Field evidence for mechanical transmission of rabbit haemorrhagic disease virus (RHDV) by flies (Diptera : Calliphoridae) among wild rabbits in Australia

Field collected flies were screened for the presence of rabbit haemorrhagic disease virus (RHDV) by applying reverse transcriptase PCR (RT-PCR) in which primers specific to the capsid protein of the virus were used. The virus was detected in flies from locations where rabbit haemorrhagic disease (RHD) was reported and also soon after the release of RHDV in a 'clean' area. Oral and/or anal excretions of flies (flyspots) were found to contain viable virus and oral inoculation of rabbits revealed that a single flyspot was able to cause RHD. We conclude that flyspots are a major potential source of the virus for oral or conjunctival transmission of the virus to rabbits. (C) 1998 Elsevier Science B.V. All rights reserved.

Retinal and lenticular ultrastructure in the aestivating salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) with special reference to a new type of photoreceptor mosaic

The salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) is endemic to southwestern Australia and inhabits shallow, freshwater pools which evaporate during the hot summer months. Burrowing into the substrate in response to falling water levels allows these fish to aestivate for extended periods of time while encapsulated in a mucous cocoon even when the pools contain no water. Only a few minutes after a major rainfall, these fish emerge into relatively clear water which subsequently becomes laden with tannin, turning the water black and reducing the pH to approximately 4.3. As part of a large study of the visual adaptations of this unique species, the retinal and lenticular morphology of the aestivating salamanderfish is examined at the level of the light and electron microscopes. The inner retina is highly vascularised by a complex system of vitreal blood vessels, while the outer retina receives a blood supply by diffusion from a choriocapillaris. This increased retinal blood supply may be an adaptation for reducing the oxygen tension during critical periods of aestivation. Large numbers of Muller cells traverse the thickness of the retina from the inner to the outer limiting membranes. The ganglion cells are arranged in two ill-defined layers, separated from a thick inner nuclear layer containing two layers of horizontal cells by a soma-free inner plexiform layer. The photoreceptors can be divided into three types typical of many early actinopterygian representatives; equal double cones, small single cones and large rods (2:1:1). These photoreceptors are arranged into a unique regular square mosaic comprising a large rod bordered by four equal double cones with a small single cone located at the corner of each repeating unit. The double cones may optimise perception of mobile prey which it tracks by flexion of its head and neck and the large rods may increase sensitivity in the dark tannin-rich waters in which it lives. Each single cone also possesses a dense collection of polysomes and glycogen (a paraboloid) beneath its ellipsoid, the first such finding in teleosts. The retinal pigment epithelium possesses melanosomes, pha,oocytes and a large number of mitochondria. The anatomy of the retina and the photoreceptor mosaic is discussed in relation to the primitive phylogeny of this species and its unique life history.

Mucosal immunisation with papillomavirus virus-like particles elicits systemic and mucosal immunity in mice

It has been shown previously that recombinant virus-like particles (VLPs) of papillomavirus can induce VLP-specific humoral and cellular immune responses following parenteral administration. To test whether mucosal administration of bovine papillomavirus type 1 (BPV1) VLPs could produce mucosal as well as systemic immune responses to VLPs, 50 mu g chimeric BPV1 VLPs containing an HPV16 E7 CTL epitope (BPVL1/E7 VLP) was administered intranasally to mice. After two immunisations, L1-specific serum IgG and IgA were observed. L1-specific IgG and IgA were also found in respiratory and vaginal secretions. Both serum and mucosal antibody inhibited papillomavirus VLP-induced agglutination of RBC, indicating that the antibody induced by mucosal immunisation may recognize conformational determinants associated with virus neutralisation. For comparison, VLPs were given intramuscularly, and systemic and mucosal immune responses were generally comparable following systemic or mucosal delivery. However, intranasal administration of VLP induced significantly higher local IgA response in lung, suggesting that mucosally delivered HPV VLP may be more effective for mediating local mucosal immune responses. Intranasal immunisation with HPV6b L1 VLP produced VLP-specific T proliferative responses in splenocytes, and immunisation with BPVL1 VLP containing an HPV16 E7 CTL epitope induced E7-specific CTL responses. We conclude that immunisation with papillomavirus VLPs via mucosal and intramuscular routes, without adjuvant, can elicit specific antibody at mucosal surfaces and also systemic VLP epitope specific T cell responses. These findings suggest that mucosally delivered VLPs may offer an alternative HPV VLP vaccine strategy for inducing protective humoral immunity to anogenital HPV infection, together with cell-mediated immune responses to eliminate any cells which become infected. (C) 1998 Academic Press.

Sequence analysis of rabbit haemorrhagic disease virus (RHDV) in Australia: alterations after its release

Liver samples from rabbits killed by RHDV, collected from five States in Australia in 1996 and 1997 were analysed by RT-PCR. A 398 bp fragment of the capsid protein (VP60) gene was amplified by PCR and directly sequenced. The alignment of the nucleotide and amino acid sequences and their comparison with the original strain of the virus released in Australia indicated genetic changes after two years have been small with 98.2% to 100% identity. The constructed phylogenetic tree suggests slight differences in nucleotide substitutions in various States but there is no clear evidence of clustering of sequences according to their geographic origin. In practical terms, sequencing of viral RNA provides a means of testing the efficacy of further releases and subsequent spread of the virus if such a strategy is employed as a means of enhancing RHD as a biological control of the wild rabbit in Australia.

The foveal photoreceptor mosaic in the pipefish, Corythoichthyes paxtoni (Syngnathidae, Teleostei)

The foveal and non-foveal retinal regions of the pipefish, Corythoichthyes paxtoni (Syngnathidae, Teleostei) are examined at the level of the light and electron microscopes. The pipefish possesses a deep, pit (convexiclivate) fovea which, although lacking the displacement of the inner retinal layers as described in other vertebrate foveae, is characterised by the exclusion of rods, a marked increase in the density of photoreceptors and a regular square mosaic of four double cones surrounding a central single cone. In the perifoveal and peripheral retinal regions, the photoreceptor mosaic is disrupted by the insertion of large numbers of rods, which reduce spatial resolving power but may uniformly increase sensitivity for off-axis rays. In addition to a temporal fovea subtending the frontal binocular field, there is also a central area centralis subtending the monocular visual field. Based on morphological comparisons with other foveate teleosts, four foveal types are characterised and foveal function discussed with respect to the theoretical advantage of a regular square mosaic.

Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Angstrom resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

Molecular characterization of Drosophila C virus isolates

Reverse transcription coupled with polymerase chain reaction and restriction enzyme analysis was used to characterize 12 Drosophila C virus isolates from geographically different regions. A 1.2-kb fragment was amplified from cDNA and profiles from digestion with 20 restriction enzymes were generated. Analysis of the restriction fragment data gave estimates of nucleotide divergence of 0-10% between isolates. The isolates were grouped on the basis of genetic distance estimates derived from the restriction data. For the isolates from which a single genotype could be purified, a geographical pattern in the distribution of viral genotypes was identified. The 4 Moroccan isolates were very closely related to each other, differing in only 1 restriction profile. The 2 Australian isolates were each other's closest relatives, as were the 2 isolates first recovered in France. The PCR-RFLP technique used in this study has provided us with a simple procedure which can be used to characterize DCV isolates. A single enzyme, Tag I, generated 5 distinct and diagnostic restriction fragment patterns, which allowed easy assignment of isolates to one of the five viral genotypes identified in this study. (C) 1999 Academic Press.

Ontogenetic changes in the retinal photoreceptor mosaic in a fish, the black bream, Acanthopagrus butcheri

The morphological development of the photoreceptor mosaic was followed by light and electron microscopy in a specific region of dorsal retina of the black bream, Acanthopagrus butcheri (Sparidae, Teleostei), from hatching to eight weeks of age. The retina was differentiated when the larvae reached a total length of 3 mm (3-5 days posthatch). Single cones, arranged in tightly packed rows, were the only morphologically distinct type of photoreceptor present until the larvae were 6 mm (day 15) in standard length (SL). At this time, the rad nuclei had become differentiated and the ellipsoids of selected cones began to form subsurface cisternae along neighbouring cone membranes. In this way, double, triple, quadruple, and occasionally photoreceptor chains of up to 10 cones were formed. At 8 mm SL, there was little apparent order in the photoreceptor mosaic. However, concomitant with subsequent growth, quadruple and other multiple cone receptors disappeared, with the exception of the triple cones, which gradually reduced in both number and retinal coverage to be restricted to central retina by 15 mm SL (days 40-55). Following this stage, the arrangement of double and single cones peripheral to the region of triple cones in dorsal retina was transformed into the adult pattern of a regular mosaic of four double cones surrounding a single cone. These results demonstrate that an established photoreceptor mosaic of rows of single cones can be reorganised to form a regular square mosaic composed of single and double cones. J. Comp. Neural. 412:203-217, 1999. (C) 1999 Wiley-Liss, Inc.

HPV6b virus like particles are potent immunogens without adjuvant in man

Subjects with genital warts were immunized three times or more with HPV6b VLPs without adjuvant. All immunized subjects had DTH to HPV6b L1 protein. Of 32 subjects, nine had HPV6b specific antibody prior to immunization and 22 acquired antibody with immunization. VLP specific antibody increased following a single immunization in 6 of 8 subjects with low level antibody at recruitment. Complete regression of genital warts was observed in 25 of 33 evaluable subjects over the 20-week observation period. We conclude that immunization with HPV6b L1 VLPs without adjuvant induces immunity to the L1 protein epitopes recognised during natural infection, and may accelerate regression of warts. (C) 2000 Elsevier Science Ltd. All rights reserved.

Functional implications of the human T-lymphotropic virus type 1 transmembrane glycoprotein helical hairpin structure

Retrovirus entry into cells follows receptor binding by the surface exposed envelope glycoprotein (Env) subunit (SU), which triggers the membrane fusion activity of the transmembrane (TM) protein. TM protein fragments expressed in the absence of SU adopt helical hairpin structures comprising a central coiled coil, a region of chain reversal containing a disulfide-bonded loop, and a C-terminal segment that packs onto the exterior of the coiled coil in an antiparallel manner. Here we used in vitro mutagenesis to test the functional role of structural elements observed in a model helical hairpin, gp21 of human T-lymphotropic virus type 1. Membrane fusion activity requires the stabilization of the N and C termini of the central coiled coil by a hydrophobic N cap and a small hydrophobic core, respectively. A conserved Gly-Gly hinge motif preceding the disulfide-bonded loop, a salt bridge that stabilizes the chain reversal region, and interactions between the C-terminal segment and the coiled coil are also critical for fusion activity. Our data support a model whereby the chain reversal region transmits a conformational signal from receptor-bound SU to induce the fusion-activated helical hairpin conformation of the TM protein.

Papillomavirus virus-like particles for the delivery of multiple cytotoxic T cell epitopes

Chimeric papillomavirus (PV) virus-like particles (VLPs) based on the bovine papillomavirus type 1 (BPV-1) L1 protein were constructed by replacing the 23-carboxyl-terminal amino acids of the BPV1 major protein L1 with an artificial polytope minigene, containing known CTL epitopes of human PV16 E7 protein, HIV IIIB gp120 P18, Nef, and reverse transcriptase (RT) proteins, and an HPV16 E7 linear B epitope. The CTL epitopes were restricted by three different MHC class 1 alleles (H-2(b), H-2(d), HLA-A*0201). The chimeric L1 protein assembled into VLPs when expressed in SF-9 cells by recombinant baculovirus. After immunization of mice with polytope VLPs in the absence of adjuvant, serum antibodies were detected which reacted with both polytope VLPs and wild-type BPV1L1 VLPs, in addition to the HPV16E7 linear B cell epitope. CTL precursors specific for the HPV16 E7, HIV P18, and RT CTL epitopes were also detected in the spleen of immunized mice. Polytope VLPs can thus deliver multiple B and T epitopes as immunogens to the MHC class I and class II pathways, extending the utility of VLPs as self-adjuvanting immunogen delivery systems. (C) 2000 Academic Press.

Differences in the post-translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system

Virus-like particles (VLPs) are being currently investigated in vaccines against viral infections in humans. There are different recombinant-protein-expression systems available for obtaining the necessary VLP preparation for vaccination. However, the differences in post-translational modifications of the recombinant proteins obtained and their differences in efficacy in eliciting an anti-viral response in vaccines are not well established. In this study we have compared the posttranslational modifications of human papillomavirus type-6b major capsid protein L1 (HPV 6bL1) expressed using recombinant baculovirus (rBV) in Sf9 (Spodoptera frugiperda) insect cells, with the protein expressed using recombinant vaccinia virus (rVV) in CV-1 kidney epithelial cells, Two-dimensional gel electrophoresis of biosynthetically labelled rBV-expressed HPV 6bL1 showed several post-translationally modified variants of the protein, whereas rVV-expressed HPV 6bL1 showed only a few variants. Phosphorylations were detected at threonine and serine residues for the L1 expressed from rBV compared with phosphorylation at serine residues only for the L1 expressed from rVV. HPV 6bL1 expressed using rBV incorporated [H-3]mannose and [H-3]galactose, whereas HPV 6bL1 expressed using rVV incorporated only [H-3]galactose. We conclude that post-translational modification of recombinant HPV 6bL1 can differ according to the system used for its expression. Since recombinant L1 protein is a potential human-vaccine candidate, the implication of the observed differences in post-translational modifications on immunogenicity of L1 VLPs warrants investigation.

Multiple conformational epitopes are recognized by natural and induced immunity to the E7 protein of human papilloma virus type 16 in man

The reactivity of sera from patients with cervical cancer with the E7 protein of human papilloma virus type 16 (HPV16) was estimated using a novel non-radioactive immunoprecipitation assay and four established protein-and peptide-based immunoassays. Six of 14 sera from patients with cervical cancer and 1 of 10 sera from healthy laboratory staff showed repeated reactivity with E7 in at least one assay. Four of the 7 reactive sera were consistently reactive in more than one assay, but only one was reactive in all four assays. Following immunization with E7, 2 of 5 patients with cervical cancer had increased E7-specific reactivity, measurable in one or more assays. No single assay was particularly sensitive for E7 reactivity, or predictive of cervical cancer. Mapping of E7 reactivity to specific E7 peptides was unsuccessful, suggesting that natural or induced E7 reactivity in human serum is commonly directed to conformational epitopes of E7, These results suggest that each assay employed with is study measures a different aspect of E7 reactivity, and that various reactivities to E7 may manifest following HPV infection or immunization. This finding is of significance for monitoring of E7 immunotherapy and for serological screening for cervical cancer. Copyright (C) 2000 S.Karger, AG. Basel.

Trichomonas vaginalis is associated with pelvic inflammatory disease in women infected with human immunodeficiency virus

We assessed the association between the causative agents of vaginal discharge and pelvic inflammatory disease (PID) among women attending a rural sexually transmitted disease clinic in South Africa; the role played by coinfection with human immunodeficiency virus type 1 (HIV-1) was studied. Vaginal and cervical specimens were obtained to detect Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, and bacterial vaginosis. HIV-1 infection was established by use of serum antibody tests. A total of 696 women with vaginal discharge were recruited, 119 of whom had clinical PID. Patients with trichomoniasis had a significantly higher risk of PID than did women without trichomoniasis (P = .03). PID was not associated with any of the other pathogens. When the patients were stratified according to HIV-1 status, the risk of PID in HIV-1-infected patients with T. vaginalis increased significantly (P = .002); no association was found in patients without HIV-1. T. vaginalis infection of the lower genital tract is associated with a clinical diagnosis of PID in HIV-1-infected women.

Sequence analysis and expression of a virus-like particle protein,VLP2, from the parasitic wasp Venturia canescens

Endoparasitoid wasps produce maternal protein secretions, which are transported into the body of insect hosts at oviposition to regulate host physiology for successful development of their offspring. Venturia canescens calyx fluid contains so-called virus-like particles (VLPs) that are essential for immune evasion of the developing parasitoid inside the host. VLPs consist of four major proteins. In this paper, we describe the isolation and molecular cloning of a gene (vlp2) that is a constituent of VLPs and discuss its possible role in VLP structure and function.