Samuel Cordey

New Respiratory Enterovirus and Recombinant Rhinoviruses among Circulating Picornaviruses

Increased genomic diversity of these viruses is demonstrated.

New complete genome sequences of human rhinoviruses shed light on their phylogeny and genomic features

BackgroundHuman rhinoviruses (HRV), the most frequent cause of respiratory infections, include 99 different serotypes segregating into two species, A and B. Rhinoviruses share extensive genomic sequence similarity with enteroviruses and both are part of the picornavirus family. Nevertheless they differ significantly at the phenotypic level. The lack of HRV full-length genome sequences and the absence of analysis comparing picornaviruses at the whole genome level limit our knowledge of the genomic features supporting these differences.ResultsHere we report complete genome sequences of 12 HRV-A and HRV-B serotypes, more than doubling the current number of available HRV sequences. The whole-genome maximum-likelihood phylogenetic analysis suggests that HRV-B and human enteroviruses (HEV) diverged from the last common ancestor after their separation from HRV-A. On the other hand, compared to HEV, HRV-B are more related to HRV-A in the capsid and 3B-C regions. We also identified the presence of a 2C cis-acting replication element (cre) in HRV-B that is not present in HRV-A, and that had been previously characterized only in HEV. In contrast to HEV viruses, HRV-A and HRV-B share also markedly lower GC content along the whole genome length.ConclusionOur findings provide basis to speculate about both the biological similarities and the differences (e.g. tissue tropism, temperature adaptation or acid lability) of these three groups of viruses.

Spread of Measles Virus D4-Hamburg, Europe, 2008–2011

TOC Summary: More than 24,300 cases were identified in 22 countries.

Identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus 71 infection.

Enterovirus 71 (EV71) is one of the most virulent enteroviruses, but the specific molecular features that enhance its ability to disseminate in humans remain unknown. We analyzed the genomic features of EV71 in an immunocompromised host with disseminated disease according to the different sites of infection. Comparison of five full-length genomes sequenced directly from respiratory, gastrointestinal, nervous system, and blood specimens revealed three nucleotide changes that occurred within a five-day period: a non-conservative amino acid change in VP1 located within the BC loop (L97R), a region considered as an immunogenic site and possibly important in poliovirus host adaptation; a conservative amino acid substitution in protein 2B (A38V); and a silent mutation in protein 3D (L175). Infectious clones were constructed using both BrCr (lineage A) and the clinical strain (lineage C) backgrounds containing either one or both non-synonymous mutations. In vitro cell tropism and competition assays revealed that the VP197 Leu to Arg substitution within the BC loop conferred a replicative advantage in SH-SY5Y cells of neuroblastoma origin. Interestingly, this mutation was frequently associated in vitro with a second non-conservative mutation (E167G or E167A) in the VP1 EF loop in neuroblastoma cells. Comparative models of these EV71 VP1 variants were built to determine how the substitutions might affect VP1 structure and/or interactions with host cells and suggest that, while no significant structural changes were observed, the substitutions may alter interactions with host cell receptors. Taken together, our results show that the VP1 BC loop region of EV71 plays a critical role in cell tropism independent of EV71 lineage and, thus, may have contributed to dissemination and neurotropism in the immunocompromised patient.

New Molecular Detection Tools Adapted to Emerging Rhinoviruses and Enteroviruses

ABSTRACT Human rhinoviruses (HRV), and to a lesser extent human enteroviruses (HEV), are important respiratory pathogens. Like other RNA viruses, these picornaviruses have an intrinsic propensity to variability. This results in a large number of different serotypes as well as the incessant discovery of new genotypes. This large and growing diversity not only complicates the design of real-time PCR assays but also renders immunofluorescence unfeasible for broad HRV and HEV detection or quantification in cells. In this study, we used the 5′ untranslated region, the most conserved part of the genome, as a target for the development of both a real-time PCR assay (Panenterhino/Ge/08) and a peptide nucleic acid-based hybridization oligoprobe (Panenterhino/Ge/08 PNA probe) designed to detect all HRV and HEV species members according to publicly available sequences. The reverse transcription-PCR assay has been validated, using not only plasmid and viral stocks but also quantified RNA transcripts and around 1,000 clinical specimens. These new generic detection PCR assays overcame the variability of circulating strains and lowered the risk of missing emerging and divergent HRV and HEV. An additional real-time PCR assay (Entero/Ge/08) was also designed specifically to provide sensitive and targeted detection of HEV in cerebrospinal fluid. In addition to the generic probe, we developed specific probes for the detection of HRV-A and HRV-B in cells. This investigation provides a comprehensive toolbox for accurate molecular identification of the different HEV and HRV circulating in humans.

Astrovirus Infection in Hospitalized Infants with Severe Combined Immunodeficiency after Allogeneic Hematopoietic Stem Cell Transplantation

Infants with severe primary combined immunodeficiency (SCID) and children post-allogeneic hematopoietic stem cell transplantation (HSCT) are extremely susceptible to unusual infections. The lack of generic tools to detect disease-causing viruses among more than 200 potential human viral pathogens represents a major challenge to clinicians and virologists. We investigated retrospectively the causes of a fatal disseminated viral infection with meningoencephalitis in an infant with gamma C-SCID and of chronic gastroenteritis in 2 other infants admitted for HSCT during the same time period. Analysis was undertaken by combining cell culture, electron microscopy and sequence-independent single primer amplification (SISPA) techniques. Caco-2 cells inoculated with fecal samples developed a cytopathic effect and non-enveloped viral particles in infected cells were detected by electron microscopy. SISPA led to the identification of astrovirus as the pathogen. Both sequencing of the capsid gene and the pattern of infection suggested nosocomial transmission from a chronically excreting index case to 2 other patients leading to fatal infection in 1 and to transient disease in the others. Virus-specific, real-time reverse transcription polymerase chain reaction was then performed on different stored samples to assess the extent of infection. Infection was associated with viremia in 2 cases and contributed to death in 1. At autopsy, viral RNA was detected in the brain and different other organs, while immunochemistry confirmed infection of gastrointestinal tissues. This report illustrates the usefulness of the combined use of classical virology procedures and modern molecular tools for the diagnosis of unexpected infections. It illustrates that astrovirus has the potential to cause severe disseminated lethal infection in highly immunocompromised pediatric patients.

Rhinovirus genome evolution during experimental human infection.

Human rhinoviruses (HRVs) evolve rapidly due in part to their error-prone RNA polymerase. Knowledge of the diversity of HRV populations emerging during the course of a natural infection is essential and represents a basis for the design of future potential vaccines and antiviral drugs. To evaluate HRV evolution in humans, nasal wash samples were collected daily for five days from 15 immunocompetent volunteers experimentally infected with a reference stock of HRV-39. In parallel, HeLa-OH cells were inoculated to compare HRV evolution in vitro. Nasal wash in vivo assessed by real-time PCR showed a viral load that peaked at 48–72 h. Ultra-deep sequencing was used to compare the low-frequency mutation populations present in the HRV-39 inoculum in two human subjects and one HeLa-OH supernatant collected 5 days post-infection. The analysis revealed hypervariable mutation locations in VP2, VP3, VP1, 2C and 3C genes and conserved regions in VP4, 2A, 2B, 3A, 3B and 3D genes. These results were confirmed by classical sequencing of additional samples, both from inoculated volunteers and independent cell infections, and suggest that HRV inter-host transmission is not associated with a strong bottleneck effect. A specific analysis of the VP1 capsid gene of 15 human cases confirmed the high mutation incidence in this capsid region, but not in the antiviral drug-binding pocket. We could also estimate a mutation frequency in vivo of 3.4×10−4 mutations/nucleotides and 3.1×10−4 over the entire ORF and VP1 gene, respectively. In vivo, HRV generate new variants rapidly during the course of an acute infection due to mutations that accumulate in hot spot regions located at the capsid level, as well as in 2C and 3C genes.

The cis-acting replication elements define human enterovirus and rhinovirus species

Replication of picornaviruses is dependent on VPg uridylylation, which is linked to the presence of the internal cis-acting replication element (cre). Cre are located within the sequence encoding polyprotein, yet at distinct positions as demonstrated for poliovirus and coxsackievirus-B3, cardiovirus, and human rhinovirus (HRV-A and HRV-B), overlapping proteins 2C, VP2, 2A, and VP1, respectively. Here we report a novel distinct cre element located in the VP2 region of the recently reported HRV-A2 species and provide evolutionary evidence of its functionality. We also experimentally interrogated functionality of recently identified HRV-B cre in the 2C region that is orthologous to the human enterovirus (HEV) cre and show that it is dispensable for replication and appears to be a nonfunctional evolutionary relic. In addition, our mutational analysis highlights two amino acids in the 2C protein that are crucial for replication. Remarkably, we conclude that each genetic clade of HRV and HEV is characterized by a unique functional cre element, where evolutionary success of a new genetic lineage seems to be associated with an invention of a novel cre motif and decay of the ancestral one. Therefore, we propose that cre element could be considered as an additional criterion for human rhinovirus and enterovirus classification.

Rhinovirus Genome Variation during Chronic Upper and Lower Respiratory Tract Infections

Routine screening of lung transplant recipients and hospital patients for respiratory virus infections allowed to identify human rhinovirus (HRV) in the upper and lower respiratory tracts, including immunocompromised hosts chronically infected with the same strain over weeks or months. Phylogenetic analysis of 144 HRV-positive samples showed no apparent correlation between a given viral genotype or species and their ability to invade the lower respiratory tract or lead to protracted infection. By contrast, protracted infections were found almost exclusively in immunocompromised patients, thus suggesting that host factors rather than the virus genotype modulate disease outcome, in particular the immune response. Complete genome sequencing of five chronic cases to study rhinovirus genome adaptation showed that the calculated mutation frequency was in the range observed during acute human infections. Analysis of mutation hot spot regions between specimens collected at different times or in different body sites revealed that non-synonymous changes were mostly concentrated in the viral capsid genes VP1, VP2 and VP3, independent of the HRV type. In an immunosuppressed lung transplant recipient infected with the same HRV strain for more than two years, both classical and ultra-deep sequencing of samples collected at different time points in the upper and lower respiratory tracts showed that these virus populations were phylogenetically indistinguishable over the course of infection, except for the last month. Specific signatures were found in the last two lower respiratory tract populations, including changes in the 5′UTR polypyrimidine tract and the VP2 immunogenic site 2. These results highlight for the first time the ability of a given rhinovirus to evolve in the course of a natural infection in immunocompromised patients and complement data obtained from previous experimental inoculation studies in immunocompetent volunteers.

Clinical features and viral kinetics in a rapidly cured patient with Ebola virus disease: a case report

BACKGROUND A detailed description of viral kinetics, duration of virus shedding, and intraviral evolution in different body sites is warranted to understand Ebola virus pathogenesis. Patients with Ebola virus infections admitted to university hospitals provide a unique opportunity to do such in-depth virological investigations. We describe the clinical, biological, and virological follow-up of a case of Ebola virus disease. METHODS A 43-year-old medical doctor who contracted an Ebola virus infection in Sierra Leone on Nov 16, 2014 (day 1), was airlifted to Geneva University Hospitals, Geneva, Switzerland, on day 5 after disease onset. The patient received an experimental antiviral treatment of monoclonal antibodies (ZMAb) and favipiravir. We monitored daily viral load kinetics, estimated viral clearance, calculated the half-life of the virus in plasma, and analysed the viral genome via high-throughput sequencing, in addition to clinical and biological signs. FINDINGS The patient recovered rapidly, despite an initial high viral load (about 1 × 10(7) RNA copies per mL 24 h after onset of fever). We noted a two-phase viral decay. The virus half-life decreased from about 26 h to 9·5 h after the experimental antiviral treatment. Compared with a consensus sequence of June 18, 2014, the isolate that infected this patient displayed only five synonymous nucleotide substitutions on the full genome (4901A→C, 7837C→T, 8712A→G, 9947T→C, 16201T→C) despite 5 months of human-to-human transmission. INTERPRETATION This study emphasises the importance of virological investigations to fully understand the course of Ebola virus disease and adaptation of the virus. Whether the viral decay was caused by the effects of the immune response alone, an additional benefit from the antiviral treatment, or a combination of both is unclear. In-depth virological analysis and randomised controlled trials are needed before any conclusion on the potential effect of antiviral treatment can be drawn. FUNDING Geneva University Hospitals, Swiss Office of Public Health, Swiss Agency for Development and Cooperation, and Swiss National Science Foundation.