Ercole Cattaneo

Evolutionary rate and genetic heterogeneity of human T-cell lymphotropic virus type II (HTLV-II) using isolates from European injecting drug users

Abstract. Seven new Italian and two new British HTLV-II isolates were obtained from injecting drug users and the entire long terminal repeat (LTR) region was sequenced. Restriction analysis showed that all the Italian isolates are of the IIb subtype, whereas the British isolates are of the IIa subtype. To understand whether the further differentiation of each two principal HTLV-II subtypes in several subgroups could be statistically supported by phylogenetic analysis, the neighbor-joining, parsimony, and maximum likelihood methods were used. The separation between IIa and IIb is very well supported by all three methods. At least two phylogenetic subgroups exist within the HTLV-IIa and at least three within the HTLV-IIb subtype. In the present analysis, no statistical support was obtained for additional phylogroups. Two particular subgroups seem interesting because they include all European and North American injecting drug user strains within the IIa and IIb subtypes, respectively. These data confirm that European HTLV-II infection among drug users is probably derived from North America. They also suggest that though a certain differentiation by restriction analysis in different subgroups is possible, carefully interpreted phylogenetic analyses remain necessary. Using the likelihood ratio test, a molecular clock for the drug user strains was calibrated. A fixation rate between 1.08 × 10−4 and 2.7 × 10−5 nucleotide substitutions per site per year was calculated for the IIa and IIb injecting drug user strains. This is the lowest fixation rate so far reported for RNA viruses, including for HIV, which typically range between 10−2 and 10−4.

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.

Complete nucleotide sequence of the Italian human T-cell lymphotropic virus type II isolate Gu and phylogenetic identification of a possible origin of South European epidemics

The complete nucleotide sequence of a human T-cell lymphotropic virus type II isolate (HTLV-II-Gu) from an Italian injecting drug user was obtained, representing the first entire sequence of a European HTLV-II isolate. The HTLV-II-Gu genome was more similar to the HTLV-IIb-NRA isolate (98.4%) and HTLV-IIb-G12 (98.2%) than to HTLV-IIa-Mo (95.2%). The classification of HTLV-II-Gu as subtype IIb was confirmed by restriction analysis. Just as for HTLV-IIa strain Mo, HTLV-IIb-Gu cultured lymphocytes produce two additional mRNAs generated through alternative splicing in the pX region. A phylogenetic analysis was performed by using the methods of neighbour-joining and parsimony with bootstrapping, and maximum likelihood. The different gene regions were analysed separately, comparing Gu with all other HTLV-II strains presently available. In the LTR, as well as in other genome regions, a clear separation between IIa and IIb was evident, and within the IIb subtype three clusters were present of which two were well supported; one contained exclusively Amerindian strains and the other included all Italian and Spanish strains together with two strains obtained from New York drug users. All data clearly showed that HTLV-IIa and IIb subtypes are closely related and are equidistant from HTLV-I, suggesting that both groups evolved simultaneously. The results suggest that HTLV-II-Gu and other IIb South European isolates were probably derived from North American IIb isolates. The data also indicate that sequence analysis is necessary to further classify IIa and IIb subtypes.

Identification of IIa and IIb molecular subtypes of human T-cell lymphotropic virus type II among Italian injecting drug users.

The molecular characterization of two human T-cell lymphotropic virus type II (HTLV-II) isolates, Gu and Va, obtained from Italian injecting drug users (IDUs) has indicated that these isolates belong to the HTLV-IIb subtype. To establish whether Italian IDUs are also infected by the HTLV-IIa variant, sequencing of the gp21 env gene of proviral DNA from further patients was carried out. Two new isolates, Bo and Md, were found, which presented a divergence of 0.4-0.7% from the IIa prototype HTLV-II-Mo, thus indicating that they belong to the HTLV-IIa subtype. The results strongly support the existence of two distinct molecular subtypes of HTLV-II infecting Italian IDUs and demonstrate that, although the IIb subtype appears to be prevalent, there is the same variability for this virus in Europe as found in the United States. The Italian IIb isolates were also seen to encode an additional 25 amino acids at the C-terminal end of tax protein, as already shown for other IIb isolates. The identification of two HTLV-II molecular subtypes among Italian drug addicts will be useful in tracing the worldwide distribution of this virus and in further understanding its molecular structure and biology.

Antigenic and Biological Relationships between Human Coronavirus OC43 and Neonatal Calf Diarrhoea Coronavirus

Monospecific antisera were prepared in mice to human coronavirus OC43 and neonatal calf diarrhoea coronavirus (NCDCV) which had been previously adapted to growth in suckling mouse brain. Brain suspension from infected suckling mice was used as immunogen. The antigenic relationship between OC43 and NCDCV was studied by the indirect immunoperoxidase antibody technique, by the haemagglutination-inhibition (HI) test and a new infectious centre-reduction neutralization test. In mouse immune sera, a two-way cross-reaction between OC43 and NCDCV was detected. However, the antigenic relationship appeared to be closer for internal (as shown by immunoperoxidase staining) as compared to surface antigens (as shown by HI and neutralization). In primary infections of natural hosts there was a high degree of cross-reactivity between the two coronavirus strains for both surface and internal antigens, and homologous and heterologous titres were consistently within an eightfold dilution difference by all tests. Most human adults and calves had antibody to both OC43 and NCDCV and geometric mean titres of homologous antibody were higher than titres of heterologous antibody. Although OC43 and NCDCV share antigenic determinants, they possessed several different biological properties, including plaque morphology by the infectious centre assay, agglutination of 1-day-old chick erythrocytes and resistance of haemagglutinin to physical and chemical treatments.

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.

Utilization of a DNA enzyme immunoassay for the detection of proviral DNA of human immunodeficiency virus type 1 by polymerase chain reaction

BACKGROUND The detection of proviral DNA by Polymerase Chain Reaction (PCR) is regarded as an important tool in the diagnosis of HIV-1 infection, specially among adults at risk of AIDS and children born to seropositive mothers. However, application of PCR in routine testing is hampered by the need to use radioactive probes. OBJECTIVES In this study, a non-radioactive test based on a microtiter plate (DNA Enzyme ImmunoAssay, DEIA) was used for the detection of proviral sequences of HIV-1 in peripheral blood cells of different patients. The results of the PCR-DEIA assay were compared to those obtained by liquid hybridization (PCR-LH), virus isolation (VI) and Western blot (WB). STUDY DESIGN The study population included 92 patients belonging to three different groups: seropositive subjects with a well-defined clinical status and WB profile; adults at risk of infection with negative or indeterminate WB; children born to seropositive mothers with still unestablished HIV-1 infection. RESULTS In the seropositive subjects, both PCR-LH and PCR-DEIA confirmed infection and gave the same results as WB. In adults at risk of infection, PCR with both methods anticipated the seroconversion in one patient with indeterminate WB and confirmed the absence of infection among seronegative and other indeterminate patients. In children born to seropositive mothers, both PCR systems as well as VI permitted an early diagnosis of infection, as confirmed by the clinical follow-up. CONCLUSION This study has shown that in subjects at risk of AIDS and in children born to seropositive mothers, the non-isotopic DEIA method presents the same sensitivity and specificity for the detection of HIV-1 infection as the radioactive procedure. The DEIA method appears to be particularly useful for the detection of PCR products in routine diagnostic analyses.