Immunocontraceptive effects on female rabbits infected with recombinant myxoma virus expressing rabbit ZP2 or ZP3.

Recombinant myxoma viruses expressing rabbit zona pellucida 2 (rZP2) or rabbit zona pellucida 3 (rZP3) glycoproteins were constructed and tested in domestic rabbits to assess their potential to induce autoimmune infertility. The recombinant virus expressing rZP2 had no effect on fertility or ovarian histology, despite all animals developing antibodies against the rZP2 antigen. However, recombinant viruses expressing rZP3 induced infertility in 70% of animals at the first breeding. Serum antibodies were relatively short-lived, but antibody was bound to zona pellucida of all rabbits from Day 10 onward. There was no obvious correlation between infertility and rZP3 antibody titer. There was a transient inflammatory response in the ovaries of rZP3-immunized rabbits at Day 15 but no T-cell response to rZP3 could be detected at any time. Dysfunctional follicular formation was present in ovaries from rabbits infected with rZP3-expressing viruses 15-40 days postinfection but this had disappeared at later time points. A recombinant myxoma virus expressing a modified rZP3 antigen with the C-terminal hydrophobic putative anchor sequence deleted was also tested. This virus did not induce either infertility or an antibody response against the zona pellucida. Thus, the context of antigen presentation was crucial for an autoimmune response.

Expression of rabbit IL-4 by recombinant myxoma viruses enhances virulence and overcomes genetic resistance to myxomatosis.

Rabbit IL-4 was expressed in the virulent standard laboratory strain (SLS) and the attenuated Uriarra (Ur) strain of myxoma virus with the aim of creating a Th2 cytokine environment and inhibiting the development of an antiviral cell-mediated response to myxomatosis in infected rabbits. This allowed testing of a model for genetic resistance to myxomatosis in wild rabbits that have undergone 50 years of natural selection for resistance to myxomatosis. Expression of IL-4 significantly enhanced virulence of both virulent and attenuated virus strains in susceptible (laboratory) and resistant (wild) rabbits. SLS-IL-4 completely overcame genetic resistance in wild rabbits. The pathogenesis of SLS-IL-4 was compared in susceptible and resistant rabbits. The results support a model for resistance to myxomatosis of an enhanced innate immune response controlling virus replication and allowing an effective antiviral cell-mediated immune response to develop in resistant rabbits. Expression of IL-4 did not overcome immunity to myxomatosis induced by immunization.

The complete cDNA sequences of IL-2, IL-4, IL-6 AND IL-10 from the European rabbit (Oryctolagus cuniculus).

The cDNAs for four rabbit cytokine genes [interleukin 2 (IL-2), IL-4, IL-6 and IL-10] have been cloned from primary lymphocytes by polymerase chain reaction (PCR) methods. IL-2 and IL-10 are both highly conserved between rabbit and other species. IL-4 and IL-6 are less strongly conserved, at both nucleotide and amino acid levels, and exhibit structural differences. An extension of the coding region of rabbit IL-6 relative to all other reported IL-6 genes results from a mutation in the usual stop codon which allows translation to continue for a further 27 amino acids. Analysis of IL-6 from four other lagomorph species suggests that this mutation is specific to the European rabbit. Sequence and structural differences of IL-4 and IL-6, while presumably not altering function, may render them highly species-specific. Several alternatively spliced variants of IL-2 and IL-4 are also reported.

The structure, sequence and developmental pattern of expression of the white gene in the blowfly Lucilia cuprina.

We have sequenced the complete coding region of the white gene of Lucilia cuprina. Strong sequence identity exists between this gene and its homologue from Drosophila melanogaster at both nucleotide and derived amino acid levels (68% and 78% respectively). The exon/intron structure of the two genes is also largely conserved, although the Lucilia gene contains one extra intron. Expression of the gene peaks during mid-pupal stage, with secondary peaks in late larval and early adult stages. Comparisons between this and other white genes will contribute to a better understanding of ATP-binding transmembrane transport proteins. The white gene should also serve as a useful marker gene in the development of a gene transformation system for the sheep blowfly.

The hermit transposable element of the Australian sheep blowfly, Lucilia cuprina, belongs to the hAT family of transposable elements.

We report the cloning of hermit, a member of the hAT family of transposable elements from the genome of the Australian sheep blowfly, Lucilia cuprina. Hermit is 2716 bp long and is 49% homologous to the autonomous hobo element, HFL1, at the nucleic acid level. Hermit has 15 bp terminal inverted repeats that share 10 bp with the terminal inverted repeats of HFL1. Conceptual translation reveals a 583 residue open reading frame (ORF) that is 64% similar and 42% identical to the HFL1 ORF. However, the sequence of the hermit element contains two frameshifts within the putative ORF, indication that hermit is an inactive element. Analysis of L. cuprina strains from within and outside Australia suggested that hermit is present as a single copy in all the genomes analysed.

Genomic sequences with homology to the P element of Drosophila melanogaster occur in the blowfly Lucilia cuprina.

We have cloned two DNA elements (Lu-P1 and Lu-P2) from the Australian sheep blowfly Lucilia cuprina that are similar to the transposable P element of Drosophila melanogaster in both structure and sequence but have diverged from it and from each other considerably. Hybridization studies indicate that a third related element probably exists in another, as yet unsequenced, clone. Neither Lu-P1 nor Lu-P2 appears to be active in terms of mobility, and it is not known whether any transposition-competent copies of other related elements occur in the genome of the blowfly. However, the isolation of any P-like sequences from a species outside of the family Drosophilidae allows comparisons to be made of more widely divergent P-related elements than has been possible previously. We are unaware of any report of the presence of multiple P-like family members within a single species. The discovery of Lu-P1 and Lu-P2 in the blowfly fuels the possibility that similar elements may be widespread in insects, and perhaps in other orders of animals.

Characterization and chromosomal distribution of a tandemly repeated DNA sequence from the Australian sheep blowfly, Lucilia cuprina.

In the course of making a Lucilia cuprina genomic DNA library, a ladder of bands was seen in partial Sau3A digests. Complete digestion reduced this ladder to predominantly monomer units of approximately 190 bp. Nine independently isolated copies of this repeat were cloned and sequenced. Only two of these isolates are identical in sequence, the most divergent being 71% homologous. This satellite DNA occurs in all three wild-type strains tested, and, for the single case examined, in the embryonic, larval, pupal, and adult DNA. It represents approximately 3%-4% of the genome. Data obtained from in situ chromosome hybridizations indicate that this sequence is concentrated around the centromeric regions of the autosomes and over most of the sex chromosomes. Labelling is much stronger in mitotic compared with polytene chromosomes showing directly that this centromeric satellite DNA is grossly under-replicated during polytenization. This under-replication is even more pronounced on the sex chromosomes compared with the autosomes.