more than 10-fold inhibition. Core protein variants with mutations in all phosphorylation sites exhibited dominant-negative effects on RNA encapsidation by wild-type protein. The results suggest that the presence of phosphoserine at position 162 of HBV core protein is required for pregenomic-RNA encapsidation, whereas phosphoserine at position 170 optimizes the process and serine might be preferable in position 155. Examination of the pregenomic-RNA-encapsidating capacities of DHBV core protein variants, in which four phosphorylation sites were jointly mutated to alanine or aspartic acid, suggests that phosphorylation of DHBV core protein at these sites may optimize pregenomic-RNA encapsidation but that its impact is much less profound than in the case of HBV. The possible mechanisms by which RNA encapsidation may be modulated by core protein phosphorylation are discussed in the context of the observed differences between the two viruses.The DNA genome of hepadnaviruses is replicated through reverse transcription of an RNA intermediate, the pregenomic RNA (reviewed in reference 4). Replication begins with encapsidation of pregenomic RNA, a process which requires two viral proteins, core (capsid) protein and polymerase; however, the enzymatic activities of the polymerase are not essential. Sequential synthesis of a minus-strand DNA by reverse transcription and synthesis of the second DNA strand are carried out by the polymerase inside the assembled nucleocapsid.

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Hepatitis E virus (HEV) is a major cause of acute hepatitis in many developing countries. Based on data from nonendemic regions, an animal reservoir of HEV has been proposed; however, data from HEV-endemic regions are limited. We tested sera from 200 pigs, 98 chickens, 86 goats, 58 sheep and 30 buffaloes for anti-HEV IgG using two different enzyme immunoassays. Specificity of the detected antibodies was confirmed using inhibition assays. Stool specimens from 210 pigs, 94 piglets and 37 sheep were tested for HEV-RNA using nested amplification methods; the polymerase chain reaction products were sequenced and compared with known human and swine HEV sequences. Of the 200 swine sera, 193 and 195, respectively, tested positive in the two assays. All goat sera showed anti-HEV reactivity in both the assays. Inhibition studies confirmed the HEV specificity of antibodies detected in swine and goat sera using both the assays. Sera from sheep, buffalo and chickens also showed high rates of apparent reactivity, but inhibition studies were unable to confirm the specificity of reactions in these species. One faecal specimen showed amplification using Indian swine HEV-specific primers. The genomic sequence of the amplicon from this isolate had only 76-79% nucleotide and 93% amino acid homology with human HEV isolates reported from India and other parts of the world, and most closely resembled swine HEV isolates from other parts of India. Infection with HEV or a related agent is widespread among animals in northern India. However, the swine HEV in India differs genetically from human HEV isolates, indicating that pigs may not play an important role in the spread of human hepatitis E in endemic regions.

Antibody to the capsid (PORF2) protein of hepatitis E virus (HEV) isHepatitis E virus (HEV) is responsible for epidemic and sporadic cases of enterically transmitted viral hepatitis, particularly in the developing world (17, 31). HEV is a singlestranded, positive-sense RNA virus, with the genome encoding three open reading frames (ORFs), of which ORF2 encodes the major structural or capsid protein, PORF2. Antibody is sufficient to confer immunity to HEV infection (38), but little is known of the structure of the viral particle or of the antibody specificities which contribute to humoral immunity, which could pose a major hurdle in the development and clinical evaluation of effective vaccines.The use of peptide scanning has led to the identification of a number of linear epitopes within the capsid protein of HEV, with many of these being type specific (4, 12, 13, 15). More recently, the use of larger overlapping peptides has revealed some conformational epitopes which are reactive with acutephase sera (16). Linkage of a number of such peptide epitopes from different strains of HEV into an artificial "mosaic" protein improves the detection of acute-phase HEV antibody (5, 14), but the antibodies induced with this protein do not appear to be neutralizing in a cell culture system which measures virus-cell binding (26). In addition, it is not known whether antibodies to any of these linear and conformational peptide epitopes can bind to intact viral particles, or indeed whether this antibody repertoire is maintained during the convalescent phase after HEV infection and thus contributes to humoral immunity.Expression of PORF2 with an N-terminal truncation of 111 amino acids (aa) in the baculovirus system results in the production of virus-like particles (VLPs), which, in contrast to synthetic peptides or full-length PORF2, appear to mimic the antigenicity and immunogenicity of the native virus (24,25,32). Most significantly, immunization of macaques with VLPs confers immunity to both homologous and heterologous virus challenge (37,38,43). The improved antibody reactivity and immunogenicity of VLPs have been attributed to conformational epitopes which are not presented by synthetic peptides, full-length PORF2, and most HEV proteins expressed in Escherichia coli (24,25), but the relevant epitopes have not been identified.Expression of the ORF2.1 fragment of PORF2 (aa 394 to 660) in E. coli also results in the presentation of conformational epitopes (2,22,23). Since antisera from animals immunized with ORF2.1 are able to inhibit the reactivity of HEV patient sera against VLPs by as much as 97% (21), it appears likely that the major epitopes within VLPs and the ORF2.1 antigen expressed in E. coli may be the same or closely overlapping.In this study we have used monoclonal antibodies (MAbs) to study the antigenic structure of HEV in more detail. We show that the conformational ORF2.1 epitope involving aa residues 394 to 457 and a linear epitope in the region of aa 434 to 457 are not only present on the surface of VLPs, but ...

The original SuperPave asphalt binder specification criterion for fatigue, G* sin δ, has received considerable criticism. Recently, a time sweep using the dynamic shear rheometer (DSR) has been proposed as an alternative test method for developing load-associated fatigue information for asphalt binders. This proposed test method is examined with respect to a phenomenon called edge fracture. Edge fracture is reported in the literature for steady state and oscillatory flow in DSR, but it has not been reported for asphalt binders. The modulus, when plotted versus number of cycles generated in a time sweep test, has the appearance typical of fatigue behavior; however, the actual response of the material depends markedly on the initial modulus of the material. The development of the modulus with repeated shearing is described with respect to flow of the asphalt binder at its circumference. The data are examined with respect to their validity as a measure of fatigue, and recommendations with respect to the use of time sweep data in a binder specification are presented.