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Great advances in technology produce unique challenges. Every technology also has a dual use, which needs to be understood and managed to extract maximum benefits for mankind and the development of civilization. The achievements of physicists in the mid-20th century resulted in the nuclear technology, which gave us the destructive power of the atomic bomb as also a source of energy. Towards the later part of the 20th century, information technology empowered us with fast, easy and cheap access to information, but also led to intrusions into our privacy. Today, biotechnology is yielding life-saving and life-enhancing advances at a fast pace. But, the same tools can also give rise to fiercely destructive forces. How do we construct a security regime for biology? What have we learnt from the management of earlier technological advances? How much information should be in the public domain? Should biology, or more broadly science, be regulated? Who should regulate it? These and many other ethical questions need to be addressed.

Genital infection with Chlamydia trachomatis results in both the local recruitment of protective immune responses and an inflammatory infiltrate that may also participate in tubal pathology. As a beginning to understanding the etiology of immune system-mediated tubal pathology, we evaluated the regional recruitment of lymphocyte subsets to different areas of the female genital tract (GT) over the course of a murine infection with the mouse pneumonitis agent of Chlamydia trachomatis (MoPn). Using flow cytometric techniques we found that the CD4 lymphocyte subset was preferentially recruited to the upper GT (oviduct and uterine horn) over the lower GT (cervical-vaginal region) throughout the course of MoPn infection. The influx of CD4 cells also correlated with the expression of endothelial cell adhesion molecules (ECAMs) and in vitro lymphocyte adherence in the upper GT. Interestingly, the expression of ECAMs in the lower GT was not maintained longer than 7 days after infection, even in the presence of viable chlamydiae. Taken together, these data suggest that regulatory mechanisms of lymphocyte recruitment differ between the upper and lower regions of the GT and may influence the clearance of chlamydiae and the development of tubal pathology.

ABSTRACTHepatitis E virus (HEV) is an important etiological agent of epidemic and sporadic hepatitis, which is endemic to the Indian subcontinent and prevalent in most of the developing parts of the world. The infection is often associated with acute liver failure and high mortality, particularly in pregnant women. In order to develop methods of intervention, it is essential to understand the biology of the virus. This is particularly important as no reliable in vitro culture system is available. We have constructed a cDNA clone encompassing the complete HEV genome from independently characterized subgenomic fragments of an Indian epidemic isolate. Transfection studies were carried out with HepG2 cells using in vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of negative-sense RNA, indicative of viral replication, was demonstrated in the transfected cells by strand-specific reverse transcription-PCR and slot blot hybridization. The viral proteins pORF2 and pORF3 and processed components of the pORF1 polyprotein (putative methyltransferase, helicase, and RNA-dependent RNA polymerase) were identified in the transfected cells by metabolic pulse-labeling with [35S]methionine-cysteine, followed by immunoprecipitation with respective antibodies. The expression of viral proteins in the transfected cells was also demonstrated by immunofluorescence microscopy. Viral replication was detected in the transfected cells up to 33 days posttransfection (six passages). The culture supernatant from the transfected cells was able to produce HEV infection in a rhesus monkey (Macaca mulatta) following intravenous injection, indicating the generation of viable HEV particles following transfection of cells with in vitro-synthesized genomic RNA. This transient cell culture model using in vitro-transcribed RNA should facilitate our understanding of HEV biology.

Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a positive-strand RNA virus with a 7.5-kb polyadenylated genome consisting of three open reading frames (ORFs). In the absence of an in vitro culture system, the replication and expression strategy of HEV and the nature of its encoded polypeptides are not well understood. We have expressed the two ORFs constituting the structural portion of the HEV genome in COS-1 cells by using simian virus 40-based expression vectors and in vitro by using a coupled transcription-translation system. We show here that the major capsid protein, encoded by ORF2, is an 88-kDa glycoprotein which is expressed intracellularly as well as on the cell surface and has the potential to form noncovalent homodimers. It is synthesized as a precursor (ppORF2) which is processed through signal sequence cleavage into the mature protein (pORF2), which is then glycosylated (gpORF2). The minor protein, pORF3, encoded by ORF3 is a 13.5-kDa nonglycosylated protein expressed intracellularly and does not show any major processing. pORF3 interacts with a cellular protein of about 18 kDa which we call 3IP, the pORF3-interacting protein. The significance of these findings are discussed in light of an existing model of HEV genome replication and expression.

Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information exists on the basic biology of the virus. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. We expressed pORF3 in transiently transfected COS-1 and Huh-7 cells and showed that it is a phosphoprotein which is modified at a serine residue(s). Deletion and site-directed mutants were created to establish Ser-80 as the phosphorylation site. This residue is present within a conserved primary sequence that showed consensus sites for phosphorylation by p34cdc2 kinase (cdc2K) and mitogen-activated protein kinase (MAPK). In vitro experiments with hexahistidine-tagged pORF3 expressed either in Escherichia coli or in COS-1 cells showed efficient phosphorylation with exogenously added MAPK. The pORF3 mutants also exhibited an in vitro phosphorylation profile with MAPK which was identical to that observed in vivo. In its primary sequence, pORF3 possesses two highly hydrophobic N-terminal domains. On subcellular fractionation, pORF3 was found to partition with the cytoskeletal fraction, and this association with the cytoskeleton was lost on deletion of hydrophobic domain I (amino acid residues 1 to 32). These results suggest that HEV pORF3 is a cytoskeleton-associated phosphoprotein and are discussed in terms of a possible function for pORF3 within the HEV replicative cycle.

A series of recombinant plasmid vectors containing hepatitis B virus (HBV) DNA sequences was constructed to study the biosynthesis of the hepatitis B virus surface antigen (HBsAg) RNA and to locate transcriptional control elements involved in the regulation of the S and pre-S DNA sequences. We examined the transcription of the HBsAg gene in permanent cell lines that were developed by transfecting with recombinant vectors containing HBV sequences and the neomycin gene followed by G418 selection. We further defined the promoter activities upstream of and within the pre-S sequences using the assayable chloramphenicol acetyltransferase gene. Results obtained from S1 nuclease digestion and primer extension suggest that HBsAg transcripts are initiated at multiple sites in the pre-S region and from a site upstream of the pre-S region. Chloramphenicol acetyltransferase assays indicate that DNA sequences within and upstream of the pre-S region contain promoter activities and that the "TATA" sequence-containing promoter and the internal promoter show similar levels of activities in CV-1 cells and several other cell lines tested.

We studied the expression of the core region of the hepatitis B virus genome in mammalian cells with recombinant plasmid vectors. Stably transformed rat fibroblast cell lines were established by transfection with vectors containing subgenomic and genome-length hepatitis B virus DNA, followed by G418 selection. The RNA transcripts directed by the core region were characterized by Northern blot hybridization and S1 nuclease mapping. Using the chloramphenicol acetyltransferase gene expression system, the promoter activity located upstream of the core open reading frame was confirmed. The synthesis of core and e polypeptides was studied with a commercial radioimmunoassay. These studies show that partial deletion of the precore sequences abolished secretion of the e antigen, but there was pronounced synthesis of the core antigen in transfected cells.

The activity of the hepatitis B viral enhancer element was studied in various cell lines. This enhancer shows strict host and tissue specificity in that it is functional only in liver cells of human origin. Further, it requires trans-acting factor(s) present in liver cells for activity, and this activity is independent of hepatitis B virus gene products in the cell lines tested.

The hepatitis B virus X gene product trans activates transcription from a variety of viral and cellular regulatory elements. We expressed the complete, nonfused X protein in Escherichia coli and showed it to be active in trans activating a human immunodeficiency virus long terminal repeat-linked chloramphenicol acetyltransferase reporter gene.

The hepatitis B virus (HBV) DNA contains an open reading frame (ORF) designated X, which has the capacity to encode a protein of 16,560 Da (subtype adw). Such a protein has not been identified in either HBV particles or infected human livers, and therefore its role in the viral life cycle remains unknown. We report here the expression of the HBV X ORF in cultured cells using recombinant vectors. A protein of 16 kDa was identified by means of an antiserum prepared against a synthetic peptide and with human antisera from hepatitis B patients as well as those with hepatocellular carcinoma. Cell fractionation and immunofluorescence studies suggest a probable association with cytoskeletal components. Our studies further located a promoter sequence upstream of the X ORF, which directs the transcription of a 0.7- to 0.8-kilobase X-specific RNA in transfected human hepatoma cells.

Hepatitis E virus (HEV) is responsible for a majority of sporadic and epidemic viral hepatitides in India and other developing countries. Even though the genomes of four geographically distinct strains of HEV have been cloned and sequenced, the Indian strain of HEV remains largely uncharacterized. We have cloned and sequenced about 2.2 kb of the HEV genome constituting the structural region from an Indian strain of HEV. The nucleotide and amino acid sequences show a high degree of conservation with sequences from other HEV strains. Open reading frames (ORF) 2 and 3 were expressed in Escherichia coli as N-terminal hexahistidine epitope fusions. The purified proteins were then used in an immunoblot assay to evaluate the antibody status in sera from individuals from an area of high-level HEV endemicity. The anti-ORF2 antibodies were found to be nonspecific and could not be correlated to clinical disease. The immunoglobulin M anti-ORF3 was found to be specific for the presence of acute disease. The implications of these findings in HEV diagnosis and vaccine development are discussed.

We have previously established that the tissue-specific activity of the hepatitis B virus (HBV) enhancer is mediated by trans-acting cellular factors. Here we have studied in vitro the interactions between the HBV enhancer DNA and cellular factors present in nuclear extracts from both liver and nonliver cell types. The results presented in this study imply the involvement of several distinct, ubiquitous, and liver-specific cellular factors with the HBV enhancer. Sequence analysis of the binding sites for these proteins on HBV DNA showed homologies to sequence motifs known to bind other previously characterized and purified transcription factors including CAAT/enhancer-binding protein. Thus, all of these binding sites may function in concert to activate liver-specific transcription of HBV genes from their respective promoters.

The presence of hepatitis B virus DNA in the sera of individuals is the most definitive marker of an active viral infection. We have used polymerase chain reaction detection of hepatitis B virus DNA directly on whole blood dried as a spot on filter paper. The method is rapid, specific, and sensitive and has the ability to detect as little as 10 virus particles by ethidium bromide staining of the polymerase chain reaction-amplified products. The method is cost-effective, and the stability of the spots makes the collection and transportation of potentially infectious blood safe.

Enterovirus-like particles have been reported in the acute phase of both epidemic and sporadic non-A, non-B (NANB) hepatitis. To examine whether these particles were the causative agent in the two types of disease, 29 patients with acute viral hepatitis in a north Indian epidemic outbreak and 9 with sporadic acute disease were investigated. 25 (86%) of 29 patients with epidemic hepatitis and 5 (56%) of 9 with sporadic disease were diagnosed as having enterically-transmitted-NANB hepatitis by exclusion. Virus-like particles (VLP) of 30-34 nm were detected in stool of 1 patient with epidemic and 1 with sporadic hepatitis. The VLPs crossreacted serologically and a specific IgM response was seen in acute epidemic and sporadic serum samples. After inoculation with infected stool rhesus monkeys had a mild rise in liver enzymes, and bile samples contained VLPs. These results suggest that the aetiological agent in epidemic and sporadic disease is the same.