Community-based Study On Molecular Epidemiology Of Hepatitis B Virus In Luohe, Henan Province And Zhaodong, Heilongjiang Province

Hepatitis B virus (HBV) chronic infection is a global health problem and the most common causes lead to chronic liver diseases in the world. Globally, there are around 400 million people with chronic hepatitis B (CHB) infection. Of these, more than 75% of CHB patients are Asians.There is increasing evidence that HBV genotype correlates with clinical outcomes of chronic HBV infection and response to treatment. The evidence for a clinical difference is stronger between genotypes B and C. The exact reason(s) why HBV genotype may be related to clinical outcomes is not clear. It is possible that different genotypes may be associated with differences in replication fitness and expression of immune epitopes. There is also a clear association between HBV genotypes and precore and basic core promoter mutations.Genotyping should remain a research tool until the time when knowledge of the HBV genotype can be used to predict the risk of adverse outcomes (fulminant hepatitis, cirrhosis, or HCC) or to guide treatment decisions (choice or duration of therapy). Genotypes B and C were the two most common HBV genotypes in China. Prevalence of HBV in Luohe, Henan province and Zhaodong, Heilongjiang Province were common in China. According to the serum epidemiology investigation, we did a research on the prevalence of HBV genotype and basic core promoter, precore mutation in the two areas. A scientific basis was tend to provided for hepatitis B prevention and control in the two regions.The study included two parts:1 Community-based Study on Molecular Epidemiology of Hepatitis B Virus genotype in Luohe, Henan Province Objective: To determine the distribution of hepatitis B virus (HBV) genotypes and serotypes in community-based Luohe, Henan province and to evaluate the relationship between genotypes and serotypes; To study the relationship between the genotypes and the clinical types; To study the relationship between the genotypes and the status of HBeAg/Anti-HBe; To study the relationship between the genotypes and alanine aminotransferase(ALT) levels; To analyze the spectra of mutations of the S domin of HBV.Methods: In 2005, 3 counties Wuyang Linying and Zhaolin in Luohe, Henan province were selected by cluster-random design and vein blood were collected in all people. HBsAg,anti-HBs, anti-HBc, HBeAg and anti-HBe were tested by SPRIA with kits. According to standard of"Chronic Hepatitis B virus prevention and therapy guide"and"Hepatitis B diagnostic criteria", together with the results of ALT, B-ultrasonic and physical examination, 254 sera samples were selected by clinical diagnosis. 144 sera samples were from asymptomatic HBV carriers (ASC), 110 from chronic hepatitis (CH). The mean age was 35 years. 136 were Male and 118 were female. Nested polymerase chain reaction was used for amplication of HBV DNA. HBV DNA was extracted from 200μl serum by QIAamp DNA Blood Mini Kit, according to the manufacture's recommendations. Two pairs of oligonucletide primers were used to amplify HBV S gene. The PCR products were subjected to electrophoresis on 1% agarose gel and visualized by using an ultraviolet transalluminator. DNA sequencing was performed by a commercial company using the ABI PRISMTM 3730XL DNA Analyzer. The nucleotide sequences were compared with reference sequence of each genotype of A-H obtained from Genbank. Sequences were aligned, and phylogenetic tree was established using the DNASTAR software to determine the genotype. Serotype was determined by the deduced amino acid sequence from nucleotide sequence. Statistical analyses were performed using Chi-square test, Fisher's exact test, Wilcoxon Two-Sample Test with SPSS 13.0 software. P<0.05 was considered statistically significant.Result: Overall, HBV DNA was detectable in 219(86.22%) of the 254 samples. A total of 219 samples of the S genes of HBV were analysed in our study. Genotype B was 22 (10.05%), and C was 179 (89.95%). Adr was 192 (87.67%), adw2 was 2 3(10.50%), ayr was 3 (1.37%) and ayw1 was 1 (0.46%). In 22 genotype B, there were adw2 21(95.45%) and ayw1 1(4.55%). In 197 genotype C, there were adr 192(97.46%), adw2 2 (1.02%) and ayr 3 (1.52%). There was a statistically significance among genotypes and serotypes (P=0.0000).Among 22 cases with genotype B, 15 cases (68.18%) were ASC and 7 (31.82%) were CH. Among 197 cases with genotype C, 109 cases (55.33%) were ASC and 88 (44.67%) were CH. There was no statistically significance among genotypes and clinical types (P=0.2487).There was no statistically significance in HBsAg/anti-HBe status among patients with genotype B and patients with genotype C (P>0.05).There was no statistically significance in ALT level among patients with genotype B and patients with genotype C (P>0.05).Comparing the 219 nucleotide sequences, we found 184 had substitutions, 49 had samesense mutations and 133 had missense mutations and 3 had nonsense mutations. In the major hydrophilic region (MHR, aa100-160), there were 60 substitutions. 35 had samesense mutations and 36 had missense mutations. There were 2 substitutions led to sense mutations- Q129H (2), the changes concentrated in the first loop of the'a'determinant; G145R(1) ,the changes concentrated in the second loop of the'a'determinant. The frequency of nucleotide substitution of S gene was 0.83/100nt. The frequency of amino acids substitution of S gene was 1.00/100nt. Conclusion:1 The predominant genotype of HBV in community-based, Luohe, Henan province was genotype C, and the second common was genotype B.2 Serotype adr predominated and had other serotypes such as adw2, ayr and ayw1.3 Different genotypes had different serotypes. Most of genotype B were adw2 and others were ayw1, and most of genotype C were adr and others were adw2 and ayr, which revealed that genotype and serotype had certain relationship.4. Patients with genotype B may have a higher chance of earlier HBeAg seroconversion compared to patients with genotype C at >30 years group.5 There was no differcnce in the ALT levels between patients with genotype B and patients with genotype C.6 There were many nucleotide substitutions of S gene, about 2/3 were missense mutations. There were mutations of S gene in the community people. Vaccine-associated mutation was found. The frequency of nucleotide substitution of community-based HBV S gene was low, which revealed that S gene was relatively conserved.2 Community-based Study on Molecular Epidemiology of hepatitis B virus basic core promoter and precore mutations of chronic hepatitis B infection in Luohe, Henan Province and Zhaodong, Heilongjiang ProvinceObjective: To determine the prevalence of hepatitis B virus (HBV) basic core promoter (BCP) and precore mutations in Luohe, Henan province and Zhaodong, Heilongjiang Province. To study the relationship between the BCP, precore mutations and genotypes; To study the relationship between the BCP, precore mutations and the status of HBeAg/Anti-HBe; To study the relationship between the BCP, precore mutations and the clinical types; To study the relationship between the BCP, precore mutations and alanine aminotransferase(ALT) levels.Methods: 219 sera samples with the S train positive were selected from the first part. 124 sera samples were from asymptomatic HBV carriers (ASC), 95 from chronic hepatitis (CH). The mean age was 38 years. 121 were male and 98 were female. In 2005, 2 villages Shuangsheng and Xianfeng in Zhaodong, Heilongjiang province were selected by cluster-random design and vein blood were collected in all people. HbsAg,anti-HBs and anti-HBc were tested by SPRIA with kits. HBeAg and anti-HBe were measured by ELISA test kit. According to the serum epidemiology data of 1986 and the standard of"Chronic Hepatitis B virus prevention and therapy guid", together with the results of ALT, B-ultrasonic and physical examination, 97 sera samples were selected by clinical diagnosis. 78 sera samples were from asymptomatic HBV carriers (ASC), 12 from chronic hepatitis (CH), 3 from liver cirrhosis (LC), 1 from hepatocellular carcinoma (HCC) and 3 from convalescene. The mean age was 34 years. 50 were male and 45 were female. Nested polymerase chain reaction was used for amplication of HBV DNA. HBV DNA was extracted from 200μl serum by QIAamp DNA Blood Mini Kit, according to the manufacture's recommendations. Two pairs of oligonucletide primers were used to amplify HBV C gene. The PCR products were subjected to electrophoresis on 1% agarose gel and visualized by using an ultraviolet transalluminator. DNA sequencing was performed by a commercial company using the ABI PRISMTM 3730XL DNA Analyzer. The nucleotide sequences were compared with reference sequence of each genotype of A-H obtained from Genbank. Sequences were aligned, and basic core promoter, precore mutations were determined by the DNASTAR software. Statistical analyses were performed using Chi-square test, Fisher's exact test, Wilcoxon Two-Sample Test with SPSS 13.0 software. P<0.05 was considered statistically significant.Result:The prevalence of mutations at nucleotide (nt) T1762/A1764 and A1896 were 40.22% and 52.51% respectively in Luohe, Henan province. The prevalence of mutations at nucleotide (nt) T1762/A1764 and A1896 were 28.57% in Zhaodong, Heilongjiang Province.Patients with genotype C had higher mutations at nucleotide (nt) T1762/A1764 (55.28%vs.27.78%, P=0.0267) compared to patients with genotype B in Luohe, Henan province. There was no statistically significance in mutations at nucleotide (nt) A1896 (50.00%vs.39.13%, P=0.3724) between genotype C- and genotype B-infected patients.Patients with genotype C had higher mutations at nucleotide (nt) T1762/A1764 (36.67%vs.0.00%, P=0.0231) compared to patients with genotype C in Zhaodong, Heilongjiang Province. There was no statistically significance in mutations at nucleotide (nt) A1896 (33.33%vs.29.85%, P=0.8598) between genotype C- and genotype B-infected patients.The prevalence of hepatitis B virus (HBV) BCP double mutations (T1762/A1764) in Luohe, Henan province was higher compared to the prevalence in Zhaodong, Heilongjiang Province (55.51%vs.33.33% , P=0.0076). There was no statistically significance in the prevalence of HBV precore mutations between Luohe, Henan province and Zhaodong, Heilongjiang. The double mutations (T1762/A1764) in the BCP was more frequently found at anti-HBe positive group than at HBeAg positive group in Luohe, Henan province (76.67%vs.29.11%,P=0.0000). The precore mutations (A1896) was more frequently found at anti-HBe positive group than at HBeAg positive group (70.21%vs.2.53%,P=0.0000).There was no statistically significance in the double mutations (T1762/A1764) in the BCP between HBeAg positive group and anti-HBe positive group in Zhaodong, Heilongjiang province (P=0.4092). The precore mutations (A1896) was more frequently found at anti-HBe positive group than at HBeAg positive group (P=0.0000). There was no statistically significance in the ALT levels between patients with double mutations (T1762/A1764) and wild-type in Luohe, Henan province (P=0.4387). Patients with double mutations (T1762/A1764) had higher ALT levels compared to patients with wild-type at HBeAg positive group (P=0.0000).There was no statistically significance in the ALT levels between patients with double mutations (T1762/A1764) and wild-type at anti-HBe positive group (P=0.4171).Patients with precore mutations (A1896) had lower ALT levels compared to patients with wild-type in Luohe, Henan province (P=0.0013). There was no statistically significance in the ALT levels between patients with precore mutations and wild-type at HBeAg positive group (P=0.7578) Patients with precore mutations (A1896) had lower ALT levels compared to patients with wild-type at anti-HBe positive group (P=0.0082).The double mutations (T1762/A1764) in the BCP was more frequently found in CH than in ASC in Luohe, Henan province (63.16%vs.45.92%,P=0.0237). The precore mutations (A1896) was more frequently found in ASC than in CH (47.47%vs.31.25%,P=0.0277).There was no statistically significance in the double mutation (T1762/A1764) in the BCP between ASC and CH in Zhaodong, Heilongjiang province (P=0.3183). The precore mutations (A1896) was more frequently found in CH than in ASC (71.43%vs.24.24%,P=0.0133).Conclusion 1 The prevalence of hepatitis B virus (HBV) BCP mutations in Luohe, Henan province was higher compared to the prevalence in Zhaodong, Heilongjiang Province. There was no differcnce in the prevalence of HBV precore mutations between Luohe, Henan province and Zhaodong, Heilongjiang province.2 Patients with anti-HBe positive have a higher mutations at nucleotide (nt) T1762/A1764 and A1896 compared to patients with HBeAg positive respectively in Luohe, Henan province.3 Patients with anti-HBe positive have a higher mutations at nucleotide (nt) A1896 compared to patients with HBeAg positive in Zhaodong, Heilongjiang province4 Patients with double mutation (T1762/A1764) had higher ALT levels compared to patients with wild-type at HBeAg positive group.5 Patients with precore mutations (A1896) had lower ALT levels compared to patients with wild-type in Luohe, Henan province. Patients with precore mutations (A1896) had lower ALT levels compared to patients with wild-type at anti-HBe positive group. There was no difference in the ALT levels between patients with precore mutations and wild-type at HBeAg positive group.6 Patients with genotype C had higher mutations at nucleotide (nt) T1762/A1764 compared to patients with genotype B in Luohe, Henan province and Zhaodong, Heilongjiang province. 7 CH had higher mutations at nucleotide (nt) T1762/A1764 compared to ASC, ASC had higher mutations at nucleotide (nt) A1896 compared to CH in Luohe, Henan province.8 There was no difference in the mutations at nucleotide (nt) T1762/A1764 between ASC and CH in Zhaodong, Heilongjiang province. CH had higher mutations at nucleotide (nt) A1896 compared to ASC...