- Volume 68, Issue 2, 1987
Volume 68, Issue 2, 1987
- Animal
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Proteins Antigenically Related to Peptides Encoded by the Mouse Mammary Tumour Virus Long Terminal Repeat Sequence Are Associated with Intracytoplasmic A Particles
More LessSUMMARYIntracytoplasmic A particles (CAP), previously identified as cytoplasmic nucleo-capsid precursors to mouse mammary tumour virus (MMTV), reacted strongly in immunodiffusion tests with polyclonal antibodies raised against synthetic oligopeptides derived from the open reading frame (ORF) in the long terminal repeat (LTR) of MMTV. In Western blots, several CAP proteins (p80, p72–68, p36, p32, p 18–12) were reactive with polyclonal antibodies raised against three separate LTR ORF synthetic peptides. Disrupted MMTV virions did not react with the anti-LTR ORF peptides suggesting that ORF proteins were excluded from mature virions during maturation. Serial dilution of anti-LTR ORF antibody demonstrated that the most reactive CAP proteins in Western blots migrated as a doublet band with estimated molecular weights of 68000 and 72000. Reactivity of anti-LTR ORF serum with these and other CAP proteins was removed upon preincubation with free synthetic peptide. Absorption with LTR synthetic peptides did not affect the reactivity of antibodies directed against MMTV gag proteins with similarly sized CAP polyproteins. LTR ORF-related proteins with molecular weights similar to those associated with CAP were also detectable in Western blots of total cytoplasmic extracts of MMTV-infected mammary tumour cells.
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Differences in the pI Heterogeneity of Virion and Intracellular Moloney Murine Leukaemia Virus p30s
More LessSUMMARYAt least three different p30 forms which vary in isoelectric point (pI) were previously shown by two-dimensional (2D) gel electrophoresis to be present in purified virions obtained from several strains of murine leukaemia virus (MLV). This heterogeneity which had been identified by Coomassie Brilliant Blue staining has been further characterized by immunological techniques. Using as substrates two Moloney (M) MLV chronically infected cell lines (M JD-54 and clone 2 cells), we found that (i) all p30s had the antigenicity of M-MLV p30, when analysed by immunoblotting of virion proteins with anti-p30 sera, and (ii) when cells were labelled with p5S]methionine, a 14C-amino acid mixture, or [l4C]serine and lysates of purified virions were immunoprecipitated with goat anti-p30 sera, four p30 spots (pI 6·0, 6·1, 6·3 and 6·6) could be clearly identified. These results strongly support the viral origin of the heterogeneous p30 spots. We next examined infected cell lysates in an attempt to pinpoint the molecular basis of this heterogeneity. When we immunoprecipitated p30s from labelled cell lysates utilizing goat anti-p30 sera it was observed that (i) in contrast to the four virion p30s, there were only three intracellular p30s (pI 6·0, 6·3 and 6·6), (ii) there was a threefold greater amount of the intracellular compared to the virion form of p30 with pI 6·0, (iii) tryptic peptide maps of both virion and intracellular p30s labelled either with [35S]methionine or 125I showed basically similar patterns with only slight differences in intensity among certain peptides for the p30s with pI 6·1, 6·3 and 6·6, and (iv) the intracellular p30 with pI 6·0 had a peptide that was not present in any of the other p30s. These results suggest that due to some as yet uncharacterized modification(s) of p30 and/or some structural differences between different p30s, a heterogeneity in pi exists. This may be important for assembly of the virion capsid. However, it is also possible that the p30 heterogeneity reflects the presence of multiple M-MLV proviruses within each of the infected cell clones.
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Expression of a Provirus of Human T Cell leukaemia Virus Type I by DNA Transfection
SUMMARYWe isolated the full length provirus of human T cell leukaemia virus type I (HTLV-I) from MT-2, a lymphoid cell line producing HTLV-I. In three non-lymphoid cell lines (COS7, human osteosarcoma HOS cells, and HeLa) this provirus expressed a transacting activity after co-transfection with a recombinant plasmid carrying a bacterial chloramphenicol acetyltransferase gene under the control of a long terminal repeat of HTLV-I provirus. The trans-acting protein p40 was detected by immunoprecipitation in transfected HOS cells. Structural proteins of HTLV-I, the gag and env products, were also formed and processed in the same manner as observed in MT-2 cells. In transfected HeLa cells, the p40 protein was mainly localized in the nucleus, while other structural proteins were detected in the cytoplasm and/or the membrane by indirect immunofluorescence. Syncytium formation was observed in HeLa cells after transfection. These results demonstrated that non-lymphoid cells could produce the major proteins of HTLV-I after DNA transfection of the cloned provirus.
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Suppression of in vitro Neutrophil Function by Feline Leukaemia Virus (FeLV) and Purified FeLV-p15E
More LessSUMMARYFeline neutrophils (PMN) were isolated and exposed to ultraviolet light-inactivated feline leukaemia virus (UV-FeLV) and purified envelope component pl5E (FeLV- pl5E). Functional capacity of exposed PMN was measured in vitro utilizing the chemiluminescence (CL) response. PMN exposed to UV-FeLV demonstrated depressed CL responses to Ca2+-ionophore A23187 and latex particles. However, FeLV-pl5E produced significant suppression in the CL response to A23187 but failed to produce significant alterations in response to latex particles. The data indicate that FeLV-pl5E may, in part, be responsible for increased morbidity and mortality among FeLV-infected cats through suppression of the PMN population.
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Synthetic Vaccines against Friend Murine Leukaemia Virus-induced Erythroleukaemia: in vivo and in vitro Studies with Synthetic Oligopeptides and Sequence-specific Antisera
More LessSUMMARYBiological activities of antisera against synthetic oligopeptides were examined. The peptide antisera were directed against amino acids 6 to 12 (pep1), 124 to 131 (pep2), 256 to 262 (pep3), 283 to 290 (pep4) and 434 to 441 (pep5) of the viral envelope glycoprotein (gp70). Peptide-specific antisera did not neutralize viral infectivity. However, antibodies to pep4 and pep5, which bound to the hydrophobic part of gp70, mediated the complement-dependent lysis of Friend murine leukaemia virus (FLV)-infected cells. STU mice were immunized against FLV-induced erythroleukaemia with synthetic oligopeptides. Vaccines containing only one of these peptides (single-peptide vaccines) as well as multi-peptide vaccines containing pep1, −4, −5 or pep1, −2, −3, −4, −5 were used. Whereas the immunizations with single-peptide vaccines did not protect the immunized mice from FLV-induced erythroleukaemia, multi-peptide vaccines enhanced the survival rate and incubation period after FLV challenge. These results revealed that immunological reactions distinct from neutralizing antibodies can be evoked by immunization with synthetic peptides and can confer limited protection against FLV-induced erythroleukaemia.
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Construction and Characterization of Deletion Mutants of Pseudorabies Virus: a New Generation of ‘Live’ Vaccines
More LessSUMMARYVarious deletions were introduced into a cloned subgenomic fragment (BamHI-1), located in the unique short (Us) region of the DNA from the virulent Northern Ireland Aujeszky-3 (NIA-3) strain of pseudorabies virus (PRV). In the cloned Hin dIII-B fragment, the MluI -BglII fragment was replaced by different MluI-BglII fragments of the deleted BamHI-7clones. Transfection of the deleted HindIII-B fragments together with the HindIII-A fragment of either the NIA-3 or the non-virulent NIA-4 strain yielded replication-competent deletion mutants. The region in Us in which sequences were deleted specified several mRNAs. Some of the mRNAs present in cells infected with NIA-3 were absent from cells infected with the deletion mutants, whereas other differently sized mRNAs were generated. The mutants were examined with respect to their biological properties in cell culture, mice and pigs. The results showed that (i) the type of cytopathic effect induced in cell culture seemed to be determined by the ULregion, (ii) using the mean time to death in mice as a parameter, markers for virulence were present in the Us and UL regions and (ii) the introduction of deletions in Usstrongly reduced the virulence of PRV for pigs. Despite the impaired capacity of the deletion mutants to induce high titres of neutralizing antibodies in the serum, inoculation with mutants derived from NIA-3 prevented clinical disease in pigs upon challenge with the virulent parent strain. These deletion mutants provide a good basis for the production of bioengineered live PRV vaccines.
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Epitope Mapping of the Major Epstein-Barr Virus Outer Envelope Glycoprotein gp350/220
More LessSUMMARYTo understand the complete immunochemical structure of the Epstein-Barr virus (EBV) major membrane glycoprotein gp350/220, monoclonal antibodies (MAbs) reacting with this important viral antigen were isolated. Through competitive inhibition binding studies, it was determined that a group of 18 IgG MAbs recognized seven distinct epitopes on the gp350/220 molecule. Eight of these MAbs fell into a single epitope group with four of those MAbs, as well as a single MAb from another epitope group, being capable of neutralizing EBV strain B95–8 transformation of umbilical cord lymphocytes.
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Demonstration and Mapping of Highly Carbohydrate-dependent Epitopes in the Herpes Simplex Virus Type 1-Specified Glycoprotein C
SUMMARYThe carbohydrate dependence of epitopes in the herpes simplex virus type 1-specified glycoprotein C (gC) was studied using a new solid-phase assay procedure. Glycoprotein C, coated on 96-well microtitre plates, was treated with sialidase and increasing concentrations of periodate. A sequential removal of peripheral monosaccharides from the oligosaccharides of gC was ascertained by an enzyme-linked lectin assay. By using a panel of gC-specific monoclonal antibodies in ELISA, it was found that gC contained two types of epitopes differing in their dependence on terminal galactose and sialic acid for expression. Control experiments indicated that the carbohydrate-dependent epitopes were peptide structures and that the carbohydrates did not directly participate in the antibody-binding reaction. The carbohydrate-dependent epitopes were mapped to antigenic site II, according to the proposed nomenclature, whereas those expressed also in the absence of peripheral sugars were located mainly in antigenic site I. These results were compatible with the relative distribution of oligosaccharides in the gC molecule.
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Restricted Replication of Herpes Simplex Virus Type 1 in Murine Embryonal Carcinoma Cells
More LessSUMMARYHerpes simplex virus type 1 (HSV-1) has a broad host range but the KOS strain of HSV-1 did not replicate efficiently in murine embryonal carcinoma (EC) cells. The yield of infectious HSV-1 from EC cells was 100- to 1000-fold lower than that from fibroblast cell lines of mouse, monkey or human origin. The thymidine kinase (TK) gene of HSV-1 is expressed early during the infectious cycle. The levels of TK mRNA and of TK activity in infected EC cells were only two- to threefold lower than levels from infected fibroblast cells. Infected EC cells supported replication of about half as much HSV-1 DNA as did fibroblast cells. The reduced yield of infectious virus was consistent with a paucity of virions in infected EC cells examined by electron microscopy, suggesting a major block late during the HSV-1 infectious cycle. We isolated a variant strain of HSV-1, called KOSEC, which replicated as efficiently in EC cells as in mouse fibroblasts. KOSEC infected EC and fibroblast cells, synthesized more TK mRNA, more TK enzyme, and more HSV-1 DNA than did the same cells infected with the KOS stain. Both HSV-1 strains induced similar levels of synthesis of gD, an early viral glycoprotein. By co-infection of EC cells with the KOS and KOSEC virus, both the elevated virus yield and the elevated TK synthesis seen in KOSEC-infected cells appeared to be recessive. Apparently a viral mutation that affects expression of some early viral functions can also overcome the EC cell restriction to HSV-1 replication.
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Macaque Monkey Type D Retrovirus Replicates in vitro in a Distinct Subpopulation of B Lymphocytes
More LessSUMMARYType D retroviruses have recently been shown to induce a wasting syndrome with associated lymphadenopathy, thymic atrophy and transient decreased peripheral blood lymphocyte blastogenic responsiveness in juvenile macaque monkeys. The replication in vitro of D/New England virus was assessed in various lymphocyte subpopulations to determine the possible pathogenesis of the immune dysfunction induced by this virus. While D/New England did not replicate in cultured T lymphocytes or induce any demonstrable dysfunction of T cells in vitro, it did grow in the cells of the B lymphocyte lineage. D/New England growth occurred in vitro in African Burkitt’s lymphoma and pre-B cell lines, but not in Epstein-Barr virus-transformed normal B lymphocytes. The infection of a restricted B lymphocyte population by this primate type D retrovirus may play a role in the aetiology of the immune abnormalities which it induces.
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Ultrastructural Studies of Kunjin Virus-infected Aedes albopictus Cells
More LessSUMMARYUltrastructural changes in Aedes albopictus cells infected with Kunjin virus were characterized from 12 to 72 h post-infection. Early in infection (16 h), there were no prominent ultrastructural changes except for an increase in the number of vacuoles in the cytoplasm. As the infection progressed the rough endoplasmic reticulum appeared to lengthen and whorls of fibres were observed within some vacuoles. Virus particles were observed in small numbers scattered in the cytoplasm between 24 to 30 h after infection. However, by 48 h, large numbers of morphologically mature virus particles were visible, usually in association with the rough endoplasmic reticulum, with the clusters of vesicles or with convoluted smooth membranes. Although no definite evidence of precursor particles or of budding of virions was obtained, the clusters of vesicles and the various membranous structures appeared to play a role in the morphogenesis of this virus. The results are compatible with the hypothesis that maturation of virus particles occurs within all these virus-induced structures.
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Integration and Transcription of Human Papillomavirus Type 16 and 18 Sequences in Cell Lines Derived from Cervical Carcinomas
More LessSUMMARYFive cell lines, SKG-I, SKG-II, SKG-IIIb, QG-U and QG-H derived from cervical carcinomas of Japanese patients, were examined for the presence of human papillomavirus (HPV) DNA and the expression of viral mRNA. The DNA of HPV type 16 was shown to be linked covalently with SKG-IIIb, QG-U and QG-H cell DNA, and HPV 18 DNA with SKG-I and SKG-II cell DNA. Although different regions of the HPV genome were integrated in these cell lines, the non-coding region and an early region including the E6 and E7 open reading frames (ORFs) were conserved in all cell lines. The complete genome of HPV 16 was found in QG-H cells by digestion of the DNA with a single-cut restriction enzyme. The other early region ORFs E1, E2, E4 and E5 were interrupted by flanking host cell DNA, suggesting that the integration into host cell DNA occurs preferentially in this region. HPV-specific mRNA species were detected in all five cell lines. In the three cell lines containing the HPV 16 genome, mRNAs hybridized with the early region of the genome, covering the entire E6 and E7 ORFs and a minor part of the E1 ORF, although the amount and size of the major mRNAs varied in these cell lines. These mRNAs did not hybridize with the late region of the HPV genome containing the L1 and L2 ORFs. In SKG-II, SKG-IIIb and QG-H cells we also detected c-myc and c-Ha-ras mRNA expression at about nine times the level of that in normal cells.
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Role of Prostaglandins and Non-steroid Anti-inflammatory Drugs in the Pathogenicity of Vaccinia Virus
More LessSUMMARYThe effect of prostaglandins (PGs) of the A series (A1 and dimethyl PGA2), E1, D2, F2α and PGI2 (prostacyclin) and of inhibitors of PG synthesis (aspirin and indomethacin) on the pathogenicity of vaccinia virus was studied in BALB/c mice. PGs of the A series, D2 and F2α conferred little or no protection to mice against the lethal effects of vaccinia virus. Mice treated with PGE1 showed a dramatic increase in mortality after viral infection. However, when mice were treated with PGI2, their survival was greatly enhanced. Mice treated with aspirin and indomethacin showed a marked increase in mortality. Increased mortality correlated with higher virus yields in target tissues (spleen) and with inhibition of antibody response, whereas the increase in survival correlated with lower virus yields and with normal antibody responses. The significance of our findings is that PGI2 can block the outcome of the disease caused by vaccinia virus whereas other PGs and their inhibitors not only worsen the disease, but may activate and enhance viral infections through immune suppression.
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- Plant
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Small Circular Single-stranded DNA Associated with Foliar Decay Disease of Coconut Palm in Vanuatu
More LessSUMMARYThe single-stranded DNA previously reported to be associated with foliar decay disease of coconut palm appears to be predominantly circular, on the basis of its behaviour in two-dimensional polyacrylamide gel electrophoresis, electron microscopy, and its resistance to end-labelling following treatment with alkaline phosphatase and polynucleotide kinase. The DNA sedimented at between 12S and 15S and had a contour length for the circular molecules consistent with the predominant DNA having a molecular weight of about 0·43 × 106, and comprising approximately 1300 nucleotides. These properties differ from the genomic DNAs of known plant viruses.
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Wheat Streak Mosaic Virus Cylindrical Inclusion Body Protein
More LessSUMMARYA protein of apparent molecular weight 66000 was purified from wheat plants infected with wheat streak mosaic virus. Antiserum to this protein, labelled with gold, specifically stained cylindrical inclusions in ultrathin sections of virus-infected cells. Antiserum to the M r 66000 protein did not react with capsid protein in Western blots, nor did antiserum to capsid protein react with the M r 66000 protein. Both antisera reacted with homologous antigens. The concentration of the M r 66000 protein in extracts from infected leaves was about 100 μg per g of leaves, which is higher than the usual concentration of virions.
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Replication of Red Clover Necrotic Mosaic Virus RNA in Cowpea Protoplasts: RNA 1 Replicates Independently of RNA 2
More LessSUMMARYInoculation of cowpea mesophyll protoplasts with unseparated RNA 1 and RNA 2 from red clover necrotic mosaic virus in the presence of polyethylene glycol resulted in virus RNA replication, the synthesis of virus capsid polypeptide and the formation of virus particles; 75 to 85% of the viable protoplasts became infected and the yield of virus particles or virus RNA after 72 h incubation corresponded to about 3 × 106 virus genomes per infected protoplast. In contrast, no replication could be detected when protoplasts were inoculated with RNA 2 alone. However, inoculation of protoplasts with RNA 1 alone resulted in its replication and the formation of virus particles, with a yield similar to that obtained after inoculation with both RNAs. Since infection of plants requires both RNA 1 and RNA 2 to be present, the demonstration of the independent replication of RNA 1 in single cells strengthens the hypothesis that RNA 2 plays a role in the cell-to-cell transmission of the virus.
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Nucleotide Sequence of the Capsid and Nuclear Inclusion Protein Genes from the Johnson Grass Strain of Sugarcane Mosaic Virus RNA
More LessSUMMARYThe nucleotide sequence of the 3′ terminal 1782 nucleotides of sugarcane mosaic virus (SCMV) genome has been determined. There is an open reading frame, from the 5′ end, of 1307 nucleotides upstream from a 475 nucleotide 3′ non-coding region that is polyadenylated. The open reading frame encodes a polypeptide of 435 amino acids.The segment of the genome encoding the viral capsid protein (mol. wt. 34200) is adjacent to the 3′ non-coding region. The predicted capsid protein is similar in sequence to the capsid protein sequence predicted for tobacco etch virus (TEV). Part of another protein encoded in the same reading frame, similar to the predicted nuclear inclusion protein from TEV, has been identified upstream from the coat protein gene.The results indicate that the genome of SCMV encodes one or more large proteins that are processed to the mature proteins.
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