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）。     

Development of a plaque assay for a cytopathic, rapidly replicating isolate of hepatitis A virus

Most hepatitis A virus (HAV) replication in cell culture has been reported to be nonlytic and relatively slow. A rapidly replicating isolate of strain HM-175 from persistently infected, serially passed cell cultures (pHM-175) was found to induce a cytopathic effect. This observation allowed the development of a classic plaque assay for pHM-175 in FRhK-4 cells. The plaques were neutralized by polyclonal and monoclonal antisera to HAV.     

A simple method for clonal selection of hepatitis A virus based on recovery of virus from radioimmunofocus overlays

Hepatitis A virus (HAV), a non-cytopathic picornavirus, has been quantitated in cell culture by autoradiographic detection of foci of viral replication developing beneath an agarose overlay following fixation and 'staining' of the cell sheet with radiolabelled antibody (radioimmunofocus assay). Using a modification of this basic technique, a clonal variant of HM-175 strain HAV was isolated from agarose overlying individual radioimmunofoci. Virus recovered from the agarose was amplified in small volume cultures of BS-C-1 cells and identified in supernatant culture fluids by cDNA-RNA hybridization. No virus was recovered from agarose which did not overlie a focus of viral replication. This method offers a simple, yet relatively rapid and certain means of selecting clonal variants of non-plaquing viruses such as hepatitis A virus.     

Comparative evaluation of new TaqMan real-time assays for the detection of hepatitis A virus

Three novel real-time TaqMan RT-PCR assays targeting the 5′-UTR, the viral protease and the viral polymerase regions of the hepatitis A virus (HAV) were developed, evaluated and compared against a new published 5′-UTR TaqMan assay (JN) and a widely used conventional RT-PCR assay (HAVc). All conventional RT-PCR (HAV, SH-Prot and SH-Poly systems) and TaqMan (SH-Prot, SH-Poly, JN and SH-5U systems) assays evaluated were consistent for the detection of the three different HAV strains (HM-175, HAS-15 and LSH/S) used and reproducible for both RNA duplicates with the exception of two reproducibility discrepancies observed with both 5′-UTR real-time systems (JN and SH-5U). Limits of detection for conventional HAV, SH-Prot and SH-Poly RT-PCR systems were found to be equivalent when tested with serially diluted suspensions of the HM-175 strain. Although the four real-time RT-PCR TaqMan assays evaluated herein produced similar and consistent quantification data down to the level of one genomic equivalent copy with their respectively cloned amplicons, significant and important differences were observed for the detection of HAV genomic RNA. Results showed that the new real-time TaqMan SH-Poly and SH-Prot primer and probe systems were more consistent and sensitive by 5 logs as compared to both 5′-UTR designs (JN and SH-5U) used for the detection of HAV genomic RNA as well as for the detection in cell culture by cytopathic effect. Considering their higher analytical sensitivity, the proposed SH-Poly and SH-Prot amplification systems could therefore represent valuable tools for the detection of HAV in clinical, environmental and food samples.     

Pathogenesis of hepatitis A in orally inoculated owl monkeys (Aotus trivirgatus)

The pathogenesis of hepatitis A virus (HAV) infection was studied in owl monkeys following oral administration of the wild-type HM-175 strain of HAV. Stools were collected daily and blood and pharyngeal swabs twice weekly for viral isolation, and animals were necropsied at various intervals after inoculation. Organs were examined for the presence of virus by isolation in cell culture and for viral antigens by immunofluorescence. Monkeys excreted HAV in the stools for 1–4 days after inoculation, presumably due to the residual unabsorbed inoculum. No virus was found in stools for the next 2–3 days. HAV re-appeared on days 4–7 and then persisted through day 39. Viremia occurred on the 10th day and continued until day 35. Virus was isolated occasionally from throat swabs 1 or 2 weeks after it was detected in stools and blood, and there was no evidence that HAV replicated in the pharyngeal tissues. Animals acquired anti HAV antibody by the 4th week, and alanine aminotransferase (ALT) was elevated 5–5.5 weeks after inoculation. HAV was isolated from liver 5 days after inoculation; however, viral antigens were first detected in Kupffer cells of the liver at 14 days and in hepatocytes at 21 days. HAV antigen was detected in epithelial cells of the intestinal crypts and in the cells of the lamina propria of the small intestine 3 days postinoculation and thereafter until the 5th week, suggesting that these cells might represent an additional site of HAV replication. © Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Experimental infection of the New World owl monkey (Aotus trivirgatus) with hepatitis A virus

Epidemiological studies have demonstrated the susceptibility of the owl monkey (Aotus trivirgatus) to hepatitis A virus, but have not shown an association between infection and histopathological or chemical evidence of liver disease. Therefore, 12 seronegative, colony-bred monkeys were inoculated intravenously with a fecal suspension containing either PA33 strain hepatitis A virus (a strain recovered from a naturally infected Aotus sp.) or HM-175 virus (recovered from a human). Viral antigen was detected by radioimmunoassay in the feces of six monkeys 6 to 17 days after inoculation with PA33 virus, and by 9 to 21 days serum aminotransferase activities were significantly elevated in each. Antibody to the virus developed in each monkey by 28 days after inoculation. Similar findings were noted in five of six monkeys inoculated with HM-175 virus, although the incubation period preceding aminotransferase elevations was somewhat longer (25 to 39 days). Liver biopsies obtained from the 11 infected monkeys demonstrated mild to moderate portal inflammation, as well as random areas of focal necrosis and inflammation extending outward from the portal region. These data confirm the susceptibility of Aotus sp. to hepatitis A virus and indicate that the infection of this primate provides a useful animal model of human hepatitis A.     

Propagation of hepatitis A virus in human embryo fibroblasts

human diploid fibroblasts and human primary liver cell carcinoma cells (PLC/PRF/5) were infected with hepatitis A virus (HAV) adapted to growth in cell culture or derived directly from human stool. Viral antigen was expressed in PLC/PRF/5 cells 28 days after infection with cell culture-adapted HAV, and 50 days after infection with virus from human stool. In human fibroblasts the periods until first expression of viral antigen were 90 and 210 days, respectively. During further passages of HAV in fibroblasts the time of first appearance of antigen decreased to about 28 days. Biophysical properties of HAV extracted from infected fibroblasts were comparable to those of HAV derived directly from human stool. Immunofluorescence studies showed that the antigen was located exclusively within the cytoplasm of the infected fibroblasts. Kinetics of antigen production indicated that an equilibrium between virus multiplication and cell metabolism was reached in persistently infected fibroblasts.     

Continuous production of hepatitis A virus in PLC/PRF/5 cell cultures: use of antigen for serology

The strain CF53 of hepatitis A virus (HAV) previously adapted to growth in PLC/PRF/5 cells was grown in 175 cm2 flasks, at different passages. After infection, cells were incubated at 32 degrees C in RPMI 1640 medium supplemented with 2.5% foetal calf serum (FCS) for 6-12 months. HAV which was released continuously in the culture medium was harvested weekly. Hepatitis A virus antigen (HAAg) and infectious virus production was stable during each passage. The antigen titre, determined by radioimmunoassay, was about 50 for each passage whereas the infectious virus titre increased from 10(3.7) (passage 7) to 10(6.0) TCID50/ml (passage 13). Virus production was not influenced by the FCS concentration (0-2.5%) in the maintenance medium. The cell culture produced HAAg was used for detection of total anti-HAV antibodies, anti-HAV titration and IgM antibody capture assay and the results were identical to those obtained with commercial kits. HAAg produced by this practical and cheap method could easily replace primate derived antigen for the detection of anti-HAV antibodies.     

Four chimpanzee monoclonal antibodies isolated by phage display neutralize hepatitis a virus

Chimpanzee immunoglobulins are virtually identical to human immunoglobulins and may have clinically useful applications. Four chimpanzee monoclonal antibodies (MAbs) to the hepatitis A virus (HAV) capsid were isolated from a combinatorial cDNA library of gamma1/kappa antibody genes using phage display. Competition assays indicated that three of the MAbs recognized the same or overlapping epitopes, whereas the fourth recognized a different, nonoverlapping epitope on the HAV capsid. All four MAbs neutralized the homologous HAV strain, HM-175, in a radioimmunofocus assay and two of the four MAbs neutralized a heterologous simian HAV strain, AGM-27. From these data, we conclude that the MAbs must recognize at least three epitopes on the HAV capsid. Furthermore, competition assays performed with neutralizing murine MAbs suggested that three of the chimpanzee MAbs recognized epitopes on the HAV capsid which have not been defined previously.     

Hepatitis A in Macaca fascicularis and M. arctoides infected by the Java monkey-55 strain of hepatitis A virus

The results are presented dealing with experimental inoculation of M. fascicularis and M. arctoides with a strain of hepatitis A virus (HAV), YaM-55, isolated from a M. fascicularis with spontaneous hepatitis A, and parallel experiments on inoculation of these monkey species with HAV preparations (strain HAS-15) obtained as a result of the strain propagation in FRhK-4 cell culture and with specimens from human hepatitis A patients containing HAV particles. The YaM-55 strain of HAV was found to be capable of producing an infectious process quite similar to HA in the inoculated seronegative M. fascicularis and M. arctoides. Two different isolates of HAV derived from humans and the HAS-15 strain of HAV propagated for a long time in FRhK-4 cell culture failed to induce a disease in these monkey species. The classification of the YaM-55 strain with HAV was verified by specific serological studies and by molecular hybridization with cloned cDNA of HAV.     

Complete nucleotide sequence of a cell culture-adapted variant of hepatitis A virus: comparison with wild-type virus with restricted capacity for in vitro replication

To determine the molecular changes associated with adaptation of hepatitis A virus (HAV) to growth in cell culture, the genome of a cell culture-adapted variant of HM175 strain HAV (p16 HM175, 16th in vitro passage level) was molecularly cloned and the complete nucleotide sequence of the virus was determined. Compared with wild-type virus, p16 HM175 replicates efficiently in monkey kidney (BS-C-1) cells (approximately 58 RNA-containing particles per one infectious unit, compared with 2.4 x 10(5) for wild-type HM175). The nucleotide sequence of p16 HM175 revealed a total of 19 mutations from the wild-type genome, including 5 mutations in the 5' nontranslated region, 1 mutation in the 3' nontranslated region, and 13 mutations predicting 8 changes in the amino acid sequences of HAV proteins. Only one amino acid substitution occurred among the capsid proteins (VP2), while others involved proteins 2A, 2B, 2C, VPg, and 3Dpol. When the sequence of p16 virus was compared with that reported previously for an independently isolated, cell culture-adapted variant of HM175 virus (J.I. Cohen et al., (1987). Proc. Natl. Acad. Sci. USA 84, 2497-2501), there were three identical mutations in nontranslated regions of the RNA, and four mutations involving identical amino acids in proteins VP2, 2B, and 3Dpol. The distribution of these mutations within the genome suggests that changes in RNA replication may be of primary importance in adaptation of the virus to growth in vitro. These data are thus helpful in understanding the molecular basis of adaptation of HAV to cell culture and, since attenuation frequently accompanies adaptation of virus to growth in cell culture, may be of benefit in planning for attenuated vaccine development.     

Accumulation of the infectious virus and the viral antigen during the multiplication of the hepatitis A virus in an embryonic kidney cell culture of the rhesus macaque (FRhK-4)

Growth characteristics of the HAS-15 strain of human hepatitis A virus (HAV) in rhesus monkey foetal kidney cell line (FRhK-4) are described. The conditions optimal for the accumulation of infectious HAV and viral antigen (HAAg) in the infected cells and tissue culture fluids were studied. The production of infectious HAV occurred in the first stage while in the second stage predominantly HAAg was accumulating intracellularly. Serological properties of the cultivated HAV proved to be very similar to those of the HAV occurring in hepatitis A patients.     

A microcarrier cell culture system for large scale production of hepatitis A virus

Hepatitis A virus (HAV) was isolated from human faeces using a fetal rhesus monkey kidney cell line (Frhk-4). Infectious medium from passage 12 was used to inoculate a large (5000 cm2) microcarrier cell culture maintained in suspension. The microcarriers used were swollen, collagen-coated dextran beads on which it was easy to propagate Frhk-4 cells. Intra- and extra-cellular virus levels were assayed and compared with conventional cultures in 25 cm2 plastic flasks. The results show that virus production per cell was similar in both systems. The number of cells per area unit in confluent cultures was initially lower in the microcarrier culture but subsequently increased. Two to three weeks post inoculation the virus yield per area unit in the microcarrier system was half of that of the conventional culture. The lower cell density per area unit in the microcarrier system was compensated by the large growth area that could be maintained in a single vessel and the total production of virus was substantial. Weekly harvests of medium with HAV antigen titres around 10(-2) contained antigenic material sufficient for several thousands of anti-HAV IgM tests. Propagation of HAV in microcarrier cell cultures thus seems a safe and simple way to produce large amounts of HAV.     

Electron microscopy of buffalo green monkey kidney cells persistently infected with hepatitis A virus and immunolocalization of HAV antigens

Studies were carried out to analyse the ultrastructural changes and the distribution of hepatitis A virus (HAV)/antigens at subcellular level in buffalo green monkey kidney (BGMK) cells persistently infected with HM-175 strain of HAV. HAV infected BGMK cells showed distinct abnormalities in the endoplasmic reticulum and cytoplasmic membrane as compared to uninfected cells. The abnormalities were characterized by wavy arrays, structures like myelin, annulate lamellae, cytoplasmic inclusion bodies and vesicles. The wavy arrays within the cytoplasm of the host cells appeared to represent degenerating membranes. A complex myelin like body was found in close association with a group of virus like particles. Annulate lamellae like structures involving single paired membrane were detected infrequently whereas the cytoplasmic vesicles were numerous in these cells. An indirect immunogold technique was utilized to localize the HAV antigenin infected cells. A high density immunogold label for HIV like particles was predominantly detected in cytoplasmic vesicles. These results suggest a strong association of membrane substructure in vesicle forms with the compartmentalized replication of HAV within persistently infected host cells.     

Enhancement of hepatitis A propagation in tissue culture with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole

The adenosine analog 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was found to increase the production of hepatitis A (HAV) antigen in two monkey kidney cell lines (Frhk-4 and Vero cells). DRB, a known inhibitor of the synthesis of messenger RNA, caused moderate changes in cell morphology. However, Frhk-4 cells could be maintained for several weeks at 80 microM of DRB, the concentration that caused maximal enhancement on HAV. DRB should be present from about the time of virus inoculation and its strongest effect was seen at low multiplicities of infection. Using radioimmunofocus assay it could be shown that DRB increased the amount of infectious virus. DRB treatment was applied in primary isolation of HAV from feces. In nine of ten strains HAV antigen expression was strongly increased and in six of the ten strains infectivity of harvested material increased by one 10log or more. DRB thus seems to be a useful enhancer of HAV growth in tissue culture.     

应用痘苗病毒载体表达的重组甲肝抗原特性的研究

目的 探讨以痘苗病毒载体表达的甲肝病毒培养合适的细胞系和重组病毒灭活以及甲肝病毒抗原保存条件.方法 将构建成功的表达甲肝抗原的重组痘苗病毒分别接种于K4、143、HEL、Hep-2和Vero等细胞,用ELISA测定重组甲肝抗原的表达产量和不同方法灭活重组病毒后的抗原活性.结果 在K4、143和HEL细胞表达重组甲肝抗原的产量略优于Hep-2和Vero细胞,重组病毒经β-丙内酯和甲醛灭活后,甲肝抗原活性不变,制备的重组甲肝抗原稳定性好.结论 痘苗病毒表达载体可以提高甲肝抗原表达效率,具有十分广阔的应用前景.     

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.     

Radioimmunofocus assay for quantitation of hepatitis A virus in cell cultures

A new method is described for the quantitation of hepatitis A virus in cell cultures, based on the immune autoradiographic detection of foci of infected cells (radioimmunofoci) developing beneath an agarose overlay 14 days after the inoculation of petri dish cultures of continuous African green monkey kidney cells (BS-C-1). The number of foci developing in each culture was linearly related to the dose of hepatitis A virus (either HM-175 or PA-21 strain) inoculated. Focus development was prevented by prior incubation of virus with specific antisera, and the specificity of the radiolabeled antibody reaction was confirmed in competitive blocking experiments. This new assay method retains many of the advantages of conventional plaque assays for virus. Compared with existing end-dilution methods for the quantitation of hepatitis A virus, the radioimmunofocus assay offers greatly improved accuracy and comparable sensitivity, yet is relatively rapid and highly conservative of reagents.