Anja Globig

Highly pathogenic avian influenza virus (H5N1) in experimentally infected adult mute swans.

Adult, healthy mute swans were experimentally infected with highly pathogenic avian influenza virus A/Cygnus cygnus/Germany/R65/2006 subtype H5N1. Immunologically naive birds died, whereas animals with preexisting, naturally acquired avian influenza virus–specific antibodies became infected asymptomatically and shed virus. Adult mute swans are highly susceptible, excrete virus, and can be clinically protected by preexposure immunity.Adult, healthy mute swans were experimentally infected with highly pathogenic avian influenza virus A/Cygnus cygnus/Germany/R65/2006 subtype H5N1. Immunologically naive birds died, whereas animals with preexisting, naturally acquired avian influenza virus-specific antibodies became infected asymptomatically and shed virus. Adult mute swans are highly susceptible, excrete virus, and can be clinically protected by preexposure immunity.

Highly pathogenic) avian influenza as a zoonotic agent.

Zoonotic agents challenging the world every year afresh are influenza A viruses. In the past, human pandemics caused by influenza A viruses had been occurring periodically. Wild aquatic birds are carriers of the full variety of influenza virus A subtypes, and thus, most probably constitute the natural reservoir of all influenza A viruses. Whereas avian influenza viruses in their natural avian reservoir are generally of low pathogenicity (LPAIV), some have gained virulence by mutation after transmission and adaptation to susceptible gallinaceous poultry. Those so-called highly pathogenic avian influenza viruses (HPAIV) then cause mass die-offs in susceptible birds and lead to tremendous economical losses when poultry is affected. Besides a number of avian influenza virus subtypes that have sporadically infected mammals, the HPAIV H5N1 Asia shows strong zoonotic characteristics and it was transmitted from birds to different mammalian species including humans. Theoretically, pandemic viruses might derive directly from avian influenza viruses or arise after genetic reassortment between viruses of avian and mammalian origin. So far, HPAIV H5N1 already meets two conditions for a pandemic virus: as a new subtype it has been hitherto unseen in the human population and it has infected at least 438 people, and caused severe illness and high lethality in 262 humans to date (August 2009). The acquisition of efficient human-to-human transmission would complete the emergence of a new pandemic virus. Therefore, fighting H5N1 at its source is the prerequisite to reduce pandemic risks posed by this virus. Other influenza viruses regarded as pandemic candidates derive from subtypes H2, H7, and H9 all of which have infected humans in the past. Here, we will give a comprehensive overview on avian influenza viruses in concern to their zoonotic potential.

Pathology of Natural Infections by H5N1 Highly Pathogenic Avian Influenza Virus in Mute (Cygnus olor) and Whooper (Cygnus cygnus) Swans

Mortality in wild aquatic birds due to infection with highly pathogenic avian influenza viruses (HPAIV) is a rare event. During the recent outbreak of highly pathogenic avian influenza in Germany, mortality due to H5N1 HPAIV was observed among mute and whooper swans as part of a rapid spread of this virus. In contrast to earlier reports, swans appeared to be highly susceptible and represented the mainly affected species. We report gross and histopathology and distribution of influenza virus antigen in mute and whooper swans that died after natural infection with H5N1 HPAIV. At necropsy, the most reliable lesions were multifocal hemorrhagic necrosis in the pancreas, pulmonary congestion and edema, and subepicardial hemorrhages. Major histologic lesions were acute pancreatic necrosis, multifocal necrotizing hepatitis, and lymphoplasmacytic encephalitis with neuronal necrosis. Adrenals displayed consistently scattered cortical and medullary necrosis. In spleen and Peyers patches, mild lymphocyte necrosis was present. Immunohistochemical demonstration of HPAIV nucleoprotein in pancreas, adrenals, liver, and brain was strongly consistent with histologic lesions. In the brain, a large number of neurons and glial cells, especially Purkinje cells, showed immunostaining. Occasionally, ependymal cells of the spinal cord were also positive. In the lungs, influenza virus antigen was identified in a few endothelial cells but not within pneumocytes. The infection of the central nervous system supports the view that the neurotropism of H5N1 HPAIV leads to nervous disturbances with loss of orientation. More investigations are necessary to clarify the mechanisms of the final circulatory failure, lung edema, and rapid death of the swans.

Epidemiological and ornithological aspects of outbreaks of highly pathogenic avian influenza virus H5N1 of Asian lineage in wild birds in Germany, 2006 and 2007.

In Germany, two distinct episodes of outbreaks of highly pathogenic avian influenza virus of subtype H5N1 (HPAIV H5N1) in wild birds occurred at the beginning of 2006, and in summer 2007. High local densities of wild bird populations apparently sparked clinically detectable outbreaks. However, these remained restricted in (i) number of birds, (ii) species found to be affected, (iii) time, and (iv) location despite the presence of several hundred thousands of susceptible wild birds and further stressors (food shortage, harsh weather conditions and moulting). Northern and southern subpopulations of several migratory anseriform species can be distinguished with respect to their preference for wintering grounds in Germany. This corroborates viral genetic data by Starick et al. (2008) demonstrating the introduction of two geographically restricted virus subpopulations of Qinghai-like lineage (cluster 2.2.A and 2.2.B) into northern and southern Germany, respectively, in 2006. The incursion of virus emerging in 2007, found to be distinct from the clusters detected in 2006 (Starick et al., 2008), may have been associated with moulting movements. Intensive past-outbreak investigations with negative results of live and dead wild birds and of terrestrial scavengers excluded continued circulation of virus on a larger scale. However, persistence of virus in small pockets of local wild bird populations could not be ruled out resiliently. 1.5% of investigated sera originating from cats sampled at the epicentres of the Ruegen 2006-outbreak contained H5-antibodies. Passive monitoring was found to be highly superior to live bird surveillance when aiming at the detection of HPAIV H5N1 in wild birds (P < 0.0001).

Rapid molecular subtyping by reverse transcription polymerase chain reaction of the neuraminidase gene of avian influenza A viruses.

Accurate identification of hemagglutinin (HA) and neuraminidase (NA) subtypes of influenza A viruses is an integral part of monitoring programs targeting avian influenza viruses (AIV). Use of highly sensitive molecular screening methods such as pan influenza-specific real-time RT-PCR (rRT-PCR) yields an increasing number of samples which are positive for AIV RNA but negative by virus isolation and, therefore, require molecular, instead of serological, subtyping. We developed specific RT-PCR assays for all known nine AIV NA subtypes. Validation using 43 reference isolates from different animal species revealed good performance characteristics regarding sensitivity and specificity. On basis of serial tenfold dilution series of reference isolates a benchmark value of C(t) 32 in an M gene-specific rRT-PCR became evident below which all nine NA subtypes were readily detectable by the subtype-specific RT-PCRs. For subtypes N1, N2, N4 and N6 detection was extended to dilutions with C(t) values of up to 35. Diagnostic applicability of the whole set of conventional NA-specific RT-PCRs was evaluated by analysis of 119 different diagnostic samples from wild birds which proved to be positive for AIV by M gene-specific rRT-PCR. Diagnostic sensitivity and specificity was confirmed by sequencing NA amplicons from 41 field isolates generated from this set and by NA inhibition assays. A universal molecular HA/NA subtyping algorithm for rRT-PCR positive avian influenza virus monitoring samples is proposed which may complement classical serological subtyping of influenza A virus isolates.

Pathogenicity of Highly Pathogenic Avian Influenza Virus (H5N1) in Adult Mute Swans

Adult, healthy mute swans were experimentally infected with highly pathogenic avian influenza virus A/Cygnus cygnus/Germany/R65/2006 subtype H5N1. Immunologically naive birds died, whereas animals with preexisting, naturally acquired avian influenza virus–specific antibodies became infected asymptomatically and shed virus. Adult mute swans are highly susceptible, excrete virus, and can be clinically protected by preexposure immunity.Adult, healthy mute swans were experimentally infected with highly pathogenic avian influenza virus A/Cygnus cygnus/Germany/R65/2006 subtype H5N1. Immunologically naive birds died, whereas animals with preexisting, naturally acquired avian influenza virus-specific antibodies became infected asymptomatically and shed virus. Adult mute swans are highly susceptible, excrete virus, and can be clinically protected by preexposure immunity.

Highly pathogenic avian influenza virus (H5N1) in frozen duck carcasses, Germany, 2007.

Article summary line: Phylogenetic and epidemiologic evidence shows incursion of HPAIV into the food chain.

Avian influenza virus monitoring in wintering waterbirds in Iran, 2003-2007

BackgroundVirological, molecular and serological studies were carried out to determine the status of infections with avian influenza viruses (AIV) in different species of wild waterbirds in Iran during 2003-2007. Samples were collected from 1146 birds representing 45 different species with the majority of samples originating from ducks, coots and shorebirds. Samples originated from 6 different provinces representative for the 15 most important wintering sites of migratory waterbirds in Iran.ResultsOverall, AIV were detected in approximately 3.4% of the samples. However, prevalence was higher (up to 8.3%) at selected locations and for certain species. No highly pathogenic avian influenza, including H5N1 was detected. A total of 35 AIVs were detected from cloacal or oropharyngeal swab samples. These positive samples originated mainly from Mallards and Common Teals.Of 711 serum samples tested for AIV antibodies, 345 (48.5%) were positive by using a nucleoprotein-specific competitive ELISA (NP-C-ELISA). Ducks including Mallard, Common Teal, Common Pochard, Northern Shoveler and Eurasian Wigeon revealed the highest antibody prevalence ranging from 44 to 75%.ConclusionResults of these investigations provide important information about the prevalence of LPAIV in wild birds in Iran, especially wetlands around the Caspian Sea which represent an important wintering site for migratory water birds. Mallard and Common Teal exhibited the highest number of positives in virological and serological investigations: 43% and 26% virological positive cases and 24% and 46% serological positive reactions, respectively. These two species may play an important role in the ecology and perpetuation of influenza viruses in this region. In addition, it could be shown that both oropharyngeal and cloacal swab samples contribute to the detection of positive birds, and neither should be neglected.

Ducks as sentinels for avian influenza in wild birds

To determine the effectiveness of ducks as sentinels for avian influenza virus (AIV) infection, we placed mallards in contact with wild birds at resting sites in Germany, Austria, and Switzerland. Infections of sentinel birds with different AIV subtypes confirmed the value of such surveillance for AIV monitoring.

Outbreaks among Wild Birds and Domestic Poultry Caused by Reassorted Influenza A(H5N8) Clade 2.3.4.4 Viruses, Germany, 2016

In November 2016, an influenza A(H5N8) outbreak caused deaths of wild birds and domestic poultry in Germany. Clade 2.3.4.4 virus was closely related to viruses detected at the Russia–Mongolia border in 2016 but had new polymerase acidic and nucleoprotein segments. These new strains may be more efficiently transmitted to and shed by birds.