| Objective TTV (transfusion transmitted virus) is a novel DNA virus which was isolated from the serum of a patient suffered from posttransfusion hepatitis. TTV infection is common in the different populations. It can be found in the health persons without any symptom. TTV superinfecion also prevails in other hepatitis virus infection. It is indeed the only etiology of some cryptogenic liver disease and some fulminant hepatitis without any markers of hepatitis A-G virus infection. There is controversy on the pathogenicity of TTV presently. Some scholars showed TTV was no pathogenicity, some had oppositional viewpoint. The remainder discovered there was an association between TTV pathogenicity and its genotype or gene variation. Therefore, It is important to investigate the TTV infection status in all kinds of population, especially whether TTV superinfection is responsible for clinical exacerbation of chronic hepatitis B patients; To study the pathogenicity of TTV and whether it has influence on the replication of HBV; To discuss the relationship between gene variation or quasispecies and pathogenicity of TTV.Methods (1) A nested polymerase chain reaction (nested-PCR) assay with specific primers from open reading frame one (ORF1) of TTV genome was established to detect TTV DNA in the serum sample from the blood donors, the patients with HBV infection and non A-G hepatitis patients. The PCR products were sequenced. The difference of mean value of ALT and TBIL was compared between the patients with andwithout TTV infection.(2)Asymmetric PCR was established to get single strand DNA in the TTV DNA positive cases, then single strand conformation polymorphism ( SSCP ) and melt curve were applied to analysis TTV gene variation. (3) A nested polymerase chain reaction (nested-PCR) assay with specific primers from the high variable region ( HVR ) of open reading frame one (ORF1) of TTV genome was established to amplificate TTV DNA in the serum sample from the chronic severe hepatitis B patient and asymptom carrier. The 10 positive clones of TTV DNA fragment in each patient were sequenced by T7 primer after PCR products cloning, the sequences of clones were compared with TA278 to show their homology. Feasibility of TTV quasispecies complexity analysis by DNA melt curve was tried.Results ( 1 ) TTV DNA was detected in the sera from 4 of 40 (10%)blood donors , 7 of 64 (11%) chronic HBV carriers (ASC ), 39 of 140(27%) chronic hepatitis B patients (CHB ) , and 76 of 160 (48%) chronic severe hepatitis B patients ( CSHB ). TTV DNA positivity rate in non-A-G hepatitis patients typed moderation; severity and graveness was 33%(4/12), 43%(6/14) and 60%(6/10) respectively. Compared with blood donors, the TTV DNA positivity rate in non-A-non-G hepatitis patients was significantly higher (PO.01) . There were significantly different TTV DNA frequence among the non-A-non-G hepatitis patients typed moderation, graveness and severity (P<0.05); It could also be seen there were higher frequence of TTV infection in the CHB and CSHB than that in ASC and blood donors (P<0.01). In the meantime ,the rate of TTV infection was higher in CSHB than that in CHB(P<0.05). The ALT and TBIL mean values were significantly higher in the CHB and CSHB patients with TTV than those without TTV infection (P <0.05) ; In thevnnon-A-non-G patients, the ALT levels were significantly higher in the patients with TTV than those without TTV infection (P <0.05) , but the TBIL levels were not significantly different between the patients with and without TTV infection (P >0.05) . The positive rate of HBV replication markers was not significantly different between the hepatitis B patients with and without TTV infection (P >0.05 ) . ( 2 ) SSCP bands with 1.25± 0.50, 2.50± 0.58, 3.0±0.63, 4.3±1.21 were detected in the blood donors, non-A-non-G patients typed moderation, graveness and severity, respectively, and the mean SSCP bands were significantly different among them. SSCP bands in ASC, moderate CHB, grave CHB and CSHB were 1.60 ± 0.55, 3.25 ± 1.06, 3.
|
PDF Full Text Request