Competitive ELISA for detection of HHV-6 antibody: seroprevalence in a Danish population

Journal of Virological Methods 56 (1996) 221-230

Journal of Virological Methods

Competitive ELISA for detection of HHV-6 antibody: seroprevalence in a Danish population

Lene Nielsen*, Bent Faber Vestergaard

Department of Virology, Statens Seruminstitut, Artillerivej 5, DK 2300 Copenhagen S Denmark

Accepted 7 September 1995

Abstract

A competitive HHV-6 enzyme-linked immunosorbent assay (ELISA) was established, utilizing HHV-6 rabbit polyclonal antibodies and HHV-6 cellular antigen. In total, 285 sera from Danish children aged l-6 years were screened for HHV-6 antibodies. The lowest seropositive rate, 78% was found in the youngest age group from 1 year to 15 months. All 120 (100%) children aged 3 years and above were seropositive. The ELISA was compared with indirect immunofluorescence assay (IFA) using a group of 83 serum samples from 65 Danish patients (aged 4 weeks to 72 years). All but one of the sera (100% of patients) were positive in the ELISA, but only 71% of sera (65% of patients) were positive with both assays. The ELISA detected one seroconversion in a baby, which was not detected by IFA. The ELISA was found to be more sensitive than IFA.

Keywords: HHV-6; ELISA; IFA; Seroprevalence in Denmark

1. Introduction

Human herpes virus 6 (HHV-6) was first isolated by Salahuddin et al. (1986) and was initially named HBLV. The virus has the characteristic morphology of a herpesvirus and was shown to be immunologically and genetically distinct from other known human herpesviruses (Salahuddin et al., 1986; Josephs et al., 1986). However, HHV-6 has been shown to share some DNA homology with cytomegalovirus (CMV); the degree of relat- edness being comparable to that observed between herpes simplex virus (HSV) and varicella zoster virus (VZV) (Lawrence et al., 1990).

Variations in the antigenic and replicative prop-

erties of different HHV-6 isolates have been de-

scribed. Some isolates grow preferentially in stimulated freshly isolated lymphocytes, while others grow readily in various T-cell lines, such as HSB-2 (Wyatt et al., 1990).

HHV-6 is now known to cause exanthema subi- turn (Yamanishi et al., 1988; Asano et al., 1989; Asano et al., 1991a) and has also been associated with a mononucleosis-like disease (Krueger et al., 1988; Niederman et al., 1988; Irving and Cun- ningham, 1990; Steeper et al., 1990). HHV-6 may also cause disease after organ or bone marrow transplantation (Okuno et al., 1990; Sutherland et

~___ al., 1991; Carrigan et al., 1991; Asano et al., * Corresponding author. 1991b; Ward et al., 1991; Cone et al., 1993).

Ol66-0934/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved

SSDI Ol66-0934(95)01964-2

222 L. Nielsen, B.F. Vestergaard / Journal of Virological Methods 56 (1996) 221-230

Studies of HHV-6 have found serological adapted from Nielsen et al. (1987). Cells were prevalences ranging from 8- 100% in various pop- harvested 8-12 days after infection, when more ulations around the world (Pellett et al., 1992; than 70% of the cells showed HHV-6 CPE. The Levine et al., 1992b). This variation in seropositiv- cells were washed twice in PBS, counted and ity may be due partly to the use of different end resuspended in double distilled water containing point titers and the use of assay systems with 2% fetal calf serum (FCS) to a concentration of different sensitivities. 10 000 000 cells/ml.

Most seroepidemiological studies of HHV-6 have used various types of immunofluorescence assays (Pellett et al., 1992; Levine et al., 1992b). The aim of this study was to establish an ELISA to enable large scale studies and routine diagnosis of the HHV-6 infection, and to investigate the prevalence of HHV-6 antibodies in a Danish population.

The mixture was transferred to a homogenizer and left in ice water (4°C) for - 15 min.

2. Materials and methods

2.1. Virus

The G&strain of HHV-6 and HSB-2 cells, were kindly donated by Dr. Robert C. Gallo (LTCB, NCI, NIH). HHV-6 was propagated in HSB-2 cells, in RPM&medium supplemented with 2.5% or 10% FCS, 1% neomycin, 1% gentamycin, 1% amphotericin B and 1% glutamine. Uninfected HSB-2 cells were grown in the same medium but always using 10% FCS.

Twenty-five ~1 of 20% NP-40 in PBS was added per ml of cell suspension and the number of free nuclei at time zero was counted in crystal violet with citric acid. Cells were homogenized carefully 5-20 times until all nuclei were free from cyto- plasm. The nuclei were separated on 22.5% Ficoll- 400 by centrifugation at 1500 x g for 20 min. The pellet was washed once with PBS, frozen and thawed and then resuspended in PBS to - 100 000000 nuclei in 1.5 ml. The suspension was sonicated until opalescent and then centrifuged for 30 min at 1500 x g. The supernatant constitutes the HHV-6 nuclear antigen and was stored at - 80°C until required.

Both HHV-6 and HSB-2 cells were free from contamination with mycoplasma.

A cellular antigen was also prepared from HHV-6 infected HSB-2 cells. The cells were harvested, washed twice in PBS and resuspended to 10% w/v in PBS with 0.5% v/v Triton X-100, and sonicated until opalescent. The suspension was centrifuged for 30 min at 1500 x g. The supernatant constitutes the HHV-6 antigen and was kept at - 80°C.

2.2. Immqnojluorescence Nuclear antigen and cellular antigen were pre-

pared similarly from uninfected HSB-2 cells.

HHV-6 immunofluorescence was done on HSB- 2 cells 8-12 days after infection with HHV-6 (Salahuddin et al., 1986). All sera were also tested on uninfected HSB-2 cells.

Serum was diluted 1:20 in PBS-Tween 20 (0.05%) with 1% bovine serum albumin (BSA). Secondary antibody (FITC-conjugated F(ab), fragment of rabbit anti-human IgG (DAK0 F 313) was diluted 1:30.

2.4. Polyclonal antibodies

2.3. Antigens

The method for the preparation of nuclear antigen from HHV-6 infected HSB-2 cells was

Each of 3 rabbits was immunized with 0.25 ml HHV-6 nuclear antigen in 0.3 ml Freund’s incom- plete adjuvant biweekly and bled 7 days after the fifth injection. The serum with the highest reactivity against HHV-6 was absorbed twice serially on a 6% (w/v) agarose activated with divinyl sulfone (Mini-Leak Medium, Kern-en-tee), coupled with uninfected HSB-2 cellular antigen (1 mg/ml gel) and a similar column coupled with FCS (35 ml/ml gel), both in a final concentration of 10% polyethylene glycol 20 000 (PEG), modified according to Lihme et al. (1986). Rabbit HHV-6

L. Nielsen, B.F. Vestergaard / Journal of Virological Methods 56 (1996) 221-230 223

antiserum (4 ml/ml gel) was absorbed on the gel, washed with washing buffer and the E-280 peak of the non-absorbed fraction was collected. After absorption the peak of the polyclonal antibody preparation did not react with cellular antigen from uninfected cells. The absorbed antibody preparation was purified on a protein-A column, modified according to Hjelm et al. (1972).

Biotinylation of the antibody preparation was modified according to Kendall et al. (1983). Two- hundred pg (5 ~1) biotin in solution (N-hydroxy- succinimidobiotin in dimethylformamide) was added per 1 mg of antibody preparation.

The final solution of biotinylated antibody was kept at - 20°C in a final solution of 0.5 mg/ml in 50% glycerol.

2.5. ELISA

Polyclonal anti-HHV-6 rabbit IgG in PBS 0.8 pg/ml was adsorbed overnight at room temperature to the wells of a 96-well microtitre plate (Nunc 269787). The plates were washed 5 times with washing buffer, consisting of PBS, 0.5 M NaCl pH 7.2 plus 1% Triton X-100. Test serum (50 ~1) at a dilution of l/5 was added to each well followed by 50 ~1 cellular antigen dilution. Both serum and antigen were diluted in ELISA buffer (washing buffer with 1% BSA and 0.2% phenol red added). Control wells contained no antigen. The plates were left overnight at room temperature then washed 5 times before adding 100 ,~l biotinylated polyclonal anti-HHV-6 rabbit IgG to each well. The biotinylated antibody was diluted in ELISA buffer containing 5% newborn calf serum. The plates were left for 2 h at room temperature, washed and then horseradish peroxidase-labelled avidin (DAK0 ~347) was added at a concentration of 1: 1000 in ELISA buffer and the plates incubated for 1 h at room temperature. After washing, bound peroxidase was detected by adding substrate (0.012% H,O, and 0.04% (w/v) o-phenylene-diamine) in 1 M citrate buffer (pH 5.0). The reaction was blocked with 1 M H,SO, and read at 490 nm.

All human sera were tested in 4 wells at a final concentration of l/10, 2 wells containing HHV-6 cellular antigen and 2 control wells without anti-

gen. The mean OD of the 2 control wells was taken as the background and subtracted from the mean OD of the 2 wells containing HHV-6 antigen. Since truly negative sera are difficult to obtain, the average OD-value of 4 wells on each plate without human serum was used as a negative reference (buffer-reference). The concentra- tions of the different reagents were adjusted to obtain a buffer reference of _ 1000 milliOD values (mOD). The background for the buffer reference was taken as the average of 4 wells containing no antigen and no human serum. This background never exceeded 120 mOD.

The inhibition coefficient was calculated as fol- lows:

OD(buffer - reference) - OD(sera) I x I

OD(buffer - reference)

Titrations were done either at 2-fold or IO-fold steps, with a start dilution of l/l0 and an end dilution of either l/l000 or l/l0 240 using 1 well for each dilution. Sera which had a titer above the end dilution were further titrated until a dilution above the end titer was obtained. Before further analysis, the mean OD of the 2 control wells was subtracted from the OD values of the wells containing HHV-6 antigen.

2.6. Serum samples

In total, 285 sera from 285 children l-6 years were tested by competitive ELISA to determine the prevalence of HHV-6 IgG antibody in early childhood. The sera were collected during 1983 from children throughout Denmark hospitalized for various reasons; 117 sera were from children aged l-2 years (14 from the 1st quarter, 50 from the 2nd quarter, 30 from the 3rd quarter and 23 from the 4th quarter), 48 sera were from children aged 2.1-3 years, and 90 sera were from children aged 3.1-7 years (30 sera for each year)

Eighty-three consecutive sera from 65 patients sent for HHV-6 testing were used for comparison of the immunofluorescence assay and the competitive assay. The age range of the patients was from 4 weeks to 72 years (mean age 35 years), 37 were female and 28 male. The sera were from patients with different symptoms including fever

224 L. Nielsen, B.F. Vestergaard / Journal of’ Virological Methods 56 (1996) 221-230

25

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.6 0.85 0.9 0.95 1

Inhibition coefficient.

Fig. 1. Distribution of inhibition coefficients with sera from 285 Danish children aged 1-6 years.

of unknown origin, liver affection, lymphadenitis, exanthema, neurological manifestations, chronic fatigue, mononucleosis, stomatitis and other infec- tious diseases. Thirteen sera from 12 patients were sent for routine examination before or after liver transplantation. Twelve sera were from 10 patients with unknown disease.

Sera (201 samples) from adult healthy blood donors were titrated. The titer was found by calculating the corresponding dilution to an inhibition coefficient of 45% using linear extrapolation of the inhibition coefficient on each side of 45% inhibition and the logarithm of the corresponding dilution. The 45% cut-off was chosen after analysis of the 285 sera from children.

The 201 sera from adult donors were tested for CMV-antibodies using the routine procedure of Statens Seruminstitut, described earlier by Nielsen et al. (1987) and so selected that half were CMV positive and half were CMV negative.

3. Results

Analysis of the 285 sera from children by the competitive assay identified 2 groups of sera. One group with inhibition coefficients < 0.4 and 1 group with inhibition coefficients > 0.5 (Fig. 1). Normally by competitive assays a zero inhibition coefficient is assigned to a group or pool of human negative sera. In this study such sera were not available and consequently the buffer reference was used to define zero inhibition. Any serum used in the assay at l/5 dilution caused some inhibition in comparison with the buffer reference (no serum). It is, therefore likely that the sera with inhibition coefficients < 0.4 actually represent negative sera. The average of the inhibition coefficients from the negative sera was 0.322 with a standard deviation (SD.) of 0.041. The cut off was consequently chosen as the average inhibition coefficient of negative sera + 3 x SD. = 0.45.

L. Nielsen, B.F. Vestergaard 1 Journal of Virological Methods 56 (1996) 221 p23O 225

30- r 25-

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.6 0.65 0.9 1’0.95 i’ 1

Inhibition coefficient

m Pos. IF m Neg. IF

Fig. 2. Correlation between indirect immunofluorescence and competitive ELISA with sera from 83 Danish patients.

The group containing the positive sera could be divided into 2 sub-groups: one with high ( 2 0.7) inhibition coefficients, which comprised most of the sera, and one, consisting of 13 sera with inhibition coefficients between 0.5 and 0.7. Nine of the sera in the latter group were from children between 1 and 2 years, and of the remaining 4, one came from each age group from 3-6 years.

In the group of children aged 1 year to 15 months, 11 of 14 (78%) were seropositive and 46 of 50 (92%) in the age group 15-18 months were found seropositive. From 18 months to 3 years, 97 of 101 sera (96%) were seropositive, and from 3.1 years upwards all the children were positive for HHV-6 antibodies.

3.1. Comparison between ELISA and IFA

Eighty-three sera from 65 patients were tested by both assays to compare ELISA with IFA (Fig. 2).

In the competitive ELISA, 82 (99O/,) sera were positive for HHV-6 antibodies. The only negative serum was the first of two sera from a 7-month old baby. The second serum was obtained 20 days later and was clearly positive with an inhibition coefficient of 0.8; both sera were however negative in the IFA. The baby was admitted to hospital with febrile convulsions, and became afebrile 3 days later and developed a rash clinically similar to exanthema subitum.

Fifty-nine (71%) sera from 42 patients (1.5-65 years) were positive for HHV-6 IgG antibodies and 24 (29%) sera from 23 patients (4 weeks to 72 years) were negative in the IFA.

The divergence between ELBA and IFA was due to a higher score of positive sera by ELISA, 82 sera were positive in ELBA compared to 59 positive sera by IFA. All sera positive for HHV-6 by IFA were also positive by ELISA. The probability that a given serum scored positive by the

226 L. Nielsen, B.F. Vestergaard 1 Journal of Virological Method 56 (1996) 221-230

T

log titer

Fig. 3. Distribution of log(HHV6-titer) with sera from 201 Danish donors.

immunofluorescence test was correlated to the level of the inhibition coefficient obtained by ELISA (Fig. 2).

3.2. HHV-6 antibody levels in 201 adult donor sera and 83 patient sera

All donor sera were positive for HHV-6 antibodies. The distribution of log(titer) of donor sera was close to a normal distribution (Fig. 3).

The distribution of 102 CMV-positive donor sera and 99 CMV-negative donor sera was different and significant at the 1% level (t-test).

There was no correlation between the CMV- titer and HHV-6 titer of CMV-positive sera (Fig.

4). The difference between the mean log(titer) of

patient sera excluding the one negative serum and the mean lo&titer) of donor sera was high and significant at the 1% level (approximated t-test), and 14% (11) patient sera had a titer > 10 000,

compared to only 0.5% (1) donor sera, a difference which was highly significant (P < 0.00005). The two sera with very high titers came from the same patient who had a pseudolymphoma proba- bly induced by tegretol. The patient also had liver affection and exanthema. The remaining 9 sera were from 8 patients with different symptoms. Altogether 3 of 9 patients with titer > 10000 had liver affection, compared to 7 out of 56 patients with known disease (not shown).

There was no correlation between the level of HHV-6-titer and the age of the patients.

4. Discussion

Many studies have shown that HHV-6 infection appears early in life, and that the highest inci- dence of HHV-6 infection is found between the ages of 6-12 months (Briggs et al., 1988; Saxinger et al., 1988; Okuno et al., 1989; Asano et al.,

L. Nielsen, B.F. Vestergaard / Journal of Virological Methods 56 (1996) 221-230

1

X

2 X

X

8 c X

X

X X

Ti :: X

8 se #

X

94

X

Fig. 4. Correlation of CMV-titer and HHV-6 titer with sera from 102 CMV-positive Danish donors.

r/ IKU

22-l

loo

1990; Farr et al., 1990; Huang et al., 1992; Pruk- sanonda et al., 1992). This is in accordance with our finding that 79% of children at the age of 12- 15 months have already acquired antibodies towards HHV-6 in Denmark. The rate of seropositivity among the Danish children is similar to that reported by others. Studies using immunofluorescence techniques, IFA or anti-comple- ment immunofluorescence (ACIF), found a seroprevalence ranging from 60-83% in l-year old children from Britain, Japan, Australia and Taiwan, respectively (Briggs et al., 1988; Okuno et al., 1989; Farr et al., 1990; Huang et al., 1992). One study (Asano et al., 1990) which used an indirect ELISA found a gradual increase in seroprevalence from 90% to 100% during the second year of life in children from Japan, a result similar to our finding that all children from 3-6 years had antibodies to HHV-6.

In contrast to children, a wide range of HHV-6 seropositivity in adults has been reported, and it is therefore important to consider the tests used in the various studies. IFA studies which tend to find a low seroprevalence (lo-64%) have defined the limiting titre of seropositivity to be 1:40 or even 1:50 (Biberfeld et al., 1988; Levine et al., 1992b; Krueger et al., 1988; Briggs et al., 1988; Cermelli et al., 1992). Studies defining a limiting titre of 1: 10 or 1:20 tend to find a seroprevalence from 70-100% (Levine et al., 1992b; Linde et al., 1988; Okuno et al., 1989; Levy et al., 1990; Levine et al., 1992a; Ranger et al., 1991).

Some investigators have reported lower sero- prevalences in selected populations (Levine et al., 1992a; Ranger et al., 1991). These studies still find a high prevalence rate in population groups from other parts of the world, using the same assay, thus there may be a genuine variation in antibody prevalence in various countries.


The starting dilution of sera appears to be less critical when studying seroprevalence in children (Briggs et al., 1988; Okuno et al., 1989; Farr et al., 1990; Huang et al., 1992). This may be due to a higher level of HHV-6 antibody content in sera from children compared to adults, reported by some authors (Briggs et al., 1988; Cermelli et al., 1992). Once positive, we did not find any correlation between the level of antibodies and age in our group of patients.

The observation of a 71% seroprevalence in a Danish population using IFA is comparable to that found in other Scandinavian studies. Linde et al. (1988) reported 85% seroprevalence in Sweden and Levy et al. (1990) 88%.

With ELISA a 100% seroprevalence was found in the Danish patient population compared to 71% observed with IFA, and the probability that a serum scored positive in the immunofluorescence test was correlated to the level of inhibition obtained with the competitive ELISA. The ELISA also detected a seroconversion, which was not found using IFA. These results demonstrate that ELISA is more sensitive than IFA, a fact that also has been shown in other studies (Asano et al., 1990; Dahl et al., 1990; Lyall, 1994).

Studies of HHV-6 seroprevalence using indirect ELISA report high seroprevalence (from 80P 97%), in healthy blood donors (Saxinger et al., 1988; Asano et al., 1990; Iyengar et al., 1991; Dahl et al., 1990). We found a seroprevalence of 100% both in a group of healthy blood donors and in patients. The mean log(titer) of patients was higher than the mean log(titer) of donors, the rate of patient sera with titer above 10000 was almost 20 times higher than the rate found in blood donors. This finding is in line with Iyengar et al. (1991) who reported a higher seroprevalence in adult patient groups with different symptoms.

Many investigators have reported simultaneous increases in specific IgG to both CMV and HHV- 6, most frequently in primary CMV-infection (Linde et al., 1988; Sutherland et al., 1991; Ward et al., 1991; Adler et al., 1993), but whether this is due to virus cross-reactivity or virus interaction still remains unclear. Our finding of a higher mean log(HHV-6-titer) in CMV-positive than in CMV-negative donors is in accordance with these

studies. The lack of correlation between the level of CMV-titer and HHV-6-titer speaks in favour of virus interaction rather than cross-reactivity.

It is concluded that the competitive ELISA is a sensitive test for detection of HHV-6 antibodies.

Acknowledgements

We thank Dr Mads Melby for initiating this study, Dr C.H. Mordhorst for providing sera from children l-7 years old, Hillar Kangro for his comments on the manuscript, Dr Robert C. Gallo for supplying us with HHV-6 and HSB-2 cells and special thanks to Bettina Schwenn for technical assistance. The work was supported by a research grant from the Danish ‘Statens Viden- skabelige Forskningsrad’.

References

Adler, S.P., McVoy, M., Chou, S., Hemplfling, S., Yamanishi,

K. and Britt. W. (1993) Antibodies induced by a primary

cytomegalovirus infection react with human herpesvirus 6

proteins. J. Infect. Dis. 168, 1119~1126.

Asano, Y., Yoshikawa, T., Suga, S., Yazaki, T., Hata, T.,

Nagai, T., Kajita, Y., Ozaki, T. and Yoshida, S. (1989)

Viremia and neutralizing antibody response in infants with

exdnthem subitum. J. Pediatr. 114, 5355539.

Asano, Y., Yoshikawa, T., Suga, S., Yazaki, T., Ozaki, T.,

Saito, Y., Hatano, Y. and Takahashi, M. (1990) Enzyme-

linked immunosorbent assay for detection of IgG antibody

to human herpesvirus 6. J. Med. Virol. 32 119- 123.

Asano, Y., Nakashima, T., Yoshikawa, T., Suga, S. and

Yazaki, T. (1991a) Severity of human herpesvirus-

viremia and clinical findings in infants with exanthem

subitum. J. Pediatr. 118, 891-895.

Asano, Y., Tetsushi, Y., Suga, S., Toshihiko, N., Yazaki, T.,

Fukuda, M., Kojima, S. and Matsuyama, T. (1991b) Reac-

tivation of herpesvirus type 6 in children receiving bone

marrow transplants for leukemia. N. Engl. J. Med. 324,

634635.

Biberfeld, P.. Pet& A.L., Eklund, A., Lindemalm, C.,

Barkhem, T., Ekman, M., Ablashi, D. and Salahuddin, Z.

(1988) Human herpesvirus- (HHV-6, HBLV) in sar-

coidosis and lymphoproliferative disorders. J. Virol. Meth-

ods 21, 49 59.

Briggs, M., Fox, J. and Tedder, R.S. (1988) Age prevalence of

antibody to human herpesvirus 6. Lancet 1058-1059.

Carrigan, D.R., Drobyski, W.R., Russler, S.K., Tapper, M.A.,

Knox, K.K. and Ash, R.C. (1991) Interstitial pneumonitis

associated with human herpesvirus- infection after mar-

row transplantation. Lancet 338, 147- 149.

L. Nielsen, B.F. Vestergaard / Journal of Virological Methods 56 (1996) 221-230 229

Cermelli, C., Moroni, A., Pietrosemoli, P., Pecorari, M. and

Portolani, M. (1992) IgG antibodies to human herpesvirus-

6 (HHV6) in Italian people. Microbiologica 15, 57-64.

Cone, R.W., Hackman, R.C., Huang, M.-L.W., Bowden,

R.A., Meyers, J.D., Metcalf, M., Zeh, J., Ashley, R. and

Corey, L. (1993) Human herpesvirus 6 in lung tissue from

patients with pneumonitis after bone marrow transplanta-

tion. N. Engl. J. Med. 329, 156&161.

Dahl, H., Linde. A., Sundqvist, V. and Wahren, B. (1990) An

enzyme-linked immunosorbent assay for IgG antibodies to

human herpes virus 6. J. Virol. Methods 29, 313-324.

Farr, T.J., Harnett, G.B., Pietroboni, G.R. and Bucens, M.R.

(1990) The distribution of antibodies to HHV-6 compared

with other herpes viruses in young children. Epidemiol.

Infect. 105, 603-607.

Hjelm. H., Hjelm, K. and Sjsquist, J. (1972) Protein A from

Staphylococcus aureus. Its isolation by affinity chromatog-

raphy and its use as an immunosorbent for isolation of

immunoglobulins. FEBS Lett. 28, 73-76.

Huang, L., Lee, C., Chen, J., Yang, C., Wang, J., Chang, M.,

Hsu, C. and Kuo, P. (1992) Primary human herpesvirus 6

infections in children: a prospective serologic study. J.

Infect. Dis. 165, 1163~1164.

Irving. W.L. and Cunningham, A.L. (1990) Serological diag-

nosis of infection with human herpesvirus type 6. Br. Med.

J. 300. 156-159.

Iyengar, S., Levine, P.H., Ablashi, D., Neequaye, J. and

Pearson, G.R. (1991) Sero-epidemiological investigations

on human herpesvirus 6 (HHV-6) infections using a newly

developed early antigen assay. Int. J. Cancer 49, 551-557.

Josephs. SF., Salahuddin, S.Z.. Ablashi, D.V., Schachter, F.,

Wong-Staal, F. and Gallo, R.C. (1986) Genomic analysis

of the human B-lymphotropic virus (HBLV). Science 234,

601~ 603.

Kendall, C.. lonescu-Matiu, 1. and Dreesman, G.R. (1983)

litilization of the biotinjavidin system to amplify the sensi-

tivity of the enzyme-linked immunosorbent (ELISA). J.

Immunol. Methods 56, 329-339.

Krueger, G.R.F., Koch, B.. Ramon, A., Ablashi, D.V.,

Salahuddin, S.Z., Josephs, SF., Streicher, H.Z., Gallo,

R.C. and Habermann. U. (1988) Antibody prevalence to

HBLV (human herpesvirus-6, HHV-6) and suggestive

pathogenicity in the general population and in patients

with immune deficiency syndromes. J. Viral. Methods 21,

175 131.

Lawrence. G.L., Chee, M., Craxton, M.A., Gompels. U.A.,

Honess, R.W. and Barrel], B.C. (1990) Human herpesvirus

6 is closely related to human cytomegalovirus. J. Virol. 64,

287 299.

Levine, P.H., Neequaye, J.. Yadav, M. and Connelly, R.

( I992a) Geographic/ethnic dilferences in human her-

pesvirus-6 antibody patterns. Microbial. Immunol. 36.

169 -172.

Levine, P.H., Jarrett, R. and Clark, D.A. (1992b) The epi-

demiology of human herpesvirus-6. In: D.V. Ablashi.

G.R.F. Krueger and S.Z. Salahuddin (Eds), Human Her-

pesvirus-6, Epidemiology, Molecular Biology and Clinical

Pathology. Perspectives in Medical Virology, Vol. 4, El-

sevier, Amsterdam, pp. 9-23.

Levy, J.A., Ferro, F., Greenspan, D. and Lennette, E.T. (1990)

Frequent isolation of HHV-6 from saliva and high sero-

prevalence of the virus in the population. Lancet 335,

1047~1050.

Lihme, A., Schafer-Nielsen, C., Larsen, K.P., Miller, KG.

and B&-Hansen. T.C. (1986) Divinylsulphone-activated

agarose formation of stable and non-leaking affinity ma-

trices by immobilization of immunoglobulins and other

proteins. J. Chromatogr. 376, 299-305.

Linde, A., Dahl, H., Wahren, B., Fridell, E., Salahuddin, Z.

and Biberfeld, P. (1988) IgG antibodies to human her-

pesvirus-6 in children and adults in primary Epstein-Barr

virus and cytomegalovirus infections. J. Virol. Methods 21.

117-123.

Lyall, E.G.H. (1994) Serological response of paediatric oncol-

ogy patients to human herpesvirus-6. J. Med Viral. 43,

373-379.

Niederman, J.C., Liu, C., Kaplan, M.H. and Brown, N.A.

(1988) Clinical and serological features of human her-

pesvirus-6 infection in three adults. Lancet 817-819.

Nielsen. CM., Hansen, K., Andersen. H.M.K., Gerstoft, J..

Vestergaard. B.F. (1987) A biotin-avidin-amplified inhibi-

tion enzyme immunoassay for detection of CMV antibod-

ies in human serum. J. Viral. Methods 16, 195 ~208.

Okuno, T.. Takahashi, K., Balachandra, K.. Shiraki, K.,

Yamanishi, K., Takahashi, M. and Baba, K. (1989)

Seroepidemiology of human herpesvirus 6 infection in nor-

mal children and adults. J. Clin. Microbial. 27, 651-653.

Okuno, T., Higashi, K., Shiraki. K., Yamanishi, K., Taka-

hashi, M.. Kokado, Y.. Ishibashi. M.. Takahara. S.,

Sonoda. T., Tanaka, K., Baba, K.. Yabuuchi, H. and

Kurdta, T. (1990) Human herpesvirus 6 infection in renal

transplantation. Transplantation 49. 519-522.

Pellett, P.E., Black, J.B. and Yamamoto, M. (1992) Human

herpesvirus 6: the virus and the search for its role as a

human pathogen. Adv. Virus Res. 41, I -51. Pruksanonda, P.. Hall, C.B., Insel, R.A., McIntyre. K., Pellett.

P.E., Long, C.E., Schnabel, K.C., Pincus. P.H., Stamey,

F.R.. Dambaugh. T.R. and Stewart J.A. (1992) Primary

human herpesvirus 6 infection in young children. N. Engl.

J. Med. 326, 1445-1450.

Ranger, S., Patillaud, S., Denis, F.. Himmich, A., Sangare, A.,

M’Boup, S.. Itoua-N’Gaporo, A.. Prince-David, M..

Chout, R., Cevallos, R. and Agut, H. (1991) Seroepidemi-

ology of human herpesvirus- in pregnant women from

different parts of the world. J. Med. Viral. 34, 194&198.

Salahuddin. S.Z., Ablashi. D.V., Markham, D.M., Josephs,

S.F.. Sturzenegger, S., Kaplan. M., Halligan. G.,

Biberfield, P., Wong-Staal. F., Kramarsky. B. and Gallo,

R.C. (1986) Isolation of a new virus. HBLV, in patients

with lymphoproliferative disorders. Science 234, 596-601.

Saxinger, C., Polesky, H.. Eby. N., Grufferman, S., Murphy,

R.. Tegtmeir, G., Parekh, V., Memon, S. and Hung, C.

(1988) Antibody reactivity with HBLV (HHV-6) in US

populations. J. Virol. Methods 21, 199 ~208.


Steeper, T.A., Horwitz, CA., Ablashi, D.V., Salahuddin, S.Z., Saxinger, C., Saltzman, R. and Schwartz, B. (1990) The spectrum of clinical and laboratory findings resulting from human herpesvirus- (HHV-6) in patients with mononucleosis-like illness not resulting from Epstein-Barr virus or cytomegalovirus. Am. J. Clin. Pathol. 93, 776-783.

Sutherland, S., Christofinis, G., O’Grady, J. and Williams, R. (1991) A serological investigation of human herpesvirus 6 infections in liver transplant recipients and the detection of cross-reacting antibodies to cytomegalovirus. J. Med. Vi- rol. 33, 172-176.

Ward, K.N., Sheldon, M.J. and Gray, J.J. (1991) Primary and recurrent cytomegalovirus infections have different effects on human herpesvirus- antibodies in immunosuppressed organ graft recipients: absence of virus cross-reactivity and evidence for virus interaction. J. Med. Virol. 34, 258267.

Wyatt, L.S., Balachandran, N. and Frenkel N. (1990) Varia- tions in the replication and antigenic properties of human herpesvirus 6 strains. J. Infect. Dis. 162, 8522857.

Yamanishi, K., Okuno, T., Shiraki, K., Takahashi, M., Kondo, T., Asano, Y. and Kurata, T. (1988) Identification of human herpesvirus- as a causal agent for exanthem subitum. Lancet 1065-1067.

Documents

Competitive ELISA for detection of HHV-6 antibody: seroprevalence in a Danish population