- Volume 51, Issue 1, 1980

Several phages infecting Bacillus megaterium QM B1551 have been isolated from the soil and partially characterized. These phages, designated MP9 to MP50, were tested for host-range on several strains of B. megaterium and 13 other Bacillus species. All the phages only infected B. megaterium and on the basis of host-range patterns, 23 groups could be distinguished. The phage patterns also distinguished subgroups of B. megaterium strains within the species and should be useful in phage typing. The phages have varying sensitivities to heat, salts and organic solvents and are all double-stranded DNA phages. Thirty-two have been examined by electron microscopy and are Bradley types A, B and C. This is the first large collection of B. megaterium phages that has been characterized.

A mammalian cell-adapted mutant of the Dobson strain of fowl plague virus (FPV-B) was characterized. Genetic analyses of recombinants between a ts mutant of this virus and either the non-adapted Dobson strain or the Rostock strain of FPV showed that the gene coding for the P3 protein of the adapted Dobson strain was sufficient to enable any recombinant to grow in L cells.

The abortive cycle of wild-type Dobson strain (FPV+) was compared to the productive cycle of the mutant. By using 100 p.f.u./cell, no quantitative difference could be detected in infected L cells between polypeptides and cRNAs induced by FPV+ and FPV-B. However, the maturation of virions at the plasma membrane did not proceed correctly. At a lower m.o.i. the amounts of virus polypeptides decreased with the m.o.i. This decrease was not the same for all polypeptides and cRNA segments: HA, M and NA and their mRNAs decreased to a greater extent than the others. These results are discussed in relation to a possible biological activity of polypeptide P3.

Micrococcal nuclease digestion was used to probe the structures in which herpes simplex virus type 1 (HSV-1) DNA is found during virus replication. Parental DNA, progeny DNA and DNA in nucleocapsids were analysed. Parental DNA was examined after infection of Vero cells with 32P- or 3H-thymidine-labelled HSV-1. Progeny DNA was examined after HSV-1-infected Vero cells were pulse-labelled with 3H-thymidine during HSV-1 DNA synthesis. In both cases, nuclei were isolated and digested with micrococcal nuclease. Digestion products were analysed by agarose or polyacrylamide gel electrophoresis (PAGE). Most parental DNA remained as intact molecules. However, a small amount was degraded into fragments which were heterogeneous in size or the size of nucleosomal cell DNA. These two classes of fragments were also produced upon digestion of progeny DNA. The heterogeneous fragments and nucleosomal fragments comprised major and minor fractions, respectively, of digested progeny DNA. When digested DNA from HSV-1-infected cells was transferred from composite polyacrylamide-agarose gels to diazobenzyloxymethyl paper, nucleosomal fragments hybridized to 32P-labelled HSV-1 DNA as well as to 32P-labelled Vero cell DNA. Therefore, nucleosomal fragments contained HSV-1 DNA sequences. HSV-1 DNA in nucleocapsids was analysed by micrococcal nuclease digestion after nucleocapsids were disrupted with pH 9.3 buffer, pyridine, Sarkosyl or NaCl/urea. Only fragments of heterogeneous size were produced. Thus, HSV-1 DNA is found predominantly in structures other than nucleosomes during virus replication.

12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent tumour promoter, was tested for its effects on the proliferation of the human Burkitt lymphoma cell line, Namalwa, and the synthesis of interferon by these cells. At nanomolar concentrations, TPA blocked thymidine incorporation into cellular DNA by more than 90% within 24 h. TPA-treated cells produced about 20-fold more interferon in response to Sendai virus than did untreated controls and simultaneous treatment with TPA and sodium n-butyrate gave a further two- to three-fold enhancement. Neither of these effects of TPA was reversed on removal of the compound; furthermore, exposure of Namalwa cells to TPA for only 1 h was sufficient for full activity.

4-O-methyl-TPA, a compound only marginally active as a tumour promoter, showed effects similar to TPA, but only at concentrations 300-fold higher. In contrast to its effects on Namalwa cells, TPA did not affect synthesis of interferon in response to Sendai virus in two other Burkitt lymphoma lines (Raji and Daudi) nor in the Epstein-Barr virus (EBV)-negative lymphoma line, BJAB; it inhibited interferon production in the human myeloid cell line, HL-60.

Two strains of fixed rabies virus were examined for their ability to regenerate defective interfering (DI) particles and for possible correlation of DI particle production with the expression of virulence. A plaque-purified stock of the attenuated ERA strain (ERApp), which characteristically caused an auto-interfering death response in adult mice inoculated i.c., was serially passed at a high m.o.i. in BHK-21 cells. By the sixth passage, DI particles were regenerated that corresponded in sedimentation velocity and DI/RNA size to the smallest of three sizes of DI particles produced by the parental stock virus. The regeneration of ERA DI particles in vivo was not detected during 15 serial high or low m.o.i. passages of infected newborn mouse brain, though the passaged virus consistently elicited an auto-interfering-type death response when assayed in adult mice. The attenuated Flury HEPpp strain regenerated up to three unique size classes of DI particles during serial passage in BHK-21 or murine neuroblastoma C1300 clone NA cells compared with the one band of DI particles produced by the parental Flury HEP stock virus. The BHK-21 cell-adapted Flury HEPpp virus failed to kill adult mice when inoculated at high concentrations after two serial passages in NA cells. However, the virus became fully virulent and a single band of regenerated DI particles was visible. Additional bands of defective particles were visible following the third serial passage in NA cells. Single-stranded RNA with a mol. wt. of 0.62 × 106 was extracted from the first DI particle population to be regenerated. This corresponded in mol. wt. to the DI/ssRNA characteristic of the parental attenuated Flury HEP virus. However, in the parental type DI/RNA, partially dsRNA could be isolated in addition to ssRNA. Double-stranded RNA could not be detected in the regenerated DI particles derived from the virulent NA cell-propagated Flury HEPpp virus. These results suggest that the virulence phenotype of fixed rabies viruses does not depend on the presence or absence of DI particles.

The kinetics of replication of five strains of human cytomegalovirus (CMV) were studied to determine the influence of (i) temperature, (ii) virus strain, (iii) m.o.i. and (iv) cell type. Relative to growth at 37°C (m.o.i. = 3 to 9) eclipse periods were extended from 24 to 48 h at 33 °C and to 72 h at 40.5 °C. Yields were reduced at 33°C and almost eliminated at 40.5 °C. No replication occurred in most instances at 40.5 °C and with 0.05 p.f.u./cell. Temperature shift studies (40.5 to 37 °C) indicated that the block to replication at 40.5 °C occurred about 12 to 16 h p.i. resulting in little synthesis of CMV DNA or late antigens. The degree of inhibition of late functions at 40.5 °C is virus strain and m.o.i. dependent, but is not dependent on the type of fibroblastic cell used. These data suggest that persistent CMV infections are favoured at 40.5 °C.

A new strain of simian haemorrhagic fever (SHF) virus was isolated from chronically infected patas monkey no. 248 (P-248) in USU-104 cells. The P-248 isolate had the same size, morphology and cytoplasmic site of replication as the prototype LVR strain. However, the P-248 isolate caused a persistent infection without noticeable cytopathology in USU-104 cells rather than the strongly lytic infection produced by prototype LVR virus. The capacity of P-248 virus to produce a persistent, non-lytic infection of USU-104 cells was a very stable characteristic of the isolate. Extensive serial passage of this isolate through USU-104 cells (over 50 passages) and rhesus monkeys (six passages) failed to unmask virus with lytic properties for USU-104 cells. Culture medium from persistently infected cultures assayed in rhesus monkey peritoneal mononuclear phagocytes, where measurable cytopathology occurs, was found to contain about 105 to 106 TCID50/ml of cell-free P-248 virus. Immunolabelling techniques showed only a low percentage of infected cells in persistently infected cultures. The mechanism of persistence of the P-248 isolate in USU-104 cells has not been determined but evidence suggests it does not involve interferon or defective interfering particles.

Cross-reactions between two strains of foot-and-mouth disease virus (FMDV) belonging to different serotypes (A and O) were studied with intact virus and virus subunits and antisera produced against the isolated structural proteins. Anti-VP1 type O serum showed cross-reactive neutralizing activity, in contrast to the sera raised against intact virus type O, whereas anti-VP1 type A serum only neutralized homologous virus. Anti-VP2, VP3 and VP4 did not show neutralizing activity. In the enzyme-linked immunosorbent assay and radio-immunoassay anti-VP1, -VP2 and -VP3 sera reacted with the 12S virus subunits of both serotypes. No activity was obtained against VP4. Competition experiments with virus subunits of virus type A and O show that anti-VP1 serum is the most type-specific. Anti-VP2 serum is completely cross-reactive, while anti-VP3 serum reacted in an intermediate way. The identical reactions obtained with anti-VP2 sera and the homologous and heterologous virus subunits suggest that the exposed VP2 antigens are identical.

Newly synthesized interferon and its mRNA were membrane-associated in a human lymphoblastoid cell (Namalwa) that had been induced with Sendai virus. Treatment with zinc, which acts as an inhibitor of proteolytic cleavage, prevented interferon production. When cytoskeletal function was disrupted by adding both colchicine and cytochalasin B to the induced Namalwa cells, secretion of interferon was inhibited. It is concluded that after translation of the interferon mRNA, the newly synthesized interferon polypeptide is discharged into the lumen of the endoplasmic reticulum, and undergoes a proteolytic cleavage before it is secreted by a process involving the cytoskeleton.

Animals were immunized with purified D-antigen or C-antigen of type 3 poliovirus to produce specific antisera which were used to analyse the antigenic characteristics of the progeny virus in harvests from poliovirus type 3-infected cells.

An examination of the virus progeny present at 24 h p.i. of cells with Sabin type 3 vaccine strain virus revealed a large population of particles sedimenting at a slightly lower rate (130S) than infectious virus (155S) in addition to slowly sedimenting (80S) empty capsids. Such 130S particles were not detected in the progeny from cells infected with strains genetically unrelated to the Sabin vaccine strains. They were non-infectious, contained RNA in an RNase-resistant form unless heated, and lacked the virion protein VP4. They expressed C-antigen rather than the D-antigen of infectious virus, and, therefore, had the properties previously described for poliovirus particles eluted from cells. The amount of incorporation of radio-isotope into the proteins or nucleic acids of such particles varied from 15 to 20% to 300% of the amount incorporated into infectious virus depending on the cells and virus strains studied. Virus strains genetically related to Sabin type 3 vaccine virus which were isolated from cases of paralytic poliomyelitis produced the particles in either low or undetectable quantities.

The reactions of polioviruses in single-radial-immunodiffusion (SRD) tests were investigated with a view to developing accurate and sensitive antigen assay systems. In direct SRD tests, employing high concentrations of immune poliovirus serum in agarose gels, poliovirus D-antigens produced clear reaction zones demonstrated by protein staining. The reactions were type-specific for polioviruses of types 1, 2 and 3 but the tests were of low sensitivity, being applicable only to the assay of virus concentrates.

A novel autoradiographic zone size enhancement (ZE) test was developed which increased the sensitivity of the SRD assay 40- to 100-fold. The ZE test was dependent upon the ability of unlabelled poliovirus to co-migrate with the radioactive marker virus and so enhance the zone size detected autoradiographically. The areas of the autoradiographic zones were directly proportional to the concentration of unlabelled antigen. The ZE test was capable of detecting poliovirus D antigens in diluted cell culture fluid harvests in amounts corresponding to 103.3 to 104.3 TCID50 of infectious virus.

Studies with poliovirus type 3 strains indicated that the ZE test was narrowly strain-specific for the D-antigen of poliovirus type 3 strains when homologous type 3 D-antigen was used as radioactive marker, but broadly cross-reactive for the D-antigen of type 3 viruses when heterologous poliovirus type 3 D-antigen was used as marker.

Varicella-zoster virus infection of primary hamster embryo cells resulted in oncogenic transformation. These transformed cells exhibited virus-specific antigens by immunofluorescence and developed surface Fc receptors. They induced aggressive fibrosarcomas when injected into inbred hamsters. The tumour-bearing hamsters develop antibodies to varicella-zoster antigens.

The replication of infectious agent has been studied in brain, thoracic cord, and lumbar cord of Compton white mice (Sinc s7) infected with the 139A strain of scrapie. Nine experiments were carried out using four different peripheral routes of injection. A highly consistent pattern of results was obtained in which replication in the CNS started in the thoracic cord after about 35% of the total incubation period had elapsed (range 25 to 42%). This was followed by the simultaneous onset of replication in brain and lumbar cord which occurred 2 to 4 weeks later. It is difficult to explain these results on the basis of haematogenous spread of infection from peripheral sites of replication (e.g. in spleen) to the CNS. However, the data are consistent with spread of infection along peripheral nerves and, in particular, along nerves of the sympathetic nervous system. It is suggested, therefore, that this may be the major route by which scrapie agent invades the CNS.

The nature of tubular structures (TS) specifically found in cells infected with herpes simplex virus type 2 (HSV-2) was investigated by studying the appearance of TS in mixed infections with herpes simplex virus type 1 (HSV-1) and HSV-2 in Vero cells. Mixed infections with HSV-1 and HSV-2 resulted in a notable reduction in TS appearance. Accumulation of TS in cells infected with u.v.-irradiated HSV-2 was decreased by superinfection with HSV-1. The majority of progeny viruses obtained from mixed infection with HSV-1 and HSV-2 was neutralized by type-specific anti-HSV-2 serum, but not by type-specific anti-HSV-1 serum. Analysis of the genotype of the progeny revealed that the yield from mixed infection contained both HSV-1 and HSV-2 genotypes. These observations indicate that the majority of HSV-1 progeny of mixed infection is phenotypically mixed and make it possible to propose that TS are materials related to the type-specific antigen for HSV-2 which can be utilized by superinfection with HSV-1, producing phenotypically HSV-2 particles.

The nuclear and cytoplasmic RNAs from Kirsten murine sarcoma virus (KiMuSV)-transformed non-producer mink cells were studied for the species of virus-specific RNA by fractionation in agarose gels, transfer to diazotized aminophenylthioether paper and hybridization to complementary DNA probe. In both nuclei and cytoplasm, only genome-length KiMuSV-specific RNA was detected. No subgenomic virus RNA species was detected in poly(A+) or poly(A-) RNA fractions. The same observations were made in KiMuSV-transformed mink cells superinfected with feline leukaemia viruses. The significance of these findings is discussed.

Human skin fibroblasts derived from a healthy individual and from a child with the genetic disorder ataxia telangiectasia were infected with herpes simplex virus type 1 (HSV-1). The virus infection did not affect the synthesis of procollagen but inhibited its release from the cells.

The uncleaved (HN0) and the cleaved (HN) forms of the haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus (NDV), strain Ulster, were analysed by polyacrylamide gel electrophoresis under reducing and non-reducing conditions. When HN0 is converted into HN, a glycopeptide with an apparent mol. wt. of about 8000, which is not found in the mature spike, is removed.