- Volume 37, Issue 2, 1977

The distribution pattern of actin-containing structures in BHK21 cells and the changes which they undergo upon infection with Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV) were studied by means of immunofluorescence.

Double labelling with antibodies conjugated with fluorescein (for actin) and rhodamine (for virus antigens) has shown that the progressive cytopathic effects after virus infection are accompanied by extensive alterations of the structures demonstrable by antiactin antibodies. In NDV-infected BHK21 cells the number of actin filaments increases, some zones which contain virus antigens apparently being in close association with the actin structures. By contrast, infection with VSV results in a strong reduction of actin-containing fibres.

The results indicate that in the genesis of morphologically detectable alterations of a cell after virus infection — the ‘cytopathic changes’ — alterations of those structural elements are involved which are also probably responsible for maintenance of cell shape and motility.

The base sequence homology between all eight 32P-labelled RNA segments of fowl plague virus (FPV) and the complementary RNA (cRNA) of an influenza B virus (B-Mass), and between segment 8 of virus N and the cRNA of the same influenza B strain has been determined. All segments of FPV and segment 8 of virus N show a significant base sequence homology, ranging from 18 to 50% suggesting that influenza A and B viruses have a common ancestor. The conserved regions in segments 4, 6, and 8 of the influenza A strains are identical to corresponding regions of the influenza B virus tested.

Tissues of the upper respiratory tract of ferrets are more susceptible to influenza virus infection than those of the lower respiratory tract. An organ culture technique has been standardized sufficiently to examine the basis for these differences.

In experiments with an Asian and two Hong Kong influenza viruses maintenance of organ cultures for 24 h before inoculation consistently and markedly increased the susceptibility of lung, the least susceptible tissue. The susceptibility of the most susceptible tissue, nasal turbinates, was increased significantly (but to a lesser extent than lung) in some experiments but not others. Tracheal organ cultures showed two patterns of susceptibility; about half were poorly susceptible but showed a marked increase with maintenance. The remainder were moderately susceptible and showed no increase with maintenance.

The increased susceptibility of lung with maintenance was not due to fibroblast outgrowth, to loss of inhibitory material easily removed by washing, to killing of commensal bacteria by the bactericidal medium nor to changes in mucus distribution. Although central necrosis occurred in the maintained tissue pieces it was not correlated with increased susceptibility and virus replicated only in peripheral cells which remained healthy throughout culturing. The increase in susceptibility was demonstrated after maintenance for 6 h and increased to a maximum by 24 h.

The reason for the low susceptibility of the lung in vivo may be established by comparing organ cultures of fresh lung and nasal turbinates and by analysing the basis for the increased susceptibility of the lung with maintenance.

In investigating the reasons for the greater susceptibility of upper respiratory tissue of ferrets to influenza virus infection, comparisons were made of the early events in virus replication in organ cultures of fresh lung (low susceptibility) and fresh nasal turbinates and maintained lung (high susceptibility). The lower susceptibility of fresh lung was reflected in a higher minimal infectious dose (MID50) and lower virus yields at 24 h except with very high inocula when yields for the three tissues were similar.

Fresh and maintained lung had similar quantities of alveolar surfactant. Extracts of fresh lung had only weak inhibitory activity on virus replication in organ cultures of all three tissues. Virus adsorption to organ cultures was similar for the three tissues and equal to that shown by frozen and thawed tissue. Estimates of virus particles adsorbed to a standard length of cell membrane in electron micrographs, indicated some differences in numbers of virus receptors on different cells and an increase of receptors with maintenance. However, this occurred for nasal turbinates as well as for lung; thus the differences in receptors did not correlate with the differences in susceptibilities between the tissues. The proportions and numbers of susceptible cells were similar for the various tissues in cell suspensions prepared by two methods from organ cultures inoculated with high virus inocula and stained with fluorescent antibody. With small inocula, which resulted in large differences in virus yield at 24 h from the three tissues, the numbers of cells initially infected were only slightly different. This indicated that the differences in susceptibility were not primarily determined by differences in initiation of infection of susceptible cells. It is suggested that the quantity or quality of virus released at the first cycle, in relation to the ease of initiating the second cycle of infection, is the dominant factor in tissue susceptibility.

A comparison has been made of the RNAs of the seven serotypes of foot-and-mouth disease virus (FMDV) by competition hybridization. Homology among the three European serotypes A, O, C and the Asia 1 serotype was 60 to 70%. Similar homologies were found among the three Southern African Territories serotypes (SAT 1, SAT 2, SAT 3), but homology between the two groups was much lower (25 to 40%). Homology between the RNAs of subtypes within serotypes A and O was greater than 70%. Double competition experiments with the European serotypes indicate sharing of nucleotide sequences.

The molecular heterogeneity of acid-stable (Type I) mouse interferons induced in C243 cells by Newcastle disease virus was analysed by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions, and the profiles of antiviral activities obtained were characterized biologically in mouse cells and in heterologous (guinea-pig) cells. Two bands of activity, A and B, were consistently present in all interferon preparations tested: under reducing conditions, the activity in all fractions of band A (with a peak of activity at about 38000 daltons) was uniformly increased, while that of band B (with a peak at about 22000 daltons) was uniformly diminished. All the active fraction in band A had only slight activity (less than 10% of homologous titres) on guinea-pig cells, whereas all those in band B were significantly more active on guinea-pig cells than on homologous L cells. Thus, mouse type I interferon preparations contain two molecular populations of interferons that can be distinguished physically (by size), biochemically (by the effect of reduction on reactivation from SDS) and biologically (by activity in heterologous cells).

The effect of mouse interferon (ITF) on the expression of Friend leukaemia virus (FLV) and on dimethyl sulphoxide (DMSO)-stimulated haemoglobin synthesis in Friend erythroleukaemic cells (FLC) was studied. Immunofluorescent staining was used to detect intracellular antigens, and incorporation of 3H-uridine into virions to detect extracellular virus release. Interferon markedly inhibited haemoglobin synthesis and FLV production, but enhanced accumulation of virus antigens in the cytoplasm; on the cell surface, however, FLV antigens were present to the same extent whether ITF was present or not. When ITF was removed, virus production rose and intracellular virus antigens fell to the levels of untreated controls.

The proteins of five herpesviruses — herpes simplex types 1 and 2 (HSV-1 and HSV-2), bovine mammillitis virus (BMV), pseudorabies virus (PRV) and equine abortion virus (EAV) — have been compared by polyacrylamide gel electrophoresis of purified virus and by antigenic analysis using virus neutralization and agar-gel immunodiffusion tests. Although there were general similarities in the number, size range and overall complexity of polypeptides separated by polyacrylamide gel electrophoresis from purified particles of the five viruses, no single component of identical mobility was present in all viruses. The major capsid polypeptide from each of the five viruses comprised about 10% of virion protein mass but had distinctive mol. wt. of 158000 to 160000 (HSV-2 and BMV), 145000 (EAV) and 140000 (PRV) relative to 155000 for HSV-1.

Despite differences of apparent mol. wt. there was a one-for-one correspondence for the majority of structural polypeptides of HSV-1 and HSV-2 and many polypeptides of BMV were recognizably analogous to those of HSV-1 and HSV-2. In contrast the polypeptides of PRV and, more particularly, EAV differed markedly from those of all the other viruses. The antigenic analysis complemented these results; HSV-1, HSV-2 and BMV all cross-neutralized, whereas PRV and EAV were only neutralized by homologous antisera. Agar gel immunodiffusion tests with extracts of infected cells demonstrated at least one antigen common to all five viruses and showed BMV to be more closely related to HSV-1 and HSV-2 than were PRV or EAV although less closely related than HSV-1 to HSV-2. PRV and EAV share more antigens with each other than with the other viruses.

The secondary structure of encephalomyocarditis (EMC) virus RNA has been studied in situ and in free solution by absorbance-temperature relationships and by circular dichroism (CD). Extracted EMC virus RNA melts reversibly and has a hypochromicity of about 20%; analysis of CD spectra and formaldehyde treatment suggests that approx. 60% of the nucleotides are involved in base-pairing at 25 °C. It is shown that the RNA within the virus particle is less structured than when it exists in free solution, being partially stabilized by capsid protein against melting until the virion is disrupted to release the intact RNA. Upon clarification to remove denatured capsid protein, the released RNA gives a melting profile identical with that of phenol-extracted virus RNA. The results suggest that the intact structure of the virus is dependent upon intimate non-covalent bonds between RNA and protein together with hydrophobic bonds between the protein subunits.

We have isolated 38 viruses capable of infecting Caulobacter crescentus. Twenty-eight of these isolates were DNA phages with a prolate cylindrical head and a long flexible tail. Each of them infected only one of the Caulobacter cell types, the swarmer cell, and many proved to be capable of establishing a lysogenic relationship with their host. Despite structural similarity and host cell specificity, the majority of these DNA phages appeared to be genetically distinct as shown by EcoR1 restriction patterns of their DNA and by host range on phage resistant and lysogenic strains. Two isolates were small RNA phages, structurally similar to the Escherichia coli RNA phages, which specifically adsorbed to pili on the Caulobacter swarmer cell. The remaining eight isolates were DNA phages with polyhedral heads ranging from 60 to 160 nm in diameter. Six had contractile tails ranging from 50 to 160 nm in length, while two had long flexible non-contractile tails. Each of these isolates was capable of attaching to all Caulobacter cell types and two were capable of mediating generalized transduction between strains of Caulobacter (Ely & Johnson, 1977).

Purified lipopolysaccharide (LPS) from the bacteriocin sensitive strain Rhizobium lupini 16-2 was shown to neutralize the killing activity of the bacteriocin. In the electron microscopical preparation the phage tail-like bacteriocin appears to be adsorbed to the LPS; the tail sheath is contracted and the fibres are oriented towards the LPS ribbon. In contrast, no interaction was observed between the bacteriocin and the LPS of two resistant strains of Rhizobium (16-2/I1 and 16-3). The inactivation of the bacteriocin by LPS depends on salt concentration, pH, and temperature. The receptor activity of LPS was destroyed by mild acid hydrolysis and by treatment with deoxycholate, which indicates that the micellar structure of the LPS is necessary for bacteriocin adsorption. The chemical composition of the 16-2 LPS was compared to that of the LPS of two resistant strains. In the case of 16-2/I1 LPS minor modifications suffice to confer resistance against the bacteriocin.

We have studied several aspects of the inhibition of DNA synthesis in Mengovirus-infected cells. In mouse L-929 cells at 5 h after infection at a multiplicity of 200 p.f.u./cell, there was a decrease in DNA synthesis. However, the rate of DNA replication fork movement, measured by equilibrium sedimentation of DNA sequentially labelled with BrdUrd and 3H-thymidine, was not significantly reduced in infected cells despite an 84% inhibition of DNA synthesis. Enzymic assay of the total cellular dTTP pool size showed it to be increased 37% in infected cells compared to controls. This was not accounted for by enhanced entry of thymidine nucleotides into the pool from either exogenous or endogenous sources, since the buoyant density of DNA pulse-labelled with 3H-BrdUrd was the same in infected and uninfected cells and there was no evidence for breakdown of DNA in infected cells. There was also a moderate decrease in the uptake of exogenous thymidine into the cell, shown by a reduction in the Vmax for transport with little change in the K m. By 6 h after infection, the dTTP pool size was slightly smaller in infected cells than in the controls and there was a marked inhibition in the rate of replication fork movement. These results show that Mengovirus infection blocks the entry of dTTP into DNA. At 5 h after infection, this block is manifested by an inhibition of initiation of new DNA chain synthesis. By 6 h, replication fork movement is also retarded and there is a more generalized derangement in DNA synthesis.

A spectroscopic characterization is given of the isometric RNA-containing chicory yellow mottle virus. The circular dichroism of the virus is close to the sum of that of the native RNA and protein components. The nucleic acid structure is formed by regions of single-chain stacked-base helices similar to that of poly(A) at neutral pH, and short double-helical loops, while the conformation of the capsid is largely accounted for by regions of β-form and random-chain structures.

The heat-induced dissociation of virus nucleoprotein occurs concomitantly with destabilization of the orderly structures of the protein moiety, indicating that only minor interactions between RNA and protein occur. It is suggested that interactions between RNA and protein could involve some aromatic residues of the capsid and the heterocyclic bases of the nucleic acid.

Absorbance-temperature profiles, at 400 nm, of plant viruses with one or more kinds of isometric particle, were determined. Two temperatures were determined, Td (the temperature at which E 400 begins to increase) and Tf (the temperature at the inflexion point of the curve). The behaviour on heating at these temperatures was of two main types. Heating in vitro appears to be a useful means of making a gross subdivision of isometric plant viruses into two groups typified by cucumber mosaic virus and turnip yellow mosaic virus, respectively, in agreement with Kaper’s (1971a) suggestion.

Exposure of chicken embryo fibroblasts (CEF) to trypsin solubilizes cell surface components, including the initial attachment site for avian tumour viruses (ATV). This soluble attachment site activity appears to interact directly with the ATV in vitro thereby interfering with the binding of at least two ATV subgroups to both intact CEF and CEF plasma membranes. A result of this interaction in vitro is reduced ATV infectivity, demonstrated by reduced transforming capacity of RSV (RAV-1).

RNA synthesis induced by a temperature-sensitive RNA negative mutant, ts-4, of Semliki Forest virus was studied both at the permissive (28 °C) and restrictive temperature (39 °C). At 28 °C ts-4 directed RNA synthesis was qualitatively similar to the wild type but overall synthesis remained somewhat lower. Only about 3% of RNA was synthesized compared to the wild type virus when ts-4 was maintained at 39 °C throughout the infection. The sedimenting radioactivity was almost exclusively in 42S RNA. When ts-4 infected cultures were shifted from 28 to 39 °C at 5 h post-infection, the synthesis of 26S RNA was shut off while 42S RNA synthesis continued. The synthesis of 26S RNA started again even in the presence of cycloheximide if the cultures were shifted back to 28 °C. Our results suggest that 26S RNA synthesis is controlled by a virus-specific protein, the function of which is not required for the synthesis of 42S RNA, In ts-4 this protein is denatured at 39 °C and becomes non-functional, but renatures and becomes functional upon shift down to 28 °C.

Poxviruses isolated from captive elephants in Germany have been characterized. Although related to vaccinia and even more closely to cowpox virus, the separate identity of elephantpox virus was established by both biological and serological methods. Elephantpox virus produces A-type inclusions in infected cells, as did cowpox, but had a lower ceiling temperature, was more heat resistant and affected rabbits differently. Cross neutralization tests on absorbed sera indicated that elephantpox, cowpox and vaccinia viruses shared one surface antigen, that elephantpox and vaccinia shared an antigen absent from cowpox, and that vaccinia virus had a surface antigen absent from elephantpox and cowpox viruses.

Examination by scanning electron microscopy (SEM) of PK-15 cells infected with each of the two cytopathogenic types of porcine enterovirus revealed changes similar to those observed by light microscopy. However, differences between the two types could be detected at an earlier stage of the infection. Cells infected with T80 virus (type 1) were rounded, with surface microridges, while V13 (type II) infected cells were characterized by cytoplasmic protrusions.

When searched for by standard virological methods, murine cytomegalovirus (MCMV) becomes undetectable by 4 months after inoculation of mice. However, a 2-week regimen of immunosuppression with anti-lymphocyte serum and corticosteroid results in reactivation and dissemination of the virus in virtually all animals. This system should be useful in defining the pathogenesis of generalized cytomegalic disease resulting from reactivation of latent virus in immunocompromised individuals.