Neural dysfunction following respiratory viral infection as a cause of chronic cough hypersensitivity

Respiratory viral infections are a common cause of acute coughing, an irritating symptom for the patient and an important mechanism of transmission for the virus. Although poorly described, the inflammatory consequences of infection likely induce coughing by chemical (inflammatory mediator) or mechanical (mucous) activation of the cough-evoking sensory nerves that innervate the airway wall. For some individuals, acute cough can evolve into a chronic condition, in which cough and aberrant airway sensations long outlast the initial viral infection. This suggests that some viruses have the capacity to induce persistent plasticity in the neural pathways mediating cough. In this brief review we present the clinical evidence of acute and chronic neural dysfunction following viral respiratory tract infections and explore possible mechanisms by which the nervous system may undergo activation, sensitization and plasticity.

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV) several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question, and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. Additionally, endo-lysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable over-expression of p62 significantly suppressed DENV replication suggesting a novel role for p62 as viral restriction factor. Overall our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an anti-viral role, which is countered by DENV.

IMPORTANCE: Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we employed high-content, imaging-based flow cytometry to quantify autophagic flux and endo-lysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endo-lysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a pro- to an antiviral cellular process, which is counteracted by the virus.

Metagenomic characterisation of the viral community of lough neagh, the largest freshwater lake in Ireland

Lough Neagh is the largest and the most economically important lake in Ireland. It is also one of the most nutrient rich amongst the world's major lakes. In this study, 16S rRNA analysis of total metagenomic DNA from the water column of Lough Neagh has revealed a high proportion of Cyanobacteria and low levels of Actinobacteria, Acidobacteria, Chloroflexi, and Firmicutes. The planktonic virome of Lough Neagh has been sequenced and 2,298,791 2×300 bp Illumina reads analysed. Comparison with previously characterised lakes demonstrates that the Lough Neagh viral community has the highest level of sequence diversity. Only about 15% of reads had homologs in the RefSeq database and tailed bacteriophages (Caudovirales) were identified as a major grouping. Within the Caudovirales, the Podoviridae and Siphoviridae were the two most dominant families (34.3% and 32.8% of the reads with sequence homology to the RefSeq database), while ssDNA bacteriophages constituted less than 1% of the virome. Putative cyanophages were found to be abundant. 66,450 viral contigs were assembled with the largest one being 58,805 bp; its existence, and that of another 34,467 bp contig, in the water column was confirmed. Analysis of the contigs confirmed the high abundance of cyanophages in the water column.

Portraying persons who inject drugs recently infected with hepatitis C accessing antiviral treatment : a cluster analysis

Objectives. To empirically determine a categorization of people who inject drug (PWIDs) recently infected with hepatitis C virus (HCV), in order to identify profiles most likely associated with early HCV treatment uptake. Methods.The study population was composed of HIV-negative PWIDs with a documented recent HCV infection. Eligibility criteria included being 18 years old or over, and having injected drugs in the previous 6 months preceding the estimated date of HCV exposure. Participant classification was carried out using a TwoStep cluster analysis. Results. FromSeptember 2007 to December 2011, 76 participants were included in the study. 60 participants were eligible for HCV treatment. Twenty-one participants initiated HCV treatment.The cluster analysis yielded 4 classes: class 1: Lukewarm health seekers dismissing HCV treatment offer; class 2: multisubstance users willing to shake off the hell; class 3: PWIDs unlinked to health service use; class 4: health seeker PWIDs willing to reverse the fate. Conclusion. Profiles generated by our analysis suggest that prior health care utilization, a key element for treatment uptake, differs between older and younger PWIDs. Such profiles could inform the development of targeted strategies to improve health outcomes and reduce HCV infection among PWIDs.

Sustained drug use changes following hepatitis C screening and counseling among recently infected persons who inject drugs: a longitudinal study

BACKGROUND: Notification of hepatitis C virus (HCV) positive status is known to have short-term impacts on subsequent alcohol, drug use and injection behaviors among persons who inject drugs (PWID). It remains to be established whether post-screening behavioral changes extend over time for PWID and whether screening test notification has behavioral impacts among HCV-negative PWID. This study sought to longitudinally assess substance use and injection behaviors after HCV status notification among HCV seroconverters and HCV-negative PWID. METHODS: Initially HCV-seronegative PWID (n = 208) were followed prospectively between 2004 and 2011 in Montreal, Canada. Semi-annual screening visits included blood sampling and an interview-administered questionnaire assessing substance use and injection behaviors. Multivariable generalized estimating equation analyses were conducted to assess substance use and behavior changes over time and compare changes between HCV seroconverters and HCV-seronegative participants while adjusting for baseline characteristics. RESULTS: Of the 208 participants (83% male; mean age, 34.7 years, mean follow-up time, 39 months), 69 (33.2%) seroconverted to HCV. A linear decrease in syringe sharing behavior was observed over time after HCV and status notification, whereas a 10% decrease for each additional 3 months of follow-up was observed for injection cocaine and heroin use among HCV seroconverters but not among HCV-seronegative PWID (P < .05). No significant changes were observed in alcohol use. CONCLUSIONS: Our results indicate that notification of HCV-positive status is associated with reduced injection drug use among seroconverters. Among PWID deemed seronegative after screening, there is no sustained trend for change in risk behavior.

Development of non-viral vectors for gene therapy for pathologies of the retina

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

New method for the simultaneous identification of pathogenic human viruses using multiplex PCR

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2014

RT-Bst: an integrated approach for reverse transcription and enrichment of cDNA from viral RNA

The synthesis of cDNA from RNA is challenging due to the inefficiency of reverse transcription (RT). In order to address this, a method was developed known as RT-Bst for sequential RT of RNA and Bst DNA polymerase amplification for enrichment of cDNA in a single tube reaction. Using genomic RNA from bacteriophage MS2, the yield of cDNA produced by RT alone and RT-Bst were compared by analysis of PCR-amplified products. Using random primers a superior performance was observed when amplifying MS2 RNA following RT-Bst compared to RT alone, indicating that greater quantities of cDNA were present after RT-Bst. RT-Bst was also compared with RT alone for their relative ability to produce sufficient cDNA to amplify 8 target regions spanning the respiratory syncytial virus (RSV) genome. Six out of 8 targets were amplified consistently by PCR subsequent to RT-Bst amplification whereas only 3 out of 8 targets could be amplified after RT alone. RSV sequences were selectively amplified using RSV specific primers from a mixed template containing an excess of MS2 RNA in a RT-Bst reaction without amplifying MS2 sequences. This suggests that RT-Bst can be used to amplify RNA sequences non-specifically using random primers and specifically using sequence specific primers and enhances the yield of cDNA when compared to RT alone.

The risk of viral dispersal and aerosolization by different hand-drying methods

Background World Health Organization hand hygiene guidelines state that if electric hand dryers are used, they should not aerosolize pathogens. Previous studies have investigated the dispersal by different hand-drying devices of chemical indicators, fungi and bacteria on the hands. This study assessed the aerosolization and dispersal of virus on the hands to determine any differences between hand-drying devices in their potential to contaminate other occupants of public washrooms and the washroom environment. Methods A suspension of MS2, an Escherichia coli bacteriophage virus, was used to artificially contaminate the hands of participants prior to using three different handdrying devices: jet air dryer, warm air dryer, paper towel dispenser. Virus was detected by plaque formation on agar plates layered with the host bacterium. Vertical dispersal of virus was assessed at a fixed distance (0.4 m) and over a range of different heights (0.0 – 1.8 m) from the floor. Horizontal dispersal was assessed at different distances of up to three metres from the hand-drying devices. Virus aerosolization and dispersal was also assessed at different times up to 15 minutes after use by means of air sampling at two distances (0.1 and 1.0 m) and at a distance behind and offset from each of the hand-drying devices. Results Over a range of heights, the jet air dryer was shown to produce over 60 times greater vertical dispersal of virus from the hands than a warm air dryer and over 1300 times greater than paper towels; the maximum being detected between 0.6 and 1.2 metres from the floor. Horizontal dispersal of virus by the jet air dryer was over 20 times greater than a warm air dryer and over 190 times greater than paper towels; virus being detected at distances of up to three metres. Air sampling at three different positions from the hand-drying devices 15 minutes after use showed that the jet air dryer produced over 50-times greater viral contamination of the air than a warm air dryer and over 110-times greater than paper towels. Conclusions Due to their high air speed, jet air dryers aerosolize and disperse more virus over a range of heights, greater distances, and for longer times than other hand drying devices. If hands are inadequately washed, they have a greater potential to contaminate other occupants of a public washroom and the washroom environment. Main messages: Jet air dryers with claimed air speeds of over 600 kph have a greater potential than warm air dryers or paper towels to aerosolize and disperse viruses on the hands of users. The choice of hand-drying device should be carefully considered. Jet air dryers may increase the risk of transmission of human viruses, such as norovirus, particularly if hand washing is inadequate.

Ultra-deep sequencing provides insights into the virology of hepatitis C super-infections in a case of three sequential infections with different genotypes

The current epidemic of Hepatitis C infection in HIV-positive men who have sex with men is associated with increasing use of recreational drugs. Multiple HCV infections have been reported in haemophiliacs and intravenous drug users. Using ultra-deep sequencing analysis, we present the case of an HIV-positive MSM with evidence of three sequential HCV infections, each occurring during the acute phase of the preceding infection, following risk exposures. We observed rapid replacement of the original strain by the incoming genotype at subsequent time points. The impact of HCV super-infection remains unclear and UDS may provide new insights.

Avaliação da infecção de megacariócitos e plaquetas pelo VHC e sua influência na fisiopatologia da hepatite C

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

IFN stimulated gene expression in the liver is a better predictor of treatment response in chronic hepatitis c than the IL28b (IFN lambda 3) genotype

Background: Therapy of chronic hepatitis C (CHC) with pegIFNa/ribavirin achieves sustained virologic response (SVR) in ~55%. Pre-activation of the endogenous interferon system in the liver is associated non-response (NR). Recently, genome-wide association studies described associations of allelic variants near the IL28B (IFNλ3) gene with treatment response and with spontaneous clearance of the virus. We investigated if the IL28B genotype determines the constitutive expression of IFN stimulated genes (ISGs) in the liver of patients with CHC. Methods: We genotyped 93 patients with CHC for 3 IL28B single nucleotide polymorphisms (SNPs, rs12979860, rs8099917, rs12980275), extracted RNA from their liver biopsies and quantified the expression of IL28B and of 8 previously identified classifier genes which discriminate between SVR and NR (IFI44L, RSAD2, ISG15, IFI22, LAMP3, OAS3, LGALS3BP and HTATIP2). Decision tree ensembles in the form of a random forest classifier were used to calculate the relative predictive power of these different variables in a multivariate analysis. Results: The minor IL28B allele (bad risk for treatment response) was significantly associated with increased expression of ISGs, and, unexpectedly, with decreased expression of IL28B. Stratification of the patients into SVR and NR revealed that ISG expression was conditionally independent from the IL28B genotype, i.e. there was an increased expression of ISGs in NR compared to SVR irrespective of the IL28B genotype. The random forest feature score (RFFS) identified IFI27 (RFFS = 2.93), RSAD2 (1.88) and HTATIP2 (1.50) expression and the HCV genotype (1.62) as the strongest predictors of treatment response. ROC curves of the IL28B SNPs showed an AUC of 0.66 with an error rate (ERR) of 0.38. A classifier with the 3 best classifying genes showed an excellent test performance with an AUC of 0.94 and ERR of 0.15. The addition of IL28B genotype information did not improve the predictive power of the 3-gene classifier. Conclusions: IL28B genotype and hepatic ISG expression are conditionally independent predictors of treatment response in CHC. There is no direct link between altered IFNλ3 expression and pre-activation of the endogenous system in the liver. Hepatic ISG expression is by far the better predictor for treatment response than IL28B genotype.