[The virus isolated from patient TT (TTV): still an orphan 2 years after its discovery]. / Le virus isolé du patient TT (TTV): toujours orphelin deux années après sa découverte.

TTV is the acronym for a virus isolated two years ago from a patient whose initials were T.T. It is a naked virus probably belonging to the Circoviridae family. TTV has a particle size of 30-50 nm and possesses a single-strand circular DNA. Epidemiologic data are derived from studies looking for the viral DNA by the polymerase chain reaction (PCR). Important differences between early and recent studies appear to be due to the use of PCR assays based on primers located in different regions of the genome. Based on the most recent studies, the prevalence of TTV infections seems very high in the general population. TTV is present in the feces and would be transmitted through the fecal-oral route. It appears to be a ubiquitous virus, also present in various animal species, from chickens to chimpanzees. No association to any pathology has been identified so far, and TTV infection does not have a significant effect on liver disease.

[Hepatitis viruses]. / Les virus des hépatites.

Five different hepatitis viruses that cause acute and chronic hepatitis in human beings have been identified. Hepatitis viruses are designated by the letters A, B, C, D and E and abbreviated as HAV, HBV, HCV, HDV, and HEV, respectively. Although each belongs to a distinctly different virologic group, they share the feature of hepatotropism, which remains poorly understood. HAV and HEV are transmitted through a fecal-oral route. HDV, also referred to as the delta agent, is a defective, parenterally transmitted virus that uses the hepatitis B surface antigen (HBsAg) of HBV as its envelope. Thus, HDV infection occurs only in patient with HBV infection. Application of molecular biologic techniques has been instrumental in elucidating the genomic sequences of the individual hepatitis viruses and in understanding viral gene expression and mechanisms of viral replication. Knowledge of the genomic structure of these viruses and perfection of molecular biological techniques have also led to clinical applicability of molecular diagnostic techniques. Indeed, these molecular diagnostic techniques have rapidly become the standards against which serologic tests are compared.

[Viral hepatitis and morphology of hepatitis viruses]. / A virushepatitis és a hepatitis virusok morfológiája.

Our knowledge has increased significantly about the hepatitis viruses and the disease caused by them. The most important hepatitis inducing viruses are the hepatitis A, B, C, D and E viruses, however, probably new members will be added soon. The possibility of differentiation and detection of these viruses was based on methods of molecular biology and serology.

Identification of enterically transmitted hepatitis virus particles by solid phase immune electron microscopy.

Small 'featureless' viruses (less than 50 nm) are difficult to identify by routine immune electron microscopy techniques, particularly when they are mixed with debris from stool or cell culture extracts. A combination of conventional immune electron microscopy (IEM) and solid phase IEM (SPIEM) methodologies was used to identify hepatitis A virus (HAV) in stool and cell culture extracts and non-A non-B hepatitis (hepatitis E) in stool extracts. Compared with conventional IEM, the modified SPIEM method resulted in a significant increase in the number of particles observed. Several small aggregates, each containing 2-20 particles, were observed scattered randomly within most grid squares. Similar results were seen with stool extracts from hepatitis E (HEV) infections. The SPIEM method is a simple, highly sensitive specific assay that facilitates rapid identification of enteric hepatitis viruses. Several experiments were done to characterize the effects of altered physical environment within the assay and to evaluate potential modifications.

Virus-like particles in the liver of a patient with fulminant hepatitis and antibody to hepatitis E virus.

In earlier studies, hepatitis E virus (HEV) particles were detected in the stools of patients with enterically transmitted non-A, non-B (ENANB) hepatitis, and HEV was etiologically associated with this disease. Such particles have not been observed in the liver, however. We describe the pathological findings in the liver of a young pregnant woman from Nepal who died as a result of fulminant NANB hepatitis. IgM antibody to HEV was detected in the patient's serum by immune electron microscopy, suggesting that she was acutely infected with that virus. On light microscopic examination of the liver we observed cholestatic hepatitis with proliferation of bile ductules and pseudoglandular arrangement of hepatocytes around distended bile canaliculi. Three types of virus-like particles were detected by electron microscopy. The most frequently observed particles were in cells lining small bile ductules; they measured 32-37 nm and were enclosed by a membrane. Particles of a second type were seen in clusters in the sinusoidal cells; they were uniform in size, without a membrane, and measured about 32 nm in diameter. Particles of a third type (65 nm) were found in epithelial cells of the small bile ductules. Among the particles we detected, the 32 nm particles most closely resembled those of HEV.

Expression of infectious viral particles by primary chimpanzee hepatocytes isolated during the acute phase of non-A, non-B hepatitis.

Liver wedge biopsies were obtained from chimpanzees during the acute phase of experimental non-A, non-B hepatitis infections. Primary chimpanzee hepatocytes were maintained for over 4 weeks in vitro with a serum-free medium supplemented with growth factors and hormones. The de novo synthesis and secretion of plasma proteins characteristic for differentiated primate hepatocytes were sustained under these culture conditions. Immunocytochemical staining for a non-A, non-B hepatitis-associated antigen revealed expression of this cytoplasmic marker during the culture period, indicating a persistence of the infection in vitro. Tissue culture medium derived from the hepatocyte cultures was used to inoculate a nonimmune chimpanzee. The animal subsequently displayed an increase in the serum levels of alanine aminotransferase, the development of histopathologic alterations indicative of viral hepatitis, and the appearance of liver cell cytoplasmic tubules diagnostic for non-A, non-B hepatitis. Concentrated tissue culture medium examined by electron microscopy contained virus-like particles with an average diameter of 39-46 nm, which exhibited an envelope and inner 37-nm core structure.

Epidemic transmission of enterically transmitted non-A, non-B hepatitis in Mexico, 1986-1987.

Outbreaks of acute hepatitis occurred in Huitzililla and Telixtac, two rural villages 70 miles south of Mexico City, Mexico, in late 1986. The first outbreak began in Huitzililla in June of that year, 1 month after the start of the rainy season. A census revealed 94 icteric case subjects, for an attack rate of 5%; two women died. Attack rates were higher for persons older than 15 years (10%) than for younger persons. A case-control study showed that illness was highly associated with water-related factors. The second outbreak began in August 1986 in Telixtac. There were 129 case subjects, for an attack rate of 6%; one woman died. Epidemiologic findings were similar to those in Huitzililla, except that most disease transmission was not linked to unsafe water sources. None of 62 case subjects in Huitzililla and only 2 of 53 case subjects in Telixtac tested had serological evidence for recent infection with hepatitis A or B. Two of eight stool samples from Huitzililla and one of the eight stool samples from Telixtac were positive by immune electron microscopy for 32- to 34-nm viruslike particles similar to those seen in cases of enterically transmitted non-A, non-B hepatitis from Asia. To our knowledge, these investigations document for the first time the epidemic transmission of enterically transmitted non-A, non-B hepatitis virus in the Americas.

Enterically-transmitted non-A, non-B hepatitis.

More than 50% of acute viral hepatitis occurring in some developing countries appears to be unrelated to infection by HAV or HBV and accumulating evidence suggests that a high proportion of this non-A, non-B hepatitis (NANB) is enterically transmitted. Epidemics or outbreaks of enterically-transmitted NANB (ET-NANB) have been documented in the Soviet Union, Nepal, Burma, Pakistan, India, Borneo, Somalia, Sudan, Ivory Coast, Algeria, and Mexico. These outbreaks primarily affect young to middle-age adults and are often associated with a high mortality rate in infected pregnant women, approaching 20% in most reported epidemics. Several investigators have reported finding 27 to 34 nm virus-like particles (VLPs) in stools of acutely infected cases. Stools containing these small, non-enveloped VLPs have been shown to cause NANB in experimentally infected cynomolgus macaques, African green monkeys, chimpanzees, rhesus monkeys, and Saguinus mystax monkeys (tamarins). Infected primates have been shown to seroconvert to 27-34 nm VLPs recovered from stools of cases occurring in the Soviet Union, India, Nepal, Burma, Pakistan and/or Mexico, suggesting that ET-NANB is caused by one virus or class of serologically related viruses. The morphological features and physicochemical properties of one candidate virus are very similar to those of some human caliciviruses, a group of viruses that is normally associated with outbreaks of severe diarrhoea.

Animal transmission of enteric non-A, non-B hepatitis infection to Macaca mulatta by faeco-oral route.

M. mulatta monkeys were inoculated faeco-orally by enteric non-A, non-B virus to study the development of clinical, biochemical, histopathological and serological changes in the blood and liver. Pooled stool samples positive for putative non-A, non-B viral antigen by micro-ELISA and aggregated viral particles by immune electron microscopy, were administered in two M. mulatta monkeys. Biochemical, histopathological and serological changes were seen in the blood and liver and excretion of 27 nm virus like particles around 27 days of inoculation in the experimental monkey but not in the control animal.

Serial transmission of a putative causative virus of enterically transmitted non-A, non-B hepatitis to Macaca fascicularis and Macaca mulatta.

In order to establish an animal model and to identify a causative virus of enterically transmitted non-A, non-B hepatitis, Macaca fascicularis was inoculated with a fecal extract obtained from Myanmar patients with acute sporadic non-A, non-B hepatitis. The primates developed acute hepatitis exhibited by a transient elevation of aminotransferases in the sera and occurrence of hepatic necroinflammation between 2 and 4 weeks postinoculation. Subsequent second passage of the fecal extract made from first-passage primates into another Macaca fascicularis and Macaca mulatta induced acute hepatitis. Likewise, third passage was also successfully performed. Immune electron microscopy of the stool extract incubated with the primate serum at the acute phase of hepatitis showed an aggregation of virus-like particles. These particles consisted of full and empty round particles without an envelope, measuring approximately 27 nm in diameter. A dispersion of similar particles was found ultrastructurally in the hyaloplasm of hepatocytes surrounding the focal necrosis. This putative causative virus appears to be a new hepatitis virus.

Occurrence and character of a putative causative virus of enterically-transmitted non-A, non-B hepatitis in bile.

The present investigation confirms the possibility that the etiological agent of enterically-transmitted non-A, non-B (ET-NANB) hepatitis (type E hepatitis), multiplied in hepatocytes, is excreted into the feces via bile. The fecal extract was inoculated into 7 cynomolgus monkeys. Bile juice was collected directly from the gallbladder by needle puncture after abdominal operation 3 to 6 times during the experimental course. All 7 monkeys developed elevated serum aminotransferases, which began gradually approximately 2 weeks postinoculation and reached a peak at 3 to 5 weeks. In parallel with this elevation, both in time and magnitude, necroinflammation was observed in the livers. The virus-like particles (VLPs) were found in the bile juice of all 7 monkeys and the serial occurrence of VLPs was typified as follows: the VLPs were negative on day 7, appeared on day 10 after inoculation, and were present until the 3rd week when the subjects were sacrificed. While the particles were individually dispersed on day 10, they started to exhibit spontaneous aggregation on and after week 2. Also, empty particles were very rare at first, but increased in ratio compared to full ones over time. Thus, the putative causative virus of ET-NANB hepatitis was demonstrated to be excreted through bile. The spontaneous aggregation of VLPs might be due to the specific antibody secreted into the bile juice and was closely correlated with hepatitis activity. The increase in empty particles might indicate an increase in disorganized assembly of the nucleic acid and protein during virus proliferation.

Prevalence of intranuclear particles in liver pathologies.

An ultrastructural study of the prevalence of electron dense 23-27 nm intranuclear particles was carried out on liver biopsies from patients with NANB chronic active hepatitis (CAH), Delta + CAH, HBsAg + CAH, nonviral liver pathologies and in one healthy volunteer. The particles were classified according to aggregation pattern and were found to be correlated with NANB CAH and Delta + CAH. No particles were observed in nonviral liver pathologies. A close antigenic relationship has been shown between the cytoplasmic alterations observed in NANB and delta hepatitis in chimpanzees. Our data indicate that there is a structural similarity between the intranuclear particles seen in both Delta and NANB hepatitis, thus reinforcing the hypothesis that the NANB and Delta agents are closely related.

Characterization of the major duck hepatitis B virus core particle protein.

The amino acid composition of the major duck hepatitis B virus (DHBV) core particle proteins was determined. The results of this analysis indicated that cores are composed of a single major protein that initiates translation from the second available AUG in the DHBV core gene. Proteins isolated from core particles purified from the cytoplasm of DHBV-infected duck hepatocytes exhibited heterogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, independent of the stage of viral DNA maturation. Incubation of native cores with alkaline phosphatase removed this heterogeneity, indicating that phosphorylation of external amino acids was responsible. Core protein isolated from mature DHBV purified from serum of infected animals did not display heterogeneity, suggesting a possible role for dephosphorylation in virus maturation.

Viruslike particles in liver in sporadic non-A, non-B fulminant hepatitis.

In a patient who followed the typical clinical course of fulminant hepatitis attributable to "sporadic" non-A,non-B (NANB) hepatitis and who finally received treatment by orthotopic liver grafting, three, apparently separate, virus-like agents (26, 45, and 80 nm) and cytoplasmic, reticular tubular structures (CTS) were identified in collapsed and regenerating areas of liver using electron microscopy. The 80-nm particles present within vacuoles, together with the finding of intranuclear rods in association with the smaller particles (26 nm), are similar to those found in the nuclei of cells infected with several different arboviruses. The third type of particle, existing as 45-nm spheres and rods, is similar in morphology only to some form of polyoma virus, which, hitherto, has not been reported as affecting the liver. Unlike typical polyoma virus, replication of the virus "cores" (25-26 nm) was extranuclear and appeared to be occurring in vacuoles. Although analysis for serological markers against a representative panel for arboviruses, flaviviruses, phleboviruses, arenavirus, and nairovirus was negative, an insect vector was implicated in the clinical history.

An enzyme-linked immunoassay for the possible detection of non-A, non-B viral antigen in patients with epidemic viral hepatitis.

An enzyme-linked immunosorbent assay has been developed by using IgM antibodies from the acute stage as a source to capture the antigen in stools of patients with epidemic non-A, non-B (NANB) viral hepatitis. 29/69 (42.3%) of the patients and 3/9 (33.3%) contacts were positive for a suspected NANB viral antigen. However, only 1/27 (3.7%) of the negative controls drawn from amongst the patients with amoebiasis, giardiasis, hepatitis due to virus A and healthy individuals was positive for NANB antigen in the stool. The suspected NANB viral antigen was more frequently detected in stools collected between the 14th and 18th day of icteric hepatitis. The study suggests that IgM antibodies from patients with acute viral NANB hepatitis react with an antigen present in the stools of a high proportion of patients with epidemic NANB viral hepatitis. This serological test may be useful to establish the etiological diagnosis of non-A, non-B (fecal-oral) viral hepatitis. ELISA-positive stools contained 27 nm viral particles.