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PubMed1980

Persistence and variation of Mangosta street rabies virus antigens during adaptation into mice.

A Mangosta street rabies virus strain antigen not shared by the challenge virus standard (CVS) was identified by counterimmunoelectrophoresis. This strain antigen persisted following nine mouse passages of the virus, during which the CVS strain antigen was not acquired. These observations suggest that the antigen composition of the two virus strains is different. Antigenic differences during the course of adaptation of Mangosta virus were also revealed by crossed serum neutralization tests. The findings are discussed in terms of the occurrence of variations in the number and localizaion of antigen sites in the virus and of the possible existence of antigenic competition by non-infective viral antigens in serum neutralization tests for rabies.

Animals↗
PubMed1987

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine virus diarrhea virus in sera from border disease virus-infected sheep.

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific antibodies against the causative agent of border disease in ovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified bovine virus diarrhea virus was used as test antigen. The optimal amount of antigen was 0.5 microgram/well, and the optimal concentration of conjugate was at 1/4,000 dilution. A total of 20 ovine serum samples, which had been collected from animals with or without border disease, were compared by ELISA and serum neutralization test for the detection of border disease-specific antibodies. ELISA was shown to be equally specific but less time-consuming and easier to perform than serum neutralization test. A positive correlation (r = 0.60) between the two tests was found.

Animals↗
PubMed1982

Antibody responses to human diploid cell vaccine for rabies with and without human rabies immune globulin.

One hundred one volunteers with no exposure to rabies were given human diploid cell vaccine (HDCV) for rabies with or without 20 international units of human rabies immune globulin (HRIG)/kg of body weight to evaluate schedules for therapy with HDCV and HRIG after exposure. All of the volunteers who received three or more doses of HDCV alone or four or more doses of HDCV with HRIG developed high titers of neutralizing antibodies by day 35, which persisted for at least 60 days. By day 7, of the 61 volunteers given HRIG and HDCV, 53% had neutralizing antibodies by a mouse neutralization test and 67% had neutralizing antibodies by a rapid fluorescent focus inhibition test. Similar antibody levels were found in volunteers given HRIG alone, a finding which suggests that low or undetectable early titers after administration of HDCV and HRIG were due to inadequate HRIG dosage rather than any interaction between the passive antibody (HRIG) and the vaccine antigen. These results suggest that trials with 30 or 40 international units of HRIG/kg in combination with HDCV are warranted.

Antibodies, Viral↗
PubMed1979

Complement-enhanced neutralizing antibody response to varicella-zoster virus.

Humoral immunity following infection with varicella-zoster virus (VZV) was evaluated by a complement-enhanced neutralization test. The specificity of the test was examined and its sensitivity compared with that of the assay which measures antibody to VZV-induced membrane antigen (FAMA). Generally, the titer of neurtalizing antibody was two- to fourfold higher than the FAMA titer. The absence of neutralizing activity at a serum dilution of 1:4 indicated susceptibility of the donor to VZV infection and correlated with an absence of FAMA (titer, less than 1:2). A survey of susceptible leukemic children exposed to chicken pox revealed that several recipients of zoster immune globulin had a subclinical infection, as manifested by seroconversion and persistence of neutralizing antibody to VZV. Results of these studies indicate that the complement-enhanced neutralization test is a sensitive and specific assay for determination of humoral immune status with regard to VZV in healthy and immunosuppressed individuals.

Adult↗
PubMed2003

Occlusive vasculitis in a patient with concomitant West Nile virus infection.

PURPOSE: To describe a patient with occlusive, retinal vasculitis and concomitant, confirmed, acute West Nile virus (WNV) infection. DESIGN: Observational case report. METHODS: Main outcome measures included comprehensive ophthalmic examination with fluorescein angiography, color photography, and serologic testing for WNV and St. Louis encephalitis (SLE) virus including plaque reduction neutralization testing (PRNT). RESULTS: A 46-year-old woman developed a sudden decrease in vision in her left eye 2 weeks after confirmed WNV infection and demonstrated multiple, small, patchy areas of retinal edema with scattered microaneurysms. Fluorescein angiography showed multiple branch artery occlusions with extensive nonperfusion. Serologic titers for WNV were positive for acute infection. Plaque reduction neutralization testing confirmed WNV infection and excluded St. Louis encephalitis virus infection. Other etiologies of occlusive vasculitis were not present. CONCLUSIONS: Occlusive, retinal vasculitis may occur in the setting of acute WNV infection.

Acute Disease↗
PubMed2000

Large cortical lesions produce enduring forelimb placing deficits in un-treated rats and treatment with NMDA antagonists or anti-oxidant drugs induces behavioral recovery.

Previous studies have utilized a lesion model of cortical injury that produces transient behavioral impairments to investigate the recovery of function process. To better understand the recovery process, it would be beneficial to use a lesion model that produces more severe, enduring, behavioral impairments. The purpose of experiment 1 was to validate whether large lesions of the sensorimotor cortex (SMC), which included the rostral forelimb and caudal forelimb regions, produced enduring behavioral deficits. Rats were given large unilateral electrolytic lesions of the SMC, administered either the N-methyl-D-aspartate (NMDA) antagonist, MK-801 or saline 16 h after injury, and tested on a battery of behavioral tests. Enduring behavioral deficits were observed, for at least 6 months, on two tests of forelimb placing while transient deficits were observed on the foot-fault and somatosensory neutralization tests. Administration of MK-801 facilitated recovery on the somatosensory neutralization test; however, it did not induce recovery on either forelimb placing test. A second experiment was performed to determine if earlier administration of MK-801, the NMDA antagonist magnesium chloride (MgCl(2)), or the anti-oxidant N-tert-butyl-alpha-phenylnitrone (PBN) could induce behavioral recovery in this chronic model. Treatment with these drugs induced behavioral recovery on the forelimb placing tests, whereas, the saline-treated rats did not show any signs of behavioral recovery for at least 3 months. Anatomical analysis of the striatum showed that MK-801 and MgCl(2) but not PBN reduced the extent of lesion-induced striatal atrophy. These results suggest that administration of MK-801, MgCl(2), or PBN shortly after cortical injury can induce recovery of function when recovery is otherwise not expected in un-treated rats.

Animals↗
PubMed1992

Protective role of antigenic sites on the envelope protein of Hantaan virus defined by monoclonal antibodies.

To investigate the role of Hantaan virus envelope glycoprotein in infection, a panel of monoclonal antibodies (MAbs) was examined in vitro with several serological tests and in vivo by passive transfer experiments in mice. An antigenic site, specific for the inhibition of infected cell focus was detected with the focus inhibition neutralization test (FINT), in addition to the neutralization related antigenic sites, which were revealed by the ordinary focus reduction neutralization test (FRNT). Suckling mice were given the MAbs by passive transfer followed by lethal Hantaan virus challenge. All neutralizing MAbs detected by either FRNT or FINT protected all mice from lethal infection, confirming the importance of the antigenic sites as a protective antigen. Mice given non-neutralizing MAbs by passive transfer, however, began to die earlier than the control group; mean time to death (18.2 +/- 2.1 to 21.5 +/- 2.8 days) being significantly shorter than that of the control group (25.8 +/- 1.8, p less than 0.01, Mann-Whitney, U probability test). Virus titers in brains of mice which died early, were about 10 times higher than those of control mice. These results indicated the early death phenomenon of mice which was mediated by the anti-virus antibody.

Animals↗
PubMedDate not supplied

Tick-borne encephalitis diagnosis in patients with inflammatory changes in the cerebrospinal fluid in a region with very low prevalence.

Tick-borne encephalitis (TBE)-IgG antibodies are used for the serologic detection of antigen contact caused by TBE infection or immunization. In the present study, enzyme-linked immune sorbent assay (ELISA) results from a group of patients with inflammatory changes in the cerebrospinal fluid (CSF) were re-examined using Western blot technology. The result of the TBE-IgG-ELISA was positive in 47 of the 904 sera samples tested. Retesting the sera with a Western blot confirmed this result in only 31.8% of the positive cases. In 134 of the 904 sera, the ELISA result was borderline. In 5.5% of these sera, the Western blot reacted specifically. The remaining 723 sera samples tested negative with the ELISA. Of these sera, 15 were selected randomly and retested with the Western blot; none of them tested positive. The high number of false positive ELISA results can be explained by the highly selected group of patients and the low prevalence of TBE in the region studied. In patients with meningitis or encephalitis with positive ELISA results and uncharacteristic clinical symptoms, the treating physician should consider the possibility of nonspecific reactions involving inflammatory mediators or cross-reactivity with other flaviviruses. The ELISA-mediated diagnosis of TBE should therefore be verified by means of the patient's history and clinical symptoms, as well as further serologic tests including the Western blot, the hemagglutination test and the neutralization test.

Blood↗
PubMed1984

Characterization of Barmah forest virus: an alphavirus with some unusual properties.

Barmah Forest virus has been characterized in a number of ways including electron microscopy of infected cells; physical studies of the virion, its RNA, and associated proteins; N-terminal sequence analysis of the two envelope glycoproteins; studies of macromolecular species present in infected cells; and serological cross-reactions with alphaviruses and bunyaviruses. From these results Barmah Forest virus is clearly an alphavirus since the structure of the virion, the mode of replication, and the macromolecular species present in infected cells are typical of alphaviruses. The N-terminal regions of the two glycoproteins E1 and E2 show extensive sequence homology (approximately 50%) with those of other alphaviruses. Barmah Forest virus cross-reacts in hemagglutination inhibition tests, although not in complement fixation tests or infectivity neutralization tests, with other alphaviruses. In some of its properties Barmah Forest virus is unusual, however. It cross-reacts in complement fixation and hemagglutination inhibition tests with Umbre virus, a bunyavirus, which originally led it to be classified as a bunyavirus; the glycosylation pattern of E2 of Barmah Forest virus appears to differ from that of other alphaviruses; and the sedimentation coefficient of the virion appears to be slightly less than that of other alphaviruses.

Aedes↗
PubMed1990

Analysis of a glycoprotein of human herpesvirus 6 (HHV-6) using monoclonal antibodies.

The virus-specific polypeptides of human herpesvirus 6 (HHV-6) were studied. Six hybridoma clones secreting monoclonal antibodies were established. Localized cytoplasmic antigen and surface antigens were found in and on the infected cells, respectively, by using the immunofluorescent test with those antibodies. In the neutralization test, two clones (OHV3 and OHV9) neutralized HHV-6, but the other five monoclonal antibodies did not. When infected cells were labeled with [35S]methionine and the antigens were immunoprecipitated by OHV3 and OHV9 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, two polypeptides with molecular weights of 98,000 (98K) and 92K were detected. Furthermore, it was found that these polypeptides were glycosylated, because they were labeled with [14C]mannose. Pulse-chase studies revealed that precursor molecules were cotranslationaly glycosylated to produce 98K glycoprotein and they were replaced by 92K glycoprotein. Endo-beta-N-acetylglucosaminidases H and F reduced those glycoproteins to putative precursor molecules of 75 kDa. This indicates that N-linked high-mannose sugars associate to 98K and 92K glycoproteins.

Animals↗
PubMed1993

Time course of hepatitis A antibody production after active, passive and active/passive immunisation: the results are highly dependent on the antibody test system used.

Two commercially available automated test systems for hepatitis A antibody, HAVAB IMX (Abbott) and ENZYMUN Anti-HAV (Boehringer) were evaluated in a study of active, passive and active/passive immunisation against hepatitis A. The inactivated hepatitis A vaccine Epaxal Berna and the hepatitis A immunoglobulin preparation Globuman were products of the Swiss Serum and Vaccine Institute. Although both hepatitis A antibody test kits were standardised with the same international WHO standard hepatitis A immunoglobulin preparation, divergent results were obtained for the level of circulating hepatitis A antibody after vaccination. One month after the vaccination the mean geometric antibody titres were 315 mIU/ml after active, 253 mIU after active/passive and 22 mIU after passive immunisation when measured with the Enzymun assay. In the same sera 70 mIU/ml after active, 60 mIU after active/passive and 18 mIU after passive immunisation could be detected with the IMX test. Antibody avidity studies could not explain the differences obtained by the two test methods. The neutralization test is the standard method for the estimation of protection against hepatitis A. This test is not suitable for large series of serum samples, and enzyme immunoassays are indispensable for vaccination studies. To be suitable for monitoring antibody development in phase I and II clinical trials as well as in postmarketing studies, EIA tests for hepatitis A antibodies must be commercially available and of known sensitivity. The Enzymun anti-HAV test developed by Boehringer Mannheim (Germany) offers the possibility to measure antibody titres around the protective level of 20 mIU/ml which is reached by the passive immunisation with immunoglobulin preparations or within two weeks after active vaccination with an inactivated hepatitis A vaccine. The Abbott IMX test system is more useful for the detection of natural infections by the hepatitis A virus.

Adult↗
PubMed1992

A randomized phase II study of a new tick-borne encephalitis vaccine using three different doses and two immunization regimens.

A new, highly purified inactivated tick-borne encephalitis (TBE) vaccine (FSME-Vaccine Behring, BI 71.061) was recently registered in Germany. A multinational phase II study was performed in seven centres located in areas endemic for TBE. A total of 379 healthy adults were randomly allocated into three dosage groups (1.0, 1.5 and 2.0 micrograms antigen per dose, respectively) and into two immunization schedules [vaccination with one dose of 0.5 ml intramuscularly on days 0, 7 and 21 (abbreviated schedule), or on days 0, 28 and 300 (conventional schedule)]. Antibody response to vaccination was assayed by enzyme-linked immunosorbent assay (ELISA), haemagglutination inhibition test (HIT) and neutralization test (NT). Seroconversion rates in the different groups 28 days after one single dose were 75.3-83.5% in ELISA, 35.8-50.6% in HIT, and 100% in NT. All vaccinees showed seroconversion in all tests on day 42 in the conventional schedule and on day 35 in the abbreviated schedule, with the exception of one subject, who remained seronegative in HIT only. Geometric mean titres (GMT) of about 3000 in ELISA were achieved by two vaccinations in the conventional schedule and showed a booster increase to 5500-8000 GMT after revaccination on day 300. Overall frequency of adverse events (related and unrelated) was 37% (conventional schedule) and 46% (abbreviated schedule) after the first, 9% and 21% after the second, and 5% and 15% after the third vaccination, respectively. Generally, side effects were mild and transient, including mainly headache, fever, malaise and local irritation. Serious, vaccine-related side effects did not occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Induction of feline leukaemia virus-neutralizing antibodies by immunization with synthetic peptides derived from the FeLV env gene.

The surface glycoprotein, gp70, of feline leukaemia virus (FeLV) contains sites which are important in inducing neutralizing antibodies. Using synthetic peptides corresponding to nucleotide sequence 729-957 (amino acid sequence 243-319) of FeLV-A/Glasgow-1, the antigenicity and immunogenicity of this part of the viral surface glycoprotein were investigated. The region contains two highly conserved domains separated by a variable region (VR4), when compared with FeLV of subgroups B and C, and an epitope known to be involved in virus neutralization is located in the N-terminal conserved domain. Five murine monoclonal antibodies generated by immunization with virus were found to be directed to this domain and four were virus-neutralizing. Polyclonal mouse, rabbit and cat anti-FeLV antisera, which were virus-neutralizing, were directed to B-cell epitopes in the peptides. To determine if those synthetic peptides could induce neutralizing antibodies, rabbits were immunized with the peptides, singly or in combination. Antibody responses were measured by ELISA for anti-peptide, anti-FeLV and anti-gp70 activity, by immunoblotting, by membrane immunofluorescence and by virus-neutralization tests. Virus-neutralizing antibodies were induced by FeLV gp70 peptides and there was a synergistic effect on antibody production when a combination of peptides covering amino acid sequence 243-319 of FeLV-A was used. In a second experiment, six rabbits and six cats were immunized with a combination of two peptides, which covered the above-defined FeLV gp70 sequence. Comparisons were made of the responses to these peptides incorporated into immunostimulating complexes (ISCOMs) via myristic acid tails, inoculated with 'empty' ISCOMs, or with Al(OH)3. Complete Freund's adjuvant (CFA) had a very strong potentiating effect on the induction of antibodies and immunization with peptides incorporated into ISCOMs was superior to immunization using adjuvants other than CFA. It is very promising that not only in rabbits, but also in the natural host of FeLV, the cat, anti-FeLV gp70 (peptides) antibodies could be induced by FeLV gp70 peptides.

Amino Acid Sequence↗
PubMed1995

Roe deer as sentinels for endemicity of tick-borne encephalitis virus.

The suitability of serological surveys of roe deer (Capreolus capreolus) in determining the spread of tick-borne encephalitis virus (TBEV) was tested in a south German area with a low risk of TBEV infection to humans. Sera obtained from 192 hunted roe were screened by an haemagglutination-inhibition test (HAI) and in an ELISA developed in our laboratory. Those found positive were tested in a neutralization test (NT). Fifty (26.0%) sera reacted positive by ELISA and 43 (86.0%) of these were confirmed by HAI or NT. Forty-seven (24.5%) samples were positive by HAI, 44 (93.6%) of which were also positive in NT or ELISA. Only insignificant increase of the antibody prevalence with age (P = 0.17 for HAI antibodies) suggests that most infections occur at an early age in scattered natural foci. The antibody prevalence in females was lower than in males (OR = 0.63; P = 0.02 for HAI antibodies). In determining the distribution of seropositive roe we increased the sample size to 235 sera. No antibodies were detected in 56 (23.8%) sera collected in the eastern third of the county. The areas of high antibody prevalence in roe match those in which humans have been infected. We conclude that serosurveys of roe deer are useful in marking out areas in which humans face the risk of infection, provided that an adequate number of sera, preferably from males, is available.

Age Distribution↗
PubMedDate not supplied

Specificity of an assay for antibodies to hepatitis B surface antigen.

Eight percent of blood donors were found to have antibodies to hepatitis B surface antigen when tested by radioimmunoassay using commercial test kits. Four percent of positive reactions proved nonspecific when tested by the neutralization test using pooled surface antigen. The risks in relying solely on the reactivity in commercial-kit suggested that the specificity of the assay must be confirmed.

Antibodies, Viral↗
PubMed1996

A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.

Cell Line↗
PubMed1995

Decay of maternally derived measles antibody in a highly vaccinated population in southern Israel.

The introduction of live attenuated measles vaccine in Israel during 1967 dramatically decreased the incidence of measles. However, cases still occur in periodic outbreaks and epidemics, with an increasing proportion of infants and children younger than 2 years of age. We examined the decay of maternally derived measles antibody during the first year of life in the Jewish population of Israel which represents a highly vaccinated population with immunization rates exceeding 90%. We used sera of healthy full term infants born in 1988 and 1989. Fifty specimens for each of the following age groups were used: 0 (cord blood), 2, 4, 6, 7 and 12 months. Three assays for each specimen were used: enzyme-linked immunosorbent assay (ELISA); hemagglutination-inhibition test (HI); and neutralization test (NT). Good correlation among all 3 tests was found. All cord blood specimens were positive by at least 2 assays. Seropositivity rates declined rapidly with age. Fifty percent of all 4-month-old infants and < 30% of all 6-month-old infants were positive by 1 test or more; at 12 months of age none of the tested specimens was positive by HI or NT and only 1 of 50 infants was positive by ELISA. In infants younger than 6 months of age, 5 (22%) of 23 specimens negative both by ELISA and by HI were positive by NT, but in 6-month-olds, only 2 (7%) of 28 negative by ELISA and HI were positive by NT, and in 12-month-olds none was positive. The results from southern Israel are similar to those obtained in North America and provide evidence that infants older than 6 months of age in a well-immunized population may be poorly protected against measles. On the basis of this information and epidemiologic data, the Israel Ministry of Health has recommended lowering the immunization age for measles, mumps and rubella from 15 months to 12 months.

Antibodies, Viral↗
PubMed1993

Neutralizing human monoclonal antibodies against Puumala virus, causative agent of nephropathia epidemica: a novel method using antigen-coated magnetic beads for specific B cell isolation.

Human monoclonal antibodies (MAbs) against Puumala (PUU) virus were generated and characterized. Human spleen B lymphocytes were preselected for specific surface immunoglobulin (Ig) using magnetic beads coated with the viral glycoproteins, and subsequently immortalized by Epstein-Barr virus transformation. Four IgG-positive monoclonal lymphoblastoid cell lines (LCLs) were established and have remained stable MAb secretors for over 12 months. Analyses of the antigen and epitope specificities recognized by the MAbs showed overlapping binding patterns of four anti-glycoprotein 2-specific clones. Identical isotypes (IgGl lambda) and isoelectric points (9.2) of the four MAbs suggested that they were derived from the same original clone. The MAbs reacted with eight PUU virus-like strains, but were negative for Hantaan, Seoul, and Prospect Hill viruses in an immunofluorescence assay, indicating binding to a conserved epitope unique for strains associated with the European form of haemorrhagic fever with renal syndrome, nephropathia epidemica. The MAbs neutralized all investigated PUU virus-like strains in a focus reduction neutralization test. The MAb neutralizing activity was significantly enhanced in the presence of human or guinea-pig complement. To stabilize and increase antibody secretion and to reduce the demand for culture medium supplements (e.g. fetal calf serum), three of the monoclonal LCLs were fused with the non-secreting human x mouse partner SPAM-8. Several of the established human x (human x mouse) monoclonal triomas grew faster and produced larger amounts of MAbs when compared with the original LCLs.

Animals↗