- Volume 69, Issue 6, 1988

Non-cytopathic strains of bovine viral diarrhoea virus (BVDV) readily establish persistent infections in cells of bovine origin. The involvement of endogenous interferon (IFN) on the maintenance of the infection level, as well as the effect of exogenous IFN and tumour necrosis factor alpha (TNF-α), was studied. Although exogenous IFN suppressed the spread and replication of virus, it did not cure the infection, even when continuously present over many cell passages. TNF-α alone had no antiviral effect in this system. However, both TNF-α and IFN enhanced the cytopathic effect of cytopathic BVDV, and induced a BVDV-like cytopathic effect in cells infected with non-cytopathic BVDV. These data are discussed with regard to possible mechanisms of pathogenesis during fatal BVDV disease in cattle.

A cDNA clone derived from the mitochondrial cytochrome oxidase I gene has been used to show that the level of mitochondrially encoded RNA species declines during herpes simplex virus type 2 infection in a manner similar to that for RNA species derived from nuclear genes. In contrast to the situation for nuclear genes, however, no change in the transcription rate of the mitochondrial genome during infection was detected, indicating that post-transcriptional processes alone are responsible for the decline in the levels of mitochondrial RNA species during infection. Two stages in this post-transcriptional degradation have been defined, only one of which is dependent upon viral protein synthesis in the infected cell.

Virus envelope glycoprotein gVI (gp50) of Aujeszky’s disease virus (ADV) was purified from a Nonidet P40-solubilized lysate of ADV-infected Vero cells by immunoaffinity chromatography using a monoclonal antibody against gVI. Mice immunized by the purified gVI produced virus-neutralizing antibody and were successfully protected against subsequent challenge with ADV.

The titre of defective interfering (DI) influenza virus measured by an assay based on the inhibition of cytopathology caused by A/WSN (H1N1) influenza virus in MDCK cells was 320000-fold greater than titres measured by inhibition of infectious centre formation. Interference was less in other types of cell. By electron microscopy, we have shown that the ratio between physical particles and DI units in preparations of the DI virus was approximately unity, which suggested that one or few DI particles is/are required to confer resistance of a MDCK cell to viral cytopathology. This human DI virus interfered heterotypically with an avian H7N1 influenza virus.

Electron microscopic examination of ultrathin sections of a continuous cell line of ovine kidney (OK) origin, infected by bovine respiratory syncytial virus (BRSV), revealed the presence of well defined bridges between virus particles. This is the first report of this novel structure. Observation of ultrathin sections of human RSV Long strain also grown on OK cells did not show inter-particle bridges and therefore suggested that this structure could be specific to BRSV. The biological significance of these bridges is not clear at this time; a possibility is that the bridges are formed by the fusion protein of BRSV which is known to cause cell fusion. Besides the structural implications, the importance is in relation to purification strategies for this virus, which must now take into account that most of the viral particles occur in large aggregates.

The ultrastructure of human immunodeficiency virus type 2 (HIV-2) was determined by negative stain and thin section electron microscopy (EM). Some virus particles had surface projections about 10 nm in length which were evenly spaced. Nonidet P40-treated particles which were penetrated by stain revealed a distinctive off-centre cone-shaped core and, in addition, free-lying cores were also seen in detergent-treated preparations. The surface of the cores was composed of a layer of small subunits. The structure of HIV-2 determined by thin section EM was the same as that deduced by negative stain EM.

Human papillomavirus type 16 (HPV 16) DNA integrated within the cell DNA in cervical carcinomas is frequently defective, but commonly retains its putative transcriptional control region and E6 and E7 open reading frames (ORFs) intact. One clone of such a partially deleted HPV 16 DNA was tested and found to be transforming for rat 3Y1 cells. The result indicates that HPV 16 DNA containing E6 and E7 ORFs with the homologous promoter is sufficient for inducing focal transformation of immortalized rat fibroblasts.

The quantitative contribution of vertical transmission to the prevalence rate of Junin virus infection in subsequent generations of its natural reservoir, Calomys musculinus, was analysed. Data on mortality and reproduction of C. musculinus infected at birth with a wild strain of Junin virus were used to estimate the infection-dependent relative survival rate (β = 0.4849) and relative fertility of the infected host (α = 0.2088). Prevalence rates of infection, obtained by mathematical simulation in optimal conditions of vertical transfer, dropped steadily to zero in a few generations. Vertical transmission was found to be insufficient to overcome the effect of highly depressed survival and fertility of the infected host and maintain a stabilized prevalence of Junin virus infection in successive generations; this suggested that viral maintenance is mainly dependent upon horizontal transmission.

Single-stranded circular DNA associated with foliar decay disease of coconut palm in Vanuatu (FDD-DNA) has been purified and three fragments have been cloned in plasmid pUC19. Clones labelled with 32P by nick translation were used as specific probes for FDD-DNA in dot blot and Southern transfer hybridization assays. These assays were more sensitive than the polyacrylamide gel electrophoresis assay developed previously for diagnosis of FDD. Hybridization tests showed that FDD-DNA had no detectable sequence similarity to the geminivirus infecting Digitaria sanguinalis which grows alongside FDD-infected coconut palms; that Hibiscus tiliaceus, which is the host plant of the vector of FDD, Myndus taffini, is not infected with FDD-DNA; and that the symptomless coconut variety, Vanuatu Tall, is susceptible to infection with FDD-DNA. Native FDD-DNA migrates as two distinct bands in polyacrylamide gels, and both hybridize to the probe. Previous estimates and data presented here show that FDD-DNA is approximately half the size of the D. sanguinalis geminivirus DNA (approx. 2350 nucleotides) and support the view that FDD is caused by a virus not typical of any plant virus taxonomic group.

The infectivity of a DNA copy of RNA3 from strain S of alfalfa mosaic virus inserted in a pT7-1 plasmid vector (clone 3pT7-1) was tested as circular DNA by co-inoculation with RNAs 1, 2 and 4 from strain B (mix I0) on young tobacco leaves. Virus multiplication was monitored by serological detection of the coat protein. Progeny RNA3 derived from circular plasmid DNA 3pT7-1 was of the same length as naturally occurring RNA3-S. Analysis by primer extension demonstrated that it had the 5′-terminal sequence of RNA3-S and not that of RNA3-B. Mix I0 also became infectious when mixed with M13 single-stranded DNA carrying RNA3-S inserted in the (-) strand orientation, probably because traces of RNA3-B present in mix I0 were protected from degradation by base-pairing and were therefore active.

A subgenomic DNA has been isolated from wheat tissue infected with a Swedish isolate of wheat dwarf virus and cloned. Restriction endonuclease analysis and nucleotide sequence determination indicated that the subgenomic DNA (1472 nucleotides) was derived from the genomic DNA (2749 nucleotides) by two separate deletions. The subgenomic DNA had lost open reading frames (ORFs) encoding the virus coat protein and a putative protein of M r 10146, but retained an ORF and an open reading region encoding putative proteins of M r 30156 and 17292, respectively, and two structural features thought to be important for virus DNA replication i.e. a potentially stable stem-loop structure containing the conserved TAATATTAC sequence and a putative primer initiation site.

DNA isolated from purified preparations of wheat dwarf virus (WDV) has been shown to contain tightly bound small DNA molecules which can act as primers for the synthesis of full-length complementary DNA in vitro. The small DNA molecules are bound in the terminating intergenic region of the WDV genome between the end of an open reading frame encoding a putative protein of M r 17292 and a conserved ‘A-T’ box containing putative transcriptional polyadenylation signals. Evidence that the small DNA molecules contain ribonucleotides at their 5′ termini is presented and their possible role in the priming of virus DNA synthesis in vivo is discussed.

An open reading frame, designated AR1, on DNA component A of tomato golden mosaic virus has been identified as the virus coat protein gene on the basis of the molecular weight and amino acid composition of the virus capsid polypeptide, the mass spectra and N-terminal sequences of peptides produced by cyanogen bromide cleavage of the capsid polypeptide and by the binding of antibodies, induced by immunization of a rabbit with a β-galactosidase-AR1 fusion protein, to the capsid polypeptide in a Western blot.

Polyadenylated RNA was isolated from Nicotiana benthamiana plants infected with tomato golden mosaic virus (TGMV). Northern hybridization with a strand-specific probe of cloned TGMV DNA A revealed a 0.9 kb transcript with the same orientation as virion DNA. The positions of the 5′ and 3′ termini of the transcript, which were mapped by nuclease protection and primer extension techniques, indicated that it corresponded to the virus coat protein mRNA and enabled the likely functional promoter and polyadenylation signals to be identified.

The transport protein of tobacco mosaic virus (TMV) (M r 30000, 30K non-structural protein) was detected on Western blots, using an antiserum to a synthetic C-terminal nonapeptide. Accumulation of this protein in subcellular fractions of inoculated leaves was measured during TMV infection of Nicotiana tabacum cv. Samsun and cv. Samsun NN. In cv. Samsun, a systemic host, the 30K protein appeared transiently in a crude membrane fraction but accumulated more stably in cell walls. In cv. Samsun NN, which is a hypersensitive host giving only localized infection, the early accumulation (up to 40 h; before any necrosis was visible) was the same as in cv. Samsun. However, as soon as necrosis was visible, the amount of 30K detected in the cell wall fraction decreased sharply and coat protein synthesis stopped. This drop in the amount of 30K protein is most easily interpreted as a side-effect of the hypersensitive reaction and may explain why TMV infection becomes localized in leaves of cv. Samsun NN.