Typing of Isolates of Herpes Simplex Virus by Mixed Agglutination

Abstract

The characteristics of herpes simplex virus (HSV) type 1 surface antigen(s) appearing on infected cells have been studied in our laboratory by the method of mixed agglutination (MA) (1). Since it has been demonstrated by membrane immunofluorescence (2, 3) that type specificity may be shown for surface antigens, our studies were extended to characterization of HSV type 2 surface antigen and the use of MA for typing HSV isolates. The results of this investigation are the subject of this communication.Materials and Methods. Cell cultures. BIRK, a continuous line derived from rabbit kidney cells, and HEp-2, were used for detection of HSV surface antigen (1). Cells were grown in Eagle's basal medium (BME) in a base of Hank's balanced salt solution supplemented with 10% fetal calf serum, penicillin (200 units/ml) and streptomycin (200 pg/ml) . After virus inoculation, cultures were maintained in BME in a base of Earle's saline (sodium bicarbonate 1.8 mg/ml), 3% fetal calf serum, and antibiotics. BGM, a continuous line derived from African green monkey kidney cells, was used for assay of infectivity (4). Cells were grown in Eagle's minimal essential medium (MEM) in Earle's saline and maintained in MEM with 3% fetal calf serum following virus inoculation.Viruses. Herpes simplex virus (HSV) type 1 strains were: MacIntyre VR# 3 ( 1 ), F and MP (refer Results) supplied by Dr. €3. Roizman, University of Chicago, Chicago, Ill., and H F propagated in this laboratory. Type 2 strains were: MS ( l ) , and 69-375 from the Erie County Virology Laboratory.Isolates of HSV were supplied by the Erie 41 County Virology Laboratory. Laboratory strains were plaque-purified twice in BGM and passaged once in BIRK. Most HSV isolates were employed after 1-3 passages in BIRK or HEp-2.Sera. Rabbit antisera to HSV type 1 (VR#3), #284, and type 2 (MS), #274 have been previously described ( 1).Absorption of sera. Sera were absorbed with BGM cells infected with either type 1 or type 2. Cells were infected with approximately 0.1-0.3 PFU/cell and incubated at 36" for 24-48 hr. Infected cells were collected by scraping, and after washing with phosphate buffered saline (PBS) pH 7.2, a cell pack was obtained by centrifugation at 2,000 rpm for 15 min. One ml of a 1 : 20 dilution of antiserum was mixed with approximately 5 X lo7 packed infected cells in a volume of approximately 0.1 ml and incubated at 36" for 2 hr followed by further incubation overnight at 5". The serum was removed following centrifugation and absorption was repeated a second time. The absorbed serum was heated at 56" for 20 min to inactivate any infectious virus. Mixed agglutination ( M A ) .The method used has been reported in detail elsewhere (1, 5 ) . The attachment of rabbit anti-HSV antibodies to infected cell cultures was revealed by the indicator system. In the present experiments the indicator cell suspension was prepared with sheep erythrocytes (SRBC) + rabbit anti-SRBC serum + sheep anti-rabbit globulin serum, Grand Island Biological Company, Grand Island, N. Y. The sheep anti...

Identification and typing of herpes simplex virus by enzyme immunoassay with monoclonal antibodies

“…A variety of techniques have been used for typing herpes simplex virus (HSV) isolates. Serological techniques include microneutralization (27,32), immunofluorescence (IF) (11,13,23), immunoperoxidase staining (3,12), indirect hemagglutination (4), mixed agglutination (14), radioimmunoassay (10), enzyme immunoassay (36), immunoelectroosmophoresis (15), counterimmunoelectrophoresis (16), and complement-dependent cytotoxicity (26). Several nonserological techniques have been developed which distinguish the viruses by restriction endonuclease (RE) analysis of virus DNA (6,20), by nucleic acid hybridization analysis (5), or on the basis of various biological and biochemical differences in virus replication (18,21,25), including sensitivity to bromovinyl deoxyuridine (22).…”

Comparison of biological, biochemical, immunological, and immunochemical techniques for typing herpes simplex virus isolates

“…A variety of serological techniques, including microneutralization (20), immunofluorescentantibody staining (5,14), immunoperoxidase staining (2), enzyme-linked immunosorbent assay (12), indirect hemagglutination (3), mixed agglutination (7), counterimmunoelectrophoresis (9), immunoelectroosmophoresis (8), radioimmunoassay (19), complement-dependent cytotoxicity (16), and monoclonal antibody analysis (17) has been used to distinguish between the two types of herpes simplex virus (HSV). Other than the monoclonal antibody analysis, all procedures require special adsorption of sera to remove cross-reacting antibodies; thus, high-titer type-specific antisera are not easily obtained.…”

Rapid typing of herpes simplex virus isolates by deoxyribonucleic acid:deoxyribonucleic acid hybridization

“…Other typing methods have utilized such serological techniques as indirect hemagglutination inhibition (2), microneutralization (34,37), mixed agglutination (13), immunofluorescence (10,23,27,28), solid-phase radioimmunoassay (9), immunoelectroosmophoresis (14), indirect immunoperoxidase staining (1), enzyme-linked immunosorbent assay (25), and complement-dependent cytotoxicity (33). The widely used immunofluorescence techniques are difficult to quantitate and are subject to observer bias, and all of the serological assays are complicated by the heterologous activity of "type-specific" antisera, a problem which is not unexpected in light of the many antigens shared by HSV-1 and HSV-2 (10,29,30,35).…”