- Volume 71, Issue 3, 1990
Volume 71, Issue 3, 1990
- Animal
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Passive immunization protects the mouse eye from damage after herpes simplex virus infection by limiting spread of virus in the nervous system
More LessMice were treated with serum containing antibodies to herpes simplex virus type 1 (HSV-1) or normal serum, 1 day before inoculation on the cornea with HSV-1 strain McKrae. As expected, without passive immunization, mice developed high levels of serum neutralizing antibody. By contrast, in passively immunized animals, such antibody became undetectable by 29 days after inoculation of serum, in spite of the virus infection. There was no difference between passively immunized mice and those given normal serum in the duration of shedding of virus in tears and the duration and severity of corneal epithelial disease. However, non-immunized mice had a high incidence of mortality and developed disease of the iris, corneal stroma and lids, and their corneas became opaque and vascularized. In non-immunized animals, the timing of isolation of virus from nervous tissues and the sequence of appearance of virus antigens in ocular tissues indicate that the disease of deeper eye tissue was caused by virus spreading from the nervous system back to the eye. Restriction of such spread in passively immunized animals seems the likely explanation for their protection from death and severe ocular damage. Despite this restricted spread, passively immunized animals had a high incidence of latent infection in the ophthalmic part of the trigeminal ganglion. However, in comparison with mice given normal serum, there was a far lower incidence of such infection in the other two parts of this ganglion and in the superior cervical ganglion. Since passively immunized animals have a high incidence of latent infection in the ophthalmic part of the trigeminal ganglion and their eyes are normal, they will prove useful in studies involving induction of recurrent disease.
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Expression from cloned DNA of biologically active glycoprotein C of herpes simplex virus type 1 in mammalian cells
More LessA DNA fragment of the herpes simplex virus type 1 genome encoding glycoprotein C (gC-1) has been cloned into different eukaryotic expression vectors for transient and stable expression of the glycoprotein in a number of cell lines. All of these expression vectors use a non-HSV promoter, such as the adenovirus major late promoter or murine leukaemia virus long terminal repeat promoter to express gC-1 in COS and CHO cells or 3T3 cells. The gC-1 protein synthesized was fully glycosylated with both N- and O-linked oligosaccharides. Synthesis of the mature 120K gC-1 glycoprotein involved partially glycosylated 100K and 105K proteins and the non-glycosylated 70K protein as intermediate molecules. Immunofluorescence studies showed that the expressed gC-1 was localized intracellularly in the nuclear envelope as well as on the cell surface. The expressed gC-1 was biologically active and could act as a receptor for the complement component C3b in the absence of other HSV proteins.
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Biochemical and immunological characterization of the major structural proteins of feline immunodeficiency virus
Feline immunodeficiency virus (FIV) structural proteins were identified using sera obtained from experimentally inoculated cats. Proteins analysed by both radioimmunoprecipitation and Western blotting were specific for FIV infection and failed to cross-react with either antisera to feline leukaemia virus of feline syncytium-forming virus. Western blot analysis of purified virus revealed immunoreactive proteins with apparent Mr of 65K, 50K, 40K, 32K, 24K, 15K and 10K. The major core structural proteins of the virus were isolated by reverse phase HPLC and the amino- terminal sequences of plO and p24 were determined.
Monoclonal antibodies specific for p24 suggested the presence of a precursor protein that could be detected in 3S[S]methionine/cysteine-labelled, virus-infected cell extracts. This putative precursor protein possessed an apparent Mr of 50K (Pr50 gag ). Further analysis revealed the presence of two additional proteins of 130K and 40K. Experiments utilizing tunicamycin, endoglycosi- dase H and glycopeptidase F revealed that pi30 and p40 exhibited properties characteristic of glycoproteins. Our studies also indicated that FIV is immunologically related to other lentiviruses.
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Intracellular proteins of feline immunodeficiency virus and their antigenic relationship with equine infectious anaemia virus proteins
Feline immunodeficiency virus (FIV) grown in cat lymphocyte and thymocyte cultures was labelled with l-[35S]methionine or [3H]glucosamine and virus-coded proteins were identified using immunoprecipitation. Polypeptides with apparent M r values of 15K, 24K, 43K, 50K, 120K and 160K were detected. An additional polypeptide of 10K was detected by Western blot analysis. The two highest M r species sometimes appeared as one band, of which only the 120K polypeptide was glycosylated. In the presence of tunicamycin gp120 was no longer detectable and a non-glycosylated precursor of 75K was found instead. Pulse-chase experiments suggested that the smaller polypeptides p24 and p15 are cleavage products of both p160 and p50. Western blot analysis using a rabbit serum directed against p26 of equine infectious anaemia virus (EIAV) and an anti-EIAV horse serum from a field case of infection revealed a cross-reactivity with p24 of FIV. Cat sera collected late after experimental FIV infection recognized p26 of EIAV, indicating a reciprocal cross-reactivity.
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Passive protection studies in mice with monoclonal antibodies directed against the non-structural protein NS3 of dengue 1 virus
More LessAntibody-mediated enhancement of dengue virus replication is thought to be a mechanism contributing to the pathogenesis of dengue haemorrhagic fever and dengue shock syndrome. Enhancement is associated with antibodies to structural components of the virus. To circumvent the problem of immune enhancement, studies to identify protective antigens of dengue virus have involved non-structural proteins. Passive and active protection against lethal dengue virus infection in mice have been demonstrated with the nonstructural protein NS1. In this study, the dengue virus non-structural protein NS3 was examined in passive protection studies with monoclonal antibodies prepared against NS3 of dengue 1 virus (Hawaiian). Five monoclonal antibodies that were authenticated to be reactive to NS3 were used to immunize 13- to 14-day old mice intraperitoneally. Thereafter, the mice were challenged intracerebrally with 100 LD50 of neurotropic dengue 1 virus and the survival indices of the mice were calculated. Significant decreases in survival indices (P< 0·05), indicating increases in survival times were observed with four of five monoclonal antibodies tested. Monoclonal antibodies to NS3 of dengue 1 virus are able to increase the survival time of mice challenged with a lethal dose of dengue 1 virus, although the mechanism remains to be defined.
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- Plant
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In vitro transcription of the double-stranded RNA genome of maize rough dwarf virus (Reoviridae)
More LessAn RNA-dependent RNA polymerase associated with particles of maize rough dwarf virus, a Fijivirus, was characterized using two in vitro assays differing in their energy regeneration systems. Optimum reaction rates occurred at pH 8·0 to 8·5 at 20 °C. The presence of virus and Mn2+ or Mg2+ was essential for enzyme activity; Mn2+ stimulated more incorporation events than Mg2+, at optimum concentrations of 2 to 4 mm and 4 mm, respectively. Incorporation was not affected by a-amanitin, actinomycin D or rifampicin. The products synthesized in vitro were single-stranded RNAs which hybridized specifically with the doublestranded genomic RNAs of the template virus, but not with genomic RNAs of five other reoviruses. The in vitro transcripts were also used to detect maize rough dwarf virus RNA in plants and in vector insects.
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Accumulation of different types of raspberry ringspot nepovirus particle in infected Nicotiana protoplasts
O. Acosta and M. A. MayoNicotiana tabacum protoplasts inoculated with raspberry ringspot virus accumulated top component (T), middle component (M) and bottom component (B) particles progressively from about 20 h post-infection (p.i.) until at least 73 h p.i.; the ratio between the amounts of T and B particles changed little during the multiplication cycle of the virus. In contrast, in raspberry ringspot virus-infected N. clevelandii protoplasts although the accumulation of T particles continued up to at least 91 h p.i., the accumulation of B and M particles decreased greatly or ceased after about 60 h p.i. In either species of protoplast there was no evidence that T particles arose from B or M particles although some physical treatments did cause an apparent loss of nucleic acid from B and M particles.
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Nucleotide sequences of an Australian and a Canadian isolate of potato leafroll luteovirus and their relationships with two European isolates
More LessThe genomes of an Australian and a Canadian isolate of potato leafroll virus have been cloned and sequenced. The sequences of both isolates are similar (about 93%), but the Canadian isolate (PLRV-C) is more closely related (about 98% identity) to a Scottish (PLRV-S) and a Dutch isolate (PLRV-N) than to the Australian isolate (PLRV-A). The 5′-terminal 18 nucleotide residues of PLRV-C, PLRV-A, PLRV-N and beet western yellows virus have 17 residues in common. In contrast, PLRV-S shows no obvious similarity in this region. PLRV-A and PLRV-C genomic sequences have localized regions of marked diversity, in particular a 600 nucleotide residue sequence in the polymerase gene. These data provide a world-wide perspective on the molecular biology of PLRV strains and their comparison with other luteoviruses and related RNA plant viruses suggests that there are two major subgroups in the plant luteoviruses.
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Sequence analysis and product assignment of segment 7 of the rice dwarf virus genome
More LessThe complete nucleotide sequence of segment 7 of the rice dwarf virus (RDV) genome was determined. The segment was 1696 bp long and its plus-strand terminal sequence, 5′ GGCAAA – – – UGAU 3′, was in agreement with the consensus sequence previously found in other segments of RDV. A 10 bp inverted repeat was found adjacent to the termini. A single long open reading frame extended for 1518 bp from the first AUG triplet (positions 26 to 28), and encoded a polypeptide of 506 amino acids (M r 55 339). This protein had 32% identity in the amino acid sequence to the 57K protein encoded by segment 7 of the wound tumour virus genome. The translation product of transcript RNA made from ‘tailored’ cDNA of RDV segment 7 comigrated with the 60K core protein of RDV in 10% polyacrylamide gel and reacted with antiserum against the 60K core protein of RDV. Segment 7 of the RDV genome therefore codes for the 60K core protein.
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Electron microscopical demonstration of different binding sites for monoclonal antibodies on particles of beet necrotic yellow vein virus
More LessBy means of the immunoelectron microscopical decoration test and the immunogold technique three different binding sites for monoclonal antibodies (MAbs) were identified on the surface of particles of beet necrotic yellow vein virus. One group of MAbs reacted with antigenic determinants along the entire length of the particles, whereas a second and a third group of MAbs reacted with determinants on the opposite extremities of the particles.
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The primary structure of the 24K protease from red clover mottle virus: implications for the mode of action of comovirus proteases
More LessWe have determined the nucleotide sequence of the region of red clover mottle virus (RCMV) bottom component RNA which encodes the RCMV equivalent of the cowpea mosaic virus (CPMV) 24K protease. From the alignment of the deduced amino acid sequence of the RCMV 24K protein with that of the homologous protein from CPMV, we speculate on the relative importance of the various amino acid residues which have been implicated in the catalytic mechanism of comovirus proteases.
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