Rapid protocol for sequencing RNA virus using delta Taq version 2.0 DNA polymerase.
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Base Sequence↗
Biomedical subjects
A C Lai
Publications and source records attributed to A C Lai.
Genetic and antigenic analysis of the influenza virus responsible for the 1992 Hong Kong equine influenza epizootic.
An outbreak of influenza occurred among thoroughbred racehorses in Hong Kong in November-December 1992, with morbidity of 37%. All horses involved had been vaccinated against equine-1 and equine-2 influenza viruses but not against the virus responsible for the 1989 equine influenza outbreak in northern China (influenza A/equine/Jilin/89, subtype H3N8). Therefore the source and nature of the virus causing the Hong Kong outbreak was investigated. Virus isolated from a horse infected during the outbreak was used for genetic analysis. All the viral gene segments were similar to those of equine-2 (H3N8) influenza viruses and unrelated to those of equine/Jilin/89 virus. The nucleotide sequence of the viral hemagglutinin gene showed high homology (99.4%) to that of influenza A/equine/Suffolk/89 (H3N8) virus which has circulated extensively in Europe. However, these viruses differed in their antigenic reactivity to a panel of monoclonal antibodies. Preliminary epizootiological information plus the concordance of amino acid sequence between hemagglutinins of the Hong Kong isolate and a contemporaneous equine-2 influenza virus isolate from the United Kingdom indicated that the probable source of the Hong Kong outbreak was horses recently imported from England or Ireland.
Amino Acid Sequence↗
Removal of the zona pellucida and parthenogenetic activation affect rates of survival of ultrarapidly frozen mouse oocytes.
Survival rates on thawing were assessed for ultrarapidly frozen mouse oocytes following the removal of zonae pellucidae using an acid Tyrode's solution, pronase or a mechanical dissection technique. Significantly higher rates of survival were observed for zona-free oocytes than for zona-intact control oocytes (303/684, 44% v. 130/498, 26%; P < 0.001). The rates of survival observed for pronuclear stage embryos (72-76%) were much greater than those observed for oocytes and were not influenced by zona removal techniques. Parthenogenetic activation of oocytes by exposure to a 7% (v/v) ethanol solution was also shown to increase survival rates of ultrarapidly frozen oocytes (155/185, 84% v. 19/102, 19%) indicating that fusion of sperm to the plasma membrane or formation of a male pronucleus are not directly responsible for the increased survival rates of pronuclear stage embryos compared with oocytes. These data support the hypothesis that increased survival of ultrarapidly frozen pronuclear stage embryos is the result of changes to the plasma membrane and/or to the zona pellucida that occur following fertilization.
Animals↗
Attenuated deletion mutant of vaccinia virus IHD-W recovered virulence by reinsertion of a terminal restriction fragment.
Since vaccinia virus is being considered as a potential immunization vector, it is important to identify genes responsible for pathogenicity. One approach to identify virulence genes is the use of attenuated deletion mutants with a null background to reintroduce regions of the deleted genome material. We have previously described an attenuated deletion mutant of vaccinia virus strain IHD-W (Z-19) that lost 21 kb from the left terminus comprising fragments HindIII C and N, displays rearrangements at the right terminus and is unable to grow in vivo. To establish whether the loss of genes present at the left terminus is the basis for the attenuated phenotype, rescue experiments were performed to reintroduce fragment HindIII C, using growth in vivo as a selection system to isolate recombinant viruses. Several recombinants were isolated and molecularly and biologically characterized. The results indicate that recovery of virulence in mice was correlated with the presence and expression of two genes: vaccinia growth factor and C4b binding protein, which are located at the left terminus. Serpin I and II genes, located at the right terminus, were not affected in Z-19 and seem not to play a role in virulence in the IHD-W strain; interestingly, a copy of the serpin I gene was found at the left terminus. Moreover, a gene involved in virulence in the WR strain, the 13.8 kDa virokine gene (N1L) was found to be located in fragment HindIII G in the IHD-W strain, and therefore did not contribute to the attenuated phenotype.
Animals↗
Changes in cell gene expression in human leukemic cells persistently infected with vaccinia virus.
Persistent viral infections in vitro are useful systems to study the coevolution of virus and cell populations. Persistent infection of mouse Friend erythroleukemic cells (FEL) with vaccinia virus results in profound changes of the virus as well as of the cells. To investigate phenotypic changes of other cell types, we have established a persistent infection with vaccinia virus in a human leukemic cell line (K562). This cell line can be induced to differentiate along the erythroid pathway synthesizing embryonic and fetal globins, thus providing a system in which specific genes can be stimulated. After serial passage, the persistently infected cells (K562vac) became spontaneously differentiated, as shown by the increase in the number of cells producing hemoglobin (benzidine positive cells), and resistant to superinfection. These phenotypic changes of the cells were not accompanied by changes in the viral population. Hybridization of cellular RNA with cloned embryonic and fetal globin genes indicated that uninduced K562 cells do not express these genes, whereas cells induced by hemin or butyrate express G gamma (fetal globin) epsilon and zeta (embryonic globins) genes. By contrast vaccinia infected cells spontaneously express the G gamma gene. These results demonstrate that persistent infection with vaccinia virus elicited phenotypic changes in the infected cell population; in this case the constitutive expression of fetal hemoglobin.
Cell Line↗
A rapid method for screening vaccinia virus recombinants.
A rapid and small-scale method for screening vaccinia virus recombinants employing micrococcal nuclease is described. This protocol utilizes the differential sensitivity of cellular and viral DNA to the nuclease, which can be selectively activated by addition of Ca2+ and inactivated by elimination of Ca2+. Two to five micrograms of viral DNA can be obtained from one infected L cell plate (50 mm) after overnight incubation.
Animals↗
Characterization of vaccinia virus deletion mutants isolated from persistently infected Friend erythroleukemia cells.
Persistent viral infections in vitro are useful to study the evolution of virus populations in the absence of immunological pressure. Several deletion mutants have been isolated in this laboratory from Friend erythroleukemia cells persistently infected with vaccinia virus strain IHD-W, designated SQAvac. Two of the mutants, which remain stable after serial passage in L cells, have been characterized. The deletion which range between 20 to 22 kb, has been localized at the left terminus comprising HindIII fragments C and N. In addition, HindIII B fragment lost the sequences that hybridize to pAG5, a plasmid containing the 3.5 kb terminal sequences and acquired different restriction sites. Phenotypic characterization of these mutants revealed that they were not replication defective since they grew in all cell lines tested and produced plaques of the same size as the wild-type. However, they were less effective in suppressing host protein synthesis. The LD50 for the mutants titered in NIH Swiss female mice was greater than 10(9) PFU as compared to 10(6) PFU for the wild-type, indicating reduced virulence in vivo. These mutants, which display different properties to previously described mutants with deletions at the left terminus, provide another valuable system to study the molecular basis of virulence of vaccinia.
Animals↗
Attenuated deletion mutants of vaccinia virus lacking the vaccinia growth factor are defective in replication in vivo.
Understanding the molecular basis of virulence in poxvirus is of great importance for the development of recombinant vaccines using vaccinia virus as a vector. We have previously described mutants of vaccinia virus with deletions ranging from 20 to 21 kb at the left terminus and with attenuated phenotype. The virulence of these mutants was studied, using different routes of inoculation, for protection from wild-type challenge in mice and for replication in vivo. Regardless of the route of inoculation, the LD50 of the deletion mutants is at least 1000-fold higher than that of the wild-type. Results from protection experiments using viable and ultraviolet-inactivated viruses, and from determination of infectivity in different organs, indicated that the mutants were unable to replicate in vivo. Southern blot hybridization of viral DNA with pSC16, a plasmid containing the vaccinia growth factor (VGF) gene, revealed that in the IHD-W strain of vaccinia virus this gene is localized at the left terminus exclusively and that the mutants lack this gene. The results suggest that absence of the VGF gene is correlated with inability to replicate in vivo and decreased virulence.
Animals↗
Differences in the susceptibility of human blood cell lines to vaccinia virus.
The replication of vaccinia virus in human hematopoietic cell lines was studied, to ascertain whether the cell type and the stage of differentiation can influence the outcome of the infection. Lymphocytic NB104 cells and myelogenous leukemic K562 cells can be productively infected with vaccinia. Whereas NB104 cells were readily lysed, cells from the K562 line stayed persistently infected. Infection of promyelocytic leukemia HL60 cells results in an abortive infection in which the virus is uncoated, does not replicate, remains latent and can be rescued by cocultivation with permissive cells. Upon addition of phorbol esters to HL60 cell cultures, viral replication resumed. These results suggests that, although vaccinia virus replication is almost independent of host-specific functions, the outcome of the infection may depend on the cell type and its stage of differentiation.
Cell Differentiation↗
Characterization of variable-region genes and shared crossreactive idiotypes of antibodies specific for antigens of various influenza viruses.
Several syngeneic monoclonal anti-idiotypic antibodies were obtained against PY206, a monoclonal antibody specific for X-31 (H3N2) influenza virus hemagglutinin. This idiotype was found in the sera of BALB/c mice immunized with various influenza viruses. Adsorption experiments indicated that the PY206 Id was borne by antibodies specific for viral hemagglutinin (HA) and/or neuraminidase (NA). This idiotype was identified on other monoclonal antibodies specific for various influenza HAs (H3 and H1). Study of the variable-region (V) genes of these monoclonal antibodies showed that its expression is independent of variable kappa (VK)21 light-chains and that the heavy-chains of the strongly idiotype-positive hybridomas derive from either the variable heavy (VH) J558 or VH 7183 family. Finally, Western blot analysis demonstrated that PY206 idiotypic determinants are located exclusively on the heavy chain.
Animals↗
Retrieval of a Greenfield IVC filter displaced to the right brachiocephalic vein.
During insertion of a central venous sheath an inferior vena cava stainless steel Greenfield filter was dislodged to the right brachiocephalic vein without a free end. Successful retrieval was achieved by using a combination of a guidewire and a snare. Percutaneous retrieval of this vena cava filter is feasible with minimal risk using this method.
Aged↗