Giorgio Achilli

Molecular characterization of two isolates of human T cell leukaemia virus type II from Italian drug abusers and comparison of genome structure with other isolates

The human T cell leukaemia virus type II (HTLV-II), whose pathogenicity is as yet unclear, was recently found to be associated with intravenous drug abuse in North America and Europe. HTLV-II was isolated from two Italian drug abusers belonging to the same cohort and coinfected with human immunodeficiency virus type 1. Two new isolates, HTLV-II Gu and Va, were established in a culture of BJAB cells, a continuous B cell line (Epstein-Barr virus-negative), and characterized by nucleotide sequence analysis of the long terminal repeat (LTR) and portions of the gag, env and X regions. These sequences were compared to those of the HTLV-II Mo isolate reported in the literature. No major variations were observed in important regulatory elements of LTR nor in the stem-bulge-loop configuration known to be essential for binding of rex protein. The results obtained from the sequence of the 1988 nucleotides examined indicated a 1.6% variability between the Gu and Va isolates and about 6% with respect to Mo. Notable differences were found in the structure of putative open reading frames of the X region when compared to those reported for the Mo isolate. Restriction analysis of proviral DNA of two isolates and comparison with the physical map of the Mo isolate confirmed the existence of genetic heterogeneity in the HTLV-II group and demonstrated that the new isolates Gu and Va belong to the HTLV-IIb subtype. The results of this study show that the new isolates have distinct features with respect to the Mo isolate though all important regulatory elements of the LTR appear to be well conserved.

Human Coronavirus OC43 Serum Inhibitor and Neutralizing Antibody by a New Plaque-Reduction Assay

Summary A plaque-reduction assay for the determination of neutralizing antibody to human coronavirus OC43 has been set up, using as a plaquing factor the fetal calf serum containing an OC43 inhibitor. A similar inhibitor has also been found in human sera both negative and positive for OC43 antibody. The inhibitor can be removed by treatment of sera with phospholipase C and is likely to be a lipoprotein of the high-density class. The new plaque-reduction assay appears to be a very sensitive test for both OC43 antibody determination and serodiagnosis of OC43 infections. It may also be very useful for studies of antigenic relationships between human and animal coronaviruses. However, before testing, sera must be routinely treated for removal of viral inhibitor.

Determination of coronavirus 229E antibody by an immune‐adherence hemagglutination method

An immune‐adherence hemagglutination (IAHA) method for coronavirus 229E antibody determination has been developed both for diagnosis of recent infections and for detection of long‐past infections. Results have been compared with those obtained by complement fixation (CF), neutralization (Nt), and indirect hemagglutination (IHA) tests. The IAHA method has been shown to be as sensitive as the CF, Nt, and IHA tests in detecting cases of acute 229E infection. However, in a seroepidemiological survey of 343 healthy people of all ages, IAHA detected 229E antibody in 254 individuals (74.0%), Nt in 166 (48.3%), IHA in 89 (25.9%), and CF in 30 (8.7%). A study of the prevalence of coronavirus 229E IAHA antibody in the different age groups has shown that during the second decade of life nearly 100% of the population acquire this type of antibody, whereas only 50% are positive at the end of the first decade. In the older age groups, the high frequency of CF antibody (“marker” of recent infection) indirectly confirms the high rate of 229E reinfections and the nonprotective nature of IAHA antibody. CF titer ⩾ 1:8 in 90% of cases corresponded to IAHA titers ⩾ 1:64. However, sera with IAHA titers of ⩾ 1:128 were often CF‐negative. Recent 229E infections (or reinfections), as determined by the presence of CF antibody, were more frequent in April‐May than in October‐November. Three cases of acute infection showing 229E seroconversion (two adults and one child) were observed during the winter‐spring season. IAHA appears to be the test of choice for seroepidemiological surveys.

Quantification of HIV-1 proviral DNA in patients with undetectable plasma viremia over long-term highly active antiretroviral therapy

OBJECTIVES To assess the prognostic role of proviral DNA in peripheral blood mononuclear cells (PBMC) of patients with undetectable viremia over long-term highly active antiretroviral therapy (HAART). METHODS Eighty-two human immunodeficiency virus (HIV)-1-infected patients, free of acquired immunodeficiency syndrome (AIDS), received zidovudine plus lamivudine plus indinavir. Levels of plasma HIV-RNA, and PBMC proviral DNA and RNA unspliced (US) transcripts were evaluated by using competitive polymerase chain reaction (cPCR) assays, every 3 months over 1 year. RESULTS Among patients with undetectable viremia at baseline, 13 of 18 with CD4 cell count 350/mm3 or less and 12 of 16 with CD4 between 351 and 700/mm3, constantly maintained undetectable RNA levels; in these patients, a mean proviral DNA decrease of 0.67 6 0.7 and 1.03 6 0.53 log (P < 0.001), respectively, a significant decrease of RNA-US transcripts (P < 0.001), and significant correlations between decreases of proviral DNA and RNA-US transcripts (P = 0.008 and P < 0.001, respectively) were observed. CONCLUSIONS Proviral DNA quantitation permits the continued monitoring of HAART in patients with undetectable viremia.

Distinct Mutational Drug Resistance Profiles of HIV-1 RNA in Plasma and Culture Isolates of Patients Receiving Antiretroviral Therapy

The differences in profiles of HIV mutational drug resistance between paired plasma samples and culture isolates were analyzed in treated and untreated HIV-infected patients. The DNA sequence of the entire protease gene and codons 1–320 of the reverse transcriptase gene was analyzed from the paired plasma samples and viral isolates. Differences in the distribution of resistance mutations from the two sources were detected in 13 of 14 patients receiving antiretroviral therapy. Some excess mutations, observed in plasma samples but not in culture isolates, are reportedly associated with resistance to antiretroviral drugs. These results emphasize the need for standardization and clinical laboratory use of phenotypic assays that avoid extensive cocultivation.