Journal of Medical Virology 51:225–230 (1997)

Analysis of Mother-to-Infant Transmission of

Hepatitis C Virus: Quasispecies Nature and Buoyant
Densities of Maternal Virus Populations
Toyoichiro Kudo,1,2* Youichiro Yanase,2 Makoto Ohshiro,2 Mitsuaki Yamamoto,2 Makoto Morita,2
Motohiro Shibata,2 and Tsuneo Morishima2
1
Department of Pediatrics, Meijo Hospital, Nagoya, Japan
2
Department of Pediatrics, Nagoya University School of Medicine, Nagoya, Japan

Mother-to-infant transmission of hepatitis C vi- blood donors have a history of transfusion. On the
rus (HCV) was analyzed by sequencing of viral other hand, at least 35% of adult patients infected with
RNA and semiquantitative polymerase chain re- HCV have no identifiable source [A-Kader and Balis-
action following ultracentrifugation of maternal treri, 1993].
sera. In two mother-infant pairs, the hypervari- Several authors [Inoue et al., 1991; Thaler et al.,
able region 1 (HVR1) and carboxyl terminus of 1991; Nagata et al., 1992; Kuroki et al., 1993; Meisel et
envelope 1 (E1) were sequenced. Both viral se- al., 1995] have reported on mother-to-infant transmis-
quences in the infants were less diverse than sion of HCV. The transmission rate seems to be 5–10%
those of their mothers. Although the E1 se- if the mothers are infected with HCV alone [Ohto et al.,
quences were almost identical in each mother- 1994; Ni et al., 1994; Giacchino et al., 1995]. Coinfec-
infant pair, the HVR1 sequences of the infants
tion with human immunodeficiency virus (HIV) is
were related, but not identical, to those of the
known to increase the transmission rate of HCV
mothers. Serial examinations of one infant re-
[Zanetti et al., 1995]. Maternal viral load has been re-
vealed that the HVR1 nucleotide sequence did
not change from 10 days to 3 months of age. In ported to correlate with the risk of transmission [Ohto
six mothers with uninfected infants, all of the et al., 1994], although the exact mechanism of mother-
dense fractions of sera contained significant to-infant transmission of HCV has not yet been clari-
amounts of HCV RNA, whereas in six mothers fied.
with infected infants, only two of those fractions The N-terminal region of the putative envelope pro-
contained significant amounts of HCV RNA. tein 2 (E2) of HCV shows marked variability in nucleo-
These results indicate that the strains of HCV de- tide and deduced amino acid sequences. This region
tected in the infants were not dominant in the has been designated hypervariable region 1 (HVR1)
mothers, but were still transmissible to the in- [Hijikata et al., 1991]. Analyses of the HVR1 have in-
fants. As dense fractions are known to contain dicated that multiple subpopulations of HCV exist in a
antibody-bound HCV particles, maternal anti- chronically infected patient [Tanaka et al., 1992], and
bodies against HCV may inhibit viral transmis- these multiple subpopulations are termed quasispecies
sion. J. Med. Virol. 51:225–230, 1997. [Domingo et al., 1985]. The formation of quasispecies is
© 1997 Wiley-Liss, Inc. influenced by the host humoral immune response [Ku-
mar et al., 1994; Kudo et al., 1995], and tentative neu-
KEY WORDS: flaviviridae; vertical transmis- tralizing antibodies are thought to play an important
sion; sequencing of viral RNA; role in their formation [Kato et al., 1993; Yamaguchi et
differential flotation centrifuga- al., 1994; Yoshioka et al., 1996].
tion Recently, HCV has been reported to form immune
complexes in the sera of chronically infected patients,
and ultracentrifugation studies suggest that the den-
sities of the circulating viral particles become higher if
INTRODUCTION

Hepatitis C virus (HCV) is known to be transmitted

most efficiently by transfusion of HCV-contaminated *Correspondence to: Toyoichiro Kudo, M.D., Ph.D., Depart-
blood or blood products and by sharing of contaminated ment of Pediatrics, Nagoya University School of Medicine, 65,
needles among intravenous drug users [Alter, 1994]. In Tsuruma-Cho, Showa-Ku, Nagoya 466, Japan.
general, 20–40% of HCV-infected adult patients or Accepted 8 October 1996

© 1997 WILEY-LISS, INC.

226 Kudo et al.

they are bound to antibodies [Miyamoto et al., 1992;

Tsai et al., 1995]. A relationship between the density of
the circulating HCV particles and infectivity of the se-
rum also has been reported [Hijikata et al., 1993].
We identified HCV-infected infants in a prospective
setting and then analyzed partial HCV sequences in
two mother-infant pairs. In addition, we determined
the densities of virus in the sera of mothers who did or
did not transmit HCV to their offspring.
MATERIALS AND METHODS
Patients
Since 1991, infants born to HCV-infected mothers
have been enrolled in a prospective study of mother-to-
infant transmission of HCV and evaluated at five cen-
ters in the Nagoya district every 3 months. In 1994, 71
infants born to 71 mothers with positive second-
Fig. 1. The analyzed portions of E1 and HVR1 of HCV. They are
generation HCV antibody tests during pregnancy were amplified by the indicated oligonucleotide primer pairs. bp, base
followed for 6–40 months. None of the infants had a pairs; E2/NS1, envelope 2/nonstructural 1 region.
history of transfusion. HCV infection in the infants was
diagnosed by detecting serum HCV RNA. Seven in- nal oligonucleotide probe corresponding to the target
fants were found to have been infected. Four of seven sequence. The hybridized DNA was detected using the
infants had persistent HCV RNA in their sera for more insoluble substrate of 5-bromo-4-chloro-3-indolyl-
than 6 months. The remaining three infants had HCV phosphate/4-nitro-blue tetrazolium chloride (BCIP/
RNA detected only transiently in their sera. Two pairs NBT) (Sigma, Tokyo). The sensitivity of this method for
of mothers and infants with persistent infection were detecting HCV RNA was estimated to be as high as 102
analyzed for HCV sequences in their sera according to copies/ml of serum using limiting dilution of the cloned
the method described below. In addition, sera from six PCR product. For semiquantitation of HCV RNA in
of seven mothers who transmitted HCV to their off- fractionated sera, the result was described as a strong
spring were analyzed for the density of the virus and signal, or ‘‘++’’, if the HCV RNA was detected following
compared with sera from six mothers who did not ethidium bromide staining of the gel, and as a weak
transmit HCV. signal, or ‘‘+’’, if detected only after Southern hybrid-
The study was approved by the Ethics Committee of ization.
Nagoya University School of Medicine. The care pro-
viders of the infants were informed and gave consent to Quantitation of HCV RNA in the Sera
participate in the study.
Quantitative detection of HCV RNA [Urdea et al.,
Anti-HCV Assay 1990; Lau et al., 1993] was undertaken using the
Second-generation anti-HCV antibodies were as- branched DNA signal amplification assay according to
sayed using an anti-HCV assay kit (Abotto, Tokyo) or the manufacturer’s instructions (Chiron, Emeryville,
Immucheck (Int. Reagents Corp, Kobe) at each center. CA).

Reverse transcription Polymerase Chain Cloning and Sequencing of the PCR Products
Reaction (PCR) Viral sequences were analyzed in the 38 region of
The PCR assay for detecting the 58 noncoding region envelope 1 (E1) and the 58 region of envelope 2/non-
of HCV was carried out as described previously [Shi- structural 1 region (E2/NS1) (Fig. 1). The target region
bata et al., 1991]. In brief, total RNA was extracted was amplified once, and the products were cloned fol-
from 100 ml of serum by a modified acid-guanidine- lowing purification by agarose gel-electrophoresis and
phenol-chloroform method [Chomczynski and Sacchi, ligation to the plasmid vector pCR (Stratagene, La
1987] and dissolved in 7 ml of RNase-free water. cDNA Jolla, CA). Recombinant clones were identified by re-
was synthesized using avian myeloblastosis virus re- striction enzyme digestion, and purified plasmids were
verse transcriptase (BRL, Gaithersberg, MD) in a 20 ml subjected to DNA sequencing. Thirty cycles of a cycle-
reaction mixture. The cDNA was amplified by 40 cycles sequencing reaction were carried out using Dye-primer
of thermal reaction (consisting of denaturation for 1 (ABI, Tokyo) or Dye-terminator (ABI, Tokyo), both
minute at 94°C, annealing for 2 minutes at 55°C, and based on the dideoxynucleotide chain termination
extension for 3 minutes at 72°C). A 10 ml aliquot of the method [Sanger et al., 1977]. The reaction products
amplified PCR product was subjected to agarose gel were ethanol precipitated and analyzed by a 373A DNA
electrophoresis and stained with ethidium bromide. Af- sequencer (ABI, Tokyo). Sequences were analyzed us-
ter transferring to a nylon membrane, the DNA was ing Genetyx (6.2.0) and Homology (2.2.2) software
hybridized with an alkaline phosphatase labeled inter- packages (S.D.C., Tokyo). HCV-BK was selected as a
Mechanisms of Vertical Transmission of HCV 227

TABLE I. Characteristics of HCV-Infected Mothers and Semiquantitation of HCV RNA in Fractionated Sera
Maternal history Mode Quantity Signal of HCV RNA
of of Breast- of
Case no. transfusion delivery feeding HCV RNAa Top fraction Bottom fraction
A. Mothers with Infected Infants
1 + Vaginal + 1.1 + +
2 − Vaginal + 1.3 − +
3 + C-section − <0.5 + +
4 − Vaginal − <0.5 − +
5 − Vaginal − 2.1 − ++
6 − Vaginal − 7.9 + ++
B. Mothers with Uninfected Infants
7 − Vaginal + <0.5 + ++
8 unknown Vaginal + 16.0 + ++
9 − Vaginal + <0.5 − ++
10 − C-section + 3.1 + ++
11 + Vaginal − 1.5 − ++
12 − Vaginal + 2.9 + ++
a
Assayed by bDNA method (Meq/ml [mega equivalents/ml]).

reference sequence, with base numbers 1295–1481 boxyl terminus of the E1 region showed significant ho-
used as a control for E1, and base numbers 1482–1562 mology between the mother and infant. Sequences
for the HVR1. from both cases were distinct from other HCV se-
quences reported in the GenBank, including HCV-BK,
Genotyping of HCV RNA which was selected as a reference (Table II).
Genotyping was carried out as described previously In contrast, each pair showed marked differences in
by Okamoto et al. [1992], and the results were reported the HVR1, which is adjacent to the carboxyl terminus
according to the method of Simmonds et al. [1993]. of E1, in both nucleotide and deduced amino acid se-
quences.
Differential Flotation Centrifugation
Differential flotation centrifugation was carried out Serial Examination of the HVR1 in One Infant
according to the methods described originally by Havel Serial examinations of the HVR1 in the infant from
et al. [1955] as modified by Hijikata et al. [1993]. case 3 showed no changes in nucleic and amino acid
Twenty microliters of each serum sample were loaded sequences until the infant was 5 months old, when al-
on 1 ml of a sodium chloride solution with a density of terations were observed in two nucleic acids, and in the
1.063 g/ml, and were centrifuged in a Beckman two deduced amino acids (Fig. 2). His anti-HCV titers
TLA100.4 rotor at 139500g for 22 hours at 14°C. Fol- had fallen from birth until 5 months of age, when they
lowing centrifugation, 100 ml of the top and bottom began to rise (data not shown).
fractions were collected, and assayed both by PCR to
detect HCV RNA semiquantitatively and by refracto- Comparison of the Densities of Circulating
metry to determine the respective densities. Virus in Maternal Sera

Statistical Analysis Twelve serum samples collected during the perinatal

period from 12 mothers were subjected to quantifica-
Fisher’s exact probability test and Student’s t test tion of total HCV RNA, differential flotation centrifu-
were applied for statistical evaluation. A value of P < gation, and semiquantitation of HCV RNA in the frac-
.05 was accepted as statistically significant. tionated sera. Six of the mothers had transmitted the
virus to their infants, and the remaining six had not.
RESULTS
The characteristics of the mothers are shown in Table
Characteristics of the mothers analyzed are shown in I. There was no significant difference in the quantity of
Table I. total HCV RNA detected between the mothers with
infected infants and those with uninfected infants in
Comparison of Partial HCV RNA Sequences of the sera tested prior to fractionation by ultracentrifu-
Mothers and Infants gation. Four of six mothers with infected infants had
Mother-infant pairs for cases 1 and 3 (Table I) were HCV RNA of type 1b, and the remaining two had type
analyzed. In both infants, HVR1 nucleic acid sequences 2a. Five of six mothers with uninfected infants had
were less diverse than in their mothers. Homology type 1b and one had type 2a.
within the HVR1 in the mother from case 1 was 83– The densities of the top fractions ranged from 1.07 to
100%, while that in HVR1 in the infant was 99–100%. 1.08 g/ml, whereas those of the bottom fractions were
The mother from case 3 showed 95–100% homology, all greater than 1.15 g/ml. In the bottom fractions, all
and her infant showed 99–100% homology. six serum samples from mothers with uninfected in-
In both pairs, nucleic acid sequence from the car- fants showed strong signals for HCV RNA. In contrast,
228 Kudo et al.

TABLE II. HCV Sequence Homology Between Mothers and Infants*

Comparison with the mother Comparison with HCV-BK
Case no. E1 HVR1 E1 HVR1
1 96–99 (95–100) 74–80 (52–60) 91–94 (92–97) 65–66 (56–59)
3 98–100 (98–100) 84–86 (70–74) 91–93 (95) 64 (48)
*Data presented as the range of sequence homology in nucleotides (in deduced amino acids, in parentheses). HCV-BK was used as the reference
for HCV genotype 1b. HVR1, hypervariable region 1.

Fig. 3. Signals of HCV RNA from each fraction of maternal sera.

Case number, see Table I; T, top fraction; B, bottom fraction; P.C.,
positive control; N.C., negative control.

vidual, differences in the V3 loop sequences from three

mother-infant pairs also have been reported [Wolinsky
et al., 1992].
Second, no nucleic or amino acid changes were ob-
served in the case 3 infant from 10 days to 3 months of
age, which argues against the possibility that the high
mutation rate of HCV accounts for the differences in
HVR1 between the mother and infant. Our results also
emphasize the role of the humoral immune response in
the formation of HCV quasispecies, and they are con-
Fig. 2. Serial examination of HVR1 in an HCV-infected infant from
case 3. Deduced amino acid sequences from the major clones of the sistent with an earlier observation regarding the HVR1
mother are indicated by single-letter codes. Letters in infant’s se- in an agammaglobulinemic patient, whose envelope
quences denote amino acids different from those in the mother. The
nucleotide or amino acid sequence of the infant is composed of a single
glycoprotein remained unchanged for 2.5 years [Kumar
strain until 3 months of age, and shows no change. et al., 1994].
Third, the mothers with uninfected infants more fre-
quently had high amounts of HCV RNA in the high-
only two of the six serum samples from mothers with density fractions of their sera than the mothers with
infected infants revealed strong signals for HCV in the infected infants. Densities of the virus are believed to
bottom fractions (P < .05). Signals for the PCR products increase in reaction to elements of the humoral im-
are shown in Figure 3. mune response, some of which may be caused by neu-
DISCUSSION tralizing antibodies, and by the production of naked
nucleocapsids [Kanto et al., 1994, 1995a,b]. Hijikata et
This study provides evidence for the existence of an al. [1993, 1995] have reported that low-density virus is
infective subpopulation among quasispecies of mater- more highly infective to chimpanzees than high-
nal HCV, and for the possible influence of antibodies density virus, and that the latter is associated with the
against HCV on perinatal transmission. HCV-specific immunoglobulin. Our results support
First, the marked difference in the HVR1 sequences these findings. However, we could not recover from the
within each mother-infant pair and the identification top fractions of maternal sera, the infectious subpopu-
of less diverse populations in infants than in mothers lation found in the infants (data not shown).
suggest that a minor but infective subpopulation of vi- Although our system was not sufficiently sensitive to
rus in the mothers was transmitted. Weiner et al. detect these infectious subpopulations in fractionated
[1993] have described similar observations in mother- sera, it is possible that the maternal immune response
to-infant transmission of HCV. In the case of HIV, resulted in inhibition of mother-to-infant transmission
which also forms quasispecies within an infected indi- by reducing the amount of infectious viral particles in
Mechanisms of Vertical Transmission of HCV 229

the circulation. This is consistent with the observation Kanto T, Hayashi N, Takehara T, Hagiwara H, Mita E, Naito M,
Kasahara A, Fusamoto H, Kamada T (1994): Buoyant density of
that the dominant populations of maternal virus were hepatitis C virus recovered from infected hosts: Two features in
not transmitted. sucrose equilibrium density-gradient centrifugation related to de-
Two mothers with infected infants showed high lev- gree of liver inflammation. Hepatology 19:296–302.
els of HCV RNA in the bottom fractions of their sera. Kanto T, Hayashi N, Takehara T, Hagiwara H, Mita E, Oshita M,
Katayama K, Kasahara A, Fusamoto H, Kamada T (1995a): Serial
This suggests not only that the density of circulating density analysis of hepatitis C virus particle populations in
virus is a key factor in transmission, but that other chronic hepatitis C patients treated with interferon-a. Journal of
factors probably play a role as well. Medical Virology 46:230–237.
Kanto T, Hayashi N, Takehara T, Hagiwara H, Mita E, Naito M,
In Table I, two of six mothers of infected infants and Kasahara A, Fusamoto H, Kamada T (1995b): Density analysis of
five of six mothers of uninfected infants have breast- hepatitis C virus particle population of infected hosts: Implica-
fed. In our recent series, however, there was no signifi- tions for virus neutralization or persistence. Journal of Hepatology
22:440–448.
cant difference in the rates of breast-fed infants be-
Kato N, Sekiya H, Ootsuyama Y, Nakazawa T, Hijikata N, Ohkoshi S,
tween those who are infected and those who are not (4 Shimotohno K (1993): Humoral immune response to hypervariable
[50%] of 8 infected infants were breast-fed, and 56 region 1 of putative envelope glycopratein (gp70) of hepatitis C
[73%] of 77 uninfected infants were breast-fed P 4 .18, virus. Journal of Virology 67:3923–3930.
Fisher’s exact test, unpublished data). Breast-feeding Kudo T, Morishima T, Shibata M, Miwata M, Matsushima M, Tsuzuki
K (1995): Low humoral responses to hepatitis C virus among pe-
did not seem to affect mother-to-infant transmission of diatric renal transplant recipients. Acta Paediatrica 84:677–682.
HCV, although breast milk was supposed to contain Kuhn L, Stein ZA (1995): Mother-to-infant HIV transmission: Timing,
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