Stefano Menzo

A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates

A method is described for the absolute quantification by polymerase chain reaction (PCR) of nucleic acids present in low abundance. The method entails the addition to the sample of competitor DNA molecules that share the same sequence as the amplified target (including primer recognition sites), except for a 20-bp insertion in the middle, which allows easy resolution by gel electrophoresis (competitive PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial rate of targets, rendering the reaction independent of the number of amplification cycles. An easy and reliable method for the construction and quantification of competitive templates obtained as recombinant PCR products was developed. The technique was used for the absolute quantification of human genomic DNA with primers from a single copy, subtelomeric region of chromosome 19.

Synthetic full-length and truncated RANTES inhibit HIV-1 infection of primary macrophages.

Objective: To evaluate the role of the selective forces exerted by the host on the HIV-1 structures involved in viral entry. Design and methods: The V3 region of the env gene was analysed in cell-free HIV-1 RNA from 17 infected subjects: 11 long-term non-progressors (LTNP) and six symptomless, typical progressor patients. To evaluate the potential biological significance of one of the rare variants detected in the LTNP, it was reproduced by recombinant PCR into a HIV-1 molecular clone. Results: The intrapatient divergence of the V3-loop sequences averaged 8.62% in LTNP and 5.29% in progressors, although LTNP displayed lower divergence from the clade B consensus than progressors (16.65 and 19.76%, respectively). The analysis of non-synonymous and synonymous substitutions indicated that selective pressure was exerted in this region in both LTNP and progressors. Individual peculiarities (unique and rare V3-loop variants) emerged, however, in most sequences from LTNP, and variants bearing mutations in a domain crucial for the V3-loop structure were more prevalent in LTNP (P = 0.0012). The pNL4-3-derived mutant reproducing a V3-loop variant detected in a LTNP was efficiently expressed upon transfection, but the mutant virus was nearly completely unable to infect CD4+ cell lines, activated primary peripheral blood lymphocytes, or monocyte-derived macrophages, suggesting that a defect impaired the entry phase of the replication cycle. Conclusions: The results indicate that host factors impose selective constraints on the evolution of the HIV-1 structures involved in viral entry. In LTNP, these factors are likely to force the virus into attenuated variants.

Rules-based HIV-1 genotypic resistance interpretation systems predict 8 week and 24 week virological antiretroviral treatment outcome and benefit from drug potency weighting

OBJECTIVES To test retrospectively the ability of four freely available rules-based expert systems to predict short- and medium-term virological outcome following an antiretroviral treatment switch in pre-treated HIV-1 patients. METHODS The HIV-1 genotype interpretation systems (GISs) HIVdb, ANRS, Rega and AntiRetroScan were tested for their accuracy in predicting response to highly active antiretroviral therapy using 8 week (n = 765) and 24 week (n = 634) follow-up standardized treatment change episodes extracted from the Italian Antiretroviral Resistance Cohort Analysis (ARCA) database. A genotypic sensitivity score (GSS) was derived for each genotype-treatment pair for the different GISs and tested as a predictor of virological treatment outcome by univariable and multivariable logistic regression as well as by receiver operating characteristic curve analysis. The two systems implementing drug potency weights (AntiRetroScan and Rega) were evaluated with and without this correction factor. RESULTS All four GSSs were strong predictors of virological treatment outcome at both 8 and 24 weeks after adjusting for baseline viro-immunological parameters and previous drug exposure (odds ratios ranging from 2.04 to 2.43 per 1 unit GSS increase; P < 0.001 for all the systems). The accuracy of AntiRetroScan and Rega was significantly increased by drug potency weighting with respect to the unweighted versions (P <or= 0.001). HIVdb and ANRS also increased their performance with the same drug potency weighting adopted by AntiRetroScan and Rega, respectively (P < 0.001 for both analyses). CONCLUSIONS Currently available GISs are valuable tools for assisting antiretroviral treatment choices. Drug potency weighting can increase the accuracy of all systems.

Competitive polymerase chain reaction and analysis of viral activity at the molecular level

Due to the high sensitivity level (which can be pushed to the limit of one molecule) and its extraordinary flexibility, the polymerase chain reaction (PCR) is the method of choice for the detection of nucleic acids present in very low concentration in biological samples. Since the qualitative features of PCR amplification have limited its use, several PCR-based approaches for the quantitation of low-abundance nucleic acid species have been planned and proposed in the last few years. Recently, different lines of evidence have indicated that competitive PCR and competitive reverse-transcription-PCR share several advantages over other quantitative approaches. This evidence opens up unexpected possibilities in many biological fields, including virology; in fact, availability of reliable techniques for the absolute quantitation of DNA and RNA species may be the key to a better understanding of the pathogenic steps of most viral diseases and for a more precise monitoring of patients treated with specific antiviral compounds. In this review article, we summarize the procedures adopted for this quantitative molecular approach; additionally, several important technical aspects to plan novel competitive PCR-based applications are analyzed, and early results obtained using cPCR for the direct evaluation of viral activity in vivo are discussed.

Sequence analysis of the hepatitis B virus pre-C region in hepatocellular carcinoma [HCC] and nontumoral liver tissues from HCC patients

We investigated whether replication-competent pre-C/C defective mutants of hepatitis B virus (HBV) are detectable in primary human hepatocellular carcinoma (HCC) tissues from patients of a geographic area endemic for such mutants. DNAs extracted from formalin-fixed paraffin-embedded HCC samples were checked for the presence of specific HBV DNA sequences using the polymerase chain reaction (PCR). Amplified pre-C regions from nine HCC samples were directly sequenced as were samples of nontumoral liver tissues from five of these patients. The data show that hypervariable distal pre-C sequences were present in all nine HCC samples; this high variability was dependent on point mutations, which led to amino acid substitutions in nearly all cases. Interestingly, seven of the nine HBV DNA-positive samples from HCC tissues (but not samples from peritumoral liver tissue) showed mutations leading to amino acid substitution at the level of a distal cysteine residue. No mutation generating a translationally defective pre-C/C region was detectable in the tumor samples. Otherwise, in four of the six nontumoral liver tissues available from the same patients, a pre-C sequence with an in-frame TAG stop codon was detectable, although in three cases as a component of mixed population.

Identification of Six Putative Novel Human Papillomaviruses (HPV) and Characterization of Candidate HPV Type 87

ABSTRACT Six putative novel human papillomavirus (HPV) types were detected by using general primers for a conserved L1 HPV region in patients examined in gynecologic centers. One of the isolates, detected in samples from 4 patients with koilocytic atypia at cervical cytology (3 of whom were also infected with human immunodeficiency virus type 1), was completely sequenced, identified as a new HPV genotype, and designated candidate HPV87 (candHPV87) by the Reference Center for Human Papillomavirus. candHPV87 shows the classic HPV genome organization and the absence of a functional E5 coding region. Phylogenetic analysis documented that thecandHPV87 genome clusters within the A3 group of HPVs, together with HPV61, HPV72, HPV83, HPV84 and candHPV86, which have been completely sequenced, and a number of other putative novel genotypes (two of which are described in this work), which have been partially characterized. To address the growth-enhancing potential of candHPV87, the E6 and E7 putative coding regions were cloned and expressed in tissue cultures. The data indicate that both proteins stimulate cell division in tissue cultures more than those of low-risk HPVs, though not as much as those of HPV16. Taken together, the clinical, molecular, and biological data suggest that the novel papillomavirus characterized in the present study is a low- to intermediate-risk HPV.

A single-step DNA extraction procedure for the detection of serum hepatitis B virus sequences by the polymerase chain reaction

A rapid single-step procedure for the isolation of low molecular weight DNA using guanidinium thiocyanate and phenol as protein denaturants is described and applied for the detection of specific hepatitis B virus (HBV) DNA sequences from serum samples by the polymerase chain reaction (PCR). The novel technique is efficient and, when compared to the standard proteinase K/phenol/chloroform method has the advantage of being faster and easily adaptable to the routine processing of a high number of clinical samples by PCR and spot hybridization techniques.

Detection of human immunodeficiency virus type 1 transcripts in peripheral blood lymphocytes by the polymerase chain reaction

A simplified application of the polymerase chain reaction (PCR) to the routine detection of human immunodeficiency virus type 1 (HIV-1) transcripts from peripheral lymphocytes of infected subjects is described. This technique is simpler than previously described assays and was shown to be highly sensitive after ethidium bromide staining of polyacrylamide gel electrophoresis of amplified material. The method can be used for the virologic evaluation of HIV-1-infected subjects, thus allowing early identification of seropositive patients with signs of active infection.

Quantitative evaluation of the recombinant HIV-1 phenotype to protease inhibitors by a single-step strategy

ObjectiveTo develop and optimize a fast and quantitative recombinant strategy for evaluating the HIV-1 phenotype to protease inhibitors (PI). Design and methodsA non-replicative HIV-1 molecular vector (designated pΔpro Δenv) capable of expressing exogenous HIV-1 protease-encoding sequences was developed in this study. The HIV-1 protease sequences were amplified from either viral isolates or plasma samples (both from 21 HIV-1-infected individuals, 19 of whom were failing different anti-HIV-1 combination treatments) and cloned in the pΔpro Δenv backbone. The HIV-1 recombinant phenotype to PI was determined directly after transfection of viral chimeric clones by measuring protease activity and calculating a percentage sensitivity index (SI%; the ratio between the results from each clone and those from a PI-sensitive reference strain). ResultsThe SI% values obtained from the recombinant clones paralleled the IC50 results of the viral isolates and documented different degrees of resistance and cross-resistance to PI, compatible, with few exceptions, with the respective genotype. Interestingly, an inverse correlation between SI% values and the presence of primary mutations for resistance to PI (P  = 0.0038 and P  = 0.0414, for indinavir and ritonavir, respectively) and a difference in SI% between samples harbouring an increasing number of mutations (indinavir, P  = 0.022; ritonavir, P  = 0.0466) were observed. ConclusionThe data substantiate the reliability of the novel strategy for a fast (5 day) quantitative evaluation of HIV-1 phenotype to PI, and indicate that this method may contribute to the understanding of mechanisms of virus resistance to PI.

Analysis of the functional relationship between V3 loop and gp120 context with regard to human immunodeficiency virus coreceptor usage using naturally selected sequences and different viral backbones.

The human immunodeficiency virus type 1 (HIV-1) gp120 V3 loop plays a predominant role in chemokine receptor usage; however, other linear and nonlinear gp120 domains are involved in this step of the HIV-1 replication cycle. At present, the functional relationship between V3 and these domains with regard to coreceptor usage is unclear. To gain insights into the nature of this relationship in naturally selected viral variants, we developed a recombinant strategy based on two different gp120 backbones derived from CXCR4 (X4)- and CCR5 (R5)-tropic viral strains, respectively. Using this recombinant model system, we evaluated the phenotype patterns conferred to chimeric viruses by exogenous V3 loops from reference molecular clones and samples from infected subjects. In 13 of 17 recombinants (76%), a comparable phenotype was observed independently of the gp120 backbone, whereas in a minority of the recombinant viruses (4/17, 24%) viral infectivity depended on the gp120 context. No case of differential tropism using identical V3 sequence in the two gp120 contexts was observed. Site-directed mutagenesis experiments were performed to evaluate the phenotypic impact of specific V3 motifs. The data indicate that while the interaction of HIV-1 with chemokine receptors is driven by V3 loop and influenced by its evolutionary potential, the gp120 context plays a role in influencing the replication competence of the variants, suggesting that compensatory mutations occurring at sites other than V3 are necessary in some cases.