The hepatitis B virus pregenome: prediction of RNA structure and implications for the emergence of deletions.

The terminally redundant pregenomic RNA of human hepatitis B virus (HBV) comprises some 3,330 nucleotides and is a replicative intermediate in the production of the circular DNA genome. Deletions are known to arise in the HBV genome during the course of chronic infection and are sometimes associated with interferon therapy. These deletions are limited to small parts of the genome such as the 357-nucleotide pre-S1 region. Long RNA molecules such as the HBV pregenome have considerable structural flexibility and will undergo secondary structure shifts between energetically favourable states in a continuous and semi-random fashion. Since prediction of structure elements that are highly conserved in different forms of one RNA molecule is now feasible by computer modelling, we have analysed the whole HBV pregenome by two different RNA structure prediction algorithms and by new methods that exploit these algorithms. Significantly, the ends of pregenomic RNA were predicted to undergo both short-range and long-range interactions, which has relevance to our knowledge of the virus replicative cycle. By incorporating phylogenetic information relating to the 6 recognised genotypes of HBV, it was possible to highlight short secondary structures that may be common to all HBV strains. For example, although the pre-S1 region was predicted to undergo local folding of a loosely defined nature, most observed pre-S1 deletions mapped to all or part of an arm carrying a better-defined structure. The loss of such sequences may be mechanistically attributable to polymerase skipping during reverse transcription, and the possible advantages of such deletions are considered.

Recent high incidence of fulminant hepatitis in Samara, Russia: molecular analysis of prevailing hepatitis B and D virus strains.

Until 1991, the Russian city of Samara was largely isolated from other parts of Russia and the rest of the world. Very recently, Samara has seen an alarming increase in the incidence of hepatitis. The proportion of fulminant cases is unusually high. We wanted to assess the roles of hepatitis B virus (HBV) and hepatitis D virus (HDV) in acute viral hepatitis in this region by analyzing the prevailing strains of both and by determining their genotypes and possible origin. Serum samples were screened for different serological markers and by PCR followed by direct sequencing. Of the 94 HBV-positive samples (80% of which were acute infections), 37 (39%) were also HDV positive. Sixty-seven percent of the patients had anti-HCV antibodies. Twenty-five percent of all patients in the study had fulminant hepatitis. Statistically significant sex differences were found among fulminant cases. For HBV, the core promoter sequences of 62 strains were determined and all but one were found to be of genotype D. None of these had any deletions. Only one strain, from a patient with fulminant fatal hepatitis, showed multiple mutations. The pre-S2 region sequences of 31 HBV strains were also compared. Phylogenetically, these fell into two distinct groups within genotype D, suggesting different origins. For HDV, part of the region encoding the delta-antigen was sequenced from four strains. All proved to be of genotype I and were similar to Far Eastern and Eastern European strains. The contribution of intravenous drug use to the sharp increase in viral hepatitis in this unique setting is discussed.

Initial interactions of subgenus D adenoviruses with A549 cellular receptors: sialic acid versus alpha(v) integrins.

Selected members of the adenovirus family have been shown to interact with the coxsackie adenovirus receptor, alpha(v) integrins, and sialic acid on target cells. Initial interactions of subgenus D adenoviruses with target cells have until now been poorly characterized. Here, we demonstrate that adenovirus type 8 (Ad8), Ad19a, and Ad37 use sialic acid as a functional cellular receptor, whereas the Ad9 and Ad19 prototypes do not.

An epitope on the adenovirus fibre tail is common to all human subgroups.

A group specific linear epitope with the sequence -FNPVY- was detected in the tail region of the adenovirus fibre by using a monoclonal antibody (Mab) and selection with a hexapeptide phage expression library. A synthetic peptide with sequence DTFNPVYPYDTE from adenovirus type 2 (Ad2) was shown by preincubation with the Mab to block its binding to fibre. A biotinylated form of this peptide bound to the monomeric fibre and not to the dimeric and trimeric forms. On the other hand the monoclonal antibodies bound to the monomeric, dimeric and trimeric forms of the fibre.

Adenovirus type 37 uses sialic acid as a cellular receptor.

Two cellular receptors for adenovirus, coxsackievirus-adenovirus receptor (CAR) and major histocompatibility complex class I (MHC-I) alpha2, have recently been identified. In the absence of CAR, MHC-I alpha2 has been suggested to serve as a cellular attachment protein for subgenus C adenoviruses, while members from all subgenera except subgenus B have been shown to interact with CAR. We have found that adenovirus type 37 (Ad37) attachment to CAR-expressing CHO cells was no better than that to CHO cells lacking CAR expression, suggesting that CAR is not used by Ad37 during attachment. Instead, we have identified sialic acid as a third adenovirus receptor moiety. First, Ad37 attachment to both CAR-expressing CHO cells and MHC-I alpha2-expressing Daudi cells was sensitive to neuraminidase treatment, which eliminates sialic acid on the cell surface. Second, Ad37 attachment to sialic acid-expressing Pro-5 cells was more than 10-fold stronger than that to the Pro-5 subline Lec2, which is deficient in sialic acid expression. Third, neuraminidase treatment of A549 cells caused a 60% decrease in Ad37 replication in a fluorescent-focus assay. Moreover, the receptor sialoconjugate is most probably a glycoprotein rather than a ganglioside, since Ad37 attachment to sialic acid-expressing Pro-5 cells was sensitive to protease treatment. Ad37 attachment to Pro-5 cells occurs via alpha(2-->3)-linked sialic acid saccharides rather than alpha(2-->6)-linked ones, since (i) alpha(2-->3)-specific but not alpha(2-->6)-specific lectins blocked Ad37 attachment to Pro-5 cells and (ii) pretreatment of Pro-5 cells with alpha(2-->3)-specific neuraminidase resulted in decreased Ad37 binding. Taken together, these results suggest that, unlike Ad5, Ad37 makes use of alpha(2-->3)-linked sialic acid saccharides on glycoproteins for entry instead of using CAR or MHC-I alpha2.

Adenovirus type 41 lacks an RGD alpha(v)-integrin binding motif on the penton base and undergoes delayed uptake in A549 cells.

Human adenovirus (Ad) types 2, 3 and 12 are known to interact with cell surface integrins alpha(v)beta(3) and alpha(v)beta(5) through an RGD motif carried by the penton base. This interaction is thought to augment virus entry after initial contact between the fiber and specific receptor(s). Ad40 and Ad41 are the only members of the human subgroup F adenoviruses. The penton base protein sequence of one Ad40 strain is known to carry the motif RGAD rather than RGD, suggesting that not all human adenoviruses use the above integrins for cell entry. We confirmed that different genomic variants of Ad40 all carry an RGAD motif on the penton base, and found that the Ad41 prototype and several other genomic variants of Ad41 carry the motif IGDD in place of RGAD or RGD. This region is most likely exposed on the Ad41 particle, but attempts to block Ad41 infectivity using a homologous peptide were unsuccessful. Infectivity of an Ad41 preparation as measured by fluorescent focus assay in A549 cells was highly dependent on the length of the adsorption period, indicating that fiber-mediated attachment is inefficient in these cells. Moreover, Ad41 virions adsorbed for 1 h were internalized in a semi-linear fashion over 8 h. This inefficient uptake may be a direct consequence of independence of subgroup F adenoviruses from alpha(v)beta(3) and alpha(v)beta(5) integrin-mediated endocytosis. Ad40 and Ad41 may thus have lost or may never have developed a dependence on the penton base RGD motif for entry.

Adenovirus core protein VII displays a linear epitope conserved in a range of human adenoviruses.

A monoclonal antibody (MAb) which recognized a linear epitope on polypeptide VII of human adenovirus (Ad) serotype 4 also interacted with polypeptides VII of Ad serotypes 2, 5, 7 and 10, but not with 12 and 40, in Western blotting. Utilizing a hexapeptide phage display library, the MAb was found to recognize the consensus sequence RXYXPX. A peptide based on a similar sequence from Ad2, viz. VEEARNYTPTPPPV, was synthesized and shown to inhibit binding of the MAb to polypeptide VII. Direct sequencing of the Ad4 polypeptide VII gene validated these observations, the sequence RNYTPA being detected. Comparison with gene sequences from other Ads indicates that this sequence is preserved in polypeptide VII of types 2 and 5 but in types 12 and 40 insertion of another residue disrupts this motif.

Characteristics and motives of adolescent volunteers in wildlife education.

The characteristics and motives of 63 suburban adolescents (20 boys, 43 girls) who are concerned with learning how to care for and make significant contributions to wildlife and the environment were assessed by telephone interviews. The data confirm studies of adults in that significantly more girls than boys became volunteers, significantly more volunteers' families than average families experienced caring interactions with animal life through pet ownership, and significantly more volunteers reported that concern for wildlife arose during early childhood rather than later. The data also indicate that early childhood experiences with pets, with adults acting as role models and providing social approval, and having instruction in wildlife care with peers all contributed to their positive attitudes toward wildlife and the pursuit of their volunteer work. Over-all, the results suggest that adolescents, wildlife, and the environment might benefit if wildlife care programs could be established for other youth such as inner city teenagers.

Hepatitis B virus X gene 1751 to 1764 mutations: implications for HBeAg status and disease.

A translational stop in the hepatitis B virus (HBV) precore codon 28 and specific changes in the core promoter region of the X gene have been suggested to influence the level of circulating HBeAg in patients. We analysed the core promoter region and precore sequences from 59 HBV strains (including 14 from the databank) of different genotypes and from patients with different HBeAg/anti-HBe patterns. The initiator and TATA elements for transcription of precore and pregenomic RNA were highly conserved. The majority of X gene deletions in the core promoter region would lead to translational frame-shifts and stops, truncating the C-terminal end of the X protein. We found significant associations between specific changes in core promoter positions 1762 to 1764, or in precore codon 28, and absence of circulating HBeAg. For the core promoter mutations alone, this association was related to the apparent degree of liver damage (as estimated by alanine aminotransferase levels) at the time of sampling. Mutations at nucleotides 1762 and/or 1764 were often accompanied by point mutations at positions 1751 to 1755. Since mutations at nucleotide positions 1762 and 1764 have recently been shown by in vitro studies to suppress HBeAg production with a concomitant enhancement of virus production, disappearance of the HBeAg-positive phenotype associated with 1762 to 1764 mutations may thus have at least as much significance for the course of infection as HBeAg absence associated with precore codon 28 stop mutations. These observations are considered against a secondary structural model for the 3' end of HBV pregenomic RNA which also predicts enhancement of virus replication after mutation at positions 1762 and 1764.

Aquarium visitors' perceptions and attitudes toward the importance of marine biodiversity.

To study people's perceptions and attitudes toward the importance of marine biodiversity, 102 visitors to San Francisco's UnderWater World aquarium were interviewed about their perceptions, attitudes, and knowledge about marine life and the environment needed for survival. They were asked about any changes in perceptions and attitudes after their visit. The data indicate that most visitors had very little education in marine biology, that 84% had previously visited other aquariums, that 98% had maintained or increased their positive attitudes toward marine life, and that only 20% financially or emotionally supported marine animal protection or preservation societies. These data, especially from people who had previously visited aquariums, indicated a need for school and college classes and texts to include more substantial and interest-stimulating information about the encompassing importance of the oceans for the survival of all terrestrial and oceanic species.

A revised secondary structure model for the 3'-end of hepatitis B virus pregenomic RNA.

The polymerase encoded by human hepatitis B virus, which has reverse transcriptase and RNase H activity, binds to its pregenomic RNA template in a two-step process involving a terminal redundancy. Both first strand and second strand DNA synthesis involve primer translocation and second strand synthesis involves a template jump. Three parts of the genome, including the so-called core promoter, are known to show deletions in strains usually arising after long-standing HBV infection, but also in some patients treated with interferon. A computer-based study of RNA template folding in the core promoter region, accommodating well-known point mutations, has generated a model for the 3' DR1 primer binding site as being part of a superstructure encompassing an already well-established stem-loop. Depending on the identity of nucleotides 1762 and 1764, the DR1 region may assume two alternative secondary structures which stabilize it as a primer binding site to different extents. Remarkably, one of these structures includes a pronounced loop which coincides with at least 12 related deletions seen in HBV DNA from different patients. Thus according to the model, the 5'- and 3'-ends of pregenomic RNA, which share primary sequences but have separate functions, are not structural equivalents. An RNA superstructure near the 3'-end of all HBV transcripts could have far-reaching implications for the modulation of both genome replication and post-transcriptional processing.

Adenovirus 41 replication: cell-related differences in viral gene transcription.

Adenovirus 41 infection of human embryo fibroblasts (HEF cells) leads to an abortive replication cycle whereas semi-permissive infection of Chang cells and permissive infection of 293 cells leads to the production of infectious particles. The aim of this study was to delineate where in the viral life cycle the block in replication occurs in non-permissive cells. DNA replication marks the onset of the late stage of the replication cycle but synthesis of DNA could only be detected when cultures were co-infected with Ad2, suggesting an early block in Ad41 replication. In order to map Ad41-specific transcripts produced following infection of HEF, Chang and 293 cells, tentative transcription units (determined by alignment with the Ad2 and Ad40 transcription maps) were first assigned to various plasmids carrying Pstl fragments. These plasmids were used as probes to detect Ad41 transcripts that map to these regions. Only transcripts mapping to the region between 0 and 12 map units were detected in Ad41 infected HEF cells. The level of late transcription was found to be low even in Chang and 293 cells and we therefore employed a more sensitive method to detect major late transcripts in Ad41 infected HEF cells. Transcripts carrying 59 kDa-fibre gene-specific sequences could be detected using RT-PCR at earlier times in 293 cells when compared to Chang cells but were present over a much longer time period in the latter cells, and could not be detected in HEF cells. These results are in agreement with the results from DNA synthesis in Ad41 infected HEF cells and mapping of transcripts to specific regions of the Ad41 genome, confirming that the Ad41 block in replication occurs within the early phase of the infectious cycle.

Characteristics and motives of volunteers in wildlife rehabilitation.

Although wildlife rehabilitation relies heavily on volunteers, the characteristics and motivations of such volunteer workers have not been studied. 80 volunteers from three San Francisco Bay Area Rehabilitation Centers, were interviewed about their preferences in working with animals and people, their methods of dealing with grief and failure over animals' deaths, and the onset of their interest in and motives for working in a difficult field. A significant number had owned pets in childhood as well as currently. 40% preferred working only with animals to working with both animals and people. 34% stayed in rehabilitation because they loved and wanted to help animals; 23% enjoyed hands-on nurturing; and the remainder primarily felt they were giving back to Nature a part of what people have taken from it.

Rapid subgenus identification of human adenovirus isolates by a general PCR.

In most clinical situations involving adenovirus infection, subgenus (subgroup) identification of an adenovirus isolate is as informative as a finer identification by serotype. A PCR method which allows the identification of human adenovirus isolates as members of subgenera A, B:1, B:2, C, D, E, or F is described. It is based on a simple (nonnested) PCR using primers which bind to regions immediately flanking the VA RNA-encoding regions of human adenovirus genomes. The PCR allows amplification of DNA from all 49 human adenovirus prototype strains so far described. Since there are differences in the lengths of the VA RNA-encoding regions in adenoviruses of different subgenera, it is possible to differentiate some subgenera according to the size of the PCR product determined by electrophoresis. This forms the basis of an initial broad categorization of isolates as belonging to either (i) subgenus B:1, C, D, or E or (ii) subgenus A, B:2, or F. Subgenus identification is completed by a one-step restriction enzyme digestion and gel electrophoresis. The method was assessed by blind subgenus identification of 200 miscellaneous primate adenovirus isolates prepared by the reference laboratory at Bilthoven, The Netherlands. Identification at the subgenus level by PCR correlated 91.5% with the results of serotyping. A further 5.5% of isolates were correctly identified as belonging to one of two specified subgenera. Six of the 200 identifications (3%) were unsuccessful for various reasons, including weak PCR products, intermediate strains, and mistaken primate host. The method should serve as a rapid means of confirming adenovirus cytopathic effects in laboratories performing virus culture, with simultaneous subgenus identification of the isolate. It will also have relevance as an aid to conventional serotyping for epidemiological purposes, since for all adenoviruses except those belonging to subgenus D, neutralization tests need only involve a maximum of four type-specific antisera.

Antigenic sites on the receptor-binding domain of human adenovirus type 2 fiber.

The trimeric fiber of adenovirus type 2 (Ad2) mediates the first stage of virus-cell attachment, and the distal head region of the fiber has been implicated as the receptor-binding domain. To locate regions on the primary polypeptide sequence of the fiber which may be involved in virus-cell interaction, peptide-based epitope mapping was performed using (1) polyclonal antibodies prepared against both native Ad2 fiber and Ad2 head protein expressed in Escherichia coli and (2) 18 monoclonal antibodies prepared against trimeric Ad2 head protein expressed in baculovirus. The approach using polyclonal antibodies revealed eight domains on the primary sequence of the head which contain one or more continuous epitopes. At least two of these regions were also recognized by monoclonal antibodies reacting against both monomeric and trimeric fiber head protein. The majority of monoclonal antibodies which did not recognize Ad2 head-specific peptides in ELISA were also nonreactive against the monomeric form of protein in Western blot, suggesting that their recognition of trimer is due to the existence of as yet undefined discontinuous epitopes or to alterations in monomer configuration. Our results correspond well with the recently published X-ray crystallographic model of Ad5 fiber head (D. Xia, L.J. Henry, R.D. Gerard, and J. Deisenhofer, Structure 2, 1259-1270, 1994), since most antigenic determinants containing linear epitopes mapped to the outer loops or uppermost beta-sheets in this structure. Four of five neutralizing monoclonal antibodies recognized trimer only and none recognized linear peptides. This might suggest that the trimeric form of fiber is necessary for making contact with the receptor(s) and that discontinuous epitopes on the head domain may be involved in fiber-cell interaction.

The hepatitis B virus X gene: analysis of functional domain variation and gene phylogeny using multiple sequences.

The hepatitis B virus (HBV) X gene shares sequences with both the polymerase and precore genes, carries several regulatory signals critical to the replicative cycle, and its product has a transactivating function. In this study, the X gene sequences of 29 HBV strains from 14 different countries were characterized and compared to all corresponding databank sequences where the origin of the strain was stated. The X gene and its product are relatively well conserved. However, several rare or unique point mutations in the predicted X protein are described which further define regions on the primary sequence which may be of structural and/or functional significance. Phylogenetic analysis of the 29 X genes and their predicted proteins in this study using unrooted trees indicates that a common ancestral sequence gave rise to two main groups of X genes, represented by HBV strains found predominantly either in the Western or Eastern Hemisphere. In turn, each of these two main groups of sequences appear to have branched into two main lineages. Introduction of 33 additional DNA sequences from the databank has further verified these inferences and confirmed the groupings as previously described subgroups A to D. Whilst the split of X gene lineages into subgroups A and D seems feasible on geographical/anthropological grounds, the corresponding split of Eastern Hemisphere lineages into B and C may require an alternative hypothesis. Additionally, there was a correlation between the HBeAg/anti-HBeAg status of our patients and nucleotide identity at two positions in the core promoter, 52 and 50 bases upstream from the precore start codon. This finding, also shown recently by others, suggests that control of HBeAg secretion may involve mutations affecting transcription and not only precore/core translation.

Children's drawings and attachment to pets.

To help confirm the concept that distances placed between the self and other figures in children's drawings represent emotional distances, 242 pet-owning and 35 nonpet-owning kindergartners through eighth graders drew pictures of themselves, a pet, and/or a family member. Owners drew pets significantly closer than family-figures although the younger the child, the greater the distance between self and pet. Older children drew themselves holding pets significantly more often, but younger children placed the family-figure between the self and the pet significantly more often. There were no significant gender differences in self-figure/pet-figure distances, but cats, dogs, caged animals, and farm animals were placed significantly closer to self-figures than were fish. Over-all, owners were clearly emotionally closer to pets than to family members, but nonowners were as close emotionally to family members as were owners.

Human and simian adenoviruses: phylogenetic inferences from analysis of VA RNA genes.

Adenovirus VA RNA genes have primary sequence constraints due to internal promoter regions and a high degree of secondary structure in the RNA product. To determine the relationships between human and simian adenoviruses, the VA RNA genes of several primate adenoviruses were characterized and compared to those sequences already published. Human adenoviruses of subgenera A, B:2, and F have only one VA RNA gene, whereas human adenoviruses of subgenera B:1, C, D, and E have two. The genomes of 12 monkey adenoviruses were found to have only one VA RNA gene, whereas the genomes of six representative chimpanzee adenoviruses were each found to have two VA RNA genes. Phylogenetic analysis of representative VA RNA gene sequences individually, irrespective of their strain of origin or partnering VA RNA gene, gave the following inferences. (1) The single VA RNA genes of human adenovirus subgenera A and F are most closely related to those of monkey adenoviruses. (2) The VA RNAI genes of human adenoviruses in subgenera B:1, D, and E, and also the single VA RNA genes of subgenus B:2 probably diverged from a common ancestral VA RNA gene. (3) This ancestral gene most likely reduplicated to give the precursor of all VA RNAII genes, the evidence for which has been almost totally lost in subgenus B:2 adenoviruses. (4) The two VA RNA genes of human subgenus C adenoviruses are relatively distant from each other phylogenetically. Since the Ad2 and Ad5 VA RNAI genes have a higher identity to the single VA RNA gene of SAV13 (SV36) than to those of any of the other human adenoviruses, these genes may have entered the human subgenus C adenovirus genome by substitution involving recombination with a simian adenovirus. The results of this study suggest that a renewed appraisal of VA RNA function in adenoviruses other than Ad2 and Ad5 may be necessary.

Loneliness and pet ownership among single women.

Relationships among loneliness, pet ownership, and attachment were studied in a sample of 148 adult female students, 59 pet owners and 89 nonowners. No significant differences were found on the loneliness reported by pet owners and nonowners. A two by two analysis of variance showed that women living entirely alone were significantly more lonely than those living with pets only, with both other people and pets, and with other people but without pets. No associations were found between loneliness and pet attachment. Also, no significant differences were found in loneliness or pet attachment scores between dog and cat owners; however, women living only with a dog were significantly more attached to the dog than those living with both a dog and other people. Conversely, women living only with a cat were significantly less attached to the cat than those living with both a cat and other people. These findings indicate that having a pet can help to diminish feelings of loneliness, particularly for women living alone, and compensate for the absence of human companionship.