An analysis of the plaque assay of herpes simplex virus in chick embryo monolayers

Japanese Journal of Microbiology

1974

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Herpes simplex virus strains isolated from genital and nongenital sites were classified into type 1 (HSV-1) and type 2 (HSV-2) by endpoint neutralization tests using IgM of rabbits hyperimmunized with either HF (HSV-1) or UW-268 (HSV-2) strain. It was found that about one-third of the genital isolates belonged to type 1, in contrast to the general concept that HSV-2 represents genital herpes strains. These HSV-1 strains, differing from HSV-2, were mostly isolates from acute herpetic lesions of female patients with constitutional symptoms. On the other hand, all nongenital isolates except one were determined to be HSV-1. There was no intermediate type equally neutralizable by both types of IgM. A majority of the HSV-2 strains produced large plaques in chick embryo (CE) cells before passage through avian cells. In contrast, all HSV-1 strains failed to produce such large CE plaques even after serial passages through avian hosts.Herpes simplex virus (HSV) can be classified into two distinct types, HSV-1 and HSV-2, on the basis of antigenical analyses [2,6,8,9,15]. The correlation between the type and the site of isolation adds importance from the clinical and epidemiological viewpoints. It has been documented that HSV-1 is isolated mainly from nongenital and HSV-2 from genital lesions. For the latter, a mode of venereal transmission has been implied [9]. It was further suggested that HSV-2 might be associated with uterine cervical cancer [11][12][13].The primary purpose of the present study was to determine whether and how frequently HSV-2 could be isolated from genital herpes infections, notably of females, in this country. As the criterion on which to determine the types, Hampar et al's neutralization technique using type-specific hyperimmune rabbit IgM and complement [4] was employed.An additional purpose was to find a simple method for type determination as a substitute for the cumbersome serological procedure. The chick embryo (CE) cell plaque marker of Figueroa and Rawls [3] and Lowry et al [7] was thought suitable for this purpose, and all the isolates were tested for their ability to form plaques in CE cells. Results of these experiments are described in this communication. MATERIALS AND METHODSViruses. Specimens for virus isolation were collected in the gynecological ward of the University of Tokyo Hospital, Tokyo, the National Cancer Center Hospital, Tokyo, and the Department of Dermatology of Toranomon Hospital, Tokyo, by applying swabs to lesions, which were immediately rinsed in a maintenance medium containing appropriate concentrations of penicillin and streptomycin. The rinse was inoculated into Vero cell cultures. The maintenance medium used was Eagle's minimum essential medium (MEM) supplemented with 2% calf serum. In cases that typical cytopathic effects were noted the culture fluid was harvested and Requests for reprints should be addressed to Dr.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Typing of Herpes Simplex Virus Strains of Genital and Nongenital Origins

Kawana

Shinkai

Japanese Journal of Microbiology

1974

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“…Other strains used for typing experiments included fresh isolates from dermatological wards of several hospitals tested earlier (25) and those isolated by Dr. T. Kawana in the gynecological ward of the University of Tokyo Hospital. Quantitation of the above representative strains was done by plaque assay in primary chick embryo cells (26) and titers were expressed as plaque-forming units (PFU).…”

Section: Methodsmentioning

confidence: 99%

A New Microplate Neutralization Test for Typing of Herpes Simplex Virus

Tada

Microbiology and Immunology

1978

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“…Virus infectivity was assayed by plaque titration in primary chick embryo cells [27] and referred to as plaqueforming units (PFU).…”

Section: Methodsmentioning

confidence: 99%

Studies on the Neutralization of Herpes Simplex Virus

Japanese Journal of Microbiology

Morishima

1972

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A portion of the seed virus remained active even in the presence of excess antibody. With an early serum or relatively low concentrations of a hyperimmune serum, the presence of such unneutralizable virus was uncovered by the addition of complement. This unneutralizable persistent fraction (PF) was distinguished from sensitized virus surviving in insufficient antibody, because the latter was inactivated by a high concentration of antibody as quickly as the control virus. The level of the PF was constant regardless of the serum species, serum lot, dilution of serum, antibody class and complement requirement of antibody, being approximately 0.1% of the seed virus. Millipore filtration of the seed virus lowered this PF level to varying degrees depending upon the porosity. However, when filtered virus-serum mixtures were incubated at 37 C for a sufficient time and then filtered again, a portion of the unneutralized virus did pass a 0.22 membrane. Furthermore, virus sensitized with insufficient antibody showed no increase in resistance to complement after 10 hr incubation at 4 C. These results proved that viral aggregation was not the essential cause of the PF. It was confirmed, on the other hand, that the neutralization of virus by hyperimmune IgG followed a one-hit curve. Analysis of these data appeared to support Rappaport's postulation that the PF represented antibody-coated virus whose critical antigenic sites were all left unbound.