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

Simple Summary Highly pathogenic avian influenza is a virus-induced contagious disease that has killed a large number of poultry and wild birds in Europe in the recent decade and is an increasing problem worldwide. In the winter of 2020-2021, Sweden experienced its worst period to date when the disease was diagnosed on 15 commercial poultry farms and over 2.2 million birds died or were euthanised. The disease was also diagnosed in 130 wild birds and nine flocks of hobby, game or zoo birds between 1 October 2020 and 30 September 2021. The aim of this article was to describe the influenza situation in Sweden during this period and to add to the knowledge related to the alarming situation with highly pathogenic influenza in birds. The disease caused animal suffering and death in wild and domestic birds and incurred high costs due to losses and extensive measures to stop spread. The outbreak investigations, where contacts were traced and virus strains were compared, concluded that the virus was brought to poultry farms by wild birds in most cases. More research is needed to obtain knowledge on risk factors, biosecurity, and wild bird presence on poultry farms to prevent future disease outbreaks. 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

The first Swedish H1N2 swine influenza virus isolate represents an uncommon reassortant

The European swine influenza viruses (SIVs) show considerable diversity comprising different types of H1N1, H3N2, and H1N2 strains. The intensifying full genome sequencing efforts reveal further reassortants within these subtypes. Here we report the identification of an uncommon reassortant variant of H1N2 subtype influenza virus isolated from a pig in a multisite herd where H1N2 swine influenza was diagnosed for the first time in Sweden during the winter of 2008-2009. The majority of the European H1N2 swine influenza viruses described so far possess haemagglutinin (HA) of the human-like H1N2 SIV viruses and the neuraminidase (NA) of either the European H1N2 or H3N2 SIV-like viruses. The Swedish isolate has an avian-like SIV HA and a H3N2 SIV-like NA, which is phylogenetically more closely related to H3N2 SIV NAs from isolates collected in the early '80s than to the NA of H3N2 origin of the H1N2 viruses isolated during the last decade, as depicted by some German strains, indicative of independent acquisition of the NA genes for these two types of reassortants. The internal genes proved to be entirely of avian-like SIV H1N1 origin. The prevalence of this SIV variant in pig populations needs to be determined, as well as the suitability of the routinely used laboratory reagents to analyze this strain

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

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

Strong protection induced by an experimental DIVA subunit vaccine against bluetongue virus serotype 8 in cattle

AbstractBluetongue virus (BTV) infections in ruminants pose a permanent agricultural threat since new serotypes are constantly emerging in new locations. Clinical disease is mainly observed in sheep, but cattle were unusually affected during an outbreak of BTV seroype 8 (BTV-8) in Europe. We previously developed an experimental vaccine based on recombinant viral protein 2 (VP2) of BTV-8 and non-structural proteins 1 (NS1) and NS2 of BTV-2, mixed with an immunostimulating complex (ISCOM)–matrix adjuvant. We demonstrated that bovine immune responses induced by this vaccine were as good or superior to those induced by a classic commercial inactivated vaccine. In this study, we evaluated the protective efficacy of the experimental vaccine in cattle and, based on the detection of VP7 antibodies, assessed its DIVA compliancy following virus challenge. Two groups of BTV-seronegative calves were subcutaneously immunized twice at a 3-week interval with the subunit vaccine (n=6) or with adjuvant alone (n=6). Following BTV-8 challenge 3 weeks after second immunization, controls developed viremia and fever associated with other mild clinical signs of bluetongue disease, whereas vaccinated animals were clinically and virologically protected. The vaccine-induced protection was likely mediated by high virus-neutralizing antibody titers directed against VP2 and perhaps by cellular responses to NS1 and NS2. T lymphocyte responses were cross-reactive between BTV-2 and BTV-8, suggesting that NS1 and NS2 may provide the basis of an adaptable vaccine that can be varied by using VP2 of different serotypes. The detection of different levels of VP7 antibodies in vaccinated animals and controls after challenge suggested a compliancy between the vaccine and the DIVA companion test. This BTV subunit vaccine is a promising candidate that should be further evaluated and developed to protect against different serotypes

Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006

<p>Abstract</p> <p>Background</p> <p>The analysis of the nonstructural (NS) gene of the highly pathogenic (HP) H5N1 avian influenza viruses (AIV) isolated in Sweden early 2006 indicated the co-circulation of two sub-lineages of these viruses at that time. In order to complete the information on their genetic features and relation to other HP H5N1 AIVs the seven additional genes of twelve Swedish isolates were amplified in full length, sequenced, and characterized.</p> <p>Results</p> <p>The presence of two sub-lineages of HP H5N1 AIVs in Sweden in 2006 was further confirmed by the phylogenetic analysis of approximately the 95% of the genome of twelve isolates that were selected on the base of differences in geographic location, timing and animal species of origin. Ten of the analyzed viruses belonged to sub-clade 2.2.2. and grouped together with German and Danish isolates, while two 2.2.1. sub-clade viruses formed a cluster with isolates of Egyptian, Italian, Slovenian, and Nigerian origin. The revealed amino acid differences between the two sub-groups of Swedish viruses affected the predicted antigenicity of the surface glycoproteins, haemagglutinin and neuraminidase, rather than the nucleoprotein, polymerase basic protein 2, and polymerase acidic protein, the main targets of the cellular immune responses. The distinctive characteristics between members of the two subgroups were identified and described.</p> <p>Conclusion</p> <p>The comprehensive genetic characterization of HP H5N1 AIVs isolated in Sweden during the spring of 2006 is reported. Our data support previous findings on the coincidental spread of multiple sub-lineage H5N1 HPAIVs via migrating aquatic birds to large distance from their origin. The detection of 2.2.1. sub-clade viruses in Sweden adds further data regarding their spread in the North of Europe in 2006. The close genetic relationship of Swedish isolates sub-clade 2.2.2. to the contemporary German and Danish isolates supports the proposition of the introduction and spread of a single variant of 2.2.2. sub-clade H5N1 avian influenza viruses in the Baltic region. The presented findings underline the importance of whole genome analysis.</p

Non-structural protein 1 of avian influenza A viruses differentially inhibit NF-κB promoter activation

<p>Abstract</p> <p>Background</p> <p>Influenza virus infection activates NF-κB and is a general prerequisite for a productive influenza virus infection. On the other hand, non-structural protein 1 (NS1) suppresses this viral activated NF-κB, presumably to prevent expression of NF-κB mediated anti-viral response. NS1 proteins of influenza A viruses are divided into two groups, known as allele A and allele B. The possible functional relevance of this NS1 division to viral pathogenicity is lacking.</p> <p>Findings</p> <p>The ability of NS1 protein from two avian influenza subtypes, H6N8 and H4N6, to inhibit NF-κB promoter activation was assessed. Further, efforts were made to characterize the genetic basis of this inhibition. We found that allele A NS1 proteins of H6N8 and H4N6 are significantly better in preventing dsRNA induced NF-κB promoter activation compared to allele B of corresponding subtypes, in a species independent manner. Furthermore, the ability to suppress NF-κB promoter activation was mapped to the effector domain while the RNA binding domain alone was unable to suppress this activation. Chimeric NS1 proteins containing either RNA binding domain of allele A and effector domain of allele B or vice versa, were equally potent in preventing NF-κB promoter activation compared to their wt. NS1 protein of allele A and B from both subtypes expressed efficiently as detected by Western blotting and predominantly localized in the nucleus in both A549 and MiLu cells as shown by <it>in situ </it>PLA.</p> <p>Conclusions</p> <p>Here, we present another aspect of NS1 protein in inhibiting dsRNA induced NF-κB activation in an allele dependent manner. This suggests a possible correlation with the virus's pathogenic potential.</p

Enhanced Pathogenesis Caused by Influenza D Virus and Mycoplasma bovis Coinfection in Calves: a Disease Severity Linked with Overexpression of IFN-gamma as a Key Player of the Enhanced Innate Immune Response in Lungs

Bovine respiratory disease (BRD) is a major disease of young cattle whose etiology lies in complex interactions between pathogens and environmental and host factors. Despite a high frequency of codetection of respiratory pathogens in BRD, data on the molecular mechanisms and pathogenesis associated with viral and bacterial interactions are still limited. In this study, we investigated the effects of a coinfection with influenza D virus (IDV) and Mycoplasma bovis in cattle. Naive calves were infected by aerosol with a French IDV strain and an M. bovis strain. The combined infection shortened the incubation period, worsened the disease, and led to more severe macroscopic and microscopic lesions compared to these parameters in calves infected with only one pathogen. In addition, IDV promoted colonization of the lower respiratory tract (LRT) by M. bovis and increased white cell recruitment to the airway lumen. The transcriptomic analysis highlighted an upregulation of immune genes in the lungs of coinfected calves. The gamma interferon (IFN-gamma) gene was shown to be the gene most statistically overexpressed after coinfection at 2 days postinfection (dpi) and at least until 7 dpi, which correlated with the high level of lymphocytes in the LRT. Downregulation of the PACE4 and TMPRSS2 endoprotease genes was also highlighted, being a possible reason for the faster clearance of IDV in the lungs of coinfected animals. Taken together, our coinfection model with two respiratory pathogens that when present alone induce moderate clinical signs of disease was shown to increase the severity of the disease in young cattle and a strong transcriptomic innate immune response in the LRT, especially for IFN-gamma.IMPORTANCE Bovine respiratory disease (BRD) is among the most prevalent diseases in young cattle. BRD is due to complex interactions between viruses and/or bacteria, most of which have a moderate individual pathogenicity. In this study, we showed that coinfection with influenza D virus (IDV) and Mycoplasma bovis increased the severity of the respiratory disease in calves in comparison with IDV or M. bovis infection. IDV promoted M. bovis colonization of the lower respiratory tract and increased white cell recruitment to the airway lumen. The transcriptomic analysis highlighted an upregulation of immune genes in the lungs of coinfected calves. The IFN-gamma gene in particular was highly overexpressed after coinfection, correlated with the disease severity, immune response, and white cell recruitment in the lungs. In conclusion, we showed that IDV facilitates coinfections within the BRD complex by modulating the local innate immune response, providing new insights into the mechanisms involved in severe respiratory diseases

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