Biomedical subjects
Publications and source records attributed to N Passarani.
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Seven neutralizing monoclonal antibodies (MAbs) to the rotavirus simian agent 11 were produced. Although displaying variable degrees of haemagglutination-inhibiting activity, they were shown by radioimmunoprecipitation and Western blot analyses to react with the major outer capsid glycoprotein (VP7). In competition binding assays, MAbs defined two distinct VP7 epitopes, which appeared to be close to each other or partially overlapping. In addition, MAbs of the two epitope groups enhanced binding of a broadly reactive, non-neutralizing, MAb specific for rotavirus group antigen.
Using solid-phase immune electron microscopy, 126 of 129 human rotavirus (HRV) strains could be serotyped directly in stools collected in Italy during the period 1981-1985. Prevalence was 70.5 per cent for serotype 1, and 13.2 per cent for each of serotypes 2 and 4. No serotype 3 strain was detected. In parallel, for 39 of 61 HRV strains tested the electropherotype of genomic RNA was successfully determined. Different electropherotypes were detected among strains of the same serotype, whereas the same electropherotype was found in HRV strains of different serotypes. Serotyping and electropherotyping of HRV strains appear to be complementary to each other, and both should be used in conjunction for epidemiological surveys.
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Using solid-phase immune electron microscopy (SPIEM) as a reference test, we examined 151 stool specimens from infants and young children with acute gastroenteritis for rotavirus detection by a one-step commercial enzyme-linked immunosorbent assay (ELISA) with labeled monoclonal antibody. Of the 83 samples determined to be positive for rotavirus by SPIEM, 82 were detected as positive by the monoclonal antibody ELISA (sensitivity, 98.7%), while 67 of the 68 specimens determined to be negative by SPIEM were correctly detected as negative by the ELISA (specificity, 98.5%). The diagnostic accuracy of the ELISA kit was 98.6%. Thus, the one-step monoclonal antibody ELISA, which can be completed in less than 90 min, appears to be highly suitable for the rapid and reliable detection of rotavirus in stools.
An electrophoretic analysis of rotavirus RNA segments was carried out on 522 faecal specimens, obtained from children hospitalized in Sicily in the period 1981/85. One hundred and one viral isolates could be characterized with respect to the electrophoretic pattern of their genomic RNAs. This analysis revealed that in 1981/82 different electropherotypes cocirculated in the infant population. In 1983 one of the patterns became prevalent; in 1984/85 only one electropherotype was detected, both in Palermo and Catania specimens. The serotyping showed that all viral strains with the prevalent electropherotype were subgroup II and serotype 1. These results contrast with the extensive genome variability of rotavirus strains observed in urban areas.
In a group of infants with acute nonbacterial gastroenteritis, examination of paired sera for antibody to human coronavirus (HCV) OC43 and neonatal calf diarrhea coronavirus showed a peculiar pattern of serological response, restricted only to HCV OC43 surface antigens, in a significantly higher proportion than among age-matched controls. In another group of infants and young children with acute nonbacterial gastroenteritis, fecal excretion of coronavirus-like particles was detected by electron microscopy in 34 (16.3%) of 208 patients as compared with three (1.6%) of 182 controls (P less than .01). Two strains of human enteric coronavirus (HECV) were purified from stools of two patients, and immune sera were raised in mice and guinea pigs. Immune electron microscopy showed a two-way cross-reactivity between HECV and HCV OC43 when tested with immune sera and convalescent-phase sera from patients with infection due to HECV or HCV OC43.
Serotyping of human rotaviruses (HRVs) by neutralization requires the previous adaptation of strains to growth in cell cultures, which is often unsuccessful. By using the solid-phase immune electron microscopy (SPIEM) technique with protein A and type-specific, cross-adsorbed, polyclonal immune sera, we divided 40 previously typed culture-adapted strains into the same four serotypes distinguished by neutralization, but HRV strains could also be typed directly on stool extracts. Of 171 HRV strains tested by SPIEM, 163 were typed as a single serotype, two were shown to be mixed serotypes, three could not be typed since they were partially antibody-coated, and three were lacking the common group antigen (atypical rotaviruses or pararotaviruses). In addition, strains of serotype 4 could be classified by SPIEM into two subtypes, 4A and 4B. Overall, strains of serotype 1 were detected in nearly 50% of the 171 HRV-positive stools examined by SPIEM, strains of either serotype 2 or 4 in about 20%, and strains of serotype 3 in nearly 10%. Although the data were too scarce to allow for definite conclusions, the prevalence of the four serotypes appeared to change with the geographic area and the year.
Complement fixation (CF), indirect double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) and solid-phase immune electron microscopy (SPIEM) were compared for their ability to subgroup 73 human rotavirus (HRV) strains from infants and young children with gastroenteritis admitted to one or the other of two different hospitals of Northern Italy. By both indirect DAS ELISA and SPIEM all 73 HRV strains were classified into one or the other of two subgroups. By CF only 67 strains could be subgrouped, as six HRV-positive stools showed anticomplementary activity which could not be eliminated. Indirect DAS ELISA required subgroup-specific, unabsorbed antisera from two different animal species. For SPIEM two antisera from a single animal species were needed, but they had to be absorbed with single-shelled bovine rotavirus for HRV subgrouping to be reliable. Indirect DAS ELISA appeared to be the technique most suitable for extensive application in epidemiological studies of HRV infections by different subgroups. However, SPIEM allowed rapid subgrouping of HRV in stool specimens showing anticomplementary activity in the CF test or non-specific reactions in the ELISA test. In one area of Northern Italy the prevalence of subgroup I HRV infections was 7.8 per cent, while in another it reached 68.1 per cent in the same period.
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Nine human rotavirus strains from stools of infants with gastroenteritis were serially propagated in MA-104 cell cultures. All strains were identified as subgroup 2 rotaviruses by RNA gel electrophoresis, complement fixation, and enzyme-linked immunosorbent assay. The human rotavirus strains were propagated for 15 to 20 passages and then used for immunization of guinea pigs and rabbits. Animal antisera were also raised against a subgroup 1 human strain purified from stools and against the cell culture-adapted Wa strain, a reference subgroup 2 rotavirus of human origin. Cross-neutralization studies revealed the existence of two distinct serotypes within the cell culture-adapted subgroup 2 human rotaviruses: strains related and unrelated to strain Wa were classified as serotypes 1 and 3, respectively. Results with convalescent-phase sera from infants with primary rotavirus infections confirmed the existence of two serotypes within subgroup 2, and the serotypes responsible for primary subgroup 2 infections could be determined on the basis of the neutralizing reactivity of convalescent sera.
Nine cell culture-adapted, as well as 30 clinical, human rotavirus (HRV) strains from fecal extracts of children with primary HRV infection were typed by rapid solid-phase immune electron microscopy with protein A and absorbed DS-1 (HRV serotype 2), Wa (serotype 1), and VA70 (assumed serotype 3) rabbit immune sera. As a reference typing test for cell culture-adapted strains, the neutralization assay was used, whereas for noncultivatable strains typing was done for comparison, indirectly, based upon the differential neutralization reactivity of convalescent-phase serum samples from patients with primary HRV infection versus the three reference HRV serotypes. Typing results by solid-phase immune electron microscopy for all strains examined were in complete agreement with those obtained by the neutralization assay, both on cell culture-adapted strains with the three reference rabbit antisera and on three reference HRV strains with human convalescent-phase serum samples. Since adaptation to growth in cell cultures of clinical HRV strains from stool specimens is a time-consuming procedure and is often unsuccessful, solid-phase immune electron microscopy is preferred over the neutralization assay, giving results in about 16 h and also allowing typing of HRV strains from stool specimens low in virus particles. In addition, HRV strains reacting differently from the three reference serotypes may be easily selected by solid-phase immune electron microscopy for further characterization, as was the case for one strain in this study.
A peculiar pattern of antibody response to human coronavirus (HCV) OC43 and neonatal calf diarrhea coronavirus (NCDCV) was observed in a significantly higher proportion of children with gastroenteritis (GE) than controls. It was directed only to OC43 surface antigens and appeared to be quite different form that observed in OC43 respiratory infections, in which an antibody response to both OC43 and NCDCV surface and internal antigens was found. Subsequently, fecal excretion of CV-like particles was detected by electron microscopy in a significantly higher proportion of infants and young children with acute GE as compared to controls. Immune electron microscopy showed a two-way cross-reactivity between HCV OC43 and human enteric coronavirus (HECV), both using immune sera (raised in animals immunized with HECV strains purified from stools of two patients) and patients' convalescent sera. These data show that HECVs are antigenically related to HCV OC43 and could be somewhat involved in the etiology of GE in infants and young children.
In 127 infants and young children suffering from acute non-bacterial gastroenteritis, diagnosis of rotavirus infection was done by virus detection and serology. Human rotavirus (HRV) detection was performed by direct electron microscopy (EM), conventional immune electron microscopy (IEM) and/or solid phase immune electron microscopy ( SPIEM ); rotavirus antigens were detected by indirect double-antibody sandwich (DAS) ELISA and HRV isolation was attempted in MA-104 or LLC-MK2 cell cultures. HRV serology was done on paired sera from all the patients by the indirect immunoperoxidase antibody (IPA) technique for HRV IgG determination, and by an indirect ELISA method using a purified HRV Wa strain as a solid phase. HRV particles were detected by EM and/or IEM in 53 cases (41.7%) and by SPIEM in 5 additional cases; HRV antigens were demonstrated by indirect DAS ELISA in the same 53 cases, whereas 40 cases (31.4%) were positive for HRV isolation in cell cultures. Sixty-four patients (50.3%) seroconverted by IPA and ELISA, including all the cases (58) positive for rotavirus detection in stools and 6 additional cases. Thus, SPIEM appears to be the most sensitive technique for detecting a few virus particles in stool specimens, but HRV serology is the most sensitive method for diagnosing HRV infections retrospectively, when paired sera are drawn at an appropriate time. However, EM possess the great advantage of detecting in fecal specimens viral agents other than rotaviruses, such as adenoviruses, enteric coronaviruses, small round viruses, astroviruses and others.
Human embryonic lung fibroblast cultures and Vero cell cultures infected with cell culture-adapted strains of human coronavirus (HCV) OC43 or neonatal calf diarrhoea coronavirus (NCDCV) were shown to possess highly cross-reactive membrane-associated antigens (MAA) by the indirect fluorescent antibody technique (IFAMA). MAA appeared 3 h post-infection, concurrently with the appearance of cytoplasmic antigens. Electron microscopic observations of cell cultures infected with either coronavirus strain and labelled with the immunoperoxidase antibody (IPA) technique for MAA detection showed that MAA consisted mainly of a strongly labelled, discontinuous, brush-like layer of amorphous material, strictly associated with the infected cell membrane. By light microscopy, reactivity of MAA with homologous and heterologous immune serum was similar to that of antigens detected by IPA in ethanol-fixed infected cells. IPA and IFAMA, but not haemagglutination-inhibiting (HI) and neutralizing (Nt) antibody, were strongly decreased by absorption of immune sera with trypsin-treated glutaraldehyde-fixed cell cultures infected with homologous virus. MAA IgG antibodies were detected by IFAMA in both human and animal sera. Sera from infants showing an HI and Nt, but not an IPA, antibody response to HCV OC43 were also free of detectable IFAMA antibody to HCV OC43.
A new complement fixation test for toxoplasmosis has been compared with the classical test indirect immunofluorescence test (IFAT) and indirect haemoagglutination test (IHAT). Neither false positive or negative results were obtained with LBCF-H100-TTE. Notably these values correlate well with IFA titers. Furthermore LBCF-H100-TTE shows the highest titers in acute cases and its time course is practically superimposable to IFAT. The differences obtained performing these different tests on the preferential detection of different antigen antibodies systems by these 3 tests are discussed.
The results of immuno-electron microscopic reaction obtained with anti-IgG and/or anti-IgM ferritin conjugated sera after the inter-reaction of RH strain with specific IgG and/or IgM immunosera, confirm: 1) antigenic sites for specific immunoglobulins both IgG and IgM localization on superficial membrane of pellicular complex; 2) the saturation of common antigenic surface sites due to the competition between IgG and IgM; 3) the competitive prevalence of IgG even if IgM concentration is relatively high in unfractioned immunosera. The practical application of such results is discussed in relation to the specific tests for IgM evidentiation during primary response.