| Background and objectiveHepatitis B virus (HBV) is a main factor which causes acute or chronic hepatitis, cirrhosis,liver failure and hepatocellular carcinoma. In China, there are about 23 million chronic hepatitis B patients and 130 million carriers, and approximately 50 percent of hepatocellular carcinoma occurred in China. Hepatitis B virus (HBV) has been classified into eight genotypes (A-H) based on an intergroup divergence of 8% or more in the complete nucleotide sequence. In China, there are four genotypes---- A, B, C and D, and the percentage of each of them is 1.2%, 39.3%, 50.2%and 8.1%respectively. Genotypes have different geographic distribution: genotype C is primary found in the north of China (about 81.6%) and in the south of China,genotype B is the main one. The investigation of HBV subgenotype has been carried out in recent years. Huy analyzed the HBV sequences of genotype C patients from Southeast Asia and the Far East and divided them into two subtypes: C1 (Cs) and C2 (Ce). C1 subtype is from Southeast Asia, like Vietnam and Thailand. C2 subtype is from the Far East, like China, Japan and Republic of Korea. Genotype B can also be divided into two subgenotypes: Ba and Bj. The researches in and out our country indicated that HBV genotype possibly closely correlate with disease proceeding, clinical manifestation, prognosis and antiviral therapy response. Researchers now generally consider that the inflammatory necrosis and fibrosis conditions are more serious in patients with genotype C than those with genotype B, and HBeAg positive rate is higher in patients with genotype C; genotype C is related with cirrhosis and hepatocellular carcinoma; genotype B has higher accumulation survival rate than genotype C. But some studies in our country indicated that the differences of hepatitic functional lesion, inflamatory necrosis and fibrosis have no statistically significant between the two genotypes. Based on this, we chose patients who infected with HBV and were in different phases in Southwest as objects for investigation, HBV DNA level, HBeAg, anti-HBe, HBV genotypes and subtypes were monitored. To be first, we studied the distribution of the age and HBV DNA level of patients who suffered from hepatocellular carcinoma (HCC), liver cirrhosis (LC) and chronic severe hepatitis (CSH) by retrospect, and to explore the effect of HBV DNA level on the pathogenesis of HCC, LC and CSH. Then we determined the genotype and subgenotype of some patients of HCC, LC and CSH to study the effect of HBV genotype and subgenotype on the pathogenesis of HCC, LC and CSH in the region of Chongqing.MethodsPatients of HCC (226 examples) who hospitalized and treated for the first time in the Department of Infectious Diseases of Southwest Hospital from 2000, 1 to 2006, 9 were enrolled. There were only 1 item of HBV-M (at least) was positive in all patients, and diagnostic code corresponded with the HCC diagnostic code which revised in 1999. Control group was patients of LC who have had type B hepatitis, and diagnostic code corresponded with the viral hepatitis prevention and cure plan which revised in 2000 and HBsAg (+). All the patients didn't receive antiviral therapy before the admission. Clinic indicatory, laboratory examination consequences was imputed into Excel table and established database after reform to case, and then was disposed by SPSS10.0. In addition, patients of HCC (78 samples), hepatic cirrhosis (58 samples), chronic hepatitis B (20 samples) and chronic severe hepatitis (42 samples) who hospitalized and treated in the Department of Infectious Diseases of Southwest Hospital from 2002 to 2007 were also enrolled. All patients were HBsAg(+) and HBV DNA>4×102copies/ml, the serum samples were stockpiled on admission. HBeAg and anti-HBe were measured by Roche's methods. HBV genotype and its subtype were determined by multiple primers PCR and PCR-RFLP, respectively.Results1. Serous HBV-M (including HBsAg, HBsAb, HBeAg, anti-HBe and HBcAb) and DNA level were retrospectively analyzed in 226 HCC patients, 51 LC and 285 CSH patients. The positive rate of HBsAg was 96.9% in HCC patients. The quantification of HBV DNA was finished by PCR in 168 HCC patients and 51liver cirrhosis patients, the positive rate of HBV DNA were 85.1%(143/168), 88.2%(45/51), respectively. HBV DNA was positive in all CSH patients, The mean value of serous HBV DNA in HCC patients, LC and CSH patients were 105.49±1.49 copies/ml, 106.15±1.38 copies/ml and 105.70±1.66 copies/ml, respectively. The level of serum HBV DNA in LC patients was higher than that in HCC patients (P<0.05). The difference among the other groups was not statistically significant. There was significant correlation between the positive rate of HBeAg and the clinical type of hepatopathy (P<0.001). The positive rate of HBeAg in LC and CSH patients were higher than that in HCC patients.2. 168 HCC patients were retrospectively analyzed. The average age of 120 HCC patients with cirrhosis was 47.91±11.95 years, and the average age of 47 HCC patients without cirrhosis was 43.40±9.07 years. The former was statistically older (P=0.021). In 143 HCC patients with positive HBV DNA, HBV DNA level of patients with cirrhosis and without cirrhosis were 105.55±1.49 copies /ml and 105.30±1.54copies/ml, respectively. The difference between them was not statistically significant.3. The quantification of HBV DNA level and HBeAg were analyzed in 78 HCC patients, 58 LC patients, 20 CHB patients and 42 CSH patients, whose level of HBV DNA were more than 4×10~2copies/ml. HBV DNA is normal distribution. In HCC, LC, CHB and CSH patients, HBV DNA level were 105.50±1.39copies/ml, 106.18±1.10 copies/ml, 106.20±1.10 copies/ml and 105.83±1.54copies/ml, respectively. HBV DNA level of HCC was obviously lower than LC and CHB(both P<0.05). But between HCC patients and CSH patients, the difference of HBV DNA was no statistically significant. HBeAg positive rate in HCC patients was obviously lower than that in LC and CHB patients (P>0.05). The difference of HBeAg quantification among HCC, LC and CHB patients was statistically significance (P<0.05). HBeAg quantification was closely related with HBV DNA level (P<0.001). The similar results were also found in anti-HBe quantification of HCC, LC and CHB patients(P<0.05).4. HBV genotypes and subgenotypes were investigated in 78 HCC patients, 58 LC patients, 20 CHB patients and 42 CSH patients (HBV DNA>4×10~2 copies/ml). In HCC patients, there are 40 persons with genotype B(51.3%), 37 with genotype C (47.4%), 1 with genotype B/C. In LC patients, there were 35 patients with genotype B(60.3%), 23 with genotype C (39.7%). In CHB patients, there 11 patients with genotype B(55%), 8 with genotype C (40%), 1 with genotype B/C. In CSH patients, there were 26 patients with genotype B(55%), 12 with genotype C (40%), 4 with genotype B/C. The difference of HBV genotypes in the four groups were not statistically different(P>0.05). First, genotype B subtype was analyzed in four groups. In HCC patients, there are 40 persons with subgenotype Ba. In LC patients, there were 32 with subgenotype Ba and 3 failures. In CHB patients, there were 10 patients with subgenotype Ba and 1 with subgenotype Bj. In CSH patients, there were 32 patients with subgenotype Ba. Secondly, the subtypes of genotype C were analyzed in all groups. In HCC patients, 24 patients with subtype Ce(64.9%), 10 with genotype Cs (27.0%), 3 failure. In LC patients, there were 18 patients with subtype Ce(78.3%), 4 with genotype Cs (17.4%) and 1 failure. In CHB patients, there were 5 patients with subgenotype Ce (62.5%) and 3 failure. In CSH patients, there were 6 patients with subgenotype Ce, 4 patients with subtype Cs and 1 failure. The difference of subgenotype C among four groups was not statistically different(P>0.05).Conclusion1. HBV level from high to low. with CHB in Chongqing were liver cirrhosis, severe hepatitis and HCC in turn.2. There is no significant correlation with HBV DNA level between HCC patients with or without LC. HCC patients with cirrhosis are obviously elder than the others .3. Loss of HBeAg is related with degradation of HBV DNA.4. Genotypes of Hepatitis B virus in Chongqing is mainly B, then to C. There is no significant correlation between HBV Genotype and the pathogenesis of HCC. And there were more mixed infections by different genotypes of HBV in severe hepatitis.5. Subgenotypes of genotype B are mainly Ba. Subgenotypes of genotype C are mainly Ce, Cs is the next in Chongqing. There is no significant correlation between HBV subgenotypes and HBV- associated liver diseases pathogenesis.
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