Henri Agut

Ancient Recombination Events between Human Herpes Simplex Viruses

Abstract Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are seen as close relatives but also unambiguously considered as evolutionary independent units. Here, we sequenced the genomes of 18 HSV-2 isolates characterized by divergent UL30 gene sequences to further elucidate the evolutionary history of this virus. Surprisingly, genome-wide recombination analyses showed that all HSV-2 genomes sequenced to date contain HSV-1 fragments. Using phylogenomic analyses, we could also show that two main HSV-2 lineages exist. One lineage is mostly restricted to subSaharan Africa whereas the other has reached a global distribution. Interestingly, only the worldwide lineage is characterized by ancient recombination events with HSV-1. Our findings highlight the complexity of HSV-2 evolution, a virus of putative zoonotic origin which later recombined with its human-adapted relative. They also suggest that coinfections with HSV-1 and 2 may have genomic and potentially functional consequences and should therefore be monitored more closely.

Update on infections with human herpesviruses 6A, 6B, and 7

Human herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, HHV-7) are genetically related to cytomegalovirus. They belong to the Roseolovirus genus and to the Betaherpesvirinae subfamily. They infect T cells, monocytes-macrophages, epithelial cells, and central nervous system cells. These viruses are ubiquitous and are responsible for lifelong chronic infections, most often asymptomatic, in the vast majority of the general adult population. HHV-6B is responsible for exanthema subitum, which is a benign disease of infants. HHV-6A and HHV-6B also cause opportunistic infections in immunocompromised patients: encephalitis, hepatitis, bone marrow suppression, colitis, and pneumonitis. Their etiological role in chronic diseases such as multiple sclerosis, cardiomyopathy, and thyroiditis is still controversial. The pathogenicity of HHV-7 is less clear and seems to be much more restricted. Chromosomal integration of HHV-6A and HHV-6B is transmissible from parents to offspring and observed in about 1% of the general population. This integration raises the question of potential associated diseases and can be a confounding factor for the diagnosis of active infections by both viruses. The diagnosis of HHV-6A, HHV-6B, and HHV-7 infections is rather based on gene amplification (PCR), which allows for the detection and quantification of the viral genome, than on serology, which is mainly indicated in case of primary infection. Ganciclovir, foscarnet, and cidofovir inhibit the replication of HHV-6A, HHV-6B, and HHV-7. Severe infections may thus be treated but these therapeutic indications are still poorly defined.

Human cytomegalovirus (CMV) susceptibility to currently approved antiviral drugs does not impact on CMV terminase complex polymorphism.

Currently approved anti-human cytomegalovirus (CMV) drugs, all targeting the viral DNA polymerase, are associated with significant toxicities and emergence of drug resistance. In this context, CMV terminase complex constitutes a promising target for novel antiviral compounds. In this study, we describe the low natural polymorphism (interstrain identity >97.7% at both nucleotide and amino acid levels) of the terminase subunits pUL56 and pUL89, and the portal protein pUL104, among 63 CMV clinical strains, and we show that the CMV resistance profile to current DNA polymerase inhibitors has no impact on the natural polymorphism of CMV terminase complex. These results support the idea that both CMV clinical strains exhibiting either susceptibility or resistance to current CMV DNA polymerase inhibitors are comparably sensitive to novel inhibitors of CMV terminase complex, such as letermovir.

Genetic Diversity within Alphaherpesviruses: Characterization of a Novel Variant of Herpes Simplex Virus 2

ABSTRACT Very low levels of variability have been reported for the herpes simplex virus 2 (HSV-2) genome. We recently described a new genetic variant of HSV-2 (HSV-2v) characterized by a much higher degree of variability for the UL30 gene (DNA polymerase) than observed for the HG52 reference strain. Retrospective screening of 505 clinical isolates of HSV-2 by a specific real-time PCR assay targeting the UL30 gene led to the identification of 13 additional HSV-2v isolates, resulting in an overall prevalence of 2.8%. Phylogenetic analyses on the basis of microsatellite markers and gene sequences showed clear differences between HSV-2v and classical HSV-2. Thirteen of the 14 patients infected with HSV-2v originated from West or Central Africa, and 9 of these patients were coinfected with HIV. These results raise questions about the origin of this new virus. Preliminary results suggest that HSV-2v may have acquired genomic segments from chimpanzee alphaherpesvirus (ChHV) by recombination. IMPORTANCE This article deals with the highly topical question of the origin of this new HSV-2 variant identified in patients with HIV coinfection originating mostly from West or Central Africa. HSV-2v clearly differed from classical HSV-2 isolates in phylogenetic analyses and may be linked to simian ChHV. This new HSV-2 variant highlights the possible occurrence of recombination between human and simian herpesviruses under natural conditions, potentially presenting greater challenges for the future.

Complementary assays for monitoring susceptibility of varicella-zoster virus resistance to antivirals

The emergence of varicella-zoster virus (VZV) resistance to current antivirals as acyclovir (ACV) constitutes a hindrance to antiviral treatment effectiveness of VZV infections, especially in immunocompromised patients. The molecular mechanisms of VZV resistance reported so far rely on the presence of mutations within thymidine kinase (TK, ORF36) and DNA polymerase (ORF28) viral genes. The aim of this work was to develop reliable and complementary diagnostic methods to detect VZV antiviral resistance: (i) a genotypic assay based on TK and DNA polymerase genes sequencing, (ii) a plaque reduction assay to determine antiviral 50% effective concentrations, and (iii) a functional assay to evaluate in vitro phosphorylation activity of recombinant TKs. As a whole, this study included the analysis of 21 VZV clinical isolates and 62 biological samples from patients experiencing VZV infection. Genetic analysis revealed 3 and 9 new amino acid changes that have not been previously described within the highly conserved TK and DNA polymerase, respectively. Then, VZV isolates bearing newly identified mutations considered as natural polymorphisms were characterized as susceptible to ACV using plaque-reduction assay in MeWo cells. In parallel, the impact of TK changes on ACV phosphorylation activity was examined using a nonradioactive in vitro enzymatic assay.

Recurrent herpetic keratitis despite antiviral prophylaxis: A virological and pharmacological study.

ABSTRACT Recurrent herpes simplex keratitis (HSK) is a leading infectious cause of blindness in industrialized countries. Antiviral prophylaxis (AVP) may fail to prevent recurrence of HSK due to viral resistance, inadequate dosing, or poor patient compliance. In this prospective multicenter study, we enrolled immunocompetent patients with recurrent HSK despite AVP. Ocular samples were tested by PCR for herpes simplex virus 1 (HSV‐1). HSV‐1 drug resistance was assessed with a genotypic assay based on UL23 and UL30 gene sequencing. After curative full dose valacyclovir (VACV) treatment was started, peak and trough acyclovir (ACV) plasma concentrations were measured, and patient compliance to AVP was assessed with a questionnaire. The study sample was comprised of 43 patients. Six (14%) patients were positive for HSV‐1 using PCR, of whom 5 (83%) harbored genotypically ACV‐resistant (ACVR) virus, due to mutations in UL23 (n = 4) or UL30 (n = 1). Disease duration was statistically significantly longer in patients with viral resistance compared to other HSK patients [35.5 ± 23.4 years (range, 6.8–68.4 years) versus 11.1 ± 12.3 years (range, 0.8–56.3 year) respectively; Mann‐Whitney p = 0.01)]. While patients were treated with full dose VACV, trough ACV plasma concentrations were below the threshold for ACV sensitivity in 9.5% of cases, and compliance was poor in 5.3% of cases. To summarize, HSV‐1 resistance to ACV seems to be a significant cause of failure of prophylaxis in patients with HSK and is associated with longer disease duration. Most PCR‐positive samples contained genotypically ACVR virus and identification may aid in adapting treatment. Incomplete 24‐h drug coverage may also explain some cases of failure of prophylaxis. HighlightsIn patients with recurrent herpes keratitis despite antiviral prophylaxis, most PCR‐positive samples contain ACVR virus.In these patients, HSV‐1 resistance is associated with a longer disease duration.Incomplete 24‐h drug coverage may also explain some cases of failure of prophylaxis.

Human Herpesviruses 6A, 6B, and 7.

Human roseoloviruses include three different species, human herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, HHV-7), genetically related to human cytomegalovirus. They exhibit a wide cell tropism in vivo and, like other herpesviruses, induce a lifelong latent infection in humans. In about 1% of the general population, HHV-6 DNA is covalently integrated into the subtelomeric region of cell chromosomes (ciHHV-6). Many active infections, corresponding to primary infections, reactivations, or exogenous reinfections, are asymptomatic. They also may cause serious diseases, particularly in immunocompromised individuals, including hematopoietic stem-cell transplant (HSCT) and solid-organ transplant recipients, and acquired immunodeficiency syndrome (AIDS) patients. This opportunistic pathogenic role is formally established for HHV-6 infection and less clear for HHV-7. It mainly concerns the central-nervous system, bone marrow, lungs, gastrointestinal tract, skin, and liver. As the best example, HHV-6 causes both exanthema subitum, a benign disease associated with primary infection, and severe encephalitis associated with virus reactivations in HSCT recipients. Diagnosis using serologic and direct antigen-detection methods currently exhibits limitations. The most prominent technique is the quantification of viral DNA in blood, other body fluids, and organs by means of real-time polymerase-chain reaction (PCR). The antiviral compounds ganciclovir, foscarnet, and cidofovir are effective against active infections, but there is currently no consensus regarding the indications of treatment or specifics of drug administration. Numerous questions about HHV-6A, HHV-6B, HHV-7 are still pending, concerning in particular clinical impact and therapeutic options in immunocompromised patients.

High conservation of herpes simplex virus UL5/UL52 helicase-primase complex in the era of new antiviral therapies

The emergence of herpes simplex virus (HSV) resistance to current antiviral drugs, that all target the viral DNA polymerase, constitutes a major obstacle to antiviral treatment effectiveness of HSV infections, especially in immunocompromised patients. A novel and promising class of inhibitors of the HSV UL5/UL52 helicase-primase (HP) complex has been reported to hinder viral replication with a high potency. In this study, we describe the low natural polymorphism (interstrain identity >99.1% at both nucleotide and amino acid levels) of HSV HP complex subunits pUL5 and pUL52 among 64 HSV (32 HSV-1 and 32 HSV-2) clinical isolates, and we show that the HSV resistance profile to the first-line antiviral drug acyclovir (ACV) does not impact on the natural polymorphism of HSV HP complex. Genotypic tools and polymorphism data concerning HSV HP complex provided herein will be useful to detect drug resistance mutations in a relevant time frame when HP inhibitors (HPIs), i.e., amenamevir and pritelivir, will be available in medical practice.

Cytokine and cellular responses to human herpesvirus-6B in patients with B-acute lymphoblastic leukemia

Primary infection with human herpesvirus‐6 (HHV‐6), is followed by its lifelong persistence in the host. Most T‐cell responses to HHV‐6 have been characterized using peripheral blood from healthy adults; however, the role of HHV‐6 infection in immune modulation has not been elucidated for some diseases. Therefore, in this study the immune response to HHV‐6 infection in patients with B‐acute lymphoblastic leukemia (B‐ALL) was analyzed. HHV‐6 load was quantified in blood samples taken at the time of diagnosis of leukemia and on remission. The same concentrations of anti‐ and pro‐inflammatory cytokines (IL‐4, IL‐1, IL‐6, IL‐8, IL‐12p70, IL‐17a, TNF‐α and IFN‐γ) were detected in plasma samples from 20 patients with and 20 without detectable HHV‐6 virus loads in blood. Characterization of T‐cell responses to HHV‐6 showed low specific T‐cells frequencies of 2.08% and 1.46% in patients with and without detectable viral loads, respectively. IFN‐γ‐producing T cells were detected in 0.03%–0.23% and in 0%–0.2% of CD4+T cells, respectively. Strong production of IL‐6 was detected in medium supernatants of challenged T‐cells whatever the HHV‐6 status of the patients (973.51u2009±u2009210.06 versus 825.70u2009±u2009210.81u2009pg/mL). However, concentrations of TNF‐α and IFN‐γ were low. Thus, no association between plasma concentrations of cytokines and detection of HHV‐6 in blood was identified, suggesting that HHV‐6 is not strongly associated with development of B‐ALL. The low viral loads detected may correspond with latently infected cells. Alternatively, HHV‐6B specific immune responses may be below the detection threshold of the assays used.

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