Blocking NS3-NS4B interaction inhibits dengue virus in non-human primates.

Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

Transcription factor expression repertoire basis for epigenetic and transcriptional subtypes of colorectal cancers.

Colorectal cancers (CRCs) form a heterogenous group classified into epigenetic and transcriptional subtypes. The basis for the epigenetic subtypes, exemplified by varying degrees of promoter DNA hypermethylation, and its relation to the transcriptional subtypes is not well understood. We link cancer-specific transcription factor (TF) expression alterations to methylation alterations near TF-binding sites at promoter and enhancer regions in CRCs and their premalignant precursor lesions to provide mechanistic insights into the origins and evolution of the CRC molecular subtypes. A gradient of TF expression changes forms a basis for the subtypes of abnormal DNA methylation, termed CpG-island promoter DNA methylation phenotypes (CIMPs), in CRCs and other cancers. CIMP is tightly correlated with cancer-specific hypermethylation at enhancers, which we term CpG-enhancer methylation phenotype (CEMP). Coordinated promoter and enhancer methylation appears to be driven by downregulation of TFs with common binding sites at the hypermethylated enhancers and promoters. The altered expression of TFs related to hypermethylator subtypes occurs early during CRC development, detectable in premalignant adenomas. TF-based profiling further identifies patients with worse overall survival. Importantly, altered expression of these TFs discriminates the transcriptome-based consensus molecular subtypes (CMS), thus providing a common basis for CIMP and CMS subtypes.

Bioassay Development for Ultrasensitive Detection of Influenza A Nucleoprotein Using Digital ELISA.

Flu is caused by the influenza virus that, due to mutations, keeps our body vulnerable for infections, making early diagnosis essential. Although immuno-based diagnostic tests are available, they have low sensitivity and reproducibility. In this paper, the prospect of detecting influenza A virus using digital ELISA has been studied. To appropriately select bioreceptors for this bioassay, seven commercial antibodies against influenza A nucleoprotein were methodically tested for their reactivity and binding affinity. The study has been performed on two markedly different platforms, being an enzyme-linked immunosorbent assay and a surface plasmon resonance system. The selected antibodies displayed completely different behavior on the two platforms and in various assay configurations. Surprisingly, the antibodies that showed overall good reactivity on both platforms had the highest dissociation constant among the tested antibodies, suggesting that, although important, binding affinity is not the only parameter to be considered when selecting antibodies. Moreover, only one antibody had the capacity to capture the nucleoprotein directly in lysis buffer used for releasing this viral protein, which might pose a huge advantage when developing assays with a fast time-to-result. This antibody was implemented on an in-house developed digital ELISA platform for ultrasensitive detection of recombinant nucleoprotein, reaching a detection limit of 4 ± 1 fM in buffer and 10 ± 2 fM in 10-fold diluted nasopharyngeal swabs, which is comparable to currently available fast molecular detection techniques. These results point to a great potential for ultrasensitive immuno-based influenza detection.

Sampling variability between two mid-turbinate swabs of the same patient has implications for influenza viral load monitoring.

BACKGROUND: With the clinical development of several antiviral intervention strategies for influenza, it becomes crucial to explore viral load shedding in the nasal cavity as a biomarker for treatment success, but also to explore sampling strategies for sensible and reliable virus collection. FINDINGS: In this study, 244 patients suffering from Influenza like Illness and/or acute respiratory tract infection were enrolled. Sampling was done using mid-turbinate flocked swabs and two swabs per patient were collected (one swab per nostril). The influenza A viral loads of two mid-turbinate flocked swabs (one for each nostril) per patient were compared and we have also assessed whether normalization for human cellular DNA in the swabs could be useful. The Influenza mid-turbinate nasal swab testing resulted in considerable sampling variability that could not be normalized against co-isolated human cellular DNA. CONCLUSIONS: Influenza viral load monitoring in nasal swabs could be very valuable as virological endpoints in clinical trials to monitor treatment efficacy, in analogy to HIV, HBV & HCV viral load monitoring. However, the differences between left and right nostrils, as observed in this study, highlight the importance of proper sampling and the need for standardized sampling procedures.

Comparison of the FilmArray RP, Verigene RV+, and Prodesse ProFLU+/FAST+ multiplex platforms for detection of influenza viruses in clinical samples from the 2011-2012 influenza season in Belgium.

Respiratory tract infections (RTIs) are caused by a plethora of viral and bacterial pathogens. In particular, lower RTIs are a leading cause of hospitalization and mortality. Timely detection of the infecting respiratory pathogens is crucial to optimize treatment and care. In this study, three U.S. Food and Drug Administration-approved molecular multiplex platforms (Prodesse ProFLU+/FAST+, FilmArray RP, and Verigene RV+) were evaluated for influenza virus detection in 171 clinical samples collected during the Belgian 2011-2012 influenza season. Sampling was done using mid-turbinate flocked swabs, and the collected samples were stored in universal transport medium. The amount of viral RNA present in the swab samples ranged between 3.07 and 8.82 log10 copies/ml. Sixty samples were concordant influenza A virus positive, and 8 samples were found to be concordant influenza B virus positive. Other respiratory viruses that were detected included human rhinovirus/enterovirus, respiratory syncytial virus, parainfluenza virus type 1, human metapneumovirus, and coronavirus NL63. Twenty-five samples yielded discordant results across the various assays which required further characterization by sequencing. The FilmArray RP and Prodesse ProFLU+/FAST+ assays were convenient to perform with regard to sensitivity, ease of use, and low percentages of invalid results. Although the limit of sensitivity is of utmost importance, many other factors should be taken into account in selecting the most convenient molecular diagnostic assay for the detection of respiratory pathogens in clinical samples.

Quantitative prediction of integrase inhibitor resistance from genotype through consensus linear regression modeling.

BACKGROUND: Integrase inhibitors (INI) form a new drug class in the treatment of HIV-1 patients. We developed a linear regression modeling approach to make a quantitative raltegravir (RAL) resistance phenotype prediction, as Fold Change in IC50 against a wild type virus, from mutations in the integrase genotype. METHODS: We developed a clonal genotype-phenotype database with 991 clones from 153 clinical isolates of INI naïve and RAL treated patients, and 28 site-directed mutants.We did the development of the RAL linear regression model in two stages, employing a genetic algorithm (GA) to select integrase mutations by consensus. First, we ran multiple GAs to generate first order linear regression models (GA models) that were stochastically optimized to reach a goal R2 accuracy, and consisted of a fixed-length subset of integrase mutations to estimate INI resistance. Secondly, we derived a consensus linear regression model in a forward stepwise regression procedure, considering integrase mutations or mutation pairs by descending prevalence in the GA models. RESULTS: The most frequently occurring mutations in the GA models were 92Q, 97A, 143R and 155H (all 100%), 143G (90%), 148H/R (89%), 148K (88%), 151I (81%), 121Y (75%), 143C (72%), and 74M (69%). The RAL second order model contained 30 single mutations and five mutation pairs (p < 0.01): 143C/R&97A, 155H&97A/151I and 74M&151I. The R2 performance of this model on the clonal training data was 0.97, and 0.78 on an unseen population genotype-phenotype dataset of 171 clinical isolates from RAL treated and INI naïve patients. CONCLUSIONS: We describe a systematic approach to derive a model for predicting INI resistance from a limited amount of clonal samples. Our RAL second order model is made available as an Additional file for calculating a resistance phenotype as the sum of integrase mutations and mutation pairs.

Study of genotypic and phenotypic HIV-1 dynamics of integrase mutations during raltegravir treatment: a refined analysis by ultra-deep 454 pyrosequencing.

BACKGROUND: The dynamics of raltegravir-resistant variants and their impact on virologic response in 23 HIV-1-infected patients, who started a salvage raltegravir-containing regimen, were investigated. METHODS: Integrase population sequencing and Ultra-Deep-454 Pyrosequencing (UDPS) were performed on plasma samples at baseline and at raltegravir failure. All integrase mutations detected at a frequency ≥1% were considered to be reliable for the UDPS analyses. Phylogenetic and phenotypic resistance analyses were also performed. RESULTS: At baseline, primary resistance mutations were not detected by both population and UDPS genotypic assays; few secondary mutations (T97A-V151I-G163R) were rarely detected and did not show any statistically association either with virologic response at 24-weeks or with the development of resistant variants at failure. At UDPS, not all resistant variants appearing early during treatment evolved as major populations during failure; only specific resistance pathways (Y143R-Q148H/R-N155H) associated with an increased rate of fitness and phenotypic resistance were selected. CONCLUSIONS: Resistance to raltegravir in integrase strand transfer inhibitor-naive patients remains today a rare event, which might be changed by future extensive use of such drugs. In our study, pathways of resistance at failure were not predicted by baseline mutations, suggesting that evolution plus stochastic selection plays a major role in the appearance of integrase-resistance mutations, whereas fitness and resistance are dominant factors acting for the late selection of resistant quasispecies.

A phylogenetic study of dengue virus in urban Vietnam shows long-term persistence of endemic strains.

Dengue virus (DENV) causes repeated outbreaks of disease in endemic areas, with patterns of local transmission strongly influenced by seasonality, importation via human movement, immunity, and vector control efforts. An understanding of how each of these interacts to enable endemic transmission (continual circulation of local virus strains) is largely unknown. There are times of the year when no cases are reported, often for extended periods of time, perhaps wrongly implying the successful eradication of a local strain from that area. Individuals who presented at a clinic or hospital in four communes in Nha Trang, Vietnam, were initially tested for DENV antigen presence. Enrolled positive individuals then had their corresponding household members invited to participate, and those who enrolled were tested for DENV. The presence of viral nucleic acid in all samples was confirmed using quantitative polymerase chain reaction, and positive samples were then whole-genome sequenced using an amplicon and target enrichment library preparation techniques and Illumina MiSeq sequencing technology. Generated consensus genome sequences were then analysed using phylogenetic tree reconstruction to categorise sequences into clades with a common ancestor, enabling investigations of both viral clade persistence and introductions. Hypothetical introduction dates were additionally assessed using a molecular clock model that calculated the time to the most recent common ancestor (TMRCA). We obtained 511 DENV whole-genome sequences covering four serotypes and more than ten distinct viral clades. For five of these clades, we had sufficient data to show that the same viral lineage persisted for at least several months. We noted that some clades persisted longer than others during the sampling time, and by comparison with other published sequences from elsewhere in Vietnam and around the world, we saw that at least two different viral lineages were introduced into the population during the study period (April 2017-2019). Next, by inferring the TMRCA from the construction of molecular clock phylogenies, we predicted that two of the viral lineages had been present in the study population for over a decade. We observed five viral lineages co-circulating in Nha Trang from three DENV serotypes, with two likely to have remained as uninterrupted transmission chains for a decade. This suggests clade cryptic persistence in the area, even during periods of low reported incidence.

The neurology of carbonic anhydrase type II deficiency syndrome.

Carbonic anhydrase type II deficiency syndrome is an uncommon autosomal recessive disease with cardinal features including osteopetrosis, renal tubular acidosis and brain calcifications. We describe the neurological, neuro-ophthalmological and neuroradiological features of 23 individuals (10 males, 13 females; ages at final examination 2-29 years) from 10 unrelated consanguineous families with carbonic anhydrase type II deficiency syndrome due to homozygous intron 2 splice site mutation (the 'Arabic mutation'). All patients had osteopetrosis, renal tubular acidosis, developmental delay, short stature and craniofacial disproportion with large cranial vault and broad forehead. Mental retardation was present in approximately two-thirds and varied from mild to severe. General neurological examinations were unremarkable except for one patient with brisk deep tendon reflexes and two with severe mental retardation and spastic quadriparesis. Globes and retinae were normal, but optic nerve involvement was present in 23/46 eyes and was variable in severity, random in occurrence and statistically correlated with degree of optic canal narrowing. Ocular motility was full except for partial ductional limitations in two individuals. Saccadic abnormalities were present in two, while half of these patients had sensory or accommodative strabismus, and seven had congenital nystagmus. These abnormalities were most commonly associated with afferent disturbances, but a minor brainstem component to this disorder remains possible. All internal auditory canals were normal in size, and no patient had clinically significant hearing loss. Neuroimaging was performed in 18 patients and repeated over as long as 10 years. Brain calcification was generally progressive and followed a distinct distribution, involving predominantly basal ganglia and thalami and grey-white matter junction in frontal regions more than posterior regions. At least one child had no brain calcification at age 9 years, indicating that brain calcification may not always be present in carbonic anhydrase type II deficiency syndrome during childhood. Variability of brain calcification, cognitive disturbance and optic nerve involvement may imply additional genetic or epigenetic influences affecting the course of the disease. However, the overall phenotype of the disorder in this group of patients was somewhat less severe than reported previously, raising the possibility that early treatment of systemic acidosis with bicarbonate may be crucial in the outcome of this uncommon autosomal recessive problem.

Evolution of inflammation and immunity in a dengue virus 1 human infection model.

Dengue virus (DENV) infections are major causes of morbidity and mortality throughout the tropics and subtropics. More than 400 million infections are estimated to occur every year, resulting in nearly 100 million symptomatic infections and more than 20,000 deaths. Early immune response kinetics to infection remain unclear, in large part due to the variable incubation period exhibited by the DENVs after introduction into a susceptible host. To fill this knowledge gap, we performed a comprehensive virologic and immunologic analysis of individuals experimentally infected with the underattenuated DENV-1 strain 45AZ5. This analysis captured both the kinetics and composition of the innate, humoral, and cellular immune responses elicited by experimental DENV-1 infection, as well as virologic and clinical features. We observed a robust DENV-specific immunoglobulin A (IgA) antibody response that manifested between the appearance of DENV-specific IgM and IgG in all challenged individuals, as well as the presence of a non-neutralizing/NS1-specific antibody response that was delayed relative to the appearance of DENV virion-specific humoral immunity. RNA sequencing analysis revealed discrete and temporally restricted gene modules that correlated with acute viremia and the induction of adaptive immunity. Our analysis provides a detailed description, in time and space, of the evolving matrix of DENV-elicited human inflammation and immunity and reveals several previously unappreciated immunological aspects of primary DENV-1 infection that can inform countermeasure development and evaluation.

Combining rapid diagnostic tests to estimate primary and post-primary dengue immune status at the point of care.

BACKGROUND: Characterising dengue virus (DENV) infection history at the point of care is challenging as it relies on intensive laboratory techniques. We investigated how combining different rapid diagnostic tests (RDTs) can be used to accurately determine the primary and post-primary DENV immune status of reporting patients during diagnosis. METHODS AND FINDINGS: Serum from cross-sectional surveys of acute suspected dengue patients in Indonesia (N:200) and Vietnam (N: 1,217) were assayed using dengue laboratory assays and RDTs. Using logistic regression modelling, we determined the probability of being DENV NS1, IgM and IgG RDT positive according to corresponding laboratory viremia, IgM and IgG ELISA metrics. Laboratory test thresholds for RDT positivity/negativity were calculated using Youden's J index and were utilized to estimate the RDT outcomes in patients from the Philippines, where only data for viremia, IgM and IgG were available (N:28,326). Lastly, the probabilities of being primary or post-primary according to every outcome using all RDTs, by day of fever, were calculated. Combining NS1, IgM and IgG RDTs captured 94.6% (52/55) and 95.4% (104/109) of laboratory-confirmed primary and post-primary DENV cases, respectively, during the first 5 days of fever. Laboratory test predicted, and actual, RDT outcomes had high agreement (79.5% (159/200)). Among patients from the Philippines, different combinations of estimated RDT outcomes were indicative of post-primary and primary immune status. Overall, IgG RDT positive results were confirmatory of post-primary infections. In contrast, IgG RDT negative results were suggestive of both primary and post-primary infections on days 1-2 of fever, yet were confirmatory of primary infections on days 3-5 of fever. CONCLUSION: We demonstrate how the primary and post-primary DENV immune status of reporting patients can be estimated at the point of care by combining NS1, IgM and IgG RDTs and considering the days since symptoms onset. This framework has the potential to strengthen surveillance operations and dengue prognosis, particularly in low resource settings.

Secondary integrase resistance mutations found in HIV-1 minority quasispecies in integrase therapy-naive patients have little or no effect on susceptibility to integrase inhibitors.

The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.

Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans.

This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

HIV-1 resistance patterns to integrase inhibitors in antiretroviral-experienced patients with virological failure on raltegravir-containing regimens.

BACKGROUND: Our aim was to study the in vivo viral genetic pathways for resistance to raltegravir, in antiretroviral-experienced patients with virological failure (VF) on raltegravir-containing regimens. METHODS: We set up a prospective study including antiretroviral-experienced patients receiving raltegravir-based regimens. Integrase (IN) genotypic resistance analysis was performed at baseline. IN was also sequenced at follow-up points in the case of VF, i.e. plasma HIV-1 RNA>400 copies/mL at month 3 and/or >50 copies/mL at month 6. For phenotyping, the IN region was recombined with an IN-deleted HXB2-based HIV-1 backbone. A titrated amount of IN recombinant viruses was used for antiviral testing against raltegravir and elvitegravir. RESULTS: Among 51 patients, 11 (21.6%) had VF. Four different patterns of IN mutations were observed: (i) emergence of Q148H/R with secondary mutations (n=5 patients); (ii) emergence of N155H, then replaced by a pattern including Y143C/H/R (n=3); (iii) selection of S230N (n=1); and (iv) no evidence of selection of IN mutations (n=2). The median raltegravir and elvitegravir fold changes (FCs) were 244 (154-647) and 793 (339-892), respectively, for the Q148H/R pattern, while the median raltegravir and elvitegravir FCs were 21 (6-52) and 3 (2-3), respectively, with Y143C/H/R. The median plasma raltegravir Cmin was lower in patients with selection of the N155H mutation followed by Y143C/H/R compared with patients with Q148H/R and with patients without emerging mutations or without VF. CONCLUSIONS: Diverse genetic profiles can be associated with VF on raltegravir-containing regimens, including the dynamics of replacement of mutational profiles. Pharmacokinetic parameters could be involved in this genetic evolution.

Droplet digital PCR is an accurate method to assess methylation status on FFPE samples.

Most tissue samples available for cancer research are archived as formalin-fixed paraffin-embedded (FFPE) samples. However, the fixation process and the long storage duration lead to DNA fragmentation and hinder epigenome analysis. The use of droplet digital PCR (ddPCR) to detect DNA methylation has recently emerged. In this study, we compare an optimized ddPCR assay with a conventional qPCR assay by targeting a dilution series of control DNA. In addition, we compare the ddPCR technology with results from Infinium arrays targeting two separate CpG sites on a set of colon adenoma FFPE samples. Our data demonstrate that qPCR and ddPCR assess methylation status equally well on dilution controls with a high DNA input. However, the methylation detection on low-input samples is more accurate using ddPCR. The proposed primer design (methylation-independent primers with amplification of solely the converted DNA target) will allow for methylation detection, independent of bisulfite conversion efficiency. Those data show that ddPCR can be used for methylation analysis on FFPE samples with a wide range of DNA input and that the precision of the assay depends largely on the total amount of amplifiable DNA fragments. Due to accessibility of the ddPCR technology and its accuracy on high- as well as low-DNA input samples, we propose the use of this approach for studies involving degraded FFPE samples.

Sampling and detection of airborne influenza virus towards point-of-care applications.

Airborne transmission of the influenza virus contributes significantly to the spread of this infectious pathogen, particularly over large distances when carried by aerosol droplets with long survival times. Efficient sampling of virus-loaded aerosol in combination with a low limit of detection of the collected virus could enable rapid and early detection of airborne influenza virus at the point-of-care setting. Here, we demonstrate a successful sampling and detection of airborne influenza virus using a system specifically developed for such applications. Our system consists of a custom-made electrostatic precipitation (ESP)-based bioaerosol sampler that is coupled with downstream quantitative polymerase chain reaction (qPCR) analysis. Aerosolized viruses are sampled directly into a miniaturized collector with liquid volume of 150 µL, which constitutes a simple and direct interface with subsequent biological assays. This approach reduces sample dilution by at least one order of magnitude when compared to other liquid-based aerosol bio-samplers. Performance of our ESP-based sampler was evaluated using influenza virus-loaded sub-micron aerosols generated from both cultured and clinical samples. Despite the miniaturized collection volume, we demonstrate a collection efficiency of at least 10% and sensitive detection of a minimum of 3721 RNA copies. Furthermore, we show that an improved extraction protocol can allow viral recovery of down to 303 RNA copies and a maximum sampler collection efficiency of 47%. A device with such a performance would reduce sampling times dramatically, from a few hours with current sampling methods down to a couple of minutes with our ESP-based bioaerosol sampler.

Lessons from osteopetrotic mutations in animals: impact on our current understanding of osteoclast biology.

Throughout life, bone tissue is in a constant state of turnover. This process of bone remodeling is the result of a combination of sequential removal of bone tissue by osteoclasts and new bone deposition by osteoblasts. The osteopetroses are a heterogeneous group of skeletal disorders characterized by a generalized increase in bone mass caused by decreased bone resorption. Over the past decade, major contributions to our current knowledge on bone resorption have been made by studies of osteopetrotic mutations in animals. A considerable heterogeneity among the various osteopetrotic animals is observed, showing that a multiplicity of mutations may cause osteopetrosis. This review focuses on the spontaneous and experimentally induced osteopetrotic mutations in animals. We will discuss their impact on our current understanding of osteoclast biology and we will correlate, when possible, the animal models of osteopetroses to diseases in humans.

A Downward Trend of the Ratio of Influenza RNA Copy Number to Infectious Viral Titer in Hospitalized Influenza A-Infected Patients.

Background. Efficacy endpoints in influenza clinical trials may include clinical symptoms and virological measurements, although virology cannot serve as the primary endpoint. We investigated the relationship between influenza A RNA copy number and quantity of infectious viruses in hospitalized influenza patients. Methods. One hundred fifty influenza-infected, hospitalized patients were included in this prospective cohort study spanning the 2012-2013 influenza season. Daily nasopharyngeal samples were collected during hospitalization, and influenza A RNA copy number and infectious viral titer were monitored. Results. The decay rate for 50% tissue culture infectious dose (TCID50) was 0.51 ± 0.14 log10 TCID50/mL per day, whereas the RNA copy number decreased at a rate of 0.41 ± 0.04 log10 copies/mL per day (n = 433). The log ratio of the RNA copy number to the infectious viral titer within patient changes significantly with -0.25 ± 0.09 units per day (P = .0069). For a 12-day observation period, the decay corresponds to a decline of this ratio of 3 log influenza RNA copies. Conclusions. Influenza RNA copy number in nasal swabs is co-linear with culture, although the rate of decay of cell culture-based viral titers was faster than that observed with molecular methods. The study documented a clear decreasing log ratio of the RNA copy number to the infectious viral titer of the patients over time.

Localization of the gene causing the osteopetrotic phenotype in the incisors absent (ia) rat on chromosome 10q32.1.

UNLABELLED: The incisors absent rat is an osteopetrotic animal model. Segregation analysis in 37 affected animals from an outcross enabled us to assign the disease causing gene to a 4.7-cM interval on rat chromosome 10q32.1. Further analysis of the genes mapped in this region will provide more insight into the underlying pathogenesis. INTRODUCTION: Many of the insights into the factors that regulate the differentiation and activation of osteoclasts are gained from different spontaneous and genetically induced osteopetrotic animal models. The osteopetrotic incisors absent (ia) rat exhibits a generalized skeletal sclerosis and a delay of tooth eruption. Although the ia rat has well been studied phenotypically, the genetic defect still remains unknown. MATERIAL AND METHODS: To map the ia locus, we outcrossed the inbred ia strain with the inbred strain Brown Norway. Intercrossing F1 animals produced the F2 generation. Thirty-one mutant F2 animals and six mutant F4 animals were available for segregation analysis. RESULTS: Segregation analysis enabled us to assign the disease causing gene to rat chromosome 10q32.1. Homozygosity for the ia allele was obtained for two of the markers analyzed (D10Rat18 and D10Rat84). Key recombinations delineate a candidate region of 4.7 cM flanked by the markers D10Rat99 and D10Rat17. CONCLUSION: We have delineated a 4.7-cM region on rat chromosome 10q32.1 in which the gene responsible for the osteopetrotic phenotype of the ia rat is located. Although the sequence of this chromosomal region is not complete, over 140 known or putative genes have already been assigned to this region. Among these, several candidate genes with a putative role in osteoclast functioning can be identified. However, at this point, it cannot be excluded that one of the genes with a currently unknown function is involved in the pathogenesis of the ia rat. Further analysis of the genes mapped in this region will provide us more insight into the pathogenesis of this osteopetrotic animal model.

Localization of the gene causing autosomal dominant osteopetrosis type I to chromosome 11q12-13.

The osteopetroses are a heterogeneous group of genetic conditions characterized by increased bone density due to impaired bone resorption by osteoclasts. Within the autosomal dominant form of osteopetrosis, the radiological type I (ADOI) is characterized by a generalized osteosclerosis, most pronounced at the cranial vault. The patients are often asymptomatic but some suffer from pain and hearing loss. ADOI is the only type of osteopetrosis not associated with an increased fracture rate. Linkage analysis in two families with ADOI from Danish origin enabled us to assign the disease-causing gene to chromosome 11q12-13. A summated maximum lod score of +6.54 was obtained with marker D11S1889 and key recombinants allowed delineation of a candidate region of 6.6 cM between markers D11S1765 and D11S4113. Previously, genes causing other conditions with abnormal bone density have been identified from this chromosomal region. The TCIRG1 gene was shown to underly autosomal recessive osteopetrosis (ARO), and, recently, mutations in the LRP5 gene were found both in the osteoporosis-pseudoglioma syndrome and the high bone mass trait. Because both genes map within the candidate region for ADOI, it can not be excluded that ADOI is caused by mutations in either the TCIRG1 or the LRP5 gene.