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The structural and functional proteins expressed by Varicella-Zoster virus (VZV), the agent that causes chicfcenpox and shingles, wereexamined at the infected cell level and the virion level. To accomplishthis a new cell line of human foreskin fibroblasts was employed for thegrowth of the virus. In addition a unique virion isolation techniquewas developed using sucrose zonal centrifugation followed by a combinationof equilibrium-viscosity sedimentation with potassium tartrateglycerolgradients.

The shut-off of protein simthesis in virus-infected cells is a phenomenon that has attracted the attention of several investigators. In an attempt to provide an explanation at the molecular level for the mechanism of shut-off, studies were initially carried out with virus-infected cells. Since the results of this kind of studies are extensively discussed in the proceeding chapter of this book, we will concentrate on a particular aspect, namely the role of initiation factors on the virus-induced inhibition of host protein synthesis.

Interferon messenger RNA activity demonstrable in intact heterogenous cells could be extracted from GD-7 virus infected L cells which had been primed by interferon but not from unprimed GD-7 virus infected cells.

We have recently applied the fluorescent antibody technique for rapid detection of viral antigens in several organs of infected trouts as well as in the supernatant of infected cell cultures (Enzmann, 1978, 1979). For diagnosis of nonacute infections of trout with VHS virus a method was developed which allows the rapid detection of viral antigens in leukocytes after isolation of the white blood cells. Leukocytes have been shown also to become infected during an acute VHS infection and may be functionally impaired during the disease. Fluorescent antibody studies on the persistence of infection and on the detection of antigens in acute infections will be described.

Summary The fate of parental SV40 DNA in monkey kidney cells was investigated by infecting the cells with purified virus labelled with [3H]-thymidine. 5-Bromodeoxyuridine (200 µg/ml) was added to the cells at 2 h after infection to label virus progeny DNA. At 72 h post-infection the cells were harvested and virus DNA was extracted and fractionated by isopycnic sedimentation in CsCl solution. The following DNAs with characteristic densities were found: light (LL), 1.70 g/ml; hybrid (HL), 1.75 g/ml and heavy DNA (HH), 1.80 g/ml. Of the total cell-associated [3H]-radioactivity (derived from parental virus), more than 90% was recovered in unreplicated parental DNA (LL DNA), about 2% was recovered in the HL DNA and about 0.7% was associated with the HH DNA. The unreplicated parental DNA was present as uncoated intact DNA complexed with proteins present in the infected cell. The HL DNA contains one light parental and one heavy progeny DNA strand. The nature of the radioactivity present in the HH DNA remains to be determined.

Infection of Drosophila cells with Cricket paralysis virus in the presence of Actinomycin D results in virtual complete inhibition of host cell protein synthesis by four hours post-infection. Using 35S-methionine or 14C-amino acids to pulse infected cells three major classes of viral induced proteins can be detected, (A) high molecular weight precursor proteins, (B) viral structural proteins and (C) low molecular weight cleavage products. The large number of high molecular weight proteins found in the infected cells suggests that a multiple cleavage cascade mechanism is partially utilized to produce virus structural proteins. In infected cells, even with short pulses, the largest viral induced protein obtained has a molecular weight of 144,000. However with pretreatment of the infected cells with iodoacetamide before pulsing, two further proteins are obtained with molecular weights of 205,000 and 190,000. Other changes occur in viral protein precursors in the presence of iodoacetamide.

Adenoviruses have been isolated from a number of different species including man (33 serotypes), monkeys, mice, dogs, cows, birds (Green, 1970), and frogs (Clark et al., 1973). The most extensively studied adenoviruses are those of human origin. In particular, we now know a great deal about the structure and replication of the DNA from human adenoviruses types 2 and 5. For this reason this review will be restricted to a discussion of these two viruses.

Summary Structural and physiological properties of two mutants of mengovirus, 205 and 280, were compared to those of wild-type virus to understand the molecular basis of changes exhibited in their biological function. Two dimensional gel electrophoresis of wild-type and mutant structural proteins revealed alterations in the isoelectric character of the alpha (1 D) protein of both mutant 205 and 280. These data suggest that alterations in the alpha (1 D) protein may be responsible for the phenotypic changes by the mutants. A delay in detectable virus-specified protein synthesis was exhibited in mutant-infected cells in comparison to wild-type. The amount of RNA synthesized in mutant- and revertant-infected cells was less than that synthesized in wild-type infected cells. Changes in virus-specified macro-molecular synthesis in mutant and revertant-infected cells reflected a decrease in the ability of the viruses to attach to cells.

Using hyperimmune rabbit sera to BHV1, ID and CIEP tests detected one antigen in BHV1 infected cell culture harvests. In nasal and ocular secretions collected from experimentally infected cattle during the early course of clinical disease two and one BHV1 antigens were detected by the ID and CIEP tests respectively. In specimens collected from eight cattle for 10 days after the onset of pyrexia the ID test demonstrated precipitating antigens in 22/35 ocular secretions and 43/60 nasal secretions, whereas the CIEP detected antigens in 40/64 ocular secretions and 44/69 nasal secretions. The antigens were thermostable, being detectable by CIEP 14 days and seven days after storage at 4 degrees C and room temperature respectively.

Summary A persistent infection by human coronavirus 229E (HCV/229E) was established in a human continuous cell line (L132). Following the initial infection with stock HCV/229E, several cultures were established of which two (HV1 and HV4) have been maintained by continuous passage for two years. These cultures have shed high titres of infectious virus continuously into the supernatant fluid since their initiation. The persistently infected cells were resistant to homologous super-infection but supported polio virus replication to normal titres. Preliminary tests indicated that 50–100 percent of the cells contain virus. Neither interferon nor reverse transcriptase could be detected in these cultures and the presence of defective interfering particles could not be demonstrated. VH1 and VH4 coronaviruses, isolated from these persistently infected cultures (HV) and identified by 229E antiserum neutralization, were more cytocidal than the parent virus as judged by plaque characteristics and CPE however, they were indistinguishable on the basis of density, EM morphology, and genome size. Present evidence indicates that temperature plays an important but as yet undetermined role in the establishment and maintenance of stable 229E persistently infected cell cultures.

The activity of the nine known adenovirus promoter sites has been studied in vitro in a whole cell extract system. Six sites (four early, one intermediate (PIX) and the major late promoters) functioned with comparable efficiency in uninfected extracts. The other early promoter (for early region II) was utilized only poorly. Two intermediate promoters (leftward at 15.9 and 72.0 map units) which function in vivo only at intermediate or late stages in the lytic cycle, were inactive in uninfected extracts. Although extracts prepared from late infected cells did not show transcription of these two promoters, these extracts did show an early to late shift in vitro . In late infected extracts the late and PIX promoters were enhanced five-to ten-fold relative to early promoters. DNA titration and extract mixing experiments indicated the presence in uninfected and infected extracts of factors which could distinguish between early and late promoter classes.

A study was made on the effect of human interferon (HuIFN) on the synthesis and release of type D viruses (MPMV and SMRV) by chronically infected human lines. Interferon at concentrations of 50 IU/ml lead to an inhibition of the extracellular virus production by about 50%-80% as measured by virus-associated reverse transcriptase activity, by metabolic labeling of the virus with (3H) uridine or (3H) amino acids, or by electron microscopy counting of particles. The profiles of intracellular viral proteins, as visualized by radioimmunoprecipitation and electrophoresis, were not different in control or IFN-treated cells and the yield of intracellular protein p27 stayed unchanged or was slightly increased. Electron microscopy of thin sections taken from the control and IFN-treated cells revealed no difference in any subcellular structures including that of the viruses. However, electron microscopic examination of IFN-treated cells showed an increased number of either budding particles and intracytoplasmic precursors (two- to five-fold) or mature virus particles (four- to ten-fold) on the cell surface of the IFN-treated cells. These results indicate that IFN inhibits further developments of MPMV and SMRV replication as was described for type C and B retroviruses.

Three-dimensional structures of the core of Marek's disease virus and herpesvirus of turkey were examined by the tilting apparatus of an electron microscope. Various types of the core found in the infected cells were considered to represent developmental stages of the viruses. The basic structure of the core consisted of a toroid surrounding a cylindrical mass, which was clearly demonstrated by tilting the core in two directions. A cylindrical mass spooled by more than two toroids, which seemed to constitute a spiral band of 10 to 20 nm, was demonstrated. The maturation process of the cores of the viruses was also discussed.

Monoclonal antibodies 35S and 55S, specific for herpes simplex virus type 1 (HSV-1) glycoproteins gB and gD respectively, were found to react with infected cell membranes but not cell-free virions. This indicates that the spectrum of viral determinants detectable on the infected cell surface is not identical to that detectable on the virion envelope. The monoclonal antibody 35S could promote recovery from ocular HSV-1 infection in mice. Thus, antibody reactive with infective cell-specific determinants can play a significant role in host resistance to HSV-1 infection.

Cytoecetes phagocytophila , a neutrophilic rickettsia which parasitizes sheep and cattle, was propagated transiently at 37°C in cultures of heparinized whole blood of sheep supplemented with Medium 199 containing HEPES buffer. Significant increases in the number of infected cells and the number of rickettsias per infected cell were observed within 24 h. Back-passage into sheep produced typical tick-borne fever.

The infection of cells by viruses is often followed by specific morphological alterations (Schrom and Bablanian, 1981). These alterations have been studied both with the light and the electron microscope (EM). Early investigations described them as cellular injury (Ackermann et al., 1954), cellular lesion (Barski et al., 1955), or cytopathic change (Dunnebacke, 1956).

The nature of nucleic acids present in the nucleoids of Epstein-Barr virus (EBV) was investigated with cytochemical methods. EBV infected cell cultures were embedded in glycol-methacrylate and thin sections were exposed to DNase and RNase and examined in the electron microscope. The results showed disappearance of viral nucleoids after DNase treatment, which provided evidence that EBV contains DNA, as do other herpes viruses.

Sindbis virus has been adapted to L cells in the course of serial passages until titre of infectivity and degree of cytopathic effect remained constant. The growth curve shows a latent period of 1 to 2 hours followed by an exponential rise of virus multiplication. Peak titre is reached 7 to 8 hours after infection.

The first version of this paper was written to introduce new students and fellows of my laboratory to the mysteries of herpesviruses. Consonant with this design sections dealing with well documented data were trimmed to the bone whereas many obscure phenomena, controversial data and seemingly trivial observations were discussed generously and at length. There is some doubt as to whether it was meant to be published, but it was not a review. The objective of reviews is frequently to bring order. But alas, even the most fluent summation of credible data frequently makes dull reading and too much plausible order, like very little entropy in chemical reactions, is not the most suitable environment on which to nurture the urge to discover. This version is more charitable but not less inbalanced. The bibliography reflects the intent of the paper and was updated last in December of 1968. It should be obvious without saying that no single account such as this can do justice or injustice, as the case may be, to the several hundred papers published on herpesviruses each year or to the many thousand papers published on herpesviruses since the first of the members of the family was experimentally transmitted to a heterologous host more than half a century ago (Gruter, 1924).

Summary The polypeptides of cells which had been infected with adenovirus and pulse labelled with high specific activity [35S]-methionine have been examined by polyacrylamide gel electrophoresis followed by autoradiography. Five virus-particle polypeptides and at least five other polypeptides which appeared to be specific for the infected cell could be discerned. One of the latter polypeptides could be detected very early in infection and is shown to be one of the major components of the previously described P antigen. The experiments also show that in the absence of arginine in the tissue culture medium, the infected cells fail to synthesize the arginine-rich core polypeptide.

Rubella virus was identified morphologically in infected cell cultures, and at the surface of rubella-agglutinated pigeon erythrocytes. The agglutinated cells possessed rubella infectivity for cell cultures, and the specificity of the haemagglutinin produced in these cultures was established serologically. Rubella virus was also concentrated efficiently by adsorption and elution using pigeon erythrocytes. The morphology of the virion and its mode of development, the identity of methods for the production and titration of haemagglutinin, and other chemical and biological similarities all indicate close affinities between rubella and arboviruses.

In its widest sense, virus morphogenesis denotes the de novo synthesis of structural virion components and their assembly into mature virus particles. The study of this process requires(a) an understanding of the nature of thesc components, (b) an awareness of the manner in which they are formed and (c) an understanding of the principles that govern their interaction. Rapid advances concerning the first two of these requirements, as they apply to animal viruses, have been registered during the past decade. The na­ ture of the genomes of most, though not all, of the major groups of animal viruses is now known, at least as far as their size, form, and overall composi­ tion are concerned. Similarly, the principal features of the polypeptide con­ stitution of many groups of animal viruses have recently been elucidated, al­ though many details still remain to be filled in. Furthermore, a great deal of information has now accumulated concerning the replication of animal virus genomes and the synthesis of the structural components that comprise the shells that encapsidate them. Considerably less is known concerning the nature of the interactions in­ volved in the morphopoiesis (1) of animal viruses. This process can be studied under two sets of conditions. The first of these is the natural one, which exists within the infected cell. The object here is to isolate and characterize the various successive intermediate stages as the simple virus components are assembled into virus particles. This is not always feasible, since the time necessary for an individual virus particle to be formed is always very much shorter than the time period during which the progeny virus population as a whole is assembled, so that cells always contain virus particles at all stages of morphogenesis. Occasionally some of the intermediate stages are suf­ ficiently stable to permit their isolation and detailed study, but this is usually not the case. There are two ways of overcoming this. First inhib­ itors capable of arresting morphopoiesis at specific intermediate stages can be used ; however such inhibitors are rare, although some are now beginning to become available. A more powerful aid is provided by suitable virus mutants. Such mutants have made possible the far-reaching series of studies on the morphopoiesis of the T-even bacteriophages (2-6) , the complexity of which exceeds that of most animal viruses. Temperature-sensitive mu

Groman, Neal B. (University of Washington, Seattle), and Grace Suzuki . Effect of spermine on lysis and reproduction by bacteriophages φX174, λ, and f 2 . J. Bacteriol. 92: 1735–1740. 1966.—A test was made of the hypothesis that lysis by all bacteriophages shares as a common and critical step an alteration in the osmotic stability of the infected cell. This was done by examining the effect of spermine on lysis. Spermine is one of a number of compounds which can stabilize spheroplasts and protoplasts to lysis in distilled water. Spermine stabilized both φX174- and f 2 -infected cells at concentrations ranging from 2 × 10 −3 to 4 × 10 −2 m , but failed to stabilize λ-infected cells at concentrations up to 8 × 10 −2 m . Stabilization was reflected both in optical density measurements and in the retention of mature phage in structures sedimentable at low speeds. At optimal concentration, over 90% of the phage was retained in these structures. These data suggest that the mechanism of lysis by φX174 and f 2 differs sharply from that caused by λ, and other observations suggest that there are differences in the lytic process of φX174 and f 2 as well. Spermine also displayed a differential effect on phage reproduction. The reproduction of λ and f 2 was inhibited by spermine, though the data do indicate that maturation occurs in its presence. The reproduction of φX174 was enhanced by spermine.

The interferon-inducing activity of herpes simplex virus strains, belonging to different antigenic groups was studied in chick fibroblast cultures. Only the initial variant of strain K and its plaque-forming clones were found to possess the ability to induce interferon. The optimal conditions for interferon formation were studied and the intensity of interferon synthesis was shown to be directly proportional to the multiplicity of infection. Clarification of “opaque” plaques formed by clones of strain K in the presence of 5-bromodeoxyuridine and during incubation of the infected cell culture at 28° C strongly suggested the correlation between interferon production and the ability of the clones of strain K to form opaque plaques.

SummaryThe synthesis of adenovirus T and V antigens in infected cell cultures incubated at different temperatures was studied. At low temperature (24°C) development of V antigens was limited, while significantly larger amounts of T antigen were produced. Raising the incubation temperature from 24 to 37°C led to a rapid increase in V antigen. Highest titers of T antigen for adenovirus types 12, 18, and 31 were obtained in Vero and RK-1 cells incubated at 30°C. The antigen produced in the cultures was found to be unusually cross-reactive with type 7 tumor antibody. Thermal separation of the synthesis of adenovirus T and V antigens in cell cultures should provide a method for the production of adenovirus T an tigens.

METHODS at present available for the detection and quantification of infectious Newcastle disease virus (NDV) particles include plaque assays1,2, the development of haemagglutinating activity and/or death of fowl embryos3, the appearance of cytopathic effects4, and the enumeration of virus infected cells by fluorescent antibody staining5. With the exception of the last technique, all these methods require a minimum of 3 days for adequate evaluation. The adsorption of erythrocytes to infected cell monolayers as a means of detecting certain non-cytopathic myxoviruses6 suggested to us a method for titrating NDV. This communication reports an assay for infectious NDV based on the ability of this virus to initiate foci of haemadsorbing cells. Results from such a titration can be accurately determined 24 h after inoculation of the virus on to susceptible monolayers. The rapidity and reproducibility of this assay technique have made it exceptionally useful in our studies on the synthesis and properties of NDV.

A series of five 1-(arylsulfonyl)-3,5-dialkyl-s-triazine-2,4,6-(1H,3H,5H)-triones was synthesized by the base-catalyzed reaction of arylsulfonamides with alkyl isocyanates. The compounds were tested for apomorphine antagonism in mice, diuretic activity in fasted rats, antiviral activity in mice, antiviral activity in infected cell cultures, CNS stimulant activity in mice, and hypoglycemic activity in rats. The only biological activity exhibited by these compounds was a slight tendency to lower blood sugar levels in glucose-primed, fasted rats. This activity may be attributable to their structural similarity to the sulfonylurea hypoglycemics.

Chromatographie an Ecteola-Cellulose ermoglicht die Trennung von 5 «loslichen» immunoprazipitierenden Antigenen aus einer mit Tollwut-Virus infizierten Zell-Kultur-Flussigkeit. Gel-Filtration derselben an Sephadex G-200 fuhrt zur Abtrennung von zwei Antigen-Klassen, die sich in ihrer Grosse unterscheiden. Die Kombination beider Verfahren ermoglicht eine weitgehende Reinigung der Antigene.

The nucleocapsid fraction derived either from Sindbis virions or from infected cell cultures was studied with respect to its ability to adsorb to susceptible cells as well as to initiate stages in viral replication. Neither preparation was adsorbed to BHK 21 cells under conditions where virion adsorption could be demonstrated. In addition parental RNA from virions was shown to be converted to an RNAase-resistant 16–20 S form, whereas no evidence for the penetration and conversion of RNA present in viral cores was observed. These studies emphasize the role of envelope proteins in facilitating entry of intact virus into susceptible cells.

An investigation of polypeptide synthesis in fowl plague virus infected chick embryo fibroblast cells is reported. The fowl plague virion contains seven types of polypeptide. The synthesis of six of these virion polypeptides was readily detected in infected cells; synthesis of the seventh, the neuraminidase polypeptide, was not detected. The virion polypeptides were synthesized in approximately the same unequal proportions as they were present in the virus particle and the rate of their synthesis was, therefore, controlled. In addition to the virion polypeptides three others were specifically detected in infected cells. One of these, of molecular weight 23,000 was shown to be synthesized before at least one of the virion polypeptides and was, therefore, suggested to be an early product of infection. Another was the smallest infected cell specific polypeptide of molecular weight 11,000. The third was a glycopeptide precursor of the virion haemagglutinin component. Apart from this glycopeptide it was not possible to detect other precursor polypeptides under conditions which allowed the clear detection of such molecules in picornavirus-infected cells. It was therefore proposed that all of the infected cell specific polypeptides with the exception of the haemagglutinin components were translated from monocistronic messenger RNA molecules.

A temperate phage, phage PS20, which originated in a lysogenic strain of Serratia marcescens was established to be able to transduce various auxotrophic markers in S. marcescens strains at an equal frequency which ranged from 10–6 to 10–7 per infected cell. Electron microscopic observations show that phage PS20 has a head (550 A) hexagonal in outline and a tail (1000 A × 150 A) with a contractile sheath which is attached to the head.

Information on the metabolism of the nucleic acids and proteins in virus-infected cells is fundamental to an understanding of the mechanisms of virus synthesis and assembly; it may also aid in the understanding of some of the causes that underlie cellular pathology and may provide a rational approach to the chemotherapy of virus infection.

Serratia marcescens HY was cured of two native prophages, ψ and y. Curing occurred after infection with temperate phage ϰ, but the cured strains did not become ϰ-lysogenic. One of them has been simultaneously cured of both Ψ and y. Recognition of cured colonies was based on their loss of immunity towards the respective phage. Whereas ϰ plates on the singly cured strains, it does not plate on the doubly cured strain. However, infection of it with ϰ leads to a limited phage multiplication, the average burst size being low and only part of the infected cells producing phage at all. The ability of the strain to serve as indicator for ϰ can be restored by relysogenization with either Ψ or y. In addition, some further relations between ϰ, Ψ, and y are reported in this paper.

In Escherichia coli cultures infected with the filamentous single-stranded DNA bacteriophage M13, mature phages cannot be detected intracellularly at any time after infection, even though as many as 1000 extracellular phages may be found per infected cell. Even at very late stages after infection, no more than about 300 intracellular infectious phage DNA molecules and no more than about 110 phage coat equivalents per infected cell can be detected.

Publisher Summary This chapter focuses on nutrient media used to support the growth of mammalian cell cultures which also provide excellent nutrition for bacteria and fungi. Routine sterility cultures saves time and expense to use multiple culture media and incubation temperatures at the outset. At the time the cell culture is opened for re-feeding or subculture, the spent media is removed and it is centrifuged for 20 minutes, and smears and cultures of the sediment are prepared. It is recommended that infected cell cultures should be autoclaved at once and start over again with a frozen stock of clean cells stored previously in liquid nitrogen. Because elimination of bacterial and fungal contamination is so unsatisfactory and detection is quite laborious, it is recommended that emphasis be placed on prevention. Prevention is practical and offers other fringe benefits. Remove antibiotics from tissue culture media. This encourages the practice of meticulous aseptic techniques and permits contaminants to grow promptly. Pretest all culture medium components including serum as described above and observe for 14 days before use.

Until recently the rod 280–300 mμ in length and 15 mμ in diameter has been considered an elementaryparticle of the tobacco mosaic virus (TMV). The composition of the rod is fairly complex. It has a two-component structure consisting of a protein casing and a ribonucleic acid axis. It is quite obvious that the rod-like particle with this complex structure must be formed from units of lesser size and must have its own history of development. However, there have been no morphological investigations undertaken and published, which would reveal the formation process of the TMV particle.

BK polyomavirus (BKPyV; hereafter referred to as BK) causes a lifelong chronic infection and is associated with debilitating disease in kidney transplant recipients. Despite its importance, aspects of the virus life cycle remain poorly understood. In addition to the structural proteins, the late region of the BK genome encodes for an auxiliary protein called agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to virion infectivity. Here, we demonstrate an essential role for agnoprotein in BK virus release. Viruses lacking agnoprotein fail to release from host cells and do not propagate to wild-type levels. Despite this, agnoprotein is not essential for virion infectivity or morphogenesis. Instead, agnoprotein expression correlates with nuclear egress of BK virions. We demonstrate that the agnoprotein binding partner α-soluble N-ethylmaleimide sensitive fusion (NSF) attachment protein (α-SNAP) is necessary for BK virion release, and siRNA knockdown of α-SNAP prevents nuclear release of wild-type BK virions. These data highlight a novel role for agnoprotein and begin to reveal the mechanism by which polyomaviruses leave an infected cell.

We analyzed lymphocytes of patients with chronic fatigue syndrome (CFS) for the presence of human herpesvirus 6 (HHV-6) and HHV-7 DNA. HHV-7 was present in over 80% of CFS patients and healthy controls, while the prevalence of HHV-6 variant A increased significantly in CFS cases (22 versus 4%; P = 0.05).

Both promastigotes and amastigotes of Leishmania enriettii were readily ingested by mouse peritoneal macrophages (MPM). Promastigotes after their entry within MPM were rapidly immobilized and their multiplication was never observed. Microscopic examination revealed that ingested promastigotes were degraded with MPM. Nonmotile amastigotes of L. enriettii taken up by MPM, on the other hand, multiplied intracellularly and eventually destroyed the infected cells.

Delayed hypersensitivity, as determined by the wattle skin reaction, to turkey herpesvirus developed in chickens sensitised with the infected cell antigen. A significant increase in skin thickness occurred in the sensitised wattles as compared with the controls.

Les etudes de virus peuvent mettre en lumiere et la regulation normale des genes cellulaires et la transformation cancereuse

Labeling of antibodies with I131 with subsequent autoradiography was used to detect the viral antigen in the cell.