Publications by authors named "Patricia Gil"

27 Publications

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A library preparation optimized for metagenomics of RNA viruses.

Mol Ecol Resour 2021 Mar 13. Epub 2021 Mar 13.

ASTRE, Cirad, INRAE, University of Montpellier, Montpellier, France.

Our understanding of the viral communities associated to animals has not yet reached the level attained on the bacteriome. This situation is due to, among others, technical challenges in adapting metagenomics using high-throughput sequencing to the study of RNA viromes in animals. Although important developments have been achieved in most steps of viral metagenomics, there is yet a key step that has received little attention: the library preparation. This situation differs from bacteriome studies in which developments in library preparation have largely contributed to the democratisation of metagenomics. Here, we present a library preparation optimized for metagenomics of RNA viruses from insect vectors of viral diseases. The library design allows a simple PCR-based preparation, such as those routinely used in bacterial metabarcoding, that is adapted to shotgun sequencing as required in viral metagenomics. We first optimized our library preparation using mock viral communities and then validated a full metagenomic approach incorporating our preparation in two pilot studies with field-caught insect vectors; one including a comparison with a published metagenomic protocol. Our approach provided a fold increase in virus-like sequences compared to other studies, and nearly-full genomes from new virus species. Moreover, our results suggested conserved trends in virome composition within a population of a mosquito species. Finally, the sensitivity of our approach was compared to a commercial diagnostic PCR for the detection of an arbovirus in field-caught insect vectors. Our approach could facilitate studies on viral communities from animals and the democratization of metagenomics in community ecology of viruses.
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http://dx.doi.org/10.1111/1755-0998.13378DOI Listing
March 2021

The Genome Segments of Bluetongue Virus Differ in Copy Number in a Host-Specific Manner.

J Virol 2020 12 9;95(1). Epub 2020 Dec 9.

CIRAD, UMR ASTRE, Montpellier, France

Genome segmentation is mainly thought to facilitate reassortment. Here, we show that segmentation can also allow differences in segment abundance in populations of bluetongue virus (BTV). BTV has a genome consisting in 10 segments, and its cycle primarily involves periodic alternation between ruminants and biting midges. We have developed a reverse transcription-quantitative PCR (RT-qPCR) approach to quantify each segment in wild BTV populations sampled in both ruminants and midges during an epizootic. Segment frequencies deviated from equimolarity in all hosts. Interestingly, segment frequencies were reproducible and distinct between ruminants and biting midges. Beyond a putative regulatory role in virus expression, this phenomenon could lead to different evolution rates between segments. The variation in viral gene frequencies remains a largely unexplored aspect of within-host genetics. This phenomenon is often considered to be specific to multipartite viruses. Multipartite viruses have segmented genomes, but in contrast to segmented viruses, their segments are each encapsidated alone in a virion. A main hypothesis explaining the evolution of multipartism is that, compared to segmented viruses, it facilitates the regulation of segment abundancy, and the genes the segments carry, within a host. These differences in gene frequencies could allow for expression regulation. Here, we show that wild populations of a segmented virus, bluetongue virus (BTV), also present unequal segment frequencies. BTV cycles between ruminants and biting midges. As expected from a role in expression regulation, segment frequencies tended to show specific values that differed between ruminants and midges. Our results expand previous knowledge on gene frequency variation and call for studies on its role and conservation beyond multipartite viruses.
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http://dx.doi.org/10.1128/JVI.01834-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737730PMC
December 2020

Identification of Umbre Orthobunyavirus as a Novel Zoonotic Virus Responsible for Lethal Encephalitis in 2 French Patients with Hypogammaglobulinemia.

Clin Infect Dis 2020 Jun 9. Epub 2020 Jun 9.

Pathogen Discovery Laboratory, Institut Pasteur, Paris, France.

Background: Human encephalitis represents a medical challenge from a diagnostic and therapeutic point of view. We investigated the cause of 2 fatal cases of encephalitis of unknown origin in immunocompromised patients.

Methods: Untargeted metatranscriptomics was applied on the brain tissue of 2 patients to search for pathogens (viruses, bacteria, fungi, or protozoans) without a prior hypothesis.

Results: Umbre arbovirus, an orthobunyavirus never previously identified in humans, was found in 2 patients. In situ hybridization and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) showed that Umbre virus infected neurons and replicated at high titers. The virus was not detected in cerebrospinal fluid by RT-qPCR. Viral sequences related to Koongol virus, another orthobunyavirus close to Umbre virus, were found in Culex pipiens mosquitoes captured in the south of France where the patients had spent some time before the onset of symptoms, demonstrating the presence of the same clade of arboviruses in Europe and their potential public health impact. A serological survey conducted in the same area did not identify individuals positive for Umbre virus. The absence of seropositivity in the population may not reflect the actual risk of disease transmission in immunocompromised individuals.

Conclusions: Umbre arbovirus can cause encephalitis in immunocompromised humans and is present in Europe.
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http://dx.doi.org/10.1093/cid/ciaa308DOI Listing
June 2020

Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus.

Infect Genet Evol 2019 10 11;74:103917. Epub 2019 Jun 11.

Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, United Kingdom.

Several Avian paramyxoviruses 1 (synonymous with Newcastle disease virus or NDV, used hereafter) classification systems have been proposed for strain identification and differentiation. These systems pioneered classification efforts; however, they were based on different approaches and lacked objective criteria for the differentiation of isolates. These differences have created discrepancies among systems, rendering discussions and comparisons across studies difficult. Although a system that used objective classification criteria was proposed by Diel and co-workers in 2012, the ample worldwide circulation and constant evolution of NDV, and utilization of only some of the criteria, led to identical naming and/or incorrect assigning of new sub/genotypes. To address these issues, an international consortium of experts was convened to undertake in-depth analyses of NDV genetic diversity. This consortium generated curated, up-to-date, complete fusion gene class I and class II datasets of all known NDV for public use, performed comprehensive phylogenetic neighbor-Joining, maximum-likelihood, Bayesian and nucleotide distance analyses, and compared these inference methods. An updated NDV classification and nomenclature system that incorporates phylogenetic topology, genetic distances, branch support, and epidemiological independence was developed. This new consensus system maintains two NDV classes and existing genotypes, identifies three new class II genotypes, and reduces the number of sub-genotypes. In order to track the ancestry of viruses, a dichotomous naming system for designating sub-genotypes was introduced. In addition, a pilot dataset and sub-trees rooting guidelines for rapid preliminary genotype identification of new isolates are provided. Guidelines for sequence dataset curation and phylogenetic inference, and a detailed comparison between the updated and previous systems are included. To increase the speed of phylogenetic inference and ensure consistency between laboratories, detailed guidelines for the use of a supercomputer are also provided. The proposed unified classification system will facilitate future studies of NDV evolution and epidemiology, and comparison of results obtained across the world.
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http://dx.doi.org/10.1016/j.meegid.2019.103917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876278PMC
October 2019

Identification of Eilat virus and prevalence of infection among Culex pipiens L. populations, Morocco, 2016.

Virology 2019 04 12;530:85-88. Epub 2019 Feb 12.

Department of animal pathology and public health. Hassan II Agronomy & Veterinary Medicine Institute, Rabat, Morocco. Electronic address:

Eilat virus (EILV) is described as one of the few alphaviruses restricted to insects. We report the record of a nearly-complete sequence of an alphavirus genome showing 95% identity with EILV during a metagenomic analysis performed on 1488 unblood-fed females and 1076 larvae of the mosquito Culex pipiens captured in Rabat (Morocco). Genetic distance and phylogenetic analyses placed the EILV-Morocco as a variant of EILV. The observed infection rates in both larvae and adults suggested an active circulation of the virus in Rabat and its maintenance in the environment either through vertical transmission or through horizontal infection of larvae in breeding sites. This is the first report of EILV out of Israel and in Culex pipiens populations.
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http://dx.doi.org/10.1016/j.virol.2019.02.007DOI Listing
April 2019

Can genotype mismatch really affect the level of protection conferred by Newcastle disease vaccines against heterologous virulent strains?

Vaccine 2018 06 26;36(27):3917-3925. Epub 2018 May 26.

CIRAD, UMR ASTRE, F-34398 Montpellier, France; CIRAD, UMR ASTRE, F-97170 Petit-Bourg, Guadeloupe, France. Electronic address:

Newcastle disease (ND), caused by virulent class II avian paramyxovirus 1 (Newcastle disease virus, NDV), occurs sporadically in poultry despite their having been immunized with commercial vaccines. These vaccines were all derived from NDV strains isolated around 70 years ago. Since then, class II NDV strains have evolved into 18 genotypes. Whether the vaccination failure results from genotype mismatches between the currently used vaccine strains and field-circulating velogenic strains or from an impaired immune response in the vaccination remains unclear. To test the first hypothesis, we performed a heterologous genotype II vaccine/genotype XI challenge in one-day old specific pathogen free (SPF) chicks and reproduced viral shedding. We then produced two attenuated strains of genotype II and XI by reverse genetics and used them to immunize two-week old SPF chickens that were subsequently challenged with velogenic strains of genotypes II, VII and XI. We found that both vaccines could induce antibodies with hemagglutination inhibition titers higher than 6.5 log. Vaccination also completely prevented disease, viral shedding in swabs, and blocked viral replication in tissues from different genotypes in contrast to unvaccinated chickens that died shortly after challenge. Taken together, our results support the hypothesis that, in immunocompetent poultry, genotype mismatch is not the main reason for vaccination failure.
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http://dx.doi.org/10.1016/j.vaccine.2018.05.074DOI Listing
June 2018

Human Usutu Virus Infection with Atypical Neurologic Presentation, Montpellier, France, 2016.

Emerg Infect Dis 2018 05;24(5):875-878

Infection with Usutu virus (USUV) has been recently associated with neurologic disorders, such as encephalitis or meningoencephalitis, in humans. These findings indicate that USUV is a potential health threat. We report an acute human infection with USUV in France putatively associated with a clinical diagnosis of idiopathic facial paralysis.
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http://dx.doi.org/10.3201/eid2405.171122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5938765PMC
May 2018

Emergence of two Usutu virus lineages in Culex pipiens mosquitoes in the Camargue, France, 2015.

Infect Genet Evol 2018 07 26;61:151-154. Epub 2018 Mar 26.

Institut Pasteur, Biology of Infection Unit, Inserm U1117, Pathogen Discovery Laboratory, Paris, France.

Usutu virus (USUV) was detected in 11 Culex pipiens mosquito pools collected in 2015 in Camargue (France) using quantitative real-time RT-PCR assays. Phylogenetic analysis of recovered virus sequences identified USUV lineages Africa 2 and Africa 3, demonstrating the simultaneous occurrence of different strains within the mosquito population. This is the first report on USUV in mosquitoes from France that concurrently accompanied the emergence of Usutu virus in blackbirds and a human case in France during 2015/2016.
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http://dx.doi.org/10.1016/j.meegid.2018.03.020DOI Listing
July 2018

Cleavage site of Newcastle disease virus determines viral fitness in persistent infection cells.

Vet Microbiol 2018 Mar 9;216:123-131. Epub 2018 Feb 9.

CIRAD, UMR ASTRE, F-97170 Petit-Bourg, Guadeloupe, France; ASTRE, Univ Montpellier, CIRAD, INRA, Montpellier, France. Electronic address:

Newcastle disease, caused by infection with virulent strains of Newcastle disease virus (NDV), poses a risk for the poultry industry. The virulence of NDV is mainly determined by the cleavage site of F protein. Lentogenic NDV can become velogenic after passages in SPF chicken brain and air sac based on some strains isolated from water birds, because the proportion of virulent-related strains gradually increases. In contrast, this proportion remains unchanged if NDV is passaged via 10-day-old SPF chicken embryos. This information suggests that environmental conditions rather than mutation affect NDV fitness in quasispecies. However, it is unknown how the environment selects virulent-related strains from a viral population. In this study, velogenic and lentogenic NDV marked by green or red fluorescence were used to establish persistent infection (PI) in BHK-21 cells. Monitoring viruses by different methods, we found that, without competition, persistently infected cells harbored lentogenic and velogenic NDV strains similarly in terms of viral release, viral spread and the period of persistent viral infection. In contrast, under competitive co-infection, velogenic NDV became dominant in quasispecies from the fifth passage of PI cells, which resulted in the progressive disappearance of the lentogenic NDV strain. This domination was concomitant with a short-term reduction in the superinfection exclusion and supernatant interference in PI cells resulting in a velogenic virus rebound. We concluded that virulent-related F protein cleavage site facilitates the spread and replication of NDV in conditions under which cells do not secret trypsin-like proteases and do not inhibit free virus infection, resulting in a gradual increase in virulent strains in quasispecies with the number of passages.
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http://dx.doi.org/10.1016/j.vetmic.2018.02.006DOI Listing
March 2018

Comparison of the efficiency of different newcastle disease virus reverse genetics systems.

J Virol Methods 2017 11 1;249:111-116. Epub 2017 Sep 1.

CIRAD, UMR ASTRE, F-97170 Petit-Bourg, Guadeloupe, France; INRA, UMR1309 ASTRE, F-34398 Montpellier, France. Electronic address:

Rescue of negative-sense single-stranded RNA viruses ((-)ssRNA virus), generally requires the handling of a large number of plasmids to provide the virus genome and essential components for gene expression and genome replication. This constraint probably renders reverse genetics of (-)ssRNA virus more complex and less efficient. Some authors have shown that the fewer the plasmids, the more efficient reverse genetics is for segmented RNA virus. However, it is not clear if the same applies for (-)ssRNA, such as Newcastle disease virus (NDV). To address this issue, six variants of NDV reverse genetic systems were established by cloning combinations of NP, P and L genes, mini-genome or full-genome in 4, 3, 2 and 1 plasmid. In terms of mini-genome and full-genome rescue, we showed that only the 2-plasmid system, assembling three support plasmids together, was able to improve the rescue efficiency over that of the conventional 4-plasmid system. These results may help establish and/or improve reverse genetics for other mononegaviruses.
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http://dx.doi.org/10.1016/j.jviromet.2017.08.024DOI Listing
November 2017

Two-plasmid system to increase the rescue efficiency of paramyxoviruses by reverse genetics: The example of rescuing Newcastle Disease Virus.

Virology 2017 09 6;509:42-51. Epub 2017 Jun 6.

CIRAD, UMR ASTRE, F-34398 Montpellier, France; INRA, UMR1309 ASTRE, F-34398 Montpellier, France.

Within paramyxoviruses, conventional reverse genetics require the transfection of a minimum of four plasmids: three to reconstruct the viral polymerase complex that replicates and expresses the virus genome delivered by a fourth plasmid. The successful transfection of four or more plasmids of different sizes into one cell and the subsequent generation of at least one viable and replicable viral particle is a rare event, which explains the low rescue efficiency, especially of low virulent viruses with reduced replication efficiency in cell lines. In this study, we report on an improved reverse genetics system developed for an avian paramyxovirus, Newcastle Disease Virus (NDV), in which the number of plasmids was reduced from four to two. Compared to the conventional method, the 2-plasmid system enables earlier and increased production of rescued viruses and, in addition, makes it possible to rescue viruses that it was not possible to rescue using the 4-plasmid system.
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http://dx.doi.org/10.1016/j.virol.2017.06.003DOI Listing
September 2017

Diverse detected in wild birds from Madagascar.

Eur J Wildl Res 2015 22;61(4):635-639. Epub 2015 May 22.

2CIRAD, UMR CMAEE, Montpellier, France.

To date, infectious bronchitis virus (IBV) is potentially found in wild birds of different species. This work reports the survey of coronaviruses in wild birds from Madagascar based on the targeting of a conserved genome sequence among different groups of CoVs. Phylogenetic analyses revealed the presence of in different species of Gruiformes, Passeriformes, Ciconiiformes, Anseriformes, and Charadriiformes. Furthermore, some sequences were related to various IBV strains. Aquatic and migratory birds may play an important role in the maintenance and spread of coronaviruses in nature, highlighting their possible contribution in the emergence of new coronavirus diseases in wild and domestic birds.
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http://dx.doi.org/10.1007/s10344-015-0931-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7087566PMC
May 2015

New avian paramyxoviruses type I strains identified in Africa provide new outcomes for phylogeny reconstruction and genotype classification.

PLoS One 2013 18;8(10):e76413. Epub 2013 Oct 18.

CIRAD, UMR CMAEE, Montpellier, France ; INRA, UMR1309 CMAEE, Montpellier, France.

Newcastle disease (ND) is one of the most lethal diseases of poultry worldwide. It is caused by an avian paramyxovirus 1 that has high genomic diversity. In the framework of an international surveillance program launched in 2007, several thousand samples from domestic and wild birds in Africa were collected and analyzed. ND viruses (NDV) were detected and isolated in apparently healthy fowls and wild birds. However, two thirds of the isolates collected in this study were classified as virulent strains of NDV based on the molecular analysis of the fusion protein and experimental in vivo challenges with two representative isolates. Phylogenetic analysis based on the F and HN genes showed that isolates recovered from poultry in Mali and Ethiopia form new groups, herein proposed as genotypes XIV and sub-genotype VIf with reference to the new nomenclature described by Diel's group. In Madagascar, the circulation of NDV strains of genotype XI, originally reported elsewhere, is also confirmed. Full genome sequencing of five African isolates was generated and an extensive phylogeny reconstruction was carried out based on the nucleotide sequences. The evolutionary distances between groups and the specific amino acid signatures of each cluster allowed us to refine the genotype nomenclature.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076413PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3799739PMC
August 2014

Serological and molecular investigation of Newcastle disease in household chicken flocks and associated markets in Eastern Shewa zone, Ethiopia.

Trop Anim Health Prod 2013 Mar 11;45(3):705-14. Epub 2012 Oct 11.

Faculty of Veterinary Science, Department of Production Animal Studies, University of Pretoria, Private Bag X04, Onderstepoort, 0110, Pretoria, South Africa.

Cross-sectional survey for Newcastle disease (ND) were conducted in nonvaccinated household flocks of village chickens to assess serological and virological ND status in households and associated live bird markets. In total, 1,899 sera and 460 pools of cloacal and tracheal swabs were sampled and tested using a commercial enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcriptase polymerase chain reaction (rRT-PCR), respectively. Additionally, paired cloacal and tracheal swabs from 1,269 individual chickens were collected from markets and tested using RT-PCR. The prevalence of households with at least one seropositive chicken was higher during the dry season (27.4 %) than during the wet season (17.4 %) (P = 0.003). Viral genome was detected in 14.2 % of households during the wet season using a fusion (F) gene assay and in 24.2 % of households during the dry season using a polymerase (L) gene assay that targets both class I and class II viruses. At the markets sampled, overall bird level prevalence was 4.9 % for period 1 (F gene assay), and 38.2 % and 27.6 % for periods 2 and 3, respectively (L gene assay). Partial sequencing of the F gene (239 bp) cleavage site indicated that the majority of the circulating strains exhibited motifs specific to virulent strains. Seroepidemiology coupled with molecular analysis can be a useful tool to assess the status of NDV infection. The village chicken population in Ethiopia is endemically infected with virulent NDV that pose a significant threat to emerging small- and medium-scale commercial poultry production.
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http://dx.doi.org/10.1007/s11250-012-0278-yDOI Listing
March 2013

Investigating avian influenza infection hotspots in old-world shorebirds.

PLoS One 2012 28;7(9):e46049. Epub 2012 Sep 28.

CIRAD-ES, UPR AGIRS, Montpellier, France.

Heterogeneity in the transmission rates of pathogens across hosts or environments may produce disease hotspots, which are defined as specific sites, times or species associations in which the infection rate is consistently elevated. Hotspots for avian influenza virus (AIV) in wild birds are largely unstudied and poorly understood. A striking feature is the existence of a unique but consistent AIV hotspot in shorebirds (Charadriiformes) associated with a single species at a specific location and time (ruddy turnstone Arenaria interpres at Delaware Bay, USA, in May). This unique case, though a valuable reference, limits our capacity to explore and understand the general properties of AIV hotspots in shorebirds. Unfortunately, relatively few shorebirds have been sampled outside Delaware Bay and they belong to only a few shorebird families; there also has been a lack of consistent oropharyngeal sampling as a complement to cloacal sampling. In this study we looked for AIV hotspots associated with other shorebird species and/or with some of the larger congregation sites of shorebirds in the old world. We assembled and analysed a regionally extensive dataset of AIV prevalence from 69 shorebird species sampled in 25 countries across Africa and Western Eurasia. Despite this diverse and extensive coverage we did not detect any new shorebird AIV hotspots. Neither large shorebird congregation sites nor the ruddy turnstone were consistently associated with AIV hotspots. We did, however, find a low but widespread circulation of AIV in shorebirds that contrast with the absence of AIV previously reported in shorebirds in Europe. A very high AIV antibody prevalence coupled to a low infection rate was found in both first-year and adult birds of two migratory sandpiper species, suggesting the potential existence of an AIV hotspot along their migratory flyway that is yet to be discovered.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046049PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460932PMC
March 2013

Circulation of avian influenza viruses in wild birds in Inner Niger Delta, Mali.

Influenza Other Respir Viruses 2012 Jul 15;6(4):240-4. Epub 2011 Dec 15.

CIRAD-ES, UR AGIRS, Montpellier, France.

Background: Avian influenza viruses (AIV) have been detected in wild birds in West Africa during the northern winter, but no information is available on a potential year-round circulation of AIV in West Africa. Such year-round circulation would allow reassortment opportunities between strains circulating in Afro-tropical birds and strains imported by migratory birds wintering in West Africa.

Objective And Method: A 2-year longitudinal survey was conducted in the largest continental wetland of West Africa, the Inner Niger Delta in Mali, to determine the year-round circulation of AIV in wild birds.

Results And Conclusions: Avian influenza virus RNA was detected during all periods of the year. Very low prevalence was detected during the absence of the migratory wild birds. However, a year-round circulation of AIV seems possible in West Africa, as shown in other African regions. West Africa may hence be another potential site of reassortment between AIV strains originating from both Afro-tropical and Eurasian regions.
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http://dx.doi.org/10.1111/j.1750-2659.2011.00314.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5779802PMC
July 2012

Genetic data from avian influenza and avian paramyxoviruses generated by the European network of excellence (EPIZONE) between 2006 and 2011--review and recommendations for surveillance.

Vet Microbiol 2012 Jan 25;154(3-4):209-21. Epub 2011 Aug 25.

Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy.

Since 2006, the members of the molecular epidemiological working group of the European "EPIZONE" network of excellence have been generating sequence data on avian influenza and avian paramyxoviruses from both European and African sources in an attempt to more fully understand the circulation and impact of these viruses. This review presents a timely update on the epidemiological situation of these viruses based on sequence data generated during the lifetime of this project in addition to data produced by other groups during the same period. Based on this information and putting it all into a European context, recommendations for continued surveillance of these important viruses within Europe are presented.
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http://dx.doi.org/10.1016/j.vetmic.2011.08.018DOI Listing
January 2012

Newcastle disease virus in Madagascar: identification of an original genotype possibly deriving from a died out ancestor of genotype IV.

PLoS One 2010 Nov 15;5(11):e13987. Epub 2010 Nov 15.

FOFIFA-DRZV, Antananarivo, Madagascar.

In Madagascar, Newcastle disease (ND) has become enzootic after the first documented epizootics in 1946, with recurrent annual outbreaks causing mortality up to 40%. Four ND viruses recently isolated in Madagascar were genotypically and pathotypically characterised. By phylogenetic inference based on the F and HN genes, and also full-genome sequence analyses, the NDV Malagasy isolates form a cluster distant enough to constitute a new genotype hereby proposed as genotype XI. This new genotype is presumably deriving from an ancestor close to genotype IV introduced in the island probably more than 50 years ago. Our data show also that all the previously described neutralising epitopes are conserved between Malagasy and vaccine strains. However, the potential implication in vaccination failures of specific amino acid substitutions predominantly found on surface-exposed epitopes of F and HN proteins is discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0013987PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2981552PMC
November 2010

Quantitative one-step real-time RT-PCR for the fast detection of the four genotypes of PPRV.

J Virol Methods 2010 May 29;165(2):168-77. Epub 2010 Jan 29.

CIRAD, UMR CIRAD/INRA Contrôle des Maladies, F-34398 Montpellier Cedex 5, France.

A one-step real-time Taqman RT-PCR assay (RRT-PCR) for peste des petits ruminants virus (PPRV) was developed to detect the four lineages of PPRV by targeting the nucleoprotein (N) gene of the virus. This new assay was compared to a conventional RT-PCR on reference strains and field materials. Quantitation was performed against a standard based on a synthetic transcript of the NPPR gene for which a minimum of 32 copies per reaction were detected with a corresponding C(t) value of 39. Depending on the lineage involved, the detection limit of RRT-PCR was decreased by one to three log copies relative to the conventional method. The lower stringency occurred with lineage III because of minor nucleotide mismatches within the probe region. The assay did not detect phylogenetically or symptomatically related viruses of ruminants (such as rinderpest, bluetongue, and bovine viral diarrhea viruses). However, it was capable of detecting 20% more positive field samples with low viral RNA loads compared to the conventional PCR method. When compared on a proficiency panel to the method developed by Bao et al. (2008), the sensitivity of the in-house assay was slightly improved on lineage II. It proved significantly faster to perform and hence better adapted for monitoring large numbers of at risk or diseased animals.
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http://dx.doi.org/10.1016/j.jviromet.2010.01.014DOI Listing
May 2010

Avian influenza in backyard poultry of the Mopti region, Mali.

Trop Anim Health Prod 2010 Jun 13;42(5):807-9. Epub 2009 Nov 13.

CIRAD, UPR AGIRs, Bamako, 1813, Mali.

This study reports the first evidence of circulation of avian influenza viruses (AIV) in domestic poultry in Mali. In the Mopti region, where AIV have already been isolated in migratory water birds, we sampled 223 backyard domestic birds potentially in contact with wild birds and found that 3.6% had tracheal or cloacal swabs positive by real-time reverse transcription PCR (rRT-PCR) for type A influenza viruses (IVA) and that 13.7% had sera positive by commercial ELISA test detecting antibodies against IVA. None of the birds positive by rRT-PCR for IVA was positive by rRT-PCR for H5 and H7 subtypes, and none showed any clinical signs therefore indicating the circulation of low pathogenic avian influenza. Unfortunately, no virus isolation was possible. Further studies are needed to assess the temporal evolution of AIV circulation in the Mopti region and its possible correlation with the presence of wild birds.
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http://dx.doi.org/10.1007/s11250-009-9497-2DOI Listing
June 2010

Evidence of infection by H5N2 highly pathogenic avian influenza viruses in healthy wild waterfowl.

PLoS Pathog 2008 Aug 15;4(8):e1000127. Epub 2008 Aug 15.

Centre de Coopération Internationale en Recherche Agronomique pour le Développement, Montpellier, France.

The potential existence of a wild bird reservoir for highly pathogenic avian influenza (HPAI) has been recently questioned by the spread and the persisting circulation of H5N1 HPAI viruses, responsible for concurrent outbreaks in migratory and domestic birds over Asia, Europe, and Africa. During a large-scale surveillance programme over Eastern Europe, the Middle East, and Africa, we detected avian influenza viruses of H5N2 subtype with a highly pathogenic (HP) viral genotype in healthy birds of two wild waterfowl species sampled in Nigeria. We monitored the survival and regional movements of one of the infected birds through satellite telemetry, providing a rare evidence of a non-lethal natural infection by an HP viral genotype in wild birds. Phylogenetic analysis of the H5N2 viruses revealed close genetic relationships with H5 viruses of low pathogenicity circulating in Eurasian wild and domestic ducks. In addition, genetic analysis did not reveal known gallinaceous poultry adaptive mutations, suggesting that the emergence of HP strains could have taken place in either wild or domestic ducks or in non-gallinaceous species. The presence of coexisting but genetically distinguishable avian influenza viruses with an HP viral genotype in two cohabiting species of wild waterfowl, with evidence of non-lethal infection at least in one species and without evidence of prior extensive circulation of the virus in domestic poultry, suggest that some strains with a potential high pathogenicity for poultry could be maintained in a community of wild waterfowl.
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http://dx.doi.org/10.1371/journal.ppat.1000127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2503949PMC
August 2008

Vaccination with Trypanosoma rangeli reduces the infectiousness of dogs experimentally infected with Trypanosoma cruzi.

Vaccine 2007 May 8;25(19):3855-8. Epub 2007 Mar 8.

Facultad de Ciencias Médicas, Universidad Nacional de Córdoba & Servicio Nacional de Chagas, Córdoba, Argentina.

The goal of this work was to test the efficacy of the vaccination with Trypanosoma rangeli in dogs. Mongrel dogs received three subcutaneous injections of fixed T. rangeli epimastigotes at 6-week intervals. Such immunisation induced antibodies against Trypanosoma cruzi. While both control and immunised dogs developed detectable parasitemia, this was lower and shorter in vaccinated animals. Interestingly, feeding of Triatoma infestans nymphs on vaccinated and chronically infected dogs led to a sharp reduction in the rate of bug infection. These results suggest that it might be possible to reduce the vectorial parasitemia through vaccination of dogs. As dogs are known to play a major role in the domestic cycle of T. cruzi, this might represent a strategy to reduce parasite transmission to humans.
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http://dx.doi.org/10.1016/j.vaccine.2007.01.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127752PMC
May 2007

Novel Vip3-related protein from Bacillus thuringiensis.

Appl Environ Microbiol 2005 Oct;71(10):6276-81

Cirad, TA 30/D, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France.

A novel vip3-related gene was identified in Bacillus thuringiensis. This novel gene is 2,406 bp long and codes for a 91-kDa protein (801 amino acids). This novel protein exhibits between 61 and 62% similarity with Vip3A proteins and is designated Vip3Ba1. Vip3Ba1 has several specific features. Differences between Vip3Ba1 and the Vip3A proteins are spread throughout the sequence but are more frequent in the C-terminal part from amino acid 456 onward. The regions containing the two proteolytic processing sites, which are highly conserved among the Vip3A toxins, are markedly different in Vip3Ba1. The pattern DCCEE (Asp Cys Cys Glu Glu) is repeated four times between position 463 and 483 in Vip3Ba1, generating the sequence 463-DCCEEDCCEEDCCEEDCCEE-483. This sequence, which is rich in negatively charged amino acids, also contains 73% of the cysteines present in Vip3Ba1. This repeated sequence is not present in Vip3A proteins. The Vip3Ba1protein was produced in Escherichia coli and tested against Ostrinia nubilalis and Plutella xylostella, and it generated significant growth delays but had no larvicidal effect, indicating that its host range might be different than that of Vip3A proteins.
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http://dx.doi.org/10.1128/AEM.71.10.6276-6281.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1265940PMC
October 2005

Analysis of the function of the photoreceptors phytochrome B and phytochrome D in Nicotiana plumbaginifolia and Arabidopsis thaliana.

Plant Cell Physiol 2005 May 7;46(5):790-6. Epub 2005 Mar 7.

Albert-Ludwigs-Universität Freiburg, Institut für Biologie II/ Botanik, Schänzlestrasse 1, 79104 Freiburg, Germany.

To investigate the mechanism of phytochrome action in vivo, NtPHYB, AtPHYB and phyD:green fluorescent protein (GFP) were overexpressed in Nicotiana plumbaginifolia and Arabidopsis thaliana. The expression of 35S:NtPHYB:GFP and 35S:AtPHYB:GFP complemented the tobacco hgl2 and Arabidopsis phyB-9 mutations, whereas the 35S:AtPHYD:GFP only rescued the hgl2 mutant. All three fusion proteins are transported into the nucleus in all genetic backgrounds. These data indicate that AtPHYD:GFP is biologically active and functions as the main red light receptor in transgenic tobacco, and establish an experimental system for the functional analysis of this elusive photoreceptor in vivo.
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http://dx.doi.org/10.1093/pcp/pci073DOI Listing
May 2005

The crystal structures of four peptide deformylases bound to the antibiotic actinonin reveal two distinct types: a platform for the structure-based design of antibacterial agents.

J Mol Biol 2002 Jul;320(5):951-62

Drug Innovation & Approval, Aventis Pharma, 13 Quai Jules Guesde, BP.14, F-94403, Vitry-sur-Seine, France.

Bacterial peptide deformylase (PDF) belongs to a sub-family of metalloproteases that catalyse the removal of the N-terminal formyl group from newly synthesised proteins. PDF is essential in prokaryotes and conserved throughout the eubacteria. It is therefore considered an attractive target for developing new antibacterial agents. Here, we report the crystal structures of four bacterial deformylases, free or bound to the naturally occurring antibiotic actinonin, including two from the major bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The overall tertiary structure is essentially conserved but shows significant differences, namely at the C terminus, which are directly related to the deformylase type (i.e. I or II) they belong to. The geometry around the catalytic metal ion exhibits a high level of similarity within the different enzymes, as does the binding mode of actinonin to the various deformylases. However, some significant structural differences are found in the vicinity of the active site, highlighting the structural and molecular requirements for the design of a deformylase inhibitor active against a broad spectrum of bacterial strains.
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http://dx.doi.org/10.1016/s0022-2836(02)00549-1DOI Listing
July 2002

Nucleocytoplasmic partitioning of the plant photoreceptors phytochrome A, B, C, D, and E is regulated differentially by light and exhibits a diurnal rhythm.

Plant Cell 2002 Jul;14(7):1541-55

Albert-Ludwigs-Universität Freiburg, Institut für Biologie II/Botanik, Schänzlestrasse 1, 79104 Freiburg, Germany.

The phytochrome family of plant photoreceptors has a central role in the adaptation of plant development to changes in ambient light conditions. The individual phytochrome species regulate different or partly overlapping physiological responses. We generated transgenic Arabidopsis plants expressing phytochrome A to E:green fluorescent protein (GFP) fusion proteins to assess the biological role of intracellular compartmentation of these photoreceptors in light-regulated signaling. We show that all phytochrome:GFP fusion proteins were imported into the nuclei. Translocation of these photoreceptors into the nuclei was regulated differentially by light. Light-induced accumulation of phytochrome species in the nuclei resulted in the formation of speckles. The appearance of these nuclear structures exhibited distinctly different kinetics, wavelengths, and fluence dependence and was regulated by a diurnal rhythm. Furthermore, we demonstrate that the import of mutant phytochrome B:GFP and phytochrome A:GFP fusion proteins, shown to be defective in signaling in vivo, is regulated by light but is not accompanied by the formation of speckles. These results suggest that (1) the differential regulation of the translocation of phytochrome A to E into nuclei plays a role in the specification of functions, and (2) the appearance of speckles is a functional feature of phytochrome-regulated signaling.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC150705PMC
http://dx.doi.org/10.1105/tpc.001156DOI Listing
July 2002