Publications by authors named "Yannick Blanchard"

49 Publications

Characterization of a Cell Culture System of Persistent Hepatitis E Virus Infection in the Human HepaRG Hepatic Cell Line.

Viruses 2021 Mar 4;13(3). Epub 2021 Mar 4.

UMR 1161 Virologie, INRAE, ANSES, Ecole Nationale Vétérinaire d'Alfort, Université Paris-Est, 94700 Maisons-Alfort, France.

Hepatitis E virus (HEV) is considered as an emerging global health problem. In most cases, hepatitis E is a self-limiting disease and the virus is cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals can develop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. The lack of efficient and relevant cell culture system and animal models has limited our understanding of the biology of HEV and the development of effective drugs for chronic cases. In the present study, we developed a model of persistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture system is based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering from acute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after seven successive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, we found that the virus was still infectious after oral inoculation into pigs. We also showed that ribavirin had an inhibitory effect on HEV replication in HepaRG. In conclusion, this system represents a relevant and efficient in vitro model of HEV replication that could be useful to study HEV biology and identify effective antiviral drugs against chronic HEV infection.
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http://dx.doi.org/10.3390/v13030406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001476PMC
March 2021

Comparison of the Staphylococcal Chromosome Cassette (SCC) in Methicillin-Resistant (MRSA) and Non- Staphylococci (MRNAS) from Animals and Humans.

Antibiotics (Basel) 2021 Mar 4;10(3). Epub 2021 Mar 4.

Bacteriology, Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine and Institute for Fundamental and Applied Research in Animals and Health (FARAH), University of Liège, Quartier Vallée 2, Avenue de Cureghem 6, 4000 Liège, Belgium.

Methicillin-resistant (MRSA) and non- staphylococci (MRNAS) cause different infections in animals, including mastitis, in livestock and humans. This study aimed to identify and compare the staphylococcal chromosome cassette (SCC) types of MRSA or MRNAS isolated from several animal species and humans in different countries. Of 1462 and non- staphylococci, 68 grew on Chrom MRSA ID agar, were phenotypically resistant to cefoxitin and tested positive with the PCR for the gene. These 60 MRSA and 8 MRNAS were isolated in Belgium mainly from cows (livestock-associated (LA) MRS) and humans (community-acquired (CA) MRS) and in Japan from dogs and cats. The SCC cassettes were identified by multiplex PCR in 52 MRSA and 7 MRNAS and by whole genome sequencing (WGS) in 8 additional MRSA. The SCC types IV and V were the most frequent in Belgian LA-MRS and CA-MRS, while the SCC type II was identified in four of the five Japanese MRSA. The remaining isolate was a bovine in which no SCC was identified. These results confirm the high prevalence of the SCC types IV and V in LA-MRS and CA-MRS in Belgium, emphasizing the possible public health hazard of the former, and the absence of SCC in some MRNAS.
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http://dx.doi.org/10.3390/antibiotics10030256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998684PMC
March 2021

Can shellfish be used to monitor SARS-CoV-2 in the coastal environment?

Sci Total Environ 2021 Mar 8;778:146270. Epub 2021 Mar 8.

Ifremer, laboratoire de Microbiologie, SG2M/LSEM, BP 21105, 44311 Nantes, France. Electronic address:

The emergence and worldwide spread of SARS-CoV-2 raises new concerns and challenges regarding possible environmental contamination by this virus through spillover of human sewage, where it has been detected. The coastal environment, under increasing anthropogenic pressure, is subjected to contamination by a large number of human viruses from sewage, most of them being non-enveloped viruses like norovirus. When reaching coastal waters, they can be bio-accumulated by filter-feeding shellfish species such as oysters. Methods to detect this viral contamination were set up for the detection of non-enveloped enteric viruses, and may need optimization to accommodate enveloped viruses like coronaviruses (CoV). Here, we aimed at assessing methods for the detection of CoV, including SARS-CoV-2, in the coastal environment and testing the possibility that SARS-CoV-2 can contaminate oysters, to monitor the contamination of French shores by SARS-CoV-2 using both seawater and shellfish. Using the porcine epidemic diarrhea virus (PEDV), a CoV, as surrogate for SARS-CoV-2, and Tulane virus, as surrogate for non-enveloped viruses such as norovirus, we assessed and selected methods to detect CoV in seawater and shellfish. Seawater-based methods showed variable and low yields for PEDV. In shellfish, the current norm for norovirus detection was applicable to CoV detection. Both PEDV and heat-inactivated SARS-CoV-2 could contaminate oysters in laboratory settings, with a lower efficiency than a calicivirus used as control. Finally, we applied our methods to seawater and shellfish samples collected from April to August 2020 in France, where we could detect the presence of human norovirus, a marker of human fecal contamination, but not SARS-CoV-2. Together, our results validate methods for the detection of CoV in the coastal environment, including the use of shellfish as sentinels of the microbial quality of their environment, and suggest that SARS-CoV-2 did not contaminate the French shores during the summer season.
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http://dx.doi.org/10.1016/j.scitotenv.2021.146270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938784PMC
March 2021

Highly Pathogenic Avian Influenza A(H5N8) Virus Spread by Short- and Long-Range Transmission, France, 2016-17.

Emerg Infect Dis 2021 Feb;27(2):508-516

We detected 3 genotypes of highly pathogenic avian influenza A(H5N8) virus in France during winter 2016-17. Genotype A viruses caused dramatic economic losses in the domestic duck farm industry in southwestern France. Our phylogenetic analysis suggests that genotype A viruses formed 5 distinct geographic clusters in southwestern France. In some clusters, local secondary transmission might have been started by a single introduction. The intensity of the viral spread seems to correspond to the density of duck holdings in each production area. To avoid the introduction of disease into an unaffected area, it is crucial that authorities limit the movements of potentially infected birds.
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http://dx.doi.org/10.3201/eid2702.202920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853534PMC
February 2021

Molecular characterization of encephalomyocarditis virus strains isolated from an African elephant and rats in a French zoo.

J Vet Diagn Invest 2021 Mar 8;33(2):313-321. Epub 2020 Dec 8.

Animal Health Laboratory, UMR1161 Virology, INRAE, ANSES, ENVA, Paris-Est University, Maisons-Alfort, France.

In November 2013, a fatal encephalomyocarditis virus (EMCV) case in a captive African elephant () occurred at the Réserve Africaine de Sigean, a zoo in the south of France. Here we report the molecular characterization of the EMCV strains isolated from samples collected from the dead elephant and from 3 rats () captured in the zoo at the same time. The EMCV infection was confirmed by reverse-transcription real-time PCR (RT-rtPCR) and genome sequencing. Complete genome sequencing and sequence alignment indicated that the elephant's EMCV strain was 98.1-99.9% identical to the rat EMCV isolates at the nucleotide sequence level. Phylogenetic analysis of the ORF, P1, VP1, and 3D sequences revealed that the elephant and rat strains clustered into lineage A of the EMCV 1 group. To our knowledge, molecular characterization of EMCV in France and Europe has not been reported previously in a captive elephant. The full genome analyses of EMCV isolated from an elephant and rats in the same outbreak emphasizes the role of rodents in EMCV introduction and circulation in zoos.
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http://dx.doi.org/10.1177/1040638720978389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953090PMC
March 2021

Genetic and Antigenic Evolution of European Swine Influenza A Viruses of HA-1C (Avian-Like) and HA-1B (Human-Like) Lineages in France from 2000 to 2018.

Viruses 2020 11 13;12(11). Epub 2020 Nov 13.

Swine Virology Immunology Unit, Ploufragan-Plouzané-Niort Laboratory, ANSES, BP53, 22440 Ploufragan, France.

This study evaluated the genetic and antigenic evolution of swine influenza A viruses (swIAV) of the two main enzootic H1 lineages, i.e., HA-1C (H1) and -1B (H1), circulating in France between 2000 and 2018. SwIAV RNAs extracted from 1220 swine nasal swabs were hemagglutinin/neuraminidase (HA/NA) subtyped by RT-qPCRs, and 293 virus isolates were sequenced. In addition, 146 H1Ny and 105 H1Ny strains were submitted to hemagglutination inhibition tests. H1N1 (66.5%) and H1N2 (25.4%) subtypes were predominant. Most H1 strains belonged to HA-1C.2.1 or -1B.1.2.3 clades, but HA-1C.2, -1C.2.2, -1C.2.3, -1B.1.1, and -1B.1.2.1 clades were also detected sporadically. Within HA-1B.1.2.3 clade, a group of strains named "Δ146-147" harbored several amino acid mutations and a double deletion in HA, that led to a marked antigenic drift. Phylogenetic analyses revealed that internal segments belonged mainly to the "Eurasian avian-like lineage", with two distinct genogroups for the M segment. In total, 17 distinct genotypes were identified within the study period. Reassortments of H1/H1 strains with H1N1pdm virus were rarely evidenced until 2018. Analysis of amino acid sequences predicted a variability in length of PB1-F2 and PA-X proteins and identified the appearance of several mutations in PB1, PB1-F2, PA, NP and NS1 proteins that could be linked to virulence, while markers for antiviral resistance were identified in N1 and N2. Altogether, diversity and evolution of swIAV recall the importance of disrupting the spreading of swIAV within and between pig herds, as well as IAV inter-species transmissions.
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http://dx.doi.org/10.3390/v12111304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697621PMC
November 2020

Controlled Heat and Humidity-Based Treatment for the Reuse of Personal Protective Equipment: A Pragmatic Proof-of-Concept to Address the Mass Shortage of Surgical Masks and N95/FFP2 Respirators and to Prevent the SARS-CoV2 Transmission.

Front Med (Lausanne) 2020 20;7:584036. Epub 2020 Oct 20.

Université de Tours, Tours, France.

The coronavirus infectious disease-2019 (COVID-19) pandemic has led to an unprecedented shortage of healthcare resources, primarily personal protective equipment like surgical masks, and N95/filtering face piece type 2 (FFP2) respirators. Reuse of surgical masks and N95/FFP2 respirators may circumvent the supply chain constraints and thus overcome mass shortage. Methods, design, setting, and measurement: Herein, we tested the effects of dry- and moist-air controlled heating treatment on structure and chemical integrity, decontamination yield, and filtration performance of surgical masks and FFP2 respirators. We found that treatment in a climate chamber at 70°C during 1 h with 75% humidity rate was adequate for enabling substantial decontamination of both respiratory viruses, oropharyngeal bacteria, and model animal coronaviuses, while maintaining a satisfying filtering capacity. Further studies are now required to confirm the feasibility of the whole process during routine practice. Our findings provide compelling evidence for the recycling of pre-used surgical masks and N95/FFP2 respirators in case of imminent mass shortfall.
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http://dx.doi.org/10.3389/fmed.2020.584036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7607499PMC
October 2020

The (VHSV) Markers of Virulence in Rainbow Trout ().

Front Microbiol 2020 20;11:574231. Epub 2020 Oct 20.

Virologie et Immunologie Moléculaires (VIM), Université Paris-Saclay, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE), Université de Versailles Saint-Quentin-en-Yvelines, Jouy-en-Josas, France.

(VHSV) is a highly contagious virus leading to high mortality in a large panel of freshwater and marine fish species. VHSV isolates originating from marine fish show low pathogenicity in rainbow trout. The analysis of several nearly complete genome sequences from marine and freshwater isolates displaying varying levels of virulence in rainbow trout suggested that only a limited number of amino acid residues might be involved in regulating the level of virulence. Based on a recent analysis of 55 VHSV strains, which were entirely sequenced and phenotyped in rainbow trout, several amino acid changes putatively involved in virulence were identified. In the present study, these amino acid changes were introduced, alone or in combination, in a highly-virulent VHSV 23-75 genome backbone by reverse genetics. A total of 35 recombinant VHSV variants were recovered and characterized for virulence in trout by bath immersion. Results confirmed the important role of the NV protein (R116S) and highlighted a major contribution of the nucleoprotein N (K46G and A241E) in regulating virulence. Single amino acid changes in these two proteins drastically affect virus pathogenicity in rainbow trout. This is particularly intriguing for the N variant (K46G) which is unable to establish an active infection in the fins of infected trout, the main portal of entry of VHSV in this species, allowing further spread in its host. In addition, salmonid cell lines were selected to assess the kinetics of replication and cytopathic effect of recombinant VHSV and discriminate virulent and avirulent variants. In conclusion, three major virulence markers were identified in the NV and N proteins. These markers explain almost all phenotypes (92.7%) observed in trout for the 55 VHSV strains analyzed in the present study and herein used for the backward validation of virulence markers. The identification of VHSV specific virulence markers in this species is of importance both to predict the phenotype of viral isolates with targeted diagnostic tests and to improve prophylactic methods such as the development of safer live-attenuated vaccines.
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http://dx.doi.org/10.3389/fmicb.2020.574231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606196PMC
October 2020

Contrasted Epidemiological Patterns of West Nile Virus Lineages 1 and 2 Infections in France from 2015 to 2019.

Pathogens 2020 Oct 30;9(11). Epub 2020 Oct 30.

UMR 1161 Virology, ANSES, INRAE, ENVA, ANSES Animal Health Laboratory, EURL for Equine Diseases, 94704 Maisons-Alfort, France.

Since 2015, annual West Nile virus (WNV) outbreaks of varying intensities have been reported in France. Recent intensification of enzootic WNV circulation was observed in the South of France with most horse cases detected in 2015 ( = 49), 2018 ( = 13), and 2019 ( = 13). A WNV lineage 1 strain was isolated from a horse suffering from West Nile neuro-invasive disease (WNND) during the 2015 episode in the Camargue area. A breaking point in WNV epidemiology was achieved in 2018, when WNV lineage 2 emerged in Southeastern areas. This virus most probably originated from WNV spread from Northern Italy and caused WNND in humans and the death of diurnal raptors. WNV lineage 2 emergence was associated with the most important human WNV epidemics identified so far in France (n = 26, including seven WNND cases and two infections in blood and organ donors). Two other major findings were the detection of WNV in areas with no or limited history of WNV circulation (Alpes-Maritimes in 2018, Corsica in 2018-2019, and Var in 2019) and distinct spatial distribution of human and horse WNV cases. These new data reinforce the necessity to enhance French WNV surveillance to better anticipate future WNV epidemics and epizootics and to improve the safety of blood and organ donations.
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http://dx.doi.org/10.3390/pathogens9110908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692118PMC
October 2020

Viral variant visualizer (VVV): A novel bioinformatic tool for rapid and simple visualization of viral genetic diversity.

Virus Res 2021 01 17;291:198201. Epub 2020 Oct 17.

Agence National de Sécurité Sanitaire, de l'environnement et du travail (ANSES) Laboratory of Ploufragan-Plouzané-Niort, Virology, Immunology and Parasitology in Poultry and Rabbit (VIPAC) Unit, Université Bretagne Loire (UBL), France. Electronic address:

Here a bioinformatic pipeline VVV has been developed to analyse viral populations in a given sample from Next Generation Sequencing (NGS) data. To date, handling large amounts of data from NGS requires the expertise of bioinformaticians, both for data processing and result analysis. Consequently, VVV was designed to help non-bioinformaticians to perform these tasks. By providing only the NGS data file, the developed pipeline generated consensus sequences and determined the composition of the viral population for an avian Metapneumovirus (AMPV) and three different animal coronaviruses (Porcine Epidemic Diarrhea Virus (PEDV), Turkey Coronavirus (TCoV) and Infectious Bronchitis Virus (IBV)). In all cases, the pipeline produced viral consensus genomes corresponding to known consensus sequence and made it possible to highlight the presence of viral genetic variants through a single graphic representation. The method was validated by comparing the viral populations of an AMPV field sample, and of a copy of this virus produced from a DNA clone. VVV demonstrated that the cloned virus population was homogeneous (as designed) at position 2934 where the wild-type virus demonstrated two variant populations at a ratio of almost 50:50. A total of 18, 10, 3 and 28, viral genetic variants were detected for AMPV, PEDV, TCoV and IBV respectively. The simplicity of this pipeline makes the study of viral genetic variants more accessible to a wide variety of biologists, which should ultimately increase the rate of understanding of the mechanisms of viral genetic evolution.
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http://dx.doi.org/10.1016/j.virusres.2020.198201DOI Listing
January 2021

VHSV Single Amino Acid Polymorphisms (SAPs) Associated With Virulence in Rainbow Trout.

Front Microbiol 2020 27;11:1984. Epub 2020 Aug 27.

Unit for Fish and Shellfish Diseases, EURL for Fish and Crustacean Diseases, National Institute of Aquatic Resources, Technical University of Denmark (DTU), Kongens Lyngby, Denmark.

The Viral Hemorrhagic Septicemia Virus (VHSV) is an OIE notifiable pathogen widespread in the Northern Hemisphere that encompasses four genotypes and nine subtypes. In Europe, subtype Ia impairs predominantly the rainbow trout industry causing severe rates of mortality, while other VHSV genotypes and subtypes affect a number of marine and freshwater species, both farmed and wild. VHSV has repeatedly proved to be able to jump to rainbow trout from the marine reservoir, causing mortality episodes. The molecular mechanisms regulating VHSV virulence and host tropism are not fully understood, mainly due to the scarce availability of complete genome sequences and information on the virulence phenotype. With the scope of identifying molecular markers for VHSV virulence, we generated an extensive dataset of 55 viral genomes and related mortality data obtained from rainbow trout experimental challenges. Using statistical association analyses that combined genetic and mortality data, we found 38 single amino acid polymorphisms scattered throughout the complete coding regions of the viral genome that were putatively involved in virulence of VHSV in trout. Specific amino acid signatures were recognized as being associated with either low or high virulence phenotypes. The phylogenetic analysis of VHSV coding regions supported the evolution toward greater virulence in rainbow trout within subtype Ia, and identified several other subtypes which may be prone to be virulent for this species. This study sheds light on the molecular basis for VHSV virulence, and provides an extensive list of putative virulence markers for their subsequent validation.
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http://dx.doi.org/10.3389/fmicb.2020.01984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493562PMC
August 2020

Recombination at the emergence of the pathogenic rabbit haemorrhagic disease virus Lagovirus europaeus/GI.2.

Sci Rep 2020 09 2;10(1):14502. Epub 2020 Sep 2.

Unité de Virologie, Immunologie, Parasitologie, Aviaires et Cunicoles, Laboratoire de Ploufragan-Plouzané-Niort, Agence nationale de sécurité sanitaire, de l'alimentation, de l'environnement et du travail (Anses), Ploufragan, France.

Rabbit haemorrhagic disease is a viral disease that emerged in the 1980s and causes high mortality and morbidity in the European rabbit (Oryctolagus cuniculus). In 2010, a new genotype of the rabbit haemorrhagic disease virus emerged and replaced the former circulating Lagovirus europaeus/GI.1 strains. Several recombination events have been reported for the new genotype Lagovirus europaeus/GI.2, with pathogenic (variants GI.1a and GI.1b) and benign (genotype GI.4) strains that served as donors for the non-structural part while GI.2 composed the structural part; another recombination event has also been described at the p16/p23 junction involving GI.4 strains. In this study, we analysed new complete coding sequences of four benign GI.3 strains and four GI.2 strains. Phylogenetic and recombination detection analyses revealed that the first GI.2 strains, considered as non-recombinant, resulted from a recombination event between GI.3 and GI.2, with GI.3 as the major donor for the non-structural part and GI.2 for the structural part. Our results indicate that recombination contributed to the emergence, persistence and dissemination of GI.2 as a pathogenic form and that all described GI.2 strains so far are the product of recombination. This highlights the need to study full-genomic sequences of lagoviruses to understand their emergence and evolution.
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http://dx.doi.org/10.1038/s41598-020-71303-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468141PMC
September 2020

Evaluation of un-methylated DNA enrichment in sequencing of African swine fever virus complete genome.

J Virol Methods 2020 11 21;285:113959. Epub 2020 Aug 21.

Viral Genetic and Biosecurity Unit, Ploufragan-Plouzané-Niort Laboratory, ANSES, Ploufragan, France. Electronic address:

African swine fever is a febrile hemorrhagic fever disease that is caused by the African swine fever virus (ASFV) and is lethal for domestic pigs and wild boar. ASFV also infects soft ticks of the genus Ornithodoros, some species of which can act as a vector for ASFV. Whole genome sequencing of ASFV is a challenge because, due to the size difference of the host genome versus the viral genome, the higher proportion of host versus virus DNA fragments renders the virus sequencing poorly efficient. A novel approach of DNA enrichment, based on the separation of methylated and un-methylated DNA, has been reported but without an evaluation of its efficacy. In this study, the efficiency of the un-methylated DNA enrichment protocol was evaluated for pig and tick samples infected by ASFV. As expected, fewer reads corresponding to ASFV were found in the methylated fraction compared to the un-methylated fraction. However, the sequencing coverage of the un-methylated fraction was not improved compared to the untreated DNA. In our hands, the ASFV DNA enrichment was inefficient for tick samples and very limited for pig samples. This enrichment process represents extra work and cost without a significant improvement of ASFV genome coverage. The efficiency of this enrichment approach and the cost/benefit ratio are discussed.
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http://dx.doi.org/10.1016/j.jviromet.2020.113959DOI Listing
November 2020

Porcine epidemic diarrhea virus: Viral RNA detection and quantification using a validated one-step real time RT-PCR.

J Virol Methods 2020 09 31;283:113906. Epub 2020 May 31.

Anses, Laboratory of Ploufragan-Plouzané-Niort, BP53, 22440, Ploufragan, France.

Since 2014, porcine epidemic diarrhea virus (PEDV) has reemerged in Europe. RT-PCR methods have been described for the detection of PEDV, but none have been validated according to a norm. In this study we described the development and validation of a SYBR™ Green one-step RT-qPCR according to the French norm NF U47-600, for the detection and quantification of PEDV viral RNA. The method was validated from sample preparation (feces or jejunum) through to nucleic acid extraction and RT-qPCR detection. Specificity and sensitivity, limit of detection (LoD), limit of quantification (LQ), linearity, intra and inter assay variability were evaluated using transcribed RNA and fecal and jejunum matrices spiked with virus. The analytical and diagnostic specificities and sensitivities of this RT-qPCR were 100% in this study. A LoD of 50 genome copies/5 μl of extract from fecal matrices spiked with virus or RNA transcript and 100 genome copies/5 μl of extract from jejunum matrices spiked with virus were obtained. The Lower LQ (LLQ) was 100 genome copies/5 μl and the Upper LQ (ULQ) 10 copies/5 μl. This method is the first, validated according a norm for PEDV and may serve as a global reference method to harmonize detection and quantification of PEDV viral RNA in both field and experimental settings.
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http://dx.doi.org/10.1016/j.jviromet.2020.113906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261358PMC
September 2020

Coding-Complete Genome Sequence of an African Swine Fever Virus Strain Liv13/33 Isolate from Experimental Transmission between Pigs and Ornithodoros moubata Ticks.

Microbiol Resour Announc 2020 Apr 23;9(17). Epub 2020 Apr 23.

Swine Virology and Immunology Unit, ANSES Ploufragan-Plouzané-Niort Laboratory, Ploufragan, France

Here, we report the coding-complete genome sequence of African swine fever (ASF) virus strain Liv13/33, isolated from experimentally infected pigs and ticks. The 11 sequences that we obtained harbored no notable differences to each other, and all of them were closely related to the genome sequence of the Mkuzi 1979 strain of genotype I.
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http://dx.doi.org/10.1128/MRA.00185-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180279PMC
April 2020

Sequencing of animal viruses: quality data assurance for NGS bioinformatics.

Virol J 2019 11 21;16(1):140. Epub 2019 Nov 21.

Department of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), viale dell'Università 10, 35120, Legnaro (PD), Italy.

Background: Next generation sequencing (NGS) is becoming widely used among diagnostics and research laboratories, and nowadays it is applied to a variety of disciplines, including veterinary virology. The NGS workflow comprises several steps, namely sample processing, library preparation, sequencing and primary/secondary/tertiary bioinformatics (BI) analyses. The latter is constituted by a complex process extremely difficult to standardize, due to the variety of tools and metrics available. Thus, it is of the utmost importance to assess the comparability of results obtained through different methods and in different laboratories. To achieve this goal, we have organized a proficiency test focused on the bioinformatics components for the generation of complete genome sequences of salmonid rhabdoviruses.

Methods: Three partners, that performed virus sequencing using different commercial library preparation kits and NGS platforms, gathered together and shared with each other 75 raw datasets which were analyzed separately by the participants to produce a consensus sequence according to their own bioinformatics pipeline. Results were then compared to highlight discrepancies, and a subset of inconsistencies were investigated more in detail.

Results: In total, we observed 526 discrepancies, of which 39.5% were located at genome termini, 14.1% at intergenic regions and 46.4% at coding regions. Among these, 10 SNPs and 99 indels caused changes in the protein products. Overall reproducibility was 99.94%. Based on the analysis of a subset of inconsistencies investigated more in-depth, manual curation appeared the most critical step affecting sequence comparability, suggesting that the harmonization of this phase is crucial to obtain comparable results. The analysis of a calibrator sample allowed assessing BI accuracy, being 99.983%.

Conclusions: We demonstrated the applicability and the usefulness of BI proficiency testing to assure the quality of NGS data, and recommend a wider implementation of such exercises to guarantee sequence data uniformity among different virology laboratories.
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http://dx.doi.org/10.1186/s12985-019-1223-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868765PMC
November 2019

Virus persistence in pig herds led to successive reassortment events between swine and human influenza A viruses, resulting in the emergence of a novel triple-reassortant swine influenza virus.

Vet Res 2019 Oct 7;50(1):77. Epub 2019 Oct 7.

Swine Virology Immunology Unit, Ploufragan-Plouzané-Niort Laboratory, ANSES, BP53, 22440, Ploufragan, France.

This report describes the detection of a triple reassortant swine influenza A virus of H1N2 subtype. It evolved from an avian-like swine H1N1 that first acquired the N2 segment from a seasonal H3N2, then the M segment from a 2009 pandemic H1N1, in two reassortments estimated to have occurred 10 years apart. This study illustrates how recurrent influenza infections increase the co-infection risk and facilitate evolutionary jumps by successive gene exchanges. It recalls the importance of appropriate biosecurity measures inside holdings to limit virus persistence and interspecies transmissions, which both contribute to the emergence of new potentially zoonotic viruses.
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http://dx.doi.org/10.1186/s13567-019-0699-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781375PMC
October 2019

Bidirectional Human-Swine Transmission of Seasonal Influenza A(H1N1)pdm09 Virus in Pig Herd, France, 2018.

Emerg Infect Dis 2019 10;25(10):1940-1943

In 2018, a veterinarian became sick shortly after swabbing sows exhibiting respiratory syndrome on a farm in France. Epidemiologic data and genetic analyses revealed consecutive human-to-swine and swine-to-human influenza A(H1N1)pdm09 virus transmission, which occurred despite some biosecurity measures. Providing pig industry workers the annual influenza vaccine might reduce transmission risk.
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http://dx.doi.org/10.3201/eid2510.190068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759248PMC
October 2019

Proficiency Testing of Virus Diagnostics Based on Bioinformatics Analysis of Simulated High-Throughput Sequencing Data Sets.

J Clin Microbiol 2019 08 26;57(8). Epub 2019 Jul 26.

Robert Koch Institute, Centre for Biological Threats and Special Pathogens 1, Berlin, Germany.

Quality management and independent assessment of high-throughput sequencing-based virus diagnostics have not yet been established as a mandatory approach for ensuring comparable results. The sensitivity and specificity of viral high-throughput sequence data analysis are highly affected by bioinformatics processing using publicly available and custom tools and databases and thus differ widely between individuals and institutions. Here we present the results of the COMPARE [llaborative anagement latform for Detection and nalyses of (e-)emerging and Foodborne Outbreaks in urope] virus proficiency test. An artificial, simulated data set of Illumina HiSeq sequences was provided to 13 different European institutes for bioinformatics analysis to identify viral pathogens in high-throughput sequence data. Comparison of the participants' analyses shows that the use of different tools, programs, and databases for bioinformatics analyses can impact the correct identification of viral sequences from a simple data set. The identification of slightly mutated and highly divergent virus genomes has been shown to be most challenging. Furthermore, the interpretation of the results, together with a fictitious case report, by the participants showed that in addition to the bioinformatics analysis, the virological evaluation of the results can be important in clinical settings. External quality assessment and proficiency testing should become an important part of validating high-throughput sequencing-based virus diagnostics and could improve the harmonization, comparability, and reproducibility of results. There is a need for the establishment of international proficiency testing, like that established for conventional laboratory tests such as PCR, for bioinformatics pipelines and the interpretation of such results.
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http://dx.doi.org/10.1128/JCM.00466-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6663916PMC
August 2019

In vitro, in cellulo and structural characterizations of the interaction between the integrase of Porcine Endogenous Retrovirus A/C and proteins of the BET family.

Virology 2019 06 16;532:69-81. Epub 2019 Apr 16.

INRA, Université Lyon 1, UMR754, Viral Infections Compared Pathology, 69007, Lyon, France; Université de Lyon, 69000, Lyon, France; UMSl3444 Biosciences Gerland Lyon Sud, 69007, Lyon, France. Electronic address:

Retroviral integrase (IN) proteins catalyze the permanent integration of the viral genome into host DNA. They can productively recruit cellular proteins, and the human Bromodomain and Extra-Terminal domain (hBET) proteins have been shown to be co-factors for integration of gamma-retroviruses such as Murine Leukemia Virus (MLV) into human cells. By using two-hybrid, co-immunoprecipitation and in vitro interaction assays, we showed that IN of the gamma- Porcine Endogenous Retrovirus-A/C (PERV IN) interacts through its C-terminal domain (CTD) with hBET proteins. We observed that PERV IN interacts with the BRD2, BRD3 and BRD4 proteins in vitro and that the BRD2 protein specifically binds and co-localizes with PERV IN protein in the nucleus of cells. We further mapped the interaction sites to the conserved Extra-Terminal (ET) domain of the hBET proteins and to several amino acids of the of the C-terminal tail of the PERV IN CTD. Finally, we determined the first experimental structure of an IN CTD - BET ET complex from small-angle X-ray scattering data (SAXS). We showed that the two factors assemble as two distinct modules linked by a short loop which confers partial flexibility. The SAXS-restrained model is structurally compatible with the binding of the PERV intasome to BRD2. Altogether, these data confirm the important role of host BET proteins in the gamma-retroviruses' targeting site and efficiency of integration.
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http://dx.doi.org/10.1016/j.virol.2019.04.002DOI Listing
June 2019

A Field Recombinant Strain Derived from Two Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-1) Modified Live Vaccines Shows Increased Viremia and Transmission in SPF Pigs.

Viruses 2019 03 23;11(3). Epub 2019 Mar 23.

Agence Nationale de Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail (Anses), Unité Virologie Immunologie Porcines, BP 53, 22440 Ploufragan, France.

In Europe, modified live vaccines (MLV) are commonly used to control porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, they have been associated with safety issues such as reversion to virulence induced by mutation and/or recombination. On a French pig farm, we identified a field recombinant strain derived from two PRRSV-1 MLV (MLV1). As a result, we aimed to evaluate its clinical, virological, and transmission parameters in comparison with both parental strains. Three groups with six pigs in each were inoculated with either one of the two MLV1s or with the recombinant strain; six contact pigs were then added into each inoculated group. The animals were monitored daily for 35 days post-inoculation (dpi) for clinical symptoms; blood samples and nasal swabs were collected twice a week. PRRS viral load in inoculated pigs of recombinant group was higher in serum, nasal swabs, and tonsils in comparison with both vaccine groups. The first viremic contact pig was detected as soon as 2 dpi in the recombinant group compared to 10 and 17 dpi for vaccine groups. Estimation of transmission parameters revealed fastest transmission and longest duration of infectiousness for recombinant group. Our in vivo study showed that the field recombinant strain derived from two MLV1s demonstrated high viremia, shedding and transmission capacities.
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http://dx.doi.org/10.3390/v11030296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466261PMC
March 2019

Targeted Next Generation Sequencing to study insert stability in genetically modified plants.

Sci Rep 2019 02 19;9(1):2308. Epub 2019 Feb 19.

Anses, Plant Health Laboratory, Bacteriology Virology GMO Unit, 7 rue Jean Dixméras, 49044, Angers cedex 01, France.

The EU directive 2001/18/EC requires any genetically modified (GM) event to be stable. In the present work, a targeted Next-Generation Sequencing (NGS) approach using barcodes to specifically tag each individual DNA molecules during library preparation was implemented to detect mutations taking into account the background noise due to amplification and sequencing errors. The method was first showed to be efficient in detecting the mutations in synthetic samples prepared with custom-synthesized mutated or non-mutated P35S sequences mixed in different proportions. The genetic stability of a portion of the P35S promoter targeted for GM detection was then analyzed in GM flour samples. Several low frequency mutations were detected in the P35S sequences. Some mutated nucleotides were located within the primers and probes used in the P35S diagnostic test. If present not as somatic mutations but as the consensus sequence of some individuals, these mutations could influence the efficiency of the P35S real time PCR diagnostic test. This methodology could be implemented in genetic stability studies of GM inserts but also to detect single nucleotide mutant GM plants produced using "new breeding techniques".
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http://dx.doi.org/10.1038/s41598-019-38701-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381221PMC
February 2019

Identification of a Divergent Avian Influenza H3N2 Virus from Domestic Ducks in France.

Microbiol Resour Announc 2018 Dec 13;7(23). Epub 2018 Dec 13.

Anses, Unité VIPAC-LNR Influenza Aviaire, Ploufragan, France.

An avian influenza H3N2 virus was isolated from domestic ducks in France in 2016. Although this French H3N2 virus possesses traits of an avian virus, the genetic distances observed for hemagglutinin (HA) and neuraminidase (NA) show that these two genes most likely evolved independently from other avian influenza sequences.
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http://dx.doi.org/10.1128/MRA.00943-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298543PMC
December 2018

Lleida Bat Lyssavirus isolation in Miniopterus schreibersii in France.

Zoonoses Public Health 2019 03 20;66(2):254-258. Epub 2018 Nov 20.

ANSES-Nancy Laboratory for Rabies and Wildlife, Malzéville, France.

Bat rabies cases are attributed in Europe to five different Lyssavirus species of 16 recognized Lyssavirus species causing rabies. One of the most genetically divergent Lyssavirus spp. has been detected in a dead Miniopterus schreibersii bat in France. Brain samples were found positive for the presence of antigen, infectious virus and viral RNA by classical virological methods and molecular methods respectively. The complete genome sequence was determined by next-generation sequencing. The analysis of the complete genome sequence confirmed the presence of Lleida bat lyssavirus (LLEBV) in bats in France with 99.7% of nucleotide identity with the Spanish LLEBV strain (KY006983).
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http://dx.doi.org/10.1111/zph.12535DOI Listing
March 2019

Lessons learnt from a porcine epidemic diarrhea (PED) case in France in 2014: Descriptive epidemiology and control measures implemented.

Vet Microbiol 2018 Nov 29;226:9-14. Epub 2018 Sep 29.

Anses, Laboratory of Ploufragan/Plouzané, BP53, 22440, Ploufragan, France; Université Bretagne Loire, Cité internationale, 1 place Paul Ricoeur CS 54417, 35044 Rennes, France. Electronic address:

An acute epidemic of porcine epidemic diarrhea (PED) has affected the USA since 2013 and spread all around the world. In France, the immune status of the pig population against PED virus (PEDV) was expected to be low due to the absence of circulation of the virus since the 80's and a compulsory notification of PED was set up in 2014. Here, we reported the first case of a PED outbreak in December 2014 in the North of France after a long absence of the disease, the monitoring of the excretion and the control measure implementation. The isolated strain in France in December 2014 was a PEDV "S-InDel" strain which was close to the "S-InDel" German PEDV strain isolated in May 2014. The individual shedding duration of PEDV in feces was estimated around 20 days for pigs of different ages. Biosecurity measures implemented allowed the limitation of PEDV spread to fattening and farrowing rooms without dissemination to the nursery block. Using strict biosecurity measures, direct shipment of infected fatteners to the slaughterhouse, strict decontamination protocols with a quarantine of 6 weeks for replacement gilts without voluntary contamination helped PEDV fade out within the herd and avoided the spread to other herds. PEDV presence in manure was investigated as well as the inactivation treatment of the virus present in the liquid manure. An increase to a pH 12 of liquid manure by liming led to the absence of PEDV detection by RT-PCR after seven days.
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http://dx.doi.org/10.1016/j.vetmic.2018.09.023DOI Listing
November 2018

Characterization of plasmids harboring bla genes in Escherichia coli from French pigs.

Vet Microbiol 2018 Oct 4;224:100-106. Epub 2018 Aug 4.

ANSES, Laboratoire de Ploufragan-Plouzané, Ploufragan, France; Université Bretagne Loire, France. Electronic address:

Resistance to extended-spectrum cephalosporins is prevalent in French pig E. coli isolates. The aim of this study was to characterize the plasmids and genes present in pathogenic and commensal extended-spectrum cephalosporins -resistant isolates. The resistance plasmids of 26 strains were sequenced and then analyzed to identify resistance and virulence genes. Results showed that resistance to extended-spectrum cephalosporins in French pig E. coli isolates is-as in other food animals in France-mainly carried by highly similar bla IncI1/ST3 plasmids. These plasmids very often bear other resistance genes such as resistance to sulphonamides (sul2), trimethoprim (dfrA17) and aminoglycosides (aadA5), and occasionally to tetracycline (tet(A)), macrolides (mph(A) and erm genes), phenicols (floR) or streptomycin (strA, strB). Few virulence genes were detected, including colicins, heat-stable enterotoxins, adhesins or temperature-sensitive hemagglutinins. The other cefotaximases detected were bla and bla, the latter being on an IncF plasmid which showed very close identity to a human epidemic plasmid. Importantly, resistance genes for quinolones or polymyxins were never detected on the extended-spectrum cephalosporins resistance plasmids. These results are helpful to evidence the risk of co-selecting cephalosporins -resistance using antibiotics outside this group. They also highlight the occasional presence in pigs of human epidemic plasmids.
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http://dx.doi.org/10.1016/j.vetmic.2018.08.005DOI Listing
October 2018

Spatiotemporal Distribution and Evolution of the A/H1N1 2009 Pandemic Influenza Virus in Pigs in France from 2009 to 2017: Identification of a Potential Swine-Specific Lineage.

J Virol 2018 12 27;92(24). Epub 2018 Nov 27.

ANSES, Ploufragan-Plouzané Laboratory, Swine Virology Immunology Unit, BP53, Ploufragan, France

The H1N1 influenza virus responsible for the most recent pandemic in 2009 (H1N1pdm) has spread to swine populations worldwide while it replaced the previous seasonal H1N1 virus in humans. In France, surveillance of swine influenza A viruses in pig herds with respiratory outbreaks led to the detection of 44 H1N1pdm strains between 2009 and 2017, regardless of the season, and findings were not correlated with pig density. From these isolates, 17 whole-genome sequences were obtained, as were 6 additional hemagglutinin (HA)/neuraminidase (NA) sequences, in order to perform spatial and temporal analyses of genetic diversity and to compare evolutionary patterns of H1N1pdm in pigs to patterns for human strains. Following mutation accumulation and fixation over time, phylogenetic analyses revealed for the first time the divergence of a swine-specific genogroup within the H1N1pdm lineage. The divergence is thought to have occurred around 2011, although this was demonstrated only through strains isolated in 2015 to 2016 in the southern half of France. To date, these H1N1pdm swine strains have not been related to any increased virulence in swine herds and have not exhibited any antigenic drift compared to seasonal human strains. However, further monitoring is encouraged, as diverging evolutionary patterns in these two species, i.e., swine and humans, may lead to the emergence of viruses with a potentially higher risk to both animal and human health. Pigs are a "mixing vessel" for influenza A viruses (IAVs) because of their ability to be infected by avian and human IAVs and their propensity to facilitate viral genomic reassortment events. Also, as IAVs may evolve differently in swine and humans, pigs can become a reservoir for old human strains against which the human population has become immunologically naive. Thus, viruses from the novel swine-specific H1N1pdm genogroup may continue to diverge from seasonal H1N1pdm strains and/or from other H1N1pdm viruses infecting pigs and lead to the emergence of viruses that would not be covered by human vaccines and/or swine vaccines based on antigens closely related to the original H1N1pdm virus. This discovery confirms the importance of encouraging swine IAV monitoring because H1N1pdm swine viruses could carry an increased risk to both human and swine health in the future as a whole H1N1pdm virus or gene provider in subsequent reassortant viruses.
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http://dx.doi.org/10.1128/JVI.00988-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258933PMC
December 2018

Emergence of bluetongue virus serotype 4 in mainland France in November 2017.

Transbound Emerg Dis 2018 Oct 8;65(5):1158-1162. Epub 2018 Jun 8.

UMR 1161 ANSES/INRA/ENVA, Université Paris-Est ANSES Maisons-Alfort, Maisons-Alfort, France.

In November 2017, a 15-day-old calf located in France (Haute-Savoie department) was found positive for bluetongue virus (BTV) RNA by RT-PCR. Laboratory investigations allowed the isolation and identification of the serotype: BTV-4. The analysis of the full viral genome showed that all the 10 genome segments were closely related to BTV-4 strains involved in a large BT outbreak in the Balkan Peninsula, in Italy since 2014 and in Corsica since the end of October 2016. These results together with epidemiological data suggest that BTV-4 has been introduced to mainland France from Corsica or Italy where BTV-4 outbreaks have been reported in summer and autumn 2016. This is the first report of the introduction of BTV-4 in mainland France.
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http://dx.doi.org/10.1111/tbed.12919DOI Listing
October 2018

Full-length genome sequences of porcine epidemic diarrhoea virus strain CV777; Use of NGS to analyse genomic and sub-genomic RNAs.

PLoS One 2018 1;13(3):e0193682. Epub 2018 Mar 1.

DTU National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark.

Porcine epidemic diarrhoea virus, strain CV777, was initially characterized in 1978 as the causative agent of a disease first identified in the UK in 1971. This coronavirus has been widely distributed among laboratories and has been passaged both within pigs and in cell culture. To determine the variability between different stocks of the PEDV strain CV777, sequencing of the full-length genome (ca. 28kb) has been performed in 6 different laboratories, using different protocols. Not surprisingly, each of the different full genome sequences were distinct from each other and from the reference sequence (Accession number AF353511) but they are >99% identical. Unique and shared differences between sequences were identified. The coding region for the surface-exposed spike protein showed the highest proportion of variability including both point mutations and small deletions. The predicted expression of the ORF3 gene product was more dramatically affected in three different variants of this virus through either loss of the initiation codon or gain of a premature termination codon. The genome of one isolate had a substantially rearranged 5´-terminal sequence. This rearrangement was validated through the analysis of sub-genomic mRNAs from infected cells. It is clearly important to know the features of the specific sample of CV777 being used for experimental studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193682PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832266PMC
June 2018

Infectious bursal disease virus in Algeria: Detection of highly pathogenic reassortant viruses.

Infect Genet Evol 2018 06 1;60:48-57. Epub 2018 Feb 1.

Avian and Rabbit Virology Immunology and Parasitology Unit (VIPAC), French Agency for Food, Environmental and Occupational Heath Safety (ANSES), Zoopole - rue des Fusillés BP 53, 22440 Ploufragan, France. Electronic address:

Infectious bursal disease (IBD) is an immunosuppressive viral disease, present worldwide, which causes mortality and immunosuppression in young chickens. The causative agent, the Avibirnavirus IBDV, is a non-enveloped virus whose genome consists of two segments (A and B) of double-stranded RNA. Different pathotypes of IBDV exist, ranging from attenuated vaccine strains to very virulent viruses (vvIBDV). In Algeria, despite the prophylactic measures implemented, cases of IBD are still often diagnosed clinically and the current molecular epidemiology of IBDV remains unknown. The presence of the virus and especially of strains genetically close to vvIBDV was confirmed in 2000 by an unpublished OIE report. In this study, nineteen IBDV isolates were collected in Algeria between September 2014 and September 2015 during clinical outbreaks. These isolates were analyzed at the genetic, antigenic and pathogenic levels. Our results reveal a broad genetic and phenotypic diversity of pathogenic IBDV strains in Algeria, with, i) the circulation of viruses with both genome segments related to European vvIBDV, which proved as pathogenic for specific pathogen-free chickens as vvIBDV reference strain, ii) the circulation of viruses closely related - yet with a specific segment B - to European vvIBDV, their pathogenicity being lower than reference vvIBDV, iii) the detection of reassortant viruses whose segment A was related to vvIBDV whereas their segment B did not appear closely related to any reference sequence. Interestingly, the pathogenicity of these potentially reassortant strains was comparable to that of reference vvIBDV. All strains characterized in this study exhibited an antigenicity similar to the cognate reference IBDV strains. These data reveal the continuous genetic evolution of IBDV strains in Algerian poultry through reassortment and acquisition of genetic material of unidentified origin. Continuous surveillance of the situation as well as good vaccination practice associated with appropriate biosecurity measures are necessary for disease control.
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http://dx.doi.org/10.1016/j.meegid.2018.01.029DOI Listing
June 2018