Doris C. Wong

Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population.

Hepatitis E virus (HEV) is a very important public health concern in many developing countries where epidemics of hepatitis E are common. Sporadic cases of clinical hepatitis E not only occur in these countries but also occur uncommonly in patients with no known epidemiological exposure to HEV in industrialized countries. The source of infection in industrialized countries is unknown but it has been suggested that animals might serve as a reservoir for HEV in both settings. We recently identified and characterized an HEV strain (swine HEV) that infects large numbers of pigs in the United States. To assess the potential of pigs to serve as a global reservoir of HEV, we measured the prevalence of HEV antibodies in pigs in two countries where hepatitis E is endemic and two countries where it is not. Swine herds in all four countries contained many pigs that were seropositive for IgG anti‐HEV, although the percentage of seropositive pigs varied greatly from herd to herd. A very limited number of pig handlers in the two endemic countries were also tested and most of them were found to be seropositive for HEV. The results from this study suggest that hepatitis E is enzootic in pigs regardless of whether HEV is endemic in the respective human population. J. Med. Virol. 59:297–302, 1999. Published 1999 Wiley‐Liss, Inc.

Distribution of Antibody to Hepatitis A Antigen in Urban Adult Populations

To investigate the prevalence and distribution of antibody to hepatitis A antigen we tested 947 randomly selected people in the Greater New York City area; 45 per cent were antigen positive, as determined by the immune adherence method. Antibody was detected two to three times more frequently in lower social classes (72 to 80 per cent) than in middle and upper-middle classes (18 to 30 per cent). The rate of antibody detection was strongly correlated with age; the prevalence gradually increased throughout adulthood and reached its peak level in people 50 years of age and older. Those with serologic evidence of past exposure to hepatitis B virus were significantly more often antibody positive than those without such evidence (61 vs. 40 per cent; P less than 0.001). Very few of the positive subjects had had hepatitis. The prevalence of this antibody varies among different population groups, increases with age, decreases with rise in socioeconomic status, is independent on sex and race, and correlates with serologic evidence of hepatitis B virus infections.

Pre-clinical immunogenicity and efficacy trial of a recombinant hepatitis E vaccine

We have demonstrated that recombinant hepatitis E vaccine suitable for clinical evaluation was highly immunogenic and efficacious in preventing hepatitis E and even infection in rhesus macaques following intravenous challenge with three different genotypes of hepatitis E virus (HEV). Two doses of vaccine were essential for optimal protection; the two-dose regimen was more important than the formulation of the vaccine for achieving efficacy. The titers of anti-HEV that were protective in this study were quantified against a World Health Organization (WHO) standard. This permits direct comparison of the results with other studies. The results of this pre-clinical trial of a candidate hepatitis E vaccine strongly suggest that it will be highly efficacious for preventing hepatitis E in the field trial of this vaccine that is currently in progress in Nepal.

INFECTION OF A CHIMPANZEE WITH HEPATITIS C VIRUS GROWN IN CELL CULTURE

Culture supernatant harvested from Daudi cells, a lymphoplastoid cell line, after 58 days of infection with the H77 strain of hepatitis C virus (HCV), was inoculated into a chimpanzee. HCV RNA, as detected by RT-PCR, first appeared in the serum and liver 5 and 6 weeks, respectively, after inoculation. Peripheral blood mononuclear cells (PBMC) collected on week 7 were also positive for HCV RNA. The major sequences of hypervariable region 1 (HVR1) of the viral genome recovered from the inoculated chimpanzee were the ones which were the majority in the original H77 inoculum and not those which were in the majority in the culture supernatant. Only the sequence recovered from PBMC was the same as the major one found in the cell culture.

Experimental Infection of Chimpanzees with Hepatitis C Virus of Genotype 5a: Genetic Analysis of the Virus and Generation of a Standardized Challenge Pool

Six major genotypes (genotypes 1-6) of hepatitis C virus (HCV) have been identified. These genetic variants are being transmitted to chimpanzees, the only recognized animal model for the study of HCV. Genotype 5a (strain SA13), a variant found primarily in South Africa, has been transmitted to chimpanzees for the first time. Experimental infection of 2 chimpanzees was characterized by early appearance of viremia and peak virus titers of 10(5)-10(6) genome equivalents/mL. The HCV infection was resolved by week 15 after inoculation in 1 chimpanzee and persisted in the other. Both chimpanzees became anti-HCV-positive by week 14 after inoculation. Both chimpanzees developed viral hepatitis. The infectivity titer of a genotype 5a challenge pool prepared from the first passage of HCV in a chimpanzee was approximately 10(4) infectious doses/mL. Finally, sequence analysis of strain SA13 confirmed that genotype 5a is genetically distinct from other genotypes of HCV.

SIGNIFICANCE OF ANTIBODY TO MYCOPLASMA AS MEASURED BY METABOLIC-INHIBITION TECHNIQUES

A problem central to the study of mycoplasma has been the classification of strains and the identification of isolates from clinical material. Several serologic approaches to the identification of mycoplasma have been employed by laboratories in recent years. These approaches have permitted the recognition of new species of mycoplasma which were indistinguishable from established species in biologic properties. Recently we developed a new serologic technique which can be used for identification of mycoplasrna and for serodiagnosis and seroepidemiology. It is based upon Jensen’s observation that specific antiserum inhibited the reduction of tetrazoliurn by Mycoplasma pneumoniae in broth medium.’ We modified Jensen’s method by incorporating substrates such as glucose, arginine, or urea in the medium as indicators of mycoplasma growth.In this manner, antibody for all of the human mycoplasma species and many of the animal strains can now be measured by metabolic-inhibition (MI) techniques. Metabolic-inhibition Techniques. Strains of “classical” mycoplasma can be divided into two groups on the basis of whether or not they produce acid from carbohydrates. Acid production causes an acid shift in the pH of the medium. Almost all of the non-acid-producing strains metabolize the amino acid arginine with the release of ammonia into the medium and a concomitant alkaline shift in the pH. Acid or alkaline pH shift can be visualized by the incorporation of phenol red in the medium. In our tests, Haflick’s medium6 supplemented with glucose was adjusted initially to pH 7.8, whereas medium supplemented with arginine was adjusted to pH 7.0. Specific antiserum inhibited metabolism of the mycoplasma and prevented an acid or alkaline shift in pH. The highest dilution of antiserum which prevented pH change (color change) was considered 8s the MI antibody end point. For maximum sensitivity, it was necessary to record the MI antibody titer when controls containing mycoplasma but not antiserum had changed approximately 0.5 pH unit, The MI titer of immune serum was not related to the size of the mycoplasma inoculum if the titer was recorded when

Experimental Transmission of Hepatitis C Virus-Associated Fulminant Hepatitis to a Chimpanzee

Hepatitis C virus (HCV) was transmitted from a patient with fulminant hepatitis C to a chimpanzee. The patient had developed two episodes of fulminant hepatitis C, each occurring after a separate liver transplantation. Serial serum and liver samples from the patient and the chimpanzee were analyzed for HCV replication, genotype, quasispecies heterogeneity, and antibodies. In the patient, the levels of HCV replication in serum and liver correlated with the degree of hepatocellular necrosis and the clinical expression of fulminant hepatitis. The same HCV strain, genotype 1a, was recovered from both episodes of fulminant hepatitis. An unusually severe acute hepatitis was also observed in the chimpanzee. The viruses recovered from the patient and the chimpanzee were almost identical and displayed relatively little quasispecies heterogeneity. Thus, the same HCV strain induced two episodes of fulminant hepatitis in a single patient and severe hepatitis in a chimpanzee, suggesting that the pathogenicity or virulence of a specific HCV strain may be important in the pathogenesis of fulminant hepatitis C.

Relative Infectivity of Hepatitis A Virus by the Oral and Intravenous Routes in 2 Species of Nonhuman Primates

Hepatitis A virus (HAV) is naturally transmitted by the fecal-oral route but can also be transmitted intravenously. To determine the relative infectivity of these 2 routes, an infectivity titration of a standard challenge pool of virulent HAV was performed in tamarins and chimpanzees. In both species, 1 oral dose of HAV was equivalent to 10(4.5) intravenous doses. These findings have relevance for attempts to develop live, attenuated HAV vaccines that can be administered orally.

Serodiagnosis of Viral Hepatitis A by a Solid-phase Radioimmunoassay Specific for IgM Antibodies

A solid-phase radioimmunoassay for detecting specific IgM antibodies to hepatitis A virus (HAV) was developed and characterized. The test utilized microtiter plates coated with anti-IgM to specifically absorb the IgM antibodies from the test serum. The anti-hepatitis A IgM antibodies are measured by the specific consecutive binding of hepatitis A antigen and radiolabelled anti-hepatitis A antibodies (anti-HA). In 6 chimpanzees infected with HAV, IgM anti-HA was detected from about the first date of elevated transaminases and was positive for about 3 months. The usefulness of the test was confirmed by testing acute phase sera of 30 patients from a common source outbreak of epidemic hepatitis, and negative sera from 2 control groups. A collection of serum specimens from 190 patients with sporadic HBsAg-negative hepatitis in Brazil was also tested and an etiologic association with HAV was confirmed in the majority of these cases.

Serologic and animal inoculation studies of a communal outbreak of viral hepatitis, type A.

Sera from individuals in an outbreak of viral hepatitis in a multifamily household, probably spread by contaminated food, were studied for antibodies to hepatitis A virus (anti-HAV), and selected acute phase sera were inoculated into marmosets. Significant rises in anti-HAV titers between acute and convalescent sera occurred in all of 15 individuals in the outbreak who experienced serum enzyme elevations and in one of 14 individuals whose serum enzyme levels remained normal. The remaining 13 individuals in the latter group had antibody levels in both early and late sera compatible with residual immunity from prior HAV infections and correlating with resistance to reinfection. Groups of marmosets were infected with acute phase sera from two of the cases; in both instances the inoculated sera contained substantial levels of anti-HAV. The marmosets developed specific anti-HAV seroconversions as well as enzyme elevations.