- Volume 56, Issue 2, 1981

In order to investigate whether defective phages of Bacillus subtilis killed sensitive bacteria by a lysis from without mechanism, the minimal number of phages required for killing was determined. This figure was found to vary with the m.o.i., giving a value of 1 on extrapolation to an m.o.i. of 0. This excluded lysis from without as the only killing mechanism, although it might play a role at high m.o.i.s. This was confirmed by experiments on leakage of ATP and u.v.-absorbing material, the uptake of oxygen and the effect of the phages on the membrane potential. Apart from a short, initial leakage of ATP, the cell membrane was not affected at low m.o.i.s. These results lead to the conclusion that at low m.o.i.s the phages acted on a cytoplasmic component. Treatment of defective phages for 10 min at pH 2.5 resulted in breakdown of the phages without complete abolition of the killing activity. The active component, which was shown not to be DNA, could not be isolated from the mixture, but SDS gel electrophoresis of PBS X and a non-killing mutant of this phage suggested that a protein with a mol. wt. of 85000 was involved in killing.

φ6R mutants of Pseudomonas phaseolicola HB10Y were isolated and 12 out of 129 spontaneous mutants were found to produce phages during cellular growth. Following mitomycin C treatment 39 out of 82 isolated resistant mutants were phage-producing ones. The decrease in growth rate of these mutants corresponded roughly to the number of phages liberated into the medium. Prolonged storage of the mutants resulted in loss of production which could be regained by growing them with a high multiplicity of phages. The phage production phenomenon was independent of phage adsorption since both adsorbing and non-adsorbing phage-producing mutants were found. Occasionally, the phage-producing strains showed abnormally high numbers of intracellular phage particles in sectioned material. The producers were found to be 2 to 5 times more resistant than the normal host or non-producing φ6R strains to the lytic enzyme associated with the φ6 virion, indicating that they had an altered cell wall structure. The lytic enzyme of φ6, needed both in penetration and progeny release, was less active on the altered cell wall, leading to diminished infection efficiency and deficient plaque formation.

The thermolability of transmissible gastroenteritis virus (TGEV) was studied between 31 and 55 °C using two different strains. The loss of infectivity followed first order kinetics except at the highest temperature. The values of the thermodynamic parameters indicated that the mechanisms involved above and below 45 °C are clearly distinct. The rates of inactivation were greater at alkaline than at neutral pH, yet the nature of the reaction appeared unchanged. Using four independent stocks of mutagenized virus, we failed to select thermal-resistant mutants by survivor selection at 38 °C. In contrast, thermal-resistant mutants were consistently obtained at 54 °C. However, the latter did not show any increased stability at 38 °C, confirming the fact that a different inactivation process takes place at high and at physiological temperatures.

The immunosuppression in dengue type 2 virus (DV)-infected mice is mediated through secretion of a soluble suppressor factor (SF) produced by the T lymphocytes of the spleen. Activity of the suppressor cells was abrogated by pretreatment with indomethacin, indicating that suppression is mediated through prostaglandin (PG) or products of the PG synthetase pathway. The present study was undertaken to resolve the relationship between SF and PG. Treatment of mice with indomethacin did not affect production of SF. Therefore, PG is different from SF and the presence of PG is not essential for its production. Normal mouse spleen cells treated in vitro with SF produced PG which mediated suppression. Production of PG by these cells was abolished by pretreatment with anti-Thy 1.2 antibody and complement. It is concluded, therefore, that immunosuppression occurs in two phases: in the first step DV stimulates a subpopulation of T lymphocytes to produce SF, and in the second step SF induces a subpopulation of T lymphocytes to produce PG or products of the PG synthetase pathway which finally mediate suppression.

Confluent monolayers of BHK-21/C13 cells excreted increasing amounts of polyamines into the extracellular medium with time. Excretion was specifically of spermidine and was not the result of cell death or lysis. Herpes simplex virus type 1 (HSV-1) completely prevented excretion of polyamines from 2 h after infection of the cells. The virus specifically inhibited the release of free spermidine but not of conjugated polyamines.

Mice infected with herpes simplex virus (HSV) were treated (separately) with the nucleoside analogues acyclovir or bromovinyldeoxyuridine by incorporating the drugs in the drinking water. This method of treatment was found to be effective for both drugs and compared favourably with intraperitoneal injection. Prompt treatment with either compound could prevent the establishment of latent infections but latent infections once established were intractable using prolonged courses of oral administration.

The genome and polypeptides of influenza A virus H3N2 strains isolated during the epidemic of 1979–1980 in the U.S.S.R. and G.D.R. have been analysed. Five varieties of H3N2 strains differing in a number of genes have been found. The isolates of the first group were similar to the A/Texas/1/77 strain in all the genes; the isolates of the second group were similar to the A/Bangkok/1/79 strain in all the genes; the strain representative of the third variety, contained all the genes except gene 4 close to those of the A/Bangkok/1/79 strain; the isolates of the fourth group contained genes 7 and 8 similar to those of the A/Bangkok/1/79 strain while the other genes corresponded to those of no strains under comparison; the viruses of the fifth group contained gene 3 similar to that of A/Moscow/406/76 strain, gene 7 was similar to that of A/Texas/1/77 strain and the other genes differed from all other strains compared. The data obtained indicate that during an influenza epidemic occurring in a certain region several influenza virus varieties of the same serotype can circulate simultaneously, differing not only in the antigenic specificity of the haemagglutinin, but also in other genes.

The major virus-specific proteins (HA, NA, NP, NS1 and M) of five different isolates of influenza B virus (B/Lee/40, B/Osaka/2/70, B/Yamagata/1/73, B/Aomori/1/76 and B/Yamagata/26/77) were compared by limited proteolysis with Staphylococcus aureus V8 protease and subsequent polyacrylamide gel electrophoresis. The peptide patterns of matrix (M) proteins from all five strains were virtually identical. The nucleoproteins (NP) as well as the non-structural proteins (NS1) were also very similar among strains although the peptides of B/Lee/40 could be distinguished from those of the strains isolated from 1970 to 1977. In contrast, the peptides from haemagglutinin (HA) glycoproteins were largely different even among the strains isolated later than 1970. It therefore appears that the HA glycoproteins of influenza B virus are more changeable than any of the non-glycosylated proteins. Furthermore, it was found that the maps of HA1 were markedly different among strains while the maps of HA2 were very similar, which suggests that the structural changes in the HA polypeptide occur preferentially in the HA1 portion. The neuraminidase (NA) glycoproteins also showed strain-dependent differences in their mapping patterns.

The composition and topography of the structural polypeptides of bovine rotavirus was studied by polyacrylamide gel electrophoresis of radioactively labelled virus grown in LLC-MK2 cells and by lactoperoxidase-catalysed iodination of single- and double-capsid particles. Bovine rotavirus was found to possess at least six structural polypeptides, three of them associated with the inner capsid (p102K, p91K and p45K) and the others with the outer capsid (p84K, p37K and p34K). The most abundant polypeptide of the inner capsid was p45K, which accounted for approx. 80% of the protein mass, followed by p91K (approx. 20% of the protein mass) and p102K (approx. 1% of the protein mass). Polypeptide 45K is not readily available for iodination, indicating that it is partially covered by p91K, which is the most exposed polypeptide of the inner capsid. The number of polypeptide molecules per single capsid particle (calculated on the basis of an RNA content of 16%) was estimated to be approx. 6 molecules of p102K, 140 of p91K and 989 of p45K. The stoichiometry and degree of exposure of outer capsid polypeptides was more difficult to establish, even when it appears that p84K and p34K are the most exposed components.

Poliovirus isolates of serotypes 2 and 3 from patients whose paralytic poliomyelitis cases were classified as oral vaccine-associated were analysed by oligonucleotide mapping of the virus genomes and by polyacrylamide gel electrophoresis of the virus proteins. Oligonucleotide maps of all isolates were similar to the maps of the corresponding oral vaccine strain. No two isolates gave identical maps. Most maps differed from that of the vaccine strain by at least one oligonucleotide spot. Maps of some isolates revealed numerous differences, indicating that multiple (> 100) genetic changes had occurred in the vaccine virus genomes during replication in one or two individuals. In contrast, maps of some neural tissue isolates showed minimal differences from the reference vaccine maps, raising the possibility that neuro-virulence may be restored by a small number of genetic changes. For many isolates, changes were also detected in the mobilities and processing rates of the virus proteins.

Several temperature-sensitive (ts) mutants of Western equine encephalitis virus (WEEV) have been isolated previously from persistently infected cultures of mosquito cells and divided into three groups: early passage RNA− mutants, early passage RNA+ mutants and late passage RNA− mutants (Maeda et al., 1979). The growth patterns of these groups, as well as of several ts mutants isolated after chemical mutagenesis and of wild-type (wt) WEEV, have been compared in BHK cells and in two strains of mosquito cells. The late passage ts mutants grew much better in mosquito cells than either the wt WEEV or the chemically induced mutants. When mosquito cells were co-infected with a late passage mutant (A125) and wt WEEV, infectious virions of both parental types as well as phenotypically mixed particles were produced. Infection of mosquito cells with WEEV resulted in a slight suppression of host DNA and protein synthesis during the acute stage of the infection (the first 1 or 2 days). Virus growth in a line of cloned mosquito cells in which WEEV produced a cytopathic infection (c.p.e.) was analysed with the result that the viruses could be divided into two groups: one in which wt WEEV, chemically induced ts mutants and early passage RNA+ mutants all induced maximal c.p.e., and another in which late passage RNA− mutants and one early passage RNA− mutant induced very little c.p.e., but released much more infectious virus into the culture fluid. Electron microscopy showed that in these cloned mosquito cells infected with a virus of the first group, large amounts of virus accumulated on or in the plasma membrane.

Two preparations of yellow fever vaccine (17D) were studied by clonal analysis. From one (17D-England) three types of clones were isolated, each differentiated by plaque size in Vero cells and virulence for intracerebrally inoculated mice: small plaque (SP) and large plaque (LP) clones were lethal, whereas medium plaque (MP) clones failed to kill at doses up to 106 p.f.u. Analysis of 24 randomly selected clones showed the original vaccine to be a mixture of predominantly MP and SP variants; 58% and 42% respectively. A single passage in suckling mice modified this composition to 90% SP and 10% LP variants. The response of six inbred strains of mice to intracerebral inoculation of the MP variant varied from complete resistance to complete susceptibility. From another 17D substrain (17D-South Africa) two types of variants were isolated (a SP avirulent type and a LP type of intermediate virulence), both different from the previously described variant.

Young adult mice were inoculated in the hind limb with rabies virus or Sindbis virus. Rabies 1820B virus antigen was detected in leg sections by immunofluorescence at 1 h post-inoculation at sites comparable in form and distribution to cholinesterase-positive sites, which represent motor end-plates (MEPs). Sites which were rabies virus antigen-positive by immunofluorescence were also cholinesterase-positive on double-stained slides. Rabies CVS virus detected by autoradiography was similarly distributed at 6 h post-inoculation. Uptake of rabies virus at motor nerve endings was confirmed by the detection of rabies antigen by immunofluorescence in ventral horn cells in the spinal cord at 20 h post-inoculation before involvement of dorsal root ganglia. Rabies virus antigen could not be detected at MEPs if the virus had been inactivated by beta propiolactone or mixed with antibody prior to injection or if the sciatic nerve had been cut 7 days earlier; similarly treated groups of mice survived for the observation period of 6 weeks. Rabies virus antigen was found at MEPs in mice given antibody 24 h before virus injection, but virus antigen was not found in the spinal cord, and mice similarly treated survived. Sindbis virus strain Ar86, which like rabies virus is neurotropic in adult mice, was also found at MEPs and in peripheral nerves by autoradiography at 6 h post-inoculation. In contrast to results with rabies virus-infected mice, stimulation of the sciatic nerve for the first hour post-inoculation prevented mortality. Sindbis virus strain Ar339, which is not neurotropic in adult mice, could not be detected at MEPs by immunofluorescence or autoradiography and mice injected with virus survived. The results presented here suggest that rabies virus and perhaps other neurotropic viruses can use the motor axon terminal at the neuromuscular junction as a site of entry into the nervous system.

The relationship between the in vitro phosphorylation of vesicular stomatitis virus (VSV) proteins and virion uncoating was examined. Activation of the VSV virion kinase with low concentrations of melittin, the active peptide component of bee venom, in the presence of γ-[32P]ATP resulted in the phosphorylation of virion proteins. Following the in vitro phosphorylation of VSV proteins in the presence of melittin and deoxyadenosine triphosphate, the virion envelope was disrupted based on the accessibility of the internal ribonucleoprotein core (RNP) to the heavy metal stain, uranyl acetate, as determined by electron microscopic observation. The RNP structure was not observed in unphosphorylated virions treated with melittin and uranyl acetate. Phosphorylated virions treated with uranyl acetate subsequently lost the capacity for transcription whereas unphosphorylated virions treated with the stain retained transcriptase activity. These observations suggest that phosphorylation of VSV proteins may contribute to virion uncoating by disrupting the virus envelope.

Eight different hybridoma cell lines producing monoclonal antibodies against the major antigens of human adenovirus type 5 have been obtained. They were selected by screening initial hybridomas by the fluorescent antibody technique followed by radioimmune precipitation and they reacted with hexon, penton, fibre and 100K polypeptides. Five apparently different epitopes against the hexon antigen were detected showing a spectrum of activity against the hexons of other serotypes, suggesting that the hexon contained a variety of subgroup specificities as well as the previously described group and type specificities.

Previously, we have shown a common antigen of several herpesviruses (pseudorabies virus, equine abortion virus and bovine mammillitis virus) to be antigenically related to the major DNA-binding proteins of herpes simplex virus types 1 and 2. In this study we have purified the cross-reacting polypeptide from cells infected with pseudorabies virus, equine abortion virus and bovine mammillitis virus and shown the cross-reacting protein to be a major DNA-binding protein for each virus. Tryptic peptide analysis of the cross-reacting DNA-binding proteins of all five viruses has shown structural similarities. The proteins thus were shown to share common antigenic sites, to have similar biological properties and to have a highly conserved amino acid sequence. This unexpected similarity between proteins from diverse herpesviruses suggests an essential and fundamental role of the major DNA-binding protein in herpes virus replication.

Mouse L cells lacking the enzyme thymidine kinase (Ltk−) were infected with varicella-zoster virus (VZV). Even though virus did not replicate in Ltk− cells, the presence of virus antigen could be observed by use of an anti-complement immunofluorescent technique at 4 h post-infection and the VZV-specific thymidine kinase could be detected in VZV-infected Ltk− cells. Ltk− cells were converted to a tk+ phenotype (Ltk+) by infection with cell-associated VZV. Clones possessing the ability to grow in selective medium were isolated and cultured successfully for more than 20 passages. One of the clones grew very slowly, but other clones showed almost the same growth rate as that of the parental Ltk− cells. The chromosome analyses of Ltk− cells and transformed cells revealed that the isolated clones were of mouse origin. VZV-specific antigen could be detected in the nuclei of Ltk+ cell clones by an immunofluorescent test, while tk activity was greatly enhanced in extracts prepared from transformed cells and its activity was neutralized by hyperimmune serum against VZV.

Intratypic electrophoretic mobility differences in high resolution SDS-polyacrylamide gels were detected between corresponding matrix (M) proteins, nucleoproteins (NP), haemagglutinin (HA) and the non-structural polypeptides NS1 and NS2 induced in Vero cells by human influenza A viruses of the antigenic subtypes H1N1 and H3N2. Such phenotypic differences were distinguishable in both H1N1 and H3N2 viruses isolated in single school and city outbreaks. Additional intratypic variation was detected in the biological property of virus plaquing in MDCK cells. Although the biochemical basis is not established, phenotypic variation could represent an additional factor influencing the epidemiology of influenza A viruses.