Blind Passage Research Articles

Characteization and distribution of two new foamy viruses isolated from chimpanzees.

Pan 1 and Pan 2 viruses are persistent viruses recoverable from many organs, including brain, of most chimpanzees; both may be present in the same organ of a given animal. These viruses induce a vacuolated, foamy syncytia without inclusion bodies both in chimpanzee explant cultures and in primary human embryo kidney (HEK) cells. Both viruses are not inhibited by IUDR. are chloroform and pH 3.0 sensitive and are inactivated following exposure to 45° C for 30 minutes. Pan 2 virus, the only one tested, is sensitive to visible light. Neither virus has yielded a detectable hemagglutinin, nor has hemadsorption of guinea pig RBC been observed. Direct and blind passage inoculations have not induced infection or evidence of viral replication in small laboratory animals. By electron microscopy they are indistinguishable from each other and are approximately 125 nm in diamter. Viral antigens are demonstrable inin vitro cultures of chimpanzee tissues as well as infected HEK cells by direct fluorescent antibody staining.

Phlebotomus fever in Egypt. Isolation of phlebotomus fever viruses from Phlebotomus papatasi.

Nineteen strains of Sicilian-type and two strains of Naples-type phlebotomus fever virus were recovered from wild-caught Phlebotomus papatasi collected in Cairo. Egypt. Maximum virus transmission appears to occur in August and September, with some activity extending to November. In a comparison of source materials for virus isolation, nonengorged specimens were superior to those containing blood. Suckling mice were a suitable host for primary isolation of both virus types; blind passages and lengthy courses of adaptation to this host were not required. The recovery of Sicilian-type virus from male sandflies was interpreted as evidence for transovarial passage of the virus.

Surgical management of diverticulum of the canaliculus.

DIVERTICULUM of the canaliculus with the development of purulent inflammation has been previously described.¹The following case not only represents this condition, but a rational, yet simple surgical approach based on a known general surgical principle, ie, that of eliminating a blind passage. A 22-year-old white woman stated she had had epiphora of the right eye since birth. At age 12 years, she had her first probing of the right nasolacrimal duct. She states that the epiphora did not improve, and over the next ten years she had many (approximately 50) repeated attempts by probing to cure her epiphora. Examination in our clinic was normal in all respects with the following exceptions: (1) Epiphora was not present. (2) Pressure on the right lower lid resulted in a white creamy material being expressed from the right lower canaliculus. (3) Irrigation of the right lower canaliculus resulted in regurgitation of material.

A study on sampling and isolation methods for the detection of virus in sewage

Dip samples were found inferior to swab samples especially for detecting virus in sewage plant effluents. Sponges exposed for 24 hours were found at least as efficient as 48 hours' swabs and as gauze swabs. Double sampling revealed many virus mixtures. The two-hour adsorption technique by the inoculation did not reveal more virus positive samples than one fifth the volume, which was not removed. Two blind passages were found useful, and as single celltype for isolations HeLa cells were found superior to monkey kidney cells.

A Naturally Occurring Latent Bedsonia Infection in Guinea-Pigs

SUMMARY By serial blind passage in chick embryo yolk sacs, a Bedsonia infection was demonstrated in four out of fifteen animals from a closed conventional guinea-pig colony. Serological examination by the complement fixation test failed to detect infected animals. The implications of these findings on the use of guinea-pigs for the isolation of Bedsonia in diagnosis and the possible source of the infection are discussed.

Susceptibility of chickens, mice and chick embryos to Sindbis, Murray Valley encephalitis and Getah viruses

The sensitivity of various host systems to Murray Valley encephalitis virus (MVE), Sindbis virus and Getah virus was compared using viruses passaged in mice and chick embryos. The methods used were intracerebral inoculation of suckling mice and day-old chicks, and inoculation by the chorio-allantoic membrane, allantoic cavity and yolk sac of chick embryos. The most sensitive system for mouse-passaged Sindbis and MVE viruses was inoculation of 10-day-old chick embryos onto the chorio-allantoic membrane. Mouse-passaged Getah virus had no infectivity for chick embryos inoculated by the yolk sac or allantoic cavity, or for chickens inoculated intracerebrally, but focal lesions were produced on the chorio-allantoic membrane after inoculation by this route. The most sensitive system for this virus was the inoculation of mice intracerebrally. Egg-passaged Sindbis and MVE viruses gave results similar to those obtained with mouse-passaged viruses. Getah virus could not be recovered from the allantoic fluid after 5 blind passages by the allantoic cavity route in chick embryos and does not appear to multiply in this site.

Interaction between human and simian adenoviruses in simian cells: Complementation, phenotypic mixing and formation of monkey cell “adapted” virions

Prototype strains of human adenoviruses types 1–3, 5–8 do not multiply in GMK cells and could not be adapted to them during several blind passages. Simian adenoviruses SA7(C8), SA7(B105), SV32, and SV38 can serve as helpers for replication of human adenoviruses in this system. Simultaneous infection of GMK cells with human and simian adenoviruses leads to the replication of both viruses. As a result of phenotypic mixing homologous and heterologous antigenic determinants are incorporated in the capsid of most virions. In the virus yield obtained after mixed infection, the appearance of virions with human adenovirus capsids capable of replicating in GMK cells was demonstrated. Such virions seem to be formed as a result of “hybridization” of original adenoviruses.

Induction of bone marrow colony-stimulating activity by a filterable agent in leukemic and normal mouse serum.

1. Leukemic Swiss mice of ICR/Ha strain which had been injected at birth with a lymphoid-leukemia-inducing virus preparation yielded sera which produced elevations of serum colony-stimulating activity within 16 hr and significant plasma-LDH-enzyme elevation at 4 days when injected intraperitoneally into normal ICR/Ha Swiss mice. Colony-stimulating activity was assayed in vitro by the stimulation of hemopoietic colony formation by DBA/1 bone marrow cells. 2. The inducing agent in leukemic serum was passageable, filterable, sedimentable, and heat-, ether-, and UV-labile. 3. A similar agent was recovered from normal Swiss serum after blind serial passages through normal mice. 4. LDH elevating virus induced a similar elevation of serum colony-stimulating activity when injected at high titers, and cross-resistance was demonstrated between LDH virus and the passaged leukemic serum agent.

Fluorescent Antibody, Complement Fixation Method for Detection of Dengue Viruses in Mice

Summary The complement fixation fluorescent antibody (CF-FA) test was used for the detection of several types of dengue viruses, using serum of types 1 through 4, TH-36 and TH-Sman, in dilutions comparable to those of the conventional CF tests employed in this laboratory. Two-way cross-reaction experiments were done to determine the extent of CF-FA staining among dengues 1 and 3, Japanese B encephalitis, Murray Valley encephalitis and Eastern equine encephalomyelitis arboviruses and their respective antisera. Although cross-reactions could be observed among certain group B agents they did not cross with the representative of group A. It was also shown that the CF-FA test could detect inapparent dengue infection in two early (blind) passages of the unadapted dengue type 3 prototype strain.

Microbial persistence. I. The capacity of tubercle bacilli to survive sterilization in mouse tissues.

A previously reported form of microbial persistence whereby large populations of tubercle bacilli can be made to "vanish" uniformly from the tissues of mice has been shown to occur generally throughout each group of animals subjected to the experimental procedure; it does not reflect the eradication of the bacilli in the majority of animals with their persistence and ultimate revival in only a minority. The one demonstrable alteration of the tubercle bacilli while "vanished" is that they are sterile. Thus, they are undetectable by cell-free culture, tissue culture, and blind animal passage, i.e. by any method based on microbial multiplication. Whether they have also undergone alteration in morphology and persist in some unconventional form cannot be stated. Acid-fast forms similar to tubercle bacilli can be detected in small numbers by intensified microscopic search of tissue homogenates but the relationship of these forms to the sterile bacilli that ultimately revive is unclear. Thus, the persisting tubercle bacilli are more correctly designated as being in a "sterile state" than one of true latency. The uniform induction of the sterile state is a specific phenomenon requiring the participation of both the nicotinamide derivative, pyrazinamide, and isoniazid. Once assumed, this sterile state is relatively stable and the time required for revival of the tubercle bacilli in the spleens in one-half the animals is seven months. This process can be speeded up by the administration of large doses of cortisone in the third or fourth month after sterilization but revival is not significantly affected by the administration of cortisone earlier.

MORPHOLOGICAL AND BIOLOGICAL STUDIES ON A VIRUS IN CULTURED LYMPHOBLASTS FROM BURKITT'S LYMPHOMA.

Lymphoblasts of two tissue culture strains (EB1 and EB2) from different biopsy specimens of Burkitt's lymphoma have been examined in thin sections by electron microscopy, and have each been found to carry a morphologically identical virus. The virus was observed in samples taken over many months, being present in about 1 to 2 per cent of the cells in two forms: Immature particles about 75 mmicro in diameter which were seen in both the nucleus and cytoplasm; and larger mature particles with a diameter of 110 to 115 mmicro, which were either within membrane-bounded cytoplasmic spaces or at the cell surface. There was some indication that the particles matured by budding through the cytoplasmic membranes. Both types of particle occurred in dead degenerating cells or, less frequently, in intact altered cells. The characteristic alterations of the latter included margination of the chromatin, fragmentation of the nuclear envelope, beaded opaque material in the mitochondria, and, with one of the cell strains (EB1), sheaves of altered spindle tubules. All attempts to isolate and identify the virus carried by the two strains of lymphoblasts failed. No pathological effects were caused in 8-day chick embryos inoculated either with whole lymphoblasts or extracts of disrupted lymphoblasts, using the intraallantoic, amniotic, and chorioallantoic routes, and the extraembryonic fluids of such chicks were without haemagglutinating activity for human, chicken, guinea pig, or monkey erythrocytes. Whole lymphoblasts or lymphoblast extracts were likewise without effect when inoculated intraperitoneally into newborn hamsters or two strains of newborn mice. Similar lymphoblast inocula did not cause detectable changes in 9 different test tissue culture systems even after 8 blind passages. The nature of the unknown, unidentified virus in the cultured lymphoblasts from Burkitt's lymphomas is considered and its possible relationship to the cells discussed.

THE ECHO 28-RHINOVIRUS-CORYZAVIRUS (ERC) GROUP OF VIRUSES.

During the past two years (1-5) we have been studying the properties of a new group of viruses which we isolated from the respiratory secretions of adults with common colds and from children with colds or lower respiratory tract involvement. The strains were designated as coryzavirus. This advance was made possible by employing the newly established WI -26 strain of diploid human fetal lung cells developed by Hayflick and Moorhead (6) of the Wistar Institute of Anatomy and Biology. All the agents of the coryzavirus group propagated well and caused marked cytopathology in the WI -26 strain of diploid human cells. All strains were found also to propagate readily and to cause cytopathic changes in KB and in HeLa cell cultures. Indeed, we have found recently that KB cells are as good as, and possibly are superior to, WI-26 cells for primary isolation of these agents. However, varying numbers of blind passages must be made in KB cells before cytopathic effects become obvious. Only one of the coryzaviruses (type 30) propagated and caused cytopathic changes in cell cultures of grivet monkey kidney. Antiserum was prepared in guinea pigs against 35 strains of coryzavirus recovered from adults and children. The strains were separated into 20 distinct immunologic types, based on crossneutralization tests, which have been designated types 11 through 30. The immunologic typing with guinea pig antiserum was confirmed, for all of 10 of the types tested, using paired serum from humans with coryzaviral infection. None of the 20 coryzaviral types were related immunologically to any of the six serologic types of rhinovirus reported by Taylor-Robinson and Tyrrell and their associates (7-9)., or to any of the prototype ECHO, Coxsackie, or reoviruses. Although unrelated immunologically, it was shown that type 28 ECHO virus, the rhinoviruses, and the coryzaviruses share certain biochemical and biophysical properties which provide a basis

Anomalous clinical pictures of varicella.

THE clinical picture of varicella, like that of other infectious diseases, presents wide variation. Besides the known irregularities in rash distribution, anomalies of the prodromal period and of the exanthem have been described (Schamberg and Kolmer, 1928a; Ronaldson and Kelleher, 1938). In the present paper, the relevant features of five cases are presented and the apparent association of the anomaly with a previous skin condition is pointed out in three cases. All five cases were observed between 1954 and 1955 when no laboratory technique was available for identification of varicella virus or antibody. In three cases, fluid collected from vesicles was inoculated on the chick-embryo chorioallantois after treatment with a mixture of penicillin and streptomycin. Reinoculations of the original inocula and serial blind passages were made to eliminate the possibility of variola virus being the agent involved since this virus may sometimes fail to provoke typical lesions (MacCallum, I954). Besides, the failure to find chorioallantoic lesions supported the clinical diagnosis since varicella virus does not provoke lesions (Irons, Bohls, Cook and Murphy, I941; MacCallum, 1954). Biopsies of representative skin lesions were made in two cases and the histological findings were interpreted in the light of recent reviews of the pathology of viral pocks (Lever, 1949; McNair Scott, Blank, Coriell and Crouse, I950).

Appearance of Persistently Cytopathic Eastern and Western Encephalitis Viruses after “Blind” Passage in Cultures of L Mouse Fibroblasts

Summary Eastern and Western encephalitis viruses (EE and WE) were successfully passed serially in cultures of L mouse fibroblasts in 11 of 16 and 7 of 8 attempts, respectively. “Blind” passage of virus was often required for development of a persistently cytopathic effect since although mouse brain suspensions usually destroyed cells initially, cytonecrosis was commonly not evident during second to fourth serial passages even though mouse infectious virus was produced. Thereafter, each virus became persistently cytopathic and readily propagated through 7–20 (EE) and 7–37 (WE) serial passages. These findings emphasize the potential value of blind passage of specimens in cell cultures during attempts to isolate viruses from natural sources. Selection of cytopathic virus from the original mouse brain suspension is considered the most probable explanation of these observations, although other possibilities are discussed. The occasional failure of EE virus to propagate and the common disappearance of cytopathic virus effect during second to fourth serial passages of either virus remain unexplained; they seemed unrelated to the serum (rabbit, human, chicken or sheep) used to maintain cells, to duration of the interval between virus inoculation and harvest, and to autointerference. The latter phenomenon did not explain adequately the absence of definitive cytonecrosis during blind passages of virus, since interference could not be regularly produced upon simultaneous inoculation of L cell cultures with infectious and heat- or ultra-violet-inactivated WE virus.

Properties of a transmissible agent capable of inducing marked DNA degradation and thymine catabolism in human cell.

SummaryThe appearance of a transmissible cytopathic agent during the first blind passage of pooled human “liver” cultures previously infected with 4 acute infectious hepatitis bloods is described. The infectivity of this agent in the “liver” culture was cell-associated. The infective degenerated “liver” cells retained their infectivity for at least 16 weeks at 4°C and 8 weeks at 36°C. Heating for 1 hour at 56°C failed to destroy completely the infectivity of infected mouse fibroblasts. One cycle of freeze-thawing or homogenizing, however, abolished the infectivity with occasional exceptions. Characteristic cytopathology consisted of cytoplasmic granularity, cell rounding followed by diminution of cell volume leading to morphologic picture of shrunken cells. During an intermediate stage of degeneration, characteristic intranuclear basophilic globules were seen regularly; the globules were Feulgen positive. The established cell line was more susceptible to the destructive action of this agent than primary c...

Trachoma viruses isolated in United States. I. Growth in embryonated eggs.

SummaryFrom 6 active trachoma cases in the United States strains of“trachoma virus”have been isolated. Conjunctival scrapings suspended in broth-saline containing 1-10 μg/ml streptomycin were inoculated into yolk sac of embryonated eggs. From 1 to 6 blind passages were required for establishment of strains which subsequently reached titers of 106.3-107.4 egg LD50/ml. The essential nature of ill-defined egg“quality”for isolation of“trachoma viruses”is discussed. The isolated agents produced unequivocal eye infections in M. Cynomolgus and in a human volunteer.

Darstellung einer Variante des Encephalomyelitis-Virus (Theiler) durch Adaptation an extraneurales Gewebe

A variant of Theiler Encephalomyelitis Virus (TEM) was obtained by adaptation to embryo mouse fibroblasts. After the 22nd passage three serial plaquepurifications were performed and the amount of the virus increased by a further passage. The variant is characterized by a remarkable decrease in pathogenicity, by a change in the symptoms of the disease and by a prolongation of the period of latency. The identity of the adapted virus strain with the original one could be proved by neutralization tests. Further propagation of the adapted virus under the conditions of the original environment by serial brain passages showed, that the new properties developed in tissue culture remained constant. This as well as the cytopathogenic effect, suddenly arising after 9 blind passages, indicated the existence of a genotypic adaptation.

Epidemic neuromyasthenia; clinical syndrome.

Viral and Bacteriologic StudiesThe nature of these illnesses has suggested to most a viral etiology, but, despite intensive efforts to implicate such an agent, results have been meager. Cerebrospinal fluid, feces, throat washings, acute-phase serums and blood clots have been inoculated by a variety of routes into many different laboratory animals and various tissue-culture systems. Despite the frequent use of blind passages, attempts to infect the following animals have thus far proved futile: sucklingl,8,10,14,15,26,29–31,34 and adult mice8,14,26,29,31; guinea pigs with8 and without cortisone10,26,31; hamsters8,10,14,26; rabbits26,31; rats with and without cortisone15; ferrets8; and cynomologus . . .

Viruses in health and disease.

VIRUSES IN HEALTH AND DISEASE C. H. ANDREWES, M.D., F.R.C.P., F.R.S.* Ofthe legion ofbacterial species in man's external and internal environment , only comparatively few ever cause disease. This is something familiar to all. Most of the bacteria in question are saprophytes or harmless commensals. Some in invertebrates may be symbiotes, often living in specialized organs; ifthey are eliminated, the host may die. Many ofthem have an intracellular habitat. Viruses differ from most bacteria in that they cannot multiply except within cells. They can, therefore, hardly be imagined to function as saprophytes or commensals. They are essentially parasites and can exist within a healthy animal only if their parasitism has come so completely under control that they can do no harm which we can detect. It is accepted by most people that, on a long-term basis, violence ofreaction between host and parasite benefits neither and that evolution tends toward a state of mutual tolerance. It is the purpose of this essay to consider whether, despite their obligate parasitism, there may not be, in fact, far more harmless viruses in animals and plants than pathogenic ones. As the great bulk ofan iceberg is under water, so the vast majority ofviruses may be submerged , too. Until lately, all the viruses we knew ofhad been discovered as a result of research directed to revealing the causes of disease. It is still true that all the viruses we know have come to light because ofthe work ofanimal and plant pathologists. No longer, however, do we believe that all the viruses we unearth in our researches are the ones we are looking for. Too many people have found "incidental" viruses which have led them astray. I. Terminology The subject of latency of virus infections has been confused because different workers have used the words "latent," "inapparent," "masked," * The National Institute for Medical Research, Mill Hill, London, N.W. 7. 211 and so forth in different senses. A symposium (i) was held in Madison, Wisconsin, in September, 1957, to clarify ideas in this fieldand, more especially , to establish uniform meanings ofkey words. There was good agreement concerning the suggestions which follow. Inapparent infections embrace all infections whose signs and symptoms are not overt. It was recognized that many workers, particularly in human medicine, would continue to use "subclinical" as an alternative to "inapparent ." Latent infections are inapparent infections which are chronic, with some measure ofequilibrium established between host and parasite. The term "latent virus" is undesirable; if the adjective is used to qualify both "virus" and "infection," it will inevitably be used in different senses. "Masked" and "masking" have been used in so many different ways that continued use ofthe terms should not be encouraged. Instead, occult virus can describe virus which cannot be recognized by direct tests and the cause ofwhose elusiveness is at present not known; specific terms can describe the cause when it has been elucidated. Meanwhile, we can call a virus occult when it fails to be revealed in a host because it is either qualitatively altered, present in minute quantity, or mixed with antibody or inhibitor —and we do not know which explanation applies. "Moderate" was suggested , at the cellular level, to describe a virus which, in aparticular system, infects a cell without killing it or preventing its multiplication; in contrast, cytocidal describes a virus lethal for a cell. The words would roughly correspond to "temperate" and "virulent" phages. Other terms that were discussed are not relevant to the subject ofthis essay. II. The Story of Virus III The writer had the advantage ofbeing concerned with one ofthe first "incidental" animal viruses to be discovered. Like many another virus in later years, it was brought to light by workers using the technique of making serial passages of organ suspensions inoculated into the same species. Rivers and Tillett (2) were trying to transmit chicken pox to rabbits. They inoculated varicella material into rabbit testes and after a few days removed these and injected suspensions ofthe ground-up organs into testes of other rabbits. After a few such "blind passages," they obtained a filterable agent which produced acute orchitis and fever, transmissible in series. Histological study...

The Propagation and Cytopathogenic Effect of an Egg-Adapted Strain of Infectious Bronchitis Virus in Tissue Culture

NFECTIOUS bronchitis (IB) is an acute and highly infectious respiratory disease of chickens. Economic losses are due to mortality and retardation of growth in young chicks and loss of production and egg quality in layers. Shalk and Hawn first described the disease and Bushnell and Brandly2 demonstrated its viral etiology. Beaudette and Hudsonl propagated the virus in embryonated eggs and Jungherr and Terrell7 demonstrated parental immunity in chicks. Jungherr et al.8 showed the existence of two antigenically different types of IB virus. Fahey and Crawley5 propagated IB virus in chicken-embryo tissues and monkeykidney cells but could not demonstrate a cytopathogenic effect (CPE). Buthala and Mathews3 could not show any CPE on a single passage of IB virus in chicken-embryo-kidney cells. In this work the growth of IBV virus in tissue culture was investigated by using serial blind passages in an attempt to produce CPE. This characteristic of the virus would be an added tool in the study of the virus.