Rhabdomyosarcoma is a mesenchymal highly malignant neoplasm. RMS is the most common soft tissues tumor of childhood,but its prenatal occurence is very rare, but also can have a genetic underlay associated with some rare familial “cancer syndromes”.We present a case of 27-year old primigravida with the singleton pregnancy. She was referred to our department at 30 weeks of gestation with the suspected facial tumor. Anomaly scan at the first and second trimester were both normal. Ultrasound showed mostly solid mass 77x52 mm with the small cystic portion arising from the right orbit and dislocating the bulb. The third part of the tumor expanding intracranially. Doppler flow in the mass was present. MRI confirm our finding, amniocentesis showed normal karyotype 46 XX. Differential diagnosis we consider teratoma or rhabdomyosarcoma. Due to the rapid growth of the tumor, its inauspicious location and 31+5 gestational age parents decided to the late termination of the pregnancy. The fetocide was done and at the same day we vaginally delivered baby girl 2370g and 46cm without no maternal injury. Postnatal autopsy confirmed rhabdomyosarcoma. We would like to point out that the fetal occurrence of head-and-neck RMS is very rare but possible. The prognosis of RMS tumor finally depends on staging and histological subtype. We consider the prognosis of our fetus in such circumstances highly adverse.

A spectrophotometric method for the quantitative determination of protein and 1-dimethylamininaphthalene-5-sulphonic acid in fluorescent antibodies was elaborated. It was shown that after exhaustive dialysis the conjugates do not contain free dye which would disturb the analysis. According to the conditions used during conjugation the average weight ratio of protein to fluorescent dye in the labelled γ-globulin was 76–245 corresponding to 3–9 molecules of dye bound to one molecule of globulin.

Light-scattering measurements were made on a preparation of polyglucose isolated from HeLa cells. The molecular weight was determined, from a Zimm plot of the data, to be 4.6" lO 6 . The character of the angular dependence of the particlescattering factor suggests the shape of the particle in solution with 779 3~ gyration radius approximates that of a rigid rod, 27oo A in length with a thickness of 5o A. By quantitative electron microscopy, using reference latex spheres, the molecular weight was estimated to be 1.35"1o 6. Electron micrographs show the preparation when air-dried on a collodion membrane to be composed of relatively uniform spheroid particles, approx. 30o A in diameter.

The structural polypeptides of purified viper virus range in molecular weight from 11,000 to 97,000 daltons and consist of 3 major and about 13 minor polypeptides. The virus contains both protein kinase and reverse transcriptase activities. Several of the structural polypeptides are phosphorylatedin vitro by the virus-associated protein kinase. However, most (possibly all) of the viral structural polypeptides are not phosphorylatedin vivo. DeoxyATP is as efficient as ATP in donating phosphate forin vitro phosphorylation of viral proteins.In vitro protein phosphorylation always precedes transcription and the virus-associated protein kinase and reverse transcriptase activities can be partially separated by sedimentation in a sucrose gradient.

Thermal inactivation of rabies and several other rhabdoviruses was studied using virus suspended in several different diluents. Rabies serogroup viruses were more stable than Kern Canyon or vesicular stomatitis viruses. Limited studies of two fish rhabdoviruses requiring low temperatures (less than 33 C) for replication indicated that they were not markedly more thermolabile than rabies virus. Bovine serum protein components in complex cell culture media stabilized virus at 56 C, but at temperatures of less than or equal to 37 C, sodium tris (hydroxymethyl)-aminomethane (NT) buffer containing ethylenediaminetetraacetic acid (EDTA) (NTE) was a much more efficient stabilizer of virus infectivity. Chelating agents EDTA and ethyleneglycol-bis-(beta-aminoethyl ether)tetraacetic acid were equally efficient in protection of rabies virus infectivity; the effect of each was lost when excess Ca2+ was added. Bovine serum in NT or NTE buffers produced a thermostabilizing effect at 37 C not provided by the same serum concentration in complex cell culture media. Bovine serum was more efficient than EDTA in stabilizing virus infectivity during repeated cycles of freezing and thawing.

Recently accumulated knowledge allows more precise comparison of the structural (and possibly evolutionary) relationships of several different animal rhabdoviruses: vesicular stomatitis virus, rabies virus, Kern Canyon virus, and spring viremia of carp virus. Each virus is composed primarily of a glycoprotein, an RNA-associated nucleoprotein, and one or two membrane proteins. Vesicular stomatitis virus group viruses contain lesser amounts of two additional distinct polypeptides, NS and L. The separate viruses undergo structural polypeptide phosphorylation in vivo according to characteristic patterns. In vesicular stomatitis virus the NS protein is selectively phosphorylated. In rabies group viruses and in spring viremia of carp virus, the nucleoprotein is the predominant phosphoprotein; in these viruses only the phosphorylated moiety is selectively cleaved off with trypsin. In Kern Canyon virus, only membrane protein and glycoprotein are weakly phosphorylated. Each virus possesses a virion-bound protein kinase. Vesicular stomatitis virus group viruses, Kern Canyon virus, and spring viremia of carp virus only contain virion-bound transcriptases of respectively decreasing levels of activity demonstrable in vitro. Vesicular stomatitis and Kern Canyon viruses replicate efficiently in enucleated cells; rabies virus does not. Based upon these observations, it is suggested that vesicular stomatitis virus may represent the most highly evolved of these rhabdoviruses, whereas spring viremia of carp and Kern Canyon viruses may represent "evolutionary links" between the vesicular stomatitis and rabies virus groups.

The fate of parental SV40 DNA in monkey kidney cells was investigated by infecting the cells with purified virus labelled with [3-H]-thymidine. 5-Bromodeoxyuridine (200 mug/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 [3-H]-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 1-7% 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.

SUMMARY Regardless of the input multiplicity used in propagating simian virus 40 in monkey kidney cells, virus populations essentially devoid of defective viruses still contain three types of particles: heavy virus (ρ = 1.35 g/ml), light virus (ρ = 1.325 g/ml) and ‘empty’ capsids (ρ = 1.30 g/ml). Neither light nor heavy virus fractions contain lipids. The protein moiety of the three particle forms is composed of the same polypeptides in similar proportions. DNA extracted from the light virus fraction sediments at a rate identical to that of DNA from heavy virus both in neutral and alkaline sucrose density gradients. The two DNAs hybridize with the same efficiency with the DNA from heavy virus and band together after isopycnic centrifuging in CsCl solution containing ethidium bromide, indicating structural similarity. The sedimentation rates of heavy and light virus particles and their sizes as determined by electron microscopy, are also similar. However, the protein to DNA ratio in the light virus fraction is twice that in the heavy virus fraction. Thus, either heavy virus particles contain two double-stranded, circular DNA molecules of identical molecular size, and light particles contain one, or the light virus fraction contains aggregates of heavy virus and particles devoid of DNA in equal proportion. The specific infectivity of heavy virus is about ten times larger than that of light virus, but the specific infectivity of virus DNA isolated from these virus forms is similar.

SummaryThe phosphorylation of structural proteins of ten rhabdoviruses has been studied. Each virus preparation has been found to possess at least one phosphoprotein which, in the case of Chandipura, Cocal, Piry viruses or vesicular stomatitis virus (VSV), Indiana and New Jersey serotypes, is the minor NS protein. In rabies virus the sole phosphoprotein appears to be the N protein of the nucleocapsid. For Mokola and Lagos bat viruses, two or three phosphoproteins have been observed, one of which is the N protein. Spring viraemia of carp virus (SVCV) preparations contain two phosphoproteins one of which is, or has a mobility close to, that of the N protein. Kern Canyon virus preparations also possess two phosphoproteins, one with the same electrophoretic mobility as (and possible identity to), the virus glycoprotein while the other has a mobility slightly slower than the N protein. Antigenically the core proteins of rabies, Mokola and Lagos bat viruses have been shown to be related to each other but distinct from those of SVCV or VSV.

Semliki forest virus and Sindbis virus (Alphaviruses belonging to the togavirus group) grown in BHK-21 cells possessed very low levels of virion-associated protein kinase activity. For comparison, vesicular stomatitis virus, also grown in BHK-21 cells, contained a virion-bound protein kinase which had a specific activity 80 times greater than that of the Alphaviruses. The Alphavirus protein kinase was unmasked by the nonionic detergent Nonidet P-40 but was not activated by cyclic nucleotides. Phosvitin was the best exogenous phosphate acceptor for assaying the viral enzyme in vitro. Phosphoprotein phosphatase activity was also detected in the Alphaviruses. Both in vivo and in vitro, all of the viral structural polypeptides were phosphorylated, and the phosphorylated amino acids were found to be serine and threonine. The viral nucleocapsid protein was about four times more efficient as a phosphate acceptor than were the envelope proteins. From 33 to 50% of the total protein kinase was bound to the viral nucleocapsid, and the specific activity of this enzyme was 4 to 10 times greater than that associated with the viral envelope.

Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses.

SummaryIncubation in medium lacking arginine of monkey kidney cells infected with simian virus 40 resulted in a marked inhibition of production of both infectious virus and empty capsids. Virus DNA synthesis and formation of a virus DNA-protein complex were unaffected. All the virus structural polypeptides were made, although in reduced amounts (42%) when compared with infected cells incubated in medium containing arginine. Protein synthesis was inhibited in uninfected cells deprived of arginine. All the newly synthesized virus proteins were found in the nuclei of arginine-deprived cells. Addition of arginine to arginine-deprived cells resulted in the assembly of pre-synthesized virus components into infectious virus and empty capsids, as well as the production of virus from virus proteins and DNA synthesized after the addition of arginine. Studies with inhibitors of protein and DNA synthesis suggested that two late virus functions were affected in the absence of arginine: the assembly of capsids and the encapsidation of DNA by capsids to form infectious virus.

Uninfected African green monkey kidney cells contain a cyclic GMP-stimulated protein kinase. The level or specific activity of the enzyme was not significantly increased after infection of the cells with simian virus 40. This enzyme probably catalyzes the phosphorylation of the structural proteins of the virus.

A procedure was developed for the separation of cellular DNA of productively infected monkey kidney cells from free simian virus 40 DNA. The application of this procedure allowed the investigation of progeny viral DNA integration into the host cell DNA by nucleic acid hybridization techniques. The purification consisted of precipitation of the cellular DNA by Hirt's (1967) method, velocity centrifugation in alkaline sucrose gradients, equilibrium centrifugation in ethidium bromide/CsCl solution, and an additional velocity centrifugation in an alkaline sucrose gradient. The efficiency of each step of the procedure was determined by monitoring the amount of contaminating free viral DNA. Purified cellular DNA, isolated from cells late after infection, contained approximately 0/sd006% free viral DNA, but as much as 2% integrated simian virus 40 DNA. This corresponds to more than 20,000 integrated virus genome equivalents per cell, as determined by DNA-DNA reassociation kinetics. Integration of simian virus 40 DNA into the cellular DNA became detectable at 24 hours after infection, and increased with the increase in the rate of viral DNA synthesis.

We have shown previously that all the structural proteins of simian virus 40 (SV40) are phosphoproteins. Virus phosphorylated in vivo could be further phosphorylated with exogenous cellular protein kinases in a cell-free system containing gamma-(32)P-ATP as phosphate donor. In intact infectious virus only polypeptides 1 and 2 (mol wt 49,000 and 40,800, respectively) were further phosphorylated in vitro. However, when infectious SV40 was partially disrupted, treated with nucleases, and then phosphorylated in vitro, all five structural polypeptides accepted additional phosphate groups. Similarly, all polypeptides of intact empty capsids, derived from infected cells, were further phosphorylated in vitro. Phosphorylation of empty capsids and infectious SV40 in vitro was enhanced from 4- to 11-fold after prior treatment of virus with alkali. The phosphate group was linked only to serine residues of the viral polypeptides phosphorylated both in vitro and in vivo.

Polypeptides derived from purified rabies, vesicular stomatitis, and Kern Canyon viruses, which were labeled with 3H-amino acids and 32P-phosphate, were subjected to electrophoretic fractionation in order to determine which of the structural proteins is phosphorylated. In the rabies virion, the nucleocapsid protein (N protein) was found to be phosphorylated, whereas the other three virus proteins were not. N protein of free viral nucleocapsids isolated from rabies virus-infected cells was also phosphorylated. The phosphorylation of the N protein of rabies virus is confined to a relatively small terminal segment of the polypeptide chain which can be specifically cleaved off by treatment of the nucleocapsid with trypsin. In vesicular stomatitis virus the minor NS protein component is the only phosphoprotein. In Kern Canyon virus the envelope glycoprotein and the N protein are both phosphorylated, but to a lesser extent than either the N protein of rabies virus or the NS protein of vesicular stomatitis virus. All three rhabdoviruses contain a virion-bound protein kinase. Free nucleocapsids derived from rabies virus-infected cells and purified by equilibrium centrifugation in CsCl solution also contain a protein kinase. Intracellular nucleocapsids of vesicular stomatitis or Kern Canyon viruses do not exhibit protein kinase activity.

The envelope glycoprotein of rabies virus was shown to be the antigen responsible for the induction of virus neutralizing (VN) antibody formation and for the protection of animals against subsequent challenge with rabies virus. Preparation of two other envelope proteins and of the nucleocapsid protein, derived from disrupted virions, induced the formation of only low levels of VN antibody and protection of animals against rabies, which could be explained by the amount of residual glycoprotein present in these preparations. Purified preparations of free viral nucleocapsid, isolated from infected cells, did not induce VN antibody formation, but elicited, in immunized animals, the formation of antibodies demonstrable by complement fixation or fluorescent antibody tests.

Simian virus 40 uptake by the nuclei of permissive monkey kidney cells was enhanced by infecting in the presence of MgSO4, DEAE-dextran, or arginine-rich histones, but not in the presence of lysine-rich histones. Nuclear uptake of virus was found to be more efficient in aged monolayer cultures than in freshly formed cell monolayers. Inhibition of cellular protein, RNA, or DNA synthesis did not prevent virus uptake. “Empty” capsids, which are essentially devoid of viral DNA, were taken up by the nuclei of permissive cells even more efficiently than infectious virus. Undegraded polypeptides of parental virus could be detected in the nuclei up to 24 hr after infection.

A simple alphabetical classification is offered as a means to codify the proteins of rhabdoviruses, using vesicular stomatitis virus as the prototype.