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.

Entry of H5N1 highly pathogenic avian influenza virus into Europe through migratory wild birds: a qualitative release assessment at the species level.

AIMS: To estimate qualitatively the probabilities of release (or entry) of Eurasian lineage H5N1 highly pathogenic avian influenza (HPAI) virus into Great Britain (GB), the Netherlands and Italy through selected higher risk species of migratory water bird. METHODS AND RESULTS: The probabilities of one or more release events of H5N1 HPAI per year (Pre(lease)) were estimated qualitatively for 15 avian species, including swans, geese, ducks and gulls, by assessing the prevalence of H5N1 HPAI in different regions of the world (weighted to 2009) and estimates of the total numbers of birds migrating from each of those regions. The release assessment accommodated the migration times for each species in relation to the probabilities of their surviving infection and shedding virus on arrival. Although the predicted probabilities of release of H5N1 per individual bird per year were low, very low or negligible, Pre(lease) was high for a few species reflecting the high numbers of birds migrating from some regions. Values of Pre(lease) were generally higher for the Netherlands than for GB, while ducks and gulls from Africa presented higher probabilities to Italy compared to the Netherlands and GB. CONCLUSIONS: Bird species with high values of Pre(lease) in GB, the Netherlands and Italy generally originate from within Europe based on data for global prevalence of H5N1 between 2003 and 2009 weighted to 2009. Potential long-distance transfer of H5N1 HPAI from North Asia and Eurasia to GB, the Netherlands and Italy is limited to a few species and does not occur from South-East Asia, an area where H5N1 is endemic. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach accommodates biogeographical conditions and variability in the estimated worldwide prevalence of the virus. The outputs of this release assessment can be used to inform surveillance activities through focusing on certain species and migratory pathways.

The evolution of pigeon paramyxovirus type 1 (PPMV-1) in Great Britain: a molecular epidemiological study.

Newcastle disease (ND), caused by virulent strains of avian paramyxovirus type 1 (APMV-1), is considered throughout the world as one of the most important animal diseases. For over three decades now, there has been a continuing panzootic caused by a variant virulent APMV-1 strain, so-called pigeon paramyxovirus type 1 (PPMV-1), primarily in racing pigeons, which has also spread to wild birds and poultry. PPMV-1 isolations have been made in Great Britain every year since 1983. In this study, we have completed a comparative phylogenetic analysis based on a 374 nucleotide section of the fusion protein gene of 63 isolates of PPMV-1 that were isolated over a 26-year period; 43 of these were sequenced for this study. Phylogenetic analysis of these sequences revealed that all were closely related and placed in the genetic sublineage 4b (VIb), subdivision 4biif.

Investigation into sampling strategies in response to potential outbreaks of low pathogenicity notifiable avian influenza initiated in commercial duck holdings in Great Britain.

The aim of this study was to evaluate potential sampling strategies for detection of infected flocks that could be applied during an outbreak of low pathogenicity notifiable avian influenza (LPNAI) initiated in duck holdings, following initial detection. A simulation model of avian influenza virus transmission and spread within and between holdings, respectively, was used to predict the impact on the size and duration of an outbreak of (i) changing the tracing window within which premises that might be the source of infection or that may have been infected by the index premises were sampled and (ii) changing the number of birds sampled in the flock being tested. It has shown that there is potential benefit in increasing the tracing window in terms of reducing the likelihood of a large outbreak. It has also shown that there is comparatively little benefit from increasing the number of birds sampled per flock.

First reported detection of a low pathogenicity avian influenza virus subtype H9 infection in domestic fowl in England.

In December 2010, infection with a H9N1 low pathogenicity avian influenza (LPAI) virus was detected in a broiler breeder flock in East Anglia. Disease suspicion was based on acute drops in egg production in two of four sheds on the premises, poor egg shell quality and evidence of diarrhoea. H9N1 LPAI virus infection was confirmed by real-time reverse transcription PCR. Sequencing revealed high nucleotide identity of 93.6 per cent and 97.9 per cent with contemporary North American H9 and Eurasian N1 genes, respectively. Attempted virus isolation in embryonated specific pathogen free (SPF) fowls' eggs was unsuccessful. Epidemiological investigations were conducted to identify the source of infection and any onward spread. These concluded that infection was restricted to the affected premises, and no contacts or movements of poultry, people or fomites could be attributed as the source of infection. However, the infection followed a period of extremely cold weather and snow which impacted on the biosecurity protocols on site, and also led to increased wild bird activity locally, including waterfowl and game birds around the farm buildings. Analysis of the N1 gene sequence suggested direct introduction from wild birds. Although H9 infection in poultry is not notifiable, H9N2 LPAI viruses have been associated with production and mortality episodes in poultry in many parts of Asia and the Middle East. In the present H9N1 outbreak, clinical signs were relatively mild in the poultry with no mortality, transient impact on egg production and no indication of zoonotic spread. However, this first reported detection of H9 LPAI virus in chickens in England was also the first H9 UK poultry case for 40 years, and vindicates the need for continued vigilance and surveillance of avian influenza viruses in poultry populations.

Development and validation of RT-PCR tests for the detection and S1 genotyping of infectious bronchitis virus and other closely related gammacoronaviruses within clinical samples.

Two tests were developed that allow the detection and genotyping of infectious bronchitis virus (IBV) and other closely related gammacoronaviruses. The first test employs a one-step, reverse transcription-polymerase chain reaction (RT-PCR) assay in which the amplification is monitored in real time using a TaqMan(®) probe. This real-time RT-PCR test was used to examine a panel of field samples and its performance compared to virus isolation in embryonated fowls' eggs. A total of 323 field samples were tested; 176 samples were positive using the real-time RT-PCR method, but only three were positive by virus isolation. Sequencing was used to confirm the positive real-time RT-PCR results for a subset of samples. The test is suitable for swabs and post-mortem samples and has been shown to be highly sensitive and specific. The second test, a genotyping method, was developed for identification of the strain of IBV present in field samples based on nucleotide variations within the gene encoding the S1 subunit of the surface spike (S) glycoprotein. This method was developed to provide a tool to inform vaccination decisions and for ongoing surveillance to detect new and emerging strains of IBV within the UK. The performance of the test was evaluated using laboratory isolates of IBV and field samples. Both tests are suitable for use in a high-throughput diagnostic laboratory.

First reported incursion of highly pathogenic notifiable avian influenza A H5N1 viruses from clade 2.3.2 into European poultry.

This study reports the first incursion into European poultry of H5N1 highly pathogenic notifiable avian influenza A (HPNAI) viruses from clade 2.3.2 that affected domestic poultry and wild birds in Romania and Bulgaria, respectively. Previous occurrences in Europe of HPNAI H5N1 in these avian populations have involved exclusively viruses from clade 2.2. This represents the most westerly spread of clade 2.3.2 viruses, which have shown an apparently expanding range of geographical dispersal since mid-2009 following confirmation of infections in wild waterfowl species in Mongolia and Eastern Russia. During March 2010, AI infection was suspected at post-mortem examination of two hens from two backyard flocks in Tulcea Country, Romania. HPNAI of H5N1 subtype was confirmed by reverse transcription polymerase chain reaction (RT-PCR). A second outbreak was confirmed 2 weeks later by RT-PCR, affecting all hens from another flock located 55 km east of the first cluster. On the same day, an H5N1 HPNAI virus was detected from a pooled tissue sample collected from a dead Common Buzzard found on the Black Sea coast in Bulgaria. Detailed genetic characterization of the haemagglutinin gene revealed the cleavage site of the isolates to be consistent with viruses of high pathogenicity belonging to clade 2.3.2 of the contemporary Eurasian H5N1 lineage. Viruses from a clade other than 2.2 have apparently spread to wild birds, with potential maintenance and spread through such populations. Whilst the scale of threat posed by the apparent westward spread of the clade 2.3.2 viruses remains uncertain, ongoing vigilance for clinical signs of disease as part of existing passive surveillance frameworks for AI, and the prompt reporting of suspect cases in poultry is advised.

A molecular epidemiological investigation of avian paramyxovirus type 1 viruses isolated from game birds of the order Galliformes.

The partial (370 nucleotides) fusion gene sequences of 55 avian paramyxovirus type 1 (APMV-1) isolates were obtained. Included were 41 published sequences, of which 16 were from strains of APMV-1 of previously determined lineages included as markers for the data analysed and 25 were from APMV-1 viruses isolated from game birds of the order Galliformes. In addition, we sequenced a further 14 game bird isolates obtained from the repository at the Veterinary Laboratories Agency. The game bird isolates had been obtained from 17 countries, and spanned four decades. Earlier studies have shown that class II APMV-1 viruses can be divided into at least 15 lineages and sub-lineages. Phylogenetic analysis revealed that the 39 game bird isolates were distributed across 12 of these sub-lineages. We conclude that no single lineage of Newcastle disease viruses appears to be prevalent in game birds, and the isolates obtained from these hosts reflected the prevailing, both geographically and temporally, viruses in poultry, pigeons or wild birds.

Infection dynamics of highly pathogenic avian influenza and virulent avian paramyxovirus type 1 viruses in chickens, turkeys and ducks.

A range of virus doses were used to infect 3-week-old chickens, turkeys and ducks intranasally/intraocularly, and infection was confirmed by the detection of virus shedding from the buccal or cloacal route by analysis of swabs collected using real-time reverse transcriptase-polymerase chain reaction assays. The median infectious dose (ID(50)) and the median lethal dose (LD(50)) values for two highly pathogenic avian influenza (HPAI) viruses of H5N1 and H7N1 subtypes and one virulent Newcastle disease virus (NDV) were determined for each virus and host combination. For both HPAI viruses, turkeys were >100-fold more susceptible to infection than chickens, while both these hosts were >10-fold more susceptible to H5N1 virus than the H7N1 virus. All infected chickens and turkeys died. Ducks were also much more readily infected with the H5N1 virus (ID(50)< or =10(1) median embryo infective dose [EID(50)]) than the H7N1 virus (ID(50)=10(4.2) EID(50)). However, the most notable difference between the two viruses was their virulence for ducks, with a LD(50) of 10(3) EID(50) for the H5N1 virus, but no deaths in ducks being attributed to infection with H7N1 virus even at the highest dose (10(6) EID(50)). For both HPAI virus infections of ducks, the ID(50) was lower than the LD(50), indicating that infected birds were able to survive and thus excrete virus over a longer period than chickens and turkeys. The NDV strain used did not appear to establish infection in ducks even at the highest dose used (10(6) EID(50)). Some turkeys challenged with 10(6) EID(50), but not other doses, of NDV excreted virus for a number of days (ID(50)=10(4.6) EID(50)), but none died. In marked contrast, chickens were shown to be extremely susceptible to infection and all infected chickens died (ID(50)/LD(50)=10(1.9) EID(50)).

IgA in the horse: cloning of equine polymeric Ig receptor and J chain and characterization of recombinant forms of equine IgA.

As in other mammals, immunoglobulin A (IgA) in the horse has a key role in immune defense. To better dissect equine IgA function, we isolated complementary DNA (cDNA) clones for equine J chain and polymeric Ig receptor (pIgR). When coexpressed with equine IgA, equine J chain promoted efficient IgA polymerization. A truncated version of equine pIgR, equivalent to secretory component, bound with nanomolar affinity to recombinant equine and human dimeric IgA but not with monomeric IgA from either species. Searches of the equine genome localized equine J chain and pIgR to chromosomes 3 and 5, respectively, with J chain and pIgR coding sequence distributed across 4 and 11 exons, respectively. Comparisons of transcriptional regulatory sequences suggest that horse and human pIgR expression is controlled through common regulatory mechanisms that are less conserved in rodents. These studies pave the way for full dissection of equine IgA function and open up possibilities for immune-based treatment of equine diseases.

Role of real-time RT-PCR platform technology in the diagnosis and management of notifiable avian influenza outbreaks: experiences in Great Britain.

Diagnosis and management of avian influenza outbreaks now include the use of validated real-time reverse transcription PCR (RRT-PCR) methods in many countries, including all member states of the European Union. Two outbreaks in poultry of notifiable avian influenza (H5 and H7 subtypes) that occurred in Great Britain during 2007 will serve as examples in which RRT-PCR demonstrated its value in 1) rapid diagnosis and confirmation of disease by sensitive and specific laboratory testing of samples derived from the index cases and 2) high-volume, rapid testing of surveillance samples. The two poultry outbreaks followed the incursion of a H7N2 low-pathogenicity notifiable avian influenza (LPNAI) virus (May-June 2007) and a Eurasian lineage H5N1 highly pathogenic notifiable avian influenza (HPNAI) virus (November 2007). Coupled with the use of high-throughput, robotic RNA extraction methods, a total of approximately 9300 and 20,300 field samples were tested by appropriate, validated RRT-PCR assays during the 4- and 5-wk duration of the H7N2 LPNAI and H5N1 HPNAI outbreaks, respectively. Fundamental features of the validated RRT-PCR assays used included their high degree of sensitivity, specificity, and rapidity, attributes that were invaluable in providing timely and accurate information for notifiable AI outbreak management.

Initial incursion of pandemic (H1N1) 2009 influenza A virus into European pigs.

The initial incursion of pandemic (H1N1) 2009 influenza A virus (pH1N1) into a European pig population is reported. Diagnosis of swine influenza caused by pandemic virus was made during September 2009 following routine submission of samples for differential diagnosis of causative agents of respiratory disease, including influenza A virus. All four pigs (aged six weeks) submitted for investigation from a pig herd of approximately 5000 animals in Northern Ireland, experiencing acute-onset respiratory signs in finishing and growing pigs, were positive by immunofluorescence for influenza A. Follow-up analysis of lung tissue homogenates by real-time RT-PCR confirmed the presence of pH1N1. The virus was subsequently detected on two other premises in Northern Ireland; on one premises, detection followed the pre-export health certification testing of samples from pigs presumed to be subclinically infected as no clinical signs were apparent. None of the premises was linked to another epidemiologically. Sequencing of the haemagglutinin and neuraminidase genes revealed high nucleotide identity (>99.4 per cent) with other pH1N1s isolated from human beings. Genotypic analyses revealed all gene segments to be most closely related to those of contemporary pH1N1 viruses in human beings. It is concluded that all three outbreaks occurred independently, potentially as a result of transmission of the virus from human beings to pigs.