- Volume 32, Issue 2, 1976

The development of measles virus (Edmonston) and SSPE measles virus (Horta-Barbosa) has been examined in enucleate BSC 1 cells. New antigen synthesis in measles virus infected enucleate cells has been demonstrated by fluorescent antibody, by the formation of extensive syncytia from enucleate cells alone and by analysis of polypeptide formation by polyacrylamide gel electrophoresis. All polypeptides formed in nucleate cells were also present in enucleate cells but the amount synthesized was reduced to around 20% of that in nucleate cells. There was also a significant reduction in the amount of antigen detected by fluorescent antibody in enucleate as compared to nucleate preparations. Examination of RNA synthesis in infected enucleate cells revealed only a marginal increase in acid-insoluble material. Titration of the output of infectious virus from enucleate cells infected at both 37 and 31 °C indicated a consistent reduction of almost two log units compared to nucleate cells. That the enucleate cells were capable of replicating input genome at these times was demonstrated by the successful growth of respiratory syncytial virus, both at 37 and 31 °C.

SSPE measles virus grew to higher yield in nucleate BSC 1 than measles virus but there was again a reduction of more than two log units in enucleate cells. All polypeptides synthesized in SSPE infected nucleate cells were apparent in enucleate cells.

A circular dichroism comparative study of isolated and in situ phage R17 RNA reveals in both cases the same degree of base pairing. However, thermal circular dichroism melting profiles exhibit the presence of free energy of interaction between RNA and capsid protein. It is apparent that the capsid stabilizes the RNA structure with and without the addition of Mg2+. A close RNA capsid association is also derived from pH titration circular dichroism studies. The pH melting of the RNA in situ starts to occur about 0.5 pH unit higher with and without the addition of Mg2+ than the acid denaturation of isolated RNA. A direct correlation between bathochromic CD peak shift of the main position band and loss of survivors is noted for the thermal melting as well as pH titration experiments. It is suggested that the heat and pH induced conformational alterations of R17 RNA in situ coinciding with loss of infectivity occur after an in situ alteration of nucleic acid-capsid protein interaction.

The nature of the DNA molecules synthesized in nuclei of herpes simplex virus (HSV)-infected cells in vivo and in vitro was studied by chromatography on BND-cellulose columns after shearing to DNA fragments of 10 to 20 × 106 daltons. The incorporation of labelled precursors occurs in the DNA fragments containing single-stranded regions, presumably the replication forks. Prolongation of DNA synthesis leads to the accumulation of labelled DNA fragments that lack single-stranded sequences. Analysis of the isolated DNA fragments by density centrifugation in CsCl gradients revealed that most of the labelled DNA molecules are of virus specificity and the minority are cellular DNA fragments. Double-stranded virus DNA fragments and virus DNA fragments containing single-stranded sequences band in CsCl gradients at a density of 1.718 g/ml, the density of virion DNA. This suggests that the replicating HSV DNA molecules have the same density as the virion DNA and contain relatively little single-stranded DNA. The synthesis of HSV DNA molecules under in vitro conditions in isolated nuclei occurs by incorporation of the precursors into DNA fragments with single-stranded regions. The synthesis of cellular DNA in nuclei from hydroxyurea and cytosine arabinoside treated cells also occurs by elongation of nascent DNA chains.

When prepared from tissue frozen with liquid nitrogen, tobacco mosaic virus replicative form RNA (TMV RF) was uniform in size but when prepared by high-speed homogenization, or when TMV RF prepared with liquid nitrogen was homogenized, 80 to 90% of the RF broke into relatively discrete pieces. The unbroken RF was not fragmented by additional homogenization. The TMV RF components susceptible and resistant to breakage, respectively, were synthesized with similar kinetics in relation to length of labelling period, but the slightly more resistant component was synthesized during the early infection period. Both components were produced by different strains of TMV but leaves infected with cowpea chlorotic mottle or southern bean mosaic viruses yielded only RF resistant to breakage. TMV replicative intermediate RNA was also broken by homogenization. The occurrence of the two RF components may be of significance in the replication of RNA viruses.

Ortho- and parainfluenza viruses isolated from the cloacas of migrating feral ducks shot on the Mississippi flyway included three strains of influenza A virus (Hav6 Nav1, Hav6 Nl, Hav7 Neq2) as well as Newcastle disease virus. One influenza virus, A/duck/Memphis/546/74, possessed Hav3 haemagglutinin, but the neuraminidase was not inhibited by any of the known influenza reference antisera. The neuraminidase on this virus was related to the neuraminidases on A/duck/GDR/72 (H2 N?), A/turkey/Ontario/7732/66 (Hav 5 N?), A/duck/Ukraine/1/60 (Hav3 N?) and A/turkey/Wisconsin/68. We therefore propose that the neuraminidase on this group of influenza viruses be designated Nav6.

The A/duck/Memphis/546/74 influenza virus caused an ocular discharge in 1 of 5 ducks and was shed in faeces for 10 days; it was stable in faecal samples for up to 3 days at 20 °C. These results suggest that ecological studies on influenza in avian species should include attempts to isolate virus from faeces. Faecal-oral transmission is an attractive explanation for the spread of influenza virus from feral birds to other animals.

Interferon inhibits the replication of simian virus 40 (SV40) in monkey cells and reduces markedly the formation of both early virus protein (i.e. SV40 T antigen) and early SV40 RNA. This suggests that in SV40 infection interferon acts primarily by inhibiting transcription. To test this conclusion further, we examined alternative mechanisms which might explain these results and made the following observations. (1) The quantity of input SV40 DNA in the nucleus 24 h post infection (p.i.) was the same in interferon-treated and control cells. Thus interferon does not appear to diminish the quantity of SV40 DNA template available for transcription. (2) Chemical inhibitors of protein synthesis did not mimic the selective inhibition of early SV40 RNA formation induced by interferon, indicating that the transcription of early SV40 RNA is not dependent upon the prior synthesis of any virus-induced protein. Thus a block in translation cannot readily explain the reduced formation of early SV40 RNA in interferon-treated cells. (3) Fractionation of SV40 infected cells after a one-hour labelling period showed that interferon produced a comparable reduction in the quantity of early SV40 RNA in the nucleus and the cytoplasm. Thus the observed inhibition of early SV40 RNA is not due solely (if at all) to enhanced cytoplasmic degradation. These results indicate that the primary effect of interferon in SV40 infected monkey cells is either to inhibit the transcription of early virus RNA or to enhance its turnover in the nucleus.

The Xf phage coat protein associated with infected cells could not be removed by washing with antiserum and tris-EDTA buffer. Although the infected cells were consecutively washed 6 times with tris-EDTA buffer, the ratios of parental phage 3H-DNA to 14C-protein were not changed. A considerable amount of the parental 14C-protein and 3H-DNA in the original ratio were detected in the membrane and the soluble cytoplasmic fractions of infected cells. The studies of the change in Xf 14C-protein and 3H-DNA incorporation into the host cells and their release showed that DNA and protein penetrate together into the host cells during the first 60 min after infection (p.i.). While virtually all parental DNA was conserved, re-utilized and released from the infected cell 60 min p.i., no apparent release of parental protein was observed. Approx. 40% of the parental protein became degraded and could be washed from the infected cell after 90 min; the rest of the parental protein remained and probably was re-utilized by the host.

An electron microscopic study of the lipid-containing bacteriophage φ6 revealed an electron dense compact inner core of 30 nm in diam., which apparently contains the nucleic acid of the virus. This inner particle is surrounded by a complex poly-hedral capsid with an outer diam. of 50 nm. Outside this is the envelope, which gives the virus a total diam. of 65 to 75 nm. The envelope, which has a thickness of a unit membrane, could be removed by treating the phage with Triton X-100. A definite structure is seen inside the envelope of the phage tail.

In infection, phages are attached by their tails to the host cell pili. Occasional pili with a few attached phages were seen in a phage resistant mutant. In the course of the infection phages were also seen attached to the outer membrane of the cell. In a phage-tolerant mutant many normal-looking pili with adsorbed phages were visible, but we could never see phage-cell membrane associations. The membrane of the phage appears to fuse with the bacterial outer membrane and 50 nm virus particles could be seen in the periplasmic space of the bacterium, probably attached to the cytoplasmic membrane. Newly formed 50 nm particles appeared 45 min post infection (p.i.) centrally in the host cell. Assembly of the envelope also began at this time and by 80 minutes p.i. all the 50 nm particles were covered by the virus membrane. At no stage were phages seen in the periphery of the bacterium. Mature phages were finally released by a rupture of the host cell without spheroplast formation.

All eukaryotic mRNA species show a characteristic individual translational efficiency under conditions of restricted polypeptide chain initiation caused by an increase in the osmolarity of the growth medium. In vaccinia virus infected L cells or HeLa cells virus mRNAs can be grouped into classes on the basis of their relative labelling under standard and hypertonic conditions. Under the latter conditions, most of the ‘early’ mRNAs possess very high translational efficiencies, most of the ‘intermediate’ mRNAs show an intermediate efficiency and the most prominent ‘late’ mRNAs show a translational efficiency which is lower than that of other virus mRNAs but still higher than the average cellular mRNA. Late in the infection cycle virus mRNAs with a relative low translational efficiency are preferentially translated under standard growth conditions whereas ‘early’ virus mRNAs which are still present and which show a higher translational resistance to hypertonic conditions are not translated. These results indicate a unique translational control operating late in the growth cycle of vaccinia virus.

Earlier results indicating that vaccinia virus entered L cells by a process of direct fusion between the virus envelope and the plasma membrane of the cell have been confirmed and extended using immuno-ferritin conjugates to locate virus antigens on the host cell surface. After fusion, components of the virus envelope become rapidly dispersed in the plasma membrane. Fusion has also been observed as the predominant mode of entry of vaccinia virus into HeLa cells.

Exposure of purified transmissible gastroenteritis virus, a porcine coronavirus, to non-ionic detergents resulted in the removal of the surface projections and > 98% of the virus lipid. Virus RNA was associated with a subviral particle which had a sedimentation coefficient of 650S, compared with 495S for the intact virion, and which banded in Cs2SO4 gradients at 1.295 g/ml. Negatively stained preparations of subviral particles were shown by electron microscopy to contain spherical particles of 60 to 70 nm diam., similar in appearance to those derived from oncornaviruses.

Polyacrylamide gel electrophoresis of the polypeptides from isolated subviral particles showed that these structures contained three of the four major virus structural proteins, the arginine-rich polypeptide VP2 and the two membrane glycopolypeptides VP3 and 4. The detergent-liberated surface projections, composed of a single species of sulphated glycopolypeptide, VP1, were isolated by rate-zonal centrifugation through sucrose gradients followed by precipitation with ammonium sulphate in the presence of bovine serum albumin.

A cross-neutralization study of 22 alphaviruses disclosed two major antigenic complexes and two viruses that were distinct from all the others. Cross-reactions were common but were restricted within the complexes. Sub-groupings within a complex were also shown, and some viruses proved to be indistinguishable by neutralization testing. The results generally paralleled previously reported data. Investigations were carried out, using BHK 21 cells in a micro tissue-culture system, on all available alphaviruses.

C-type particles secreted in vivo by MOPC-315 myeloma cells were characterized. These particles localize at a density of 1.16 g/ml in sucrose and possess a 60 to 70S RNA and an RNA-instructed DNA polymerase. Endogenous enzyme activity requires manganese and is inhibited by ribonuclease or by the omission of any of the deoxynucleoside triphosphates. The enzyme utilizes the virus 60 to 70S RNA as a template to synthesize DNA molecules which specifically hybridize to the homologous RNA.

When tris-HCL was used as the buffer for inoculation of tomato protoplasts with tobacco mosaic virus, a greater proportion became infected than when phosphate was used. Using 0.05 m-tris-HCl buffer, pH 8.0, in the presence of poly-l-ornithine or poly-d-lysine, 50 to 80% infection was obtained.

Human cytomegalovirus (CMV) induces nuclear antigens resembling the Epstein-Barr nuclear antigen (EBNA) as early as 3 h after infection. These early antigens can be detected only with the anti-complement immunofluorescence staining (ACIF) technique. Synthesis of these new antigens is not influenced by cytosine arabinoside (ara-C).

Centrifugation through CsCl was used to isolate 32P-labelled RNA in a one-step purification procedure. The method is suitable for quantitative as well as preparative studies and appears to have considerable advantages over conventional methods of RNA extraction. We have used this procedure to investigate the RNA synthesized in Vero cells infected with canine distemper virus (CDV). We show that the combination of CsCl centrifugation and affinity chromatography on poly-U Sepharose provides a rapid method for isolating messenger RNA from virus infected cells.

Virazole or Ribavirin (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) inhibits the growth of vaccinia virus at a concentration of 50 µg/ml. Although vaccinia virus DNA and polypeptides are made to a certain extent in the presence of Virazole, the DNA fails to acquire resistance to deoxyribonuclease and virus particles are not formed. Reversibility of the antiviral effect occurs when the drug is washed out from the infected cultures or when guanosine at an equimolar concentration is added.

Electrophoretic heterogeneity in preparations of arabis mosaic virus (AMV) was due to differences in net surface charge among the sedimenting components. Bottom component migrated more rapidly than top component in polyacrylamide gels and on cellulose acetate strips. When nucleic acid was removed from bottom component the nucleic acid-free protein shell showed sedimentation and electrophoretic properties similar to those of top component.