Direct evidence of H7N7 avian influenza virus mutation from low to high virulence on a single poultry premises during an outbreak in free range chickens in the UK, 2008.

H5 and H7 subtypes of low pathogenicity avian influenza viruses (LPAIVs) have the potential to evolve into highly pathogenic avian influenza viruses (HPAIVs), causing high mortality in galliforme poultry with substantial economic losses for the poultry industry. This study provides direct evidence of H7N7 LPAIV mutation to HPAIV on a single poultry premises during an outbreak that occurred in June 2008 in free range laying hens in Oxfordshire, UK. We report the first detection of a rare di-basic cleavage site (CS) motif (PEIPKKRGLF), unique to galliformes, that has previously been associated with a LPAIV phenotype. Three distinct HPAIV CS sequences (PEIPKRKKRGLF, PEIPKKKKRGLF and PEIPKKKKKKRGLF) were identified in the infected sheds suggesting molecular evolution at the outbreak premises. Further evidence for H7N7 LPAIV preceding mutation to HPAIV was derived by examining clinical signs, epidemiological descriptions and analysing laboratory results on the timing and proportions of seroconversion and virus shedding at each infected shed on the premises. In addition to describing how the outbreak was diagnosed and managed via statutory laboratory testing, phylogenetic analysis revealed reassortant events during 2006-2008 that suggested likely incursion of a wild bird origin LPAIV precursor to the H7N7 HPAIV outbreak. Identifying a precursor LPAIV is important for understanding the molecular changes and mechanisms involved in the emergence of HPAIV. This information can lead to understanding how and why only some H7 LPAIVs appear to readily mutate to HPAIV.

Evaluation of ELISA and haemagglutination inhibition as screening tests in serosurveillance for H5/H7 avian influenza in commercial chicken flocks.

Avian influenza virus (AIV) subtypes H5 and H7 can infect poultry causing low pathogenicity (LP) AI, but these LPAIVs may mutate to highly pathogenic AIV in chickens or turkeys causing high mortality, hence H5/H7 subtypes demand statutory intervention. Serological surveillance in the European Union provides evidence of H5/H7 AIV exposure in apparently healthy poultry. To identify the most sensitive screening method as the first step in an algorithm to provide evidence of H5/H7 AIV infection, the standard approach of H5/H7 antibody testing by haemagglutination inhibition (HI) was compared with an ELISA, which detects antibodies to all subtypes. Sera (n = 1055) from 74 commercial chicken flocks were tested by both methods. A Bayesian approach served to estimate diagnostic test sensitivities and specificities, without assuming any 'gold standard'. Sensitivity and specificity of the ELISA was 97% and 99.8%, and for H5/H7 HI 43% and 99.8%, respectively, although H5/H7 HI sensitivity varied considerably between infected flocks. ELISA therefore provides superior sensitivity for the screening of chicken flocks as part of an algorithm, which subsequently utilises H5/H7 HI to identify infection by these two subtypes. With the calculated sensitivity and specificity, testing nine sera per flock is sufficient to detect a flock seroprevalence of 30% with 95% probability.

Antigenic and genetic analyses of isolate APMV/wigeon/Italy/3920-1/2005 indicate that it represents a new avian paramyxovirus (APMV-12).

Isolate wigeon/Italy/3920-1/2005 (3920-1) was obtained during surveillance of wild birds in November 2005 in the Rovigo province of Northern Italy and shown to be a paramyxovirus. Analysis of cross-haemagglutination-inhibition tests between 3920-1 and representative avian paramyxoviruses showed only a low-level relationship to APMV-1. Phylogenetic analysis of the whole genome and each of the six genes indicated that while 3920-1 grouped with APMV-1 and APMV-9 viruses, it was quite distinct from these two. In the whole-genome analysis, 3920-1 had 52.1 % nucleotide sequence identity to the closest APMV-1 virus, 50.1 % identity to the APMV-9 genome, and less than 42 % identity to representatives of the other avian paramyxovirus groups. We propose isolate wigeon/Italy/3920-1/2005 as the prototype strain of a further APMV group, APMV-12.

Demonstration of Ornithobacterium rhinotracheale in pheasants (Phasianus colchicus) with pneumonia and airsacculitis.

Outbreaks of respiratory disease were investigated in reared pheasants (Phasianus colchicus) aged approximately 18 to 32 weeks, released into the semi-wild on four shooting estates in southern England. The clinical signs in the affected birds included swelling of the face and eyes, loss of condition, gasping respirations and coughing. The gross pathology findings included sinusitis, airsacculitis, pleural oedema and lung lesions. The histopathological findings in the affected lungs were characterized by a granulomatous pneumonia. Ornithobacterium rhinotracheale (ORT) was isolated from respiratory tract tissues, and 16S rRNA gene sequencing on three isolates revealed two distinct genotypes, one previously associated with some electrophoretic type (ET) 1 strains and the other a novel genotype that clustered among sequences previously associated with ET 3, ET 4, ET 5 and ET 6 isolates. The localization of ORT within the lung tissue was demonstrated by fluorescent in-situ hybridization in the bronchial exudate of three cases, although not within the granulomatous lesions themselves. In each case, ORT was identified as part of a complex of other respiratory agents including avian paramyxovirus type 2, avian coronavirus, Mycoplasma gallisepticum, Mycoplasma synoviae and other Mycoplasma species, Escherichia coli, Pasteurella multocida, other Pasteurellaceae and Syngamus trachea, suggesting synergism with other agents. Exposure to other intercurrent factors, including adverse weather conditions and internal parasitism, may also have exacerbated the severity of disease.

Phylogenetic and molecular characteristics of Eurasian H9 avian influenza viruses and their detection by two different H9-specific RealTime reverse transcriptase polymerase chain reaction tests.

Avian influenza viruses (AIVs) of the H9 haemagglutinin subtype are endemic in many Asian and Middle-East countries, causing mortality and morbidity in poultry. Consequently there is a need for accurate and sensitive detection of Eurasian H9 subtype viruses. Two H9 RealTime reverse transcriptase polymerase chain reaction (RRT-PCR) tests, developed by Monne et al. (2008) and Ben Shabat et al. (2010), were originally validated with a limited number of H9 specimens. In the present study, the two tests have been assessed using 66 diverse H9 isolates and 139 clinical specimens from six H9 poultry outbreaks in four geographically disparate Eurasian countries. The Monne et al. (2008) test was modified and successfully detected all H9 viruses from all three Eurasian H9 lineages. Bayesian analysis of the clinical specimens' results revealed this test to be more sensitive (97%) than the Ben Shabat et al. (2010) test (31%). The latter test detected most H9 isolates of the G1 lineage, but no isolates from other H9 lineages. Mismatches in the primer/probe binding sequences accounted for sensitivity differences between the two H9 RRT-PCRs. Genetic analysis of 34 sequenced H9 haemagglutinin genes showed the South Asian and Middle-East H9 isolates to belong to the H9 G1 lineage, and possessed residues that appear to preferably bind alpha 2,6-linked sialic acid receptors which indicate a potential for human infection. European H9s clustered phylogenetically in a broader geographical group that includes recent North American H9 wild bird isolates and contemporary Asian viruses in the Y439 H9 lineage.

The ecology and age structure of a highly pathogenic avian influenza virus outbreak in wild mute swans.

The first UK epizootic of highly pathogenic (HP) H5N1 influenza in wild birds occurred in 2008, in a population of mute swans that had been the subject of ornithological study for decades. Here we use an innovative combination of ornithological, phylogenetic and immunological approaches to investigate the ecology and age structure of HP H5N1 in nature. We screened samples from swans and waterbirds using PCR and sequenced HP H5N1-positive samples. The outbreak's origin was investigated by linking bird count data with a molecular clock analysis of sampled virus sequences. We used ringing records to reconstruct the age-structure of outbreak mortality, and we estimated the age distribution of prior exposure to avian influenza. Outbreak mortality was low and all HP H5N1-positive mute swans in the affected population were <3 years old. Only the youngest age classes contained an appreciable number of individuals with no detectable antibody responses to viral nucleoprotein. Phylogenetic analysis indicated that the outbreak strain circulated locally for ~1 month before detection and arrived when the immigration rate of migrant waterbirds was highest. Our data are consistent with the hypothesis that HP H5N1 epizootics in wild swans exhibit limited mortality due to immune protection arising from previous exposure. Our study population may represent a valuable resource for investigating the natural ecology and epidemiology of avian influenza.

Outbreak of Newcastle disease due to pigeon paramyxovirus type 1 in grey partridges (Perdix perdix) in Scotland in October 2006.

In October 2006, following an initially non-statutory disease investigation affecting 12-week-old grey partridges (Perdix perdix), an outbreak of Newcastle disease due to infection with the avian paramyxovirus type 1 virus responsible for the current panzootic in pigeons (PPMV-1) was confirmed in Scotland. Two pens of partridges were affected by signs including loss of condition, diarrhoea, progressive neurological signs and mortality totalling approximately 24 per cent, and laboratory evidence of the infection was obtained only in these groups. The premises had approximately 17,000 poultry including a collection of 375 birds of rare breeds, containing endangered breeds of significant conservation value, which were not culled but subjected to a health monitoring and testing programme. Investigations suggested that a population of feral pigeons living above the affected pens of partridges was the likely source of the outbreak. Laboratory and genetic analyses confirmed that the isolate recovered from the clinically affected partridges was PPMV-1, belonging to genetic lineage 4b. However, the virus could not be isolated from or detected in dead pigeons collected from the affected buildings.

Recent epidemiology and ecology of influenza A viruses in avian species in Europe and the Middle East.

There have been at least ten distinct outbreaks of LPAI or HPAI in poultry caused by H5 or H7 viruses in the last eight years in Europe and the Middle East. There appears to be an increased occurrence of such episodes consistent with global trends. As a result, surveillance systems have been enhanced to facilitate early detection of infection in poultry, together with active surveillance of wild bird populations. These complementary activities have resulted in the detection of a number of viruses in wild bird populations, including some with high genetic similarity to newly detected viruses in poultry, for example, H7N3 in Italy and H7N7 in the Netherlands. Furthermore, there is evidence for continued circulation of H5 and H7 viruses in wild Anseriformes, thereby presenting a real and current threat for the introduction of viruses to domestic poultry, especially those reared in outdoor production systems. Viruses of H9N2 subtype continue to circulate widely in the Middle East and are associated with significant disease problems in poultry. The epidemiology has the potential to be complicated further by introduction of novel viruses through illegal importation of captive birds, such as was detected with H5N1 in Belgium in 2004. Continual genetic exchange in the avian virus gene pool and independent evolution of all gene segments either within an individual host species or among wild bird hosts suggests that these viruses are not in evolutionary stasis in the natural reservoir.

Strains of avian paramyxovirus type 1 of low pathogenicity for chickens isolated from poultry and wild birds in Denmark.

Twenty-one strains of avian paramyxovirus type 1 of low virulence for chickens were isolated in Denmark between 1996 and the beginning of 2003. The low virulence of the strains was demonstrated by sequencing the fusion (F) gene at the cleavage site motif and in some cases by determining the intracerebral pathogenicity index in day-old chicks. By using a panel of monoclonal antibodies it was shown that the isolates belonged to four different antigenic groups (five C2 isolates, six E isolates, six H isolates and four G/Q isolates). They were placed in three distinguishable genetic groups by phylogenetic analysis of a partial sequence of the F gene. The origin of the six E isolates was probably contaminated vaccines; the other viruses were isolated from wild birds and from poultry which probably came into contact with wild birds.

Retrospective characterization of Newcastle Disease Virus Antrim '73 in relation to other epidemics, past and present.

In November 1973 Newcastle disease suddenly appeared in Northern Ireland, where the viscerotropic disease had not been seen in 3 1/2 years and the two Irelands had been regarded as largely disease free for 30 years. It was successfully controlled with only 36 confirmed affected layer flocks, plus 10 more slaughtered as 'dangerous contacts'. Contemporary investigations failed to reveal the source of the Irish epidemic. Using archival virus samples from most of the affected flocks, RT PCR was conducted with primers selected for all six NDV genes. Phylogenetic analyses of three genes, HN, M and F, confirmed vaccine as the cause of one of the outbreaks. The other six samples were identical and closely related to previous outbreaks in the United States and western Europe initiated by infected imported Latin American parrots. The probable cause of the epidemic followed from the importation from The Netherlands of bulk feed grains contaminated with infected pigeon faeces.

Pathogenesis of H7 influenza A viruses isolated from ostriches in the homologous host infected experimentally.

Infections of ostriches with avian influenza A viruses are generally associated with clinical disease, but the occasional high mortality in young birds does not appear to be related directly to virus pathotype. In this study we investigated the pathogenesis of two H7 viruses for 11-wk-old ostriches inoculated intranasally, and clinical symptoms, virus excretion, and immune response were studied. One of the viruses (A/Ostrich/Italy/1038/00) was highly pathogenic for chickens, whereas the other (A/Ostrich/South Africa/1609/91) was of low pathogenicity for chickens. Clinical signs in ostriches receiving virulent virus were slight depression and hemorrhagic diarrhea, while the group receiving avirulent virus was clinically normal except for green diarrhea. Both viruses were transmitted to in-contact sentinel birds housed with the infected groups 3 days postinfection. Postmortem examination of the birds infected (including the sentinel bird) with virus highly pathogenic for chickens were grossly normal except for localized pneumonic lesions. The results of the study are presented and discussed.

Avian paramyxoviruses and influenza viruses isolated from mallard ducks (Anas platyrhynchos) in New Zealand.

A comprehensive study using virological and serological approaches was carried out to determine the status of live healthy mallard ducks (Anas platyrhynchos) in New Zealand for infections with avian paramyxoviruses (APMV) and influenza viruses (AIV). Thirty-three viruses isolated from 321 tracheal and cloacal swabs were characterized as: 6 AIV (two H5N2 and four H4N6), 10 APMV-1 and 17 APMV-4. Of 335 sera samples tested for AIV antibodies, 109 (32.5%) sera were positive by nucleoprotein-blocking ELISA (NP-B-ELISA). Serum samples (315) were examined for antibody to APMV-1, -2, -3, -4, -6, -7, -8, -9 by the haemagglutination inhibition test. The largest number of reactions, with titres up to > or =1/64, was to APMV-1 (93.1%), followed by APMV-6 (85.1%), APMV-8 (56%), APMV-4 (51.7%), APMV-7 (47%), APMV-9 (15.9%), APMV-2 (13.3%) and APMV-3 (6.0%). All of the H5N2 isolates of AIV and the APMV-1 isolates from this and earlier New Zealand studies had low pathogenicity indices assessed by the Intravenous Pathogenicity Index (IVPI) with the result 0.00 and Intracerebral Pathogenicity Index (ICPI) with results 0.00-0.16. Partial genomic and antigenic analyses were also consistent with the isolates being non-pathogenic. Phylogenetic analysis of the 10 APMV-1 isolates showed 9 to be most similar to the reference APMV-1 strain D26/76 originally isolated in Japan and also to the Que/66 strain, which was isolated in Australia. The other isolate was very similar to a virus (MC 110/77) obtained from a shelduck in France.

Phylogenetic analysis of avian paramyxovirus 1 strains isolated in Finland.

Eight strains of avian paramyxovirus type 1 (PMV-1) isolated in Finland during the last 3 decades were studied with reverse transcriptase polymerase chain reaction (RT-PCR) and subsequent sequence analysis of the region of 208 nucleotides covering the fusion (F) protein cleavage site. Both genetic and antigenic heterogeneity of the strains was significant. Direct epidemiological links between strains isolated during successive outbreaks, and also between strains isolated from wild fauna and from poultry or captive birds, were seen in this study. These results also support the previously published view that wild waterfowl serve as a reservoir for the apathogenic or low-pathogenic strains, allowing them to evolve further into pathogenic strains.