Search

The nucleic acids of two amoebal viruses were studied by several independent methods. The filamentous virus, V(ABRM), was shown to be inhibited by bromodeoxyuridine, iododeoxyuridine, and cytosine arabinoside. With acridine orange staining, V(ABRM) inclusions appeared greenish-yellow, indicating that these contained double-stranded nucleic acid. The polyhedral virus, V(301), was also inhibited by bromodeoxyuridine, iododeoxyuridine, and cytosine arabinoside. In addition, nucleic acid hybridization showed that a new DNA species was synthesized in infected amoebal cultures. The intracellular localization of this new DNA was consistent with previous electron microscope studies of the cytoplasmic maturation of V(301).

The polyhedral amoebal virus V(301) presents sectioned profiles consistent with its having icosahedral (532) symmetry. The DNA appears to be packed in an orderly, folded arrangement of lesser symmetry.

This and a companion report deal with the identification and morphogenesis of viruses in axenized cultures of Entamoeba histolytica. There are probably two different types of virus each producing a different pathological picture in different amoebal strains, or, less likely, there is one type of agent having widely different morphological and morphogenetical pictures in different strains of E. histolytica. Both types of agent produce a lytic response in axenized amoebae and have been serially passaged to an extent assuring their replicating nature. One appears to replicate in the nucleus as multiple clusters of fine filaments which ultimately lyse the nucleus, causing cell death. The second type of agent appears to be a typical polyhedral virus, seen only in the cytoplasm and also resulting in lysis of the cell. A particle morphologically indistinguishable from this second agent is also found in late passages of the agent producing the nuclear pathology.

The intracellular development of two morphologically different amoebal viruses has been studied by electron microscopy. One is a polyhedral agent which was observed as early as 24 hr after infection in the perinuclear cytoplasm. Subsequently, cell lysis occurred and particles were found in large number bound to membranes of disrupted amoebae. Other particles were found in phagocytic vacuoles suggesting a possible portal of entry into amoebae. The other virus is a filamentous particle which is first seen in small clusters in the nucleus after 24 hr of infection. The number of particles increases such that by 72 hr massive whorls of particles occupy a substantial part of the nucleus. After rupture of the nuclear membrane, clusters of filaments are widely dispersed throughout the cytoplasm. Still later, the cytoplasmic membrane disintegrates and clusters of filaments are found extracellularly, but free of cell membranes. The morphology of these agents is discussed in comparison with a variety of plant, animal, and bacterial viruses.

Some biological characteristics and bioassay of a polyhedral virus isolated from normal-appearing strain HB-301 of Entamoeba histolytica are described. The virus produced lysis of susceptible strains of E. histolytica, yet it caused no cytopathological effect in the host strain, HB-301. The virus and host amoeba existed in an equilibrium; approximately 10(4) mean infective dose units of virus were produced per million HB-301 amoebae. Superinfection and antiviral antiserum treatment failed to disturb this equilibrium permanently. The mechanism of viral persistence in strain HB-301 amoebae remains to be determined. Purification of the virus was attempted. Ninety-nine percent of the viral infectivity was associated with a low-speed pellet which consisted of complexes of virus and cellular membranes. Various treatments of this low-speed pellet failed to release virus. Biologically active, membrane-free virus of low titer was prepared by differential centrifugation of supernatant solutions and employed in electron microscopy and other studies.

Treatment with boticin S5(1), a bacteriocin produced by a nontoxigenic organism closely related to Clostridium botulinum type E, caused extensive changes in the structure of a sensitive C. botulinum type E strain. Nucleoid deoxyribonucleic acid, normally seen as fine filaments scattered throughout the cell, was aggregated into dense deoxyribonucleic acid masses. Mesosomes appeared to undergo structural rearrangement from lamellar to vesicular configuration. Eventual dissolution of cell contents left bacterial ghosts composed of seemingly intact cell walls with remnants of the cytoplasmic membrane and internal structures. The morphological changes observed in boticin-treated strain 070 cells were very similar to those produced by a bacteriocin-like substance from phage type 71 Staphylococcus aureus on sensitive beta-hemolytic streptococci. A similarity in the mode of action of the two bactericidal agents is suggested.