Yoshiyuki Maki

Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

A method for the isolation of genomic fragments of RNA virus based on cDNA representational difference analysis (cDNA RDA) was developed. cDNA RDA has been applied for the subtraction of poly(A)+ RNAs but not for poly(A)− RNAs, such as RNA virus genomes, owing to the vast quantity of ribosomal RNAs. We constructed primers for inefficient reverse transcription of ribosomal sequences based on the distribution analysis of hexanucleotide patterns in ribosomal RNA. The analysis revealed that distributions of hexanucleotide patterns in ribosomal RNA and virus genome were different. We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA. A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides. In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells. Using non-ribosomal primers, the cDNA fragments of severe acute respiratory syndrome coronavirus and bovine parainfluenza virus 3 were efficiently amplified by subtracting the cDNA amplicons derived from uninfected cells from those that were derived from virus-infected cells. The results suggest that cDNA RDA with non-ribosomal primers can be used for species-independent detection of viruses, including new viruses.

Reactivity of Synthetic SAG1 (p30) Peptide Sequences with RH, S273 and Beverley Strain-Induced Anti- Toxoplasma gondii Antibodies

Objectives: We compared the reactivity of IgG1 and IgG2a antibodies in mouse sera after infection with virulent RH and low-virulent S273 and Beverley strains of Toxoplasma gondii against RH SAG1 recombinant p30 (rp30) and synthetic SAG1 peptides. Methods: Infected mouse serum samples were collected 9 days after infection, and the level of total IgG, IgG1 and IgG2a against the RH SAG1 rp30 protein and twenty peptides of the RH SAG1 protein were assessed. The glycosylphosphatidylinositol (GPI) modification site, the hydrophilic-hydrophobic structure, the transmembrane region and the secondary structure of the SAG1 sequence of virulent and low-virulent strains were analyzed using software. Results: The virulent strain-infected mice produced a higher level of IgG1 but a lower IgG2a against the rp30 antigen, while the low-virulent strain-infected mice produced a higher level of IgG2a than the virulent strain. The difference in the secondary structure of SAG1 protein between the virulent and low-virulent strain was largely confined to amino acid positions 291–336, showing mutations and GPI anchor site. Conclusion:The difference in the reactivity of IgG against the rp30 antigen and synthetic peptides between virulent and low-virulent strains points to the importance of the primary and secondary structure assumed by antigens in the activation of Th cells and, subsequently, in the induction of IgG and its subclasses.

Expression of SAG-1 of Toxoplasma gondii in transgenic mice.

Abstract We describe the expression of SAG-1 cDNA in B6C3F1 mice by microinjecting a 3.3 kbp DNA fragment, consisting of the cytomegalovirus enhancer-chicken β-actin hybrid promoter and SAG-1 into the pronucleus of a fertilized egg at the one-cell stage. Offspring derived from this microinjection were analyzed for the integration and functional expression of the SAG-1 transgene. Steady-state expressions of both the mRNA for SAG-1 and SAG-1 protein product were detected in the brain, thymus, spleen and liver. Approximately 50% of F1 and F2 progeny inherited the SAG-1 transgene from SAG-1 transgenic mice in Mendelian fashion. These results indicated that SAG-1 transgenic lines were established. Transgenic mice harboring the SAG-1 gene will contribute a critical tool of defining the molecular mechanisms of SAG-1 in pathogenesis and host immune response.

Toxoplasma gondii Antigens GRA1 (p24) and SAG1 (p30): A Comparison of Their Stimulatory Influence on T-Cell Activation and Cytokine Expression in in vitro Cultures

The influence of recombinant cell surface SAG1 (rp30) and secretory GRA1 (rp24) antigens (Ag) on T-cell activation and cytokine induction in vitro was compared. T-cell activity and the level of IFN-γ, IL-10 and IL-12 expression in rp30-immunized T cells were considerably increased in the presence of rp30 Ags. IgG2a and IgG1 antibodies (Ab) were detected in sera of rp24- and rp30-immunized mice, with the secretory rp24 Ag having induced significantly higher titer of IgG1 Ab. In vitro, the greater antigenicity of surface rp30 Ag was notable based on the level of T-cell activation, and cytokine synthesis suggestive of the participation of Th1 cells. Although, IFN-γ expression by rp24 Ag was lower compared to rp30 Ag, the synthesis of both IgG2a and IgG1 Abs reflects the protective nature of rp24 Ag. We have generated two recombinant Toxoplasma gondii Ags that demonstrated differences in antigenicity in vitro. It would be interesting to evaluate the mechanism(s) of immunity induced by SAG1 (p30) and GRA1 (p24) Ags against infection with T. gondii in vivo.

Increased susceptibility to Toxoplasma gondii infection in SAG-1 transgenic mice.

SAG-1, one of the major surface proteins of Toxoplasma gondii, has been reported to play an important role in immune and pathogenic mechanisms of the parasites but its exact function is still unclear. We investigated the time courses of T. gondii infection in B6C3F1 transgenic mice carrying the SAG-1 gene. SAG-1 transgenic mice were infected intraperitoneally with a high virulent RH strain or a low virulent Beverley strain of T. gondii. When infected with RH strain tachyzoites, no significant differences in time courses of survivals between SAG-1 transgenic and wild-type mice were observed. Both groups succumbed to an acute infection within 8 days after infection. However, a lower survival rate (20%) was observed in SAG-1 transgenic mice than in wild-type (80%), when infected with Beverley strain cysts. This result indicates that SAG-1 transgenic mice are more susceptible to T. gondii infection as compared with their wild-type counterpart. ELISA using recombinant SAG-1 protein indicates that SAG-1 transgenic mice do not produce antibodies to the SAG-1 molecule. These findings may provide a critical tool for analysing the molecular mechanisms of pathogenesis and host immune responses during toxoplasmosis.

Unresponsiveness to Surface Antigen 1 Modifies Cytokine Profiles in Acute Toxoplasma gondii Infection

Resistance to Toxoplasma gondii involves the development of a highly polarized Th1-type cytokine expression. SAG1 transgenic mice are highly susceptible to T. gondii infection due to their non-reactivity to SAG1 of the protozoan parasite. Here we describe cytokine profiles during the acute phase of T. gondii infection, which are associated with the susceptibility of SAG1 transgenic mice. SAG1 transgenic mice showed a 4.5-fold increase in susceptibility upon inoculation with a sublethal dose of the Beverley strain of T. gondii compared to their wild-type counterparts (mortality: 81 vs. 18%, respectively). When analysis of the most important cytokines involved in the mediation of resistance to infection was carried out, SAG1 transgenic mice exhibited low production levels of IL-12, IFN-γ and TNF-α in sera during the acute phase of T. gondii infection. Antibody and T cells specific for SAG1 were not mounted upon SAG1 stimulation in SAG1 transgenic mice. Moreover, in vitro studies indicated that in SAG1 transgenic mice IFN-γ and IL-12 production was lower than in their wild-type counterparts, although levels of TNF-α increased in SAG1 transgenic mice on day 9 after infection. Low IgG2a levels were detected in SAG1 transgenic mouse sera. Unresponsiveness to SAG1 of T. gondii renders SAG1 transgenic mice unable to develop a strong Th1-based protection against T. gondii infection. These results provide evidence that SAG1 is a pivotal antigen involved in the induction of immune responses towards the development of Th1-protective immunity during T. gondii infection.