Membrane-containing virus particles exhibit the mechanics of a composite material for genome protection.

The protection of the viral genome during extracellular transport is an absolute requirement for virus survival and replication. In addition to the almost universal proteinaceous capsids, certain viruses add a membrane layer that encloses their double-stranded (ds) DNA genome within the protein shell. Using the membrane-containing enterobacterial virus PRD1 as a prototype, and a combination of nanoindentation assays by atomic force microscopy and finite element modelling, we show that PRD1 provides a greater stability against mechanical stress than that achieved by the majority of dsDNA icosahedral viruses that lack a membrane. We propose that the combination of a stiff and brittle proteinaceous shell coupled with a soft and compliant membrane vesicle yields a tough composite nanomaterial well-suited to protect the viral DNA during extracellular transport.

Increased risk of virologic failure to the first antiretroviral regimen in HIV-infected migrants compared to natives: data from the ICONA cohort.

Migrant and Italian HIV-infected patients (n = 5773) enrolled in the ICONA cohort in 2004-2014 were compared for disparities in access to an initial antiretroviral regimen and/or risk of virologic failure (VF), and determinants of failure were evaluated. Variables associated with initiating antiretroviral therapy (ART) were analysed. Primary endpoint was time to failure after at least 6 months of ART and was defined as: VF, first of two consecutive virus loads (VL) >200 copies/mL; treatment discontinuation (TD) for any reason; and treatment failure as confirmed VL >200 copies/mL or TD. A Poisson multivariable analysis was performed to control for confounders. Migrants presented significantly lower CD4 counts and more frequent AIDS events at baseline. When adjusting for baseline confounders, migrants presented a lower likelihood to begin ART (odds ratio 0.80, 95% confidence interval (CI) 0.67-0.95, p 0.012). After initiating ART, the incidence VF rate was 6.4 per 100 person-years (95% CI 4.8-8.5) in migrants and 2.7 in natives (95% CI 2.2-3.3). Multivariable analysis confirmed that migrants had a higher risk of VF (incidence rate ratio 1.90, 95% CI 1.25-2.91, p 0.003) and treatment failure (incidence rate ratio 1.16, 95% CI 1.01-1.33, p 0.031), with no differences for TD. Among migrants, variables associated with VF were age, unemployment and use of a boosted protease inhibitor-based regimen versus nonnucleoside reverse transcriptase inhibitors. Despite the use of more potent and safer drugs in the last 10 years, and even in a universal health care setting, migrants living with HIV still present barriers to initiating ART and an increased risk of VF compared to natives.

Structures of respiratory syncytial virus nucleocapsid protein from two crystal forms: details of potential packing interactions in the native helical form.

Respiratory syncytial virus (RSV) is a frequent cause of respiratory illness in infants, but there is currently no vaccine nor effective drug treatment against this virus. The RSV RNA genome is encapsidated and protected by a nucleocapsid protein; this RNA-nucleocapsid complex serves as a template for viral replication. Interest in the nucleocapsid protein has increased owing to its recent identification as the target site for novel anti-RSV compounds. The crystal structure of human respiratory syncytial virus nucleocapsid (HRSVN) was determined to 3.6 Å resolution from two crystal forms belonging to space groups P2(1)2(1)2(1) and P1, with one and four decameric rings per asymmetric unit, respectively. In contrast to a previous structure of HRSVN, the addition of phosphoprotein was not required to obtain diffraction-quality crystals. The HRSVN structures reported here, although similar to the recently published structure, present different molecular packing which may have some biological implications. The positions of the monomers are slightly shifted in the decamer, confirming the adaptability of the ring structure. The details of the inter-ring contacts in one crystal form revealed here suggest a basis for helical packing and that the stabilization of native HRSVN is via mainly ionic interactions.

Crystallization and preliminary X-ray analysis of the human respiratory syncytial virus nucleocapsid protein.

Human respiratory syncytial virus (HRSV) has a nonsegmented negative-stranded RNA genome which is encapsidated by the HRSV nucleocapsid protein (HRSVN) that is essential for viral replication. HRSV is a common cause of respiratory infection in infants, yet no effective antiviral drugs to combat it are available. Recent data from an experimental anti-HRSV compound, RSV-604, indicate that HRSVN could be the target site for drug action. Here, the expression, purification and preliminary data collection of decameric HRSVN as well as monomeric N-terminally truncated HRSVN mutants are reported. Two different crystal forms of full-length selenomethionine-labelled HRSVN were obtained that diffracted to 3.6 and approximately 5 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 133.6, b = 149.9, c = 255.1 A, and space group P2(1), with unit-cell parameters a = 175.1, b = 162.6, c = 242.8 A, beta = 90.1 degrees , respectively. For unlabelled HRSVN, only crystals belonging to space group P2(1) were obtained that diffracted to 3.6 A. A self-rotation function using data from the orthorhombic crystal form confirmed the presence of tenfold noncrystallographic symmetry, which is in agreement with a reported electron-microscopic reconstruction of HRSVN. Monomeric HRSVN generated by N-terminal truncation was designed to assist in structure determination by reducing the size of the asymmetric unit. Whilst such HRSVN mutants were monomeric in solution and crystallized in a different space group, the size of the asymmetric unit was not reduced.

Hepatotoxicity of antiretroviral drugs.

The use of highly active antiretroviral therapy (HAART) has significantly slowed the HIV disease progression. However, adverse effects are now a limiting cause of HAART benefit in a substantial proportion of patients. Particularly hepatotoxicity which is a common complication occurring during every HAART regimen. All antiretroviral (ARV) drugs classes, Nucleoside/Nucleotide reverse transcriptase inhibitors (NRTI), non-Nucleoside reverse transcriptase inhibitors (nNRTI) and Protease Inhibitors (PI) may cause hepatotoxicity but in different pathways. Many risk factors have been identified for developing antiretroviral-related hepatotoxicity, however severe hepatitis remains very uncommon in patients receiving HAART, also if the incidence of hepatotoxicity is rather high. That being the case, means that every new available antiretroviral drug strongly necessities studies which can evaluate its hepatotoxicity and drug-drug interactions, to define the potential risk factors and the outcome of any side effects. This report will review the risk factors, the epidemiology and the pathogenic mechanisms of hepatotoxicity caused in every antiretroviral drug.

Quality of life assessment in HIV-positive persons: application and validation of the WHOQOL-HIV, Italian version.

QoL assessment is currently considered essential for clinical trials in HIV infection, as commonly used end-points (CD4 level, viral load, opportunistic diseases) are inadequate to catch the complexity of treatment outcomes. The World Health Organization has recently developed a standardized set of instruments to assess subjective quality of life (QoL) in different medical conditions, including HIV infection. Here we report evidence for the acceptability, reliability and validity of the Italian version of the WHOQOL-HIV. The Italian version of WHOQOL-HIV has been administered in a sample of 151 HIV-positive persons, consecutively attending the largest infectious diseases hospital in southern Italy. Mean time of administration and percentage of missing responses, Cronbach alpha, Pearson coefficient and oneway ANOVA were applied to assess, respectively, acceptability, reliability, convergent and disciminant validity, and sensitivity to change. Mean time of administration was 28 minutes; only 2 questionnaires showed more than 20% of missing responses. Cronbach alpha was above 0.70 in 22 of the 28 sections of the WHOQOL-HIV; it ranged between 0.53 and 0.68 in the remaining 6 sections. Each of the 7 QoL principal domains correlated with overall QoL at a significance level p < 0.001. Moreover, correlation between principal domains were always statistically significant (p < 0.01) with only two exceptions. Finally, mean scores in each QoL domain were in the expected direction (worse in AIDS patients as compared to asymptomatic and symptomatic persons). The Italian version of WHOQOL-HIV is a valid and reliable instrument to assess subjective QoL in HIV-positive persons. It seems potentially useful to assess patients' life satisfaction, and to calibrate standards of care in different stages of the infection.

Extra-helical guanine interactions in DNA.

We review the extra-helical guanine interactions present in many oligonucleotide crystals. Very often terminal guanines interact with other guanines in the minor groove of neighboring oligonucleotides through N2 x N3 hydrogen bonds. In other cases the interaction occurs with the help of Ni2+ ions. Guanine/netropsin stacking in the minor groove has also been found. From these studies we conclude that guanine may have multiple extra-helical interactions. In particular it may be considered a very effective minor groove binder, which could be used in the design of sequence selective binding drugs. Interactions through the major groove are seldom encountered, but might be present when DNA is stretched. Such interactions are also analyzed, since they might be important for homologous chromosome pairing during meiosis.

Sexual behaviour of women living with HIV/AIDS naïve for antiretroviral therapy: the ICONA-BEHEPI Study.

This study describes the sexual behaviours of women living with HIV, and assesses differences by history of drug use. Its general aim is to contribute in the design of programmes to help people with HIV/AIDS (PWH/A) adopt and maintain safe sexual behaviours. A self-administered questionnaire on sexual and drug use behaviours was distributed to study participants. Between 1997 and 1999, 573 women with HIV infection naive to antiretroviral therapies completed the questionnaire (of whom 234 reported a history of injection drug use (IDU) and were enrolled in the study. Non-IDU women reported fewer sexual partners, both in their lifetime and in the preceding month, than IDU women: 19% of IDU and 4% of non-IDU women reported more than 25 lifetime sexual partners (p < 0.001). Interestingly, 83% of non-IDU women were infected by their regular partners: these women reported the lowest number of sexual partners. No difference emerged between IDU and non-IDU women in terms of number of sexual intercourse in the two weeks preceding the interview or in terms of condom use in the last intercourse (reported, overall, by 54% of these 573 women). Among women who had sex partners at the time of interview, more non-IDU (65%) than IDU (43%) women reported HIV-positive partners (p < 0.001). Overall, these findings stress a marked heterogeneity in the levels of past and recent sexual promiscuity according to history of drug use. It suggests the need to differentiate and individualize messages about self-protection and behaviours that may prevent further spread of HIV infection.

Stacking interaction of guanine with netropsin in the minor groove of d(CGTATATACG)2.

We present the structure of the decanucleotide d(CGTATATACG) determined by single crystal X-ray diffraction at 1.58 A resolution. A netropsin drug is found in the minor groove with guanine stacked on a pyrrole ring of the drug, a feature described here for the first time. The stacked guanine is an extra-helical base coming from the end of a neighbour oligonucleotide. This observation may open the way to the development of minor groove binding drugs with a higher sequence selectivity. The oligonucleotide is in the B-conformation, but the terminal base-pairs are disrupted: the cytosine residues are disordered while the guanine residues penetrate into the minor groove of neighbouring duplexes. Four hydrated Ni ions with octahedral co-ordination are found associated with the N7 atoms of each guanine. The high affinity of these ions with guanine suggests that they may be used as probes for specific guanine residues.

Structure of the oligonucleotide d(CGTATATACG) as a site-specific complex with nickel ions.

In this paper we explore the application of Ni2+to the crystallization of oligonucleotides. We have determined in this way the structure of a fully alternating (Y-R) decanucleotide d(CGTATATACG) by single crystal X-ray diffraction. This is the first oligonucleotide crystal structure with an alternating 5'-(TA)3-3' central part. Alternating oligonucleotides have a particular interest since they often have a unique structure. In this case the general conformation is B-like with an alternating twist and an end-to-end interaction which involves terminal guanines. The crystal belongs to space group P41212 with a = b = 52.46, c = 101.49 A. This packing imposes a 90 degrees crossing of the symmetry related helices. This is a new way of packing for decamers. The oligonucleotide structure is characterized by the specific association with seven nickel ions, involving the N7 atom of every guanine. One of the Ni2+ions is shared between two guanines of symmetry related molecules. Until now no oligonucleotide has been crystallized in the presence of this metal ion. A novel C.A.T triplet structure has also been tentatively identified.

Insulin-degrading activity in experimental liver cirrhosis of the rat.

Liver cirrhosis in man is often associated with hyperinsulinemia but its pathogenesis is still unexplained. To investigate whether insulin degradation is impaired in cirrhotic liver, the specific insulin-degrading enzyme activity (EC 3.4.22.11) was assayed in liver cytosol of rats with CCl4-induced liver cirrhosis. No difference was found between liver cytosol of cirrhotic and control rats. The results show that experimental CCl4-induced liver cirrhosis does not damage the specific insulin-degrading activity and support the hypothesis that impaired hepatic insulin handling is not an important cause of hyperinsulinemia in liver cirrhosis.

[Demonstration of the interchangeability of 2 types of recombinant anti-hepatitis-B vaccine]. / Dimostrazione dell'interscambiabilita' dei due tipi di vaccino anti-epatite B DNA-ricombinante.

A vaccination cycle cannot always be completed with the same type of vaccine with which it was initiated because the same type of vaccine is not always available at the vaccination centers. For this reason we have verified the possibility of substituting one of two anti hepatitis B DNA-recombinant vaccines [Engerix B (SK&F); Recombivax HB (MSD)] both available in Italy in the vaccination protocol. A study was performed on 480 subjects using both types of DNA recombinant vaccine and on 160 using only one type of vaccine. Our data show that there is no difference in immunogenic potency of the two DNA recombinant vaccines available in Italy and that a cycle begun with one vaccine can be completed with the other with no effect on the percentage of seroconversion or the mean anti-HBs titre.

[Adaptability of the anti-hepatitis-B vaccine Recombivax HB to the Piazza protocol for the mass vaccination of newborn infants]. / Adattabilità del vaccino anti-epatite B Recombivax HB allo "schema Piazza" per la vaccinazione di massa dei nuovi nati.

27 healthy babies born to HBsAg, antiHBs and antiHBc negative mothers were given three doses of hepatitis B vaccine "Recombivax HB" (5 micrograms/dose/0.5 ml) at 3, 5 and 11 months of age (Piazza's protocol). AntiHBs response was highly satisfactory. Since both in terms of seroconversion rate and of mean antiHBs titre immunogenicity of other hepatitis B vaccines given at 3, 5 and 11 months of age was already demonstrated, it is possible to conclude that Piazza's protocol is valid for all hepatitis B vaccines available in Italy and will certainly facilitate the compulsory hepatitis B vaccination in infants in Italy.

A modified immunisation schedule for the hepatitis B vaccine Recombivax HB suitable for mass vaccination in childhood.

Following the demonstration of a fully satisfactory immunogenic activity of a hepatitis B vaccination protocol consisting of three doses of Hevac B Pasteur vaccine given at 3,5 and 11 months of age, it was possible to administer this vaccine at the same times as the vaccinations for diphtheria, tetanus and polio which are mandatory in Italy at those ages. We have also shown that both another plasma-derived vaccine, H-B-VAX (MSD), as well as the DNA-recombinant Engerix B (SK&F) are highly immunogenic when given at the same times as the mandatory childhood vaccinations. In this paper we demonstrate that the same schedule can be used for another hepatitis B vaccine prepared by a DNA-recombinant technique, Recombivax HB (MSD) recently introduced in Italy. In fact two doses of this vaccine, the first given at three months of age and the second two months later, resulted in a 100% seroconversion rate and a mean anti-HBs titre of 440 mUI/ml. Although the date are incomplete since the third dose will be given at 11 months of age, we conclude that this hepatitis B vaccine can also be used in the mass vaccination campaigns of infants in Italy, the first of which was initiated in January 1987 in an hyperendemic area near Naples (HBsAg prevalence about 14%). We underline that this mass vaccination campaign is the first in Europe.

Immunogenicity of a DNA-recombinant hepatitis B vaccine (Engerix B) given at 3, 5 and 11 months of age with a new schedule suitable for mass vaccination programmes.

Following the demonstration of a fully satisfactory immunogenic activity of a hepatitis B vaccination protocol consisting of three doses given at the 3rd and 5th months of age with a booster at 11, it was possible to administer this vaccine at the same times as the vaccinations for diphtheria, tetanus and polio which are mandatory in Italy at those ages. A field trial of this protocol in a hyperendemic area near Naples (prevalence of HBsAg about 14%) started on January 1987. The French vaccine, Hevac B, Pasteur, was used. At this time compliance is 99%, and fully satisfactory results both in terms of seroconversion rate (96.3%) and of mean anti-HBs titre (4,352 mIU/ml) two months after the booster dose have been obtained. In this paper we demonstrate that even for a new hepatitis B vaccine prepared by a DNA-recombinant technique (Engerix B, SK & F) recently introduced in Italy, the same schedule can be used. In fact two doses of this vaccine, the first given at three months of age and the second two months later, resulted in a 100% seroconversion rate and a mean anti-HBs titre of 560 mIU/ml. Two months after the booster given at 11 months of age the mean anti-HBs titre was 12,100.

Prevalence of intranuclear particles in liver pathologies.

An ultrastructural study of the prevalence of electron dense 23-27 nm intranuclear particles was carried out on liver biopsies from patients with NANB chronic active hepatitis (CAH), Delta + CAH, HBsAg + CAH, nonviral liver pathologies and in one healthy volunteer. The particles were classified according to aggregation pattern and were found to be correlated with NANB CAH and Delta + CAH. No particles were observed in nonviral liver pathologies. A close antigenic relationship has been shown between the cytoplasmic alterations observed in NANB and delta hepatitis in chimpanzees. Our data indicate that there is a structural similarity between the intranuclear particles seen in both Delta and NANB hepatitis, thus reinforcing the hypothesis that the NANB and Delta agents are closely related.

Immunologic abnormalities in patients with chronic delta infection.

Data reported in this paper emphasize the existence of close relationships between hepatitis delta patients and subjects affected by other viral diseases, first of all AIDS and related conditions. Some immunologic abnormalities characterizing initial stages of HIV infection (reduced CD4/CD8 ratio, based on increased CD8+ cells; B-cell polyclonal activation with the presence of circulating immune complexes) were found, in fact, also in delta patients. No statistically significant difference was observed by comparing delta vs no delta subjects.