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PubMed2004

Walker 256 tumor MHC class I expression during the shift from A variant to the immunogenic AR variant.

Novel tumor cell variants can be obtained by serially passaging tumor cells in different media and/or environments. Serial intraperitoneal (ip) passages of the Walker 256 tumor A variant was followed for studying the generation of its regressive AR variant. MHC class I molecule expression was assessed since variations in this molecule would explain changes in tumor cell immunogenicity and therefore, the shift from progressive A variant to the regressive AR variant. Within 25 ip passages all serial repetitions shifted from A to AR variant, which was characterized by a significant increase in red blood cell (RBC) osmotic fragility with marked spleen hypertrophy in the host. In one serial repetition AR tumor cells were rejected (ip passage number 36) and immunity against the AR and A variants was conferred. Flow cytometry analysis showed a significant increase in the number MHC class I positive cells in AR variant (n = 15, 14.21 +/- 1.32) compared with A variant (n = 10, 9.10 +/- 1.22). These data provide evidence that the generation of the AR variant could result from factors present in the ip environment leading to an increase in the number of Walker 256 MHC class I positive tumor cells, probably due to immune selection of MHC class I negative tumor cells.

Animals↗

PubMed1981

Interactions among clonal subpopulations affect stability of the metastatic phenotype in polyclonal populations of B16 melanoma cells.

Analysis of the metastatic properties of clones isolated from mouse B16 melanoma cell lines (B16-F1 and F10) shows extensive cellular heterogeneity and the presence of subpopulations that have widely differing metastatic abilities. This pattern of metastatic heterogeneity is maintained during serial passage in vitro and in vivo. In contrast, even a short serial passage of individual clones isolated from these heterogeneous parent lines results in rapid emergence of variant subclones that have different metastatic properties. If several clones are mixed and cocultivated, this instability is not expressed. These data suggest that, in polyclonal populations, the various clonal subpopulations somehow interact with one another to "stabilize" their relative proportions within the population. Restriction of clonal diversity by selective killing of the majority of clones in a polyclonal population eliminates the stabilizing restraints and stimulates rapid emergence of new subpopulations to create heterogeneous populations containing a new panel of phenotypically diverse subpopulations that then reach stable proportions until the next selection pressure(s) is encountered.

Animals↗

PubMed1991

Quantitative immunocytochemical staining for recombinant tissue-type plasminogen activator in transfected Chinese hamster ovary cells.

Tissue-type plasminogen activator (tPA) is a serine protease which cleaves plasminogen to its active form, plasmin. tPA plays a physiologic role in hemostasis, wound healing, and embryogenesis. Therapeutically, recombinant human tPA is used as a thrombolytic in myocardial infarction. Although production of therapeutic quantities of tPA in Chinese hamster ovary (CHO) cells transfected with the human gene for tPA is practical, production costs remain high. One important factor which determines the ultimate cost of tPA (or any other recombinant protein expressed in mammalian cells) is its production level on a per cell basis. We have used postembedding immunocytochemical staining with colloidal gold to study the subcellular localization of tPA in CHO cells expressing recombinant tPA (rCHO) in an effort to understand the factor(s) which might limit secretion. Staining for tPA was evaluated visually and by morphometric analysis and was specific and reproducible. Serially passaged rCHO showed no significant change in staining density over 31 serial passages. Staining density was greatest over dilated cisternae of the rough endoplasmic reticulum and nuclear envelope. Golgi stacks and large acid phosphatase-positive vacuoles (probably lysosomes) were also heavily stained. Staining of lysosomal vacuoles suggested that rCHO might be degrading nascent tPA. Incubation of rCHO with 125I-tPA showed that the cells were not internalizing tPA from the media. These results suggest that rCHO fail to secrete a portion of the tPA they synthesize and that it is degraded in lysosomes. This observation may have important implications on the choice of expression systems for efficient production of large quantities of recombinant proteins.

Animals↗

PubMed2001

Combination of benzo[a]phenothiazines with acyclovir against herpes simplex virus.

The combined antiviral effects of some benzo[a]phenothiazines and 9-[2-hydroxy(ethoxy)methyl]guanine (acycloguanosine, acyclovir, ACV) on the multiplication of herpes simplex virus type 2 (HSV-2) were studied using Vero cells. The antiviral effect of ACV on a wild strain of HSV-2 was enhanced in the presence of 5-oxo-5H-benzo[a]phenothiazine and 6-methyl-5-oxo-5H-benzo[a]phenothiazine in a yield reduction test. A mathematical formula was used to interpret the drug interaction and a synergistic effect was found with a combination of ACV and benzo[a]phenothiazines. The effect of simultaneous application of two benzo[a]phenothiazines on the multiplication of HSV-2 strain during serial passages was also investigated. The combinations of 5-oxo-5H-benzo[a]phenothiazine or 6-methyl-5-oxo-5H-benzo[a]phenothiazine with ACV at a low concentration using serial passages of a plaque-purified ACV sensitive HSV-2 strain, reduced the infective virus population. A similar effect was also found on the activity of other benzo[a]phenothiazine derivatives. When the two most effective derivatives of 5-oxo-5H-benzo[a]phenothiazine or 6-methyl-5-oxo-5H-benzo[a]phenothiazine were simultaneously used with ACV against a wild type HSV-2 strain during consecutive passages, the infective virus titres were decreased, but their effect was only moderate. These results suggest that a combination of some benzo[a]phenothiazines with ACV might enhance their antiviral activity probably by reduction of the mutagenic rate in the virus populations.

Acyclovir↗

PubMed2004

Expansion of quasispecies diversity but no evidence for adaptive evolution of SHIV during rapid serial transfers among seronegative macaques.

Four successive, rapid serial passages of the nonpathogenic, CCR5-tropic simian-human immunodeficiency virus SHIV(SF162) in rhesus macaques resulted in an increase in acute plasma viremia with each passage and the emergence of a pathogenic isolate SHIV(SF162P3) in one of the passage three transfer animals (macaque T353). To explore the mechanism(s) underlying increased virulence of SHIV(SF162) upon in vivo passage, the evolution of the HIV-1 envelope gene was characterized in plasma and PBMC samples obtained from animals before (week 1) and after (week 3) the time of virus transfer. We found no evidence in support of adaptive evolution of the HIV gp120 during rapid serial passage; however, the animals which later received passage virus had more diverse quasispecies. SHIV(SF162P3)-like gp120 sequences were first detected in macaque T353 at week 6, after seroconversion. These sequence changes increased in frequency and number at later time points. The first sequence change conferred neutralization escape but not an increase in viral infectivity that could account for the apparent increase in replicative capacity of the later passage viruses. Collectively, our data argue against any host-specific adaptation of the HIV-1 envelope gp120 as the basis for the generation of more aggressive SHIV variants during rapid serial transfers in seronegative macaques, and support the model of quasispecies diversity as a predictor of pathogenesis. Envelope sequence changes accumulate principally in response to immune pressure exerted by the host, generating viral variants that can persist in the presence of a strong host immune response.

Amino Acid Sequence↗

PubMed2003

Surface glycoprotein PSA (GP46) expression during short- and long-term culture of Leishmania chagasi.

The mRNAs encoding promastigote surface antigen (PSA) of Leishmania chagasi have previously been shown to increase about 30-fold as in vitro cultured parasites progress from logarithmic to stationary phase, growth phases that are, respectively associated with parasites having low and high infectivity to mammals. Experiments reported here establish by western blot analysis that PSA proteins of 44 and 66 kDa also increase about 30-fold as parasite cultures reach stationary phase. Serial passage of parasite cultures resulted in a progressive reduction in PSA protein and RNA abundance to levels less than 3% that of cultures newly-initiated with parasites derived from a parasitized rodent. Loss of PSA mRNA abundance in serially passaged cells was not due to reduced PSA gene transcription rates, as determined by nuclear run-on assays. Neither was the loss associated with a marked decrease in PSA mRNA stability. Analysis of PSA RNA stability in the presence of actinomycin D, an inhibitor of transcription elongation, failed to detect a difference in fully processed cytosolic PSA mRNA stability regardless of the number of times a culture was passaged or the growth phase of the culture. Based on the lack of detectable difference in (cytosolic) mature PSA mRNA stability during promastigote development, the data indirectly suggest that the regulated expression of PSA in cells from low-passage cultures and the loss of PSA expression in high-passage cultures may be mediated by nuclear events that occur after transcription of the PSA genes and before arrival of the mature mRNAs in the cytoplasm.

Animals↗

PubMed1981

Enhanced neutral protease activity in proliferating rheumatoid synovial cells.

Proteolytic enzymes associated with rheumatoid synovial cells (RSC) have been implicated in the degradation of articular cartilage. Proteases have been measured in proliferating rheumatoid synovial cells (P-RSC), proliferating nonrheumatoid synovial cells (P-NSC), and their nonproliferating counterparts (NP-RSC and NP-NSC). The P-RSC and P-NSC exhibit enhanced total surface-associated plus secreted neutral protease activity, as compared with NP-RSC and NP-NSC. The P-RSC exhibited significantly higher protease activity in this category compared to P-NSC. The RSC also secreted higher levels of secreted proteases alone compared to NSC. The secreted protease activity alone was not related to the proliferative state of the cells. Extractable protease activity was measured in early-passaged and serially-passaged P-RSC, NP-RSC, P-NSC, and NP-NSC. Extractable cellular protease activity measured at pH ranges from 5.0 through 8.0 was not significantly different between P-RSC and NP-RSC or between P-NSC and NP-NSC. The RSC contained elevated extractable cellular protease activity measured at pH ranges from 5.0 through 8.0 compared to extracts from later-passaged cells. The neutral protease activity in the early-passaged RSC was also higher than that measured at pH 6.0 or 8.0. In synovial cells cellular protease activity was related to the proliferative state and the origin of the cells. The P-RSC exhibited the highest levels of surface associated plus secreted neutral protease activity. The RSC also possessed the highest levels of extractable protease activity compared to NSC.

Arthritis, Rheumatoid↗

PubMed1981

Identification of two strains of cultured canine renal epithelial cells (MDCK cells) which display entirely different physiological properties.

Cultured monolayers of Madin-Derby canine kidney (MDCK) cells grown upon permeable filter supports display many features of in vivo epithelia. Measurements of transmonolayer resistance, open-circuit p.d., dilution and bi-ionic p.d.s have been made of MDCK monolayers mounted in Ussing-type chamber (0.75 cm window radius). Previously reported values of transmonolayer resistance of 100 omega cm2 (Cereijido, Robbins, Dolan, Rotunno & Sabatini, 1978; Misfeldt, Hamamoto & Pitelka, 1976) indicate a 'leaky' epithelium. The major finding of this work is that MDCK monolayers can also display values of transmonolayer resistance similar to 'tight' epithelia (mean value = 4116 omega cm2). The reason for such differences is the existence of separate strains of MDCK cells. High-resistance monolayers originate from cell stocks of sixty serial passages whilst low-resistance monolayers originate from cell stocks of 109 serial passages. Measurements of transmonolayer dilution, and bi-ionic p.d.s together with Na ion tracer fluxes and electron microscope observations of La3+ penetration through the apical tight junctions, substantiate the finding of high-resistance properties in MDCK monolayers and confirm the existence of two separate MDCK cell strains displaying entirely different physiological properties. Differences extend to cellular morphology and cellular volume in the two strains of MDCK cells.

Animals↗

PubMed1972

Foot-and-mouth disease virus: selection by homogenized calf kidney adsorption and cell culture passage.

Foot-and-mouth disease virus was passaged 20 times by alternately adsorbing the virus with homogenized calf kidney and propagating the adsorption-resistant fraction of virus in cell cultures. Passage of the virus was accompanied by a marked decrease in pathogenicity for 35-day-old mice and a reduction in its ability to adsorb to homogenized kidney from such mice. Adsorption by homogenized calf kidney and infant mouse kidney also decreased with passage, but pathogenicity for cattle and infant mice remained high. The passaged virus did not seem to change antigenically. When the 20th-passage virus was serially passaged 10 times in cell cultures, no reversion in pathogenicity for 35-day-old mice was observed.

Adsorption↗

PubMed1994

A cultured malignant B-1 line serves as a model for Richter's syndrome.

Human chronic lymphocytic leukemia (CLL) is a malignancy of B-1 cells characterized by the accumulation of mature appearing, long lived, slow growing B-1 cells in peripheral blood. CLL occasionally evolves into an aggressive large cell lymphoma termed Richter's syndrome. NZB mice can be used to model the early stage of CLL because aged NZB mice can spontaneously develop slow growing malignant B-1 cell clones. The malignant NZB B-1 clones fail to grow in culture and are typically carried in vivo as passaged lines. During serial passage, an aggressive lymphoma developed as a result of a continued transformation of the original B-1 clone, similar to the development of Richter's syndrome. An in vitro cell line was established from the aggressive lymphoma, which was stromal dependent and could rapidly metastasize when passaged into recipient animals. Analysis of adhesion molecules did not reveal any consistent characteristics that could account for the metastatic potential of the Richter's-like cells. In addition, the aggressive in vitro line had the identical heavy chain sequence as the slow growing NZB malignant B-1 clones. The in vitro and in vivo aggressive B-1 cells had very high levels of IL-10 message, and underwent more apoptosis in response to anti-IgM than did nonaggressive B-1 clones. Taking these characteristics together, we have composed a comprehensive animal model system for human CLL that includes both the aged NZB mice for the early stage and the recipients of the in vitro B-1 line for the late stage or Richter's syndrome. This model system can be used to study, not only the ontogeny and genetic linkage of CLL, but also the regulatory factors involved in transformation and growth both in vivo and in vitro.

Animals↗

PubMed1994

Nonsense mutations in the vpr gene of HIV-1 during in vitro virus passage and in HIV-1 carrier-derived peripheral blood mononuclear cells.

Long-term, persistent infection by HIV-1 is a prerequisite for the development of AIDS. However, little is known of the determinants required for HIV-1 to cause persistence. We have reported previously that persistent infection of a T cell line by a cytopathogenic strain of HIV-1 became increasingly likely with in vitro serial passage of the virus. DNA sequencing of the persistent strains revealed a nonsense mutation in the vpr gene in all isolates tested. Here, we report the development and use of a semi-quantitative PCR method to detect the vpr nonsense mutation within populations of virus. Our results show that vpr mutants also arise in cells during acute infection and increase progressively with serial passage of the virus. In addition, HIV-1-seropositive individuals were examined and found to carry the same vpr nonsense mutation at high frequency in virus-infected PBMC. These data are consistent with a mechanism of HIV-1 persistence in vivo and in vitro in which virus cytopathogenic potential is lost by the build up of nonsense mutations in vpr.

AIDS-Related Complex↗

PubMed2003

Emergence of phenotypic resistance to ciprofloxacin and levofloxacin in methicillin-resistant and methicillin-sensitive Staphylococcus aureus strains.

The emergence of phenotypic resistance to ciprofloxacin and levofloxacin in methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MRSA) strains was studied. Twenty MRSA and 77 methicillin-sensitive S.aureus (MSSA) strains susceptible to both quinolones were investigated for resistance after single step or serial passages. No growth of 20 MRSA strains was observed at 4xMIC of levofloxacin after 48 h incubation, but 4 of 77 (5%) MSSA strains grew at the same concentration. At 4xMIC concentration of ciprofloxacin, 10 MSSA (13%) and five MRSA (25%) strains were grown. In the serial passages of MRSA strains, resistance to ciprofloxacin was 75 and 5% for levofloxacin by the third passage. In the seventh passage this resistance was 100 and 15%, respectively. In MSSA strains, resistance to ciprofloxacin was 75 and 19% to levofloxacin at the third passage and at the seventh passage, 100 and 61%, respectively. Emergence of ciprofloxacin resistance was more common and developed more rapidly than resistance to levofloxacin in both MRSA and MSSA strains.

Ciprofloxacin↗

PubMed1991

The inability of human immunodeficiency virus to infect chimpanzee monocytes can be overcome by serial viral passage in vivo.

Studies of lentivirus infection in ruminants, nonhuman primates, and humans suggest that virus infection of macrophages plays a central role in the disease process. To investigate whether human immunodeficiency virus type 1 (HIV-1) can infect chimpanzee macrophages, we recovered monocytes from peripheral blood mononuclear cells of HIV-1-negative animals and inoculated these and control human monocytes with a panel of four human-passaged monocytotropic virus strains and one chimpanzee-passaged isolate. HIV-1 infected human monocytes synthesized proviral DNA, viral mRNA, p24 antigen, and progeny virions. In contrast, except for the chimpanzee-passaged HIV-1 isolate, chimpanzee monocytes failed to support HIV-1 replication when cultured under both identical and a variety of other conditions. Proviral DNA was demonstrated only at background levels in these cell cultures by polymerase chain reaction for gag- and env-related sequences. Interestingly, the chimpanzee-passaged HIV-1 isolate did not replicate in human monocytes; viral p24 antigens and progeny virions were not detected. The same monocytotropic panel of HIV-1 strains replicated in both human and chimpanzee CD4+ T lymphoblasts treated with phytohemagglutinin and interleukin-2. The failure of HIV-1 to infect chimpanzee monocytes, which can be overcome by serial in vivo viral passage, occurs through a block early in the viral life cycle.

Animals↗

PubMed2005

Genetic complementation to identify DNA elements that influence complement resistance in Leishmania chagasi.

Past studies showed that Leishmania spp. promastigotes exhibit differential sensitivity to complement mediated lysis (CML) during development in vitro and in vivo. Leishmania chagasi promastigotes in cultures during logarithmic and stationary growth phases are CML-sensitive or CML-resistant when exposed to human serum, respectively, but only in cultures recently initiated with parasites from infected animals; serially passaged cultures become constitutively CML-sensitive regardless of growth phase. Building on these observations, a genetic screen was conducted to identify novel complement resistance factors of L. chagasi. A cosmid library containing genomic DNA was transfected into a promastigote line previously subjected to >50 serial passages. Selection with human serum for CML resistance yielded 12 transfectant clones. Cosmids isolated from 7 of these clones conferred CML resistance when transfected into an independent, high-passage promastigote culture; at 12% human serum, the mean survival of transfectants was 37% (+/- 11.6%), and that of control transfectants was about 1%. Inserts within the 7 cosmids were unique. Determination of the complete DNA sequence for 1 cosmid indicated that its 32-kilobase insert was 89% identical (overall) to a 31-kilobase region of Leishmania major chromosome 36, which is predicted to encode 6 genes, all of which encode hypothetical proteins.

Animals↗

PubMed1991

De novo generation of defective interfering RNAs of tomato bushy stunt virus by high multiplicity passage.

Defective interfering (DI) RNAs were generated de novo in each of 12 independent isolates of tomato bushy stunt virus (TBSV) upon serial passage at high multiplicities of infection (m.o.i.) in plants, but not in any of 4 additional isolates after 11 serial passages at low m.o.i. The DI RNAs were detected in RNA isolated from virus particles and in 2.3 M LiCl-soluble RNA fractions isolated from inoculated leaves. Symptom attenuation leading to persistent infections was closely correlated with the passage in which DIs first developed. Comparisons of nucleotide sequences of 10 cDNA clones from 2 DI RNA populations and with a previously characterized TBSV DI RNA revealed the same four regions of sequence from the TBSV genome were strictly conserved in each of the DI RNAs: the virus 5' leader sequence of 168 bases; a region of approximately 200-250 bases from the viral polymerase gene; approximately 70 bases from the 3' terminus of the viral p19 and p22 genes; and approximately 130 bases from the 3' terminal noncoding region. Conservation of the sequence motif present in all of the DIs suggests that there might be a common mechanism of DI formation as well as selection pressure to maintain sequences essential for replication and encapsidation.

Base Sequence↗

PubMed2026

Comparative dynamics of Japanese encephalitis virus adaptation in porcine macrophages and insect cells.

BACKGROUND: Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne Orthoflavivirus that circulates primarily in birds and pigs. Previous observations of vector-free transmission between pigs indicates the possibility of single-host cycling in swine. Therefore, the aim of this work was to investigate the evolutionary pressure of single host cycling using a relevant primary cell culture model. METHODS: To investigate whether such single-host cycles affect viral infectivity, fitness and genomic adaptations, two strains and a reverse genetic cDNA-derived clone of JEV were serially passaged 12 times in primary porcine monocyte-derived macrophages (MDMs), in Aedes albopictus-derived C6/36 cells, and alternately between both cell types. Next-generation sequencing analysis was used to identify selected single nucleotide variants (SNVs) and haplotypes. Phenotype-to-genotype connections were confirmed using reverse genetics. RESULTS: For all viruses, serial passaging in MDMs - but not in C6/36 cells - led to a rapid increase in relative infectivity toward MDMs, accompanied by reduced plaque sizes in porcine endothelial cells. In contrast to C6/36 cells, MDM imposed a strong selective pressure, rapidly favoring selection of many SNVs and viral haplotypes. In addition, we identified a dominant selection of mutants with glutamic acid to lysine substitutions at positions 49 or 138 in the E protein, which explained the small plaque phenotype and caused viral sensitivity to heparin-mediated inhibition of attachment, indicating enhanced virus binding to glycosaminoglycans (GAG). The E138K mutant also explained the increased relative infectivity for MDM. CONCLUSION: This work demonstrates a high evolutionary pressure on JEV in MDM causing rapid selections of minor haplotypes. Furthermore, the efficient selection of E49K and E138K SNV, which were responsible for the phenotype, are likely caused by a selective pressure for GAG binding, observed in vitro with other mammalian cells.

Animals↗

PubMed1996

A major rearrangement of the VP6 gene of a strain of rotavirus provides replication advantage.

During coinfection of BSC-1 cells with bovine rotavirus B223 and human rotavirus 69M and subsequent serial passages at low multiplicity of infection (0.1 m.o.i.), a reassortant virus (BMR) with a rearranged VP6 gene became the predominant strain. At passage 24 virus extracted from 50 of 51 plaques (98%) contained the rearranged gene 6, which had been first observed in passage 19. The analyses of the clones obtained from passages before the appearance of the rearranged VP6 gene (passage 15) and after (passage 20) indicated that the B223 VP6 gene was the origin of the rearranged VP6 gene. To test whether the rearranged VP6 gene was responsible for the selection advantage observed, reassortant C11 was generated with BMR and WA rotavirus, containing the rearranged VP6 gene and the other 10 genes from WA. Coinfection of WA rotavirus and reassortant C11 and subsequent serial passages at low m.o.i. resulted in 100% of virus from clones extracted at passage 18 being identical to reassortant C11; demonstrating that the rearranged VP6 gene was once again selected over the normal VP6 gene. The selection advantage of the rearranged VP6 gene could not be explained by comparison of the growth curves of the viruses, as there was no significant difference between the growth cycles of rotavirus B223 and reassortant BMR, nor between rotavirus Wa and reassortant C11. However, the plaque and electropherotype analysis at passage 1 of Wa and C11 coinfection revealed that 85% of the progeny viruses contained the rearranged gene 6. These data show that the gene 6 rearrangement resulted in selection of the relevant reassortant, possibly by suppression of competitive strains, and may indicate a new mechanism for the evolution of rotavirus.

Animals↗

PubMed1993

Encapsidation of genetically engineered poliovirus minireplicons which express human immunodeficiency virus type 1 Gag and Pol proteins upon infection.

The use of recombinant viruses for the expression of a wide array of foreign proteins has become commonplace during the last few years. Recently, we have described the construction and characterization of chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes in which the gag and pol genes of HIV-1 have been substituted for the VP2 and VP3 capsid genes of the P1 capsid precursor region of poliovirus. Transfection of these RNAs into tissue culture cells results in replication of the RNA genome and expression of HIV-1-P1 fusion proteins (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Here we report on the encapsidation and amplification of the minireplicons to obtain sufficient quantities for biological characterization. To do this, HIV-1-poliovirus minireplicon genomes containing the gag or pol gene were transfected into cells previously infected with a recombinant vaccinia virus (VV-P1) which expresses the poliovirus capsid precursor protein, P1 (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). The chimeric minireplicons replicated and expressed the appropriate HIV-1-P1 fusion proteins as determined by immunoprecipitation with HIV-1-specific antibodies. The encapsidated genomes were isolated by ultracentrifugation. Reinfection of cells with the encapsidated chimeric RNA genomes resulted in expression of the HIV-1-Gag-P1 or HIV-1-Pol-P1 fusion protein. Serial passaging of the encapsidated chimeric HIV-1-poliovirus genomes was accomplished by coinfecting cells with the encapsidated minireplicons and VV-P1, resulting in stocks of the encapsidated minireplicons. Northern (RNA) blot analysis of passaged material revealed that no detectable deletions of the chimeric genomes occurred during 14 serial passages. Infection of cells by the encapsidated minireplicons was blocked by antipoliovirus antibodies. Coinfection of cells with encapsidated minireplicons and type 1 Sabin poliovirus resulted in encapsidation of the chimeric genomes by wild-type poliovirus as measured by immunoprecipitation of the HIV-1-P1 fusion proteins with HIV-1-specific antibodies. The results of this study demonstrate the encapsidation of poliovirus minireplicons which express foreign proteins and point to the future use of this system as a potential vaccine vector.

Capsid↗