Sergio Grutadauria

A novel human adenovirus hexon protein of species D found in an AIDS patient

To date, human adenoviruses are classified into 53 types (types 1–51 and types 53 and 54), which have been grouped into six species named A through F, and the recently identified type 52 has been proposed as member of a new species, G. Type classification is based on type-specific epitopes within loop 1 (L1) and loop 2 (L2) of the hexon protein, which contain seven hypervariable regions that are responsible for type specificity. In this paper, we present the characterization of an adenovirus strain isolated from a male AIDS patient in Cordoba, Argentina. This strain was found to be a member of species D by genomic Sma I restriction analysis. Sequencing of the L1 and L2 regions of the hexon gene and immunological characterization by virus neutralization revealed this hexon to be unique and distinct from the previously identified hexons of types within species D. A seroepidemiologic study in the human population of Cordoba showed that this strain was not endemic in the local human population.

Evaluation of Antibodies against a Rubella Virus Neutralizing Domain for Determination of Immune Status

ABSTRACT The protective immune responses against rubella virus (RV) are related to its neutralizing epitopes, an issue that is important to consider when assessing the immune status of patients with remote infection. In the present paper, we compare the antibodies detected by a synthetic-peptide-based enzyme immunoassay (EIA) with antibodies detected by the traditional technique of hemagglutination inhibition (HIA) in patients with remote RV infection. The synthetic peptide used as an antigen (SP15) represents a neutralizing epitope that corresponds to amino acids 208 to 239 of the E1 glycoprotein. The SP15-EIA was developed, all variables that affected the assay were standardized, and the test was validated using reference sera. Serum samples (n = 129) from patients with remote RV infection were tested by HIA and SP15-EIA. Discrepant sera were assayed by MEIA (IMX/Abbot). The comparison between HIA and SP15-EIA, taking HIA as the standard methodology for determining immune status, showed that SP15-EIA is very specific and sensitive for detecting protecting antibodies (specificity, 100%; sensitivity, 98.20%). This study demonstrates that antibodies against the neutralizing domain represented by SP15 would be important in the memory response after natural infection and may be a good tool in the determination of the true immune status of patients with remote infection with regard to RV.

Cell-fusion assay for the detection of rubella virus in Vero cells

BACKGROUND AND OBJECTIVES Rubella virus (RV) produces a subtle and slow-developing cytopathic effect in Vero cells that is difficult to recognize, especially at low multiplicities of infection. In order to facilitate the detection of RV in cell culture, we standardized a low-pH virus-mediated cell-fusion assay. STUDY DESIGN The incubation periods, temperatures, pH and multiplicity of infection were established. The specificity of the method was tested by immunofluorescence assay and cell-fusion inhibition by specific sera. RESULTS Six days post infection, Vero cells were treated for 5 min with fusion medium. After that, monolayers were incubated with medium at neutral pH for 16 h and then stained. Gigantic cells with multiple nuclei were observed. CONCLUSIONS The method allowed the observation of unequivocal images that are easier to recognize than the cytopathic effect caused by RV in the same cell line. At the same time, the method is simple, accessible and shown to be specific to demonstrate the replication of several strains and isolates of RV in Vero cells.

Analysis of viral glycoproteins by glycosidic digestion inside a polyacrylamide gel.

We adapted the method described by Cleveland et al. (1977); (Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102-1106) to study the glycosidic residues linked to the viral glycoproteins of two enveloped viruses: Junin virus (JV) and rubella virus (RV). Radioiodinated glycoproteins were obtained from purified virions, isolated from SDS-polyacrylamide gels and then hydrolysed by specific glycosidases inside a second gel. N-linked oligosaccharides, mannose and galactose were found as terminal residues in the JV-G1 glycoprotein. Mannose and N-glycans of complex hybrid type were present on RV glycoproteins.