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.

A new cluster of hepatitis A infection in hemophiliacs traced to a contaminated plasma pool.

Recently, several clusters of hepatitis A have been observed among hemophiliacs linked to factor VIII concentrates treated for virus inactivation solely with the solvent/detergent (S/D) method, a procedure that does not affect nonenveloped viruses such as the hepatitis A virus (HAV). A new outbreak of hepatitis A in six hemophiliacs treated with the same lot of a factor VIII preparation occurred recently in Germany. The objective of the study was to clarify whether these diseases were caused by the administration of the S/D-treated plasma product, rather than a community-acquired infection. Polymerase chain reactions designed to detect HAV nucleic acid have been carried out in the implicated factor VIII lots, in the corresponding plasma pools, and in serum samples of four out of six infected individuals. The nucleic acid sequences were determined in samples that resulted in positive amplification products. HAV sequences were found in one of the two plasma pools used for manufacture of the incriminated product, in the incriminated lot itself, and in all recipient sera tested so far, although the latter were collected up to 7 weeks after the onset of jaundice. The sequences obtained were completely identical, revealing a unique HAV strain of genotype IA. This study provides conclusive evidence that hepatitis A can be transmitted by factor VIII concentrates treated solely by the S/D procedure for virus inactivation. This inactivation method is not effective against nonenveloped viruses. Since a number of hepatitis A transmission episodes have been described with such preparations during the past 10 years, their continued use seems to be questionable unless additional virus removal or inactivation steps are introduced to prevent the transmission of nonenveloped viruses. Molecular approaches again proved to be reliable tools for elucidating the chain of virus transmission.

Genetic variability of hepatitis E virus within and between three epidemics in India.

Hepatitis E virus (HEV) is an important cause of epidemic and sporadic acute viral hepatitis in many developing countries, including India. We evaluated the genetic variability within two regions (a 476-nt long ORF1 segment and a 304-nt long ORF2 segment) from specimens collected during three outbreaks in the cities of Karnal (1987), Yamunanagar (1989), and Meerut (1996), India, and from one patient, residing in Lucknow, India, who had a case of sporadic hepatitis (1996). Within an outbreak, sequences in the ORF1 and ORF2 regions were 99.3-100.0% identical. However, when strains were compared between outbreaks, identity in the ORF1 and ORF2 region was 97.1-99.2 and 96.4-100.0%, respectively. A comparison of these sequences to previously published Indian ORF1 and ORF2 sequences revealed even lower similarities, 95.2-98.5 and 95.1-98.7%, respectively. One patient in the Meerut outbreak had genomic sequences that differed substantially from the other patients affected during this outbreak and probably reflected a sporadic infection. The sporadic hepatitis E strain from Lucknow clustered with a previously described HEV strain from a patient with fulminant hepatic failure (FHF). Our data suggest that the ORF1 and ORF2 segments can be used to study the molecular epidemiology of HEV infection and indicate that much remains to be determined about the genetic variability of Indian HEV strains.

Hepatitis A virus sequence detected in clotting factor concentrates associated with disease transmission.

Since the early 1990s hepatitis A virus (HAV) infections among recipients of solvent-detergent treated factor VIII concentrates have occurred in Europe, South Africa and the United States. A review of the epidemiological and laboratory-based investigations of the outbreaks in Germany and Ireland were consistent with transmission by factor concentrates but limited information about transmission based upon nucleic acid sequences was obtained, and no clear chain of transmission could be established. Within the United States, hepatitis A infections associated with solvent detergent concentrate occurred in a single patient in 1993, and a cluster of cases in 1995. Although the 1993 factor concentrate was positive for virus, samples from the patient were not available. The virus present in the cluster of 1995 factor VIII patients, the factor concentrate they received, and the original plasma pool was identical, while the virus identified in the factor IX patient differed by a single base.

Hepatitis E virus RNA detection in serum and feces specimens with the use of microspin columns.

This report describes the use of microspin columns for extraction of hepatitis E virus (HEV) RNA from stool and serum specimens for reverse transcription-polymerase chain reaction (RT-PCR) and compares this method with the glass powder method. The microspin column method was found to be 1- to 2-log more sensitive in detecting HEV RNA than the glass powder method and had better reproducibility. The microspin column method also detected HEV RNA in a larger number of specimens than the glass powder method from among a panel of serum and stool specimens. Use of this method may allow better assessment of viremia and fecal excretion in patients and experimental animals infected with HEV.

Hepatitis A virus infections associated with clotting factor concentrate in the United States.

BACKGROUND: Two cases of hepatitis A among persons exposed to the same lot of solvent/detergent-treated antihemophilic factor VIII concentrate were reported to a surveillance system. An investigation was conducted to find additional cases and determine the source of infection. STUDY DESIGN AND METHODS: A seroprevalence study was conducted among persons with exposure to the suspect lot for serologic evidence of recent infection with hepatitis A virus (HAV). RESULTS: Six cases of recent HAV infection were discovered: four of the patients had been infused with material from the suspect lot of factor VIII, and two had received infusions of factor IX concentrate made from plasma pools common to the suspect factor VIII lot. HAV was identified in one of the plasma pools, in the factor VIII product, and in serum or stool from two factor VIII recipients and one factor IX recipient. The genetics sequence of the virus in the plasma pool, the factor VIII lot, and the factor VIII recipients were identical, while that of the virus in the factor IX recipient differed by a single base. CONCLUSION: These data document the transmission of HAV by a factor VIII concentrate and implicate factor IX products manufactured from a common source-plasma pool.

[Chimpanzee model of hepatitis E virus infection].

For the study of the hepatitis E virus infection animal model, two chimpanzee were inoculated with hepatitis E virus. After one month of the inoculation, the animal got typical hepatitis profile. ALT, anti virus-IgM, antivirus-IgG, were detected in detail. The results showed solid HEV infection evidence of typical hepatitis within one month after the inoculation.

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).

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.

Expression of a hepatitis E virus (HEV)-trpE fusion protein containing epitopes recognized by antibodies in sera from human cases and experimentally infected primates.

A 1700 base cDNA fragment coding for the putative structural gene(s) of hepatitis E virus (HEV) was inserted into the pATH 10 expression vector. The fusion protein (C2) expressed by this plasmid was found to contain epitopes recognized by anti-HEV antibodies. C2 protein was used in a Western blot format to examine its usefulness in detecting anti-HEV antibodies in well documented human cases of HEV and non-human primates infected with HEV. Both IgM and IgG anti-HEV could be detected in our Western blot assay. This Western blot assay was found not to detect antibodies from acute-phase sera from patients with either HAV or HBV. The C2 protein contains broadly cross-reactive epitopes, and the Western blot assay was able to detect anti-HEV antibodies in patient sera from Asia, Africa, and North America. The optimum serum dilution for the detection of both IgM and IgG was 1:25.

Application of two RNA extraction methods prior to amplification of hepatitis E virus nucleic acid by the polymerase chain reaction.

Amplification of the enterically-transmitted non-A, non-B hepatitis virus (HEV) RNA using conventional reverse transcriptase reactions followed by the polymerase chain reaction (PCR) of the cDNA has not been successful. However, after application of two different RNA capture/extraction methods we were able to amplify HEV nucleic acid from clinical samples and specimens from experimentally infected animals. The first procedure, adapted from an immune electron microscopy (IEM) technique, incorporated an immunocapture step with concentration of the virus-antibody complexes by pelleting in a Beckman airfuge. In the second method, glass powder (or size-fractionated silicon dioxide) was used to capture the RNA from its surrounding milieu by adsorption of the nucleic acid to the silicate particles. Since conventional immunoassays for HEV antigen or antibody are not currently available, the use of these RNA extraction methods, coupled with PCR techniques, will be valuable in screening clinical specimens and in further defining the course of disease using animal infectivity studies.

Hepatitis E virus: identification of type-common epitopes.

Large epidemic outbreaks of enterically transmitted non-A, non-B viral hepatitis (ET-NANBH) have been documented in developing countries. A molecular clone derived from the causative agent, the hepatitis E virus (HEV), has recently been described (G.R. Reyes, M.A. Purdy, J.P. Kim, K.-C. Luk, L.M. Young, K.E. Fry, and D. Bradley, Science 247:1335-1339, 1990). We now report the isolation, by serologic screening, of two cDNA clones derived from a fecal sample collected during a 1986 outbreak of ET-NANBH in Telixtac, Mexico. The cDNA clones encode epitopes that specifically reacted with acute- and convalescent-phase sera collected during five different ET-NANBH epidemics and represent the initial cloning of the Mexico strain of HEV. Recombinant fusion proteins expressed from these clones were also recognized by antibodies from cynomolgus macaques experimentally infected with HEV. The cDNA clones were shown to be derived from HEV by their specific hybridization to the previously recognized full-length genomic RNA transcript of approximately 7.5 kb. In addition, however, subgenomic polyadenylated transcripts of approximately 2.0 and approximately 3.7 kb were also identified in HEV-infected cynomolgus monkey liver. Sequences homologous to the epitope clones were isolated from the Burma strain of the virus, and these demonstrated reactivity comparable to that seen with the Mexico strain epitopes. When compared with the available full-length sequence of the Burma strain of HEV, it was discovered that the cDNA clones were encoded in different open reading frames (ORFs). The comparison between Mexico and Burma HEV strains indicated amino acid homologies of 90.5 and 73.5% for these epitope-encoding clones derived from ORF2 and ORF3, respectively. The identification of these clones not only has provided insight into the expression strategy of HEV but has also resulted in a source of recombinant protein useful in the diagnosis of HEV-induced hepatitis.

Parenterally transmitted non-A, non-B hepatitis: virus-specific antibody response patterns in hepatitis C virus-infected chimpanzees.

An established chimpanzee model of parenterally-transmitted non-A, non-B hepatitis was used to define virus-specific immune response patterns in acutely and persistently infected animals. Serial bleedings were obtained from 23 chimpanzees that had been experimentally infected with an isolate of hepatitis C virus, originally recovered from contaminated lots of factor VIII (antihemophilic) materials. Sera were assayed for the presence of antihepatitis C virus by a newly developed radioimmunoassay procedure that incorporated recombinant DNA-expressed viral antigen as a reagent. Twenty-one of 23 hepatitis C virus infected animals were shown to acquire antihepatitis C virus, most within 2-8 weeks after the major peak of alanine aminotransferase activity. All chimpanzees with biochemical, electron microscopic, and histological evidence of chronic disease clearly acquired antibody; 14 of 16 animals observed through the acute phase of disease were also shown to acquire antibody. A booster effect or anamnestic response was noted in two chimpanzees (one of which was negative for antihepatitis C virus following the acute phase of disease) after challenge with hepatitis C virus. Antihepatitis C virus was not neutralizing, because some animals with high levels of antibody were also shown to have high titers of circulating hepatitis C virus. The development and maintenance of anti-hepatitis C virus appears to reflect concomitant virus replication and high potential for infectivity.

Enterically transmitted non-A, non-B hepatitis: serial passage of disease in cynomolgus macaques and tamarins and recovery of disease-associated 27- to 34-nm viruslike particles.

An experimental model of enterically transmitted non-A, non-B hepatitis (ET-NANBH) was established in tamarins (Saguinus mystax mystax) and cynomolgus macaques (Macaca fascicularis). First-passage animals were inoculated with two different stool suspensions obtained from human patients with well-defined ET-NANBH that originated from Burma and Pakistan, where epidemics of ET-NANBH occur. Both inocula contained 27- ato 34-nm-diameter viruslike particles (VLPs) that were specifically aggregated by acute-phase ET-NANBH sera. ET-NANBH was subpassaged in both tamarins and cynomolgus macaques by using pools of stool suspensions from first-passage animals. One additional passage of disease in cynomolgus macaques resulted in a significantly shortened incubation period and increased severity of disease. VLPs similar to those found in the human inocula were observed in stool specimens of first-, second-, and third-passage cynomolgus macaques and in first- and second-passage tamarins. Our findings indicate that cynomolgus macaques are particularly suitable experimental models for studies of human ET-NANBH. The 27- to 34-nm VLPs found in infected human and primate stools appear to be etiologically linked to disease.

Biochemically silent posttransfusion non-A, non-B hepatitis interferes with superinfection by hepatitis A virus.

Superinfection of a silently non-A, non-B (NANB)-infected chimpanzee with hepatitis A virus (HAV) resulted in minimal liver enzyme elevations, lack of detectable HAV in stool, and questionable presence of HAV antigen in liver biopsy specimens obtained during the expected period of virus replication. Our findings indicate that even biochemically silent NANB hepatitis can strongly interfere with infection by at least one other hepatotrophic virus.

Posttransfusion non-A, non-B hepatitis in chimpanzees. Physicochemical evidence that the tubule-forming agent is a small, enveloped virus.

Posttransfusion non-A, non-B hepatitis associated with the formation of hepatocyte cytoplasmic tubules was experimentally transmitted to chimpanzees by intravenous inoculation of a proven-infectious plasma that had been pelleted and microfiltrated, or purified by a combination of pelleting and rate-zonal banding. The results of these studies indicate that a factor VIII-derived non-A, non-B tubule-forming agent will pass through an 80-nm membrane filter and that it can be recovered from infected plasma by use of a purification procedure that assumes the non-A, non-B tubule-forming agent is a small, enveloped virus. Our findings, in combination with the known sensitivity of the non-A, non-B tubule-forming agent to chloroform and its apparent lack of nucleic acid homology with hepatitis B virus, further suggest that at least one etiologic agent of human posttransfusion non-A, non-B hepatitis may be a small, enveloped RNA virus.

Hepatitis A virus: growth characteristics of in vivo and in vitro propagated wild and attenuated virus strains.

Serial passage of the MS-1 strain hepatitis A virus (HAV) in marmosets was shown to increase the yield of virus and to shorten the incubation period from approximately 55 days in the first passage to 3-7 days in the ninth and higher passages. Intravenous inoculation of susceptible chimpanzees with MS-1 HAV was found to result in a typical course of disease in two animals who had received eighth marmoset-passage virus, including the occurrence of elevated ALT activity, presence of HAV antigen in liver and stool, and seroconversion to anti-HAV. Two chimpanzees inoculated with 20th passage MS-1 HAV (M001 liver homogenate) exhibited normal or nearly normal ALT activity and had no demonstrable or significant HAV in weekly liver biopsy specimens or in serial stool suspensions obtained during 64 days of observation. However, both animals seroconverted to anti-HAV within 2 weeks after inoculation, as did the animals who had received eighth passage MS-1 HAV. These findings suggest that subpassage of the MS-1 strain of HAV in marmosets resulted in the generation of an attenuated virus strain that was still capable of inducing a vigorous antibody response in intravenously infected chimpanzees. Serial propagation of wild and attenuated strains of HAV (HAS-15 and MS-1/M001, respectively) in FRhK-4 cells was associated with a significant decrease in the growth period for both viruses. Our studies have also shown that HAS-15 HAV can be recovered in maximum yield in later passages as early as 2 to 3 days after inoculation.

Posttransfusion non-A, non-B hepatitis: physicochemical properties of two distinct agents.

Two separate and distinct episodes of non-A, non-B hepatitis were induced in each of two chimpanzees by two inocula: one containing a chloroform-resistant agent and the other containing a chloroform-sensitive agent. Both agents were recovered from liver tissue and plasma obtained from a single chimpanzee during the acute and chronic phases of infection with a factor VIII concentrate, respectively. The chloroform-resistant agent did not cause unique changes in hepatocytes; in contrast, the chloroform-sensitive agent did induce the formation of cytoplasmic tubules, convoluted endoplasmic reticulum, and dense reticular inclusion bodies. The latter changes are similar in character to those induced in infected cells by some enveloped mammalian RNA viruses.