Surveillance and investigative diagnosis of a poultry flock in Great Britain co-infected with an influenza A virus and an avirulent avian avulavirus type 1.

A detailed veterinary and laboratory investigation revealed an unusual case of concurrent avian avulavirus type 1 (AAvV-1, formerly called avian paramyxovirus type 1) and low pathogenicity avian influenza (LPAI) virus infections of chickens during March 2010 in a mixed poultry and livestock farm in Great Britain. Respiratory signs and daily mortality of 5-6 birds in a broiler flock 8-weeks of age prompted submission of two carcasses to an Animal and Plant Health Agency (APHA) regional laboratory. Infectious bronchitis virus infection was suspected initially and virus isolation in SPF embryonated fowls' eggs was attempted at APHA-Weybridge. Avirulent AAvV-1 was detected in the first sampling. Both in vitro nucleotide sequencing of the fusion gene and in vivo pathotyping by intracerebral pathogenicity index revealed an avirulent AAvV-1 not definitively ascribed to licensed vaccine. Upon initial detection of the AAvV-1 virus, statutory restrictions were placed on the farm, an official veterinary visit was performed and further samples were submitted to APHA-Weybridge for official statutory disease investigation. An H2N3 LPAI virus was subsequently isolated from tissue samples and swabs submitted from the follow-up statutory investigation. The subtype was confirmed by haemagglutination inhibition test (HAIT) and neuraminidase inhibition (NI) tests on egg-amplified virus. As neither virus was notifiable according to the internationally recognized EU and OIE standards, and/or definitions of disease, statutory farm restrictions were lifted. Veterinary investigations identified the broiler flock to be free-range, next to a river and duck pen, reinforcing the suspicion of wild bird origin for both viruses which may have been co-circulating in ducks. It could not, however, be established as to whether there were separate introductions of the two viruses or whether there had been a single co-introduction of the viruses. The described case highlights the value of integrated surveillance and laboratory approaches, including veterinary field investigations, international standards and definitions of notifiable avian disease, validated RRT-PCR assays, and virus isolation in achieving rapid and accurate diagnostic results.

Antibody responses to avian influenza viruses in wild birds broaden with age.

For viruses such as avian influenza, immunity within a host population can drive the emergence of new strains by selecting for viruses with novel antigens that avoid immune recognition. The accumulation of acquired immunity with age is hypothesized to affect how influenza viruses emerge and spread in species of different lifespans. Despite its importance for understanding the behaviour of avian influenza viruses, little is known about age-related accumulation of immunity in the virus's primary reservoir, wild birds. To address this, we studied the age structure of immune responses to avian influenza virus in a wild swan population (Cygnus olor), before and after the population experienced an outbreak of highly pathogenic H5N1 avian influenza in 2008. We performed haemagglutination inhibition assays on sampled sera for five avian influenza strains and show that breadth of response accumulates with age. The observed age-related distribution of antibody responses to avian influenza strains may explain the age-dependent mortality observed during the highly pathogenic H5N1 outbreak. Age structures and species lifespan are probably important determinants of viral epidemiology and virulence in birds.

Lack of virological and serological evidence for continued circulation of highly pathogenic avian influenza H5N8 virus in wild birds in the Netherlands, 14 November 2014 to 31 January 2016.

In 2014, H5N8 clade 2.3.4.4 highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/1996 lineage emerged in poultry and wild birds in Asia, Europe and North America. Here, wild birds were extensively investigated in the Netherlands for HPAI H5N8 virus (real-time polymerase chain reaction targeting the matrix and H5 gene) and antibody detection (haemagglutination inhibition and virus neutralisation assays) before, during and after the first virus detection in Europe in late 2014. Between 21 February 2015 and 31 January 2016, 7,337 bird samples were tested for the virus. One HPAI H5N8 virus-infected Eurasian wigeon (Anas penelope) sampled on 25 February 2015 was detected. Serological assays were performed on 1,443 samples, including 149 collected between 2007 and 2013, 945 between 14 November 2014 and 13 May 2015, and 349 between 1 September and 31 December 2015. Antibodies specific for HPAI H5 clade 2.3.4.4 were absent in wild bird sera obtained before 2014 and present in sera collected during and after the HPAI H5N8 emergence in Europe, with antibody incidence declining after the 2014/15 winter. Our results indicate that the HPAI H5N8 virus has not continued to circulate extensively in wild bird populations since the 2014/15 winter and that independent maintenance of the virus in these populations appears unlikely.

The Detection of a Low Pathogenicity Avian Influenza Virus Subtype H9 Infection in a Turkey Breeder Flock in the United Kingdom.

In April 2013, an H9N2 low pathogenicity avian influenza (LPAI) virus was isolated in a turkey breeder farm in Eastern England comprising 4966 birds. Point-of-lay turkey breeding birds had been moved from a rearing site and within 5 days had shown rapid onset of clinical signs of dullness, coughing, and anorexia. Three houses were involved, two contained a total of 4727 turkey hens, and the third housed 239 male turkeys. Around 50% of the hens were affected, whereas the male turkeys demonstrated milder clinical signs. Bird morbidity rose from 10% to 90%, with an increase in mortality in both houses of turkey hens to 17 dead birds in one house and 27 birds in the second house by day 6. The birds were treated with an antibiotic but were not responsive. Postmortem investigation revealed air sacculitis but no infraorbital sinus swellings or sinusitis. Standard samples were collected, and influenza A was detected. H9 virus infection was confirmed in all three houses by detection and subtyping of hemagglutinating agents in embryonated specific-pathogen-free fowls' eggs, which were shown to be viruses of H9N2 subtype using neuraminidase inhibition tests and a suite of real-time reverse transcription PCR assays. LPAI virus pathotype was suggested by cleavage site sequencing, and an intravenous pathogenicity index of 0.00 confirmed that the virus was of low pathogenicity. Therefore, no official disease control measures were required, and despite the high morbidity, birds recovered and were kept in production. Neuraminidase sequence analysis revealed a deletion of 78 nucleotides in the stalk region, suggesting an adaptation of the virus to poultry. Hemagglutinin gene sequences of two of the isolates clustered with a group of H9 viruses containing other contemporary European H9 strains in the Y439/Korean-like group. The closest matches to the two isolates were A/turkey/Netherlands/11015452/11 (H9N2; 97.9-98% nucleotide identity) and A/mallard/Finland/Li13384/10 (H9N2; 97% nucleotide identity). Both PB2 partial sequences were a 100% nucleotide identity with A/mallard/France/090360/09, indicating a European origin of the causative virus. Furthermore, partial sequencing analysis of the remaining genes revealed the virus to be genotypically of European avian origin and therefore of lower risk to public health compared with contemporary viruses in Central and Eastern Asia. Occupational health risks were assessed, and preventative measures were taken.

Phylogenetic analysis of the nucleotide sequences for the HN gene of 22 avian paramyxovirus type 2 viruses reveals marked heterogeneity.

The nucleotide sequence of the HN gene was determined for 21 isolates of avian paramyxovirus type 2 virus and compared with the published HN gene of APMV-2/chicken/California/Yucaipa/56. The HN gene of the 22 viruses had five different lengths in the range of 1737 to 1755 nucleotides coding for 579 to 585 amino acids. Phylogenetic analysis of a corresponding 1734-nucleotide sequence from the HN gene of each virus established five genetic groups (I to V), two of which (II and IV) could be divided into two sub-groups (IIa and IIb; and IVa and IVb). Although there were some exceptions, generally isolates placed in the same genetic group had >80% similarity in nucleotide sequence and <80% with the other isolates; while those in the same sub-group had >90% nucleotide sequence similarity.

Overview of incursions of Asian H5N1 subtype highly pathogenic avian influenza virus into Great Britain, 2005-2008.

Since 2005 there have been five incursions into Great Britain of highly pathogenic avian influenza (HPAI) viruses of subtype H5N1 related to the ongoing global epizootic. The first incursion occurred in October 2005 in birds held in quarantine after importation from Taiwan. Two incursions related to wild birds: one involved a single dead whooper swan found in March 2006 in the sea off the east coast of Scotland, and the other involved 10 mute swans and a Canada goose found dead over the period extending from late December 2007 to late February 2008 on or close to a swannery on the south coast of England. The other two outbreaks occurred in commercial poultry in January 2007 and November 2007, both in the county of Suffolk. The first of these poultry outbreaks occurred on a large turkey farm, and there was no further spread. The second outbreak occurred on a free-range farm rearing turkeys, ducks, and geese and spread to birds on a second turkey farm that was culled as a dangerous contact. Viruses isolated from these five outbreaks were confirmed to be Asian H5N1 HPAI viruses; the quarantine outbreak was attributed to a clade 2.3 virus and the other four to clade 2.2 viruses. This article describes the outbreaks, their control, and the possible origins of the responsible viruses.

High level of protection induced by two fowlpox vector vaccines against a highly pathogenic avian influenza H5N1 challenge in specific-pathogen-free chickens.

The objective of the study was to compare efficacy of two fowlpox (FP) vector vaccines (FP-AI) against H5N1 highly pathogenic avian influenza (HPAI): one (vFP89) expressing the native hemagglutinin (HA) gene from H5N8 A/turkey/ Ireland/1378/83 and the other (vFP2211) expressing a modified synthetic HA gene from H5N1 A/chicken/Indonesia/7/2003. Four groups of 20 1-day-old specific-pathogen-free chickens were made: Groups 1 and 2 were immunized with 3 log10 tissue-culture infectious dose 50% (TCID50) of vFP89 and vFP2211, respectively, whereas group 3 was immunized with vFP89, but received a booster immunization at 2 wk of age with an inactivated vaccine containing A/turkey/Wisconsin/68 H5N9 virus (inH5N9); group 4 was left unvaccinated. Ten birds from each group were challenged on day 21 with A/turkey/Turkey/1/2005 clade 2.2 H5N1 HPAI virus. The 10 other chickens from each group were put in contact with their groupmates on day 22. FP-AI induced low hemagglutination inhibition (HI) titers before challenge (GMT < 4 log2) and an HI titer boost was observed 1 wk after the inH5N9 boost. All directly challenged and 9/10 nonvaccinated contact chickens died after challenge (mean death time of 2.3 and 6.1 days, respectively) and most of them shed virus before death via cloacal and buccal routes. All vaccinated birds were clinically protected from HPAI challenge. One (vFP2211), 2 (vFP89+inact.), or 3 (vFP89) out of the 10 directly challenged vaccinated chickens shed virus via the buccal route 2-5 days postinfection. No shedding was detected in the contact-challenged vaccinated birds. Altogether, these data show excellent levels of protection in all three vaccinated groups, and therefore no detectable effect of the origin of the inserted H5 gene on protection under these tested conditions.

Status of wild birds in Bulgarian zoos with regard to orthomyxovirus and paramyxovirus type 1 infections.

Newcastle disease virus (NDV) and avian influenza virus (AIV) are pathogens of major economic and social importance, and the diseases they cause are often devastating, particularly in domestic poultry. Both viruses are naturally found in a wide variety of wild birds, particularly aquatic species, where asymptomatic infection typically occurs. Wild birds are therefore considered to be a natural reservoir for both viruses. Wild birds kept in captivity are in an environment that promotes transmission of infection with both influenza and Newcastle disease viruses. This report describes a survey for the detection of antibodies against Newcastle disease and avian influenza A viruses using the hemagglutination inhibition test in samples from 88 wild birds from 38 species in four Bulgarian zoos. Samples with positive results against NDV were also tested against avian paramyxovirus type 3 (APMV-3). Real-time reverse-transcriptase PCR was also performed to detect viral RNA of NDV and AIV among 127 wild birds from 57 species from the same zoos. In 13 samples from seven avian species (ten birds from the family Phasianidae, two from the family Numidae, and one from the family Columbidae), antibodies against APMV-1 were detected. Seven birds, whose sera were APMV-1 positive, had been vaccinated. The other six birds (five Phasianidae representatives and one of the Columbidae family) had no immunization history. No antibodies against both H5 and H7 AIV and against APMV-3 were detected, and no RNA of NDV and AIV were detected.

Characterisation of a highly pathogenic influenza A virus of subtype H5N2 isolated from ostriches in South Africa in 2004.

OBJECTIVES: The HPAI H5N2 strain that caused an outbreak in ostriches of the Eastern Cape Province, South Africa in 2004 was characterized. DESIGN: Haemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) were performed on sera from ostrich farms in the outbreak region, and intravenous pathogenicity (IVPI) tests, reverse-transcriptase-polymerase-chain reaction (RT-PCR), nucleic acid sequencing and phylogenetic comparisons were performed on the HPAI H5N2 virus isolated during the outbreak. RESULTS: The deduced amino acid sequence at the HA0 cleavage site determined by RT-PCR and nucleotide sequencing was PQREKRRKKRGLF and thus the virus fell within the definition of a highly pathogenic virus, but in an IVPI test in chickens on the virus isolated from the index case and a value of 0.63 was recorded, which is below the criterion for highly pathogenic viruses in this in vivo test. After a further passage in embryonated eggs a second IVPI was carried out and an elevated value of 1.19 was obtained. Cloacal swabs were taken from the initial IVPI birds, inoculated into embryonated chickens eggs and a third IVPI was then performed on the resulting haemagglutinating, infective allantoic fluid. An index of 2.73 was recorded. CONCLUSIONS: HI tests appeared to be the more sensitive test compared to AGID when testing for antibodies to avian influenza in sera. An ostrich-derived virus with a virulent HA0 cleavage site was not initially virulent in chickens but after passage in the latter the virulence increased. Phylogenetic analyses demonstrated the link between AI viruses carried by wild ducks and those infecting ostriches.

Reovirus infections associated with high mortality in psittaciformes in The Netherlands.

In The Netherlands between January 2002 and December 2004, numerous psittaciformes died showing severe splenomegaly and hepatomegaly with multifocal acute necrosis. At the start of the outbreaks mostly parakeets were affected, but later larger parrots were also involved. Seventy-eight birds showed the same features and six were examined completely, including a virological examination. Tests for polyomavirus, Pacheco's disease (herpesvirus) and circovirus psittacine beak and feather disease (PBFD) viruses and Chlamydophila psittaci were carried out. All results were negative, except for two cases of circovirus infection. Many concurrent bacterial and parasitic infections were seen. Immunohistochemistry revealed reovirus antigen in intralesional mononuclear cells, and reovirus-like particles could be observed by negative contrast electron microscopy. A reovirus was grown and the isolates reacted with polyclonal reovirus antiserum but did not react with monoclonal antibodies against chicken reovirus. The virus was therefore considered a psittacine reovirus. Because reoviruses were seen consistently, they seemed to be the most probable cause of the outbreaks. Climate, the introduction of new birds and the transportation of birds might be other factors involved in the disease seen in The Netherlands. No regional influence could be seen; therefore, we suggested that the virus might be widespread and carriers could be a source of re-introduction.

A sensitive retroviral pseudotype assay for influenza H5N1-neutralizing antibodies.

BACKGROUND: The World Health Organisation (WHO) recommended the development of simple, safe, sensitive and specific neutralization assays for avian influenza antibodies. We have used retroviral pseudotypes bearing influenza H5 hemagglutinin (HA) as safe, surrogate viruses for influenza neutralization assays which can be carried out at Biosafety Level 2. RESULTS: Using our assay, sera from patients who had recovered from infection with influenza H5N1, and sera from animals experimentally immunized or infected with H5 tested positive for the presence of neutralizing antibodies to H5N1. Pseudotype neutralizing antibody titers were compared with titers obtained by hemagglutinin inhibition (HI) assays and microneutralization (MN) assays using live virus, and showed a high degree of correlation, sensitivity and specificity. CONCLUSIONS: The pseudotype neutralization assay is as sensitive as horse erythrocyte HI and MN for the detection of antibodies to H5N1. It is safer, and can be applied in a high-throughput format for human and animal surveillance and for the evaluation of vaccines.

Retention of 1.2 kbp of 'novel' genomic sequence in two European field isolates and some vaccine strains of Fowlpox virus extends open reading frame fpv241.

The emergence of variant fowlpox viruses (FWPVs) and increasing field use of recombinants against avian influenza H5N1 emphasize the need to monitor vaccines and to distinguish them from field strains. Five commercial vaccines, two laboratory viruses and two European field isolates were characterized by PCR and sequencing at 18 loci differing between attenuated FP9 and its pathogenic progenitor. PCR failed to discriminate between the viruses and sequence determination revealed no significant differences at any locus, except for a polymorphic locus encompassed by deletion 24 (9.3 kbp) in FP9. Surprisingly, 'novel' previously unreported sequence (spanning 1.2 kbp) was found in both European field isolates and three of the vaccines. It was absent from the other two vaccines, removed by a 1.2 kbp deletion identical to that surprisingly also observed in the completely sequenced genome of FPV USDA. This locus (H9) adds a potentially useful tool for discriminating between FWPV field isolates and vaccines.

An outbreak of low pathogenic avian influenza in a mixed-species aviculture unit in Dubai in 2005.

This case describes an outbreak of low pathogenic hemagglutinin 9 neuraminidase 2 avian influenza virus (AIV) in two white-bellied bustards (Eupodotis senegalensis), one stone curlew (Burhinus oedicnemius), and a blacksmith plover (Antibyx armatus) in a private zoologic collection in Dubai, United Arab Emirates. The four birds showed signs of respiratory disease, and all died as a result of disease or euthanasia. Attention has been paid to the diagnostic process and common differential diagnosis for upper respiratory tract disease in bustards, curlews, and plovers. To the knowledge of the authors, AIV has not been previously described in these species.

Avipoxvirus phylogenetics: identification of a PCR length polymorphism that discriminates between the two major clades.

Avipoxvirus infections have been observed in an extensive range of wild, captive and domesticated avian hosts, yet little is known about the genome diversity and host-range specificity of the causative agent(s). Genome-sequence data are largely restricted to Fowlpox virus (FWPV) and Canarypox virus (CNPV), which have been sequenced completely, showing considerable divergence between them. It is therefore proving difficult, by empirical approaches, to identify pan-genus, avipoxvirus-specific oligonucleotide probes for PCR and sequencing to support phylogenetic studies. A previous preliminary study used the fpv167 locus, which encodes orthologues of vaccinia virus core protein P4b (A3). PCR per se did not discriminate between viruses, but restriction-enzyme or sequence analysis indicated that the avipoxviruses clustered either with FWPV or with CNPV. Here, further study of the P4b locus demonstrated a third cluster, from psittacine birds. A newly identified locus, flanking fpv140 (orthologue of vaccinia virus H3L), confirms the taxonomic structure. This locus is particularly useful in that viruses from the fowlpox-like and canarypox-like clusters can be discriminated by PCR on the basis of fragment size, whilst sequence comparison allows discrimination for the first time between Pigeonpox virus and Turkeypox virus. Except within the psittacines, virus and avian host taxonomies do not show tight correlation, with viruses from the same species located in very different clades. Nor are all the existing recognized avipoxvirus species, defined primarily by avian host species (such as CNPV and Sparrowpox virus), resolved within the present structure.

Newcastle disease virus (strain Herts 33/56) in tissues and organs of chickens infected experimentally.

Six-week-old susceptible specific pathogen free chickens were infected intranasally with the virulent Newcastle disease virus strain Herts 33/56 and the levels of virus present in blood, faeces, breast muscle, leg muscle and a pool of heart/kidney/spleen were estimated in birds killed humanely at each day post inoculation. Highest titres were recorded at day 4 post inoculation when titres of virus were 10(6) median egg infectious doses (EID50)/g in the heart/kidney/spleen pool, 10(4.2) EID50/g in the leg muscle and 10(4) EID50/g in the breast muscle and faeces. A median oral infectious dose of Newcastle disease virus strain Herts 33/56 for 3-week-old chickens was estimated to be equivalent to 10(4) EID50.

Antigenic and genotypical characterization of Newcastle disease viruses isolated in Taiwan between 1969 and 1996.

Three major epidemics of Newcastle disease (ND) occurred in Taiwan over the past three decades (in 1969, 1984, and 1995). In order to gain a better understanding of the relationships between past ND epizootics in Taiwan, 36 ND viruses (NDVs) isolated between 1969 and 1996 were characterized antigenically and genotypically. The antigenicity of these viruses was analysed by their ability to cause binding of mouse monoclonal antibodies (mAbs) to cell cultures infected with the isolate. Using a panel of 22 mAbs to divide NDVs into subgroups, a total of 18 binding patterns were revealed. The sequences covering the cleavage site of the fusion protein gene of these isolates were also determined. The results of the phylogenetic analysis placed 36 NDVs into I, II, VIb, VIIa, VIII and two novel genotypes (provisionally termed X and VIh). The 1969 velogenic isolates were of genotypes X and VIh; the 1984-1985 velogenic isolates were genotyped VIb, VIh, VIIa, and X; while the 1995-1996 velogenic isolates were genotyped VIIa or VIII. Some 1969 and 1984 velogenic isolates were of the same mAbs binding pattern and genotype, and the mAbs binding patterns of the 1995-1996 isolates have not been seen before. It is concluded that velogenic NDVs of different genotype and antigenic type have co-circulated in Taiwan at least since 1969. Also there were epizootiological links between strains isolated in 1969 and 1984, whereas the 1995-1996 epidemic was caused by new antigenic variants.

Heat inactivation of Newcastle disease virus (strain Herts 33/56) in artificially infected chicken meat homogenate.

Heat inactivation curves were constructed for Newcastle disease virus strain Herts 33/56 in artificially infected meat homogenate at 60 degrees C, 65 degrees C, 70 degrees C, 74 degrees C and 80 degrees C. For the four higher temperatures the time taken to reduce the infectivity by 90% (1 log10) at the specified temperature (Dt) were estimated as: D65=120 sec, D70=82 sec, D74=40 sec and D80 29=sec.

Recombination resulting in virulence shift in avian influenza outbreak, Chile.

Influenza A viruses occur worldwide in wild birds and are occasionally associated with outbreaks in commercial chickens and turkeys. However, avian influenza viruses have not been isolated from wild birds or poultry in South America. A recent outbreak in chickens of H7N3 low pathogenic avian influenza (LPAI) occurred in Chile. One month later, after a sudden increase in deaths, H7N3 highly pathogenic avian influenza (HPAI) virus was isolated. Sequence analysis of all eight genes of the LPAI virus and the HPAI viruses showed minor differences between the viruses except at the hemagglutinin (HA) cleavage site. The LPAI virus had a cleavage site similar to other low pathogenic H7 viruses, but the HPAI isolates had a 30-nucleotide insert. The insertion likely occurred by recombination between the HA and nucleoprotein genes of the LPAI virus, resulting in a virulence shift. Sequence comparison of all eight gene segments showed the Chilean viruses were also distinct from all other avian influenza viruses and represent a distinct South American clade.