Yukio Itoh

Species-specific TT viruses and cross-species infection in nonhuman primates.

ABSTRACT Viruses resembling human TT virus (TTV) were searched for in sera from nonhuman primates by PCR with primers deduced from well-conserved areas in the untranslated region. TTV DNA was detected in 102 (98%) of 104 chimpanzees, 9 (90%) of 10 Japanese macaques, 4 (100%) of 4 red-bellied tamarins, 5 (83%) of 6 cotton-top tamarins, and 5 (100%) of 5 douroucoulis tested. Analysis of the amplification products of 90 to 106 nucleotides revealed TTV DNA sequences specific for each species, with a decreasing similarity to human TTV in the order of chimpanzee, Japanese macaque, and tamarin/douroucouli TTVs. Full-length viral sequences were amplified by PCR with inverted nested primers deduced from the untranslated region of TTV DNA from each species. All animal TTVs were found to be circular with a genomic length at 3.5 to 3.8 kb, which was comparable to or slightly shorter than human TTV. Sequences closely similar to human TTV were determined by PCR with primers deduced from a coding region (N22 region) and were detected in 49 (47%) of the 104 chimpanzees; they were not found in any animals of the other species. Sequence analysis of the N22 region (222 to 225 nucleotides) of chimpanzee TTV DNAs disclosed four genetic groups that differed by 36.1 to 50.2% from one another; they were 35.0 to 52.8% divergent from any of the 16 genotypes of human TTV. Of the 104 chimpanzees, only 1 was viremic with human TTV of genotype 1a. It was among the 53 chimpanzees which had been used in transmission experiments with human hepatitis viruses. Antibody to TTV of genotype 1a was detected significantly more frequently in the chimpanzees that had been used in transmission experiments than in those that had not (8 of 28 [29%] and 3 of 35 [9%], respectively; P = 0.038). These results indicate that species-specific TTVs are prevalent in nonhuman primates and that human TTV can cross-infect chimpanzees.

An autoantibody cross-reactive to hepatitis C virus core and a host nuclear antigen

GOR, an epitope borne by the amino acid sequence, GRRGQKAKSNPNRPL, is recognized by anti-GOR antibodies specifically found in patients with non-A, non-B hepatitis (NANBH). The epitope is not coded for by the hepatitis C virus (HCV), the presumed causative agent for NANBH, but by a single copy gene of the host. Anti-GOR antibodies, distinct from anti-HCV (c100-3) antibodies, were revealed to have dual specificities; they target both the presumed core gene product of HCV and a host component. This cross recognition is probably derived from homologous regions between the GOR epitope and a viral epitope on the core protein in HCV. It is therefore suggested that anti-GOR is an autoantibody induced by HCV infection. This may explain the autoimmune disease like aspect of NANBH pathogenesis.

Non-A, Non-B (Type 1) Hepatitis Agent Capable of Inducing Tubular Ultrastructures in the Hepatocyte Cytoplasm of Chimpanzees: Inactivation by Formalin and Heat

We have reported two kinds of viruslike particles derived from human sera that induced morphologically and serologically different types of non-A, non-B hepatitis in chimpanzees. A chimp serum containing one such agent capable of inducing a type of hepatitis with cytoplasmic tubular ultrastructures (non-A, non-B, type 1) was titrated for its infectivity in chimps. Two chimps who received 1 ml of a 10(-2) dilution of the original serum developed the characteristic morphological changes in the liver together with elevated serum transaminase levels, while the other two who received 1 ml of a 10(-4) dilution failed to show such changes. The two who escaped the infection were proven to be susceptible to the agent, because they developed non-A, non-B, type 1 hepatitis when they were challenged by 1 ml of a 10(-1) dilution of the same serum on the 23rd week after the first inoculation. One milliliter of a 10(-1) dilution containing more than 10 chimp infecting units were inactivated in the presence of 1/2000 formalin or by heating at 100 degrees C for 5 min and then given to four other chimps. None of them developed clinical or histologic evidence of non-A, non-B, type 1 hepatitis, thereby indicating that both formalin and heat could destroy the ability of the agent to induce this type of hepatitis.

Demonstration of hepatitis B e antigen in hepatitis B core particles obtained from the nucleus of hepatocytes infected with hepatitis B virus.

Liver tissue infected with hepatitis B virus was homogenized and nuclei were separated by centrifugation. Hepatitis B core particles were obtained from the nucleus by the digestion with pronase followed by ultracentrifugation in a sucrose density gradient. Hepatitis B core particles were then treated with sodium dodecyl sulphate and 2 mercaptoethanol and tested for hepatitis B e antigen (HBeAg) by the haemagglutination method. The antigenicity of HBeAg was clearly demonstrated in hepatitis B core particles so treated, although untreated core particles did not reveal any detectable HBeAg activity. The localization of HBeAg in hepatitis B core particles was further supported by the results of a fluorescent antibody technique. When a frozen section of the liver infected with hepatitis B virus was stained with the specific rabbit antibody against HBeAg labelled with fluorescent isothiocyanate, only nuclei of hepatocytes were stained, in a similar distribution to hepatitis B core antigen visualized by fluorescent antibody against hepatitis B core antigen in a nearby section.

Lack of detectable reverse transcriptase activity in human and chimpanzee sera with a high infectivity for non-A, non-B hepatitis.

A serum sample from a patient with hepatitis and samples from two experimentally infected chimpanzees, all with a high infectivity for non-A, non-B hepatitis, were tested for reverse transcriptase. Biopsy confirmed that the hepatocytes of the chimpanzees that received these sera contained the characteristic tubular structures associated with non-A, non-B hepatitis. None of these three sera revealed detectable enzyme activity. We have not been able to confirm the association of reverse transcriptase activity with non-A, non-B hepatitis reported recently.

Nude Mice Bearing Human Primary Hepatocellular Carcinoma That Produces Hepatitis B Surface, Core, and e Antigens, as well as Deoxyribonucleic Acid Polymerase

A primary hepatocellular carcinoma, from a patient carrying hepatitis B virus, was transplanted to athymic nude mice, and maintained through eight passages involving 39 mice. Hepatitis B surface and e antigens were detected in the circulation of tumor-bearing mice. Hepatitis B surface, core, and e antigens were demonstrated in tumor cells. Hepatitis B core particles were visualized in the tumor extract by immune electron microscopy, and they exhibited an activity of deoxyribonucleic acid polymerase. In the tumor tissue, deoxyribonucleic acid of hepatitis B virus was present both in integrated and extrachromosomal forms. Nude mice carrying the tumor would provide opportunities for studying the replication of hepatitis B virus and the expression of its various proteins, and also for evaluating the efficacy of virustatic drugs in interrupting the persistent infection.

Application of hepatitis B core particles produced by human primary hepatocellular carcinoma (PLC/342) propagated in nude mice to the determination of anti-HBc by passive hemagglutination

Human primary hepatocellular carcinoma (PLC/342), carried by nude mice, produces hepatitis B core particles as well as hepatitis B surface antigen particles. Core particles purified form PLC/342 tumors displayed epitopes of hepatitis B core antigen (HBcAg) but not epitopes of hepatitis B e antigen (HBeAg) on their surface, unlike core particles prepared from Dane particles, derived from plasma of asymptomatic carriers, that expressed epitopes of both HBcAg and HBeAg. Core particles obtained from PLC/342 tumors were applied to the determination of antibody to HBcAg (anti-HBc) by passive hemagglutination. The assay detected anti-HBc not only in individuals with persistent infection with hepatitis B virus and in those who had recovered from transient infection, but also in patients with acute type B hepatitis, indicating that it can detect anti-HBc of either IgG or IgM class. A liberal availability of core particles from tumors carried by nude mice, taken together with an easy applicability of the method, would make the passive hemagglutination for anti-HBc a valuable tool in clinical and epidemiological studies, especially in places where sophisticated methods are not feasible.