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.

Risk factors for avian influenza in Danish poultry and wild birds during the epidemic from June 2020 to May 2021.

Exploring the risk factors of avian influenza (AI) occurrence helps us to monitor and control the disease. Since late 2020, the number of avian influenza outbreaks in domestic and wild birds has increased in most European countries, including Denmark. This study was conducted to identify potential risk factors for wild birds and poultry during the epidemic in 2020/2021 in Denmark. Using Danish AI surveillance data of actively surveyed poultry and passively surveyed wild birds from June 2020 to May 2021, we calculated geographical attributes for bird locations and assessed the potential risk factors of AI detections using logistic regression analyses. 4% of actively surveyed poultry and 39% of passively surveyed wild birds were detected with AI circulating or ongoing at the time. Of these, 10 and 99% tested positive for the H5/H7 AI subtypes, respectively. Our analyses did not find any statistically significant risk factors for actively surveyed poultry within the dataset. For passively surveyed wild birds, bird species belonging to the Anseriformes order had a higher risk of being AI virus positive than five other taxonomic bird orders, and Galliformes were of higher risk than two other taxonomic bird orders. Besides, every 1 km increase in the distance to wetlands was associated with a 5.18% decrease in the risk of being AI positive (OR (odds ratio) 0.95, 95% CI 0.91, 0.99), when all other variables were kept constant. Overall, bird orders and distance to wetlands were associated with the occurrence of AI. The findings may provide targets for surveillance strategies using limited resources and assist in risk-based surveillance during epidemics.

Transmission dynamics and pathogenesis differ between pheasants and partridges infected with clade 2.3.4.4b H5N8 and H5N1 high-pathogenicity avian influenza viruses.

During the UK 2020-2021 epizootic of H5Nx clade 2.3.4.4b high-pathogenicity avian influenza viruses (HPAIVs), high mortality occurred during incursions in commercially farmed common pheasants (Phasianus colchicus). Two pheasant farms, affected separately by H5N8 and H5N1 subtypes, included adjacently housed red-legged partridges (Alectoris rufa), which appeared to be unaffected. Despite extensive ongoing epizootics, H5Nx HPAIV partridge outbreaks were not reported during 2020-2021 and 2021-2022 in the UK, so it is postulated that partridges are more resistant to HPAIV infection than other gamebirds. To assess this, pathogenesis and both intra- and inter-species transmission of UK pheasant-origin H5N8-2021 and H5N1-2021 HPAIVs were investigated. Onward transmission to chickens was also assessed to better understand the risk of spread from gamebirds to other commercial poultry sectors. A lower infectious dose was required to infect pheasants with H5N8-2021 compared to H5N1-2021. However, HPAIV systemic dissemination to multiple organs within pheasants was more rapid following infection with H5N1-2021 than H5N8-2021, with the former attaining generally higher viral RNA levels in tissues. Intraspecies transmission to contact pheasants was successful for both viruses and associated with viral environmental contamination, while interspecies transmission to a first chicken-contact group was also efficient. However, further onward transmission to additional chicken contacts was only achieved with H5N1-2021. Intra-partridge transmission was only successful when high-dose H5N1-2021 was administered, while partridges inoculated with H5N8-2021 failed to shed and transmit, although extensive tissue tropism was observed for both viruses. Mortalities among infected partridges featured a longer incubation period compared to that in pheasants, for both viruses. Therefore, the susceptibility of different gamebird species and pathogenicity outcomes to the ongoing H5Nx clade 2.3.4.4b HPAIVs varies, but pheasants represent a greater likelihood of H5Nx HPAIV introduction into galliforme poultry settings. Consequently, viral maintenance within gamebird populations and risks to poultry species warrant enhanced investigation.

Characterization of high pathogenicity avian influenza H5Nx viruses from a wild harbor seal and red foxes in Denmark, 2021 and 2022.

In 2021 and 2022, clade 2.3.4.4b H5Nx high pathogenicity avian influenza viruses were detected in one harbor seal and in one adult and three fox cubs in Denmark. The viruses were closely related to contemporary viruses found in Europe, and some had obtained amino acid substitutions related to mammalian adaptation. Notably, the virus distribution appeared to have been different in the infected fox cubs, as one exclusively tested positive for the presence of HPAIV in the brain and the other two only in the lung. Collectively, these findings stress the need for increased disease surveillance of wild and farmed mammals.

Using surveillance data for early warning modelling of highly pathogenic avian influenza in Europe reveals a seasonal shift in transmission, 2016-2022.

Avian influenza in wild birds and poultry flocks constitutes a problem for animal welfare, food security and public health. In recent years there have been increasing numbers of outbreaks in Europe, with many poultry flocks culled after being infected with highly pathogenic avian influenza (HPAI). Continuous monitoring is crucial to enable timely implementation of control to prevent HPAI spread from wild birds to poultry and between poultry flocks within a country. We here utilize readily available public surveillance data and time-series models to predict HPAI detections within European countries and show a seasonal shift that happened during 2021-2022. The output is models capable of monitoring the weekly risk of HPAI outbreaks, to support decision making.

Mass mortality among colony-breeding seabirds in the German Wadden Sea in 2022 due to distinct genotypes of HPAIV H5N1 clade 2.3.4.4b.

Mass mortality was observed among colony-breeding seabirds in the German Wadden Sea area of the North Sea during the summer months of 2022. Several species' colonies were affected, most notably sandwich terns (Thalasseus sandvicensis), common terns (Sterna hirundo) and Germany's only northern gannet (Morus bassanus) colony on the island of Heligoland. Mortality in some tern colonies reached 40%, while other colonies were almost spared. In all cases, infections with the high-pathogenicity avian influenza virus (HPAIV) subtype H5N1 of clade 2.3.4.4b were identified to have caused the epidemic. Phylogenetic analysis of whole-genome sequences revealed that the outbreaks were dominated by two genotypes, Ger-10-21 N1.2 and Ger-10-21 N1.5, previously identified in Germany. Spatiotemporal analyses of phylogenetic data suggested that these viruses could have entered the continental North Sea coastal region via the British Isles. A close linkage of viruses from tern colonies in the German Wadden Sea was evident with further connections to breeding colonies in Belgium and the Netherlands, and further spread to Denmark and Poland. Several of the affected species are endangered, such that negative effects of epizootic HPAIV infections on populations are feared, with uncertain long-term consequences.

Severe Human Case of Zoonotic Infection with Swine-Origin Influenza A Virus, Denmark, 2021.

During routine surveillance at the National Influenza Center, Denmark, we detected a zoonotic swine influenza A virus in a patient who became severely ill. We describe the clinical picture and the genetic characterization of this variant virus, which is distinct from another variant found previously in Denmark.

Pathogenesis and infection dynamics of high pathogenicity avian influenza virus (HPAIV) H5N6 (clade 2.3.4.4b) in pheasants and onward transmission to chickens.

High pathogenicity avian influenza viruses clade 2.3.4.4 H5 have spread among wild birds worldwide during recent years causing annual die-offs among wild birds and outbreaks in poultry in multiple European countries. The outbreaks significantly impact the poultry and game bird sectors. Infected game birds may act as a bridging species potentially enabling spread of virus into commercial and backyard premises. In this study, the pathogenesis and transmission of a HPAIV clade 2.3.4.4b H5N6 virus was investigated in pheasants and chickens. Efficient virus transmission was detected between pheasants over multiple rounds of naïve pheasant introductions and onwards to chickens. Mortality of up to 100% was observed for both infected pheasants and chickens. Intra-species transmission from chicken to chicken was less efficient. The study confirmed that clade 2.3.4.4b H5N6 HPAIV is highly virulent in pheasants and emphasises the role of pheasants as a bridging host for the infection of commercial poultry.

Experimental infection of high health pigs with porcine circovirus type 2 (PCV2) and Lawsonia intracellularis.

Background: Porcine circovirus type 2 (PCV2) and Lawsonia intracellularis infections can cause enteritis in pigs. A Danish study showed a significantly higher probability of detecting PCV2 without concurrent L. intracellularis infection, indicating that one of these pathogens has an impact on the dynamics of the other. Therefore, a delayed co-infection model was set up, initially aiming at investigating the interaction between PCV2 and L. intracellularis in pigs challenged with PCV2 and 2 weeks later with L. intracellularis. But due to PCV2 contamination of the L. intracellularis inoculum the aim was revisited to describing the infection dynamics and pathogenesis of pigs infected with PCV2 followed by delayed simultaneous exposure to PCV2 and L. intracellularis. Twenty-four high-health piglets were divided into three groups of eight pigs (A, B, C) and inoculated at experimental day (EXD) 0 with mock (groups A and B) or PCV2 (group C), and at EXD 14 with mock (group A) or L. intracellularis/PCV2 (groups B and C). The pigs underwent daily clinical examination, and were necropsied at EXD 51-52. Furthermore, histology, immunohistochemistry, serology and PCR for PCV2 and L. intracellularis, and measurement of C-reactive protein were carried out. Results: Group A remained negative for PCV2 and L. intracellularis. Following inoculation with L. intracellularis/PCV2, no significant differences were observed between group B and C, however pigs already infected with PCV2 (group C) showed milder clinical signs and exhibited milder intestinal lesions, less shedding of L. intracellularis and developed higher L. intracellularis antibody titers than the pigs in group B that only received the combined infection. Though the differences between group B and C were non-significant, all results pointed in the same direction, indicating that the pigs in group B were more affected by the L. intracellularis infection compared to the pigs in group C. Conclusions: Previous exposure to PCV2 had limited impact on the subsequent exposure to a combined L. intracellularis/PCV2 inoculation. However, there was a tendency that the infection dynamics of PCV2 and development of antibodies to PCV2 and L. intracellularis were altered in pigs previously exposed to PCV2. These differences should be confirmed in further experimental trials.

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.

Characterization of Influenza D Virus in Danish Calves.

Influenza D virus (IDV) was first described in 2011 and has been found to mainly circulate among cattle and swine populations worldwide. Nasal swab samples were collected from 100 Danish calf herds (83 dairy and 17 veal herds) from 2018-2020. Influenza D virus was detected in 12 of the herds. Samples with the lowest cycle quantification value were selected for full genome sequencing. A hemagglutinin-esterase fusion (HEF) gene sequence from a Danish IDV collected in 2015 was also included in this study. Phylogenetic analysis showed that viruses from seven of the IDV-positive herds belonged to the D/OK lineage and clustered together in the HEF tree with the IDV collected in 2015. Viruses from the four other herds belonged to the D/660 lineage, where three of the viruses clustered closely together, while the fourth virus was more phylogenetically distant in all gene segments. The high level of genetic similarity between viruses from two different herds involved in calf trading suggests that transmission occurred through the movement of calves. This study is, to our knowledge, the first to describe the characterization of IDV in calves in Denmark.

Landscape effects and spatial patterns of avian influenza virus in Danish wild birds, 2006-2020.

Avian influenza (AI) is a contagious disease of birds with zoonotic potential. AI virus (AIV) can infect most bird species, but clinical signs and mortality vary. Assessing the distribution and factors affecting AI presence can direct targeted surveillance to areas at risk of disease outbreaks, or help identify disease hotspots or areas with inadequate surveillance. Using virus surveillance data from passive and active AIV wild bird surveillance, 2006-2020, we investigated the association between the presence of AIV and a range of landscape factors and game bird release. Furthermore, we assessed potential bias in the passive AIV surveillance data submitted by the public, via factors related to public accessibility. Lastly, we tested the AIV data for possible hot- and cold spots within Denmark. The passive surveillance data was biased regarding accessibility to areas (distance to roads, cities and coast) compared to random locations within Denmark. For both the passive and active AIV surveillance data, we found significant (p < .01) associations with variables related to coast, wetlands and cities, but not game bird release. We used these variables to predict the risk of AIV presence throughout Denmark, and found high-risk areas concentrated along the coast and fjords. For both passive and active surveillance data, low-risk clusters were mainly seen in Jutland and northern Zealand, whereas high-risk clusters were found in Jutland, Zealand, Funen and the southern Isles such as Lolland and Falster. Our results suggest that landscape affects AIV presence, as coastal areas and wetlands attract waterfowl and migrating birds and therefore might increase the potential for AIV transmission. Our findings have enabled us to create risk maps of AIV presence in wild birds and pinpoint high-risk clusters within Denmark. This will aid targeted surveillance efforts within Denmark and potentially aid in planning the location of future poultry farms.

Reassortant Influenza A(H1N1)pdm09 Virus in Elderly Woman, Denmark, January 2021.

A case of human infection with influenza A(H1N1)pdm09 virus containing a nonstructural gene highly similar to Eurasian avian-like H1Nx swine influenza virus was detected in Denmark in January 2021. We describe the clinical case and report testing results of the genetic and antigenic characterizations of the virus.

Molecular Characterization of Highly Pathogenic Avian Influenza Viruses H5N6 Detected in Denmark in 2018-2019.

Beginning in late 2017, highly pathogenic avian influenza (HPAI) H5N6 viruses caused outbreaks in wild birds and poultry in several European countries. H5N6 viruses were detected in 43 wild birds found dead throughout Denmark. Most of the Danish virus-positive dead birds were found in the period from February to April 2018. However, unlike the rest of Europe, sporadic HPAI H5N6-positive dead wild birds were detected in Denmark in July, August, September, and December 2018, with the last positive bird being found in January 2019. HPAI viruses were not detected in active surveillance of apparently healthy wild birds. In this study, we use full genome sequencing and phylogenetic analysis to investigate the wild bird HPAI H5N6 viruses found in Denmark. The Danish viruses were found to be closely related to those of contemporary HPAI H5N6 viruses detected in Europe. Their sequences formed two clusters indicating that at least two or more introductions of H5N6 into Denmark occurred. Notably, all viruses detected in the latter half of 2018 and in 2019 grouped into the same cluster. The H5N6 viruses appeared to have been maintained undetected in the autumn 2018.

Co-circulation of multiple influenza A reassortants in swine harboring genes from seasonal human and swine influenza viruses.

Since the influenza pandemic in 2009, there has been an increased focus on swine influenza A virus (swIAV) surveillance. This paper describes the results of the surveillance of swIAV in Danish swine from 2011 to 2018. In total, 3800 submissions were received with a steady increase in swIAV-positive submissions, reaching 56% in 2018. Full-genome sequences were obtained from 129 swIAV-positive samples. Altogether, 17 different circulating genotypes were identified including six novel reassortants harboring human seasonal IAV gene segments. The phylogenetic analysis revealed substantial genetic drift and also evidence of positive selection occurring mainly in antigenic sites of the hemagglutinin protein and confirmed the presence of a swine divergent cluster among the H1pdm09Nx (clade 1A.3.3.2) viruses. The results provide essential data for the control of swIAV in pigs and emphasize the importance of contemporary surveillance for discovering novel swIAV strains posing a potential threat to the human population.

Novel Clade 2.3.4.4b Highly Pathogenic Avian Influenza A H5N8 and H5N5 Viruses in Denmark, 2020.

Since late 2020, outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses belonging to clade 2.3.4.4b have emerged in Europe. To investigate the evolutionary history of these viruses, we performed genetic characterization on the first HPAI viruses found in Denmark during the autumn of 2020. H5N8 viruses from 14 wild birds and poultry, as well as one H5N5 virus from a wild bird, were characterized by whole genome sequencing and phylogenetic analysis. The Danish H5N8 viruses were found to be genetically similar to each other and to contemporary European clade 2.3.4.4b H5N8 viruses, while the Danish H5N5 virus was shown to be a unique genotype from the H5N5 viruses that circulated at the same time in Russia, Germany, and Belgium. Genetic analyses of one of the H5N8 viruses revealed the presence of a substitution (PB2-M64T) that is highly conserved in human seasonal influenza A viruses. Our analyses showed that the late 2020 clade 2.3.4.4b HPAI H5N8 viruses were most likely new incursions introduced by migrating birds to overwintering sites in Europe, rather than the result of continued circulation of H5N8 viruses from previous introductions to Europe in 2016/2017 and early 2020.

Objective pathogen monitoring in nursery and finisher pigs by monthly laboratory diagnostic testing.

BACKGROUND: Infectious diseases are of great economic importance in commercial pig production, causing both clinical and subclinical disease, with influence on welfare, productivity, and antibiotic use. The causes of these diseases are often multifactorial and laboratory diagnostics are seldom routinely performed. The aim of the present study was to explore the benefits of monthly pathogen monitoring in nursery and finisher herds and to examine association between laboratory results and observed clinical signs, including coughing and diarrhoea. Three monthly samplings were conducted in three different age groups in six nursery and four finisher production units. For each unit, two pens were randomly selected in each age group and evaluated for coughing and diarrhoea events. Furthermore, faecal sock and oral fluid samples were collected in the selected pens and analysed for 18 respiratory and enteric viral and bacterial pathogens using the high-throughput real-time PCR BioMark HD platform (Fluidigm, South San Francisco, USA). RESULTS: In total, 174 pens were sampled in which eight coughing events and 77 diarrhoeic events were observed. The overall findings showed that swine influenza A virus, porcine circovirus 2, porcine cytomegalovirus, Brachyspira pilosicoli, Lawsonia intracellularis, Escherichia coli fimbria types F4 and F18 were found to be prevalent in several of the herds. Association between coughing events and the presence of swine influenza A virus, porcine cytomegalovirus (Cq ≤ 20) or a combination of these were found. Furthermore, an association between diarrhoeic events and the presence of L. intracellularis (Cq ≤ 24) or B. pilosicoli (Cq ≤ 26) was found. CONCLUSIONS: The use of high-throughput real-time PCR analysis for continuous monitoring of pathogens and thereby dynamics of infections in a pig herd, provided the veterinarian and farmer with an objective knowledge on the distribution of pathogens in the herd. In addition, the use of a high-throughput method in combination with information about clinical signs, productivity, health status and antibiotic consumption, presents a new and innovative way of diagnosing and monitoring pig herds and even to a lower cost than the traditional method.

Development of a high-throughput real-time PCR system for detection of enzootic pathogens in pigs.

Respiratory and intestinal diseases in pigs can have significant negative influence on productivity and animal welfare. A wide range of real-time PCR (rtPCR) assays are used in our laboratory (National Veterinary Institute, Technical University of Denmark) for pathogen detection, and PCR analyses are performed on traditional rtPCR platforms in which a limited number of samples can be analyzed per day given limitations in equipment and personnel. To mitigate these restrictions, rtPCR assays have been optimized for the high-throughput rtPCR BioMark platform (Fluidigm). Using this platform, we developed a high-throughput detection system that can be used for simultaneous examination of 48 samples with detection specificity for 18 selected respiratory and enteric viral and bacterial pathogens of high importance to Danish pig production. The rtPCR assays were validated and optimized to run under the same reaction conditions using a BioMark 48.48 dynamic array (DA) integrated fluidic circuit chip, and the sensitivity and specificity were assessed by testing known positive samples. Performance of the 48.48DA was similar to traditional rtPCR analysis, and the specificity of the 48.48DA was high. Application of the high-throughput platform has resulted in a significant reduction in cost and working hours and has provided production herds with a new innovative service with the potential to facilitate the optimal choice of disease control strategies such as vaccination and treatment.

Subtyping of Swine Influenza Using a High-Throughput Real-Time PCR Platform and a Single Microfluidics Device.

Reverse transcription real-time PCR (RT-qPCR) is one of several techniques used to determine the presence and level of infectious veterinary pathogens in diagnostic laboratories. Here we describe how automation of PCR reactions using integrated fluidic circuits (IFCs), an IFC controller MX and a Biomark HD instrument allows for the testing of 48 field samples with swine influenza for up to 48 different subtypes simultaneously in nanoliter volumes.