Highly Pathogenic Avian Influenza A(H5N1) Virus in a Harbor Porpoise, Sweden.

We found highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b associated with meningoencephalitis in a stranded harbor porpoise (Phocoena phocoena). The virus was closely related to strains responsible for a concurrent avian influenza outbreak in wild birds. This case highlights the potential risk for virus spillover to mammalian hosts.

Screening antivirals with a mCherry-expressing recombinant bovine respiratory syncytial virus: a proof of concept using cyclopamine.

Bovine respiratory syncytial virus (BRSV) is a pathogenic pneumovirus and a major cause of acute respiratory infections in calves. Although different vaccines are available against BRSV, their efficiency remains limited, and no efficient and large-scale treatment exists. Here, we developed a new reverse genetics system for BRSV expressing the red fluorescent protein mCherry, based on a field strain isolated from a sick calf in Sweden. Although this recombinant fluorescent virus replicated slightly less efficiently compared to the wild type virus, both viruses were shown to be sensitive to the natural steroidal alkaloid cyclopamine, which was previously shown to inhibit human RSV replication. Our data thus point to the potential of this recombinant fluorescent BRSV as a powerful tool in preclinical drug discovery to enable high throughput compound screening.

Evaluating the potential of whole-genome sequencing for tracing transmission routes in experimental infections and natural outbreaks of bovine respiratory syncytial virus.

Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in cattle. Genomic sequencing can resolve phylogenetic relationships between virus populations, which can be used to infer transmission routes and potentially inform the design of biosecurity measures. Sequencing of short (<2000 nt) segments of the 15 000-nt BRSV genome has revealed geographic and temporal clustering of BRSV populations, but insufficient variation to distinguish viruses collected from herds infected close together in space and time. This study investigated the potential for whole-genome sequencing to reveal sufficient genomic variation for inferring transmission routes between herds. Next-generation sequencing (NGS) data were generated from experimental infections and from natural outbreaks in Jämtland and Uppsala counties in Sweden. Sufficient depth of coverage for analysis of consensus and sub-consensus sequence diversity was obtained from 47 to 20 samples respectively. Few (range: 0-6 polymorphisms across the six experiments) consensus-level polymorphisms were observed along experimental transmissions. A much higher level of diversity (146 polymorphic sites) was found among the consensus sequences from the outbreak samples. The majority (144/146) of polymorphisms were between rather than within counties, suggesting that consensus whole-genome sequences show insufficient spatial resolution for inferring direct transmission routes, but might allow identification of outbreak sources at the regional scale. By contrast, within-sample diversity was generally higher in the experimental than the outbreak samples. Analyses to infer known (experimental) and suspected (outbreak) transmission links from within-sample diversity data were uninformative. In conclusion, analysis of the whole-genome sequence of BRSV from experimental samples discriminated between circulating isolates from distant areas, but insufficient diversity was observed between closely related isolates to aid local transmission route inference.

Gizzard erosions in broiler chickens in Sweden caused by fowl adenovirus serotype 1 (FAdV-1): investigation of outbreaks, including whole-genome sequencing of an isolate.

The present paper describes the investigation of the first outbreaks of adenoviral gizzard erosions (AGE) in Sweden, in five broiler flocks. The investigation included whole viral genome sequencing and investigation of genomic organization and sequence relationships with other adenoviruses. All five flocks had a history of decreased growth and uneven size of birds since 9-10 days of age. Macroscopically, lesions consistent with AGE (detached koilin layers, discolouration, bleeding, erosions) were identified in gizzards in all five flocks. In four flocks histology was performed, and degeneration and inflammation of the koilin layer and gizzard mucosa were identified in all four. In one flock, intranuclear inclusion bodies typical for fowl adenovirus (FAdV) were detected in trapped epithelial cells in the koilin layer. In four flocks in situ hybridization was performed, and cells positive for FAdV serotype 1 (FAdV-1) were demonstrated in the koilin layer and gizzard mucosa. FAdV species A (FAdV-A) was detected in gizzard, liver, caecal tonsils and bursa of Fabricius by polymerase chain reaction (PCR) and sequencing. Ten out of ten examined parent flocks of the affected chickens were seropositive for FAdV, indicating former or on-going infection. However, FAdV was not detected in embryos from seropositive parent flocks and thus vertical transmission was not demonstrated. The entire nucleotide sequence of one sample was determined and found to be 43,856 base pairs (bp) in length. The genome sequence and organization were found to be similar to that of the reference apathogenic avian adenovirus "chicken embryo lethal orphan" (CELO). RESEARCH HIGHLIGHTSAGE in Swedish broilers: necropsy, histopathology, ISH, PCR, whole-genome sequencing.Whole FAdV-genome analysed: 43,856 bp, found to be most similar to CELO (U46933.1).Multiple point mutations, site insertions and deletions identified compared to CELO.Paper adds knowledge about European disease situation and pathogenic FAdV-strains.

Pathogenesis, Host Innate Immune Response, and Aerosol Transmission of Influenza D Virus in Cattle.

The recently discovered influenza D virus (IDV) of the Orthomyxoviridae family has been detected in swine and ruminants with a worldwide distribution. Cattle are considered to be the primary host and reservoir, and previous studies suggested a tropism of IDV for the upper respiratory tract and a putative role in the bovine respiratory disease complex. This study aimed to characterize the pathogenicity of IDV in naive calves as well as the ability of this virus to transmit by air. Eight naive calves were infected by aerosol with a recent French isolate, D/bovine/France/5920/2014. Results show that IDV replicates not only in the upper respiratory tract but also in the lower respiratory tract (LRT), inducing moderate bronchopneumonia with restricted lesions of interstitial pneumonia. Inoculation was followed by IDV-specific IgG1 production as early as 10 days postchallenge and likely both Th1 and Th2 responses. Study of the innate immune response in the LRT of IDV-infected calves indicated the overexpression of pathogen recognition receptors and of chemokines CCL2, CCL3, and CCL4, but without overexpression of genes involved in the type I interferon pathway. Finally, virological examination of three aerosol-sentinel animals, housed 3 m apart from inoculated calves (and thus subject to infection by aerosol transmission), and IDV detection in air samples collected in different areas showed that IDV can be airborne transmitted and infect naive contact calves on short distances. This study suggests that IDV is a respiratory virus with moderate pathogenicity and probably a high level of transmission. It consequently can be considered predisposing to or a cofactor of respiratory disease.IMPORTANCE Influenza D virus (IDV), a new genus of the Orthomyxoviridae family, has a broad geographical distribution and can infect several animal species. Cattle are so far considered the primary host for IDV, but the pathogenicity and the prevalence of this virus are still unclear. We demonstrated that under experimental conditions (in a controlled environment and in the absence of coinfecting pathogens), IDV is able to cause mild to moderate disease and targets both the upper and lower respiratory tracts. The virus can transmit by direct as well as aerosol contacts. While this study evidenced overexpression of pathogen recognition receptors and chemokines in the lower respiratory tract, IDV-specific IgG1 production as early as 10 days postchallenge, and likely both Th1 and Th2 responses, further studies are warranted to better understand the immune responses triggered by IDV and its role as part of the bovine respiratory disease complex.

Comparison of 2016-17 and Previous Epizootics of Highly Pathogenic Avian Influenza H5 Guangdong Lineage in Europe.

We analyzed the highly pathogenic avian influenza (HPAI) H5 epizootic of 2016-17 in Europe by epidemiologic and genetic characteristics and compared it with 2 previous epizootics caused by the same H5 Guangdong lineage. The 2016-17 epizootic was the largest in Europe by number of countries and farms affected and greatest diversity of wild birds infected. We observed significant differences among the 3 epizootics regarding region affected, epidemic curve, seasonality, and outbreak duration, making it difficult to predict future HPAI epizootics. However, we know that in 2005-06 and 2016-17 the initial peak of wild bird detections preceded the peak of poultry outbreaks within Europe. Phylogenetic analysis of 2016-17 viruses indicates 2 main pathways into Europe. Our findings highlight the need for global surveillance of viral changes to inform disease preparedness, detection, and control.

Overcoming species barriers: an outbreak of Lagovirus europaeus GI.2/RHDV2 in an isolated population of mountain hares (Lepus timidus).

BACKGROUND: Prior to 2010, the lagoviruses that cause rabbit hemorrhagic disease (RHD) in European rabbits (Oryctolagus cuniculus) and European brown hare syndrome (EBHS) in hares (Lepus spp.) were generally genus-specific. However, in 2010, rabbit hemorrhagic disease virus 2 (RHDV2), also known as Lagovirus europaeus GI.2, emerged and had the distinguishing ability to cause disease in both rabbits and certain hare species. The mountain hare (Lepus timidus) is native to Sweden and is susceptible to European brown hare syndrome virus (EBHSV), also called Lagovirus europaeus GII.1. While most mountain hare populations are found on the mainland, isolated populations also exist on islands. Here we investigate a mortality event in mountain hares on the small island of Hallands Väderö where other leporid species, including rabbits, are absent. RESULTS: Post-mortem and microscopic examination of three mountain hare carcasses collected from early November 2016 to mid-March 2017 revealed acute hepatic necrosis consistent with pathogenic lagovirus infection. Using immunohistochemistry, lagoviral capsid antigen was visualized within lesions, both in hepatocytes and macrophages. Genotyping and immunotyping of the virus independently confirmed infection with L. europaeus GI.2, not GII.1. Phylogenetic analyses of the vp60 gene grouped mountain hare strains together with a rabbit strain from an outbreak of GI.2 in July 2016, collected approximately 50 km away on the mainland. CONCLUSIONS: This is the first documented infection of GI.2 in mountain hares and further expands the host range of GI.2. Lesions and tissue distribution mimic those of GII.1 in mountain hares. The virus was most likely initially introduced from a concurrent, large-scale GI.2 outbreak in rabbits on the adjacent mainland, providing another example of how readily this virus can spread. The mortality event in mountain hares lasted for at least 4.5 months in the absence of rabbits, which would have required virus circulation among mountain hares, environmental persistence and/or multiple introductions. This marks the fourth Lepus species that can succumb to GI.2 infection, suggesting that susceptibility to GI.2 may be common in Lepus species. Measures to minimize the spread of GI.2 to vulnerable Lepus populations therefore are prudent.

Spatiotemporal Analysis of the Genetic Diversity of Seal Influenza A(H10N7) Virus, Northwestern Europe.

UNLABELLED: Influenza A viruses are major pathogens for humans, domestic animals, and wildlife, and these viruses occasionally cross the species barrier. In spring 2014, increased mortality of harbor seals (Phoca vitulina), associated with infection with an influenza A(H10N7) virus, was reported in Sweden and Denmark. Within a few months, this virus spread to seals of the coastal waters of Germany and the Netherlands, causing the death of thousands of animals. Genetic analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of this seal influenza A(H10N7) virus revealed that it was most closely related to various avian influenza A(H10N7) viruses. The collection of samples from infected seals during the course of the outbreak provided a unique opportunity to follow the adaptation of the avian virus to its new seal host. Sequence data for samples collected from 41 different seals from four different countries between April 2014 and January 2015 were obtained by Sanger sequencing and next-generation sequencing to describe the molecular epidemiology of the seal influenza A(H10N7) virus. The majority of sequence variation occurred in the HA gene, and some mutations corresponded to amino acid changes not found in H10 viruses isolated from Eurasian birds. Also, sequence variation in the HA gene was greater at the beginning than at the end of the epidemic, when a number of the mutations observed earlier had been fixed. These results imply that when an avian influenza virus jumps the species barrier from birds to seals, amino acid changes in HA may occur rapidly and are important for virus adaptation to its new mammalian host. IMPORTANCE: Influenza A viruses are major pathogens for humans, domestic animals, and wildlife. In addition to the continuous circulation of influenza A viruses among various host species, cross-species transmission of influenza A viruses occurs occasionally. Wild waterfowl and shorebirds are the main reservoir for most influenza A virus subtypes, and spillover of influenza A viruses from birds to humans or other mammalian species may result in major outbreaks. In the present study, various sequencing methods were used to elucidate the genetic changes that occurred after the introduction and subsequent spread of an avian influenza A(H10N7) virus among harbor seals of northwestern Europe by use of various samples collected during the outbreak. Such detailed knowledge of genetic changes necessary for introduction and adaptation of avian influenza A viruses to mammalian hosts is important for a rapid risk assessment of such viruses soon after they cross the species barrier.

Accurate Detection of Avian Respiratory Viruses by Use of Multiplex PCR-Based Luminex Suspension Microarray Assay.

A novel oligonucleotide suspension microarray (Luminex microsphere system) was developed for the rapid detection of avian respiratory viruses of major clinical importance. This test was optimized and validated with 70 clinical samples. The developed tool was accurate for high-throughput detection and differentiation of the most important avian respiratory viruses: avian influenza virus (AIV), Newcastle disease virus (NDV), infection bronchitis virus (IBV), and infectious laryngotracheitis virus (ILTV) in single- and mixed-virus infections. A multiplex reverse transcriptase PCR (RT-PCR), followed by a monoplex or a multiplex Luminex assays, were realized using a Luminex 200 analyzer instrument. The sensitivity, specificity, and reproducibility of the multiplex DNA suspension microarray system were evaluated. The results showed no significant differences in the median fluorescence intensity (MFI) value in monoplex and multiplex Luminex assays. The sensitivity and specificity proved to be completely concordant with monoplex real-time RT-PCR. We demonstrated that the multiplex DNA suspension microarray system is an accurate, high-throughput, and relatively simple method for the rapid detection of the main respiratory viruses of poultry.

Highly pathogenic avian influenza A(H5N8) outbreaks: protection and management of exposed people in Europe, 2014/15 and 2016.

Introduction of highly pathogenic avian influenza (HPAI) virus A(H5N8) into Europe prompted animal and human health experts to implement protective measures to prevent transmission to humans. We describe the situation in 2016 and list public health measures and recommendations in place. We summarise critical interfaces identified during the A(H5N1) and A(H5N8) outbreaks in 2014/15. Rapid exchange of information between the animal and human health sectors is critical for a timely, effective and efficient response.

Proteomic and Lipidomic Profiling of Calves Experimentally Co-Infected with Influenza D Virus and Mycoplasma bovis: Insights into the Host-Pathogen Interactions.

The role of Influenza D virus (IDV) in bovine respiratory disease remains unclear. An in vivo experiment resulted in increased clinical signs, lesions, and pathogen replication in calves co-infected with IDV and Mycoplasma bovis (M. bovis), compared to single-infected calves. The present study aimed to elucidate the host-pathogen interactions and profile the kinetics of lipid mediators in the airways of these calves. Bronchoalveolar lavage (BAL) samples collected at 2 days post-infection (dpi) were used for proteomic analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, lipidomic analyses were performed by LC-MS/MS on BAL samples collected at 2, 7 and 14 dpi. Whereas M. bovis induced the expression of proteins involved in fibrin formation, IDV co-infection counteracted this coagulation mechanism and downregulated other acute-phase response proteins, such as complement component 4 (C4) and plasminogen (PLG). The reduced inflammatory response against M. bovis likely resulted in increased M. bovis replication and delayed M. bovis clearance, which led to a significantly increased abundance of oxylipids in co-infected calves. The identified induced oxylipids mainly derived from arachidonic acid; were likely oxidized by COX-1, COX-2, and LOX-5; and peaked at 7 dpi. This paper presents the first characterization of BAL proteome and lipid mediator kinetics in response to IDV and M. bovis infection in cattle and raises hypotheses regarding how IDV acts as a co-pathogen in bovine respiratory disease.

Prevalence of avian paramyxovirus type 1 in Mallards during autumn migration in the western Baltic Sea region.

BACKGROUND: Newcastle disease virus (NDV) is the causative agent of the Newcastle disease, a severe disease in birds associated with substantial economic losses to the poultry industry worldwide. Sweden is situated along the Western European waterfowl flyway and applies a non-vaccination policy combined with directives of immediate euthanisation of NDV infected flocks. During the last decades there have been several outbreaks with NDV in poultry in Sweden. However, less is known about the virus prevalence in the wild bird population including waterfowl, a well-established reservoir of avian paramyxovirus type 1 (APMV-1), the paramyxovirus serotype that include pathogenic NDV. METHODS: The survey constituted of 2332 samples from Mallards (Anas platyrhynchos), trapped in the southern part of Sweden during autumn migration in 2010. These samples were screened for APMV-1 by real-time reverse transcription PCR, and viral strains from positive samples were isolated and characterized by sequence analysis of the fusion gene and by phylogenetic analysis. CONCLUSIONS: Twenty of these samples were positive for APMV-1, hence a virus prevalence of 0.9% (95% Confidence Interval [95% CI]=0.54%, 1.35%). The highest APMV-1 prevalence was detected in juvenile Mallards sampled in November (n=887, prevalence 1.24% ([95% CI])=0.67%, 2.24%). Sequence analysis and evaluation of phylogenetic relatedness indicated that isolated APMV-1 strains were lentogenic, and phylogenetically most closely related to genotype Ib strains within the clade of class II viruses. The sampling system employed enabled us to follow APMV-1 infections and the shedding of one particular viral strain in one individual bird over several days. Furthermore, combining previous screening results with the APMV-1 detections in this study showed that more than 50% of Mallards that tested positive for APMV-1 RNA were co-infected with influenza A virus.

Genetic diversity of Newcastle disease virus in Pakistan: a countrywide perspective.

BACKGROUND: Newcastle disease (ND) is one of the most deadly diseases of poultry around the globe. The disease is endemic in Pakistan and recurrent outbreaks are being reported regularly in wild captive, rural and commercial poultry flocks. Though, efforts have been made to characterize the causative agent in some of parts of the country, the genetic nature of strains circulating throughout Pakistan is currently lacking. MATERIAL AND METHODS: To ascertain the genetics of NDV, 452 blood samples were collected from 113 flocks, originating from all the provinces of Pakistan, showing high mortality (30-80%). The samples represented domesticated poultry (broiler, layer and rural) as well as wild captive birds (pigeons, turkeys, pheasants and peacock). Samples were screened with real-time PCR for both matrix and fusion genes (1792 bp), positive samples were subjected to amplification of full fusion gene and subsequent sequencing and phylogenetic analysis. RESULTS: The deduced amino acid sequence of the fusion protein cleavage site indicated the presence of motif (112RK/RQRR↓F117) typical for velogenic strains of NDV. Phylogenetic analysis of hypervariable region of the fusion gene indicated that all the isolates belong to lineage 5 of NDV except isolates collected from Khyber Pakhtunkhwa (KPK) province. A higher resolution of the phylogenetic analysis of lineage 5 showed the distribution of Pakistani NDV strains to 5b. However, the isolates from KPK belonged to lineage 4c; the first report of such lineage from this province. CONCLUSIONS: Taken together, data indicated the prevalence of multiple lineages of NDV in different poultry population including wild captive birds. Such understanding is crucial to underpin the nature of circulating strains of NDV, their potential for interspecies transmission and disease diagnosis and control strategies.

Molecular and genetic characterization of bovine parainfluenza type 3 European field and vaccine strains.

Bovine Parainfluenza Type 3 virus (BPIV-3) is an enveloped, non-segmented single-stranded, negative-sense RNA virus belonging to the Paramyxoviridae family (genus Respirovirus) with a well-known role in Bovine Respiratory Disease (BRD) onset. Being isolated for the first time in 1959, BPIV-3 currently circulates worldwide in cattle herds and is routinely tested in suspected BRD cases. Different commercial vaccines are available to prevent infection and/or to reduce the clinical signs associated with BPIV-3 infection, which are essential to prevent secondary infections. Despite years of molecular surveillance, a very limited number of complete genome sequences were made publicly available, preventing thus the understanding of the genetic diversity of the circulating strains in the field. In addition, no data about the genetic identity between field and vaccine strains is currently available. In this study, we sequenced the full-genome and genetically characterized BPIV-3 strains isolated from animals displaying respiratory illness in France and Sweden, as well as the vaccine strains contained in three different commercialized vaccines. Our results show that the sequences from France and Sweden belong to genotype C. However, a third sequence from Sweden from 2017 clustered within genotype A. The sequencing of vaccine strains revealed that two of the vaccine strains clustered within genotype C, whereas the third vaccine strain belonged to genotype A. Altogether, our findings suggest that both genotypes A and C circulate in Europe and that BPIV-3 field and vaccine strains are genetically divergent. Our sequencing results could be useful to better understand the genetic differences between the circulating field and vaccine BPIV-3 strains. This is crucial for a correct interpretation of diagnostic findings and for the assessment of BPIV-3 prevalence in cattle population.

Detection of Influenza D-Specific Antibodies in Bulk Tank Milk from Swedish Dairy Farms.

Influenza D virus (IDV) has been detected in bovine respiratory disease (BRD) outbreaks, and experimental studies demonstrated this virus's capacity to cause lesions in the respiratory tract. In addition, IDV-specific antibodies were detected in human sera, which indicated that this virus plays a potential zoonotic role. The present study aimed to extend our knowledge about the epidemiologic situation of IDV in Swedish dairy farms, using bulk tank milk (BTM) samples for the detection of IDV antibodies. A total of 461 and 338 BTM samples collected during 2019 and 2020, respectively, were analyzed with an in-house indirect ELISA. In total, 147 (32%) and 135 (40%) samples were IDV-antibody-positive in 2019 and 2020, respectively. Overall, 2/125 (2%), 11/157 (7%) and 269/517 (52%) of the samples were IDV-antibody-positive in the northern, middle and southern regions of Sweden. The highest proportion of positive samples was repeatedly detected in the south, in the county of Halland, which is one of the counties with the highest cattle density in the country. In order to understand the epidemiology of IDV, further research in different cattle populations and in humans is required.

A novel array of real-time RT-PCR assays for the rapid pathotyping of type I avian paramyxovirus (APMV-1).

Newcastle disease (ND) caused by virulent avian paramyxovirus type I (APMV-1) is a WOAH and EU listed disease affecting poultry worldwide. ND exhibits different clinical manifestations that may either be neurological, respiratory and/or gastrointestinal, accompanied by high mortality. In contrast, mild or subclinical forms are generally caused by lentogenic APMV-1 and are not subject to notification. The rapid discrimination of virulent and avirulent viruses is paramount to limit the spread of virulent APMV-1. The appropriateness of molecular methods for APMV-1 pathotyping is often hampered by the high genetic variability of these viruses that affects sensitivity and inclusivity. This work presents a new array of real-time RT-PCR (RT-qPCR) assays that enable the identification of virulent and avirulent viruses in dual mode, i.e., through pathotype-specific probes and subsequent Sanger sequencing of the amplification product. Validation was performed according to the WOAH recommendations. Performance indicators on sensitivity, specificity, repeatability and reproducibility yielded favourable results. Reproducibility highlighted the need for assays optimization whenever major changes are made to the procedure. Overall, the new RT-qPCRs showed its ability to detect and pathotype all tested APMV-1 genotypes and its suitability for routine use in clinical samples.

Genomic and biological characterization of a velogenic Newcastle disease virus isolated from a healthy backyard poultry flock in 2010.

BACKGROUND: Newcastle disease virus (NDV) causes severe and economically important disease in poultry around the globe. None of NDV strains in Pakistan have been completely characterized and the role of rural poultry in harbouring NDV is unclear. Since they have a very important role for long-term circulation of the virus, samples were collected from apparently healthy backyard poultry (BYP) flocks. These samples were biologically analyzed using mean death time (MDT) and intracerebral pathogenicity index (ICPI), whereas genotypically characterized by the real-time PCRs coupled with sequencing of the complete genome. FINDINGS: Despite of being non-pathogenic for BYP, the isolate exhibited MDT of 49.6 h in embryonated chicken eggs and an ICPI value of 1.5. The F gene based real-time PCR was positive, whereas M-gene based was negative due to substantial changes in the probe-binding site. The entire genome of the isolate was found to be 15192 nucleotides long and encodes for six genes with an order of 3'-NP-P-M-F-HN-L-5'. The F protein cleavage site, an indicative of pathogenicity, was 112RRQKRF117. Complete genome comparison indicated that the RNA dependent RNA polymerase gene was the most and the phosphoprotein was least conserved gene, among all the genes. The isolate showed an Y526Q substitution in the HN protein, which determines neuraminidase receptor binding and fusion activity of NDV. Phylogenetic analysis, based on F and HN genes, classified this isolate into genotype VII, a predominant genotype responsible for ND outbreaks in Asian countries. However, it clustered well apart from other isolates in this genotype to be considered a new subgenotype (VII-f). CONCLUSIONS: These results revealed that this isolate was similar to virulent strains of NDV and was avirulent in BYP either due to resistance of local breeds or due to other factors such as substantial mutations in the HN protein. Furthermore, we have characterized the first isolate of NDV, which could act as domestic reference strain and could help in development and selection of appropriate strain of NDV for vaccine in the country.

Sequencing and analysis of the complete genome of Newcastle disease virus isolated from a commercial poultry farm in 2010.

Newcastle disease virus (NDV) infects wild and domestic birds but causes contagious and lethal disease in domestic poultry. ND is currently endemic in Pakistan, but no complete genome sequence of a Pakistani NDV isolate has been reported. An NDV strain isolated from a commercial poultry farm was completely sequenced. Phylogenetic analysis revealed that the isolate is closely related to genotype VII and, more specifically, to subgenotype VIIb, yet with substantial enough differences to be regarded as new subgenotype (VIIf). These findings provide insight into the genetic nature of NDV circulating in Pakistan and are useful for both laboratory diagnosis and vaccine development for NDV.

Highly Pathogenic Avian Influenza (HPAI H5Nx, Clade 2.3.4.4.b) in Poultry and Wild Birds in Sweden: Synopsis of the 2020-2021 Season.

Highly pathogenic avian influenza (HPAI, Gs/Gd lineage) was introduced to Europe in 2005 and has since caused numerous outbreaks in birds. The 2020-2021 season was the hitherto most devastating when considering bird numbers and duration in Europe. Surveillance data, virologic results and epidemiologic investigations from the 2020-2021 outbreaks in Sweden were analysed. Subtypes H5N8 and H5N5 were detected on 24 farms with poultry or other captive birds. In wild birds, subtypes H5N8, H5N5, H5N1, H5N4, H5Nx were detected in 130 out of 811 sampled birds. There was a spatiotemporal association between cases in wild birds and poultry. Based on phylogeny and epidemiology, most of the introductions of HPAI to commercial poultry were likely a result of indirect contact with wild birds. A definite route of introduction to poultry could not be established although some biosecurity breaches were observed. No spread between farms was identified but airborne spread between flocks on the same farm was suspected. Our findings exemplify the challenges posed by the continuously changing influenza viruses that seem to adapt to a broader species spectrum. This points to the importance of wild bird surveillance, compliance to biosecurity, and identification of risk factors for introduction on poultry farms.

Evolutionary and temporal dynamics of emerging influenza D virus in Europe (2009-22).

Influenza D virus (IDV) is an emerging influenza virus that was isolated for the first time in 2011 in the USA from swine with respiratory illness. Since then, IDV has been detected worldwide in different animal species, and it was also reported in humans. Molecular epidemiological studies revealed the circulation of two major clades, named D/OK and D/660. Additional divergent clades have been described but have been limited to specific geographic areas (i.e. Japan and California). In Europe, IDV was detected for the first time in France in 2012 and subsequently also in Italy, Luxembourg, Ireland, the UK, Switzerland, and Denmark. To understand the time of introduction and the evolutionary dynamics of IDV on the continent, molecular screening of bovine and swine clinical samples was carried out in different European countries, and phylogenetic analyses were performed on all available and newly generated sequences. Until recently, D/OK was the only clade detected in this area. Starting from 2019, an increase in D/660 clade detections was observed, accompanied by an increase in the overall viral genetic diversity and genetic reassortments. The time to the most recent common ancestor (tMRCA) of all existing IDV sequences was estimated as 1995-16 years before its discovery, indicating that the virus could have started its global spread in this time frame. Despite the D/OK and D/660 clades having a similar mean tMRCA (2007), the mean tMRCA for European D/OK sequences was estimated as January 2013 compared to July 2014 for European D/660 sequences. This indicated that the two clades were likely introduced on the European continent at different time points, as confirmed by virological screening findings. The mean nucleotide substitution rate of the hemagglutinin-esterase-fusion (HEF) glycoprotein segment was estimated as 1.403 × 10-3 substitutions/site/year, which is significantly higher than the one of the HEF of human influenza C virus (P < 0.0001). IDV genetic drift, the introduction of new clades on the continent, and multiple reassortment patterns shape the increasing viral diversity observed in the last years. Its elevated substitution rate, diffusion in various animal species, and the growing evidence pointing towards zoonotic potential justify continuous surveillance of this emerging influenza virus.