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ABSTRACT Phylogenetic evidence from the recent resurgence of high-pathogenicity avian influenza (HPAI) virus subtype H5N1, clade 2.3.4.4b, observed in European wild birds and poultry since October 2021, suggests at least two different and distinct reservoirs. We propose contrasting hypotheses for this emergence: (i) resident viruses have been maintained, presumably in wild birds, in northern Europe throughout the summer of 2021 to cause some of the outbreaks that are part of the most recent autumn/winter 2021 epizootic, or (ii) further virus variants were reintroduced by migratory birds, and these two sources of reintroduction have driven the HPAI resurgence. Viruses from these two principal sources can be distinguished by their hemagglutinin genes, which segregate into two distinct sublineages (termed B1 and B2) within clade 2.3.4.4b, as well as their different internal gene compositions. The evidence of enzootic HPAI virus circulation during the summer of 2021 indicates a possible paradigm shift in the epidemiology of HPAI in Europe.

Abstract To improve understanding of the pathobiology of highly pathogenic avian influenza virus (HPAIV) infections in wild birds, pathogenicity and transmissibility of HPAIV H5N8 subtype clade 2.3.4.4b was evaluated in ~ 8-week-old herring gulls (Larus argentatus) divided into 3 groups: naïve birds (group A), birds previously exposed to low pathogenic avian influenza virus (LPAIV) H5N1 (group B) and LPAIV H13N6 (group C). The HPAIV H5N8 virus was highly virulent for naïve gulls, that showed early morbidity, high mortality, a broad spectrum of clinical signs, including violent neurological disorders, systemic distribution of the virus in organs accompanied by high level of shedding and transmission to contact birds. Pre-exposure to homologous and heterologous LPAIV subtypes conferred only partial protection: we observed increased survival rate (statistically significant only in group B), nervous signs, pantropic distribution of virus in organs, shedding (significantly reduced in gulls of group C in the early phase of disease and asymptomatic shedding in the late phase), transmission to contact gulls (more pronounced in group B) and near-complete seroconversion in survivors. Histopathological and immunohistochemical results indicate virus tropism for the neural, respiratory and myocardial tissues. In conclusion, we demonstrate that HPAIV H5N8 clade 2.3.4.4b is highly virulent and lethal for fully susceptible herring gulls and that pre-exposure to homo- and heterosubtypic LPAIV only partially modulates the disease outcome.

Abstract Defective interfering particles (DIPs) of influenza virus are generated through incorporation of highly truncated forms of genome segments, mostly those coding polymerase complex proteins (PB2, PB1, PA). Such particles are able to replicate only in the presence of a virus with the complete genome, thus DIPs may alter the infection outcome by suppressing production of standard virus particles, but also by stimulating the immune response. In the present study we compared the clinical outcome, mortality and transmission in chickens and turkeys infected with the same infectious doses of H7N7 low pathogenic avian influenza virus containing different levels of defective gene segments (95/95(DVG-high) and 95/95(DVG-low)). No clinical signs, mortality or transmission were noted in SPF chickens inoculated with neither virus stock. Turkeys infected with 95/95(DVG-high) showed only slight clinical signs with no mortality, and the virus was transmitted only to birds in direct contact. In contrast, more severe disease, mortality and transmission to direct and indirect contact birds was observed in turkeys infected with 95/95(DVG-low). Apathy, lower water and food intake, respiratory system disorders and a total mortality of 60% were noted. Shedding patterns in contact turkeys indicated more efficient within- and between-host spread of the virus than in 95/95(DVG-high) group. Sequencing of virus genomes showed no mutations that could account for the observed differences in pathogenicity. The results suggest that the abundance of DIPs in the inoculum was the factor responsible for the mild course of infection and disrupted virus transmission.

We report detection of a highly pathogenic avian influenza A(H5N8) clade 2.3.4.4b virus in Europe. This virus was generated by reassortment between H5N8 subtype virus from sub-Saharan Africa and low pathogenicity avian influenza viruses from Eurasia.

In this study, we investigated the clinical response, viral shedding, transmissibility, pathologic lesions, and tropism of HPAIV Gs/Gd H5N8 subtype (clade 2.3.4.4b), following experimental infection of three groups of captive mallards (Anas platyrhynchos): (i) fully susceptible, (ii) pre-exposed to low pathogenic avian influenza virus (LPAIV) H5N1 subtype, and (iii) pre-exposed to LPAIV H3N8 subtype. Infection of naïve mallards with HPAIV H5N8 resulted in ~60% mortality, neurological signs, abundant shedding, and transmission to contact ducks, who also became sick and died. High amounts of viral RNA were found in all collected organs, with the highest RNA load recorded in the brain. The IHC examinations performed on tissues collected at 4 and 14 days post-infection (dpi) revealed tropism to nervous tissue, myocardium, respiratory epithelium, and hepatic and pancreatic cells. The mallards pre-exposed to LPAIV H5N1 and challenged with HPAIV H5N8 were asymptomatic and showed a significant reduction of viral RNA shedding, yet still sufficient to cause infection (but no disease) in the contact ducks. The AIV antigen was not detected in organs at 4 and 14 dpi, and microscopic lesions were mild and scarce. Similarly, mallards previously inoculated with LPAIV H3N8 remained healthy after challenge with HPAIV H5N8, but viral RNA was detected in large quantities in swabs and organs, particularly in the early phase of infection. However, in contrast to mallards from group I, the IHC staining yielded negative results at the selected timepoints. The virus was transmitted to contact birds, which remained symptomless but demonstrated low levels of viral RNA shedding and mild- to moderate tissue damage despite negative IHC staining. The results indicate that naïve mallards are highly susceptible to HPAIV H5N8 clade 2.3.4.4b and that homo- and heterosubtypic immunity to LPAIV can mitigate the clinical outcomes of infection.

Bornaviruses are a diverse family of viruses infecting various hosts, including birds. Aquatic bird bornavirus 1 (ABBV-1) and aquatic bird bornavirus 2 (ABBV-2) have been found in wild waterfowl but data on their prevalence are scarce. To gain knowledge on the occurrence of ABBVs in Poland, samples originating from dead birds of the Anseriformes order collected in 2016–2021 were tested with a real time RT-PCR method targeting the ABBVs genome. A total of 514 birds were examined, including 401 swans, 96 ducks and 17 geese. The presence of ABBV-1 RNA was detected in 52 swans (10.1% of all tested birds) from 40 different locations. No positive results were obtained for ducks and geese. Sequences of about 2300 bases were generated for 18 viruses and phylogenetic analysis was performed. A relatively low genetic diversity of the examined ABBV-1 strains was observed as all were gathered in a single cluster in the phylogenetic tree and the minimum nucleotide identity was 99.14%. The Polish strains were closely related to ABBV-1 identified previously in Denmark and Germany, but a limited number of sequences from Europe hinders the drawing of conclusions about interconnections between Polish and other European ABBVs. The results of the present study provide new insights into the distribution and genetic characteristics of ABBVs in wild birds in Europe.

Avian influenza virus (AIV) is a highly diverse and widespread poultry pathogen. Its evolution and adaptation may be affected by multiple host and ecological factors, which are still poorly understood. In the present study, a turkey-origin H9N2 AIV was used as a model to investigate the within-host diversity of the virus in turkeys, quail and ducks in conjunction with the clinical course, shedding and seroconversion. Ten birds were inoculated oculonasally with a dose of 106 EID50 of the virus and monitored for 14 days. Virus shedding, transmission and seroconversion were evaluated, and swabs collected at selected time-points were characterized in deep sequencing to assess virus diversity. In general, the virus showed low pathogenicity for the examined bird species, but differences in shedding patterns, seroconversion and clinical outcome were noted. The highest heterogeneity of the virus population as measured by the number of single nucleotide polymorphisms and Shannon entropy was found in oropharyngeal swabs from quail, followed by turkeys and ducks. This suggests a strong bottleneck was imposed on the virus during replication in ducks, which can be explained by its poor adaptation and stronger selection pressure in waterfowl. The high within-host virus diversity in quail with high level of respiratory shedding and asymptomatic course of infection may contribute to our understanding of the role of quail as an intermediate host for adaptation of AIV to other species of poultry. In contrast, low virus complexity was observed in cloacal swabs, mainly from turkeys, showing that the within-host diversity may vary between different replication sites. Consequences of these observations on the virus evolution and adaptation require further investigation.