Virus mutation during 'slow infection': temporal development and characterization of mutants of visna virus recovered from sheep.

Visna virus could be recovered from peripheral blood leukocytes of sheep for years after intracerebral inoculation. Viruses recovered from sheep prior to and several months after development of antibody were antigenically identical to the parental strain used for inoculation. Subsequently, mutant viruses which were not neutralized by the animals' sera were obtained. Longitudinal studies of leukocyte viruses collected from two infected sheep showed that more than one strain of virus could co-exist in the animal. Virus neutralization tests using sequentially collected sera and the viruses recovered from leukocytes revealed a sequential development of antibody to parental and then to each strain of mutant virus. Characterization of two of the mutant viruses showed that they were antigenically stable, virulent in cell culture and when inoculated into new sheep, elicited antibodies which cross reacted with the parental virus from which they were derived. This continuous mutation of Visna virus in persistently infected sheep may be a mechanism for the production of chronic disease.     

Wild-type temperature-sensitive and -resistant visna viruses: Isolation and biological comparison

Summary The plaques formed by wild-type (WT) populations of strain K485 visna (V-K485) virus, strain K796 visna (V-K796) virus, and of progressive pneumonia virus in sheep choroid plexus (SCP) cell cultures are heterogeneous in size. Plaque purification procedures showed that this heterogeneity was due to the presence of two biologically different viruses, large plaque-forming (Lpf) virus and small plaque-forming (Spf) virus. The V-K796 Lpf virus, the V-K796 Spf virus, and the V-K796 WT virus are antigenically similar to each other. Although the V-K796 Spf virus is less cytopathic than the V-K796 Lpf virus, both viruses have a 14- to 16-hour latent period in SCP cells and have similar rates of synthesis for the initial 24 to 30 hours after infection. The V-K796 Spf virus is considered a temperature-sensitive visna virus since, unlike the V-K796 Lpf virus, it does not produce plaques at 41° C and it is more thermosensitive than the V-K796 Lpf variant. Population analyses of two strains of wild-type visna virus and one strain of progressive pneumonia virus demonstrated that these populations contain variants with the phenotypes of V-K796 Spf virus and V-K796 Lpf virus, that the Spf virus was not selected against in sheep, and that cultivation of the wild type visna viruses at 37° C selected against the temperature-sensitive variants. The existence of the temperature-sensitive Spf viruses in these wild-type virus populations suggests that the Spf variants maintain the persistent visna virus infection in sheep and delay the development of the host's cell-mediated immune response to the viral proteins.With 4 Figures     

Comparative studies of Visna and Maedi viruses as antigens.

Rabbits were immunized with purified visna and maedi viruses, using complete Freund adjuvant, in footpad and intramuscular sites. The resulting antisera and their isolated immunoglobulin G (IgG) and M (IgM) classes were evaluated by tanned-cell passive hemagglutination (PHA), complement fixation, gel diffusion, and virus neutralization tests. Early, intermediate, and late bleedings showed increasingly high antibody activities by the PHA, complement fixation, and gel diffusion tests. The activities were associated mainly with the IgG class, although low, but significant activities were also found in the IgM class, as detected by PHA and complement fixation tests. Both antibody classes appeared at the same time during the course of immunization. The viruses, when tested against specific rabbit anti-visna and anti-maedi sera in gel diffusion tests, showed the presence of one to two precipitin lines depending upon the antibody concentration of the sera. A low amount of neutralizing activity was demonstrated in late bleedings but was not seen in earlier bleedings. The neutralizing activity against visna virus in immunized rabbit sera was found to be associated only with the IgG class. Visna and maedi viruses appeared to be immunologically identical when examined in gel diffusion tests and showed the same degree of inhibition when compared in passive hemagglutination inhibition test using antisera made specific for each virus.     

Immunodominance and antigenic variation of the principal neutralization domain of HIV-1

The principal neutralization domain (PND) of HIV-1 is located in the third variable region (V3) of the envelope glycoprotein gp 120. Cross-reactivity of experimental and natural sera with recombinant proteins containing the V3 region of four HIV-1 variants showed that a group of viruses (among which HIV-1 MN) had antigenically similar V3 regions. The V3 regions of HIV-1 IIIB and HIV-1 RF were antigenically distinct. Antibodies raised to V3 domains of two isolates from the "MN group" neutralized HIV-1 MN (and not HIV-1 IIIB), thus confirming the antigenic similarity of V3 of these isolates to that of HIV-1 MN. Human antibodies to the V3 region were shown to be mainly directed to the central area in V3, where the neutralization domain is. In addition, antibody reactivities in sera of 397 Dutch and 39 Tanzanian HIV-1-infected individuals show that the V3 neutralization domain is highly antigenic, and that viruses from the MN group predominate in The Netherlands and to a lesser extent in Tanzania. Thus, if the protective value of antibodies to the PND can be established, then PND (poly)peptides derived from the MN group may be important components of a subunit vaccine against HIV-1.     

Characteristics of a novel lentivirus derived from South African sheep with pulmonary adenocarcinoma (jaagsiekte)

A novel lentivirus was isolated from South African sheep with experimentally transmitted lung adenocarcinoma. Similar to visna virus and caprine arthritis encephalitis virus, this new strain induced cytopathic effects on ovine plexus choroid cultures. In contrast to a recent Israeli isolate from sheep with adenocarcinoma, the South African lentivirus could not transform fibroblast cultures. The antigenic relatedness between the new isolate and visna virus was assessed by immunoprecipitation of radiolabeled viral proteins, using monospecific antisera against visna virus proteins. The results indicate that the new virus contains four major structural proteins of sizes similar to those of visna virus (i.e., gp135, p30, p16, and p14) and have some common antigenic determinants (about 90% in the major core antigen p30). However, the nucleotidic sequences of the novel lentivirus were found to be only 16.5 to 27.4% homologous to visna virus and 8.3 to 15% homologous to caprine arthritis encephalitis virus, by means of liquid hybridization under stringent conditions. The genetic divergence indicated by this last result was confirmed by the dissimilar restriction endonuclease cleavage map of the new virus in comparison to those of visna virus and three caprine arthritis encephalitis virus strains. The demonstration of a third type of ovine lentivirus supports the concept of an important genetic variation among the lentiviruses infecting one animal species.     

Isolation of mutants of Aujeszky's disease virus with antigenically altered glycoprotein E by affinity chromatography using monoclonal antibodies

An efficient method for isolation of virus mutants with antigenically altered proteins is described. The method is based on the separation of viruses with wild-type and antigenically altered proteins by affinity chromatography using monoclonal antibodies (MAbs). A nonessential glycoprotein E (gE) of Aujeszky's disease virus (ADV) was chosen as a model for introducing the antigenic changes. The ADV strain Ka mutagenised with 5-bromo-2'-deoxyuridine was used for the selection of mutants that do not bind to gE-specific MAb conjugated to resin. After three rounds of isolation by affinity chromatography, the resulting viruses that escape the binding to MAb were plaque-purified by plating at limiting dilution, and virus isolates were tested by the gE-specific sandwich ELISA in which the selecting MAb was used as a capture antibody. About 70% of the ADV isolates tested were not recognised by the sandwich gE-ELISA. The analysis of some of virus isolates in indirect ELISA with a panel of 16 gE-specific MAbs revealed that at least several of the generated virus isolates were mutants expressing gE with alterations in the epitope of the selecting MAb 75/7, as well as in the majority of other conformation-dependent epitopes of gE. The method for the production of antigenically altered viruses by affinity chromatography using MAbs is simple and convenient, and can be utilised with MAbs irrespective of their virus-neutralising activity.     

Antigenic relationships of murine coronaviruses

Two serological tests were used to examine the antigenic relationships between murine hepatitis viruses that cause different diseases in mice. Antisera prepared by immunization of mice with the individual viruses were tested for their ability to neutralize both the homologous immunogen and the other viruses. By a plaque reduction neutralization test, each antiserum was found to be specific for the immunizing virus; however, there was substantial cross-reactivity, indicating the viruses were closely related. By kinetic neutralization, two of the viruses tested, MHV-JHM and MHV-2, were found to be antigenically distinct. MHV-3 and MHV-A59 were found to be antigenically very similar but distinct. These data show that kinetic neutralization is a more precise method for determining the antigenic relationships between murine coronaviruses.     

Pathogenesis of visna. I. Sequential virologic, serologic, and pathologic studies.

A total of 56 Icelandic sheep were infected with visna virus by intracerebral injection of strain 1514 and the course of infection was followed for 12 months. Virus was isolated from more than 90 per cent of the animals, primarily from central nervous system and lymphoid tissues. However, titers of free infectious virus were minimal and virus isolation often required the use of tissue explants. All sheep raised serum-neutralizing and complement-fixing antibodies beginning 1 to 3 months after infection. Differences in neutralization titers against the infecting strain (1514) and a reference strain (796) suggested that antigenic drift might occur during prolonged infection. High cerebrospinal fluid neutralization titers in the spinal fluid indicated local antibody production in the central nervous system. Although the incidence of clinical disease during the 1st year of infection was less that 10 per cent, approximately 80 per cent of the sheep examined had central nervous system histologic lesions of variable severity, which were marked 1 month after infection with little progression during the subsequent year. There was a striking correlation between the severity of central nervous system lesions and the frequency of virus isolations from all tissues. These observations provide detailed base line data on visna infection, suggest some of the mechanisms responsible for the persistence of infection and for the slowness and irregularity of disease occurrence, and form the basis for further experiments on the role of immunologic mechanisms in the pathogenesis of this slow infection.     

Antigenic relationships between strains of influenza B virus.

An immunological relationship between strains of influenza B virus, considerably differing from one another in haemagglutination inhibition (HI) and virus neutralization (VN) tests, was established. The relationships were also evaluated based on the ability of influenza B viruses to replicate in the lungs of mice immunized with strains possessing antigenically distinct haemagglutinin. There was no substantial difference in the protection of animals immunized with homologous or heterologous strains. Studies on the character of the immunological response of men convalescent after influenza B infection or after vaccination showed an antibody increase to both the epidemic virus and chronologically remote viruses considerably differing in antigenic properties. The data obtained suggest that influenza B viruses isolated from 1940 to 1975 belong to one antigenic subtype.     

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.     

Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy

The prevalence of hepatitis B virus vaccine escape mutants has increased as a consequence of the introduction of global vaccination programs. Furthermore and as a consequence of the organization of the genome of hepatitis B virus (HBV) into overlapping reading frames, the selection of polymerase mutants during long-term lamivudine therapy can select viruses with changes in the overlapping S gene coding for the hepatitis B small antigen (HBsAg). We have investigated the role of lamivudine in selecting HBV mutants with antigenically altered HBsAg protein using pooled human vaccine sera in enzyme immunosorbent assays and radioimmunoassays. HBsAg proteins containing the vaccine escape mutations G145R and D144E/G145R demonstrated markedly reduced binding to anti-HBs antibody. HBsAg mutants including E164D, W196S, I195M, M198I, and E164D/I195M (corresponding to the polymerase protein changes of V519L, M550I, L526M/M550V V553I, and V519L/L526M/M550V) selected during lamivudine treatment also demonstrated reduced binding to anti-HBs antibody. These findings raise the possibility of lamivudine-resistant mutants arising that possess antigenically distinct HBsAg proteins.     

Serologic differences among nondefective reticuloendotheliosis viruses

Summary Antigenic relationships among 26 isolates of reticuloendotheliosis virus (REV) obtained from several avian species were compared by cross neutralization tests with polyclonal chicken sera and by immunofluorescent assays with monoclonal antibodies to REV strain T. The isolates were all strongly related by neutralization assays and thus probably constitute a single serotype. However, 3 antigenic subtypes were suggested by minor but distinct differences in neutralization titers. The validity of these 3 subtype designations was confirmed by differential reactivity of viral isolates to selected monoclonal antibodies. Subtype-associated differences in serum antibody titers were noted following the inoculation of chickens with the REV isolates.     

Antigenic and structural characterization of multiple subpopulations of H3N2 influenza virus from an individual

Influenza viruses grown in embryonated chicken eggs frequently possess antigenically distinguishable hemagglutinin (HA) compared to virus from the same source grown in mammalian cell culture. To further investigate the extent of variation among viruses from an individual, viruses were isolated from throat washes collected over a 48-hr period during infection with influenza virus designated A/Mem/6/86 (H3N2). Viruses were isolated from limit dilutions in eggs and mammalian Madin-Darby canine kidney (MDCK) cells and the antigenic, structural, and receptor-binding properties of these viruses were determined. Viruses which could be isolated in MDCK cells were present at 10- to 100-fold higher frequency in the original sample than viruses which could be isolated in eggs. The HA of virus clones isolated in MDCK cells were antigenically and structurally identical. In contrast, viruses from the same source, selected at limit dilution in eggs, could be divided into three distinct subpopulations based on the distinguishable antigenic and structural characteristics of their HA molecules. The three groups of egg-grown viruses could be distinguished from each other, and from MDCK cell-grown viruses, not only by a panel of anti-HA monoclonal antibodies, but also by immune ferret sera raised to H3N2 virus strains of recent years and sera raised to the different egg-grown clones themselves. Of these groups, group 1 and group 2 egg-grown viruses each represented a minor subpopulation of viruses which could be isolated in eggs, while viruses of the third antigenic phenotype were the most frequently isolated in eggs. Amino acid substitutions in the HA of egg-grown viruses occurred in antigenic and receptor-binding sites of the molecule. Group 1 viruses each possessed two amino acid substitutions in their HA molecules at residues 193 and 229 in HA1. Group 3 viruses, which displayed altered receptor specificities compared to MDCK cell-grown viruses and other egg-grown viruses, possessed a single amino acid substitution at residue 145 in HA1. The HA of the group 2 egg-grown viruses appeared structurally identical, yet displayed marked differences in antigenic and receptor-binding properties, compared to viruses isolated in MDCK cells. These results demonstrate that multiple, distinct subpopulations of virus can be isolated from a single patient during an infection with influenza and highlights the potential problems in selecting the most appropriate virus for epidemiological and vaccine purposes since selection could result in the use of viruses that are not representative of those which predominate in a human population.     

Use of hyperimmune mouse ascitic fluids for arbovirus differentiation by indirect immunofluorescence and conventional serology

The cross-reactivity of 22 arbovirus species (alphaviruses; tick- and mosquito-borne flaviviruses; and selected bunyaviruses) was tested with monovalent immune mouse ascitic fluids by indirect immunofluorescence (IIF) in comparison with classical serological reactions (virus neutralization -- VN; haemagglutination inhibition -- HI; and complement fixation -- CF -- reactions). Known relationships within the virus groups studied were confirmed. As to the differentiation limits, the VN test was followed by IIF. Evaluation of the ratio of heterologous to homologous antibody activities showed that with the exception of antigenically closely related arboviruses (Western equine encephalomyelitis -- Sindbis; Japanese encephalitis -- Murray Valley encephalitis; dengue viruses; California -- Tahyna), most arboviruses within the antigenic complexes tested could be relatively reliably differentiated by IIF.     

Antigenic structure of the E2 glycoprotein from transmissible gastroenteritis coronavirus

The antigenic structure of transmissible gastroenteritis (TGE) virus E2 glycoprotein has been defined at three levels: antigenic sites, antigenic subsites and epitopes. Four antigenic sites (A, B, C and D) were defined by competitive radioimmunoassay (RIA) using monoclonal antibodies (MAbs) selected from 9 fusions. About 20% (197) of the hybridomas specific for TGE virus produced neutralizing MAbs specific for site A, which was one of the antigenically dominant determinants. Site A was differentiated in three antigenic subsites: a, b and c, by characterization of 11 MAb resistant (mar) mutants, that were defined by 8, 3, and 3 MAbs, respectively. These subsites were further subdivided in epitopes. A total of 11 epitopes were defined in E2 glycoprotein, eight of which were critical for virus neutralization. Neutralizing MAbs were obtained only when native virus was used to immunize mice, although to produce hybridomas mice immunizations were made with antigen in the native, denatured, or mixtures of native and denatured form. All neutralizing MAbs reacted to conformational epitopes. The antigenic structure of the E2-glycoprotein has been defined with murine MAbs, but the antigenic sites were relevant in the swine, the natural host of the virus, because porcine sera reacted against these sites. MAbs specific for TGE virus site C reacted to non-immune porcine sera. This reactivity was not directed against porcine immunoglobulins. These results indicated that TGE virus contains epitope(s) also present in some non-immunoglobulin component of porcine serum.     

Radioimmunoassay for Quantitation of Antibodies to Alphaviruses with Staphylococcal Protein A

A radioimmunoassay (RIA) procedure is described for measuring antibodies to alphaviruses in human and other mammalian sera. The test employed protein Abearing Staphylococcus aureus as a solid-phase immunoadsorbent for (3)H-labeled viruses complexed with immunoglobulin G. Using antibodies produced in humans and guinea pigs, the RIA procedure clearly differentiated among antibodies to Venezuelan, western, and eastern equine encephalomyelitis viruses. Sensitivity of the RIA depended on the concentrations of labeled viruses employed. The dilution of serum that effected binding of 50% of the (3)H-labeled virus (determined by probit analysis) was consistently higher than the neutralizing antibody titer determined by a conventional plaque reduction neutralization test using 80% plaque reduction end points. In addition, sera from 73 individuals were screened for seroconversion following live attenuated Venezuelan equine encephalomyelitis virus vaccine (strain TC-83) inoculation, by RIA using a single serum dilution (1:80); results were identical with seroconversions identified by plaque reduction neutralization test. Hyperimmune Venezuelan equine encephalomyelitis virus sera from a number of mammalian species were successfully titrated by RIA; the species tested were human, guinea pig, white rat, rabbit, burro, dog, monkey, sheep, and cotton rat. The protein A-mediated RIA is a rapid, sensitive, specific, and precise serological tool for measuring antibodies to surface antigens of alphaviruses, and should allow the subsequent development of a competitive binding RIA to measure antigenic potency of inactivated alphavirus vaccines.     

Characterization of foot-and-mouth disease serotype Asia1 viruses grown in the presence of polyclonal antisera in serology and nucleotide sequence analysis

Summary. Foot-and-mouth disease viruses (FMDV) have a high rate of mutation and spontaneous mutants can be readily isolated in the laboratory. In this study, plaque purified FMDV Asia1 vaccine strains (IND 63/72 and IND 491/97) were passaged in-vitro in Baby Hamster Kidney-21 cell monolayers in the presence of sub-neutralizing levels of antiviral polyclonal sera (APS), raised in guinea pigs against the purified and inactivated whole virus particles of IND 63/72, IND 491/97 and IND 13/01. After serial passages under selective immune pressure, the viruses starts growing in the presence of undiluted sera and showed certain characteristics like an increased resistance to neutralization by APS and reduction in plaque counts on titration in plaque assay. Cross-neutralization of these viruses with above-mentioned APS revealed selection of three complete and one partial polyclonal antibody resistant (PAR) viruses based on the r value in micro neutralization test. Alterations were detected at several amino acid residues in the structural protein-coding P1 region. Many of the residues inferred to be positively selected sites in other serotypes of this virus were also prone to substitution under immune selection pressure in Asia1 virus. The present work extends the finding that selection exerted by host antibody also plays a major role in the rapid evolution of FMDV Asia1, as observed in other serotypes.     

Antibodies Produced by Rabbits Immunized with Visna Virus

Immunization of New Zealand White rabbits with purified visna virus elicited antibody activity demonstrated by passive hemagglutination (PHA), complement fixation (CF), and indirect immunofluorescent tests. The antibody activities of hyperimmune sera and Sephadex G-200 fractions of the sera were studied. It was found that the PHA test was 10 to 100 times more sensitive than the CF test in detecting visna antibodies in rabbits. It was also found that the immunoglobulin M fractions from Sephadex G-200 filtration displayed greater PHA activity than did the immunoglobulin G fractions. Although neutralizing antibody was demonstrated in the serum of the natural host (sheep), our attempts to demonstrate neutralizing antibody in the sera from hyperimmunized rabbits (non-natural host) so far have failed.     

Serological investigation of an outbreak of simian varicella in Erythrocebus patas monkeys.

An epizootic of simian varicella occurring in a colony of Erythrocebus patas monkeys was studied serologically by using radioimmunoassay and neutralization tests against (i) a virus strain isolated from an animal that died during the epizootic, (ii) a simian varicella virus strain from an earlier outbreak of simian varicella-like disease at another facility, and (iii) human varicella-zoster virus. Serological tests detected more cases of infection among the animals exposed to virus during the epizootic than were evidenced by clinical findings; only 6 of the 26 animals with seroconversion developed a rash. Good correlation was seen between antibody responses demonstrated by radioimmunoassay and by the neutralization tests. Specificity of the radioimmunoassay was evidenced by the complete agreement with neutralization results for 17 animals which failed to show an antibody response over the course of the outbreak and were assumed not to have been infected. Thus radioimmunoassay is a reliable, rapid, and relatively economical method which could be used for serological screening of primates entering experimental colonies to identify those which might be potential sources of outbreaks through activation of latent simian varicella virus infection. Close correlation was seen between antibody responses to the virus strain from the current outbreak and the one from another epizootic, indicating that the two outbreaks were caused by antigenically similar viruses. Animals showing neutralizing antibody responses to the simian varicella viruses also showed responses to human varicella-zoster virus, which further substantiates the close antigenic relationship between human and simian varicella viruses.     

Resistance of recent measles virus wild-type isolates to antibody-mediated neutralization by vaccinees with antibody

The neutralization capacity of sera from Luxembourgian adolescent vaccinees and from Nigerian women with measles-induced immunity to a number of measles virus strains was compared. Although both cohorts were matched for their hemagglutination inhibition and standard neutralization titers, 12 of the 22 late convalescent sera, and only 6 of 24 vaccinees neutralized all viruses. Similarly, only 2 of 20 viruses were not neutralized by at least 75% of late convalescent sera, in comparison to 10 of 20 viruses that resisted neutralization by at least 75% of the vaccinees. The more resistant viruses were not limited to a certain clade. One Nigerian virus was resistant to neutralization by 30% of the late convalescent women and by 75% of vaccinees. These results suggest that qualitative differences in neutralizing antibodies may reduce further protection of infants by passively acquired immunity against wild-type viruses when vaccinated girls become mothers.