Valérie Caro

Emergence of Zaire Ebola Virus Disease in Guinea

In March 2014, the World Health Organization was notified of an outbreak of a communicable disease characterized by fever, severe diarrhea, vomiting, and a high fatality rate in Guinea. Virologic investigation identified Zaire ebolavirus (EBOV) as the causative agent. Full-length genome sequencing and phylogenetic analysis showed that EBOV from Guinea forms a separate clade in relationship to the known EBOV strains from the Democratic Republic of Congo and Gabon. Epidemiologic investigation linked the laboratory-confirmed cases with the presumed first fatality of the outbreak in December 2013. This study demonstrates the emergence of a new EBOV strain in Guinea.

Chikungunya virus, southeastern France.

In September 2010, autochthonous transmission of chikungunya virus was recorded in southeastern France, where the Aedes albopictus mosquito vector is present. Sequence analysis of the viral genomes of imported and autochthonous isolates indicated new features for the potential emergence and spread of the virus in Europe.

Clinical features and viral diagnosis of two cases of infection with Middle East Respiratory Syndrome coronavirus: a report of nosocomial transmission

Summary Background Human infection with a novel coronavirus named Middle East Respiratory Syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia and the Middle East in September, 2012, with 44 laboratory-confirmed cases as of May 23, 2013. We report detailed clinical and virological data for two related cases of MERS-CoV disease, after nosocomial transmission of the virus from one patient to another in a French hospital. Methods Patient 1 visited Dubai in April, 2013; patient 2 lives in France and did not travel abroad. Both patients had underlying immunosuppressive disorders. We tested specimens from the upper (nasopharyngeal swabs) or the lower (bronchoalveolar lavage, sputum) respiratory tract and whole blood, plasma, and serum specimens for MERS-CoV by real-time RT-PCR targeting the upE and Orf1A genes of MERS-CoV. Findings Initial clinical presentation included fever, chills, and myalgia in both patients, and for patient 1, diarrhoea. Respiratory symptoms rapidly became predominant with acute respiratory failure leading to mechanical ventilation and extracorporeal membrane oxygenation (ECMO). Both patients developed acute renal failure. MERS-CoV was detected in lower respiratory tract specimens with high viral load (eg, cycle threshold [Ct] values of 22·9 for upE and 24 for Orf1a for a bronchoalveolar lavage sample from patient 1; Ct values of 22·5 for upE and 23·9 for Orf1a for an induced sputum sample from patient 2), whereas nasopharyngeal specimens were weakly positive or inconclusive. The two patients shared the same room for 3 days. The incubation period was estimated at 9–12 days for the second case. No secondary transmission was documented in hospital staff despite the absence of specific protective measures before the diagnosis of MERS-CoV was suspected. Patient 1 died on May 28, due to refractory multiple organ failure. Interpretation Patients with respiratory symptoms returning from the Middle East or exposed to a confirmed case should be isolated and investigated for MERS-CoV with lower respiratory tract sample analysis and an assumed incubation period of 12 days. Immunosuppression should also be taken into account as a risk factor. Funding French Institute for Public Health Surveillance, ANR grant Labex Integrative Biology of Emerging Infectious Diseases, and the European Communitys Seventh Framework Programme projects EMPERIE and PREDEMICS.

Natural Genetic Exchanges between Vaccine and Wild Poliovirus Strains in Humans

ABSTRACT In a previous study of poliovirus vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis (VAPP) (9, 11), we reported that a high proportion (over 50%) of viruses had a recombinant genome. Most were intertypic vaccine/vaccine recombinants. However, some had restriction fragment length polymorphism (RFLP) profiles different from those of poliovirus vaccine strains. We demonstrate here that five such recombinants, of 88 VAPP strains examined, carried sequences of wild (nonvaccine) origin. To identify the parental wild donor of these sequences, we used RFLP profiles and nucleotide sequencing to look for similarity in the 3D polymerase-coding region of 61 wild, cocirculating poliovirus isolates (43 type 1, 16 type 2, and 2 type 3 isolates). In only one case was the donor identified, and it was a wild type 1 poliovirus. For the other four vaccine/wild recombinants, the wild parent could not be identified. The possibility that the wild sequences were of a non-poliovirus-enterovirus origin could not be excluded. Another vaccine/wild recombinant, isolated in Belarus from a VAPP case, indicated that the poliovirus vaccine/wild recombination is not an isolated phenomenon. We also found wild polioviruses (2 of 15) carrying vaccine-derived sequences in the 3′ moiety of their genome. All these results suggest that genetic exchanges with wild poliovirus and perhaps with nonpoliovirus enteroviruses, are also a natural means of evolution for poliovirus vaccine strains.

Human Skin Microbiota: High Diversity of DNA Viruses Identified on the Human Skin by High Throughput Sequencing

The human skin is a complex ecosystem that hosts a heterogeneous flora. Until recently, the diversity of the cutaneous microbiota was mainly investigated for bacteria through culture based assays subsequently confirmed by molecular techniques. There are now many evidences that viruses represent a significant part of the cutaneous flora as demonstrated by the asymptomatic carriage of beta and gamma-human papillomaviruses on the healthy skin. Furthermore, it has been recently suggested that some representatives of the Polyomavirus genus might share a similar feature. In the present study, the cutaneous virome of the surface of the normal-appearing skin from five healthy individuals and one patient with Merkel cell carcinoma was investigated through a high throughput metagenomic sequencing approach in an attempt to provide a thorough description of the cutaneous flora, with a particular focus on its viral component. The results emphasize the high diversity of the viral cutaneous flora with multiple polyomaviruses, papillomaviruses and circoviruses being detected on normal-appearing skin. Moreover, this approach resulted in the identification of new Papillomavirus and Circovirus genomes and confirmed a very low level of genetic diversity within human polyomavirus species. Although viruses are generally considered as pathogen agents, our findings support the existence of a complex viral flora present at the surface of healthy-appearing human skin in various individuals. The dynamics and anatomical variations of this skin virome and its variations according to pathological conditions remain to be further studied. The potential involvement of these viruses, alone or in combination, in skin proliferative disorders and oncogenesis is another crucial issue to be elucidated.

Nucleic Acid Amplification Tests for Diagnosis of Bordetella Infections

The global epidemiology of pertussis has recently been reviewed (12, 15). Bordetella pertussis continues to circulate even in populations where high vaccination coverage of infants and children is achieved (15, 23), because the protection after natural infection wanes after 10 to 15 years and protection after vaccination lasts for 6 to 10 years (15). A significant increase of pertussis cases was observed in the United States, in Europe, and in other countries with high vaccination coverage, making pertussis a reemerging disease. Transmission of the disease in highly vaccinated populations occurs mainly from adolescents and adults to infants or among older vaccinated children, adolescents, and adults (15). Thus, most cases of pertussis are now observed in unvaccinated infants, older schoolchildren, adolescents, and adults. In outbreak situations asymptomatic carriage has been observed in up to ∼50% (12).

Polymorphism of Bordetella pertussis Isolates Circulating for the Last 10 Years in France, Where a Single Effective Whole-Cell Vaccine Has Been Used for More than 30 Years

ABSTRACT We compared Bordetella pertussis isolates collected in France over the last 10 years, the vaccine strains used for more than 30 years, and isolates collected before the introduction of generalized vaccination. The analysis included serotyping, pulsed-field gel electrophoresis of chromosomal DNA after digestion withXbaI and SpeI, and sequencing of thept S1 gene, encoding the S1 subunit of pertussis toxin, and the prn gene, encoding the adhesin pertactin. We found that the incidence of infection increases every 3 years. Ninety-five per cent of the isolates analyzed express type 3 fimbriae. Most of the isolates circulating since 1991, unlike the vaccinal strains, express a type A pertussis toxin and a type 2 pertactin. The isolates could be classified into five major groups by pulsed-field gel electrophoresis. Most of these groups correlated with the pertactin type expressed by the isolates. Pulsed-field gel electrophoresis is more discriminative than sequencing particular genes since it could differentiate isolates expressing type 2 pertactin into two subgroups: those circulating in 1993 to 1997 and those circulating in 1997 to 2001. This observation suggests that there has been continuous evolution of the B. pertussis population.

Genomic Features of Intertypic Recombinant Sabin Poliovirus Strains Excreted by Primary Vaccinees

ABSTRACT The trivalent oral poliomyelitis vaccine (OPV) contains three different poliovirus serotypes. It use therefore creates particularly favorable conditions for mixed infection of gut cells, and indeed intertypic vaccine-derived recombinants (VdRec) have been frequently found in patients with vaccine-associated paralytic poliomyelitis. Nevertheless, there have not been extensive searches for VdRec in healthy vaccinees following immunization with OPV. To determine the incidence of VdRec and their excretion kinetics in primary vaccinees, and to establish the general genomic features of the corresponding recombinant genomes, we characterized poliovirus isolates excreted by vaccinees following primary immunization with OPV. Isolates were collected from 67 children 2 to 60 days following vaccination. Recombinant strains were identified by multiple restriction fragment length polymorphism assays. The localization of junction sites in recombinant genomes was also determined. VdRec excreted by vaccinees were first detected 2 to 4 days after vaccination. The highest rate of recombinants was on day 14. The frequency of VdRec depends strongly on the serotype of the analyzed isolates (2, 53, and 79% of recombinant strains in the last-excreted type 1, 2, and 3 isolates, respectively). Particular associations of genomic segments were preferred in the recombinant genomes, and recombination junctions were found in the genomic region encoding the nonstructural proteins. Recombination junctions generally clustered in particular subgenomic regions that were dependent on the serotype of the isolate and/or on the associations of genomic segments in recombinants. Thus, VdRec are frequently excreted by vaccinees, and the poliovirus replication machinery requirements or selection factors appear to act in vivo to shape the features of the recombinant genomes.

Evaluation of High-Throughput Sequencing for Identifying Known and Unknown Viruses in Biological Samples

ABSTRACT High-throughput sequencing furnishes a large number of short sequence reads from uncloned DNA and has rapidly become a major tool for identifying viruses in biological samples, and in particular when the target sequence is undefined. In this study, we assessed the analytical sensitivity of a pipeline for detection of viruses in biological samples based on either the Roche-454 genome sequencer or Illumina genome analyzer platforms. We sequenced biological samples artificially spiked with a wide range of viruses with genomes composed of single or double-stranded DNA or RNA, including linear or circular single-stranded DNA. Viruses were added at a very low concentration most often corresponding to 3 or 0.8 times the validated level of detection of quantitative reverse transcriptase PCRs (RT-PCRs). For the viruses represented, or resembling those represented, in public nucleotide sequence databases, we show that the higher output of Illumina is associated with a much greater sensitivity, approaching that of optimized quantitative (RT-)PCRs. In this blind study, identification of viruses was achieved without incorrect identification. Nevertheless, at these low concentrations, the number of reads generated by the Illumina platform was too small to facilitate assembly of contigs without the use of a reference sequence, thus precluding detection of unknown viruses. When the virus load was sufficiently high, de novo assembly permitted the generation of long contigs corresponding to nearly full-length genomes and thus should facilitate the identification of novel viruses.

Natural genetic recombination between co-circulating heterotypic enteroviruses

Natural recombination in poliovirus is a frequent phenomenon. In practice, whenever different genotypes have the opportunity to infect the same individual, a high proportion of viruses with recombinant genomes are excreted. To determine whether enteroviruses other than poliovirus can naturally produce viable virions with recombinant genomes, we studied the molecular features of two distant regions of the viral genomes - the VP1 coding region and the 3D polymerase coding region - of the echovirus serotypes associated with a large outbreak of aseptic meningitis. Nucleotide sequences of nine epidemic strains [belonging to echovirus serotypes 4 (E4), 7 (E7) and 30 (E30)] in the two genomic regions (300 nt of VP1 and 520 nt of 3D polymerase) were compared to prototype and field strains, and phylogenetic trees were generated from alignments. In the VP1 region, each of the three epidemic serotypes clustered with the homotypic prototype strain, whereas in the 3D polymerase region, E7 and E30 grouped as a single cluster, distant from the two corresponding prototype strains. This suggests that one of these two E7 and E30 strains has evolved through recombination with the other or that both have acquired the 3D polymerase coding region from a common ancestor. Our results suggest that such genetic recombinations between different echovirus serotypes are possible when multiple epidemic strains are circulating simultaneously.