Download Two groups of human herpesvirus 6 identified by sequence

Journal of General Virology (1993), 74, 613 622. Printed in Great Britain
613
Two groups of human herpesvirus 6 identified by sequence analyses of
laboratory strains and variants from Hodgkin's lymphoma and bone
marrow transplant patients
U. A. Gompels,~*t D. R. Carrigan, 2 A. L. Carss ~ and J. Arno 3
University of Cambridge Departments of 1Medicine and 3 Pathology, Addenbrooke's Hospital, Hills Road, Cambridge
CB2 2QQ, U.K. and 2 Department of Pathology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, U.S.A.
Fifteen human herpesvirus 6 (HHV-6) strain variants
were analysed by PCR amplifications, restriction enzyme
site polymorphism and sequence analyses. Three DNA
regions were chosen for study: a fragment of a variable
glycoprotein gene (210 bp), the conserved glycoprotein
H (gH) gene complete with intergenic sequences
(2381 bp) and the 5' intergenic region with the Nterminal coding sequence of gH up to a polymorphic
BamHI site (427 bp). Infected cell DNA from five
laboratory reference strains including GS, Ul102, AJ,
Z29 and KF were examined together with DNA from
peripheral blood lymphocytes infected with HHV-6
reactivated from blood and/or marrow from five bone
marrow transplant (BMT) patients. Separate blood and
marrow isolates were obtained from four BMT patients.
In addition, HHV-6 sequences were examined directly
from one of six Hodgkin's lymphomas and six B cell
proliferations which contained HHV-6 DNA as detected
by PCR amplification. The results show two groups of
very closely related but heterogeneous strains which
correlate with previous groupings by antigenic and
restriction site differences. These are variant A strains
(including laboratory strains GS, Ul102 and AJ) and
variant B strains (including laboratory strains Z29 and
KF, the Hodgkin's lymphoma strain, and the nine BMT
patient isolates). Variations between the groups were 4
to 6 % in nucleotide sequence and 5 to 8.5 % in amino
acid sequence. Within each group maximum heterogeneity was observed in different genes. Variant A
strains differed by 2-0% in the variable glycoprotein
gene sequence whereas variant B strains were identical
in this region; conversely, variant B strains differed by 2
to 3 % in the gH N-terminal and intergenic sequences
whereas variant A strains differed there by less than
0'2%. There was evidence for sequence drift independent of selection: relationships between the groups
were shown by analyses of amino acid sequence, coding
nucleotide sequence as well as intergenic sequence, and
the B variant-specific BamHI site in the gH gene was due
to a non-coding nucleotide substitution. There was little
evidence for in vivo or in vitro variation: the gH nucleotide
sequence from the uncultured lymphoma strain (first
variant B gH gene identified) was almost identical to the
gH sequence from four BMT isolates, and matched
BMT isolates from blood and marrow were identical or
with a single nucleotide substitution. The overall
variation observed between the HHV-6 strain groups
was similar or less than that seen between human
cytomegalovirus strains such as AD169 or Towne, but
deafly distinct from the much greater divergence
between currently designated herpesvirus species.
Introduction
number of cell types of lymphoid and non-lymphoid
lineage (Lusso et al., 1988; Takahashi et al., 1989;
Tedder et al., 1987). A possible site of latent virus DNA
has been identified in monocytes or macrophages (Kondo
et al., 1991) and initial infection may occur through
epithelial cells in salivary glands which can harbour the
virus (Fox et al., 1990; Levy et al., 1990a). Primary
infection is inapparent or causes a mild skin rash in
infants, 'exanthem subitum' (Yamanishi et al., 1988;
Dewhurst et al., 1992); seroconversion (60 % to 90% of
Human herpesvirus 6 (HHV-6) is a recent isolate in the
human herpesvirus family (Salahuddin et al., 1986;
Downing et al., 1987; Tedder et al., 1987). This virus has
a tropism in vitro and in vivo for CD4 + T lymphocytes,
although limited replication in vitro has been shown in a
J- Presentaddress: Viral PathogenesisUnit, Departmentof Clinical
Sciences,LondonSchoolof Hygieneand TropicalMedicine,University
of London,Keppel Street, LondonWC1E 7HT, U.K.
0001-1373 © 1993 SGM
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614
U. A. Gompels and others
the population) occurs usually before 1 year of age
(Briggs et al., 1988; Okuno et al., 1989).
As with other herpesviruses, HHV-6 infection remains
for the lifetime of the host and may be reactivated to
cause secondary infections during immunosuppression.
As HHV-6 has a tropism similar to that of human
immunodeficiency virus (HIV) in infections of CD4 ÷ T
lymphocytes and monocytes/macrophages (Yamanishi
et al., 1988; Okuno et al., 1989; Levy et al., 1990a;
Kondo et al., 1991; Wrzos et al., 1990), it has been
suggested that HHV-6 may reactivate and participate in
AIDS or cause complications in immunodeficient transplant or cancer patients (Lusso et al., 1989, 1991 ; Morris
et al., 1989; Ward et al., 1989; Okuno et al., 1990;
Carrigan et al., 1991). Complications include fatal
fulminant hepatitis (Asano et al., 1990) and HHV-6associated interstitial pneumonitis in bone marrow transplant (BMT) patients (Carrigan et al., 1991). Furthermore, a marrow-suppressive role for HHV-6 has
been proposed (Knox & Carrigan, 1992; Drobyski et al.,
1992). There has been conflicting evidence for both
positive and negative interactions with HIV, although
the basis for this discrepancy may lie in strain differences
(Ensoli et al., 1989; Carrigan et al., 1990; Levy et al.,
1990c; Lusso et al., 1991).
Two groups of HHV-6 strains, A and B or ' U l l 0 2 like' and 'Z29-1ike', have been proposed on the basis of
antigenic differences in reactivity to a subset of HHV-6specific monoclonal antibodies (MAbs) and variations in
restriction enzyme sites (Ablashi et al., 1991; Aubin et
al., 1991; Schirmer et al., 1991; Chandran et al., 1992).
Laboratory strains GS and U1102 are included in group
A, whereas Z29, SF and KF are in group B. Most
exanthem subitum isolates from infants have been group
B, but strains of both groups, A and B, have been isolated
from two patients (Dewhurst et al., 1992). Strains from
either group, including GS, Ul102, Z29 and SF, have
been isolated from adults infected with HIV (Salahuddin
et al., 1986; Downing et al., 1987; Lopez et al., 1988;
Levy et al., 1990b). Human sera are broadly crossreactive and cannot distinguish strain groups in immunofluorescence tests or radioimmunoassays, although the
groups can be differentiated in reactions with certain
MAbs, in particular those recognizing envelope glycoproteins (Ablashi et al., 1991; Chandran et al., 1992).
There appear to be some growth differences between the
groups, although these may be strain-specific. Comparative studies show growth of some isolates in different
CD4 + T leukaemic cell lines: group A isolates in HSB2
cells, and group B isolates in MOLT3 cells (Wyatt et al.,
1990; Ablashi et al., 1991; Schirmer et al., 1991;
Chandran et al., 1992; Dewhurst et al., 1992). It has been
suggested that the HHV-6 strain variant groups be
reclassified (Schirmer et al., 1991), but the molecular
basis of the grouping has not been determined and
therefore it is not clear whether this represents variation
between strains or new species.
We are working on the Ul102 strain of HHV-6
(Downing et al., 1987), for which the only complete
restriction enzyme fragment linkage map and set of
clones is available (Martin et al., 1991). Sequencing
studies of this virus DNA have shown that the
organization of conserved genes and their encoded amino
acid sequences are more related to those of human
cytomegalovirus (HCMV) than those of the other human
herpesviruses (Lawrence et al., 1990; Neipel et aL, 1991).
However, these viruses are distant relations: with the
exception of the sequence around the conserved spliced
gene (66% identity) (Efstathiou et al., 1988), little
nucleotide sequence similarity exists that can be detected
by Southern blot hybridization. A similar relationship is
observed between varicella-zoster virus (VZV) and
herpes simplex virus (HSV) with amino acid identities
between aligned sequences of 15 % to 59 % (McGeoch et
al., 1988; Davison & Taylor, 1987). These four virus
species have different overall structures, base composition and sizes. In contrast, the prototypes of HHV-6
group A (U 1102) and group B (Z29) show in addition to
extensive DNA cross-hybridization an identical genome
structure, base composition and size. These are AT-rich,
162 kbp genomes bounded by direct terminal repeats
(Martin et al., 1991; Lindquester & Pellett, 1991).
In this study we investigated the molecular basis for
differences between HH¥-6 strain groups by PCR and
sequence analysis of 15 variants, including laboratory
reference strains from both groups. We also studied, by
PCR, strains in two potential patient groups: firstly, low
passage isolates from BMT patients and secondly, DNA
directly from Hodgkin's lymphoma, because previous
studies have detected virus sequences in this tissue from
some patients with this disease (Torrelli et al., 1991).
Both variable and conserved genes are examined and the
results show overall identity between strain groups of 94
to 96%, which is similar to or less than variation
between strains of HCMV or Epstein-Barr virus (EBV)
(Chou & Dennison, 1991; Sample et al., 1990).
Methods
Cells. Human T cell lines, JJhan and HSB2, and phytohaemagglutinin
(PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from
an HHV-6 seronegative normal donor were grown in suspension
cultures and used to propagate virus isolates. Cells were grown in
RPMI-1640 medium (Flow Laboratories) supplemented with 10%
fetal calf serum (Myoclone, Gibco-BRL), glutamine and antibiotics.
Viruses. Fourteen HHV-6 isolates were used for this study. HHV-6
(U1102) was cultivated as described (Martin et aL, 1991; Downing et
al., 1987). HHV-6 (AJ) (Tedder et aL, 1987) was a gift from R. Tedder
(Middlesex School of Medicine, London, U.K.). HHV-6 (Z29) (Lopez
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HHV-6
et al., 1988) was a gift from P. Pellett (Centers for Disease Control,
Atlanta, Ga., U.S.A.). HHV-6 (GS) was a gift from R. Gallo (National
Cancer Institute, Bethesda, Md., U.S.A.). HHV-6 (KF) known as DC
in Chandran et al. (1992) was isolated from peripheral blood
lymphocytes of a patient with chronic lymphocytopenia, propagated as
described (Russler et al., 1991). HHV-6 isolates C1, C2, C3, C4, C7, C8
and C9 were isolated from BMT patients with evidence of marrow
suppression as described (Drobyski et al., 1992). Isolates C5 and C6
(BA) were from a BMT patient having engraftment failure with
disseminated HHV-6 infection (Carrigan et al., 1991). C1 and C2 (UPN
410), C3 and C4 (UPN 399), C5 and C6 (BA strain), and C7 and C8
(NB, UPN 356) were matched sets of marrow and blood HHV-6
isolates from patients after BMT (Drobyski et al., 1992). Patients KF,
UPN 410 and BA in addition appeared to have HHV-6-associated
pneumonitis (Russler et al., 1991 ; Carrigan et al., 1991 ; Drobyski et al.,
1992). Isolates C1 to C8 were from patients in the BMT Program,
Medical College of Wisconsin, Milwaukee, Wis., U.S.A. ; BMT patient
JM (isolate C9) was from the Sloan Kettering BMT Program, New
York, U.S.A.
Tissue samples. Biopsy samples from 12 B cell proliferations or
Hodgkin's lymphoma were collected and stored at - 7 0 °C (Histopathology Department, Addenbrooke's Hospital, Cambridge,
U.K.).
DNA extractions. Uninfected JJhan, infected T cell lines or PHA
blasts were treated with proteinase K and SDS, then extracted with
phenol-chloroform as described (Gompels et al., 1992). Tissue samples
were minced, treated with Pronase or proteinase K S D S and extracted
with phenol~hloroform.
PCR amplifications. Two primer sets were used to amplify sequences
from the genes for conserved glycoprotein gH or a non-conserved
glycoprotein (BHLF2) (Gompels et al., 1992): gH, 5' ATA AGA TCT
GTT TAT GGA TCC TCA 3", 5' CGG CGT TTA GCT GGA TCC
GGA CAA 3'; BHLF2, 5' GAT GGA TCC TCC AAA GGA AGT
GGT AAC 3', 5' GAA GGA TCC TTG CGG ATG GCA ATG AGC 3'.
These are at positions 6386 and 4006 (gH) and 3319 and 3110
(BHLF2) of the BamHI H sequence (Gompels et al., 1992). The primers
were modified at the underlined nucleotides to create BamHI sites.
Amplification reactions with these primers used Taq polymerase
(Promega) and deoxynucleoside triphosphates (Boehringer Mannheim)
with standard buffer conditions and 1-5 to 20 mM-MgCI~.PCR thermal
cycling reaction conditions were 93 °C for 4 min, then 30 cycles at
93 °C for 30 s, 43 °C for 30 s, 72 °C for 120 s. The gH primers amplified
a 2381 bp product; the BHLF2 primers, a 210 bp product. Each PCR
reaction included both positive (HHv-6 strain AJ-infected JJhan cell
DNA) and negative (uninfected JJhan cells) controls. No evidence of
HHV-6 (AJ) DNA PCR contamination was detected; all negative
controls were negative and PCR-positive for cellular genes only (not
shown).
Sequence analysis. Amplified DNA fragments were digested with
BamHI and separated by electrophoresis in an agarose gel. The
relevant DNA fragments were purified, then ligated with BamHI-
digested, phosphatase-treated M 13rap 18 vector DNA (Messing, 1983).
The sequence was determined from single-stranded M 13 clones by the
dideoxynucleotide chain termination method (Sanger et al., 1977) using
[3~S]dATP (Amersham) as radioactive label (Biggin et al., 1983). For
gH PCR products from the C1 and C4 isolates, the sequence was
determined from both strands. The sequence from all BHLF2 PCR
products was determined from both strands. For the 210 bp BHLF2
and 382 bp N-terminal gH fragments, sequences were derived from an
M13 universal primer (Promega). For the complete sequence of gH,
internal 17-mer primers were used from the following positions in the
amplified sequence: 194, 390, 573, 751, 928, 1107, 1296, 1516, 1735,
1938 and 2161 (Fig. 1).
strain g r o u p s
615
Results and Discussion
T o e x a m i n e H H V - 6 strain v a r i a t i o n , three regions o f
D N A were c h o s e n for P C R a n d s u b s e q u e n t sequence
analyses. T h e first, a c o n s e r v e d gene (gH), was c h o s e n
because it is one o f the least c o n s e r v e d o f a subset o f
c o n s e r v e d genes in herpesviruses ( D a v i s o n & T a y l o r ,
1987; Chee et al., 1990). A s such it w o u l d be suitable
b o t h to identify v a r i a t i o n a n d to c o m p a r e the degree o f
v a r i a t i o n f o u n d to t h a t o c c u r r i n g b e t w e e n o t h e r herpesviruses a n d their strains. In a d d i t i o n , this is a n i m p o r t a n t
c o n s e r v e d gene b e c a u s e the e n c o d e d g l y c o p r o t e i n has
roles in the infectivity a n d i m m u n o g e n i c i t y o f the viruses
( G o m p e l s et al., 1988, 1992). F u r t h e r m o r e , v a r i a t i o n in
g H correlates with v a r i a t i o n d i s t r i b u t e d t h r o u g h o u t the
genome. I n t e r g e n i c regions were also amplified with
c o d i n g sequences to c o m p a r e the overall drift to possible
c o d i n g sequence selection. T h e second region focused o n
was the v a r i a b l e N - t e r m i n a l intergenic a n d c o d i n g
sequence o f g H e x t e n d i n g to a B group-specific B a m H I
site. T h e third r e g i o n o f D N A was f r o m a n o n - c o n s e r v e d
g l y c o p r o t e i n gene ( B H L F 2 ) . This sequence is n o t
c o n s e r v e d in H C M V , the closest r e l a t i o n to H H V - 6 ;
a l t h o u g h H C M V has a ' p o s i t i o n a l h o m o l o g u e ' which
also e n c o d e s a g l y c o p r o t e i n , the sequences have diverged
b e y o n d d e t e c t a b l e similarity. B o t h g H a n d the v a r i a b l e
g l y c o p r o t e i n are e n c o d e d by the H H V - 6 strain U1102
BamHI
H sequence a n d c o m p a r i s o n s to n u c l e o t i d e
sequences f r o m strain G S also show this n o n - c o n s e r v e d
g l y c o p r o t e i n gene to be h y p e r v a r i a b l e b e t w e e n strains.
T h e r e is 5-0% difference in a n overall c o n s e r v e d
sequence, which has less t h a n 0"5 % v a r i a t i o n ( G o m p e l s
et al., 1992).
Reference l a b o r a t o r y strains were collected f r o m b o t h
the A a n d B g r o u p s which h a d been identified b y
antigenic differences a n d restriction e n z y m e site p o l y m o r p h i s m s ( S c h i r m e r et al., 1991 ; A b l a s h i et al., 1991).
These are U l l 0 2 - 1 i k e strains, o r g r o u p A , including
U l 1 0 2 , G S a n d AJ, a n d Z29-1ike strains, or g r o u p B,
including Z29 a n d K F . T o a n a l y s e f u r t h e r strain variants,
two sources o f D N A were u s e d : one f r o m a H o d g k i n ' s
l y m p h o m a a n d the s e c o n d f r o m infected P B M C s (less
t h a n two passages) f r o m r e a c t i v a t e d H H V - 6 in B M T
p a t i e n t s ' b l o o d a n d m a r r o w after t r a n s p l a n t a t i o n . T h e
use o f these sources a v o i d s c o m p l i c a t i o n s o f P C R
c o n t a m i n a t i o n because relatively large a m o u n t s o f virus
D N A were s h o w n to be p r e s e n t in selected H o d g k i n ' s
l y m p h o m a s (three o f 25 were H H V - 6 - p o s i t i v e ) (Torrelli
et al., 1991) a n d H H V - 6 v i r a e m i a in B M T p a t i e n t s
( C a r r i g a n et al., 1991). I n a d d i t i o n , analysis o f the
H o d g k i n ' s l y m p h o m a s a m p l e allows identification o f
H H V - 6 v a r i a t i o n w i t h o u t a n y influence o f p a s s a g e in
c u l t u r e d cells. M o r e o v e r , analyses o f the B M T specimens
f r o m m a t c h e d b l o o d a n d m a r r o w isolates allow the
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U. A. Gompels and others
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F D A
I N
T T B S L
TTAC T C G G C T C C G A T A T C A C A T A C C A C C T G T T T G A T G C C A T C A A C A C G A C A G A A T C G T T A
. . . . . . . . A . . . . . . . . . . . G ..... T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
480
L9
L
H
I
V
Y
S
L N
M Y
P S Q G
I Y Y V
R V V
E V
G T G T A T T C C T I ~ A A C A T G T A C C CI'I~ACAGC-GAATTTATTACGTCAGGGTCGTAGAAGTT
660
--C . . . . . . . . . . . . C . . . . . . . . . . . . . C . . . . . . . . . . . . A . . . . . . . T - - G ..... C L9
R
Q M
Q Y
D • V
~ C K
L P N
S
L K
E L I
CGACAGATGCAATACGACAACGTTI~CTGTAAGCTGCCTAAT~CTCTCAAGGAACTAATA
720
........................ C ................................... AJi
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C L9
A
•
P
V
Q
V
R
C
A
K
I
T
R
•
V
E
D
I
•
T
T ~'~CCAGTC CAAGTCAGATCCC-CTAAAATTACC/2GCTAT~TGGGCGAAC~%CATCT~TACC
. . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . A ....... C ....... C . . . . . . . . . . .
780
G
M
Y
@
N
T
T
I
N
•
K
&
I' Y
K
K
S
F
K
~
T
L
T
D
D
L
L
L
I
V
E
K
D V
M
I
900
L9
R
D
ATATTCAAACAGACATTGACAC, ACGATTTAC TATTGATAGTCGAAAAAGACGTAATAGAT
960
. . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . G C G - L9
D
V
Q Y
R
F
I
S D A
T • V
D E T L N
D V D
G T A C A A T A C CG"FFIV.ATA T C A G A T G C G A C A T T C G T A G A C C ~ % A A C G T T G A A T G A C G T A G A T
1020
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . @ . . . . . . . . . . . L9
E
V
E
ALL
L
K
•
N
N
L
G
I
Q
T
S
•
V
I
*. L
K
@
V
8
L
T
I
T
T
T
I
V
K
Y
A
G
Q D
L L V
L R M
I I S Q
T ¢ E F
A A A T A T G C A G G A C A A G A T C I ~ T I D 9 3 C TACCgtAACATC TCATCTCAAACATGCU.AGTTC
2040
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . T L9
Q
B
V
V
M
B
•
D
D
I
D
@
P
L
Q
Y
I
•
I
TGTCAC4%GCG TAGTCATGGAATATC.A ~ T A T C G A O G G T
C CC T T A C A A T A C A T I T A C A T A
2100
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C ...... L9
K N
I D E
L
K
T
L
T D P N
N N
5 L V P N
AAAAACATAGACGAACTAAAAACATTGACAGATCCCAACAACAATTTACTTGT~CC CAAC
2160
. . . . . . . . . . . . . . . . . . . . . . . . C .... C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L9
A
K
N
Q
S
V
F l
M
S
E
V
CAAAAACGGCTCT~'FI"FI'IC~A~TGTCTC~AAGTC
2220
V
G
I D
I D
Q
v
s
I I L V
I I T I L I A
GGAATCGATATAGACCAAGTGTCTATCATATTGGTTATCATTTATATTCICJ~TCGCAATA
.................... A ........................ G ..............
2280
L9
S
A T G A T G T A C G C ~ A A CACCACCAGCATAAACI'ITAAAC-CCCCITATAAGAAAAGT~CATTC
. . . . . . . . T ..... T ..... -G . . . . . . . . . . . . . . . . . . . . . . . -G . . . . . . . . . . . . .
X
P
T R T K Y L
L
L
ACCAGGACC, C A C T A T C ~ A G C
R
S
S
L9
H
E
H
F
F
T
P D • M
I L
Y
I Q N
P A
G D
L T
C A T S • C'I'VgACT C C G G A C T'fTA T G A T A C T G T A C A T C C A G A A T C C C G O G G G A G A T C T G A C T
840
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C ........ A - A . . . . . . . . . . 59
M
I
Y
A
T
l
I
I Z T A
I P
L
N
~ T
C V
S T N
Y
G C T A C G A G T A T A A T A A T C A C A G C C A T A C CTC T C A A T T C C A ~ C C A A C T A T
1980
........................................... A ................ AJi,AJii
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . A - - T - - - L9
C
H
G
V
R
I
A
L
F
G
L
T
R
L
I
R
L
e
*
ATTIC TTTAT~X~.ATTATATACe~CTTATCAGA~TAAACGTTTTATTTATG
2340
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C AJi, A J i i
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A L9
~ A G G T A T T A G A T C ~ A A I"F~GTCCGTATACAGCTAAACGC CG
2381
- - T- - C . . . . . . . A G A . . . . . . . .
L9
@
GAAGTAGAAGCTCTACTACTCAAATTTAATAACCTAGGAATCCAAACCCTATTAAGAGGA
1080
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C---- L9
D C K
K
P N
Y A
G
I P Q N M
P L ~ G
I V
GACTGTAAAAAACC CAACTATGCCGGCATAC CGCAGATGATGTTTCTTTACGGTATCGTA
1140
H
• ~ Y
S T
K N
T
@
P M
P ~ L R V
L K T
C A T T T C T C A T A T A G C A C A A A A A A C A C A G G A C C A A T G C C C G T G T TAAGAG-I~TFAAAGACA
1200
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . L9
F
H
E
N
L L
S
X D
S • V N
R
~ V N
V
S
E G
CACGAAAATCTCCTGTCCATCGACTCATT~GTCAACCGATGTGTGAACGTATCGGAAGGT
............ T-T ................................... C .........
1260
L9
I
T
L
Q
•
P
K
M
K
E
F
L
K
Y
E
P
S
D
Y
S
Y
ACGTTACAATACCCAAAAATC4%AGGAATITI~AAAATACGAGCCCTCGGACTATAGCTAC
---A ........................................................
1320
L9
I
T K
N
K
S
! S V
S T
L L T • L A
T A
Y
ATAAC CAAAAACAAATCeATTTCCGTATCTACGCTG~TCACGTACT~AGO~ACAGCGTAC
1380
Fig. 1. Complete HHV-6 gH coding sequence and intergenic sequences
of strain U1102 compared to laboratory strains GS and A J, and to the
variant sequence from Hodgkin's lymphoma, sample 9 (L9). The
U1102 sequence is from Gompels et al. (1992) and a PCR-amplified
product. The GS sequence is from Josephs et al. (1991). AJi and AJii
are the sequences of strain AJ from two separate PCR amplification
products. Amino acid sequences in bold represent conserved residues in
HCMV. The HHV-6 strain sequences are 5.0 % variant, but are 75 %
divergent from the HCMV amino acid sequence. Primers used are
overlined; N-linked glycosylation sites and hydrophobic N-terminal
and C-terminal sequences are underlined; conserved glycosylation sites
are also overlined; coding changes are shown by a star.
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H H V - 6 strain groups
GCAAAC
~ C ~ , = A C
L
G
T
L
ATGTI~.TFfCAC
8
~ G A C A C G G A C ,
AA T I ' I ~
C
120
N
............................................................
...........................................................
C-~
.%.7
.........................
-G .....
C-- . . . . . . .
C-G ......
T .........
KF
.........................
-G .....
G ........
C-G ......
T .........
C1
.........................
-G .....
G- .......
C-G ......
T .........
C2
..........................
G .... AG-
.........
G ................
L9
............
A .............
G--
-AG-
.........
G ................
C3
............
A .............
G .... AG-
.........
G ................
C4
............
A .............
G .... AG ..........
G ................
C6
............
A .............
G .... AG ..........
G ................
C9
AATIL~kI~GTI~
CA~FfACACA
TCA'F F1"~'1GTAATC TATTTAA
T A ~ T C A A A A A G
............................................................
180
..........................................................
AJ
............................................................
KF
...............
A ...........................................
CI
...............
A ............................................
C2
............................................................
L9
...............
A ............................................
C3
...............
A ............................................
C4
...............
A ............................................
...............
A ............................................
C6
C9
M
~ C
~ ' q * i ~ . ~ G , A A T ~ . ~ C A G ~ C C G R ~ f C A CAC T I X 2 A T C ' G A ~ A A C ~ C A A C q ? A ' I X 3 240
.......
.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C~
...........................................................
...........................
~
...........................
TC ...............................
C1
..........................
TC
C2
...........................
..............................
I~
..............................
59
C ...............................
............................
C
..............................
C3
............................
C ...............................
C6
............................
C ...............................
C9
I
F
L
L
S
R
L
CTCC~L'CGACTC
M
V
F
S
V
L
T.
T
~
C
Y
0
M
R
P
W
T
T.
N
~ Q G G I ~ C T G T T G A C T C C C ~ T E A C C ~ G A C C G T T C ~ C
300
............................................................
GS
............................................................
AJ
---T
...................
T ...............................
---T
...................
---T
...............
---T
.................
T ..................
---T
..................
---T
..................
---T
---T
KF
G--T-
C1
...............................
G--T-
C2
A ............
G--C-
L9
T ..................
A ............
G--C-
C3
T ..................
A ............
G--C-
C4
...................
T ..................
A ............
G--C-
C6
...................
T ..................
A ............
G--C-
C9
E
I
G--T-
T ...............................
C---T
8
N
K
8
B
H
C
R
S
N
G
N
7
E
N
P
I
V
R
ATATCGAACTCGAGCCATTGTAGAAAT~AA~TCCAA~
...........................................................
............................................................
P
g
CCGGC 3 6 0
GS
AJ
.........
C~ ...........
A ...........
C ............
T ........
G---
.........
GA ...........
A ...........
C ............
T ........
G---
C1
.........
GA ...........
A ...........
C ............
T ........
G---
C2
.........
GA ...........
A ...........
C ............
T ........
G---
L9
.........
GA ...........
A ...........
C ............
T ........
G---
C3
........
...........
A ...........
C ............
T ........
G---
C4
.........
GA ...........
A ...........
C ............
T ........
G---
C6
.........
GA ...........
A ...........
C ............
T ........
G---
C9
F
I
~
KF
T
F
N
TI'T~ff~%ACATITAAC'
F
•
T
X
J.'lT I ~ , T A C A ~ C G A C A C
N
D
Y
R
I
~ ' / ~
Y
Q
TATCAAGTC
V
P
K
~ T G C
C
420
............................................................
GS
...........................................................
AJ
--C .....
T ..................................................
KF
--C ....
T ...................................................
C1
--C .....
T
..................................................
C2
........
T ...................................................
L9
........
T ...................................................
C3
........
T .................................................
C4
........
T ...................................................
C6
........
T ...................................................
C9
L
L
G
S
TII%CTCGC-CI~C
430
............
GS
............
AJ
........
A---
KF
........
A---
C1
........
A---
........
A---
L9
........
A---
C3
C2
........
A---
C4
........
A---
C6
--C-- ....
A---
C9
Fig. 2. N-terminal gH coding and intergenicsequences of strain U1102
compared to laboratory strains (GS, AJ and KF) and variants from a
Hodgkin's lymphoma (L9) and BMT patients (C1 to C9). C1 with C2
and C3 with C4 are matched marrow and blood isolates from BMT
patients UPN 410 (C1 and C2) and UPN 399 (C3 and C4). The
sequence is shown extending to the polymorphicBamHI site, which is
underlined; coding changes are shown by a star.
617
identification of variation in vivo in viruses isolated from
separate sites.
H H V - 6 sequences in Hodgkin's lymphoma
In an ongoing study to be described elsewhere, Hodgkin's
lymphomas and other lymphoproliferations were analysed for the presence of herpesvirus sequences (U.
Gompels, S. Efstathiou, J. Arno & E. English, unpublished results). One of the 12 samples (sample L9, a mixed
cellularity Hodgkin's tymphoma) analysed by PCR for
the gH gene was positive for HHV-6-specific sequences.
Sample L9 contained a restriction site polymorphism in
the gH gene such that B a m H I digestion of the PCR
product yielded two products of 427 bp (N-terminal and
intergenic region) and 1954 bp (C-terminal and intergenic region). Both these fragments were cloned and
sequenced. The sequence was compared to the sequence
derived from multiple PCR/sequencing reactions for the
same region in group A strains U1102, GS and AJ. The
error rate in this procedure was only a single point
mutation (AJi, Fig. 1) in 2381 bp (0.04%). Overall
variation between laboratory strains was less than 0-2 %
whereas variation from the L9 sequence was 4 % for the
nucleotide sequence and 5 % for the amino acid sequence
(Fig. 1). The same level of variation was observed in the
intergenic sequences, indicating an overall sequence drift.
The L9 gH sequence is the first complete herpesvirus
gene sequence determined directly from a sample
obtained in vivo, without any virus tissue culture passage.
As such, it was important to determine whether the
variation observed was due to the effects from culturing
the laboratory strains, the lymphoma or whether the L9
sequence represented another HHV-6 strain variant
group. To examine this question, a sequence representing
the more variable N-terminal region of gH was analysed
in other HHV-6 isolates.
H H V - 6 sequences in B M T patients
Preliminary analyses of isolates C1 to C9 from BMT
patients show antigenic and restriction site differences to
HHV-6 group A laboratory strains U l 1 0 2 or GS
(Carrigan et al., 1991; Drobyski et al., 1992). These
isolates had characteristics more similar to those of
group B laboratory strains Z29 or KF. In PCR reactions
with the gH primers, a 2"3 kb fragment could be amplified
from all infected cell D N A preparations, including those
from infections with group B laboratory strains Z29 or
KF. U p o n digestion with B a m H I all these isolates and
group B laboratory strains yielded two fragments of
1.95 kb and 0"4 kb, like the L9 sequence but unlike group
A laboratory strains GS, U 1102 or AJ (not shown). The
0.4 kb fragments were cloned and sequenced for strain
KF and isolates C1, C2, C3, C4, C6 and C9. These
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618
U. A. Gompels and others
g
E
S
L
X
It
&
II
8
K
F
S
8
II
W
L
T
R
8
L
GAAC~Aq~2CI'IV~GGATGGCAATGAGCAAAq~TCCAACTCGAATCTGACACGG~
60
............................................................
GS
............................................................
AJ
............................................................
C9
..........................................................
............................................................
T
a
p
T
P
E
I
8
K
II
Ie
r
II
Y
T
ACP'i'~"T'I~CG ~ ~ A A T ' P A C ~ . C
$
P
V
Z29
r
•
L
~.GCT ' P I ~ ' ~ A ~
L
•
TCI'AT 120
..... G .........
G ............................................
GS
..... G .........
G---T
AJ
........................................
..... G ..... TC-TG
............................................
CI9
..... G ..... TC-TG
............................................
KF
..... G ..... TC-TG
............................................
Z29
II
T
T
$
C
I
R
V
P
$
II
D
Q
¥
F
E
H
K
Q
$
AACACAACATCATGCGTCCCITCAAATGATCAATAq~FI~TAAACAGTCGC
P
K
CAA~AC
P
I
CTATA
180
......................................
T .....................
GS
......................................
T ....................
A]
...............
A---G
..................
-G .... T ...............
C9
...............
A---G
..................
-G .... T ...............
KF
...............
A---G
...................
G .... T ...............
7-*29
I
N
V
T
T
8
AATGq~ACC_ACTTC
.......
F
~
O
R
G
&
i
CCATC
T .....................
210
GS
..............................
AJ
..............................
C9
..............................
KF
..............................
Z29
Fig. 3. Portion of the variable glycoprotein gene coding sequence
(BHLF2) from laboratory strains (U 1102, GS, AJ, Z29 and KF) and a
BMT patient isolate (C9). The U1102 sequence is from Gompels et al.
(1992) and PCR amplification products. The GS sequence is from
Josephs et al. (1991) and PCR amplificationproducts. The primers used
are underlined and coding changes are shown by a star.
U1102
I
I
AJ
GS
Z29 KF
0.96 t
96%
0.98
98%
1-00
100%
C9
Fig. 4. Dendrogram showing relationships between nucleotide
sequences from the variable HHV-6 (BHLF2) glycoprotein gene in
HHV-6 strains. The dendrograms in Fig. 4, 5 and 6 are produced by
the PILEUP program of the GCG package (Version 7.1-UNIX, June,
1992; Devereux et al., 1984). This is based on the multiple alignment
program of Feng & Doolittle (1987). The similarity scores for amino
acid sequences are indicated on the vertical axis and are based on a
weight matrix from amino acid substitutions in related protein families.
Overall identities are also shown at branch points. Nucleic acid
sequence identities are shown (1.0 = 100%). The vertical branch
lengths are proportional to the similarity between nucleotide or amino
acid sequences. These are unrooted trees.
results, in comparison to the results for strains Ul102,
AJ and GS and the L9 sequence, are shown in Fig. 2.
The results show clearly that the L9 sequence
resembles sequences from group B strain variants and is
distinct from group A, U 1102-like viruses. Therefore, the
L9 gH sequence is the first group B gH gene identified.
However the group B strains appear heterogeneous.
Although all have a 5 to 6 % difference as compared to
the group A, Ull02-1ike, nucleotide sequences, the
group B strains segregate further into KF-, C1- and C2related strains and L9-, C3-, C4-, C6- and C9-related
sequences which are 2 to 3 % different from each other.
In the isolates examined here, this variation is not
produced in vivo, because matched m a r r o w and blood
isolates C 1 and C2 are identical except for one nucleotide
substitution and these differ f r o m another matched pair
(C3 and C4) which have identical sequences. Thus, in
these two examples virus reactivating in marrow appears
to be the same (as far as this sequence can indicate) as
virus circulating in the blood after the transplant. Here
we show that the sequence from a region that varies
between strains shows little or no variation between these
matched isolates.
Although the N-terminal coding and intergenic regions
of H H V - 6 gH show strain variation in one group of
H H V - 6 sequences, the other group, the Ull02-1ike
sequences U1102, GS and AJ, cannot be distinguished.
As the non-conserved glycoprotein gene B H L F 2 was
hypervariable between strains GS and Ul102, with an
overall difference of 5"0% (Gompels et al., 1992), a
portion of this gene was used in P C R amplification and
sequence analysis to test strain variation. Across a
210 bp sequence group B laboratory strains Z29 and K F
and B M T patient isolate C9 were identical, but distinct
from group A strains U1102, AJ and GS. In addition,
each of the group A strains could be differentiated by
several nucleotide substitutions. These substitutions were
found in multiple P C R reactions and showed strains GS
and AJ both differed from U1102 (Fig. 3). Overall the
U1102-like strains were 2 % variant but differed by 4 %
from the Z29-1ike strains for the nucleotide sequence and
8'5 % for the amino acid sequence. Therefore the 210 bp
sequence differentiated between group A strains and the
gH and intergenic sequences differentiated between
group B strains (Fig. 1, 2 and 3).
Relationships between H H V - 6 strain variants and with
other herpesviruses
The relationships between the two groups of H H V - 6
sequences can be summarized in dendrograms (Fig. 4
and 5). Clearly there are at least two groups of strain
variants, group A or U1102-like and group B or Z29- or
KF-like, in agreement with earlier findings on re-
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H H V - 6 strain groups
0-95o 95%
FL_,
0-98 -
98%
0.99 -
99%
1.0- 100%
C2 C1 KF L9 C9 C6 C4 C3 AJ GS U1102
Fig. 5. Dendrogram showing relationships between nucleotide
sequences from the HHV-6 gH N-terminal and intergenicsequences.
Details as for Fig. 4. The same relationships were shown using
intergenic, coding or amino acid sequences. The relationships shown
here are for overallnucleotidesequence.
striction enzyme polymorphisms and antigenic differences (Schirmer et al., 199t ; Ablashi et al., 1991). These
clusters contain very closely related strains showing
nucleotide differences of only 4 to 6 %. Each group
shows at least 2 to 3 % heterogeneity but at different
genes. This is similar to or less than the degree of
variation between variant and conserved genes compared
for strains of HCMV or EBV (Chou & Dennison, 1991;
Sample et al., 1990). EBV sequences with differences of
4 % in the intergenic regions of groups of strains are
referred to as 'homologous', in comparison to the more
variable EBNA 2, 3A, 3B, 3C and EBER genes.
Conserved EBV genes have not been analysed to
determine whether this intergenic sequence variation of
4 % occurs throughout the genome. There are two
groups of EBV strain variants called types 1 and 2, more
recently designated variants A and B (Sample et al.,
1990; Roizman et al., 1992). In contrast, HCMV strains
have been identified by analysis of conserved genes gB
and gH. Variation in gH distinguishes two groups, strain
AD169-1ike and strain Towne-like, which differ by 5.0 %
(Pachl et al., 1989; Chou, 1992), and more heterogeneity
between these groups has been shown for the gB sequence
(Chou & Dennison, 1991).~
For EBV strain groups (variants A or B) or HCMV
strains, it is not clear whether there is consistent grouping
by restriction fragment polymorphisms across the entire
genome as has been shown for some HHV-6 isolates. If
this is not the case, EBV and HCMV strains may vary
more overall than HHV-6 strains. Another possibility,
although not mutually exclusive, is that EBV and HCMV
strains appear more variable in restriction enzyme
analyses owing to genome structure differences such as
internal repetitive sequences. Mutation rates are higher
at these sites, presumably mediated by homologous
recombination, and an example has been shown for the
HHV-6 terminal repeats (Lindquester & Pellett, 1991).
However, unlike EBV and HCMV HHV-6 does not
contain extensive internal repeats (Martin et al., 1991).
619
PCR amplification products from conserved genes
without repeats in HCMV strains or selected regions in
EBV strains can be grouped by restriction enzyme
polymorphisms (Chou & Dennison, 1991; Sample et al.,
1990). Studies of virus isolated by cell culture may also
distort views of overall variation. For example, the
growth properties of the EBV strain groups differ in B
lymphocytes and may favour group A isolations.
It has been proposed that in HCMV, gB and gH strain
variation may be due to immune selection (Chou &
Dennison, 1991; Chou, 1992), but there is only limited
evidence to support this. A variant epitope in HCMV gH
has been identified at the N terminus which distinguishes
sera reacting with the AD169-1ike or Towne-like strain
variants. This site elicits MAbs and polyclonal antibodies
that can neutralize virus infectivity (Urban et al., 1992).
In contrast, neutralization epitopes in gB are conserved
among strain variants (Meyer et al., 1990; Lehner et al.,
1991). In HSV gH there is a single example: of 66 HSV1 clinical isolates only one was resistant to neutralization
by a MAb, and had the same nucleotide and resulting
amino acid substitution (although overall 0-4 % variance
existed between four laboratory strains) as an antibodyselected variant isolated in the laboratory (Gompels et
al., 1991). Thus, it is possible that immune selection may
operate on HHV-6 gH, giving rise to variants. But if it
does so, it is relatively infrequent and variation is at a low
level. For example, the variation observed is far less than
has been recorded for HIV gpl20 (to 39 %), a proposed
target for immune selection (Myers, 1990). Furthermore,
HHV-6 undergoes sequence drift clearly independent of
immune selection. The dendrogram in Fig. 5 shows
relationships determined from nucleotide sequences, but
the same result is obtained for the intergenic sequence as
well as the encoded amino acid sequence. In addition,
the variant B a m H I site, a genetic polymorphism which
appears to be maintained in isolates of the Z29-1ike
group, is a non-coding change. These aspects of
nucleotide sequence drift have not been examined for
HCMV. Moreover, further study is required to determine
whether the 2 to 3 % heterogeneity observed within
HHV-6 strain groups is also distributed throughout the
genome.
How do these HHV-6 variant groups relate to the
other herpesvirus species? In Fig. 6, relationships
between the amino acid sequences of the gH protein
family are shown for different herpesvirus species,
prototypes for the alpha-, beta- and gammaherpesvirus
subfamilies. Nucleotide sequences could not be compared because they are not conserved between herpesvirus species. The 5 % difference between HHV-6 strain
groups represented by strains U1102 (HHV-6A) and L9
(HHV-6B) is identical to the variation observed between
HCMV (HHV-5) strains AD169 and Towne (Pachl et al.,
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620
U. A. Gompels and others
i
I
0.3 B 15%
0.5 - I - 20%
i
1.0 - I -
I
,
Gamma
I
I
,
~,.
Beta
1-45 -4- 95%
1.5 B
I
t,q
Alpha
Fig. 6. Dendrogram showing relationships between herpesvirus species
and strains in the gH protein family. Details as for Fig. 4. The
relationships between gH amino acid sequences were examined for
prototypes of herpesvirus species. Alphaherpesviruses are represented
by HSV-1 (or HHV-1; alpha-l, GC-rich base composition) and VZV
(or HHV-3; alpha-2, AT-rich); betaherpesviruses by HCMV (or HHV5; beta- 1, GC-rich) and HHV-6 (beta-2, AT-rich); gammaherpesviruses
by EBV (or HHV-4; gamma-1, GC-rich) and herpesvirus saimiri (HVS
or SHV-2; gamma-2, AT-rich). Strains of HCMV AD169 (HCMV-A)
and Towne (HCMV-T) are shown with strains of HHV-6 variant A
(U1102) and variant B (L9).
1989) but clearly different from the diversity of 75 to
85 % between other herpesvirus species prototypes (Fig.
1 and 6). Among the most closely related distinct
herpesvirus species are the alphaherpesviruses equine
herpesvirus 1 and 4 (EHV-1 and EHV-4) and HHV-1
and -2 (or HSV-1 and HSV-2) (Roizman et al., 1992).
HHV-1 and HHV-2 are 16 to 33 % variant in sequences
examined across their genomes (McGeoch et al., 1987).
The HHV-2 gH sequence has not been determined, but
the gH sequences from EHV-1 and EHV-4 are available
for comparison; the amino acid sequences are 15%
variant and of different length, 848 and 855 residues,
respectively (Nicolson et al., 1990). Again this is distinct
from variation between strains of HCMV and HHV-6.
Thus, in conclusion, on the basis of sequence analysis of
selected genes and intergenic regions, HHV-6 strains
form at least two groups which appear to be strain
variants.
A consensus has been agreed (XVII International
Herpesvirus Workshop, Edinburgh, August 1992) that
the HHV-6 strain variant groups be termed 'variant A'
for Ull02-1ike viruses and 'variant B' for Z29-1ike
viruses, similar to a precedent for naming EBV strain
variant groups (Sample et al., 1990; Roizman et al.,
1992). The International Committee on Taxonomy of
Viruses (ICTV) herpesvirus study group recommends
designation of separate species if the viruses differ across
their entire genome and have distinct biological and
epidemiological characteristics (Roizman et al., 1992).
The naming involves the host and serial arabic numbers;
for example if HHV-6 strain groups should be regarded
as separate species the next available nomenclature is
human herpesvirus 8 or HHV-8 (Roizman et al., 1992).
However, as summarized in the previous sections, no
difference in epidemiology has been identified between
the HHV-6 strain groups. Furthermore, the level of
nucleotide sequence variation found here between the
groups was similar to that found between current strains
of other herpesvirus species; the groups are more closely
related to each other than any other pairs of designated
herpesvirus species. Within the ICTV guidelines for
herpesviruses (Roizman et al., 1992) there exists a
recommendation for naming groups of strains of HHV4 (EBV). These are designated variants A and B
(previously termed types 1 and 2; Sample et al., 1990).
The term' groups' cannot be used as this denotes genome
structures with respect to their repetitive and unique
sequence organization (groups A to F; Roizman et al.,
1992). Therefore the decision has been made to designate
HHV-6 strain groups variants A and B, and the data
presented here would support this nomenclature.
After the completion of these studies, sequence
analyses of PCR products were presented for 10 isolates
(including laboratory strains GS, U1102 and Z29) of two
other regions of the genome, the tegument gene and
major capsid gene (Aubin et al., 1993). The variation
observed between strain groups is 5-0% and in
agreement with the studies we have presented here.
Preliminary PCR and sequence analyses of the gH gene
and intergenic region of the isolates of Aubin et al. (1993)
agree with their grouping by studies of the tegument and
major capsid genes. We found similar variation between
HHV-6 variant groups (5.0 %) and heterogeneity (2"0 %)
in group B variants in the N-terminal gH sequence.
Furthermore, Chou & Marousek (1992) have recently
shown that sequences for the HHV-6 glycoprotein B
homologues have little variation, of only 4.0 %, between
strains GS (variant A) and Z29 (variant B) as compared
to the greater variation, 11-0%, observed between gB
sequences of HCMV (Chou & Dennison, 1991). In studies
of restriction enzyme polymorphisms of geographically
distinct HSV-1 strain groups, 3'0 % sequence variation
has been calculated (Sakaoka et al., 1987). The geographical distributions of HCMV and HHV-6 strain
variants have not been determined. Continued sequencing studies on representative laboratory strains and
analysis of further isolates or direct examination by PCR
of HHV-6 sequences from tissue samples may determine
the distribution and possible overlap or additional
distinctions between HHV-6 strains. Further study is
required to determine how these distinctions relate to
biological properties or epidemiology.
We thank Christine Lelliott for expert technical assistance with cell
culture and growth of HHV-6, Drs Henri Agut and Helene Collandre
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On: Tue, 01 Aug 2017 19:52:18
H H V - 6 strain groups
for providing DNA samples of the HHV-6A (SIE) and HHV-6B
(BOU, BLE, MBE, MAR) isolates described by Aubin et al. (1991,
1993) and Suzanne Distort for professional typing of this manuscript.
This work was supported by the Wellcome Trust, U.K., U.A.G. is a
Wellcome Trust Senior Research Fellow in Basic Biomedical Sciences.
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(Received 22 September 1992; Accepted 1 December 1992)
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