First insights into Puumala orthohantavirus circulation in a rodent population in Alsace, France.

In-depth knowledge on the mechanisms that maintain infection by a zoonotic pathogen in an animal reservoir is the key to predicting and preventing transmission to humans. The Puumala orthohantavirus (PUUV), the most prevalent orthohantavirus in Western Europe, causes a mild form of haemorrhagic fever with renal syndrome (HFRS) in humans. In France, this endemic illness affects the north-eastern part of the country. We conducted a 4-year capture-mark-recapture study in a bank vole population, combined with molecular analyses, to explore the epidemiological situation of PUUV in Alsace, a French region where human cases have occurred, but for which no studies have been conducted on this reservoir host. PUUV-infected bank voles were detected in the 2 years that showed high bank vole density with a prevalence of 4%. The individual PUUV sequences identified in this study were similar from year to year and similar to other French sequences. On a very small spatial scale, the distribution of seropositive bank voles was very heterogeneous in time and space. The short distances travelled on average by bank voles resulted in spatial clusters of seropositive rodents, which spread only very gradually throughout the year.

Bank vole immunoheterogeneity may limit Nephropatia Epidemica emergence in a French non-endemic region.

Ecoevolutionary processes affecting hosts, vectors and pathogens are important drivers of zoonotic disease emergence. In this study, we focused on nephropathia epidemica (NE), which is caused by Puumala hantavirus (PUUV) whose natural reservoir is the bank vole, Myodes glareolus. We questioned the possibility of NE emergence in a French region that is considered to be NE-free but that is adjacent to a NE-endemic region. We first confirmed the epidemiology of these two regions and we demonstrated the absence of spatial barriers that could have limited dispersal, and consequently, the spread of PUUV into the NE-free region. We next tested whether regional immunoheterogeneity could impact PUUV chances to circulate and persist in the NE-free region. We showed that bank voles from the NE-free region were sensitive to experimental PUUV infection. We observed high levels of immunoheterogeneity between individuals and also between regions. Antiviral gene expression (Tnf and Mx2) reached higher levels in bank voles from the NE-free region. During experimental infections, anti-PUUV antibody production was higher in bank voles from the NE-endemic region. These results indicated a lower susceptibility to PUUV for bank voles from this NE-free region, which might limit PUUV persistence and therefore, the risk of NE.

Molecular diagnostic and genetic characterization of highly pathogenic viruses: application during Crimean-Congo haemorrhagic fever virus outbreaks in Eastern Europe and the Middle East.

Several haemorrhagic fevers are caused by highly pathogenic viruses that must be handled in Biosafety level 4 (BSL-4) containment. These zoonotic infections have an important impact on public health and the development of a rapid and differential diagnosis in case of outbreak in risk areas represents a critical priority. We have demonstrated the potential of a DNA resequencing microarray (PathogenID v2.0) for this purpose. The microarray was first validated in vitro using supernatants of cells infected with prototype strains from five different families of BSL-4 viruses (e.g. families Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae and Paramyxoviridae). RNA was amplified based on isothermal amplification by Phi29 polymerase before hybridization. We were able to detect and characterize Nipah virus and Crimean-Congo haemorrhagic fever virus (CCHFV) in the brains of experimentally infected animals. CCHFV was finally used as a paradigm for epidemics because of recent outbreaks in Turkey, Kosovo and Iran. Viral variants present in human sera were characterized by BLASTN analysis. Sensitivity was estimated to be 10(5) -10(6) PFU/mL of hybridized cDNA. Detection specificity was limited to viral sequences having ~13-14% of global divergence with the tiled sequence, or stretches of ~20 identical nucleotides. These results highlight the benefits of using the PathogenID v2.0 resequencing microarray to characterize geographical variants in the follow-up of haemorrhagic fever epidemics; to manage patients and protect communities; and in cases of bioterrorism.

[Long-term seroprotection against Japanese encephalitis using an inactivated vaccine (Jevax)]. / Séroprotection à long terme avec le vaccin inactivé contre l'encéphalite japonaise (Jevax).

Japanese encephalitis vaccine (Jevax) is an inactivated vaccine using the Nakayama viral strain. Until 2007, Jevax was the only Japanese encephalitis vaccine available in France but the duration of seroprotection after vaccination and exact timing of booster injections was unclear for travelers from non-endemic areas. The purpose of this report is to describe the results of a retrospective study in which neutralizing antibody levels were measured in 71 subjects previously vaccinated with Jevax. All subjects underwent testing at the Pasteur Institute Medical Center as part of preparation for humanitarian missions to endemic Japanese encephalitis areas in 2005-2006. A neutralizing antibody level greater than or equal to 20 was considered as protective. Findings showed that 49 of the 71 subjects (69%) still had protective antibody levels at a median of 4 years after the last Jevax immunization. In multivariate analysis, the only factor correlated with long-term seroprotection was the total number of vaccinations received. Based on these findings, it was concluded that long-term seroprotection after Jevax vaccination requires repeated booster injections even in subjects frequently exposed to the virus. No correlation was found between seroprotection and the interval between the booster injections.

West Nile virus in wild resident birds, Southern France, 2004.

An equine West Nile virus (WNV) outbreak occurred in 2004 in the Camargue, a wetland area in the south of France where the virus was first reported in 1962 and re-emerged in 2000. WNV neutralizing antibodies were detected in resident birds and two isolates from a House Sparrow (Passer domesticus) and a Common Magpie (Pica pica) were completely sequenced. Phylogenetic analyses revealed that these isolates are closely related to strains previously found in horses in southern Europe and North Africa. More extensive investigation is required to determine whether WNV has been re-introduced or has become endemic in the Camargue.

Evolutionary relationship between Old World West Nile virus strains. Evidence for viral gene flow between Africa, the Middle East, and Europe.

Little is known about the genetic relationships between European and other Old-World strains of West Nile virus (WNV) and persistence of WNV North of Mediterranean. We characterized the complete genomes of three WNV strains from France (horse-2000), Tunisia (human-1997) and Kenya (mosquito-1998), and the envelope, NS3 and NS5 genes of the Koutango virus. Phylogenetic analyses including all available full-length sequences showed that: (1) Koutango virus is a distant variant of WNV; (2) the three characterized strains belong to lineage 1, clade 1a; (3) the Tunisian strain roots the lineage of viruses introduced in North America. We established that currently available partial envelope sequences do not generate reliable phylogenies. Accordingly, establishing a large WNV sequence database is pivotal for the understanding of spatial and temporal epidemiology of this virus. For rapid completion of that purpose, colinearized E-NS3-NS5 gene sequences were shown to constitute a valuable surrogate for complete sequences.

West Nile outbreak in horses in southern France, 2000: the return after 35 years.

On September 6, 2000, two cases of equine encephalitis caused by West Nile (WN) virus were reported in southern France (Hérault Province), near Camargue National Park, where a WN outbreak occurred in 1962. Through November 30, 76 cases were laboratory confirmed among 131 equines with neurologic disorders. The last confirmed case was on November 3, 2000. All but three cases were located in a region nicknamed "la petite Camargue," which has several large marshes, numerous colonies of migratory and resident birds, and large mosquito populations. No human case has been confirmed among clinically suspected patients, nor have abnormal deaths of birds been reported. A serosurvey has been undertaken in horses in the infected area, and other studies are in progress.

West Nile in the Mediterranean basin: 1950-2000.

Recent West Nile virus (WNV) outbreaks have occurred in the Mediterranean basin. In Algeria in 1994, about 50 human cases of WN encephalitis were suspected, including 8 fatal cases. In Morocco in 1996, 94 equines were affected of which 42 died. In Tunisia in 1997, 173 patients were hospitalized for encephalitis or meningoencephalitis. West Nile serology performed on 129 patients was positive in 111 cases (87%) including 5 fatal cases. In Italy in 1998, 14 horses located in Tuscany were laboratory confirmed for WNV infection; 6 animals died. In Israel in 1998, serum samples from horses suffering from encephalomyelitis had WNV antibodies and virus was isolated from the brain of a stork; in 1999 WNV was identified in commercial geese flocks, and in 2000 hundreds of human cases have been reported. In September 2000, WNV infection was detected in horses located in southern France, close to the Camargue National Park where a WNV outbreak occurred in 1962. By November 30, 76 cases were laboratory confirmed among 131 equines presenting with neurological disorders. No human case has been laboratory confirmed among clinically suspect patients. The virus isolated from a brain biopsy is closely related to the Morocco-1996 and Italy-1998 isolates from horses, to the Senegal-1993 and Kenya-1998 isolates from mosquitoes, and to the human isolate from Volgograd-1999. It is distinguishable from the group including the Israel-1998 and New York-1999 isolates, as well as the Tunisia-1997 human isolate.

[West Nile viral meningo-encephalitis in Tunisia]. / Méningo-encéphalite à arbovirus West Nile en Tunisie.

In autumn 1997 an epidemic outbreak of meningoencephalitis was observed in two coastal districts of Tunisia. A total of 173 cases were recorded with 8 deaths. Detection with IgM capture and indirect IgG ELISAs demonstrated West Nile virus infection in 86% of patients from whom specimens were collected. West Nile is endemic in Asia and Sub-Saharan Africa. Epidemics in humans and horses have also been reported in the Mediterranean region and southern European countries. However this is the first report in Tunisia. Special West Nile virus surveillance is necessary especially in countries at high-risk for repeated introduction of this arbovirus.

Identification of Ebola virus sequences present as RNA or DNA in organs of terrestrial small mammals of the Central African Republic.

The life cycle of the Ebola (EBO) virus remains enigmatic. We tested for EBO virus in the organs of 242 small mammals captured during ecological studies in the Central African Republic. EBO virus glycoprotein or polymerase gene sequences were detected by reverse transcription PCR in RNA extracts of the organs of seven animals and by PCR in DNA extract of one animal. Neither live virus nor virus antigen was detected in any organ sample. Direct sequencing of amplicons identified the virus as being of the Zaire/Gabon subtype. Virus-like nucleocapsids were observed by electron microscopy in the cytoplasm of the spleen cells of one animal. The animals belonged to two genera of rodents (Muridae; Mus setulosus, Praomys sp1 and P. sp2) and one species of shrew (Soricidae; Sylvisorex ollula). These preliminary results provide evidence that common terrestrial small mammals living in peripheral forest areas have been in contact with the EBO virus and demonstrate the persistence of EBO virus RNA and DNA in the organs of the animals. Our findings should lead to better targeting of research into the life cycle of the EBO virus.