Biomedical subjects
R Wigand
Publications and source records attributed to R Wigand.
Adenine arabinoside inhibition of adenovirus replication enhanced by an adenosine deaminase inhibitor.
The inhibition of adenovirus multiplication by adenine arabinoside was determined by yield reduction in one-step multiplication cycle. Inhibition was greatly enhanced by an adenosine deaminase inhibitor (2-deoxycoformycin) in concentrations down to 10 ng/ml. Adenovirus types from four subgroups showed similar results. However, the enhancing effect of adenosine deaminase inhibitor was great in HeLa cells, moderate in human fibroblasts, and negligible in Vero cells. This difference could be explained by different concentrations of adenosine deaminase found in cell homogenates.
[Laboratory procedures in adenoviruses. VI. Neutralization in HeLa cell cultures by macro and micro methods (author's transl)].
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[Virological diagnosis of epidemic keratoconjunctivitis (author's transl)].
Among 180 patients with proved adenovirus infection of the eye, 144 infections with adenovirus 8 (Ad 8) were found. 11 or 7 patients were infected with Ad 19 or Ad 4 respectively; further 18 cases were associated with 6 other serotypes. The main symptoms of epidemic keratoconjunctivitis (EKC) (pronounced inflammatory swelling of the plica and caruncula, nummular corneal infiltations) are not confined to Ad 8 infections. For the virological diagnosis, a combination of virus isolation and serology is recommended. Virus isolation is mostly successful from conjunctival swabs obtained during the first week of the disease. For serological investigation, the first blood specimen should be taken as early as possible, the second not earlier than 15 days after onset of disease. The mere application of the group-specific adenovirus complement-fixation is insufficient; it should be supplied by neutralization and hemagglutination-inhibition with Ad 8 (or Ad 19) virus, although these reactions are not invariably type-specific. A swifter diagnosis of the adenovirus infection by immunofluorescence performed in cell cultures inoculated with conjunctival material, 3 or 7 days after inoculation, was often successful; however, this procedure proved to be less sensitive than virus isolation.
Relationship of human adenoviruses 12, 18, and 31 as determined by hemagglutination inhibition.
Adenoviruses 12 and 31, but not Ad18, agglutinate rat blood cells at high titer, providing suitable blood cells be available and a prolonged contact period of virus with the erythrocytes is allowed. Purified virus particles show direct, and virus-free supernatants show direct and indirect, hemagglutination, ie, enhancement of HA by heterologous antiserum. Hemagglutination inhibition with rabbit antisera shows cross-reactions between Ad12 and Ad31 with titers 4--32 times lower than with homologous antigens; Ad18 antisera react with antigens from both of the other serotypes. No cross-reactions were seen with antisera from other adenoviruses. This suggests an antigenic relationship of the three viruses of subgroup IV in their fiber antigen gamma, in addition to the known relation in the hexon (epsilon), which is apparent in cross-neutralization.
The susceptibility of Vero cell cultures for human adenoviruses.
Human adenoviruses 1 to 28 were shown to produce a cytopathic effect in Vero cell cultures. Viruses of subgroups III and IV (Ad 1, 2, 5, 6, 12, and 18) were readily passaged in Vero cell cultures and were produced in high amounts. This was also found for Ad 11, 16, and 21, while Ad 3, 4, and 7 showed a lower degree of multiplication and Ad 14 could not be passed serially. For Ad 8, 26, 27, 20, 25, and 28, a multiplication in Vero cells could not be proved, while the remaining serotypes of subgroup II showed a moderate degree of multiplication. The sensitivity of Vero cells to small amounts of virus was lower than that of HeLa cells. No adaption of adenoviruses to Vero cells after 5 Vero passages was observed. Attempts to enhance virus multiplication by coinfection with SV40 failed.
Re: "the Seattle Virus Watch. VII. Observations of adenovirus infections".
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Amino acid requirement of adenovirus multiplication.
The absence from the medium of any of the 13 amino acids essential for cell growth has an inhibiting effect on the multiplication of adenovirus 9-15 and adenovirus I in HeLa cell cultures. The inhibition is accentuated by previous amino acid starvation of the cultures. Whereas with arginine deprivation, the arginine pool inside the cells is at a minimum within 30 min, the cells are assumed to adapt slowly to the new metabolic state, which is characterized by an increased 'turnover' of protein synthesis. With arginine deficiency and in Hanks' BSS some synthesis of virus and capsid proteins takes place. Quantitative and possibly qualitative differences between the influence of the various deficient media were observed. The experiments rule out DNA synthesis as a primary cause of the amino acid deficiency effect. They lead to the hypothesis that arginine deficiency inhibits the formation of an essential protein which is synthesized very late in the infectious cycle under complete MEM.
Combined antiviral effect of DNA inhibitors on adenovirus multiplication.
9 purine or pyrimidine analogues or other inhibitors of DNA synthesis were studied on their single and combined inhibitory effect on the multiplication of adenovirus 9--15 in HeLa cell cultures. The criterium of inhibition was the total suppression of the formation of infectious virus. Inhibiting concentrations were derived from isobolograms. 34 of 36 drug pairs acted in a synergistic manner, one pair showed additive and one no interaction.
[Laboratory procedures in adenoviruses. V. A comparison of three group-specific serological reactions (author's transl)].
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Biological and biophysical characteristics of mouse adenovirus, strain FL.
Mouse adenovirus, strain FL (MAV), is best propagated in primary rather than secondary cultures of mouse kidney cells. Virus release from the cells into the medium is fairly efficient; the average yield per cell is about 1000 TCID50. Morphological and other biophysical characteristics are those of a typical adenovirus: the icosahedral shell with a diameter of 74 nm, fiber projections of 29 nm length, its localization inside the infected kidney cells, a buoyant density in CsC1 of 1.34 g/ml, inhibition of multiplication by inhibitors of DNA synthesis. The thermostability is higher than that of human adenoviruses, whereas, in contrast to these, MAV is inactivated to a great extent by trypsin. Complete or incomplete hemagglutinin or toxin-like activity were not detected. The virus shows no cross-neutralization with human adenoviruses and a one-side cross-reaction with another murine adenovirus strain K-87. Soluble complement-fixing antigen of MAX exhibits a sedimentation rate of 12S identical to the hexon component of human adenoviruses; both antigens show a partial antigenic relationship in tests with appropriate antisera.
The kinetics of adenovirus infection and spread in cell cultures infected with low multiplicity.
The cause for the long "incubation period" required till the appearance of the CPE in cell cultures inoculated with small amounts of adenovirus was investigated with adenovirus type 5 in HeLa cell cultures. The spread of virus in a culture from cell to cell is minimal, as shown in cultures with antiserum in the medium. The spread by spontaneously released virus via the medium is more important. It can be accelerated by repeated subcultivation after freezing and thawing the cells in 2 or 3 days' intervals. The quantity of virus produced by one infected HeLa cell was found to be 200 TCID50 within 48 hours, independent of the MOI. The growth cycle too is largely independent of the virus dose. The data suggest that the long duration of the "incubation period" is fully explained by a burst size of 200, a cycle length of 40 to 48 hours and the assumption of a slow and steadily working process of spontaneous release of virus into the medium. Some other possible causes, like impurities in the inoculum or slow and asynchronous early stages of infection, have been ruled out by appropriate experiments.
[Laboratory procedures in adenoviruses. IV. The influence of antiglobulin and complement on neutralization and hemagglutination-inhibition (author's transl)].
The neutralization of adenovirus 3,7, and 19 by rabbit antisera is enhanced by additional application of antirabbit globulin. All sera investigated, obtained early or late after the start of immunization, showed two- to sixfold titer rises. Complement also enhances the neutralization; the titer is raised to a greater extent in early than in late sera. To achieve maximal effects, antiglobulin and complement had to be used in fairly high concentration (ca. 10%). In hemagglutination-inhibition the same set of sera showed a titer increase mediated by anti-rabbit globulin (mostly by the factor 2 to 4), while complement was without influence. The neutralization titer of human sera from patients with adenovirus infections, as opposed to the rabbit antisera, was only slightly increased by addition of anti-human globulin or complement. These reagents had likewise negligible influence on the hemagglutination-inhibition of adenovirus 8 by human sera.
Pardoxical cytotoxic effect of antisera used in diagnostic virology. Brief report.
Rabbit antisera prepared against adenovirus-infected or uninfected HEK cells showed a high degree of cytotoxicity towards HeLa and other human cell cultures, while antisera prepared against infected HeLa cells did not. The toxic activity was independent of complement; it could be removed by adsorption to HEK monolayers.
Does adenovirus subtype 7a exist?
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Adenoviridae.
The elevation of the genus Adenovirus to a family named Adenoviridae is described. This family at the present stage contains two genera, mammalian adenoviruses--Mastadenovirus--and avian adenoviruses--Aviadenovirus. The two genera are separated primarily on the basis of difference in immunological properties of the virions.
[A comparison of two tests for rheumatoid factor: latex test and l-agglutination (author's transl)].
Positive and negative reactions of latex test (LX) and streptococcal L-agglutination (LA) were correlated significantly with one another. However, in approximately 70% of the sera tested only one test was positive, LX at a much higher rate than LA. Postive rheumatoid factor tests were found in 117 patients with rheumatoid arthritis and in 595 patients with other diseases, exclusive of connective tissue diseases. Among the seropositive patients with rheumatoid arthritis, a positive reaction was seen in 85% and 71% by LX or LA respectively. Patients with nonrheumatic diseases showed positive reactions at a rate of 3,1%, with 2,3% and 1,7% in LX or La respectively. This shows that LA is less sensitive, but more specific as compared with LX. With increasing age of the patients, the rate of "nonspecific positive" reactors in patients with nonrheumatic diseases increases, more strongly for LX than for LA. The distribution of positive tests (LX positive, LA positive, or both positive) was analyzed in various groups of patients. The rate of positive reactions in both tests is significantly higher than patients with rheumatoid arthritis; it further increases with the severity of the disease. In cases with positive CRP, the rate of sera positive in both tests is higher, equally in patients with and without rheumatoid arthritis. In patients with nonrheumatic diseases an elevated serum gamma-globulin is associated with positive reactions in both tests and with positive LX. In all other grouping of patients, the rheumatoid factor tests did not yield significant deviations from chance distribution, as determined by chi2 analysis.
Laboratory procedures in adenoviruses. III. Sensitivity and specificity of serological tests (author's transl).
84 paired sera from patients with adenoviral infections were tested in neutralization and hemagglutination-inhibition (HI) against the homologous as well as against heterologous virus types of the respective subgroup. Only titer rises are considered as positive reactions. Sera of patients being infected with adenoviruses of subgroup I viruses (52 pairs, types 7, 3, 4, 14) exhibit high rates of positive reactions in the group-specific complementfixation (CF) and in homologous neutralization, lower rates in homologous HI. On the other hand, heterologous reactions are seen more frequently in neutralization than in HI. Patients with type 4 infections often show antibody responses with viruses of subgroup III (types 1, 2, 5, 6). In infections with these virus types, homologous antibody response is seen in approximately half of the cases only; heterologous reactions are observed rarely. Patients with type 8-infections (25 pairs) almost invariably show positive homologous neutralization, while HI or CF tests often fail to show rising titers. These patients, as well as 2 patients with type 19-infection, exhibit a remarcable high rate and range of cross-reactions in neutralization as well as in HI with numerous types if subgroup II and, to a lesser extent, with viruses outside of subgroup II. Although the relatively late appearance of type-specific antibodies (usually between the 10 th and 14 th days after onset of disease) is advantageous for the demonstration of antibody rises, the type-specific diagnosis of adenoviral infections should preferably be performed by isolation and typing of the virus. The heterologous reactions appear to be based on the presence of overlapping intertypic antigenic determinants rather than on an anamnestic antibody response.