Mariette F. Ducatez

High pathogenicity and low genetic evolution of avian paramyxovirus type I (Newcastle disease virus) isolated from live bird markets in Uganda

BackgroundNewcastle disease is still a serious disease of poultry especially in backyard free-range production systems despite the availability of cross protective vaccines. Healthy-looking poultry from live bird markets have been suspected as a major source of disease spread although limited studies have been conducted to ascertain the presence of the virulent strains in the markets and to understand how they are related to outbreak strains.MethodsThis study evaluated the occurrence of Newcastle disease virus in samples collected from poultry in live bird markets across Uganda. The isolates were pathoyped using standard methods (mean death time (MDT), intracelebral pathogenicity index (ICPI), and sequencing of the fusion protein cleavage site motif) and also phylogenetically analysed after sequencing of the full fusion and hemagglutin-neuraminidase genes. The isolates were classified into genotypes and subgenotypes based on the full fusion protein gene classification system and compared with other strains in the region and world-wide.ResultsVirulent avian paramyxovirus type I (APMV-1) (Newcastle disease virus) was isolated in healthy-looking poultry in live bird markets. The viruses belonged to a new subgenotype, Vd, in genotype V, and clustered together with Tanzania and Kenya strains. They harbored low genetic diversity.ConclusionThe occurrence of virulent AMPV-1 strains in live bird markets may serve as sources of Newcastle disease outbreaks in non-commercial farms.

First outbreaks and phylogenetic analyses of avian influenza H9N2 viruses isolated from poultry flocks in Morocco

BackgroundH9N2 avian influenza viruses continue to spread in poultry and wild birds worldwide. Morocco just faced its first H9N2 influenza virus outbreaks early 2016 affecting different types of poultry production. After its introduction, the virus spread very rapidly throughout the country.MethodsSamples were collected from 11 chicken flocks with high morbidity and mortality rates. Four viruses were successfully isolated from broiler chickens and one from broiler breeders and fully sequenced.ResultsPhylogenetic and molecular markers analyses showed the Moroccan viruses belonged to the G1 lineage and likely originated from the Middle East. As known for H9N2 viruses, the Moroccanisolates possess several genetic markers that enhance virulence in poultry and transmission to humans.ConclusionThe present study demonstrated that under field conditions H9N2 could have a devastating effect on egg production and mortalities and highlighted a lack of surveillance data on the pathogen in the region.

Diversity of avipoxviruses in captive-bred Houbara bustard

Implementation of conservation breeding programs is a key step to ensuring the sustainability of many endangered species. Infectious diseases can be serious threats for the success of such initiatives especially since knowledge on pathogens affecting those species is usually scarce. Houbara bustard species (Chlamydotis undulata and Chlamydotis macqueenii), whose populations have declined over the last decades, have been captive-bred for conservation purposes for more than 15 years. Avipoxviruses are of the highest concern for these species in captivity. Pox lesions were collected from breeding projects in North Africa, the Middle East and Central Asia for 6 years in order to study the diversity of avipoxviruses responsible for clinical infections in Houbara bustard. Molecular and phylogenetic analyses of 113 and 75 DNA sequences for P4b and fpv140 loci respectively, revealed an unexpected wide diversity of viruses affecting Houbara bustard even at a project scale: 17 genotypes equally distributed between fowlpox virus-like and canarypox virus-like have been identified in the present study. This suggests multiple and repeated introductions of virus and questions host specificity and control strategy of avipoxviruses. We also show that the observed high virus burden and co-evolution of diverse avipoxvirus strains at endemic levels may be responsible for the emergence of novel recombinant strains.

Prevalence and molecular characterization of avian infectious bronchitis virus in poultry flocks in Morocco from 2010 to 2014 and first detection of Italy 02 in Africa

The aim of this study was to investigate the prevalence and diversity of infectious bronchitis virus (IBV) genotypes in poultry flocks in 16 areas of Morocco between 2010 and 2014. A total of 360 chicken flocks suspected of being infected by IBV were screened for the IBV N gene using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Flocks were classified into four groups according to their IBV vaccination programme. Group 1 contained unvaccinated birds. Group 2 received a single application of live H120 vaccine. Groups 3 and 4 birds received one or two booster vaccination(s), respectively, mostly using the H120 vaccine. The real-time RT-PCR results showed that 51.7% of the flocks were positive for the IBV genome with geographical disparities. Molecular characterization of IBV was performed on 50 RT-PCR positive samples by partially sequencing the S1 gene, including the hypervariable regions (nucleotides 705–1097). Two predominant genotypes were detected, with the Massachusetts type dominating (66%), among which 25% of the samples were identical to the H120 vaccine. The second most common genotype (present in 32% of the flocks) was surprisingly Italy 02, revealing the first detection of this genotype in Morocco and also in Africa. 793B, the predominant genotype in the late 1990s in Morocco, was only detected on one occasion and was identical to the 4/91 vaccine strain. This study highlights the high prevalence of IBV in poultry farms in Morocco and confirms its continuous dynamic changes and evolution.

Low pathogenic avian influenza (H9N2) in chicken: Evaluation of an ancestral H9-MVA vaccine.

Modified Vaccinia Ankara (MVA) has proven its efficacy as a recombinant vector vaccine for numerous pathogens including influenza virus. The present study aimed at evaluating a recombinant MVA candidate vaccine against low pathogenic avian influenza virus subtype H9N2 in the chicken model. As the high genetic and antigenic diversity of H9N2 viruses increases vaccine design complexity, one strategy to widen the range of vaccine coverage is to use an ancestor sequence. We therefore generated a recombinant MVA encoding for the gene sequence of an ancestral hemagglutinin H9 protein (a computationally derived amino acid sequence of the node of the H9N2 G1 lineage strains was obtained using the ANCESCON program). We analyzed the genetics and the growth properties of the MVA vector virus confirming suitability for use under biosafety level 1 and tested its efficacy when applied either as an intra-muscular (IM) or an oral vaccine in specific pathogen free chickens challenged with A/chicken/Tunisia/12/2010(H9N2). Two control groups were studied in parallel (unvaccinated and inoculated birds; unvaccinated and non-inoculated birds). IM vaccinated birds seroconverted as early as four days post vaccination and neutralizing antibodies were detected against A/chicken/Tunisia/12/2010(H9N2) in all the birds before challenge. The role of local mucosal immunity is unclear here as no antibodies were detected in eye drop or aerosol vaccinated birds. Clinical signs were not detected in any of the infected birds even in absence of vaccination. Virus replication was observed in both vaccinated and unvaccinated chickens, suggesting the MVA-ancestral H9 vaccine may not stop virus spread in the field. However vaccinated birds showed less histological damage, fewer influenza-positive cells and shorter virus shedding than their unvaccinated counterparts.

Full genome sequence of guinea fowl coronavirus associated with fulminating disease

Guinea fowl coronavirus (GfCoV), a recently characterized avian coronavirus, was identified from outbreaks of fulminating disease (peracute enteritis) in guinea fowl in France. The full-length genomic sequence was determined to better understand its genetic relationship with avian coronaviruses. The full-length coding genome sequence was 26,985 nucleotides long with 11 open reading frames and no hemagglutinin–esterase gene: a genome organization identical to that of turkey coronavirus [5′ untranslated region (UTR)—replicase (ORFs 1a, 1ab)—spike (S) protein—ORF3 (ORFs 3a, 3b)—small envelop (E or 3c) protein—membrane (M) protein—ORF5 (ORFs 4b, 4c, 5a, 5b)—nucleocapsid (N) protein (ORFs N and 6b)—3′ UTR]. This is the first complete genome sequence of a GfCoV and confirms that the new virus belongs to group gammacoronaviruses.

Development of an improved polykaryon-based influenza virus rescue system

BackgroundVirus rescue from transfected cells is an extremely useful technique that allows defined viral clones to be engineered for the purpose of rational vaccine design or fundamental reverse genetics studies. However, it is often hindered by low primary rescue success rates or yields, especially with field-derived viral strains.ApproachWe investigated the possibility of enhancing influenza virus rescue by eliciting cell fusion to increase the chances of having all necessary plasmids expressed within the same polykaryon. To this end we used the Maedi-Visna Virus envelope protein which has potent fusion activity in cells from a wide range of different species.ResultsCo-transfecting cells with the eight plasmids necessary to rescue influenza virus plus a plasmid expressing the Maedi-Visna Virus envelope protein resulted in increased rescue efficiency. In addition, partial complements of the 8-plasmid rescue system could be transfected into two separate populations of cells, which upon fusion led to live virus rescue.ConclusionThe simple modification described here has the potential to improve the efficiency of the virus rescue process and expand the potential applications for reverse genetic studies.

Co-infection of turkeys with Escherichia coli (O78) and H6N1 avian influenza virus

ABSTRACT Respiratory diseases are responsible for major economic losses in poultry farms. While in most cases a single pathogen is not alone responsible for the clinical outcome, the impact of co-infections is not well known, especially in turkeys. The purpose of this study was to assess the possible synergism between Escherichia coli (O78) and low pathogenic avian influenza virus (LPAIV, H6N1), in the turkey model. Four-week-old commercial turkeys were inoculated with either H6N1, O78 or both agents simultaneously or three days apart. We have established an experimental infection model of turkeys using aerosolization that better mimics field infections. Birds were observed clinically and swabbed on a daily basis. Necropsies were performed at 4 and 14 days post single or dual inoculation and followed by histological and immunohistochemical analyses. Combined LPAIV/E. coli infections resulted in more severe clinical signs, were associated with higher mortality and respiratory organ lesions (mucous or fibrinous exudative material in lungs and air sacs), in comparison with the groups given single infections (P < 0.05). The time interval or the sequence between H6N1 and E. coli inoculation (none or three days) did not have a significant effect on the outcome of the dual infection and disease although slightly greater (P > 0.05) respiratory signs were observed in turkeys of the E. coli followed by H6N1 inoculated group. Microscopic lesions and immunohistochemical staining supported clinical and macroscopic findings. Efficient virus and bacteria replication was observed in all inoculated groups. E. coli and H6N1 thus exercise an additive or synergistic pathogenic effect in the reproduction of respiratory disease.

Recommendations for a standardized avian coronavirus (AvCoV) nomenclature: outcome from discussions within the framework of the European Union COST Action FA1207: “towards control of avian coronaviruses: strategies for vaccination, diagnosis and surveillance”*

ABSTRACT Viruses within the Coronaviridae family show variations within their genome sequences, especially within the major structural protein the Spike (S) glycoprotein gene. Therefore, many different antigenic forms, serotypes or variant strains of avian coronaviruses (AvCoV) exist worldwide. Only a few of them, the so called protectotypes, cross protect against different serotypes. New serotypes arise by recombination or spontaneous mutations. From time to time, antigenic virus variants appear, which differ significantly from known serotypes. The result of this variability is an inconsistent nomenclature and classification of virus strains. Furthermore, there are currently no standard classification methods defined. Within the framework of the COST Action FA1207 “Towards control of avian coronaviruses: strategies for diagnosis, surveillance and vaccination” (working groups “Molecular virology” and “Epidemiology”), we aimed at defining and developing a unified and internationally standardized nomenclature and classification of AvCoVs. We recommend the use of “CoV Genus/AvCov/host/country/specimen id/year” to refer to AvCoV strains.

Identification of a novel coronavirus from guinea fowl using metagenomics.

While classical virology techniques such as virus culture, electron microscopy, or classical PCR had been unsuccessful in identifying the causative agent responsible for the fulminating disease of guinea fowl, we identified a novel avian gammacoronavirus associated with the disease using metagenomics. Next-generation sequencing is an unbiased approach that allows the sequencing of virtually all the genetic material present in a given sample.