ABSTRACT
<p><b>OBJECTIVE</b>Tupaia belangeri (tree shrew) has a close phylogenetic relationship with primates and has been shown to be susceptible to a variety of human viruses. This study was conducted to investigate whether or not hepatitis C virus (HCV) could infect primary tupaia hepatocytes (PTHs) in vitro.</p><p><b>METHODS</b>Serum-derived HCV was cultivated with PTHs, and then positive and negative strand HCV RNA in PTHs, as well as the encapsidated HCV RNA in the culture medium were detected to evaluate the infection. Virus from the culture medium of the infected PTHs was passed to naïve PTHs, and the quasispecies of HCV were compared among the inoculum and PTHs after infection and passage.</p><p><b>RESULTS</b>Both positive and negative strand HCV RNA were detected in PTHs after infection. The negative strand RNA was detectable from day 5 to day 10 after infection, while the positive strand RNA was positive up to day 14. HCV RNA, which was RNase resistant, could be detected from the culture medium of the infected PTHs from day 3 to day 14. Production of infectious virons of PTH were demonstrated by passage HCV to naïve PTHs. Compared analysis of HCV quasispecies after infection and passage showed that PTHs were selectively infected with defined HCV quasispecies, and new quasispecies emerged in PTHs after passage.</p><p><b>CONCLUSION</b>The present study strongly indicates that PTHs could be infected by HCV and support HCV replication in vitro. Our results would be helpful for the establishment of a tupaia model of HCV infection.</p>
Subject(s)
Animals , Cells, Cultured , Hepacivirus , Virulence , Physiology , Hepatocytes , Virology , Tupaia , Virus ReplicationABSTRACT
<p><b>OBJECTIVE</b>Viral hepatitis remains a major public health problem and the most common type of liver disease worldwide. There are an increasing number of patients with chronic hepatitis B who develop acute hepatitis on chronic condition (AOC) and die of acute hepatic failure both as a result of lack of understanding of the pathogenesis of the disease and lack of effective treatment. The hallmark of AOC is the extreme rapidity of the necromicroinflammatory process resulting in widespread or total hepatocellular necrosis in weeks or even days. Our previous studies have shown in an experimental animal model of fulminant viral hepatitis caused by murine hepatitis virus strain 3, the importance of macrophage activation, and expression of a unique gene mfgl 2 which encodes a serine protease capable of directly cleaving prothrombin to thrombin, resulting in widespread fibrin deposition within the liver and hepatocyte necrosis. The undergoing study in this report is designed to identify the role of hfgl 2 (human fibrinogen like protein 2) /fibroleukin in patients with viral hepatitis.</p><p><b>METHODS</b>Liver tissues were obtained from 23 patients with AOC hepatitis B, and from 13 patients with inactive chronic hepatitis B (CHB) and 14 patients with chronic hepatitis B with cirrhosis during the year of 1995 to the end of 2001. Liver biopsies were performed within 30 min after the patients were diagnosed with death as a result of acute hepatic failure. Liver samples were also obtained from 4 liver donors as normal controls. In addition, peripheral blood mononuclear cells (PBMC) were isolated from 30 patients (unpaired) with AOC hepatitis B and 10 patients with CHB during the May of 2001 to March of 2002 and 10 healthy volunteer as negative control. PBMCs were freshly isolated and smeared on slides and kept at -80 degree C for further use. Histological sections were stained with hemotoxylin and eosin. A 169 bp of hfgl 2 cDNA probe and a polyclonal or monoclonal antibody against hfgl 2 were used to detect the expression of hfgl 2 mRNA and protein in liver samples as well as PBMC by immune histochemistry separately.</p><p><b>RESULTS</b>Liver tissues from the patients with acute on chronic hepatitis had classical pathological features of acute necroinflammation. Hfgl 2 was detected by immune histochemistry in 21 of 23 patients (91.3%) in liver sections from patients with acute on CHB, while only 1 of 13 patients (7.7%) with CHB and cirrhosis and no evidence of active disease had hfgl 2 mRNA or protein expression. 28 of 30 patients (93.3%) with acute on CHB and 1 of 10 with CHB were detected with hfgl 2 expression in PBMC. There was no hfgl 2 expression in either the liver tissue or the PBMC from the normal donors. There was positive correlation of hfgl 2 expression and the severity of the disease displayed by the value of bilirubin and PT.</p><p><b>CONCLUSION</b>The molecular and cellular results reported here in patients with acute on chronic hepatitis and who died of acute hepatic failure correlates with previous report in 8 patients with fulminant hepatic failure (FHF) and mimic closely the changes observed in the murine model of fulminant viral hepatitis in which the pathogenesis of the disease has been studied in a stepwise fashion. This study further suggests that virus induced hfgl 2 prothrombinase/fibroleukin expression and the potent function of the protein it encodes plays a pivotal role in initiating acute severe hepatitis on the baseline of chronic hepatitis. The measurement of hfgl 2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of disease in patients with the AOC hepatitis B and a target for therapeutic intervention.</p>