Expression of P23 of Cryptosporidium parvum in Toxoplasma gondii and evaluation of its protective effects.

In this study, P23 of Cryptosporidium parvum sporozoites, an immunodominant surface protein, was stably expressed in Toxoplasma gondii (Tg/P23) and its protective effects were evaluated in a mouse model. The molecular weight and antigenic property of P23 expressed by Tg/P23 were similar to those of the native P23. Mice immunized with lysed Tg/P23 tachyzoites produced specific neutralizing antibodies against C. parvum. These findings indicate that the T. gondii vector may provide a new tool for the production of a recombinant vaccine against cryptosporidiosis in animals.

Mutation hot spots in the canine herpesvirus thymidine kinase gene.

The guanine and cytosine content (GC-content) of alpha-herpesvirus genes are highly variable despite similar genome structures. It is known that drug resistant HSV, which has the genome with a high GC-content (approximately 70%), commonly includes frameshift mutations in homopolymer stretches of guanine (G) and cytosine (C) within the thymidine kinase (TK) gene. However, whether such mutation hotspots exist in the TK gene of canine herpesvirus (CHV) which has a low GC-content was unknown. In this study, we investigated mutations in the TK gene of CHV. CHV was passaged in the presence of iodo-deoxyuridine (IDU), and IDU-resistant clones were isolated. In all IDU-resistant virus clones, mutations in the TK gene were observed. The majority of these mutations were frameshift mutations of an adenine (A) insertion or deletion within either of 2 stretches of eight A's in the TK gene. It was demonstrated that CHV TK mutations frequently occur at a limited number of hot spots within long homopolymer nucleotide stretches.

Immunization with pseudorabies virus harboring Fc domain of IgG makes a contribution to protection of mice from lethal challenge.

To enhance the efficacy of an inactivated vaccine against pseudorabies virus (PRV), we evaluated the adjuvant properties of Fc domain of IgG. A cell line expressing mouse IgG Fc chimera on its surface was established. We found that when PRV was propagated in the cells expressing the Fc chimera, PRV virion incorporated the Fc. Immunization of BALB/c mice with inactivated PRV harboring Fc, which had been propagated in the cells expressing Fc on its surface, induced higher antibody production against PRV and protected mice more effectively from lethal challenge of virulent strain, comparing to the immunization with normal inactivated virus. Virus harboring Fc has a great potential as a new inactivated vaccine.

Pseudorabies virus propagated in rabbit kidney-derived RK13 cells is neutralized by natural IgM antibodies in normal swine serum which specifically lyse host cells.

Pseudorabies virus (PRV) propagated in rabbit kidney-derived RK-13 cells (PRV-RK) was neutralized by serum obtained from specific pathogen-free pigs through the activation of complement. The virus-neutralizing activity of swine serum was lost after treatment with ethylene glycol-bis-aminoethylether-N,N,N',N'-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA). Anti-C1q and anti-IgM antibodies also inhibited virus-neutralizing activity. Though IgG-depleted swine serum neutralized PRV, IgM and IgG-free swine serum lost virus-neutralizing activity. Pre-incubation of swine serum with RK-13 cells, but not with swine kidney-derived CPK cells, at 4 degrees C eliminated the virus-neutralizing activity to PRV-RK. Results indicated that swine serum contained natural IgM against an antigen(s) on the RK-13 cell surface and that this surface antigen was integrated into the PRV envelope during the budding process. Thus the natural IgM in swine serum reacted with the RK-13 antigen on the viral envelope, activated the complement cascade and neutralized the PRV-RK.

Restriction of bovine herpesvirus 1 (BHV-1) growth in non-permissive cells beyond the expression of immediate early genes.

Mouse BALB/3T3-A31-1-1 (A31) cells are non-permissive to bovine herpes virus-1 (BHV-1) but permissive to pseudorabies virus (PrV). The promoter activity of the immediate early gene of BHV-1 (BICP4) was very weak when compared with that of PrV in A31 cells. Infectious BHV-1 genomic DNA co-transfected into A31 cells with plasmids expressing BICP4 and BICP0 by a strong promoter failed to yield any progeny virus. Growth of BHV-1 in non-permissible A31 cells is restricted in many phases of the growth. The fact that expression of BICP4 and/or BICP0 in A31 cells does not improve the yield of progeny virus from infectious BHV-1 genomic DNA suggests that some more growth restrictions exist beyond the expression of BHV-1 immediate early proteins.

The neutralization of pseudorabies virus by anti-alpha-galactocyl natural antibody in normal serum.

Pseudorabies virus (PrV), a member of herpesviridae alphaherpes subfamily, can infect human cells in vitro. However, the transmission to Old World primates including humans is strongly restricted. In this study, we report the neutralizing activity of normal human serum against PrV grown in CPK cells derived from pig. PrV grown in all Old World primates-derived cells, which was tested in this study, were not neutralized by normal human serum. The virion of PrV grown in CPK cells harbored Galalpha1-3Galbeta1-4GlcNAc-R (alpha-gal epitope) on its surface, while PrV grown in Vero cells did not. Depletion of antibodies reacting to surface antigens of CPK cells negated the neutralization activity of human serum. Blockade of anti-alpha-gal antibodies by adding soluble Galalpha1-3Gal to normal human serum also prevent the inactivation of PrV grown in CPK cells. Although normal swine serum did not neutralize PrV grown in CPK cells, swine serum supplemented with exogenous anti-alpha-gal antibodies did. These results indicate that anti-alpha-gal antibodies in normal human serum contribute to the neutralization of PrV. Anti-alpha-gal antibodies in normal human serum may prevent transmission of PrV into humans.

Applications of recombinant Leishmania amazonensis expressing egfp or the beta-galactosidase gene for drug screening and histopathological analysis.

Leishmania amazonensis recombinants expressing the enhanced green fluorescent protein (egfp) gene or beta-galactosidase gene (lacZ) were constructed for drug screening and histopathological analysis. The egfp or lacZ in a leishmanial transfection vector, p6.5, was introduced into L. amazonensis promastigotes, and egfp or lacZ-carrying recombinant L. amazonensis, La/egfp and La/lacZ, respectively, were obtained. Expression of egfp or lacZ in both promastigotes and amastigotes could be clearly visualized by fluorescence microscopy or by light microscopy with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), respectively. Fluorescence signal and beta-galactosidase activity measured by a colorimetric reaction with chlorophenol red beta-D-galactopyranoside (CPRG) were well correlated to the numbers of these parasites. The inhibitory concentration (IC50) of a leishmanicidal drug, amphotericin B, in L. amazonensis promastigotes measured using La/egfp and La/lacZ was similar to that measured by conventional methods such as cell counting, thymidine incorporation and colorimetric assay. Furthermore, the fluorescence signal and absorbance of CPRG correlated well with the numbers of La/egfp and La/lacZ amastigotes in macrophages, respectively, suggesting La/egfp and La/lacZ can be a convenient and useful tool for drug screening not only in promastigotes, but also in amastigotes of L. amazonensis. La/lacZ collected from mouse tissues four weeks after the parasite infection were stained well with X-Gal. La/lacZ allowed parasite detection at high sensitivity in the tissues of infected mice and will be useful for following infections in macrophages in vivo. Thus, the marker-transfected Leishmania parasites constructed in this study will be useful for analyses of Leishmania parasites, especially at the intracellular stage.

Recombinant bovine herpesvirus-1 expressing p23 protein of Cryptosporidium parvum induces neutralizing antibodies in rabbits.

In order to develop a vaccine against cryptosporidiosis in cattle, we constructed a recombinant bovine herpesvirus-1 (BHV-1) expressing an immunodominant surface protein, p23, of Cryptosporidium parvum sporozoites. In the recombinant virus, the p23 gene under the control of a CAG promoter and a gene coding for an enhanced green fluorescent protein were integrated into the gG gene of BHV-1. Despite a low frequency of homologous recombination, cloning of the recombinants was easy because of the specific fluorescence of the plaques formed by recombinants. These plaques were among the plaques of the nonfluorescent parental virus. All clones selected for fluorescence also contained the p23 gene. In MDBK cells infected with the recombinant BHV-1, the antibody against the p23 protein recognized the p23 protein as an approximately 23-kDa specific band in Western blotting analysis. Rabbits immunized with the recombinant produced IgG against the p23 protein. It was also demonstrated that the sera of immunized rabbits reduced infection of C. parvum sporozoites in HCT-8 cells. The serum of an immunized rabbit reduced infection compared with the normal rabbit serum control. These results indicate that the recombinant BHV-1 induces neutralizing antibodies in rabbits.

Bovine herpesvirus 1 U(S) ORF8 protein induces apoptosis in infected cells and facilitates virus egress.

The bovine herpesvirus 1 (BHV-1) U(S) ORF8 protein with homology to the Us9 protein of other alphaherpesviruses induces apoptosis in rabbit kidney (RK13) cells without the presence of other BHV-1-encoded proteins. In this article, we have characterized the cytotoxicity and growth behavior of a BHV-1 recombinant, BHV-1/D8, which fails to express the U(S) ORF8 protein in infected cells. BHV-1/D8 exhibited a reduced cytotoxicity to RK13 cells when compared to the cytotoxicity of control BHV-1 strains. In RK13 cells, the onset of apoptosis was not observed during the infection with BHV-1/D8, and the virus multiplication of BHV-1/D8 was markedly greater than that of control viruses. However, virus release of progeny viruses from the infected RK13 cells into culture supernatant was significantly decreased by the loss of the U(S) ORF8 protein. These data demonstrate that the U(S) ORF8 protein activates the apoptotic process and facilitates virus release from the BHV-1-infected cells.

Pretreatment of leishmania homologue of receptors for activated C kinase (LACK) promotes disease progression caused by Leishmania amazonensis.

A cDNA coding Leishmania homologue of receptors for activated C kinase (LACK), which was known to play an important role in the early phase of Leishmania infection, was molecularly cloned from Leishmania amazonensis promastigote by using reverse transcription and nested polymerase chain reaction, and was sequenced. The L. amazonenis LACK cDNA showed 97.3 to 99.3% homology and its deduced amino acid sequence showed 98.7 to 99.7% identity in comparison with LACK sequences from five other species. The amino acid sequences in the immunodominant peptide region were completely conserved among Leishmania spp. tested. Intravenous pretreatment of the recombinant L. amazonensis LACK into BALB/c mice showed progressive lesion development compared to PBS (-) injected control mice, suggesting the important role of LACK in the early phase of L. amazonensis infection.

Bovine herpesvirus-1 (BHV-1) recombinant expressing pseudorabies virus (PrV) glycoproteins B and C induces type 1 immune response in BALB/c mice.

Bovine herpesvirus 1 (BHV-1) attached poorly and penetrated into a mouse cell line, BALB 3T3/A31, but a recombinant BHV-1/TF7-6, which expresses pseudorabies virus (PrV) gB and gC genes, did attach and penetrated into cells more efficiently. In this study the gene green fluorescent protein (GFP) has been integrated into genome of BHV-1/TF7-6 and its parental line of BHV-1. When the mouse mesenteries were incubated in vitro and infected with BHV-1/TF7-6/GFP, strong fluorescence was observed while BHV-1/GFP infection hardly demonstrated fluorescence, suggesting that BHV-1 recombinant expressing PrV gB and gC can infect mouse tissue cells more efficiently than the parental BHV-1 does. When BALB/c mice were inoculated with purified BHV-1/TF7-6 or its parental BHV-1, the former induced lower level of anti-BHV-1 immunoglobulin G (IgG) than the latter did. When sub-classes of anti-BHV-1 IgG were analyzed, it was found that mice immunized with BHV-1/TF7-6 or the parental BHV-1 demonstrated the same level of IgG2a. Since anti-BHV-1 IgG1 level was lower in mice inoculated with BHV-1/TF7-6, the IgG2a:IgG1 ratio was higher in BHV-1/TF7-6 inoculated mice than in the parental BHV-1 inoculated ones. These results indicate that BHV-1/TF7-6 induces type 1 predominant immune to BALB/c mice.

Mechanisms of apoptosis in murine fibroblasts by two intracellular protozoan parasites, Toxoplasma gondii and Neospora caninum.

Studies to clarify the mechanisms of apoptosis in host cells, A31 (BALB/3T3 clone A31 fibroblasts), caused by two intracellular protozoan parasites, Toxoplasma gondii and Neospora caninum, were carried out in an in vitro system. The viability of N. caninum-infected cells was significantly reduced following treatment with mouse interferon (IFN)-gamma. By contrast, mouse IFN-gamma treatment had no significant effect on the induction of apoptosis in T. gondii-infected cells. Apoptosis of N. caninum-infected and mouse IFN-gamma-treated cells was shown to be associated with increased DNA fragmentation, and increased caspase-3 and -8 activity, and the administration of caspase-3 and -8 inhibitors inhibited cell death. FasL expression was clearly induced by N. caninum-infection and IFN-gamma treatment compared to the T. gondii-infected cells and the uninfected control with or without IFN-gamma treatment. The reduction in host-cell viability was prevented with the addition of antimouse FasL monoclonal antibody (mAb). Moreover, TUNEL analyses indicated that apoptosis was induced by the treatment with Fas mAb in both T. gondii and N. caninum-infected cells. These results suggest that the Fas/FasL pathway may play a crucial role in the induction of apoptosis in N. caninum- and T. gondii-infected cells mediated by IFN-gamma.

Canine herpesvirus ORF2 is a membrane protein modified by N-linked glycosylation.

Canine herpesvirus (CHV) ORF2, located downstream of the glycoprotein C (gC) gene, has homologues with some of the alphaherpesviruses. To characterize CHV OFR2, a recombinant CHV carrying a LacZ gene in the ORF2 locus, and recombinant vaccinia virus expressing ORF2 protein were constructed. Northern blot analysis revealed ORF2 and a gamma2 class late gene, and its protein product was detectable in CHV-infected cells reacted with ORF2 protein antiserum. Tunicamycin and N-glycosidase F treatment revealed that the ORF2 protein was modified by N-linked glycosylation. Fractionation and immune fluorescence analyses of the CHV-infected cells showed the ORF2 as a membrane protein transportable to the surface of infected cells. In vitro, the ORF2 protein did not affect viral replication and cell-to-cell viral spreading. Present findings represent the first evidence pointing to the CHV ORF2 as a membrane protein modified by an N-linked glycosylation.

Induction of apoptosis in rabbit kidney cell under high-level expression of bovine herpesvirus 1 U(s) ORF8 product.

OBJECTIVE: The open reading frame 8 (ORF8) within the short unique (U(s)) region of the bovine herpesvirus 1 (BHV-1) genome was predicted to encode a protein with homology to U(s)9 protein of other alpha herpesviruses. The aim of this study is to identify the protein encoded by the U(s) ORF8 and to examine the effect of its expression in mammalian cells. METHODS: A polyclonal antiserum was raised against U(s) ORF8 protein expressed in Escherichia coli. A recombinant baculoviurs designated as Ac/CA8 was created by integrating U(s) ORF8 under the control of the mammalian CAG promoter into the baculovirus genome. U(s) ORF8 protein was expressed in rabbit kidney (RK13) cells transduced by Ac/CA8. RESULTS: The antiserum reacted specifically with 27-and 32-kD polypeptides from the BHV-1-infected and Ac/CA8-transduced RK13 cells. High-level expression of U(s) ORF8 protein in RK13 cells transduced by Ac/CA8 led to a distinct cytopathic effect and a reduction in cell viability; the onset of apoptotic cell death was induced as judged by DNA laddering and chromatin condensation analyses. CONCLUSIONS: These data represent the identification of BHV-1 U(s )ORF8 protein, which directly induces apoptosis in RK13 cells.

Pseudorabies virus (PRV) is protected from complement attack by cellular factors and glycoprotein C (gC).

Swine kidney derived CPK cells were resistant to swine complement attack in vitro while rabbit kidney derived RK13 cells were destroyed by swine complement. To rabbit complement, RK13 cells were resistant but CPK cells were sensitive. This phenomenon was known as homologous restriction (Proc. Natl. Acad. Sci. USA 78 (1981) 5118). The gC deletion mutant of pseudorabies virus (PRVdlgC) grown in CPK cells was resistant to swine complement while the same virus grown in RK13 cells was neutralized by swine complement. PRVdlgC grown in RK13 cells was more resistant to rabbit complement than the virus grown in CPK cells. Hence, the sensitivity of PRVdlgC to swine or rabbit complement was similar to that of the cells in which the virus was grown. It would appear that cell derived factors were present on the virion and they were protective against homologous complement but not against heterologous complement. The expression of gC rendered PRV more resistant to swine or rabbit complement, but the protective effect of gC was much less than that of cell derived factors. The best protection against complement was obtained when gC and cell derived factors were coexistent on the virion.

Bovine herpesvirus 1 glycoprotein G is necessary for maintaining cell-to-cell junctional adherence among infected cells.

Glycoproteins gE and gG of bovine herpesvirus 1 (BHV-1) are involved in viral cell-to-cell transmission. We have compared the subcellular localizations of gE and gG and examined the cell-to-cell adherence of bovine kidney (MDBK) cells infected with BHV-1 mutants lacking gE or gG. In BHV-1-infected MDBK cells, gE was observed at cell junctions but did not localize at apical or basal plasma membranes. BHV-1 gG was primarily found in the cytoplasm and was also observed at boundaries among infected cells. During the infection with wild-type or gE-negative BHV-1, the filamentous actin and the adherent junctional proteins accumulated at the cell junctions. In contrast, cell junctions of MDBK cells infected with gG-negative BHV-1 were loosened, and the junctional proteins and BHV-1 gE were distributed in the cytoplasm. These data indicate that BHV-1 gG facilitates viral cell-to-cell spread by maintaining the cell-to-cell junctions among the infected cells.