Serological Evidence for Influenza A Virus Exposure in Wild Birds in Trinidad & Tobago.

Migratory waterfowl and shorebirds are known to be important reservoirs for influenza A viruses (IAV) and they have been repeatedly implicated as causing avian influenza virus (AIV) outbreaks in domestic poultry flocks worldwide. In recent years, wild birds have been implicated in spreading zoonotic H5 influenza viruses to many countries, which has generated high levels of public health concern. Trinidad and Tobago (T&T) is positioned along the wintering route of migratory birds from the Americas; every year, many species of wild birds stopover on the islands of T&T, potentially carrying AIVs and exposing local populations of wild and domestic birds, including commercial poultry, to infection. The aim of this study was to trap, sample, and test as many wild bird species as possible to see whether they were actively infected or previously exposed to AIV. A total of 38 wild birds were trapped, sampled, and tested for IAV RNA, antibodies specific for influenza A nucleoprotein (NP) and antibodies that were specific for H5 and H7 subtypes. Five of the samples tested antibody positive for IAV, while three of these samples had positive titres (≥16) for the H5 subtype, indicating that they were likely to have been previously infected with an H5 IAV subtype. One of the samples tested positive for IAV (M gene) RNA. These results highlight the potential threat that is posed by wild birds to backyard and commercial poultry in T&T and emphasise the importance of maintaining high levels of biosecurity on poultry farms, ensuring that domestic and wild birds are not in direct or indirect contact. The results also underline the need to carry out routine surveillance for AIV in domestic and wild birds in T&T and the wider Caribbean region.

Virus pathotype and deep sequencing of the HA gene of a low pathogenicity H7N1 avian influenza virus causing mortality in Turkeys.

Low pathogenicity avian influenza (LPAI) viruses of the H7 subtype generally cause mild disease in poultry. However the evolution of a LPAI virus into highly pathogenic avian influenza (HPAI) virus results in the generation of a virus that can cause severe disease and death. The classification of these two pathotypes is based, in part, on disease signs and death in chickens, as assessed in an intravenous pathogenicity test, but the effect of LPAI viruses in turkeys is less well understood. During an investigation of LPAI virus infection of turkeys, groups of three-week-old birds inoculated with A/chicken/Italy/1279/99 (H7N1) showed severe disease signs and died or were euthanised within seven days of infection. Virus was detected in many internal tissues and organs from culled birds. To examine the possible evolution of the infecting virus to a highly pathogenic form in these turkeys, sequence analysis of the haemagglutinin (HA) gene cleavage site was carried out by analysing multiple cDNA amplicons made from swabs and tissue sample extracts employing Sanger and Next Generation Sequencing. In addition, a RT-PCR assay to detect HPAI virus was developed. There was no evidence of the presence of HPAI virus in either the virus used as inoculum or from swabs taken from infected birds. However, a small proportion (<0.5%) of virus carried in individual tracheal or liver samples did contain a molecular signature typical of a HPAI virus at the HA cleavage site. All the signature sequences were identical and were similar to HPAI viruses collected during the Italian epizootic in 1999/2000. We assume that the detection of HPAI virus in tissue samples following infection with A/chicken/Italy/1279/99 reflected amplification of a virus present at very low levels within the mixed inoculum but, strikingly, we observed no new HPAI virus signatures in the amplified DNA analysed by deep-sequencing.

Quantifying transmission of highly pathogenic and low pathogenicity H7N1 avian influenza in turkeys.

Outbreaks of avian influenza in poultry can be devastating, yet many of the basic epidemiological parameters have not been accurately characterised. In 1999-2000 in Northern Italy, outbreaks of H7N1 low pathogenicity avian influenza virus (LPAI) were followed by the emergence of H7N1 highly pathogenic avian influenza virus (HPAI). This study investigates the transmission dynamics in turkeys of representative HPAI and LPAI H7N1 virus strains from this outbreak in an experimental setting, allowing direct comparison of the two strains. The fitted transmission rates for the two strains are similar: 2.04 (1.5-2.7) per day for HPAI, 2.01 (1.6-2.5) per day for LPAI. However, the mean infectious period is far shorter for HPAI (1.47 (1.3-1.7) days) than for LPAI (7.65 (7.0-8.3) days), due to the rapid death of infected turkeys. Hence the basic reproductive ratio, [Formula: see text] is significantly lower for HPAI (3.01 (2.2-4.0)) than for LPAI (15.3 (11.8-19.7)). The comparison of transmission rates and [Formula: see text] are critically important in relation to understanding how HPAI might emerge from LPAI. Two competing hypotheses for how transmission rates vary with population size are tested by fitting competing models to experiments with differing numbers of turkeys. A model with frequency-dependent transmission gives a significantly better fit to experimental data than density-dependent transmission. This has important implications for extrapolating experimental results from relatively small numbers of birds to the commercial poultry flock size, and for how control, including vaccination, might scale with flock size.

Selection of variant viruses during replication and transmission of H7N1 viruses in chickens and turkeys.

The influence of different glycosylation patterns of the haemagglutinin glycoprotein of H7N1 avian influenza viruses on virus replication in vivo was examined. Experimental infection of chickens and turkeys was carried out with H7N1 avian influenza viruses with alternative sites of glycosylation in the haemagglutinin and infected birds were sampled daily by swabbing the buccal and cloacal cavities. cDNAs of the HA1 coding region of the HA gene were prepared from the swabs and cloned into plasmids. Sequencing multiple plasmids made from individual swabs taken over the period of virus shedding showed that viruses with specific patterns of glycosylation near the receptor binding site were stable when birds were infected with a single variant, but when presented with a mixed population of viruses encoding differing patterns of glycosylation a specific variant was rapidly selected in the infected host.

First reported detection of influenza A (H1N1)pdm09 in turkeys in the United Kingdom.

We report the first occurrence of pandemic (H1N1) 2009 virus [A(H1N1)pdm09] infection on two epidemiologically linked turkey breeder premises in the United Kingdom during December 2010 and January 2011. Clinically, the birds showed only mild signs of disease, with the major presenting sign being an acute and marked reduction in egg production, leading to the prompt reporting of suspected avian notifiable disease for official investigation. Presence of A(H1N1)pdm09 infection in the United Kingdom turkey breeder flocks was confirmed by detailed laboratory investigations including virus isolation in embryonated specific pathogen-free fowls' eggs, two validated real-time reverse transcription-PCR tests, and nucleotide sequencing of the hemagglutinin and neuraminidase genes. These investigations revealed high nucleotide identity with currently circulating human A(H1N1)pdm09 strains, suggesting that human-to-poultry transmission (reverse zoonosis) was the most likely route of infection. Peak levels of human influenza-like illness community transmission also coincided with the onset of clinical signs in both affected turkey breeder flocks. This case demonstrated the value of the existing passive surveillance framework and associated veterinary and laboratory infrastructure that enables the detection and management of both exotic and new and emerging disease hazards and risks. The case also presents further evidence of the susceptibility of turkeys to infection with influenza A viruses of nonavian origin.

Reassortant Pandemic (H1N1) 2009 virus in pigs, United Kingdom.

Surveillance for influenza virus in pigs in the United Kingdom during spring 2010 detected a novel reassortant influenza virus. This virus had genes encoding internal proteins from pandemic (H1N1) 2009 virus and hemagglutinin and neuraminidase genes from swine influenza virus (H1N2). Our results demonstrate processes contributing to influenza virus heterogeneity.

Within-host variation of avian influenza viruses.

The emergence and spread of H5N1 avian influenza viruses from Asia through to Europe and Africa pose a significant animal disease problem and have raised concerns that the virus may pose a pandemic threat to humans. The epizootological factors that have influenced the wide distribution of the virus are complex, and the variety of viruses currently circulating reflects these factors. Sequence analysis of the virus genes sheds light on the H5N1 virus evolution during its emergence and spread, but the degree of virus variation at the level of an individual infected bird has been described in only a few studies. Here, we describe some results of a study in which turkeys, ducks and chickens were infected with either one of two H5N1 or one of three H7N1 viruses, and the degree of sequence variation within an individual infected avian host was examined. We developed 'deep amplicon' sequence analysis for this work, and the methods and results provide a background framework for application to disease outbreaks in the field.

Significant rising antibody titres to influenza A are associated with an acute reduction in milk yield in cattle.

Sporadic cases of an acute fall in milk production, "milk drop", were investigated in a Holstein Friesian dairy herd in Devon. The investigation was a case control study with two controls per case. Paired blood samples demonstrated that rising antibody titres to human influenza A/England/333/80 (H1N1) and human influenza A/Eng/427/88 (H3N2) were associated with an acute fall in milk production. Rising titres to bovine respiratory syncytial virus (BRSV), bovine virus diarrhoea virus (BVD), infectious bovine rhinotracheitis (IBR) and parainfluenza virus 3 (PI3) were not associated with an acute fall in milk production. Cases with rises in antibody to influenza A had significantly higher respiratory scores and rectal temperatures than their controls. The mean loss of milk production for the cases with rises in antibody to influenza A compared to their controls was 159.9L. This study provides further evidence that influenza A persists in cattle and causes clinical disease.

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.