Pier Egisto Valensin

Antiretroviral Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Protease from Paired Cerebrospinal Fluid and Plasma Samples

Twenty-four adults infected with human immunodeficiency virus type 1 (HIV-1) with central nervous system symptoms were studied for antiretroviral resistance mutations in HIV-1 RNA obtained from paired cerebrospinal fluid (CSF) and plasma samples. Paired sequences were obtained from 21 and 13 patients for reverse transcriptase (RT) and for protease, respectively. Mutations conferring resistance to the RT inhibitors zidovudine, lamivudine, or nevirapine were detected in 14 patients, including 11 pretreated and 3 drug-naive subjects. The mutation patterns in the 2 compartments were different in most patients. Genotypic resistance to protease inhibitors was detected in both plasma and CSF from 1 patient treated with multiple protease inhibitors. However, accessory protease inhibitor resistance mutations at polymorphic sites were different in plasma and CSF in several patients. Partially independent evolution of viral quasispecies occurs in plasma and CSF, raising the possibility that compartmentalization of drug resistance may affect response to antiretroviral treatment.

Detection of neurotropic viruses circulating in Tuscany: the incisive role of Toscana virus.

Acute meningitis is perhaps the most frequent among central nervous system infections. We report a study considering 277 cases of meningitis hospitalized in the southern Tuscany area (Italy) during the period from 1995 to 1998 investigated by tissue culture and polymerase chain reaction (PCR) methods. The cytochemical analysis of the cerebrospinal fluid samples suggested the diagnosis of aseptic meningitis, recognized as viral meningitis in 104 cases by detection of viral DNA or RNA. The results collected by tissue culture technique, available for 95 clinical samples, reported a positive isolation for only 12 cases. The viruses identified in the neurological infection were Toscana virus (81%), enterovirus (12%), mumps virus (3%), measles virus (1%), and herpes virus type 1 (3%). These data demonstrate the incisive role of the RNA viruses as the cause of meningitis, and overall the relevance of Toscana virus. J. Med. Virol. 60:86–90, 2000.

Serological Survey of Toscana Virus Infections in a High-Risk Population in Italy

ABSTRACT Toscana virus is the most important agent responsible for meningitis in central Italy. We report a serosurveillance study, using an immunoenzymatic assay, of 360 serum samples harvested from a high-risk population occupationally exposed to Toscana virus in two regions of Italy, Tuscany and Piedmont. The results indicates a seroprevalence of Toscana virus of 77.2% in the forestry workers, particularly in the Tuscany region. This fact is strictly correlated with the ecological niches specific for the survival of Toscana virus arthropod vector.

Localization of a new neutralizing epitope on the mumps virus hemagglutinin–neuraminidase protein

Four protein fragments which span the entire hemagglutinin-neuraminidase protein (HN) of mumps virus were expressed in HeLa cells and cell extracts were tested for their capability to induce neutralizing antibodies in mice. Fragment HN3 (aa 213-372) was able to induce the production of hemagglutination-inhibiting and neutralizing antibodies. When a subfragment of HN3, the synthetic peptide NSTLGVKSAREF (aa 329-340 of HN) was used for immunization, hemagglutination-inhibiting and neutralizing antibodies against mumps wild type virus but not against the Urabe Am9 vaccine virus were raised. The peptide could, therefore, contain a new epitope, which may be critical for protective host humoral immune response.

A Mediterranean arbovirus: The Toscana virus

Toscana virus (Bunyaviridae family, Phlebovirus genus) is a sandfly fever virus responsible for human neurological infections. Sandfly viruses are transmitted by insect vectors (Phlebotomus species) and the infection is present in climatic areas that allow the life cycle of the vector. The arthropode-borne Toscana virus is the etiologic agent of meningitis, meningoencephalitis, and encephalitis. The frequency of this neuropathic infection increases in the summer months, peaking in August in the endemic Mediterranean areas (Italy, Portugal, Spain, and Cyprus). Infection diagnosis is carried out by molecular assays and immunoenzymatic tests, which are rapid and sensitive. Recent studies have investigated the antigenic properties of the viral proteins (nucleoprotein N and surface glycoproteins G1 and G2), to better understand their immunogentic role.

Antigenicity and immunogenicity of the V3 domain of HIV type 1 glycoprotein 120 expressed on the surface of Streptococcus gordonii.

Five different V3 domains of HIV-1 gp120 were expressed on the surface of the gram-positive bacterium Streptococcus gordonii, a model live vector for vaccine delivery. Sera of HIV-1-infected individuals and human monoclonal antibodies specifically recognized the gp120 sequences on the bacterial surface. Recombinant V3 from the reference HIV-1 strain MN was also shown to retain a conformation that allowed reaction with a conformation-specific monoclonal antibody. A V3-specific serum antibody response was detected in mice immunized both by subcutaneous injection and by vaginal colonization. V3-specific IgG2a antibodies, suggestive of a Th1 response, were found in the sera of mice colonized by recombinant bacteria.

Evaluation of the presence of 2-LTR HIV-1 unintegrated DNA as a simple molecular predictor of disease progression.

In a preliminary cross‐sectional analysis of 109 human immunodeficiency virus type 1 (HIV‐1)‐infected subjects the presence of 2‐long terminal repeat (LTR) unintegrated circular HIV‐1 DNA in peripheral blood mononuclear cells (PBMC) was found to be associated with both symptomatic infection (P = 0.0037) and low CD4 counts (P = 0.0004). To investigate the prognostic significance of the presence of 2‐LTR HIV‐1 DNA, a subset of 23 2‐LTR‐negative and 25 2‐LTR‐positive asymptomatic individuals were followed up for 12–24 months. The two groups did not differ in terms of baseline CD4 counts, zidovudine (ZDV) therapy, and duration of HIV‐1 infection. Longitudinal analysis of CD4 values did not indicate a significantly different CD4 outcome between the two groups. However, when only ZDV‐treated subjects were considered, a significant (P = 0.042) decrease in CD4 counts was found at month 24 with respect to baseline in 2‐LTR‐positive (n = 12) but not in 2‐LTR‐negative (n = 11) patients. Moreover, when >40% CD4 loss from baseline and/or development of CDC stage B or C symptoms were considered as indicators of disease progression, there was a significantly higher number of events in the whole 2‐LTR‐positive group than in the whole 2‐LTR‐negative group (P = 0.0197 at month 12, P = 0.0299 at month 18, P = 0.0373 at month 24). Thus, the presence of 2‐LTR‐HIV‐1 DNA in PBMC merits further investigation as a simple, qualitative, molecular predictor of disease progression and decreased response to antiretroviral therapy. J. Med. Virol. 52:20–25, 1997.

Antiretroviral therapy with protease inhibitors in human immunodeficiency virus type 1– and human herpesvirus 8–coinfected patients

Human herpesvirus 8 (HHV‐8) is believed to play a role in the pathogenesis of Kaposis sarcoma (KS) and possibly in other proliferative disorders often associated with human immunodeficiency virus type 1 (HIV‐1) infection. Recent case reports have indicated resolution of KS and clearance of HHV‐8 DNA from peripheral blood mononuclear cells (PBMC) in HIV‐1–infected subjects following highly effective antiretroviral therapy, including HIV‐1 protease inhibitors (PI), suggesting a possible activity for these compounds on HHV‐8 replication. In the present study, the time course of PBMC HHV‐8 DNA levels, plasma HIV‐1 RNA load, and CD4+ T‐cell counts were followed up in six coinfected subjects (four with and two without KS) under antiretroviral therapy with PI. A specific anti–HHV‐8 role for PI was not consistently found, since fluctuation of HHV‐8 viral load over time appeared to be independent of treatment. Nevertheless, our data support the hypothesis that KS patients may significantly benefit from PI therapy as an indirect consequence of partial restoration of immune functions following effective anti–HIV‐1 combination therapy. J. Med. Virol. 57:140–144, 1999.

Development and significance of the HIV-1 reverse transcriptase M184V mutation during combination therapy with lamivudine, zidovudine, and protease inhibitors.

To analyze the emergence and role of the lamivudine (3TC)-selected HIV-1 reverse transcriptase (RT) M184V mutation under triple therapy, we performed a retrospective study of 40 nucleoside RT inhibitor-pretreated and 16 drug-naive patients who were switched to combined treatment with zidovudine (ZDV) plus 3TC plus a protease inhibitor (PI). Plasma viral load and pol genotype were analyzed at baseline and after 24 and 48 weeks of combination therapy. Emergence of the M184V RT mutation at week 48 was detected in 3 of 16 (18.7%) initially drug-naive subjects as opposed to 21 of 40 (52.5%) ZDV-pretreated patients. Multivariate logistic analysis detected HIV-1 RNA load at week 24 as the best predictor of subsequent selection of the M184V mutant (p = .0121). Among ZDV-resistant study subjects at week 24 (n = 17), those with mutant RT M184V codon had a more favorable HIV-1 RNA slope than those with wild-type RT 184M codon (p = .0551). This trend was observed, although in a less evident manner, even in pretreated ZDV-sensitive patients. These findings suggest that development of the 3TC-resistance M184V mutation under triple therapy with 3TC, ZDV, and a PI may have unexpected beneficial effects in vivo in addition to those associated with resensitization of ZDV-resistant virus to ZDV.

Detection of genotypically drug-resistant HIV-1 variants and non-B subtypes in recently infected antiretroviral-naive adults in Italy

Sezione di Microbiologia, Dipartimento di Biologia Molecolare, Universita di Siena, Siena, Italy; U.O. di Malattie Infettive, Azienda Ospedaliera di Careggi, Firenze, Firenze, Italy; U.O. di Malattie Infettive, Azienda Ospedaliera di Prato, Prato, Italy; U.O. di Malattie Infettive, Azienda Ospedaliera di Grosseto, Grosseto, Italy; and Servizio di Microbiologia e Virologia, Azienda Ospedaliera Senese, Siena, Italy.