Samuel J. Martin

Persistent infection of tissue culture cells by RNA viruses

In this paper, the characteristics of cultured cells persistently infected with RNA viruses, other than leuko viruses are described. The roles that the host cell, interferon, virus mutants and defective interfering particles may play in the establishment and maintenance of persistent infection, are discussed.It is proposed that the interaction of viruses with certain types of host cells can lead to persistent infection. The differences in virus-host interactions may be attributable to differences in membrane properties of various cells. Defective interfering particles may play a role in the establishment of persistent infections in cells which normally undergo lytic virus development. Mutant types of virus appear to be prominent in the virus released from persistently infected cells, but the role that various mutants play in the maintenance of persistent infections remains unclear.

Defective interfering particles produced during the replication of measles virus

The RNA species present in purified measles virus preparations after 10−3 diluted passage (DP), three undiluted passages (UP3) and eight undiluted passages (UP8) were analysed by sedimentation on sucrose gradients. The particles contained RNAs sedimenting at 52S and 4S, 52S, 18S and 4S, and 52S, 30S, 18S, 11S and 4S respectively. Measles virus particles produced by UP3 were separated into two homogenous populations by rate zonal centrifugation on sucrose gradients. The rapidly sedimenting population contained encapsidated 52S RNA and was infectious virus. The slowly sedimenting particles contained encapsidated 18S RNA, were non-infectious, and they interfered with the replication of infectious virus.

An immunological study of infection of hamsters with large and small plaque canine distemper viruses

SummaryThe small plaque virus (SPV), derived from the Onderstepoort strain of canine distemper virus (CDV) does not cause a lethal encephalitis in weanling hamsters. When we immunosuppressed hamsters infected with this virus they developed an acute disease, similar to that produced by the large plaque virus (LPV). Passive transfer of maternal antibody from SPV infected mothers to their offspring was effective in preventing acute disease following LPV infection. Co-infection of animals with both LPV and SPV resulted in increased hamster survival, associated with high titres of serum antibody. Similarly, heat inactivated SPV, present during infection with LPV, increased the survival rate. Heat inactivated LPV did not inhibit acute disease, although hamsters had high titres of neutralizing antibody. A small number of animals developed a delayed or reoccurring paralysis after immunosuppression, exposure to maternal antibody or co-infection. It would appear that the neurovirulence of CDV for hamsters can be modified by altering the levels of circulating antibody early in infection.

Structure and function relationships of the envelope of measles virus

The glycoproteins and lipids of NP-40 solubilised measles virus envelopes were separated by equilibrium centrifugation in CsCI. Four virus phospholipids were tested for their ability to re-aggregate with the glycoproteins to form active haemolytic activity. Phosphatidylethanolamine was the most effective, phosphatidylserine was ineffective, and phosphatidylcholine and sphingomyelin were effective only at high concentrations. Re-assembly of the glycoproteins with cholesterol did not result in the formation of haemolytic activity but its presence could enhance the activity of the phosphatidylethanolamine—assembled haemolysins.Treatment of measles virus with 5% Triton-X-100 and 1.0 M-KCl solubilised the virus glycoproteins and the membrane protein. Removal of the KCl by dialysis resulted in the precipitation of the membrane protein, which was identified by gelelectrophoresis as virus protein 6. Sedimentation of the two soluble glycoproteins on sucrose gradients containing 1% Triton-X-100 and 1.0 M-KCl allowed the separation of two components having sedimentation coefficients of 6 to 7S and 9S respectively. When these components were resedimentated under identical conditions a pure preparation of the larger virus glycoprotein was obtained from the 9S material. A pure preparation of the smaller glycoprotein could not be isolated from the 6 to 7S material. The larger glycoprotein contained all of the virus haemagglutinating activity. Re-assembly of the partially purified glycoproteins with virus lipids demonstrated that the biologically active haemolysin required both virus glycoproteins and envelope lipids. Inhibition studies on the haemolytic activity indicate that a proteolytic enzyme is responsible for measles virus haemolysis.

Comparison of morbillivirus proteins by limited proteolysis

Proteins of a number of measles and SSPE virus strains have been compared by limited proteolysis and they appear to be largely conserved amongst the various strains. Viruses derived from SSPE cannot be distinguished from other measles viruses by this technique. Small differences in the digest patterns of the M proteins have been observed between the Edmonston and other measles virus strains. Furthermore, in some strains where the M proteins migrate slower in SDS-PAGE the limited proteolysis patterns are slightly different from those in other MV and SSPE virus strains. The limited proteolysis pattern of some canine distemper virus (CDV) proteins have been determined and nucleocapsid break-down products have been identified in infected cells. Comparisons of proteins of four strains of CDV have shown that these, too, are largely conserved, although the digest of proteins of CDV appear to show more pronounced differences than those present in the MV and SSPE virus group. Limited proteolysis can be used readily to distinguish MV from CDV isolates.

A simple method for the removal of mycoplasma contamination from paramyxovirus stocks

A simple and quick method of detecting mycoplasmas in virus stocks using the fluorochrome Hoechst 33258 is described. Different methods of removal of mycoplasmas from stocks are discussed. The simple but effective method using gentamicin (0.2 mg/ml) or chloramphenicol (5 micrograms/ml) is demonstrated and chosen as the most efficient as judged using both the Hoechst stain and the direct assessment of mycoplasma RNA species labelled with [5-3H]uridine.