- Volume 86, Issue 1, 2005

The interaction between human papillomavirus (HPV) particles and cell surface heparan sulfate requires intact conformation of the HPV particles. Type-specific HPV serology is currently based on virus-like particles (VLPs) with intact conformation. Presence of incorrectly folded VLPs in VLP preparations is recognized as an important cause of cross-reactivity in HPV serology. Heparin-coated microtitre plates were evaluated for capturing conformationally correct VLPs and improving the type specificity of HPV serology. Hybrid VLPs between HPV16 and HPV11, which had been found to have significant reactivity with children's sera and a batch of HPV18 VLPs that had failed the quality control because of significant reactivity with sera from virginal women, were tested in parallel with heparin ELISA, ordinary ELISA and type-specific mAb capture ELISA. Control sera from children that had detectable reactivity with HPV16/11 hybrid VLPs in ordinary ELISA did not react in heparin-based ELISA, but some hybrid VLPs also had background reactivity in capture ELISAs. Control sera from virginal women that had some reactivity with a poor quality HPV18 VLP preparation in ordinary ELISA had no reactivity in heparin or capture ELISA, suggesting that certain VLP preparations expose cross-reactive epitopes that are not exposed on VLPs with heparin-binding ability. As the sensitivity was similar or only marginally affected by the use of heparin plates, use of heparin-coated plates may improve the type specificity of VLP-based ELISAs and reduce interassay variability attributable to variable quality of different VLP batches.

Binding of hepatitis B virus nucleocapsids to mouse B cells leads to production of nucleocapsid-specific antibodies, class II presentation of peptides and the generation of T helper-1 immunity. This T-cell-independent activation of B cells is thought to result from cross-linking of cell-surface immunoglobulin molecules, if these contain a specific motif in the framework region 1–complementarity determining region 1 junction. In the present study, it was observed that nucleocapsids bound to different B-cell lines, an interaction that was not dependent on cell-surface-expressed immunoglobulins. Furthermore, binding to several non-B-cell lines was observed. Capsids that lacked the carboxy-terminal protamine-like domains did not bind to cells. Treatment of nucleocapsids with ribonucleases enhanced the attachment of nucleocapsids to cells. Various soluble glycosaminoglycans inhibited attachment of nucleocapsids, while treatment of cells with heparinase I also reduced binding. These observations demonstrated that the arginine-rich protamine-like regions of the core proteins are responsible for the attachment of nucleocapsids to glycosaminoglycans expressed on the plasma membranes of cells.

The proline residue at position 306 in hepatitis B virus (HBV) reverse transcriptase (rtP306) has been suggested to constrain the conformation of the α-helices in the thumb subdomain that interacts with the viral DNA template-primer. To study the impact of residue rt306 in HBV replication further, 11 site-directed mutants were constructed that substituted rtP306 with different amino acids. The replicative competencies of these mutants were assayed by HepG2 cell transfection and real-time PCR. When rtP306 was substituted with glycine or threonine, the replication competency of these mutants was drastically reduced to 1·96 and 4·51 % of the wild-type HBV level, respectively. When rtP306 was substituted with glutamic acid, the replicative competency of the mutant increased up to 9·4-fold compared with wild-type virus. The results also showed that changes in the replicative competency of these constructed mutants were not associated with functional changes of HBV enhancer I. These results indicate the importance of amino acid(s) at the interface between the thumb and palm subdomains in modulating the replicative competency of HBV isolates. This regulatory residue(s) could serve as a new target for the development of anti-HBV drugs.

A second genotype of Mamestra configurata nucleopolyhedrovirus-A (MacoNPV-A), variant 90/4 (v90/4), was identified due to its altered restriction endonuclease profile and reduced virulence for the host insect, M. configurata, relative to the archetypal genotype, MacoNPV-A variant 90/2 (v90/2). To investigate the genetic differences between these two variants, the genome of v90/4 was sequenced completely. The MacoNPV-A v90/4 genome is 153 656 bp in size, 1404 bp smaller than the v90/2 genome. Sequence alignment showed that there was 99·5 % nucleotide sequence identity between the genomes of v90/4 and v90/2. However, the v90/4 genome has 521 point mutations and numerous deletions and insertions when compared to the genome of v90/2. Gene content and organization in the genome of v90/4 is identical to that in v90/2, except for an additional bro gene that is found in the v90/2 genome. The region between hr1 and orf31 shows the greatest divergence between the two genomes. This region contains three bro genes, which are among the most variable baculovirus genes. These results, together with other published data, suggest that bro genes may influence baculovirus genome diversity and may be involved in recombination between baculovirus genomes. Many ambiguous residues found in the v90/4 sequence also reveal the presence of 214 sequence polymorphisms. Sequence analysis of cloned HindIII fragments of the original MacoNPV field isolate that the 90/4 variant was derived from indicates that v90/4 is an authentic variant and may represent approximately 25 % of the genotypes in the field isolate. These results provide evidence of extensive sequence variation among the individual genomes comprising a natural baculovirus outbreak in a continuous host population.

The genetic variability and population structure of a collection of 45 Grapevine leafroll-associated virus 3 (GLRaV-3) isolates were studied by single-stranded conformation polymorphism (SSCP) and sequence analysis of the RNA-dependent RNA polymerase (RdRp), heat-shock protein 70 homologue (HSP-70) and coat protein (CP) genes. The distribution of SSCP profiles was not correlated with the geographical origin of the isolates, indicating that GLRaV-3 is a single, undifferentiated population. The majority of the isolates showed an SSCP profile and a population structure that were composed of a single predominant variant. However, 10 % of the isolates for the RdRp and HSP-70 genes and 15 % for the CP gene were composed of a combination of two or more variants. Estimation of genetic diversity and phylogenetic analysis disclosed the possible existence of vines with mixed infections by diverging sequence variants, showing, in some cases, possible recombination events. Furthermore, differences in the genetic diversity and constraints existing in the three regions analysed indicated a higher variability in the CP gene. The epidemiological and biological implications of this finding are discussed.

The RNA-binding properties of the cell-to-cell movement protein (MP) of Apple chlorotic leaf spot virus were analysed. MP was expressed in Escherichia coli and was used in UV-crosslinking analysis, using a digoxigenin–UTP-labelled RNA probe and gel-retardation analysis. The analyses demonstrated that MP bound cooperatively to single-stranded RNA (ssRNA). When analysed for NaCl dependence of the RNA-binding activity, the majority of the MP could bind ssRNA even in binding buffer with 1 M NaCl. Furthermore, competition binding experiments showed that the MP bound preferentially to ssRNA and single-stranded DNA without sequence specificity. MP deletion mutants were used to identify the RNA-binding domain by UV-crosslinking analysis. Amino acid residues 82–126 and 127–287 potentially contain two independently active, single-stranded nucleic acid-binding domains.

Until June 2004, thirty-eight scrapie cases with unusual features, designated Nor98, have been diagnosed in Norway. This study investigated the distribution of PrP genotypes among Nor98 cases, their flock-mates and a random sample of Norwegian slaughtered sheep. The PrP genotype distribution of Nor98 cases differed markedly from that of previous cases of classical scrapie. A leucine/phenylalanine polymorphism at codon 141 with hitherto unknown significance to scrapie was strongly associated with Nor98 cases. Twenty of 38 (52·6 %) cases were either homozygous or heterozygous for phenylalanine at codon 141. In contrast, this allele was present in only 10·5 % of the flock-mates and 4·5 % of the random sample of slaughtered sheep. Moreover, the H154 allele was represented in 24 of 38 (63·2 %) of Nor98 cases, as opposed to 27·0 % of Nor98 flock-mates and 17·0 % of the slaughtered sheep.

In Creutzfeldt–Jakob disease (CJD), the type (type 1 or 2) of abnormal isoform of the prion protein (PrPSc) in the brain and the genotype at codon 129 of the PrP gene are major determinants of clinicopathological phenotype. Little is known about the difference in biochemical properties between the two types of PrPSc, except for the different proteinase K cleavage sites. To investigate the size of aggregates formed by PrPSc types 1 and 2, brain homogenates from various cases of CJD with the same genotype (homozygous for methionine at codon 129) were passed through filters with a mean pore size of 72±4 nm. Type 2 PrPSc was efficiently removed from the filtrates by the filters, in contrast to type 1. Even type 2 PrPSc from a patient without amyloid plaques was removed more efficiently than type 1 from patients with amyloid plaques. These results indicate that type 2 PrPSc has a larger aggregation size than type 1, irrespective of the existence of amyloid plaques.

Transmissible spongiform encephalopathies (TSEs), sometimes known as prion diseases, are caused by an infectious agent whose molecular properties have not been determined. Traditionally, different strains of TSE diseases are characterized by a series of phenotypic properties after passage in experimental animals. More recently it has been recognized that diversity in the degree to which an abnormal form of the host protein PrP, denoted PrPSc, is glycosylated and the migration of aglycosyl forms of PrPSc on immunoblots may have some differential diagnostic potential. It has been recognized that these factors are affected by the strain of TSE agent but also by other factors, e.g. location within the brain. This study shows in some cases, but not others, that host PrP genotype has a major influence on the degree of PrPSc glycosylation and migration on gels and provides further evidence of the effect of brain location. Accordingly both the degree of glycosylation and the apparent molecular mass of PrPSc may be of some value for differential diagnosis between TSE strains, but only when host effects are taken into account. Furthermore, the data inform the debate about how these differences arise, and favour hypotheses proposing that TSE agents affect glycosylation of PrP during its biosynthesis.