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Effect of rolling on foci development and viral replication for Herpes Simplex Virus (HSV)
95
Journal of Virological Methods, 20 (1988) 95-100 Elsevier
JVM 00720
Effect of rolling on foci development and viral replication for herpes simplex virus (HSV)” Charis T. Mavromoustakis’,
Donald T. Witiak’ and John H. Hughes1,3
‘Department of Medical Microbiology and Immunology, College of Medicine, Ohio State University, 2Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Ohio State University, Columbus, Ohio, U.S.A.; 3The Children’s Hospital Viral Diagnostic Laboratory, Columbus, Ohio, U.S.A. (Accepted
10 February
1988)
Summary The effect of rolling on the number of herpes simplex virus (HSV) foci and yield of infectious virus was examined for cultures inoculated with clinical specimens. Inoculated cultures were either rolled at 2 revolutions per minute (rpm) or held stationary. Cultures that were rolled at 2 rpm showed a significantly greater number of foci and plaque forming units (PFU) over stationary cultures. The geometric mean fold increase in PFU between rolled and stationary cultures was 8-fold. Rolling of inoculated cultures should be used in the clinical virology laboratory to aid in the rapid detection of HSV. HSV; Rolling
culture;
Viral replication
Introduction Several factors have increased the need for rapid, practical, accurate and inexpensive techniques for detecting herpes simplex virus (HSV). Among them are the prevalence of HSV infections, the importance of active infections in pregnant women, disseminated disease in immunocompromised patients (Kilbrick, 1980; King et al., 1982) as well as the availability of antiviral therapy (Hirsch, 1980; * This research
was supported
Correspondence U.S.A.
to: Dr. John H. Hughes,
0166-0934/88/$03.50
0
in part by the National
1988 Elsevier
700 Children’s
Science
Publishers
Heart
and Lung Institute
grant
HL 12740
Drive, 415 Ross Hall. Columbus.
B.V. (Biomedical
Division)
Ohio 43205,
Hirsch, 1983). Recent advances in the rapid laboratory diagnosis of HSV infections have reduced detection turnaround times from an average of 5 to 7 days to less than 24 h by using direct antigen detecting methods. An advantage to direct antigen detection methods is that viable virus does not have to be present. Although many of the direct techniques yield results within 4 to 24 h (Gleaves. 1985; Landry, 1986). the sensitivity of the detection method is usually less than that of the viral isolation (Morgan, 1984) and technical variables are involved in distinguishing a positive from a negative specimen. Thus, tissue culture is still the standard by which other detection methods are compared (Hughes, 1986; Landry. 1986). In this study we examined the effect that rolling had on the number of HSV foci. Foci formation was determined in cultures that were either rolled at 2 revolutions per minute (rpm) or held stationary. Our objective was to determine if rolling would enhance the number of HSV cell foci in cultures inoculated with clinical specimens. If more foci were produced with rolling conditions, then HSV detection should be easier. In addition to investigating foci formation, we also determined the effect that rolling had on viral yield at 23-96 h post inoculation for cultures inoculated with clinical specimens of HSV.
Materials
and Methods
For this study, specimens from both genital and non-genital sites were collected on swabs, placed in transport medium, and stored at refrigerated temperatures until delivered to the laboratory within 24 h. After receiving the specimens in the laboratory, 0.2 ml aliquots were inoculated into duplicate culture tubes of human diploid fibroblast cells (Flow 6000). One culture was held stationary at 35°C while the other was rolled at 2 rpm. All cultures were observed daily for HSV cytopathic effect (CPE) and the number of HSV foci was determined. Four clinical specimens, two below the waist and two above the waist were randomly chosen and serial dilutions were prepared. One hundred ~1 of each viral preparation were inoculated into F6000 cells and scored daily for CPE. After 24, 48, 72 and 96 h post-inoculation, infected cultures were frozen at -70°C. The same serial dilutions for each test condition were then used for a viral yield assay so a comparison could be made between rolled and stationary cultures for the number of plaque forming units (PFU) produced. For viral yield assays, 12-well plates were seeded with African green monkey kidney cells (BGM 70) and incubated at 37°C in 5% CO* and 95% relative humidity. Monolayers were then infected with serial dilutions from the two conditions (2 rpm and stationary). At the end of a 2 h adsorption period, agarose overlay was added and cultures were incubated again for 48 h. At the end of the incubation period, cultures were fixed with 10% formalin buffered with PBS and then stained with 10% crystal violet. Plates were examined microscopically and the number of PFUs was determined.
97
Results Thirty-four specimens inoculated into F6000 were examined for the number of infected cells foci. Out of 34 specimens, 27 resulted in cultures with countable foci. Forty-four percent of all inoculated cultures were positive for foci by day one and by day two 94% of the cultures were positive. All of the rolled cultures with TABLE Effect
1 of rolling
Specimen
Number
on herpes
simplex
virus (HSV) Number
foci formation
in Flow 6000 cells
of Foci
Fold Increase
Rolling
Stationary
6-3543 6-3639 6-3659 6-3693 6-4543 6-4913 6-4917 6-5193 6-5340 6-5347 6-5413 6-5441 6-5697 6-5740 6-5746 6-5757 6-5765 6-5786 6-5807 6-6003 6-6113 6-6283 6-6318
18 12 9 13 13 12 90 35 17 3+” 2+ 14 12 5 10 ND” 28 28 2 49 18 27 TNC’
6-6336 6-6415 6-642 1 6-644 1 7-0046 7-0063 7-0108 7-0115 7-0611 7-0725 7-1323
18 12 TNC 11 15 13 12 16 TNC TNC 29 15.5”
8 4 2 2 6 6 40 16 8 20 0 7 8 1 6 8 10 16 0 36 6 14 TNC 11 7 TNC 2 7 8 8 11 4 11 9 6.7
” l-4+ represents 25-1000/o infected cells. h ND-Not Done or Not Determined. ’ TNC-Too Numerous to Count, but less than ’ Geometric mean.
1+
in Foci
2.2 3.0 4.5 6.5 2.2 2.0 2.2 2.2 2.1 ND ND 2.0 1.5 5.0 1.6 ND 2.8 1.8 ND 1.4 3.0 1.9 ND 1.6 1.7 ND 5.5 2.1 1.6 1.5 1.4 ND ND 3.2 2.3
98 TABLE
2
Mean fold increase tionary cultures Specimen”
1 2 3 4
in PFU
for herpes
simplex
virus isolates
Mean fold increase
for cultures
rolled
at 2 rpm versus
sta-
in
Foci
PFU
5.0h 2.1 2.4 3.2
7.2’ 6.6 10.5 10.5
a Results from four clinical specimens obtained from above the waist (oral-1,2) and below the waist (genital-3,4). ’ This represents the mean fold increase of infected foci between rolled and stationary conditions for 4 infected cultures. ’ This represents the mean fold increase of PFU for 4 infected cultures using 3 wells for each plaque assay.
countable foci had a greater number of foci when compared to companion stationary cultures with a geometric mean fold increase in foci of 2.3 (Table 1). A significant enhancement in foci numbers was seen between rolled and stationary cultures (P < 0.01, t-test). Four frozen clinical specimens were diluted, inoculated into Flow 6000 cells and scored for CPE, at 24 and 48 h post-infection. Specimens that were rolled showed a greater number of foci when compared to the control (stationary) cultures. The difference in foci development was more dramatic at 72 and 96 h. Specimens that were rolled showed a geometric mean fold increase in foci of 3.0 when compared to stationary cultures (Table 2). Plaque assay results correlated with foci results. Significantly more PFUs were produced in cultures that were rolled than in stationary cultures. The geometric mean fold increase in PFU between rolled and stationary cultures was 8.7.
Discussion The use of centrifugation to inoculate monolayers has been studied previously (Sharp, Smith 1960; Padgett, Walker 1962; Osborn, Walker 1968; Hudson et al. 1976; Tenser 1978; Tenser, Dunstan 1980; Gleaves et al. 1984) and recent studies (Gleaves et al, 1985; Salmon et al, 1986) demonstrated that low speed centrifugation increased the diagnostic sensitivity for HSV. Tenser (Tenser 1978) reported almost a lo-fold increase in HSV sensitivity, with laboratory strains, after low speed centrifugation (1100 x g for 10 min) and a nearly 100-fold increase with ultracentrifugation (28 000-45 000 X g for 1.5-2.3 h). Gleaves et al. (1985) reported that low speed centrifugation (700 x g for 40 min) probably enhances adsorption of HSV onto monolayers, and when centrifugation was used in conjunction with immunofluorescence, positive detection and serotyping of HSV was possible 16 h postinoculation. Salmon et al. (1986) have recently demonstrated an increase in the
99
number of HSV foci when low speed centrifugation (3500 x g for 15 min) was used but observed no significant difference in sensitivity of HSV detection with clinical specimens. For murine cytomegalovirus (MCMV) Osborn and Walker (1968) detected 10 to 100-fold more PFU when assayed by centrifugation at 1900 x g for 10 min compared to inocula assayed without centrifugation. Similar results were not observed for HSV or pseudorabies virus preparations. Human cytomegalovirus also undergoes centrifugal enhancement of infectivity (Hudson et al., 1976) and detection of human CMV by using low speed centrifugation has diagnostic applications (Gleaves et al., 1984). To our knowledge no reports have been published comparing the foci formation and viral yields produced by HSV when cultures were continuously rolled at 2 rpm speed. In this study we demonstrated that rolling at 2 rpm increased the number of HSV foci as well as the viral yields. This should aid in the earlier and easier detection of HSV in cultures inoculated with clinical specimens. The enhancement in the number of HSV foci may be attributed to several factors such as: 1) the physical transfer of virus or virus-infected cells to noninfected cells by rolling, 2) an increase in viral adsorption by rolling, or 3) activation of host cells for enhanced viral replication. In recent experiments, high speed rolling (96 rpm) was superior to slow speed rolling (2 rpm) and stationary conditions, not only for increasing the number of HSV foci but for decreasing the detection time as well. Cultures that were rolled at 96 rpm became positive sooner than cultures that were rolled at 2 rpm or stationary. There was also a decrease in the time required for rolled cultures to reach maximum CPE development. At the present time it is not known how much force or for how long it is necessary to enhance viral replication. We suggest that rolling of inoculated cultures should be used in the clinical virology laboratory to aid in the rapid detection of HSV and possibly other viral pathogens.
References Gleaves, C.A., Smith, T.F., Shuster, E.A. and Pearson, G.R. (1984) Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen. J. Clin. Microbial. 19, 917-919. Gleaves, C.A., Wilson, D.J., Weld, A.D. and Smith, T.F. (1985) Detection and serotyping of herpes simplex virus in MRC-5 cells by use of centrifugation and monoclonal antibodies 16 h postinoculation. J. Clin. Microbial. 21, 29-32. Hirsch, MS. and Swartz, M.N. (1980) Antiviral agents. N. Engl. J. Med. 302, 903-907; 949-953. Hirsch, M.S. and Schooley, R.T. (1983) Treatment of herpesvirus infections. N. Engl. J. Med. 309, 963-970;103+1039. Hudson, J.B., Misra, V. and Mosmann, T.R. (1976) Cytomegalovirus infectivity: analysis of the phenomenon of centrifugal enhancement of infectivity. Virology 72, 235-243. Hughes, J.H., Mann, D.R. and Hamparian, V.V. (1986) Viral isolation versus immune staining of infected cell cultures for the Laboratory Diagnosis of Herpes Simplex Virus infections. J. Clin. Microbiol. 24, 487-489. Kilbrick, S. (1980) Herpes simplex infections at term. J. Am. Med. Assoc. 243, 157-160.
100 King, D.H. and Galasso, G. (eds.) (1982) Proceedings of a symposium on a cyclovir. Am. J. Med. 73. l-329. Landry, M.L., Zibello, T.A. and Hsiung, G.D. (1986) Comparison of in situ hybridization and immunologic staining with cytopathology for detection and identification of herpes simplex virus infection in cultured cells. J. Clin. Microbial. 24, 968971. Morgan, M.A. and Smith, T.F. (1984) Evaluation of an enzyme-linked immunosorbent assay for the detection of herpes simplex virus antigen. J. Clin. Microbial. 19, 730-732. Osborn, J.E. and Walker, D.L. (1968) Enhancement of infectivity of murine cytomegalovirus in vitro by centrifugal inoculation. J. Virol. 2, 853-858. Padgett, B.L. and Walker, D.L. (1962) Use of centrifugal force to promote adsorption of myxoma virus to cell monolayers. Proc. Sot. Biol. Med. 111, 364-367. Salmon, V.C., Turner, R.B., Speranza, M.J. and Overall, J.C. (1986) Rapid detection of herpes simplex virus in clinical specimens by centrifugation and immunoperoxidase staining. J. Clin. Microbiol. 23, 683-686. Sharp, D.G. and Smith, K.O. (1960) Rapid adsorption of vaccinia virus on tissue culture cells by ccntrifugal force. Proc. Sot. Biol. Med. 104, 167-169. Tenser, R.B. (1978) Ultracentrifugal inoculation of herpes simplex virus. Infec. Immun. 21. 281-285. Tenser, R.B. and Dunstan, M.E. (1980) Mechanisms of herpes simplex virus infectivity enhanced by ultracentrifugal inoculation. Infec. Immun. 30, 193-197.
Journal of Virological Methods, 20 (1988) 95-100 Elsevier
JVM 00720
Effect of rolling on foci development and viral replication for herpes simplex virus (HSV)” Charis T. Mavromoustakis’,
Donald T. Witiak’ and John H. Hughes1,3
‘Department of Medical Microbiology and Immunology, College of Medicine, Ohio State University, 2Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Ohio State University, Columbus, Ohio, U.S.A.; 3The Children’s Hospital Viral Diagnostic Laboratory, Columbus, Ohio, U.S.A. (Accepted
10 February
1988)
Summary The effect of rolling on the number of herpes simplex virus (HSV) foci and yield of infectious virus was examined for cultures inoculated with clinical specimens. Inoculated cultures were either rolled at 2 revolutions per minute (rpm) or held stationary. Cultures that were rolled at 2 rpm showed a significantly greater number of foci and plaque forming units (PFU) over stationary cultures. The geometric mean fold increase in PFU between rolled and stationary cultures was 8-fold. Rolling of inoculated cultures should be used in the clinical virology laboratory to aid in the rapid detection of HSV. HSV; Rolling
culture;
Viral replication
Introduction Several factors have increased the need for rapid, practical, accurate and inexpensive techniques for detecting herpes simplex virus (HSV). Among them are the prevalence of HSV infections, the importance of active infections in pregnant women, disseminated disease in immunocompromised patients (Kilbrick, 1980; King et al., 1982) as well as the availability of antiviral therapy (Hirsch, 1980; * This research
was supported
Correspondence U.S.A.
to: Dr. John H. Hughes,
0166-0934/88/$03.50
0
in part by the National
1988 Elsevier
700 Children’s
Science
Publishers
Heart
and Lung Institute
grant
HL 12740
Drive, 415 Ross Hall. Columbus.
B.V. (Biomedical
Division)
Ohio 43205,
Hirsch, 1983). Recent advances in the rapid laboratory diagnosis of HSV infections have reduced detection turnaround times from an average of 5 to 7 days to less than 24 h by using direct antigen detecting methods. An advantage to direct antigen detection methods is that viable virus does not have to be present. Although many of the direct techniques yield results within 4 to 24 h (Gleaves. 1985; Landry, 1986). the sensitivity of the detection method is usually less than that of the viral isolation (Morgan, 1984) and technical variables are involved in distinguishing a positive from a negative specimen. Thus, tissue culture is still the standard by which other detection methods are compared (Hughes, 1986; Landry. 1986). In this study we examined the effect that rolling had on the number of HSV foci. Foci formation was determined in cultures that were either rolled at 2 revolutions per minute (rpm) or held stationary. Our objective was to determine if rolling would enhance the number of HSV cell foci in cultures inoculated with clinical specimens. If more foci were produced with rolling conditions, then HSV detection should be easier. In addition to investigating foci formation, we also determined the effect that rolling had on viral yield at 23-96 h post inoculation for cultures inoculated with clinical specimens of HSV.
Materials
and Methods
For this study, specimens from both genital and non-genital sites were collected on swabs, placed in transport medium, and stored at refrigerated temperatures until delivered to the laboratory within 24 h. After receiving the specimens in the laboratory, 0.2 ml aliquots were inoculated into duplicate culture tubes of human diploid fibroblast cells (Flow 6000). One culture was held stationary at 35°C while the other was rolled at 2 rpm. All cultures were observed daily for HSV cytopathic effect (CPE) and the number of HSV foci was determined. Four clinical specimens, two below the waist and two above the waist were randomly chosen and serial dilutions were prepared. One hundred ~1 of each viral preparation were inoculated into F6000 cells and scored daily for CPE. After 24, 48, 72 and 96 h post-inoculation, infected cultures were frozen at -70°C. The same serial dilutions for each test condition were then used for a viral yield assay so a comparison could be made between rolled and stationary cultures for the number of plaque forming units (PFU) produced. For viral yield assays, 12-well plates were seeded with African green monkey kidney cells (BGM 70) and incubated at 37°C in 5% CO* and 95% relative humidity. Monolayers were then infected with serial dilutions from the two conditions (2 rpm and stationary). At the end of a 2 h adsorption period, agarose overlay was added and cultures were incubated again for 48 h. At the end of the incubation period, cultures were fixed with 10% formalin buffered with PBS and then stained with 10% crystal violet. Plates were examined microscopically and the number of PFUs was determined.
97
Results Thirty-four specimens inoculated into F6000 were examined for the number of infected cells foci. Out of 34 specimens, 27 resulted in cultures with countable foci. Forty-four percent of all inoculated cultures were positive for foci by day one and by day two 94% of the cultures were positive. All of the rolled cultures with TABLE Effect
1 of rolling
Specimen
Number
on herpes
simplex
virus (HSV) Number
foci formation
in Flow 6000 cells
of Foci
Fold Increase
Rolling
Stationary
6-3543 6-3639 6-3659 6-3693 6-4543 6-4913 6-4917 6-5193 6-5340 6-5347 6-5413 6-5441 6-5697 6-5740 6-5746 6-5757 6-5765 6-5786 6-5807 6-6003 6-6113 6-6283 6-6318
18 12 9 13 13 12 90 35 17 3+” 2+ 14 12 5 10 ND” 28 28 2 49 18 27 TNC’
6-6336 6-6415 6-642 1 6-644 1 7-0046 7-0063 7-0108 7-0115 7-0611 7-0725 7-1323
18 12 TNC 11 15 13 12 16 TNC TNC 29 15.5”
8 4 2 2 6 6 40 16 8 20 0 7 8 1 6 8 10 16 0 36 6 14 TNC 11 7 TNC 2 7 8 8 11 4 11 9 6.7
” l-4+ represents 25-1000/o infected cells. h ND-Not Done or Not Determined. ’ TNC-Too Numerous to Count, but less than ’ Geometric mean.
1+
in Foci
2.2 3.0 4.5 6.5 2.2 2.0 2.2 2.2 2.1 ND ND 2.0 1.5 5.0 1.6 ND 2.8 1.8 ND 1.4 3.0 1.9 ND 1.6 1.7 ND 5.5 2.1 1.6 1.5 1.4 ND ND 3.2 2.3
98 TABLE
2
Mean fold increase tionary cultures Specimen”
1 2 3 4
in PFU
for herpes
simplex
virus isolates
Mean fold increase
for cultures
rolled
at 2 rpm versus
sta-
in
Foci
PFU
5.0h 2.1 2.4 3.2
7.2’ 6.6 10.5 10.5
a Results from four clinical specimens obtained from above the waist (oral-1,2) and below the waist (genital-3,4). ’ This represents the mean fold increase of infected foci between rolled and stationary conditions for 4 infected cultures. ’ This represents the mean fold increase of PFU for 4 infected cultures using 3 wells for each plaque assay.
countable foci had a greater number of foci when compared to companion stationary cultures with a geometric mean fold increase in foci of 2.3 (Table 1). A significant enhancement in foci numbers was seen between rolled and stationary cultures (P < 0.01, t-test). Four frozen clinical specimens were diluted, inoculated into Flow 6000 cells and scored for CPE, at 24 and 48 h post-infection. Specimens that were rolled showed a greater number of foci when compared to the control (stationary) cultures. The difference in foci development was more dramatic at 72 and 96 h. Specimens that were rolled showed a geometric mean fold increase in foci of 3.0 when compared to stationary cultures (Table 2). Plaque assay results correlated with foci results. Significantly more PFUs were produced in cultures that were rolled than in stationary cultures. The geometric mean fold increase in PFU between rolled and stationary cultures was 8.7.
Discussion The use of centrifugation to inoculate monolayers has been studied previously (Sharp, Smith 1960; Padgett, Walker 1962; Osborn, Walker 1968; Hudson et al. 1976; Tenser 1978; Tenser, Dunstan 1980; Gleaves et al. 1984) and recent studies (Gleaves et al, 1985; Salmon et al, 1986) demonstrated that low speed centrifugation increased the diagnostic sensitivity for HSV. Tenser (Tenser 1978) reported almost a lo-fold increase in HSV sensitivity, with laboratory strains, after low speed centrifugation (1100 x g for 10 min) and a nearly 100-fold increase with ultracentrifugation (28 000-45 000 X g for 1.5-2.3 h). Gleaves et al. (1985) reported that low speed centrifugation (700 x g for 40 min) probably enhances adsorption of HSV onto monolayers, and when centrifugation was used in conjunction with immunofluorescence, positive detection and serotyping of HSV was possible 16 h postinoculation. Salmon et al. (1986) have recently demonstrated an increase in the
99
number of HSV foci when low speed centrifugation (3500 x g for 15 min) was used but observed no significant difference in sensitivity of HSV detection with clinical specimens. For murine cytomegalovirus (MCMV) Osborn and Walker (1968) detected 10 to 100-fold more PFU when assayed by centrifugation at 1900 x g for 10 min compared to inocula assayed without centrifugation. Similar results were not observed for HSV or pseudorabies virus preparations. Human cytomegalovirus also undergoes centrifugal enhancement of infectivity (Hudson et al., 1976) and detection of human CMV by using low speed centrifugation has diagnostic applications (Gleaves et al., 1984). To our knowledge no reports have been published comparing the foci formation and viral yields produced by HSV when cultures were continuously rolled at 2 rpm speed. In this study we demonstrated that rolling at 2 rpm increased the number of HSV foci as well as the viral yields. This should aid in the earlier and easier detection of HSV in cultures inoculated with clinical specimens. The enhancement in the number of HSV foci may be attributed to several factors such as: 1) the physical transfer of virus or virus-infected cells to noninfected cells by rolling, 2) an increase in viral adsorption by rolling, or 3) activation of host cells for enhanced viral replication. In recent experiments, high speed rolling (96 rpm) was superior to slow speed rolling (2 rpm) and stationary conditions, not only for increasing the number of HSV foci but for decreasing the detection time as well. Cultures that were rolled at 96 rpm became positive sooner than cultures that were rolled at 2 rpm or stationary. There was also a decrease in the time required for rolled cultures to reach maximum CPE development. At the present time it is not known how much force or for how long it is necessary to enhance viral replication. We suggest that rolling of inoculated cultures should be used in the clinical virology laboratory to aid in the rapid detection of HSV and possibly other viral pathogens.
References Gleaves, C.A., Smith, T.F., Shuster, E.A. and Pearson, G.R. (1984) Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen. J. Clin. Microbial. 19, 917-919. Gleaves, C.A., Wilson, D.J., Weld, A.D. and Smith, T.F. (1985) Detection and serotyping of herpes simplex virus in MRC-5 cells by use of centrifugation and monoclonal antibodies 16 h postinoculation. J. Clin. Microbial. 21, 29-32. Hirsch, MS. and Swartz, M.N. (1980) Antiviral agents. N. Engl. J. Med. 302, 903-907; 949-953. Hirsch, M.S. and Schooley, R.T. (1983) Treatment of herpesvirus infections. N. Engl. J. Med. 309, 963-970;103+1039. Hudson, J.B., Misra, V. and Mosmann, T.R. (1976) Cytomegalovirus infectivity: analysis of the phenomenon of centrifugal enhancement of infectivity. Virology 72, 235-243. Hughes, J.H., Mann, D.R. and Hamparian, V.V. (1986) Viral isolation versus immune staining of infected cell cultures for the Laboratory Diagnosis of Herpes Simplex Virus infections. J. Clin. Microbiol. 24, 487-489. Kilbrick, S. (1980) Herpes simplex infections at term. J. Am. Med. Assoc. 243, 157-160.
100 King, D.H. and Galasso, G. (eds.) (1982) Proceedings of a symposium on a cyclovir. Am. J. Med. 73. l-329. Landry, M.L., Zibello, T.A. and Hsiung, G.D. (1986) Comparison of in situ hybridization and immunologic staining with cytopathology for detection and identification of herpes simplex virus infection in cultured cells. J. Clin. Microbial. 24, 968971. Morgan, M.A. and Smith, T.F. (1984) Evaluation of an enzyme-linked immunosorbent assay for the detection of herpes simplex virus antigen. J. Clin. Microbial. 19, 730-732. Osborn, J.E. and Walker, D.L. (1968) Enhancement of infectivity of murine cytomegalovirus in vitro by centrifugal inoculation. J. Virol. 2, 853-858. Padgett, B.L. and Walker, D.L. (1962) Use of centrifugal force to promote adsorption of myxoma virus to cell monolayers. Proc. Sot. Biol. Med. 111, 364-367. Salmon, V.C., Turner, R.B., Speranza, M.J. and Overall, J.C. (1986) Rapid detection of herpes simplex virus in clinical specimens by centrifugation and immunoperoxidase staining. J. Clin. Microbiol. 23, 683-686. Sharp, D.G. and Smith, K.O. (1960) Rapid adsorption of vaccinia virus on tissue culture cells by ccntrifugal force. Proc. Sot. Biol. Med. 104, 167-169. Tenser, R.B. (1978) Ultracentrifugal inoculation of herpes simplex virus. Infec. Immun. 21. 281-285. Tenser, R.B. and Dunstan, M.E. (1980) Mechanisms of herpes simplex virus infectivity enhanced by ultracentrifugal inoculation. Infec. Immun. 30, 193-197.