Noroviruses are global agents of acute gastroenteritis, but the development of control strategies has been hampered by the absence of a robust animal model. Studies in chimpanzees have played a key role in the characterization of several fastidious hepatitis viruses, and we investigated the feasibility of such studies for the noroviruses. Seronegative chimpanzees inoculated i.v. with the human norovirus strain Norwalk virus (NV) did not show clinical signs of gastroenteritis, but the onset and duration of virus shedding in stool and serum antibody responses were similar to that observed in humans. NV RNA was detected in intestinal and liver biopsies concurrent with the detection of viral shedding in stool, and NV antigen expression was observed in cells of the small intestinal lamina propria. Two infected chimpanzees rechallenged 4, 10, or 24 mo later with NV were resistant to reinfection, and the presence of NV-specific serum antibodies correlated with protection. We evaluated the immunogenicity and efficacy of virus-like particles (VLPs) derived from NV (genogroup I, GI) and MD145 (genogroup II, GII) noroviruses as vaccines. Chimpanzees vaccinated intramuscularly with GI VLPs were protected from NV infection when challenged 2 and 18 mo after vaccination, whereas chimpanzees that received GII VLPs vaccine or a placebo were not. This study establishes the chimpanzee as a viable animal model for the study of norovirus replication and immunity, and shows that NV VLP vaccines could induce protective homologous immunity even after extended periods of time.

An infectious cDNA clone of hepatitis E virus was mutated in order to prevent synthesis of either open reading frame 2 (ORF2) protein or ORF3 protein. HuH-7 cells transfected with an ORF2-null mutant produced ORF3, and those transfected with an ORF3-null mutant produced ORF2. Silent mutations introduced into a highly conserved nucleotide sequence in the ORF3 coding region eliminated the synthesis of both ORF2 and ORF3 proteins, suggesting that it comprised a cis-reactive element. A mutant that was not able to produce ORF3 protein did not produce a detectable infection in rhesus macaques. However, a mutant that encoded an ORF3 protein lacking a phosphorylation site reported to be critical for function was able to replicate its genome in cell culture and to induce viremia and seroconversion in rhesus monkeys, suggesting that phosphorylation of ORF3 protein was not necessary for genome replication or for production of infectious virions.Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in many developing countries, where it is responsible for massive waterborne epidemics, as well as sporadic cases of hepatitis E (10). Although originally thought to be virtually absent from most industrialized countries, sporadic cases are becoming more commonly recognized, probably due to increased awareness and better diagnostic tests. The relatively high seroprevalence of anti-HEV in normal populations in the industrialized countries coupled with a very high seroprevalence in numerous animal species, including rodents, swine, and chickens, has lent credence to the hypothesis put forward by Balayan many years ago that hepatitis E is a zoonotic disease (7). Recently, convincing evidence that HEV might be transmitted by eating raw or undercooked meat was obtained (15,21).HEV is a nonenveloped virus with a single-stranded RNA genome of 7.2 kb (10). The genome is positive sense and contains three open reading frames (ORFs). The 5Ј two-thirds of the genome contains ORF1, which encodes nonstructural proteins involved in viral RNA synthesis. ORF2, the capsid gene, occupies the 3Ј-terminal part of the genome and ORF3, which overlaps both ORF1 and ORF2, encodes a very small protein containing only 123 amino acids.HEV has been very difficult to grow in cell culture, so only the most basic knowledge of its molecular biology has been obtained. The genome is capped and polyadenylated, and the first step of infection after uncoating and release of the viral genome into the cytoplasm is expected to be translation of ORF1. ORF2 and ORF3 proteins are believed to be encoded by separate but 3Ј-coterminal subgenomic RNAs (14,20), which would need to be synthesized by the newly translated viral RNA-dependent RNA polymerase (RdRp): their termini have not yet been mapped, and it is not known how their synthesis or translation is regulated.ORF2 protein is the major, if not only, viral capsid protein and as such would be required for virion production and infectivity. In vitro vector-based expression studies suggest recombinant ORF2 protei...

Hepatitis B virus (HBV)-associated acute liver failure (ALF) is a dramatic clinical syndrome due to a sudden loss of hepatic cells leading to multiorgan failure. The mechanisms whereby HBV induces ALF are unknown. Here, we show that liver tissue collected at the time of liver transplantation in two patients with HBVassociated ALF is characterized by an overwhelming B cell response apparently centered in the liver with massive accumulation of plasma cells secreting IgG and IgM, accompanied by complement deposition. We demonstrate that the molecular target of these antibodies is the hepatitis B core antigen (HBcAg); that these antibodies display a restricted variable heavy chain (V H ) repertoire and lack somatic mutations; and that these two unrelated individuals with ALF use an identical predominant V H gene with unmutated variable domain (IGHV1-3) for both IgG and IgM anti-HBc antibodies, indicating that HBcAg is the target of a germline human V H gene. These data suggest that humoral immunity may exert a primary role in the pathogenesis of HBV-associated ALF.

The chimpanzee is the only animal model for investigating the pathogenesis of viral hepatitis types A through E in humans. Studies of the host response, including microarray analyses, have relied on the close relationship between these two primate species: chimpanzee samples are commonly tested with human-based reagents. In this study, the host responses to two dissimilar viruses, hepatitis E virus (HEV) and hepatitis C virus (HCV), were compared in multiple experimentally infected chimpanzees. Affymetrix U133 ؉ 2.0 human microarray chips were used to assess the entire transcriptome in serial liver biopsies obtained over the course of the infections. Respecting the limitations of microarray probes designed for human target transcripts to effectively assay chimpanzee transcripts, we conducted probe-level analysis of the microarray data in conjunction with a custom mapping of the probe sequences to the most recent human and chimpanzee genome sequences. Time points for statistical comparison were chosen based on independently measured viremia levels. Regardless of the viral infection, the alignment of differentially expressed genes to the human genome sequence resulted in a larger number of genes being identified when compared with alignment to the chimpanzee genome sequence. This probably reflects the lesser refinement of gene annotation for chimpanzees. In general, the two viruses demonstrated very distinct temporal changes in host response genes, although both RNA viruses induced genes that were involved in many of the same biological systems, including interferoninduced genes. The host response to HCV infection was more robust in the magnitude and number of differentially expressed genes compared to HEV infection.

Hepatitis E virus is a nonenveloped RNA virus. However, the single capsid protein resembles a typical glycoprotein in that it contains a signal sequence and potential glycosylation sites that are utilized when recombinant capsid protein is overexpressed in cell culture. In order to determine whether these unexpected observations were biologically relevant or were artifacts of overexpression, we analyzed capsid protein produced during a normal viral replication cycle. In vitro transcripts from an infectious cDNA clone mutated to eliminate potential glycosylation sites were transfected into cultured Huh-7 cells and into the livers of rhesus macaques. The mutations did not detectably affect genome replication or capsid protein synthesis in cell culture. However, none of the mutants infected rhesus macaques. Velocity sedimentation analyses of transfected cell lysates revealed that mutation of the first two glycosylation sites prevented virion assembly, whereas mutation of the third site permitted particle formation and RNA encapsidation, but the particles were not infectious. However, conservative mutations that did not destroy glycosylation motifs also prevented infection. Overall, the data suggested that the mutations were lethal because they perturbed protein structure rather than because they eliminated glycosylation.Hepatitis E virus (HEV) is transmitted via the fecal-oral route, predominantly through contaminated water. HEV causes acute self-limiting hepatitis and in much of the developing world is responsible for sporadic infections, as well as large epidemics, of acute hepatitis E, especially in Asia and Africa.Four genotypes comprising a single serotype of mammalian HEV have been identified (26,28). HEV was recently classified as the sole member of the genus Hepevirus, family Hepeviridae (3); sequence analysis suggests it is most closely related to rubella virus, an enveloped virus in the family Togaviridae. However, HEV does not contain a lipid envelope (26). The virion is 27 to 30 nm in diameter (1, 26) and has a sedimentation coefficient of 183S (2). The HEV genome is a positivesense RNA, approximately 7.2 kb in length, with a short, capped 5Ј noncoding region and a 3Ј noncoding region preceding a poly(A) tail. Viral genomic RNA is infectious for some cultured cells and nonhuman primates: transfection with capped recombinant genomes results in production of infectious virions in vitro and acute hepatitis and/or seroconversion in vivo (6-8). The coding region of HEV contains three partially overlapping open reading frames (ORFs): ORF1 encodes the nonstructural proteins, ORF2 encodes the capsid protein, and ORF3, which overlaps the N terminus of ORF2, encodes a small protein of 114 amino acids (aa) (13, 15) that might be a regulatory protein (7,19,33,44). The ORF2 and ORF3 proteins are encoded by a bicistronic subgenomic RNA (13,35,45).HEV does not grow sufficiently well either in cell culture or in nonhuman primates to permit direct biochemical analysis. Therefore, various in vitro systems have been utili...

The recombinant truncated ORF2 (capsid) antigen derived from the Meng strain of swine hepatitis E virus (HEV) differs from that of the Sar-55 strain of human HEV by approximately 5% at the amino acid level. Serial serum samples from two chimpanzees and six rhesus monkeys experimentally infected with HEV were tested with one enzyme immunoassay (EIA) based on the Sar-55 antigen and with a second EIA based on the Meng antigen. We obtained 98% agreement ( ‫؍‬ 0.952) by direct comparison. The virtually identical results obtained with these antigens in detecting seroconversion following infection with HEV suggests that they were reacting with antibodies that detect the same or very similar epitopes of HEV. We then tested human and swine serum samples for anti-HEV in EIAs that utilized one or the other of the two ORF2 antigens and showed that these results were also virtually identical. The specimens tested included swine sera from the United States, Canada, China, Korea, and Thailand and sera from veterinarians, U.S. and non-U.S. volunteer blood donors, and U.S. and non-U.S. animal handlers. We tested 792 swine sera and obtained 93% agreement ( ‫؍‬ 0.839). We similarly tested 882 human sera and obtained 99% agreement ( ‫؍‬ 0.938). Moreover, we found virtually no difference in the levels of prevalence of anti-HEV as measured by the two tests, again suggesting that the antigens derived from human and swine HEV contain the same immunodominant epitopes.Hepatitis E, which was previously described as epidemic waterborne hepatitis (2, 5), is an acute, self-limiting viral disease. The etiological agent is a single-stranded positive-sense RNA virus that is not enveloped (30). The viral RNA is approximately 7.2 kb in size and contains three partially overlapping open reading frames (ORFs) (30). ORF1 encodes nonstructural proteins, while ORF2 encodes the capsid protein (35) and ORF3 encodes a cytoskeleton-associated phosphoprotein (42). The virus was originally placed within the family Caliciviridae but is presently unclassified (4).Hepatitis E is an important public health problem in developing countries and a common cause of epidemics. The virus is transmitted primarily via the fecal-oral (20,21,30), and large epidemics of hepatitis E have been linked to contaminated water and poor public health conditions (2,20,40). In general, onset of symptoms occurs about 28 to 36 days postinfection (3,36).Hepatitis E virus is similar to hepatitis A virus with regard to transmission, virulence, and epidemiology, although some reports attribute higher morbidity and mortality to hepatitis E in developing countries (5,20,22). Thus, hepatitis E in most individuals is a mild disease, except in pregnant women (particularly in the third trimester), for whom mortality rates as high as 20% have been reported (3,6,16).Hepatitis E is endemic in parts of Asia and northern Africa, and one epidemic was reported in Mexico (7,10,12,17,22,41,43). Hepatitis E has been recognized with increasing frequency in industrialized countries and other areas where inf...

The measurement of antibodies to hepatitis E virus (anti-HEV) has been essential for understanding the epidemiology of hepatitis E. Studies to determine the prevalence of HEV infections require a reliable serologic assay that is sensitive and specific. It is also important to distinguish the acute from the convalescent phase of an infection; this usually requires the detection of the immunoglobulin M (IgM) class of antibody. Few enzyme immunoassays (EIAs) that measure IgM anti-HEV have been described, and most have utilized the sandwich method. The present study describes an EIA that detects IgM anti-HEV by antibody class capture methodology. The assay was validated by using serum and/or plasma panels from experimentally infected nonhuman primates. It was used to demonstrate an anamnestic response and the reappearance of IgM anti-HEV in a chimpanzee experimentally challenged with HEV at two different times 45 months apart. The class capture method was more sensitive than the sandwich EIA when used to test clinical samples from two hepatitis E epidemics in Pakistan; it also had the advantage of distinguishing IgM anti-HEV in the presence of high titers of IgG anti-HEV.

The effect of action potentials on elimination of mouse neuromuscular junctions (NMJ) was studied in a three-compartment cell culture preparation. Axons from superior cervical ganglion or ventral spinal cord neurons in two lateral compartments formed multiple neuromuscular junctions with muscle cells in a central compartment. The loss of synapses over a 2-7-day period was determined by serial electrophysiological recording and a functional assay. Electrical stimulation of axons from one side compartment during this period, using 30-Hz bursts of 2-s duration, repeated at 10-s intervals, caused a significant increase in synapse elimination compared to unstimulated cultures (p < 0.001). The extent of homosynaptic and heterosynaptic elimination was comparable, i.e., of the 226 functional synapses of each type studied, 111 (49%) of the synapses that had been stimulated were eliminated, and 87 (39%) of unstimulated synapses on the same muscle cells were eliminated. Also, simultaneous bilateral stimulation caused significantly greater elimination of synapses than unilateral stimulation (p < 0.005). These observations are contrary to the Hebbian hypothesis of synaptic plasticity. A spatial effect of stimulus-induced synapse elimination was also evident following simultaneous bilateral stimulation. Prior to stimulation, most muscle cells were innervated by axons from both side compartments, but after bilateral stimulation, muscle cells were predominantly unilaterally innervated by axons from the closer compartment. These experiments suggest that synapse elimination at the NMJ is an activity-dependent process, but it does not follow Hebbian or anti-Hebbian rules of synaptic plasticity. Rather, elimination is a consequence of postsynaptic activation and a function of location of the muscle cell relative to the neuron. An interaction between spatial and activity-dependent effects on synapse elimination could help produce optimal refinement of synaptic connections during postnatal development.