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 neutralization of influenza and other viruses by homologous immune serum. Studies in roller tube tissue cultures

Summary Neutralization tests using influenza virus, poliovirus, and adenovirus in various tissue cultures showed a linear relationship between the log virus concentration and log serum concentration at the end point of neutralization, the slope of the neutralization line being steep. The effect on this slope of variables in the serum, virus seed, and tissue cultures used and in the method of performing the test was observed.The neutralization slope was reduced in all the systems when the serum was washed out of the culture tubes 2 hours after inoculating the mixtures.When influenza virus and antiserum reacted in a concentrated solution more neutralization occurred than when they reacted in a dilute solution.A small amount of true reactivation on dilution of influenza virus/ serum mixtures was possibly demonstrated.It was concluded that many of the results supportedDulbecco's theory of neutralization.     

Immunological relationship among human adenoviruses of subgenus D

Summary Antigenic relationships between adenoviruses of subgenus D were determined by neutralization tests in HeLa cell cultures by CPE inhibition. For cross-testing, several antisera of the same species were tested against the prototype viruses 39 wild strains belonging to 12 different virus species were also studied. Marked variation in the degree of cross-neutralization between individual sera of the same species was often observed. However, virus strains within a species mostly showed identical serological reactions. Hence, antigenic specificity appears to be a fairly constant property of any one species.Strong cross-neutralizations between species are presumably due to a relationship of the (hexon) antigen, whereas weak cross-neutralizations found between viruses related by hemagglutination-inhibition are due to the (fiber) antigen.Viruses related to adenovirus 15(Mastadenovirus h 15) showed a variety of cross-reactions in neutralization tests. In view of the new species definitions of adenoviruses and to facilitate identification, changes in the classification of Ad 15, 25, 29, and 15/H9 are proposed. The prototypes of Ad 13, 15, 25, 29, and 30 have been cloned by terminal dilution.Aided by a grant from the Bundesministerium für Jugend, Familie und Gesundheit.     

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.     

Characterization and distribution of two new foamy viruses isolated from chimpanzees

Summary Pan 1 and Pan 2 viruses are persistent viruses recoverable from many organs, including brain, of most chimpanzees; both may be present in the same organ of a given animal. These viruses induce a vacuolated, foamy syncytia without inclusion bodies both in chimpanzee explant cultures and in primary human embryo kidney (HEK) cells. Both viruses are not inhibited by IUDR. are chloroform and pH 3.0 sensitive and are inactivated following exposure to 45° C for 30 minutes. Pan 2 virus, the only one tested, is sensitive to visible light. Neither virus has yielded a detectable hemagglutinin, nor has hemadsorption of guinea pig RBC been observed. Direct and blind passage inoculations have not induced infection or evidence of viral replication in small laboratory animals. By electron microscopy they are indistinguishable from each other and are approximately 125 nm in diamter. Viral antigens are demonstrable inin vitro cultures of chimpanzee tissues as well as infected HEK cells by direct fluorescent antibody staining.Pan 1 and Pan 2 viruses do not share common antigens detectable by neutralization or fluorescent antibody staining. The viruses are not neutralized by reference reagent antisera specific for simian foamy virus types 1, 3, 4, 5, measles, canine distemper or other myxo- and pseudomyxoviruses tested. Antiserum prepared against foamy virus type 2, at a dilution of 110, neutralizes 50 TCID₅₀ of Pan 2 virus, whereas antiserum prepared against Pan 2 virus does not neutralize foamy virus, type 2. Homologous antibody is present in the serum of each chimpanzee from whose tissue the virus has been recovered. Antibodies to the Pan foamy viruses have been detected in uninoculated control chimpanzees and in those bled in the African bush.This work was submitted as partial fulfillment for the Ph.D. degree at The Catholic University of America, Washington, D.C.     

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.     

Two types of morphological transformation of bovine kidney cells infected in vitro with SV40

Summary Two types of morphological transformation of bovine kidney cells were obtained after inoculation with SV₄₀. Primary cultures inoculated were transformed into cultures with epithelioid cells growing mainly in monolayer. When the kidney cells were subcultivated and infected at the 6th passage, another type of transformation, characterized by epithelioid and fibroblastic cells growing in a disorganized multilayer, was seen.Cell lines were obtained from both types of transformed cultures. The epithelioid cell cultures were found to be free of infectious SV₄₀ whereas the cultures composed of epithelioid as well as fibroblastic cells yielded virus even at high passage levels. Both types of transformed cultures contained the complement-fixing tumor antigen. A comparison between the cultures as regards their susceptibility to various viruses showed that the epithelioid cell cultures were more susceptible to parainfluenza virus type 3 than the cultures with both epithelioid and fibroblastic cells. There were no differences in susceptibility to foot-and- mouth disease virus, bovine enterovirus, infectious bovine rhinotracheitis virus, pseudorabies virus, Newcastle disease virus or bovine viral diarrhoea virus.This investigation was supported by grants from the Swedish Cancer Society.     

Antibodies to hepatitis B virus x-protein in sera of patients with acute and chronic acitve hepatitis

Sera of patients with acute (AH) and chronic active hepatitis (CAH) were tested for anti-hepatitis B virus (HBV) x-protein (HBx) by immunoblotting, using recombinant MS2- and gal-HBx fusion proteins as substrate. Antibodies against HBx were detected in 5 out of 17 patients with AH at an early stage of infection, and in 13 out of 35 patients with CAH. Positive sera from AH patients showed a relatively weak anti-HBx reactivity when compared to sera from CAH patients. In follow up studies we tested serial serum samples from patients positive for anti-HBx. Patients with AH were observed for 3 to 6 weeks and CAH patients for up to 51 months. In general anti-HBx reactivities appeared to be stable although significant differences in apparent antibody levels were noted when sera from individual patients were compared. Our data further support an early expression of HBx-antigen in HBV-infected individuals. There was no correlation between HBe-antigen and anti-HBx in CAH.     

Influence of prostaglandin E2 and indomethacin on interferon-γ production by cultured peripheral blood leukocytes of multiple sclerosis patients and healthy donors

The addition of indomethacin to concanavalin A (Con A)-induced cultures of human peripheral blood leukocytes (PBL) caused an increase in interferon response, regardless of whether the PBLs were derived from multiple sclerosis (MS) patients or from control donors. Specifically the response rates increased from 71 to 100% in controls and from 24 to 53% in MS patient-derived cultures. The amounts of interferon produced also increased in both groups by 0.8 log U/ml. However, interferon yields of nonresponsive cultures becoming interferon-producing only after indomethacin treatment remained relatively low. In control cultures, maximal increases of interferon production were obtained with doses of 0.05 to 0.1 µg/ml indomethacin; for MS patients higher doses were needed—0.1 to 0.5 µg/ml. Conversely, a relatively low dose (0.05 µg/ml) of exogenous prostaglandin E₂ (PGE₂) was able to inhibit interferon production completely in MS patient-derived cultures, whereas in control cultures higher doses were needed (0.1 to 1.0 µg/ml). Analysis of endogenous PGE₂ levels in the PBL cultures revealed that PGE₂ production was similar in nonresponder MS cultures and responder control cultures but that MS leukocytes were more sensitive to the inhibitory effect of PGE₂ on interferon production. We conclude that in a minor percentage of MS patient-derived PBL cultures, the deficiency in interferon- (IFN-) production can be (partially) overcome by treatment of the cells with indomethacin. However, in the major part of nonresponder MS cultures, indomethacin has no effect, indicating that the PG system is not the major cause for the defective interferon response in MS.     

Cross-neutralization tests with swine enterovirus strain S 180/4, and Teschen,Talfan, and swine polio-encephalomyelitis viruses

Summary The enterovirus S 180/4, which produces encephalomyelitis and pneumonitis by inoculation in antibody-free pigs, was used in neutralization tests in swine kidney tissue culture with antisera of Teschen, Talfan, swine polio-encephalomyelitis strains T 80-T 52 A, benign enzootic paresis (Denmark), ECHO strain 20 and canine hepatitis. Similarly, S 180/4 antiserum was tested against viruses of Teschen, Talfan, swine polio-encephalomyelitis T 80 and T 52 A. Significant cross-neutralization was only obtained between S 180/4 and T 80-T 52 A.Aided by grants from Exportmedelsfonden, Royal Academy of Agriculture, Stockholm.     

Characterization of a small Porcine DNA virus

Summary Characteristics of a small DNA virus isolated from kidney cell cultures of healthy 3 week-old pigs are described.The virus isolate multiplies in kidney cell cultures of pig origin, produces intranuclear inclusion bodies, and hemagglutinates guinea pig, human group 0, chicken, cat, rat and mouse red blood cells. It multiplies in pigs resulting in antibody production, but is not pathogenic for newborn hamsters and mice.The virus particle is 20–22 m in size, hexagonal in shape and without a lipid containing envelope. Buoyant density is between 1.37 and 1.38g/ml. This virus is stable within a wide range of pH, resistant to heat (56°C), against treatment with trypsin, and lipid solvents.The porcine virus was proposed as a member of the picodna virus group, and named Porcine Picodna Virus (PPV).Supported by a grant of the Common Market Research Program for control of European and African Swine Fever.     

Isolation and characterization of two new herpes-like viruses from capuchin monkeys.

Two herpes-like viruses were isolated from capuchin monkey (Cebus apella) brain and (Cebus albifrons) spleen cell cultures, respectively. Both isolates induced similar cytopathic effects consisting of rounded and ballooned cells in the original monkey cell cultures and in a wide range of permissive cell types. Neutralizing antibody to each virus was present in serum from the capuchin monkey from which it was isolated, but the two viruses did not cross-react by neutralization. Fluorescein isothiocyanate conjugates of hyperimmune rabbit serum to one of the isolates showed an antigenic cross relationship between the two isolates. By electron microscopy, herpes-like virus particles were observed in the nucleus and cytoplasm of infected human diploid fibroblast cell cultures. Virus-infected cell cultures stained with acridine orange revealed small deoxyribonucleic acid-containing intranuclear inclusion bodies. Both viruses were inhibited by 5-fluorodeoxyuridine and inactivated by chloroform or exposure to 56 degrees C for 30 min. Antisera prepared against 16 prototype herpesviruses and cytomegaloviruses did not neutralize approximately 100 50% tissue culture infective doses of either capuchin isolate. Neutralizing antibody to the capuchin isolates was detected in sera from 8 of 17 capuchin monkeys but not in sera from 16 humans, 15 chimpanzees, and 10 spider, 6 rhesus, and 5 squirrel monkeys.     

Correlation Between Interferon Production and Clinical Disease Activity in Patients with Multiple Sclerosis

We determined the interferon (IFN) serum levels and in vitro activated IFN production in eight patients with relapsing/remitting multiple sclerosis (MS), using a whole-blood test system and the mitogen concanavalin A and the viral antigen Newcastle disease virus for induction of the IFN production. During the overall study period of 12 months we observed, in relation to clinical disease progression, a biphasic increase in the individual IFN and IFN production. While mitogen-induced IFN synthesis showed a significant augmentation prior to the onset of a new relapse (P < 0.05), virus-induced IFN production showed a temporal delayed increase which was related to clinical remission (P < 0.01). The observed fluctuations in the individual production of both IFN subtypes were not reflected in the sera of the patients. Although the reason for the temporal different imbalance in the production of both IFN subtypes remains unknown, the observed association between increased IFN production and clinical remission emphasizes a possible role for type 1 IFNs in the resolution of the MS relapse.     

Identification of Different Measles Virus-Specific Antibodies in the Serum and Cerebrospinal Fluid from Patients with Subacute Sclerosing Panencephalitis and Multiple Sclerosis

Matched serum and cerebrospinal fluid (CSF) samples from eight cases of subacute sclerosing panencephalitis (SSPE) and 15 cases of multiple sclerosis (MS) were characterized in neutralization, hemolysis-inhibition (HLI), hemagglutination-inhibition (HI) with Tween 80—ether-treated antigen, complement-fixation (CF), and immunodiffusion tests. CF tests were carried out with crude virus material, purified nucleocapsids, and small particle hemagglutinin as antigens. A certain diversity in the relative content of antibodies against different virus products in various sera was found. There was a high degree of correlation between titers of neutralizing and HLI antibodies, but a less strict correlation between titers of HLI and HI antibodies. Serum samples from two cases of MS and one case of SSPE contained high titers of HLI and neutralizing antibodies in the presence of only low titers of HI antibodies demonstrable with Tween 80—ether-treated antigen. The major fraction of antibodies detected in CF and immunodiffusion tests reacted with nucleocapsids. There was a tendency of nucleocapsid CF antibody titers, as compared to neutralization and HLI antibody titers, to be higher in samples from patients with SSPE than from cases of MS. No significant differences were found between antibody titers recorded in neutralization, HLI, and HI tests carried out with two different measles virus strains, Edmonston and a strain (LEC) derived from a case of SSPE. Comparison of antibodies against measles virus products and, as a reference, against a group-specific vertex capsomer antigen of adenovirus in matched serum and CSF samples revealed a production of measles virus-specific antibodies within the central nervous system of all cases of SSPE and 8 out of 15 cases of MS.     

Neutralization of adenovirus infectivity and cytotoxin in various cell cultures

The neutralization of human adenovirus 5 and 11 by homologous and heterologous rabbit antisera was determined by CPE inhibition in various cell cultures (HeLa, HEL, Vero, secondary kidney cells from cercopithecus, rabbit, mouse), or in HeLa cells made impermissive by IUdR inhibition. The results concerning sensitivity and specificity were similar in all cases. Crude and purified virus showed similar neutralization. Immunofluorescence neutralization in HeLa cell cultures gave similar results; this method is suitable for demonstrating subtle immunological relations between adenovirus types.The neutralization of the early cytopathic factor (‘cytotoxin’) showed a pattern of cross-reactivity different from the virion; the cytotoxin was found to be active in part of the cell cultures only. It is concluded from the results that the virus function(s) blocked by antibody appear to be identical for the replicative cycle in infection and for the initiation of the abortive infection in non-permissive cells. Hence, either kind of cells may be used for neutralization tests.     

Characterization of African horsesiekness virus

Summary Purified African horsesickness virus was shown to possess an icosahedral shape, measure approximately 55m in diameter and probably consist of 32 capsomeres. Electron microscopic evidence indicates a close morphological relationship between the virus and bluetongue virions. African horsesickness virus has a double-stranded RNA genome which was resolved into five components by sucrose gradient sedimentation analysis and into six segments in four size groups by means of polyacrylamide gel electrophoresis. The remarkably close relationship between African horsesickness and bluetongue viruses has led to the suggestion that these two viruses be classified in one sub-group of the newly proposed diplorna-virus group.     

“Karshi” virus,a new flavivirus (Togaviridae) isolated fromOrnithodoros papillipes (Birula, 1895) ticks in Uzbek S.S.R.

Summary Three identical strains of an arbovirus were isolated from 475Ornithodoros papillipes ticks collected in June, 1972, in burrows of the great gerbil (Rhombomys opimus Licht., 1882) in the environs of Beshkent, Karshinsk steppe, Uzbek S.S.R. The isolate was found to range among flaviviruses. Complement-fixation, agar diffusion precipitation and neutralization tests is tissue culture and mice indicated a one-way antigenic relationship between the isolate and West Nile virus. However, the pattern of differences between them made it possible to consider the isolated agent as a new virus, Karshi virus. The results of electron microscopic studies of this virus are presented.With 3 Figures     

The kinetics of adenovirus infection and spread in cell cultures infected with low multiplicity

Summary 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 much 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.With 9 FiguresAided by a grant from the Deutsche Forschungsgemeinschaft.     

Studies on in vitro neutralization of adenoviruses

Summary The incubation time titration method has been applied to a study of the neutralization of adenovirus by antibody. Following dilution of mixtures of virus and serum a marked reactivation phenomenon was apparent. The results indicate that this phenomenon was partly caused by remaining reactive antibody in the medium, partly by a faster and more extensive reaction between virus and serum in higher concentrations. Whether or not a real reactivation also contributed to the dilution effect cannot be definitely decided upon.The results indicated, furthermore, that antibody may influence the virus in another way than by a complete inactivation. This effect was correlated to the dose of inoculum. Some possibilities regarding the mechanism of the phenomenon are discussed.With suitable concentrations of virus the incubation time titration method has been found applicable for routine work.     

On the nature of the cystine-dependent phenotype of poliovirus

Summary 18 of 39 adenovirus strains of group II were found to be serologically intermediate: they were related to one serotype in hemagglutinationinhibition and to another in neutralization. They have been designated with two type numbers, the first indicating the type related in HA-inhibition, the second that related in neutralization. The strains were classified into ten different serological categories. Three or possibly two more of the type halves were hitherto unknown new antigens. A further strain was found to have antigenic constituents of both types 26 and 27, demonstrable in both serological tests.Six of the prototypes of group II were also classified by two type numbers, indicating the close relationship between the respective types: 8 and 9, 10 and 19, 15 and 22 in hemagglutination-inhibition, 15 and 25, 15 and 29, 13 and 30 in neutralization.Aided by a grant from the Deutsche Forschungsgemeinschaft.