David Boutolleau

Utilization of Microsatellite Polymorphism for Differentiating Herpes Simplex Virus Type 1 Strains

The herpes simplex virus type 1 (HSV-1) genome is a linear double-stranded DNA of 152 kpb. It is divided into long and short regions of unique sequences termed UL and US, respectively, and these are flanked by regions of inverted internal and terminal repeats. Microsatellites are short tandem repeats of 1- to 6-nucleotide motifs; they are often highly variable and polymorphic within the genome, which raises the question of whether they may be used as molecular markers for the precise differentiation of HSV-1 strains. In this study, 79 different microsatellites (mono-, di-, and trinucleotide repeats) in the HSV-1 complete genome were identified by in silico analysis. Among those microsatellites, 45 were found to be distributed in intergenic or noncoding inverted repeat regions, while 34 were in open reading frames. Length polymorphism analysis of the PCR products was used to investigate a set of 12 distinct HSV-1 strains and allowed the identification of 23 polymorphic and 6 monomorphic microsatellites, including two polymorphic trinucleotide repeats (CGT and GGA) within the UL46 and US4 genes, respectively. A multiplex PCR method that amplified 10 polymorphic microsatellites was then developed for the rapid and accurate genetic characterization of HSV-1 strains. Each HSV-1 strain was characterized by its own microsatellite haplotype, which proved to be stable over time in cell culture. This relevant innovative tool was successfully applied both to confirm the close relationship between sequential HSV-1 isolates collected from patients with multiple recurrent infections and to investigate putative nosocomial infections.

Length variability of telomeric repeat sequences of human herpesvirus 6 DNA

The telomeric repeat sequences (TRS) located near both ends of human herpesvirus 6 (HHV-6) genome are unique structures of unknown function among human herpesviruses. The goal of the present study was to investigate the variability of TRS copy number among different laboratory strains and HHV-6-infected clinical specimens regarding the two variants A and B of HHV-6. DNA obtained from infected cells was submitted to a PCR assay designed to amplify the part of genome containing TRS specifically either for HHV-6A or HHV-6B. Amplicons were analyzed by electrophoresis on agarose gel with ethidium bromide staining and nucleotide sequencing. The number of TRS copies was highly variable among the distinct laboratory strains and clinical specimens studied, ranging from 15 up to more than 180. However, this number was constant for a given strain after serial propagation in cell cultures as well as in different samples from the same subject. This permitted to detect a mixed infection with two distinct strains of HHV-6A within the same patient. The PCR-based analysis of HHV-6 TRS has a limited sensitivity but is highly specific, which provides the opportunity to include it in the set of molecular tools dedicated to the study of HHV-6 epidemiology.

Characterization of a cidofovir-resistant HHV-6 mutant obtained by in vitro selection.

Cidofovir (CDV) was used for in vitro selection of a human herpesvirus 6 (HHV-6) mutant with decreased susceptibility to this drug. The resulting mutant was highly resistant to CDV as compared to its sensitive counterpart (inhibitory concentration 50% (IC50): 213 microM versus 1.8 microM). Its replication fitness was not impaired. Genotypic characterization of the resistant virus revealed a mutation in the U38 gene encoding the viral DNA polymerase. The resulting R798I amino acid change was located in the conserved domain VII close to the highly conserved motif KKRY interacting with the DNA primer-template duplex, and is likely responsible for the high-level resistance to CDV, even though a definite virological and/or biochemical confirmation is required. The possible emergence of such changes in HHV-6 DNA polymerase in patients receiving CDV therapy should be taken into account in the treatment of HHV-6 infections.

Human NKG2A overrides NKG2C effector functions to prevent autoreactivity of NK cells

To the editor: In April 2009, we saw a 64-year-old woman with persistent indolent proliferation of CD3−CD56+ large granular lymphocytes.[1][1] She had no other clinical symptoms and no autoimmune events, and her clinical course has remained benign over 22 months. Neither clinical nor imaging

Temporal and Spatial Compartmentalization of Drug-Resistant Cytomegalovirus (CMV) in a Child with CMV Meningoencephalitis: Implications for Sampling in Molecular Diagnosis

ABSTRACT We describe a case of antiviral-resistant cytomegalovirus meningoencephalitis occurring after hematopoietic stem cell transplantation. Antiviral-resistant cytomegalovirus was identified in blood 16 months earlier. However, wild-type cytomegalovirus was evidenced in blood when the meningoencephalitis was diagnosed. Treatment of meningoencephalitis should be adapted to all previously identified resistance mutations in any compartment.

Microsatellite analysis of HSV-1 isolates: from oropharynx reactivation toward lung infection in patients undergoing mechanical ventilation.

BACKGROUND According to recent reports, herpes simplex virus type 1 (HSV-1) induces bronchopneumonitis (BPn) in immunocompetent patients undergoing prolonged mechanical ventilation (MV), whose respiratory functions deteriorate with a poor outcome. HSV-1 BPn is associated with HSV symptomatic or symptomless reactivation in the oropharynx. OBJECTIVES We sought to systematically and genetically characterize HSV-1 strains isolated from immunocompetent patients receiving prolonged MV and to characterize the genetic relationship of strains sequentially isolated from oropharyngeal samples (OPS) and broncho-alveolar liquids (BAL) to determine the natural course of HSV BPn. STUDY DESIGN In this molecular epidemiological study, microsatellite technology was used to determine genetic relationships between 211 HSV-1 strains isolated from OPS and/or BAL from 106 patients receiving MV. RESULTS Microsatellite haplotypes of HSV-1 strains sequentially isolated from the same individual were identical, and HSV-1 isolates from the lung were genetically indistinguishable from strains isolated from the oral cavity. Each patient was characterized by their own HSV-1 microsatellite haplotype, and no nosocomial transmission of strains between patients was observed. CONCLUSION Our results demonstrate that, in patients who receive MV, the HSV-1 pulmonary infection results from the reactivation of genetically related HSV-1 in the oropharynx, which progressively infects the lower respiratory tract.

Clinical and microbiological evaluation of travel-associated respiratory tract infections in travelers returning from countries affected by pandemic A(H1N1) 2009 influenza.

Abstract Background Although acute respiratory tract infections (RTI) have been recognized as a significant cause of illness in returning travelers, few studies have specifically evaluated the etiologies of RTI in this population. Methods This prospective investigation evaluated travelers returning from countries with endemic influenza A(H1N1) 2009, and who were seen in our department at the onset of the outbreak (April–July 2009). Patients were included if they presented with signs of RTI that occurred during travel or less than 7 days after return from overseas travel. Patients were evaluated for microbial agents with RespiFinder plus assay, and throat culture according to clinical presentation. Results A total of 113 travelers (M/F ratio 1.2:1; mean age 39 y) were included. They were mainly tourists (n = 50; 44.2%) mostly returning from North America (n = 65; 58%) and Mexico (n = 21; 18.5%). The median duration of travel was 23 days (range 2–540 d). The median lag time between return and onset of illness was 0.2 days (range 10 d prior to 7 d after). The main clinical presentation of RTI was influenza‐like illness (n = 76; 67.3%). Among the 99 microbiologically evaluated patients, a pathogen was found by polymerase chain reaction (PCR) or throat culture in 65 patients (65.6%). The main etiological agents were influenza A(H1N1) 2009 (18%), influenza viruses (14%), and rhinovirus (20%). A univariate analysis was unable to show variables associated with influenza A(H1N1) 2009, whereas rhinorrhea was associated with viruses other than influenza (p = 0.04). Conclusion Despite the A(H1N1) 2009 influenza pandemic, rhinovirus and other influenza viruses were also frequent causes of RTI in overseas travelers. Real‐time reverse transcription‐PCR and nasopharyngeal swab cultures are useful diagnostic tools for evaluating travelers with RTI.

Les infections à herpèsvirus humain 6 (HHV-6) : un vaste domaine encore à explorer

Resume L′herpesvirus humain 6 (HHV-6) est un beta-herpesvirus proche du cytomegalovirus et de l′herpesvirus humain 7. Il montre un tropisme cellulaire etendu in vivo , incluant en particulier les lymphocytes T CD4-positifs et les cellules du systeme nerveux central. L′infection a HHV-6 est tres frequente dans la population generale. Le HHV-6 est l′agent responsable de l′exantheme subit ou sixieme maladie. Il est implique aussi dans des infections opportunistes, parfois gravissimes, des sujets immunodeprimes. Son role dans certaines maladies chroniques telle que la sclerose en plaques reste controverse. Le diagnostic virologique de l′infection se fonde en particulier sur l′amplification genique. Un traitement specifique par les medicaments antiherpetiques actifs contre le cytomegalovirus est indique dans les formes d′infection les plus graves. Les nombreuses questions encore en suspens justifient de poursuivre les investigations sur ce virus de connaissance recente.

Antivirales (a excepción del VIH y la hepatitis)

Los antivirales son en la actualidad un sector esencial de la farmacopea antiinfecciosa. Incluso si no se tienen en cuenta los antirretrovirales y los antivirales dirigidos contra los virus de la hepatitis B y C, que constituyen una parte muy importante, existen varias moléculas que se utilizan en la práctica clínica que permiten luchar eficazmente contra las infecciones por virus herpes, adenovirus, poxvirus, virus del papiloma y virus de la gripe. La mayoría de estas moléculas se dirigen contra las enzimas virales implicadas en la replicación de los genomas virales. La mayoría de los análogos nucleosídicos (de los que el arquetipo es el aciclovir) y los análogos nucleotídicos (cuyo arquetipo es el cidofovir) requieren una fosforilación previa para inhibir, por un mecanismo de competición y, en ocasiones de terminación, la actividad de una polimerasa de ADN (ácido desoxirribonucleico). El foscarnet, análogo de pirofosfato, ejerce esta inhibición directamente sin modificación. En la actualidad, se dispone de menos antivirales para los virus ARN (ácido ribonucleico) que para los de ADN, aunque los inhibidores de la neuraminidasa han demostrado su eficacia contra los virus de la gripe. La especificidad de los antivirales suele ser estrecha, limitada por lo general para cada molécula a unos pocos virus relacionados. Las otras limitaciones del uso actual de los antivirales son la imposibilidad de erradicar las infecciones virales latentes, la aparición de resistencia, los efectos indeseables relacionados en gran parte con la toxicidad celular relativa de las moléculas y su coste. Se esperan avances tanto en la eficacia de los antivirales como en su tolerabilidad clínica y el número de las enfermedades virales tratadas. Es esencial que las exigencias económicas no restrinjan la dinámica de uno de los ámbitos más innovadores de la medicina contemporánea.

Microbiological evaluation of respiratory tract infections in pilgrims returning from countries affected by Middle East respiratory syndrome coronavirus (MERS-CoV)

no: 127 Presentation at ESCV 2016: Poster 66 Microbiological evaluation of respiratory tract infections in pilgrims returning from countries affected by Middle East respiratory syndrome coronavirus (MERS-CoV) S. Burrel 1,∗, S. Jaureguiberry 2, E. Caumes 2, A. Aubry 3, H. Agut 1, D. Boutolleau 1 1 Sorbonne Université, UPMC Univ Paris 06, CR7, CIMI-Paris, INSERM U1135, and AP-HP, Hôpitaux Universitaires La Pitié-Salpêtrière – Charles Foix, Service de Virologie, Paris, France 2 Sorbonne Université, UPMC Univ Paris 06, CR7, CIMI-Paris, INSERM U1135, and AP-HP, Hôpitaux Universitaires La Pitié-Salpêtrière – Charles Foix, Service de Maladies Infectieuses et Médecine Tropicale, Paris, France 3 Sorbonne Université, UPMC Univ Paris 06, CR7, CIMI-Paris, INSERM U1135, and AP-HP, Hôpitaux Universitaires La Pitié-Salpêtrière Charles Foix, Service de Bactériologie-Hygiène, Paris, France Since September 2012, the World Health Organization (WHO) has been notified of 1728 laboratory-confirmed cases of infection with Middle East respiratory syndrome coronavirus (MERS-CoV), including at least 624 related deaths (disease outbreak news of April 26, 2016). Although MERS-CoV appears to be transmitted through respiratory droplets between humans with close contact, dromedary camels are likely to be a zoonotic source of MERS-CoV infection in humans. Early detection of MERS-CoV infection among international travelers exposed to camels or healthcare facilities in the Middle East remains essential. All travelers returning from MERS-CoV-affected areas to Paris (France) are given particular attention and those with fever and/or respiratory symptoms are referred to a dedicated infectious disease unit as the Infectious Disease Department of La Pitié-Salpêtrière University Hospital in Paris. The aim of this study was to investigate the microbiological etiologies of respiratory tract infections (RTI) among these specific travellers from the beginning of the 2015 Hajj and Umrah pilgrimage period (September 2015) to April 2016. Upon admission, patients were isolated and nasopharyngeal swabs, sputum samples and, for persons on ventilators, bronchoalveolar lavage specimens were collected by trained nurses. We examined which etiological respiratory pathogens were identified during screening for MERS-CoV in symptomatic travellers returning to Paris during September 2015 to April 2016 period, from MERS-CoV endemic regions (published WHO bulletins). Firstly, samples were screened with a specific MERS-CoV realtime reverse transcription PCR targeting region upstream of the E gene (upE), as recommended by WHO. The second step of the etiologic diagnosis entailed an investigation for other respiratory viruses (influenza A/B viruses, respiratory syncytial virus, metapneumovirus, rhinovirus-enterovirus, parainfluenza viruses, other human coronaviruses) using Respiratory MWS r-gene® kits (bioMérieux) and for bacteria using standardized culture procedures. A total of 31 symptomatic travellers mainly returning from Saudi Arabia (mean age 63.1 years, range 21–92 years; 58% male) were included during the study period and overall 48 respiratory clinical specimens were collected. None of the tested specimens were positive for MERS-CoV. Since a negative result should not absolutely rule out the possibility of MERS-CoV infection, notably if specimen is collected late or very early in the illness, some patients were screened twice. The vast majority of viral RTI, sometimes associated with bacteria superinfection, in these pilgrims returning home, were due to seasonal influenza A viruses (29%), rhinoviruses (23%), and other coronaviruses (7%) distinct from the MERS-CoV. Four patients were presenting acute lobar pneumonia, none were formally diagnosed. However, all were cured with antibiotics, as the presentation suggested pneumococcal infection. One case of Q fever, another known zoonosis transmitted by dromedary camels, and one case of Legionnella pneumophilia-associated disease were diagnosed among tested pilgrims. Continuous surveillance should be implemented to ensure the timely detection of possible imported cases of MERS-CoV and their immediate isolation in order to avoid secondary cases. However, clinicians should be aware that influenza viruses and rhinoviruses are the most commonly identified pathogens in returning pilgrims with acute RTI. http://dx.doi.org/10.1016/j.jcv.2016.08.106 Abstract no: 136 Presentation at ESCV 2016: Poster 67no: 136 Presentation at ESCV 2016: Poster 67 Determination of genotype distribution and the various polymorphisms in cytomegalovirus (CMV) strains Saliha Gökçe Alagöz 1,∗, Tekin Karslıgil 2, Mehmet Ozaslan 1 1 Gaziantep University Faculty of Science Biology Department, Turkey 2 Gaziantep University Faculty of Medicine Microbiology Department, Turkey