Jpn. J. Infect. Dis., 53, 67‑69, 2000

Short Communication

Viral Load in Indonesian Patients with Chronic Liver Disease and in Blood

Donors Infected with Different Subtypes of Hepatitis C Virus
Maria I. Lusida, SoetJIPtO, Retno Handajani, Chairul A. Nidom, Soemarto, Siswanto Darmadi,
Pangestu Adi, Motoko Fujiil, Tsunenori Fujital, Satoshi Ishidol and Hak Hottal･2*
TTy10Pical Disease Center; A irlangga UniversiOJ, JI. Mulyorejo, Surabqya, Indonesia and/Department ofMicy10biology and2Internationa/ Center for Medical Research, Kobe UniversitySchool of Medicine,
Kusunoki‑cho 7‑5‑I, Chuo‑ku, Kobe 650‑001 7, Japan(Received Febmary 7, 2000. Accepted March 1 0, 2000)

SUMMARY: The viral load of different hepatitis CviruS (HCV) subtypes, including the globally distributed
HCV‑ 1 b and the unique Indonesian subtype HCV‑ lc, was analyzed using serum samples obtained from Indone‑
sian blood donors and patients with chronic liver disease. The mean viral load of HCV･ lc was comparable withthat orHCV‑lb, suggestlng that HCV‑lc is as pathogenic as HCV‑lb. On the other hand, the mean viral load orHCV‑2a was lower than that ofHCV‑ lb or HCV‑ lc, with this result being consistent with previous obseⅣations.

Interestingly, some HCV‑2a strains were associated with a high viral load that was almost equlValent to that or

HCV‑ 1b and HCVl 1 c･ This result implies the possibilitythat there exists a minor fraction ofHCV‑2a strains thatcause higher levels of viremla COmParedwith the majorityof ordinary HCV12a strains.
Hepatitis C virus (HCV) readily establishes chronic persis‑tent infection that often results in chronic hepatitis (CH) andtranscription‑polymerase chain reaction (RTIPCR) that wasdesigned to amplify a portion of the NS5B reg10n Ofthe HCV

more deteriorating diseases such as liver cirrhosis (LC) andhepato.cellular carcinoma (HCC)･ HCV is classified into atleast six Clades (formerly called genotypes), each of which

genome (5, 6). Nucleotide sequences of the amplified NS5B
fragments were determined, and based on these sequences asubtype was asslgned to each HCV strain, as describedcan be further divided into a number of subtypes (1‑3). The

previously (5･ 6)･ Levels ofHCV viral load were assessed byprevalence of each HCV subtype has been reported to vary

uslng a COmmerCially available kit (Amplicor HCV Monitor

in different geographical areas (1 , 3‑6). In addition, viral

subtype among various populations, Including healthy blood

Test, ver･ I ･0; Roche Diagnostic Systems, Inc., Branchburg,N.J., USA) according to the manufacturer's instructions. In
addition, serum alamine aminotransferase (ALT) levels weredetermined by using the Granutest ALAT (Merck, Darmstadt,Germany). Normal ALT levels were <23 U/liter for menand

donors, patients with liver disease,and patients on maintenance<19 U/liter fわr women when tested at 25oC.

pathogenicity and sensitivity to interferon (IFN) treatment

appear to vary among the subtypes. We previously reported on
the prevalence of anti‑HCV antibodies and that of each HCV

hemodialysis, in Surabaya, Indonesia (5‑7). In the course ofWe analyzed the possible relationship between HCV viral

the study, We identified a unlque Indonesian subtype, HCV‑

load and age, HCV subtypes, or ALT levels in blood donors.
Blood donors under 40 years of age were all infected withHCV‑2a, with a mean ALT level of29.4 U/liter and meanlc (formerly referred to as HCVl1d), which has been foundalmost exclusively in Indonesia (4‑6).

In addition to viral subtypes, the HCV RNA level in theHCV viral load of3.4 logaritlmtiter/ml (Table 1). In contrast,

serum is an important factor. It has been reported to correlatenearly half of the blood donors over 40 were infected withwellwith liver damage. Moreover, lower pretreatment serum

HCV‑1b or HCV‑lc, with a meanAIJ level or40.4 U/literand mean HCV viral load or 5.6 logarithm titer/ml. The
difference in the mean viral load between the two groups wasHCV RNA levels have been shown to be associated with abetter response to IFN therapy (8). Therefore, the determina‑tion or HCV viral load and HCV subtypes has a clinicalstatistically significant (P< 0.005). Blood donors over 40withimportance. In the present paper, We describe the possible

HCV‑2a showed a mean ALT level andviraHoad practicallyrelationship between viral load and HCV subtypes in healthy

the same as those of donors under 40. These results suggest

blood donors and in patients with CH, LC, and HCC in

the possibility that HCV genotype 1 strains, such as HCV‑ lband HCV‑lc, have the capaclty tO Cause higher degrees ofSurabaya, Indonesia.

Sera were obtained from the Red Cross Blood Transfusion

viremia and, Consequent)y, more severe liver damage, which

Center and Dr. Soetomo Hospital, Faculty or Medicine,is indicated by elevated ALT levels. A similar observation,
reported previously, was that patients infected with HCV‑ laor HCV‑1b were more viremic than thoseinfected with HCV‑
3a (9)･ Our result also implies a possible shift of the prevalent

Airlangga University, Surabaya, between August 1 995 andJune 1 997. These samples were tested for anti‑HCV antibodies
by enzyme‑linked immunosorbent assay (UBI HCV EIA,
United BiologlCals, Inc., New York, N.Y, USA; Ortho HCV
Ab ELISA Test II, Ortho Diagnostics, Inc., Tokyo),and positive
sera samples were further tested for HCV RNA by a reverse

HCV subtypes among blood donors in Surabaya,斤om HCV･lb and HCV‑lc to HCV‑2a. It appears that HCV｣2a has been
more recently introduced, and is currently prevailing, almostexclusive)y, among blood donors. Such a changlng Pattem Of*Corresponding author: Tel: +81 ‑78‑382‑5500, Fax: +81 ‑78‑382‑

HCV subtype prevalence over time has also been reported
for patients on maintenance hemodialysIS and for kidney

55 1 9, E‑mail: hotta@kobe‑u.ac.jp

67

Table 1. HCVviral load and AIJ levels in blood donors infectedwithHCV‑lb, HCV‑1c or HCV‑2aAge(ygroupear)HCVsub<40No.

typesamp2a≧40ofleALTslevel(U/li6Ⅵral loadl) (range)ter)

29.4±19.9

1b or lc62a8

3.4±0.6 (<3.0‑4.4)

40.4±15.8

5.6±0.32) (5.3‑6.0)

27.7±1 5.2

3.7±0.7 (<3.0‑4.8)

1)Mean titers (Log1｡HCV RNA copies/ml) ± SD.
2)p<0.005, compared with age group <40, Or age group ≧40, HCV‑2a.

recipients (10). If this is the case, the prevalence ofHCV‑2aamong patients with chronic liver disease in Surabaya will

increase in the future.
To confirm the possibly higherviral load observed withHCV‑lb and HCV‑lc, the viral load of each subtype in all(IuJ al!lVN∝>3工)OL601･l七･I‑一6竜,StJ+

patient groups was analyzed. The results obtained clearly

demonstrated that the mean viral titers ofHCV‑ lb and HCV‑l c were significantly higher than that of HCV‑2a (Figure).
Table 2 also shows a general tendency of HCV‑lb and HCV‑

5･iH]J

lc to be associated with higher viral load than仇at ofHCV‑

2ainpatients withchromic liver disease;the difference observed

among groups in CH was statistically slgnificant while thedifferences observed in LC and HCC were not, probably due

4to the small numbers ofsamples tested. It should also be notedthat the mean viralload ofHCV‑ 1c was practically the sameas that ofHCV‑lb.

While most HCV‑2a strains are associated with a low viralload, Our present result also implies the possibility that there
exists a distinct,仇ough minor, group orHCV‑2a strains thatare associated with a high viral load (Figure). It is tempting

to speculate that this particular group or HCV‑2a is more
pathogenic, and is more responsible than the majorlty Ofordinary HCVl2a strainswith a low viral load for the occur‑
rence ofCH, LC, and HCC. Follow‑up study of blood donors
infected with HCVl2a of high viral titers and those of low

1btiters is necessary to investlgate Whether this speculation islc

HCV subtype

co汀eCt. Also, a sequence comparison between HCV‑2aisolates of high titers and those or low titers, as well as acomparison between HCV‑2a isolates obtained from long‑term

Figure. HCV viral load of different HCV subtypes in sera ofblood

healthy carriers and those obtained from patients with LCanddonors and patients with CH, LC, and HCC. Each dot represents theHCC, may help us understand the molecular mechanism(S)that underlies the pathogeniclty Of HCV. Such sequence

deviations are shown by longand short horizontal bars, respectively.

viral load of a partlClpant in this study. Mean values and standard

Table 2. HCV viralloadand ALT levels in blood donors, CH, LC, and HCC infected with different HCV subtypes
No.
Patientgroup

Subtypen^MJ八一ofBlood donorl 1 2 1 1 2 1 1 2 1 1 2b C a 也 c a b c a b C aAge′:.:三二､samples

4 2̲ 4 6 4 3 6 6 5 9 4 3

ALT′T(years)Tlevel/.;.A̲､

ViralloadI)(range)(U/liter)

5 1.7±2.6

31.4±5.6

5.7±0.3 (5.3‑6.0)

51.5±4.958.4±1 3.4

5.4±0.2 (5.3‑5.6)

44.0±13.828.5±16.762.3±3. 1

30.0±14.2

5. 1±0.7 (4.4‑6. 1)

3.5±0.62) (<3.0‑4.8)44.3±21.4

29.0±8.4

5. 1±0.7 (4.1‑5.7)

66.0±3.035.7±25.4

3.8±0.43) (3.5‑4.3)

62.0±4.651.3±73.2

4.9±1. 1 (<3.0‑6.0)

62.5±5.7

51.7±31.8

5.1±1.1 (<3.0‑6.0)

61.8±4.3

35.4±14.2

4.0±1.1 (<3.0‑5.6)58.3±10.6

46.4±22.3

4.6±1.0 (<3.0‑5.8)58.3±1.362.6±32.7

4.3±1.1 (<3.0‑5.7)63.3±5.824.3±15.3

3.9±0.5 (3.4‑4.3)

りMean liters (Log‑｡HCV RNA copies/ml) ± SD.

2)p<0.005, comparedwith HCV‑lb and HCV‑ lc.
3)p<0.05, Compared withHCV‑lb and HCV‑I c.

68comparisons are currently underway ln Our laboratory.

M･I･, Soemarto, W･ and Homma, M･ (1994): SequeヮceanalysIS Of hepatitis C virus obtained from Indoneslan
patients and identification of novel sequence variants.

ACKNOWLEDGMENTS

p.3 1013 13. In K. Nishioka, H. Suzuki, S. Mishiro and T.

We are grateful to Dr. T. Rahayu and Mrs. Koen Poedjiati

Oda (eds.), Viral Hepatitis and Liver Disease. Springer‑Verlag, Hong Kong.

for the collection of serum samples.

This work was camied out during a large scale cooperative

5. Hotta, H., Handajani, R., Lusida, M. Ⅰ., Soemarto, W.,study between SoutheastAsian countries and Japan, conducted

Doi, H., Miyajima, H. and Homma, M. (1994): Subtype

by the Japan Society for the Promotion of Science. This work

analysIS Ofhepatitis C virus in Indonesia on the basis ofwas also supported in part by a grant‑in‑aid from the Minis什yNS5b reglOn Sequences. ∫. Clin. Microbio1., 32, 3049‑of Education, Science, Sports and Culhre or Japan, and a3051.

research grant from the Mitsui Life Social Welfare Founda‑

6. Soetjipto, Handajani, R., Lusida, M. I., Darmadi, S., Adi,P., Soemarto, Ishido, S., Katayama, Y and Hotta, H.

tion.

( 1 996): Differential prevalence ofhepadtis C virussubtypes
in healthy blood donors, patients on maintenance hemo‑

REFERENCES

dialysIS, and patients wi仇hepatocellular carcinoma in

1. Doi, H., Apichartpiyakul, C., Ohba, K., Mizokami, M.
and Hotta, H. (1996): Hepatitis C virus (HCV) subtype

Surabaya, Indonesia. ∫. Clin. Microbio1., 34, 2875‑2880.

7. Darmadi, S., SoetJ)pto, Handajani, R., Lusida, M. I.,
Soemarto, Sakugawa, H., Ishido, S. and Hotta, H. (1996):
Hepatitis C virus infection‑associated markers in sera

prevalence in Chiang Mai, Thailand, and identification
ornovel subtypes orHCV major type 6. ∫. Clin.

Microbio1., 34, 569‑574.
2. Robertson, B., Myers, G., Howard, C., Brettin, T., Bukh,

from blood donors in Surabaya, Indonesia. Microbiol.
Immuno1., 40, 401‑405.

J., Gaschen, B., Gojobori, T., Maertens, G., Mizokami,

8. Wilber, ∫. C., Wuestehube, L ∫. andUrdea, M. S. (1997):M., Nainan, 0., Netesov, S., Nishioka, K., Shin‑I, T.,Simmonds, P., Smith, D., Stuyver, L. and Weiner, A.

Ha汀ison and AJ. Zuckeman (eds.), The Molecular

Quantification of hepatitis C viraemia. p. 1 531 166. In T.J.

Medicine ofViralHepatitis. JolmWiley & Sons, Singapore.
9. Garcia, F., Roldan, C., Hemandez‑Quero, J., Bemal, M.
C., Martinez, M. A., Lopez, M. A., Piedrola, G. andMaroto, M. C. ( 1996): Relationship between viral geno‑(1998): Classification, nomenclature, and databasedevelopment fわr hepatitis C virus (HCV) and related

viruses: proposals for standardization. Arch. Viro1., 143,2493‑2503.
3. Simmonds, P., Holmes, E. C., Cha, T.A., °han, S. W.,

type and viral load in patients with chronic hepatitis C.

McOmish, F., Irvine, B., Beall, E., Yap, P. L., Kolberg, J.
and Urdea, M. S. (1993): Classification of hepatitis C

10. Pol, S., Thiers, V., Nousbaum, ∫. B., Legendre, C.,virus into six Tajor genotypes and a series of subtypes

Berthelot, P., Kreis, Il. and Brechot, C. (1995): The

Eur. J. Clin. MicrobioI. Infect. Dis., 15, 8841887.

by phylogenetlC analysts Of the NS‑5 reglOn. ∫. Gen.

changlng relative prevalence of hepatitis C vims geno‑

Viro1., 74, 2391‑2399.

types: evidence in hemodialyzed patients and kidney

4. Hotta, H., Doi, H., Hayashi, T., Purwanta, M., Lusida,

reclplentS. Gastroenterology, 1 08, 58 1 ‑583.

69