Biomedical subjects
Publications and source records attributed to N J Schmidt.
High-titered, sensitive, and stable complement-fixing antigens for human cytomegalovirus were consistently produced from human diploid fibroblast cells infected at a high multiplicity of virus, harvested after 7 days of incubation, and sonically treated immediately in 0.1 M glycine buffer, pH 9.5.
This paper describes the preparation of seven combination pools of equine antisera, designated J though P, for identification of 19 coxsackievirus A immunotypes. Each pool is composed of 4 to 6 antisera; the serotypes included are A1-6, 8, 10-15, and 17-22. These pools, unlike the previously prepared A-H enterovirus pools, were lyophilized from volumes of 0.5 ml dispensed into 5-ml vials, and when rehydrated with 5 ml of diluent provide 50-antibody-unit material ready for use in identification tests without further dilution. Procedures for using the antiserum pools are given, and guidance is provided for interpreting the results of serum neutralization tests in identifying field isolates.
Acute hemorrhagic conjunctivitis (AHC), a new disease entity first reported in 1969, has since become pandemic throughout the world. In Tunisia during an epidemic in 1972 to 1973 we studied 25 cases of AHC, which were characterized by explosive onset of lid edema, chemosis, conjunctival hemorrhages, follicular hypertrophy, and epithelial keratitis. Clinical signs peaked in 48 hours and cleared without sequelae in five to seven days. Paired acute- and convalescent-phase sera from six of the patients showed a rising titer of neutralizing antibody to the prototype strain of AHC virus (J6 70/71), which was isolated in Japan. Two viral isolates from Tunisian patients were also antigenically related to the Japanese strain, indicating that a single etiologic agent (a new member of the picornavirus group) is probably responsible for the current pandemic of AHC.
Systematic studies on the replication of varicella-zoster virus in infected human fetal diploid lung cells have defined more optimal conditions for infection and harvesting of cultures and have led to the production of cell-free virus preparations with infectivity titers of greater than or equal to 10(6) plaque-forming units per ml. The highest yields of cell-free virus were obtained by (i) sonic treatment of the cellular phase of cultures inoculated with trypsin-dispersed infected cells at ratios of 1 infected cell to 6 to 10 uninfected cells in the monolayer and (ii) harvesting cells after 24 to 36 h of incubation at 36 degrees C. At this time the cultures showed minimal viral cytopathic effect. Spread of infectivity occurred much more rapidly in cultures inoculated with whole infected cells than in those infected with cell-free virus. Complement-fixing antigens with improved titers of greater than or equal to 1:128 were prepared from varicella-zoster virus-infected cell cultures in the same manner as cell-free virus, but harvested after 3 to 4 days of incubation when the cultures showed an advanced cytopathic effect.
Hamsters immunized with human cytomegalovirus (CMV) concentrated and purified by polyethylene glycol precipitation and density gradient centrifugation produced antisera with high titers of specific viral antibody, and which showed no significant reactivity with human host cell components. The antisera had high titers of CMV antibody in complement fixation, indirect fluorescent-antibody (FA), and neutralization tests, but titers obtained by indirect radioimmunoassay (RIA) were markedly higher. The antisera were used to follow the development of CMV antigen in infected host cells by indirect RIA and indirect FA staining. Virus-specific antigen was first detectable by RIA at 8 h after infection, and by FA staining at 16 h; cells contained optimal amounts of antigen for RIA and FA assays at 72 to 100 h postinfection. Immune globulins from the antisera were labeled with 125I for use in direct RIA. The labeled globulins gave highly specific reactions with CMV-infected cells, including those infected with low-passage isolates, and showed no reactivity with cells infected with other human herpesviruses or certain other human viruses.
Foci of cells infected with human cytomegalovirus were noted to stain more intensely than uninfected cells with neutral red, and this provided the basis for development of a plaque assay and plaque reduction neutralization test for cytomegalovirus. Plaques demonstrable by neutral red staining could be counted at 8 days after infection; thus, results could be obtained earlier than for plaque assay systems based upon the viral cytopathic effect, a fewer manipulations were required for staining cell monolayers to demonstrate plaques. Certain variables affecting plaque size and numbers and antibody titers were defined. Addition of fresh guinea pig complement to the reaction mixtures markedly enhanced cytomegalovirus-neutralizing antibody titers of hyperimmune animal sera, but titers of human sera were enhanced only two-or fourfold.
An indirect solid-phase radioimmunoassay (RIA) was applied to titration of serum and cerebrospinal fluid (CSF) antibodies against a variety of viruses including rubella, mumps, measles, herpes simplex, varicella-zoster, and vaccinia. The test used fixed, virus-infected cells as a source of antigen, and conditions for optimal production of viral antigen were determined for each virus-host cell system. In acute, uncomplicated viral infections, sera taken 2 to 5 days after onset generally had low homotypic RIA titers ranging from less than 1:100 to 1:500, whereas convalescent-phase titers ranged from 1:128,000 to 1:512,000. Rubella and measles antibody titers as high as 1:256,000 were demonstrated by RIA in CSF from patients with chronic panencephalitis, whereas homologous antibody titers of 1:4,000 were detected in CSF from acute mumps, herpes simplex, and varicella-zoster virus infections with central nervous system involvement. Some heterotypic antibody was demonstrable by RIA in CSF, but, with the exception of herpes simplex antibody in a mumps virus infection, titers were markedly lower than those to the infecting virus type. RIA generally demonstrated titers at least 1,000 times higher than those obtained by conventional assays such as complement fixation, hemagglutination inhibition, neutralization, and immunofluorescent staining.
The BGM line of African green monkey kidney cells was less sensitive than primary rhesus monkey kidney cells or human fetal diploid kidney cells for isolation of certain echoviruses and adenoviruses from fecal specimens.
The Delta herpesvirus (DHV) which produced outbreaks of exanthematous disease in patas monkeys was shown to have a close immunological relationship to varicella-zoster (V-Z) virus of man. Immunization of rhesus monkeys with DHV or V-Z virus resulted in the development of neutralizing antibodies to both viruses and also in the production of complement-fixing antibodies to V-Z virus. Immunoglobulin M neutralizing antibody to V-Z virus was demonstrated in the serum of a rhesus monkey immunized with DHV, suggesting a primary antibody response rather than recall of antibody to a related virus. Convalescent-phase sera from human zoster cases had comparable levels of neutralizing antibody for both DHV and V-Z virus. Patas monkeys involved in an outbreak of DHV infection showed seroconversion to both DHV and V-Z virus by neutralization tests and to V-Z virus by complement fixation tests. The demonstration of the close antigneic relationship between DHV and V-Z virus suggests that DHV may be useful in an animal model system for studies on the latency and reactivation of V-Z virus.
Neutralization of varicella-zoster (V-Z) virus by human sera and immune rhesus monkey sera was enhanced by fresh guinea pig complement. There was no marked difference in the degree to which complement enhanced neutralization by sera from current V-Z virus infections and sera from long-past varicella infections. Immunoglobulin G neutralizing antibody in sera from varicella cases was enhanced by complement to a slightly higher degree than was immunoglobulin M (IgM) antibody, and immunoglobulin G neutralizing antibody in immune monkey sera was enhanced to a much greater degree than was IgM antibody. There was a rapid decline in the complement requirement of IgM neutralizing antibodies over the course of immunization of the rhesus monkeys. V-Z neutralizing antibody titers in the presence of complement were higher than complement-fixing titers of the same sera in all groups of individuals studied. IgM neutralizing antibody for V-Z virus was demonstrable in all cases of varicella but in only 1 of 22 zoster cases, and V-Z IgM neutralizing antibody was not detectable in primary herpes simplex virus infections in which heterotypic antibody titer rises occurred to V-Z virus. Complement-fixing antibody for V-Z virus was absent in 19S serum fractions which contained IgM neutralizing antibody for the virus.
Studies comparing the enhancing effect of guinea pig complement on homotypic and heterotypic neutralizing antibodies produced in initial herpes simplex virus (HSV) infections of humans indicated that antibodies to HSV type 1 and HSV type 2 were enhanced to about the same extent, and there was no signigicant difference in the degree to which complement enhanced homotypic and heterotypic HSV-neutralizing antibody. Homotypic and heterotypic immunoglobulin G neutralizing antibodies were enhanced by complement to as great, or greater, an extent as immunoglobulin M (IgM) HSV antibodies in the same sera. In patients with initial HSV type 1 infections, the IgM neutralizing antibody response was type specific. On the other hand, patients with initial HSV type 2 infections produced both homotypic and heterotypic IgM neutralizing antibody. An initial HSV type 2 infection in an individual previously infected with HSV type 1 elicited the production of IgM neutralizing antibody to both HSV type 1 and HSV type 2. However, patients with recurrent HSV type 1 infections failed to produce IgM antibody to either HSV type during reactivation of the virus.
Antibody titers to cytomegalovirus were determined in 204 single-serum specimens and in 138 serum pairs, utilizing as antigen extracts of infected cells prepared by either freezing and thawing cells (FT antigen) or extracting them with a glycine buffer (GE antigen). With the serum pairs, 40% of the early sera and 44% of the late sera showed a fourfold or higher antibody titer with the GE antigen compared with those with the FT antigen, whereas 39% of the single sera had a fourfold or higher titer with the GE antigen. Sucrose density gradient centrifugation of the two antigen preparations indicated that the GE antigen contained two peaks of complement-fixing activity, one at a density of 1.18 to 1.22 g/cm3 and one at 1.06 to 1.10 g/cm3. The FT antigen had only one discernible peak of complement-fixing activity at 1.04 to 1.10 g/cm3. On electron microscopy, the more dense peak contained mainly nucleocapsids with some dense bodies and occasional enveloped virions, and the lighter peak was composed of amorphous material. Antibody titers obtained with the crude GE antigen and the more dense peak (designated viral antigen) were in agreement, whereas titers with the FT antigen agreed with those obtained with the lighter peak (soluble antigen). The antibody detected with both antigens was mainly immunoglobulin G.
The RD cell line, derived from a human rhabdomyosarcoma, supported replication of a number of group A coxsackieviruses, including types A5 and A6 which heretofore have been propagable only in suckling mice. A number of the group A coxsackievirus types which replicated in RD cells had higher titers in this cell line than in other cell culture systems. In tests on a limited number of clinical specimens, RD cells were slightly less sensitive than suckling mice for isolation of group A coxsackieviruses, but they did permit the recovery of certain virus types which previously could be isolated only in suckling mice. Group B coxsackieviruses replicated poorly or not at all in RD cells.
An indirect solid phase radioimmunoassay (RIA) was developed for typing antibody to herpes simplex virus (HSV) types 1 and 2 (HSV-1 and HSV-2) in human sera. The test is based upon absorption of sera with uninfected, HSV-1-infected cells and testing for residual antibody. The high sensitivity of the RIA method for detecting HSV antibody permits examination of sera at high dilutions, and thus relatively small volumes of virus-infected cells are required for cross-absorption of antibodies. Results obtained in RIA typing of HSV antibodies showed good agreement with those obtained by microneutralization and inhibition of passive hemagglutination. The HSV antibody type(s) determined by RIA also showed good correlation with the virus type isolated from the individual, either from clinical specimens or sensory nerve ganglia. The technique was very sensitive for detection and typing of HSV antibdodies in cerebrospinal fluids. The RIA method was highly suitable for detecting two types of HSV antibody in the same serum specimen, and it was possible to show that a marked, type-specific antibody response to HSV-2 does occur in individuals with a primary HSV-2 infection who have experienced a prior infection with HSV-1.
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Indirect hemagglutinating (IHA) antibody responses to Herpesvirus hominis types 1 and 2 (HVH-1 and HVH-2) were compared to complement-fixing and neutralizing antibody responses in immunized laboratory animals (rabbits, guinea pigs, and hamsters) and in natural infections of man. With the immunized animals, type specificity was seen only in the IHA test and only with antisera produced in hamsters and in the rabbits immunized with HVH-2. In human nongenital infections (considered to be caused predominately by HVH-1), IHA and neutralizing antibodies developed at about the same rate and reached approximately the same levels for HVH-1 and HVH-2. IHA titers tended to be higher than neutralizing antibody titers for both virus types. In genital infections (considered to be caused predominately by HVH-2), there was a rapid IHA antibody response to HVH-2, and the early HVH-2 antibody demonstrable by IHA, but not by neutralization tests, was found to be immunoglobulin M in nature. In genital infections, IHA titers for HVH-2 were markedly higher than neutralization titers, but there was no pronounced difference in neutralizing the IHA antibody titers for HVH-1. Several patients with genital infections fialed to develop IHA antibody for HVH-1. The IHA test possessed no greater sensitivity than did complement fixation or neutralization tests for serodiagnosis of HVH infections. Despite the fact that a number of patients with genital infections produced IHA antibody only for HVH-2, the test was no more effective than the neutralization test in providing a type-specific serodiagnosis of infection, due largely to the fact that the rapid IHA antibody response to HVH-2 prevented demonstration of a further, significant antibody titer increase in a number of cases.
Inhibition of the indirect hemagglutination reaction (IHA inhibition) was compared to several other methods for type-specific identification of Herpesvirus hominis (HVH) antibodies and isolates. The method appears to have the greatest value for typing antibodies for HVH type 1 and HVH type 2 in human sera; identification of antibody type was relatively simple and results were definitive. The IHA-inhibition test permitted serological diagnosis of HVH type 2 infection in three young adults with meningoencephalitis, thus extending the mounting evidence that nervous system involvement with this virus type is not limited to neonatal infections. II/I indexes of neutralizing or IHA antibody gave an accurate indication of the presence of HVH type 2 antibody in those sera containing type 2 antibody by IHA inhibition, but they indicated the presence of HVH type 2 antibody in one-half or more of the sera shown to contain only HVH type 1 antibody by IHA inhibition. For typing HVH isolates, the IHA-inhibition test gave results identical to those obtained by direct fluorescent-antibody staining using cross-absorbed conjugates, but the IHA-inhibition test was much more cumbersome and time-consuming to perform than was direct fluorescent-antibody staining. A microneutralization technique for virus typing also gave results identical to those obtained with direct fluorescent-antibody staining and IHA inhibition. However, typing HVH isolates by plaque size or the differential effect of incubation temperature was found to be less definitive and accurate.