Guidelines for preventing and slowing myopia progression in Brazilian children.

This document on myopia control is derived from a compilation of medical literature and the collective clinical expertise of an expert committee comprising members from the Brazilian Society of Pediatric Ophthalmology and the Brazilian Society of Contact Lenses and Cornea. To manage myopia in children, the committee recommends corneal topography and biannual visits with cycloplegic refraction, along with annual optical biometry. For fast-progressing myopia, biannual biometry should be considered. Myopic progression is defined as an annual increase in spherical equivalent greater than 0.50 D/year or in axial length greater than 0.3 mm (until 10 years old) or 0.2 mm (above 11 years). The proposed treatments for myopia progression include environmental control, low concentration atropine, defocus glasses, contact lenses, or Ortho-K lenses, and combinations of these methods may be necessary for uncontrolled cases. Treatment should be sustained for at least 2 years. This document serves as a comprehensive guideline for diagnosing, treating, and monitoring pre-myopic and myopic children in Brazil.

Preservatives and pH in low-dose atropine formulations for clinical trials.

Over a century ago, atropine has been tested to arrest myopia progression with good results. In recent years, many randomized clinical trials have tested different concentrations against placebo. Three recent such studies with low-dose atropine showed that it was less effective than previous studies, even the last one showing no difference in myopia progression between the treated and control group. Previous randomized studies had been performed in Asian populations, and these last three were extended to Western Caucasian populations, based on the initial observation that differences in iris pigmentation could be a factor for a difference in effectiveness. We have noticed that the three last studies in the West have used the same patented formulation, while previous studies have preferred compounded low-dose atropine. Here we review how the power of hydrogen (pH) and preservatives could account for differences in drug penetration to the eye, possibly explaining the differences between studies.

Guidelines for preventing and slowing myopia progression in Brazilian children / Diretrizes brasileiras para prevenir e retardar a progressão da miopia em crianças brasileiras

ABSTRACT This document on myopia control is derived from a compilation of medical literature and the collective clinical expertise of an expert committee comprising members from the Brazilian Society of Pediatric Ophthalmology and the Brazilian Society of Contact Lenses and Cornea. To manage myopia in children, the committee recommends corneal topography and biannual visits with cycloplegic refraction, along with annual optical biometry. For fast-progressing myopia, biannual biometry should be considered. Myopic progression is defined as an annual increase in spherical equivalent greater than 0.50 D/year or in axial length greater than 0.3 mm (until 10 years old) or 0.2 mm (above 11 years). The proposed treatments for myopia progression include environmental control, low concentration atropine, defocus glasses, contact lenses, or Ortho-K lenses, and combinations of these methods may be necessary for uncontrolled cases. Treatment should be sustained for at least 2 years. This document serves as a comprehensive guideline for diagnosing, treating, and monitoring pre-myopic and myopic children in Brazil.

RESUMO Esta revisão foi baseada na literatura médica e na experiência clínica de um comitê de especialistas membros da Sociedade Brasileira de Oftalmologia Pediátrica e da Sociedade Brasileira de Lentes de Contato e Córnea. Rotineiramente as crianças devem ser submetidas a topografia da córnea no primeiro exame e visitas semestrais com refração cicloplegiada e biometria óptica anual. A progressão da miopia foi definida como um aumento anual no equivalente esférico maior que 0,50 D/ano ou do comprimento axial maior que 0,3 mm (até 10 anos) ou 0,2 mm (mais de 11 anos). Os tratamentos propostos para a progressão são controle ambiental, atropina em baixa concentração, óculos com defocus, lentes de contato ou ortoceratologia, devendo-se considerar associações para casos não controlados. O tratamento deve ser realizado por pelo menos 2 anos. O presente documento é uma diretriz para diagnóstico, tratamento e acompanhamento de crianças pré-míopes e míopes no Brasil.

Genomic characterization of the rotavirus G3P[8] strain in vaccinated children, reveals possible reassortment events between human and animal strains in Manhiça District, Mozambique.

Mozambique introduced the rotavirus vaccine (Rotarix®; GlaxoSmithKline Biologicals, Rixensart, Belgium) in 2015, and since then, the Centro de Investigação em Saúde de Manhiça has been monitoring its impact on rotavirus-associated diarrhea and the trend of circulating strains, where G3P[8] was reported as the predominant strain after the vaccine introduction. Genotype G3 is among the most commonly detected Rotavirus strains in humans and animals, and herein, we report on the whole genome constellation of G3P[8] detected in two children (aged 18 months old) hospitalized with moderate-to-severe diarrhea at the Manhiça District Hospital. The two strains had a typical Wa-like genome constellation (I1-R1-C1-M1-A1-N1-T1-E1-H1) and shared 100% nucleotide (nt) and amino acid (aa) identities in 10 gene segments, except for VP6. Phylogenetic analysis demonstrated that genome segments encoding VP7, VP6, VP1, NSP3, and NSP4 of the two strains clustered most closely with porcine, bovine, and equine strains with identities ranging from 86.9-99.9% nt and 97.2-100% aa. Moreover, they consistently formed distinct clusters with some G1P[8], G3P[8], G9P[8], G12P[6], and G12P[8] strains circulating from 2012 to 2019 in Africa (Mozambique, Kenya, Rwanda, and Malawi) and Asia (Japan, China, and India) in genome segments encoding six proteins (VP2, VP3, NSP1-NSP2, NSP5/6). The identification of segments exhibiting the closest relationships with animal strains shows significant diversity of rotavirus and suggests the possible occurrence of reassortment events between human and animal strains. This demonstrates the importance of applying next-generation sequencing to monitor and understand the evolutionary changes of strains and evaluate the impact of vaccines on strain diversity.

Drug Repurposing for COVID-19: A Review and a Novel Strategy to Identify New Targets and Potential Drug Candidates.

In December 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) was first identified in the province of Wuhan, China. Since then, there have been over 400 million confirmed cases and 5.8 million deaths by COVID-19 reported worldwide. The urgent need for therapies against SARS-CoV-2 led researchers to use drug repurposing approaches. This strategy allows the reduction in risks, time, and costs associated with drug development. In many cases, a repurposed drug can enter directly to preclinical testing and clinical trials, thus accelerating the whole drug discovery process. In this work, we will give a general overview of the main developments in COVID-19 treatment, focusing on the contribution of the drug repurposing paradigm to find effective drugs against this disease. Finally, we will present our findings using a new drug repurposing strategy that identified 11 compounds that may be potentially effective against COVID-19. To our knowledge, seven of these drugs have never been tested against SARS-CoV-2 and are potential candidates for in vitro and in vivo studies to evaluate their effectiveness in COVID-19 treatment.

Molecular Epidemiology of Rotavirus Strains in Symptomatic and Asymptomatic Children in Manhiça District, Southern Mozambique 2008-2019.

Group A rotaviruses remain the leading cause of diarrhoea in children aged <5 years. Mozambique introduced rotavirus vaccine (Rotarix®) in September 2015. We report rotavirus genotypes circulating among symptomatic and asymptomatic children in Manhiça District, Mozambique, pre- and post-vaccine introduction. Stool was collected from enrolled children and screened for rotavirus by enzyme-immuno-sorbent assay. Positive specimens were genotyped for VP7 (G genotypes) and VP4 (P genotypes) by the conventional reverse transcriptase polymerase chain reaction. The combination G12P[8] was more frequently observed in pre-vaccine than in post-vaccine introduction, in moderate to severe diarrhoea (34%, 61/177 vs. 0, p < 0.0001) and controls (23%, 26/113 vs. 0, p = 0.0013) and mixed genotypes (36%, 24/67 vs. 7% 4/58, p = 0.0003) in less severe diarrhoea. We observed changes in post-vaccine compared to pre-vaccine introduction, where G3P[4] and G3P[8] were prevalent in moderate to severe diarrhoea (10%, 5/49 vs. 0, p = 0.0002; and 14%, 7/49 vs. 1%, 1/177, p < 0.0001; respectively), and in less severe diarrhoea (21%, 12/58 vs. 0, p = 0.003; and 24%, 14/58 vs. 0, p < 0.0001; respectively). Our surveillance demonstrated the circulation of similar genotypes contemporaneously among cases and controls, as well as switching from pre- to post-vaccine introduction. Continuous surveillance is needed to evaluate the dynamics of the changes in genotypes following vaccine introduction.

The role of muscle in the susceptibility and progression of axial Spondyloarthritis: The MyoSpA Study Protocol.

BACKGROUND: Axial Spondyloarthritis (axSpA) is a chronic, inflammatory rheumatic disease that affects the axial skeleton, causing pain, stiffness, and fatigue. Genetics and environmental factors such as microbiota and microtrauma are known causes of disease susceptibility and progression. Murine models of axSpA found a decisive role for biomechanical stress as an inducer of enthesitis and new bone formation. Here, we hypothesize that muscle properties in axSpA patients are compromised and influenced by genetic background. OBJECTIVES: To improve our current knowledge of axSpA physiopathology, we aim to characterize axial and peripheral muscle properties and identify genetic and protein biomarker that might explain such properties. METHODS: A cross-sectional study will be conducted on 48 participants aged 18-50 years old, involving patients with axSpA (according to ASAS classification criteria, symptoms duration < 10 years) and healthy controls matched by gender, age, and levels of physical activity. We will collect epidemiological and clinical data and perform a detailed, whole body and segmental, myofascial characterization (focusing on multifidus, brachioradialis and the gastrocnemius lateralis) concerning: a) Physical Properties (stiffness, tone and elasticity), assessed by MyotonPRO®; b) Strength, by a dynamometer; c) Mass, by bioimpedance; d) Performance through gait speed and 60-second sit-to-stand test; e) Histological and cellular/ molecular characterization through ultrasound-guided biopsies of multifidus muscle; f) Magnetic Resonance Imaging (MRI) characterization of paravertebral muscles. Furthermore, we will perform an integrated transcriptomics and proteomics analysis of peripheral blood samples. DISCUSSION: The innovative and multidisciplinary approaches of this project rely on the elucidation of myofascial physical properties in axSpA and also on the establishment of a biological signature that relates to specific muscle properties. This hitherto unstudied link between gene/protein signatures and muscle properties may enhance our understanding of axSpA physiopathology and reveal new and useful diagnostic and therapeutic targets.

Hepatitis delta: In vitro evaluation of cytotoxicity and cytokines involved in PEG-IFN therapy.

The treatment for hepatitis Delta virus (HDV) still consists of Pegylated interferon (PEG-IFN) combined with inhibitors of Hepatitis B virus (HBV) replication. In some patients may be occur a virological response, which means a negative HDV RNA 6 months after stopping treatment. In this study it was conducted an in vitro approach with the aim to mimic possible immunological events that are observed in patients responding to PEG-IFN therapy. Jurkat cells (human T lymphocyte cell line) were employed alone or co-cultured with THP-1 (human monocytic cell line) and stimulated with controls and HBV Surface Antigen (HBsAg), Small-Delta Antigen (SHDAg), and HBsAg + SHDAg combined. Twenty-four hours stimulation with SHDAg and/or HBSAg led to a toxic profile in a co-culture condition and cell supernatants were collected for cytokines quantification. PEG-IFN was added and cells were incubated for additional 24 h. Co-cultured cells incubated with the association (SHDAg + PEG-IFN) significantly produced levels of IFN-γ, IL-2 and IL-12. On the other hand, the HBsAg alone was able to inhibit the production of IFN-γ, suggesting that this antigen may hinder the treatment exclusively with PEG-IFN.

Hepatitis delta virus interacts with splicing factor SF3B155 and alters pre-mRNA splicing of cell cycle control genes.

Hepatitis delta virus (HDV) is the agent responsible for the most severe form of human viral hepatitis. The HDV genome consists of a single-stranded circular RNA molecule that encodes for one single protein, the delta antigen. Given its simplicity, HDV must make use of several host cellular proteins to accomplish its life cycle processes, including transcription, replication, post-transcriptional, and post-translational modifications. Consequently, identification of the interactions established between HDV components and host proteins assumes a pivotal interest in the search of novel therapeutic targets. Here, we used the yeast three-hybrid system to screen a human liver cDNA library to identify host proteins that interact with the HDV genomic RNA. One of the identified proteins corresponded to the splicing factor SF3B155, a component of the U2snRNP complex that is essential for the early recognition of 3' splice sites in the pre-mRNAs of human genes. We show that the interaction between the HDV genomic RNA and SF3B155 occurs in vivo and that the expression of HDV promotes changes in splicing of human genes whose alternative splicing is SF3B155-dependent. We further show that expression of HDV triggers alterations in several constitutive and alternative splicing events in the tumor suppressor RBM5 transcript, with consequent reduction of its protein levels. This is the first description that HDV expression promotes changes in the splicing of human genes, and we suggest that the HDV-induced alternative splicing changes, through SF3B155 sequester, may contribute for the early progression to hepatocellular carcinoma characteristic of HDV-infected patients.

Diminuição da progressão da miopia com atropina 0, 025% / Decrease of myopia progression with 0. 025% atropine

Resumo Objetivo: Demonstrar a eficácia do uso do colírio de atropina 0,025% em crianças míopes, no Brasil, para a diminuição da progressão da miopia. Métodos: Realizou-se estudo prospectivo em 60 pacientes do Hospital Geral Universitário e Oftalmocenter Santa Rosa - Cuiabá - MT, com idades entre 6 e 12 anos, com equivalente esférico da refração entre -1,00 a -6,00 DE, refração cilíndrica < -1,00 DC e taxa de progressão anual de 0,50 DE (ou maior). Efetuou-se exame oftalmológico geral, topografia corneana e a medida do diâmetro anteroposterior do globo ocular (DAP). Os pacientes foram divididos em dois grupos: em que o Grupo 1 recebeu colírio de atropina 0,025%, todas as noites, e prescreveu-se a refração total com lentes com antirreflexo de multicamadas; e, no Grupo 2, somente a refração total. Nova avaliação foi realizada dois anos após. O teste T Student pareado foi utilizado para comparações das refrações, DAP e ceratometrias, medidas no exame inicial e no exame com 2 anos de seguimento. Resultados: Das 60 crianças, 30 eram do Grupo 1 com idade média de 8,21 ± 1,72 anos, e as do grupo controle com idade média de 8,17 ± 1,73 anos. Quatorze (46,66%) e 16 (53,33%) eram do sexo masculino nos Grupos 1 e 2, respectivamente. O Grupo 1 revelou menor progressão da miopia (Grupo 1: 0,43 ± 0,19D, Grupo 2: 1,24 ± 0,37D) e menor crescimento do DAP em relação ao grupo controle (Grupo 1: 0,19 ± 0,09mm, Grupo 2: 0,48 ± 0,12mm). Houve diferença estatisticamente significativa (P<0,05) entre o grupo tratado e o controle em relação à refração e ao crescimento DAP. A topografia não teve mudança estatisticamente significativa. Conclusão: A atropina em baixas concentrações foi eficaz em diminuir a progressão da miopia em 65% desta população estudada, por 2 anos. No entanto estudos com maior número de participantes e em diversas regiões do Brasil poderiam demonstrar melhor esse fato.

Abstract Purpose: To demonstrate the efficacy of 0.025% atropine eyedrops in myopic children in Brazil for decreasing myopia progression Methods: This was a prospective study with 60 children from Hospital Geral Universitário and Oftalmocenter Santa Rosa in Cuiabá, MT, Brazil, aged between 6 to 12 years, with spherical equivalent refractive error of -1.00 to -6.00 diopters (D) and astigmatism of -1.00 D or smaller. They underwent a complete ophthalmological examination, corneal topography and optical biometry. Children were assigned into two groups: group 1 used 0.025% atropine drop, once-nightly dosing, and it was prescribed total refraction in anti-reflective coating lens; and group 2 was prescribed just total refraction. A new evaluation was conducted 2 years after that. Paired student's t-test was used to compare refractions, axial length and keratometry which were measured in an initial exam and after a two-year follow-up. Results: Of the 60 children, the 30 in group 1 had an age mean and SD 8.21 +/- 1.72, and of the control group were 8.17 +/- 1.73 years. Fourteen (46,66%) and 16 (53,33%) were male, respectively. Myopic progression was significantly lower in group 1 (-0.43 +/- 0.19 D) than in group 2 (-1.24 +/- 0.37 D) and axial length increase was also significantly smaller in group 1(0.19 +/- 0.09 mm) than in group 2 (0.48 +/- 0.12 mm). There were no significant statistical differences regarding keratometry between groups. Conclusions: Low dose atropine eyedrops were effective in decreasing myopia progression in 65% of this population studied for 2 years. Furthermore, a larger scale randomized controlled study with longer follow-up seems warranted.

Correção óptica e evolução da hipermetropia / Optical correction and evolution of hipermetropy

Resumo Objetivo: Comparar as alterações da refração e da biometria ocular na população infantil hipermetrópica com e sem correção óptica total. Métodos: Realizou-se estudo prospectivo longitudinal não randomizado em 41 pacientes com hipermetropia, entre 3 e 6 dioptrias ou/e com esotropia acomodativa pura nos ambulatórios do Hospital Geral Universitário e Oftalmocenter Santa Rosa, com idade inicial entre 4 e 6 anos. Os pacientes foram divididos em dois grupos, em que o Grupo 1 compôs-se pelos pacientes hipermétropes que não necessitavam usar sua correção óptica ou poderiam usá-la parcialmente, e o Grupo 2 por pacientes com esotropia acomodativa pura e pelos hipermétropes que necessitavam usar toda sua correção óptica. Os pacientes submeteram-se a exame oftalmológico completo, incluindo refração objetiva em autorrefrator com cicloplegia, biometria óptica e topografia corneana em uma medida inicial e outra 3 anos mais tarde. Comparou-se a refração e parâmetros biométricos com teste T student. Resultados: A média da idade inicial foi de 5,23 ± 0,81 e 5,36 ± 0,74 anos, a refração inicial foi +3,99 ± 0,92 e +4,27 ± 0,85 D, o diâmetro anteroposterior do globo ocular foi de 21,42 ± 0,84 e 21,22 ± 0,86 mm, e a ceratometria foi de 42,55 ± 1,24 e 42,39 ± 1,22 D, para os Grupos 1 e 2, respectivamente. Em relação à refração, houve redução significativa do poder esférico no Grupo 1, em 3 anos; e não houve no Grupo 2 (p<0,05). Com relação ao diâmetro anteroposterior do globo ocular, ocorreu aumento significativo no Grupo 1 e não houve no Grupo 2 (p<0,05 ). Não se verificou diferença significativa na comparação das ceratometrias em 3 anos nos Grupos 1 e 2. Conclusão: Estes dados permitiram concluir que a correção total da hipermetropia pode prejudicar a emetropização natural em crianças.

Abstract Objective: To compare changes in refraction and ocular biometric parameters in hyperopic children with and without full optical correction. Methods: Non-randomized prospecting study with 41 subjects (21 males and 20 females) aged 4 to 6 years with accommodative esotropia and or hyperopia between 3 to 6 diopters, select in Hospital Geral Universitário and Oftalmocenter Santa Rosa. The patients were divided in two groups: group 1 for hyperopic patients that did not need to use optical correction or could use partial correction, and group 2 for patients with accommodative esotropia or hyperopia who needed to use full optical correction all the time. The patients were examined to a complete ophthalmological examination, including objective cycloplegic refraction with auto refractometer, optical biometry and corneal topography, in baseline measurements and 3 years after that. Refraction and ocular biometric parameters were compared using T student test. Results: The mean initial age was 5.23 ± 0.81 and 5.36 ± 0.74 years; the initial refractive error in average was +3.99 ± 0.92 e +4.27 ± 0.85 D, the initial axial length was 21.42 ± 0.84 and 21.22 ± 0.86 mm, and initial keratometry was 42.55 ± 1.24 e 42.39 ± 1.22 D for group 1 and 2, respectively. In relation to refractive error, there was a significant decrease in group 1 and there was not in group 2 (p < 0.05). In relation to axial length, there was significant increase in group1 and there was not in group 2 (p<0.05). The 3-year comparison showed no statistically significant differences in keratometry for both groups. Conclusion: This study suggests that full optical correction of hyperopia may inhibit natural emmetropization during early and late childhood.

Structural and nucleic acid binding properties of hepatitis delta virus small antigen.

AIM: To further characterize the structure and nucleic acid binding properties of the 195 amino acid small delta antigen, S-HDAg, a study was made of a truncated form of S-HDAg, comprising amino acids 61-195 (∆60HDAg), thus lacking the domain considered necessary for dimerization and higher order multimerization. METHODS: Circular dichroism, and nuclear magnetic resonance experiments were used to assess the structure of ∆60HDAg. Nucleic acid binding properties were investigated by gel retardation assays. RESULTS: Results showed that the truncated ∆60HDAg protein is intrinsically disordered but compact, whereas the RNA binding domain, comprising residues 94-146, adopts a dynamic helical conformation. We also found that ∆60HDAg fails to multimerize but still contains nucleic acid binding activity, indicating that multimerization is not essential for nucleic acid binding. Moreover, in agreement with what has been previously reported for full-length protein, no apparent specificity was found for the truncated protein regarding nucleic acid binding. CONCLUSION: Taken together these results allowed concluding that ∆60HDAg is intrinsically disordered but compact; ∆60HDAg is not a multimer but is still capable of nucleic acid binding albeit without apparent specificity.

Hepatitis delta virus: A fascinating and neglected pathogen.

Hepatitis delta virus (HDV) is the etiologic agent of the most severe form of virus hepatitis in humans. Sharing some structural and functional properties with plant viroids, the HDV RNA contains a single open reading frame coding for the only virus protein, the Delta antigen. A number of unique features, including ribozyme activity, RNA editing, rolling-circle RNA replication, and redirection for a RNA template of host DNA-dependent RNA polymerase II, make this small pathogen an excellent model to study virus-cell interactions and RNA biology. Treatment options for chronic hepatitis Delta are scarce and ineffective. The disease burden is perhaps largely underestimated making the search for new, specific drugs, targets, and treatment strategies an important public health challenge. In this review we address the main features of virus structure, replication, and interaction with the host. Virus pathogenicity and current treatment options are discussed in the light of recent developments.

Distância interpupilar e convergência na população présbita / Interpupillary distance and convergence in the presbyopic population

Objetivo:Determinar as medidas da distância interpupilar (DIP) e convergência ocular na população présbita com pupilômetro de reflexo corneano.Métodos:Cento e sessenta pacientes, com idades entre 41 e 85 anos, sendo 66 do sexo masculino e 94 do sexo feminino, foram avaliados com medida da DIP longe e de perto com PRC.Resultados:A média da idade foi de 55,9 ± 11,5 anos, com DIP média de 65,02 ± 2,78 e 62,47 ± 3,15mm para o sexo masculino e feminino, respectivamente. A média da convergência foi de 5,00 ± 0,5mm. Houve forte correlação direta entre o aumento da DIP e a convergência.Conclusão:Este estudo sugere que a DIP média é pouco menor na população présbita feminina e que existe variação da convergência de acordo com a DIP do paciente.

Objective:To determine the measurements of interpupillary distance (IPD) and ocular convergence in the presbyopic population with corneal reflex pupillometer.Methods:160 subjects (66 males and 94 females) aged 41 to 85 years have their measurements of IPD done - far and near - by CRP method.Results:The mean age was 55.9 ± 11.5 years; the average IPD was 65.02 ± 2.78 and 62.47 ± 3.15 mm for male and female respectively. Also, the average convergence was 5.00 ± 0.5 mm. There was a strong direct correlation between the increase in IPD and convergence.Conclusions:This study suggests that the average IPD is slightly smaller in presbyopic female population, and there is variation in the convergence according to the IPD of the subject.

Detection of Wolbachia in Dirofilaria infected dogs in Portugal.

Wolbachia pipiens, an intracellular endosymbiont bacteria of filarial nematodes, has been implicated in the pathogenesis of filarial diseases, in particular in heavy Dirofilaria spp. infections. Antibiotic therapy (doxycycline) against Wolbachia has been proven to be suitable adjunct therapy, prior to adulticide treatment of canine dirofilariosis. Despite its importance, investigation on the Wolbachia/Dirofilaria complex in Portugal had not been undertaken so far. This study reports the first detection of Wolbachia in Dirofilaria spp. infected dogs in the context of an ongoing epidemiological survey in central-south regions in the country. Wolbachia DNA was detected by PCR in 52.6% (20/38) of canine blood samples positive for Dirofilaria immitis based on parasitological (Knott's and Acid Phosphatase) and serological (Witness(®)Dirofilaria) methods. No Wolbachia DNA could be detected in samples from dogs with occult infections (parasite negative but antigen positive). The lack of Wolbachia detection in some microfilaremic dogs was somewhat unexpected and needs to be elucidated in further studies, as the presence or absence of these bacteria in association with microfilaria is of importance for veterinarians in the management and control of canine dirofilariosis.

HIV-2 integrase polymorphisms and longitudinal genotypic analysis of HIV-2 infected patients failing a raltegravir-containing regimen.

To characterize the HIV-2 integrase gene polymorphisms and the pathways to resistance of HIV-2 patients failing a raltegravir-containing regimen, we studied 63 integrase strand transfer inhibitors (INSTI)-naïve patients, and 10 heavily pretreated patients exhibiting virological failure while receiving a salvage raltegravir-containing regimen. All patients were infected by HIV-2 group A. 61.4% of the integrase residues were conserved, including the catalytic motif residues. No INSTI-major resistance mutations were detected in the virus population from naïve patients, but two amino acids that are secondary resistance mutations to INSTIs in HIV-1 were observed. The 10 raltegravir-experienced patients exhibited resistance mutations via three main genetic pathways: N155H, Q148R, and eventually E92Q - T97A. The 155 pathway was preferentially used (7/10 patients). Other mutations associated to raltegravir resistance in HIV-1 were also observed in our HIV-2 population (V151I and D232N), along with several novel mutations previously unreported. Data retrieved from this study should help build a more robust HIV-2-specific algorithm for the genotypic interpretation of raltegravir resistance, and contribute to improve the clinical monitoring of HIV-2-infected patients.

Support of the infectivity of hepatitis delta virus particles by the envelope proteins of different genotypes of hepatitis B virus.

UNLABELLED: This study examined how the envelope proteins of 25 variants of hepatitis B virus (HBV) genotypes A to I support hepatitis delta virus (HDV) infectivity. The assembled virions bore the same HDV ribonucleoprotein and differed only by the HBV variant-specific envelope proteins coating the particles. The total HDV yields varied within a 122-fold range. A residue Y (position 374) in the HDV binding site was identified as critical for HDV assembly. Virions that bound antibodies, which recognize the region that includes the HBV matrix domain and predominantly but not exclusively immunoprecipitate the PreS1-containing virions, were termed PreS1*-HDVs. Using in vitro infection of primary human hepatocytes (PHH), we measured the specific infectivity (SI), which is the number of HDV genomes/cell produced by infection and normalized by the PreS1*-MOI, which is the multiplicity of infection that reflects the number of PreS1*-HDVs per cell in the inoculum used. The SI values varied within a 160-fold range and indicated a probable HBV genotype-specific trend of D > B > E > A in supporting HDV infectivity. Three variants, of genotypes B, C, and D, supported the highest SI values. We also determined the normalized index (NI) of infected PHH, which is the percentage of HDV-infected hepatocytes normalized by the PreS1*-MOI. Comparison of the SI and NI values revealed that, while a particular HBV variant may facilitate the infection of a relatively significant fraction of PHH, it may not always result in a considerable number of genomes that initiated replication after entry. The potential implications of these findings are discussed in the context of the mechanism of attachment/entry of HBV and HDV. IMPORTANCE: The study advances the understanding of the mechanisms of (i) attachment and entry of HDV and HBV and (ii) transmission of HDV infection/disease.

Envelope proteins derived from naturally integrated hepatitis B virus DNA support assembly and release of infectious hepatitis delta virus particles.

UNLABELLED: A natural subviral agent of human hepatitis B virus (HBV), hepatitis delta virus (HDV), requires only the envelope proteins from HBV in order to maintain persistent infection. HBV surface antigens (HBsAgs) can be produced either by HBV replication or from integrated HBV DNA regardless of replication. The functional properties of the integrant-generated HBsAgs were examined using two human hepatocellular carcinoma-derived cell lines, Hep3B and PLC/PRF/5, that contain HBV integrants but do not produce HBV virions and have no signs of HBV replication. Both cell lines were able to support HDV replication and assembly/egress of HDV virions. Neither of the cell lines was able to produce substantial amounts of the pre-S1-containing HDV particles. HDV virions assembled in PLC/PRF/5 cells were able to infect primary human hepatocytes, while Hep3B-derived HDV appeared to be noninfectious. These results correlate with the findings that the entire open reading frame (ORF) for the large (L) envelope protein that is essential for infectivity is present on HBV RNAs from PLC/PRF/5 cells, while an L protein ORF that was truncated and fused to inverted precore sequences was found using RNAs from Hep3B cells. This study demonstrates for the first time that at least some of the HBV DNA sequence naturally integrated during infection can produce functional small and large envelope proteins capable of the formation of infectious HDV virions. Our data indicate that in vivo chronic HDV infection can persist in the absence of HBV replication (or when HBV replication is profoundly suppressed) if functional envelope proteins are supplied from HBV integrants. IMPORTANCE: The study addresses the unique mechanism of HDV persistence in the absence of ongoing HBV replication, advances our understanding of HDV-HBV interactions, and supports the implementation of treatments directly targeting HDV for HDV/HBV-infected individuals.

Hepatitis delta virus: a peculiar virus.

The hepatitis delta virus (HDV) is distributed worldwide and related to the most severe form of viral hepatitis. HDV is a satellite RNA virus dependent on hepatitis B surface antigens to assemble its envelope and thus form new virions and propagate infection. HDV has a small 1.7 Kb genome making it the smallest known human virus. This deceivingly simple virus has unique biological features and many aspects of its life cycle remain elusive. The present review endeavors to gather the available information on HDV epidemiology and clinical features as well as HDV biology.

Searching for nuclear export elements in hepatitis D virus RNA.

AIM: To search for the presence of cis elements in hepatitis D virus (HDV) genomic and antigenomic RNA capable of promoting nuclear export. METHODS: We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid pDM138. Twenty cDNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pDM138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by real-time polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export. RESULTS: Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter mRNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway. CONCLUSION: A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.