Immunogenicity and protective efficacy of a multivalent herpesvirus vectored vaccine against H9N2 low pathogenic avian influenza in chicken.

The application of recombinant herpesvirus of turkey, expressing the H9 hemagglutinin gene from low pathogenic avian influenza virus (LPAIV) H9N2 and the avian orthoavulavirus-1 (AOAV-1) (commonly known as Newcastle Disease virus (NDV)) fusion protein (F) as an rHVT-H9-F vaccine, is an alternative to currently used classical vaccines. This study investigated H9- and ND-specific humoral and mucosal responses, H9-specific cell-mediated immunity, and protection conferred by the rHVT-H9-F vaccine in specific pathogen-free (SPF) chickens. Vaccination elicited systemic NDV F- and AIV H9-specific antibody response but also local antibodies in eye wash fluid and oropharyngeal swabs. The ex vivo H9-specific stimulation of splenic and pulmonary T cells in the vaccinated group demonstrated the ability of vaccination to induce systemic and local cellular responses. The clinical protection against a challenge using a LPAIV H9N2 strain of the G1 lineage isolated in Morocco in 2016 was associated with a shorter duration of shedding along with reduced viral genome load in the upper respiratory tract and reduced cloacal shedding compared to unvaccinated controls.

Protection conferred by an H5 DNA vaccine against highly pathogenic avian influenza in chickens: The effect of vaccination schedules.

H5 highly pathogenic avian influenza (HPAI) viruses of the Asian lineage (A/goose/Guangdong/1/96) belonging to clade 2.3.4.4 have spread worldwide through wild bird migration in two major waves: in 2014/2015 (clade 2.3.4.4c), and since 2016 up to now (clade 2.3.4.4b). Due to the increasing risk of these H5 HPAI viruses to establish and persist in the wild bird population, implementing vaccination in certain sensitive areas could be a complementary measure to the disease control strategies already applied. In this study, the efficacy of a novel DNA vaccine, encoding a H5 gene (A/gyrfalcon/Washington/41088-6/2014 strain) of clade 2.3.4.4c was evaluated in specific pathogen-free (SPF) white leghorn chickens against a homologous and heterologous H5 HPAI viruses. A single vaccination at 2 weeks of age (1 dose), and a vaccination at 2 weeks of age, boosted at 4 weeks (2 doses), with or without adjuvant were characterized. The groups that received 1 dose with or without adjuvant as well as 2 doses with adjuvant demonstrated full clinical protection and a significant or complete reduction of viral shedding against homologous challenge at 6 and 25 weeks of age. The heterologous clade 2.3.4.4b challenge of 6-week-old chickens vaccinated with 2 doses with or without adjuvant showed similar results, indicating good cross-protection induced by the DNA vaccine. Long lasting humoral immunity was observed in vaccinated chickens up to 18 or 25 weeks of age, depending on the vaccination schedule. The analysis of viral transmission after homologous challenge showed that sentinels vaccinated with 2 doses with adjuvant were fully protected against mortality with no excretion detected. This study of H5 DNA vaccine efficacy confirmed the important role that this type of so-called third-generation vaccine could play in the fight against H5 HPAI viruses.

RNA sequencing of avian paramyxovirus (Paramyxoviridae, Avulavirinae) isolates from wild mallards in Belgium, 2021: complete genomes and coinfections.

We used untargeted RNA sequencing to characterize three Avulavirinae isolates from pooled samples obtained from wild mallards in Belgium in 2021. The complete genome sequences of two avian Orthoavulavirus-1 (AOAV-1) strains and one avian Paraavulavirus-4 (APMV-4) strain were determined confirming hemagglutination inhibition testing of the virus isolates. In addition, the applied sequencing strategy identified an avian influenza virus (AIV) coinfection in all three virus isolates, confirming weak-positive AIV realtime RT-PCR results from the original sample material. In one AOAV-1 isolate, partial sequences covering all genome segments of an AIV of subtype H11N9 could be de novo assembled from the sequencing data. Besides an AIV coinfection, RNA metagenomic data from the APMV-4 isolate also showed evidence of Alpharetrovirus and Megrivirus coinfection. In total, two AOAV-1 of Class II, genotype I.2 and one APMV-4 complete genome sequences were assembled and compared to publicly available sequences, highlighting the importance of surveillance for poultry pathogens in wild birds. Beyond the insights from full genome characterization of virus isolates, untargeted RNA sequencing strategies provide additional insights in the RNA virome of clinical samples as well as their derived virus isolates that are particularly useful when targeting wild avifauna reservoirs of poultry pathogens.

Evaluation of NS4A, NS4B, NS5 and 3'UTR Genetic Determinants of WNV Lineage 1 Virulence in Birds and Mammals.

West Nile virus (WNV) is amplified in an enzootic cycle involving birds as amplifying hosts. Because they do not develop high levels of viremia, humans and horses are considered to be dead-end hosts. Mosquitoes, especially from the Culex genus, are vectors responsible for transmission between hosts. Consequently, understanding WNV epidemiology and infection requires comparative and integrated analyses in bird, mammalian, and insect hosts. So far, markers of WNV virulence have mainly been determined in mammalian model organisms (essentially mice), while data in avian models are still missing. WNV Israel 1998 (IS98) is a highly virulent strain that is closely genetically related to the strain introduced into North America in 1999, NY99 (genomic sequence homology > 99%). The latter probably entered the continent at New York City, generating the most impactful WNV outbreak ever documented in wild birds, horses, and humans. In contrast, the WNV Italy 2008 strain (IT08) induced only limited mortality in birds and mammals in Europe during the summer of 2008. To test whether genetic polymorphism between IS98 and IT08 could account for differences in disease spread and burden, we generated chimeric viruses between IS98 and IT08, focusing on the 3' end of the genome (NS4A, NS4B, NS5, and 3'UTR regions) where most of the non-synonymous mutations were detected. In vitro and in vivo comparative analyses of parental and chimeric viruses demonstrated a role for NS4A/NS4B/5'NS5 in the decreased virulence of IT08 in SPF chickens, possibly due to the NS4B-E249D mutation. Additionally, significant differences between the highly virulent strain IS98 and the other three viruses were observed in mice, implying the existence of additional molecular determinants of virulence in mammals, such as the amino acid changes NS5-V258A, NS5-N280K, NS5-A372V, and NS5-R422K. As previously shown, our work also suggests that genetic determinants of WNV virulence can be host-dependent.

Combined Phylogeographic Analyses and Epidemiologic Contact Tracing to Characterize Atypically Pathogenic Avian Influenza (H3N1) Epidemic, Belgium, 2019.

The high economic impact and zoonotic potential of avian influenza call for detailed investigations of dispersal dynamics of epidemics. We integrated phylogeographic and epidemiologic analyses to investigate the dynamics of a low pathogenicity avian influenza (H3N1) epidemic that occurred in Belgium during 2019. Virus genomes from 104 clinical samples originating from 85% of affected farms were sequenced. A spatially explicit phylogeographic analysis confirmed a dominating northeast to southwest dispersal direction and a long-distance dispersal event linked to direct live animal transportation between farms. Spatiotemporal clustering, transport, and social contacts strongly correlated with the phylogeographic pattern of the epidemic. We detected only a limited association between wind direction and direction of viral lineage dispersal. Our results highlight the multifactorial nature of avian influenza epidemics and illustrate the use of genomic analyses of virus dispersal to complement epidemiologic and environmental data, improve knowledge of avian influenza epidemiologic dynamics, and enhance control strategies.

Concomitant NA and NS deletion on avian Influenza H3N1 virus associated with hen mortality in France in 2019.

An H3N1 avian influenza virus was detected in a laying hens farm in May 2019 which had experienced 25% mortality in Northern France. The complete sequencing of this virus showed that all segment sequences belonged to the Eurasian lineage and were phylogenetically very close to many of the Belgian H3N1 viruses detected in 2019. The French virus presented two genetic particularities with NA and NS deletions that could be related to virus adaptation from wild to domestic birds and could increase virulence, respectively. Molecular data of H3N1 viruses suggest that these two deletions occurred at two different times.

Metagenomic sequencing determines complete infectious bronchitis virus (avian Gammacoronavirus) vaccine strain genomes and associated viromes in chicken clinical samples.

Infectious bronchitis virus (IBV, genus Gammacoronavirus) causes an economically important and highly contagious disease in chicken. Random primed RNA sequencing was applied to two IBV positive clinical samples and one in ovo-passaged virus. The virome of a cloacal swab pool was dominated by IBV (82% of viral reads) allowing de novo assembly of a GI-13 lineage complete genome with 99.95% nucleotide identity to vaccine strain 793B. In addition, substantial read counts (16% of viral reads) allowed the assembly of a near-complete chicken astrovirus genome, while lower read counts identified the presence of chicken calicivirus and avian leucosis virus. Viral reads in a respiratory/intestinal tissue pool were distributed between IBV (22.53%), Sicinivirus (Picornaviridae, 24%), and avian leucosis virus (37.04%). A complete IBV genome with 99.95% nucleotide identity to vaccine strain H120 (lineage GI-1), as well as a near-complete avian leucosis virus genome and a partial Sicinivirus genome were assembled from the tissue sample data. Lower read counts identified chicken calicivirus, Avibirnavirus (infectious bursal disease virus, assembling to 98.85% of segment A and 69.66% of segment B closely related to D3976/1 from Germany, 2017) and avian orthoreovirus, while three avian orthoavulavirus 1 reads confirmed prior real-time RT-PCR result. IBV sequence variation analysis identified both fixed and minor frequency variations in the tissue sample compared to its in ovo-passaged virus. Metagenomic methods allow the determination of complete coronavirus genomes from clinical chicken samples while providing additional insights in RNA virus sequence diversity and coinfecting viruses potentially contributing to pathogenicity.

The Expression of Hemagglutinin by a Recombinant Newcastle Disease Virus Causes Structural Changes and Alters Innate Immune Sensing.

Recombinant Newcastle disease viruses (rNDV) have been used as bivalent vectors for vaccination against multiple economically important avian pathogens. NDV-vectored vaccines expressing the immunogenic H5 hemagglutinin (rNDV-H5) are considered attractive candidates to protect poultry from both highly pathogenic avian influenza (HPAI) and Newcastle disease (ND). However, the impact of the insertion of a recombinant protein, such as H5, on the biological characteristics of the parental NDV strain has been little investigated to date. The present study compared a rNDV-H5 vaccine and its parental NDV LaSota strain in terms of their structural and functional characteristics, as well as their recognition by the innate immune sensors. Structural analysis of the rNDV-H5 demonstrated a decreased number of fusion (F) and a higher number of hemagglutinin-neuraminidase (HN) glycoproteins compared to NDV LaSota. These structural differences were accompanied by increased hemagglutinating and neuraminidase activities of rNDV-H5. During in vitro rNDV-H5 infection, increased mRNA expression of TLR3, TLR7, MDA5, and LGP2 was observed, suggesting that the recombinant virus is recognized differently by sensors of innate immunity when compared with the parental NDV LaSota. Given the growing interest in using NDV as a vector against human and animal diseases, these data highlight the importance of thoroughly understanding the recombinant vaccines' structural organization, functional characteristics, and elicited immune responses.

Molecular and virological characterization of the first poultry outbreaks of Genotype VII.2 velogenic avian orthoavulavirus type 1 (NDV) in North-West Europe, BeNeLux, 2018.

After two decades free of Newcastle disease, Belgium encountered a velogenic avian orthoavulavirus type 1 epizootic in 2018. In Belgium, 20 cases were diagnosed, of which 15 occurred in hobby flocks, 2 in professional poultry flocks and 3 in poultry retailers. The disease also disseminated from Belgium towards the Grand Duchy of Luxembourg by trade. Independently, the virus was detected once in the Netherlands, almost simultaneously to the first Belgian detection. As such Newcastle disease emerged in the entire BeNeLux region. Both the polybasic sequence of the fusion gene cleavage site and the intracerebral pathotyping assay demonstrated the high pathogenicity of the strain. This paper represents the first notification of this specific VII.2 subgenotype in the North-West of Europe. Time-calibrated full genome phylogenetic analysis indicated the silent or unreported circulation of the virus prior to the emergence of three genetic clusters in the BeNeLux region without clear geographical or other epidemiological correlation. The Dutch strain appeared as an outgroup to the Belgian and Luxembourgian strains in the time-correlated genetic analysis and no epidemiological link could be identified between the Belgian and Dutch outbreaks. In contrast, both genetic and epidemiological outbreak investigation data linked the G.D. Luxembourg case to the Belgian outbreak. The genetic links between Belgian viruses from retailers and hobby flocks only partially correlated with epidemiological data. Two independent introductions into the professional poultry sector were identified, although their origin could not be determined. Animal experiments using 6-week- old specific pathogen-free chickens indicated a systemic infection and efficient transmission of the virus. The implementation of re-vaccination in the professional sector, affected hobby and retailers, as well as the restriction on assembly and increased biosecurity measures, possibly limited the epizootic and resulted in the disappearance of the virus. These findings emphasize the constant need for awareness and monitoring of notifiable viruses in the field.

Effectiveness of a Simultaneous rHVT-F(ND) and rHVT-H5(AI) Vaccination of Day-Old Chickens and the Influence of NDV- and AIV-Specific MDA on Immune Response and Conferred Protection.

The recombinant herpesvirus of turkey (rHVT) vaccines targeting Newcastle disease (ND) and H5Nx avian influenza (AI) have been demonstrated efficient in chickens when used individually at day-old. Given the practical field constraints associated with administering two vaccines separately and in the absence of a currently available bivalent rHVT vector vaccine expressing both F(ND) and H5(AI) antigens, the aim of this study was to investigate whether interference occurs between the two vaccines when simultaneously administered in a single shot. The studies have been designed to determine (i) the ND and AI-specific protection and antibody response conferred by these vaccines inoculated alone or in combination at day-old, (ii) the influence of maternally-derived antibodies (MDA), and (iii) the potential interference between the two vaccine. Our results demonstrate that their combined administration is efficient to protect chickens against clinical signs of velogenic Newcastle disease virus (vNDV) and H5-highly pathogenic avian influenza virus (HPAIV) infections. Viral shedding following co-vaccination is also markedly reduced, while slightly lower NDV- and AIV-specific antibody responses are observed. NDV- and AIV-specific MDA show negative effects on the onset of the specific antibody responses. However, if AIV-specific MDA reduce the protection against H5-HPAIV induced by rHVT-H5(AI) vaccine, it was not observed for ND.

Atypical Pathogenicity of Avian Influenza (H3N1) Virus Involved in Outbreak, Belgium, 2019.

In 2019, an outbreak of avian influenza (H3N1) virus infection occurred among commercial poultry in Belgium. Full-genome phylogenetic analysis indicated a wild bird origin rather than recent circulation among poultry. Although classified as a nonnotifiable avian influenza virus, it was associated with reproductive tropism and substantial mortality in the field.

Cytokine production and phenotype of Histomonas meleagridis-specific T cells in the chicken.

The protozoan parasite Histomonas meleagridis is the causative agent of the re-emerging disease histomonosis of chickens and turkeys. Due to the parasite's extracellular occurrence, a type-2 differentiation of H. meleagridis-specific T cells has been hypothesized. In contrast, a recent study suggested that IFN-γ mRNA+ cells are involved in protection against histomonosis. However, the phenotype and cytokine production profile of H. meleagridis-specific T cells still awaits elucidation. In this work, clonal cultures of a virulent monoxenic strain of H. meleagridis were used for infecting chickens to detect IFN-γ protein and IL-13 mRNA by intracellular cytokine staining and PrimeFlow™ RNA Assays, respectively, in CD4+ and CD8ß+ T cells. Infection was confirmed by characteristic pathological changes in the cecum corresponding with H. meleagridis detection by immunohistochemistry and H. meleagridis-specific antibodies in serum. In splenocytes stimulated either with H. meleagridis antigen or PMA/ionomycin, IFN-γ-producing CD4+ T cells from infected chickens increased in comparison to cells from non-infected birds 2 weeks and 5 weeks post-infection. Additionally, an increase of IFN-γ-producing CD4-CD8ß- cells upon H. meleagridis antigen and PMA/ionomycin stimulation was detected. Contrariwise, frequencies of IL-13 mRNA-expressing cells were low even after PMA/ionomycin stimulation and mainly had a CD4-CD8ß- phenotype. No clear increase of IL-13+ cells related to H. meleagridis infection could be found. In summary, these data suggest that H. meleagridis infection induces a type-1 differentiation of CD4+ T cells but also of non-CD4+ cells. This phenotype could include γδ T cells, which will be addressed in future studies.

Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus.

Several Avian paramyxoviruses 1 (synonymous with Newcastle disease virus or NDV, used hereafter) classification systems have been proposed for strain identification and differentiation. These systems pioneered classification efforts; however, they were based on different approaches and lacked objective criteria for the differentiation of isolates. These differences have created discrepancies among systems, rendering discussions and comparisons across studies difficult. Although a system that used objective classification criteria was proposed by Diel and co-workers in 2012, the ample worldwide circulation and constant evolution of NDV, and utilization of only some of the criteria, led to identical naming and/or incorrect assigning of new sub/genotypes. To address these issues, an international consortium of experts was convened to undertake in-depth analyses of NDV genetic diversity. This consortium generated curated, up-to-date, complete fusion gene class I and class II datasets of all known NDV for public use, performed comprehensive phylogenetic neighbor-Joining, maximum-likelihood, Bayesian and nucleotide distance analyses, and compared these inference methods. An updated NDV classification and nomenclature system that incorporates phylogenetic topology, genetic distances, branch support, and epidemiological independence was developed. This new consensus system maintains two NDV classes and existing genotypes, identifies three new class II genotypes, and reduces the number of sub-genotypes. In order to track the ancestry of viruses, a dichotomous naming system for designating sub-genotypes was introduced. In addition, a pilot dataset and sub-trees rooting guidelines for rapid preliminary genotype identification of new isolates are provided. Guidelines for sequence dataset curation and phylogenetic inference, and a detailed comparison between the updated and previous systems are included. To increase the speed of phylogenetic inference and ensure consistency between laboratories, detailed guidelines for the use of a supercomputer are also provided. The proposed unified classification system will facilitate future studies of NDV evolution and epidemiology, and comparison of results obtained across the world.

Co-circulation of genetically distinct highly pathogenic avian influenza A clade 2.3.4.4 (H5N6) viruses in wild waterfowl and poultry in Europe and East Asia, 2017-18.

Highly pathogenic avian influenza (HPAI) H5 clade 2.3.4.4 viruses were first introduced into Europe in late 2014 and re-introduced in late 2016, following detections in Asia and Russia. In contrast to the 2014-15 H5N8 wave, there was substantial local virus amplification in wild birds in Europe in 2016-17 and associated wild bird mortality, with evidence for occasional gene exchange with low pathogenic avian influenza (LPAI) viruses. Since December 2017, several European countries have again reported events or outbreaks with HPAI H5N6 reassortant viruses in both wild birds and poultry, respectively. Previous phylogenetic studies have shown that the two earliest incursions of HPAI H5N8 viruses originated in Southeast Asia and subsequently spread to Europe. In contrast, this study indicates that recent HPAI H5N6 viruses evolved from the H5N8 2016-17 viruses during 2017 by reassortment of a European HPAI H5N8 virus and wild host reservoir LPAI viruses. The genetic and phenotypic differences between these outbreaks and the continuing detections of HPAI viruses in Europe are a cause of concern for both animal and human health. The current co-circulation of potentially zoonotic HPAI and LPAI virus strains in Asia warrants the determination of drivers responsible for the global spread of Asian lineage viruses and the potential threat they pose to public health.

Study of the underlying mechanisms and consequences of pathogenicity differences between two in vitro selected G1-H9N2 clones originating from a single isolate.

The G1-H9N2 avian influenza virus (AIV) has caused significant economic losses in the commercial poultry industry due to reduced egg production and increased mortality. The field observations have shown that H9N2 viruses circulate and naturally mix with other pathogens and these simultaneous infections can exacerbate disease. To avoid an incorrect virus characterization, due to co-infection, isolates were purified by in vitro plaque assays. Two plaque purified G1-H9N2 clones, selected on different cell types, named MDCK-and CEF-clone in regards to the cell culture used, were studied in vivo, revealing two different virulence phenotypes. Subsequently, the underlying mechanisms were studied. Specifically, the phenotypical outcome of SPF bird infection by the two clones resulted in completely different clinical outcomes. These differences in clinical outcome were used to study the factors behind this output in more detail. Further studies demonstrated that the more severe disease outcome associated with the MDCK-clone involves a strong induction of pro-inflammatory cytokines and a lack of type I interferon production, whereas the mild disease outcome associated with the CEF-clone is related to a greater antiviral cytokine response. The immunosuppressive effect of the MDCK-clone on splenocytes was further demonstrated via ChIFN-γ lack production after ex vivo mitogenic stimulation. Genome sequencing of the two clones identified only four amino acid differences including three in the HA sequence (HA-E198A, HA-R234L, HA-E502D-H9 numbering) and one in the NA sequence (NA-V33M). In the present study, valuable insights on the mechanisms responsible for AI pathogenicity and molecular mechanisms of H9N2 infections in chicken were obtained while highlighting the impact of the cells viruses are grown on their virulence.

Domestic canaries (Serinus canaria forma domestica) are susceptible to low pathogenic avian influenza virus infections.

Avian influenza viruses have been isolated from many bird species; however, little is known about the susceptibility of pet birds to low pathogenic avian influenza (LPAI) viruses. To address this research gap, domestic canaries (Serinus canaria forma domestica) were experimentally infected with H5 and H7 LPAI viruses to determine susceptibility and to evaluate samples for diagnostic purposes. Clinical evidence of infection (e.g. ruffled plumage and apathy) and mortality were noted for the canaries inoculated with chicken-adapted LPAI viruses. Real-time reverse transcription-polymerase chain reaction (RRT-PCR) demonstrated higher viral RNA levels in buccal compared to faecal samples. No clinical signs or mortality were observed in canaries inoculated with LPAI virus originating from wild birds; however, the canaries in this group did have evidence of viral RNA in buccal and faecal samples. Overall, this study showed that domestic canaries are susceptible to LPAI virus infections and that they can shed large amounts of viral RNA, primarily through the respiratory route. Thus, buccal swabs might be better samples than faeces for efficient detection of some LPAI virus infections in these birds. Although canaries have not been identified as a significant reservoir for LPAI viruses, they may be infected by LPAI viruses. Thus, the importance of the control of domestic canaries for detection of LPAI viruses should not be underestimated, especially in the contexts of international commercial exchange and outbreaks. RESEARCH HIGHLIGHTS Canaries are susceptible to infection with H5/H7 LPAI viruses. Canaries inoculated with LPAI viruses excrete large amounts of viral RNA. Buccal swabs may be appropriate specimens for AI virus detection in canaries. The control of canaries for LPAI virus detection should not be overlooked.

Usutu Virus Epizootic and Plasmodium Coinfection in Eurasian Blackbirds ( Turdus merula) in Flanders, Belgium.

At the end of the summer of 2016, unusually high levels of mortality were detected in Passeriformes and Strigiformes in Flanders, Belgium, mainly in Eurasian Blackbirds ( Turdus merula). A passive surveillance program demonstrated a widespread Usutu virus outbreak and revealed a coinfection with Plasmodium in 99% of the dead passerine birds that were necropsied.

Complete coding sequence of a novel picorna-like virus in a blackbird infected with Usutu virus.

Using random high-throughput RNA sequencing, the complete coding sequence of a novel picorna-like virus (a 9,228-nt contig containing 212,202 reads) was determined from a blackbird (Turdus merula) infected with Usutu virus. This sequence shares only 36% amino acid sequence identity with its closest homolog, arivirus 1, (an unclassified member of the order Picornavirales), and shares its dicistronic genome arrangement. The new virus was therefore tentatively named "blackbird arilivirus" (ari-like virus). The nearly complete genome sequence consists of at least 9,228 nt and contains two open reading frames (ORFs) encoding the nonstructural polyprotein (2235 amino acids) and structural polyprotein (769 amino acids). Two TaqMan RT-qPCR assays specific for ORF1 confirmed the presence of high levels of this novel virus in the original sample. Nucleotide composition analysis suggests that blackbird arilivirus is of dietary (plant) origin.

Early immune responses and profiling of cell-mediated immunity-associated gene expression in response to rHVT-IBD vaccination.

Infectious bursal disease (IBD) remains a major threat to the poultry industry. Recombinant herpesvirus of turkey (rHVT)-IBD vaccines have been successfully used to induce a protective immune response against IBD. However, the capacity for rHVT-IBD vaccines to induce early protection without detectable antibodies, and the underlying mechanisms mediating specific cell-mediated responses in the early stages following vaccination, have been poorly investigated. Therefore, in this study, specific pathogen-free (SPF) chickens were vaccinated with rHVT-IBD and T-cell subsets were analyzed. Both splenic and circulating CD8+ cell populations increased at 7 days postvaccination (dpv). Next, the expression of adaptive immunity-related genes was analyzed in the spleen and lung of rHVT-IBD-vaccinated chickens. Upregulation of CD8 expression was observed at 7 dpv. Interestingly, a parallel increase in the transcription of granzymes A and K was also detected from 7 dpv. To our knowledge, the latter result is the first to be reported, and it suggests that cytotoxic activity of CD8+ T lymphocytes is activated. In contrast, expression of the innate genes examined remained largely unchanged following vaccination. To further investigate the IBD virus (IBDV)-specific responses triggered by rHVT-IBD vaccination, vaccinated chickens were inoculated with an attenuated IBDV strain with the aim of restimulating induced immune responses in vivo. The expression profiles of various genes associated with adaptive immune responses were subsequently analyzed in lung, spleen, and bursa of Fabricius samples. Significant upregulation of CD4, CD8, perforin, and IFNγ expression were observed in the bursa samples 7 days postinoculation (dpi). In the lung, transcript levels of CD8, granzymes and perforin were also significantly higher in the rHVT-IBD-vaccinated chickens at 7 dpi, thereby suggesting that specific cellular immune responses were activated. Overall, these results support the hypothesis that stimulation of specific CD8+ cell-mediated immunity contributes to the response against IBDV in rHVT-IBD-vaccinated chickens.

Complete Coding Sequence of Usutu Virus Strain Gracula religiosa/U1609393/Belgium/2016 Obtained from the Brain Tissue of an Infected Captive Common Hill Myna (Gracula religiosa).

The complete and annotated coding sequence and partial noncoding sequence of an Usutu virus genome were sequenced from RNA extracted from a clinical brain tissue sample obtained from a common hill myna (Gracula religiosa), demonstrating close homology with Usutu viruses circulating in Europe.