RESUMO
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.
Assuntos
Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Vírus da Influenza A Subtipo H5N2/genética , Vírus da Influenza A Subtipo H5N2/patogenicidade , Influenza Aviária/virologia , Animais , Aves , Galinhas , Surtos de Doenças , Patos , França/epidemiologia , Genes Virais/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus da Influenza A Subtipo H5N2/classificação , Vírus da Influenza A Subtipo H5N2/isolamento & purificação , Influenza Aviária/epidemiologia , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Aves Domésticas/virologia , Doenças das Aves Domésticas/virologia , RNA Viral/genética , Análise de Sequência de DNARESUMO
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.
Assuntos
Anticorpos Antivirais/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Doenças das Aves Domésticas/prevenção & controle , Vacinas de DNA/imunologia , Animais , Proteção Cruzada , Patos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A Subtipo H5N2/genética , Vírus da Influenza A Subtipo H5N2/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Influenza Aviária/imunologia , Doenças das Aves Domésticas/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
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.
Assuntos
Avulavirus/genética , Aves/virologia , Genoma Viral , Animais , Sequência de Bases , DNA Complementar , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , Análise de Sequência de RNA , Proteínas Virais/genéticaRESUMO
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.
Assuntos
Anticorpos Antivirais/sangue , Aves/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/classificação , Influenza Aviária/sangue , Neuraminidase/imunologia , Animais , França/epidemiologia , Influenza Aviária/epidemiologia , Neuraminidase/classificação , Testes de Neutralização/veterinária , Áreas AlagadasRESUMO
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.
Assuntos
Patos , Virus da Influenza A Subtipo H5N1/classificação , Influenza Aviária/virologia , Animais , Encéfalo/virologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/mortalidade , Influenza Aviária/patologia , Rim/virologia , Pâncreas/virologia , RNA Viral/análise , Traqueia/virologia , Carga Viral , Eliminação de Partículas ViraisRESUMO
Infections with H5/H7 low-pathogenic avian influenza (LPAI) viruses are now notifiable because such viruses can mutate into highly pathogenic avian influenza viruses, leading to serious problems for both animal and public health. Domestic ducks can play a crucial role in the transmission of H5 LPAI viruses to other poultry. Although prime boost vaccination using, respectively, a recombinant vaccine and an inactivated vaccine was shown to be protective in ducks against H5N1 highly pathogenic avian influenza, vaccination of domestic ducks against H5 LPAIV is poorly documented. However, substituting inactivated vaccines with subunit vaccines might be more advantageous. In this context, we generated a triple recombinant baculovirus composed of HA and NA proteins derived from a French H5N3 LPAI virus strain and the M protein derived from an Italian H7N1 LPAI virus strain. We describe a molecular construction strategy that enabled the development of virus-like particles (VLPs). Western blot analyses and neuraminidase inhibition assay of cell supernatants purified by sucrose density gradient ultracentrifugation showed that HA, NA and M1 proteins were expressed and co-released. Electron microscopy examination revealed VLPs that were morphologically identical to wild-type virus. Immunogold electron microscopy demonstrated that H5 and N3 proteins were present on the VLP surface, and haemagglutination and neuraminidase assays showed that the H and N proteins, respectively, were biologically active. In addition, VLP immunogenicity (induction of haemagglutination-inhibiting antibodies) was demonstrated in specific pathogen free Muscovy ducks. According to our successful previous experimental results of protection in ducks following vaccination with the three baculovirus-expressed proteins, the present results make feasible the reliable use of H5N3 VLPs as a subunit vaccine in this species.
Assuntos
Patos/imunologia , Vírus da Influenza A/patogenicidade , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Animais , Anticorpos Antivirais , Baculoviridae , Linhagem Celular , Relação Dose-Resposta Imunológica , Regulação Viral da Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Influenza Aviária/virologia , Insetos , Plasmídeos/genética , Recombinação GenéticaRESUMO
In February 2006, a highly pathogenic avian influenza (HPAI) H5N1 virus was isolated from Common Pochards (Aythia ferina) in the Dombes region of France, an important migrating and wintering waterfowl area. Thereafter, HPAI H5N1 virus was isolated from 39 swab pools collected from dead waterfowl found in the Dombes, but only from three pooled samples collected outside of this area but located on the same migration flyway. A single turkey farm was infected in the Dombes. The epizootic lasted 2 mo and was restricted to the Dombes area. Virus-positive pools were detected in 20 of 1,200 ponds and infected Mute Swans (Cygnus olor) represented 82% of the virus-positive pools. Other infected species included Common Pochard (n=4), Grey Heron (Ardea cinerea, n=1), Eurasian Buzzard (Buteo buteo, n=1), and Greylag Goose (Anser anser, n=1). Despite intensive monitoring during and after the outbreak, HPAI H5N1 virus was not isolated from healthy wild birds. Our results are consistent with an HPAI H5N1-virus introduction into the Dombes via migrating ducks. These birds could have been pushed west by a severe cold spell in central Europe where the virus had already been detected. The Mute Swan served as an excellent epidemiologic sentinel during this outbreak; swans appear to be highly sensitive to infection with these viruses and swan mortality was easy to detect. During the outbreak, the mortality rates for wild birds remained moderate and the virus affected a limited number of species.
Assuntos
Anseriformes/virologia , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/epidemiologia , Influenza Aviária/virologia , Migração Animal , Animais , Animais Domésticos/virologia , Animais Selvagens/virologia , Surtos de Doenças/veterinária , Patos/virologia , Feminino , França/epidemiologia , Influenza Aviária/transmissão , Masculino , Vigilância da População , Estações do Ano , Especificidade da Espécie , Perus/virologiaRESUMO
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 70â¯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.
Assuntos
Anticorpos Antivirais/imunologia , Pareamento Incorreto de Bases/imunologia , Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle/genética , Doenças das Aves Domésticas/prevenção & controle , Vacinas Virais/genética , Animais , Anticorpos Antivirais/sangue , Linhagem Celular , Galinhas/imunologia , Cricetinae , Ensaio de Imunoadsorção Enzimática/veterinária , Genótipo , Doença de Newcastle/terapia , Vírus da Doença de Newcastle/imunologia , Doenças das Aves Domésticas/terapia , Vacinação/veterinária , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Eliminação de Partículas ViraisRESUMO
In Asia, domestic ducks have been shown to play a pivotal role in H5 high-pathogenicity avian influenza virus transmission. We have also observed that the same situation may exist for H5 low-pathogenicity avian influenza (LPAI) virus. No data are available regarding the protection afforded by commercial inactivated vaccines against H5 LPAI virus infection in ducks, and two preliminary experiments using commercial inactivated vaccines gave poor results. Virus-like particles (VLPs) have been shown to be immunogenic in different species. With regard to the influenza model, the matrix (M) protein has been shown to be necessary for the formation of VLPs. In order to attempt to develop a VLP influenza vaccine expressing hemagglutinin and neuraminidase (NA) of interest, we generated a triple recombinant baculovirus (rB) expressing three structural proteins: H5, N3, and M, derived from a recent French LPAI virus strain. Although the three proteins were successfully expressed in rB-infected cells and displayed the expected biological activity, no VLPs were observed. Despite this result, the protection afforded to ducks by rB-infected cell lysates was assessed and was compared with the protection afforded by an inactivated commercial H5N9 vaccine. For this purpose, specific-pathogen-free Muscovy ducks (15 per group) received rB-infected cell lysates (3 wk apart), while a second group received the H5N9 vaccine. Ten days after the boost, a homologous virus challenge was implemented. Both vaccines induced positive hemagglutination inhibition titers and M immune response, whereas lysates of rB-infected cells elicited NA immune response. Tracheal and cloacal sheddings were measured using M-based real-time-reverse transcription-polymerase chain reaction and were compared with the sheddings of vaccinated and unvaccinated infected controls. Lysates of rB-infected cells afforded a significant decrease of cloacal shedding and a delayed peak of tracheal shedding, whereas the inactivated commercial vaccine afforded a significant decrease of tracheal shedding only.
Assuntos
Patos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/classificação , Vacinas contra Influenza/imunologia , Proteínas da Matriz Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Baculoviridae , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/patogenicidade , Influenza Aviária/prevenção & controle , Proteínas Recombinantes , Organismos Livres de Patógenos Específicos , Proteínas da Matriz Viral/metabolismo , Proteínas não Estruturais Virais/metabolismoAssuntos
Charadriiformes/virologia , Reações Falso-Negativas , Vírus da Influenza A Subtipo H5N2/genética , Vírus da Influenza A Subtipo H5N2/isolamento & purificação , Influenza Aviária/diagnóstico , Influenza Aviária/virologia , Animais , Cloaca/virologia , Análise por Conglomerados , França , Técnicas de Diagnóstico Molecular/métodos , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de DNA , Virologia/métodosRESUMO
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.
Assuntos
Anticorpos Antivirais/sangue , Técnicas de Laboratório Clínico/métodos , Vírus da Influenza A/imunologia , Influenza Aviária/diagnóstico , Neuraminidase/imunologia , Medicina Veterinária/métodos , Proteínas Virais/imunologia , Animais , Galinhas , Patos , Ensaio de Imunoadsorção Enzimática/métodos , Curva ROC , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Doença de Newcastle/virologia , Vírus da Doença de Newcastle/classificação , Vírus da Doença de Newcastle/genética , Doenças das Aves Domésticas/virologia , Animais , Animais Selvagens/virologia , Aves , Códon/genética , Genótipo , Proteína HN/genética , Doença de Newcastle/transmissão , Vírus da Doença de Newcastle/isolamento & purificação , Vírus da Doença de Newcastle/patogenicidade , Filogenia , Aves Domésticas , Doenças das Aves Domésticas/transmissão , Proteínas Virais de Fusão/genéticaRESUMO
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.
Assuntos
Genoma Viral/genética , Metapneumovirus/genética , Sequência de Aminoácidos , Sequência de Bases , Códon/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
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.
Assuntos
Patos/virologia , Vírus da Influenza A/fisiologia , Influenza Aviária/transmissão , Influenza Aviária/virologia , Animais , Vírus da Influenza A/patogenicidade , Influenza Aviária/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Organismos Livres de Patógenos EspecíficosRESUMO
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.
Assuntos
Patos/virologia , Varíola Aviária/genética , Imunização Secundária/métodos , Virus da Influenza A Subtipo H5N1/patogenicidade , Vacinas contra Influenza/farmacologia , Influenza Aviária/prevenção & controle , Vírus da Doença de Newcastle/genética , Animais , Encéfalo/virologia , Varíola Aviária/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Imunidade Humoral , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Aviária/mortalidade , Influenza Aviária/virologia , Vírus da Doença de Newcastle/imunologia , Resultado do Tratamento , Vacinas Sintéticas , Eliminação de Partículas Virais/imunologiaRESUMO
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.
RESUMO
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.
Assuntos
Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática/veterinária , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/classificação , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , Animais , Anticorpos Monoclonais , Anticorpos Antivirais/isolamento & purificação , Galinhas , Ensaio de Imunoadsorção Enzimática/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/metabolismo , Influenza Aviária/sangue , Influenza Aviária/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Organismos Livres de Patógenos EspecíficosRESUMO
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.
Assuntos
Anticorpos Antivirais/sangue , Técnicas de Laboratório Clínico/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Influenza Aviária/diagnóstico , Medicina Veterinária/métodos , Animais , Teorema de Bayes , Patos , Ensaio de Imunoadsorção Enzimática/métodos , Sensibilidade e Especificidade , Soro/imunologiaRESUMO
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.
Assuntos
Regiões 5' não Traduzidas , Metapneumovirus/genética , Vírus da Doença de Newcastle/genética , RNA Viral/genética , Análise de Sequência de DNA/métodos , Reação em Cadeia da Polimerase , RNA Viral/química , Transcrição ReversaRESUMO
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.