High pathogenic avian influenza A(H5) viruses of clade 2.3.4.4b in Europe-Why trends of virus evolution are more difficult to predict.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

Outbreak of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in cats, Poland, June to July 2023.

BackgroundOver a 3-week period in late June/early July 2023, Poland experienced an outbreak caused by highly pathogenic avian influenza (HPAI) A(H5N1) virus in cats.AimThis study aimed to characterise the identified virus and investigate possible sources of infection.MethodsWe performed next generation sequencing and phylogenetic analysis of detected viruses in cats.ResultsWe sampled 46 cats, and 25 tested positive for avian influenza virus. The identified viruses belong to clade 2.3.4.4b, genotype CH (H5N1 A/Eurasian wigeon/Netherlands/3/2022-like). In Poland, this genotype was responsible for several poultry outbreaks between December 2022 and January 2023 and has been identified only sporadically since February 2023. Viruses from cats were very similar to each other, indicating one common source of infection. In addition, the most closely related virus was detected in a dead white stork in early June. Influenza A(H5N1) viruses from cats possessed two amino acid substitutions in the PB2 protein (526R and 627K) which are two molecular markers of virus adaptation in mammals. The virus detected in the white stork presented one of those mutations (627K), which suggests that the virus that had spilled over to cats was already partially adapted to mammalian species.ConclusionThe scale of HPAI H5N1 virus infection in cats in Poland is worrying. One of the possible sources seems to be poultry meat, but to date no such meat has been identified with certainty. Surveillance should be stepped up on poultry, but also on certain species of farmed mammals kept close to infected poultry farms.

Homo- and Heterosubtypic Immunity to Low Pathogenic Avian Influenza Virus Mitigates the Clinical Outcome of Infection with Highly Pathogenic Avian Influenza H5N8 Clade 2.3.4.4.b in Captive Mallards (Anas platyrhynchos).

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.

Pathogenicity of highly pathogenic avian influenza H5N8 subtype for herring gulls (Larus argentatus): impact of homo- and heterosubtypic immunity on the outcome of infection.

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.

Disentangling the role of poultry farms and wild birds in the spread of highly pathogenic avian influenza virus in Europe.

In winter 2016-7, Europe was severely hit by an unprecedented epidemic of highly pathogenic avian influenza viruses (HPAIVs), causing a significant impact on animal health, wildlife conservation, and livestock economic sustainability. By applying phylodynamic tools to virus sequences collected during the epidemic, we investigated when the first infections occurred, how many infections were unreported, which factors influenced virus spread, and how many spillover events occurred. HPAIV was likely introduced into poultry farms during the autumn, in line with the timing of wild birds' migration. In Germany, Hungary, and Poland, the epidemic was dominated by farm-to-farm transmission, showing that understanding of how farms are connected would greatly help control efforts. In the Czech Republic, the epidemic was dominated by wild bird-to-farm transmission, implying that more sustainable prevention strategies should be developed to reduce HPAIV exposure from wild birds. Inferred transmission parameters will be useful to parameterize predictive models of HPAIV spread. None of the predictors related to live poultry trade, poultry census, and geographic proximity were identified as supportive predictors of HPAIV spread between farms across borders. These results are crucial to better understand HPAIV transmission dynamics at the domestic-wildlife interface with the view to reduce the impact of future epidemics.

Has Epizootic Become Enzootic? Evidence for a Fundamental Change in the Infection Dynamics of Highly Pathogenic Avian Influenza in Europe, 2021.

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.

Highly Pathogenic Avian Influenza H5Nx in Poland in 2020/2021: a Descriptive Epidemiological Study of a Large-scale Epidemic.

Introduction: Highly pathogenic avian influenza (HPAI) outbreaks caused by the Gs/Gd lineage of H5Nx viruses occur in Poland with increased frequency. The article provides an update on the HPAI situation in the 2020/2021 season and studies the possible factors that caused the exceptionally fast spread of the virus. Material and Methods: Samples from poultry and wild birds delivered for HPAI diagnosis were tested by real-time RT-PCR and a representative number of detected viruses were submitted for partial or full-genome characterisation. Information yielded by veterinary inspection was used for descriptive analysis of the epidemiological situation. Results: The scale of the epidemic in the 2020/2021 season was unprecedented in terms of duration (November 2020-August 2021), number of outbreaks in poultry (n = 357), wild bird events (n = 92) and total number of affected domestic birds (approximately ~14 million). The major drivers of the virus spread were the harsh winter conditions in February 2020 followed by the introduction of the virus to high-density poultry areas in March 2021. All tested viruses belonged to H5 clade 2.3.4.4b with significant intra-clade diversity and in some cases clearly distinguished clusters. Conclusion: The HPAI epidemic in 2020/2021 in Poland struck with unprecedented force. The conventional control measures may have limited effectiveness to break the transmission chain in areas with high concentrations of poultry.

Avian Bornaviruses in Wild Aquatic Birds of the Anseriformes Order in Poland.

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.

Highly Pathogenic Avian Influenza H5N8 in Poland in 2019-2020.

INTRODUCTION: Repeated incursions of highly pathogenic avian influenza virus (HPAIV) H5 subtype of Gs/GD lineage pose a serious threat to poultry worldwide. We provide a detailed analysis of the spatio-temporal spread and genetic characteristics of HPAIV Gs/GD H5N8 from the 2019/20 epidemic in Poland. MATERIAL AND METHODS: Samples from poultry and free-living birds were tested by real-time RT-PCR. Whole genome sequences from 24 (out of 35) outbreaks were generated and genetic relatedness was established. The clinical status of birds and possible pathways of spread were analysed based on the information provided by veterinary inspections combined with the results of phylogenetic studies. RESULTS: Between 31 December 2019 and 31 March 2020, 35 outbreaks in commercial and backyard poultry holdings and 1 case in a wild bird were confirmed in nine provinces of Poland. Most of the outbreaks were detected in meat turkeys and ducks. All characterised viruses were closely related and belonged to a previously unrecognised genotype of HPAIV H5N8 clade 2.3.4.4b. Wild birds and human activity were identified as the major modes of HPAIV spread. CONCLUSION: The unprecedentedly late introduction of the HPAI virus urges for re-evaluation of current risk assessments. Continuous vigilance, strengthening biosecurity and intensifying surveillance in wild birds are needed to better manage the risk of HPAI occurrence in the future.

Low pathogenic avian influenza virus isolates with different levels of defective genome segments vary in pathogenicity and transmission efficiency.

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.

Sub-Saharan Africa and Eurasia Ancestry of Reassortant Highly Pathogenic Avian Influenza A(H5N8) Virus, Europe, December 2019.

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.

A Turkey-origin H9N2 Avian Influenza Virus Shows Low Pathogenicity but Different Within-Host Diversity in Experimentally Infected Turkeys, Quail and Ducks.

Avian influenza virus (AIV) is a highly diverse and widespread poultry pathogen. Itsevolution and adaptation may be affected by multiple host and ecological factors, which are stillpoorly understood. In the present study, a turkey-origin H9N2 AIV was used as a model toinvestigate the within-host diversity of the virus in turkeys, quail and ducks in conjunction with theclinical course, shedding and seroconversion. Ten birds were inoculated oculonasally with a doseof 106 EID50 of the virus and monitored for 14 days. Virus shedding, transmission andseroconversion were evaluated, and swabs collected at selected time-points were characterized indeep sequencing to assess virus diversity. In general, the virus showed low pathogenicity for theexamined bird species, but differences in shedding patterns, seroconversion and clinical outcomewere noted. The highest heterogeneity of the virus population as measured by the number of singlenucleotide 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 duringreplication in ducks, which can be explained by its poor adaptation and stronger selection pressurein waterfowl. The high within-host virus diversity in quail with high level of respiratory sheddingand asymptomatic course of infection may contribute to our understanding of the role of quail asan intermediate host for adaptation of AIV to other species of poultry. In contrast, low viruscomplexity was observed in cloacal swabs, mainly from turkeys, showing that the within-hostdiversity may vary between different replication sites. Consequences of these observations on thevirus evolution and adaptation require further investigation.

Estimation of the number of exposed people during highly pathogenic avian influenza virus outbreaks in EU/EEA countries, October 2016-September 2018.

We estimated that more than 11,000 people were exposed to highly pathogenic avian influenza viruses in EU/EEA countries over the outbreak period October 2016-September 2018 by cross-linking data submitted by Member States to European Food Safety Authority and EMPRES-i. A stronger framework for collecting human exposure data is required.

Ultrasensitive electrochemical genosensor for direct detection of specific RNA sequences derived from avian influenza viruses present in biological samples.

An electrochemical genosensor based on an epoxy-phenanthroline-Fe(III)-NH2-ssDNA layer for the detection of RNA derived from Avian Influenza is presented. The biosensor preparation consists of: (I) modification of gold electrodes with aminoethanethiol, (II) modification of the self-assembled monolayer of aminoethanethiol with 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline using "click" chemistry, (III) a first step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (IV) a second step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (V) immobilization of the single stranded amino-DNA probe via "click" chemistry between epoxy and amino groups. The interactions between the ssDNA probe and RNA targets were explored with Osteryoung Square Wave Voltammetry. The genosensor showed a remarkable detection limit of 3 copies/µL (5 aM) for RNA extracted from A/swan/Poland/305/06 (H5N1) containing a fully complementary sequence. A linear dynamic range for this sequence was observed from 3.0×103 to 3.0×105 [copies/µl]. RNA extracted from A/mallard/Poland/446/09 (H7N7), containing a non-complementary sequence, generated a much weaker response. Moreover, the developed genosensor allows to distinguish RNA present in biological samples having 2, 3 and 4 mismatches. This biosensing approach can become a potential alternative tool for detecting RNA samples in biomedical research and early clinical diagnosis of avian influenza viruses.

Evolution of H9N2 low pathogenic avian influenza virus during passages in chickens.

The process of avian influenza virus (AIV) evolution in a new host was investigated in the experiment in which ten serial passages of a turkey-derived H9N2 AIV were carried out in specific pathogen free chickens (3 birds/group) inoculated by oculonasal route. Oropharyngeal swabs collected 3 days post infection were used for inoculation of birds in the next passage and subjected to analysis using deep sequencing. In total, eight mutations in the consensus sequence were found in the viral pool derived from the 10th passage: four mutations (2 in PB1 and 2 in HA) were present in the inoculum as minority variants while the other four (2 in NP, 1 in PA and 1 in HA) emerged during the passages in chickens. The detected fluctuations in the genetic heterogeneity of viral pools from consecutive passages were most likely attributed to the selective bottleneck. The genes known for bearing molecular determinants of the AIV host specificity (HA, PB2, PB1, PA) contributed most to the overall virus diversity. In some cases, a fast selection of the novel variant was noticed. For example, the amino-acid substitution N337K in the haemagglutinin (HA) cleavage site region detected in the 6th passage as low frequency variant had undergone rapid selection and became predominant in the 7th passage. Interestingly, detection of identical mutation in the field H9N2 isolates 1-year apart suggests that this substitution might provide the virus with a selective advantage. However, the role of specific mutations and their influence on the virus adaptation or fitness are mostly unknown and require further investigations.

Phylogenetic and molecular analysis of highly pathogenic avian influenza H5N8 and H5N5 viruses detected in Poland in 2016-2017.

Sixty-five poultry outbreaks and sixty-eight events in wild birds were reported during the highly pathogenic H5N8/H5N5 avian influenza epidemic in Poland in 2016-2017. The analysis of all gene segment sequences of selected strains revealed cocirculation of at least four different genome configurations (genotypes) generated through reassortment of clade 2.3.4.4 H5N8 viruses detected in Russia and China in mid-2016. The geographical and temporal distribution of three H5N8 genotypes indicates separate introductions. Additionally, an H5N5 virus with a different gene configuration was detected in wild birds. The compilation of the results with those from studies on the virus' diversity in Germany, Italy and the Netherlands revealed that Europe was affected by at least eight different H5N8/H5N5 reassortants. Analysis of the HA gene sequence of a larger subset of samples showed its diversification corresponding to the genotype classification. The close relationship between poultry and wild bird strains from the same locations observed in several cases points to wild birds as the primary source of the outbreaks in poultry.

Genetic characterization of H9N2 avian influenza viruses isolated from poultry in Poland during 2013/2014.

The study presents molecular characterization of H9N2 avian influenza (AI) isolates from field outbreaks in turkeys that occurred in Poland in 2013-2014. Sequences of all gene segments of one isolate from 2013 (A/turkey/Poland/14/2013(H9N2)) and two isolates from 2014 (A/turkey/Poland/08/2014(H9N2), A/turkey/Poland/09/2014(H9N2)) were obtained and analyzed in search of the phylogenetic relationship and molecular markers of zoonotic potential or increased pathogenicity. All gene segments were shown to originate from the wild bird reservoir and the close relationship of the analyzed isolates proved the link between the outbreaks in 2013 and 2014. However, remarkable molecular differences between isolates from 2013 to 2014 were identified, including mutation in the HA cleavage site (CS) leading to conversion from the PAASNR*GLF to the PAASKR*GLF motif and truncation of the PB1-F2 protein. Additionally, T97I substitution in the PA protein in A/turkey/Poland/08/2014 was detected which can be responsible for enhanced activity of viral polymerase in mammalian cells. However, experimental infection of mice with both isolates from 2014 showed their low pathogenicity, and no statistically significant differences in virus replication were observed between the viruses. Nevertheless, these findings indicate the dynamic evolution of H9N2 in the field emphasizing the need for monitoring of the situation in terms of H9N2 AI in Europe.

Genetic Changes in Pigeon Paramyxovirus Type-1 Induced by Serial Passages in Chickens and Microscopic Lesions Caused by the Virus in Various Avian Hosts.

INTRODUCTION: Genotype VI of avian avulavirus 1 (AAvV-1) has pigeons and doves as its reservoir and is often termed pigeon paramyxovirus type-1 (PPMV-1). The pathogenesis of PPMV-1 infections in poultry is largely obscure. It is known that PPMV-1 requires a series of passages in chickens before it becomes adapted to gallinaceous poultry. MATERIAL AND METHODS: Changes in the genome of PPMV-1 were analysed after serial passages in specific pathogen free (SPF) chickens, using high-throughput sequencing. Additionally, histopathological lesions induced by PPMV-1 in experimentally inoculated pigeons, chickens, and turkeys were evaluated. RESULTS: Following six passages of PPMV-1 in chickens, 10 nonsynonymous substitutions were found including one (in the NP protein) which dominated the genetic pool of viral quasispecies. Histopathological changes induced by the post-passage PPMV-1 strain were more prominent than changes wrought by the pre-passaged PPMV-1 strain and the lesions were most intense in pigeons followed by chickens and turkeys. CONCLUSION: PPMV-1 is highly adapted to pigeons and passaging through chickens results in the acquisition of novel amino acids in the polymerase complex, which may alter the pathogenic potential of the virus.

SURVEILLANCE FOR AVIAN INFLUENZA VIRUS IN WILD BIRDS IN POLAND, 2008-15.

We tested wild birds in Poland during 2008-15 for avian influenza virus (AIV). We took 10,312 swabs and feces samples from 6,314 live birds representing 12 orders and 84 bird species, mostly from orders Anseriformes and Charadriiformes, for testing and characterization by various PCR methods. From PCR-positive samples, we attempted to isolate and subtype the virus. The RNA of AIV was detected in 1.8% (95% confidence interval [CI], 1.5-2.1%) of birds represented by 48 Mallards ( Anas platyrhynchos ), 11 Mute Swans ( Cygnus olor ), 48 Common Teals ( Anas crecca ), three Black-headed Gulls (Chroicocephalus ridibundus), one Common Coot ( Fulica atra ), one Garganey (Spatula querquedula), and one unidentified bird species. Overall, the prevalence of AIV detection in Mallards and Mute Swans (the most frequently sampled species) was 2.0% (95% CI, 1.4-2.5%) and 0.5% (95% CI, 0.2-0.8%), respectively; the difference was statistically significant (P=0.000). Hemagglutinin subtypes from H1 to H13 were identified, including H5 and H7 low pathogenic AIV subtypes. Mallards and Common Teals harbored the greatest diversity of subtypes. We observed seasonality of viral detection in Mallards, with higher AIV prevalence in late summer and autumn than in winter and spring. In addition, two peaks in AIV prevalence in summer (August) and autumn (November) were demonstrated for Mallards. The prevalence of AIV in Mute Swans did not show any statistically significant seasonal patterns.

Phylogenetic Study of H5 Low Pathogenic Avian Influenza Viruses Detected in Wild Birds in Poland in 2010-2015.

INTRODUCTION: The genomes of nine H5 subtypes of low pathogenic avian influenza virus (LPAIV) strains identified in wild birds in Poland between 2010 and 2015 were sequenced, and their phylogenetic relationship was determined. MATERIAL AND METHODS: AIV genome segments were amplified by RT-PCR and the PCR products were sequenced using Sanger method. Phylogenetic trees were generated in MEGA6 software and digital genotyping approach was used to visualise the relationship between analysed strains and other AIVs. RESULTS: High genetic diversity was found in the analysed strains as multiple subgroups were identified in phylogenetic trees. In the HA tree, Polish strains clustered in two distinct subclades. High diversity was found for PB2, PB1, PA and NP, since 5-8 sublineages could be distinguished. Each strain had a different gene constellation, although relationship of as much as six out of eight gene segments was observed between two isolates. A relationship with poultry isolates was found for at least one segment of each Polish strain. CONCLUSION: The genome configuration of tested strains indicates extensive reassortment, although the preference for specific gene constellation could be noticed. A significant relationship with isolates of poultry origin underlines the need for constant monitoring of the AIV gene pool circulating in the natural reservoir.