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Coinfections affecting the porcine respiratory system have often been overlooked, in favor of mono-infections, even though they are significantly more common in the field. In pigs, the term 'porcine respiratory complex' is used to describe coinfections involving both viruses, such as, for example, the swine influenza type A virus (swIAV), the porcine respiratory and reproductive syndrome virus (PRRSV), and the porcine circovirus type 2 (PCV-2), as well as bacteria. Until recently, most studies were primarily focused on clinical aspects and paid little attention to the molecular consequences of coinfections. This narrative review addresses the consequences of coinfections in the porcine respiratory system involving viruses. When possible, interactions that can occur between viruses are briefly presented. Conversely, research involving bacteria, protozoa, and fungi has not been considered at all. Finally, the main limitations complicating the interpretation of results from coinfection/superinfection studies are considered, and prospects in this exciting field of health research are presented.

Bovine respiratory disease is still a major concern and has major economic impact. Another consequence of respiratory infections is the use of antimicrobial molecules to control bacterial pathogens. This can participate in the emergence and shedding of antimicrobial resistance that can threaten animal as well as human health. Appeasing pheromones with their capacity to reduce stress and thus their ability to preserve the functions of the immune system have been proposed to reduce the use of antimicrobial substances. In this study, we assessed the effect of appeasing pheromone administration on bovine health and performance during the fattening period. Zootechnical and health parameters and whole blood immune transcript expressions were measured over four weeks in bulls to determine the effect of the pheromone. We observed increased clinical signs on Day 8 (D8) and decreased clinical signs on D30 in bulls who received the pheromone and a higher expression of interleukin 8 transcripts in this group than in the control group on D8. Our results are overall in line with previous reports in livestock species. Further studies are needed to shed more light on the effect of appeasing pheromones and decipher their exact mechanisms of action.

Background: Group B enteroviruses are common causes of acute myocarditis, which can be a precursor of chronic myocarditis and dilated cardiomyopathy, leading causes of heart transplantation. To date, the specific viral functions involved in the development of dilated cardiomyopathy remain unclear., Methods: Total RNA from cardiac tissue of patients with dilated cardiomyopathy was extracted, and sequences corresponding to the 5' termini of enterovirus RNAs were identified. After next-generation RNA sequencing, viral cDNA clones mimicking the enterovirus RNA sequences found in patient tissues were generated in vitro, and their replication and impact on host cell functions were assessed on primary human cardiac cells in culture., Results: Major enterovirus B populations characterized by 5' terminal genomic RNA deletions ranging from 17 to 50 nucleotides were identified either alone or associated with low proportions of intact 5' genomic termini. In situ hybridization and immunohistological assays detected these persistent genomes in clusters of cardiomyocytes. Transfection of viral RNA into primary human cardiomyocytes demonstrated that deleted forms of genomic RNAs displayed early replication activities in the absence of detectable viral plaque formation, whereas mixed deleted and complete forms generated particles capable of inducing cytopathic effects at levels distinct from those observed with full-length forms alone. Moreover, deleted or full-length and mixed forms of viral RNA were capable of directing translation and production of proteolytically active viral proteinase 2A in human cardiomyocytes., Conclusions: We demonstrate that persistent viral forms are composed of B-type enteroviruses harboring a 5' terminal deletion in their genomic RNAs and that these viruses alone or associated with full-length populations of helper RNAs could impair cardiomyocyte functions by the proteolytic activity of viral proteinase 2A in cases of unexplained dilated cardiomyopathy. These results provide a better understanding of the molecular mechanisms that underlie the persistence of EV forms in human cardiac tissues and should stimulate the development of new therapeutic strategies based on specific inhibitors of the coxsackievirus B proteinase 2A activity for acute and chronic cardiac infections.

The presence of pneumoviruses in pigs is poorly documented. In this study, we used the published sequence of the nucleoprotein (N) of the recently identified Swine Orthopneumovirus (SOV) to express and purify SOV N as a recombinant protein in Escherichia coli. This protein was purified as nanorings and used to set up an enzyme-linked immunosorbent assay, which was used to analyse the presence of anti-pneumovirus N antibodies in swine sera. Sera collected from different pig farms in the West of France and from specific pathogen free piglets before colostrum uptake showed indirectly that a pneumovirus is circulating in pig populations with some variations between animals. Piglets before colostrum uptake were sero-negative for anti-pneumovirus antibodies while most of the other pigs showed positivity. Interestingly, in two farms presenting respiratory clinical signs and negative or under control for some common respiratory pathogens, pigs were detected positive for anti-pneumovirus antibodies. Globally, anti-pneumovirus N antibody concentrations were variable between and within farms. Further studies will aim to isolate the circulating virus and determine its potential pathogenicity. SOV could potentially become a new member of the porcine respiratory complex, important on its own or in association with other viral and bacterial micro-organisms.

The pig has garnered more and more interest as a model animal to study various conditions in humans. The growing success of the pig as an experimental animal model is explained by its similarities with humans in terms of anatomy, genetics, immunology, and physiology, by their manageable behavior and size, and by the general public acceptance of using pigs for experimental purposes. In addition, the immunological toolbox of pigs has grown substantially in the last decade. This development led to a boost in the use of pigs as a preclinical model for various human infections including sexually transmitted diseases (STIs) like Chlamydia trachomatis. In the current review, we discuss the use of animal models for biomedical research on the major human STIs. We summarize results obtained in the most common animal models and focus on the contributions of the pig model towards the understanding of pathogenesis and the host immune response. In addition, we present the main features of the porcine model that are particularly relevant for the study of pathogens affecting human female and male genital tracts. We also inform on the technological advancements in the porcine toolbox to facilitate new discoveries in this biologically important animal model. There is a continued need for improvements in animal modeling for biomedical research inclusive STI research. With all its advantages and the highly improved toolbox, the porcine model can play a crucial role in STI research and open the door to new exciting discoveries., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We assessed Enterovirus (EV) &Parvovirus B19 (PVB19) genomes and CD3, CD68&HLA-DR detection in dilated cardiomyopathies (DCM). EV&PVB19 genomes and CD3, CD68&HLA-DR were detected by PCR and immunohistochemistry assays in 115 endomyocardial biopsies obtained in 13 idiopathic DCM (iDCM) and 10 explained DCM (eDCM) patients. Results were compared with those of 47 atrial surgical samples (47 surgery controls) and 22 autoptic cardiac samples (11 healthy heart controls) (2008-2014, Reims, France). EV was detected in 23.1% of iDCM patients but not in eDCM and controls (P = 0.003) (viral load 803 copies/μg). PVB19 was detected in 76.9%, 80.0%, 63.6% and 78.2% of iDCM, eDCM, healthy heart and surgery controls (P = 0.99) with a mean viral load of 413, 346, 1,428, and 71 copies/μg. CD3, CD68 or HLA-DR were detected in 100 and 50% of EV and PVB19 "mono-infected" iDCM patients. EV was exclusively detected in iDCM cases in association with CD3, CD68, or HLA-DR indicating that EV could be an etiological cause in a subset of iDCM cases. By contrast the equal frequent detection of PVB19 in iDCM cases and controls without association with CD3, CD68, or HLA-DR suggested that PVB19 could be a bystander in many DCM cases. J. Med. Virol. 89:55-63, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

We performed deep sequencing analysis of the enterovirus 5' noncoding region in cardiac biopsies from a patient with dilated cardiomyopathy. Results displayed a mix of deleted and full-length coxsackievirus B3, characterized by a low viral RNA load (8.10(2) copies/μg of nucleic acids) and a low viral RNA positive-sense to RNA negative-sense ratio of 4.8.

Background: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is mainly an autosomal dominant disease characterized by fibrofatty infiltration of the right ventricle, leading to ventricular arrhythmias. Mutations in desmosomal proteins can be identified in about half of the patients. The pathogenic mechanisms leading to disease expression remain unclear., Objective: The purpose of this study was to investigate myocardial expression profiles of candidate molecules involved in the pathogenesis of ARVC/D., Methods: Myocardial messenger RNA (mRNA) expression of 62 junctional molecules, 5 cardiac ion channel molecules, 8 structural molecules, 4 apoptotic molecules, and 6 adipogenic molecules was studied. The averaged expression of candidate mRNAs was compared between ARVC/D samples (n = 10), nonfamilial dilated cardiomyopathy (DCM) samples (n = 10), and healthy control samples (n = 8). Immunohistochemistry and quantitative protein expression analysis were performed. Genetic analysis using next generation sequencing was performed in all patients with ARVC/D., Results: Following mRNA levels were significantly increased in patients with ARVC/D compared to those with DCM and healthy controls: phospholamban (P ≤ .001 vs DCM; P ≤ .001 vs controls), healthy tumor protein 53 apoptosis effector (P = .001 vs DCM; P ≤ .001 vs controls), and carnitine palmitoyltransferase 1β (P ≤ .001 vs DCM; P = 0.008 vs controls). Plakophillin-2 (PKP-2) mRNA was downregulated in patients with ARVC/D with PKP-2 mutations compared with patients with ARVC/D without PKP-2 mutations (P = .04). Immunohistochemistry revealed significantly increased protein expression of phospholamban, tumor protein 53 apoptosis effector, and carnitine palmitoyltransferase 1β in patients with ARVC/D and decreased PKP-2 expression in patients with ARVC/D carrying a PKP-2 mutation., Conclusion: Changes in the expression profiles of sarcolemmal calcium channel regulation, apoptosis, and adipogenesis suggest that these molecular pathways may play a critical role in the pathogenesis of ARVC/D, independent of the underlying genetic mutations., (Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Acute Respiratory Infections (ARIs) are responsible for considerable morbidity and mortality worldwide. Documentation of respiratory specimens can help for an appropriate clinical management with a significant effect on the disease progress in patient, the antimicrobial therapy used and the risk of secondary spread of infection. Here, we compared the performances of four commercial multiplex kits used in French University Hospital diagnostic microbiology laboratories for the detection of ARI pathogens (i.e., the xTAG Respiratory Viral Panel Fast, RespiFinder SMART 22, CLART PneumoVir and Fast Track Diagnostics Respiratory Pathogen 33 kits). We used a standardised nucleic acids extraction protocol and a comprehensive comparative approach that mixed reference to well established real-time PCR detection techniques and analysis of convergent positive results. We tested 166 respiratory clinical samples and identified a global high degree of correlation for at least three of the techniques (xTAG, RespiFinder and FTD33). For these techniques, the highest Youden's index (YI), positive predictive (PPV) and specificity (Sp) values were observed for Core tests (e.g., influenza A [YI:0.86-1.00; PPV:78.95-100.00; Sp:97.32-100.00] & B [YI:0.44-1.00; PPV:100.00; Sp:100.00], hRSV [YI:0.50-0.99; PPV:85.71-100.00; Sp:99.38-100.00], hMPV [YI:0.71-1.00; PPV:83.33-100.00; Sp:99.37-100.00], EV/hRV [YI:0.62-0.82; PPV:93.33-100.00; Sp:94.48-100.00], AdV [YI:1.00; PPV:100.00; Sp:100.00] and hBoV [YI:0.20-0.80; PPV:57.14-100.00; Sp:98.14-100.00]). The present study completed an overview of the multiplex techniques available for the diagnosis of acute respiratory infections.

Enteroviruses (EVs) are small naked single-stranded positive RNA viruses (Picornaviridae) of approximately 7,400 nucleotides divided in four species (HEV A-D) and including 120 serotypes. EVs are common human pathogens, transmitted through fecal-oral and respiratory routes. Although the majority of EV infections remains asymptomatic (90 %), these viruses are considered as one of the most common causes of acute viral illnesses in immunocompetent pediatric and adult subjects. High levels of genetic diversity allow these viruses to infect various target cells resulting in a wide spectrum of human acute pathologies including meningitis, respiratory syndromes, cutaneous syndromes, myocarditis and mother-to-child infections. During the early phases of the acute viral infection, EV can modulate the non-specific antiviral strategies developed by the infected target cell (modulation of class I MHC viral antigen presentation ; inhibition of type I interferon expression genes) and to disturb dendritic cell functions resulting in a viral immune escape. This immunological escape allows the generation of genetically modified viruses resulting from RNA genomic deletions, mutations or recombination mechanisms. Persistent replication activities of these genetically modified viruses can induce modulation of specific functions and endocellular pathways of infected cells and the development of chronic inflammatory or autoimmune mechanisms (auto-reactive T and -B cells and auto-antibodies). The persistence of these genetically modified viruses can result in direct or indirect tissue injuries that can explain a subset of chronic myocarditis and dilated cardiomyopathy (DCM), type 1 diabetes mellitus and post-polio syndrome (PPS) cases. Actually no specific and curative therapies are available against EV-induced chronic human pathologies. A better understanding of the molecular mechanisms implicated in viral persistence will stimulate the research into new therapeutic strategies to prevent and treat chronic infections caused by EVs.

Background: Retention of HAART-eligible HIV-infected patients in clinical follow-up systems are now becoming an important issue in sub-Saharan African countries., Methods: In this retrospective study (April 2008 to November 2011), we assessed the attrition rate variations in a cohort of 509 HAART-eligible patients in Chad., Results: Decrease in levels of loss to follow-up were observed during the implementation of continuous free access to HAART (72.5 vs 10%; p<0.001) and was independent of gender, age, WHO clinical stage and CD4+ T cell count at inclusion and of the time delay to initiate HAART (p>0.48)., Conclusions: These data suggest that the implementation of free access to HAART without any interruption of supply, from autumn 2009, could be the factor that potentially changed the HIV patient attrition rate in this resource-limited setting., (© The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Viruses are the leading cause of central nervous system (CNS) infections, ahead of bacteria, parasites, and fungal agents. A rapid and comprehensive virologic diagnostic testing method is needed to improve the therapeutic management of hospitalized pediatric or adult patients. In this study, we assessed the clinical performance of PCR amplification coupled with electrospray ionization-time of flight mass spectrometry analysis (PCR-MS) for the diagnosis of viral CNS infections. Three hundred twenty-seven cerebrospinal fluid (CSF) samples prospectively tested by routine PCR assays between 2004 and 2012 in two university hospital centers (Toulouse and Reims, France) were retrospectively analyzed by PCR-MS analysis using primers targeted to adenovirus, human herpesviruses 1 to 8 (HHV-1 to -8), polyomaviruses BK and JC, parvovirus B19, and enteroviruses (EV). PCR-MS detected single or multiple virus infections in 190 (83%) of the 229 samples that tested positive by routine PCR analysis and in 10 (10.2%) of the 98 samples that tested negative. The PCR-MS results correlated well with herpes simplex virus 1 (HSV-1), varicella-zoster virus (VZV), and EV detection by routine PCR assays (kappa values [95% confidence intervals], 0.80 [0.69 to 0.92], 0.85 [0.71 to 0.98], and 0.84 [0.78 to 0.90], respectively), whereas a weak correlation was observed with Epstein-Barr virus (EBV) (0.34 [0.10 to 0.58]). Twenty-six coinfections and 16 instances of uncommon neurotropic viruses (HHV-7 [n = 13], parvovirus B19 [n = 2], and adenovirus [n = 1]) were identified by the PCR-MS analysis, whereas only 4 coinfections had been prospectively evidenced using routine PCR assays (P < 0.01). In conclusion, our results demonstrated that PCR-MS analysis is a valuable tool to identify common neurotropic viruses in CSF (with, however, limitations that were identified regarding EBV and EV detection) and may be of major interest in better understanding the clinical impact of multiple or neglected viral neurological infections.

Viral detection in heart tissues has become a central issue for the diagnosis and exploration of the pathogenesis of idiopathic dilated cardiomyopathy (IDCM). In the present study, common cardiotropic viruses in 67 explanted heart samples of 31 IDCM adult patients were detected and semiquantified by using for the first time a new technology based on PCR assay coupled to electrospray ionization-time of flight mass spectrometry analysis (PCR-MS), with comparison to reference quantitative real-time PCR (RT-qPCR) assay. PCR-MS identified single or mixed enterovirus (EV) and parvovirus B19 (PVB19) infections in 27 (40.2%) of 67 samples, corresponding to 15 (48.3%) of the 31 patients, whereas RT-qPCR identified viral infections in 26 (38.8%) samples, corresponding to 16 (51.6%) of the patients. The PCR-MS results correlated well with EV and PVB19 detection by RT-qPCR (kappa = 0.85 [95% confidence interval {CI}, 0.72 to 1.00] and kappa = 0.82 [95% CI, 0.66 to 0.99], respectively). The levels of EV RNA (median, 550 [range, 178 to 3,200] copies/μg of total extracted nucleic acids) and of PVB19 DNA (median, 486 [range, 80 to 1,157] copies/μg of total extracted nucleic acids) were measured using PCR-MS and correlated with those obtained by RT-qPCR (r(2) = 0.57, P = 0.002 and r(2) = 0.64, P < 0.001 for EV and PVB19, respectively). No viruses other than EV and PVB19 strains were detected using the new PCR-MS technology, which is capable of simultaneously identifying 84 known human viruses in one assay. In conclusion, we identified single or mixed EV and PVB19 cardiac infections as potential causes of IDCM. The PCR-MS analysis appeared to be a valuable tool to rapidly detect and semiquantify common viruses in cardiac tissues and may be of major interest to better understand the role of viruses in unexplained cardiomyopathies.

Objective: To assess the etiological role and the clinical characteristics of HRV and HEV infections in pediatric patients hospitalized for acute respiratory tract infections (ARTIs)., Methods: RT-qPCR assays and molecular sequencing methods were used to identify HRV and HEV strains in nasopharyngeal aspirates of 309 hospitalized pediatric patients with microbiologically unexplained ARTIs and in 210 hospitalized pediatric patients without respiratory symptoms from September 2009 to June 2010 in France., Results: Among the 309 ARTI cases, 15 HEV and 172 HRV strains were identified whereas only 1 HEV and 37 HRV strains were observed in control patients (187 vs. 38: P < 10(-3)). HRV strains were identified in 150 of the 164 lower ARTIs whereas HEV strains were identified in only 14 of these cases. Among bronchiolitis and asthma exacerbation cases (n = 133), HEV infected cases were older (Median age (months) 36 vs. 11, P = 0.003) and were more frequently associated with a respiratory distress (P = 0.01) and a need for oxygen supply at the time of admission (P = 0.01) than cases infected by HRV strains., Conclusion: HRV and HEV strains were identified as potential etiological causes of 60.5% of microbiologically unexplained ARTIs diagnosed in hospitalized pediatric cases. A higher clinical severity was observed in HEV infected bronchiolitis or asthma exacerbation cases in comparison to HRV infected cases., (Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Few studies have evaluated the contribution of multiple virus and bacterial infections in acute exacerbation of chronic obstructive pulmonary disease. This study estimated the burden of multiple viral and bacterial respiratory infections in moderate to very severe chronic obstructive pulmonary disease patients that were prospectively followed-up during a 12-month pilot study. Clinical data were collected monthly and sputum was collected at the time of each acute exacerbation event. Classical culture techniques for bacteria and multiplex polymerase chain reaction (PCR) and microarray detection assays were performed to identify viral and atypical bacterial pathogens in the sputum. Overall, 51 patients were included and 45 acute exacerbation events were investigated clinically and microbiologically. Among the 45 acute exacerbation events, 44% had evidence of viral infection involving human rhinovirus (HRV) and metapneumovirus (hMPV) in 20% and 18%, respectively. Intracellular bacteria were not found in sputum by PCR. Common bacterial pathogens were identified in 42% of acute exacerbation patients, most frequently Branhamella catarrhalis, Streptococcus pneumoniae and Haemophilus influenzae. Viral or virus and bacteria co-infections were detected in 27% of acute exacerbation events (n = 12) with HRV and hMPV involved in 92% of cases. Patients with co-infections did not present greater clinical severity scores at exacerbation and more recurrence of acute exacerbation events at 3 and 6 months than those with single infections (P > 0.4). These results suggest that HRV and hMPV may be contributors or cofactors of AECOPD. These findings indicate that viral or virus and bacterial co-infections do not impact significantly on the clinical severity of acute exacerbation of chronic obstructive pulmonary disease and recurrence at 3 and 6 months., (Copyright © 2013 Wiley Periodicals, Inc.)

Enterovirus 68 was detected in 10 respiratory specimens from pediatric patients hospitalized for acute wheezing or bronchitis during 2009 in the northeast of France. Viral loads ranged from 2 × 10(5) to 7.2 × 10(7) copies/ml. Alignment of 5' nontranslated regions and phylogenetic analysis of partial VP1 gene sequences show that these viruses clustered and belonged to clade C.

Unlabelled: In March 2009, a new strain of influenza A/H1N1 virus was identified in Mexico, responsible for a pandemic. Worldwide, more than 13,500 patients died, most often from acute respiratory distress syndrome. Because sudden death cases were rare, involving mostly young apparently healthy persons, influenza A/H1N1 (2009)-related deaths may be misdiagnosed, which can raise medico-legal issues., Case History: we report on an unexpected out-of-hospital death involving a young male with no past medical history and no vaccination. Fever was his only symptom. Laboratory tests: histology showed patchy necrotic foci with mononuclear inflammation in the lungs. The heart was histologically normal, but virological analyses using molecular biology on frozen myocardial samples showed high virus load. In conclusion, this case report shows that influenza A/H1N1 (2009) virus can be a cause of sudden cardiac death in the young and demonstrates the importance of quantitative virological analyses for the diagnosis of myocarditis., (© 2012 American Academy of Forensic Sciences.)

Standardized one-step real-time RT-PCR assay detected enterovirus RNA in cardiac biopsy samples from 4 of 20 patients suffering from idiopathic dilated cardiomyopathy (IDCM). The median viral load was 287 copies per microgram of total extracted nucleic acids, with positive- to negative-strand RNA ratios ranging from 2 to 20. These results demonstrate enterovirus persistence in the heart of IDCM patients, characterized by low viral loads and low positive- to negative-RNA ratios.

Newly available molecular tools allow a sensitive detection of a broad panel of viruses in respiratory tract specimens. In the present study, the application of a multiplex RT-PCR DNA microarray in diagnosis and epidemiological survey of viral infections in infants hospitalized for bronchiolitis was assessed. One hundred and thirty-eight nasopharyngeal aspirates collected from October 2007 to September 2008 were tested by direct immunofluorescence and viral culture, a combination of referenced RT-PCRs and the DNA microarray. One or more viruses were detected in 96, 126 and 126 of the specimens by direct immunofluorescence and viral culture, RT-PCRs and DNA microarray, respectively (70 vs. 91 vs. 91%, P < 10(-3)). The RT-PCRs and the DNA microarray yielded concordant results for 99% of specimens and identified mixed viral infections in 85 (62%). The most common associations were: human bocavirus and respiratory syncytial virus (32%), adenovirus and respiratory syncytial virus (30%), and parainfluenza virus type 3 and respiratory syncytial virus (23%). None of the bronchiolitis severity parameters including intensive care unit admission, O(2) supply, O(2) saturation percentage, O(2) length and length of stay at the hospital appeared to be significantly increased in multiple viral infections compared to single viral infections (P > 0.1). In conclusion, the use of this DNA microarray in clinical virology practice allows rapid and accurate identification of common and uncommon viral respiratory pathogens in infants hospitalized for bronchiolitis. It should improve the clinical management, the epidemiological survey, and the prevention of the nosocomial transmission of respiratory viruses in pediatric wards., (Copyright © 2012 Wiley Periodicals, Inc.)

Herpes simplex virus 1 (HSV-1) esophagitis diagnosis is routinely based on the endoscopic findings confirmed by histopathological examination of the esophagitis lesions. Virological diagnosis is not systematically performed and restricted to viral culture or to qualitative PCR assay from esophagitis biopsy specimens. The aim of this study was to assess the interest of quantitative real-time PCR assay in HSV-1 esophagitis diagnosis by comparing the results obtained to those of histological examination associated with immunohistochemical staining, which is considered the "gold standard." From 53 esophagitis biopsy specimens, the PCR assay detected HSV-1 in 18 of 19 histologically proven to have herpetic esophagitis and in 9 of 34 that had esophagitis related to other causes, demonstrating sensitivity, specificity, positive predictive value, and negative predictive value of 94.7%, 73%, 66.7%, and 96%, respectively. Interestingly, HSV-1 was not detected in 16 specimens without the histological aspect of esophagitis. The viral loads normalized per μg of total extracted DNA in each biopsy specimen detected positive by HSV PCR were then compared and appeared to be significantly higher in histopathologically positive herpetic esophagitis (median = 2.9 × 10(6) ± 1.1 × 10(8)) than in histopathologically negative herpetic esophagitis (median = 3.1 × 10(3) ± 6.2 × 10(3)) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 10(4) copies would allow an HSV-1 esophagitis diagnosis with a sensitivity and specificity of 83.3% and 100%, respectively. In conclusion, this work demonstrated that HSV quantitative PCR results for paraffin-embedded esophageal tissue was well correlated to histopathological findings for an HSV-1 esophagitis diagnosis and could be diagnostic through viral load assessment when histopathological results are missing or uncertain.

In the present study, we comparatively assessed the pathophysiological mechanisms developed during lung infection of BALB/C female mice infected by an original wild type Klebsiella pneumoniae subsp. ozaenae strain (CH137) or by a referent subspecies K. pneumoniae. subsp. pneumoniae strain (ATCC10031). The mice infected with 2.10⁶ CFU K. p. subsp. pneumoniae (n = 10) showed transient signs of infection and all of them recovered. All of those infected with 1.10⁶ CFU K. p. subsp. ozaenae (n = 10) developed pneumonia within 24 h and died between 48 and 72 h. Few macrophages, numerous polymorphonuclear cells and lymphocytes were observed in their lungs in opposite to K. p. subsp. pneumoniae. In bronchoalveolar lavage, a significant increase in MIP-2, IL-6, KC and MCP-1 levels was only observed in K. p. subsp. ozaenae infected mice whereas high levels of TNF-α were evidenced with the two subspecies. Our findings indicated a lethal effect of a wild type K. p. subsp. ozaenae strain by acute pneumonia reflecting an insufficient alveolar macrophage response. This model might be of a major interest to comparatively explore the pathogenicity of K. p. subsp ozaenae strains and to further explore the physiopathological mechanisms of gram-negative bacteria induced human pneumonia., (Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Viruses are the main etiological cause of central nervous system (CNS) infections. A rapid molecular diagnosis is recommended to improve the therapeutic management of patients. The aim of this study was to evaluate the performances of a DNA microarray, the Clart Entherpex kit (Genomica, Coslada, Spain), allowing the rapid and simultaneous detection of 9 DNA and RNA neurotropic viruses: herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), HHV-7, HHV-8, and the human enteroviruses (HEVs). This evaluation was performed with 28 samples from the European proficiency panels (Quality Control for Molecular Diagnostics [QCMD]; Glasgow, Scotland) and then with 78 cerebrospinal fluid (CSF) specimens. The majority of the QCMD results obtained by the DNA microarray were similar to those recorded by the overall QCMD participants. The main discrepant results were observed for low concentrations of HSV-2 and HEVs. From the clinical samples, the kit detected 27 of the 28 herpesvirus CNS infections and all of the 30 HEV-positive CSF samples. No false-positive result was observed among the 20 virus-negative CSF samples. The clinical sensitivity, specificity, and negative and positive predictive values of the assay were 98.3, 100, 95.2, and 100%, respectively, when the results were compared to those of commercially available PCR assays. Interestingly, HHV-7 was detected in 11 (37%) of the 30 HEV-positive CSF samples from children suffering from aseptic meningitis causing significantly longer lengths of stay at the hospital than infection with HEVs alone (2.4 versus 1.4 days; P = 0.038). In conclusion, this preliminary study showed that this DNA microarray could be a valuable molecular diagnostic tool for single and mixed DNA and RNA virus infections of the CNS.

The early steps of echovirus 6 (E6) infection remain poorly understood and the only described receptor for haemagglutinating E6 strains is the decay accelerating factor (DAF). There is, however, accumulating evidence suggesting that E6 interaction with DAF is necessary but not sufficient for infection. In this report, we investigated the role of the coxsackie-adenovirus-receptor (CAR) as a potential DAF co-receptor during E6 infection. Using stably transfected Chinese Hamster Ovary (CHO) cells expressing CAR and DAF receptors, we found that DAF expression allowed attachment of both haemagglutinating and non-haemagglutinating E6 strains but was not sufficient for promoting E6 cell entry. Interestingly, the co-expression of DAF and CAR rendered 0.1-0.2% of cells permissive to some E6 strains' infection. Although our results did not show a major role of the CAR/DAF cooperation for E6 infection, it nevertheless indicated the use of CAR in the cell entry step of some minor E6 quasispecies. Moreover, the present report validates the use of recombinant CHO cells as valuable cellular model for the further characterisation of E6 receptors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We prospectively tested 95 nasal swabs or nasopharyngeal aspirates taken from 56 adults and 39 children visiting the Reims University Medical Centre (northern France) for influenza-like illnesses (ILI) during the early stage of the French influenza A/H1N1v pandemic (October 2009). Respiratory samples were tested using a combination of two commercially available reverse transcription-PCR (RT-PCR) DNA microarray systems allowing rapid detection of influenza A virus strains, including the new A/H1N1v strain as well as 20 other common or newly discovered respiratory viruses. Concomitantly, a generic and classical real-time RT-PCR assay was performed to detect all circulating influenza A virus strains in the same samples. Of the 95 respiratory samples tested, 30 (31%) were positive for the detection of influenza A/H1N1v virus infection by both RT-PCR DNA microarray and classical real-time RT-PCR detection assays. Among the infections, 25 (83%) were monoinfections, whereas 5 (17%) were multiple infections associating influenza A/H1N1v virus with coronavirus (CoV), human bocavirus (HBoV), respiratory syncytial virus (RSV), or human rhinoviruses (HRVs). Of the 95 respiratory samples tested, 35 (37%) were positive for respiratory viruses other than influenza A/H1N1v virus. Among these infections, we observed 30 monoinfections (HRVs [63%], parainfluenza viruses [PIVs] [20%]), influenza A/H3N2 virus [6%], coronavirus [4%], and HBoV [4%]) and 5 multiple infections, in which HRVs and PIVs were the most frequently detected viruses. No specific single or mixed viral infections appeared to be associated significantly with secondary hospitalization in infectious disease or intensive care departments during the study period (P > 0.5). The use of RT-PCR DNA microarray systems in clinical virology practice allows the rapid and accurate detection of conventional and newly discovered viral respiratory pathogens in patients suffering from ILI and therefore could be of major interest for development of new epidemiological survey systems for respiratory viral infections.

Although numerous reports have described inflammatory bowel diseases (IBDs) complicated with cytomegalovirus (CMV) infection, the virus participation as an exacerbating factor remains unclear. The aim of this study was thus to clarify the clinical significance of CMV infection complicating exacerbation and to correlate CMV detection with various characteristics in IBD patients. Sixty-seven colonic biopsies obtained from 53 patients admitted for IBD exacerbation were retrospectively analyzed by real-time PCR assay. The CMV genome was detected in seven (10.4%) colonic biopsies related to seven patients (three ulcerative colitis and four Crohn's diseases). Among the patients with IBD studied, patients with evidence of CMV infection were older (P = 0.047), were more likely male gender (relative risk [RR] 4.48; 95% confidence interval [CI] 0.94-21.36), received corticosteroids (RR 3.2; CI 0.79-13.02) or azathioprine (RR 3.17; CI 0.80-12.57) treatments, presented more extended lesions (RR for rectum-sigmoid-left colon 3.75 (0.0-69.37) and for pancolitis 2.45 (0.36-16.23)), and had a more severe disease (RR 3.3; CI 0.87-12.48) than those without CMV infection. Viral loads measured in the colonic mucosa of infected patient ranged from 5 to 236961 genome copies by microgram of total extracted DNA. No relationship was observed between the severity of the disease and the viral load level. Furthermore, CMV disappeared in five infected IBD patients in remission without antiviral agents. In conclusion, these results showed infrequent CMV detection in colonic biopsies of IBD patients during exacerbation leaving open the question of the relationship between CMV reactivation and the onset or the severity of IBD exacerbation., ((c) 2010 Wiley-Liss, Inc.)

Human Enteroviruses (HEV) (picornaviridae) are considered as one the major viral causes of childhood acute respiratory wheezing illnesses including bronchiolitis and asthma exacerbation. To identify the mechanisms that can regulate the development of airway mucosa inflammation during HEV respiratory lower tract infection, we investigated the profile and the levels of mRNA and protein secretion for CC and CXC human chemokines by HEV-infected primary human bronchial epithelial cells (SAE cells) using RT-PCR array and Bio-Plex assay. Cultures of SAE cells were infected by reference and wild-type HEV respiratory strains, demonstrating a replicative and productive viral infection. We observed that the replicative infection of the SAE cells by reference and wild-type HEV strains induced specific dose and time-dependent increases in mRNA and protein secretion only for RANTES, MCP-1 and IL-8 and not for all other CC and CXC human chemokines tested. The protein secretion of these chemokines appeared to be significantly increased at 48 or 72h post-infection in cultures treated by low-doses of IFN-gamma comparatively to mock-infected cells (P<0.001), and was correlated to the viral replication activity. In conclusion, our findings demonstrated a selective production of RANTES, IL-8 and MCP-1 released by HEV-infected epithelial cells of the small bronchioles along with mechanisms of amplification mediated by IFN-gamma., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Background: Echovirus 30 (E-30) was responsible in France for a major aseptic meningitis outbreak during 2005 summer season. However, the virological mechanisms responsible for the periodic emergence of the epidemic strains remain to be investigated., Objectives: To assess the genetic diversity of two genome regions, VP1 and 3Dpol, of echovirus 30 strains isolated during the 2005 aseptic meningitis outbreak in Champagne Ardenne (CA) area (France)., Study Design: Partial VP1 genomic region of 23 E-30 strains isolated in CA was sequenced and compared with 73 E-30 strains originating from different French areas to estimate the number and the diversity of E-30 lineages. Partial sequences for 3D polymerase (3Dpol) were analyzed to detect potential recombination events within the non-structural (NS) region of the genome of EV neurotropic strains., Results: Phylogenetic analysis of the VP1 evidenced the co-circulation of 6 distinct E-30 lineages responsible for the 2005 aseptic meningitis outbreak in France of which three had co-circulated in CA. Partial sequencing of the 3Dpol coding region showed that all of the E-30 strains exhibited different phylogenetic links between VP1 and 3Dpol genomic regions, suggesting multiple intra- or inter-serotypic recombination events within the NS part of the genome., Conclusions: Our findings revealed existence of multiple lineages and suggested frequent recombination events among E-30 strains having co-circulated in a restricted area during a short time outbreak period. Moreover, our data demonstrated that study of single VP1 genome region analysis could not accurately describe the phylogenetic origin of E-30 isolates., (Copyright 2010 Elsevier B.V. All rights reserved.)

From January to December 2007, 973 stool specimens were prospectively collected from children hospitalized for gastroenteritis signs or from neonates and premature cases who were born in two French hospital settings in the north of France. They were tested by rapid enzyme immunoassay (EIA) analyses for rotavirus and adenovirus and by two commercially available ELISA tests for the detection of norovirus and astrovirus. The overall rates of prevalence for rotavirus, norovirus, adenovirus, and astrovirus were 21, 13, 5, and 1.8%, respectively, and they did not significantly differ between the two hospital settings (P=0.12). Mixed virus infections were detected in 32 (3.3%) of the 973 study children and were associated with norovirus in 21 (66%) infants, including 5 premature cases. From fall to spring, norovirus infections accounted for 52% of documented gastroenteritidis viral infections at a time when rotavirus was epidemic, resulting in mixed norovirus and rotavirus gastrointestinal tract infections. Of the 367 documented viral gastroenteritis cases, 15 (4.1%) were identified as nosocomial infections, 5 of which occurred in premature cases. These findings highlight the need to implement norovirus and astrovirus ELISA detection assays in association with rapid EIA rotavirus and adenovirus detection assays for the clinical diagnosis and the nosocomial prevention of gastroenteritis viral infections in pediatric departments.

Aims: We examined the impact of enterovirus (EV) cardiac replication activity on the endomyocardial mitochondrial pathway in patients with acute myocarditis., Methods and Results: Levels of apoptotic cardiomyocytes were determined by TUNEL and ligation-mediated polymerase chain reaction (PCR) assays and EV replication activity was assessed by immunostaining of EV VP1 capsid protein in ventricular myocytes of patients with acute myocarditis (n = 25), and healthy heart controls (n = 15). Ratio of cytosolic/mitochondrial cytochrome c concentrations was determined by ELISA assay, levels of active caspase-9 were determined by western blot analysis and Bax/Bcl2 mRNA ratio was assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in the same cardiac tissues. Patients with EV-associated acute myocarditis (n = 15) exhibited a significantly higher number of apoptotic cardiomyocytes than those with non-EV-associated acute myocarditis (n = 10) and controls (n = 15) (P < 0.001). Endomyocardial ratio of cytosolic/mitochondrial cytochrome c concentrations and levels of active caspase-9 protein were significantly increased in EV than in non-EV-related myocarditis patients (P < 0.001). Moreover, Bax/Bcl2 mRNA ratio was significantly increased in EV than in non-EV-related myocarditis patients (P < 0.001)., Conclusion: Our findings evidence an EV-related activation of the cardiomyocyte mitochondrial apoptotic pathway in patients with acute myocarditis. Moreover, our results indicate that this EV-induced pro-apoptotic mechanism could be partly related to an up-regulation of Bax expression, and suggest that inhibition of this cell death process may constitute the basis for novel therapies.

Enteroviruses (EV) (Picornaviridae) are common infectious agents divided into 4 species, including 108 serotypes and responsible for a wide range of human pathologies including upper and lower respiratory tract infections occurring in adults and infants. Recent clinical studies indicated that these viruses are considered as the third etiological cause of bronchiolitis in young infants aged 1-12 months. Moreover, several clinical case studies reported the etiological role of the coxsackievirus A16 (CV-A16), the enterovirus 71 (EV-71) and of a newly discovered genotype (EV-104) in the development of acute or fatal pneumonia indicating that EV belonging to species A to C can be responsible for severe lower respiratory tract infections in immunocompetent infants or adults. Taking into account these recent epidemiological and clinical data and because of frequent mutations and intra-species enteroviral RNA genomic recombination events, the EV respiratory strains have to be considered as potential agents of further emerging infectious diseases in human populations.

Background: Influenza virus is one of the major viral respiratory pathogens infecting human beings., Objectives: To determine the influenza A virus variants responsible for the 2006-2007 epidemic season in Corsica Island, France., Study Design: Of 134 nasal samples of adult patients tested by culture and RT-PCR assays, 85 influenza A strains were identified; 81 (95%) were sub-typed as A/H3N2 and 4 (5%) were sub-typed as A/H1N1., Results: All of the HA sequences of the A/H3N2 viruses circulating in Corsica Island appeared to be closely related to the A/Wisconsin/67/2005 vaccine strain and segregated into two sub-groups that were genetically distinct from other viruses circulating in other countries during 2006/2007. One of these sub-groups was distinguished by the substitution H156Q whereas the second demonstrated at least one of the 3 other additional mutations (R142G, L157S and K173E) common to the HA1 sequence of A/Nepal/921/2006 reference strain. Among the 14 strains of this second sub-group, 10 viral strains had been isolated from vaccinated adult patients., Conclusion: These findings suggest that a prospective analysis of the HA sequences of influenza isolates may allow an early detection of newly evolved variants with potential epidemiological inference.

Background: Human Bocavirus (HBoV) is a newly discovered parvovirus whose role as a causative agent of respiratory disease remains unclear., Study Design: We investigated the presence of HBoV by quantitative PCR in the nasopharyngeal samples of 192 French children consecutively hospitalized for acute bronchiolitis. Other common respiratory viruses were detected using immunofluorescence assays, cell culture detection, or RT-PCR assays., Results: HBoV was detected in 24 (12.5%) of 192 study children. In 14/192 cases (7%) HBoV was the sole isolate and in 10/192 (5%) it was part of a mixed viral infection. HBoV was the third most common pathogen detected after respiratory syncytial virus (45/192; 23%) and rhinovirus (24/192; 12%). It occurred more often in infants aged 1-12 months (P=0.002). Median levels of HBoV DNA genome in respiratory samples were significantly higher in patients with single HBoV infection than in patients with mixed respiratory viral infection with HBoV (4x10(8)copies/ml vs. 2x10(3)copies/ml, P<0.001)., Conclusions: Our data suggest that HBoV at a high viral load could be an etiologic agent of respiratory tract disease, whereas the exact role of HBoV at a low viral load, as etiological cause or as pathophysiological co-factor of respiratory diseases, remains to be determined.

Human enteroviruses, which belong to the family of Picornaviridae, are common infectious viral agents transmitted by fecal-oral or airway routes. These positive RNA viruses possess a high genetic diversity and variability. They can evolve through genetic mutations or recombination mechanisms that are associated to the emergence of new potential epidemic serotypes. Human enteroviruses use different cellular receptors: receptors and co-receptors that are directly related to the tropism and the epidemiologic characteristics of some enterovirus serotypes. The receptors onto the cell-surface settle within a capsid depression, called canyon, initiating the process of viral uncoating. For some enteroviruses, a co-receptor molecule allows the crossing of cell topological barriers that is required to initiate the target cell infection. After the attachment phase, enteroviruses use the endocellar signaling pathways to support and optimize their entry into target-cells via endocytic pathways. The clathrin coated pits and the caveolae are both major ways of enterovirus entry in the cell even if "new" endocytic pathways regulated by enzymes of theADP ribosylation factors family and of the Rho family small GTPases have been recently described. The viral genetic diversity allows the human enteroviruses to simultaneously or alternatively use several distinct endocytic pathways in accordance to the infected cell lines, and allows a rapid and efficient adaptation to cellular microenvironments and to multiple immune selection pressures developed during the pathophysiological course of human infection. In conclusion, entry mechanisms used by human enteroviruses to infect target cells are various but they are closely dependent on the cellular functions that will be driven towards viral benefits. In the present time, the attachment and entry phases of the human enteroviruses into the target cell represent major viral events that may be targeted for the development of further new antiviral strategies.

Background: Enteroviruses (EVs) are considered as a major viral etiological cause of aseptic meningitis in children., Objectives: We assessed the clinical and virological features of an aseptic meningitis outbreak in North-East of France, 2005., Study Design: Classical bacteriological analysis, Herpesviridae and EV PCR assays had been prospectively performed on cerebrospinal fluid (CSF) samples taken from 80 children hospitalized for aseptic meningitis. For each EV strain identified as etiological agent, a phylogenetic comparison of partial EV VP1 capsid protein coding gene was performed., Results: The children older than 12 months (n=75) presented a typical aseptic meningitis syndrome, whereas the children aged less than 1 year (n=5) demonstrated only fever and hypotonia. Among the 80 studied children, EV was identified as the etiological cause of aseptic meningitis in 73 (91%) cases. Echovirus 30 (E30) was the most common isolated serotype (84% of 51 EV strains). VP1 phylogenetic analysis revealed that E30 strains were genetically closer to those isolated during 2000 aseptic meningitis outbreak comparatively to those identified during 2003 and 2006 non-epidemic years. Moreover, the genetic study demonstrated the co-circulation of four distinct lineages without any difference in temporal distribution or clinical features during the 2005 outbreak., Conclusions: The present report demonstrates the co-circulation of distinct E30 lineages during the same aseptic meningitis outbreak season. This E30 genetic diversity may be a prerequisite for the emergence of new strains potentially responsible for further aseptic meningitis outbreaks.