Francesca Beneduce

Detection of hepatitis A virus in shellfish by nested reverse transcription-PCR

A method for the detection of HAV in shellfish, based on the use of guanidinium isothiocyanate-containing solution for RNA extraction and purification steps, followed by nested PCR, is hereby proposed. Tests were carried out on mollusc samples spiked with HAV strain FG. Results showed that in samples subjected only to one round of PCR it was possible to detect HAV at concentrations of 10(3)-10(4) TCID50/10 g of mollusc. The use of the nested PCR renders the system more sensitive and specific enabling the identification of HAV concentrations as low as 1 TCID50/10 g of mollusc. Furthermore thus method, in addition to allowing the avoidance of confirming tests, such as hybridization, proved to be inexpensive and simple to perform.

Complete nucleotide sequence of a cytopathic hepatitis A virus strain isolated in Italy

The molecular basis of the cytopathic effect induced in cell culture by some hepatitis A virus (HAV) strains and variants has not been determined. In order to assess the molecular mechanism(s) underlying this particular phenotype the genome of an Italian cytopathic isolate (strain FG) was sequenced from cDNAs obtained by RT-PCR. Sequence analysis revealed the presence of mutations common to either adapted or cytopathic variants of HAV. In particular, amino acid deletions in proteins VP1 and 3A were detected. Expression of protein 3A in E. coli showed that the N-terminal deletion renders this protein toxic to bacteria.

Prevalence of TT virus in plasma pools and blood products

A high prevalence of TT virus (TTV), a novel virus recently identified in the serum of a patient with post‐transfusion hepatitis of unknown aetiology, has been reported in blood donors worldwide. We investigated the presence of TTV DNA in several lots of blood products and in the corresponding plasma pools. In the process, we determined, from three sets of primers, the one which was most efficient in detecting the viral nucleic acid. This set amplifies the region closest to the 3′‐end of the TTV genome which was proved, by sequence analysis, to be more conserved than the other two regions. Whereas all 10 intravenous immunoglobulin and 21 albumin batches were TTV negative, 4/5 factor VIII concentrates and 4/10 intramuscular immunoglobulin batches were TTV positive. A high prevalence of TTV DNA (70%) was found in the plasma pools that were collected from four different countries. These results confirm the worldwide distribution of this virus and show that TTV is removed with a varying efficiency during the manufacture of blood products.

Chimeric hepatitis A virus particles presenting a foreign epitope (HIV gp41) at their surface.

Hepatitis A virus (HAV) protein 2A has been demonstrated to be involved in virus morphogenesis and suggested to be located on the surface of the particle. To determine whether this protein can function as a target structure to harbor and expose foreign epitopes on HAV particles, a full-length HAV cDNA, containing a seven amino acid stretch of human immunodeficiency virus type 1 (HIV-1) envelope protein gp41, was constructed. Following vaccinia virus MVA-T7-mediated expression of the cDNA in COS7 and Huh-T7 cells, chimeric HAV particles, exposing the foreign epitope gp41 on their surface, were produced. These particles were found to be empty capsids (70S), as judged by immunospecific enzyme linked immunosorbent assay (ELISA) on sucrose gradient fractions and immunoelectron microscopy. The immunological detection of VP1-2A harboring the gp41 epitope of HIV suggests that the 2A domain of HAV is suitable to present foreign antigenic epitopes.

Application of reverse transcriptase-nested-PCR for detection of poliovirus in mussels

In order to identify polioviruses in molluscs, we hereby propose a method based on precipitation with PEG 6000 followed by the use of a commercial kit (RNAfast II-Molecular System-San Diego) for the extraction and purification of viral RNA. The RT-PCR phase is followed by a second amplification using nested primers to increase the sensitivity and specificity of the method. Tests were carried out on mollusc samples spiked with Poliovirus 1. Results showed that in samples subjected only to one round of PCR it was possible to detect Poliovirus concentrations as small as 10(3)TCID50/ml. The use of nested-PCR makes the system more sensitive and specific enabling the identification of Poliovirus concentrations as small as 1 TCID50/ml.

PREVALENCE OF TT VIRUS IN HEALTHY CHILDREN AND THALASSEMIC PEDIATRIC AND YOUNG ADULT PATIENTS

Background The recently discovered TT virus (TTV) has been shown to be highly prevalent in patients with cryptogenetic chronic liver disease and fulminant hepatitis. To study the frequency of TTV and to evaluate the possible association with liver disease, 37 pediatric and young adult patients with thalassemia, and 36 healthy children were included in the study. The sera of 100 blood donors selected randomly in the same period were also tested for TTV DNA. Methods The TTV amplification by polymerase chain reaction (PCR) was performed using a first set of primers that recognize an internal sequence into N22 and a second set of primers amplifying a sequence within 5´NCR (5´ noncoding region). Results The first set of primers revealed TTV DNA in 73% of thalassemic patients, in 8% of healthy children, and in 5% of healthy blood donors. With the second set of primers, the prevalence of TTV DNA was, respectively, 100% in thalassemic patients, 44.5% in healthy pediatric patients, and 87% in healthy blood donors. All individuals who tested positive for TTV by the first set of primers were also positive by the second primer set. The TTV infection seemed not to be the cause of altered transaminase levels. Sequencing of TTV clones from thalassemic patients showed the presence of different TTV variants in the same serum. Conclusion The prevalence of TTV in polytransfused children is similar to that detected in blood donors. Moreover, TTV can be detected in healthy children of all ages. The presence of TTV seems to have no clinical significance.

Site-directed mutagenesis of hepatitis A virus protein 3A: effects on membrane interaction.

Due to a stretch of hydrophobic amino acids, protein 3A of hepatitis A virus (HAV) has been suggested to act as a membrane anchor or a carrier of the genome-linked protein 3B (VPg) during viral RNA synthesis. Mutagenesis analysis was performed in order to elucidate the role of the N- and C-terminal tracts of protein 3A in cell membrane interaction. Expression of the mutated proteins in E. coli cells demonstrated that the presence of positively charged residues at the C-terminus is not required for membrane anchoring. Changes in the primary sequence involving charged amino acids at the N- and C-termini critically influenced the ability of the protein 3A of a cytopathic strain of HAV to change bacterial membrane permeability. This result demonstrates the strict correlation between the structure and pore-forming potential of HAV protein 3A.