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Characteristics on the adenoviruses. III. Reproductive cycle of types 1 to 4
Abstract
The strains of types 1 to 4 adenoviruses studied attained maximum combination with host cells, strain HeLa, in 5 to 6 hours. During this adsorption period approximately 75 per cent of the total virus became associated with the host cells. Following adsorption of virus to HeLa cells, these agents underwent cyclic multiplication periods similar to bacterial and other animal viruses. The latent or eclipse period of the multiplication cycle for types 1 and 2 viruses was 17 hours, whereas the types 3 and 4 agents had a latent period 14 to 15 hours in length. The rate of viral propagation during the incremental period was very similar for types 1, 2, and 4 viruses, but was slower for the type 3 agent. During the incremental period of viral propagation newly synthesized virus was not released into the fluid phase of the culture and even after 6 days of viral multiplication when marked cytopathic alterations of the infected HeLa cells had occurred, the spontaneous release was only 2 to 6 per cent of the total virus detectable.
... The effects of proflavine on the synthesis of soluble antigens and infectious adenovirus, type 5, are considered in this communication. MATERIALS 84,1962 SYNTHESIS OF ADENOVIRUS AND SOLUBLE ANTIGENS tubes as previously described (Ginsberg, 1958) (1957). In the latter case, the growth medium employed was identical with that described by Levintow and Darnell (1960), with the exception that 10% human rather than 5% horse serum was employed. ...
... Virus infectivity and toxin titrations. Infectivity titrations were carried out in tube cultures of HeLa cells, using 10-5 (1:3.2) dilution increments as previously described (Ginsberg, 1958). The toxic activity of crude, infected celllysates was also assayed in tube cultures of normal HeLa cells. ...
Wilcox, Wesley C. (University of Pennsylvania, Philadelphia) and Harold S. Ginsberg. Effect of proflavine on the synthesis of adenovirus, type 5, and associated soluble antigens. J. Bacteriol.
84
526–533. 1962.—The synthesis of type 5 adenovirus in HeLa cells was suppressed to a considerable extent by low concentrations of proflavine, an acridine dye. In comparison, the processes leading to the production of soluble complement-fixing antigens and toxin were less sensitive to the action of this chemical. Addition of proflavine to infected cells at different times during the virus growth cycle revealed that the processes leading to the synthesis of soluble antigens began prior to the first appearance of newly synthesized virus. This observation is compatible with the hypothesis that the soluble antigens may represent virus subunits or precursor materials. In addition, these data indicate that it is possible to interrupt the latter stages of the virus synthetic process by addition of proflavine late in the eclipse period.
The evolution of the intranuclear lesion produced by type 5 adenovirus in HEp-2 and HeLa cells is described as seen in the light microscope and the bodies formed in the course of the infection characterized histochemically. Some 12 hours after infection acidophilic protein bodies, without appreciable nucleic acid, first appear in the nucleus and coalesce into a network. Within or in association with this material, DNA-containing masses (viral aggregates) are formed which rapidly increase in amount and then coalesce. At the same time, a protein is produced, histochemically different from that of the acidophilic or basophilic structures mentioned, within the infected nucleus, which constitutes a matrix within which regular cytstals of a protein, (presumably non-viral) materialize. These structural and histochemical features are correlated with details which have been observed in parallel studies with the electron microscope.
Flanagan, John F. (University of Pennsylvania, Philadelphia), and Harold S. Ginsberg. Role of ribonucleic acid biosynthesis in multiplication of type 5 adenovirus. J. Bacteriol.
87
977–987. 1964.—The requirement for ribonucleic acid (RNA) biosynthesis in the multiplication of type 5 adenovirus was investigated by using radioactive phosphorus to label nucleic acids and two pyrimidine analogues, 6-azauridine and 5-fluorouracil or 5-fluorouridine, to inhibit synthesis of functional RNA. The data indicate that biosynthesis of RNA after infection is essential for production of virus-specific deoxyribonucleic acid, virus antigens, and infectious particles. The onset of essential RNA synthesis occurs 8 to 9 hr after virus infection and prior to the biosynthesis of other known virus-induced macromolecules.
The life of adenoviruses began with the investigations of the structure of the virion and the mechanism of its replication. Purification of the virus and electron microscopic examination revealed the virion to be a highly structured icosahedron with a fiber protein at each apex. It was then found that adenoviruses contain DNA and the virion was attached to susceptible cells via the fibers. Hexon, the major capsid protein, not the fiber attachment protein, contains the antigen that stimulates the development of virus-specific, neutralizing antibodies. This is essential to state because many virologists believe that neutralizing antibodies merely inhibit virion attachment to susceptible cells. The influenza virus neutralizing antibody-stimulating antigen does not prevent attachment to susceptible cells, although this critical antigen is on the stem of the hemagglutinin structure. The chapter discusses about the researcher who had done extensive work in the study of adenovirus.
Infectious tissue culture fluids of the majority of serotypes of adenovirus at low dilutions detach HeLa or KB cells from glass surfaces within a few hours after inoculation. A reproducible method for testing cell detachment was devised. The factor present in infectious tissue culture fluids and responsible for cell detachment is trypsin-sensitive and non-dialyzable; it is smaller and more resistant to the effect of heat or ultraviolet light than the infectious virus particle. Cell detachment activity was found to be temperature-dependent, and the cell-detaching titer of infectious tissue culture fluids was not affected by repeated exposure to HeLa cells. Inhibition of cell detachment by human or rabbit sera was observed only when other antibodies to adenovirus antigens were also present, but the antibody inhibiting cell detachment could not be correlated quantitatively with complement-fixing or homologous neutralizing antibody.
HeLa cells infected with types 1 or 4 adenovirus were separated into cytoplasmic and nuclear fractions by mechanical disruption and differential centrifugation and the quantity of infectious virus in each was determined. The results showed that the majority of infectious virus of both types could be isolated in the cytoplasmic fraction. It was not possible to explain the large amount of type 4 virus in the cytoplasmic fraction by the number of nuclei disrupted in the fractionation procedure, but the amount of type 1 virus in the cytoplasmic fraction could have been contributed by disrupted nuclei. This suggested that there might be a basic difference in the intracellular formation of the two types of virus. The intracellular distribution of complement-fixing antigen was similar to that of infectious virus in type 4-infected cells. Technical difficulties, inherent in cellular fractionation studies, were encountered but did not appear to explain the results obtained.
During a single cycle of multiplication of type 4 adenovirus in HeLa cells an average 2-fold increase in total DNA occurred over that measured in uninfected cells. Of the total DNA from infected cells about 23 per cent was extractable into 0.15 M NaCl, a quantity approximately four and a half times greater than that of the DNA obtained from normal cells in 0.15 M NaCl. Ninety to 99 per cent of infectious virus was also extracted into the 0.15 M NaCl fraction. Concurrently with the accumulation of DNA in virus-infected cells there occurred a 2-fold increase in total protein. The proportionate increases in protein were approximately equal in 0.15 M NaCl and water extracts of infected cells. High speed centrifugation indicated that the bulk of the DNA and protein was not directly associated with infectious viral particles. Although in virus-infected cells a markedly increased synthesis occurred of a DNA which had solubility properties different from the major portion of normal host DNA, nucleotide analysis did not reveal any other qualitative differences. However, a marked change in nucleotide molar ratios was observed in the 0.15 M NaCl-soluble DNA from virus-infected cells.
It seems apparent from the findings that the biochemical alterations found in HeLa cells infected with type 4 adenovirus are intimately related to the infectious process.
The growth of adenovirus type 5 has been investigated in HeLa cells under conditions in which a very high proportion of cells are infected. Following what appears to be an eclipse phase, the cell-associated virus titer begins to rise by the eighth hour after infection and thereafter increases rapidly until it approaches a plateau by about the thirtieth hour. Release of virus from cells is slow and only about 5% of the virus is released by the thirtieth hour. The average yield of virus per cell was 103.7 TCD50 or about 50,000 virus particles.Investigations with fluorescent antibody have shown the proportion of cells developing virus antigen and the intracellular localization of the antigen. The antigen appears as discrete granules or rings in nuclei at about the twelfth hour and increases until the nuclei appear as brightly fluorescent masses. Some cells show cytoplasmic fluorescence which is weaker than that in the nuclei. The proportion of cells containing virus antigen increases from about 20% at 12 hours to nearly 100% at 24 hours. When many cells were infected at one time all stages and degrees of nuclear fluorescence were seen 24 hours later, which suggests that there is considerable cell-to-cell variation in the length of time necessary for initiation of the infective process.
Type 4 adenovirus infection of HeLa cells effected a marked increase in synthesis of the saline-soluble DNA fraction, but not the host-cell DNA (the water-soluble fraction). This was demonstrated by the marked increase in specific activity of saline-soluble DNA but not water-soluble DNA when P(32)-inorganic phosphate or sodium formate-C(14) was employed. When these isotopes were used to label cells before viral infection rather than during the process of viral propagation, the saline-soluble DNA from infected cells had a specific activity of 10 to 20 per cent less than that of uninfected cells, indicating that the saline-soluble DNA was synthesized both from prelabeled precursors of the cell pools and unlabeled materials from the medium. Saline-soluble DNA began to increase between 10 to 12 hours after viral infection and 3 to 4 hours before appearance of newly propagated infectious virus. The specific activity of the acid-soluble pool of infected cells also increased between 10 to 12 hours after viral inoculation when sodium formate-C(14) was used as a radioisotope. When P(32)-inorganic phosphate was utilized, the specific activity of infected-cell RNA was increased approximately the same relative amount as when total RNA was determined chemically; i.e., 30 to 40 per cent. With type 5 adenovirus, not only did a 3- to 5-fold increase in saline-soluble DNA occur, but also an increase was measured in specific activity of RNA when P(32)-inorganic phosphate was used.
Adenoviruses offer a number of advantages for integrated structural and biological studies of virus products. Most of the different serotypes can be produced in various cell systems with high yields. The major fraction of the products released from infected cells are structural components not aggregated in the form of intact virions or empty capsids. These components are soluble, in the true sense of the word, have dimensions making them accessible to electron microscopic analysis and in most cases exhibit a considerable stability. Furthermore they can be readily quantified by immunological or other sensitive biological techniques.
Stages in the nuclear changes consequent to infection with type 5 adenovirus are shown and described. Viral development seems to be confined to the nucleus where characteristic particles are found. The shape of the intracellular virus depends upon the method of preservation employed, appearing spherical after osmium tetroxide or freezing-substitution, occasionally exhibiting angulated faces after formalin and often assuming an hexagonal profile after potassium permanganate. The non-viral crystals are encountered in zones of low density, and it is suggested that crystallization results from the accumulation of protein in these areas. An hypothesis is presented to explain why these crystals, in contrast to the insect polyhedra, contain few viral particles.
Biochemical synthesis in adenovirus-infected HeLa cells was studied utilizing 5-fluorodeoxyuridine (5-FUDR), a potent inhibitor of deoxyribonucleic acid production. Synthesis of saline-soluble DNA and infectious virus was completely suppressed by addition of the analogue to cells as late as 10 hours after infection. The inhibitory effect of this compound was totally reversed by addition of 10(-6)M thymidine to the culture medium. Synthesis of DNA essential for virus production began 10 hours after infection and was completed by 16 hours after infection. These data support the hypothesis that the saline-soluble DNA is a precursor of infectious virus particles. Studies of antigen production indicated that formation of virus-specific proteins was directly dependent upon synthesis of DNA.
Synthesis of specific proteins in adenovirus-infected HeLa cells was studied utilizing the amino acid analog p-fluorophenylalanine. Virus multiplication and the production of specific soluble antigens could be completely suppressed by addition of this compound. Two distinct stages were revealed within the virus eclipse period when the presence of analog prevented virus synthesis. The first or early effect of p-fluorophenylalanine appeared to result from interference with production of functional enzymes necessary for synthesis of virus structural protein. An overall picture of the temporal relationships between DNA, protein, and virus synthesis was obtained by conducting inhibition studies in parallel employing 5-fluorodeoxyuridine, an inhibitor of thymidilate synthetase, as well as p-fluorophenylalanine. These studies revealed that virus DNA synthesis preceded the production of virus-specific protein by approximately 2 hours and the synthesis of mature infectious virus by about 4 hours.
Ankudas, Milda M. (University of Illinois, Chicago) and Newton Khoobyarian. Rabbit heart cell cultures, strain RHF-1. II. Behavior of adenovirus types 1 to 4. J. Bacteriol. 84:1287-1291. 1962.-In general, the findings indicate that adsorption to RHF-1 cells of adenovirus types 1, 2, and 4, but not type 3, precedes the events leading toward virus multiplication. Adenovirus type 3 attained maximal adsorption (90%) in 2 hr, with no evidence of virus multiplication. Under optimal conditions in the present experiments, the type 1 virus appeared to be released from the infected cells at a much slower rate than types 2 and 4. No correlation seemed to exist between the extent of cytopathic changes produced by type 1 in RHF-1 cells and the rise in virus infectivity during the logarithmic phase. On the other hand, the progression of cytopathic effect of type 2- and type 4-infected cells appeared to be a direct result of virus propagation. Further-more, the relative yield of virus per host cell, though small in quantity, was more or less similar for all three types. Histopathologically, no marked difference among the cells infected with types 1, 2, and 4 was clearly evident. Upon serial subculturing of these viruses in RHF-1 cells, a concomitant decrease in infectious virus, as well as complement-fixing antigen titers at each passage level, was also noted.
SUMMARY The growth characteristics of an oncogenic simian ade- novirus, SV20, in rhesus monkey (RhMK) and hamster kidney (HamK) cell cultures were compared. The ad sorption rate of SV20 in both cell systems was rather slow, but increased steadily up to 6 hr. There was 3 to 8 times more cell-associated virus in RhMK cells than in HamK cells during the first 6 hr of infection. A complete growth cycle of SV20 was obtained in infected monkey cells. This includes sequential nuclear alterations, pro duction of T antigen, viral antigen, and infectious virus. In hamster cells, SV20 induced an abortive infection. Eosinophilic inclusions and T antigen were observed as two distinct entities in the infected hamster cells, but no viral antigen or infectious virus was found in the hamster cell system. Marked stimulation of DNA synthesis in SV20-infected hamster cells was observed.
Beta-glucuronidase activity was investigated during a 48-h period in which virus replication and changes in cell morphology occurred. Infection of an established line of chimpanzee liver cells with either nononcogenic adenovirus 5 or highly oncogenic adenovirus 12 under one-step growth conditions produced differing patterns of enzyme activity. There was an increase in total activity and also enhanced leakage of beta-glucuronidase from cells infected with adenovirus 12. In contrast, the enzymatic pattern of cells infected with adenovirus 5 was similar to that of uninfected cells. Hydrocortisone prevented the abnormal release of beta-glucuronidase from adenovirus 12-infected cells. The compound had no effect on total enzyme activity or on virus replication and the development of cytopathology.
Ankudas, Milda M. (University of Illinois, Chicago) and Newton Khoobyarian. Rabbit heart cell cultures, strain RHF-1. II. Behavior of adenovirus types 1 to 4. J. Bacteriol.
84
1287–1291. 1962.—In general, the findings indicate that adsorption to RHF-1 cells of adenovirus types 1, 2, and 4, but not type 3, precedes the events leading toward virus multiplication. Adenovirus type 3 attained maximal adsorption (90%) in 2 hr, with no evidence of virus multiplication. Under optimal conditions in the present experiments, the type 1 virus appeared to be released from the infected cells at a much slower rate than types 2 and 4. No correlation seemed to exist between the extent of cytopathic changes produced by type 1 in RHF-1 cells and the rise in virus infectivity during the logarithmic phase. On the other hand, the progression of cytopathic effect of type 2- and type 4-infected cells appeared to be a direct result of virus propagation. Further-more, the relative yield of virus per host cell, though small in quantity, was more or less similar for all three types. Histopathologically, no marked difference among the cells infected with types 1, 2, and 4 was clearly evident. Upon serial subculturing of these viruses in RHF-1 cells, a concomitant decrease in infectious virus, as well as complement-fixing antigen titers at each passage level, was also noted.
The purpose of this paper is to demonstrate an example of the actual problem of virology and to point out several unsolved problems which may be satisfactorily explained only in the light of aerosol research.
In the electron microscopic observation on adenovirus type 12 infected cells, abundant virus particles can always be recognized in the nucleus, although in some rare instances virus particles are found in the cytoplasm. Therefore, it is difficult to consider the virus formation to take place aside from the nucleus and it seems that the nucleus is really the essential site of virus multiplication.
The fact that there are two different arrangements of virus particles presents a problem. It appears that usually crystalline arrayed virus particles come into being, and then they take noncrystalline arrangement, but this sequence does not necessarilly take place. The crystalline arranged virus particles and noncrystalline arranged ones often appear simultaneously and independently of each other,
At the early stage of infection, the electron dense masses are seen in the nucleus. These masses are the matrix of virus particles, because the arrangement of virus particles in crystalline or noncrystalline array are seen with these electron dense masses.
Das Interesse an der Erforschung der Ätiologie von akuten Viruserkrankungen der Atemwege hat in der letzten Zeit stark zugenommen Diese Erkrankungen vermögen, auch wenn sie nur leicht verlaufen und nicht epidemisch auftreten, durch ihre Häufigkeit außerordentliche volkswirtschaftliche Schäden zu verursachen (Huebner, Baroyan, McDonald, Matumoto). Verbesserte und neue Methoden in der virologischen Diagnostik haben in den letzten Jahren zu der Entdeckung einer großen Anzahl von Viren geführt, die häufig als „neuere“Respirationstrakt-Viren den „klassischen“Influenza-Viren und Viren der Ornithose-Psittakose-Gruppe gegenübergestellt werden. Es steht heute fest, daß außerhalb von Influenzaepidemien und -pandemien die überwiegende Anzahl der akuten Virusinfektionen der Atemwege durch die neueren Respirationstrakt-Viren bedingt ist. In der vorliegenden Übersicht werden diejenigen dieser Viren besprochen werden, die sich bereits in wohldefinierte Gruppen einteilen lassen und/oder deren Bedeutung für die Ätiologie von Respirationstrakterkrankungen besonders groß ist.
Zu den bemerkenswerten Ergebnissen der neuen experimentellen Erforschung menschlicher Viruskrankheiten gehört die Erkenntnis, daß die weit verbreiteten undifferenzierten Erkrankungen der Luftwege äußerst verschiedenartige und dabei komplexe Probleme darstellen (Huebner u. a. (1958). Innerhalb der Erreger dieser großen, noch gar nicht vollständig bekannten Krankheitsgruppe nehmen die Infektionen durch die soge-nannten Adenoviren den größten Kaum ein.
Higher titres of the WBR-1 strain of bovine adenovirus type 3 were obtained in cultures of suspended MDBK cells than in monolayer cell sheets. The virus yield per cell was about 90 times greater in suspended culture. Highest yields were obtained when the cells were suspended at 14 × 105/ml. A pH value of 7.6 was optimum for virus production in suspended culture.
The growth of five ovine adenovirus serotypes in lamb kidney cell cultures is described. All five serotypes exhibited similar changes although nuclear enlargement and deformity was most marked with types 3 and 5. The first inclusions seen by H & E staining were irregular eosinophilic bodies. These increased and coalesced, and later refractile ‘pearl-like’ inclusions and granular basophilic inclusions were also seen. A single large basophilic inclusion was eventually formed. The first fluorescent inclusions were small and granular. Later, fluorescent rings and fluorescent reticular-like networks were observed. Electron-microscope studies with types 1 and 5 showed virus particles and associated inclusions of several types accumulating in the centre of infected nuclei. The sequence of changes observed was similar to those described for the human adenovirus 3, 4, 7 subgroup, and the Bartha group A bovine adenoviruses.
Adenoviruses generally cause mild illnesses. Conjunctivitis, nasorhinitis, and pharyngitis are known to be caused by type 5 virus. A patient is described with clinical findings of whooping cough who died during a paroxysm. Studies of postmortem tissues revealed necrotizing bronchiolitis and cytopathologic changes in lung parenchyma typical of adenovirus infection. Type 5 adenovirus was isolated from lungs, liver, and kidney. The relationship of adenovirus infection to the pertussis syndrome is briefly discussed.
The sequential cytological changes which develop in tissue culture cells infected with adenovirus types 5 and 7 are described
and compared with those produced by adenovirus types 1, 2, 3, and 4. The evidence that is presented indicates that types 1,
2, and 5 belong to one major subdivision of the adenovirus group and types 3, 4, and 7 to another. That the host cell nucleus
is the principal site of adenovirus synthesis has been confirmed by fluorescent antibody studies. They have demonstrated the
occurrence of type-specific adenovirus antigen in the characteristic intranuclear inclusions and other virus-induced structures
reported to contain virus-like particles or shown by electronmicroscopy.
Die Vermehrung von Adenovirus 19 und 26 wurde in HeLa-Zellkulturen untersucht. Die Virusadsorption verlief langsam und unvollstndig. Nach einer Latenzzeit von 20 bis 22 Stunden, in deren letztem Drittel bereits ein cytopathischer Effekt eintrat, folgte eine Vermehrungsphase bis 30 bis 36 Stunden nach Infektion. Lsliches Hmagglutinin und komplementbindendes Gruppenantigen wurden parallel zum infektisen Virus gebildet. Eine Freisetzung des Virus ins Medium erfolgte meist erst ab 72 Stunden nach Infektion. Unterschiede in der Lnge der Latenzzeit zu den Typen 4 und 5 waren nicht zu beachten.The multiplication of adenovirus types 19 and 26 was studied in HeLa cell cultures. Virus adsorption was slow and incomplete. A latent period of 20 to 22 hours was followed by virus multiplication until 30 to 36 hours after infection. The cytopathic effect started at 14 hours after infection. Soluble haemagglutinin and group-specific complement-fixing antigen were formed in parallel with the infectious virus. Virus release into the culture medium usually began at 72 hours after infection. There was no difference in the latent period between these types and type 4 and 5.
The replication of avian adeno-like virus from chickens (CELO) in chicken-kidney-tissue cultures takes essentially the same course as the replication of human adenovirus. By increasing the incubation temperature to 40° C the growth-cycle is accelerated considerably. The eclipse-phase and the exponential period as well as the time of virus release are decreased. Nevertheless the ratio of cell-associated to free virus remains the same and is always greater than 100. The yield of newly formed virus, however, undergoes no significant changes at incubation temperatures of 34° C, 37° C and 40° C. The virus reproduction can be specifically suppressed through IUDR and FPA. This suppression can be reversed by adding a surplus of thymidine and phenylalanine, respectively. Using the analogs as inhibitors the following stages in the growth-cycle can be observed: Within the 10 hour eclipse-phase the production of early protein starts 2 hours after infection, 3 hours before the formation of the complete infectious virus occurs, the synthesis of virus-specific DNA can be observed. 2 hours prior to completion of the virion the structural protein appears. During the formation of complete infectious virus the synthesis of DNA and structural protein continues.
The physical characteristics of reovirus type 3 and its interaction with L cells have been investigated. After repeated passage of the Dearing strain in L cells, a variant emerged which was characterized by decreased sensitivity to specific antibody and by diminished release from cells. The original and variant strains were not significantly different in their antigenic constitution, size, or fine structure.The amount of each which adsorbed to L cells in 2 hours was approximately 80% of that adsorbed in 4 hours. The latent period was 7 hours and the maximal yields were reached at 15 hours with both the original and the variant strains. Both caused the development of a perinuclear inclusion in infected cells. The inclusion was Feulgen negative; it contained viral antigenic material.Both strains were insensitive to inhibition by 5-fluoro-2′-deoxyuridine and 5-bromo-2′-deoxyuridine, whereas vaccinia virus was markedly inhibited. Mitomycin C and actinomycin D inhibited reovirus to a lesser extent than vaccinia virus under most conditions used. However, after pretreatment of cells with actinomycin D, their ability to produce reo or vaccinia viruses was restricted to a similar degree. 5,6-Dibromo-1-β-d-ribofuranosylbenzimidazole and aminopterin inhibited the original Dearing strain and vaccinia virus to a similar degree. The variant strain was slightly less sensitive to the inhibitors than the original. The nucleic acid of reovirus 3 appears to be of the ribose type.
A method is described for inactivation of viruses by oxidation at controlled potentials. If the applied potential is adjusted properly, it is possible to destroy the infectivity of the viruses without changing other properties. The rate of loss of infectivity is shown to be dependent upon the applied potential and the purity of the virus preparation used. Control experiments are described which indicate that the loss of infectivity is due to oxidation of the viruses at the surface of the working electrode. Contact of the viruses with this electrode is necessary for the inactivation to occur.A method is also described which relates the “resting potential” of the electrolytic cell to the number of electrons transferred per functional group on the virus particle.
The growth of measles virus in Hep-2 cultures has been investigated. Maturation of the first new virus occurred 15–18 hours after inoculation, but no virus was released into the fluid phase for 27–30 hours. Even after this time the major part of the active virus remained with the cells.At 37° the virus had a half-life of about 2 hours. Thus there was little accumulation of live virus in tissue culture fluid but during the period of active virus production there was an equilibrium between release and decay.The virus was relatively stable when suspended in media at 0° of pH 6 to 9, but titer was rapidly lost at 4.5 and lower.
Multiple infection of mass cultures of KB cells with type 2 adenovirus resulted in marked stimulation of incorporation of P32-orthophosphate into RNA mononucleotides and DNA. Studies at several time intervals revealed greatest incorporation of P32 into RNA at 9–18 hours, into DNA at both 9–18 and 18–27 hours after infection. The data are interpreted as indicating a virus-stimulated incorporation of medium P32 at 9–18 hours into the phosphate pool of the cell. At this time, the acid-soluble nucleotide and phospholipid fractions were also found to be more active in infected than in uninfected cells. The prolonged increased uptake of isotope into the DNA of the infected cell may reflect, in addition to a more highly labeled phosphate pool, a stimulated synthesis of a new DNA, perhaps virus precursor.
Studies correlating the production of infectious adenovirus (types 5 and 7) and the progression of the stages of virus-induced cytologic change in HeLa cells are presented. The results reveal a close relationship between the development of the characteristic nuclear changes and adenovirus synthesis. They suggest that cells manifesting the first stages of nuclear change, characterized by the appearance of eosinophilic, Feulgen-negative inclusions, contain little or no mature infectious virus, whereas cells in the later stages, with Feulgen-positive and basophilic inclusions, contain relatively large amounts.
Suspension of adenovirus type 5 in 2.0 ml of cell culture fluid at 37 degrees C were subjected to smoke from four cigarettes over a 4-h period. The cigarettes were smoked in a normal manner, and the inhaled smoke was exhaled through glass tubing into the virus-containing fluid. The virus suspensions were then titrated, using monolayer cultures of HEp-2 cells. Smoke from filter-tipped or regular cigarettes caused a 2- to 3-log drop in titer of tissue culture infectious doses of adenovirus type 5 per 0.1 ml of virus suspension. No reductions in titers were observed with parallel suspensions of the virus subjected to normal inhaled and exhaled air.
Products of cocultivation of seven human adenovirus serotypes in different pairwise combinations were characterized. Capsids composed of fibers (or more likely pentons) and hexons of different serotypes were identified by use of different techniques. One evidence for this was the demonstration by electron microscopy of the occurrence of fibers of different lengths, i.e., specific for different serotypes, in single virions. Capsid mosaics also could involve hexons of different types. The frequency of occurrence of mixed capsids was related to the degree of biological relatedness between the serotypes used for mixed infections.Cocultivation products also included mixed polymeric soluble components. Dodecons composed of pentons of two different serotypes or of core component(s) of one type and pentons mainly of another type were identified. Pentons of serotypes, which during single infections do not produce dodecons, e.g., types 6 and 16, were found to be capable of participating in the formation of structures of this kind.Monomeric soluble capsid components were characterized by reference to their anion exchange chromatography behavior. Cocultivation of types 3 and 11 caused the appearance of four different populations of hexons. Two of these corresponded to hexons of types 3 and 11 present in the control mixture. The two other populations of hexons contained type-specific determinants of both serotypes in varying proportions. It is proposed that these hexons represent hybrid soluble components containing polypeptides derived from the virus of two different serotypes. No evidence for a mixing between structural proteins of penton components of different serotypes was found.
CELO virus is resistant to trypsin at 37° C; it is not stabilized by 1M MgCl2 at 50° C. The multiplication of CELO is inhibited by iododeoxyuridine, a drug which selectively inhibits DNA viruses. In growth studies, it was found that the titer of the cell-associated virus is more than 100 times higher than the extra-cellular one.
Some electron microscopic pictures are presented, showing the reaction of the nuclear components to viral multiplication, the occurrence of “ring-like bodies”, small irregular bodies, and the release of virus in the extracellular space.
All here described properties of CELO are common to adenoviruses, a further evidence that CELO should be classified in the adenovirus group.
The cells of a human epithelial cancer cultivated en masse have been shown to support the multiplication of all three types of poliomyelitis virus. These cells (strain HeLa of Gey) have been maintained in vitro since their derivation from an epidermoid carcinoma of the cervix in February, 1951. As the virus multiplied it caused in from 12 to 96 hours degeneration and destruction of the cancer cells. The specific destructive effect of the virus was prevented by adding homotypic antibody to the cultures but not by adding heterotypic antibodies. Methods for the preparation of large numbers of replicate cultures with suspensions of strain HeLa cells were described. The cells in suspension were readily quantitated by direct counts in a hemocytometer. A synthetic solution that maintains cellular viability was employed for viral propagation. The experimental results demonstrate the usefulness of strain HeLa cells for (a) the quantitation of poliomyelitis virus, (b) the measurement of poliomyelitis antibodies, and (c) the production of virus.
An estimate of the accuracy of methods of titration of the herpes simplex virus on the CAM has been given. The pattern of growth of the herpes virus on the CAM has been described, and its similarity to the patterns of other animal viruses studied has been indicated.
Initial adsorption of virus onto the CAM occurs very rapidly, 99% or more occurring within 1 hour, with most of it within 10–15 minutes. Following this there is a continued decrease in residual seed virus at a slower rate for the next 7 hours.
The latent period varies in length (from 4–12 hours) inversely with the number of infectious units in the inoculum. During this period there is no detectable evidence of infectious virus in the host cells.
Following the latent period infectious virus becomes again detectable and increases at the rate of 10 fold every 2 hours for the next 10 hours. Following this, the rate levels off for another 6 hours, then rises again slowly.
For the first 12–18 hours of the incremental period more virus is obtained from within the membranes than can be demonstrated as having escaped from the infected cells. After this period, virus formation within the cells appears to come into equilibrium with the escape of virus from the cells.
No evidence of complement-fixing activity was found before the demonstration of infectious virus in high titer.
After allantoic injection of chick embryos with a known amount of influenza virus, the process of adsorption of the agent onto host cells and infection of them can be interrupted at a given time by the administration of large quantities of heterologous virus inactivated by irradiation. A sudden great increase in the amount of free virus in the allantoic fluid occurring after 6 hours in the case of the PR8 strain, and 9 hours in that of the Lee strain, indicates that the untreated virus associated with the host cells has multiplied. The length of the period preliminary to this increase remains the same even though the concentration of the original inoculum is varied over a wide range. Since administration of the irradiated virus leaves no susceptible host cells, because of the interference phenomenon, and further adsorption of active virus is minimized or entirely prevented, practically the entire new increment of virus can be found in the allantoic fluid and assayed; for every ID(50) adsorbed about 50 ID(50) are released. Homologous irradiated virus, on the other hand, when injected after infection of the allantoic sac, reduces the yield of virus to a more or less considerable extent. Some inhibitory effect can still be observed when the homologous irradiated virus is given several hours after infection. This effect is linked to the virus particle and destroyed by prolonged irradiation.
A study has been made to establish the statistical significance of results obtained in mouse infectivity titrations of influenza virus. Five titrations, each composed of five replicas, were carried out and 50 per cent end points were calculated for each titration. Three criteria for evaluating the end points were employed, namely, the presence or absence of pulmonary lesions, the occurrence of death, and a weighted composite taking into account both the extent of lung consolidation and the occurrence of death. Standard deviations of the distribution of end points obtained by each method were computed, and from these data levels of probabilities for significance in the differences between end points were determined. It was found that the chances are 19 out of 20 that differences of 0.99, 0.77, and 0.73 logarithmic units, respectively, for the lesion, the death, and the weighted end points are significant.
Representative viruses of the RI-APC group were observed with the electron microscope in thin sections of infected HeLa cells. The viral particles varied in density, were approximately 60 mmicro in diameter and had a center to center spacing when close packed of about 65 mmicro. Many of the less dense particles exhibited an internal body averaging 24 mmicro in diameter. It was suggested that within the nucleus the virus differentiated from dense granular and reticular material and formed crystals. Disintegration of the crystals and disruption of the nuclear membrane with release of virus into the cytoplasm appeared to occur at any stage. No evidence to suggest development of the virus in the cytoplasm was obtained. It was possible to deduce the structure of the viral crystal from the electron micrographs. The viral particles are packed in a cubic body-centered lattice. Correlative histochemical observations in the light microscope which are now in progress revealed that the crystals and non-crystalline aggregates of virus were strongly Feulgen-positive.
The cells of a human epithelial cancer cultivated en masse have been shown to support the multiplication of all three types of poliomyelitis virus. These cells (strain HeLa of Gey) have been maintained in vitro since their derivation from an epidermoid carcinoma of the cervix in February, 1951. As the virus multiplied it caused in from 12 to 96 hours degeneration and destruction of the cancer cells. The specific destructive effect of the virus was prevented by adding homotypic antibody to the cultures but not by adding heterotypic antibodies. Methods for the preparation of large numbers of replicate cultures with suspensions of strain HeLa cells were described. The cells in suspension were readily quantitated by direct counts in a hemocytometer. A synthetic solution that maintains cellular viability was employed for viral propagation. The experimental results demonstrate the usefulness of strain HeLa cells for (a) the quantitation of poliomyelitis virus, (b) the measurement of poliomyelitis antibodies, and (c) the production of virus.
The rate of adsorption of WEE virus onto chicken embryo cells in vitro was determined both on a cell layer and on a cell suspension.
One-step growth curves were determined in cell suspensions and on cell layers. The latent period varied between 2 and 3frac12 hours; it was shorter on cell layers and decreased with higher multiplicity of infection. The shortest period is probably the real latent period.
The growth curves of the virus showed an initial exponential rise and reached a maximal constant value after 6 to 8 hours. The maximum virus yield per cell varied between 200 and 1000 on the cell layer, and between 100 and 200 in suspended cells.
The yield of single infected cells was determined. An analysis of the distributions of the individual yields obtained after various periods of virus growth led to two main conclusions: (1) that virus is released from the same cell over a long period of time; (2) that one phase of the intracellular virus growth is exponential.
A microbial agent, presumably a virus, was recovered from throat washings from a patient with primary atypical pneumonia in an epidemic of acute respiratory illness which occurred at Fort Leonard Wood during the winter of 1952-1953. This agent multiplies in human cell tissue cultures producing obvious cytopathogenic changes but not in common laboratory hosts. The patient whose throat washings yielded the virus de veloped specific neutralizing and complement-fixing antibodies for the agent. Other patients in the epidemic with primary atypical pneumonia (PAP) or undifferentiated acute respiratory disease (ARD) also developed antibodies for the agent but those cases with proved influenza A′ did not. A portion of the population maintains an antibody level against the new agent suggesting a rather general experience with it.
1. From the present evidence it appears that an unidentified, possibly new, tissue culture cytopathogenic agent has been isolated repeatedly from human adenoids undergoing spontaneous degeneration in tissue culture. The filter ability and the inability to cultivate the agent on bacteriological media and to demonstrate organisms in stained tissue culture preparations would indicate that the agent belongs to the group of viruses or rickettsial It is tentatively proposed to designate the agent as the “adenoid degeneration agent”, abbreviated as “A.D. agent”. 2. That the agent is derived from the adenoid tissue rather than from the nutrient media is indicated by the fact that some adenoids and all human embryonic tissues cultivated in the identical media and at the same time have not undergone degeneration, although they are susceptible to infection with the agent; also, repeated attempts to isolate the agents from adenoid cultures not demonstrating degeneration have been uniformly unsuccessful. 3. Further investigation is in progress to determine the relation of the agent to the adenoids and to study their possible role in human disease; particularly upper respiratory infections.
This article has no abstract; the first 100 words appear below.
The undifferentiated respiratory diseases, the febrile catarrhs (acute respiratory disease, exudative nonstreptococcal pharyngitis, primary atypical pneumonias)¹,² and the afebrile common colds continue as unsolved omnipresent sources of human distress. About two years ago our group at the National Microbiological Institute and a group at Johns Hopkins University School of Hygiene embarked on collaborative efforts to study these ailments. The general, quite elastic plan was to observe them in a concerted manner — to study them simultaneously from clinical, epidemiologic and laboratory standpoints. A particularly important and specific part of the plan, however, was to utilize tissue-culture technics providing multi-purpose new . . .
*From the Department of Health, Education and Welfare, Public Health Service, National Institutes of Health, National Microbiological Institute, and the Department of Microbiology, School of Hygiene and Public Health, Johns Hopkins University.
Supported in part by grant from the Division of Research Grants, United States Public Health Service.
Source Information
† Medical director, United States Public Health Service; chief, Virus and Rickettsial Diseases Section, Laboratory of Infectious Diseases, National Microbiological Institute; assistant professor of infectious diseases in pediatrics, Georgetown University School of Medicine.
‡ Senior assistant surgeon, United States Public Health Service, Laboratory of Infectious Diseases, National Microbiological Institute.
§ Associate professor of microbiology, Johns Hopkins University School of Medicine.
¶ Senior assistant surgeon, Laboratory of Clinical Investigation, Clinical Center, National Microbiological Institute, National Institutes of Health; assistant clinical professor of pediatrics, Georgetown University, School of Medicine.
∥ Medical director, United States Public Health Service; chief, Epidemiology Section, National Microbiological Institute, Laboratory of Infectious Diseases.
BETHESDA, MARYLAND
The formation of plaques by Newcastle disease virus (NDV) on monolayers of chick embryo cells requires only a single infective particle and furnishes a reliable method of assay. The values so obtained agree with those found by egg titrations.Attachment of NDV to chick embryo cells requires the presence of an ionic medium. The rate of attachment is the same as the rate observed with red cells. Attachment is almost independent of temperature between the range of 0 and 27°. The pH dependence curve for attachment suggests the involvement of carboxyl groups in the binding of virus to cells.Under conditions where NDV elutes completely from red cells, there is little or no elution from host cells.One-step virus growth curves, determined with cell suspensions, exhibited a latent period of 3 to 4 hours, followed by a rise period which lasted for 4 to 8 hours. The average yield per infected cell varied between 18 and 37.
HeLa cells were infected with adenoviruses (types 1-4) and sectioned for electron microscopy after intervals of 20 to 48 hours. Clusters of virus-like particles were found within the nuclei of infected cultures but not in those of uninfected controls. The particles were often arranged in rows as if in crystalline formation. Maximal diameter of particles was approximately 65 mmicro, and internal bodies were demonstrated. Lesions of infected cells included target-like structures of the nuclear membrane, large nuclear vacuoles (type 2), and increased numbers of large irregular electron-dense granules in the cytoplasm 48 hours after infection. Examination of infected cultures by light microscopy, using the Feulgen reaction, showed intranuclear inclusion bodies and a cytopathogenic effect consisting of clumping of cells without pyknosis of nuclei. A lipide stain showed numerous cytoplasmic granules that were not identical with the large, irregular, electron-dense granules of the cytoplasm. Practically all the cells showed the viral cytopathogenic effect, but only a minority of cells were found to contain virus-like particles or intranuclear inclusion bodies.
Types 1-4 of the new respiratory viruses, termed adenoviruses, were relatively stable when exposed to temperatures ranging from 4°C to 36°C. Type 1-3 viruses were inactivated in 10 to 20 minutes at 50°C, but the infectivity titer of type 4 virus was reduced less than 2 log units (99%) in 20 minutes. The adenoviruses were also resistant to inactivation at pH 6.0 to 9.0. The patterns of inactivation of the 4 viruses by alterations of pH as well as incubation at 50°C were similar, although consistent and significant differences among the 4 types were measured.
Neutralization titrations carried out with types 1–4 APC-RI viruses and type-specific rabbit sera, as well as human convalescent sera, indicated that preliminary incubation of virus-serum mixtures at 22° or 37°C was necessary to obtain maximum titers. Prolongation of the incubation period beyond 30 min did not increase the neutralization titer appreciably; with human convalescent sera, neutralization titers were similar whether a mixture of serum and either type 3 or 4 virus was incubated 30 min at 4°C or 120 min at 37°C.
Investigation of the quantitative aspects of the neutralization reaction with the new respiratory viruses and type-specific immune sera indicates that the reaction between the two components, expressed as logarithms to the base 10, describes a linear relationship. The ratio between the two variables, the logarithm of the quantity of virus neutralized and the logarithm of the serum dilution end point, was 1.0 for types 3 and 4 viruses and 2.0 for types 1 and 2 viruses. The species of animal from which serum was obtained for the neutralization titrations did not influence the slope of the neutralization line.
The sequential morphological and cytochemical alterations in HeLa cells infected with adenovirus types 1 to 4 are described.
Each of the four viruses studied led to consistent and reproducible cytological changes not observed in uninfected control
cultures. All four agents produced striking and characteristic changes in the nuclei of infected cells. Alterations in the
cytoplasm, though present, were less marked, particularly in the early stages of infection. In cells infected with type 1
or 2 adenovirus, rounded intranuclear inclusions which progressed from eosinophilic and Feulgen-negative to basophilic and
Feulgen-positive, together with a homogeneous glassy, Feulgen-positive nuclear background, were prominent features. Cells
infected with type 3 or 4 adenovirus exhibited marked rearrangement of basophilic nuclear material and sharply defined crystal-like
inclusions, predominantly intranuclear in location, which also varied from Feulgen-negative to positive. In terms of detailed
cytological effects, therefore, the four agents could be divided into two subgroups, viz., types 1 and 2 on the one hand and types 3 and 4 on the other. Measurement of DNA in individual nuclei by means of Feulgen
microspectrophotometry revealed the values in infected cells to be increased above the levels of the uninfected controls.
A GROUP of viruses causing very peculiar changes of the infected cells in tissue cultures was recently isolated in our laboratory1. The strains were obtained from cases of either mesenteric lymphadenitis or pharyngitis with cervical lymphadenitis. An apparently identical microscopical picture1 was caused by agents described by Neva and Enders2 and Hilleman and Werner3. So far, all strains exhibiting this pattern of cytopathic activity which have been tested in our laboratory have had complement-fixing antigens in common, although by neutralization tests they could be differentiated into three types1.
SummaryA mixture of tryptose phosphate broth, 15 to 25%, maintenance solution (MS), 67.5 to 77.5%, and chicken serum, 7.5%, maintained HeLa cells in tissue culture for at least 10 days. During this period HeLa cells increased 3 to 5 fold in number. Furthermore, smaller quantities of ARD, AD and Type I poliomyelitis viruses could be detected and significantly more ARD virus could be propagated in HeLa cells maintained in this medium than in cells supported with maintenance solution and chicken serum alone.
The amino acid requirements of a human uterine carcinoma cell (HeLa strain) have been defined. The 12 compounds previously found to be essential for the growth of a mouse fibroblast proved similarly essential for this human epithelial cell. They included arginine, cyst(e)ine, histidine, and tyrosine, in addition to the eight amino acids required for nitrogen balance in man (isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine). Only the L-amino acids were active; the D-enantiomorphs had no demonstrable effect at physiologic concentrations.
The minimum concentrations required for survival and limited growth varied from 0.003 µM per ml. for L-tryptophan, to 0.1 µM per ml. for L-lysine. The concentrations permitting optimum growth similarly varied from 0.01 µM per ml. for tryptophan, to 0.1 µM per ml. for leucine, isoleucine, threonine, lysine, and valine. The latter optimum concentrations of the individual amino acids were closely correlated with their serum levels. With at least six of the amino acids, high concentrations, in the range 1 to 10 µM per ml., caused a definite growth inhibition.
In the absence of a single essential amino acid, degenerative changes occurred in the cells, culminating in their death and dissolution. In the early stages, however, these degenerative changes could be reversed by the restoration of the missing component.
A study of the multiplication of PVM in the mouse lung yielded evidence in support of the hypothesis that discrete cycles occur. The cycle is characterized by a latent period of about 15 hours, an incremental period of 10 to 15 hours and a total duration of 24 to 30 hours. The yield of virus per cycle is approximately 16-fold.
A large proportion, approximately 90 per cent, of PVM is lost on intranasal inoculation and cannot be recovered from the lung. Of the virus which reaches the lung, almost all becomes non-infective within 12 hours even though a considerable amount retains the capacity to cause hemagglutination. During the incremental period infective virus appears as soon as any increase in hemagglutination is demonstrable.
The rate of multiplication of PVM in the mouse lung is relatively constant, averaging 7.9-fold per day with but slight variations, irrespective of the amount of virus inoculated. Similarly, the rate of increase in the amount of pneumonia is relatively constant, averaging 4.7-fold per day, even though the quantity of virus inoculated is varied over a wide range. It follows that viral multiplication proceeds 1.7 times more rapidly than does extension of the pathological lesion, both reaching limiting maximal values in periods which are predictable from the amount of virus inoculated. From the concentration of virus determined at any time during the incremental period, the amount of pneumonia present earlier or later in the incremental period can be computed with considerable precision. The results support the postulate that the extent of the pathological lesion is dependent upon the degree of viral multiplication.
Influenza A virus, PR8 strain, increases in amount in the infected mouse lung at a relatively constant rate. When more than 25 M.S.50 doses of virus is inoculated, the rate of multiplication appears to be independent of the amount of virus introduced; has a value of 1,100-fold increase per day. The rate of increase in the pulmonary lesions induced by infection of the mouse lung with PR8 also appears to be relatively constant and independent of the amount of virus inoculated; has a value of 8.5-fold increase per day.
The essential variables in the PR8-mouse lung system appear to be equated satisfactorily by functions which were derived previously (4) during a similar quantitative investigation on pneumonia virus of mice (PVM). Evidence in support of the hypothesis that the processes of multiplication of PR8 and PVM are different in the mouse lung is presented.
Quantitative aspects of the multiplication of influenza A in the mouse lung. Relation between the degree of viral multiplication and the extent of pneumonia
- H S Ginsberg
- F L Horsfall
- Jr
Ginsberg, H. S., and Horsfall, F. L., Jr., Quantitative aspects of the multiplication of influenza A in the mouse lung. Relation between the degree of viral multiplication and the extent of pneumonia, J. Exp. Med., 1952, 95, 135.