Journal of Medical Virology 40343-346 (1993)

Frequent Isolation of Human Herpesvirus 7 From Saliva Samples

Yasufumi Hidaka, Ying Liu, Masahiro Yamamoto, Ryoichi Mori, Chiaki Miyazaki, Koichi Kusuhara, Kenji Okada, and Kohji Ueda Department of Virology (Y.H., Y.L., R.M.), Department of Pediatrics (Y.H., C.M., K.K., K.O., K.U.), and Department of Ophthalmology (M.Y.), Faculty of Medicine, Kyushu University, Fukuoka, Japan

Human herpesvirus 7 (HHV-7) was isolated fre- quently from saliva specimens. The isolation rates were 81% (13116) in adutts, 70% (7/10) in children over 1 year old, and none (0/7) in chil- dren less than l year old, respectively, indicating that infection of HHV-7 occurs during early in- fancy and the virus shedding rate after infection is very high. Human herpesvirus 6 (HHV-6) was not isolated from saliva specimens although some studies on isolation of HHV-6 from saliva was reported previously. o 1993 Wiley-Liss, tnc.

KEY WORDS: HHV-7, HHV-6, isolation, saliva

INTRODUCTION Human herpesvirus 6 (HHV-6) was discovered by

Salahuddin et al. [1986] and shown as the causative agent of exanthem subitum by Yamanishi et al. [19881, but the route of infection was not found. Since some investigators reported that HHV-6 was frequently iso- lated from saliva or detected by the polymerase chain reaction (PCR) [Pietroboni et al., 1988; Fox et al., 1990; Harnett et al., 1990; Jarrett et al., 1990; Kid0 et al., 1990; Levy et al., 19901, we attempted to isolate HHVS from saliva to determine the route of infection of exan- them subitum. We isolated frequently a cytopathic (CP) agent like HHV-6; however, the characteristics of the virus were not the same as those reported for HHV-6. The CP agent did not react with anti-HHV-6 mono- clonal antibodies and could not be detected by PCR amplification for HHV-6. However, the virus reacted with anti-HHV-7 monoclonal antibodies and was shown to be HHV-7. We now report the isolation and characteristics of HHV-7 from saliva.

MATERIALS AND METHODS Subjects

Saliva specimens were collected from healthy labora- tory personnel and children who visited the Depart- ment of Pediatrics of Kyushu University Hospital and its affiliated hospital. Informed consent was obtained to use saliva specimens for this study. The samples were collected in sterile tubes, diluted 1:2 with lymphocyte 0 1993 WILEY-LISS, INC.

growth medium (LGM) consisting of RPMI 1640 with 10% fetal bovine serum, 20 U of recombinant interleu- kin 2 per ml, 50 pg of gentamicin per ml, and 2.5 pg of fungizone per ml. The diluted saliva samples were cen- trifuged at 2,OOOg for 10 minutes or filtered through a 0.45-pm pore size filter before inoculation onto lympho- cytes [Harnett et al., 19901.

Cells Following the previous reports of HHV-6 [Pietroboni

et al., 1988; Levy et al., 19901, we used peripheral blood mononuclear cells (PMC) of healthy adults for virus isolation. PMC from healthy adults were separated by Ficoll-Hypaque centrifugation, washed with phos- phate-buffered saline (PBS), and stimulated with 5 pg of phytohemagglutinin (PHA) per ml for 48-72 hours. The PHA was then removed by washing, and the PMC were suspended in LGM at a concentration of 2 x lo6 cells/ml.

Virus Isolation A 1.0-ml volume of PMC suspension was transferred

to a 15-ml plastic tube and inoculated with 1 ml of diluted saliva followed by centrifugal enhancement a t 2,500g for 1 hour [Pietroboni et al., 19891. The PMC culture was then incubated at 37°C with 5% GOz. A 50% medium exchange was made every 3 days, and the cultures were monitored for the presence of a C P change. In all these experiments uninoculated PMC always served as control.

Detection of Viral Antigens Samples of infected PMC were washed in PBS, spot-

ted onto slides, dried, and fixed with 100% cold acetone for 10 minutes. They were then tested for viral antigens

Accepted for publication December 16,1992. Address reprint requests to Yasufumi Hidaka, MD, Depart-

ment of Pediatrics, Faculty of Medicine, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812, Japan.

This paper was presented in part at the 39th meeting of the Society of Japanese Virologists, October 1991, Fukuoka, Japan (Abstract 1090) and the 24th meeting of the Japanese Society for Pediatric Infectious Diseases, December 1992, Kohchi, Japan (Ab- stract A-12).

344 Hidaka et al.

by indirect immunofluorescence using (1) HHV-6 and HHV-7 positive child's serum, (2) HHV-6-specific mon- oclonal antibodies; C3 108-103 [Yamamoto et al., 19901, OHV-1, OHV-2, or OHV-3 [Okuno et al., 1990, 1992a1, or (3) HHV-7-specific monoclonal antibodies; KR3 or KR4 [Okuno et al., 1992bl. OHV-1, OHV-2, OHV-3, KR3, and KR4 were provided by Dr. K. Yamanishi and Dr. T. Okuno, Osaka, Japan.

Electron Microscopy Virus-infected PMC pellets were fixed with glutaral-

dehyde for several hours at 4°C. The fixed pellets were washed overnight in PBS and post-fixed for 1 hour in 1% osmium tetroxide. They were then rinsed in PBS and dehydrated in a series of ethanol concentrations and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate.

PCR To test for HHV-6 DNA, PCR was performed follow-

ing the method of Kondo et al. [ 19901. The sequences as primers were 5 ' -GTGTTTCCATTGTACTGAAAC- CGGT-3' and 5'-TAAACATCAATGCGTTGCATA- CAGTS'. Briefly, 100 ~1 of infected PMC suspension was treated with 1 mg of proteinase K per ml for 3 hours a t 50°C and boiled for 10 minutes. A 5 - ~ 1 sample was used for PCR. HHV-6 strain 229 and strain HST [Yamanishi et al., 19881 were used as positive controls.

RESULTS Isolation of CP Agents

The characteristics of persons examined and the re- sults of virus isolation from saliva are shown in Table I. Twenty-five CP agents were obtained from 38 saliva samples from 33 persons. One person was tested repeat- edly and the same virus-positive result was obtained, therefore 6 saliva samples from this person were counted as one sample. Sixteen saliva samples from healthy adults and 17 from children were inoculated into healthy adults' PMC. Thirteen samples from adults (81%) and 7 from children (41%) yielded CP agents. Virus isolation rates in children were 0% (017) in 0-11 months, 50% (2/4) in 12-23 months, 75% (314) in 24-35 months, 100% (212) in more than 36 months, respectively. This CP agent was not isolated from chil- dren under 12 months old.

Characterization of the CP Agents The characteristics of the 25 CP agents isolated from

saliva are summarized in Table 11. All showed balloon- ing and polykaryotic CP changes like HHV-6 8 to 12 days after inoculation of the saliva samples. Examina- tion of thin sections prepared from PMC infected with one of the CP agents designated 005YH revealed typi- cal herpesvirus virions, 200 nm in diameter and con- taining an electron-dense cylindrical core, a capsid, a tegument, and an envelope (data not shown).

Antigenic detection was carried out by immunofluo- rescence test (Table 11). Nine strains were tested with HHV-6- and HHV-7-positive child's serum and all

TABLE I. Isolation of HHV-7 From Saliva*

Age Sex 30 Y M 30 Y M 30 Y F 26 Y M 30 Y M 32 Y M 27 Y M 26 Y M

3 Y F 2 Y M

41 Y M 49 Y F 22 Y F 32 Y F 41 Y F

M F

39 Y 32 Y 31 Y M 33 Y F 27 Y M 30 Y M 30 Y M

4 m F

6 m M 1 y , l m F

10 v F 8 m F 2 Y F l y , l m M 2 Y M 2 Y F 3 m M

10 m F 8 m M 1 y , 8 m F 8 m F 6 m F

31 v M

Clinical state Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Lissencephalia PyothorA SSPE Healthy HBV vaccinee Tonsillitis URI Pneumonia Eczema Gastroenteritis Hemorrhoids Atopic dermatitis LBWI Exanthem subitum Healthv

Cells

Al" A1 A1 A1 A2 A2 A2 A2 A3 A3 A3 A3 A3 A3 A3 A3 A3 B1 Bl B1 B1 B1 C l A4 A4 A4 A5 A5 A6 A6 A6 A6 A6 B2 B2 B2 A7 A8

Isolation of HHV-7 OOIYHb 002YHb 003SH 004KT 005YHb Negative 006TY 007YS 008CH 009NH Negative OlOKU OllKO 012TH 013IM 014AU Negative 015HM Negative 016EL 017TT 018YHb 019YHb Negative Negative 020SM Negative Negative 021SA 022MM 023YN Negative Negative Negative 024YN Negative Negative 025YHb

*SSPE = subacute sclerosing panencephalitis; HBV = hepatitis B vi- rus; URI = upper respiratory infection; LBWI = low birth weight in- fant. "A, B, and C indicate different adult's PMC. Number indicates differ- ent experiments. bThese six strains were obtained from one person.

showed positive fluorescence. Twenty-two strains in- cluding the above 9 were tested with mouse monoclonal antibodies to HHV-6 but none of them reacted with the antibodies. All strains reacted with mouse monoclonal antibodies to HHV-7 and were confirmed to be HHV-7.

Eleven strains were tested for HHV-6 DNA by PCR amplification. None showed HHV-6-specific DNA am- plification although the positive control HHVB did (Table 11).

DISCUSSION We frequently isolated CP agents from human saliva

and confirmed these to be HHV-7. The CP agents from saliva did not react with anti-HHV-6 monoclonal anti- bodies and were not amplified by PCR for HHV-6. This indicates that the CP agents did not contained HHV-6.

Frenkel et al. [19901 isolated HHV-7 from peripheral mononuclear cells of a healthy adult. They showed that

Isolation of HHV-7 From Saliva 345

TABLE 11. Characteristics (Immunofluorescence and PCR) of HHV-7 Isolated From Saliva

Serum of HHV-Gb HHV-7" HHV-6 Strain child" C3 108-103 OHV-1 OHV-2 OHV-3 KR3 KR4 PCR OOlYH 002YH 003SH 004KT 005YH 006TY 007YS 008CH 009NH OlOKU OllKO 012TH 013IM 014AU 015HM 016EL 017" 018YH 019YH 020SM 021SA 022MM 023YN 024YN 025YH

+ + + + + + + + +

ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

-

ND ND ND ND ND ND

N D ~ ND -

-

- ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

ND ND + ND ND ND + ND

+ ND + ND + +

- -

- - - -

ND ND + + ND ND + ND ND ND + + ND ND + ND ND ND + ND ND ND + ND ND ND + ND ND ND + + ND ND + + ND ND + ND ND ND + ND ND ND + ND ND ND + + ND ND + +

ND + + + +

ND

+ + ND ND

+ + ND - ND ND + + ND ND + +

- -

ND ND ND ND

ND ND

-

-

- -

- - -

- -

-

-

ND ND ND ND ND ND ND ND

"Serum of child: HHV-6 and HHV-7 antibody-positive serum. bC3 108-103,OHV-l, OHV-2,OHV-3: anti-HHV-6 monoclonal antibodies. 'KR3, KR4 anti-HHV-7 monoclonal antibodies. dND: not done.

HHV-7 resembled HHVS but could be distinguished from HHV-6 by viral DNA analysis. Wyatt and Frenkel [19921 reported that HHV-7 could be isolated from sa- liva of healthy adults. In this paper, we determined HHV-7 isolation in Japan and confirmed the report of HHV-7 isolation from adults in the USA. Furthermore we showed that HHV-7 was also frequently isolated from children. This is the first report of isolation of HHV-7 from children.

HHV-6 has been shown to be the causative agent of exanthem subitum [Yamanishi et al., 1988; Ueda et al., 19891 and in many cases the infection with HHV-6 oc- curred at the age of 6 months, when maternal immuno- globulins have almost disappeared. Almost all children have acquired the antibody to HHV-6 by the age of 12 months [Takahashi et al., 19881. HHV-7 was not iso- lated from the saliva of children before they were 12 months old. This shows that infection of HHV-7 occurs later than that of HHV-6, confirming serological stud- ies by Wyatt et al. [19911. The pathogenicity of HHV-7 is not clear. We asked the mothers of HHV-7-positive children whether their children had experienced any rash or febrile illness, but we could not gain useful information about the pathogenicity of the virus.

ACKNOWLEDGMENTS We thank Dr. K. Yamanishi and Dr. T. Okuno (Re-

search Institute for Microbial Diseases, Osaka Univer-

sity, Osaka, Japan) for the gift of MAbs and Dr. Mary Louise Robbins for reviewing the manuscript.

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