G. Cammarota

Differences in hepatitis C virus quasispecies composition between liver, peripheral blood mononuclear cells and plasma

Hepatitis C virus (HCV) exists in vivo as a highly variable mixture of closely related genomes (quasispecies), but the pathogenetic significance of such heterogeneity is still largely unknown. To investigate this issue, we compared the composition of HCV quasispecies found in the liver, peripheral blood mononuclear cells (PBMC) and plasma of ten patients by single-strand conformation polymorphism analysis of the E2/NS1 region and sequencing of the variants detected. We found considerable quasispecies differences between the liver and PBMC in all the patients, involving variant numbers, relative quantities and relative electrophoretic mobilities, but no apparent tissue-specific trend. Genome variants present in the liver and/or PBMC were not detected in the corresponding plasma samples, while certain HCV variants were present only in plasma. No dominant amino acids or amino acid pattern characteristic of variants present solely in the PBMC were detected in the E2/NS1 region sequenced.

Analysis of the genetic diversity and phylogenetic relationship of Italian isolates of feline immunodeficiency virus indicates a high prevalence and heterogeneity of subtype B

The genetic diversity of 32 Italian isolates of feline immunodeficiency virus (FIV) was studied. Isolates were obtained from domestic cats living in different areas. Sequence data were obtained from a 308 bp fragment of the p25 region of the gag gene. Phylogenetic relationships among these sequences and previously published sequences were determined. All the Italian isolates could be assigned to subtype B; however, four isolates formed two separate clusters and may represent genetic outliers. The reliability of classification results was confirmed by repeating the phylogenetic analysis with DNA sequences from the entire gag genes of two isolates and from the surface glycoprotein domain of the env gene of four isolates. It is concluded that the short segment of gag used permits reliable genotyping of FIV isolates. The study also shows that subtype B is largely prevalent in Italy.

During Readaptation In Vivo, a Tissue Culture-Adapted Strain of Feline Immunodeficiency Virus Reverts to Broad Neutralization Resistance at Different Times in Individual Hosts but through Changes at the Same Position of the Surface Glycoprotein

ABSTRACT The broad resistance to antibody-mediated neutralization of lentiviruses recently isolated from infected hosts is a poorly understood feature which might contribute to the ability of these viruses to persist and to the failure of experimental vaccines to protect against virulent viruses. We studied the underlying molecular mechanisms by examining the evolution of a neutralization-sensitive, tissue culture-adapted strain of feline immunodeficiency virus upon reinoculation into specific-pathogen-free cats. Reversion to broad neutralization resistance was observed in seven of seven inoculated animals and, in individual hosts, started to develop between less than 4 and more than 15 months from infection. After comparison of the envelope sequences of the inoculum virus, of an additional 4 neutralization-sensitive in vitro variants, and of 14 ex vivo-derived variants (6 neutralization sensitive, 5 resistant, and 3 with intermediate phenotype), a Lys→Asn or →Glu change at position 481 in the V4 region of the surface glycoprotein appeared as a key player in the reversion. This conclusion was confirmed by mutagenesis of molecularly cloned virus. Analysis of viral quasispecies and biological clones showed that the intermediate phenotype was due to transient coexistence of neutralization-sensitive and -resistant variants. Since the amino acid position involved was the same in four of four recent revertants, it is suggested that the number of residues that control reversion to broad neutralization resistance in FIV might be very limited. Amino acid 481 was found to be changed only in one of three putative long-term revertants. These variants shared a Ser→Asn change at position 557 in region V5, which probably collaborated with other mutations in long-term maintenance of neutralization resistance, as suggested by the study of mutagenized virus.

Helicobacter pylori is an aetiological factor for ischaemic heart disease: the case against.

Helicobacter pylori is one of four organisms often investigated for ari association with ischaemic heart disease. The four, including Chlamydia pneumoniae, Cytomegalovirus and Herpes virus, cause low-grade, life-long infections that can produce a persistent inflammation, the kind that leads to heart disease. Several studies suggest an association, but others suggest none. Patients with poor access to medical care are more likely to become infected and also more likely to suffer from coronary artery disease. Although the literature data are provocative and interesting, the two things may not be related. Helicobacter pylori infection is quite prevalent among individuals without ischaemic heart disease and absent in many of those with ischaemic heart disease. Thus, more definite answers about whether there is any link between Helicobacter pylori and cardiovascular disease are needed. It would be essential to establish the specific mechanisms that possibly confer vulnerability or protection toward coronaropathy. But a definite answer could come from clinical trials designed to test whether antibiotics can prevent the disease. Until now, no randomised trial has suggested a positive effect of Helicobacter pylori eradication in reducing the incidence of cardiac events.

Quantitation of feline immunodeficiency proviruses in doubly infected cats using competitive PCR and a fluorescence-based RFLP

A nested polymerase chain reaction assay, which amplifies a region of the gag gene, was developed for the direct detection of feline immunodeficiency virus (FIV) DNA sequences in the blood of infected cats. This method detects as few as ten copies of a plasmid containing the whole genome of the FIV-Pet isolate on agarose gel. To distinguish two FIV isolates in double infected cats, we devised an RFLP analysis on PCR amplified products exploiting sequence differences in the gag gene of the two strains. To quantitate the two strains, a fluorescent inner-sense primer was used in the second amplification step. Amplicons were subsequently digested, heat-denatured and loaded on a polyacrylamide gel in an automated DNA sequencer. The proportion of the two isolates was determined using the laser-excited fluorescence of labelled strain specific fragments. These data were used to extrapolate the numbers of proviral genomes from the total viral load as estimated by using a competitive PCR assay. These sensitive and specific assays complement virological detection of FIV and enable superinfection studies to be evaluated; a prerequisite for the testing of live attenuated immunodeficiency virus vaccines.