Novel UL23 and UL30 substitutions in HSV1 and HSV2 viruses related to polymorphism or drug resistance.

Data on herpes simplex virus (HSV) polymorphism as well as acyclovir (ACV) and foscarnet (FOS) resistance mutations are not exhaustive and may hinder accurate diagnosis by next-generation sequencing (NGS). Here, we report novel UL23 and UL30 substitutions for HSV1 and HSV2 identified in immunocompromised patients treated for hematological malignancies during the last 6 years of HSV resistance surveillance at the University Hospital of Lyon. For HSV1, 35 novel UL23 substitutions and 52 novel UL30 substitutions were identified. For HSV2, 2 novel UL23 substitutions and 12 novel UL30 substitutions were identified. These results allow to complete the database of HSV1 and HSV2 substitutions, related either to polymorphism or to ACV and FOS resistance.

Protracted viral shedding and viral load are associated with ICU mortality in Covid-19 patients with acute respiratory failure.

BACKGROUND: Protracted viral shedding is common in hospitalized patients with COVID-19 pneumonia, and up to 40% display signs of pulmonary fibrosis on computed tomography (CT) after hospital discharge. We hypothesized that COVID-19 patients with acute respiratory failure (ARF) who die in intensive care units (ICU) have a lower viral clearance in the respiratory tract than ICU patients discharged alive, and that protracted viral shedding in respiratory samples is associated with patterns of fibroproliferation on lung CT. We, therefore, conducted a retrospective observational study, in 2 ICU of Lyon university hospital. RESULTS: 129 patients were included in the study, of whom 44 (34%) died in ICU. 432 RT-PCR for SARS-CoV-2 were performed and 137 CT scans were analyzed. Viral load was significantly higher in patients deceased as compared to patients alive at ICU discharge (p < 0.001), after adjustment for the site of viral sampling and RT-PCR technique. The median time to SARS-CoV-2 negativation on RT-PCR was 19 days [CI95 %:15-21] in patients alive at ICU discharge and 26 days [CI95 %:17-infinity] in non-survivors at ICU discharge. Competitive risk regression identified patients who died in ICU and age as independent risk factors for longer time to SARS-CoV-2 negativation on RT-PCR, while antiviral treatment was independently associated with shorter time. None of the CT scores exploring fibroproliferation (i.e., bronchiectasis and reticulation scores) were significantly associated with time to SARS-CoV-2 negativation. CONCLUSIONS: Viral load in respiratory samples is significantly lower and viral shedding significantly shorter in ICU survivors of COVID-19 associated acute respiratory failure. Protracted viral shedding is unrelated to occurrence of fibrosis on lung CT.

Letermovir breakthroughs during the French Named Patient Programme: interest of monitoring blood concentration in clinical practice.

OBJECTIVES: To analyse mechanisms of letermovir breakthrough during compassionate primary and secondary prophylaxis. METHODS: Mechanisms of letermovir breakthrough during compassionate primary and secondary prophylaxis were analysed in four patients from the French Named Patient Programme by the French National Reference Centre for Herpesviruses. RESULTS: Of three absolute resistance cases, two were associated with treatment interruption or low letermovir concentrations in blood. A fourth case of breakthrough was not associated with resistance. Next-generation sequencing (NGS) genotyping confirmed rapid emergence of resistant mutants, within 3 months of treatment initiation. CONCLUSIONS: Measurement of letermovir concentration and genotyping should be recommended for patient follow-up during letermovir therapy.

Functional balance between neuraminidase and haemagglutinin in influenza viruses.

Seasonal influenza A and B viruses are important human pathogens responsible for significant morbidity and mortality worldwide. In addition, influenza A zoonotic viruses are a constant pandemic threat. These viruses present two major surface glycoproteins: the haemagglutinin (HA) and the neuraminidase (NA). These two glycoproteins both recognize the sialic acid and have complementary activities, the HA binds the sialic acid through its receptor-binding site, the NA is a receptor-destroying enzyme that cleaves α2-3 and α2-6-linked sialic acids. Therefore, the functional HA/NA balance is a critical factor for a good viral fitness and plays a major role in overcoming the host barrier and the efficiency of sustained human-to-human transmission. Although the two glycoproteins are in constant evolution, the HA/NA balance seems to remain stable in human viruses because an optimal balance is required to maintain good viral fitness. Understanding the evolution of influenza viruses requires an in-depth exploration of the HA/NA balance.

Increased detection of Mycoplasma pneumoniae infection in children, Lyon, France, 2010 to 2011.

Recent reports from several northern European countries indicate an increase in detection of Mycoplasma pneumoniae infection in the past two years, notably in children aged 5­15 years. Analysis of our laboratory database showed a similar pattern, with a higher proportion of respiratory samples positive for M. pneumonia by real-time PCR in paediatric patients aged 5­15 years. Our data indicate that in 2010 and 2011, France experienced the first epidemic peak of M. pneumonia infection since 2005.

Pediatric neurological complications associated with the A(H1N1)pdm09 influenza infection.

BACKGROUND: Influenza-related neurological complications (INC) have been reported during seasonal flu in children. OBJECTIVES: To investigate the types, outcomes and incidence of INC occurring during the 2009 A(H1N1) pandemic, a retrospective analyze was conducted in the single French pediatric hospital of Lyon from October 2009 to February 2010. STUDY DESIGN: All children presenting with fever, influenza-like illness, respiratory distress or neurological symptoms were tested for influenza A(H1N1)pdm09 infection from respiratory specimens using real time RT-PCR. RESULTS: INC occurred in 14 A(H1N1)pdm09 positive children (7.7% of A(H1N1)pdm09 positive children admitted to hospital) with a median age of 5.1 years. Admission to the intensive care unit (ICU) was required for nine children (64.3%). Half of the children with INC had comorbidity and three had coinfection, both characteristics mainly found in children requiring the ICU. All children received oral oseltamivir treatment. Febrile seizures were observed in eight children, half of them having a chronic comorbidity (2 epilepsy, 1 nonketotic hyperglycinemia, 1 anoxic encephalopathy). Other INC, less commonly reported, included 2 cases of encephalitis, 1 encephalopathy, 1 basilar artery thrombosis, 1 myasthenic crisis and 1 coma. Eleven of the 14 children (78.6%) recovered, one had a minor disability, one child developed a locked-in syndrome and one died from complications of an acute necrotizing encephalopathy. DISCUSSION: INC can be observed even in children with no underlying disorder. It may lead to dramatic issue in a significant number of cases.

Pandemic A(H1N1)2009 influenza virus detection by real time RT-PCR: is viral quantification useful?

The emergence of the influenza A(H1N1) 2009 virus prompted the development of sensitive RT-PCR detection methods. Most are real time RT-PCRs which can provide viral quantification. In this manuscript, we describe a universal influenza A RT-PCR targeting the matrix (M) gene, combined with an RNaseP RT-PCR. These PCRs allow the detection of all influenza A virus subtypes, including A(H1N1)2009, together with a real-time assessment of the quality of the specimens tested. These PCR procedures were evaluated on 209 samples collected from paediatric patients. Viral loads determined through Ct values were corrected according to the RNaseP Ct value. The mean viral load in the collected samples was estimated to be 6.84 log RNA copies/mL. For poor quality samples (RNaseP Ct > 27), corrections resulted in +3 to +8 Ct values for the M gene RT-PCR. Corrected influenza Ct values were lower in late samples. No correlation was established between viral loads and clinical severity or duration of disease.This study shows that real time RT-PCR targeting the matrix gene is a reliable tool for quantification of type A influenza virus but emphasises the need for sample quality control assessment through cellular gene quantification for reliable estimation of the viral load. This method would be useful for disease management when repeated specimens are collected from an infected individual.

Rhinoviruses delayed the circulation of the pandemic influenza A (H1N1) 2009 virus in France.

In contrast to the experience in other European countries, the onset of the A(H1N1)2009 influenza virus epidemic was unexpectedly slow in France during the first part of autumn 2009. Our objective was to test the hypothesis that intense circulation of rhinoviruses might have reduced the probability of infection by A(H1N1)2009 virus at the beginning of autumn 2009. Systematic analysis for the detection of A(H1N1)2009 (H1N1) and human rhinovirus (HRV) was performed by RT-PCR from week 36 to week 48 on respiratory samples sent to the diagnostic laboratory by the paediatric hospital (n = 2121). Retrospective analysis of the obtained data, using 2 x 2 contingency tables with Fisher's exact test, revealed evidence of an inverse relationship between HRV and H1N1 detection. Between weeks 36 and 48 of 2009, both HRV and H1N1 were detected but in different time frames. HRV dispersed widely during early September, peaking at the end of the month, whereas the H1N1 epidemic began during mid-October and was still active at the end of this survey. During the co-circulation period of these two respiratory viruses (weeks 43-46), HRV detection appeared to reduce the likelihood of H1N1 detection in the same sample (OR = 0.08-0.24 p <0.0001). These results support the hypothesis that HRV infections can reduce the probability of A(H1N1) infection. This viral interference between respiratory viruses could have affected the spread of the H1N1 viruses and delayed the influenza pandemic at the beginning of autumn in France.

Serosurveillance study on transmission of H5N1 virus during a 2006 avian influenza epidemic.

In 2006 an outbreak of avian influenza A(H5N1) in Turkey caused 12 human infections, including four deaths. We conducted a serological survey to determine the extent of subclinical infection caused by the outbreak. Single serum samples were collected from five individuals with avian influenza whose nasopharyngeal swabs tested positive for H5 RNA by polymerase chain reaction, 28 family contacts of the cases, 95 poultry cullers, 75 individuals known to have had contact with diseased chickens and 81 individuals living in the region with no known contact with infected chickens and/or patients. Paired serum samples were collected from 97 healthcare workers. All sera were tested for the presence of neutralizing antibodies by enzyme-linked immunoassay, haemagglutination inhibition and microneutralization assays. Only one serum sample, from a parent of an avian influenza patient, tested positive for H5N1 by microneutralization assay. This survey shows that there was minimal subclinical H5N1 infection among contacts of human cases and infected poultry in Turkey in 2006. Further, the low rate of subclinical infection following contact with diseased poultry gave further support to the reported low infectivity of the virus.

Anti N1 cross-protecting antibodies against H5N1 detected in H1N1 infected people.

The A(H5N1) influenza virus pandemic may be the result of avian H5N1 adapting to humans, leading to massive human to human transmission in a context of a lack of pre-existing immunity. As A(H1N1) and A(H5N1) share the same neuraminidase subtype, anti-N1 antibodies subsequent to H1N1 infections or vaccinations may confer some protection against A(H5N1). We analysed, by microneutralization assay, the A/Vietnam/1194/04 (H5N1) anti-N1 cross-protection acquired either during A/New-Caledonia/20/99 (H1N1) infection or vaccination. In cases with documented H1N1 infection, H5N1 cross-protection could be observed only in patients born between 1930 and 1950. No such protection was detected in the sera of vaccinated individuals.

Influenza A replication and host nuclear compartments: many changes and many questions.

It is over 40 years since investigations showed that influenza A, one of the rare nuclear replicating RNA viruses, induces marked remodeling of the host nuclear architecture. Influenza modifies and/or hijacks host nuclear machinery in order to replicate, express viral proteins and interfere with host antiviral response. Numerous interactions between constitutive nuclear proteins and viral factors are now characterized but less is known concerning their functional significance and their connection with viral-induced modifications of nuclear ultrastructure. Therefore, the purpose of this review is to summarize data and hypotheses about functional interplays between host nuclear compartments and influenza A during viral replication.

[Genotyping diagnosis of acyclovir resistant herpes simplex virus]. / Détection par génotypage de la résistance des virus herpes simplex à l'aciclovir.

Herpes simplex virus resistant to acyclovir (ACV) is a major concern among immunocompromised patients. ACV resistance might be due to mutations located in one of the two genes involved in ACV mechanism of action, the thymidine kinase gene (TK, involved in 95% of the cases) and the DNA polymerase gene. TK gene mutations consist, in half of the cases, in nucleotide insertion or deletion, occurring most of the time in G or C homopolymers considered as hot spots. Half of the other cases involves nucleotide substitutions leading to amino acids substitutions. Studies of sensitive strains revealed a high degree of TK polymorphism, many mutations being not implied in ACV resistance. At the present time, resistance detection can be performed by phenotypic tests that require virus culture and results cannot be given to the physician before 7 to 10 days. Genotyping diagnosis performed directly from clinical samples would allow to detect resistance more rapidly, in order to switch quickly to an appropriate treatment by foscarnet or cidofovir.

[New targets for new anti-herpes drugs]. / Nouvelles cibles pour le développement de molécules antiherpétiques.

Although infections are often subclinical, herpes simplex virus (HSV) can cause mild to severe diseases, especially in immunocompromised patients. There are few drugs licensed for the treatment of HSV infections. Most target the viral DNA polymerase, such as acyclovir that remains the reference treatment some thirty years after its discovery! Extensive clinical use of this drug has led to the emergence of resistant strains, mainly in immunocompromised patients, these infections can be managed with only two drugs, foscarnet and cidofovir, both much more toxic than acyclovir. This highlights the crucial need for the development of new anti-herpes drugs that can inhibit infection by both wild-type viruses and drug-resistant strains. Over the last few years, significant efforts have been made to set up a range of strategies for the identification of potential new antiviral drugs. One alternative is to develop drugs with different mechanisms of action. The present article reviews potential viral and cellular targets that are now known to be involved in HSV multiplication and for which specific inhibitors with anti-HSV activity, at least in cell culture, have been identified. These drugs inhibit viral proteins involved in viral replication (DNA polymerase, ribonucleotide reductase or helicase-primase complex). Other drugs acting on cellular proteins needed for viral replication have also been described; these drugs are targetting cyclin-dependent kinases or the polyamine biosynthetic pathway.

Herpes simplex virus thymidine kinase mutations associated with resistance to acyclovir: a site-directed mutagenesis study.

Mutations in the thymidine kinase (TK) gene of herpes simplex virus (HSV) may confer resistance to acyclovir (ACV). Because of the high genetic polymorphism of this gene, discriminating between mutations related to resistance and mutations related to gene polymorphism can be difficult, especially when no sensitive strain has been previously isolated from the same patient. To assess the role of the mutations located at codons 51, 77, 83, and 175, previously detected in HSV-1 clinical isolates (F. Morfin, G. Souillet, K. Bilger, T. Ooka, M. Aymard, and D. Thouvenot, J. Infect. Dis. 182:290-293, 2000), in the acquisition of resistance to ACV, four mutants with site-directed mutations at these respective codons were constructed. The enzymatic activity of the proteins, produced using both a reticulocyte lysate system and a bacterial system, was evaluated using [(3)H]thymidine as substrate. This site-directed mutagenesis revealed that mutations at codons 51, 83, and 175 induce a loss of HSV-1 TK activity and are thus clearly involved in the acquisition of resistance to ACV. On the other hand, the mutation at codon 77 does not affect enzyme activity.

[Contribution of the laboratory in case of resistance to acyclovir of herpes simplex and varicella zoster virus]. / Apport du laboratoire en cas de résistance à l'aciclovir des virus herpes simplex et varicelle zona.

Herpes simplex virus (HSV) and varicella zoster virus (VZV) are susceptible to acyclovir which inhibits viral replication through two viral enzymes, thymidine kinase (TK) and DNA polymerase. Resistance may occur, it is a rare phenomenon among immunocompetent patients but resistance is more frequent and may be associated with serious complications among immunocompromised patients. Virological survey of these at risk patients is needed to detect resistant virus as soon as possible through phenotypic tests performed on virus isolated on cell cultures. Resistant virus may also be genetically characterised by detection of mutations within TK and DNA polymerase genes. Pharmacological parameters also have to be taken into consideration and a determination of acyclovir blood concentration should be performed in case of unexplained therapeutic failure. Improvement of immune system, when possible, may resolve these infections. Alternative treatments using drugs such as foscarnet or cidofovir which have a different mechanism of action compared to acyclovir, are recommended but these molecules are often more toxic than acyclovir.