Serologic response in human hepatitis A: detection of antibody by radioimmunoassay and immune adherence hemagglutination.

An indirect solid-phase radioimmunoassay (RIA) for detection of antibody to the hepatitis A antigen (anti-HAV) was developed using polystyrene pearls as the solid phase and hepatitis A antigen (HAAg) extracted from marmoset livers. This RIA was compared to an immune adherence hemagglutination assay (IAHA) which employed HAAg derived from the stools of chimpanzees collected during acute hepatitis A. Anti-HAV was detected in the sera of 15 humans with naturally acquired hepatitis A infection. Sensitivity and specificity were greater using the RIA, permitting the detection of anti-HAV as early as the time of onset of jaundice. Either seroconversion or a significant increase in the titer of anti-HAV was demonstrated following hepatitis A exposure in paired sera from six patients by both techniques. No significant difference in anti-HAV responses was noted between patients with icteric compared to anicteric hepatitis A or between children and adults with hepatitis A.     

Excretion of hepatitis A virus in the stools of hospitalized hepatitis patients

A study was carried out to determine whether hepatitis A virus (HAV) can be detected in the stools of patients hospitalized for HAV infection. Acute phase samples of whole blood and stool, as well as completed questionnaires, were obtained from 31 patients hospitalized at any of 13 hospitals in the Phoenix metropolitan area. Blood specimens were tested for hepatitis B surface antigen (HBsAg), IgG antibody to HAV (IgG anti-HAV), and IgM antibody to HAV (IgM anti-HAV). Stools were tested for HAV by radioimmunoassay. Five patients (16.1%) had acute hepatitis B, five (16.1%) had acute non-A/non-B hepatitis, and 21 (67.7%) had acute hepatitis A. Of these 21 patients with acute hepatitis A, 11 (52.4%) were found to have HAV in their stools. These results confirm the potential for infectivity of stools of patients hospitalized for hepatitis A and emphasizes the need for caution when dealing with such stools.     

Etiopathogenetic aspects of hepatitis A. I. Excretion of hepatitis A virus, biochemistry of liver function, and humoral immune response in patients with hepatitis A on admission to hospital

The excretion of hepatitis A virus (HAV) in stools from 30 patients with clinically overt hepatitis A infection on the day of their admission to the hospital was determined and compared with the dynamics and values of biochemical indices of hepatocyte injury as well as with the immune response to HAV. Virus was found in 16 out of 30 stools (53%) collected within 1 week after the appearance of clinical symptoms. In sera obtained on the day of hospitalization both IgM and IgA anti-HAV were detected in all of the 30 patients, while IgG anti-HAV were found in 20 (67%). There was a correlation between HAV excretion and increasing SGPT upon admission to hospital, while the level of SGPT or bilirubin as well as presence or absence of IgG anti-HAV did not correlate with excretion of HAV. HAV from stools was characterized morphologically and physicochemically. The majority of particles visualized by immune electron microscopy had electrondense appearance, while electron-lucid particles were only occasionally encountered. Isopycnic banding of HAV in CsCl revealed a broad range of densities with HAV activity. Rebanding of pooled fractions containing HAV revealed peak amounts of the virus in fractions with densities 1.32-1.33 gm/cm3.     

Detection of hepatitis A virus RNA in serum from patients with acute hepatitis

Hepatitis A virus (HAV) RNA was extracted from the sera of patients with acute hepatitis and then detected by molecular hybridization using cloned HAV complementary DNA (cDNA). HAV RNA was detected in 20 of 85 patients with acute HAV infection, mainly during the prodromal stage, or early during the icteric phase of the disease; it was detected as long as 21 days after its initial detection. Patients with HAV RNA in the serum had a significantly higher titer of anti-HAV IgM.     

Application of a solid-phase radioimmunoassay and immune electron microscopy for hepatitis A in diagnosis and research

With crude virus suspensions from stool and antibodies from hepatitis-A patients, a solid-phase radioimmunoassay (RIA) for detection of hepatitis virus A (HVA) and antibodies against hepatitis virus A (anti-HVA) has been developed. Examples for the application of this test are demonstrated. Virus particles from the stools of the two patients were further characterized. Serologically, they were identical or very similar to the MS-1 strain. Isopycnic CsCl-gradient centrifugation of both strains revealed two peaks, but the particles of different densities did not differ in size or serologically. A modification of the RIA was also useful for determination of IgM antibodies in patients' sera fractionated by sucrose-density centrifugation.The application of the RIA method for serologic epidemiology is demonstrated by a comparison of anti-HVA prevalence in German and non-German women residing in Germany.     

Hepatitis a virus infection: Pathogenesis and serodiagnosis of acute disease

The early development of immune electron microscopic (IEM) methods for the detection of HAV in acute-phase stool suspensions and antibody to HAV (anti-HAV) in serum made it possible to serologically identify cases of hepatitis A using paired acute and convalescent phase sera. Introduction of less cumbersome and time-consuming serologic test methods, including complement fixation (CF) and immune adherence hemagglutination (IAHA), made it feasible to rapidly assay larger numbers of specimens for HAV or anti-HAV. Subsequent development of sensitive immunofluorescence (IF) assays, solid-phase radioimmunoassays (RIA), and enzyme immunoassays (EIA) for HAV and anti-HAV heralded intensive laboratory studies of the biophysical and biochemical properties of the virus as well as efforts to define the pathogenesis and clinical course of disease. Results of the latter studies showed that the bulk of HAV was usually excreted in stool before the onset of clinical symptoms. Other serologic studies demonstrated that all acutely ill patients had circulating anti-HAV IgM, while all convalescent patients were positive for anti-HAV IgG. The development of sensitive serologic tests (RIA and EIA) that could differentiate between anti-HAV IgM and IgG made it possible to serodiagnose an acute case of hepatitis A using a single-phase serum specimen.     

持续感染甲型肝炎病毒细胞株的建立和应用

用甲型肝炎病毒（ＨＡＶ）ＨＭ１７５株感染恒河猴胚肾细胞系（Ｆｒｈｋ－４），经过连续传代培养，建立了一株持续感染ＨＡＶ的细胞株。通过１８６代的传代培养和制备ＨＡＶ抗原的实际应用，表明本株带毒培养细胞具有生长速度快、繁殖率高的特点，能大量表达ＨＡＶ抗原。１：４－１：６扩增培养，３～４天可以长成单层，培养７～１０天可以收获ＨＡＶ抗原，用于组装抗ＨＡＶＩｇＭ诊断试剂盒，该试剂盒与美国雅培公司（Ａｂｂｏｔｔ）试剂盒对照检测１４８份血清，总符合率９８．６５（１４６／１４８），与临床对照检测２１３６份病人血清，符合率为９８．９２％（２１１３／２１３６）。     

Serodiagnosis of viral hepatitis A: detection of acute-phase immunoglobulin M anti-hepatitis A virus by radioimmunoassay.

A modified micro solid-phase radioimmunoassay (RIA) for antibody to hepatitis A virus (anti-HAV) was developed. This double antibody procedure was performed by coating the surface of a polyvinyl microtiter plate "well" with 200 microliter of a 1:1,000 dilution of a patient's test serum. Purified HAV and 125I-labeled immunoglobulin G (IgG) anti-HAV were then sequentially added to form an antibody sandwich. The specificity and sensitivity of the RIA procedure for anti-HAV were verified by examination of coded human and chimpanzee serum specimens. Radioimmunoassay of early-acute-phase serum specimens from human cases of hepatitis A revealed the presence of anti-HAV activity. Differential examination by RIA of IgG and IgM fractions of acute-phase sera from experimentally infected chimpanzees demonstrated that IgM contained the bulk of the anti-HAV activity. A modification of the RIA procedure for anti-HAV (RIA-IgM blocking), incorporating an incubation step with anti-IgM (Mu chain specific), was further shown to differentiate acute- from convalescent-phase hepatitis A sera. This adapted RIA-IgM blocking procedure required less than 1 microliter of a single acute-phase serum specimen for the diagnosis of viral hepatitis A.     

Hepatitis A viral load in relation to severity of the infection

A correlation between hepatitis A virus (HAV) genomes and the clinical severity of hepatitis A has not been established. The viral load in sera of hepatitis A patients was examined to determine the possible association between hepatitis A severity and HAV replication. One hundred sixty‐four serum samples from 91 Japanese patients with sporadic hepatitis A, comprising 11 patients with fulminant hepatitis, 10 with severe acute hepatitis, and 70 with self‐limited acute hepatitis, were tested for HAV RNA. The sera included 83 serial samples from 20 patients. Viral load was measured by real‐time RT‐PCR. The detection rates of HAV RNA from fulminant, severe acute, and acute hepatitis were 10/11 (91%), 10/10 (100%), and 55/70 (79%), respectively. Mean values of HAV RNA at admission were 3.48 ± 1.30 logcopies/ml in fulminant, 4.19 ± 1.03 in severe acute, and 2.65 ± 1.64 in acute hepatitis. Patients with severe infection such as fulminant hepatitis and severe acute hepatitis had higher initial viral load than patients with less severe infection (P < 0.001). Viremia persisted for 14.2 ± 5.8 days in patients with severe infection and 21.4 ± 10.6 days in those with acute hepatitis after clinical onset (P = 0.19). HAV RNA was detectable quantitatively in the majority of the sera of hepatitis A cases during the early convalescent phase by real‐time PCR. Higher initial viral replication was found in severely infected patients. An excessive host immune response might follow, reducing the viral load rapidly as a result of the destruction of large numbers of HAV‐infected hepatocytes, and in turn severe disease might be induced. J. Med. Virol. 83:201–207, 2011. © 2010 Wiley‐Liss, Inc.     

Propagation of human hepatitis A virus in conventional cell lines

Fecal extracts of hepatitis A (HA) patients were selected for the presence of hepatitis A virus (HAV) by radioimmunoassay (RIA) and immune electron microscopy (IEM). When FL and Vero cells were inoculated with fecal extracts containing HAV, development of hepatitis A antigen (HAAg) was evident in the cytoplasm of the two cell lines by the indirect immunofluorescence (IF) test. The antigen was detectable in the cells 12 hr postinoculation (pi), and reached a plateau within two days pi. FL cell cultures inoculated with a specimen containing HAV were harvested and passaged four times. During the passages, efficient production of HAAg was confirmed in the infected cultures by three different serological tests: The indirect IF test, RIA using fixed cells, and RIA by the sandwich method. At the second and fourth passages, HAV particles were recovered in abundance from infected FL cell cultures by IEM. Throughout these experiments, no cytopathic effect (CPE) was discernible in the cultures.     

Duration of viremia in human hepatitis a viral infection as determined by polymerase chain reaction

Viremia in hepatitis A viral (HAV) infection is said to be limited to pre-symptomatic period. However, it is not clear how long viremia lasts in human infection due to the lack of a simple and sensitive detection system. In an attempt to find HAV genome in patients′ sera, we used the RTPCR method by setting a pair of primers in the 5′ non-coding region. While in most cases HAVRNA was detected only before alanine aminotransferase (ALT) reached peak levels with this sensitive system, the viral genome was observed in some patients′ sera even after ALT reached peak levels. Some patients also had HAV viremia after seroconversion to HAV antibody. These results show that viremia in HAV infection lasts longer than has been previously thought, and give a warning of possible secondary blood-borne infection. © 1993 Wiley-Liss, Inc.     

Combination of radioimmunological and immunoelectron microscopic methods for detecting hepatitis A virus

The possibility of combined performance of radioimmunoassay (RIA) and immune electron microscopy (IEM) in one preparation using protein A of Staphylococcus aureus for hepatitis A virus (HAV) detection in fecal specimens from hepatitis A patients within a short time (40-50 min) has been demonstrated. In the examinations of one preparation by RIA and IEM, their sensitivity was found to be approximately similar. According to RIA, a high content of HAV antigen was observed in those preparations where in addition to typical particles there were structures resembling individual fragments of empty HAV particles coated with antibody.     

The detection of hepatitis B surface antigen by radioimmunoassay.

A comparative study was performed to assess the sensitivity and specificity of counterimmunoelectrophoresis (CIEP) and radioimmunoassay (RIA) for the detection of hepatitis B surface antigen. The 8,823 sera examined included selected reference panels and sera collected from populations with low, moderate and high rates of chronic antigen carriage. Overall, hepatitis B surface antigen was detected in 265 sera by CIEP and in 376 by RIA. As well as detecting 46.4% additional positives, the RIA test detected all CIEP-positive sera; i.e., there were no false negative results. However, 150 sera (1.8% of the total tested) gave a positive result by RIA which was not repeatable on retesting. The explanation for this phenomenon appeared to lie in inadequate washing of the antibody-coated tubes.     

Lack of complement-dependent cytolytic antibodies in hepatitis A virus infection

Sera collected from patients with acute hepatitis A virus (HAV) infection and convalescent sera were examined for cytolytic activity against HAV-infected human-embryo lung fibroblasts (HAV carrier fibroblasts). Using the 51chromium release assay, no complement dependent antibody mediated cytolytic activity against HAV carrier cells could be detected. In control experiments with identical cell strains, anti-herpes simplex virus (HSV) positive sera and complement caused specific lysis of HSV type 1 infected target cells. The data presented here do not support the hypothesis that in the possible immunopathogenesis of HAV infection, complement-dependent cytolytic antibodies play an essential role.     

Rapid detection of human rotavirus strains in stools by single-sandwich enzyme-linked immunosorbent assay systems using monoclonal antibodies

Using murine monoclonal antibodies (MAbs) raised against the common antigen of group A rotavirus (RV), two single-sandwich ELISA systems were developed for detection of RV in stools: one using polyclonal antibody (PAb) as capture and a MAb as detector antibody (referred to as PAb-MAb assay); and the other based on the use of two different MAbs as capture and detector antibodies (referred to as MAb-MAb assay). In each single-sandwich ELISA system, samples and peroxidase-labeled MAb were incubated sequentially (two-step method) or simultaneously (one-step method). Using the two-step procedure on purified RV, 50 pg of protein was detected in the PAb-MAb as well as in the MAb-MAb assay, whereas the one-step method detected 0.4 ng and a conventional double-sandwich ELISA detected 3.2 ng of viral protein. Titration of RV samples from stools and cell cultures showed that single-sandwich ELISA titers were, on the average, 10-100-fold higher than those obtained by electron microscopy (EM), but 10-100-fold lower than those obtained by solid-phase immune EM (SPIEM). However, when 200 stool samples previously examined by EM or SPIEM were tested by the single-sandwich ELISA systems, specificity and sensitivity of these assays were 100%, and comparable to SPIEM. No false positive results were obtained when 54 samples of meconium and 91 stools from newborns in the first five days of life were tested. The two-step procedure appeared to be somewhat preferable over the one-step method, which, although faster, gave a marked prozone with a few samples in the MAb-MAb assay. The use of MAbs in rapid single-sandwich ELISA systems for RV detection in stools appears highly convenient, due to reliable results and short test performance times.     

Hepatitis A-virus in cell culture: I. Propagation of different hepatitis A-virus isolates in a fetal rhesus monkey kidney cell line (Frhk-4)

A fetal rhesus monkey kidney cell line (Frhk-4) was infected with different hepatitis A-virus (HAV) isolates GBG, GBM, GJA. The time-dependent adsorption of the HAV isolates to Frhk-4 cells was measured. Replication of all three isolates in these cells could be demonstrated intracellularly 8–10 weeks after infection, and release of HAV into the supernatant some 10–15 weeks after infection could be shown. The specificity of the virus determination by RIA from supernatants of HAV-infected cells from passages 1,2, and 3 in Frhk-4 cells was shown with sera that were collected from a chimpanzee infected with MS-1 both before infection as well as during convalescence. These results were subsequently compared with sera collected from human patients before the onset of hepatitis as well as during convalescence. With immunofluorence microscopy a cytoplasmic fluorescence could be shown in HAV-infected Frhk-4 cells and finally the release of 27 nm HAV particles into the supernatant of HAV-infected Frhk-4 cells could be demonstrated by immune electron microscopy.     

Solid-phase antibody capture hemadsorption assay for detection of hepatitis A virus immunoglobulin M antibodies.

A solid-phase antibody capture hemadsorption (SPACH) assay was developed to detect hepatitis A virus (HAV)-specific immunoglobulin M (IgM) antibodies in sera from humans recently infected with hepatitis. The assay is performed with microtiter plates coated with anti-human IgM antibodies to capture IgM antibodies from the test sera. HAV-specific IgM antibody is detected by the addition of HAV hemagglutinating antigen and goose erythrocytes. Hemadsorption of erythrocytes to antigen-antibody complexes attached to the solid phase indicate the presence of IgM antibodies. The SPACH assay was compared to a commercial radioimmunoassay and was found to be equally or more sensitive and specific for the detection of HAV IgM antibodies. The SPACH assay is an alternative, rapid assay that doesn't require hazardous substrates or radioactivity for the detection of HAV-specific antibodies.