Cross Complement Fixation Tests Research Articles

Viruses isolated from reptiles: identification of three new members of the family Rhabdoviridae.

The growth of four viruses isolated from lizards in Brazil (Marco, Chaco, and Timbo viruses) and Australia (Almpiwar virus) was studied in a variety of continuous cell lines of mammalian, reptilian, amphibian, and piscine origin. Although replication was found in certain cell lines derived from the coldblooded species, cytopathic effect (CPE) was absent or minimal and growth was less than or equal to that in mammalian cells. Those observations appear to limit the value of poikilothermic cells for primary isolation of viruses from field-collected, cold-blooded vertebrates or arthropods that feed upon them. The four reptilian viruses were found to be naturally occurring temperature sensitive agents, with optima for growth of approximately 30 degrees C. Electron microscope studies showed three of the viruses (Marco, Chaco, and Timbo) to be new members of the family Rhabdoviridae. Marco virus particles were conically shaped and resembled bovine ephemeral fever virus, and two lyssaviruses (Kotonkan and Obodhiang). Chaco and Timbo viruses were cylindrical viruses resembling other rhabdoviruses with particle lengths longer than the prototype VSV. No serologic relationships were found in cross complement fixation tests between these viruses, Marco virus, and 34 other rhabdoviruses.

Antigenic relationships among several simian varicella-like viruses and varicella-zoster virus

Cross-neutralization and complement fixation tests demonstrated the immunological identity of the Delta herpesvirus, the 592S virus, the Liverpool vervet monkey virus, the herpesvirus of patas monkeys, and the Medical Lake macaque virus. These viruses were isolated from diverse outbreaks of varicella-like disease in simians and from various simian species. All of the simian viruses were shown to be related to human varicella-zoster (V-Z) virus, as evidenced by the fact that immunization of monkeys with each of the simian viruses elicited the production of both neutralizing and complement-fixing antibodies to V-Z virus. However, cross-complement fixation tests indicated that the simian viruses are not so closely related to V-Z virus as they are to one another. Varicella or zoster infections in humans produced neutralizing and complement-fixing antibody responses to each of the simian viruses; the responses were more marked in zoster infections than in varicella infections but, in most patients, antibody levels produced to the simian viruses were much lower than those to the homologous V-Z virus.

Serological relatedness of monkeypox, variola, and vaccinia viruses.

Closely related human and monkey orthopoxviruses were differentiated by serologic techniques. Antiviral sera were tested by immunodiffusion for reactivity against six different viral antigens prepared from either infected cell cultures or infected chorioallantoic membranes (CAMs) of embryonated eggs. Portions of each antiserum were separately absorbed with heterologous antigens from infected CAMs to remove common reactivity. The absorbed sera formed immunodiffusion precipitates with both types of antigen preparation and revealed specific-character differences that made it possible to classify the viruses as variola, vaccinia, or monkeypox. Cross-complement fixation tests were also used to examine the immunologic reactivities of antisera to detergent-treated, purified preparations of three orthopoxviruses. Only common reactivities were detected by this method, however, and differentiating reactivities were not observed.

Purification of complement-fixing antigens of Rickettsia sennetsu by ether treatment.

Soluble and particulate complement-fixing antigens of Rickettsia sennetsu were prepared from spleen suspensions of mice infected with the rickettsia and treated with cyclophosphamide. The medium used during purification of antigens, a solution consisting of equal volumes of phosphate-glutamate-sucrose buffer and veronal-buffered saline, was suitable for obtaining antigens with high titers and no anti-complementary activity. By heat treatment, it was demonstrated that the soluble antigen was heat labile and the particulate antigen was heat stable. The soluble antigen was precipitated by 80% saturation with ammonium sulfate. Cross-complement fixation tests using both soluble and particulate antigens revealed that there was no antigenic difference among strains of R. Sennetsu. On the other hand, no cross-activity was observed between R. sennetsu and R. orientalis.

Complement-fixation tests of Rickettsia orientalis with sodium deoxycholate treated antigen.

Complement-fixation tests of Rickettsia orientalis were performed by using antigens purified with sodium deoxycholate. Cross complement-fixation tests between antigens of the Gilliam, Ohzeki, Karp and Kato strains and immune mouse sera of these four strains and of the Kaneko strain revealed difference in antigenic structure among the Gilliam, Karp and Kato strains. The Ohzeki strain was identical with the Gilliam strain and the Kaneko strain was identical with the Karp strain. In the homologous system, complement-fixing antibodies of mice infected with R. orientalis began to appear on the 15th day after infection and rose rapidly in a few days, and reached maximum in 4–6 weeks after infection and 8 weeks at latest. Subsequently, the titers declined gradually and showed low titers on the 52nd week in spite of survival of rickettsia in mice. In the heterologous system among which antigenic structures were different, the titers rose somewhat later and declined earlier than those of the homologous system. It was confirmed that the antigen treated with sodium deoxycholate was utilizable in the specific serological diagnosis of scrub typhus, because convalescent sera from all ten patients with the disease showed significant rise of complement-fixing titers against the antigen.

Oncogenicity of the simian adenoviruses.

Five of 17 adenoviruses of rhesus or cynomolgus monkey origin induced tumors in newborn hamsters. The tumors appeared between 42 and 280 days after subcutaneous inoculation and had the general characteristics of lymphomas. The tumors were specific by cross-complement fixation tests. An adenovirus recovered from Cercopithecus monkeys appeared to be highly oncogenic; all 23 inoculated hamsters developed tumors within 30 to 40 days.

STUDIES ON THE SERUM AND THE INFECTIOUS HEPATITIS VIRUSES OF MAN. II. COMPLEMENT FIXATION STUDIES WITH SERUM AND INFECTIOUS HEPATITIS VIRUSES.

Serum hepatitis (SH) virus was isolated in tissue culture from the blood of each of the five human volunteers 41 to 90 days following their inoculation with NIH icterogenic plasma pool VI. Six other strains of hepatitis viruses were isolated from patient sources; one from a case of clinical serum hepatitis and the remainder from five (5) cases of clinically diagnosed infectious hepatitis. All agents were grown in human lung cell and rabbit kidney tissue culture systems. SH 204 virus was neutralized by specific rabbit antisera prepared against viruses SH 211, SH 204, SH 210, SH‐Neib, IH‐MJ and IH‐Horst. These antisera at a final dilution of 1:40 neutralized 10 to 1,000 TCID50 units of the SH 204 virus. Cross complement fixation tests were carried out with the ten hepatitis viruses against specific rabbit antisera which was prepared against each of the virus agents. Irrespective of the source of isolation of the SH or IH virus, there were no apparent differences in the antigenic structure of the viruses, since all produced a common complement fixing antibody in rabbits immunized with the different viral agents. The complement fixation titers observed ranged from 1:200 to 1:3000. In sharp contrast, normal human serum and normal rabbit kidney and human lung cell tissue culture fluids, when tested against antisera produced to these agents as antigens, fixed complement in a range of titer of only 1:8 to 1:96.Complement fixation tests were carried out against specific antisera for many of the other human enteric viruses. SH virus did not fix complement with specific antisera prepared against Coxsackie, Group B1 virus types one to six; Coxsackie, Group A, Virus, type nine; Echo, types one, three, six, eight, 23, 26, 28; Folio viruses, types one, two and three; and Para‐influenza, type three virus. However, the SH virus was found to fix complement with antisera prepared against Echo virus, types two, seven, 24 and 25, but the titers observed ranged only from 1:64 to 1:128. Complement fixing antibodies were also demonstrated in three of 25 normal calf sera tested, but were not demonstrated in normal serum obtained from guinea pigs, rabbits, rhesus monkey, horse, human infant, sheep or cat. Antisera against infectious canine hepatitis virus did not fix complement with the SH virus. Finally, convalescent sera obtained from the volunteers who had been inoculated with the icterogenic plasma pool, demonstrated a high titer of complement fixing antibodies against the SH virus.

The antigenic structure of psittacosis and feline pneumonitis viruses: I. Isolation of complement-fixing antigens with group and species specificity

Alkali-soluble and ether-soluble complement-fixing antigens were isolated by chemical fractionation of purified psittacosis and feline pneumonitis elementary body suspensions disrupted by sonic vibration. Both group- and species-specific serological activity were demonstrated in the alkali-soluble fractions by means of cross absorption and cross complement fixation tests. The destruction of the group activity with either potassium periodate or lecithinase produced psittacosis and feline pneumonitis species-specific antigens. In comparable experiments, the ether-soluble antigens exhibited only group serological activity. The alkali-soluble and ether-soluble group-specific complement-fixing antigens are serologically related. However, they show differences in heat stability, hemagglutinating properties, and prozone manifestation.

日本腦炎ワクチンの研究に関連して調べられたマウス腦と鷄胎兒との比較

Between mouse brain (MB) and 12-day-old chick embryo body (CE) tissue, as materials for the preparation of JAPANESE ENCEPHALITIS vaccine, several differences were found:1) Total Nitrogen content of MB tissue was 2.3 times while the supernate of MB centrifuged at 2500r.p.m. for 15 minutes contained 1.2 times as much Nitrogen, 2) total solids of MB was 2.6 times, 3) mouse intracerebral titers of infected MB tissue were from 6.3 to 200 times, 4) and calculated mouse i. c. LD 50 per mg Nitrogen of MB supernate was from 5, 4 to 170 times, all the above mentioned, approximately, as compared with those of CE tissue. 5) In the cross complement fixation tests using MB and CE antigens with the corresponding immune guinea pig sera, the reaction in homologous relation were stronger than in heterologous relation. 6) Any significant differences in neutralization indices could not be found in cross neutralization test using the same immune guinea pig sera as in c. f. test, and the corresponding both viruses. 7) In cross immunization tests in mice which were vaccinated with MB and CE vaccine and challenged with both viruses, the protection in homologous relation was not necessarily stronger than that in heterologous relation. 8) The protective power of CE vaccine was disproportionnally weaker than that of MB vaccine on the basis of infective titer before inactivation of vaccine. 9) The heated infected mouse brain suspension have a tendency of lowering LD50 titer when added to MB or CE virus in infectivity titration. The same was not observed in the heated CE suspension. This inhibiting effect on virus seems to be partly nonspecific.

Studies on the Specific Soluble Proteins of Pasteurella Pestis and P. Pseudotuberculosis

Summary and Conclusions A complement fixation test has been employed in the study of the specific protein fractions of the virulent and avirulent plague and pseudotuberculosis strains in the elucidation of the antigenic structure of these organisms. The P⅓ fraction of the soluble proteins of virulent and protective plague strains grown in enriched casein hydrolysate broth specifically fixes the complement, no cross-reactions being observed with the antisera against either the protein fraction or the whole of the avirulent nonprotective plague or pseudotuberculosis organisms. The test confirms the findings of other serological tests, viz. precipitation and agglutination, carried out with these protein fractions or their antisera. The water-extractable fraction of a virulent plague strain is the most specific antigen found, but the tests fails to distinguish between the virulent and the relatively avirulent protective strains and a comparatively larger dose is required for mouse protection. Antigenic deterioration of plague strains can be detected by cross complement fixation tests. Antisera against pseudotuberculosis strains have given divergent results amongst themselves and further work with their protein fractions is indicated. Antiplague rabbit sera are preferable to anti-plague horse sera for the complement fixation test. The immunogenic values of the protein fractions and of whole vaccines prepared from various plague and pseudotuberculosis strains, have been determined. The results confirm the serological findings. Three categories of plague strains can be distinguished, viz., (1) virulent protective, (2) avirulent protective and (3) avirulent non-protective, the pseudotuberculosis strains falling under the last group. The protective power of the Haffkine plague vaccine lies in the protein fractions of its supernatant. The P⅓ fraction (antigen A) of the first or the second group of organisms is fully responsible for the mouse protection given by the vaccine. Its immunogenic values seem to depend on the quality of the medium used. The corresponding P½-⅓ fraction (antigen B), on the other hand, gives practically no protection, even in heavy doses, but further work is needed to confirm this observation. The results of mouse-protection tests with the anti-protein sera support the above findings. Certain future lines of studies on plague have been indicated, including the possibility of replacing with advantage the whole vaccine by the P½ fraction for human immunization.

Studies on Certain Viruses Isolated in the Tropics of Africa and South America. Immunological Reactions As Determined by Cross Complement-Fixation Tests

Summary Noninfectious, lipid-extracted, complement-fixing antigens, which were satisfactory for use with specific antisera prepared in guinea pigs and mice, have been made from the brains of mice infected with 27 different viruses or strains of virus. Lipids were extracted either by means of the acetone-ether extraction technique of Casals, or, in the case of 5 viruses, with benzene. There were no failures to secure an antigen when an attempt was made to do so. The sera used in this study were all obtained from guinea pigs and mice, no attempt having been made to examine human sera for the presence of complement-fixing antibody to any of the viruses under investigation. The following viruses gave no cross reactions and are considered to be distinct entities: Bwamba, Bunyamwera, Zika, Wyeomyia, Anopheles A and Anopheles B. Kumba virus is considered to be merely a strain of Semliki virus. Neither of these 2 viruses gave any other cross reactions. West Nile virus in our hands gave no cross reactions, but we have no reason to doubt the validity of cross reactions previously observed by others. Mengo virus gave no cross reactions with any of the other viruses studied, except for a one-way cross with several strains of the GDVII type of mouse encephalomyelitis—that is, by use of Mengo antiserum and GDVII type antigens. Uganda S and yellow fever gave irregular, one-way cross reactions, as did Ntaya and Ilhéus. Four viruses, namely Haemagogus A, Haemagogus B, Leucocelaenus and Sabethes, are shown to be strains of the GDVII type of mouse encephalomyelitis.

THE IMMUNOLOGICAL RESPONSE TO INFLUENZA VIRUS INFECTION AS MEASURED BY THE COMPLEMENT FIXATION TEST

A quantitative complement fixation test with influenza immune sera and virus antigens obtained from allantoic fluid is described. The method utilizes a photoelectric densitometer which provides a simple, objective, and accurate determination of the hemolytic reaction. The enhancement of the hemolytic activity of complement in the presence of serum or allantoic fluid necessitates a preliminary titration of complement in the presence of these agents. An accurate appraisal of the activity of the complement under the conditions of the actual test permits the selection of an optimal amount of complement and greatly increases the sensitivity of the test. The substance (or substances) responsible for the enhanced hemolytic activity of complement has been found in human and many animal sera and in allantoic fluids obtained from the developing chick embryo. It requires the presence of both complement and hemolysin, resists heating at 100 degrees C. for 2 hours, and is dialyzable. Allantoic fluid or mouse lung preparations of influenza virus contain a complement-fixing antigen which is intimately associated with the virus particle. It sediments in the high speed centrifuge at the same rate as the hemagglutinin and infective particle and, like the latter, is adsorbed by fowl red blood cells and eluted from the cells on standing at room temperature or 37 degrees C. It cannot be separated from the virus particle by repeated washings in the centrifuge or repeated adsorptions with red blood cells; the hemagglutinin and complement-fixing antigen titers remain roughly proportional. This antigen shows a high degree of strain specificity in cross complement fixation tests with PR8, W.S., and swine ferret antisera, and, as found with the neutralization test, it shows little or no strain specificity with human sera. A soluble antigen is also present in influenza virus preparations which can be readily separated from the virus particle by centrifugation. It is not adsorbed by red blood cells. Furthermore, it reacts in lower titer with ferret antisera and usually shows less strain specificity in cross complement fixation tests. In general, allantoic fluid virus preparations contain much less of the soluble antigen than mouse lung extracts.

STUDIES ON INFLUENZA VIRUS

Influenza complement fixation tests designed for use with ferret serum are described. Complement-fixing antigens derived from influenza ferret lungs were unsatisfactory due to their low content of soluble antigen; those prepared from mouse lungs or developing chick embryo membranes proved to be better antigenically and were reliable when the various reagents in the test were properly adjusted to eliminate non-specific fixation of complement. The results of cross complement fixation tests indicated that the soluble antigens of the PR8 and W.S. strains of influenza A virus were closely similar, if not identical. They indicated also that the soluble antigen of swine virus possessed components present in the antigens of the human strains of virus.