A Chick-Embyo Cell Microtest for Typing of Herpesvirus Hominis

Abstract

Characteristics of plaque formation in cell culture of primary chick embryo (PCE), have been found useful as a biological marker in differ en t ia t ing Herpes virus hominis (H V H ) oral type 1 from genital type 2 strains (1, 2). Type 1 strains appear to or have been reported to produce small plaques and to have lower efficiency of plaque formation than type 2 strains in PCE cell culture. We describe a simple microtest for differentiation of HVH types based on the apparent differential sensitivity of PCE cell culture in microplates. The type of cytopathic effect (CPE) and the efficiency with which the virus infects the PCE monolayer are used as criteria for typing. The results of microtyping in PCE are compared with kinetic neutralization (K) and immunofluorescence (IF) typing.Materials and Methods. Tissue Culture. Ten per cent fetal calf serum in Eagle's minimal essential medium (MEM) with 100 IU/ml of penicillin, 100 pg/ml of streptomycin, 50 pg/ml of mycostatin, 6.6 mM sodium bicarbonate and 2.0 mM L-glutamine was used throughout.Primary chick embryo cells were obtained from minced 9-to 10-day old embryos by dispersion with 0.2% trypsin. Strains of golden Syrian hamster embryo cells (HE) and rabbit kidney cells (RK), originated in our laboratories. Human diploid fetal tonsil (FT) fibroblasts, were kindly supplied by Dr. Berttina Wentworth, Seattle. The passage numbers were as follows: HE 15-17, RK 1-3; and FT 15-25.Viruses. Herpesvirus hominis strains used in these experiments included four that were of known type and 106 clinical isolates from Taipei and Seattle. The reference strains were: UW-168 (3) and KOS (4), type 1; UW-268(3) and 196-2(4), type 2. The UW-168 and UW-268 strains had been passed approximately 50 times in HEp-2, HeLa and FT cells. Of the 106 clinical isolates, 59 were genital in origin; 57 were from the cervix of patients attending venereal disease clinics, and two were urinary isolates. The nongenital isolates included, 45 from throat swabs (from a study of respiratory infection), one from typical herpes labialis, and one from a brain specimen obtained at autopsy. The previously untyped herpes strains were in the 2nd through 10th passage at the time of typing, initially isolated in FT, HEp-2 or primary monkey kidney cells and passed in FT and/or HEp-2 cells. Virus pools for all strains were prepared in HE, RK or FT cells and have titers ranging from lO4mo to TCID50/ml as assayed in a microtest system using HE or FT cells. Two of the untyped clinical isolates, NHS-17 and 23, were passed an additional nine times in RK and then either ten times in FT (NHS-17) or 21 times in HE cells .Virus rnicrotyping. Serial tenfold dilutions of the stock virus to be typed were made in MEM. Diluted virus (0.05 ml) was delivered to two wells per dilution in each of two 324

A Micro‐Neutralization System for Detection of s‐CRN Antibody to Herpes Simplex Virus

“…Earlier tests employed monolayered cells, but the use of monodispersed cells (7) further simplified the technique. Reading of results has also become easy after adoption of naked-eye observation of CPE in stained cells (7).…”

“…In order to apply the detection of s-CRN antibody to routine practice, we adopted the micro-neutralization system of Yang et al (7) with modification, in which virus-serum mixtures were incubated at 4 C for 3 days and then given C and a suspension of monodispersed Vero cells in succesion. However, differing from CRN antibody, the titer obtained was much lower than expected from the result of the plaque reduction test.…”

Treatment of Experimental Herpesvirus Infections with Phosphonoformate and Some Comparisons with Phosphonoacetate

“…With the exception of the guinea pig infection, the pathogenesis of these experimental infections has been reported in detail in previous publications (8,11,12 genital lesions. These strains were typed using a modification of the chick embryo microtest as described by Yang et al (19). The origin and preparation of pools of the Smith strain of MCMV and vaccinia virus have been previously described (4,5).…”

Rapid Identification and Typing of Herpes Simplex Virus in Clinical Specimens by a Direct Microneutralization Test

“…It is also necessary to determine the type of HSV to trace the route of infection as well as to accumulate data for epidemiological analysis. Isolation of the virus from patients followed by a microneutralization test for viral identification and typing (8,12), if performed within a short period of time, would serve these purposes. The isolation rate seems to be nearly 100% when a suitable transport medium and adequate cells are used (9).…”