- Volume 38, Issue 2, 1978

Fibroblastoid cell cultures derived from leukaemic bone marrow were successfully infected with BVV. After 2 months of subcultivation the cultures showed the appearance of foci of altered cells, suggestive of malignant transformation. Such foci were absent in non-inoculated cultures. Both control and inoculated cultures had a limited life span, i.e. neither of them could be developed into continuous transformed cell lines. The presence of at least some BVV genome functions in the inoculated cells was demonstrated (i) by immunofluorescence using a reference BVV serum, (ii) by detection in the supernatant culture fluid of sedimentable particles bearing RNA-dependent DNA polymerase activity with preference for Mg2+ ions, and (iii) by electron microscopic detection of scarce cell-associated virus particles in one of the infected cultures. Infectious BVV could not be rescued. In contrast to leukaemic bone marrow cultures, diploid human embryonic fibroblasts of various origin could not be infected with BVV.

A new set of hybrid viruses has been isolated whose closed circular genomes 5 to 6 kB in size, contain DNA sequences derived in part from adenovirus 2 and in part from SV40. The structure of these genomes is complex, but in the simplest case, analyses by restriction endonuclease digestion and hybridization indicate that the adenovirus 2 DNA is present as a continuous block, of maximum size 2.8 kB. Different hybrids contain sequences derived from different segments of the adenovirus 2 genome.

Some morphological, biological, immunological and biochemical characterizations of a virus, rat helper virus pseudotype Kirsten sarcoma virus, KiMSV-(RHHV), have been presented here. KiMSV(RHHV) has a type C virus ultrastructure. It is strictly rat tropic and is able to transform rat cells in vitro. The possibility of its being a xenotropic mouse virus has been carefully ruled out by exhaustive analyses of host range and immunological studies. Antigenically KiMSV (RHHV) demonstrates cross reactivity with an antiserum specific against rat leukaemia virus, no cross reactivity with antiserum against Moloney leukaemia virus, and only minor cross reactivity with antiserum against cat leukaemia virus. Analysis of virus proteins and glycoproteins by equilibrium acrylamide gradient gel electrophoresis showed that the virus complex possesses both a gp70 fraction and a p30 fraction. KiMSV(RHHV) sediments isopycnically in a linear sucrose gradient at 1.145 to 1.155 g/ml and possesses RNA and reverse transcriptase activity.

Ouabain markedly inhibited the growth of the mouse cell lines K3b and JLS-V9 and the production of murine leukaemia virus (MuLV) in them. The inhibition of MuLV production was abolished by exposing the cells to normal medium or by adding a high concentration (43 mm) of K+ ions to the ouabain-containing medium. MuLV production was reduced by ouabain more rapidly than host cell directed protein synthesis. After treatment of cells with ouabain (0.5 mm) for 7 h, extracellular reverse transcriptase activities were reduced by 87 to 92%. However, the intracellular level of polymerase activities remained almost unchanged (77 to 98% relative to the control). Mouse interferon inhibited the production of MuLV in K3b cells and this antiviral action was not blocked by 0.5 mm-ouabain.

Several factors were investigated in relation to infection and multiplication of clover yellow mosaic virus (CYMV) in cowpea mesophyll protoplasts. The extent of virus multiplication was determined by fluorescent antibody staining and infectivity assay. The conditions that gave the most infection (38%) were: preincubation of the virus with 0.4 µg/ml poly-l-ornithine, a virus concentration of 4 µg/ml, an inoculum containing citrate buffer at pH 6.0, and contact between inoculum and protoplasts for at least 15 min. Time-course studies revealed synchronous one-step growth of virus with rapid accumulation of virus antigen from 12 to 48 h after inoculation.

Lymphocytic choriomeningitis virus (LCM) and Pichinde virus grew readily and produced cytopathology in MDCK and PK-15 cells. It is known that in these cell lines, the synthesis or function of defective interfering (DI) virus particles is restricted. Survival curves of single MDCK cells infected with low multiplicities of LCM showed one-particle-to-kill kinetics. At high multiplicities of infection, there was a maximum degree of cell-killing, or even a reduction in the amount of cell-killing, depending on how much DI virus was present in a particular standard virus stock. DI LCM virus could completely prevent standard virus from producing c.p.e. in MDCK monolayers with one-particle-to-protect kinetics. It could still prevent killing of the cells when added within a short time after infection with standard virus, but was able to interfere with synthesis of standard virus when added even later. On passage of LCM or Pichinde virus without dilution in MDCK cells, there was no homologous auto-interference. Furthermore, there was only slight interference with the synthesis of standard virus when these cells were pre-treated with DI virus.

A long-tailed and generalized transducing Salmonella phage ES18 can recombine with a serologically unrelated phage Fels 1. When ES18 phage stocks grown on Fels 1 lysogens were plated on Fels 1 lysogens resistant to ES18, plaques of a new hybrid phage P181 were found at a frequency of about 10−8. Serological analysis of phage P181 showed that it carries the protein coat of Fels 1 and has no serological cross-reactivity with ES18. Phage P181 expresses the clear plaque morphology similar to that of ES18. ES18 was also found to carry a homology with P22 in at least the c regions. Thus, the c markers (c+, c1, c2 and c3) of P22 can be transferred to ES18. When these ES18 strains were used for isolation of P181 phage, the clear plaque morphology of P181 mimicked that of the P22c marker in the ES18 strain which gave rise to the P181 strain. Salmonella typhimurium strain Q1, lysogenic for P181, is immune to ES18 but not to Fels 1. Furthermore, ES18 lysogens are immune to P181. Therefore it is concluded that the hybrid phage P181 conserves the protein coat of Fels 1 and carries at least the c regions of ES18.

When ES18 was grown on Fels 1 lysogens of recombination-deficient (recA) mutants, P181 hybrid particles were found in the ES18 stocks at a frequency of about 10−11. This frequency is about 1000-fold lower than that found in ES18 stocks grown on the wild type hosts lysogenic for Fels 1. Therefore, it is concluded that the bacterial recombination mechanism plays an important role in the formation of P181 by recombination between the prophage Fels 1 and the superinfecting phage ES18.

Although phage ES18 belongs to B group phages, it possesses a generalized transducing capability. We have studied a possible origin of ES18.

The amino acid composition, behaviour in SDS-polyacrylamide gel electrophoresis and electrophoretic patterns of cyanogen bromide peptides were studied for the protein subunits of different preparations of potato virus X (PVX). The results indicate that the protein subunits of PVX can be partially degraded in the intact virus at the N-terminus by reducing agent-dependent proteases in crude plant sap and by trypsin, and at the C-terminus by reducing agent-independent proteases occurring in some virus preparations.

Guinea pigs immunized with hepatitis B surface antigen (HBsAg), types adw, adr and ayw, and with two major polypeptides derived from HBsAg/adw developed cell-mediated immunity as determined by the macrophage migration inhibition assay. Peritoneal exudate cells from animals immunized with a 22000- or a 25000-mol. wt. polypeptide derived from HBsAg/adw showed significant migration inhibition after challenge with either polypeptide or with purified HBsAg. Significant inhibition of macrophage migration was not observed when polypeptide-sensitized cells were challenged with normal human serum or with normal human liver extract. Similarly, a cell-mediated immune response was not observed in peritoneal exudate cells from animals sensitized to normal human serum or normal human liver extract which were challenged with either of the polypeptides. The humoral immune response to either of the polypeptides, as measured by radioimmunoassay, was substantially lower than that observed in animals immunized with intact particles. This apparent difference between cellular and humoral responses suggests that the macrophage migration assay is a sensitive indicator of the immunogenicity of the smaller mol. wt. HBsAg-derived polypeptides in guinea pigs.

The RNA species of the CAM isolate of tobacco rattle virus have been labelled at their 3′ termini and partially digested with T1 ribonuclease. The two RNAs give an identical pattern of labelled fragments following acrylamide gel electrophoresis, showing considerable structural similarity between the 3′ ends of these molecules. RNA fragments derived from the 3′ ends of short particle RNA and long particle RNA labelled in vitro with 125I were tested for their ability to anneal to complementary long particle RNA. Fragments derived from short particle RNA failed to hybridize, showing that despite their structural similarity, the 3′ sequences of these RNAs are not homologous and that the homologous sequence known to be shared by TRV RNAs are not at the 3′ ends of the molecules.

Using high resolution electron microscopic autoradiography and velocity sedimentation, RNA synthesis was examined in chick embryo cells infected with Japanese encephalitis virus (JEV). RNA was labelled with 3H-uridine for 5 min or 10 min at 15 h after infection in the presence of actinomycin D and d-glucosamine. Microautoradiography showed significant numbers of silver grains on the nuclear membranes of 5 min pulse-labelled thin cell sections.

The RNA species in membrane fractions obtained from the nucleus and cytoplasm of the infected cells were analysed by sucrose density gradient sedimentation. Radioactive 23S replicative form RNA and 8-12S RNA were obtained from the outer membrane fractions of the nuclear envelope. Labelled 42S virus RNA was obtained from the fractions containing large vesicle membranes and plasma membranes.

These results suggest that JEV-RNA synthesis is initiated in the perinuclear region in close association with the outer membranes of the nuclear envelope.

HBsAg antigenicity was found to be sensitive to periodate treatment. Antigenic determinants a and d were especially sensitive, losing almost all of their activity, while determinants r and w were found to be quite stable. Also, it is now possible to prepare the monospecific antibodies, anti-r and anti-w, by the use of this new, simpler procedure.

Aedes aegypti mosquito cells, usually cultured at 28 to 30 °C, were adapted to grow at 15°C. They were designated A. aegypti (c) cells, and had an estimated doubling time of 10 days. Sindbis virus (SV) replicated in these cells to peak titres of over 1.0 × 109 p.f.u./ml 8 to 10 days after inoculation. These, or about 10-fold lower titres, continued to be produced over a 130 day test period without causing visible cell damage. Continuous virus proliferation and the yield of uniformly large plaque forming progeny viruses are the two most important features which differentiate infection with this virus in A. aegypti (c) cells from that of A. aegypti cells grown at 28 °C (Peleg & Stollar, 1974). Absence of homologous interference vis-à-vis cell-virus coexistence suggests that homologous interference is not a prerequisite for maintaining cell-virus coexistence. Preinoculation of A. aegypti (c) cultures with a small plaque forming Sindbis virus (SV-S) leads, under certain conditions, to the establishment of homologous interference.

Virus Hepatitis and its Control, 1977, 304 pp., 110 tables, 17 figures. By Yvonne E. Cossart. Published by Baillière Tindall. Price U.K. £7.50 cased.

Although human embryo kidney (HEK), muscle (HEM) and lung (HEL) cells are capable of supporting the replication of BK virus (BKV) through passage levels 9, 12 and 12 respectively, only third, fourth and fifth passage level HEK cells were found to be satisfactory for the plaque assay of the virus. BSC-1 and VERO cells can also be used for the plaque assay of BKV. However, in HEK cells plaques can be visualized in 20 days (compared to 28 days in BSC-1 cells), and since in VERO cells the plaques are poorly defined and the titre about 1 log10 lower than in either HEK or BSC-1 cells, HEK cells were the ones chosen for use.

In 1974 an epizootic occurred among budgerigar flocks in Kunitachi, Tokyo, and a causative agent which possessed haemagglutinating, neuraminidase, and haemolytic activities was isolated from the lung of a dead budgerigar. This agent was 100 to 300 nm in diameter and pleomorphic. The width of the ribonucleoprotein was estimated to be about 20 nm. These results indicated that the virus, designated Kunitachi virus, was a member of the paramyxovirus group. The virus contained in the amniotic fluid from infected embryonated hen’s eggs, however, at times displayed no haemagglutinating activity with different erythrocytes and complete haemagglutination could only be detected in purified preparations. The Kunitachi viruses including three strains recently isolated from the same host were found to be serologically distinct from the known paramyxovirus strains and appeared to constitute a new subtype of avian paramyxovirus.

Semi-permissive mouse, rabbit and monkey cells were stably transformed by BK virus (BKV). The specificity of transformation was demonstrated by the presence of BKV tumour (T) antigen in nuclei of transformed cells and by virus rescue with Sendai virus-mediated fusion or transfection. Two out of seven BKV-transformed cell lines were oncogenic. Permissive human cells were only abortively transformed by BKV, since morphologically modified cells persisted in culture for a few passages and eventually died.

An antiviral factor from virus-infected plants (AVF) was purified in an active form on SDS-polyacrylamide gels. AVF binds to concanavalin A and is partially sensitive to α-glucosidase. It is sensitive to pronase only when incubated in conditions suitable for proteolysis of glycoproteins. Alkaline phosphatase affected the electrophoretic mobility of AVF, but did not abolish antiviral activity. AVF was insensitive to DNase, β-glucosidase and pancreatic lipase. The AVF band obtained upon electrophoresis could be stained with Coomassie blue and by the Schiff-periodate procedure for carbohydrates. AVF is considered to be a phosphoglycoprotein with a mol. wt. of about 22000.

The mutation lg of phage P2 has been located on the genetic map of P2 to the right of, and closely linked to, the del2 deletion, probably within tail gene F. The lg mutation causes larger burst sizes, compared with the wild type, especially at high incubation temperatures. The frequency of defective particles is lower in preparations of P2 lg than in those of wild type P2. It seems that the mutation lg improves the efficiency of particle assembly.