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

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 log2. 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.

Emerging highly pathogenic H5 avian influenza viruses in France during 2015/16: phylogenetic analyses and markers for zoonotic potential.

Several new highly pathogenic (HP) H5 avian influenza virus (AIV) have been detected in poultry farms from south-western France since November 2015, among which an HP H5N1. The zoonotic potential and origin of these AIVs immediately became matters of concern. One virus of each subtype H5N1 (150169a), H5N2 (150233) and H5N9 (150236) was characterised. All proved highly pathogenic for poultry as demonstrated molecularly by the presence of a polybasic cleavage site in their HA protein - with a sequence (HQRRKR/GLF) previously unknown among avian H5 HPAI viruses - or experimentally by the in vivo demonstration of an intravenous pathogenicity index of 2.9 for the H5N1 HP isolate. Phylogenetic analyses based on the full genomes obtained by NGS confirmed that the eight viral segments of the three isolates were all part of avian Eurasian phylogenetic lineage but differed from the Gs/Gd/1/96-like lineage. The study of the genetic characteristics at specific amino acid positions relevant for modulating the adaptation to and the virulence for mammals showed that presently, these viruses possess most molecular features characteristic of AIV and lack some major characteristics required for efficient respiratory transmission to or between humans. The three isolates are therefore predicted to have no significant pandemic potential.

Development and evaluation of an N9-specific enzyme-linked immunosorbent assay to detect antibodies in duck and chicken sera.

A serological test for detecting N9-specific antibodies may be useful as a DIVA strategy to differentiate vaccinated from infected animals or simply for direct serological detection of infection with N9-subtype virus. The method currently recommended for the detection of antibodies against neuraminidase is neuraminidase inhibition (NI), which is a laborious method using toxic chemicals and has low sensitivity. The present study describes the development and validation of an N9-specific ELISA. Data obtained with this N9 ELISA were compared to those obtained with nucleoprotein-based ELISA, haemagglutination inhibition test using homologous antigen and NI assay. 785 sera from ducks and chickens were used, from flocks previously determined to be AI negative or from experimentally infected or immunized flocks. Sensitivity and specificity were evaluated, and a ROC curve and kappa values, which provide a comparison between methods, were calculated. The results obtained in this study indicate that the N9 based-ELISA is effective in detecting N9-specific antibodies with high specificity and with better sensitivity than the recommended NI method; using data from 177 common sera tested with N9 ELISA and NI assay both compared to NP-based ELISA, their specificity were evaluated at 93.6% and 91.5% respectively, and sensitivity at 90.8% and 39.2% respectively.

Molecular comparisons of full length metapneumovirus (MPV) genomes, including newly determined French AMPV-C and -D isolates, further supports possible subclassification within the MPV Genus.

Four avian metapneumovirus (AMPV) subgroups (A-D) have been reported previously based on genetic and antigenic differences. However, until now full length sequences of the only known isolates of European subgroup C and subgroup D viruses (duck and turkey origin, respectively) have been unavailable. These full length sequences were determined and compared with other full length AMPV and human metapneumoviruses (HMPV) sequences reported previously, using phylogenetics, comparisons of nucleic and amino acid sequences and study of codon usage bias. Results confirmed that subgroup C viruses were more closely related to HMPV than they were to the other AMPV subgroups in the study. This was consistent with previous findings using partial genome sequences. Closer relationships between AMPV-A, B and D were also evident throughout the majority of results. Three metapneumovirus "clusters" HMPV, AMPV-C and AMPV-A, B and D were further supported by codon bias and phylogenetics. The data presented here together with those of previous studies describing antigenic relationships also between AMPV-A, B and D and between AMPV-C and HMPV may call for a subclassification of metapneumoviruses similar to that used for avian paramyxoviruses, grouping AMPV-A, B and D as type I metapneumoviruses and AMPV-C and HMPV as type II.

H5-based DNA constructs derived from selected highly pathogenic H5N1 avian influenza virus induce high levels of humoral antibodies in Muscovy ducks against low pathogenic viruses.

BACKGROUND: H5 low pathogenic avian influenza virus (LPAIV) infection in domestic ducks is a major problem in duck producing countries. Their silent circulation is an ongoing source of potential highly pathogenic or zoonotic emerging strains. To prevent such events, vaccination of domestic ducks might be attempted but remains challenging. Currently licensed vector vaccines derived from H5N1 HPAIV possess clade 0, clade 2.2 or clade 2.3.4 HA sequences: selection of the best HA candidate inducing the largest cross protection is a key issue. For this purpose, DNA immunization of specific pathogen free Muscovy ducks was performed using different synthetic codon optimized (opt) or native HA genes from H5N2 LPAIV and several H5N1 HPAIV clade 2.1, 2.2.1 and 2.3.4. Humoral cross-immunity was assessed 3 weeks after boost by hemagglutination inhibition (HI) and virus neutralization (VN) against three French H5 LPAIV antigens. FINDINGS: Vaccination with LP H5N2 HA induced the highest VN antibody titre against the homologous antigen; however, the corresponding HI titre was lower and comparable to HI titres obtained after immunization with opt HA derived from clades 2.3.4 or 2.1. Compared to the other HPAIV-derived constructs, vaccination with clade 2.3.4 opt HA consistently induced the highest antibody titres in HI and VN, when tested against all three H5 LPAIV antigens and H5N2 LPAIV, respectively: differences in titres against this last strain were statistically significant. CONCLUSION: The present study provides a standardized method to assess cross-immunity based on HA immunogenicity alone, and suggests that clade 2.3.4-derived recombinant vaccines might be the optimal candidates for further challenge testing to vaccinate domestic Muscovy ducks against H5 LPAIV.

Characterisation of a type 1 Avian Paramyxovirus belonging to a divergent group.

Newcastle disease, induced by a type 1 Avian Paramyxovirus (APMV-1), is one of the most serious poultry diseases. APMV-1 are divided into two classes based on genetic analysis: class II strains have been recovered from wild or domestic birds and include virulent and avirulent isolates whereas class I strains have been mainly isolated from wild birds and are avirulent. Within class I, a new proposed genotype has recently been reported. The only full genome strain of this group is presently characterised from the point of view of codon usage with reference to class I and class II specificities. Class-specific residues were identified on HN and F proteins that are the two major proteins involved in cell attachment and pathogenicity. Comparison of protein patterns and codon usage for this newly identified APMV-1 strain indicates it is similar to class I viruses but contains a few characteristics close to the viruses of class II. Transmission of viruses from this recently identified divergent group from wild birds to domestic birds could have a major impact on the domestic poultry industry.

Quantitative transmission characteristics of different H5 low pathogenic avian influenza viruses in Muscovy ducks.

EU annual serosurveillance programs show that domestic duck flocks have the highest seroprevalence of H5 antibodies, demonstrating the circulation of notifiable avian influenza virus (AIV) according to OIE, likely low pathogenic (LP). Therefore, transmission characteristics of LPAIV within these flocks can help to understand virus circulation and possible risk of propagation. This study aimed at estimating transmission parameters of four H5 LPAIV (three field strains from French poultry and decoy ducks, and one clonal reverse-genetics strain derived from one of the former), using a SIR model to analyze data from experimental infections in SPF Muscovy ducks. The design was set up to accommodate rearing on wood shavings with a low density of 1.6 ducks/m(2): 10 inoculated ducks were housed together with 15 contact-exposed ducks. Infection was monitored by RNA detection on oropharyngeal and cloacal swabs using real-time RT-PCR with a cutoff corresponding to 2-7 EID50. Depending on the strain, the basic reproduction number (R0) varied from 5.5 to 42.7, confirming LPAIV could easily be transmitted to susceptible Muscovy ducks. The lowest R0 estimate was obtained for a H5N3 field strain, due to lower values of transmission rate and duration of infectious period, whereas reverse-genetics derived H5N1 strain had the highest R0. Frequency and intensity of clinical signs were also variable between strains, but apparently not associated with longer infectious periods. Further comparisons of quantitative transmission parameters may help to identify relevant viral genetic markers for early detection of potentially more virulent strains during surveillance of LPAIV.

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

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.

An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies.

BACKGROUND: Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method.H5 and H7 specific monoclonal antibodies were experimentally raised and used in the development of inhibition ELISAs for detection of serological response specifically directed against AIV subtypes H5 and H7. The ELISAs were evaluated with polyclonal chicken anti-AIV antibodies against AIV subtypes: H1N2, H5N2, H5N7, H7N1, H7N7, H9N9, H10N4 and H16N3. RESULTS: Both the H5 and H7 ELISA proved to have a high sensitivity and specificity and the ELISAs detected H5 and H7 antibodies earlier during experimental infection than the HI test did. The reproducibility of the ELISA's performed at different times was high with Pearson correlation coefficients of 0.96-0.98. CONCLUSIONS: The ELISAs are a potential alternative to the HI test for screening of large amounts of avian sera, although only experimental sera were tested in this study.

Prime-boost vaccination with recombinant H5-fowlpox and Newcastle disease virus vectors affords lasting protection in SPF Muscovy ducks against highly pathogenic H5N1 influenza virus.

Vaccination protocols were evaluated in one-day old Muscovy ducklings, using an experimental Newcastle disease recombinant vaccine (vNDV-H5) encoding an optimized synthetic haemagglutinin gene from a clade 2.2.1 H5N1 highly pathogenic (HP) avian influenza virus (AIV), either as a single administration or as a boost following a prime inoculation with a fowlpox vectored vaccine (vFP89) encoding a different H5 HP haemagglutinin from an Irish H5N8 strain. These vaccination schemes did not induce detectable levels of serum antibodies in HI test using a clade 2.2.1 H5N1 antigen, and only induced H5 ELISA positive response in less than 10% of vaccinated ducks. However, following challenge against a clade 2.2.1 HPAIV, both protocols afforded full clinical protection at six weeks of age, and full protection against mortality at nine weeks. Only the prime-boost vaccination (vFP89+vNDV-H5) was still fully protecting Muscovy ducks against disease and mortality at 12 weeks of age. Reduction of oropharyngeal shedding levels was also constantly observed from the onset of the follow-up at 2.5 or three days post-infection in vaccinated ducks compared to unvaccinated controls, and was significantly more important for vFP89+vNDV-H5 vaccination than for vNDV-H5 alone. Although the latter vaccine is shown immunogenic in one-day old Muscovy ducks, the present work is original in demonstrating the high efficacy of the successive administration of two different vector vaccines encoding two different H5 in inducing lasting protection (at least similar to the one induced by an inactivated reassortant vaccine, Re-5). In addition, such a prime-boost schedule allows implementation of a DIVA strategy (to differentiate vaccinated from infected ducks) contrary to Re-5, involves easy practice on the field (with injection at the hatchery and mass vaccination later on), and should avoid eventual interference with NDV maternally derived antibodies. Last, the HA insert could be updated according to the epidemiological situation.

Evaluation of a commercial ELISA for H5 low pathogenic avian influenza virus antibody detection in duck sera using Bayesian methods.

Following the emergence of highly pathogenic avian influenza (AI), active surveillance of infections due to the H5 and H7 subtypes in poultry has increased and been made compulsory in Europe since 2002, by means of annual serological surveys using the haemagglutination inhibition (HI) test. Domestic anseriforms, particularly ducks and geese, are more frequently infected by H5 low pathogenic AI virus, often subclinically, and represent a threat for other terrestrial poultry. 1783 sera, mainly from ducks, have been used to evaluate and compare a commercial ELISA kit detecting H5 antibodies with the currently recommended HI test. Different approaches to calculating specificity and sensitivity have been used, including the original Bayesian method. Results were similar when data were analyzed at the individual and batch levels, and when using different methods of calculation. However, results showed that H5 ELISA had both a higher sensitivity and a lower specificity than the HI test. Given that sensitivity is the most important factor for a screening test, H5 ELISA could therefore be recommended for AI surveillance, followed in cases of positivity by molecular tests aimed at detecting the virus gene.

An alternative method to determine the 5' extremities of non-segmented, negative sense RNA viral genomes using positive replication intermediate 3' tailing: application to two members of the Paramyxoviridae family.

Determining the sequence of non-segmented, negative sense RNA viral genomes is far from routine and often requires the application of several techniques. In this study, an existing method used currently just for determination of the genomic 3' extremity was used to determine both the 3' and 5' sequence extremities of a Newcastle disease virus and an avian metapneumovirus. This was achieved with a single 3' nucleotide tailing reaction of both the genomic RNA and the full length, positive sense, antigenomic RNA, followed by a single reverse transcription reaction targeted to the common polynucleotide tails, and then individual PCRs specific for each extremity using PCR primers derived from the sequence of the RT primer or from neighbouring virus sequences known previously. For each virus the method was employed separately. Sequences from both viruses were in agreement with those reported previously for other paramyxoviruses, yet one extra base at the 3' and one extra base at the 5' were identified for the avian metapneumovirus. In this study, importantly, the newly determined extremities maintained the complementarity known to exist between the extremities of these viruses. The method was equally successful with both viruses and can be tailored easily to function with other non-segmented, negative sense viruses through minor modification of only the primer sequences.

Complete genome sequence of a newcastle disease virus strain belonging to a recently identified genotype.

We report the first complete genome sequence of a strain that presents some pathogenicity and that belongs to a recently characterized genotype of avian paramyxovirus type 1 (APMV-1). This virus, isolated from the common teal, presents the most divergent genome within class I of APMV-1.

Complete genome sequence of a novel avian paramyxovirus.

We report here the complete genome of a new avian paramyxovirus (APMV-11) isolated from common snipes. Sequence data from this virus showed that it has the largest genome of APMV and unusual P gene mRNA editing.

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.

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.

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

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.

Possible sources and spreading routes of highly pathogenic avian influenza virus subtype H5N1 infections in poultry and wild birds in Central Europe in 2007 inferred through likelihood analyses.

Recurrent outbreaks of H5N1 HPAIV occurred in several Central European countries in 2007. In-depth phylogenetic analyses which included full-length genomic sequences of the viruses involved were performed to elucidate possible origins of incursions and transmission pathways. Tree reconstructions as well as host-shift and ancestral area inferences were conducted in a maximum likelihood framework. All viruses belonged to a separate subgroup (termed "EMA-3") within clade 2.2, and, thus, were distinct from two lineages of HPAIV H5N1 viruses (termed "EMA-1" and "EMA-2") present in the same geographic area in 2006. Analysis of concatenated coding regions of all eight genome segments significantly improved resolution and robustness of the reconstructed phylogenies as compared to single gene analyses. At the same time, the methodological limits to establish retrospectively transmission networks in a comparatively small geographic region and spanning a short period of time became evident when only few corroborating field-epidemiological data are available. Ambiguities remained concerning the origin of the EMA-3 viruses from a region covering Southeast Germany and the Czech Republic as well as routes of spread to other European countries. AIV monitoring programmes in place for wild birds and poultry in these countries did not reveal presence of these viruses in either population. Host switches between domestic poultry and wild bird populations occurred several times. Analysis of outbreaks in Northeast Germany and nearby Northern Poland in December 2007 demonstrated that geographic and even temporal vicinity of outbreaks does not necessarily indicate a common source of incursion.

Presence of serum antibodies to influenza A subtypes H5 and N1 in swans and ibises in French wetlands, irrespective of highly pathogenic H5N1 natural infection.

Highly pathogenic (HP) avian influenza A viruses (AIVs) subtype H5N1 (subclade 2.2) were detected in wild birds during outbreaks in France during winter 2006 and summer 2007 in la Dombes wetlands (eastern France) and in Moselle wetlands (northeastern France), respectively. Blood samples from apparently healthy wild birds were collected in 2006 and 2007 from the end of the outbreak to several weeks after the influenza A outbreak inside and outside the contaminated areas, and in 2008 outside the contaminated areas. The samples were tested for the presence and/or quantitation of serum antibodies to influenza A subtypes H5 and N1 using hemagglutination inhibition tests (HITs), a commercial N1-specific enzyme-linked immunosorbent assay kit, and virus neutralization assay. In the HIT, low pathogenicity (LP) and HP H5 AIVs (belonging to H5N1, H5N2, and H5N3 subtypes) were used as antigens. One hundred mute swans were bled in the la Dombes outbreak area in 2006. During 2007, 46 mallards, 69 common pochards, and 59 mute swans were sampled in the Moselle outbreak area. For comparison, blood samples were also collected in 2007 from 60 mute swans from the Marne department where no HP H5N1 influenza A cases have been reported, and in 2008 from 111 sacred ibises in western France where no HP H5N1 influenza A infections in wild birds have been reported either. Mute swans (irrespective of their origin and time of sampling) and sacred ibises (from an area with no known outbreaks) had the highest prevalence of positive sera in the H5 HIT (49-69% and 64%, respectively). The prevalence of anti-H5 antibodies in mallards and common pochards was lower (28% and 27%, respectively). Positive H5- and N1-antibody responses were also significantly associated in swans (irrespective of their origin and time of sampling) and in sacred ibises. However, in swans from the area without outbreaks, the HIT titer against an H5N1 LPAIV was significantly higher than against an H5N1 2.2.1 HPAIV, whereas no difference could be shown for swans from the outbreak areas sampled in 2006 and 2007. These results suggest that ibises and swans from areas without declared outbreaks had acquired humoral immunity after AIV infections with subtypes H5 and N1 but independently from HP H5N1 infection. However, for swans living in outbreak areas, it cannot be excluded that this immunity might result from either a subclinical or a nonlethal infection by HP H5N1.

Experimental infection of Muscovy ducks with highly pathogenic avian influenza virus (H5N1) belonging to clade 2.2.

Highly pathogenic (HP) H5N1 avian influenza (AI) is enzootic in several countries of Asia and Africa and constitutes a major threat, at the world level, for both animal and public health. Ducks play an important role in the epidemiology of AI, including HP H5N1 AI. Although vaccination can be a useful tool to control AI, duck vaccination has not proved very efficient in the field, indicating a need to develop new vaccines and a challenge model to evaluate the protection for duck species. Although Muscovy duck is the duck species most often reared in France, the primary duck-producing country in Europe, and is also produced in Asia, it is rarely studied. Our team recently demonstrated a good cross-reactivity with hemagglutinin from clade 2.2 and inferred that this could be a good vaccine candidate for ducks. Two challenges using two French H5N1 HP strains, 1) A/mute swan/France/06299/06 (Swan/06299), clade 2.2.1, and 2) A/mute swan/France/070203/07 (Swan/070203), clade 2.2 (but different from subclade 2.2.1), were performed (each) on 20 Muscovy ducks (including five contacts) inoculated by oculo-nasal route (6 log10 median egg infectious doses per duck). Clinical signs were recorded daily, and cloacal and oropharyngeal swabs were collected throughout the assay. Autopsies were done on all dead ducks, and organs were taken for analyses. Virus was measured by quantitative reverse transcriptase-PCR based on the M gene AI virus. Ducks presented severe nervous signs in both challenges. Swan/070203 strain led to 80% morbidity (12/15 sick ducks) and 73% mortality (11/15 ducks) at 13.5 days postinfection (dpi), whereas Swan/06299 strain produced 100% mortality at 6.5 dpi. Viral RNA load was significantly lower via the cloacal route than via the oropharyngeal route in both trials, presenting a peak in the first challenge at 3.5 dpi and being more stable in the second challenge. The brain was the organ containing the highest viral RNA load in both challenges. Viral RNA load in a given organ was similar or statistically significantly higher in ducks challenged with Swan/06299 strain. Thus, the Swan/06299 strain was more virulent and could be used as a putative challenge model. Moreover, challenged ducks and contacts contained the same amounts of viral RNA load, demonstrating the rapid and efficient transmission of H5N1 HP in Muscovy ducks in our experimental conditions.