Prevalence and Clinical Significance of SEN
Virus Infection Among Volunteer Blood
Donors in Southern Taiwan
CHIA-YEN DAI, MD, MS, MING-LUNG YU, MD, PhD, ZU-YAU LIN, MD, MS, SHINN-CHERNG CHEN, MD, PhD, MING-YUH HSIEH, MD, LIANG-YEN WANG, MD, JUNG-FA TSAI, MD, PhD, WAN-LONG CHUANG, MD, PhD, and WEN-YU CHAN, MD
Of the eight different isolates of SEN virus (SENV), SENV-D and SENV-H have been suggested associated with transfusion-associated hepatitis. The prevalence and clinical significance of these two SENV strains among blood donors in southern Taiwan were investigated in this study. Sera of 223 blood donors who were negative for serum hepatitis B surface antigen (HBsAg) and third-generation HCV antibody (anti-HCV) from a blood center of southern Taiwan were tested for alanine aminotransferase (ALT), GB virus C/hepatitis G virus (GBV-C/HGV) anti-envelope protein 2 (anti-E2) antibody and RNA, and SENV-D and -H DNA. Of the 223 donors, the prevalence of SENV-D and/or -H (SENV-D/H), SENV-D, SENV-H DNA, GBV-C/HGV RNA, and anti-E2 were 24.2, 19.7, 5.8, 2.2, and 8.5%. The donors with SENV-D DNA had a significantly higher mean age than those without (31.2 ± 10.9 vs. 27.5 ± 8.3 years; P = 0.014). No association between positive SENV DNA and gender, GBV-C/HGV exposure, mean ALT level, or abnormal ALT was found. Based on multiple logistic regression analysis, the increased age was the only independent factor associated with positive SENV-D DNA (odds ratio, 1.042; 95% confidence interval, 1.01–1.08). Nearly a fourth of blood donors in southern Taiwan were infected by SENV-D/H, with SENV-D more prevalent than SENV-H. Patients with higher ages have a higher prevalence of SENV-D. SENV-D or SENV-H infection was not associated with ALT levels.
KEY WORDS: alanine aminotransferase; blood donor; GBV-C/HGV; SENV.
Taiwan is a hepatitis B endemic area (1). The prevalence rate of chronic hepatitis C (CHC), although ranging from 0.95 to 2.6% in the general population (1, 2), has been re-ported to be up to 57.9% in some communities in southern Taiwan (3). Chronic hepatitis B virus (HBV) and chronic hepatitis C virus (HCV) infections account for the major-ity of cases with abnormal liver biochemistry in Taiwan. Screening tests have been introduced into the blood banks
Manuscript accepted April 6, 2004.
From the Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
Address for reprint requests: Dr. Wang-Long Chuang, Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical Uni-versity Hospital, No. 100, Tz-You 1st Rd, Kaohsiung 807, Taiwan; [email protected]; [email protected].
routinely for HBV and HCV to avoid the transmission of hepatitis. Nevertheless, sporadic cases of posttransfusion hepatitis are still observed occasionally. More efforts have been devoted to identify potential causative viral agents responsible for these patients.
A flavivirus-like virus named GB virus C (GBV-C) and another isolate of GBV-C, hepatitis G virus (GBV-C/HGV), have been claimed to be associated with different types of hepatitis (4–6). However, other and our previous reports have indicated that GBV-C/HGV does not account for liver diseases (7–9). A novel nonenveloped, single-stranded DNA virus was found in 1997 and designated TT virus (TTV) after the initials (T.T.) of the index pa-tient (10, 11). TTV, with a circular genome that comprises 3852 bases with a particle size of 30–50 nm, resembles the
circoviridae (12). There were some reports suggesting that TTV may be associated with elevated ALT levels (10, 11, 13, 14) and may play a role in the development of chronic liver diseases of unknown etiology (10, 15). In contrast, some investigations revealed that the presence of TTV DNA was not correlated with abnormal ALT levels (16, 17) or liver injury (18, 19). Therefore, the pathogenicity and hepatopathic effects of GBV-C/HGV and TTV infec-tion remain controversial.
A new family of DNA viruses was recently isolated and designated SEN virus (SENV), after the initials of the in-fected patient (20, 21). SENV is a small, single-stranded circular DNA virus that is distantly related to the large TT virus family and eight different variants (A–H) have been shown by the phylogenetic analysis of SENV (22). Two strains of SENV (SENV-D and SENV-H; SENV-D/H) are more prevalent in patients with transfusion-associated non-AE hepatitis than in healthy blood donors, which suggested significant associations between SENV-D and SENV-H and transfusion-associated hepatitis (20, 23). However, the association of SENV infection with hep-atocyte damage or the aminotransferase level remained uncertain. In the present study, we investigated the preva-lence and clinical significance of SENV-D and SENV-H infection among blood donors from southern Taiwan.
METHODS
Subjects. Two hundred twenty-three volunteer blood donors from a blood center in Kaohsiung, the largest city in southern Taiwan, were enrolled in the study. There were 167 men and 56 women ranging in age from 18 to 64 years (mean± stan-dard deviation [SD]: 28.2 ± 8.9 years). All the serum samples were negative for both HBsAg and anti-HCV. The studies are in accordance with the Helsinki Declaration of 1995 and were approved by the Ethical Committee of Kaohsiung Medical Uni-versity Hospital.
Laboratory Tests. Third-generation HCV anitbody and HBsAg were detected with commercially available enzyme-linked immunosorbent assay (ELISA) kits (Abbott, North Chicago, IL). Alanine aminotransferase (ALT) (normal upper limit of serum ALT= 34 IU/liter) was measured on a multi-channel autoanalyzer.
Detection of SENV-D and SENV-H by Polymerase Chain Reaction (PCR). The presence of SENV-D and SENV-H DNA was determined by PCR using primers as described previ-ously with modification (22). Briefly, total DNA was extracted from 200 µL of serum with the QIAamp blood kit (QIA-GEN Ltd., Hilden, Germany) and resuspended in 50 µL of elution buffer. For the PCR, 25 µL of reaction mixture con-taining 2.5µL of the DNA sample, 1× PCR buffer (10 mM Tris–HCl [pH 9.0] , 50 mM KCl, 1.5 mM MgCl2, 0.01%
gelatin, and 0.1% Triton X-100), a 200µM concentration of each dNTP, 100 ng of each primer (sense primer for SENV-D, 5-GTAACTTTGCGGTCAACTGCC-3; sense primer for SENV-H, 5-GGTGCCCCTWGTYAGTTGGCGGTT-3 [W= A or T]; universal antisense primer, 5-CCTCGGTTKSAAAK
GTYTGATAGT-3[K= G or T, S = C or G, and Y = C or T]), and 1.25 U of Taq DNA polymerase was amplified in a thermal cycler (Perkin–Elmer Cetus) for 35 cycles consisting of denat-uration at 95◦C for 45 sec, primer annealing for 45 sec at 58◦C for SENV-D and at 62◦C for SENV-H, and extenstion at 72◦C for 45 sec, with a final extension step at 72◦C for 7 min. The amplified products (231 bp for D and 230 bp for SENV-H) were separated in 3% agarose gel electrophoresis and stained with ethidium bromide. Precautions against amplicon contami-nation were taken during the procedures.
Detection of GBV-C/HGV RNA and Anti-E2 Antibody in Serum. GBV-C/HGV RNA was detected by nested neverse transcription PCR (RT-PCR) using primers targeting the 5’ UTR as described previously (24). The anti-E2 antibody was measured by an enzyme-linked immunosorbent assay from Boehringer Mannheim (GmbH, Germany), strictly according to be manu-facturer’s instructions (25).
Statistical Analyses. Descriptive statistics such as means and proportions were calculated: Frequency was compared be-tween groups using the chi-square test or Fisher’s exact test, and group means were compared using Student’s t test. Stepwise logistic regression method was used to analyze the study data. Odds ratios (ORs) and their associated 95% confidence intervals (CIs) were used to quantify the magnitude of their associations.
P< 0.05 was considered statistically significant.
RESULTS
Prevalence of SENV-D/H DNA. Of 223 blood donors without HBsAg and anti-HCV, the PCR results showed that 44 (19.7%) were positive for SENV-D and 13 (5.8%) were positive for SENV-H. With three (1.4%) patients pos-itive for both SENV-D and SENV-H DNA (SENV-D/H co-infection), the overall prevalence of SENV-D/H was 24.2%.
GBV-C/HGV Viremia and Anti–E2 Antibodies.
GBV-C/HGV RNA was present in five (2.2%) of the stud-ied samples. With 19 (8.5%) positive for anti-E2 antibod-ies and 2 (0.4%) positive for both serum GBV-C/HGV RNA and anti-E2 antibodies, the prevalence of HGV ex-posure, defined as positive for serum GBV-C/HGV RNA and/or anti-E2, was 9.9%. There was no association be-tween the presence of GBV-C/HGV RNA and SENV-D/H or between the GBV-C/HGV exposure rate and SENV-D/H (Table 1).
Serum ALT Levels and SENV Viremia. The mean
serum ALT level of the studied samples was 14.5 ± 9.8 IU/L. Nine donors had elevated ALT levels (range: 37–73 IU/L). The mean ALT level was similar in SENV-D/H DNA-positive and -negative donors (15.0 ± 10.0 vs. 14.4 ± 9.8 IU/L) and also similar in D and SENV-H DNA-positive and -negative donors (16.0 ± 10.4 vs. 14.2 ± 9.7 and 11.2 ± 6.7 vs. 14.8 ± 10.0 IU/L, respec-tively). Also, no association between mean ALT levels and GBV-C/HGV viremia or exposure (10.8 ± 8.4 vs. 14.6 ± 9.9 and 15.9 ± 15.2 vs. 14.4 ± 9.1 IU/L, respectively) was
TABLE1. COMPARISON OFCLINICALCHARACTERITICSBETWEENINDIVIDUALSWITH ANDINDIVIDUALSWITHOUTSENV DNA AMONG
BLOODDONORS FROMSOUTHERNTAIWAN
D and/or H D H
Positive Negative Positive Negative Positive Negative
(N= 54) (N= 169) (N= 44) (N= 179) (N= 13) (N= 210) Gender (male %) 39 (72.2) 128 (75.7) 31 (70.5) 136 (76.0) 11 (84.6) 156 (74.3) Age (year) 30.2± 10.4a 27.6± 8.3a 31.2± 10.9b 27.5± 8.3b 27.0± 8.3 28.3± 8.9 ALT (IU/L) 15.0± 10.0 14.4± 9.8 16.0± 10.4 14.2± 9.7 11.2± 6.7 14.8± 10.0 GBV-C/HGV RNA (positive, %) 1 (1.9) 4 (2.4) 1 (2.3) 4 (2.2) 0 (0) 5 (2.4) GBV-C/HGV exposure (positive, %) 7 (13.0) 15 (8.9) 5 (11.4) 17 (9.5) 3 (23.1) 19 (9.1) Blood donation (first time, %) 11 (20.4) 39 (23.1) 10 (22.7) 40 (22.4) 1 (7.7) 49 (23.3)
Note. Values are given as mean± SD.aP= 0.054.bP= 0.014. ALT, alanine aminotransferase, GBV-C/HGV, GB virus C/hepatitis G virus.
observed (Table 1). Of the nine donors with elevated ALT levels, three were SENV-D viremic and all were negative for SENV-H and GBV-C/HGV RNA. The prevalence of SENV infection between normal and abnormal ALT levels was similar (23.8 vs. 33.3%; P= 0.2).
Clinical Features of SENV-D/H Infection. The
com-parison of clinical characteristics between SENV viremic and nonviremic blood donors is shown in Table 1. The mean age was higher in SENV-D/H DNA-positive (30.2 ± 10.4 years) blood donors than -negative ones (27.6 ± 8.3 years) (P = 0.054). The significantly higher mean age among subjects with positive SENV-D DNA than among those without SENV-D DNA was noted (31.2 ± 10.9 vs. 27.5 ± 8.3 years; P = 0.014). Nevertheless, the mean age was similar in SENV-H DNA-positive and -negative donors (27.0 ± 8.6 vs. 28.3 ± 8.9 years). No association between positive SENV DNA and gender or the preva-lence of GBV-C/HGV exposure was found. Based on multiple logistic regression analysis, increased age was identified to be the only independent factor associated with positive SENV-D DNA (OR and 95% CI, 1.042 and 1.01–1.08).
Times of Blood Donation and SENV Viremia. Fifty
of the 223 blood donors (22.4%) donated blood for the first time. When the SENV DNA-positive rates between individuals with first and those with repeated donations were compared, no significant difference was observed (SENV-D/H, 22.0 vs. 24.9%; SENV-D, 20.0 vs. 19.7%; SENV-H, 2.0 vs. 6.9%).
DISCUSSION
According to previous studies, the SENV-D and -H strains, of the eight different strains (A–H), were more prevalent in patients with transfusion-associated non-AE hepatitis than in healthy blood donors, which suggested their significant associations with transfusion-associated hepatitis (20, 23). The prevalence of SENV-D or -H among blood donors was reported to be 1.8% (23) in the United
States and 10 to 22% in Japan (26, 27). In the present study, the prevalence of SENV-D/H infection in healthy blood donors from southern Taiwan (24.2%) seemed sim-ilar to the finding from Japan (27) and higher than the re-ports of 15% from northern Taiwan by Kao and cowork-ers (28). It is noteworthy that a different prevalence of these two SENV isolates, D and H, between southern and northern Taiwan exists. Kao et al. reported that the preva-lence of SENV-H was two to seven times higher than that of SENV-D in different northern Taiwanese subjects in-cluding healthy adults, patients with acute or chronic hep-atitis, intravenous drug users, hemophiliacs, thalassemic patients, and patients on maintenance hemodialysis (28, 29). In their studies the prevalence of SENV-D and -H among healthy adults was 2 and 15%, respectively. We demonstrated in our study that the prevalence of SENV-D was higher than that of SENV-H among southern Taiwan blood donors (19.7 and 5.8%), which is similar to the findings of most other studies around the world showing that SENV-D is more prevalent than SENV-H (27, 30, 31). The results indicated a different geographic distribution of SENV variants in Taiwan. As observed in previous stud-ies, differences in prevalence and genotype distribution of HCV were also noted between southern and northern Taiwan (32–34). We supposed that the independent SENV-D/H outbreaks take place across southern and northern Taiwan.
There was a higher mean age among donors who were SENV-D viremic than those who were nonviremic. How-ever, no significant correlation between age and preva-lence of SENV-H observed. The cause of discrepancy be-tween trends of change in prevalence of SENV-D and -H was not clear. The possible explanations to clarify the is-sues such as different exposure rates or routes of infection as well as different rates of spontaneous clearance between these two strains make further large-scale and longitudinal studies necessary.
The clinical implications and etiological importance in association with liver diseases of SENV infection still
remain undetermined. Being more prevalent in serum of these patients and less frequent in serum samples from healthy blood donors, SENV-D and SENV-H were con-sidered to be associated with transfusion-associated non-A–E hepatitis (20, 21). However, several reports did not support that SENV-D/H was the causal agent for non-A–E fulminant hepatitis (26, 28). In the present study, we failed to show an association between infection with SENV-D/H, SENV-D, or SENV-H and abnormal ALT levels. Besides, SENV viremia was not correlated with ALT levels. Our data, comparable with other reports (26, 28), denied a role for SENV-D/H as causal agents in the development of hepatitis. Likewise, GBV-C/HGV ex-posure was demonstrated to be irrelevant to ALT levels as in previous reports (7–9). Furthermore, we showed repeated donations did not decrease the SENV viremia rate in blood donors. This depicts the fact that available screening tests for donated blood nowadays cannot pre-vent SENV infection entirely. On the other hand, be-cause the majority of SENV-infected donors have nor-mal ALT levels, there is no impact on SENV transmission even if donors with elevated ALT levels are prevented from giving further blood. Since the possible correla-tion between SENV infeccorrela-tion and liver diseases has not been determined, it is not, indeed, suggested nowadays to screen donated blood for SENV from a cost–benefit point of view.
ACKNOWLEDGMENTS
This research was supported by National Science Council, R.O.C., Grant No. NSC-91-2314-B037-344.
REFERENCES
1. Chuang WL, Chang WY, Lu SN, et al: The role of hepatitis C virus in chronic hepatitis B virus infection. Gastroenterol Jpn 28 (suppl 5):23–27, 1993
2. Chen DS, Wang JT, Chen PJ, Wan TH, Sung JL. Hepatitis C virus infection in Taiwan. Gastroenterol Jpn 26 (Suppl 3):164–166, 1991
3. Wang JH, Lu SN, Wu JC, et al: A hyperendemic community of hepatitis B virus and hepatitis C virus infection in Taiwan. Trans R Soc Trop Med Hyg 93:1–2, 1999
4. Simons JN, Leary TP, Dawson GL, et al: Isolation of novel virus-like sequences associated with human hepatitis. Nat Med 1:564–569, 1995
5. Linnen J,Wages J Jr, Zhang-Keck ZY, et al: Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent.Science 271:505–508, 1996
6. Yoshiba M, Okamoto H, Mishiro S: Detection of the GBV-C hep-atitis virus genome in serum from patients with fulminant hephep-atitis of unknown etiology. Lancet 346:1131–1132, 1995
7. Alter MJ, Gallagher M, Morris TT, et al: Acute non-A-E hepatitis in the United States and the role of hepatitis G virus infection. N Engl J Med 336:741–746, 1997
8. Alter HJ, Nakatsuji Y, Melpolder J, et al: The incidence of transfusion-associated hepatitis G virus infection and its relation to liver disease. N Engl J Med 336:747–754, 1997
9. Yu ML, Chuang WL, Dai CY, et al: The serological and molecular epidemiology of GB virus C/hepatitis G virus infection in a hepatitis C and B endemic area. J Infect 42:61–66, 2001
10. Nishizawa T, Okamoto H, Konishi K, Yoshizawa H, Miyakawa Y, Mayumi M: A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiol-ogy. Biochem Biophys Res Commun 241:92–97, 1997
11. Okamoto H, Nishizawa T, Kato N, Ukita M, Ikeda H, Iizuka H: Molecular cloning and characterization of a novel virus (TTV) as-sociated with posttransfusion hepatitis of unknown etiology. Hepatol Res 10:1–16, 1998
12. Mushahwar IK, Erker JC, Muerhoff AS, et al: Molecular and bio-physical characterization of TT virus: evidence for a new virus family infecting humans. Proc Natl Acad Sci USA 96:3177–3182, 1999
13. Itoh K, Hirakawa K, Okamoto H, et al: Infection by an unenveloped DNA virus associated with non-A to -G hepatitis in Japanese blood donors with or without elevated ALT levels. Transfusion 39:522– 526, 1999
14. Okamoto H, Nishizawa T, Ukita M: A novel unenveloped DNA virus (TT virus) associated with acute and chronic non-A to G hepatitis. Intervirology 42:196–204, 1999
15. Hohne M, Berg T, Muller AR, Schreier E: Detection of sequences of TT virus, a novel DNA virus, in German patients. J Gen Virol 79:2761–2764, 1998
16. Niel C, de Oliveira JM, Ross RS, Gomes SA, Roggendorf M, Viazov S: High prevalence of TT virus infection in Brazilian blood donors. J Med Virol 57:259–263, 1999
17. Takayama S, Miura T, Matsuo S, Taki M, Sugii S: Prevalence and persistence of a novel DNA TT virus (TTV) infection in Japanese haemophiliacs. Br J Haematol 104:626–629, 1999
18. Hsieh SY, Wu YH, Ho YP, Tsao KC, Yeh CT, Liaw YF: High preva-lence of TT virus infection in healthy children and adults and in patients with liver disease in Taiwan. J Clin Microbiol 37:1829– 1831, 1999
19. Kao JH, Chen W, Hsiang SC, Chen PJ, Lai MY, Chen DS: Prevalence and implication of TT virus infection: minimal role in patients with non-A-E hepatitis in Taiwan. J Med Virol 59:307–312, 1999 20. Mushahwar IK: Recently discovered blood-borne viruses: Are they
hepatitis viruses or merely endosymbionts? J Med Virol 62:399– 404, 2000
21. Bowden S: New hepatitis viruses: contenders and pretenders. J Gas-troenterol Hepatol 16:124–131, 2001
22. Tanaka Y, Primi D, Wang RY, et al: Genomic and molecular evolu-tionary analysis of a newly identified infectious agent (SEN virus) and its relationship to the TT virus family. J Infect Dis 183:359–367, 2001
23. Umemura T, Yeo AE, Sottini A, et al: SEN virus infection and its re-lationship to transfusion-associated hepatitis. Hepatology 33:1303– 1311, 2001
24. Mukaide M, Mizokami M, Orito E, et al: Three different GB virus C/hepatitis G virus genotypes. Phylogenetic analysis and a geno-typing assay based on restriction fragment length polymorphism. FEBS. Lett 407:51–58, 1997
25. Tacke M, Kiyosawa K, Stark K, et al: Detection of antibodies to a putative hepatitis G virus envelope protein. Lancet 349:318–320, 1997
26. Shibata M, Wang RY, Yoshiba M, Shih JW, Alter HJ, Mitamura K: The presence of a newly identified infectious agent (SEN virus) in
patients with liver diseases and in blood donors in Japan. J Infect Dis 184:400–404, 2001
27. Kobayashi N, Tanaka E, Umemura T, et al: Clinical significance of SEN virus infection in patients on maintenance haemodialysis. Nephrol Dial Transplant 18:348–352, 2003
28. Kao JH, Chen W, Chen PJ, Lai MY, Chen DS: Prevalence and Impli-cation of a newly identified infectious agent (SEN virus) in Taiwan. J Infect Dis 185:389–392, 2002
29. Kao JH, Chen W, Chen PJ, Lai MY, Chen DS: SEN virus infection in patients with chronic hepatitis C: preferential coinfection with hepatitis C genotype 2a and no effect on response to therapy with interferon plus ribavirin. J Infect Dis 187:307–310, 2003 30. Umemura T, Alter HJ, Tanaka E, et al: Association between SEN
virus infection and hepatitis C in Japan. J Infect Dis 184:1246–1251, 2001
31. Umemura T, Alter HJ, Tanaka E, et al: SEN virus: response to interferon alfa and influence on the severity and treat-ment response of coexistent hepatitis C. Hepatology 35:953–959, 2002
32. Wu JS, Lee HF, Hsiau HL, et al: Genotype distribution of hepatitis C virus infection in Taiwan. J Med Virol 44:74–79, 1994
33. Yu ML, Chuang WL, Chen SC, et al: The genotypes of hepatitis C virus in patients with chronic hepatitis C virus infection in southern Taiwan. Kaohsiung J Med Sci 12:605–612, 1996
34. Yu ML, Chuang WL, Chen SC et al: Changing prevalence of hepatitis C virus genotypes: molecular epidemiology and clin-ical implications in the hepatitis C virus hyperendemic areas and a tertiary referral center in Taiwan. J Med Virol 65:58–65, 2001
