Latent Period in Leukaemia Induction by the Radiation Leukaemia Virus

IN C57BL/6 mice, infection with the radiation leukaemia virus is age dependent and leukaemogenesis is highly effective only when the virus is inoculated shortly after birth^1,2, but several months elapse before the disease is actually manifested. Why is there such a long latent period before the leukaemic cells proliferate into a palpable tumour, especially as in the newborn mouse the target organ for the development of the disease, the thymus, is still in a proliferative stage, with abundance of immature lymphoid cells and an incompetent immunological system?     

Affinity recovery of Moloney Murine Leukaemia Virus

Lipid enveloped retroviruses such as Moloney Murine Leukaemia Virus (MoMuLV) are commonly used gene therapy vectors. Downstream processing protocols used for their purification are time consuming and a potentially generic, single step capture method for the recovery of retroviral particles is proposed that exploits streptavidin-biotin affinity chromatography. The ability of four conventional adsorbent solid phases, Fractogel, Sepharose, Magnespheres and STREAMLINE immobilised with streptavidin, to capture and recover biotinylated Moloney Murine Leukaemia Virus was studied. MoMuLV can be biotinylated whilst retaining infectivity and the biotinylated virus can be adsorbed to Streptavidin Magnespheres yielding a 2298-fold increase in titre. For optimal virus biotinylation purification using Fractogel streptavidin can yield a 1896-fold increase in cfu/mg of protein and a 1191-fold decrease in DNA/cfu. Infectious virus can be recovered from Fractogel streptavidin with a maximum recovery of 16.7%.     

Inhibition of Leukaemia Virus Replication by Polyadenylic Acid

A PREVIOUS communication from this laboratory demonstrated that the DNA polymerase of the Rauscher leukaemia virus is strongly inhibited in vitro by unprimed, single stranded polyribonucleotides¹ as a result of competition between the polymers and the active template for the same enzyme binding site. This inhibition was apparently specific, since partially purified preparations of DNA polymerase from Escherichia coli and BALB/c mouse embryos were not inhibited in the same conditions. We attempted to determine therefore whether single stranded polyribonucleotides would have any effect on the activities of oncogenic RNA viruses in cultured cells.     

Separation of Murine Cellular and Murine Leukaemia Virus DNA Polymerases

The DNA polymerase of murine leukaemia virus has been purified and can be separated physically from enzymes with some similar properties that are present in normal cells. Chromatographic and immunological methods have made it possible to identify the viral enzyme in virus-transformed cells.     

Protein Kinase and Phosphate Acceptor Proteins in Rauscher Murine Leukaemia Virus

Rauscher murine leukaemia virus contains both a protein kinase and substrate proteins. The proteins phosphorylated seem to include the major structural components of the virus. The enzyme and substrate proteins are also found in other membrane-maturing viruses, including vesicular stomatitis virus.     

Cell Line derived from Balb/3T3 that is Transformed by Murine Leukaemia Virus : a Focus Assay for Leukaemia Virus

Balb/3T3 derived cell line UC1-B transforms on MuLV infection, forming foci of transformants which can be used as an assay for MuLV. MuLV virus type C is produced as well as bizarre forms of uncertain origin.     

Elucidating and Minimizing the Loss by Recombinant Vaccinia Virus of Human Immunodeficiency Virus Gene Expression Resulting from Spontaneous Mutations and Positive Selection

While characterizing modified vaccinia virus recombinants (rMVAs) containing human immunodeficiency virus env and gag-pol genes, we detected nonexpressing mutants by immunostaining individual plaques. In many cases, the numbers of mutants increased during successive passages, indicating strong selection pressure. This phenomenon provided an opportunity to investigate the formation of spontaneous mutations in vaccinia virus, which encodes its own cytoplasmic replication system, and a challenge to reduce the occurrence of mutations for vaccine production. Analysis of virus from individual plaques indicated that loss of expression was due to frameshift mutations, mostly by addition or deletion of a single nucleotide in runs of four to six Gs or Cs, and large deletions that included MVA DNA flanking the recombinant gene. Interruption of the runs of Gs and Cs by silent codon alterations and moving the recombinant gene to a site between essential, highly conserved MVA genes eliminated or reduced frameshifts and viable deletion mutants, respectively. The rapidity at which nonexpressing mutants accumulated depended on the individual env and gag-pol genes and their suppressive effects on virus replication. Both the extracellular and transmembrane domains contributed to the selection of nonexpressing Env mutants. Stability of an unstable Env was improved by swapping external or transmembrane domains with a more stable Env. Most dramatically, removal of the transmembrane and cytoplasmic domains stabilized even the most highly unstable Env. Understanding the causes of instability and taking preemptive actions will facilitate the development of rMVA and other poxviruses as human and veterinary recombinant vaccines.Vaccinia virus (VACV), the first recombinant virus shown to induce a protective immune response against an unrelated pathogen (21, 22), is being employed as a vector for veterinary and wildlife vaccines (19). Development of recombinant VACV for human use, however, has been impeded by safety concerns. For this reason, there is interest in modified VACV Ankara (MVA), a highly attenuated smallpox vaccine with an exemplary safety profile even in immunodeficient animals (17, 26, 27). MVA is severely host range restricted and propagates poorly or not at all in most mammalian cells because of a block in virion assembly (29). Initial experiments with recombinant MVA (rMVA) demonstrated its ability to robustly express foreign proteins (29) and induce protective humoral and cell-mediated immunity (30). Currently, rMVA candidate vaccines expressing genes from a wide variety of pathogens are undergoing animal and human testing (13).While developing candidate human immunodeficiency virus (HIV) and other vaccines, we encountered a tendency for mutant rMVA that had lost the ability to express foreign proteins to arise after tissue culture passage (28, 34, 37). This instability may initially go undetected, however, unless individual plaques are isolated and analyzed. Nevertheless, once established in the population, the nonexpressors can rapidly overgrow the original rMVA. These considerations are particularly important for production of large vaccine seed stocks of rMVA. The instability of cloned genes in MVA is surprising, since MVA had already undergone genetic changes during its adaptation through hundreds of passages in chicken embryo fibroblasts (CEFs) and is now quite stable. Indeed, identical 167,000-bp genome sequences have been reported for three independent plaque isolates, accession numbers {"type":"entrez-nucleotide","attrs":{"text":"U94848","term_id":"2772662","term_text":"U94848"}}U94848, {"type":"entrez-nucleotide","attrs":{"text":"AY603355","term_id":"47088326","term_text":"AY603355"}}AY603355, and {"type":"entrez-nucleotide","attrs":{"text":"DQ983236","term_id":"115607417","term_text":"DQ983236"}}DQ983236, and by Antoine et al. (1). Although the cause of the instability of the gene inserts had not been previously investigated, harmful effects of the recombinant protein seem to play a role in the selective advantage of nonexpressing mutants. Thus, reducing the expression level of parainfluenza virus and measles virus transmembrane proteins and deleting part of the cytoplasmic tail of HIV Env improves the stability of rMVAs (28, 34, 37). Reducing expression, however, can also decrease immunogenicity and therefore may be undesirable (36).In view of the importance of understanding and overcoming this pernicious instability problem, we carried out a systematic study of HIV env and gag-pol genes that were unstable in rMVA. We also considered that the analysis would provide basic information regarding the kinds of errors that can occur during replication of the VACV genome, which encodes its own cytoplasmic replication system (20). The most common mutations, which led to loss of recombinant gene expression, were large deletions that extended deep into the MVA flanks and frameshift mutations within consecutive identical nucleotides in the insert. The frequency of viable mutations was minimized by introducing the recombinant gene between two essential, highly conserved MVA genes and by making silent codon alterations to interrupt the homonucleotide runs. In addition, we constructed a panel of recombinant viruses with chimeric and truncated env genes to determine the basis for the selection of nonexpressing mutants and to prevent their expansion during virus propagation. Understanding the causes of the instability and taking preemptive actions should facilitate the development of MVA and other poxviruses as human and veterinary vaccines. In addition, these insights may have application to other DNA expression vectors.     

Cat Interferon inhibits Feline Leukaemia Virus Infection in Cell Culture

TRANSMISSION of feline leukaemia can be accomplished with tissue extracts from cases which occur naturally¹. Virus particles which are morphologically indistinguishable from the murine and avian C-type viruses are present in cats with the transmitted disease². Feline leukaemia virus (FeLV) replicates in cat cell cultures³ and infected cells are demonstrable by the indirect immunofiuorescent antibody test which detects FeLV group-specific antigen as granular punctate fluorescence in the cytoplasm of acetone fixed cells⁴; this method allows easy quantitation of the antiviral effect of interferon. We report the production and assay of feline interferon using the fluorescent antibody test with FeLV infected cat cell cultures.     

Selective Inhibition by Streptovaricin of Splenomegaly Induced Rauscher Leukaemia by Virus

STREPTOVARICINS, inhibitors of the RNA dependent DNA polymerase of oncogenic RNA viruses¹, inhibit Moloney sarcoma virus-induced transformation². If selective inhibition of virus-mediated oncogenesis can be extended to the whole animal it could provide a mode of chemotherapy which does not depend on the non-selective action of most oncolytic drugs.     

Phenotypic Mixing of Envelope Proteins of the Parainfluenza Virus SV5 and Vesicular Stomatitis Virus

Cells mixedly infected with parainfluenza virus SV5 and vesicular stomatitis virus (VSV) yield phenotypically mixed virions, in addition to both parental types. Two types of phenotypically mixed virions have been identified: 0.6 to 1.2% of the VSV plaque formers were neutralized by SV5 antiserum, but not by VSV antiserum, suggesting the presence of a VSV genome in an SV5 envelope; 9 to 45% of the VSV plaque formers were neutralized by both antisera, indicating the presence of both SV5 and VSV antigens in their envelopes. The presence of SV5 antigen in virions with the typical bullet-shaped appearance of VSV was confirmed with ferritin-labeled anti-SV5 antibody. In contrast to standard VSV, phenotypically mixed virions adsorbed to and eluted from chicken erythrocytes, indicating that these virions contained in their envelopes SV5 hemagglutinin, and possibly neuraminidase. Thus, the VSV nucleocapsid can interact with membranes which contain SV5 proteins in the manner which leads to virus maturation, and the production of a high yield of phenotypically mixed virions with the morphology of VSV indicates that this process can function efficiently. No evidence of genetic recombination between the two viruses was found. These results raise the possibility of an evolutionary relatedness between the paramyxoviruses and the rhabdoviruses.     

Increased Transformation Efficiency of Simian Virus 40 in Rat Embryo Cells infected with Rauscher Leukaemia Virus

SIMIAN virus 40 (SV40) is oncogenic for hamsters¹ and Mastomys². In vitro studies have shown that SV40 is capable of transforming cells derived from hamster³, mouse⁴, rabbit⁴, pig⁴, cow⁵, monkey⁶, human⁷, guinea-pig⁸ and rat⁹. We have established and studied continuous lines of uninfected and Rauscher leukaemia virus (RLV) infected rat embryo (RE) cells¹⁰. Rat embryo cells exposed to RLV have produced infectious virus and complement-fixing (CF) antigen characteristic of the murine leukaemia-sarcoma virus complex for more than 18 months. This communication describes increased transformation efficiency of SV40 in RLV infected RE cells (RLV-RE) compared with uninfected RE cells.     

Resistance-Associated Loss of Viral Fitness in Human Immunodeficiency Virus Type 1: Phenotypic Analysis of Protease and gag Coevolution in Protease Inhibitor-Treated Patients

We have studied the phenotypic impact of adaptative Gag cleavage site mutations in patient-derived human immunodeficiency virus type 1 (HIV-1) variants having developed resistance to the protease inhibitor ritonavir or saquinavir. We found that Gag mutations occurred in a minority of resistant viruses, regardless of the duration of the treatment and of the protease mutation profile. Gag mutations exerted only a partial corrective effect on resistance-associated loss of viral fitness. Reconstructed viruses with resistant proteases displayed multiple Gag cleavage defects, and in spite of Gag adaptation, several of these defects remained, explaining the limited corrective effect of cleavage site mutations on fitness. Our data provide clear evidence of the interplay between resistance and fitness in HIV-1 evolution in patients treated with protease inhibitors.     

Nested PCR在检测牛白血病病毒前病毒中的应用

用ＮｅｓｔｅｄＰＣＲ检测牛白血病病毒的前病毒形式。从牛白血病病毒基因ｇｐ５１上选择两对引物进行ＮｅｓｔｅｄＰＣＲ体外扩增,产物经２％ａｇａｒｏｓｅ凝胶电泳和用生物素标记的探针鉴定,证实其特异性。结果表明该方法能从两个ＢＡＴ－ＢＬＶ细胞内检测出ＢＬＶ的前病毒ＤＮＡ形式。     

Murine Leukaemia C-type Virus associated with Functional Murine Carcinomas of Endocrine Origin

The discovery of C-type viral particles related to murine leukaemia virus in two different mouse tumour cell types is indirect support for the “oncogene” hypothesis.     

Phenotypic heterogeneity of Gross virus-induced thymic lymphomas in the rat: cellular origins and migratory properties

Neoplastic thymocytes from rat thymic lymphoma-leukemias induced by the rat-adapted Gross leukemia virus (RAGV) were analyzed for a variety of differentiation markers. The neoplasms from individual rats all expressed the antigenic phenotype MP+, W3/13+, Thy-1+, RT-1+, RT-7+, W3/25-. However, approximately two-thirds of the neoplasms were positive for the OX 8 antigen, and one-third were negative. The OX 8- neoplasms only involved the thymus, whereas approximately 40% of the OX 8+ neoplasms involved the spleen as well as the thymus. Virtually all OX 8+ and OX 8- neoplastic cells contained terminal deoxynucleotidyl transferase (TdT), and both OX 8+ and OX 8- lymphomas expressed the lactate dehydrogenase (LDH)-5' isozyme and the primary, but not the secondary, ADA isozyme. This enzymatic phenotype is characteristic of thymocyte precursors, but not thymocytes. Our results therefore indicate that RAGV-induced lymphomas arise from transformed prethymic TdT+ cells which contain the LDH-5' and the primary ADA isozymes. These preleukemic cells presumably migrate to the thymus where they express the RT-7 pan-T-cell antigen and, in some instances, the OX 8 antigen during the development of overt leukemia. The OX 8+ neoplasms, being more differentiated than their OX 8- counterparts, then migrate to peripheral lymphoid tissues.     

Genotypic and Phenotypic Characterization of Chikungunya Virus of Different Genotypes from Malaysia

Background

Mosquito-borne Chikungunya virus (CHIKV) has recently re-emerged globally. The epidemic East/Central/South African (ECSA) strains have spread for the first time to Asia, which previously only had endemic Asian strains. In Malaysia, the ECSA strain caused an extensive nationwide outbreak in 2008, while the Asian strains only caused limited outbreaks prior to this. To gain insight into these observed epidemiological differences, we compared genotypic and phenotypic characteristics of CHIKV of Asian and ECSA genotypes isolated in Malaysia.

Methods and Findings

CHIKV of Asian and ECSA genotypes were isolated from patients during outbreaks in Bagan Panchor in 2006, and Johor in 2008. Sequencing of the CHIKV strains revealed 96.8% amino acid similarity, including an unusual 7 residue deletion in the nsP3 protein of the Asian strain. CHIKV replication in cells and Aedes mosquitoes was measured by virus titration. There were no differences in mammalian cell lines. The ECSA strain reached significantly higher titres in Ae. albopictus cells (C6/36). Both CHIKV strains infected Ae. albopictus mosquitoes at a higher rate than Ae. aegypti, but when compared to each other, the ECSA strain had much higher midgut infection and replication, and salivary gland dissemination, while the Asian strain infected Ae. aegypti at higher rates.

Conclusions

The greater ability of the ECSA strain to replicate in Ae. albopictus may explain why it spread far more quickly and extensively in humans in Malaysia than the Asian strain ever did, particularly in rural areas where Ae. albopictus predominates. Intergenotypic genetic differences were found at E1, E2, and nsP3 sites previously reported to be determinants of host adaptability in alphaviruses. Transmission of CHIKV in humans is influenced by virus strain and vector species, which has implications for regions with more than one circulating CHIKV genotype and Aedes species.