Development of a microwave-based extraction for forensic biological samples.

In this study, a quick microwave-based treatment was developed as a front end for DNA analysis of forensic samples. The effect of microwave treatment is to cause cell disruption which can improve the release of DNA during direct PCR as well as with extraction methods. Exposure to microwave preprocessing improved the quality of rapid genotyping, particularly when used with low level samples. Optimal results were obtained when samples were microwaved at 300W for 40 s, resulting in improved allele detection. Overall, the addition of this simple preprocessing step improves sensitivity and allele recovery for low level DNA samples when combined with expedited DNA analysis workflows. Its main advantages include speed, low cost, compatibility with downstream DNA methods and application to a wide variety of samples.

HIV-1 diversity among inmates of Italian prisons.

In Italy, the prevalence of non-B HIV-1 subtypes ranges reportedly from 5.4% to 12.6%, yet there are no data on their circulation in prisons, where the prevalence of HIV infection is high. A retrospective study was conducted to evaluate the circulation of non-B subtypes and to characterize their determinants in five Italian prisons. To this end an aliquot of samples of blood was taken in the period 2001-2006 from all 262 HIV-positive inmates in whom antiretroviral treatment had failed. Complete HIV-1 PR and RT regions were sequenced for all samples and subjected to phylogenetic analysis; 250 (95.4%) sequences clustered with subtype B. The non-B subtype was found in 4% of Italian prison inmates and 16.7% of non-Italian prison inmates; the overall percentage increased from 1.8% for inmates infected in 1982-1990 to 4.4% in 1991-1999 and 21.9% in 2000-2006. Factors significantly associated with non-B subtypes were an exposure to other than injecting drug use and a first positive HIV test in 2000-2006. Non-B subtypes were distributed within five monophyletic clades. In all cases but one, it was possible to correlate the history of HIV-exposure to the origin of the clade, with high bootstrap values. In conclusion, although the sample may not be representative of the prison inmate population in Italy, the data suggest strongly that the circulation of non-B subtypes has apparently increased. Non-B subtypes were found to have been associated with heterosexual contact and time of the first HIV-positive test. Knowledge of the different subtypes circulating in prisons may be useful for tracking the epidemiology of HIV infection and for choosing antiretroviral therapy.

Response of feline immunodeficiency virus (FIV) to tipranavir may provide new clues for development of broad-based inhibitors of retroviral proteases acting on drug-resistant HIV-1.

The feline AIDS model for HIV-1 treatment failed in the 1990s, due to structural features resembling protease inhibitor (PI) resistant HIV-1 variants. Widespread drug-resistance to PIs now invokes the possibility of rescuing feline immunodeficiency virus (FIV) as a model for PI treatment. We here analyzed susceptibility of FIV to second generation PIs, lopinavir, atazanavir, and the structurally unrelated non-peptidic PI tipranavir. We found that FIV protease resembles HIV-1 protease drug resistance mutations limiting binding of lopinavir and atazanavir but not tipranavir. All three PIs were found to inhibit FIV replication in a concentration-dependent manner, but only tipranavir inhibited FIV similarly to HIV-1. This drug inhibited FIV synergistically with ritonavir. Inhibition of protease activity was confirmed by Western blot analysis. In molecular docking simulations, tipranavir displayed energetically favorable interactions with the catalytic cavity of the mature dimeric FIV protease. The calculated hydrogen bond network was similar to that found in HIV-1 protease/tipranavir complexes and involved atoms in the protein backbone. We also modeled the interaction of tipranavir with an immature protease monomer, suggesting that inhibition of protease dimerization may be a secondary modality for FIV inhibition by tipranavir. In conclusion, tipranavir is the first FDA-approved non-reverse transcriptase inhibitor of HIV-1 to show anti-FIV properties. The tipranavir response by FIV may 1) support the idea of using FIV as a small animal model for PI-resistant HIV-1, thus expanding access to animal AIDS models; and 2) pave the way for development of novel broad-based inhibitors for treatment of drug resistant HIV-1.

Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS.

BACKGROUND: Treatment of feline immunodeficiency virus (FIV) infection has been hampered by the absence of a specific combination antiretroviral treatment (ART). Integrase strand transfer inhibitors (INSTIs) are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. METHODS: Phylogenetic analysis of lentiviral integrase (IN) sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD) was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD). Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. RESULTS: The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. CONCLUSION: We report a drug class (other than nucleosidic reverse transcriptase inhibitors) that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound successfully tested in human clinical trials, as one of the most potent anti-FIV agents ever tested in vitro. This finding may provide new avenues for treating FIV infection and contribute to the development of a small animal model mimicking the effects of ART in humans.

H5N1 influenza virus evolution: a comparison of different epidemics in birds and humans (1997-2004).

The selection pressure acting along the entire genome sequence of H5N1 avian influenza viruses isolated from several bird species and humans infected in the 1997 and 2004 outbreaks, and on the HA1 genes from H5N1 viruses isolated during the entire study period, in eastern Asia was evaluated. According to maximum-likelihood analysis, viral genes appeared to be, in both epidemics, under strong purifying selection, with only the PB2, HA and NS1 genes under positive selection. Specific codons under positive selection were detected by using codon-based substitution models. Positive-selection analysis performed on single-codon sites might be helpful in clarifying the driving force of avian and human influenza virus evolution and in selecting specific targets for vaccines and antiviral drugs.