Recombinant Parvovirus Research Articles

Responsiveness of a new parvovirus B19 antigen produced in baculovirus expression system to B19-specific IgG and IgM antibodies in every epidemic, 1968, 1980, 1987 and 1992 in Japan

A new recombinant parvovirus B19 antigen was tested whether it was responsive to human serum antibodies in every epidemic year of erythema infectiosum for 25 years, because wild strains of B19 parvovirus were changeable genetically. The antigen was empty particles of both B19-VP1 and VP2 produced in baculovirus expression system. Specimens were 21 sera in 1968, 19 in 1980, 44 in 1987 and 33 in 1992, derived from 67 patients with erythema infectiosum, fever and/or non-specific exanthem and aplastic crisis in persons with hereditary spherocytosis. Each patient had been confirmed of B19 parvovirus infection by other methods as radio immunoassay and/or enzyme-linked immunosolvent assay for B19-IgG and IgM using other antigens and by detection of B19-genome DNA using the polymerase chain reaction. Days of the illness of every serum were confirmed including before infection to 216 days after onset. Sera from 23 patients with measles, Kawasaki disease and rubella were selected for controls, and those patients who had not been infected with B19 parvovirus. Tests were carried out by enzyme immunoassay, indirect method for IgG and IgM capture method. In a total of 103 specimens after onset of symptoms B19-IgG was positive in yearly specimens, and B19-IgM was also positive in all acute phase sera. B19-IgG in most of all sera was kept in peak level up to 216 days after onset. B19-IgM increased rapidly in acute phase and seemed to disappear within one to 5 months after onset. Thirty-seven specimens including 14 obtained at state before infection and 23 controls were completely negative for both B19-IgG and IgM.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene marking after bone marrow transplantation

50. Dillon N. Regulating gene expression in gene therapy. Trends Biotechnoll993,11,167-173. 51. Comelis JJ, Becquart P, Duponchel N, et al. Transformation of human fibroblasts by ionizing radiation, a chemical carcinogen, or simian virus 40 correlates with an increase in susceptibility to the autonomous parvoviruses H-l virus and minute virus of mice. J Viroll988,62,1679-1686. 52. Russell SJ, Brandenburger A, Flemming CL, Collins MKL, Rommelaere J. Transformation-dependent expression of interleukin genes delivered by a recombinant parvovirus. J Virol 1992, 66, 2821-2828. 53. Friedman JM, Babiss LE, Clayton DF, Damell Jr JE. Cellular promoters incorporated into the adenovirus genome: cell specificity of albumin and immunoglobulin expression. Mol Cell&o1 1986,6, 3791-3797. 54. Babiss LE, Friedman JM, DamelI Jr JE. Cellular promoters incorporated into the adenovirus genome: effect of viral DNA replication on endogenous and exogenous gene transcription. 7 Mol Bioll987,193,643-650. 57.

Recombinant LuIII autonomous parvovirus as a transient transducing vector for human cells.

Recombinants based on the genome of the autonomous parvovirus, LuIII, were constructed by replacing the viral coding sequences in an infectious clone (pGLu883) by a luciferase or beta-galactosidase reporter, which was linked to the viral P4 promoter. In cells cotransfected with either of these constructs, together with a plasmid supplying LuIII nonstructural and capsid proteins, excision and replication of the recombinant genome occurred. Transducing virions accumulated in the culture medium of the cotransfected cells, as assayed by reporter activity in recipient cells exposed to this medium. Transducing activity could be neutralized by antiserum to LuIII. Production of replicative form DNA and transducing virions were observed following cotransfection of HeLa, 293, or NB324K cells, in increasing order of efficiency. When homology existed between the recombinant genome and sequences flanking the viral genes in the helper construct, concomitant production of replication-competent, cytopathic virus was sometimes observed. This could be minimized by removal of the left end homology from the helper; by this means, preparations of luciferase transducing virus were obtained free from replication-competent virus. With such preparations, we observed luciferase expression (declining after 3 days) for up to 7 days in recipient HeLa cells. Hybridization of the recombinant viral DNA with strand-specific luciferase probes indicated packaging of both strands (as reported for LuIII), but with a several-fold excess of the (-) strand. We suggest that transducing-autonomous parvoviruses will be useful in gene transfer applications, possibly including gene therapy when only transient expression is desired.

Encapsidation of a recombinant LuIII parvovirus genome by H1 virus and the fibrotropic or lymphotropic strains of minute virus of mice.

We previously constructed a recombinant LuIII parvovirus genome lacking viral coding sequences and used it to generate luciferase-transducing virions, by cotransfection of cells with a helper plasmid expressing LuIII viral proteins. Here, we describe similar cotransfections using alternative, replication-defective helpers encoding the non-structural and capsid proteins of parvovirus H1, or of either the fibrotropic or lymphotropic parvovirus strain of minute virus of mice [MVM(p) or MVM(i)]. Each cotransfection generated transducing virus which directed luciferase expression after infection of HeLa cells. The transducing activity of virus produced using either LuIII or H1 helper plasmids could be specifically neutralized by antiserum raised against the corresponding infectious virus. When the recombinant LuIII parvovirus was pseudotyped with MVM(p) or MVM(i), the resulting virions efficiently expressed luciferase after infection in human or murine cells known to be permissive for both MVM strains. The MVM(p) pseudotyped virus also expressed this reporter efficiently when infected into the murine A9 fibroblast line. In contrast, the recombinant virus generated with an MVM(i) helper gave luciferase expression that was barely detectable after infection of A9 cells which are highly restrictive for MVM(i) productive infection. These results support the notion that the allotropic determinant of these MVM strains functions through their capsid proteins. Pseudotyping of recombinant parvovirus genomes should be useful in controlling their host range as vectors, and in studying mechanisms influencing the permissiveness of parvovirus infections.

Recombinant parvovirus B19 capsids as a new substrate for detection of B19-specific IgG and IgM antibodies by an enzyme-linked immunosorbent assay

A new enzyme-linked immunosorbent assay for the detection of B19-specific IgG and IgM antibodies was established using B19 capsids synthesized in a baculovirus expression system. These B19 capsids, consisting of either coat protein VP2 alone or of both VP1 and VP2, have been shown to be similar to native virus in size and appearance. The results obtained for the detection of B19-specific antibodies showed good correlations with a radioimmunoassay which uses native B19 virus and an immunofluorescence assay based on insect cells expressing coat protein VP1. The course of the antibody response could be followed by determining the titers of sequential serum samples taken after a recent B19 infection. Both types of recombinant capsids form an excellent source of antigen for the detection of both B19 IgG and IgM antibodies and are a very promising substitute for native virus.

Transformation-dependent expression of interleukin genes delivered by a recombinant parvovirus.

The prototype strain of minute virus of mice [MVM(p)] is an autonomous parvovirus with a tropism for cells expressing a neoplastically transformed phenotype. To generate gene transfer vectors for tumor-specific gene expression, human interleukin-2 (IL-2) and murine interleukin-4 (IL-4) genes were cloned under the control of the p38 late promoter of MVM(p). Upon transfection into permissive cells, the recombinant MVMIL2 or MVMIL4 DNA was excised, amplified, and, in the presence of a helper plasmid, packaged into recombinant viral particles. The recombinant viruses were able to transfer fully functional IL-2 and IL-4 genes to permissive target cells and retained the oncotropic host range properties of the parental virus. Following infection with MVMIL2, nontransformed fibroblasts of rodent (FR3T3) or human (MRC-5) origin produced minimal IL-2 compared with the high levels of IL-2 production observed in their transformed derivatives (FREJ4 and MRC-5V1).

Recombinant subunit canine parvovirus vaccine; dog parvovirus VP-2 protein produced during replication of recombinant nuclear-polyhedrosis virus CPV 6C2B23 in Spodoptera frugiperda insect hosts or tissue culture

Recombinant subunit canine parvovirus vaccine; dog parvovirus VP-2 protein produced during replication of recombinant nuclear-polyhedrosis virus CPV 6C2B23 in Spodoptera frugiperda insect hosts or tissue culture

Construction of a recombinant human parvovirus B19: adeno-associated virus 2 (AAV) DNA inverted terminal repeats are functional in an AAV-B19 hybrid virus.

To facilitate genetic analysis of the human pathogenic parvovirus B19, we constructed a hybrid B19 viral genome in which the defective B19 inverted terminal repeats were replaced with the full-length inverted terminal repeats from a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The hybrid AAV-B19 genome was rescued from a recombinant plasmid and then the DNA was replicated upon transfection into adenovirus 2-infected human KB cells in the presence of AAV genes coding for proteins required for AAV DNA replication (AAV-Rep proteins). In addition, in the presence of AAV genes coding for the viral capsid proteins (AAV-Cap proteins), the rescued/replicated hybrid AAV-B19 genomes were packed into mature AAV progeny virions, which were subsequently released into culture supernatants. The recombinant AAV-B19 progeny virions were infectious for normal human bone marrow cells and strongly suppressed erythropoiesis in vitro. The availability of an infectious recombinant B19 virus should facilitate the mutational analysis of the viral genome, which, in turn, may yield information on individual viral gene functions in B19-induced pathogenesis. The hybrid AAV-B19 genome may also prove to be a useful vector for gene transfer in human bone marrow cells.