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Double labeling and color microradioautography were used in a new method of hybridization in situ to identify different genes in individual cells. The method is based on the unequal penetration of 3H and 35S into two layers of nuclear track emulsion separated by a thin barrier film. Hybridization of a 35S-labeled probe specific for one kind of gene results in silver grains over cells in both layers of emulsion; a 3H-labeled probe for a second gene provides grains only in the first layer of emulsion. Silver grains are converted to magenta-colored grains in the first layer and to cyan-colored grains in the second to facilitate enumeration of grains in each layer. This technique should be widely applicable in analyses of differential gene expression in single cells or in discrete populations of cells.

Mu DNA, isolated from infected cells or minicells, has been shown to be held by proteins in twisted and open circular forms. Circularization does not require protein synthesis in the infected cells. A 64,000-dalton polypeptide is injected into the infected cell with Mu DNA and co-sediments with Mu DNA through sucrose gradients. Circularization of the infecting Mu DNA does not require removal of the Escherichia coli DNA sequences which are attached to both ends of the Mu genome in the viral particle.

Direct and indirect immunoperoxidase (IP) techniques were evaluated for their potential in identifying infectious pancreatic necrosis (IPN) virus. Both techniques were shown to offer a relatively simple, rapid and efficient means for the specific identification of IPN virus in infected cells. The direct IP method resulted in less nonspecific staining; however, the indirect method was clearly specific and utilized commercially available reagents.

Circulating antigens were detected in sera of mice experimentally infected with a high close of Trypanosoma cruzi by reaction with sera from chronically infected mice. The immunodiffusion reaction between homologous acute and chronic sera produced four precipitation lines. By reaction with chronic mouse serum, circulating antingens were detected in sera from heavily infected hamsters, dogs, rabbits and in sera from chagasic patients. A reaction was also found in urine from acutely infected mice and dogs. Trypanosoma cruzi exoantigen was detected in trypanosome culture medium and in the supernatant of infected cell cultures. Attempts to isolate the antigens are described.Antígenos circulantes foram detectados em soros de camundongos infectados experimentalmente com elevadas doses de Trypanosoma cruzi pela reação com soros obtidos de camundongos em fase crônica de infecção. A reação de imunodifusão entre soros homólogos agudo e crônico produziu quatro linhas de precipitação. Por reação com soro crônico de camundongo antígenos circulantes foram detectados em soros de crícetos, cães e coelhos infectados com doses elevadas de Trypanosoma cruzi e em soros de pacientes chagásicos. Uma reação foi também observada com urina de camundongos e cães infectados de forma aguda. Exoantígeno de Trypanosoma cruzi foi detectado em meio de cultura de tripanosomas e em sobrenadantes de culturas de células infectadas. Tentativas de isolamento dos antigenos são descritas.

The specificity of protein synthesis in reovirus-infected mouse SC-1 cells was investigated, with the following results: a, extracts from infected and control cells translated saturating levels of capped globin mRNA with the same efficiency; b, infected cell extracts did not display a virus-induced increase in the capacity to translate decapped globin mRNA; c, translation of both cellular and viral messages was equally sensitive to the cap analog, 7-methylguanosine 5'-triphosphate. These findings are consistent with a previously published model (Walden, W., Godefroy-Colburn, T., and Thach, R. E. (1981) J. Biol. Chem. 256, 11739-11746) in which translation rates in reovirus-infected SC-1 cells are regulated by competition of capped host and viral mRNAs for a component of the unaltered protein synthetic system. Similar experimental results were obtained using reovirus-infected mouse L cells, with the following exceptions: a, the shutoff of host translation was far greater in L cells than in SC-1 cells; b, extracts of infected L cells were less active than controls for translation of all mRNAs tested. The reasons for the differences between SC-1 cells and L cells in their response to reovirus infection are being investigated.

Several factors which might affect the relative proportions of genomes and antigenomes in Sendai virus have been examined. The relative amount of antigenomes assembled into mature virions was found to be independent of the time of harvest, whereas the amount of cellular RNAs incorporated into virions was found to increase late in infection. Virus strain and host cell did affect the relative amounts of antigenomes in both mature virions and the infected cell, but in all cases more antigenomes were present in the infected cell than would be expected from their function as intermediates in genome replication.

Supra-optimal temperature 42° C inhibits the multiplication of adenovirus particles in lytically infected cells although viral DNA and polypeptides are present in cytoplasm. It was demonstrated that the transport of viral polypeptides from cytoplasm, where they are synthesized, to nucleus, where viral particles are assembled, is not blocked at 42° C. The relative amounts of viral polypeptides present in nucleus were higher at 42° C than at 37° C, indicating that a thermic regulation may act during protein synthesis in cytoplasm. A particular infected cell specific polypeptide, termed NCVP-1 (non-capsid viral polypeptide-1), which migrates close to hexon polypeptide, has been detected at both temperatures 37° and 42° C. NCVP-1 has a rapid turn-over and does not seem to be metabolically related to hexon.

Cytomegalovirus-infected human fibroblasts developed large numbers of fine microvilli within 90 min after addition of virus. The structure of these microvilli gradually changed as the infection proceeded, although the infected cells were easily detected by their large number of processes. These processes may be involved in the increased transport of ions and nutrients into the infected cell.

Summary Three immuno-peroxidase techniques (direct, indirect and peroxidase-antiperoxidase) were compared for their potential in foot-and-mouth disease research. Each technique was shown to offer a simple and efficient means for the detection of the virus of foot-and-mouth disease and of virus-infection-associated (VIA) antigen in infected cells.

All RNA viruses that cause cancer under natural conditions fall into the "retro" category, meaning that the first step in their replication is the "reverse transcription" of RNA into DNA. This peculiarity of their biologic behavior has helped explain other intriguing features of these viruses and opened new directions for future research into the role of viruses in altering the infected cell's genetic material and in oncogenesis.

A rapid, simple, reproducible and semi-quantitative assay to measure neutralizing antibodies has been developed. It employs a unique cell line which is exquisitively sensitive to infection with all HIV isolates tested. The assay is amenable to microtiter formulation as well as analysis by automation.

Polyomavirus was purified from infected mouse cell lysates under mild physiological conditions. When analyzed in a sucrose gradient, a major virus peak (240S) was identified. This sucrose-isolated virus could be divided into two populations based on its stability to CsCl gradient centrifugation. Members of the unstable population were shown to eject their DNA cores when subjected to CsCl gradient centrifugation, forming empty capsids, whereas the stable population was unaffected by the same CsCl treatment. Formaldehyde fixation of the 240S virus particles stabilized the virions and prevented ejection of DNA and generation of empty capsids. When formaldehyde-fixed 240S virus was examined with the electron microscope, only full virions were observed. These results indicate that polyoma capsids are not preformed in vivo, but instead are generated when infected cell lysates are subjected to harsh CsCl purification procedures.

We have isolated a ts mutant of influenza A/FPV/Rostock/34 that induces the synthesis of a novel small polypeptide in infected cells. This polypeptide is encoded by a subgenomic virion RNA derived from RNA segment 3, apparently by internal deletion. A second polypeptide, similarly derived from RNA segment 1, was found only after in vitro translation of infected cell RNA. The subgenomic vRNAs we describe are probably similar to those found in influenza DI virus preparations. The possible role of ‘subgenomic’ polypeptides in DI virus-mediated interference is discussed.

The developing process of Plasmodiophora brassicae from the very young plasmodium to the fully matured resting spore was examined by using a scanning electron microscope on the infected cortical cells of turnip and Chinese cabbage. In the early stage of infection, the initial plasmodia in an infected cell were observed as spherical bodies of smooth surfaces with or without strand-like materials and ranged from 3 to 5μm in diameter or as a small cluster mass. The plasmodia developed rapidly. The cut surface of young plasmodia indicated that these developed into a large sponge-like structure. In some cases, the plasmodia consisted of spherical bodies. In a club-cut surface, young plasmodia and vegetative plasmodium were found in the two contiguous cells, respectively. With progressive growth of a plasmodium, a mature resting spore mass was seen in a cell and young plasmodia were noted in its contiguous cell. Young plasmodium or vegetative plasmodium and a resting spore mass were found together in a cell. Immature resting spores were observed to have filamentous connecting materials, while mature spores exhibited spines on their surfaces. At 45-60 days after sowing, the resting spores in an infected cell of club sampled perfectly matured and the cell was filled by innumerable spores. The spore mass did not appear to have an enveloping membrane. In paraffin sections of club tissue, the cell wall of the resting spore was clearly seen.

A characteristic c.p.e. is observed during growth of human adenoviruses in established cell lines. When infected cell cultures are examined by cytochemical techniques, basophilic and Feulgen-positive intranuclear inclusions are prominent features (Boyer, Denny & Ginsberg, 1959). However, the development and form of the mature inclusion differs according to the antigenic type of the infecting virus. Microscopic examination of unstained monolayers reveals a distinctive rounding and aggregation of cells into grape-like clusters. Even though infected cells show considerable c.p.e. they do not lyse and progeny virus particles are not readily released (Ginsberg, 1958; Denny & Ginsberg, 1961). Normally adenoviruses grow only in cell cultures derived from tissues of their natural hosts (Pereira et al. 1963). As adenoviruses isolated from apes grow in human cells (Rowe, Hartley & Huebner, 1958), it was of interest to investigate the replication of human adenoviruses in ape cells.

Purified hexon antigens were prepared from adenovirus infected cell cultures of types 1, 2, 5, 6, 7, and 12. Molecular size was determined by molecular sieve chromatography. Types 2, 5, 6, 7, and 12 had molecular weights ranging from 182,000–204,000 ± 10%. Type 1 hexon antigen yielded a value of 236,000 ± 10%. These data are in agreement with Valentine and Pereira's (2) calculated value of 210,000. Sedimentation constants of 10.6–11.4 were obtained with hexon antigens of types 2 and 12. The sedimentation coefficients were compatible with the molecular size determinations. The physical and serological characteristics of the hexon antigens were described and discussed.

A messenger selection process operates in infected cell extracts at the level of polypeptide chain initiation; and the agent responsible is present chiefly in the factor fraction of the ribosome.

The synthesis and accumulation of mengovirus replicative intermediate, replicative form, and single-stranded RNA proceed in an identical manner in both restrictive and productive cells for the first 4 hours after infection. Subsequent to 4-hour post-infection, total mengovirus RNA accumulation in restrictively infected MDBK ceases, whereas viral RNA accumulation in productively infected L cells and Ehrlich ascites tumor cells continues. Pulse-labeling experiments demonstrate that the synthesis of viral RNA continues beyond 4 hours after infection in MDBK at a rate equal to that in productively infected cell lines. It is concluded that the restrictive cessation in viral RNA accumulation and the limited yield of virus in MDBK result from the degradation of newly synthesized viral RNA.

Studies on the process of lysogenization was performed with the specified system of temperate phage β and KA cell (Shigella flexnerii type 5) on which concentrated researches have been made by other workers in this laboratory.In this system some of the survivals of infected cells usually form colonies with irregularly bitten edge. Replating these unentire colonies and assaying for the phage-producing ability and phage-sensitivity, it was found that these colonies consisted of both lysogenic cells and mostly sensitive cells, being partially lysed by phage liberated from their lysogenic members.The growth of infected survivals was followed to elucidate the manner with which sensitive cells originate. Thus, the decrease in the fraction of lysogenic centers to total population accompanying with the division of infected survivals was definitely shown. This result cannot be interpreted but the segregation of sensitive cells occurs in the progeny of infected survival. In other words, it is most conceivable from the characters (multiplicity and slope) of these curves that lysogenic response may be supported by each phage material infected and that the germ supporting lysogenic response may be randomly distributed into the progeny of initially infected cell, during which it does hardly any multiplication themselves. Therofore, it was pointed out that the germ to produce lysogenic state, were different from prophage in character, and pertinent to be referred to as potential prophage.The author laid emphasis on that prophage is to be established only after the period of potential prophage to be elapsed.

Bacterial viruses were discovered in 1915 when Twort (49) observed a serially transmissible agent that destroys bacteria. Twort published this finding in a brief note that remained unnoticed until, two years later, d'Herelle (20) announced his, probably independent, discovery of an entirely analogous filtrable virus, to which he gave the name "bacteriophage". D'Herelle's announcement caused an immediate sensation in the world of medical microbiology, because d'Herelle promulgated the idea that bacteriophages, rather than antibodies, are the chief agents of natural immunity against bacterial infections and should be most useful for a generalized therapy and prophylaxis. Despite holding to these, in the event quite mistaken, notions, d'Herelle managed to recognize bacterial viruses for what we know them to be today. Within two or three years of his original discovery he had invented the method of plaque counting that made possible the accurate titration of phage, and by 1923 he had outlined the true life cycle of the phage (21): the virus particle first attaches itself to the surface of the bacterium, then penetrates into the interior of the cell, where it reproduces itself to generate an issue of many progeny viruses. The progeny are liberated and ready to infect further bacteria when the infected cell finally bursts open, or undergoes lysis. Although, now in retrospect, these ideas not only seem eminently plausible but also happen to be true, they were accepted by few of d'Herelle's contemporaries. Especially the doctrine that the bacteriophage is a selfreproducing virus gave widespread offense, and such adversaries of d'Herelle as Bordet (3) and Gratia (19) preferred to think of it as a self-stimulating enzyme endogenous to the bacterium. Nevertheless, as Burnet (6) observed in 1934, "however agnostic they have been in regard to the nature of a phage, all workers manipulated and in practice thought of it as an extrinsic, virus-like agent." Phage research made a Big Leap Forward with the appearance of M. Schlesinger on the scene. From about 1930 until his death in 1935, Schlesinger was the first to train the methods of "molecular biology" on bacterial viruses. Schlesinger showed by various indirect methods, such as the adsorption capacity of the bacterial cell for phage and the sedimentation velocity of the phage, that the virus particle has a maximum linear dimension of the order of 0.1 I& and a mass of about 4 x 10 -1eg.

Only about 10% of the cells in chick embryo cultures which are infected with high concentrations of Rous sarcoma virus produce typical Rous sarcoma foci when the cells are transferred to a normal culture. The proportion of cells which produce foci may rise sharply if the plating is delayed for a day or two after infection and carried out under optimal conditions. It can be shown that this rise is not due to the infection of normal cells in the culture by virus released from infected cells. On the contrary, it appears that cells which are plated for focus formation within a few hours after infection have a low probability for initiating a focus, but this probability rises if the plating is delayed until 1 or 2 days after infection. The increased probability for focus formation by an infected cell associated with the lengthened interval between infection and plating is apparently due to the greater chance that cells infected for a day or more will either produce virus during a critical period after plating or will multiply to produce progeny cells with a characteristic morphology. The direct infectivity assay of the free virus itself is probably inefficient to about the same extent as the assay of freshly infected cells, and methods are suggested for arriving at more accurate estimates of the number of infective particles in a preparation. Cytochemical evidence supports the view that the proportion of cells actually infected is higher than that revealed by plating the cells soon after infection. The same observations suggest that early stages of infection are associated with aberrant synthesis of ribonucleic acid.

Exposure of hamster cells to large doses of polyoma virus is known to result in permanent transformation of a small proportion of cells. Experiments with cells in suspension now suggest that a much higher proportion of infected cells undergoes a temporary change, in which at least one characteristic of transformation is acquired for a few cell divisions and then lost again. Thissuggests that acquisition of the transformed phenotypes does not depend upon stable integration of the viral genome.

Treatment of rubella virus preparations by heating (50°C, 10 min.) resulted in an activation of their hemagglutinating potency, whereas the infectivity (as determined by plaque forming units) of the treated viral preparations remained unaffected.

Earlier light microscopic investigations have revealed that the endophyte ofAlnus glutinosa presents itself in three different forms. In the present study this is confirmed by electron microscopy; also, new data on the cytology of the endophyte have been obtained.

1. When detached portions of the chorioallantoic membranes of normal fertile eggs are examined in the dark-field microscope spherical and tubular cytoplasmic protrusions are seen to develop from the margins of the cells and to become detached from them. This is probably a normal physiological phenomenon which occurs to some extent in the intact egg.2. Similar cell protrusions occur from chorioallantoic membranes of eggs infected with influenza virus. The phenomenon appears to be more pronounced in the infected egg than in the normal egg and in particular tubular cytoplasmic protrusions are much more frequent in the infected egg than in the normal.3. Tubular cytoplasmic protrusions may fragment into small spherical particles or may contract into filaments. All types of cell protrusion become detached from the cells and undergo contraction in size probably as a result of loss of water. The infective elementary bodies and filaments present in the allantoic fluid of eggs infected with influenza virus are derived from the cells in this way.4. It is concluded that the infective particle of the influenza virus is a fragment of the cell cytoplasm and consists of a closely aggregated mass of virus protein enclosed in cell membrane.The author is indebted to Prof. W. T. Astbury and Dr R. Reed for the electron photomicrographs reproduced in this paper.

This is the first in a series of three papers presenting results obtained by the use of data processing machines for testing virus-genetic theories. A method is described which permits the representation of the genetic characteristics of a virus and other features, such as labelling or radiation damages of any kind by numbers (to be called genetic data) which can be inserted into the memory of a data processing computer. The genetic theory to be tested can thereafter be translated into a program for the data processing machine that will instruct the machine to perform on the “genetic data” the operations which, according to the theory, would be performed in a cell infected by the virus or viruses specified by the genetic data. The machine will give an output in the form of numbers specifying the genetic characteristics of the progeny viruses, if any, liberated by the infected cell. This output can be compared with experimental data obtained by virus-genetic experiments as a test for the theory. The method is primarily designed to simulate crossing experiments ( not chemical experiments) by a data processing machine. This method has been used to test a variety of partial replica models. The model which has been selected as the one giving the best fit to experimental data is described in this paper. The results of the testing operations, showing how the theoretic predictions obtained by the machine fit a large number of experimental results, are presented in the next two papers of this series.

Nascent f2 phage RNA can be isolated from infected Escherich a coli attached to replicating structures. The isolated replicating structures contain both single- and double-stranded RNA. We have digested away the double-stranded portions with E. coli ribonuclease III and have studied the properties of the purified nascent single strands. They resemble incomplete viral RNA by the criterion of annealing as well as by enzymatic and chromatographic tests. End group analysis of the isolated nascent phage RNA shows it to contain the same 5' end group present in intact RNA isolated from phage particles. Since this end group is the guanosine tetraphosphate, viral strands are synthesized in a 5' to 3' direction. The average size of a nascent strand was determined by measuring the ratio of 5'-terminal phosphate to internal phosphate, and comparing this ratio to that obtained from intact phage RNA. The nascent strands average 0.25 of a full length strand, shorter than the size expected were RNA synthesized at a constant rate. This finding may reflect one way in which the phage controls the availability of its cistrons for translation in the infected cell.

Sindbis virus, a group A arbovirus, is composed of a nucleocapsid and a glycolipoprotein membrane (Pfefferkorn & Hunter, 1963a; Strauss et al. 1968; Schelle & Pfefferkorn, 1969). The membrane protein is specified by the virus genome (Pfefferkorn & Clifford, 1964; Strauss et al. 1968; Strauss, Burge & Darnell, 1969) whereas the phospholipids and carbohydrate are derived from the host of origin (Pfefferkorn & Hunter, 1963b; Burge & Strauss, 1969; Strauss, Burge & Darnell, 1970) during maturation by budding through the membranes of cytopathic vacuoles (CPV) or the plasma membranes of the infected cell (Grimley, Berezesky & Friedman, 1968; Hackett et al. 1968; Holmes, Wark & Warburton, 1969; Nakai, Shand & Howatson, 1969). We have studied the maturation of Sindbis virus with the use of ferritin-conjugated antibody. After exhaustive adsorption with normal chick embryo cells, the globulin fraction precipitated from rabbit anti-Sindbis serum (Keckwick, 1940; Strauss et al. 1960) was conjugated with ferritin (Singer, 1959; Hsu, Rifkind & Zabriski, 1963; Rifkind, Hsu & Morgan, 1964).

Groman, Neal B. (University of Washington, Seattle) and Grace Suzuki . Temperature and lambda phage reproduction. J. Bacteriol. 84: 431–437. 1962.—The effect of temperature on lambda phage production in Escherichia coli K-12 was studied, and a temperature-phage yield relationship established. Phage yield declines slowly below optimal temperature and rapidly above the optimum. An extensive comparison of phage reproduction at 37 and 44 C was made when it was observed that at 44 C phage yield was reduced to 2 to 5% that at 37 C in the absence of any significant effect on bacterial growth. The reduced yield is manifest rather uniformly in every infected cell, and the reduction is due primarily to a speed-up of the lytic process. Temperature shift-up and shift-down experiments established that the temperature-affected step or process is initiated about 25 min after adsorption. This is close to the time the first intracellular phage appears. The data indicate that heat inactivation, lysogenization or major blocks in adsorption, penetration, replication, and maturation make minor contributions, if any, to the depression of the yield. Phenotypic adaptation of phage and host to the production of a greater yield at 44 C was not observed. There was no correlation observed between the enhanced thermoresistance of free λ vir, tr 1 phage and ability to reproduce at 44 C.

The replicating DNA pool contained in Escherichia coli infected with T-even bacteriophage was examined by chromatography of phenol-extracted lysates on columns of methylated albumin—kieselguhr and by zone sedimentation of unextracted lysates. The pool apparently contains few DNA molecules having the properties of DNA extracted from mature phage particles. Part of the replicating DNA resisted phenol extraction and part of the DNA that could be extracted was found to bind irreversibly to the chromatographic columns. If extraction was eliminated and the lysates examined by sedimentation, the bulk of the newly synthesized DNA sedimented considerably faster than mature phage DNA. These properties could be those of a large, fragile structure that contains regions of single-strandedness. In contrast with the newly synthesized DNA, the intrabacterial parental DNA immediately after infection could be identified as normal phage DNA molecules. These, however, seem to become modified in some way, since their recovery rapidly decreased with time after infection. A small part of the parental DNA became fragmented in the infected cell. A much smaller fraction of newly synthesized DNA was present as fragments. The difference between lysates produced by treating cells with detergent and treating cells first with lysozyme and then detergent is discussed, and some properties of the cells treated only with detergent are described.

Summary Long-term inapparent infections were established in continuously grown uncloned Aedes aegypti mosquito cells inoculated with Semliki Forest virus and incubated at 28°. This response of mosquito cells to infection, which contrasts with the outcome of infection by the same virus of mammalian cells at 37°, was due to a small proportion of virus-producing cells in the culture, and a low yield of virus per infected cell. The percentage of virus-producing cells was between 2.8 and 8% at the height of infection, and between 0.01 and 0.09% at the lowest level of infection. The yield of virus per infected cell was estimated to be between 1 and 6 p.f.u.

Experiments of Lanni showed that only about 10% of the DNA of adsorbed T5 phages is rapidly transferred to the host cells; further transfer is almost halted and follows only if protein synthesis is allowed to occur. The T5-DNA molecule is known to to have several single-strand breaks at specific loci which may serve as a kind of mechanical stop in the injection process. To find out if the specific breaks on the T5-DNA have any influence on the transfer of the DNA molecule from the head through the long narrow phage tail core, we studied T5-DNA ejection triggered by T5 receptors isolated from E. coli B cells. In experiments using equilibrium density gradients we have shown that the whole T5-DNA molecule is expelled once the tail core has been opened. The specific single breaks of the T5-DNA molecule have no influence apparently on the transfer through the tail core, since no time delay in kinetic experiments was observed. More than 70% of the DNA content of receptor-bound phages was ejected in 10 min, whereas Lanni found that only 10% of the phage DNA was transferred into bacteria in the same period when 5 × 10 9 cells/ml were used. Zone sedimentation of a phage receptor complex through sucrose gradients showed that the whole expelled DNA is concentrated in one peak of the same sedimentation constant as the intact T5-DNA. The present results show that the first-step-transfer DNA (FST-DNA) does not exist as a separate piece in the phage particle. They suggest that Lanni's observation results from an unusual uptake mechanism by the infected cell. All these findings fit the physical model of phage DNA ejection which predicts that the whole phage DNA should be expelled once the tail core has been opened ( Zarybnicky, 1969a ).

Mutagenized T4 was screened for hydroxyurea sensitivity on mutant strain Escherichia coli OK305, which was growing slowly on a synthetic medium. Most of the hydroxyurea-sensitive mutants of T4 isolated in this manner are different from those previously described by Warner et al. (1970) and Hercules et al. (1971), and several may be in new cistrons. Most of them produce fewer progeny per infected cell than wild-type T4, even in the absence of hydroxyurea. The first seven mutants studied map at seven distinct sites and affect at least five different cistrons which are scattered throughout the major early region of the T4 genetic map (between gene 38 and rI). At least one of these mutants is defective in the ability to degrade E. coli DNA.

The protein coat of the filamentous DNA-containing bacteriophages M13 and fd could be removed from the exterior of the host cell after successful infection had been initiated. M13 and fd thus behaved similarily to other known Escherichia coli bacteriophages. If the disassembled coat protein subunits were not removed from the surface of the infected cell, they were eventually ingested and utilized by the host and appeared partially in progeny phage.

A method for rapid measurement of HeLa cell destruction by cytopathic poliovirus based upon alterations of the rate of oxygen uptake of infected cell populations is presented. Alterations of the rate of respiration of infected cell populations occurred concomitantly maximal virus production. The magnitude of the alteration in rate of oxygen uptake was proportional to the virus-cell ratio within limits. Quantification of the yield of virus as well as the length of the latent period could be readily made.

Although, as is well known, the ultra-microscopic viruses are invisible in histological preparations, yet characteristic bodies occur within certain of the cells of animals infected with such organisms. The origin and nature of these so-called “virus bodies” has been the subject of much controversy. By some they have been regarded as the actual parasite, or at least as phases in its life cycle. To von Prowazek they were dual in character consisting of microorganisms embedded in material produced by the reaction of the cytoplasm of the infected cell. Still other observers regarded such bodies as products of cellular disintegration. In a former paper (Findlay and Ludford (1926)) we have referred to the various views held by writers in this field and have made a survey of the literature of the subject in the form of a pictographic review. We shall, therefore, only mention previous work in this field, in so far as it directly concerns our personal observations. A conspicuous fault of much of the earlier work has been the unsatisfactory histological technique employed, especially the nature of the fixative. The earlier cytological work was carried out with fixatives, which although satisfactory for the subsequent demonstration of nuclear structures were very destructive to the cytoplasm. This is particularly unfortunate since most of the virus bodies occur in the ground cytoplasm of cells. We have endeavoured to rectify this source of error in our work by employing fixatives, which have been proved to fix the cells in such a manner as to give an appearance as nearly as possible identical with their structure, as seen in the living cells.

The fine structure of the rat virus X14 has been studied in the electron microscope by negative staining and ultra-thin sectioning techniques. The virion appears to be a member of the T = 3 icosadeltahedral series with a markedly concave image in the electron microscope. The virus develops first in the nucleus of the infected cell and cannot be detected in the electron microscope until 3 days after inoculation. This investigation was supported in part by Public Health Service research grant CA 04600 from the National Cancer Institute and grant AI 05382 from the National Institute for Allergy and Infectious Diseases, National Institutes of Health. The authors are grateful to Dr. J. L. Melnick for his continuing support and very helpful criticism of this manuscript.

Transformation of a clonal line of BHK21 cells by type 12 adenovirus (Ad 12) is described. A transformation rate of approximately 2 × 10−5 per initially infected cell was observed after high multiplicity infection. A study of the fate of the abortively infected cells revealed, however, that only a small fraction survived the infection and initiated growth of a colony. Of these rare surviving colonies, nearly half contained transformed cells. Of three BHK21 sublines tested, one yielded transformed cells only from colonies growing in soft agar suspension, a second yielded transformants only as foci in monolayers, while the third did not yield detectable transformants by either method. The adenovirus-transformed cells were distinguished from the parental BHK21 cells by the following characteristics: The latter property is most likely due to blockage of a step late in the adenovirus replicative cycle, since 88% of the cells, which synthesized Ad2-specific structural antigens did not yield infectious virus. A similar shift from a productive to a largely abortive response to Ad 2 was seen in BHK21 cells simultaneously infected with Ad 12 and Ad2. This finding supports the view that the restriction of Ad2 replication in Ad 12-transformed cells, like the other characteristics mentioned, is associated with continued activity of at least part of the Ad12 genome.

A blue-green algal (BGA) virus (AS-1) which infects the unicellular algae, Anacystis nidulans and Synechococcus cedrorum, has been isolated from a waste stabilization pond. We propose the AS-1 virus as the prototype of a new BGA viral class. The AS-1 particle contains double-stranded DNA and appears polyhedral in structure with a contractile tail. It has a head edge-to-edge diameter of 900 A, a tail length of 2435 A, and a tail width of 225 A. Magnesium ions are not required for stabilization of the virus, while NaCl increases its adsorption rate by 34%. AS-1 has a latent period of 8.5 hr, a rise period of an additional 7.5 hr and an average burst size of 50 PFU/infected cell.

The susceptibility of JTC-3 cell incubated at 40°C and/or 36°C to poliovirus Type 1 and Coxsackie Virus B-5 has been studied.Results were as follows:1) The intracellular and extracellular viral yields were produced less in cells held at 40°C than 36°C.2) The CPE was developed more slowly in cells held at 40°C than 36°C.3) Infected cells incubated at 40°C were transferred at 36°C after 2 days, on the contratry, the another infected cells incubated at 36°C were transferred at 40°C after the same hours.In the former group the CPE was developed more rapidhy than in the latter group.4) Any inactivator of virus could not be recognixed in the infected cell incubated at 40°C.5) The high temperature prior to the infection was not responsible to the alteration of viral susceptibility of cells.

The titre of infectious phage particles in phage lysates stored at +4°C gradually fell. The inactivated particles retained their capacity for adsorption to male cell receptors, however, competing for the latter with infectious particles and thus protecting the cells from infection. The upper limit of the infected cell fraction in a F+ population fell abruptly with aging of the lysate even when the input of p.f.u. was kept constant. F2 particles inactivated by u.v. radiation behaved similarly to particles inactivated spontaneously during storage of the lysate at +4°C.

A study of the physiological effects of T2 ghosts, prepared by the method of Anderson, has been made. It was found that 75% of the ghosts can be adsorbed to host cells as measured with S35. Of these, only 10 to 35% are killers. The remainder, although adsorbed, do not prevent colony formation. A number of experiments, both in liquid culture and on agar plates, show that the effect of this latter class of particles is a temporary one. They lead to an arrest in bacterial multiplication, to a very marked inhibition of protein synthesis (as measured by S35 uptake and β-galactosidase synthesis), to exclusion of either T2 or T1 phage, to continued respiration as with a phage infected cell, and to P32 uptake approaching that to be expected from the number of cells not killed. At about 80 min., these cells reverse the action of the ghosts and resume all their normal metabolic activities.

When chick embryo cells are infected with high concentrations of Rous sarcoma virus (RSV) and cloned, most of the clones which produce RSV contain transformed cells. The remaining virus-producing clones, however, are normal in appearance. When cells from these nontransformed virus-producing clones are dispersed and recloned they give rise to clones, of which 90% are normal in appearance and produce no virus and 10% contain transformed cells and produce virus. It is suggested that nontransformed virus-producing clones arise from infected cells which grow into mixed clones. This occurs when virus is segregated to only one of the two daughter cells produced during the first divisions of a newly infected cell.

Sodium deoxycholate treatment of adenovirus-infected cell cultures was found to release rapidly sedimenting penton components with a buoyant density in CsCl of about 1.34 g/ml. The high-density penton material derived from human and simian adenovirus-infected cells occurred in monomeric form, while that derived from canine adenovirus-infected cells was in the form of non-symmetrical groupings of pentons aggregated at the vertex capsomere. Deoxyribonuclease treatment and 3H-thymidine-DNA labeling experiments revealed that the high-density of the pentons so obtained was due to a small associated segment of nuclease-sensitive material. A possible explanation of the nature of the nuclease-sensitive material is given.

Five virus isolates were obtained from pox-like lesions in swine. The respective skin lesions, infected cell cultures, virus particles and experimentally infected young swine were identical to that of swinepox by histologic examination, immunofluorescence and electron microscopy. The fluorescent antibody test provided a rapid and accurate means of diagnosis of naturally occurring lesions.

The amino acid composition was determined on the proteins of naked nucleocapsids from several isolates of herpesvirus types 1 (HSV-1) and 2 (HSV-2). The viral nucleocapsids were isolated from whole infected cell lysates and partially purified by treatment with Nonidet P-40 followed by centrifugation through a discontinuous sucrose gradient. The partially purified virions were freed of residual fragments of envelope by treatment with a combination of deoxycholate and Tween-40. Highly purified HSV-1 and HSV-2 nucleocapsids had a similar amino acid composition. Arginine was not present in unusually high levels in the HSV nucleocapsid.

1. Using living midrib cells of plants, the writer made microscopical observations on the protoplasmic changes induced by the infection of Phytophthora infestans.2. In the highly resistant variety, acceleration of the appearance of protoplasmic strands, the migration of the nucleus towards the infected part, and the rhythmic movement of the nucleus around the infected part were observed in the early stage of penetration of P. infestans. These activated visible movements in the infected cell may possibly be due to the enhanced motive force of the protoplasmic streaming in that cell. This acceleration of protoplasmic movement in the infected cell was more remarkable in the resistant variety than in the susceptible one.3. The number of protoplasmic strands in which the particles flow towards infected part is remarkably greater than that of the strands in which the particles flow in the opposite direction. So it seems that the motive force of the protoplasmic streaming is stronger in the direction towards the site of infection than in the contrary direction. The migration of the nucleus towards the site of infection may be resulted from this polarity of the motive force in the infected cell.4. It is presumed that the enhanced motive force in the infected cell of the resistant variety may be concerned with the increased energy metabolism in the infected tissues.

Stages in the development of an insect virus are shown in this study. Ultra-thin sections of diseased tissue were observed and photographed with the electron microscope. The presence of virus particles in large numbers in the infected cell nucleus is demonstrated by electron micrographs. Information on the development of polyhedral bodies is presented.

Mouse peritoneal macrophage cells suspended in a TC 199 calf serum medium and cultured in Leighton tubes, were infected with Trypanosoma (Schizotrypanum) cruzi culture forms in order to study the development of this parasite in vitro. Three cycles of development were thought to occur: 1. Epimastigotes or trypomastigotes were taken up by macrophages and transformed into amastigotes. These multiplied by binary fission and ruptured the infected cell after 4‐5 days. Released amastigotes were taken up by uninfected macrophages and the cycle was repeated. 2. A small proportion of intracellular amastigotes developed into ‘ovoid forms’ which transformed progressively into promastigotes, epimastigotes and trypomastigotes. 3. Some amastigotes became ‘round forms’ from which sphaeromastigotes developed. These transformed directly into trypomastigotes without the formation of epimastigotes.