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Biomedical subjects

G Gerna

Publications and source records attributed to G Gerna.

At least 145 records · Page 8Linked to original sources
PubMed1989

Protection of Peruvian children against rotavirus diarrhea of specific serotypes by one, two, or three doses of the RIT 4237 attenuated bovine rotavirus vaccine.

A randomized placebo-controlled double-blind field trial of RIT 4237 attenuated rotavirus vaccine in Lima, Peru, evaluated the protection against diarrheal illness by one, two, or three doses of vaccine. There were 391 children, 2-18 months old, studied for the occurrence of diarrhea during the 18 months after vaccination. Three doses of the vaccine provided 40% protection against any diarrheal illness associated with rotavirus alone but 58%-75%; protection against the more severe rotaviral illnesses. The vaccine appeared to be more efficacious when it was administered to children in the first year of life. Three doses provided up to 89% efficacy against more severe diseases due to serotype 1 rotavirus, and one dose also afforded significant protection. The protection was lower, even with three doses, against serotype 2 rotavirus. This vaccine trial has provided important insights on how such trials should be conducted and on the serotype-specificity of protection from rotavirus infection. Future vaccines should be able to protect against severe disease caused by all rotavirus serotypes and must work in developing countries where rotavirus is the most important cause of diarrheal mortality.

Antibodies, Viral↗
PubMed1989

Rapid detection of respiratory syncytial virus in nasopharyngeal secretions by immunofluorescence and ELISA does not justify discontinuation of virus isolation.

Direct fluorescent antibody assay (DFA) using monoclonal antibody and enzyme-linked immunosorbent assay (ELISA) for rapid detection of Respiratory Syncytial Virus (RSV) in nasopharyngeal secretions (NPS) were compared with conventional virus isolation and identification procedures in cell cultures. When 134 NPS were examined from infants and young children with acute respiratory tract infection, 42 (31%) were culture-positive for RSV and 31 of these were detected by the appearance of a typical cytopathic effect and identified by DFA either before or after its appearance, whereas 11 were identified as RSV-positive by DFA performed blindly on HEp-2 cell cultures 5 or 10 days after inoculation. DFA for RSV on NPS smears was positive in 33 (26%) cases, from seven of which RSV was not isolated. The same group of 134 NPS was tested for RSV detection by three commercial ELISA kits. The sensitivities of the three ELISA kits when compared with a combination of culture and DFA results, were comparable (53%, 51%, and 47% for Ortho, Kallested, and Abbott, respectively), whereas specificity was 100% for all three assays. In the group of 26 NPS detected as positive by both virus isolation and DFA, 20-22 (77-85% according to different kits) were found positive for RSV by the three ELISA assays. These data suggest that virus isolation is still critical for diagnosis of a fair number of cases of RSV infection. Of the two rapid techniques, DFA is a valuable complementary method, whereas ELISA still lacks sensitivity. However, both DFA and ELISA were able to detect RSV in 7 of 8 young patients with severe respiratory infection (pneumonia, bronchiolitis), thus permitting diagnosis of RSV infection at least two days before culturing.

Animals↗
PubMed1988

Factors affecting amplification of BK virus episomal vectors in human cells. Brief report.

Analysis of factors determining replication of BK virus (BKV) episomal vectors in human cells showed that vector copy number was related to the level of BKV T antigen expression. T antigen was synthesized efficiently, as assessed by indirect immunofluorescence, in vector-transfected primary embryonic fibroblasts undergoing neoplastic transformation. Surprisingly, transfected continuous cell lines (143 B, HeLa and KB), kept under biochemical selection or tested in transient assays, produced negligible amounts or no T antigen, revealed only by a sensitive ELISA test, suggesting that in these cells vector amplification was under the control of cellular factors. Presence or absence of BKV late region sequences, BKV strain, orientation of the inserted genes and presence or absence of selection were not relevant for vector replication. Type of biochemical selection, however, was important, since BKV vectors containing the thymidine kinase gene replicated better than those containing the neo gene. Despite great variability, vector copy number increased in transfected clones of adenovirus 5-transformed 293 cells, in the absence of immunofluorescence detectable T antigen. These cells express adenovirus immediate early proteins E1A and E1B which may directly or indirectly activate BKV origin of replication.

Antigens, Polyomavirus Transforming↗
PubMed1988

The outer capsid glycoprotein VP7 of simian rotavirus SA11 contains two distinct neutralization epitopes.

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.

Antibodies, Monoclonal↗
PubMed1988

Comparative sensitivities of solid-phase immune electron microscopy and enzyme-linked immunosorbent assay for serotyping of human rotavirus strains with neutralizing monoclonal antibodies.

Suspensions of 24 rotavirus strains, 6 for each known human rotavirus serotype, were serially diluted and titrated by (i) enzyme-linked immunosorbent assay (ELISA) for rotavirus detection, using monoclonal antibodies (MAbs) specific for group-specific sites of the VP6 inner capsid protein; (ii) ELISA for subgrouping, using MAbs reactive with subgroup-specific determinants of rotavirus VP6; (iii) ELISA for serotyping, using MAbs directed to serotype-specific sites of the VP7 outer capsid glycoprotein; and (iv) solid-phase immune electron microscopy (SPIEM) for serotyping, using VP7-specific MAbs. In addition, in each preparation the proportion of double-shelled rotavirus particles were determined by direct electron microscopy. Results showed that SPIEM was 2- to 16-fold more sensitive than ELISA for serotyping of rotavirus. The titers in VP7-specific tests correlated well with the proportion of double-shelled virus particles in each of the samples. Titers obtained by ELISA for serotyping of suspensions containing 20% or fewer complete particles were up to 4,096-fold lower than those obtained by ELISA for detection. ELISA serotyping titers of samples containing 20 to 80% double-shelled rotavirus particles were up to 128-fold lower than ELISA detection titers, whereas preparations with nearly 100% complete particles had ELISA titers that were less different from each other. ELISA subgrouping titers were four- to eightfold lower than corresponding rotavirus detection titers. It was concluded that, although SPIEM appears to be more sensitive than ELISA, the amount of complete virus particles in the specimens is of critical importance for successful serotyping of human rotavirus strains. Samples rich in single-shelled particles but containing low amounts of VP7 outer capsid glycoprotein might even be strongly reactive in assays for rotavirus detection and subgrouping but virtually unreactive in tests for serotyping.

Antibodies, Monoclonal↗
PubMed1988

Identification of two subtypes of serotype 4 human rotavirus by using VP7-specific neutralizing monoclonal antibodies.

Two distinct subtypes of human rotavirus serotype 4 were identified by using neutralizing monoclonal antibodies directed to the major outer capsid glycoprotein, VP7, of strains ST3 (subtype 4A) and VA70 (subtype 4B). Specimens containing serotype 4 rotavirus, obtained from different countries, were examined for subtyping by using solid-phase immune electron microscopy, enzyme-linked immunosorbent assay, and, for cell culture-adapted strains, neutralization assay. All 59 human rotavirus strains identified as serotype 4 by using animal antisera were classified into either subtype by monoclonal antibodies. This suggests that the antigenic difference between the two subtypes is a consequence of critical variations within the immunodominant serotype 4-specific neutralization site of rotavirus VP7. Subtype 4A (ST3-like) strains were predominant and were detected in stools from patients with gastroenteritis, as well as from healthy infants and young children.

Animals↗
PubMed1987

Association of BK virus with human brain tumors and tumors of pancreatic islets.

BK virus (BKV) DNA was detected by Southern blot hybridization in 19 out of 74 (25.6%) human brain tumors and in 4 out of 9 (44.4%) human tumors of pancreatic islets. BKV DNA was free, in an episomal state and generally in a low copy number (0.2 to 2 genome equivalents per cell). Only occasional tumors contained 10 to 20 genome copies per cell. Viral DNA sequences integrated into cellular DNA were not detected. A number of tumors expressed BKV-specific RNA and T antigen. By transfection of total tumor DNA into human embryonic fibroblasts, viruses with the biological and antigenic properties of BKV were rescued from 6 brain tumors and from 2 tumors of pancreatic islets. Restriction endonuclease mapping of the genomes of the rescued viruses showed that they differ from wild-type BKV. They are all similar to each other and to BKV-IR, a virus previously rescued from a human tumor of pancreatic islets, suggesting the possible association of a BKV variant with specific types of human neoplasms. The significance of the relationship of these BKV variants to human tumors and their possible etiologic role in human oncogenesis are discussed.

Adenoma, Islet Cell↗
PubMed1987

Electropherotype heterogeneity within serotypes of human rotavirus strains circulating in Italy. Brief report.

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.

Child, Preschool↗
PubMed1987

Expression of bovine rotavirus neutralization antigen in Escherichia coli.

A 646 bp fragment derived from a full length cDNA clone of genomic segment 9 of bovine rotavirus (NCDV strain) was inserted into Escherichia coli expression plasmid pEX1. The fragment encodes amino acids 50 to 265 of the major vital neutralization antigen VP7, a 326 amino acid long outer shell glycoprotein. Several transformed bacterial clones were isolated in which the recombinant plasmid directed the synthesis of a cro-beta-galactosidase-VP7 fusion protein that was recognized by rabbit polyclonal antibodies against NCDV rotavirus. Sera from rabbits immunized with the fusion protein specifically reacted with VP7 among NCDV virion polypeptides. The chimeric polypeptide was also specifically recognized by two monoclonal antibodies against UK strain rotavirus VP7 that exhibited virus-neutralizing activity. However, immune sera to the chimeric polypeptide showed no neutralizing activity against bovine rotavirus. These results are discussed in view of a recent report that a fusion VP7-beta-galactosidase polypeptide comprising 35 more amino acids at the carboxy terminus was able to induce neutralizing antibodies in mice to simian rotavirus SA11.

Animals↗
PubMed1987

Synergistic neutralization of rubella virus by monoclonal antibodies to viral haemagglutinin.

Using murine monoclonal antibodies (MAbs) to rubella virus haemagglutinin, five epitopes were identified in competitive ELISA binding assays: A, B, D and E by haemagglutination-inhibiting (HI) MAbs with no neutralizing (Nt) activity, and C by a MAb with neither activity. However, when HI and Nt activities were determined in the presence of anti-mouse immunoglobulins, epitopes A, B and D were defined by both HI and Nt MAbs, whereas epitopes C and E were identified by HI MAbs without Nt activity. A synergistic Nt activity, in the absence of anti-mouse immunoglobulins, was displayed by mixtures of antibodies of different epitope groups. Analysis of mixtures of MAb pairs each belonging to a different epitope class, showed that synergistic Nt activity was elicited primarily by the group A epitope, secondarily by groups B and D and only minimally by groups C and E.

Animals↗
PubMed1987

Comparative evaluation of a commercial enzyme-linked immunoassay and solid-phase immune electron microscopy for rotavirus detection in stool specimens.

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.

Antigens, Viral↗
PubMed1987

Rubella IgM antibody determination: comparison of two indirect and two capture commercial enzyme immunoassays.

A new capture ELISA kit (ETI-Rubek-M) for rubella IgM antibody determination was compared with a commercial reverse (Rubenostika IgM) and two indirect ELISAs (Rubelisa M and Rubazyme-M). Of the 89 sera from 38 patients with acute rubella infection, 70 gave identical results with the four assays (78% concordance), 74 were in agreement in the two indirect ELISAs, and 83 concorded in the reverse assays (83% and 93% concordance, respectively). On the whole, ETI-Rubek-M and Rubenostika IgM proved equally sensitive; sensitivity of the indirect ELISA kits was lower, Rubazyme-M being slightly more sensitive than Rubelisa M. Specificity of the two reverse assays was 100%, whereas Rubelisa M and Rubazyme-M showed a specificity of 88.8% and 93.3%, respectively. In indirect ELISAs, false positive IgM results were mainly observed in cytomegalovirus IgM-positive sera from patients recovering from a primary cytomegalovirus infection. Two sequential sera from one of these patients with IgM to cytomegalovirus were found to be true positive for rubella-specific IgM antibody with ETI-Rubek-M, Rubelisa M and Rubazyme-M.

Acute Disease↗
PubMed1987

Subacute sclerosing panencephalitis: detection of measles antibody in serum and cerebrospinal fluid by enzyme-linked immunosorbent assay, complement fixation and hemagglutination inhibition.

Subacute sclerosing panencephalitis (SSPE), a slowly progressive central nervous system disease, is related to the measles virus. Because laboratory immunoassays are important in the diagnosis of the disease, we compared enzyme-linked immunosorbent assay (ELISA), complement fixation (CF), and hemagglutination inhibition (HI) for the detection of measles antibody in the serum and cerebrospinal fluid of patients with SSPE and in controls. Differences in the sensitivity of the three methods were not observed in the serum and cerebrospinal fluid from patients because all specimens were positive, but ELISA and CF were more sensitive that HI for both serum and cerebrospinal fluid from controls. ELISA also distinguished between subacute sclerosing panencephalitis cases and controls with high measles antibody levels (acute measles). Thus, it appears that ELISA for measles antibody is superior to CF and HI in the laboratory diagnosis of SSPE and is the preferred method.

Antibodies, Viral↗
PubMed1986

Electropherotyping of human rotaviruses: an epidemiological survey of rotavirus infections in Sicily.

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.

Child↗
PubMed1986

Immunoglobulin M to the membrane of uninfected fibroblasts in primary human cytomegalovirus infections.

Thirty-two patients with primary human cytomegalovirus (HCMV) infection were examined for presence of antibody to the membrane (Me) of uninfected human embryonic lung fibroblasts (HELF) using an anti-complement immunofluorescence (ACIF) test. Patients were divided into three groups according to age: less than or equal to 12 months (11 cases), 2-6 years (8 cases), greater than or equal to 13 years (13 cases). Convalescent-phase sera from 13 out of 13 of the older and 6 out of 8 of the middle-aged group gave positive fluorescence staining of the membrane of living uninfected HELF monolayers, whereas only one out of 11 younger patients showed antibody reactivity to HELF Me. ACIF performed on IgM and IgG fractions of randomly selected Me antibody-positive sera, showed a reactivity to HELF Me restricted to antibody of the IgM class (Me IgM). HELF Me IgM were first detected during the second week after the onset of symptoms of primary HCMV infection and disappeared usually within 4-6 months. Me IgM were not detected in sera from patients with HCMV infections other than primary (i.e. recurrent or congenital), or with other herpesvirus infections and were not detected in sera from patients with severe immunological diseases. HELF Me IgM were shown not to be related to rheumatoid factor and their reactivity was shown to be restricted to HELF cells, for it was not observed in any of the other cell types tested. The determination of HELF Me IgM antibody appears to be a new, practical and useful serological tool for the diagnosis of primary infections in patients over 2 years.

Cell Line↗