Comparison of four-year toxicities and local control of ultra-hypofractionated vs moderate-hypofractionated image guided prostate radiation with HDR brachytherapy boost: A phase I-II single institution trial.

Purpose/Objectives: To analyze the long term efficacy and safety of an ultra-hypofractionated (UHF) radiation therapy prostate treatment regimen with HDR brachytherapy boost (BB) and compare it to moderate-hypofractionated regimens (MHF). Materials/Methods: In this single arm, prospective monocentric study, 28 patients with intermediate risk prostate cancer were recruited in an experimental treatment arm of 25 Gy in 5 fractions plus a 15 Gy HDR BB. They were then compared to two historical control groups, treated with either 36 Gy in 12 fractions or 37.5 Gy in 15 fractions with a similar HDR BB. The control groups included 151 and 311 patients respectively. Patient outcomes were reported using the International Prostate Symptom Score (IPSS) and Expanded Prostate Index Composite (EPIC-26) questionnaires at baseline and at each follow-up visit. Results: Median follow-up for the experimental arm was 48.5 months compared to 47 months and 60 months compared to the 36/12 and 37,5/15 groups respectively. The IPSS and EPIC scores did not demonstrate any significant differences in the gastrointestinal or genitourinary domains between the three groups over time. No biochemical recurrence occurred in the UHF arm as defined by the Phoenix criterion. Conclusion: The UHF treatment scheme with HDR BB seems equivalent to standard treatment arms in terms of toxicities and local control. Randomized control trials with larger cohorts are ongoing and needed to further confirm our findings.

Development of a Double-Antigen Microsphere Immunoassay for Simultaneous Group and Serotype Detection of Bluetongue Virus Antibodies.

Bluetongue viruses (BTV) are arboviruses responsible for infections in ruminants. The confirmation of BTV infections is based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs) using the BTV viral protein 7 (VP7) as antigen. The determination of the BTV serotype by serological analyses could be only performed by neutralization tests (VNT) which are time-consuming and require BSL3 facilities. VP2 protein is considered the major serotype-defining protein of BTV. To improve the serological characterization of BTV infections, the recombinant VP7 and BTV serotype 8 (BTV-8) VP2 were synthesized using insect cells expression system. The purified antigens were covalently bound to fluorescent beads and then assayed with 822 characterized ruminant sera from BTV vaccinations or infections in a duplex microsphere immunoassay (MIA). The revelation step of this serological duplex assay was performed with biotinylated antigens instead of antispecies conjugates to use it on different ruminant species. The results demonstrated that MIA detected the anti-VP7 antibodies with a high specificity as well as a competitive ELISA approved for BTV diagnosis, with a better efficiency for the early detection of the anti-VP7 antibodies. The VP2 MIA results showed that this technology is also an alternative to VNT for BTV diagnosis. Comparisons between the VP2 MIA and VNT results showed that VNT detects the anti-VP2 antibodies in an early stage and that the VP2 MIA is as specific as VNT. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple BTV serotypes can be detected simultaneously.

Japanese encephalitis in a French traveler to Nepal.

Japanese encephalitis is frequent in Asia, with a severe prognosis, but rare in travelers. Culex mosquitoes transmit Japanese encephalitis virus. Risk factors are destination, duration of stay, summer and fall seasons, outdoor activities, and type of accommodation. We report the case of a French traveler to Nepal with neutralization-based serological confirmed Japanese encephalitis. He presented classical clinical (viral syndrome before an encephalitis status with behavioral disorder, global hypotonia, mutism, movement disorders, seizure, and coma), radiological (lesions of thalami, cortico-spinal tracts, and brainstem) and biological features (lymphocytic meningitis). Nowadays, the presence of Japanese encephalitis virus in Nepal, including mountain areas, is established but Japanese encephalitis remains rare in travelers returning from this area and neurologist physicians need to become familiar with this. We recommend vaccination for travelers spending a long period of time in Nepal and having at-risk outdoor activities.

Genomic characterization of Sebokele virus 1 (SEBV1) reveals a new candidate species among the genus Parechovirus.

We determined the genomic features and the taxonomic classification of Sebokele virus 1 (SEBV1), a previously unclassified arbovirus isolated in 1972 from rodents collected in Botambi, Central African Republic. The complete genome sequence was obtained using a deep sequencing approach (Illumina technology) and dedicated bioinformatics workflows for data analysis. Molecular analysis identified SEBV1 as a picornavirus, most closely related to Ljungan viruses of the genus Parechovirus. The genome has a typical Ljungan virus-like organization, including the presence of two unrelated 2A protein motifs. Phylogenetic analysis confirmed that SEBV1 belongs to the parechovirus phylogroup and was most closely related to the Ljungan virus species. However, it appeared clearly distinct from all members of this phylogroup, suggesting that it represents a novel species of the genus Parechovirus.

Molecular diagnostic and genetic characterization of highly pathogenic viruses: application during Crimean-Congo haemorrhagic fever virus outbreaks in Eastern Europe and the Middle East.

Several haemorrhagic fevers are caused by highly pathogenic viruses that must be handled in Biosafety level 4 (BSL-4) containment. These zoonotic infections have an important impact on public health and the development of a rapid and differential diagnosis in case of outbreak in risk areas represents a critical priority. We have demonstrated the potential of a DNA resequencing microarray (PathogenID v2.0) for this purpose. The microarray was first validated in vitro using supernatants of cells infected with prototype strains from five different families of BSL-4 viruses (e.g. families Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae and Paramyxoviridae). RNA was amplified based on isothermal amplification by Phi29 polymerase before hybridization. We were able to detect and characterize Nipah virus and Crimean-Congo haemorrhagic fever virus (CCHFV) in the brains of experimentally infected animals. CCHFV was finally used as a paradigm for epidemics because of recent outbreaks in Turkey, Kosovo and Iran. Viral variants present in human sera were characterized by BLASTN analysis. Sensitivity was estimated to be 10(5) -10(6) PFU/mL of hybridized cDNA. Detection specificity was limited to viral sequences having ~13-14% of global divergence with the tiled sequence, or stretches of ~20 identical nucleotides. These results highlight the benefits of using the PathogenID v2.0 resequencing microarray to characterize geographical variants in the follow-up of haemorrhagic fever epidemics; to manage patients and protect communities; and in cases of bioterrorism.

Resequencing microarray method for molecular diagnosis of human arboviral diseases.

BACKGROUND: Resequencing DNA microarray (RMA) technology uses probes designed to identify a panel of viral sequences. It can be used for detecting emerging viruses by revealing the nucleotide polymorphisms within the target of interest. OBJECTIVES/STUDY DESIGN: As a new tool for molecular diagnosis of arbovirus infection, high density PathogenID v2.0 RMA (PID2-RMA) was assessed for the detection and genetic analysis of dengue, West Nile, and Chikungunya viruses in spiked blood samples or sera from individuals infected with dengue virus. Viral RNAs extracted from biological samples were retrotranscribed into cDNA and amplified using the Phi 29 polymerase-based method. This amplified cDNA was used for hybridization on PID2-RMA. RESULTS: A good specificity of RMA-based detection was demonstrated using a panel of arboviruses including Dengue, West Nile and Chikungunya viruses. This technology was also efficient for the detection and genetic analysis of the different serotypes of dengue virus in sera of infected patients. Furthermore, the mixing of dengue, West Nile and Chikungunya prototype viruses within a single sample of human blood did not interfere with the sensitivity of PID2-RMA. CONCLUSIONS: Our data show that high density PID2-RMA was suitable for the identification of medically important arboviruses. It appears to be particularly adapted to the genetic analysis of dengue, West Nile, and Chikungunya viruses in urgent clinical situations where the rapid identification and characterization of the pathogen is essential.

Sci-Sat AM: Brachy - 07: Plastic scintillation detector validation for kV dosimetry.

PURPOSE: To characterize the plastic scintillation detectors (PSDs) response in the diagnostic energy range. A fast and adaptable method for real-time dosimetry in superficial x-ray therapy and interventional radiology is proposed. METHOD: A PSD (1 mm diameter and 10 mm long) is coupled to a 5 m long optical fiber. Scintillation photons are guided to a polychromatic photodiode which provides an electrical current proportional to the input light signal. If the incident energy spectrum is known, the dose measured in the PSD's polystyrene sensitive volume can be converted to score dose in any other media such as air, water or soft tissues using the large cavity theory (LCT). A software simulating x-ray tube spectra and filtration has been benchmarked and is used for analysis. The method is confirmed by Monte Carlo simulations. RESULTS: PSDs cannot be assumed energy independent with low-energy photons as a factor 2 has been observed in the energy response between 80 kVp and 150 kVp. When the dose is converted to the desired medium, the PSD's energy dependence is compensated and a 2.1% standard deviation was observed upon the studied energy ranges, which is inside the measurement and calculation uncertainties. Percent depth dose (PDD) measurements are in good agreement with Monte Carlo simulations and results can be improved if the proposed method is applied to compensate beam hardening. CONCLUSION: PSDs present great potential for real-time dose measurements with radiologic photon energy.

Fast dose calculation in magnetic fields with GPUMCD.

A new hybrid imaging-treatment modality, the MRI-Linac, involves the irradiation of the patient in the presence of a strong magnetic field. This field acts on the charged particles, responsible for depositing dose, through the Lorentz force. These conditions require a dose calculation engine capable of taking into consideration the effect of the magnetic field on the dose distribution during the planning stage. Also in the case of a change in anatomy at the time of treatment, a fast online replanning tool is desirable. It is improbable that analytical solutions such as pencil beam calculations can be efficiently adapted for dose calculations within a magnetic field. Monte Carlo simulations have therefore been used for the computations but the calculation speed is generally too slow to allow online replanning. In this work, GPUMCD, a fast graphics processing unit (GPU)-based Monte Carlo dose calculation platform, was benchmarked with a new feature that allows dose calculations within a magnetic field. As a proof of concept, this new feature is validated against experimental measurements. GPUMCD was found to accurately reproduce experimental dose distributions according to a 2%-2 mm gamma analysis in two cases with large magnetic field-induced dose effects: a depth-dose phantom with an air cavity and a lateral-dose phantom surrounded by air. Furthermore, execution times of less than 15 s were achieved for one beam in a prostate case phantom for a 2% statistical uncertainty while less than 20 s were required for a seven-beam plan. These results indicate that GPUMCD is an interesting candidate, being fast and accurate, for dose calculations for the hybrid MRI-Linac modality.

First two autochthonous dengue virus infections in metropolitan France, September 2010.

In September 2010, two cases of autochthonous dengue fever were diagnosed in metropolitan France for the first time. The cases occurring in Nice, southeast France, where Aedes albopictus is established, are evidence of dengue virus circulation in this area. This local transmission of dengue calls for further enhanced surveillance, active case finding and vector control measures to reduce the spread of the virus and the risk of an epidemic.

[Live attenuated measles vaccine as a potential multivalent pediatric vaccination vector]. / Utilisation potentielle du vaccin vivant atténué contre la rougeole comme vecteur de vaccination pédiatrique multivalent.

Live attenuated RNA viruses make highly efficient vaccines. Among them, measles virus (MV) vaccine has been given to a very large number of children and shown to be highly efficacious and safe. MV vaccine induces a life-long immunity after a single or two low-dose injections. It is easily produced on a large scale in most countries and can be distributed at low cost. Reversion to pathogenicity has never been observed with this vaccine. For all these characteristics, MV vaccine might be a very promising vector to immunize children against both measles and other infectious agents such as HIV or flaviviruses, in the developing world. In this article, we describe recent data demonstrating the capacity of recombinant Schwarz measles virus to express proteins from Human Immunodeficiency or West Nile viruses, and to induce specific immune responses able, in the case of West Nile virus, to protect from an experimental challenge.

Variations in biological features of West Nile viruses.

Pathological findings in humans, horses, and birds with West Nile (WN) encephalitis show neuronal degeneration and necrosis in the central nervous system (CNS), with diffuse inflammation. The mechanisms of WN viral penetration of the CNS and pathophysiology of the encephalitis remain largely unknown. Since 1996, several epizootics involving hundreds of humans, horses, and thousands of wild and domestic bird cases of encephalitis and mortality have been reported in Europe, North Africa, the Middle East, Russia, and the USA (see specific chapters in this issue). However, biological and molecular markers of virus virulence should be characterized to assess whether novel strains with increased virulence are responsible for this recent proliferation of outbreaks.

Determinants in the envelope E protein and viral RNA helicase NS3 that influence the induction of apoptosis in response to infection with dengue type 1 virus.

One mechanism by which dengue (DEN) virus may cause cell death is apoptosis. In this study, we investigated whether the genetic determinants responsible for acquisition by DEN type 1 (DEN-1) virus of mouse neurovirulence interfere with the induction of apoptosis. Neurovirulent variant FGA/NA d1d was generated during the adaptation of the human isolate of DEN-1 virus strain FGA/89 to grow in newborn mouse brains and mosquito cells in vitro [Desprès, P. Frenkiel, M. -P. Ceccaldi, P.-E. Duarte Dos Santos, C. and Deubel, V. (1998) J. Virol., 72: 823-829]. Genetic determinants possibly responsible for mouse neurovirulence were studied by sequencing the entire genomes of both DEN-1 viruses. Three amino acid differences in the envelope E protein and one in the nonstructural NS3 protein were found. The cytotoxicity of the mouse-neurovirulent DEN-1 variant was studied in different target cells in vitro and compared with the parental strain. FGA/NA d1d was more pathogenic for mouse neuroblastoma cells and attenuated for human hepatoma cells. Changes in virus replicative functions and virus assembly may account, in a large part, for the differences in the induction of apoptosis. Our data suggest that identified amino acid substitutions in the envelope E protein and viral RNA helicase NS3 may influence DEN-1 virus pathogenicity by altering viral growth.

Alpha-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum.

We report that endoplasmic reticulum alpha-glucosidase inhibitors have antiviral effects on dengue (DEN) virus. We found that glucosidase inhibition strongly affects productive folding pathways of the envelope glycoproteins prM (the intracellular glycosylated precursor of M [membrane protein]) and E (envelope protein): the proper folding of prM bearing unprocessed N-linked oligosaccharide is inefficient, and this causes delayed formation of prME heterodimer. The complexes formed between incompletely folded prM and E appear to be unstable, leading to a nonproductive pathway. Inhibition of alpha-glucosidase-mediated N-linked oligosaccharide trimming may thus prevent the assembly of DEN virus by affecting the early stages of envelope glycoprotein processing.

Recurrence of the T666M calcium channel CACNA1A gene mutation in familial hemiplegic migraine with progressive cerebellar ataxia.

Familial hemiplegic migraine (HM) is an autosomal dominant migraine with aura. In 20% of HM families, HM is associated with a mild permanent cerebellar ataxia (PCA). The CACNA1A gene encoding the alpha1A subunit of P/Q-type voltage-gated calcium channels is involved in 50% of unselected HM families and in all families with HM/PCA. Four CACNA1A missense mutations have been identified in HM: two in pure HM and two in HM/PCA. Different CACNA1A mutations have been identified in other autosomal dominant conditions: mutations leading to a truncated protein in episodic ataxia type 2 (EA2), small expansions of a CAG trinucleotide in spinocerebellar ataxia type 6 and also in three families with EA2 features, and, finally, a missense mutation in a single family suffering from episodic ataxia and severe progressive PCA. We screened 16 families and 3 nonfamilial case patients affected by HM/PCA for specific CACNA1A mutations and found nine families and one nonfamilial case with the same T666M mutation, one new mutation (D715E) in one family, and no CAG repeat expansion. Both T666M and D715E substitutions were absent in 12 probands belonging to pure HM families whose disease appears to be linked to CACNA1A. Finally, haplotyping with neighboring markers suggested that T666M arose through recurrent mutational events. These data could indicate that the PCA observed in 20% of HM families results from specific pathophysiologic mechanisms.

[Recent knowledge on the pathogenesis of yellow fever and questions for the future]. / Connaissances récentes sur la pathogénie de la fièvre jaune et questions pour le futur.

Yellow fever is an arthropod-borne disease with symptoms ranging from mild fever to acute hepatonephritis, hemorrhages and shock often fatal. The pathophysiology of severe yellow fever in humans and in monkeys susceptible to the virus is largely unknown. Yellow fever virus replicates in Küpffer cells and in hepatocytes in the liver. The degree of severity in yellow fever disease is linked to different factors related to virus virulence and to host susceptibility. A better knowledge of the complex interactions between the virus and the host is requested before initiating new actions in prophylaxy and therapy against yellow fever.

[Apoptotic cell death in response to dengue virus infection: what are the consequences of viral pathogenesis?]. / La mort cellulaire par apoptose en réponse à l'infection par le virus de la dengue: quelles conséquences dans la pathogénie virale?

Dengue is a human disease of viral etiology which may be fatal in its hemorragic form. It is widely spread in the tropical areas of the different continents and has been dramatically expanding over the past 30 years. Although an immunological disorder is thought to be involved in dengue physiological symptoms, the pathogenesis of dengue hemorragic fever has not yet been elucidated. Whether the immune response is deleterious or beneficial to the host remains a matter of debate. Other factors, related to virus replication in specific host cells, could also contribute to the severity of the disease. Apoptotic cell death is one of the important consequences of dengue virus infection both in vitro and in vivo. Dengue replication triggers apoptotic signals in neurons and hepatocytes although the original effectors and kinetics differ. Implications of the ongoing apoptotic processes in viral pathogenesis will be further discussed.

Apoptotic cell death in response to dengue virus infection: the pathogenesis of dengue haemorrhagic fever revisited.

BACKGROUND: Dengue virus infection may be asymptomatic or lead to undifferentiated febrile illness or dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS). The major clinical manifestations of DHF/DSS are high fever, haemorrhage, hepatomegaly and circulatory failure. OBJECTIVES: The relatively high level of viraemia only a few days after infection may reflect a large number of replication sites. However, the degree of cell injury in fatal cases of DHF/DSS is not sufficient to explain death and suggests metabolic disturbance rather than tissue destruction. This theory was investigated in this study. RESULTS: We demonstrated that replication of dengue virus in infected cells induces stress leading to apoptotic cell death in vitro and in vivo. CONCLUSIONS: The elimination of apoptotic bodies by phagocytic cells is a previously unsuspected pathway of dengue virus clearance from infected tissues. However, the mechanisms of host defence involving apoptosis and phagocytic cell activation may cause local tissue injury or transient homeostasis imbalance and may trigger further deleterious events.