C57BL/6, C3H, and BALB/c mice were vaccinated with plasmids encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2, previously described as strong inducers of immunity. Seroconversion for the relevant antigen was obtained in the majority of the animals. T. gondii lysate stimulated specific T-cell proliferation and secretion of gamma interferon (IFN-␥) in spleen cell cultures from vaccinated BALB/c and C3H mice but not in those from control mice. Although not proliferating, stimulated splenocytes from DNA-vaccinated C57BL/6 mice also produced IFN-␥. No interleukin-4 was detected in the supernatants of lysate-stimulated splenocytes from DNA-vaccinated mice in any of the mouse strains evaluated. As in infected animals, a high ratio of specific immunoglobulin G2a (IgG2a) to IgG1 antibodies was found in DNA-vaccinated C3H mice, suggesting that a Th1-type response had been induced. For BALB/c mice, the isotype ratio of the antibody response to DNA vaccination was less polarized. The protective potential of DNA vaccination was demonstrated in C3H mice. C3H mice vaccinated with plasmid encoding GRA1, GRA7, or ROP2 were partially protected against a lethal oral challenge with cysts of two different T. gondii strains: survival rates increased from 10% in controls to at least 70% after vaccination in one case and from 50% to at least 90% in the other. In vaccinated C3H mice challenged with a nonlethal T. gondii dose, the number of brain cysts was significantly lower than in controls. DNA vaccination did not protect BALB/c or C57BL/6 mice. Our results demonstrate for the first time in an animal model a partially protective effect of DNA vaccination against T. gondii.

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SummaryThe complete sequence of a hemagglutinin (HA) gene of a recent human influenza A strain, A/Victoria/3/75, is 1768 nucleotides long and contains the information for 567 amino acids. It codes for a signal peptide of 16 amino acids, the HA1 chain of the mature hemagglutinin of 329 amino acids, a connecting region between HA1 and HA2 consisting of a single arginine residue and the HA2 portion of 221 amino acids. The sequence is compared with the hemagglutinin of two members of other subtypes, the human H2 strain A/Jap/305/57 and the avian Havl strain A/FPV/Rostock/34, and with one of the same H3 subtype, A/Memphis/3/72. To align the HA1 chain of different major subtypes several deletions/insertions of single amino acids must be invoked, but two more extensive differences are found at both ends, one leading to an extension of the amino terminal sequence of HA1 and the other (four residues) occurring in the region processed away between HA1 and HA2. Comparison of the HA1 of two H3 strains suggests that drift probably depends on single base mutations, some of which change antigenic determinants. The HA2 region, which apparently is not involved in the immune response, is highly conserved even between different subtypes, and single base substitutions account for all the observed diversity. A hydrophobic segment of 24 residues is present in the same position close to the carboxyl terminus of HA2 in both Victoria and FPV, and presumably functions in implantation into the lipid bilayer. The many conserved features not only in HA2 but also in HA1 suggest a rather rigid architecture for the whole hemagglutinin molecule.

We recently reported a high divergence among African subtype F strains. Three well-separated groups (F1, F2, and F3) have been shown based on the phylogenetic analysis of the p24 gag and envelope sequences with genetic distances similar to those observed for known subtypes. In this study, we characterized the near-full-length genomes of two strains from epidemiological unlinked individual belonging to each of the subgroups: F1 (96FR-MP411), F2 (95CM-MP255 and 95CM-MP257), and F3 (96CM-MP535 and 97ZR-EQTB11). Phylogenetic analysis of the near-full-length sequences and for each of the genes separately showed the same three groups, supported by high bootstrap values. Diversity plotting, BLAST subtyping, and bootstrap plotting confirmed that the divergent F strains correspond to nonrecombinant viruses. The divergence between F1 and F2 is consistently lower than that seen in any other intersubtype comparison, with the exception of subtypes B and D. Based on all the different analyses, we propose to divide subtype F into two subclades, with F1 gathering the known subtype F strains from Brazil and Finland, and our African strain (96FR-MP411), and F2 containing the 95CM-MP255 and 95CM-MP257 strains from Cameroon. The F3 strains, 97ZR-EQTB11 from the Democratic Republic of Congo and 96CM-MP535 from Cameroon, meet the criteria of a new subtype designated as K. The equidistance of subtype K to the other subtypes of HIV-1 suggests that this subtype existed as long as the others, the lower distance between B and D, and between F1 and F2 suggest a more recent subdivision for these latter strains.

Two strains of simian immunodeficiency viruses (SIV) isolated from chimpanzees (SIVCPZ-GAB and SIVCPZ-GAB2) originating from Gabon have previously been genetically characterized and shown to belong phylogenetically to the same lineage as the human immunodeficiency virus type 1 (HIV-1). We describe the sequence analysis of a third HIV-1-related virus, SIVCPZ-ANT, isolated from a wild captured chimpanzee originating from Zaire. This virus displayed the same genetic organization as HIV-1 and was found to fall on the same lineage as HIV-1 and SIVCPZ-GAB. Protein sequence identity with SIVCPZ-GAB ranged from 72% (Pol) to 48% (Env) for the structural proteins, while a particularly divergent Vpu was found (only 25% identity to SIVCPZ-GAB). The V3 regions of the SIVCPZ isolates were exceptionally conserved in contrast to the high divergence of V3 among HIV-1 isolates. However, SIVCPZ-ANT did not show a greater degree of sequence similarity with SIVCPZ-GAB than with HIV-1 isolates and represents a quite divergent outgroup of the HIV-1 lineage. Our data suggest multiple introductions of HIV-1 in the human population and shed new light on the origin of the HIV-1 pandemic.

Lentivirus infections in small ruminants represent an economic problem affecting several European countries with important sheep-breeding industries. Programs for control and eradication of these infections are being initiated and require reliable screening assays. This communication describes the construction and evaluation of a new serological screening enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (MVV) in sheep and to caprine arthritis encephalitis virus (CAEV) in goats. The solid phase is sensitized with a combination of the major core protein p25 of MVV produced in Escherichia coli and a peptide derived from the immunodominant region of the viral transmembrane protein gp46. The peptide carries an N-terminal biotin residue and is complexed with streptavidin prior to being coated. The new assay was evaluated with 2,336 sheep serum samples from different European countries with large differences in the levels of prevalence of MVV infections, and the results have been compared to those of the standard agar gel immunodiffusion test. Discrepant samples were analyzed by Western blotting with viral lysate, and most sera could be classified unambiguously. The estimated overall sensitivity of the new ELISA was 99.4% (95% confidence interval [CI], 98.4 to 99.8%) and the specificity was 99.3% (95% CI, 98.7 to 99.6%). A limited set of goat sera (n = 212) was also analyzed, with similar results. These data indicate that the new assay is a reliable tool that can be used in control and eradication programs for small ruminant lentivirus infections.

Most human immunodeficiency virus (HIV) drug susceptibility studies have involved subtype B strains. Little information on the impact of viral diversity on natural susceptibility to antiretroviral drugs has been reported. However, the prevalence of non-subtype-B (non-B) HIV type 1 (HIV-1) strains continues to increase in industrialized countries, and antiretroviral treatments have recently become available in certain developing countries where non-B subtypes predominate. We sequenced the protease and reverse transcriptase (RT) genes of 142 HIV-1 isolates from antiretroviral-naive patients: 4 belonged to group O and 138 belonged to group M (9 subtype A, 13 subtype B, 2 subtype C, 5 subtype D, 2 subtype F1, 9 subtype F2, 4 subtype G, 5 subtype J, 2 subtype K, 3 subtype CRF01-AE, 67 subtype CRF02-AG, and 17 unclassified isolates). No major mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors were detected. Major mutations linked to resistance to non-NRTI agents were detected in all group O isolates (A98G and Y181C) and in one subtype J virus (V108I). In contrast, many accessory mutations were found, especially in the protease gene. Only 5.6% of the 142 strains, all belonging to subtype B or D, had no mutations in the protease gene. Sixty percent had one mutation, 22.5% had two mutations, 9.8% had three mutations, and 2.1% (all group O strains) had four mutations. In order of decreasing frequency, the following mutations were identified in the protease gene: M36I (86.6%), L10I/V (26%), L63P (12.6%), K20M/R (11.2%), V77I (5.6%), A71V (2.8%), L33F (0.7%), and M46I (0.7%). R211K, an accessory mutation associated with NRTI resistance, was also observed in 43.6% of the samples. Phenotypic and clinical studies are now required to determine whether multidrug-resistant viruses emerge more rapidly during antiretroviral therapy when minor resistance-conferring mutations are present before treatment initiation.

Analysis of the complete sequence of a human immunodeficiency virus (HIV) isolate (Ant70) obtained from a Cameroonian patient indicates that this virus is the most divergent strain within the HIV-1 family hitherto described. Comparison of the Pol protein, usually highly conserved within the HIV-1 family, shows only about 73% similarity with the HIVmm isolate, whereas for the more variable proteins such as envelope, similarities of 50% or lower are found. The principal neutralizing determinant (V3 loop) and the immunodominant region within gp41 also contain some unusual substitutions, which may have implications for protein function as well as for serological assays based on these regions. Phylogenetic analyses show that this isolate occupies a unique position relative to the human HIV-1 isolates and the recently described SIVcpz virus, indicating that this Cameroonian isolate may provide us with new insights into the origins of the HIV-1 family.