Sviluppo e caratterizzazione di nuovi inibitori diretti contro la polimerasi dei virus dell'influenza di tipo A e B

Influenza (flu) is an airborne highly-infectious disease, characterized by high morbidity and significant mortality, especially in at-risk population (young children, elderly people, patients with chronic disease). Influenza type A (IAV) and type B (IBV) viruses, characterized by remarkable genetic instability and antigenic variability, are responsible for seasonal epidemics, which affect every year 15-20% of the world population. In addition, IAV is able to infect a wide variety of birds and mammals and as happened with the recent case of H1N1 swine flu, a genetic reassortment between viral genomic segments of different species may occur and cause pandemics. During the past century, the pandemics of Spanish flu (1918), Asian flu (1957) and Hong Kong flu (1968) caused millions of deaths worldwide. For these reasons IAV and IBV pose a large threat to public health and it is absolutely necessary have therapeutic agents effective against these viruses. The therapeutic options currently available for influenza include vaccination and two distinct classes of anti-influenza drugs represented by the NA inhibitors and the M2 ion channel blockers. However, vaccines need to be reformulated each year due to the genetic variability of the virus and are not always protective. In addition, the production of a new vaccine takes several months, so it is highly unlikely that vaccination may be effective in preventing the spread of a rapidly emerging influenza pandemic. Oseltamivir (tamiflu) and zanamivir (relenza) are NA inhibitors and act by blocking the release of the viral particle from infected cells. Amantadine (symadine, symmetrel) and rimantadine (Flumadine) block the ion channel of the M2 protein, preventing viral uncoating and thus the release of genomic segments in the cytoplasm. These drugs, however, have several drawbacks, including considerable side effects, limited efficacy, significant limitations of administration and above all they are associated with a rapid emergence of drug-resistant viral strains. In fact, there no an effective antiviral therapy against influenza and thus there is a strong need for the development of new anti-influenza compounds, possibly endowed with an innovative mechanism of action. An interesting target for the development of novel anti-influenza strategies is represented by the viral RNA polymerase; in fact, the influenza virus polymerase is a conserved element among different viral strains, as it is not subjected to genetic variability. The influenza virus RNA polymerase is a heterocomplex which consists of three proteins (polymerase acid protein [PA], polymerase basic protein 1 [PB1] and polymerase basic protein 1 [PB2]). The three polymerase subunits interact each other, in particular PB1 interacts with PB2 and PA, while at the moment there is little evidence of an interaction between PB2 and PA. It has been shown that the PA/PB1 interaction is crucial for influenza virus replication: in fact, the inhibition of this interaction result in the inhibition of viral polymerase activity. The fact that the interface of PA-PB1 interaction is highly conserved among different IAV and IBV strains and that this interaction is essential for viral replication makes it a potential target for the development of novel anti-influenza compounds with broad-spectrum activity. Thanks to an in silico screening of a virtual library of small molecules, performed by our research group, we have identified a few compounds able to dissociate the interaction between PA and PB1 in vitro, at micromolar concentrations (Muratore et al., 2012a). The same compounds showed no cytotoxicity in cell lines, but at the same time exhibited only modest antiviral activities against IAV. The purpose of this project was to evaluate the binding modes of these compounds with the target and based on this information to design structural analogues, in order to increase the antiviral activity, preserving the absence of cytotoxicity and their peculiar mechanism of action. Once synthesized, these analogues have been tested in various experiments, in order to assess their ability to inhibit viral replication of IAV in plaque reduction assays (PRA), but also to determine their inhibitory effect on PA-PB1 interaction in ELISA and to investigate their possible cytotoxicity on cell lines in MTT assays. Among these, the compounds 70, T22 and T40 showed inhibitory effects on viral polymerase activity, with EC50 values comparable or even lower than that obtained with ribavirin (RBV), used as reference drug. Then, we also evaluated the antiviral activity of these analogues over the time; what emerged is that all compounds show a maximum of activity at 12 h post-infection (p.i.), but they still maintain the antiviral effects at 48 h pi. Moreover, the most promising analogues were able to inhibit the replication of a panel of several strains of IAV and IBV, including an oseltamivir-resistant clinical isolate, thus demonstrating to have anti-influenza broad-spectrum activity. In contrast, the same compounds showed no inhibitory effect on the replication of other DNA and RNA viruses, confirming to possess a specific mechanism of action. Finally, for some promising analogues, we made predictions and simulations of virtual interaction with the cavity of PA as well as studies to assess the possible variation of inhibitory activity against mutated PA targets in some key residues as compared to the wild-type (wt) PA. This has allowed us to delineate the structure-activity relationships (SAR) and to understand the binding modes of these inhibitors, obtaining useful suggestions for the design of new analogues characterized by a better fit into the PA cavity and thus a more potent antiviral activity. In conclusion, this study demonstrates the potential of these compounds as inhibitors of influenza virus RNA polymerase, which will be tested in next future in combinations with current anti-Flu drugs in order to evaluate possible synergistic effects. In addition, this work has led to the identification of a series of new scaffolds endowed with antiviral activity that will be further studied to develop a new generation of therapeutic agents capable to fight the infection by IAV and IBV

Structural Investigations on Novel Non-Nucleoside Inhibitors of Human Norovirus Polymerase

Human norovirus is the first cause of foodborne disease worldwide, leading to extensive outbreaks of acute gastroenteritis, and causing around 200,000 children to die annually in developing countries. No specific vaccines or antiviral agents are currently available, with therapeutic options limited to supportive care to prevent dehydration. The infection can become severe and lead to life-threatening complications in young children, the elderly and immunocompromised individuals, leading to a clear need for antiviral agents, to be used as treatments and as prophylactic measures in case of outbreaks. Due to the key role played by the viral RNA-dependent RNA polymerase (RdRp) in the virus life cycle, this enzyme is a promising target for antiviral drug discovery. In previous studies, following in silico investigations, we identified different small-molecule inhibitors of this enzyme. In this study, we rationally modified five identified scaffolds, to further explore structure–activity relationships, and to enhance binding to the RdRp. The newly designed compounds were synthesized according to multiple-step synthetic routes and evaluated for their inhibition of the enzyme in vitro. New inhibitors with low micromolar inhibitory activity of the RdRp were identified, which provide a promising basis for further hit-to-lead optimization

Targeting the Complement Serine Protease MASP-2 as a Therapeutic Strategy for Coronavirus Infections

MASP-2, mannose-binding protein-associated serine protease 2, is a key enzyme in the lectin pathway of complement activation. Hyperactivation of this protein by human coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2 has been found to contribute to aberrant complement activation in patients, leading to aggravated lung injury with potentially fatal consequences. This hyperactivation is triggered in the lungs through a conserved, direct interaction between MASP-2 and coronavirus nucleocapsid (N) proteins. Blocking this interaction with monoclonal antibodies and interfering directly with the catalytic activity of MASP-2, have been found to alleviate coronavirus-induced lung injury both in vitro and in vivo. In this study, a virtual library of 8736 licensed drugs and clinical agents has been screened in silico according to two parallel strategies. The first strategy aims at identifying direct inhibitors of MASP-2 catalytic activity, while the second strategy focusses on finding protein-protein interaction inhibitors (PPIs) of MASP-2 and coronaviral N proteins. Such agents could represent promising support treatment options to prevent lung injury and reduce mortality rates of infections caused by both present and future-emerging coronaviruses. Forty-six drug repurposing candidates were purchased and, for the ones selected as potential direct inhibitors of MASP-2, a preliminary in vitro assay was conducted to assess their interference with the lectin pathway of complement activation. Some of the tested agents displayed a dose-response inhibitory activity of the lectin pathway, potentially providing the basis for a viable support strategy to prevent the severe complications of coronavirus infections

Daclatasvir plasma level and resistance selection in HIV patients with hepatitis C virus cirrhosis treated with daclatasvir, sofosbuvir, and ribavirin

ObjectivesEffective treatment with direct-acting antiviral drugs against hepatitis C virus (HCV) is a medical need in cirrhotic HIV–HCV co-infected patients.MethodsThis study investigated the plasma levels of daclatasvir (DCV) and ribavirin (RBV) in HIV–HCV co-infected subjects treated with DCV, sofosbuvir, and RBV. Drug concentrations were quantified using validated high-performance liquid chromatography methods with ultraviolet detection. The HCV non-structural protein 5A and non-structural protein 5B coding regions were analyzed by population-based sequencing.ResultsDCV was dosed at week 4 and at week 8 of treatment, and RBV at week 8. One patient had the lowest DCV level, corresponding to 32.7% of the overall median value of the other patients at week 4 and about 40% at week 8. The Y93H variant was detected in this subject at weeks 8, 16, and 20 of treatment, but not before treatment or at day 2, and the patient experienced virological failure. Another subject with the Y93H variant at baseline and appropriate DCV levels had HCV RNA <12 IU/ml at week 12 and undetectable at week 16.ConclusionsSub-optimal DCV drug levels allow the selection of resistance-associated variants and fail to contribute to antiviral activity. No definite reason for the low DCV level was found. Quantifying the drug is suggested in difficult-to-treat patients

Sviluppo e caratterizzazione di nuovi inibitori diretti contro la polimerasi dei virus dell'influenza di tipo A e B

Influenza (flu) is an airborne highly-infectious disease, characterized by high morbidity and significant mortality, especially in at-risk population (young children, elderly people, patients with chronic disease). Influenza type A (IAV) and type B (IBV) viruses, characterized by remarkable genetic instability and antigenic variability, are responsible for seasonal epidemics, which affect every year 15-20% of the world population. In addition, IAV is able to infect a wide variety of birds and mammals and as happened with the recent case of H1N1 swine flu, a genetic reassortment between viral genomic segments of different species may occur and cause pandemics. During the past century, the pandemics of Spanish flu (1918), Asian flu (1957) and Hong Kong flu (1968) caused millions of deaths worldwide. For these reasons IAV and IBV pose a large threat to public health and it is absolutely necessary have therapeutic agents effective against these viruses. The therapeutic options currently available for influenza include vaccination and two distinct classes of anti-influenza drugs represented by the NA inhibitors and the M2 ion channel blockers. However, vaccines need to be reformulated each year due to the genetic variability of the virus and are not always protective. In addition, the production of a new vaccine takes several months, so it is highly unlikely that vaccination may be effective in preventing the spread of a rapidly emerging influenza pandemic. Oseltamivir (tamiflu) and zanamivir (relenza) are NA inhibitors and act by blocking the release of the viral particle from infected cells. Amantadine (symadine, symmetrel) and rimantadine (Flumadine) block the ion channel of the M2 protein, preventing viral uncoating and thus the release of genomic segments in the cytoplasm. These drugs, however, have several drawbacks, including considerable side effects, limited efficacy, significant limitations of administration and above all they are associated with a rapid emergence of drug-resistant viral strains. In fact, there no an effective antiviral therapy against influenza and thus there is a strong need for the development of new anti-influenza compounds, possibly endowed with an innovative mechanism of action. An interesting target for the development of novel anti-influenza strategies is represented by the viral RNA polymerase; in fact, the influenza virus polymerase is a conserved element among different viral strains, as it is not subjected to genetic variability. The influenza virus RNA polymerase is a heterocomplex which consists of three proteins (polymerase acid protein [PA], polymerase basic protein 1 [PB1] and polymerase basic protein 1 [PB2]). The three polymerase subunits interact each other, in particular PB1 interacts with PB2 and PA, while at the moment there is little evidence of an interaction between PB2 and PA. It has been shown that the PA/PB1 interaction is crucial for influenza virus replication: in fact, the inhibition of this interaction result in the inhibition of viral polymerase activity. The fact that the interface of PA-PB1 interaction is highly conserved among different IAV and IBV strains and that this interaction is essential for viral replication makes it a potential target for the development of novel anti-influenza compounds with broad-spectrum activity. Thanks to an in silico screening of a virtual library of small molecules, performed by our research group, we have identified a few compounds able to dissociate the interaction between PA and PB1 in vitro, at micromolar concentrations (Muratore et al., 2012a). The same compounds showed no cytotoxicity in cell lines, but at the same time exhibited only modest antiviral activities against IAV. The purpose of this project was to evaluate the binding modes of these compounds with the target and based on this information to design structural analogues, in order to increase the antiviral activity, preserving the absence of cytotoxicity and their peculiar mechanism of action. Once synthesized, these analogues have been tested in various experiments, in order to assess their ability to inhibit viral replication of IAV in plaque reduction assays (PRA), but also to determine their inhibitory effect on PA-PB1 interaction in ELISA and to investigate their possible cytotoxicity on cell lines in MTT assays. Among these, the compounds 70, T22 and T40 showed inhibitory effects on viral polymerase activity, with EC50 values comparable or even lower than that obtained with ribavirin (RBV), used as reference drug. Then, we also evaluated the antiviral activity of these analogues over the time; what emerged is that all compounds show a maximum of activity at 12 h post-infection (p.i.), but they still maintain the antiviral effects at 48 h pi. Moreover, the most promising analogues were able to inhibit the replication of a panel of several strains of IAV and IBV, including an oseltamivir-resistant clinical isolate, thus demonstrating to have anti-influenza broad-spectrum activity. In contrast, the same compounds showed no inhibitory effect on the replication of other DNA and RNA viruses, confirming to possess a specific mechanism of action. Finally, for some promising analogues, we made predictions and simulations of virtual interaction with the cavity of PA as well as studies to assess the possible variation of inhibitory activity against mutated PA targets in some key residues as compared to the wild-type (wt) PA. This has allowed us to delineate the structure-activity relationships (SAR) and to understand the binding modes of these inhibitors, obtaining useful suggestions for the design of new analogues characterized by a better fit into the PA cavity and thus a more potent antiviral activity. In conclusion, this study demonstrates the potential of these compounds as inhibitors of influenza virus RNA polymerase, which will be tested in next future in combinations with current anti-Flu drugs in order to evaluate possible synergistic effects. In addition, this work has led to the identification of a series of new scaffolds endowed with antiviral activity that will be further studied to develop a new generation of therapeutic agents capable to fight the infection by IAV and IBV.L’influenza è un’infezione respiratoria, di origine virale, caratterizzata da elevata morbidità e mortalità soprattutto in soggetti ad alto rischio (bambini, anziani, pazienti con malattie croniche/debilitanti, ecc.). I virus influenzali di tipo A (IAV) e di tipo B (IBV), caratterizzata da notevole instabilità genetica e variabilità antigenica, sono la causa di epidemie annuali che colpiscono ogni anno il 15-20% della popolazione mondiale. Inoltre IAV è in grado di infettare una grande varietà di uccelli e mammiferi e come accaduto con il recente caso di influenza suina H1N1, un riassortimento genico tra i segmenti genomici virali di differenti specie può verificarsi e causare anche pandemie. Nel secolo scorso, la pandemie di influenza spagnola (1918), l’influenza asiatica (1957) e l’influenza di Hong Kong (1968), hanno causato effetti devastanti, determinando diverse decine di milioni di morti in tutto il mondo. Per questi motivi IAV e IBV rappresentano una grave minaccia per la salute pubblica ed è assolutamente necessario disporre di agenti terapeutici efficaci contro questi virus. Le opzioni terapeutiche attualmente disponibili contro l’influenza includono la vaccinazione e due distinte classi di farmaci rappresentati dagli inibitori di neuraminidasi (NA) e dai bloccanti del canale ionico M2. Tuttavia i vaccini devono essere riformulati ogni anno a causa della variabilità genetica del virus e non sono sempre protettivi. Inoltre, la produzione di un nuovo vaccino richiede vari mesi, quindi è del tutto improbabile che la vaccinazione possa essere efficace nell’impedire la diffusione di una pandemia influenzale rapidamente emergente. Oseltamivir (tamiflu) e zanamivir (relenza) fanno parte degli inibitori di neuraminidasi ed agiscono bloccando il rilascio delle particelle virali dalle cellule infettate. Amantadina (symadine, symmetrel) e rimantadina (flumadine) invece vanno a bloccare il canale ionico della proteina M2, prevenendo la transcapsidazione del virus e quindi il rilascio dei segmenti genomici nel citoplasma. Questi farmaci tuttavia presentano numerosi svantaggi, tra cui considerevoli effetti collaterali, efficacia ridotta, notevoli limitazioni nella somministrazione e soprattutto sono associati ad una rapida emergenza di ceppi virali farmaco-resistenti. Di fatto, non esiste ancora una terapia efficace contro l’influenza e questo rende necessario lo sviluppo di nuovi farmaci, possibilmente dotati di un innovativo meccanismo d’azione. Un interessante bersaglio per lo sviluppo di nuovi farmaci antivirali è rappresentato dall’RNA polimerasi virale, in quanto è un elemento conservato fra i diversi ceppi virali e non è soggetto a riassortimento genico. L’RNA polimerasi virale è un complesso eterotrimerico, costituito da tre proteine (la proteina acida della polimerasi [PA], la proteina basica 1 della polimerasi [PB1] e la proteina basica 2 della polimerasi [PB2]). Queste tre subunità interagiscono fisicamente tra di loro e, in particolare, la subunità PB1 interagisce sia con PB2 che con PA, mentre al momento esistono evidenze di un’interazione tra PB2 e PA. E’ stato dimostrato che l’interazione PA/PB1 è essenziale per la replicazione del virus: infatti l’inibizione di questa interazione causa l’inibizione della polimerasi virale. Il fatto che l’interfaccia di interazione PA-PB1 sia molto conservata tra differenti ceppi di IAV e IBV e che questo legame sia essenziale per la replicazione virale fa sì che esso rappresenti un potenziale bersaglio per lo sviluppo di nuovi composti anti-influenzali ad ampio spettro. Grazie ad uno screening in silico di una libreria di strutture di small molecules, condotto dal nostro gruppo di ricerca, sono stati identificati alcuni composti in grado di dissociare l’interazione tra le subunità PB1 e PA della RNA polimerasi del virus dell’influenza in vitro, a concentrazioni micromolari (Muratore et al., 2012a). Gli stessi composti non mostravano citotossicità in linee cellulari, ma allo stesso tempo esibivano solo modeste attività antivirali contro IAV. Lo scopo di questo progetto è stato valutare le modalità di interazione di questi composti con il bersaglio e sulla base di queste informazioni disegnare degli analoghi strutturali per aumentarne l’attività antivirale, preservando allo stesso tempo l’assenza di citotossicità ed il peculiare meccanismo d’azione. Una volta sintetizzati, questi analoghi sono stati saggiati in vari esperimenti al fine di valutarne appunto la loro capacità di inibire la replicazione virale di IAV mediante saggi di riduzione delle placche (plaque reduction assays, PRA), ma anche determinare il loro effetto inibitorio sull’interazione PA-PB1 in saggi ELISA ed indagare sulla loro eventuale citotossicità su linee cellulare in saggi MTT. In questi saggi, alcuni analoghi si sono messi in luce mostrando delle promettenti attività inibitorie e sono stati selezionati per essere ulteriormente caratterizzati. L’attività dei composti più interessanti è stata confermata mediante minireplicon assay, un saggio cellulare che permette di determinare l’effetto delle molecole sull’attività polimerasica di IAV. Tra questi, i composti 70, T22 e T40 hanno mostrato un effetto inibitorio sull’attività polimerasica virale con valori di EC50 comparabili o addirittura minori a quello ottenuto con la ribavirina (RBV), utilizzata come farmaco di riferimento. E’ stata poi valutata anche l’attività antivirale di questi analoghi nel tempo; quello che è emerso è che tutti i composti mostravano un massimo di attività alle 12 ore post-infezione (p.i.), senza tuttavia perdere l’effetto antivirale neanche a 48 ore p.i. Inoltre gli analoghi più promettenti erano capaci di inibire la replicazione di un pannello di diversi ceppi di IAV e di IBV, incluso un isolato clinico oseltamivir-resistente, dimostrando così di avere un’attività anti-influenzale ad ampio spettro. Al contrario, gli stessi composti non hanno evidenziato alcun effetto inibitorio sulla replicazione di altri virus a DNA e RNA, confermando di possedere uno specifico meccanismo d’azione. Infine per alcuni analoghi promettenti sono stati eseguite delle previsioni e delle simulazioni virtuali di interazione con la cavità di PA ed anche studi in cui è stata valutata l’eventuale variazione o meno di attività inibitoria nei confronti del bersaglio di PA mutato in alcuni residui chiave rispetto a quello wild-type (wt). Questo ci ha permesso di delineare delle relazioni struttura-attività (structure-activity relationships, SAR) e di ottenere preziose informazioni riguardo al modo in cui questi inibitori interagiscono con PA, ricavando di conseguenza utili suggerimenti per il disegno di nuovi analoghi caratterizzati da un miglior adattamento alla tasca di legame e da un’attività ancor più potente. In conclusione, questo studio dimostra le potenzialità di questi composti come inibitori dell’RNA polimerasi dei virus influenzali e nel prossimo futuro saranno saggiati in combinazione con i farmaci anti-influenzali attualmente in commercio per valutare possibili effetti sinergici. Inoltre questo lavoro ha permesso di identificare nuovi scaffold dotati di attività antivirale che saranno ulteriormente studiati per sviluppare una nuova generazione di agenti terapeutici capaci di combattere l’infezione da IAV e IBV

Development of a simple HPLC–UV method for the determination of the hepatitis C virus inhibitor simeprevir in human plasma

A simple high-performance liquid chromatography method for the determination of the hepatitis C virus protease inhibitor simeprevir in human plasma was developed and validated. The method involved a rapid and simple solid-phase extraction of simeprevir using Oasis HLB 1cc cartridges, isocratic reversed-phase liquid chromatography on an XTerra RP18 (150 mm × 4.6 mm, 3.5 μm) column, and ultraviolet detection at 225 nm. The mobile phase consisted of phosphate buffer (pH 6, 52.5 mM) and acetonitrile (30:70, v/v). This assay proved to be sensitive (lower limit of quantification of 0.05 μg/mL), linear (correlation coefficients ≥0.99), specific (no interference with various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of variation ≤8.3%), and accurate (deviations ranged from −8.0 to 1.2% and from −3.3 to 6.0% for intra-day and inter-day analysis, respectively). The method was applied to therapeutic monitoring of patients undergoing simeprevir treatment for hepatitis C and proved to be robust and reliable. Thus, this method provides a simple, sensitive, precise and reproducible assay for dosing simeprevir that can be readily adaptable to routine use by clinical laboratories with standard equipment

Drug Repurposing Approach Identifies Inhibitors of the Prototypic Viral Transcription Factor IE2 that Block Human Cytomegalovirus Replication

New targets for antiviral strategies are needed against human cytomegalovirus (HCMV), a major human pathogen. A cell-based screen aimed at finding inhibitors of the viral transcription factor Immediate-Early 2 (IE2) was performed in HCMV-infected cells expressing EGFP under the control of an IE2-inducible viral promoter. Screening of a library of bioactive small molecules led to the identification of several compounds able to inhibit EGFP expression and also HCMV replication with potency in the low-micromolar range. Follow-up studies with four selected hits indicated that they all block viral DNA synthesis as well as viral Early and Late gene expression. Furthermore, mechanistic studies confirmed that the compounds specifically act via inhibition of IE2 transactivating activity, thus blocking viral Early gene expression and the progression of virus replication. These results provide proof of concept for identifying small molecules that modulate the activity of a microbial transcription factor to control pathogen replication

Potent and broad-spectrum cycloheptathiophene-3-carboxamide compounds that target the PA-PB1 interaction of influenza virus RNA polymerase and possess a high barrier to drug resistance

Influenza viruses are major respiratory pathogens responsible for both seasonal epidemics and occasional pandemics worldwide. The current available treatment options have limited efficacy and thus the development of new antivirals is highly needed. We previously reported the identification of a series of cycloheptathiophene-3-carboxamide compounds as influenza A virus inhibitors that act by targeting the protein-protein interactions between the PA-PB1 subunits of the viral polymerase. In this study, we characterized the antiviral properties of the most promising compounds as well as investigated their propensity to induce drug resistance. Our results show that some of the selected compounds possess potent, broad-spectrum anti-influenza activity as they efficiently inhibited the replication of several strains of influenza A and B viruses, including an oseltamivir-resistant clinical isolate, with nanomolar or low-micromolar potency. The most promising compounds specifically inhibited the PA−PB1 binding in vitro and interfered with the influenza A virus polymerase activity in a cellular context, without showing cytotoxicity. The most active PA-PB1 inhibitors showed to possess a drug resistance barrier higher than that of oseltamivir. Indeed, no viral variants with reduced susceptibility to the selected compounds emerged after serial passages of influenza A virus under drug selective pressure. Overall, our studies identified potent PA-PB1 inhibitors as promising candidates for the development of new anti-influenza drugs

Development and validation of a simple and robust HPLC method with UV detection for quantification of the hepatitis C virus inhibitor daclatasvir in human plasma

none8nomixedNannetti, Giulio; Messa, Lorenzo; Celegato, Marta; Pagni, Silvana; Basso, Monica; Parisi, Saverio G.; Palù, Giorgio; Loregian, AriannaNannetti, Giulio; Messa, Lorenzo; Celegato, Marta; Pagni, Silvana; Basso, Monica; Parisi, Saverio G.; Palu', Giorgio; Loregian, Ariann

Near Full-Length Sequence Analysis of HIV Type 1 BF Recombinants from Italy

Abstract Recombination between HIV-1 subtypes B and F has generated several circulating and unique recombinant forms, particularly in Latin American areas. In Italy, subtype B is highly prevalent while subtype F is the most common pure non-B subtype. To investigate the recombination pattern in Italian BF recombinant viruses, we characterized full-length sequences derived from 15 adult patients, mostly Italian and infected by the heterosexual route. One of the BF mosaics was a CRF29, three sequences clustered with low bootstrap values with CRF39, CRF40, and CRF42. With the exception of the CRF29-like sequence, the other recombination patterns were unique, but two possible clusters were identified. Analysis of the gp120 V3 domain suggested a possible link with subtype F from Eastern Europe rather than from Latin America, favoring the hypothesis of local recombination between clade B and F viruses over that of import of BF recombinants from Latin America. HIV-1 subtypes B and F appear prone to generation of unique recombinants in Italy, warranting epidemiological surveillance and investigation of a possible clinical significance