Comparison of Guinea Pig Embryo Cells, Rabbit Kidney Cells, and Human Embryonic Lung Fibroblast Cell Strains for Isolation of Herpes Simplex Virus

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1 JOURNAL OF CLINICAL MICROBIOLOGY, My 1982, p /82/ $02.00/0 Vol. 15, No. 5 Comprison of Guine Pig Embryo Cells, Rbbit Kidney Cells, nd Humn Embryonic Lung Fibroblst Cell Strins for Isoltion of Herpes Simplex Virus MARIE L. LANDRY, 12* DONALD R. MAYO,"3 AND G. D. HSIUNG13 Deprtments of Lbortory Medicine' nd Medicine,2 Yle University School of Medicine, New Hven, Connecticut 06510, nd Virology Lbortory, Veterns Administrtion Medicl Center, West Hven, Connecticut Received 1 October 1981/Accepted 23 December 1981 With the recognition of the importnce nd the prevlence of herpes simplex virus infections nd the vilbility of effective chemotherpy, there hs come demnd for rpid, sensitive, nd ccurte dignosis. Virus isoltion in cell culture remins the most widely employed dignostic method, nd the cell cultures used vry gretly from lbortory to lbortory. Therefore, study ws undertken to compre severl commonly employed humn fibroblst cell strins nd two nonhumn cells, primry rbbit kidney nd guine pig embryo cells, for the primry isoltion of this vilus. Our experience in the isoltion of herpes simplex virus in the clinicl lbortory with these cell systems ws evluted, nd reisoltions from clinicl specimens were performed. We found tht guine pig embryo nd rbbit kidney cell cultures were eqully sensitive for the isoltion of herpes simplex virus from clinicl specimens nd were superior to the humn embryonic lung fibroblst cell strins tested in terms of both sensitivity nd rpidity of ppernce of cytopthic effect. The use of guine pig embryo cells for the isoltion of this virus from clinicl specimens hs not been previously reported. Herpes simplex virus (HSV) is considered to be the most frequent cuse of spordic, ftl encephlitis in this country (4). Over the pst i0 yers, this virus hs lso been recognized s n importntt cuse of venerel disese nd serious neontl infection (11) nd s cuse of chronic, severe, nd sometimes life-thretening infection in immunosuppressed ptients (6). In ddition, genitl HSV type 2 (HSV-2) infection in the femle hs been linked to cervicl cncer (8). Antivirl therpy is now vilble for some serious HSV infections (12, 13). With the recognition of the importnce nd the prevlence of HSV infections nd the vilbility of effective chemotherpy, there hs come demnd for rpid, sensitive, nd ccurte dignosis. Although number of rpid ditgnostic serologicl techniques re currently being developed, virus isoltion in cell culture remitis the most widely employed dignostic method. In recent yers, tissue culture systems hve lrgely replced nimls nd eggs for the isoltion of HSV from clinil specimens. In generl, HSV is esily isolted nd propgted in wide vriety of cell cultures, lthough rhesus monkey kidney cells hve been noted to be less sensitive thn humn cells (3). In reviewing the literture on methods used for the isoltion of HSV, it 842 becme evident tht the cell cultures used vried gretly from lbortory to lbortory (7, 10). Humn embryonic lung fibroblst (HELF) cell strins re the most common system used by clinicl lbortories (7, 10). However, it hs been reported tht primry rbbit kidney (RK) cells were 10-fold more sensitive thn WI-38 cells, one of the most commonly used HELF cell strins for the isoltion of HSV from clinicl specimens (2). Since the use of the most sensitive nd rpid system for the isoltion of 14SV in the clinicl lbortory is importnt, study ws undertken to compre severl humn fibroblst cell strins nd two nonhumn cells, primry RK nd guine pig embryo (GPE) cells, for the primry isoltion of this virus. The use of GPE cells for the isoltion of HSV hs not been previously reported. Our experience in isolting HSV from clinicl specimens during the pst 2 yers with different cell systems ws evluted, nd reisoltions from clinicl specimens which hd been frozen were performed. The results re included in the present report. MATERIALS AND METHODS Cell culture. Primry RK nd GPE cells were prepred in our lbortory s previously described (1). Primry or secondry RK cells were used; GPE cells

2 VOL. 15, 1982 GPE, RK, AND HELF CELLS FOR HSV ISOLATION 843 TABLE 1. Comprison of GPE, RK, nd HELF cell cultures for the isoltion of HSV from clinicl specimens No. of virus isoltions Cell cultures inoculted No. of specimens GPE (n = 34) RK (n = 58) HELF (n = 59)b GPE, RK, nd HELF GPE nd HELF NDc 8 RK nd HELF 35 ND GPE nd RK ND n, Number of specimens tested. b The observed differences between GPE nd RK cells were not significnt, wheres the differences between GPE nd HELF nd between RK nd HELF were significnt; P < 0.05 nd P < 0.001, respectively (t-test). c ND, Not determined. t up to four to six pssge levels were utilized. For HELF, WI-38 cells purchsed from M. A. Bioproducts (Wlkersville, Md.) were used throughout most of the study period. Flow 2000, MRC5, nd W138 cells were purchsed from Flow Lbortories (McLen, V.) nd were used interchngebly in the clinicl lbortory. HELF cells, purchsed from Americn Type Culture Collection, were propgted in our lbortory nd were utilized s they were vilble. Preprtion of clinicl specimens. For originl isoltion, clinicl specimens (throt swbs, lesion swbs, nd brin tissue) were plced in virl trnsport medium contining Hnks blnced slt solution with ntibiotics nd l1o fetl bovine serum. Brin tissue ws minced in dilute trypsin; the cells obtined were then pelleted by low-speed centrifugtion, nd the trypsin ws removed. The cells were suspended in Egle minimum essentil medium with 2% clf serum to finl concentrtion of 10% (vol/vol). All clinicl specimens were inoculted, depending upon the vilbility of cultures, into two or three of the following cell types: GPE, RK, nd HELF. After the culture medium ws removed, two tubes of given cell type were inoculted per smple (0.2 ml per tube) nd llowed to dsorb for 1 h t 33 C. Fresh miniml essentil medium ws then dded, nd the cultures were incubted t 33 C in rotting drum. Specimens suspected of contining vricell-zoster virus (VZV) were inoculted into HELF nd GPE or RK cells in similr mnner. Virus isoltion nd identifiction. During the study period, six different observers were involved in exmining virl cultures in the clinicl lbortory. Cultures were exmined 24 h fter inocultion nd then every other dy for evidence of cytopthic effect (CPE), except for brin specimens nd cervicovginl cultures obtined from pregnnt women, which were exmined dily. When CPE ws extensive, cultures were hrvested nd frozen t -70 C until further studies were performed. Presumptive identifiction of virus isoltes ws determined by chrcteristic CPE nd cell susceptibility. The finl identifiction ws done only in selected instnces by immunofluorescence or by plque reduction neutrliztion tests. Typing of HSV isoltes ws determined by selective cell susceptibility, tht is, bility to produce plques in GPE cells nd chicken embryo cells. HSV-1 produced plques only in GPE cells, wheres HSV-2 produced lrger plques in both GPE nd chicken embryo cells (1). Isoltion nd presumptive identifiction of VZV were mde on the bsis of chrcteristic CPE, rte of progression of CPE (VZV ws much slower to progress thn HSV), nd cell susceptibility (VZV produced CPE in HELF but not in RK cells, wheres HSV produced CPE in both HELF nd RK cells). Subcultures into HELF nd GPE or RK cells were lwys performed to confirm the results. In selected cses, neutrliztion tests were performed by using type-specific ntiser. Reisoltion of HSV from clinicl specimens. For reisoltion, clinicl specimens which hd been frozen t -70 C for vrious periods of time were thwed nd inoculted in seril 10-fold dilutions (0.1 ml per tube; two tubes of ech cell type per specimen) into GPE nd RK or GPE, WI-38, MRC5 nd Flow 2000 cell cultures. After dsorption for 1 h t 36 C, miniml essentil medium with 2% clf serum ws dded, nd the cultures were incubted t 36 C. Cultures were exmined for CPE dily during the study period. A number of the clinicl specimens were frozen nd thwed severl times for these comprtive studies. Serodignosis. Acute- nd convlescent-phse ser obtined from ptients with HSV or VZV infection or both were tested by complement fixtion for the presence of ntibody to HSV nd VZV. The complement fixtion test ws performed in seprte lbortory without knowledge of the clinicl dignosis. Sttistics. Sttisticl nlysis ws performed by using the Student t test, the pired t-test, nd the chisqure test, s pproprite. RESULTS Comprison of GPE, RK, nd HELF cells for the isoltion of HSV from clinicl specimens. Over 2-yer period, 1979 to 1981, HSV ws isolted from 69 clinicl specimens (45 HSV-1 nd 24 HSV-2) inoculted into t lest two of the three cell types, GPE, RK, nd HELF. The sensitivities of the three cell types for the isoltion of HSV re summrized in Tble 1. HSV ws recovered in GPE cells from 33 of 34 specimens positive for HSV, in RK cells from 58 of 58 positive specimens, nd in HELF cells from 48 of 59 positive specimens inoculted. GPE nd RK cells were eqully sensitive for the isoltion of HSV from clinicl specimens, nd both were more sensitive thn the HELF cells

3 844 LANDRY, MAYO, AND HSIUNG TABLE 2. Speed of detecting HSV CPE in GPE, RK, nd HELF cell cultures inoculted with clinicl specimens No. of times CPE noted first in: Simultneous Cell cultures inoculted No. of specimens onset of CPE GPE (n = 34)' RK (n = 58) HELF (n = 59)b (n = 69) GPE, RK nd HELF 13 5c GPE nd HELF 11 8 NDd 0 3 RK nd HELF 35 ND GPE nd RK ND 8 n, Number of specimens tested. b The observed differences between GPE nd RK cells were not significnt, wheres the differences between GPE nd HELF nd between RK nd HELF were significnt; P < (t-test). c In four of five specimens inoculted into GPE, RK, nd HELF cells, the onset of CPE occurred simultneously in GPE nd RK, but lter in HELF. In one specimen, the onset of CPE ws fster in RK thn in GPE or HELF cells. dnd, Not determined. tested (P < 0.05 nd P < 0.001, respectively). In ddition, when the rpidity of detecting HSV CPE in the different cell systems ws compred (Tble 2), significnt difference ws gin noted between GPE nd HELF nd RK nd HELF. CPE ws noted first in GPE cells in 15 of 34 specimens nd in RK cells in 21 of 58 specimens inoculted. When RK nd GPE were both inoculted with the sme clinicl specimens, there ws no significnt difference in the time of onset of CPE; it ws usully simultneous. However, of 59 specimens inoculted into HELF, the ppernce of CPE ws never noted first in HELF when compred with GPE or RK or both (P < 0.001). The men dy of onset of CPE in specimens producing CPE in ll cell types considered ws lso not significntly different when GPE nd RK cells were compred (Tble 3). However, the onset of CPE in HELF ws significntly slower thn in both GPE (P < 0.05) nd RK (P < 0.01). No difference ws noted between HSV-1 nd HSV-2. Thus, tests in the clinicl lbortory reveled tht GPE nd RK cells were eqully sensitive for the isoltion of HSV in terms of both the number of positive isoltions nd the rpidity of onset of TABLE 3. HSV isoltion from clinicl specimens: men dy of onset of CPE in different cell cultures Cell cultures inoculted Men dy of onset of No. of CPE specimens sce GPE RK HELF GPE GPE, RK nd HELF GPE nd HELF NDb 3.6 RK nd HELF 29 ND GPE nd RK ND The observed differences between GPE nd RK cells were not significnt, wheres the differences between GPE nd HELF nd between RK nd HELF were significnt; P < 0.05 nd P < 0.01, respectively (pired t-test). b ND, Not determined. J. CLIN. MICROBIOL. CPE, nd both GPE nd RK were superior to HELF. The HELF cells tested yielded fewer positive isoltions, nd positive results were not detected s quickly s in GPE nd RK cells. Comprison of GPE nd RK cells for the reisoltion of HSV from clinicl specimens. Clinicl specimens previously known to contin HSV nd which hd been frozen t -70 C for vrious periods were thwed nd ssyed in seril 10- fold dilutions in GPE nd RK cells to determine the reltive sensitivities of these two cell types. The results re included in Tble 4. Virus ws recovered from 17 specimens, nd the infectivity TABLE 4. Comprison of GPE cells nd RK cells for the reisoltion of HSV from clinicl specimens Infectivity titer Virus. (log TCID5O.1 type Specimen no. ml) GPE RKb HSV HSV Reisoltions were done fter clinicl specimens were frozen for vrious periods of time. TCID50, 50% tissue culture infective dose. b The observed differences between GPE nd RK were not significnt (pired t-test).

4 VOL. 15, 1982 GPE, RK, AND HELF CELLS FOR HSV ISOLATION 845 TABLE 5. Comprison of GPE cells with three HELF strins for the reisoltion of HSV from clinicl specimens No. of reisoltions GPE cells WI-38 MRC5 Flow 2000 More sensitive by: >10-fold dilution fold dilution <10-fold dilution Less sensitive by:.10 fold dilution <10 fold dilution Eqully sensitive The differences in sensitivity of - 10-fold dilution between GPE nd WI-38, GPE nd MRC5, nd GPE nd Flow 2000 were significnt; P < 0.01, P < 0.05, nd P < 0.01, respectively (chi-squre test). titers obtined in GPE nd RK were identicl in 13 instnces. In three specimens (1699, 1806, nd 1935), GPE cells were more sensitive thn RK cells by less thn 10-fold dilution, nd in one specimen (1877), RK ws more sensitive thn GPE by less thn 10-fold dilution. These differences were not sttisticlly significnt. Comprison of GPE cells with three HELF strins for the reisoltion of HSV from clinicl specimens. Twenty clinicl specimens previously positive for HSV nd frozen t -70 C were thwed nd inoculted in seril 10-fold dilutions into GPE, WI-38, MRC5, nd Flow 2000 cells. As summrized in Tble 5, GPE cells were found to be more sensitive thn WI-38 (P < 0.01), MRC5 (P < 0.05), nd Flow 2000 (P < 0.01) cells for the reisoltion of HSV from clinicl specimens when differences of 10-fold or greter were considered. In no instnce were GPE cells less sensitive thn either WI-38, MRC5, or Flow 2000 cells by 10-fold dilution or greter, wheres GPE cells were more sensitive by 10-fold or greter thn WI-38, MRC5, nd Flow 2000 in 11, 9, nd 10 reisoltions, respectively. In this study, Flow 2000 cells were the lest sensitive of the cells tested; in five reisoltions, they were less sensitive thn GPE cells by greter thn 10-fold. Significntly, three specimens from which HSV ws reisolted, using GPE cells, were negtive for virus in the HELF strins. Dul infection with HSV nd VZV. Over 3- month period, throt swbs s well s lesion swbs were obtined from 12 ptients with clinicl VZV infection. VZV infection ws confirmed by virus isoltion nd serology in 11 of these ptients. One ptient with clinicl zoster presented 10 dys fter the onset of his rsh; VZV ws not isolted from the lesions, but VZV ntibody titer by complement fixtion ws 1:512. In ddition, HSV ws isolted from the throts of three of these ptients in whom HSV infection ws not suspected cliniclly. In two of the ptients in whom HSV ws isolted from the throt, the virus ws isolted only in GPE or RK cells, not in HELF cells; in the third ptient, chrcteristic HSV CPE ws evident within 2 dys in GPE cells, but did not pper in HELF cells until dy 7 post-inocultion. Only in the ptients with dul infection (VZV nd HSV) ws double ntibody rise seen (Tble 6). In nine ptients from whom VZV lone ws isolted, rise in ntibody to VZV, but not HSV, ws seen. DISCUSSION The importnce of HSV in humn disese is being incresingly recognized. In mny clinicl TABLE 6. Dul infection with HSV nd VZV in ptients with clinicl VZV infection Antibody titer Type of infection Ptient no. Clinicl dignosis Virus isolted Dys fter (CF) onset of rsh HSV VZV Double infection 1 Vricell VZV HSV Zoster VZV HSV Zoster VZVb HSV Single infectionc 4 Zoster VZV Titers expressed s reciprocls of serum dilution; CF, complement fixtion test. b Ptient no. 3 presented 10 dys fter the onset of clinicl zoster; thus, VZV ws not isolted. HSV-1 ws isolted from his throt. c VZV lone ws isolted from n dditionl eight ptients in whom n ntibody rise occurred only to VZV, not to HSV.

5 846 LANDRY, MAYO, AND HSIUNG virology lbortories, HSV is the single most frequent virus isolted (10). In the Ntionl Institute of Allergy nd Infectious Diseses (NIAID) collbortive ntivirl study of herpes simplex encephlitis, denine rbinoside tretment is stopped fter 5 dys if HSV is not isolted from the brin biopsy (13). In the NIAID study of the tretment of neontl infection with denine rbinoside, dely in dignosis resulted in dely in the dministrtion of ntivirl therpy nd dversely ffected the outcome (12). According to current recommendtions (11), women determined to be t high risk for hving genitl herpes infection, which is frequently symptomtic, should hve weekly cervicovginl cultures for HSV during the lst 1 to 2 months of pregnncy. If HSV is isolted within the week before delivery, cesrin section is indicted. Thus, it is impertive tht rpid nd sensitive dignostic techniques for HSV be vilble. HELF re the most commonly used cells for the isoltion of HSV in clinicl virology lbortories (7, 10). HELF cells support the growth of wide vriety of virus types in ddition to HSV e.g., VZV, cytomeglovirus, mny enteroviruses, rhinoviruses, some denoviruses, etc.) nd thus hve brod use in clinicl lbortories. In 1970, it ws first reported tht primry RK cells were 10-fold more sensitive thn WI-38 cells, the most commonly used HELF cell line (2). In tht study, of 14 throt swbs positive for HSV with RK cells, only 9 were positive with WI-38 cells. In our study, we confirmed the results of this erlier report nd found tht RK cells were more sensitive thn the HELF cell strins tested for the isoltion of HSV from clinicl specimens. In ddition, we found tht GPE cells were s sensitive s RK cells for the primry isoltion of HSV nd offer severl dvntges. GPE cells re obtined from chep nd redily vilble niml, they grow redily, they cn be pssged t lest four to six times, nd they hve recently been found to be useful for the isoltion of severl types of group A coxsckieviruses from clinicl specimens, viruses tht generlly require the use of newborn mice for isoltion (5). Primry RK cells cn be pssged only once, nd their spectrum is generlly limited to HSV. However, the nrrow host spectr of both GPE nd RK cells my llow for more rpid presumptive dignosis of HSV-induced CPE, s opposed to tht of HELF cells, in which mny humn viruses my produce CPE. Although GPE nd RK cells hve more nrrow spectr thn tht of HELF cells for humn viruses, their greter sensitivity is n importnt dvntge. The use of more sensitive system cn result in more rpid dignosis s well s more positive J. CLIN. MICROBIOL. virus isoltions, importnt in both chemotherpy nd prophylctic cesrin section for genitl herpes in pregnncy. In our series, of 59 clinicl specimens inoculted into HELF cells, 11 (19%) were negtive for HSV in this cell system. The use of GPE nd RK cells resulted in the isoltion of HSV within 3 to 5 dys from number of importnt specimens including brin biopsy, number of genitl specimens, nd lesion swb from n immunosuppressed ptient, ll negtive for HSV with HELF cells. In ddition, comprison of GPE cells with three commonly used HELF cell strins for the reisoltion of HSV from clinicl specimens found GPE cells to be superior. Three of 20 specimens (15%) negtive for virus in the HELF strins upon reisoltion were positive with GPE cells. The use of more sensitive cell culture systems lso gve new insight into well-known phenomenon. Previous reports hve indicted tht clinicl infection with VZV my be ssocited with rise in ntibody to both HSV nd VZV (9). This "heterologous" ntibody rise hs been ttributed to possible ntigenic similrities between these two viruses. Virus isoltion ws not ttempted in these erly studies. To determine the specificity of the ntibody rise in our ptients with VZV infection, n ttempt ws mde to serch for the incidence of mixed infections with HSV s possible source of the heterologous or double ntibody rise. As noted in our study, only ptients with dul infection hd double ntibody rise in our smll series, new finding mde possible by the use of the more sensitive GPE nd RK cells. This greter sensitivity of GPE nd RK cells my reflect the fct tht these cells were primry or erly pssge cells. Primry or erly pssge humn fibroblsts my be eqully sensitive; however, they my be more difficult to obtin. In summry, from the dt presented, it is cler tht GPE nd RK cell cultures were eqully sensitive for the isoltion of HSV from clinicl specimens nd were superior to vriety of HELF strins tested in terms of both sensitivity nd rpidity of ppernce of CPE. GPE nd RK cells were eqully sensitive for the isoltion of HSV-1 nd HSV-2. The use of GPE cells for the isoltion of HSV from clinicl specimens hs not been previously reported. Thus, lthough HELF cell strins re the most commonly used cells for the isoltion of HSV in clinicl lbortories, in situtions in which the most sensitive nd rpid culture system is necessity, the use of GPE or RK cells should be strongly considered. ACKNOWLEDGMENTS This study ws supported by Public Helth Service reserch trining grnt T-32-AI from the Ntionl Institute of

6 VOL. 15, 1982 GPE, RK, AND HELF CELLS FOR HSV ISOLATION 847 Allergy nd Infectious Diseses nd by the Veterns Administrtion Reserch Fund. We would like to thnk Emmnuel Lerner for his expert help with the sttistics. LITERATURE CITED 1. Anderson, C. A., M. J. August, nd G. D. Hsiung Pthogenicity of wild-type nd temperture-sensitive mutnts of herpes simplex virus type 2 in guine pigs. Infect. Immun. 30: Dougls, G. R., nd R. B. Couch A prospective study of chronic herpes simplex virus infection nd recurrent herpes lbilis in humns. J. Immunol. 104: Hslung, G. D., F. Pinheiro, nd M. 0. Gbrielson The virus dignostic lbortory: functions nd problems bsed on three yers' experience. Yle J. Biol. Med. 36: Johnson, R. T., L. C. Olson, nd E. L. Buescher Herpes simplex virus infections of the nervous system. Arch. Neurol. 18: Lndry, M. L., H. P. Mdore, C. K. Y. Fong, nd G. D. Hsiung Use of guine pig embryo cell cultures for isoltion nd propgtion of group A coxsckieviruses. J. Clin. Microbiol. 13: Muller, S. A., E. C. Herrmnn, nd R. K. Winkelmnn Herpes simplex infections in hemtologic mlignncies. Am. J. Med. 52: Rwls, W. E Herpes simplex virus types 1 nd 2 nd herpesvirus simie, p In E. H. Lennette nd N. J. Schmidt (ed.), Dignostic procedures for: virl, rickettsil nd chlmydil infections, 5th ed. Americn Public Helth Assocition, Wshington, D.C. 8. Rwls, W. E., W. A. F. Tompkins, nd J. L. Melnick The ssocition of herpes-virus type 2 nd crcinom of the uterine cervix. Am. J. Epidemiol. 89: Schp, G. J. P., nd J. Huismn Simultneous rise in complement-fixing ntibodies ginst herpesvirus hominis nd vricell-zoster virus in ptients with chicken pox nd shingles. Arch. Gesmte Virusforsch. 25: Smith, T. F Specimen requirements, trnsport nd recovery of virus in cell cultures, p In D. A. Lennette, S. Specter, nd K. D. Thompson (ed.), Dignosis of virl infections: the role of the clinicl lbortory. University Prk Press, Bltimore, Md. 11. Visintine, A. M., A. J. Nhmis, nd W. E. Josey Genitl herpes. Perintl Cre 2: Whitley, R. J., A. J. Nhmls, S. J. Soong, G. J. Glsso, C. L. Fleming, nd C. A. Alford Vidrbine therpy of neontl herpes simplex virus infection. Peditrics 66: Whitley, R. J., S. J. Soong, M. S. Hirsch, A. W. Krchmer, R. Dolin, G. J. Glsso, J. K. Dunnick, C. A. Alford, nd the NIAID collbortive ntivirl study group Herpes simplex encephlitis: vidrbine therpy nd dignostic problems. N. Engl. J. Med. 304: Downloded from on October 11, 2018 by guest