Seroneutralization Test Research Articles

Enterovirus no polio de casos con parálisis residual. Colombia, 1992-1995

Acute flaccid paralysis (AFP) has a wide variety of primary sources and causal agents: physical, physiopathological, toxic and infectious agents. Amongst the latter, Wild Poliovirus and Enterovirus 71 (EV71) are the most frequently isolated agents. After the elimination of native wild poliovirus in Colombia in June, 1991 as the major cause of paralytic illness and having a collection of non-polio enterovirus isolates from 20.8% of the total AFP cases annually notified, there was the need to define non-polio enterovirus role in AFP incidence and the dynamics of their circulation and distribution in Colombia. During 1992-1995, the Epidemiological Surveillance System for Flaccid Paralysis notified 856 cases in children less than 15 years old, of which 69 cases were the object of the present study. 57 isolates were recovered from RD and Hep-2 C cells. Serotype identification was made by neutralisation tests using LBM mixtures (antisera A to H) and molecular characterisation by Polymerase Chain Reaction (PCR) using complementary primers (vira1 genome VPl region) followed by amplified fragment sequencing. Data analysis was done by means of descriptive analysis. No association of old circulation for a specific serotype within a determined geographical region was established, nor was there any causal association with a specific pathology.The presence of an EV 71 isolate in a Guillain-Barré Syndrome case is described.The description of 22 different serotypes of non-polio enteroviruses circulating in Colombia (19 serotypes were identified by molecular methods and 3 by seroneutralisation test) agree with the most frequently found serotypes as described in other studies.The Coxsackievirus 85, B1, 83, Echovirus 6, 7, 13, 20, 30 and Coxsackievirus A2, A10, A14, A16, A18, A21 serotypes were the most frequently found in Colombia during 1992-1995.

Acute infectious bursal disease in poultry: Immunological and molecular basis of antigenicity of a highly virulent strain

This study has confirmed, by the use of immunological and molecular tools, that the recent failures of vaccination against infectious bursal disease (IBD) encountered in Europe were not related to antigenic variation, but to increased virulence of the circulating IBD virus strains. Neutralizing monoclonal antibodies (Mabs) showed that the vaccines of intermediate virulence and the pathogenic strain 849VB had a similar pattern of reactivity in ss neutralization tests. Four distinct epitopes could be defined in seroneutralization and addition ELISA tests. All neutralizing Mabs bound to the structural VP2 protein only in its native form. Moreover, Mabs which did not neutralize some strains precipitated well the VP2 protein from extracts of cells infected with the same virus. This suggests that slight changes in the conformation of the epitope were sufficient to allow the virus to escape to neutralization. VP2 sequencing results confirmed that the neutralizing epitopes are clustered in the variable domain which is highly hydrophobic and flanked by two major hy-drophilic peaks. Three potential 'minor' antigenic sites were identified within the hydrophobic region. Comparison of the VP2 sequence of 849VB strain with other highly virulent isolates showed that they are close together and clearly distinct from 'classical' strains. Moreover, sequencing of IBD vaccines revealed that some of them had not been cloned.

Immunity to diphtheria in the 4–70 year age group in Italy

Diphtheria antibody level in serum samples obtained from 270 subjects aged 4–70 was measured by in vitro seroneutralization test on Vero cells. Of the studied population, 27.8% had an antibody titre below the protective level (<0.01 IU/ml). The prevalence of susceptible subjects showed a significant age-related increase ( p<0.01), with the highest value (53.8%) in the 31–40 age group. Mean antibody titre was maximum in individuals aged 13–14, then decreased reaching the minimum level in the 41–50 age group. These data suggest that in individuals aged 30–50, diphtheria immunity is not satisfactory, both for prevalence of immune subjects and for antibody levels. Therefore, a revaccination of adults with reduced doses of diphtheria toxoid may be advisable.

An outbreak of enterotoxaemia caused by Clostridium perfringens type D in goats in Patagonia

Forty-four of a flock of 117 angora goats in the Rio Negro province of Argentina died within four days. Most of the animals died shortly after the onset of clinical signs, but in a few the clinical course lasted for several days. Post mortem the small and large intestines were filled with watery contents, blood and fibrin clots, and there were numerous ulcers on the mucosa. Small areas of malacia were observed histologically in the brain. Clostridium perfringens type D in pure culture was isolated from the kidneys and gut contents of the affected animals. Epsilon toxin was identified by the mouse seroneutralisation test in the supernatant solution from cultures of these isolates and in the intestinal contents of the affected animals. Heavy infestations with coccidia, nutritional and environmental stress, and an anthelmintic overdose were possible predisposing factors for the outbreak.

Effect of one booster dose in antirabies vaccination

The antibody response obtained after one booster dose of rabies vaccine prepared in suckling mice brains at Serviço de Saúde Pública da Cidade do Rio de Janeiro is described. Four prime vaccinal groups were used: group I, persons who had received 16 doses 10 years before this investigation; group II, persons who had received 5 doses of the vaccine 10 years before; group III, persons who had received 9 doses 5 years before; and group IV, persons who had received 16 doses 5 years before. One booster dose of the vaccine was administered to all persons involved in the study. Blood samples were collected before vaccination (day 0), then 7 and 30 days after vaccination. Antibody titres were determined by seroneutralization test in mice (SWM). 1 The results demonstrated that all persons who had been treated with 5, 9 or 16 doses of the vaccine 5 to 10 years before had their antibody titres increased on the 7th and 30th days after one booster dose.

An ELISA for the detection of antibodies to avian nephritis virus and related entero‐like viruses

An ELISA for the detection of antibodies against avian nephritis virus (ANV) and related entero-like viruses was developed. Different antigenic preparations made from chicken kidney cells infected with the G-4260 strain of ANV were compared. Crude antigen obtained by fluorocarbon treatment of infected cells was found to be appropriate and to give reproducible results with antisera directed against ANV and three entero-like particles (ELPs): the Belgian entero PV2 and entero 3, and the Irish ELP-1. A cut-off value was determined using 30 specific pathogen-free (SPF) sera and the absence of antigenic relationships with nine different reference sera directed against avian viruses was demonstrated. The cross antigenic relationships between avian encephalomyelitis virus (AEV), ANV and the three ELPs was investigated by ELISA and confirms the classification of those fowl enteroviruses into two serotypes: the first including ANV and ELPs and the second including AEV. A strong correlation (94%) was demonstrated between ELISA and a seroneutralisation test. Using ELISA, antibodies against ANV and related ELPs were demonstrated in 13/14 breeding and 8/10 broiler flocks from Belgium.

Electron microscopy, plaque assay and preliminary serological characterization of three ectromelia virus strains isolated in Poland in the period 1986–1988

Three strains of viruses have been identified as ectromedia virus (EV) based on their origin, clinical features, morphology, size of virions (140 x 220 nm), replicative ability and specific cytoplasmatic fluorescence. The mean diameter of plaques produced by EV strains was 0.76 mm (range 0.5-1.0 mm). The neutralizing properties of the tested sera were evaluated by seroneutralization (SN) and hemagglutination inhibition (HAI) tests.

Specific detection of enteroviruses in clinical samples by molecular hybridization using poliovirus subgenomic riboprobes

Enteroviruses were specifically detected in crude clinical specimens or in cell cultures in which the viruses were amplified by dot hybridization by using poliovirus type 1-derived, subgenomic radiolabeled cRNA probes (riboprobes). The sensitivity of this test varied from 2.5 to 33%, when clinical specimens without cell culture were examined, and was about 85% in cell culture lysates. The specificity of the test was 90 to 100%. The riboprobe corresponding to the 5'-noncoding sequence specifically detected the majority of enteroviruses (56 of 57 tested); the riboprobe derived from the VPI capsid region hybridized with the three poliovirus serotypes and with some coxsackieviruses type A and with echovirus type 7. Echovirus 22 did not hybridize with any riboprobe. In stool specimens, nasal aspirates, and cerebrospinal fluids from patients with meningitis, only one type of virus was identified in different clinical samples from the same patient by the seroneutralization test. Hybridization allowed the detection of enteroviral RNAs easily in stool specimens and nasal aspirates but with a low efficiency in cerebrospinal fluids without amplification of the viruses in cell cultures.

Antigenic relationships between fowl enteroviruses

An antigenical cross-relationship was observed by immunofluorescence and seroneutralisation tests between the G-4260 strain of avian nephritis virus and three other entero-like particles, one isolated by McNulty et al. (Avian Pathology, 13: 429) and the entero PV2 and entero 3 both isolated in our laboratory. No cross reactions were observed between those viruses and the avian encephalomyelitis virus.

Seroconversion to bovine respiratory syncytial virus in goats with pneumonia in Zaire

Coupled sera were taken among goats suffering from pneumonia in Zaire. The presence of antibodies to bovine syncytial respiratory virus was investigated by a seroneutralization test. Out of 26 animals, 9 already had antibodies at the time of the first blood sampling, 9 presented a seroconversion and 8 remained seronegative during the two months of observation. These results suggest it would be useful to study the frequency of the infection of small ruminants with this virus and its clinical and economical impact in Central Africa.

SEROLOGICAL EVIDENCE OF BOVINE HERPESVIRUS 1-RELATED VIRUS INFECTION IN THE WHITE-TAILED DEER POPULATION ON ANTICOSTI ISLAND, QUEBEC

High white-tailed deer (Odocoileus virginianus) population densities and the occurrence of harsh environmental conditions are present on Anticosti Island, located in the Gulf of Saint-Lawrence (Quebec, Canada). This island is the northernmost region of white-tailed deer distribution in northeastern North America. The aim of this work was to determine whether a herpesvirus serologically related to bovine herpesvirus 1 (BHV1) may occur in a stressed white-tailed deer population. One hundred one deer sera were collected from apparently healthy animals during the hunting season from September to late November 1985. Fifty-three percent of tested deer were positive to the seroneutralization test using Colorado strain of BHV1 virus. Higher percentages of seropositivity were observed in animals of both sexes greater than 4-yr-old. Analysis of antibody titers in seropositive animals according to age suggests that BHV1-related viral infection is endemic in the Anticosti Island deer population. It is postulated that environmental stress may induce immunosuppression of certain infected and/or carrier animals in their population that shed virus for long periods of time.

Un test immunoenzymatique applique a la detection des anticorps diriges contre la peste bovine: L'interet d'utiliser un virus purifie cultive sur cellules de lignee

Two types of in vitro assays (enzyme immunoassay and sero-neutralization test) were compared for their ability to detect antibodies to rinderpest virus in field sera from West African bovines. Purified virus grown on Vero cells (Ag/Vero) or bovine kidney cells (Ag/BK), were tested as antigen in enzyme-linked immunoassays (EIA). The results of the comparative evaluation of the two antigens by EIA, prove that Ag/Vero did enhance the sensitivity and the specificity of the test in comparison to Ag/BK and offers a 94% correspondence with seroneutralization.

Cellular and Humoral Response of in Ovo-Bursectomized Chickens to Experimental Challenge with Velogenic Newcastle Disease Virus

Humorally deficient, in ovo-bursectomized (Bx) and sham-Bx chickens were vaccinated twice, 1 month apart, with Newcastle disease virus (NDV) Roakin strain and challenged with a velogenic viscerotropic NDV strain via the oronasal route. Hemagglutination-inhibition and seroneutralization tests showed that Bx chickens had reduced antibody-mediated immunity to virus infection. In contrast, they had significantly higher cell-mediated immunity (CMI) before challenge, as estimated simultaneously by determination of blastogenic capacity of peripheral blood lymphocytes induced by phytohemagglutinin and by specific antigen stimulation. After virus challenge, there was transitory inhibition of CMI based on marked reductions in levels of stimulation indices, and this impairment in CMI was supported by persistence of virus in Bx chickens for longer periods. Bx chickens resisted challenge, even though antibody titers were well below those considered predictive of resistance to challenge, suggesting that CMI provides a degree of resistance to velogenic NDV.

Isolement chez des canards mulards d'une souche hypervirulente de virus de la Peste du canard et d'un paramyxovirus aviaire de type 6

This paper describes the isolation and identification of a duck plague virus (DP) and a paramyxovirus (PMV 6), from the livers and intestines collected in 4-month old mule ducks, under fattening, exhibiting 75% mortality and necrotic-haemorrhagic gross lesions. These viruses were isolated in specific pathogen free (SPF) muscovy duck eggs and SPF chicken eggs respectively. Then the DP virus was adapted to duck and chicken fibroblasts. The disease was reproduced in 2-week old SPF muscovy ducklings, intramuscularly inoculated with the previous organs, as well as in contact ducks. From them, only the DP virus was isolated again. Experimentally the intramuscular inoculation of the duck plague French vaccinal strain, 4 h post contact, did not prevent the disease and did not decrease its severity. Regarding the DP virus, the typical signs and lesions observed in experimentally infected muscovy ducks as well as the presence of intranuclear inclusions of the epithelial cells of their oesophagus, intestines, bursa of Fabricus and liver on the one hand, and on the other hand, of the epithelial cells of the duck egg chorio-allantoïc membrane and fibroblasts inoculated with the samples first defined, allowed the characterization of the virus. Direct electron microscopy, as well as the results of seroneutralization tests with different specific avian Herpes virus antisera confirmed the DP virus identification. Moreover the DP isolate was not antigenically different from the serotype actually known. The haemagglutinating virus (PMV 6) was characterized by direct electron microscopy as well as with 18 specific avian Myxovirus antisera; its identification was confirmed too by the specific seroconversion observed 4 weeks post-inoculation of this virus, in 11 weeks old SPF muscovy ducklings. Finally an assay was carried out to appreciate the pathogenicity of theses viruses inoculated either separately or associated. It showed the high pathogenicity of the DP strain. The PMV 6 was apathogenic and no synergic effect with the DP virus was demonstrated. It appears to be the first isolation of PMV 6 in France, to our knowledge. The epidemiological circumstances related to theses isolations are discussed. The failure of the emergency vaccination in contact ducks, might be attributed to the high virulence of the DP strain.

Rapid diagnosis of echovirus 33 infection by neutralizing specific IgM antibody.

In 1982, we isolated 20 strains of echovirus type 33 (EV33) from 18 patients. We studied the humoral response to EV33 of 2,437 subjects from whom at least one serum was available during the same year. In 388 subjects with the neutralizing antibody level at 64 or more, we assayed the EV33 IgM antibodies by seroneutralization test after fractionation of sera by ion exchange chromatography. One hundred ninety-five subjects (8.0%) had a high titre (greater than or equal to 32) of EV33 IgM antibodies, which was considered as evidence of recent infection. The EV33-positive IgM fractions were assayed against five other enteroviruses. Sixty percent of the IgM fractions did not cross-react with any of the five serotypes, 8.7% cross-reacted with at least one serotype but with predominant EV33 IgM response, and 31.3% had an equivalent or greater amount of non-EV33 IgM antibodies; the type specificity of the assay was directly related to the age of the subjects. These findings suggest that determination of neutralizing specific IgM antibody is a sensitive and rapid test for the diagnosis of enterovirus infections, especially in young people.

The influence of the type of immunosorbent on rabies antibody EIA; advantages of purified glycoprotein over whole virus

Two types of in vitro assay (enzyme immunoassay and seroneutralization test) for the titration of rabies antibodies were used to assay sera from mice and humans immunized with cell culture vaccines or neural tissue vaccines. Enzyme immunoassays (EIA) were performed in plates sensitized with whole virus, purified glycoprotein or purified nucleocapsid. Neutralizing antibody titres were determined by the rapid fluorescent focus inhibition test (RFFIT) and by an in vitro seroneutralization test including a rapid enzyme immunotitration of intracellular antigens (REITICA). The results obtained with sera of immunized mice and humans showed that (1) cell culture vaccines mainly induced the synthesis of antiglycoprotein neutralizing antibodies; and (2) neural tissue vaccines induced a high synthesis of antinucleocapsid non-neutralizing antibodies and a more or less important synthesis of antiglycoprotein antibodies depending on the origin of the tissue used for their preparation. Consequently, it was emphasized that when using EIA, the antibody titration must be run in glycoprotein-coated plates rather than in whole virus-coated plates to appreciate correctly the immunizing potency of a rabies vaccine, especially neural tissue vaccine.

The use of an immunoenzymatic assay for the estimation of tetanus antitoxin in human sera: a comparison with seroneutralization and indirect haemagglutination

The ability of an enzyme-linked immunosorbent assay (ELISA) to detect tetanus antitoxin in human sera has been evaluated in comparison with the in vivo seroneutralization test. The results of this study, carried out on 171 serum samples, show that ELISA is a sensitive and specific in vitro test for immunity to tetanus in man; it reveals the minimum protective level of 0.01 IU/ml and is well correlated with seroneutralization. A comparison has also been made with indirect haemagglutination. Differences in specificity in low titered sera, although not statistically significant, have been observed. Reported data suggest that the ELISA may be used for the estimation of tetanus antitoxin in sero-epidemiological surveys and for clinical purposes with reliability equal--and perhaps superior--to that of IHA.

The determination of the potency of human tetanus immunoglobulins by an enzyme-linked immunosorbent assay

A micro enzyme-linked immunosorbent assay has been adapted to the quantitation of specific tetanus antibodies in commercial preparations of human immunoglobulins. The results of the assay are compared with those obtained from the same samples tested by seroneutralization tests in vivo. Statistical analysis of the data shows good correlation between the titres obtained with the two tests. Results obtained by indirect haemagglutination are also reported. It is proposed that all interested laboratories perform the described immunoenzymatic method in vitro for a given period in parallel with the in vivo test to gain sufficient experience of this technique with a view to its use as an alternative to the in vivo test.

Premier isolement du virus Mokola à partir d'un rongeur ( Lophuromys sikapusi)

Summary An isolate of Mokola virus (AnRB3247) was obtained from the brain of a wild rodent Lophuromys sikapusi caught in the Central African Republic. This was the first isolation of Mokola virus from a rodent and the first isolation of this virus in the Central African Republic. This isolate was identified with Mokola virus by complement fixation and seroneutralization tests. The antigenic pattern of this isolate was determined with a panel of monoclonal antibodies, and was compared to those of Mokola isolates from Nigeria and Cameroun, of Lagos bat virus and of Duvenhage virus.

Counterimmunoelectrophoresis for Identification of Infectious Pancreatic Necrosis Virus After Isolation in Cell Culture

An isolate of IPNV identified by seroneutralization test as serotype VR-299 was produced in RTG-2 cells, purified by density gradient centrifugation, and used to prepare the immune serum. Parameters of the CIE technique were adapted for the IPNV and one precipitin line was observed either with the purified antigen or with known positive specimens. A greater number of fish organ homogenates were positive after multiple passages in cell cultures than by direct CIE examination. However, the precipitin antigen was detected in cell culture fluid at early passages. The use of CIE following virus isolation in cell culture is recommended for routine identification of IPNV.