FUS-mediated blood-brain barrier disruption for delivering anti-Aß antibodies in 5XFAD Alzheimer's disease mice.

PURPOSE: Amyloid-ß (Aß) peptides, the main component of amyloid plaques found in the Alzheimer's disease (AD) brain, are implicated in its pathogenesis, and are considered a key target in AD therapeutics. We herein propose a reliable strategy for non-invasively delivering a specific anti-Aß antibody in a mouse model of AD by microbubbles-enhanced Focused Ultrasound (FUS)-mediated Blood-brain barrier disruption (BBBD), using a simple single stage MR-compatible positioning device. METHODS: The initial experimental work involved wild-type mice and was devoted to selecting the sonication protocol for efficient and safe BBBD. Pulsed FUS was applied using a single-element FUS transducer of 1 MHz (80 mm radius of curvature and 50 mm diameter). The success and extent of BBBD were assessed by Evans Blue extravasation and brain damage by hematoxylin and eosin staining. 5XFAD mice were divided into different subgroups; control (n = 1), FUS + MBs alone (n = 5), antibody alone (n = 5), and FUS + antibody combined (n = 10). The changes in antibody deposition among groups were determined by immunohistochemistry. RESULTS: It was confirmed that the antibody could not normally enter the brain parenchyma. A single treatment with MBs-enhanced pulsed FUS using the optimized protocol (1 MHz, 0.5 MPa in-situ pressure, 10 ms bursts, 1% duty factor, 100 s duration) transiently disrupted the BBB allowing for non-invasive antibody delivery to amyloid plaques within the sonicated brain regions. This was consistently reproduced in ten mice. CONCLUSION: These preliminary findings should be confirmed by longer-term studies examining the antibody effects on plaque clearance and cognitive benefit to hold promise for developing disease-modifying anti-Aß therapeutics for clinical use.

Glial Gap Junction Pathology in the Spinal Cord of the 5xFAD Mouse Model of Early-Onset Alzheimer's Disease.

Gap junctions (GJs) are specialized transmembrane channels assembled by two hemi-channels of six connexin (Cx) proteins that facilitate neuroglial crosstalk in the central nervous system (CNS). Previous studies confirmed the crucial role of glial GJs in neurodegenerative disorders with dementia or motor dysfunction including Alzheimer's disease (AD). The aim of this study was to examine the alterations in astrocyte and related oligodendrocyte GJs in association with Aß plaques in the spinal cord of the 5xFAD mouse model of AD. Our analysis revealed abundant Aß plaque deposition, activated microglia, and astrogliosis in 12-month-old (12M) 5xFAD mice, with significant impairment of motor performance starting from 3-months (3M) of age. Additionally, 12M 5xFAD mice displayed increased immunoreactivity of astroglial Cx43 and Cx30 surrounding Aß plaques and higher protein levels, indicating upregulated astrocyte-to-astrocyte GJ connectivity. In addition, they demonstrated increased numbers of mature CC1-positive and precursor oligodendrocytes (OPCs) with higher immunoreactivity of Cx47-positive GJs in individual cells. Moreover, total Cx47 protein levels were significantly elevated in 12M 5xFAD, reflecting increased oligodendrocyte-to-oligodendrocyte Cx47-Cx47 GJ connectivity. In contrast, we observed a marked reduction in Cx32 protein levels in 12M 5xFAD spinal cords compared with controls, while qRT-PCR analysis revealed a significant upregulation in Cx32 mRNA levels. Finally, myelin deficits were found focally in the areas occupied by Aß plaques, whereas axons themselves remained preserved. Overall, our data provide novel insights into the altered glial GJ expression in the spinal cord of the 5xFAD model of AD and the implicated role of GJ pathology in neurodegeneration. Further investigation to understand the functional consequences of these extensive alterations in oligodendrocyte-astrocyte (O/A) GJ connectivity is warranted.

Spectrum of Phenotypic, Genetic, and Functional Characteristics in Patients With Epilepsy With KCNC2 Pathogenic Variants.

BACKGROUND AND OBJECTIVES: KCNC2 encodes Kv3.2, a member of the Shaw-related (Kv3) voltage-gated potassium channel subfamily, which is important for sustained high-frequency firing and optimized energy efficiency of action potentials in the brain. The objective of this study was to analyze the clinical phenotype, genetic background, and biophysical function of disease-associated Kv3.2 variants. METHODS: Individuals with KCNC2 variants detected by exome sequencing were selected for clinical, further genetic, and functional analysis. Cases were referred through clinical and research collaborations. Selected de novo variants were examined electrophysiologically in Xenopus laevis oocytes. RESULTS: We identified novel KCNC2 variants in 18 patients with various forms of epilepsy, including genetic generalized epilepsy (GGE), developmental and epileptic encephalopathy (DEE) including early-onset absence epilepsy, focal epilepsy, and myoclonic-atonic epilepsy. Of the 18 variants, 10 were de novo and 8 were classified as modifying variants. Eight drug-responsive patients became seizure-free using valproic acid as monotherapy or in combination, including severe DEE cases. Functional analysis of 4 variants demonstrated gain of function in 3 severely affected DEE cases and loss of function in 1 case with a milder phenotype (GGE) as the underlying pathomechanisms. DISCUSSION: These findings implicate KCNC2 as a novel causative gene for epilepsy and emphasize the critical role of KV3.2 in the regulation of brain excitability.

Pathogenic and Low-Frequency Variants in Children With Central Precocious Puberty.

Background: Central precocious puberty (CPP) due to premature activation of GnRH secretion results in early epiphyseal fusion and to a significant compromise in the achieved final adult height. Currently, few genetic determinants of children with CPP have been described. In this translational study, rare sequence variants in MKRN3, DLK1, KISS1, and KISS1R genes were investigated in patients with CPP. Methods: Fifty-four index girls and two index boys with CPP were first tested by Sanger sequencing for the MKRN3 gene. All children found negative (n = 44) for the MKRN3 gene were further investigated by whole exome sequencing (WES). In the latter analysis, the status of variants in genes known to be related with pubertal timing was compared with an in-house Cypriot control cohort (n = 43). The identified rare variants were initially examined by in silico computational algorithms and confirmed by Sanger sequencing. Additionally, a genetic network for the MKRN3 gene, mimicking a holistic regulatory depiction of the crosstalk between MKRN3 and other genes was designed. Results: Three previously described pathogenic MKRN3 variants located in the coding region of the gene were identified in 12 index girls with CPP. The most prevalent pathogenic MKRN3 variant p.Gly312Asp was exclusively found among the Cypriot CPP cohort, indicating a founder effect phenomenon. Seven other CPP girls harbored rare likely pathogenic upstream variants in the MKRN3. Among the 44 CPP patients submitted to WES, nine rare DLK1 variants were identified in 11 girls, two rare KISS1 variants in six girls, and two rare MAGEL2 variants in five girls. Interestingly, the frequent variant rs10407968 (p.Gly8Ter) of the KISS1R gene appeared to be less frequent in the cohort of patients with CPP. Conclusion: The results of the present study confirm the importance of the MKRN3-imprinted gene in genetics of CPP and its key role in pubertal timing. Overall, the results of the present study have emphasized the importance of an approach that aligns genetics and clinical aspects, which is necessary for the management and treatment of CPP.

Altered Expression of Glial Gap Junction Proteins Cx43, Cx30, and Cx47 in the 5XFAD Model of Alzheimer's Disease.

Glial gap junction proteins, called connexins (Cxs), form gap junctions in the central nervous system (CNS) to allow the bidirectional cytosolic exchange of molecules between adjacent cells. Their involvement in inheritable diseases and the use of experimental animal models that closely mimic such diseases revealed the critical role of glial GJs in myelination and homeostasis. Cxs are also implicated in acquired demyelinating disorders, such as Multiple Sclerosis (MS) and Alzheimer's disease (AD). Animal and human studies have revealed a role of the astrocytic Cx43 in the progression of AD but the role of Cx47, which is the main partner of Cx43 in the astrocyte-oligodendrocyte GJs is still unknown. The aim of this study was to investigate the astrocytic connexins, Cx43 and Cx30 in relation to oligodendrocytic Cx47 in the cortex and thalamus of the 5XFAD mouse model of AD. The model was characterized by increased Aß deposition, gliosis, neuronal loss, and memory impairment. Compared to wild-type mice, Cx43 and Cx30 showed increased immunoreactivity in older 5XFAD mice, reflecting astrogliosis, while Cx47 immunoreactivity was reduced. Moreover, Cx47 GJ plaques showed reduced colocalization with Cx43 plaques. Oligodendrocyte precursor cells (OPCs) and mature oligodendrocyte populations were also depleted, and myelin deficits were observed. Our findings indicate reduced astrocyte-oligodendrocyte gap junction connectivity and possibly a shift in Cx43 expression toward astrocyte-astrocyte gap junctions and/or hemichannels, that could impair oligodendrocyte homeostasis and myelination. However, other factors, such as Aß toxicity, could directly affect oligodendrocyte survival in AD. Our study provides evidence that Cxs might have implications in the progression of AD, although the role of oligodendrocyte Cxs in AD requires further investigation.

Neurophysiological and Genetic Findings in Patients With Juvenile Myoclonic Epilepsy.

OBJECTIVE: Transcranial magnetic stimulation (TMS), a non-invasive procedure, stimulates the cortex evaluating the central motor pathways. The response is called motor evoked potential (MEP). Polyphasia results when the response crosses the baseline more than twice (zero crossing). Recent research shows MEP polyphasia in patients with generalized genetic epilepsy (GGE) and their first-degree relatives compared with controls. Juvenile Myoclonic Epilepsy (JME), a GGE type, is not well studied regarding polyphasia. In our study, we assessed polyphasia appearance probability with TMS in JME patients, their healthy first-degree relatives and controls. Two genetic approaches were applied to uncover genetic association with polyphasia. METHODS: 20 JME patients, 23 first-degree relatives and 30 controls underwent TMS, obtaining 10-15 MEPs per participant. We evaluated MEP mean number of phases, proportion of MEP trials displaying polyphasia for each subject and variability between groups. Participants underwent whole exome sequencing (WES) via trio-based analysis and two-case scenario. Extensive bioinformatics analysis was applied. RESULTS: We identified increased polyphasia in patients (85%) and relatives (70%) compared to controls (47%) and significantly higher mean number of zero crossings (i.e., occurrence of phases) (patients 1.49, relatives 1.46, controls 1.22; p < 0.05). Trio-based analysis revealed a candidate polymorphism, p.Glu270del,in SYT14 (Synaptotagmin 14), in JME patients and their relatives presenting polyphasia. Sanger sequencing analysis in remaining participants showed no significant association. In two-case scenario, a machine learning approach was applied in variants identified from odds ratio analysis and risk prediction scores were obtained for polyphasia. The results revealed 61 variants of which none was associated with polyphasia. Risk prediction scores indeed showed lower probability for non-polyphasic subjects on having polyphasia and higher probability for polyphasic subjects on having polyphasia. CONCLUSION: Polyphasia was present in JME patients and relatives in contrast to controls. Although no known clinical symptoms are linked to polyphasia this neurophysiological phenomenon is likely due to common cerebral electrophysiological abnormality. We did not discover direct association between genetic variants obtained and polyphasia. It is likely these genetic traits alone cannot provoke polyphasia, however, this predisposition combined with disturbed brain-electrical activity and tendency to generate seizures may increase the risk of developing polyphasia, mainly in patients and relatives.

C5aR agonist enhances phagocytosis of fibrillar and non-fibrillar Aß amyloid and preserves memory in a mouse model of familial Alzheimer's disease.

According to the amyloid hypothesis of Alzheimer's disease (AD) the deposition of prefibrillar and fibrillar Aß peptide sets off the pathogenic cascades of neuroinflammation and neurodegeneration that lead to synaptic and neuronal loss resulting in cognitive decline. Various approaches to reduce amyloid load by reducing production of the Aß peptide or enhancing amyloid clearance by primary or secondary immunization have not proven successful in clinical trials. Interfering with the normal function of secretases and suboptimal timing of Aß peptide removal have been put forward as possible explanations. Complement, an innate component of the immune system, has been found to modulate disease pathology and in particular neuronal loss in the AD mouse model but its mechanism of action is complex. C1Q has been shown to facilitate phagocytosis of Aß peptide but its Ablation attenuates neuroinflammation. Experiments in AD mouse models show that inhibition of complement component C5a reduces amyloid deposition and alleviates neuroinflammation. Phagocytes including microglia, monocytes and neutrophils carry C5a receptors. Here, a widely used mouse model of AD, 5XFAD, was intermittently treated with the oral C5a receptor agonist EP67 and several neuronal and neuroinflammatory markers as well as memory function were assessed. EP67 treatment enhanced phagocytosis, resulting in a significant reduction of both fibrillar and non-fibrillar Aß, reduced astrocytosis and preserved synaptic and neuronal markers as well as memory function. Timely and phasic recruitment of the innate immune system offers a new therapeutic avenue of treating pre-symptomatic Alzheimer disease.

A novel mutation in the erythroid transcription factor KLF1 is likely responsible for ameliorating ß-thalassemia major.

We describe the identification of a novel missense mutation in the second zinc finger of KLF1 in two siblings who, based on their genotype, are predicted to suffer from beta thalassemia major but are, in fact, transfusion-free and in good health. These individuals, as well as two additional members of the same family also carrying this KLF1 mutation, exhibit high levels of fetal hemoglobin (HbF). KLF1 is an erythroid transcription factor, which plays a critical role in the regulation of the developmental switch between fetal and adult hemoglobin by regulating the expression of a multitude of genes including that of BCL11A. The mutation appears to be the main candidate responsible for the beta thalassemia-ameliorating effect as this segregates with the observed phenotype and also exogenous expression of the KLF1 mutant protein in human erythroid progenitor cells resulted in the induction of γ-globin, without, however, affecting BCL11A levels. This report adds to the weight of evidence that heterozygous KLF1 mutations can ameliorate the severity of the ß-thalassemia major phenotype.

HIV-1 transmission networks across Cyprus (2010-2012).

A molecular epidemiology study of HIV-1 infection was conducted in one hundred diagnosed and untreated HIV-1-infected patients in Cyprus between 2010 and 2012, representing 65.4% of all the reported HIV-1 infections in Cyprus in this three-year period, using a previously defined enrolment strategy. Eighty-two patients were newly diagnosed (genotypic drug resistance testing within six months from diagnosis), and eighteen patients were HIV-1 diagnosed for a longer period or the diagnosis date was unknown. Phylogenetic trees of the pol sequences obtained in this study with reference sequences indicated that subtypes B and A1 were the most common subtypes present and accounted for 41.0 and 19.0% respectively, followed by subtype C (7.0%), F1 (8.0%), CRF02_AG (4.0%), A2 (2.0%), other circulating recombinant forms (CRFs) (7.0%) and unknown recombinant forms (URFs) (12%). Most of the newly-diagnosed study subjects were Cypriots (63%), males (78%) with median age 39 (Interquartile Range, IQR 33-48) reporting having sex with other men (MSM) (51%). A high rate of clustered transmission of subtype B drug-sensitive strains to reverse transcriptase and protease inhibitors was observed among MSM, twenty-eight out of forty-one MSM study subjects (68.0%) infected were implicated in five transmission clusters, two of which are sub-subtype A1 and three of which are subtype B strains. The two largest MSM subtype B clusters included nine and eight Cypriot men, respectively, living in all major cities in Cyprus. There were only three newly diagnosed patients with transmitted drug resistant HIV-1 strains, one study subject from the United Kingdom infected with subtype B strain and one from Romania with sub-subtype A2 strain, both with PI drug resistance mutation M46L and one from Greece with sub-subtype A1 with non-nucleoside reverse transcriptase inhibitors (NNRTI) drug resistance mutation K103N.

The molecular spectrum and distribution of haemoglobinopathies in Cyprus: a 20-year retrospective study.

Haemoglobinopathies are the most common monogenic diseases, posing a major public health challenge worldwide. Cyprus has one the highest prevalences of thalassaemia in the world and has been the first country to introduce a successful population-wide prevention programme, based on premarital screening. In this study, we report the most significant and comprehensive update on the status of haemoglobinopathies in Cyprus for at least two decades. First, we identified and analysed all known 592 ß-thalassaemia patients and 595 Hb H disease patients in Cyprus. Moreover, we report the molecular spectrum of α-, ß- and δ-globin gene mutations in the population and their geographic distribution, using a set of 13824 carriers genotyped from 1995 to 2015, and estimate relative allele frequencies in carriers of ß- and δ-globin gene mutations. Notably, several mutations are reported for the first time in the Cypriot population, whereas important differences are observed in the distribution of mutations across different districts of the island.

Genotyping of BCL11A and HBS1L-MYB SNPs associated with fetal haemoglobin levels: a SNaPshot minisequencing approach.

BACKGROUND: B-thalassaemia and sickle cell disease (SCD) are two of the most common monogenic diseases that are found in many populations worldwide. In both disorders the clinical severity is highly variable, with the persistence of fetal haemoglobin (HbF) being one of the major ameliorating factors. HbF levels are affected by, amongst other factors, single nucleotide polymorphisms (SNPs) at the BCL11A gene and the HBS1L-MYB intergenic region, which are located outside the ß-globin locus. For this reason, we developed two multiplex assays that allow the genotyping of SNPs at these two genomic regions which have been shown to be associated with variable HbF levels in different populations. RESULTS: Two multiplex assays based on the SNaPshot minisequencing approach were developed. The two assays can be used to simultaneous genotype twelve SNPs at the BCL11A gene and sixteen SNPs at HBS1L-MYB intergenic region which were shown to modify HbF levels. The different genotypes can be determined based on the position and the fluorescent colour of the peaks in a single electropherogram. DNA sequencing and restriction fragment length polymorphism (PCR-RFLP) assays were used to verify genotyping results obtained by SNaPshot minisequencing. CONCLUSIONS: In summary, we propose two multiplex assays based on the SNaPshot minisequencing approach for the simultaneous identification of SNPs located at the BCL11A gene and HBS1L-MYB intergenic region which have an effect on HbF levels. The assays can be easily applied for accurate, time and cost efficient genotyping of the selected SNPs in various populations.

Cellular HIV type 1 DNA levels are equivalent among drug-sensitive and drug-resistant strains in newly diagnosed and antiretroviral naive patients.

The emergence of resistance against current antiretroviral drugs to human immunodeficiency virus type 1 (HIV-1) is an increasingly important concern to the continuous success of antiretroviral therapy to HIV-1-infected patients. In the past decade, a number of studies reported that the prevalence of transmitted drug resistance among newly diagnosed patients has reached an overall 9% prevalence worldwide. Also, a number of studies using longitudinal HIV-1 patient study cohorts demonstrated that the cellular HIV-1 DNA level in peripheral blood mononuclear cells (PBMCs) has a prognostic value for the progression of HIV-1 disease independently of plasma HIV-1 RNA load and CD4 count. Using a previously established molecular-beacon-based real-time PCR methodology, cellular HIV-1 DNA levels were quantified in newly diagnosed and antiretroviral-naive patients in Northern Greece recruited between 2009 and 2010 using a predefined enrolling strategy, in an effort to investigate whether there is any relationship between cellular HIV-1 DNA levels and HIV-1 transmitted drug resistance. As part of the same study, DNA sequences encoding the env (C2-C5 region of gp120) were also amplified from PBMC-extracted DNA in order to determine the genotypic coreceptor tropism and genetic subtype. Cellular HIV-1 DNA levels had a median of 3.309 log10 HIV-1 copies per 10(6) PBMCs and demonstrated no correlation between cellular HIV-1 DNA levels and HIV-1 transmitted drug resistance. An absence of association between cellular HIV-1 DNA levels with plasma viral HIV-1 RNA load and CD4 levels was also found reconfirming the previously published study. Genotypic analysis of coreceptor tropism indicated that 96% of samples, independently of the presence or not of genotypic drug resistance, were CCR5-tropic. Overall, the findings reconfirmed the previously proposed proposition that transmitted drug resistance does not have an impact on disease progression in HIV-1-infected individuals. Also, CCR5 coreceptor tropism dominance suggests that both drug-resistant and drug-sensitive strains behave similarly in early infection in newly diagnosed patients.

Short communication: molecular epidemiology of HIV type 1 infection in northern Greece (2009-2010): evidence of a transmission cluster of HIV type 1 subtype A1 drug-resistant strains among men who have sex with men.

A prospective molecular epidemiology study of HIV-1 infection was conducted in newly diagnosed and antiretroviral-naive patients in Northern Greece between 2009 and 2010 using a predefined enrolling strategy. Phylogenetic trees of the pol sequences obtained in this study with reference sequences indicated that subtypes B and A1 were the most common subtypes present and accounted for 44.9% and 42.9%, respectively, followed by subtype C (3.1%), CRF02_AG (4.1%), CRF04_cpx (2.0%), and subtypes CRF01_01, F1, and G (1.0%). A high rate of clustered transmission of subtype A1-resistant strains to reverse transcriptase (RT) inhibitors was observed among men having sex with men. Indeed, 15 out of 17 study subjects (88.2%) infected with transmitted drug resistance (TDR) strains were implicated in transmission clusters, 10 of whom (66.7%) were men who have sex with men (MSM), and were also infected with subsubtype A1 strains. The main cluster within subtype A1 (I) included eight men reporting having sex with men from Thessaloniki infected with dual-class RT-resistant strains carrying both T215C and Y181C mutations.

Molecular characterization of HIV type 1 strains from newly diagnosed patients in Cyprus (2007-2009) recovers multiple clades including unique recombinant strains and lack of transmitted drug resistance.

HIV-1 evolution generates substantial genetic diversity among isolates, the majority of which are represented in areas where multiple strains cocirculate. A heterogeneous genetic HIV-1 pool has been found in Cyprus, directing us to determine the dynamics of the local HIV-1 infection by characterizing strains isolated from 74 subjects during 2007-2009, representing 88% of the known-living HIV-1-infected population, of whom 53 are newly diagnosed therapy-naive patients and 21 are chronic patients, according to the European HIV Resistance guidelines. Near full-length genome sequences were amplified by RT-nested PCR using diluted RNA from all HIV-1 seropositives and sequenced using a newly designed assay. Resistant mutations were not found among the population of the newly diagnosed therapy-naive patients either to protease, reverse transcriptase, or integrase inhibitors. Phylogenetic analyses indicated subtype B as the main subtype (48.6%), followed by subtype A (18.9%), subtype C (10.8%), CRF02_AG (8.1%), CRF11_cpx (2.7%), and (sub)subtype F1 and CRF37_cpx (1.4% each). Six HIV-1 isolates (8.1%) were not classified in any pure (sub)subtype or circulating recombinant form (CRF). Complete phylogenetic and bootscanning analyses revealed that each isolate had a new, unique recombinant pattern and is distinct from all other CRFs or unique recombinant forms (URFs) reported so far. Two of the six isolates have the same mosaic pattern. Analogous to results of the earlier epidemiological studies, this study expands on the HIV-1 sequence database and reveals the high degree of diversity of HIV-1 infection in Cyprus.

Cellular HIV-1 DNA levels in drug sensitive strains are equivalent to those in drug resistant strains in newly-diagnosed patients in Europe.

BACKGROUND: HIV-1 genotypic drug resistance is an important threat to the success of antiretroviral therapy and transmitted resistance has reached 9% prevalence in Europe. Studies have demonstrated that HIV-1 DNA load in peripheral blood mononuclear cells (PBMC) have a predictive value for disease progression, independently of CD4 counts and plasma viral load. METHODOLOGY/PRINCIPAL FINDINGS: Molecular-beacon-based real-time PCR was used to measure HIV-1 second template switch (STS) DNA in PBMC in newly-diagnosed HIV-1 patients across Europe. These patients were representative for the HIV-1 epidemic in the participating countries and were carrying either drug-resistant or sensitive viral strains. The assay design was improved from a previous version to specifically detect M-group HIV-1 and human CCR5 alleles. The findings resulted in a median of 3.32 log(10) HIV-1 copies/10(6) PBMC and demonstrated for the first time no correlation between cellular HIV-1 DNA load and transmitted drug-resistance. A weak association between cellular HIV-1 DNA levels with plasma viral RNA load and CD4(+) T-cell counts was also reconfirmed. Co-receptor tropism for 91% of samples, whether or not they conferred resistance, was CCR5. A comparison of pol sequences derived from RNA and DNA, resulted in a high similarity between the two. CONCLUSIONS/SIGNIFICANCE: An improved molecular-beacon-based real-time PCR assay is reported for the measurement of HIV-1 DNA in PBMC and has investigated the association between cellular HIV-1 DNA levels and transmitted resistance to antiretroviral therapy in newly-diagnosed patients from across Europe. The findings show no correlation between these two parameters, suggesting that transmitted resistance does not impact disease progression in HIV-1 infected individuals. The CCR5 co-receptor tropism predominance implies that both resistant and non-resistant strains behave similarly in early infection. Furthermore, a correlation found between RNA- and DNA-derived sequences in the pol region suggests that genotypic drug-resistance testing could be carried out on either template.

Near full-length genetic analysis of HIV sequences derived from Cyprus: evidence of a highly polyphyletic and evolving infection.

The molecular epidemiology of HIV-1 infection was previously studied in Cyprus but the degree of HIV-1 diversity has remained indefinable. The main objective of the present study is to examine HIV-1 strains isolated from 77 HIV-1-infected individuals representing 38% of the known infected population in Cyprus in the period 1986 to 2006. DNA of the near full-length genome encoding gag, pol, vif, vpr, vpu, tat, rev, env, and 5'-end of nef was amplified by nested PCR/RT-PCR from all HIV-1 seropositives and sequenced using a newly designed assay. Detailed phylogenetic and bootscanning analyses were performed to determine phylogenetic associations and subtype assignments. Phylogenetic analyses of the obtained viral sequences indicated that subtype B was the dominant subtype (61%), followed by subtype A (23.3%), subtype C (5.2%), CRF02_AG (3.9%), and subtype D, CRF01_AE, and CRF04_cpx (1.3% each). Two HIV-1 isolates (2.6%), originating from the Democratic Republic of Congo (DRC), were not classified in any pure (sub)subtype or circulating recombinant form (CRF). Complete phylogenetic and bootscanning analyses revealed that one of these isolates had a new, unique recombinant pattern, comprising segments of subtypes D and G, and is distinct from any other CRFs or URFs reported so far. Detailed analyses of the sequence of the second isolate, which could not be classified, reveal that it is close to subtype K reference sequences but clusters near the root of the clade. At least two epidemiologically unrelated HIV-1 seropositives with an HIV-1 variant similar to this isolate are required to designate this variant as a novel HIV-1 subtype or subsubtype of subtype K. Analogous to results of the earlier epidemiological studies, these data exhibit the extensive heterogeneity of HIV-1 infection in Cyprus, which is being fueled by a continuous entry of new strains from other countries, creating an evolving and polyphyletic infection.

Genetic analysis of HIV type 1 strains from newly infected untreated patients in cyprus: high genetic diversity and low prevalence of drug resistance.

Abstract The molecular epidemiology of HIV-1 infection was first studied in Cyprus in the mid-1990s, but the extent of HIV-1 diversity and the prevalence of drug resistance have remained elusive. In an effort to address this issue, the present study examined HIV-1 strains isolated from 37 newly diagnosed untreated HIV-1 patients, representing 72% of the total number of newly diagnosed and drug-naive patients in the period 2003 to 2006. DNA sequences encoding the gag (p17, p24, p2, p7, p1, and p6), pol (protease and reverse transcriptase), and env (gp160) regions were amplified by RT-PCR from plasma HIV-1 RNA from all patients and sequenced using a newly designed methodology. All amplified products were studied according to established genetic methodologies to determine the genetic subtype and the prevalence of drug-resistance-associated mutations to currently available antiretroviral drugs. Analyses of the obtained viral sequences indicated that subtype A was the most common subtype present and accounted for 38% of the infections followed by subtype B (35%), subtype C (13%), CRF02_AG (8%), and subtypes D and CRF01_AE (3% each). One patient (2.7%) had an M41L/M and another patient (2.7%) an M184V amino acid substitution in the reverse transcriptase (RT) associated with high-level resistance to RT inhibitors. There were no patients with resistant mutations to protease inhibitors (PI). Additionally, one patient (2.7%) had an L44M amino acid substitution within the HR1 region of gp41 conferring resistance to the enfuvirtide (T20) fusion inhibitor. Similar to results of the 1994 molecular epidemiological study, these data demonstrate the extensive heterogeneity of HIV-1 infection in Cyprus and the low prevalence of transmitted resistance to current HIV-1 antiretroviral drugs. Taken together, these findings demonstrate that HIV-1 infection in Cyprus is being replenished by a continuous influx of new strains from many countries, establishing an ever-evolving and polyphyletic infection in the island.

Implications of HIV-1 M group polymorphisms on integrase inhibitor efficacy and resistance: genetic and structural in silico analyses.

The extensive polymorphisms among HIV-1 subtypes have been implicated in drug resistance development. Integrase inhibitors represent the latest addition to the treatment of HIV-1, and their efficacy and resistance patterns among M group strains are currently under investigation. This study analyzed the intersubtype variation within 108 integrase sequences from seven subtypes. The residues associated with catalytic activity and primary resistance to raltegravir were highly conserved among all strains. Variations were observed in residues associated with secondary resistance. Molecular modeling studies indicated a two-way binding mode of raltegravir that explains the resistance pathways and the implication of nonconservative mutations in integrase-raltegravir interactions.