Hepatitis E virus infection in chimpanzees: a retrospective analysis.

Different patterns of disease were observed among 11 chimpanzees who were inoculated intravenously with hepatitis E virus (HEV) positive fecal specimens from four different outbreaks (Nepal 1981, Uzbekistan 1981, Pakistan 1985, and Mexico 1986). Five chimpanzees had marginal or no liver enzyme elevations within 70 days of inoculation. Two of the chimpanzees had limited viremia, but did not produce detectable antibody. The four remaining chimpanzees had liver enzyme elevations, viral shedding, viremia, seroconversion to anti-HEV, and detectable HEV antigen in liver biopsy specimens. These results may reflect the range of infection patterns that develop in humans after natural exposure to the HEV.

Pressure cycling technology: a novel approach to virus inactivation in plasma.

BACKGROUND: Hydrostatic-pressure virus inactivation is a novel approach to the inactivation of pathogens in plasma and blood-derived components, that retains the therapeutic properties of these products. STUDY DESIGN AND METHODS: A custom-built apparatus was used to pressurize human plasma samples spiked with lambda phage. Phage titer and plasma protein activities were monitored after pressure treatment. RESULTS: Pressure-mediated inactivation of lambda phage was found to be an effective means for virus inactivation, particularly when performed at near-zero (0 degrees C) temperatures, rather than at temperatures above 20 degrees C and below -40 degrees C. The efficiency of inactivation was improved by an increase in applied pressure and repeated cycling from atmospheric to high pressure. In contrast, activities of plasma proteins alkaline phosphatase and total amylase did not vary with temperature and remained within 29 percent and 6 percent, respectively, of starting values after the same pressure treatments. By combining cycling, near-zero temperatures, and high pressure, phage titers in serum were reduced approximately 6 log after 10 to 20 minutes of treatment. Activities of plasma proteins IgG, IgM, and factor X were at 104 percent, 89 percent, and 80 percent, respectively, of starting values after 20 minutes of the same temperature and pressure treatment. CONCLUSION: High-pressure procedures may be useful for the inactivation of viruses in blood and other protein-containing components.

Hepatitis viruses: their role in human cancer.

Hepatitis B virus (HBV) has been shown to be linked causally to the development of hepatocellular carcinoma (HCC) in humans. One of the HBV gene products, the "X" protein, has been specifically implicated in the malignant transformation of hepatocytes; mutations in one or more of the HBV structural proteins have also been linked to HCC. HBV DNA may act as an insertional mutagen in the myc family of genes. Mutations within the pre-core and core promoter regions of HBV-DNA have also been associated with the development of HCC. Patients chronically infected with hepatitis C virus (HCV) often develop cirrhosis; a significant proportion of these patients progress to HCC. Although numerous genotypes of HCV exist, type 1b is most often associated with the eventual development of HCC in chronically infected patients. The molecular mechanisms for the malignant transformation of hepatocytes by HCV have not been elucidated.

Quantification of DNA-protein interaction by UV crosslinking.

Measurement of the affinity of a protein for a promoter sequence is critical when assessing its potential to regulate transcription. Here we report that the DNA protein crosslinking (DPC) assay can be used to measure affinity, amount and molecular weight of DNA binding proteins to specific and non-specific DNA sequences. By applying a theoretical analysis to evaluate the binding data, it was shown that the affinity constants of two proteins (named DPC80 and DPC107) to the MT3 region of the mouse thymidine kinase promoter were 2 x 10(-9) M, which is 10(4) times higher than to non-specific DNA. Similar affinity constants were found when the purified proteins corresponding to DPC80 and DPC107 instead of nuclear extracts were used to assess the reliability of the DPC assay. A value for crosslinking efficiency was determined as 0.07, however, it is not needed for computation of the DNA-protein affinity, but with it the abundance of a binding protein can be estimated. In summary, the DPC assay is useful for quantifying DNA binding proteins and thereby judging their influence on transcription.

Non-A, non-B hepatitis unrelated to the hepatitis C virus (non-ABC).

The history of non-ABC hepatitis is a kaleidoscope of intriguing, but often conflicting and confounding data. Studies of transfusion-associated non-ABC hepatitis are less convincing than they originally seemed. Chimpanzee cross-challenge studies, once the bastion for the theory of multiple NANB hepatitis agents, now have an alternative explanation in the impaired immune response associated with HCV infection and the ability of this agent to reinfect individuals previously assumed to be immune. Nonetheless, there are so many cases of acute and chronic NANB hepatitis that cannot currently be attributed to HCV that it is hard to avoid the implication of at least one, and possibly more, non-ABC hepatitis agents. There are now some transmission studies in small primates to support this contention, though recent chimpanzee transmission studies have been disappointingly negative. As with the hepatitis C virus, the breakthrough in this disease will not come from classic serology or virology, but from molecular biology. Similar molecular approaches to those that elucidated HCV are in progress and are promising in preliminary experiments. It is anticipated that the pace of molecular biology is such that a great deal more will be known about non-ABC in a relatively brief time, and perhaps one or more non-ABC agents will prove to be real and clinically relevant.

Hepatitis E virus: a brief review of the biology, molecular virology, and immunology of a novel virus.

Our basic understanding of the biology, molecular virology, and immunology of hepatitis E virus (HEV) is briefly reviewed. HEV is a small, round, nonenveloped virus with morphologic and biophysical properties most similar to viruses found in the family Caliciviridae. The genome of HEV is approximately 7.5 kb in length and consists of a positive-sense, single-stranded RNA molecule that contains three distinct open reading frames (ORF1, ORF2, ORF3) that appear to encode for nonstructural and structural proteins based on the presence of well-defined consensus motifs and genomic organization similar to those of other calici- or calici-like viruses. Limited epitope mapping of the viral genome with synthetic peptides has revealed the presence of highly immunoreactive type-common and type-specific epitopes; these finding are consistent with the results of other studies that used recombinant expressed proteins from both the nonstructural and structural regions of the derived viral proteins encoded by ORFs 1, 2, and 3. Synthetic peptides and recombinant expressed proteins have been used to develop Western blot assays and enzyme immunoassays (EIAs) for the detection of IgA, IgG, and IgM anti-HEV in human and primate sera. Knowledge of the dynamics of HEV antigen and antibody expression in experimentally-infected primates is emerging, and prototype vaccines have been developed with recombinant expressed ORF2 and ORF3 proteins. Limited seroprevalence studies of anti-HEV in endemic and nonendemic regions of the world using one or more of the above assays has revealed a strong correlation between level of sanitation and incidence of disease and prevalence of anti-HEV.

Viral specificity of hepatitis E virus antigens identified by fluorescent antibody assay using recombinant HEV proteins.

A fluorescent antibody (FA) assay for hepatitis E virus antigen (HEVAg) in infected liver tissue was used to confirm the presence of virus-specific antigens in hepatocytes during the course of infection. With the cloning of the HEV genome it is now possible to determine which viral antigens are recognized by this FA assay. Recombinant HEV proteins covering the carboxyl half of HEV open reading frame 2 (ORF2) were used in this study to demonstrate that some of the most immunoreactive virus-specific antigens detected by FA are contained within this region of ORF2 (nucleotides 6169-7126).

Serum-responsive expression from the murine thymidine kinase promoter is specifically disrupted in a transformed cell line.

Thymidine kinase (TK) gene expression is controlled in normal cells at both the transcriptional and posttranscriptional levels. Together, these regulatory systems mediate the 20-50-fold induction of TK mRNA observed as cells traverse the G1-S boundary of the cell cycle. Previously, we have reported that a "Yi" protein complex was observed to bind the mouse TK promoter in a cell cycle-dependent manner in nontransformed cells (Q-P. Dou, J. L. Fridovich-Keil, and A. B. Pardee, Proc. Natl. Acad. Sci. USA, 88: 1157-1161, 1991) and bound constitutively in transformed cells (D. W. Bradley, Q-P. Dou, J. L. Fridovich-Keil, and A. B. Pardee, Proc. Natl. Acad. Sci. USA, 87: 9310-9314, 1990). Nonetheless, TK mRNA levels in these cells continue to exhibit a marked S-specific induction (> 10 fold), raising the question: what is the status of TK promoter-mediated, as opposed to posttranscriptional, gene regulation in these transformed cells? To address this question, we have used cell synchrony experiments involving both transformed and nontransformed cells stably transfected with a TK promoter-beta-globin reporter gene construct. We have found that, in marked contrast to the tight regulation of reporter gene expression observed in nontransformed cells (J. L. Fridovich-Keil, J. M. Gudas, Q-P. Dou, I. Bouvard, and A. B. Pardee, Cell Growth & Differ., 2: 67-76, 1991), reporter gene expression in the transformed cells is constitutive and, therefore, closely parallels the presence of Yi DNA-binding activity. These data are fully consistent with other recently published observations concerning differential controls of TK transcriptional and posttranscriptional regulation (J. M. Gudas, J. L. Fridovich-Keil, and A. B. Pardee, Cell Growth & Regul., 4: 421-430, 1993) and support the hypothesis that, in transformed cells, endogenous TK is regulated predominantly at the posttranscriptional level.

Preliminary evidence that a trpE-HEV fusion protein protects cynomolgus macaques against challenge with wild-type hepatitis E virus (HEV).

Immunization of two cynomolgus macaques (cynos) with trpE-C2 protein, a trpE-HEV fusion protein that represents the carboxyl two thirds of the putative capsid protein, prevented development of biochemical evidence of viral hepatitis in these primates after challenge by wild-type HEV from either a Burmese or Mexican stool isolate. Neither of the immunized animals showed any elevation of alanine aminotransferase activity after challenge with wild-type HEV in marked contrast with the unimmunized (control) cynos. In the case of the Burmese HEV challenged cyno, the protective effect was complete with the animal failing to demonstrate any evidence of HEV infection. The immunized cyno challenged with Burmese HEV did not exhibit any HEV RNA in its stools or HEV antigen in its liver. The immunized cyno (#8902) challenged with Mexican virus exhibited HEV RNA in its stools and HEV antigen in its liver; however, microscopic examination of liver biopsy specimens from this cyno failed to detect histopathologic evidence of viral hepatitis. All of the animals (naive and immunized) developed anti-HEV IgM and IgG responses after HEV challenge. Our preliminary studies indicate that the trpE-C2 protein is a promising candidate HEV vaccine.

The sequence of hepatitis E virus isolated directly from a single source during an outbreak in China.

In this study an IgM antibody-mediated antigen-capture procedure for direct extraction of hepatitis E virus (HEV) RNA from clinical specimens was developed and used with an efficient method for generating viral cDNA that was subsequently sequenced using the dideoxy chain termination method. This is the first time the complete HEV genome has been isolated directly from a single human clinical specimen obtained during an outbreak of enterically transmitted non-A, non-B hepatitis. When the Chinese-derived sequence was compared with the original isolate of Burmese HEV from an experimentally infected cynomolgus macaque, the homology between the two sequences was 94% and 98.5% at the nucleotide and amino acid levels, respectively. The methods we developed for generating and sequencing genomic HEV cDNA dramatically improved the efficiency of cloning the viral genome and should be helpful for continued analysis of this virus as well as other RNA viruses that have proven to be difficult to clone and sequence directly.

Molecular characterization of hepatitis C and E viruses.

The molecular features of each of the major viruses of non-A, non-B hepatitis, namely hepatitis C virus (HCV) and hepatitis E virus (HEV) are briefly described. The organization of the genome of each of these viruses is discussed and compared to those of other related or distantly related viruses that contain single-stranded, positive-sense RNA genomes. HCV has been tentatively classified as a separate genus within the Flaviviridae, whereas HEV has been loosely associated with caliciviruses and subsequently assigned to the Caliciviridae, although it does possess unique genetic features not found in other caliciviruses.

Molecular cloning and sequencing of the Mexico isolate of hepatitis E virus (HEV).

Hepatitis E virus (HEV) is the major causative agent of hepatitis E or what was formerly known as enterically transmitted non-A, non-B hepatitis. The disease has a worldwide distribution but occurs principally in developing countries in any of three forms: large epidemics, smaller outbreaks, or sporadic infections. Genetic variation of different HEV strains was previously noted and it will be important to determine the extent to which this variation may pose problems in the diagnosis and treatment of HEV infection. To analyze differences at the genetic level between HEV(Mexico; M) and the previously characterized HEV(Burma; B) and HEV(Pakistan; P) isolates, overlapping cDNAs were cloned from samples obtained from an infected human and an experimentally inoculated cynomolgus macaque. These cDNA clones, representing the nearly complete (7185-bp) genome of HEV(M), confirmed an expression strategy for the virus that involves the use of 3 forward open reading frames (ORFs). The HEV(M) strain has an overall 76 and 77% nucleic acid identity with the HEV(B) strain and HEV(P) strain, respectively; however, the degree of sequence variation was not uniform throughout the viral genome. A hypervariable region was identified in ORF1 that exhibited a 58 and 54% nucleic acid sequence and 13% amino acid similarity with the Burma strain and the Pakistan strain, respectively. A large number of the nucleotide differences occurred at the third codon position, with the deduced amino acid sequences similarity of 83, 93, and 87% between HEV(M) and HEV(B) isolates in ORF1, ORF2, and ORF3, respectively, and with 84, 93, and 87% amino acid identities between HEV(M) and HEV(P) isolates in ORF1, ORF2, and ORF3, respectively. The nucleotide sequences derived from the highly conserved regions of HEV genome will be useful in developing polymerase chain reaction-based tests to confirm the viral infection. Knowledge of the extent of the sequence variation encountered with HEV will not only aid in the future development of diagnostic and vaccine reagents but also further our understanding of how HEV strain variation might impact the pathological outcome of infection.

Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses.

Computer-assisted comparison of the nonstructural polyprotein of hepatitis E virus (HEV) with proteins of other positive-strand RNA viruses allowed the identification of the following putative functional domains: (i) RNA-dependent RNA polymerase, (ii) RNA helicase, (iii) methyltransferase, (iv) a domain of unknown function ("X" domain) flanking the papain-like protease domains in the polyproteins of animal positive-strand RNA viruses, and (v) papain-like cysteine protease domain distantly related to the putative papain-like protease of rubella virus (RubV). Comparative analysis of the polymerase and helicase sequences of positive-strand RNA viruses belonging to the so-called "alpha-like" supergroup revealed grouping between HEV, RubV, and beet necrotic yellow vein virus (BNYVV), a plant furovirus. Two additional domains have been identified: one showed significant conservation between HEV, RubV, and BNYVV, and the other showed conservation specifically between HEV and RubV. The large nonstructural proteins of HEV, RubV, and BNYVV retained similar domain organization, with the exceptions of relocation of the putative protease domain in HEV as compared to RubV and the absence of the protease and X domains in BNYVV. These observations show that HEV, RubV, and BNYVV encompass partially conserved arrays of distinctive putative functional domains, suggesting that these viruses constitute a distinct monophyletic group within the alpha-like supergroup of positive-strand RNA viruses.

New strategies for isolation of low abundance viral and host cDNAs: application to cloning of the hepatitis E virus and analysis of tissue-specific transcription.

The ability to clone viruses molecularly has led to dramatic advances in our understanding of this diverse group of agents at the molecular level. These insights are critical to the development of experimental strategies for the containment and control of the various viral pathogens that cause hepatitis in man. Knowledge of the genes and gene products have, for example, assisted in the design of expression systems that have been useful for the cell-free expression and production of viral constituents in the development of a subunit (recombinant) vaccine for HBV. Of particular advantage to the experimenter would be the ability to clone the virus directly from the infectious source without the need to resort to biologic amplification systems that require costly investments of money and manpower. This would obviate the need for the time-intensive development of a tissue culture propagation or the expensive cost of developing an animal model. The methods described here have applications to the discovery and isolation of low abundance transcripts from host (cellular) genes. These host encoded products may have important roles in virus replication and certainly by themselves constitute a growing area of study. The direct selection protocol has already proven its value in the isolation of novel (previously undescribed) gene sequences from complex cellular sources. The application of these methodologies should, at the single cell level, aid in the delineation of those important host-encoded gene products that are critical for the efficient in vitro propagation of hepatotropic viruses. The identification of these low abundance cellular genes will elucidate the biologic interplay between host and obligate intracellular parasite (virus) and potentially lead to the development of new strategies for virus control that take into consideration the role of the cell as host.

Acute sporadic hepatitis E in children living in Cairo, Egypt.

Seventy-three pediatric patients with acute hepatitis and 19 control patients without liver disease living in Cairo, Egypt, were evaluated with a newly developed Western blot assay for IgM antibody to hepatitis E virus (IgM anti-HEV). The mean age of acute hepatitis patients was 6.4 years (range, 1-13 years); 56% were male. Among the 73 acute cases, hepatitis A was diagnosed in 30 (41%), possible acute hepatitis B in three (4%), hepatitis E in nine (12%), and by exclusion, non-A, non-B hepatitis in 29 (40%). Two additional acute cases were positive for both IgM anti-HAV and IgM anti-HEV. None of the 19 control subjects had IgM anti-HEV. Parenteral risk factors were associated with cases of non-A, non-B hepatitis but were not associated with acute hepatitis E. Contact with a family member with jaundice was associated with acute hepatitis A. In contrast to prior epidemics of enterically-transmitted non-A, non-B hepatitis, HEV was found to be a common cause of acute hepatitis in a pediatric population. This study provides additional evidence that HEV may be a frequent cause of acute sporadic hepatitis among children living in some developing countries.

Hepatitis C virus antigen in hepatocytes: immunomorphologic detection and identification.

Hepatitis C virus (HCV) antigen was detected immunohistochemically using fluorescein isothiocyanate-labeled immunoglobulin G fractions from chimpanzee and human sera strongly reactive with recombinant hepatitis C virus structural and non-structural proteins. The antigen was localized in the cytoplasm of hepatocytes in all 9 chimpanzees with acute hepatitis C, in 5 of 10 chimpanzees with chronic HCV infection, and in 11 of 12 patients with chronic hepatitis C. The specificity of the hepatocellular HCV and FITC-labeled probes for HCV was ascertained by blocking studies with paired serum samples obtained from 8 infected and uninfected chimpanzees or from 14 patients during the acute and chronic phases of HCV infection. Absorption experiments on FITC-labeled probes with selected host proteins (normal liver homogenate, plasma proteins, red blood cells) did not indicate cross reactivity of the probes with these antigens. Direct immunomorphologic evidence for the HCV specificity of hepatocellular HCV antigen deposits and the FITC-labeled polyclonal anti-HCVAg probe was established in absorption experiments using recombinant HCV nonstructural proteins. The putative HCV NS3 protein was the most prominent component of hepatocellular HCV antigen.

Acute sporadic hepatitis E in Sudanese children: analysis based on a new western blot assay.

A newly developed Western blot assay for antibody to hepatitis E virus (anti-HEV) was used to evaluate 39 cases of acute pediatric hepatitis and 39 control patients in Khartoum, Sudan. The mean age of cases was 6.5 years (range, 2-14); 64% were male. Acute hepatitis A (IgM anti-HAV-positive) was diagnosed in 13 cases, acute hepatitis B (IgM anti-HBc-positive) in 1, and acute hepatitis E (positive for IgM anti-HEV) in 23 (59%). None of the cases with IgM anti-HAV or IgM anti-HBc had IgM anti-HEV; 3 controls had IgM anti-HEV. Acute hepatitis E was associated with recent contact with a family member or acquaintance with jaundice and the presence of indoor plumbing. The newly developed hepatitis E assay appeared to be specific for the diagnosis of acute icteric non-A, non-B hepatitis. Hepatitis E was found to be the most common cause of acute sporadic hepatitis in children living in an urban area of Africa.