Effects of freezing on African horse-sickness virus

Summary The infectivity of both neurotropic and viscerotropic African horsesickness virus decreased markedly when the viruses, suspended either in tissue-culture medium or phosphate buffered saline, were stored at temperatures between –20° C and –30° C. Using infectious tissue-culture fluids, the inactivation curves of the virus at –22° C and –30° C were compared. During the first 7 days' storage, 4.7 and 3.9 log units of infectivity were lost at the respective temperatures. It was established that salts such as NaCl, CaCl₂ and MgCl₂ contained in the solutions, were chiefly responsible for the inactivation of the virus. Without these salts, AHS virus was rather stable at –20° C. Infectivity of AHS virus was protected by adding approximately 5% lactose, sucrose, or glucose to the suspension before freezing at –20° C to –30° C. Glycerin, polyvinylpyrolidone, and a high concentration of serum also protected the virus infectivity. AHS virus was stable at –70° C even in the presence of salts.This work was undertaken at the Near East Animal Health Institute, a project established by the United Nations Development Program Special Fund through the Food and Agriculture Organization in cooperation with the Ministry of Agriculture of Iran.A summary of this work was reported at the 52nd Annual Meeting of the Federation of American Societies for Experimental Biology, April 15–20, 1968, Atlantic City, New Jersey.     

Electron microscopic study of the formation of African horse-sickness virus

Summary BHK 21/13 cells infected with African horse-sickness virus (strain 13/63, type 3) for varying periods of time have been studied with the electron microscope. Evidence is presented to show that the virus does not multiply in the nucleus, but that replication occurs in the cytoplasmic matrix. It is also indicated that the particle often leaves the host cell with an envelope derived from the cell membrane. The diameter of the particle is roughly estimated to be 70 m., excluding the envelope.     

Inactivation of African horse-sickness virus by betapropiolactone and by pH

The inactivation of several types of African horse sickness virus (AHSV) by pH and by betapropiolactone (BPL) was studied. At 19 degrees - 22 degrees C the virus was stable between pH 6.0 and 10.4, whether suspended in mouse brain or in serumfree buffer. Below pH 5.6 and above pH 10.9, more than 99 per cent of infectivity was inactivated within 15 minutes. The addition of 50 per cent serum did not influence pH stability. Disinfection in the presence of citric acid and caustic soda is briefly discussed. Inactivation by BPL was complete within 30 minutes at 37 degrees C, yet incomplete after 15 hours at 4 degrees C. Types 3 and 9 virus grown in suckling mouse brain and types 1, 3 and 9 produced in pig kidney cells were equally susceptible to 0.1 per cent BPL, more than 99.9 per cent being inactivated. The effectiveness of BPL was reduced at least 10-fold by the addition of 50 per cent serum. No infective virus was detected following incubation of either tissue culture virus with 0.2 per cent BPL or of mouse brain virus with 0.3 per cent BPL. Virus suspensions exposed to 0.3 per cent BPL required buffering with Tris of at least 0.05 molar strength in order to maintain the pH within an acceptable range. Inactivated antigens prepared with 0.4 per cent or lower concentrations of BPL were immunogenic in guinea pigs.     

Studies on the replication of African horse-sickness virus in two different cell line cultures

Summary Single-passage growth patterns of African horse-sickness (AHS) virus in two different cell lines, MS and VERO, of monkey origin were compared by titrating extracellular virus in cultures infected at high multiplicity.Both by fluorescent-antibody and acridine orange staining techniques, large antigenic bodies containing RNA were found in the cytoplasm of infected VERO cells. This was not so evident in infected MS cells.Metabolic analogs, bromodeoxyuridine (BUDR) and iododeoxyuridine (IUDR) did not inhibit the yield of AHS virus either in MS or VERO cell cultures indicating that synthesis of new DNA is not required for replication of this virus. The yield of AHS virus was not inhibited by actinomycin D and mitomycin C, suggesting that synthesis or function of DNA is not directly required for the replication of the virus. MS cells were more sensitive to the toxic effect of these chemicals than VERO cells.This work was undertaken at the Near East Animal Health Institute, a project established by the United Nations Development Program Special Fund through the Food and Agriculture Organization in cooperation with the Ministry of Agriculture of Iran.     

The multiplication of African horse-sickness virus in two species ofCulicoides (Diptera,Ceratopogonidae)

Type 9 African horse-sickness virus multiplied to a high titre in both Culicoides nubeculosus and C. variipennis after intrathoracic inoculation and in C. variipennis after oral ingestion. The orally infected C. variipennis were able to transmit the virus by biting after 13 days incubation at 26 degrees C but not after 6 days incubation. Intrathoracically inoculated C. variipennis were able to transmit the virus after 4 days incubation. It is suggested that C. variipennis can act as a biological vector of African horse-sickness virus.     

Rates of infection in, and transmission of, African horse-sickness virus by Aedes aegypti mosquitoes.

Very low infection rates (less than 3%) were obtained when Aedes aegypti mosquitoes ingested blood contained 5.8--6.5 log10 MLD50/0.02 ml African horse sickness virus (AHSV). When A. aegypti mosquitoes were inoculated intrathoracically with virus, however, high infection rates were achieved. Mosquitoes infected by inoculum failed to transmit virus to embryonated hens eggs by bite, and virus could not be detected in membrane or blood when inoculated mosquitoes were allowed to engorge on uninfected blood through a chick skin membrane. It was concluded that the mosquito A. aegypti is unlikely to be an effective vector of AHSV.     

The growth of African horse-sickness virus in embryonated hen eggs and the transmission of virus byCulicoides variipennis Coquillett (Diptera,Ceratopogonidae)

Seven-day-old embryonated hen eggs were infected with African Horse Sickness virus by the yolk sac and intravenous routes. Virus reached a high titre in the blood of infected embryos. Culicoides variipennis midges which took a blood meal from infected eggs became infected with virus, and after 7 days at 26 degrees - 27 degrees C transmitted African Horse Sickness virus to uninfected eggs. C. variipennis may therefore be considered a biological vector of African Horse Sickness virus in the laboratory.     

The neutralization of interferons by antibody. I. Quantitative and theoretical analyses of the neutralization reaction in different bioassay systems.

The highly specific ability of antibodies to inhibit the biologic activity of cytokines or other therapeutic proteins is widely used in research and a subject of increasing clinical importance. The need exists for a standardized approach to the reporting of neutralizing antibody potency soundly based on theoretical and practical considerations and tested by experimental data. Pursuant to the original studies of Kawade on the theoretical and functional aspects of neutralization of interferons (IFN), experimental data were obtained by different laboratories employing varied methodology to address two hypotheses concerning the nature of IFN neutralization reactions, based on a derived formula that allows expression of neutralizing power as the reduction of 10 laboratory units (LU)/ml to 1 LU/ml, the end point of most bioassays. Two hypotheses are posed: (1) antibody acts to neutralize a fixed amount of biologically active IFN molecules, or (2) antibody reduces IFN activity in a set ratio of added/residual biologically active IFN. The first, or fixed amount, hypothesis relates to the reactivity of high-affinity antibodies neutralizing equimolar amounts of antigen, whereas the second, or constant proportion, hypothesis postulates a reduction in the ratio of total added IFN to residual active IFN molecules, such as a low-affinity antibody might exhibit. Analyses of data of the neutralization of IFN-alpha and IFN-beta are presented, employing human polyclonal antibodies and murine monoclonal antibodies (mAb). The theoretical constructs of Kawade are extended in the Appendix and correlated with new experimental data in the text. The data clearly indicate that the low-antibody affinity, constant proportion hypothesis, rather than the high-antibody affinity, fixed amount hypothesis, is applicable, if the bioassay is sensitive to IFN. The findings presented here and in the following paper (pp. 743-755, this issue) taken together provide the basis for a standardized method of expression of neutralizing potency and substantiate the earlier operational 10/1 LU/ml approach recommended by the World Health Organization. The accompanying paper relates neutralization results to the sensitivity of the bioassay to IFN and describes the rationale for a recommended unit of antibody neutralization.     

Studies on the neutralization of Japanese encephalitis virus

Summary The neutralization reaction of Japanese encephalitis virus with early serum was compared with that with late serum. The analysis of antiserum by Sephadex G200 gel filtration indicated that the neutralizing activity in early serum was present only in the IgM fraction, while that in late serum was present only in the IgG fraction. The antibody dose response curves in early serum were characterized by the early and high appearance of a persistent fraction. This fraction was found to consist of an infectious virus antibody complex (sensitized virus) which was neutralized by anti-IgM serum. The amount of virus neutralized by anti-IgM serum varied with the concentration of antiviral antibody employed for the sensitization. In contrast, it was a characteristic of the neutralization by late serum that the residual infectivity was inversely related to the concentration of antibody in the serum, resulting in a low level of a non-neutralized virus fraction. Therefore, the maximal reduction of residual infectivity by anti-IgG serum was attained under an optimal ratio of antibody to virus.Virus sensitized with early serum had a blocking effect against a high concentration of late serum antibody, but was neutralized by anti-IgM serum. Virus sensitized with an insufficient amount of late serum antibody was neutralized not only by high concentrations of late serum antibody, but also was supersensitized by early serum antibody.Since the sensitized virus which had been adsorbed on host cells was still neutralizable by anti--globulin, aggregation seemed to be excluded as the main factor in the mechanism of neutralization by anti--globulin serum.     

Quantitative studies of blue tongue virus in mice

Summary The California BT₈ strain of egg-propagated blue tongue virus was adapted to the suckling mouse brain and its growth curve in this host studied.The possible use of a graded response (survival times) for the quantitative assay of blue tongue virus was investigated. The variances of survival times within virus dilutions were heterogeneous and dependent of the mean. Employing the reciprocals of the time responses reduced this heterogeneity more effectively than other scedasticity transformations. The regression of transformed survival times on virus dilutions was significant but linearity existed only over a limited dose range. No time metameter was found that allowed the computation of a linear time-dose regression over a larger scale of virus dilutions. Thus, a reference curve based on this graded response was not considered more advantageous for virus assay than the quantal response. The inability to find a function of the graded response which approximated a normal distribution may be due to interferon production in the mouse brain.The relationship of mouse age to virus susceptibility was influenced by the passage level of virus used. Virus of the 7th passage produced mortality in one but not in 2-week-old mice, whereas virus representing the 53rd passage killed 5-month-old mice. Peak titers were recorded at approximately the same time in the different age groups, but were inversely related to mouse age. A marked prolongation of survival times was observed in 3- to 20-week-old mice as compared to one-week-old mice.Analysis of graded response data showed the distribution of survival times for suckling mice to be approximately normal, but for mice one and one-half to 3 months of age the same distribution was positively skew     

Studies on neutralization of Japanese encephalitis virus

The effect of anti-cellular rabbit serum (ACRS) on the neutralization of sensitized Japanese encephalitis virus (JEV) by anti-rabbit IgG serum was examined to elucidate the interaction between virus-antibody complex and the surface of the host cells during the process of neutralization. ACRS had no effect on the adsorption of either sensitized or non-sensitized virus, but was able to restore the lost infectivity of sensitized virus which occurred during the process of neutralization by anti-rabbit IgG serum. This restoration of infectivity was found to take place not only by the addition of ACRS to the reaction mixtures (virus-antibody, anti-rabbit IgG complex) but also by pretreatment of the host cells with ACRS. Although the restoration of lost infectivity varied in magnitude with the concentration of ACRS used, it never exceeded the infectivity titer of the sensitized virus befor incubatio with anti-rabbit IgG serum. This result suggests that ACRS has no ability to reverse the neutralization by anti-viral serum. Since the ACRS reacted only with anti-rabbit IgG serum treated sensitized virus, resulting in an increase of the number of infectious centers, the restoration of lost infectivity was explained as being due to the enhancement of adsorption of sensitized virus to the host cells by bridge formation of anti-rabbit IgG antibody between them.     

Serological studies on the antigenic relationship between herpes simplex virus and varicella-zoster virus.

If crossreacting antibodies between varicella-zoster virus (VZV) and herpes simplex virus (HSV) exist, one would expect more positive reactions with VZV in a group of HSV positive patients than in a group of HSV negative patients. This statement can only apply to a group of individuals where positive and negative reactions with respect to HSV and VZV are evenly distributed. Such a distribution can only be found among children. Therefore, the relationship between HSV-1 and VZV was the only one which was considered in this investigation, since the incidence of HSV-2 antibodies in children is very rare. The sera from 197 children were examined using the neutralization test (NT), the complement fixation test (CFT) and the indirect immunofluorescent assay (IFT) and could be classified as either HSV positive (80) or HSV negative (117). The children's ages were similar in both groups. Approximately the same proportion of VZV positive sera was found in both groups when examined using IFT (53% in the HSV positive and 47% in the HSV negative group). However, when the CFT was applied the proportion of VZV positive sera in the two groups differed markedly (22% of the HSV positive sera and 38% of the HSV negative sera). These findings suggest that crossreactivity observed between HSV and VZV in acute HSV and VZV infections is evidently not dependent on crossreacting antibodies but is apparently confined to the cellular level of the immune response.     

The nature of neutralization reaction between effector protein and monoclonal antibody: a quantitative study of neutralization characteristics of anti-interferon antibodies

Several monoclonal antibodies to human interferon-alpha and beta were examined quantitatively for neutralization of antigenically homogeneous interferons. The extent of neutralization increased with antibody concentration in each case with a rate considerably lower than in the case of conventional (polyclonal) antibodies, and there often appeared to be a limit to the maximum interferon titre that can be neutralized, even using very high antibody concentrations (there were no such limits with conventional antibodies). This suggests that the interferon in a 1:1 interferon-antibody complex in general retains some activity, to the degree characteristic of that antibody; namely, each monoclonal antibody is considered to have a characteristic efficacy of neutralization, rather than being either neutralizing or non-neutralizing in an all-or-one fashion. The antibody dose-dependence curves were interpreted to be governed by two independent parameters of the antibody: the efficacy of neutralization and the affinity. The former is reflected by the maximum interferon titre neutralizable by high antibody doses and the latter by the minimum antibody dose that can effect detectable neutralization. Thus, quantitative neutralization tests of monoclonal antibodies to an effector protein would give useful information for classifying them as to their affinities and as to whether the epitopes they recognize are important for the biological activity of the effector.