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Journal of Clinical Microbiology, May 1999, p. 1634-1637, Vol. 37, No. 5
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
High Prevalence of GB Virus C in Brazil and
Molecular Evidence for Intrafamilial Transmission
João R. R.
Pinho,1,2,*
Paolo M. De
A.
Zanotto,3
João L. P.
Ferreira,1
Laura M.
Sumita,2
Flair J.
Carrilho,4
Luiz C.
da
Silva,5
M. Lourdes
Capacci,6
Adávio O.
Silva,6
Betty
Guz,7
Fernando L.
Gonçales Jr.,8
Neiva S. L.
Gonçales,9
Gregory A.
Buck,10
Gregory A.
Meyers,11 and
A.
Plínio
Bernardini2
Serviço de Virologia, Instituto Adolfo
Lutz,1 Laboratório
Bioquímico Jardim Paulista,2
Disciplina Doenças Infecciosas e Parasitárias,
EPM, UNIFESP,3 Departamento
Gastroenterologia, Faculdade de Medicina, USP,4
Laboratório Hepatologia, Instituto de Medicina
Tropical de São Paulo,5 Hospital
da Beneficência Portugesa,6 and
Hospital do Servidor Público
Estadual,7 São Paulo, and
Disciplina Moléstias Infecciosas, Faculdade de
Ciências Médicas,8 and
Hemocentro, UNICAMP,9 Campinas,
São Paulo, Brazil, and Medical College of Virginia,
Virginia Commonwealth University,10 and
Commonwealth Biotechnologies, Inc.,11
Richmond, Virginia
Received 2 October 1998/Returned for modification 15 December
1998/Accepted 16 February 1999
 |
ABSTRACT |
The prevalence of GB virus C (GBV-C) in candidate Brazilian blood
donors with normal and elevated alanine aminotransferase levels was
found to be 5.2% (5 of 95) and 6.5% (5 of 76), respectively. Among
Brazilian patients, GBV-C was found in 9.5% (13 of 137) of cases of
hepatitis not caused by hepatitis A virus (HAV), HBV, HCV, HDV, or HEV
(non-A-E hepatitis) and in 18.2% (8 of 44) of individuals infected
with HCV. Molecular characterization of GBV-C by partial sequencing of
the NS3 region showed clustering between members of a single family,
implying intrafamilial transmission. In conclusion, these results
together suggest that contagion mechanisms which facilitate
intrafamilial transmission of GBV-C may partially explain the high
prevalence of viremic carriers worldwide.
| |
TEXT |
Two different groups have reported
the discovery of a novel flavivirus-like agent potentially associated
with human hepatitis, designated GB virus C (GBV-C) (18) or
hepatitis G virus (HGV) (13). GBV-C transmission by blood
transfusion has been clearly demonstrated (1, 13),
explaining the high prevalence of this virus among intravenous drug
users, polytransfused individuals, hemophiliacs, and hemodialysis
patients (13). Vertical transmission from mother to infant
has also been reported (6, 21), and epidemiological data
further suggest the occurrence of horizontal transmission in confined
patients (5). Sexual transmission has been reported through
the analysis of patients infected with human immunodeficiency virus
(HIV) (8), prostitutes (9), and couples
(10). GBV-C has also been documented in cases of hepatitis
not caused by hepatitis A virus (HAV), HBV, HCV, HDV, or HEV (non-A-E
hepatitis) worldwide (13, 18), including Brazil (17); in HBV- and HCV-infected patients (13); and
in cases of aplastic anemia (18). Furthermore, despite its
high prevalence in patients with hepatitis, the association between
GBV-C and hepatitis, cirrhosis, hepatocellular carcinoma, or end-stage
liver disease is uncertain, especially because many infections also occur in healthy individuals (1, 2). The need for additional studies to address this question is clear (14).
Herein, we analyze the prevalence of GBV-C in candidate blood donors
and hepatitis patients in Brazil. Through an analysis of viral
sequences from infected family members of two patients with index
cases, we also provide evidence for intrafamilial transmission of this virus.
Patients.
Samples were obtained from five population groups.
Group 1 comprised 137 non-A-E hepatitis patients. These were
individuals who had acute or chronic hepatic disorders and for whom
viral hepatitis was considered in the differential diagnosis. These patients were always seronegative for hepatitis B surface antigen (HBsAg), antibody to hepatitis B core antigen (anti-HBc), and anti-HCV.
Patients with acute cases also tested negative for anti-HAV immunoglobulin M (IgM), anti-HBc IgM, anti-HEV, and HCV RNA. Group 2 consisted of 44 hepatitis C patients, diagnosed by reactive anti-HCV
serology and/or HCV RNA by PCR. Group 3 consisted of 95 volunteer blood
donors from the Blood Bank of Hemocentro, Campinas, São Paulo
State, Brazil, in the order of their acceptance. These blood donors
underwent routine screening tests for blood-borne infections (alanine
aminotransferase [ALT] levels, HBsAg, and anti-HBc, HCV, HIV, human
T-cell leukemia virus types 1 and 2, Treponema pallidum, and
Trypanosoma cruzi). Group 4 comprised 76 candidate volunteer
blood donors from the same blood bank, in sequential order. These
individuals were not accepted, exclusively due to the detection of ALT
levels higher than the cutoff value. Familial contacts (two wives and
four daughters) of two GBV-C patients made up group 5.
GBV-C RNA detection.
RNA was extracted from 100 µl of serum
by using guanidine isothiocyanate. All the extracted material was then
used in cDNA synthesis with Moloney murine leukemia virus reverse
transcriptase and random hexamers. The cDNA was amplified with primers
covering the NS3 region by using a nested PCR protocol. In the first
round of amplification, the primers ns3.2-a2 and ns3.2-s1 were used in
a "touchdown" PCR (18). One-tenth of the first-round
product was then used in the second amplification, with primers gbvc-s1 and gbvc-a1 in another touchdown PCR protocol (12). Positive samples were identified in a 2% agarose gel, and the specificity of
each result was confirmed by sequencing, as described below. To avoid
false-positive results, strict procedures proposed for nucleic acid
amplification diagnostic techniques were followed (11).
These procedures include (i) use of completely independent separated
areas for RNA extraction, PCR setup, and PCR product analysis; (ii) use
of dedicated reagents, equipment, pipettes, plasticware, gloves, and
lab coats for each of these areas; and (iii) control of daily flow of
personnel only from pre- to postamplification areas. At the
preamplification areas, special procedures were adopted: use of aerosol
resistance tips, constant changing of gloves, use of paper sheets for
opening tubes to avoid aerosols, and working with stock solutions and
preparing reaction mixes in separate areas before starting any
manipulation with samples on the same day. In every reaction run, one
negative control was analyzed for every three samples and a weakly
positive control serum, from a patient previously determined to be
viremic, was included (17).
Sequencing.
PCR fragments were directly sequenced with
fluorescent dye terminators on a Perkin-Elmer Applied Biosystems 377 Prism automated DNA sequencer. Reactions were performed essentially as
described by the manufacturer by using commercially available
sequencing kits. Sequencing primers were those used in the second PCR
amplification. Raw sequence data were assembled into contigs, edited
with Sequencher sequence analysis software (Gene Codes, Ann Arbor,
Mich.), and exported as ASCII formatted files for subsequent analysis.
Double-stranded edited sequences were used for all phylogenetic analyses.
Phylogenetic analysis.
A data set of 246 nucleotides in length
(including gaps), with 53 sequences, consisting of 26 GBV-C sequences,
representing a wide range of geographical locations, taken from
sequence databases and 27 sequences from Brazilian patients, was
analyzed. Clinical and epidemiological data for 23 patients are
presented in Table 1. Only patients from
whom GBV-C sequences were used in the subsequent phylogenetic analysis
are shown in Table 1. Phylogenetic trees were reconstructed by using
the neighbor-joining method from genetic distances estimated by the
HKY85 model of DNA substitution, with values for the
transition/transversion ratio (1.98), the shape parameter (
= 0.309)
of the gamma distribution which describes rate variation among sites,
and the proportion of invariant sites estimated from the empirical
data. The robustness of the groupings observed on the phylogenetic tree
was assessed by bootstrap resampling (1,000 replications). All analyses
were carried out with the PAUP package (beta version 4.0.0d52; kindly
provided by David Swofford, Sinauer Associates, Inc.).
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TABLE 1.
Geographical origins and epidemiological, clinical,
and/or histopathological data for the 23 Brazilian GBV-C-infected
patients whose GBV-C sequences were used in the phylogenetic analysis
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|
Results and discussion.
We detected GBV-C RNA in 13 of 137 (9.5%) Brazilian non-A-E hepatitis patients, in general agreement with
the preliminary data reported by our group (1 of 13; 7.5%)
(17). This prevalence is similar to those reported
elsewhere
8.3% in Europe (13), 8.7% in the United States
(6), and 8% in Japan (19)although higher
levels are observed in China (16.7% [20]) and, most
noticeably, in Italy (39% [7]). These cases remain
without etiological diagnosis, since no relationship between GBV-C and
liver diseases has been proven (1, 2) and no other known
causes of hepatitis (such as alcoholism, drug use, autoimmunity, or
hereditary diseases) were reported among these patients, with the
exception of one with a history of alcoholism.
GBV-C was also detected in 8 of 44 (18.2%) HCV-infected individuals.
This is within the range reported in other regions: 8% for Japan
(19), 8.2% for China (20), 18.7% for Europe
(13), and 20% for the United States (4). The
fact that this prevalence is higher than that observed among non-A-E
hepatitis patients probably reflects the greater level of exposure to
blood products in HCV-infected patients.
In contrast to the situation in patients with hepatitis, our data
reveal that Brazil has one of the highest prevalences of
GBV-C among
volunteer blood donors reported so far: 5 of 95 (5.2%)
and 5 of 76 (6.5%), among donors with normal and elevated ALT
levels,
respectively. A higher prevalence in blood donors was
found in Vietnam
(7.4%) (
3). In China, the GBV-C prevalence
was 7.9% in
professional blood donors but only 0.6% in healthy
volunteers
(
20). In the United States, the GBV-C prevalence
in
commercial blood donors was 12.9% (
4). Much lower
prevalences
are found in volunteer blood donor populations from the
United
States, ranging from 0.8 (
4) to 1.7% (
13)
in donors with
normal ALT levels and from 1.5 (
13) to 3.9%
(
4) in donors
with elevated ALT levels. The finding of
similar prevalence rates
among non-A-E hepatitis patients and blood
donors with normal
and elevated ALT levels reinforces the hypothesis
that GBV-C is
not pathogenic in most infected individuals (
1,
2). It should
be noted that the prevalence of this virus in
Brazil is probably
higher than that shown herein, as other authors have
since published
reports that by using 5' noncoding region-derived
primers, about
one-third more cases could be detected (
15,
22). Other studies
should be carried out to better analyze GBV-C
prevalence in different
Brazilian populations. These studies should
verify whether the
high prevalence in Brazilian candidate volunteer
blood donors
is also found in the general population, since the former
are
generally relatives or close friends of hospitalized patients
needing blood transfusions. Perhaps this fact introduces an important
bias in the selection of Brazilian blood donors and is related
to the
elevated prevalence found in this
study.
GBV-C sequences obtained from the Brazilian patients were checked for
any insertion or deletion that would alter the coding
capacity of the
NS3 gene. We did not find any mutational event
that would cause
frameshifts or premature termination in the
sequences.
It has been previously shown that the utilization of short sequence
fragments from the NS3 region did not provide relevant
information for
classification of this virus (
16). Because of
a lack of
phylogenetic information in the sequence data (reflected
in the low
bootstrap values), it was not possible to fully resolve
the
evolutionary relationships of a worldwide sample of GBV-C
sequences by
using the NS3 amplicon described here. A longer region
is clearly
required for analyses of this
kind.
However, the analysis of our sequence data from the families of two
patients with index cases provides important information
about the mode
of GBV-C transmission (Fig.
1). In one
family (F2),
no close relationship was found among the sequences
obtained from
two infected individuals, suggesting that infection in
this couple
was coincidental (not an unlikely event given the high
prevalence
of this virus). In another family (F1), the sequences
obtained
from three infected members (the father, the mother, and the
younger
of two daughters) form a tight phylogenetic cluster (100%
bootstrap
support), strongly supporting intrafamilial transmission. No
other
GBV-C risk factor was reported for any other member of the
family.
The route of transmission within this family is probably by
sexual
contact between the parents and by vertical transmission from
the mother to her second daughter. Intrafamilial transmission
of this
kind may therefore represent an important pathway for
the propagation
of GBV-C and may help to explain the high prevalence
among blood
donors.
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FIG. 1.
Neighbor-joining phylogenetic tree of GBV-C NS3
sequences. The geographical origin of each sequence is indicated by the
first letter in the sequence identifier (see the key). Encircled
lineages represent viral samples from members of the two families (F1
and F2). The boxed lineages at the top of the tree are from the same
patient. Branch lengths are proportional to the degrees of sequence
divergence, estimated by a maximum-likelihood method.
|
|
The genetic distances among the sequences obtained from family F1 were
5 to 10 times smaller (data not shown) than the distances
among
sequences from unrelated patients which appeared adjacent
in the tree
(e.g., B-MPC3 and B-69251). On the other hand, different
sequences from
the same patient show a divergence level which
is comparable to that
observed for F1 (e.g., A-GBCNS3 and A-GBC1
[Fig.
1]). Given the close
relatedness among the sequences obtained
from viruses circulating in
members of the same family (F1), intrafamilial
transmission is the most
parsimonious explanation, because otherwise
one has to postulate
several transmission events from an index
case not included in this
study, which would entail several independent
transmission
events.
In conclusion, a high prevalence of GBV-C was found among different
Brazilian populations, including non-A-E hepatitis and
hepatitis C
patients, candidate blood donors, and intrafamilial
contacts of two
patients with index cases. The high prevalence
of this virus seems to
be sustained by efficient contagion mechanisms
that facilitate
intrafamilial transmission. Blood transfusion
is also another efficient
mechanism for sustaining the high GBV-C
prevalence, as no screening
test specific for this virus is routinely
used.
Nucleotide sequence accession numbers.
The GenBank accession
numbers of the sequence data reported in this article are AF124758
through AF124784.
| |
ACKNOWLEDGMENTS |
We thank Edward C. Holmes (Department of Zoology, Oxford
University, Oxford, United Kingdom) for suggestions and comments during
the preparation of the manuscript.
This work was supported by FAPESP, São Paulo, São Paulo
State, Brazil (Proc. 97/04955-0), and by the Laboratório
Bioquímico Jardim Paulista.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Serviço de
Virologia, Instituto Adolfo Lutz, Avenida Doutor Arnaldo 355, 01246-902, São Paulo, São Paulo, Brazil. Phone:
55-11-3061-0111, ext. 2070. Fax: 55-11-853-3505. E-mail:
jrrpinho{at}usp.br.
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Journal of Clinical Microbiology, May 1999, p. 1634-1637, Vol. 37, No. 5
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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