Ultramicroscopic Virus Research Articles

Landry's rabies and paralysis

In recent years, more and more generalizations have begun to be made between clinically different diseases caused by filterable and ultramicroscopic viruses. Such Schiikrin Spatz point to the similarity of the pathological and anatomical picture between rabies and acute epidemic encephalitis, which is so great that without knowledge of the anamnesis and clinical course, it is impossible to know which disease is in question.

Regulating phage therapy: The biological master file concept could help to overcome regulatory challenge of personalized medicines.

In a note published in The Lancet in December 1915, Frederic Twort described the discovery of an infectious, filterable agent that causes the glassy transformation and eventual killing of bacteria, now identified as Staphylococcus hyicus [1]. Two years later, Felix d'Herelle independently described an invisible microbe antagonistic to dysentery bacilli in a note to the Comptes Rendus de l'Academie des Sciences [2]. These two reports are the first published descriptions of bacteriophages, a term coined by d'Herelle, who foresaw the therapeutic potential of these newly identified bacteriolytic agents. > The key issue is whether phage therapy medicinal products (PTMPs) require a marketing authorization or not. Yet, phage therapy, despite varying degrees of success during the 20th century, never made it into widespread clinical use owing to the discovery of antibiotics [3]. However, the increasing antibiotic resistance among major pathogens such as Staphylococcus or Mycobacterium has become a critical public health problem—in the European Union (EU) alone, around 25,000 patients die each year from an infection with drug‐resistant bacteria (http://ecdc.europa.eu/en/publications/publications/0909\_ter\_the\_bacterial\_challenge\_time\_to\_react.pdf). Although difficult to estimate, the annual global death toll attributable to antimicrobial resistance might range from the hundreds of thousands to as much as millions in the coming decades (https://amr-review.org/sites/default/files/AMR%20Review%20Paper%20-%20Tackling%20a%20crisis%20for%20the%20health%20and%20wealth%20of%20nations\_1.pdf). In light of this growing health crisis, phage therapy has attracted renewed interest as an alternative to antibiotics [3]. ### Regulatory challenges for phage therapy However, this reemergence raises regulatory concerns. There has been intense discussion on how to classify phage‐based therapeutics within the EU's regulatory framework [4], [5], [6] with some consensus among Members States' regulatory authorities and the European Medicines Agency that these would be regulated as biological medicinal products [7]. Nonetheless, positions still vary because the current medicinal product regulation is not well suited for this unorthodox therapeutic …

ONCE MORE ABOUT PHAGE THERAPY: WHAT IS RIGHT, AND WHAT IS NOT

A little more than 100 years ago the article “An investigation on the nature of ultra-microscopic viruses” by the English physician and researcher F. Twort was published in The Lancet. Twort described the phenomenon of transparent spots which occurred on the bacterial lawn when he worked with bacterial cultures using a petri dish. He examined the spots and found zones of dead bacteria. The spots were transmissible: they appeared on other dishes once transferred to a new lawn. Twort, failing to give a convincing explanation for this phenomenon, proposed three hypotheses: it could be an unusual manifestation of the bacterial life cycle, or an enzyme produced by the bacteria themselves or some sort of “ultra-microscopic virus” which could cause the death of bacteria. Having voiced his hypotheses, Tvort let the matter drop, but many believe that with the article began the era of bacteriophages.

Better Luck Next Time: The Tesurrection of Bacteriophage Therapy

Summary Bacteriophage therapy, which was a major treatment in the first half of the twentieth century, was largely displaced by antibiotics, which proved more effective. With increased perception of antibiotic resistance amongst bacteria as a significant problem and regulatory pressures for product with minimal environmental consequences, bacteriophages have again become fashionable. Evidence is reviewed that suggests that bacteriophage therapy,when properly understood and applied, can be effective, though formal evidence is limited. However, consideration of the economic case suggests that, at best, bacteriophages will not displace antibiotics are the therapy of choice in the short or medium terms. Conflict of Interest All three authors are employed by Fixed-Phage Ltd. a company which specializes in bacteriophage immobilization. Refe rences 1. Hankin EH (1896) L’action bactericide des eaux de la Jumna et du Gange sur le vibrion du cholera. Ann Inst. Pasteur 10: 5112. Twort FW (1915) An investigation on the nature if ultramicroscopic viruses. Lancet 11: 1241.3. D’Herelle F (1917) Sur un microbe invisible antagoniste des bacillus dysenteriques. C R Acad. Sci (Paris); 165:373-375.4. Bruynoghe R, Maisin J (1921) Essais de therapeutique au moyen dubacteriophage. C R Soc Biol. 85:1120–1121.5. Eaton MD, Bayne-Jones S (1934) Bacteriophage therapy. Review of the principles and results of the use of bacteriophage in the treatment of infections. JAMA. 23:1769–1939.6. Sulakvelidze A, Alavidze Z, Morris J G (2001). Bacteriophage therapy. Antimicrob. Agents Chemother. 45: 649-659.7. Wommack KE, Hill RT, Kessel M, Russek-Cohen E,Colwell R (1992) Distribution of viruses in Chesapeake Bay Applied and Environmental Microbiology 58: 2965-2970.8. Wihelm S, Suttle CA (1999) Viruses and nutrient cycles in the sea. BioScience 49: 81-788.9. Lanning S, and Williams ST (1982) Methods for the direct isolation and enumeration of actinophages in soil. J Gen Micro128: 2063-2071.

Biology for Survival

The very acceptance of the word survival connotes the belief that extinction is possible. As individuals we expect to become extinct; as a group of organisms we hope to survive. At best calculations it is usually conceded that there must be living today at least a million species of animal life. Yet this great number is, perhaps, but a handful compared to the vast hordes that have lived and become extinct in all the past ages of the earth. The brief notations in the earth's own fossil dairy are interpreted to us by paleontologists as indicating that there have been eras in the past, some of them millions, of years in length, when certain groups were very abundant both in number of species and number of individuals. There was the age of mollusks, the age of great reptiles, the age of fishes. Thousands of species in these larger groups were abundant enough to leave many fossils but they are now extinct. How many more may have existed but have never been found? We do not know where life came from nor when it began. We do not even know if it ever began. When that statement is made to a group of students they always look up with a puzzled expression. They say that if life exists today it must have had a beginning; everything had to have a beginning. But when asked if it is not quite possible to think of life as continuing on down through the future for eternity, they agree. We have thought of this for most of our lives. If life can exist from the present to infinity in the future, why could it not have existed from the present to infinity in the past? Most persons struggle with this idea, but only because it is new to them. So we do not know where or when life began or if it began, but we do know that it has existed in myriad forms from ultramicroscopic viruses and phages to giant saurians and whales. We do know that many more forms have become extinct than have survived, and can assume that many forms now extant will become extinct. Are there reasons to believe that man will be different from the rest? Man is an organism, composed of cellular units, and chemical compounds like other organisms. Man is a mammal, a member of a special group of organisms. He has hair and milk glands, reproduces, rears his young, carries on body funetions, and dies like the others. Man is larger than most animals, but smaller than some. Man is stronger than most animals, but weaker than some. Man's life span is longer than most animals, but shorter than others. Man has many generalizations, but a few specializations. A horse has a highly specialized foot. Only one toe remains; bones have disappeared and the nail has become a hoof. These are good weapons of defense and offense and an excellent means of rapid transit over long distances. But a horse cannot play a piano or finger the strings of a violin, or paint a picture. Man cannot claw with the tiger, or bite with the wolf, or fly with the bird, or swim with the fish. He lacks the appropriate specializations to do this although he possesses the same basic structures. But he can make a sharp headed spear that will slay the tiger and the wolf; and he * Presented before the sectonal meetings of the National Association of Biology Teachers and Detroit Biology Club at Cranbrook Institute, Michigan, November 19, 1949.

OPERATIVE TREATMENT FOR BENIGN RECTAL STRICTURE (LYMPHOGRANULOMA VENEREUM)

It is generally conceded that the large majority of nonmalignant and nontraumatic strictures involving the rectum, especially those occurring in Negro women, are caused by lymphogranuloma venereum. Many articles have appeared in the medical literature describing this condition since Frei1introduced his intracutaneous test in 1925. The causative agent is thought to be an ultramicroscopic virus transmitted during coitus, which after an incubation period of one week causes a transient, painless ulcerative lesion on the genitals. Two weeks later there is edema of the lymph nodes draining the initial lesion, associated with hyperplasia of the lymphoid tissues, fibrosis and abscess formation. These secondary manifestations vary with the sex of the patient and the location of the primary lesion. Martin and Bacon2in their detailed report of this condition stated: There exists a dense network of lymphatics in the external genitalia and anorectal region of both the male and

Sedimentation of Poliomyelitis Virus by Means of a Vacuum Ultracentrifuge.

This is a brief preliminary report of results indicating that we have been successful in sedimenting poliomyelitis virus, one of the smallest of the ultramicroscopic viruses,1 from clear aqueous suspensions by means of ultracentrifugation in vacuum. The machine employed is similar in design to that recently described by Bauer and Pickels,2Thus far we have obtained complete results on 2 experiments. In both, the material subjected to ultracentrifugation consisted of a 25% suspension of glycerinated pooled virus cords in physiological saline. The lipoids in these suspensions were removed by ether extraction and the aqueous fraction was centrifuged in an Angle centrifuge at 3000 r.p.m. for at least an hour. In the first experiment the resultant fluid was water-clear to the eye; in the second, very slightly opalescent. Eight cc. of the clear suspension were placed in each of a series of celluloid tubes seated in the rotor.In the first experiment, the rotor was in motion for a total of 4 hours. Stroboscopic de...

ZINC SULFATE PROPHYLAXIS IN POLIOMYELITIS

The increasing number of inquiries coming from physicians in various sections of the country makes it desirable to publish at this time a note on the results of recent work on chemoprophylaxis in experimental poliomyelitis and to suggest a procedure which may hold something of practical value in controlling the spread of this disease in man during epidemic periods. The more fundamental aspects of the disease which initiated these studies have been summarized by one of us in an earlier article inThe Journal.1The chief facts that need to be carried in mind to follow the logic of this new approach are briefly as follows: The disease is caused by a highly neurotropic virus, which in size is among the smallest of known ultramicroscopic viruses.2 In monkeys, and probably in man also, the virus reaches the central nervous system by way of the olfactory nerve. Complete

Control of Rabbit Infestation by the Use of a Virus

OF all the pests from which the pastoral industry in Australia has suffered, rabbit infestation is prob ably the most serious economically, and little per manent success seems to follow the methods of control in use at present, such as trapping and poisoning. One hope would appear to lie in the discovery of some infectious disease, deadly to the rabbit but innocuous to man and other animals, and easily communicable to, and spreading widely by natural means among, the rabbits. Sir Charles Martin, as a result of an experimental inquiry, suggests that a disease ‘rabbit myxomatosis’ may fulfil these requirements (Commonwealth of Australia. Council for Scientific and Industrial Research, Bull. No. 96. Melbourne, 1936). This is an infectious, highly fatal, febrile disease caused by an ultra-microscopic virus, causing mucinous discharges, swellings and tumours in affected animals, fatal within twelve days or so, transmitted sufficiently by contact, and with a fatality of almost a hundred per cent. It attacks animals of the genus Oryctolagus, to which wild rabbits in Europe and Australia, and domesticated rabbits in Europe and America, belong; but it does not affect the indigenous rabbits of America, or man and other animals and birds. Experiments were conducted by Sir Charles Martin in a pen measuring 50 yards x 10 yards, with both tame and wild rabbits. A colony having been established for three weeks or thereabouts, one or two rabbits inoculated with the disease were introduced. With colonies of 27 and 30 tame, and 55 and 44 wild, rabbits, the fatality was 99-6 per cent among the tame, and 100 per cent among the wild. The disease originally came from epizootics among tame rabbits in South America.

Further Investigations on the Nature of Ultra-Microscopic Viruses and their Cultivation

Further Investigations on the Nature of Ultra-Microscopic Viruses and their Cultivation

Sir Patrick Laidlaw, F.R.S.

SIR PATRICK LAIDLAW has been appointed by the Medical Research Council to be deputy director of the National Institute for Medical Research, and head of the Department of Pathology and Bacteriology, in succession to the late Capt. S. R. Douglas. Sir Patrick has been a member of the Council's scientific staff at the National Institute since 1922, before which he was lecturer in pathology at Guy's Hospital. He received the Royal Medal of the Royal Society in 1933, and was knighted in 1935. He has latterly been engaged chiefly in the investigation of diseases of man and animals due to infection with ultra-microscopic viruses. His successful work on the cause and prevention of dog distemper, supported by the Field Distemper Fund, is well known. More recently he and his colleagues have demonstrated the presence of a virus in human influenza, and have opened up an experimental line of attack upon this disease by which it is hoped to obtain important results.

STUDIES ON THE ETIOLOGY OF SPONTANEOUS CONJUNCTIVAL FOLLICULOSIS OF RABBITS

Before summarizing the experimental results presented herewith, a brief review is indicated of our study on follicular conjunctivitides existing in the different species of animals thus far examined. Spontaneous follicular conjunctivitis of monkeys and chimpanzees, which resembles the early, or follicular, stages of human trachoma, has been shown to be an infectious disease, and to arise from the action of a bacterium and not an ultramicroscopic virus (2, 10). A new species of organism, designated tentatively as Bacterium simiae, n.sp., and capable of reproducing granular lids in normal monkeys and apes, has been recovered from cultures of simian folliculosis tissues. This organism, although of a different species, is of the same genus as Bacterium granulosis which is associated with human trachoma (3). The studies were extended beyond folliculosis in monkeys and apes to a similar disease widespread among stock rabbits. Its clinical appearance and microscopic changes resemble the spontaneous malady in simians with some minor differences (1). Like the latter, rabbit folliculosis was also shown to be infectious and contagious. In the present paper a bacteriological investigation of the rabbit affection is described. In the experiments undertaken we followed the method used by Noguchi in his studies on trachoma, that is, the different organisms recovered from normal and affected conjunctivae were inoculated into the conjunctivae of selected, quarantined rabbits. Among the bacteria thus isolated from folliculosis tissue a new species was disclosed that brought about conjunctival reactions apparently indistinguishable either from the disease seen in nature or from the experimental infection induced by inoculation of folliculosis tissue. The organism was specifically active in rabbits whether injected subconjunctivally or instilled into the conjunctival sac. Furthermore, the special bacterium has thus far been cultivated only from folliculosis cases and not from other forms of conjunctivitis nor from normal tissues. Finally, specifically pathogenic strains of the identical microorganism have been recovered from rabbits experimentally infected with the bacterium. A distinctive feature of this bacterium is that it is of the same genus as the microorganisms heretofore designated as Bacterium granulosis and Bacterium simiae. It is clearly evident then that the three bacteria cultivated to this time form a new genus, for which we propose the name Noguchia. The diagnosis of the genus has already been described in this paper, but as was also shown in a foregoing section, certain specific characters differentiate the three. Accordingly, we suggest a change in the names of the granulosis and simiae species to the proposed binomials, Noguchia granulosis, n.sp., and Noguchia simiae, n.sp. The specific organism, having an evident causal relationship to rabbit folliculosis, can therefore be placed in this classification, with the name of Noguchia cuniculi, n.sp. In conclusion it would appear that in three types, at least, of follicular reactions in the conjunctiva-in man (the primary lesion of trachoma consisting essentially of follicles), in the simian, and in the rabbit-there is an intimate association of microorganisms having the same generic, but different specific properties.

STUDIES ON THE ETIOLOGY OF SPONTANEOUS CONJUNCTIVAL FOLLICULOSIS OF RABBITS

Spontaneous conjunctival folliculosis is widespread among various species of rabbits. It exists in two forms: Type I, in which the lesions are localized and the disease is relatively inactive, and Type II, in which the follicles are closely distributed over the entire surface of the conjunctivae and the affection is more active and characterized by extensive inflammatory reactions. One type can be converted into the other either by experimental methods or by natural processes. The disease can be transmitted from rabbit to rabbit by means of subconjunctival inoculation of suspensions of the affected tissues or by instillation of such material into the conjunctival sac, or even by mere contact of folliculosis animals with rabbits having smooth conjunctivae. It is plain that the disease is an infection. The causal agent of the infection is not filtrable through Seitz discs that retain Serratia marcescens nor through Berkefeld V candles that permit the passage of this organism. Furthermore, the lesions of the spontaneous or of the experimental disease do not exhibit the cytotropic effects or the inclusion bodies suggestive of the action of an ultramicroscopic virus. They are characterized, on the other hand, by a persistent and progressive chronicity and show certain resemblances to the granulomata. The evidence suggests that the spontaneous conjunctival folliculosis of rabbits is due to a microorganism-one having a low grade pathogenic action. In a paper shortly to be published, a bacterium capable of reproducing folliculosis in normal rabbits will be described.

A Paper on POLIOMYELITIS

A notable advance in knowledge may raise as many questions as it solves, and a period of intensive and fruitful research may at first sight seem to leave us as far as ever from the solution of the problem we are in vestigating. This might plausibly be said of poliomyelitis. Few affections of the nervous system have been more closely or successfully studied within the past few years, yet fresh pathological and epid?miological factors appear for elucidation, effective therapy remains as remote as ever, and many of our earlier certitudes have gone by the board. Yet we must necessarily be nearer a grasp of the many problems presented by poliomyelitis, and the present time is appropriate for a review of some recent advances in our knowledge. A comprehensive summary of these is not possible within the limits of my present opportunity, and I propose, therefore, to confine myself to a brief consideration of the question of the specific treatment of the disease in the light of our rapidly changing conception of its nature. By way of definition we may say that poliomyelitis is one of a small group of infections of the nervous systems due to the action of ultra-microscopic viruses, the morpho logical identities of which are unknown to us. Other examples of the group are epidemic encephalitis and rabies, and in all three it is the grey matter which is the point of attack, whence these viruses are spoken of as polioclastic. Of the characteristics they have in common perhaps the most striking are that they multiply only in contact with the living cell, within which their life and activity appear to be very brief, for after a short period they disappear. To this disappearance the name auto-sterilization has been applied, and in consequence of it virus diseases may be spoken of as self-limiting.

The photodynamic action of methylene blue on certain viruses

In a previous communication (Perdrau and Todd, 1933), dealing with the photodynamic action of methylene blue on bacteriophage, we have recorded the results of experiments confirming the observations of Clifton (1931), both as regards the part played by visible light in bringing about the reaction and also as regards the necessity for the presence of free oxygen. In the course of these experiments it was found that while the free phage is highly sensitive to the photodynamic action of methylene blue, it becomes very much more resistant to the dye in the presence of the corresponding living bacterium. This protection of the phage is only exercised by living bacteria, killed organisms being without effect. It is, moreover, not a strictly specific phenomenon, as protection is in some cases brought about by bacteria other than those for which the phage has been prepared. In view of the many similarities of behaviour shown by the bacteriophage and the ultramicroscopic viruses, it seemed not improbable that some of the latter might be susceptible to the photodynamic action of methylene blue, and tests were accordingly made with such viruses as we had at our disposal.

Prevention of Distemper

THE inquiry into the causation and prevention of dog distemper started in 1922 has now been brought to a successful issue by the Field Distemper Council, Dr. P. P. Laidlaw and Mr. G. W. Dunkin, the Medical Research Council and the staff of the Wellcome Foundation at Beckenham. The disease has now been accurately defined and distinguished from other dog illnesses with which it used to be confused; it has been shown to be due to an ultramicroscopic virus and efficient methods of prevention have been worked out in the laboratory and confirmed in large-scale practical trials on packs of foxhounds and other dogs. Dogs are first given an injection of an emulsion of the organs of an animal which has died of acute distemper in which the virus has been killed with dilute formalin; in response, the animal develops a moderate degree of resistance which makes it possible, a fortnight later, to give it a dose of live virus which stimulates the animal to become definitely immune. The blood serum of such immune animals can protect against the virus and is of use in treating cases of the disease and also in making a vaccinating mixture with live virus, which has the practical advantage of needing only one dose instead of two. These immunological reactions also make it possible to identify the disease with far greater certainty, and the new methods of prevention are available for ferrets, silver foxes, fitches, thinks, etc., in which it has been found to occur.

A Concept of the Ultramicroscopic Virus Diseases and a Classification

A Concept of the Ultramicroscopic Virus Diseases and a Classification

Microbes and Ultramicrobes: an Account of Bacteria, Viruses and the Bacteriophage

DR. GARDNER has added another good biological book to this excellent series. He does not attempt to deal with the whole of microbiology but, after a rather scrappy chapter on the structure and functions of bacteria, he gives good and stimulating accounts of bacterial variation, the ultramicro-scopic viruses and the bacteriophage, as up to date as any version of these moving topics can be and adapted for the general biologist as well as the technical specialist. There are a few mistakes; for example, distemper vaccine is not made in guinea-pigs (p. 65). Mr. de Beer adds an appendix pointing out the analogy between genes and viruses.

Recent advances in the study of poliomyelitis

Summary 1. The evidence at present indicates that the virus of poliomyelitis is a true ultramicroscopic virus, probably less than 50 μμ. in size. 2. The virus apparently does not yield to cultivation on lifeless culture media, but like other ultramicroscopic viruses is regenerated only in the presence of living tissue cells. 3. As an antigen the virus calls forth antibodies resembling antitoxins more closely than antibacterial antibodies. This suggests that virus may be an aggregate much simpler in composition than a bacterial cell. 4. The pathogenicity of the virus is restricted to man and certain species of monkeys. 5. The virus is highly selective in directing its attack, not only restricting its attack to certain hosts but to certain special cells within these hosts. After reaching the olfactory bulb from the nasopharyngeal vault, it seems to travel exclusively by nerve axons to different levels of the central nervous system, apparently remaining in all stages of the disease in very intimate association with nerve cells. 6. The immunity following recovery from poliomyelitis is of a solid and durable character. This may be determined in part by a persistence of some virus in the tissues of the recovered individual. 7. There is evidence that a large percentage of the adult population has acquired an immunity to poliomyelitis by natural means, probably as the result of a subclinical infection. 8. The value of serum therapy continues to remain an unsettled question, the general impression being that it is of some value if used early enough in the disease. 9. In addition to human convalescent serum for therapeutic purposes, there has become available the serum of certain normal human adults not known to have had the disease, and also the serum of individual poliomyelitis refractory animals which have been subjected to virus injections over a period of time. The serum of individual sheep, goats, and especially horses may attain an antibody value comparable to that of the average poliomyelitis convalescent serum. 10. Actual solution of the poliomyelitis problem seems to rest on a systematic active immunization of children and young adults by appropriate methods still to be worked out. Such preventive measures also call for some convenient, inexpensive, and reliable means of detecting those who require artificial immunization.

The Cause of Cancer

DR. W. E. GYE,* rightly we believe, thinks that the problem of the causation of cancer is at present most likely to be solved by studying the malignant tumours of fowls first described by Rous twenty years ago. Ordinary mammalian tumours, either arising spontaneously or induced by tarring or other irritating procedures, can be transmitted from one animal to another only by transplanting living tumour cells. The Rous tumours are peculiar in that they can be transmitted by means of dead cells, by cell-free extracts, and by such extracts after they have been passed through fine filters which will detain ordinary bacteria. In other words, the filtrates contain some carcinogenic agent which resembles an ultra-microscopic virus, and Drs. Gye and Purdy now give an account of the multitudinous experiments which they have made in an attempt to ascertain the nature of this agent.