- Volume 30, Issue 1, 1976

A simian type-C virus has been detected in cultures chronically infected with Mason-Pfizer monkey virus (M-PMV). Simultaneous budding of M-PMV and type-C virus particles from the same cells was observed in cultures incubated at 37 or 40 °C. However, the frequency of such cells was greater in cultures grown at 40 °C. Although clusters of type-C viral buds were seen at the surface of the cells, extracellular mature type-C particles in cell pellets or concentrated virus preparations were very rarely found. The increase in frequency of type-C buds was found to be transitory since cultures adapted to growing at the high temperature demonstrated budding type-C particles only occasionally. Cultures producing type-C buds were found to contain, in addition to M-PMV antigens, serological activity with polyvalent antisera produced against multiple structural components of endogenous baboon virus (BV) or simian sarcoma virus (SiSV). The reactivity, however, was found not to be serologically related to the major SiSV p28 core protein.

Single administrations of poly C or poly I are anti-viral against infections of encephalomyocarditis (EMC) and Semliki Forest virus (SFV) in mice but poly U and poly A are not. The degree of protection is dose-dependent and mice which die do so at a later time than untreated controls even in a strain of mouse in which the time of death is not dependent on the dose of virus given. No circulating interferon is found after treating mice with poly C or poly I even at polynucleotide doses which give the same degree of protection as the interferon inducer, poly I:C. Several additional features distinguish the protection by poly C and poly I from interferon induction: the effect is low 24 h before infection and maximal 6 h before infection, the effect is short-lived and mice do not show hypo-reactivation to repeated treatment. Limited treatment of mice with poly I:C, interferon or poly C before infection itself results in additional protection when poly C is also administered after infection, indicating that poly C has an effect after onset of virus replication. After infection poly C and poly I are both more effective by the intravenous route but before infection they are most effective when administered by the same route as the virus. Quantitative comparisons of the protective effects of poly C, poly I and the interferon inducer, poly I:C, are possible from dose response curves of the polynucleotides at different times relative to infection and by different routes of administration. The results are considered in relation to the presence of homopolyribonucleotide tracts in the viral genomes and effects on the reticulo-endothelial system of the mice.

Cells of five grass and cereal species infected with cocksfoot mottle virus, phleum mottle virus or cocksfoot mild mosaic virus were examined in ultra-thin sections. The intracellular distribution of the virus particles and their effects on cell ultrastructure appeared dependent more on the severity of the host/virus interaction than on the particular virus or host species involved. Particles of all three viruses occurred in the cytoplasm of mesophyll and phloem companion cells. Those of cocksfoot mottle virus were observed occasionally also in the nucleus. Relatively few particles, randomly distributed in the cytoplasm, were present in cells of plants with mild symptoms. In plants with severe symptoms the particles were numerous and often in near- or true-crystalline aggregates. In partially disrupted cells, virus particles occurred occasionally in membrane-bound, sometimes vesiculated, packets in the cell vacuole. These seemed to originate from the tonoplast. In more severely disrupted cells the tonoplast was lost and virus particles were scattered randomly throughout the vacuole.

Ultrastructural effects ranged from an increase in endoplasmic reticulum and vacuolation of the cytoplasm in the cells of plants with mild symptoms, to the total disintegration of all cell organelles in plants with the severest symptoms. Extrusions, which often encircled a volume of cytoplasm developed from the chloroplasts and mitochrondia; membrane-bound vesicles, containing virus particles, appeared in the stroma of chloroplasts and in the mitochondria.

The triggering mechanism for interferon synthesis in mouse peritoneal macrophages and chick embryo (CE) cells by Newcastle disease virus (NDV) exposed to hydroxylamine or homologous antiserum was investigated in relation to the intracellular fate of these agents.

Inactivation of NDV at 22 °C by 1 m-hydroxylamine proceeded with first-order kinetics, whereas the interferon-inducing capacity of hydroxylamine-treated virus in macrophages was unimpaired. In contrast to infective NDV, hydroxylamine-inactivated virus produced interferon in CE cells, and such a virus still had partial RNA-dependent RNA polymerase activity. Hydroxylamine-inactivated NDV was adsorbed to and uncoated in both normal and chloroquine diphosphate treated cells, but no viral double-stranded RNA was detected. Hydroxylamine treatment of virion-extracted RNA and neutralization of intact virions by antibody abolished the capacity of the virus to induce interferon. Infective as well as neutralized NDV interacted with macrophages to the same degree, but association between NDV and CE cells was prevented by antibody-coating. In macrophages, the RNA of neutralized NDV became more sensitive to RNase than RNA of infective NDV, but this process was inhibited in chloroquine diphosphate-treated cells. These results suggest that interferon induction by NDV involves components of the virion which are present up to the regular uncoating process.

Viral polypeptides associated with the membrane-free replication complex of poliovirus RNA were multiple in nature. The structural protein precursors [VP0, VP1, VP3] predominated, and because they were found in a cytoplasmic component with the same S value and density as the replication complex are likely to be attached to it in vivo. They were not present in the form of empty capsids. The electrophoretic polypeptide pattern of the membrane-bound replication complex was similar but showed a predominance of NCVPX or VP1, unless the cells were slightly depleted in amino acids when the non-structural polypeptide NCVP2 became important. Cystine was the only amino acid capable of reversing this depletion effect on its own.

The morphology and physico-chemical properties of a temperate phage, B33, of Pseudomonas aeruginosa have been determined. This phage is similar in size and structure to the previously described and serologically related phage B3 (Holloway, Egan & Monk, 1960), but differs from it in its plating properties on bacteria harbouring R plasmids. The plasmid RP1-1 causes a reduction in e.o.p. of B33 of 10−6, and the mechanism whereby this occurs has been studied. The interaction is a specific one since other plasmids either fail to affect plating or completely abolish it. The mechanism by which the latter occurs is different from that mediated by RP1-1, since mutants of B33 insensitive to RP1-1 nevertheless fail to plaque on these hosts.

Canaline, a pyridoxal phosphate antagonist, is shown to inhibit two distinct events in the replication of vaccinia virus in HeLa cells. Initial events proceed in the presence of the inhibitor leading to the formation of DNA-containing, cytoplasmic inclusions. However, further DNA synthesis is required for the subsequent production of infectious progeny following the reversal of canaline inhibition by pyridoxal phosphate. Inhibition of a separate, maturation event is shown by the delayed addition of canaline resulting in the failure to coat virus-specific DNA synthesized previously in the absence of the inhibitor. Thus, the replication of vaccinia virus is sensitive to inhibition by canaline at an early and a late stage in the replication cycle. Reversal is accomplished alternatively by the addition of ‘nonessential’ amino acids suggesting that the effects of canaline result from inhibition of specific protein functions.

Wild isolates of Mycobacterium tuberculosis may be divided into the three internationally recognized phage types on the basis of susceptibility to myco-bacteriophages DS6A, BK1 and D34. Strains of type A are lysed at high efficiency by DS6A only; type B is lysed by BK1 grown on Mycobacterium smegmatis ATCC607 and DS6A, while type C is lysed additionally by D34 grown on atypical Mycobacterium F130. Propagation of D34 on a C-strain (D34·C) or BK1 on a B-strain (BK1·B) has no effect on viral host-range. D34·C has an efficiency of plating (e.o.p.) of 10−5 on type B strains and 10−7 on A strains. BK1·B plates on A strains at an e.o.p. of 10−5. BK1 recovered from and repropagated on an A strain (BK1·A) has an e.o.p. of 1.0 on strains of all classes. D34·B has an e.o.p. of 1.0 on strains of type B and C, while D34·A plates with high efficiency on types B and C and displayed an e.o.p. of 10−4 on type A. Repropagation of these viruses on the M. tuberculosis strains originally lysed by them results in the restoration of their previous host range. Variations in plating efficiency cannot be explained by differences in viral absorption alone. These findings suggest that the three phage types of human tubercle bacilli are related by a hierarchical pattern of DNA restriction and modification in which the C pattern is included in the B, and both patterns are included in A-modified DNA. Viruses such as DS6A which are equally virulent for strains of all classes are not susceptible to host dependent restriction.

Bacteriophage MX-1 is a virulent DNA phage for Myxococcus. The host range includes strains of Myxococcus xanthus, M. fulvus and M. virescens. The phage has a sedimentation coefficient (s o 20,w) of 1145S and a density of 1.531 g/ml. By using SDS-polyacrylamide gel electrophoresis, 23 phage proteins with apparent mol. wt. between 10000 and 150000 were resolved. Gel filtration in the presence of nonionic detergent partially resolved the proteins. The fraction excluded from Sephadex G-100, fraction 1, contains two glycoproteins. Fraction 1 was resolved into three fractions (1.1, 1.2 and 1.3) by chromatography on Sephadex G-200. The glycoproteins were present in fraction 1.2; all the proteins from this fraction were derived from the phage tail. Comparison of the amino-acid, hexosamine and neutral-sugar compositions of the two glycoproteins showed that they are distinct molecular species; the smaller molecule is not a subunit of the larger. The significance of these findings is discussed and compared with the proteins of the tails of T-even phage of Escherichia coli.

Depending on passage history, strain WE infectious LCM virus either damages L cells more or less severely or leaves them morphologically intact. Correspondingly, the plaques which are formed on L cell monolayers are of different appearance, ranging from intensely turbid to clear. Multiplication of LCM virus in certain mouse organs profoundly affects plaque characteristics. The brain, for instance, favours lytic variants while the spleen supports the replication of virus which forms turbid plaques. This statement holds if virus taken from organs of persistently infected mice or virus passaged from mouse to mouse is analysed and is true also if the initial preparation contains virus forming predominantly either clear or turbid plaques on L cell monolayers. Selection is not rapid and not absolute. It may take months of multiplication before a final state is reached, and even then the number of characteristic plaques is usually in great excess of the rest but never reaches 100%. Cloning procedures may alter the proportions, but with our experimental conditions no plaque has ever been isolated which would retain its characteristics upon passage. Differences of plaque type morphology were not reflected in differences of pathogenic properties, and both clear and turbid variants caused persistent infection if used to infect newborn mice and led to disease with signs of neurological involvement and death if inoculated intracerebrally into adult animals.

Twenty African arboviruses, five alphaviruses, nine flaviviruses, three Bunyamwera Group viruses, two Bwamba Group viruses and one ungrouped virus were titrated in parallel in 11 cell systems, in suckling mice and adult mice. The relative sensitivities of the in vitro and in vivo systems have been compared.

The highest infectivities were obtained in suckling mice. Vero and LLC-MK2 cells produced plaques with the greatest number of viruses and Semliki Forest virus grew most readily. Ntaya virus and Dengue 1 virus were difficult to culture in vitro and Zika virus yielded better in cell culture than in adult or suckling mice.

In vitro and in vivo neutralization tests were made on human sera in groups of 50. Each group of sera was tested against one of five viruses, representatives of three of the arbovirus groups titrated. Good agreement was obtained between the two test systems with West Nile, O’nyong-nyong and Wesselsbron viruses but there were significant differences in results obtained with Germiston and Pongola viruses.

The cytotoxic effect of poly(rI).poly(rC) for interferon-treated cells is preceded by discrete ultrastructural alterations in (1) the mitochondria: swelling of the cristae and increased density of their matrix, (2) the nucleus: disorganization of the structure, and (3) the endoplasmic reticulum: slight dilatation with disorganization and depletion of the filament bundles.

Our results do not allow us to conclude whether any of these alterations is a primary effect of the treatment. Possibly they are secondary to some, as yet unknown, functional defect, and only reflect incipient cell death.

With the exception of phage Qβ, the RNAs of all the other small icosahedral RNA viruses so far examined contain a poly (A) tract. This tract has been implicated in the infectivity of poliovirus RNA. We have now shown that Nodamura virus, a divided genome virus from which infective RNA can be extracted, does not contain any poly (A) tracts. This evidence with Nodamura virus shows that poly (A) is not a necessary requirement for the infectivity of virus RNA molecules.

Adsorption of bacteriophage T4 ghosts on to Escherichia coli cells has been known to cause a dramatic change in the cellular metabolism, the effect being similar to that of colicin K. It is shown here that the inhibitory activity of ghosts is not expressed either at 0 °C in the ordinary media (like colicin K) or even at 30 °C in a medium containing EDTA, in contrast to colicin K.

Since the process of sheath contraction of T4 phage is blocked by EDTA, it is suggested that the adsorbed ghosts may not exert their inhibitory activity until sheath extraction occurs.

The DNA polymerase activity in BHK 21/C13 cells infected with pseudorabies virus is inhibited by incubation with antiserum to pseudorabies but not by incubation with pre-immune serum or by antiserum to herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2). It also differs from the cell enzyme and the enzymes in HSV-1 or HSV-2 infected cells in its requirement for KCl in the in vitro assay. It seems likely, therefore, that pseudorabies virus specifies its own DNA polymerase.

The effect of virion age on the two A and B 8S RNA conformational isomers was investigated by electrophoresis on polyacrylamide gels. The relative proportions of A and B 8S RNA components of the immature virus (rapid harvest, 30 min) are intermediate between those previously found for the mature virus and for the host cell. These results suggest that a change of 8S RNA conformation occurs during virus maturation.

Four temperature sensitive mutants of adenovirus type 12, classified into complementation group D, synthesize most or all of the late virus-specific polypeptides at the restrictive temperature, as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Infections by these mutants under restrictive conditions apparently result in some over production of the virion hexon polypeptide compared to wild type infection. Genetic recombination between the four mutants has been demonstrated. A partial linear genetic map of the D cistron is presented.