear was 2.3 cm. across and had extended through the ear when about one-tenth of it was taken for steeping. The bits steeped in Tyrode yielded no growth, whereas those exposed to virus gave rise to the large, malignant tumor just mentioned. It is common knowledge that extraneous viruses 5 can flourish in tumors. Some induce no evident change, while others cause necrosis or the formation of inclusion bodies. The papilloma virus causes no inclusions, but it stimulates tar tumors to active growth, brings about morphological alterations in many of them, frequently acts as the determining factor in their survival, and makes some take on forthwith the character of carcinomas.

Antigenicity and Infectivity of Extracts of Virus-Induced Rabbit Papillomas

The antigen which binds complement when mixed with sera neutralizing the Shope papilloma-virus bears an evident relation to the virus itself; for it is present in high titer in virus-containing extracts of papillomas, it is retained by filters which hold back the virus, and its effectiveness is destroyed by the same amount of heating which renders the virus non-pathogenic. 1 The experiments now to be reported throw further light on this relation. Tests with Extracts of Non-Infectious Papillomas and of Normal Skin. On inoculation the Shope virus gives rise to papillomas in both domestic and cottontail rabbits; yet only infrequently can it be extracted in active state from the domestic-rabbit growths, and occasionally those of cottontails fail to yield it. Papillomas from 6 cottontail and 8 domestic rabbits, which were produced by the virus yet from which it could not be recovered, have been extracted as usual and tested repeatedly for capacity to bind complement under optimal conditions. Most of these non-infectious extracts failed to bind complement, but a few bound it in a very low titer. Extracts of the normal skin of 3 cottontail rabbits also failed to bind complement, whereas the highly-infectious extracts of their naturally occurring papillomas bound it completely on concurrent test. Antigenicity of Extracts of Non-Infectious Papillomas. Shope has found that extracts of virus-induced papillomas, which yield no active virus on extraction, will elicit virus-neutralizing antibodies when injected intraperitoneally into rabbits of homologous species. 2 He has generously sent sera from 9 domestic rabbits immunized in this way for complement-fixation tests. They bind complement when tested with suitable antigens, and their capacity to do so varies directly with their ability to neutralize the virus. The sera of 3 normal domestic rabbits failed to bind complement in the same test, as have all other normal sera tested under like conditions.

A Complement-Fixation Reaction Involving the Rabbit Papilloma Virus (Shope)

Summary A coniplernent-fixation reaction is described, with extracts or filtrates of papillonias containing infective Shope virus and antisera effective against the latter. The implications of the work are being studied.

Distinct Types of Antibodies in the Blood of Rabbits Carrying the Transplanted V2 Carcinoma

Rabbits carrying the transplanted V2 carcinoma—a squamous-cell cancer derived originally from a virus-induced papilloma—regularly develop in their blood an antibody capable of neutralizing in vitro the papilloma virus (Shope) and capable also of fixing complement in mixture with it. 1 Further studies have now shown that the virus-neutralizing and complement-fixing titers of sera procured from rabbits carrying the V2 carcinoma invariably parallel one another, and that the antibody responsible for both reactions can be readily absorbed from the sera upon admixture with the virus. From the findings it appears certain that the antibody is directed against the papilloma virus per se, and that it is identical with the antiviral antibody present in the blood of rabbits carrying benign virus-induced papillomas, which is in turn identical with that present in the blood of rabbits injected with purified papilloma virus. 2 In further serum tests, carried out by the methods used in the serological study of the Brown-Pearce tumor, 3 we have recently encountered in the blood of rabbits with the transplanted V2 carcinoma two other types of antibodies, wholly distinct from one another and from the antiviral antibody just described. When the sera of normal adult rabbits and of rabbits carrying the V2 carcinoma are heated at 56°C for 30 minutes and tested for capacity to fix complement in mixture with unheated 1:20 or 1:40 saline extracts of normal and neoplastic tissues (e. g., normal rabbit liver or kidney, V2 carcinoma, Brown-Pearce carcinoma) complement-fixation takes place in nearly all of the mixtures.∗ In general the normal sera react in dilutions as high as 1:16 to 1:32 with the normal tissue extracts, and only about half as well with the neoplastic-tissue antigens. On the other hand, sera from rabbits that have carried progressively enlarging V2 carcinomas for some weeks not only react with normal tissue extracts but are vastly more potent in mixture with the homologous antigen, generally fixing 2 units of complement completely in dilutions as high as 1:128 or 1:256 or more in mixture with extracts of the V2 carcinoma.

Development of Antiviral Properties within Lymph Nodes.

Every intradermal injection is intralymphatic and infectious agents introduced by way of a skin wound may reach the lymphatic glands directly. 1 , 2 Specific bacterial agglutinins are formed in the nodes draining the ears of mice into which killed cultures of bacteria have been injected intradermally. 3 Experiments now show that the lymph nodes elaborate a principle capable of neutralizing a virus draining to them. Rabbits were inoculated intradermally in one ear with a standard amount of vaccine virus and in the other with typhoid bacterin. Thirty-two experiments were done. In half of these the ears were amputated after an hour, while in the others the ears were left intact. After later intervals of from 2 hours to 15 days the cervical lymph nodes of both sides, inguinal nodes, bone marrow and spleen were removed and extracts of them were inoculated separately into 3 or 6 normal rabbits (as was serum procured from the same animals at the same time) to test for their content of virus (titration tests). In addition neutralization tests were set up to learn whether these extracts possessed antiviral properties. Only the extracts of the ears and cervical lymph nodes of the virus-injected side gave rise to virus lesions. A few hours after the injection of standard amounts of virus in the ears equal amounts of virus were recovered from the lymph nodes. By the second or third day an increase in virus was demonstrable in the extracts of the nodes on the virus-injected side. A rapid decline took place thereafter and by the 7th to the 9th days the extracts failed to produce lesions. At no time did the serum or extracts from other nodes or organs give evidence of virus.

A Natural Antibody Reacting with Sedimentable Constituents of Normal Tissues

While making serological studies of several transplanted rabbit cancers by the methods that disclosed a distinctive substance in the Brown-Pearce carcinoma, 1 we have noted that the blood serum of normal rabbits will fix complement in mixture with saline extracts of normal rabbit tissues in dilutions far beyond any anticomplementary or summative effects. This phenomenon appears to be due to a natural antibody that reacts with sedimentable constituents of normal tissues. The antibody can be detected by means of a standardized complement fixation test in which 2 units of complement are employed and 2 hours at room temperature allowed for fixation. Antigens are prepared by extracting normal rabbit tissues (liver, kidney, brain, etc.—either fresh or preserved frozen at −22°C) with sand in a mortar, suspending the ground paste in physiological saline (1:10 to 1:40 or more) and centrifuging at 4400 rpm for 20 minutes. The unheated supernatant liquids, opalescent but free from gross particles, have proved notably effective in the tests. When the sera of 22 normal adult rabbits were mixed with antigens consisting of 1:20 or 1:40 saline extracts of normal rabbit livers, fixation invariably occurred, and it was complete with many of the sera in dilutions as high as 1:16 to 1:64. No fixation was got when the sera of 13 rabbits less than 4 weeks old from 7 litters were tested concurrently with the same antigens. None of the sera proved anticomplementary in dilutions of 1:2 or more in concurrent tests though a few showed slight anticomplementary effect when a double volume of the 1:2 dilution was tested, and the antigens were not anticomplementary when 4 and 8 times the standard amount were employed in control tests. Experiments were next undertaken to ascertain some of the properties of the substance present in the serum of normal adult rabbits which is responsible for the fixation.