- Volume 28, Issue 1, 1975

Hamsters inoculated with the TC-83 vaccine strain of Venezuelan encephalitis (VEE) virus were protected against a normally lethal challenge by virulent VEE virus, strain 68U201, inoculated 30 h after vaccination. Protection was correlated with significantly decreased levels of challenge virus in target tissues (spleen and bone marrow). Inhibition of challenge virus replication was correlated more closely with the interferon concentrations in spleen and bone marrow than with the vaccine virus infectivities in these tissues at the time of challenge. The induction of early defence mechanisms involves the efficient interaction of virus with the spleen; although this interaction is more efficient for TC-83 vaccine, virulent VEE viruses are sensitive to the induced protective mechanism. Interferon appears to be a mediator of this mechanism.

Evidence is presented that adaptation of IPN virus (strain VR 299) to FHM cells entails the selection of a variant virus type that differs significantly from the parental, and most representative, RTG-2 virus type in being able to adsorb efficiently to, and form plaques in, FHM cells.

The plaque titre of FHM-non-adapted virus stocks (RTG-2 viruses) was reduced by at least 99.99% in FHM cells, while FHM-adapted virus stocks (FHM viruses) produced plaques at equally high titres in both RTG-2 and FHM cells.

FHM viruses and RTG-2 viruses differed also in their behaviour in RTG-2 cells in respect to plaque size distribution and growth characteristics, but both virus-types were shown to be morphologically identical, and no significant difference in reactivity against specific antiserum could be detected.

Analysis of virus in individual RTG-2 plaque isolates or plaque progeny shows that a mutation of relatively high frequency (10−4 to 10−5) probably causes the ability to infect the FHM cells efficiently. Only these mutant virus-types were found in FHM plaque isolates.

A persistent infection of measles virus was established in HEp2 cells. All cells contained virus antigen when tested by specific immunofluorescence and approx. 50% were positive by haemadsorption. Infectious virus released into the supernatant medium was usually equivalent to no more than 0.001 p.f.u./cell, but between 10 and 40% of the infected cells produced plaques when plated on Vero cells.

Passage of persistently infected cultures in the presence of measles antibody had no effect on the proportion of antigen-positive cells.

Virus obtained from the supernatant medium of persistently infected cultures was temperature sensitive at 39.5 °C when tested on Vero cells whereas the original nonpersistent virus produced infections on Vero cells at 39.8 °C.

On passage of the persistently infected culture at 39.5 °C most of the surface antigens disappeared within 24 h whereas the intracellular virus antigens had not totally disappeared until the 5th passage.

l-rhamnose has been found to be useful to investigate the process of irreversible adsorption of PL-1 phage to its host bacterium, Lactobacillus casei ATCC27092. l-rhamnose inhibited phage adsorption to cells without inactivating free phages. Adsorption inhibition was correlated with the concentrations of l-rhamnose. The inhibitory effect of l-rhamnose on phage adsorption was of a competitive nature against host cells. Among other saccharides tested, l-fucose, l-mannose and d-ribose showed a slight degree of adsorption-inhibiting activity.

In early stages of phage adsorption in a tris-maleate buffer, where the binding of phages to cells was still reversible, addition of l-rhamnose resulted in the partial desorption of phages from the cells to which they had adsorbed. However, the number of infective phages desorbed by l-rhamnose treatment gradually fell off as incubation continued, showing that the phages became firmly bound to the cells. Therefore, it is possible to determine the number of phages irreversibly adsorbed to cells by using this desorption technique with l-rhamnose. The process of irreversible phage adsorption, that is, the formation of phage-cell complexes from which no more infective phages could be desorbed, was dependent on temperature and strongly inhibited at 0 °C.

Precipitating antigens present in extracts of chick embryo cells infected with the HPRS-16 attenuated strain of Marek’s disease virus (att-MDV) were separated by gel filtration on Sephadex G200 and some of their properties determined. The two main antigens detected with convalescent MD serum, referred to as ‘B’ and ‘C’ antigens, had mobilities of 0.55 and 0.25 respectively relative to phenol red on electrophoresis in 7.5% acrylamide gel. The B antigen was relatively stable and of low mol. wt. in comparison with the C antigen. B and C antigens were in some instances also detected in culture medium of infected cells, but were distinguishable from the A antigen, a major glycoprotein antigen released into the culture medium of cells infected with HPRS-16. The results of immunodiffusion studies suggest that B antigen is common to MDV and strains of herpes virus of turkeys (HVT) and that at least 2 antigens (including C) are MDV specific. The A antigen was also common to MDV and HVT strains. It was noted however that the capacity of HPRS-16/att to synthesize A antigen was considerably reduced in comparison with HPRS-16 and HVT strains, and in some preparations the A antigen could not be detected. Evidence was also obtained for the presence of HVT-specific antigens associated mainly with the cell fraction.

Adenovirus type 5 ‘cores’ prepared by heating in the presence of deoxycholate and partially purified on a glycerol density gradient could be visualized as roughly isometrical particles with a condensed centre from which twisted filaments or loops of DNA emanated. This compact structure was readily dispersed by spreading on distilled water or by treatment with EDTA, Nonidet, DNase or trypsin. Spreading with Nonidet was particularly effective in unfolding the cores and revealing long filaments about 100 Å thick presumably of the virus nucleoprotein. Subunits (about 30 to 60 Å in diam.) could be seen free in the DNase-treated cores, suggesting a particulate nature of one or both of the core proteins.

Ross River virus, an Australian group A arbovirus, was adapted by serial passage to cell cultures and to day old mice. The results of titrations in mice of different ages allowed the comparison of virulence between different stocks. Passage in cell cultures depressed the virulence of virus while passage in mice raised the level of virulence. Clones of original virus populations revealed heterogeneity with respect to virulence but none of the 41 clones was as highly virulent as virus passed 10 times in mice. Clones selected in sequence during serial passage in mice indicated that adaptation proceeded by the overgrowth of variants of increasingly higher virulence, and that clones from relatively highly passaged strains were still heterogeneous in virulence.

Two field strains of Ross River virus (RRV) which differed in virulence for laboratory mice were maintained without detectable change in virulence when passaged alternately in Aedes aegypti mosquitoes and newborn mice. No biological mechanism or selection pressure was identified to explain this suppression of the usual change to higher virulence observed when RRV is passed serially in infant mice. The maintenance of initial virulence by alternating passages appears to be related to the fact that A. aegypti can be infected only if fed on mice during the period of peak viraemia and that at this time a sub-population of higher virulence may not be present in high enough infectivity to be represented in the mosquito’s blood meal.

The poliovirus replication complex was isolated and purified from infected HeLa S3 cells. Preparations with RNA-dependent RNA polymerase activity were concentrated 200- and 1000-fold with respect to the original virus and total protein content. The enzyme activity was found to be associated with the proteins NCVP1, 2, 3, 4, (5), 6 and VP1/NCVPx. The structural proteins VP2, 3 and 4 were not present. Addition of cycloheximide to infected cells resulted in a decrease in the in vitro polymerase activity and a loss in NCVP1 content. Treatment of the infected cells with toloylsulphonyl-phenylalanine chloromethyl ketone (TPCK) and iodoacetamide (IAA) led to an inhibition of in vivo RNA synthesis. The 750 g supernatant fluids obtained from extracts of these cells were able to block RNA synthesis in vitro. Electrophoretic profiles of the respective protein compositions indicate that large virus precursor proteins are responsible for the inhibition of poliovirus RNA synthesis in vivo and in vitro.

Specific changes at the surface of HeLa cells infected with mumps virus were investigated in parallel with the scanning and transmission electron microscope.

The distribution of haemadsorption binding sites and virus-induced antigens at cell surfaces was simultaneously studied by labelling virus-specific antigens with peroxidase-conjugated antibodies after haemadsorption.

New information was obtained upon the three-dimensional aspect of the red blood cells, the topographical distribution of their binding sites on the infected cells, and the specific structures at the cell surface which are involved in the process of haemadsorption.

Three replicative forms of RNA (RF I, RF II, and RF III) have been isolated from BHK cells infected with Semliki Forest virus. Using analytical and rate-zonal sedimentation the mol. wt. of these replicative forms were estimated to be 8.5 × 106, 5.5 × 106 and 3.1 × 106 respectively. After continuous labelling from 1 to 6 h post-infection, RF I constituted more than 80% of the total replicative forms. Competition hybridization experiments showed that one strand of RF I was 42S RNA which had opposite (negative) polarity to that found in the virus particle. The positive strand of RF I was 42S RNA. The negative strand of replicative intermediate (RI) was also found to be 42S RNA. No evidence was found for an RI with a 26S negative strand. RF I was shown to contain non-hydrogen bounded poly A at or near the 3′ end of the component 42S positive strand. Isolation and analysis of the poly A tract from RF I on an acrylamide gel showed it to be of essentially the same average size as the poly A tract from virus particle RNA. About 30% of the RI molecules contained non-hydrogen bonded poly A. No poly U was detected in either RF I or RI. The kinetics of positive and negative strand synthesis were investigated during virus multiplication. These experiments showed that the rate of negative strand synthesis reaches a maximum 2½ h post-infection and thereafter rapidly falls. The rate of positive strand synthesis increases rapidly up to 3 h post-infection and then remains constant for a further 3 to 4 h.

Several adult tissues, newborns, and embryos of uninfected BALB/c mice were analysed for RNA complementary to [3H]-DNA transcripts synthesized from an endogenous type-C virus of BALB/c 3T3. The technique of RNA:DNA hybridization was used and the extent of hybridization was measured by the use of a single-strand-specific nuclease (S-1), purified from Aspergillus oryzae.

Virus-specific RNA was detected in all adult and embryonic tissues tested. However, the RNA extracted from tissues having higher proliferative activity, such as spleen, small intestine, uterus and embryos, hybridize the [3H]-DNA probe to a greater extent than the RNA from tissues with low proliferative activity, such as kidney and liver. These observations add further support to the view that the repression of the virus genome in normal cells is not complete, and suggest the existence of a correlation between a qualitative or quantitative change in the endogenous C-type virus genome transcription pattern and cell proliferation.

Physico-chemical properties of the RNA of cowpea chlorotic mottle virus (CCMV), either as extracted from or contained in virus particles, were investigated. The similarity of the hypochromicity obtained on alkali hydrolysis of isolated CCMV RNA and that of RNA in virus particles showed that the secondary structure could be conserved upon extracting the RNA from virus particles. Through the pH range in which the virus particles swell, the hypochromicity determined from the melting profiles of purified, unfractionated CCMV RNA remained constant, as did the sedimentation coefficients of the separated RNA species which comprise the multicomponent genome of CCMV. Changes in RNA conformation are, therefore, probably not responsible for the swelling of the virus particles.

Particles of cowpea chlorotic mottle virus (CCMV) underwent an abrupt structural transition as the pH was raised to near pH 6.75. At low ionic strength (I = 0.2) the transition was observed as a decrease from 83S to 73S in the sedimentation coefficient of the virus particle. At high ionic strength (I = 1.0) the virus disassembled to components sedimenting at about 40S. These observations suggest a structural role for the RNA in maintaining virus stability. As ionic strength at pH 7.5 was increased, a critical point was reached (I = 0.4) at which the virus disassembled to the 40S aggregates. The effect of raising the temperature of both unswollen and swollen virus was to gradually, but substantially, reduce the sedimentation coefficient.

Disc electrophoresis of the soluble proteins in leaf extracts failed to reveal any change induced by infection with raspberry ringspot virus either in inoculated or in systemically infected symptom-bearing or recovered leaves of Nicotiana benthamiana. Evidence is given that three of the four proteins previously thought to occur only in virus-infected or polyacrylic acid-injected Nicotiana tabacum leaves are probably normal constituents. Environmental conditions were critical for the change in protein pattern induced by alfalfa mosaic virus in tobacco leaves.

An attempt was made to hybridize the chicken (Gallus domesticus) male with Japanese quail (Coturnix coturnix japonica) female in order to study the genetic susceptibility of hybrid embryos to avian RNA tumour viruses of subgroups, A, B, D and E. In the hybrids the results supported the prevailing concept that susceptibility is dominant over resistance regardless of the dominant trait contributed by either parent. It was also observed that the Ie gene of the chicken was unable to suppress the ‘quail-coded’ susceptibility to subgroup E virus in the hybrid system, suggesting the lack of penetrance of the Ie gene. Despite the fact that some hybrids were resistant to viruses of subgroups B and D, they were susceptible to subgroup E virus, which was not expected on the basis of the concept that subgroup B-resistant cells cannot be E-susceptible. Also, the hybrids were susceptible to E virus regardless of gs antigen expression and presence of the Ie gene in the genome. This indicates that our earlier suggestion that the Ie gene is another expression of the gs antigen-determining gene is inconsistent.