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Duck hepatitis A virus (DHAV) infection in ducklings causes acute hepatitis with considerable economic losses. In this study, Pekin and Muscovy duckling flocks (n=9) suffering from high mortality and hepatic lesions were examined by RT-PCR for DVHA. 44.4 % (4/9) of samples were positive for DHAV (5' UTR region), of which 100 % (4/4) were DVHA-3 (VP1 gene). VP1 sequencing and phylogenetic analysis of an isolate originated from Muscovy ducklings showed that it shared 96.8 % -100 %, 88.5-89.2 %, and 86.5-88.2 % nucleotide similarity (ns) with the Egyptian, Korean-Vietnamese, and Chinese DVHA-3 strains, respectively. It was distinguished from the DHAV-1 vaccine (67.6 % ns). The sequenced DVHA-3 isolate was used to experimentally infect 5-day-old Pekin and Muscovy ducklings vs. control groups. No apparent clinical signs or deaths were reported in the experimentally-infected groups. Pekin ducklings showed greater cloacal viral shedding than Muscovy until the 6th dpi (P<0.05). DVHA-3 induced a significant rise in IFN-β and IL-1β serum levels, where Muscovy ducklings' levels were higher than Pekin ducklings. Among the biochemical parameters, AST was only increased on the 4th dpi in both breeds vs. control (P<0.05). Compared to Muscovy ducklings at 2, 4, and 6 day post infection (dpi), the infected Pekin group had lower lipase levels (P≥0.05, p<0.05, and p<0.05, respectively), while ALT was higher at 4 and 6 dpi (P<0.05). The histopathological lesions supported the gross lesions, and their scores were dominant at 2 and 4 dpi in both breeds. At 6 and 8 dpi, Pekin showed more severe histopathological changes compared to Muscovy for the liver, heart, brain, and intestines; the pancreas, kidney, and lung showed the opposite pattern. In conclusion, Pekin ducklings displayed distinct DHAV-3 infection results from Muscovy ducklings, and more research utilizing a variety of DHAV-3 strains has to be carried out., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Duck circovirus (DuCV) is widely recognized as a prominent virus in China's duck farming industry, known for its ability to cause persistent infections and significant immunosuppression, which can lead to an increased susceptibility to secondary infections, posing a significant threat to the duck industry. Moreover, clinical evidence also indicates the potential vertical transmission of the virus through duck embryos to subsequent generations of ducklings. However, the limited availability of suitable cell lines for in vitro cultivation of DuCV has hindered further investigation into the molecular mechanisms underlying its infection and pathogenicity. In this study, we observed that oral DuCV infection in female breeding ducks can lead to oviduct, ovarian, and follicular infections. Subsequently, the infection can be transmitted to the fertilized eggs, resulting in the emergence of virus-carrying ducklings upon hatching. In contrast, the reproductive organs of male breeding ducks were unaffected by the virus, thus confirming that vertical transmission of DuCV primarily occurs through infection in female breeding ducks. By analyzing transcriptome sequencing data from the oviduct, we focused on claudin-2 , a gene encoding the tight junction protein CLDN2 located on the cell membrane, which showed significantly increased expression in DuCV-infected oviducts of female breeding ducks. Notably, CLDN2 was confirmed to interact with the unique structural protein of DuCV, namely capsid protein (Cap), through a series of experimental approaches including co-immunoprecipitation (co-IP), GST pull-down, immunofluorescence, and adhesion-blocking assays. Furthermore, we demonstrated that the Cap protein binds to the extracellular loop structural domains EL1 and EL2 of CLDN2. Subsequently, by constructing a series of truncated bodies of the CLDN2 promoter region, we identified the transcription factor SP5 for CLDN2. Moreover, we found that DuCV infection triggers the activation of the MAPK-ERK signaling pathway in DEF cells and ducks, leading to an upregulation of SP5 and CLDN2 expression. This process ultimately leads to the transportation of mature CLDN2 to the cell surface, thereby facilitating increased virus adherence to the target organs. In conclusion, we discovered that DuCV utilizes host CLDN2 proteins to enhance adhesion and infection in oviducts and other target organs. Furthermore, we elucidated the signaling pathways involved in the interaction between DuCV Cap proteins and CLDN2, which provides valuable insights into the molecular mechanism underlying DuCV's infection and vertical transmission., Importance: Although duck circovirus (DuCV) poses a widespread infection and a serious hazard to the duck industry, the molecular mechanisms underlying DuCV infection and transmission remain elusive. We initially demonstrated vertical transmission of DuCV through female breeding ducks by simulating natural infection. Furthermore, a differentially expressed membrane protein CLDN2 was identified on the DuCV-infected oviduct of female ducks, and its extracellular loop structural domains EL1 and EL2 were identified as the interaction sites of DuCV Cap proteins. Moreover, the binding of DuCV Cap to CLDN2 triggered the intracellular MAPK-ERK pathway and activated the downstream transcription factor SP5. Importantly, we demonstrated that intracellular Cap also interacts with SP5, leading to upregulation of CLDN2 transcription and facilitating enhanced adherence of DuCV to target tissue, thereby promoting viral infection and transmission. Our study sheds light on the molecular mechanisms underlying vertical transmission of DuCV, highlighting CLDN2 as a promising target for drug development against DuCV infection., Competing Interests: The authors declare no conflict of interest.

Between 2013 and 2018, the novel A/Anhui/1/2013 (AH/13)-lineage H7N9 virus caused at least five waves of outbreaks in humans, totaling 1,567 confirmed human cases in China. Surveillance data indicated a disproportionate distribution of poultry infected with this AH/13-lineage virus, and laboratory experiments demonstrated that this virus can efficiently spread among chickens but not among Pekin ducks. The underlying mechanism of this selective transmission remains unclear. In this study, we demonstrated the absence of Neu5Gc expression in chickens across all respiratory and gastrointestinal tissues. However, Neu5Gc expression varied among different duck species and even within the tissues of the same species. The AH/13-lineage viruses exclusively bind to acetylneuraminic acid (Neu5Ac), in contrast to wild waterbird H7 viruses that bind both Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The level of Neu5Gc expression influences H7 virus replication and facilitates adaptive mutations in these viruses. In summary, our findings highlight the critical role of Neu5Gc in affecting the host range and interspecies transmission dynamics of H7 viruses among avian species.IMPORTANCEMigratory waterfowl, gulls, and shorebirds are natural reservoirs for influenza A viruses (IAVs) that can occasionally spill over to domestic poultry, and ultimately humans. This study showed wild-type H7 IAVs from waterbirds initially bind to glycan receptors terminated with N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). However, after enzootic transmission in chickens, the viruses exclusively bind to Neu5Ac. The absence of Neu5Gc expression in gallinaceous poultry, particularly chickens, exerts selective pressure, shaping IAV populations, and promoting the acquisition of adaptive amino acid substitutions in the hemagglutinin protein. This results in the loss of Neu5Gc binding and an increase in virus transmissibility in gallinaceous poultry, particularly chickens. Consequently, the transmission capability of these poultry-adapted H7 IAVs in wild water birds decreases. Timely intervention, such as stamping out, may help reduce virus adaptation to domestic chicken populations and lower the risk of enzootic outbreaks, including those caused by IAVs exhibiting high pathogenicity., Competing Interests: The authors declare no conflict of interest.

Avian migration is a global phenomenon that transcends geographical boundaries. These migratory birds serve as unwitting carriers of diverse Gammacoronaviruses (γ-CoVs) and Deltacoronaviruses (δ-CoVs). While recombination events have been documented among γ-CoVs in avian species and β-CoVs in mammals, evidence for recombination between CoVs of distinct genera remains limited. This minireview examines the prevalence of CoVs in both domestic waterfowl (ducks and geese) and wild bird populations inhabiting various regions. We investigate the dissemination patterns of γ-CoVs and δ-CoVs among these populations, highlighting their shared characteristics. Furthermore, the review explores the intricate web of cross-species transmission of δ-CoVs from wild birds to mammals, with a particular focus on pigs. Understanding the distinct features of CoVs harbored by waterfowl and wild birds and their potential for cross-species transmission is crucial for preparedness and response to future CoV epidemics., Competing Interests: The authors declare no conflict of interest.

The emergence of clade 2.3.4.4b H5N1 highly pathogenic avian influenza (HPAI) viruses in 2021 has led to unprecedented epidemics in poultry, changing epidemiological patterns of year-round infections in resident wild avifauna and more frequent spill-over events to mammals. Given this situation, it is important that we recognise that traditional HPAI management strategies are no longer sufficient, and policy changes are required. Poultry vaccination has emerged as a crucial intervention in the current control of HPAI, as evidenced by France's nationwide campaign targeting domestic ducks. However, due to the logistical challenges and potential trade implications of vaccination, broader structural reforms appear also necessary. These include a shift from farm-level to territorial-level biosecurity approaches, putting into practice the concept of 'regional biosecurity'. Given the role duck farm density has played in successive HPAI epidemics in France, there is a need to think about the spatial distribution of poultry farms as a structural component of regional biosecurity and to consider the reduction of farm concentration as a measure to prevent viral spread. The integration of regional biosecurity and poultry vaccination into prevention strategies should impact the way poultry are produced and traded in the future.

A suspected outbreak of duck astrovirus (DAstV) disease occurred in a large Muscovy duck farm in Guangdong Province, China, in June 2022, which severely affected the production performance and health of Muscovy ducks. This study aimed to investigate the prevalence of DAstV disease in Southeast China. Herein, we employed semi-nested PCR ethodto screen 5203 swab and liver samples from 11 Muscovy duck farms in 5 provinces of China for the presence of DAstV. Among them, 1356 samples (26.06%, 1356/5203) tested positive for DAstV, out of which 11 DAstV strains were isolated after 10 generations of blind transmission through Leghorn male hepatoma (LMH) cells and performed their whole-genome sequencing. The alignment results showed that the 11 DAstV isolates exhibited relatively low homology (15.4%-75%) with the astrovirus isolates from other species published in GenBank, whereas their homology (nucleotide: 90.4%-99.99%; amino acid: 94%-99.8%) with the DAstV type 1 (DAstV-1) reference strain was higher, indicating considerable homology. The results indicated that DAstV-1 was the main pathogenic factor. Herein, we successfully recreated the clinical symptoms of natural infection in 28-day-old specific-pathogen-free (SPF) ducks using the DAstV-1-GDB-2022 strain. The primary clinical manifestations included liver enlargement, hemorrhaging, and disruptions in liver function. Additionally, we confirmed the cross-species transmission potential of DAstV-1, marking the first occurrence of clinical symptoms of DAstV in 28-day-old SPF chickens. Our findings provide new perspectives on the epidemiology and pathogenicity of DAstV-1 and may help in advancing the development of DAstV vaccines., Competing Interests: DISCLOSURES No conflict of interest exits in the submission of this manuscript. All the authors listed have approved the manuscript that is enclosed., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Influenza A viruses (IAV) pose substantial burden on human and animal health. Avian, swine and human IAV bind sialic acid on host glycans as receptor, whereas some bat IAV require MHC class II complexes for cell entry. It is unknown how this difference evolved and whether dual receptor specificity is possible. Here we show that human H2N2 IAV and related avian H2N2 possess dual receptor specificity in cell lines and primary human airway cultures. Using sialylation-deficient cells, we reveal that entry via MHC class II is independent of sialic acid. We find that MHC class II from humans, pigs, ducks, swans and chickens but not bats can mediate H2 IAV entry and that this is conserved in Eurasian avian H2. Our results demonstrate that IAV can possess dual receptor specificity for sialic acid and MHC class II, and suggest a role for MHC class II-dependent entry in zoonotic IAV infections., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Short-beak and dwarfism syndrome (SBDS) is a new disease caused by a genetic variant of goose parvovirus in ducks that results in enormous economic losses for the waterfowl industry. Currently, there is no commercial vaccine for this disease, so it is urgent to develop a safer and more effective vaccine to prevent this disease. In this study, we optimized the production conditions to enhance the expression of the recombinant VP2 protein and identified the optimal conditions for subsequent large-scale expression. Furthermore, the protein underwent purification via nickel column affinity chromatography, followed by concentration using ultrafiltration tube. Subsequently, it was observed by transmission electron microscopy (TEM) that the NGPV recombinant VP2 protein assembled into virus-like particles (VLPs) resembling those of the original virus. Finally, the ISA 78-VG adjuvant was mixed with the NGPV-VP2 VLPs to be prepared as a subunit vaccine. Furthermore, both agar gel precipitation test (AGP) and serum neutralization test demonstrated that NGPV VLP subunit vaccine could induce the increase of NGPV antibody in breeding ducks. The ducklings were also challenged with the NGPV, and the results showed that the maternal antibody level could provide sufficient protection to the ducklings. These results indicated that the use of the NGPV VLP subunit vaccine based on the baculovirus expression system could facilitate the large-scale development of a reliable vaccine in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Herpesviruses-encoded microRNAs (miRNAs) have been discovered to be essential regulators in viral life cycle, participating in viral replication, latent or lytic infection, and immunological escape. However, the roles of miRNAs encoded by duck plague virus (DPV) are still unknown. Dev-miR-D28-3p is a miRNA uniquely encoded by DPV CHv strain. The aim of this study was to explore the effect of dev-miR-D28-3p on DPV replication and explore the potential mechanisms involved. Our findings demonstrated that transfection of dev-miR-D28-3p mimic into duck embryo fibroblasts (DEFs) effectively suppressed viral copies, viral titers and viral protein expressions during DPV infection, while the results above were reversed after transfection with dev-miR-D28-3p inhibitor. Subsequently, we further discovered that dev-miR-D28-3p specifically bound to DPV-encoded UL27 and inhibited its expression, suggesting that UL27 was the target gene of dev-miR-D28-3p. Finally, we investigated the role of UL27 in DPV replication and found the overexpression of UL27 increased viral copies, viral titers, and viral protein expressions; whereas the opposite results appear when knockdown of UL27. Our findings illustrated a novel mechanism that DPV regulated itself replication via dev-miR-D28-3p, paving the way for exploring the role of DPV-encoded miRNAs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Competing interests The authors declare that they have no competing interests, (Copyright © 2024 Elsevier B.V. All rights reserved.)

DHAV-1 is a highly infectious pathogen that can cause acute hepatitis in ducklings. MicroRNA (miRNA) plays an essential regulatory role in virus response. We characterized and compared miRNA and mRNA expression profiles in duck embryonic fibroblasts (DEF) and the liver of ducklings infected with DHAV-1. DHAV-1 infected DEF was divided into infection group (D group) and blank group (M group), and DHAV-1 infected duckling group was divided into infection group (H group) and blank group (N group). D vs. M have 130 differentially expressed (DE) miRNA (DEM) and 2204 differentially expressed (DE) mRNA (DEG), H vs. N have 72 DEM and 1976 DEG. By the intersection of D vs. M and H vs. N comparisons, 15 upregulated DEM, 5 downregulated DEM, 340 upregulated DEG and 50 downregulated DEG were found with both in vivo and in vitro DHAV-1 infection. In particular, we identified the same DE miRNA target genes and functional annotations of DE mRNA. We enriched with multiple gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which may have important roles in viral virulence, host immunity, and metabolism. We selected miR-155, which is co-upregulated, and found that miR-155 targets SOCS1 to inhibit DHVA-1 replication., (© 2024. The Author(s).)

RNA editing is increasingly recognized as a post-transcriptional modification that directly affects viral infection by regulating RNA stability and recoding proteins. the duck hepatitis A virus genotype 3 (DHAV-3) infection is seriously detrimental to the Asian duck industry. However, the landscape and roles of RNA editing in the susceptibility and resistance of Pekin ducks to DHAV-3 remain unclear. Here, we profiled dynamic RNA editing events in liver tissue and investigated their potential functions during DHAV-3 infection in Pekin ducks. We identified 11,067 informative RNA editing sites in liver tissue from DHAV-3-susceptible and -resistant ducklings at three time points during virus infection. Differential RNA editing sites (DRESs) between S and R ducks were dynamically changed during infection, which were enriched in genes associated with vesicle-mediated transport and immune-related pathways. Moreover, we predicted and experimentally verified that RNA editing events in 3'-UTR could result in loss or gain of miRNA-mRNA interactions, thereby changing the expression of target genes. We also found a few DRESs in coding sequences (CDSs) that altered the amino acid sequences of several proteins that were vital for viral infection. Taken together, these data suggest that dynamic RNA editing has significant potential to tune physiological processes in response to virus infection in Pekin ducks, thus contributing to host differential susceptibility to DHAV-3.

A disease called "hydrosalpinx fluid and egg drop syndrome" (HFEDS) was observed in four flocks of Jinding ducks (Anas platyrhynchos domesticus) in Northeast China during the years 2022 to 2023. Here, we investigated the possible involvement of avian metapneumovirus (AMPV) infection. Full-length genome sequencing and sequence analysis of two AMPV strains showed that they belong to Eurasian lineage of AMPV subtype C. Based on surface glycoprotein (G) sequence comparisons, the Eurasian lineage can be divided into two sublineages (E1 and E2), and sublineage E2 is circulating in Jinding duck populations in Northeast China., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

High pathogenicity avian influenza virus (HPAIV) is a rapidly evolving virus causing significant economic and environmental harm. Wild birds are a key viral reservoir and an important source of viral incursions into animal populations, including poultry. However, we lack a thorough understanding of which species drive incursions and whether this changes over time. We explored associations between the abundances of 152 avian species and outbreaks of highly pathogenic avian influenza (HPAI) in poultry premises across Great Britain between October 2021 and January 2023. Spatial generalized additive models were used, with species abundance distributions sourced from eBird. Associations were investigated at the species-specific level and across species aggregations. During autumn/winter, associations were generally strongest with waterbirds such as ducks and geese; however, we also found significant associations in groups such as non-native gamebirds and rapid change in species-specific associations over time. Our results demonstrate the value of citizen science to rapidly explore wild species as potential facilitators of disease incursions into well-monitored populations, especially in regions where viral surveillance in wild species is limited. This can be a critical step towards prioritizing targeted surveillance that could inform species-specific biosecurity measures; particularly for HPAIV, which has undergone sudden shifts in host range and continues to rapidly evolve.

Short-beak and dwarf syndrome (SBDS) is caused by novel goose parvovirus (NGPV) infection, which leads to farm economic losses. Our research aimed to investigate the potential of administering isolated lactic acid bacteria (LAB) in alleviating SBDS in ducks. Eight wild LAB strains were isolated from duck feces and their biosecurity was investigated in both duck embryo fibroblast (DEF) and live ducks. Moreover, the LAB strains exhibited no detrimental effects on bone metabolism levels and facilitated the tight junction proteins (TJPs) mRNA expression, and contributing to the mitigation of inflammation in healthy ducks. Subsequently, we conducted in vitrol and in vivo experiments to assess the impact of LAB on NGPV infection. The LAB strains significantly reduced the viral load of NGPV and downregulated the mRNA levels of pro-inflammatory factors in DEF. Additionally, LAB treatment alleviated SBDS in NGPV-infected ducks. Furthermore, LAB treatment alleviated intestinal damage, and reduced the inflammatory response, while also mitigating bone resorption in NGPV-infected ducks. In conclusion, the LAB strains isolated from duck feces have favorable biosecurity and alleviate SBDS in ducks, and the mechanism related to LAB improves intestinal barrier integrity, alleviates inflammation, and reduces bone resorption. Our study presents a novel concept for the prevention and treatment of NGPV, thereby establishing a theoretical foundation for the future development of probiotics in the prevention and treatment of NGPV., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Novel goose parvovirus (NGPV) is continuously threatening the global duck industry, as it causes short beak and dwarfism syndrome among different duck breeds. In this study, we investigated the viral pathogenesis in the tongue of affected ducks, as a new approach for deeper understanding of the syndrome. Seventy-three, 14- to 60-day-old commercial Pekin ducks were clinically examined. Thirty tissue pools of intestine and tongue (15 per tissue) were submitted for molecular identification. Clinical signs in the examined ducks were suggestive of parvovirus infection. All examined ducks had short beaks. Necrotic, swollen, and congested protruding tongues were recorded in adult ducks (37/73, 51%). Tongue protrusion without any marked congestion or swelling was observed in 20-day-old ducklings (13/73, 18%), and no tongue protrusion was observed in 15-day-old ducklings (23/73, 32%). Microscopically, the protruding tongues of adult ducks showed necrosis of the superficial epithelial layer with vacuolar degeneration. Glossitis was present in the nonprotruding tongues of young ducks, which was characterized by multifocal lymphoplasmacytic aggregates and edema in the propria submucosa. Immunohistochemical examination displayed parvovirus immunolabeling, mainly in the tongue propria submucosa. Based on polymerase chain reaction, goose parvovirus was detected in 9 out of 15 tongue sample pools (60%). Next-generation sequencing confirmed the presence of a variant goose parvovirus that is globally named NGPV and closely related to Chinese NGPV isolates. Novel insights are being gained from the study of NGPV pathogenesis in the tongue based on molecular and immunohistochemical identification., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Short beak and dwarfism syndrome (SBDS) is attributed to Novel Goose Parvovirus (NGPV), which has inflicted significant economic losses on farming in China. Despite its significant impact, limited research has been conducted on the pathogenesis of this disease. The SD strain, a parvovirus variant isolated from ducks in Shandong province, was identified and characterized in our study. Phylogenetic analysis and sequence comparisons confirmed the classification of the SD strain as a member of NGPV. Based on this information, we established an animal model of SBDS by inoculating Cherry Valley ducks with the SD strain. Our findings indicate that infection with the SD strain leads to a reduction in body weight, beak length, width, and tibia length. Notably, significant histopathological alterations were observed in the thymus, spleen, and intestine of the infected ducks. Furthermore, the SD strain induces bone disorders and inflammatory responses. To evaluate the impact of NGPV on intestinal homeostasis, we performed 16S rDNA sequencing and gas chromatography to analyze the composition of intestinal flora and levels of short-chain fatty acids (SCFAs) in the cecal contents. Our findings revealed that SD strain infection induces dysbiosis in cecal microbial and a decrease in SCFAs production. Subsequent analysis revealed a significant correlation between bacterial genera and the clinical symptoms in NGPV SD infected ducks. Our research providing novel insights into clinical pathology of NGPV in ducks and providing a foundation for the research of NGPV treatment targeting gut microbiota., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The emergence of novel avian influenza reassortants in wild birds in recent years is a public health concern. However, the viruses that circulate in migratory birds are not fully understood. In this study, we summarized and categorized global H11 avian influenza viruses and reported that waterfowl and shorebirds are the major reservoirs of the identified H11 viruses. The surveillance data of the 35,749 faecal samples collected from wild bird habitats in eastern China over the past seven years revealed a low prevalence of H11 viruses in birds, with a positive rate of 0.067% (24 isolates). The phylogenetic analysis of the twenty viruses indicated that H11 viruses have undergone complex reassortment with viruses circulating in waterfowl and shorebirds. These tested viruses do not acquire mammalian adaptive mutations in their genomes and preferentially bind to avian-type receptors. Experimental infection studies demonstrated that the two tested H11N9 viruses of wild bird origin replicated and transmitted more efficiently in ducks than in chickens, whereas the pigeon H11N2 virus isolated from a live poultry market was more adapted to replicate in chickens than in ducks. In addition, some H11 isolates replicated efficiently in mice and caused body weight loss but were not lethal. Our study revealed the role of waterfowl and shorebirds in the ecology and evolution of H11 viruses and the potential risk of introducing circulating H11 viruses into ducks or chickens, further emphasizing the importance of avian influenza surveillance at the interface of migratory birds and poultry.

High pathogenicity avian influenza (HPAI) virus H5N1 first emerged in Bangladesh in 2007. Despite the use of vaccines in chickens since 2012 to control HPAI, HPAI H5Nx viruses have continued to infect poultry, and wild birds, resulting in notable mass mortalities in house crows ( Corvus splendens ). The first HPAI H5Nx viruses in Bangladesh belonged to clade 2.2.2, followed by clade 2.3.4.2 and 2.3.2.1 viruses in 2011. After the implementation of chicken vaccination in 2012, these viruses were mostly replaced by clade 2.3.2.1a viruses and more recently clade 2.3.4.4b and h viruses. In this study, we reconstruct the phylogenetic history of HPAI H5Nx viruses in Bangladesh to evaluate the role of major host species in the maintenance and evolution of HPAI H5Nx virus in Bangladesh and reveal the role of heavily impacted crows in virus epidemiology. Epizootic waves caused by HPAI H5N1 and H5N6 viruses amongst house crows occurred annually in winter. Bayesian phylodynamic analysis of clade 2.3.2.1a revealed frequent bidirectional viral transitions between domestic ducks, chickens, and house crows that was markedly skewed towards ducks; domestic ducks might be the source, or reservoir, of HPAI H5Nx in Bangladesh, as the number of viral transitions from ducks to chickens and house crows was by far more numerous than the other transitions. Our results suggest viral circulation in domestic birds despite vaccination, with crow epizootics acting as a sentinel. The vaccination strategy needs to be updated to use more effective vaccinations, assess vaccine efficacy, and extension of vaccination to domestic ducks, the key reservoir.

Multiple genotypes of highly pathogenic avian influenza (HPAI) H5 clade 2.3.4.4b viruses have caused epizootics in wild birds and poultry. The HPAI H5N1 genotype C virus caused a modest epizootic, whereas the occurrence of the HPAI H5N1 genotype AB virus in 2021 resulted in the largest avian influenza epizootic in Europe to date. Here we studied the pathogenicity of two HPAI H5N1 viruses by experimentally infecting chickens, Pekin ducks, Eurasian wigeons and Barnacle geese. Our study demonstrates that pathogenicity of the H5N1-2021-AB virus is lower in Pekin ducks, Eurasian wigeons and Barnacle geese compared to the H5N1-2020-C virus, whereas virus shedding was high for both viruses. After inoculation with H5N1-2021-C viral antigen expression was higher in the brain of Pekin ducks, Eurasian wigeons and Barnacle geese, which caused higher mortality compared to inoculation with H5N1-2021-AB virus. Subclinical infections occurred in Pekin ducks and Eurasian wigeons and mortality was reduced in Barnacle geese after inoculation with H5N1-2021-AB virus while H5N1-2020-C virus caused high morbidity and mortality in these species. This H5N1-2021-AB virus trait may have contributed to efficient spread of the virus in wild bird populations. Therefore, high mortality, virus shedding and long-lasting viral antigen expression found in Barnacle geese may have increased the risk for introduction into poultry.

Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 10 7.5 TCID 50 /mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.

Duck hepatitis virus 1 (DHV-1) is a major threat to the global poultry industry, causing significant economic losses due to high mortality rates in young ducklings. To better understand the evolution and host adaptation strategies of DHV-1, we conducted a comprehensive codon usage analysis of DHV-1 genomes. Our phylogenetic analysis revealed three well-supported DHV-1 phylogroups (Ia, Ib, and II) with distinct genetic diversity patterns. Comparative analyses of the codon usage bias and dinucleotide abundance uncovered a strong preference for A/U-ended codons and a biased pattern of dinucleotide usage in the DHV-1 genome, with CG dinucleotides being extremely underrepresented. Effective number of codons (ENC) analysis indicated a low codon usage bias in the DHV-1 ORF sequences, suggesting adaptation to host codon usage preferences. PR2 bias, ENC plot, and neutrality analyses revealed that both mutation pressure and natural selection influence the codon usage patterns of DHV-1. Notably, the three DHV-1 phylogroups exhibited distinct evolutionary trends, with phylogroups Ia and Ib showing evidence of neutral evolution accompanied by selective pressure, while the phylogroup II evolution was primarily driven by random genetic drift. Comparative analysis of the codon usage indices (CAI, RCDI, and SiD) among the phylogroups highlighted significant differences between subgroups Ia and Ib, suggesting distinct evolutionary pressures or adaptations influencing their codon usage. These findings contribute to our understanding of DHV-1 evolution and host adaptation, with potential implications for the development of effective control measures and vaccines.

Introduction: Muscovy duck parvovirus (MDPV), Goose parvovirus (GPV), Duck circovirus, (DuCV) and Duck adenovirus 3 (DAdV-3) are important pathogens that cause high morbidity and mortality in ducks, causing huge economic loss for the duck industry., Methods: The present study, a quadruplex one-step real time quantitative PCR method for the detection of MDPV, GPV, DuCV, and DAdV-3 was developed., Results: The results showed that assay had no cross-reactivity with other poultry pathogens [Duck plague virus (DPV), Duck tembusu virus (DTMUV), H6 avian influenza virus (H6 AIV), New duck reovirus (NDRV), Newcastle disease virus (NDV), H4 avian influenza virus (H4 AIV), Escherichia coli ( E. coli ), Muscovy duck reovirus (MDRV), Egg drop syndrome virus (EDSV), Pasteurella multocida ( P. multocida )]. The sensitivity result showed that the limits of detection for MDPV, GPV, DuCV, and DAdV-3 were 10, 10, 1 and 10 copies/µl, respectively; The coefficients of variation intra- and inter-method was 1-2%; The range of linear (109 to 103 copies/µL) demonstrated the R2 values for MDPV, GPV, DuCV, and DAdV-3 as 0.9975, 0.998, 0.9964, and 0.996, respectively. The quadruplex real time quantitative PCR method efficiency was 90.30%, 101.10%, 90.72%, and 90.57% for MDPV, GPV, DuCV, and DAdV-3, respectively. 396 clinical specimens collected in some duck sausages from June 2022 to July 2023 were simultaneously detected using the established quadruplex real time quantitative PCR method and the reported assays. The detection rates for MDPV, GPV, DuCV, and DAdV-3 were 8.33% (33/396), 17.93% (71/396), 33.58% (133/396), and 29.04% (115/396), respectively. The agreement between these assays was greater than 99.56%., Discussion: The developed quadruplex real-time quantitative PCR assay can accurately detect these four viruses infecting ducks, providing a rapid, sensitive, specific and accurate technique for clinical testing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Chen, An, Chen, Wang, Zhu, Yu, Xia and Zhang.)

High pathogenicity avian influenza viruses (HPAIVs) have caused major epizootics in recent years, with devastating consequences for poultry and wildlife worldwide. Domestic and wild ducks can be highly susceptible to HPAIVs, and infection leads to efficient viral replication and massive shedding (i.e., high titres for an extended time), contributing to widespread viral dissemination. Importantly, ducks are known to shed high amounts of virus in the earliest phase of infection, but the dynamics and impact of environmental contamination on the epidemiology of HPAIV outbreaks are poorly understood. In this study, we monitored mule ducks experimentally infected with two H5N8 clade 2.3.4.4b goose/Guangdong HPAIVs sampled in France in 2016-2017 and 2020-2021 epizootics. We investigated viral shedding dynamics in the oropharynx, cloaca, conjunctiva, and feathers; bird-to-bird viral transmission; and the role of the environment in viral spread and as a source of samples for early detection and surveillance. Our findings showed that viral shedding started before the onset of clinical signs, i.e., as early as 1 day post-inoculation (dpi) or post-contact exposure, peaked at 4 dpi, and lasted for up to 14 dpi. The detection of viral RNA in aerosols, dust, and water samples mirrored viral shedding dynamics, and viral isolation from these environmental samples was successful throughout the experiment. Our results confirm that mule ducks can shed high HPAIV titres through the four excretion routes tested (oropharyngeal, cloacal, conjunctival, and feather) while being asymptomatic and that environmental sampling could be a non-invasive tool for early viral RNA detection in HPAIV-infected farms., (© 2024. The Author(s).)

As a founding member of the Src family of kinases, Src has been confirmed to participate in the regulation of immune responses, integrin signaling, and motility. Ducks are usually asymptomatic carriers of RNA viruses such as Newcastle disease virus and avian influenza virus, which can be deadly to chickens. The beneficial role of Src in modulating the immune response remains largely unknown in ducks. Here, we characterized the duck Src and found that it contains a 192-base-pair 5' untranslated region, a 1602-base-pair coding region, and a 2541-base-pair 3' untranslated region, encoding 533 amino acid residues. Additionally, duSrc transcripts were significantly activated in duck tissues infected by Newcastle disease virus compared to controls. The duSrc transcripts were notably widespread in all tissues examined, and the expression level was higher in liver, blood, lung, pancreas, and thymus. Moreover, we found the expression levels of IFN-β, NF-κB, IRF3, and Src were significantly increased in DEFs after infection with 5'ppp dsRNA, but there was no significant difference before and after treatment in DF1 cells. Furthermore, overexpression of duSrc followed by stimulation with 5'ppp dsRNA led to an elevation of IFN-β levels. The SH3 and PTKc domains of duSrc contributed to promoting the activity of IFN-β and NF-κB in DEFs stimulated by 5'ppp dsRNA.

Duck enteritis virus (DEV) may lead to vascular injury, gastrointestinal mucosal erosion, lymphoid organ injury, and Polyinosinic-polycytidylic acid (Poly I:C) has an antiviral effect by inducing low levels of interferon. The purpose of this study was to explore the pathogenesis of DEV-induced intestinal injury in ducks and to verify the therapeutic effects of different concentrations of Poly I:C. In this study, duck enteritis model was established by infecting healthy Pekin ducks with DEV. Duck intestinal tissues were extracted from normal control group, model group, and treatment group with different doses of Poly I:C. In vivo, HE and TUNEL staining were used to observe the morphological changes and apoptosis. In vitro, the proliferation and apoptosis of duck intestinal epithelial cells were evaluated by MTT assay, TUNEL staining, and flow cytometry. The results showed that Poly I:C protected ducks from DEV toxicity by improving intestinal morphology and inhibiting apoptosis. In addition, the antiviral effect of Poly I:C on DEV was found in a dose-dependent manner, with a more relatively obvious effect at a high dose of Poly I:C. All in all, these results demonstrated that Poly I:C played a vital role in the apoptosis induced by DEV in ducks and modest dose of Poly I:C treatment worked well and may provide important reference for the development of new antiviral drugs in the future., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Goose circovirus (GoCV) is a common pathogen that causes immunosuppression and promotes secondary infections with other infectious agents in geese worldwide. In the present study, we identified GoCV in 2 out of 93 duck flocks from China and successfully sequenced the complete genomes of 2 strains (AH22du and HN20du). The whole genome of the two strains shared a high identity of 90.5 to 98.63% with China GoCV reference, and low identity of 58.98% with DuCV reference, respectively. Phylogenetic tree constructed on the two and other genome sequences of GoCV revealed three main branches. Both strains sequenced in this study were distributed on different sub-branches with most other Chinese GoCV strains, and AH22du clustered into an independent sub-branch within the cluster. Recombination analysis predicted that HN20du might potentially recombine from the major parent of yk4 (Zhejiang Province, China, 2007) and minor parent of GD/YJ/g2 (Guangdong Province, China, 2020). To the best of our knowledge, this is the first report of GoCV in ducks from China. This broadened host spectrum of GoCVs requires attention from the waterfowl industry and researchers., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The mixed infection of duck hepatitis A virus 3 (DHAV-3) and novel duck reovirus (NDRV) has caused significant losses to the global duck farming industry. On-site point-of-care testing of viruses plays a crucial role in the early diagnosis, prevention, and disease control. Here, we proposed an RPA-CRISPR Cas12a/Cas13a one-pot strategy (DRCFS) for rapid and simultaneous detection of DHAV-3 and NDRV. This method integrated the reaction of RPA and CRISPR Cas12a/Cas13a in a single tube, eliminating the need to open the lid during the intermediate processes and thereby avoiding aerosol contamination. On this basis, we proposed a dual RPA-CRISPR strategy coupled with a lateral flow analysis platform (DRC-LFA). This circumvented the necessity for complex instruments, enabling direct visual interpretation of results, making the test more accessible and user-friendly. Our findings demonstrated that the DRCFS method could detect DHAV-3 and NDRV at concentrations as low as 10 0 copy/μL, while DRC-LFA achieved limit of 10 1 copies/μL within 35 min. Furthermore, when DRCFS, DRC-LFA, and qPCR were employed collectively for clinical samples analysis, all three methods yielded consistent results. The specificity, sensitivity, and user-friendly of these methods rendered them invaluable for on-site virus detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Migratory wild birds can carry various pathogens, such as influenza A virus, which can spread to globally and cause disease outbreaks and epidemics. Continuous epidemiological surveillance of migratory wild birds is of great significance for the early warning, prevention, and control of epidemics. To investigate the pathogen infection status of migratory wild birds in eastern China, fecal samples were collected from wetlands to conduct pathogen surveillance. The results showed that duck orthoreovirus (DRV) and goose parvovirus (GPV) nucleic acid were detected positive in the fecal samples collected from wild ducks, egrets, and swan. Phylogenetic analysis of the amplified viral genes reveals that the isolates were closely related to the prevalent strains in the regions involved in East Asian-Australasian (EAA) migratory flyway. Phylogenetic analysis of the amplified viral genes confirmed that they were closely related to circulating strains in the regions involved in the EAA migration pathway. The findings of this study have expanded the host range of the orthoreovirus and parvovirus, and revealed possible virus transmission between wild migratory birds and poultry., (Copyright © 2024. Published by Elsevier Inc.)

The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-β expression during virus infection, the expression level of IFN-β in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks., (Copyright © 2024 by The American Association of Immunologists, Inc.)

A Newcastle disease virus (NDV)-vectored vaccine expressing clade 2.3.4.4b H5 Hemagglutinin was developed and assessed for efficacy against H5N1 highly pathogenic avian influenza (HPAI) in specific pathogen-free (SPF) chickens, broilers, and domestic ducks. In SPF chickens, the live recombinant NDV-vectored vaccine, rK148/22-H5, achieved complete survival against HPAI and NDV challenges and significantly reduced viral shedding. Notably, the live rK148/22-H5 vaccine conferred good clinical protection in broilers despite the presence of maternally derived antibodies. Good clinical protection was observed in domestic ducks, with decreased viral shedding. It demonstrated complete survival and reduced cloacal viral shedding when used as an inactivated vaccine from SPF chickens. The rK148/22-H5 vaccine is potentially a viable and supportive option for biosecurity measure, effectively protecting in chickens against the deadly clade 2.3.4.4b H5 HPAI and NDV infections. Furthermore, it aligns with the strategy of Differentiating Infected from Vaccinated Animals (DIVA)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Avian influenza A virus (IAV) surveillance in Northern California, USA, revealed unique IAV hemagglutinin (HA) genome sequences in cloacal swabs from lesser scaups. We found two closely related HA sequences in the same duck species in 2010 and 2013. Phylogenetic analyses suggest that both sequences belong to the recently discovered H19 subtype, which thus far has remained uncharacterized. We demonstrate that H19 does not bind the canonical IAV receptor sialic acid (Sia). Instead, H19 binds to the major histocompatibility complex class II (MHC class II), which facilitates viral entry. Unlike the broad MHC class II specificity of H17 and H18 from bat IAV, H19 exhibits a species-specific MHC class II usage that suggests a limited host range and zoonotic potential. Using cell lines overexpressing MHC class II, we rescued recombinant H19 IAV. We solved the H19 crystal structure and identified residues within the putative Sia receptor binding site (RBS) that impede Sia-dependent entry., Competing Interests: Declaration of interests The A.G.-S. laboratory has received research support from GSK, Pfizer, Senhwa Biosciences, Kenall Manufacturing, Blade Therapeutics, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N-fold LLC, Model Medicines, Atea Pharma, Applied Biological Laboratories, and Merck outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, Pfizer, and Prosetta, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott, and Astrazeneca. A.G.-S. is an inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines, influenza virus vaccines, and influenza virus therapeutics, which list F.K. as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, Castlevax, to develop SARS-CoV-2 vaccines. F.K. is a co-founder and scientific advisory board member of Castlevax. F.K. has consulted for Merck, Curevac, Seqirus, and Pfizer and is currently consulting for 3rd Rock Ventures, GSK, Gritstone, and Avimex. The Krammer laboratory is also collaborating with Dynavax on influenza vaccine development., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. The genome of DTMUV is translated into a polyprotein, which is further cleaved into several protein by viral NS2B3 protease and host proteases. Crucially, the cleavage of the NS2A/2B precursor during this process is essential for the formation of replication complexes and viral packaging. Previous research has demonstrated that alanine mutations in NS2A/2B (P1P1' (AA)) result in an attenuated strain (rDTMUV-NS2A/2B-P1P1' (AA)) by disrupting NS2A/2B cleavage. In this study, we investigate the effects of the P1P1' (AA) mutation on the viral life cycle and explore compensatory mutations in rDTMUV-NS2A/2B-P1P1' (AA). Infected ducklings exhibit similar body weight gain and viral tissue loads to DTMUV-WT. Compensatory mutations E-M349E and P1(T) emerge, restoring proliferation levels to those of rDTMUV-WT. Specifically, E-M349E enhances viral packaging, while P1(T) reinstates NS2A/2B proteolysis in vitro. Thus, our findings reveal novel compensatory sites capable of restoring the attenuated DTMUV during polyprotein cleavage and packaging., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The rapid spread of highly pathogenic avian influenza (HPAI) A (H5N1) viruses in Southeast Asia in 2004 prompted the New Zealand Ministry for Primary Industries to expand its avian influenza surveillance in wild birds. A total of 18,693 birds were sampled between 2004 and 2020, including migratory shorebirds (in 2004-2009), other coastal species (in 2009-2010), and resident waterfowl (in 2004-2020). No avian influenza viruses (AIVs) were isolated from cloacal or oropharyngeal samples from migratory shorebirds or resident coastal species. Two samples from red knots (Calidris canutus) tested positive by influenza A RT-qPCR, but virus could not be isolated and no further characterization could be undertaken. In contrast, 6179 samples from 15,740 mallards (Anas platyrhynchos) tested positive by influenza A RT-qPCR. Of these, 344 were positive for H5 and 51 for H7. All H5 and H7 viruses detected were of low pathogenicity confirmed by a lack of multiple basic amino acids at the hemagglutinin (HA) cleavage site. Twenty H5 viruses (six different neuraminidase [NA] subtypes) and 10 H7 viruses (two different NA subtypes) were propagated and characterized genetically. From H5- or H7-negative samples that tested positive by influenza A RT-qPCR, 326 AIVs were isolated, representing 41 HA/NA combinations. The most frequently isolated subtypes were H4N6, H3N8, H3N2, and H10N3. Multivariable logistic regression analysis of the relations between the location and year of sampling, and presence of AIV in individual waterfowl showed that the AIV risk at a given location varied from year to year. The H5 and H7 isolates both formed monophyletic HA groups. The H5 viruses were most closely related to North American lineages, whereas the H7 viruses formed a sister cluster relationship with wild bird viruses of the Eurasian and Australian lineages. Bayesian analysis indicates that the H5 and H7 viruses have circulated in resident mallards in New Zealand for some time. Correspondingly, we found limited evidence of influenza viruses in the major migratory bird populations visiting New Zealand. Findings suggest a low probability of introduction of HPAI viruses via long-distance bird migration and a unique epidemiology of AIV in New Zealand., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Stanislawek et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Emerging pathogens can threaten human and animal health, necessitating reliable surveillance schemes to enable preparedness. We evaluated the repeatability and reproducibility of a method developed previously during a single year at one study site. Hunter-harvested ducks and geese were sampled for avian influenza virus at three discrete locations in the UK. H5N1 highly pathogenic avian influenza (HPAIV) was detected in four species (mallard [Anas platyrhynchos], Eurasian teal [Anas crecca], Eurasian wigeon [Mareca penelope] and pink-footed goose [Anser brachyrhynchus]) across all three locations and two non-HPAIV H5N1, influenza A positive detections were made from a mallard and Eurasian wigeon at two locations. Virus was detected within 1-to-4 days of sampling at every location. Application of rapid diagnostic methods to samples collected from hunter-harvested waterfowl offers potential as an early warning system for the surveillance and monitoring of emerging and existing strains of avian influenza A viruses in key avian species., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Highly pathogenic avian influenza H5N6 and H5N1 viruses of clade 2.3.4.4b were simultaneously introduced into South Korea at the end of 2023. An outbreak at a broiler duck farm consisted of concurrent infection by both viruses. Sharing genetic information and international surveillance of such viruses in wild birds and poultry is critical.

Duck Tembusu Virus (DTMUV) is a pathogen of the Flaviviridae family that causes infections in poultry, leading to significant economic losses in the duck farming industry in recent years. Ducks infected with this virus exhibit clinical symptoms such as decreased egg production and neurological disorders, along with serious consequences such as ovarian hemorrhage, organ enlargement, and necrosis. Variations in morbidity and mortality rates exist across different age groups of ducks. It is worth noting that DTMUV is not limited to ducks alone; it can also spread to other poultry such as chickens and geese, and antibodies related to DTMUV have even been found in duck farm workers, suggesting a potential risk of zoonotic transmission. This article provides a detailed overview of DTMUV research, delving into its genomic characteristics, vaccines, and the interplay with host immune responses. These in-depth research findings contribute to a more comprehensive understanding of the virus's transmission mechanism and pathogenic process, offering crucial scientific support for epidemic prevention and control.

Gyroviruses are small single-stranded DNA (ssDNA) viruses that are largely associated with birds. Chicken anemia virus is the most extensively studied gyrovirus due to its disease impact on the poultry industry. However, we know much less about gyroviruses infecting other avian species. To investigate gyroviruses infecting waterfowl, we determined six complete genome sequences that fall into three gyrovirus groups, referred to as waterfowl gyrovirus 1 (n = 3), 2 (n = 2), and 3 (n = 1), in organs from hunter-harvested waterfowl from Arizona (USA). The waterfowl gyrovirus 1 variants were identified in multiple organs of a single American wigeon and represent a tentative new species. The waterfowl gyrovirus 2 variants were identified in the livers of two American wigeons and share >70% VP1 nucleotide sequence identity with gyrovirus 9, previously identified in the spleen of a Brazilian Pekin duck (MT318123) and a human fecal sample (KP742975). Waterfowl gyrovirus 3 was identified in a northern pintail spleen sample, and it shares >73% VP1 nucleotide sequence identity with two gyrovirus 13 sequences previously identified in Brazilian Pekin duck spleens (MT318125 and MT318127). These gyroviruses are the first to be identified in waterfowl in North America, as well as in American wigeons and northern pintails., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

An outbreak of duck astrovirus (DAstV) has occurred in duck farming regions of China, causing substantial economic setbacks in the duck industry. This investigation aimed to examine the variations in DAstV pathogenicity among ducks at different age intervals. Infections were induced in ducks at distinct age groups (1, 7, 14, 21, and 28 d) utilizing the DAstv-1-GDB-2022 strain. The results indicate increased pathogenicity of the DAstv-1-GDB-2022 strain in ducklings aged 21 to 28 d, manifesting as liver and kidney enlargement, severe bleeding, and potential fatalities. Conversely, ducklings aged 1 and 14 d displayed milder symptoms postinfection. Notably, viral shedding continued in ducks of diverse age groups even 21 d postinfection (Dpi). Moreover, DAstV replicates in various tissues, predominantly affecting the liver. Immunohistochemical tests using rabbit anti-DAstV antibodies revealed robust positive signals in both the liver and kidneys, which correlated with the clinical symptom severity observed through macroscopic and microscopic examinations. Serum biochemical assays and indirect ELISA demonstrated a consistent response to DAstV infection across different age groups, with older ducklings exhibiting increased sensitivity. In conclusion, this study successfully replicated clinical symptoms similar to those of natural DAstV infection using the DAstv-1-GDB-2022 strain. Importantly, we systematically delineated the differences in susceptibility to DAstV among ducks at various ages, laying the foundation for further research into the pathogenic mechanisms of DAstV and potential vaccine development., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Endogenous retroviruses (ERV) are viral genomes integrated into the host genome and can be stably inherited. Although ERV sequences have been reported in some avian species' genome, the duck endogenous retroviruses (DERV) genome has yet to be quantified. This study aimed to identify ERV sequences and characterize genes near ERVs in the duck genome by utilizing LTRhavest and LTRdigest tools to forecast the duck genome and analyze the distribution of ERV copies. The results revealed 1,607, 2,031, and 1,908 full-length ERV copies in the Pekin duck (ZJU1.0), Mallard (CAU&#95;wild&#95;1.0), and Shaoxing duck (CAU&#95;laying&#95;1.0) genomes, respectively, with average lengths of 7,046, 7,027, and 6,945 bp. ERVs are mainly distributed on the 1, 2, and sex chromosomes. Phylogenetic analysis demonstrated the presence of Betaretrovirus in 3 duck genomes, whereas Alpharetrovirus was exclusively identified in the Shaoxing duck genome. Through screening, 596, 315, and 343 genes adjacent to ERV were identified in 3 duck genomes, respectively, and their functions of ERV neighboring genes were predicted. Functional enrichment analysis of ERV-adjacent genes revealed enrichment for Focal adhesion, Calcium signaling pathway, and Adherens junction in 3 duck genomes. The overlapped genes were highly expressed in 8 tissues (brain, fat, heart, kidney, liver, lung, skin, and spleen) of 8-wk-old Mallard, revealing their important expression in different tissues. Our study provides a new perspective for understanding the quantity and function of DERVs, and may also provide important clues for regulating nearby genes and affecting the traits of organisms., (Copyright © 2024. Published by Elsevier Inc.)

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus-like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors., (© 2024. The Author(s).)

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N -acetylneuraminic acid (NeuAc) or N -glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo . We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo , the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N -glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N -glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N -acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N -glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N -glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection., Competing Interests: The authors declare no conflict of interest.

Highly pathogenic avian influenza (HPAI) H5-viruses are circulating in wild birds and are repeatedly introduced to poultry causing outbreaks in the Netherlands since 2014. The largest epizootic ever recorded in Europe was caused by HPAI H5N1 clade 2.3.4.4b viruses in the period 2021-2022. The recent H5-clade 2.3.4.4 viruses were found to differ in their virulence for chickens and ducks. Viruses causing only mild disease may remain undetected, increasing the risk of virus spread to other farms, wild birds and mammals. We developed in ovo models to determine the virulence of HPAI viruses for chickens and ducks, which are fast and have low costs. The virulence of five contemporary H5-viruses was compared studying replication rate, average time to death and virus spread in the embryo. Remarkable differences in virulence were observed between H5-viruses and between poultry species. The H5N1-2021 virus was found to have a fast replication rate in both the chicken and duck in ovo models, but a slower systemic virus dissemination compared to three other H5-clade 2.3.4.4b viruses. The results show the potential of in ovo models to quickly determine the virulence of novel HPAI viruses, and study potential virulence factors which can help to better guide the surveillance in poultry.

Duck hepatitis B virus (DHBV) is an avian member of the hepatotropic DNA viruses, or hepadnaviridae. It shares with the human hepatitis B virus (HBV) a similar genomic organization and replication strategy via reverse transcription, but is simpler than HBV in lacking the X gene and in expressing just two coterminal envelope proteins: Large (L) and small (S). DHBV has been extensively used as a convenient and valuable animal model for study of the hepadnaviral life cycle, and for drug screening in vitro but also in vivo. Ducks and primary duck hepatocytes (PDHs) are inexpensive, easily accessible, and readily infected with DHBV. The high levels of genome replication and protein expression in duck liver and PDHs also facilitate monitoring of viral life cycle using conventional molecular biology techniques such as Southern blot for replicative DNA and covalently closed circular DNA (cccDNA), Northern blot for viral RNAs, and Western blot for viral proteins., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Importance: Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that replicates well in mosquito, bird, and mammalian cells. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in the serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and poses a threat to mammalian health. Thus, understanding the pathogenic mechanism of DTMUV is crucial for identifying potential antiviral targets. In this study, we discovered that NS3 can induce the mitochondria-mediated apoptotic pathway through the PERK/PKR pathway; it can also interact with voltage-dependent anion channel 2 to induce apoptosis. Our findings provide a theoretical basis for understanding the pathogenic mechanism of DTMUV infection and identifying potential antiviral targets and may also serve as a reference for exploring the pathogenesis of other flaviviruses., Competing Interests: The authors declare no conflict of interest.

The placenta-specific 8 (PLAC8) gene, also known as ONZIN or C15, codes for a cysteine-rich peptide originally identified in mouse placental tissue and subsequently identified in a variety of epithelial tissues and immune cells. PLAC8 is also expressed in birds, such as ducks, where its functional roles remain unknown. Here, we aimed to determine the mRNA and protein expression profiles and the functional role of duck PLAC8 during the infection of duck hepatitis A virus type 1 (DHAV-1). We found that the duck PLAC8 is also a cysteine-rich polypeptide composed of 114 amino acid residues, with no signal peptide. Duck PLAC8 is highly expressed in the immune organs of young cherry valley ducks, including the thymus, bursa fabricius, and spleen. However, it has negligible expression level in liver, brain, kidney, and heart. Additionally, PLAC8 expression was considerably induced after DHAV-1 infection both in vitro and in vivo, especially in the immune organs of ducklings. This tissue expression distribution and induction upon infection suggest that PLAC8 might play a critical role in innate immunity. Our data showed that PLAC8 significantly suppressed the expression of Toll-like receptor 7 (TLR7), leading to decreased expression of downstream signaling molecules including myeloid differentiation primary response gene 88 (MyD88) and nuclear factor kappa-B (NF-κB). This ultimately resulted in low levels of type I interferon and interleukin 6 (IL-6). Additionally, PLAC8 positively regulated DHAV-1 replication levels. RNAi against PLAC8 in duck embryo fibroblasts considerably inhibited DHAV-1 propagation, while PLAC8 overexpression significantly facilitated DHAV-1 replication., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The cytosolic DNA sensing pathway mediates innate immune defense against infection by many DNA viruses; however, viruses have evolved multiple strategies to evade the host immune response. Duck enteritis virus (DEV) causes an acute and contagious disease with high mortality in waterfowl. The mechanisms employed by DEV to block the DNA sensing pathway are not well understood. Here, we sought to investigate the role of DEV US3, a serine/threonine protein kinase, in the inhibition of DNA sensing. We found that ectopic expression of DEV US3 significantly inhibited the production of IFN-β and expression of interferon-stimulated genes induced by interferon-stimulatory DNA and poly(dA-dT). US3 also inhibited viral DNA-triggered IFN-β activation and promoted DEV replication in duck embryo fibroblasts, while knockdown of US3 during DEV infection enhances the IFN-β response and suppresses viral replication. US3 inhibited the DNA-sensing signaling pathway by targeting interferon regulatory factor 7 (IRF7), and the kinase activity of US3 was indispensable for its inhibitory function. Furthermore, we found that US3 interacts with the activation domain of IRF7, phosphorylating IRF7, blocking its dimerization and nuclear translocation, and finally leading to the inhibition of IFN-β production. These findings expand our knowledge on DNA sensing in ducks and reveal a novel mechanism whereby DEV evades host antiviral immunity. IMPORTANCE Duck enteritis virus (DEV) is a duck alphaherpesvirus that causes an acute and contagious disease with high mortality, resulting in substantial economic losses in the commercial waterfowl industry. The evasion of DNA-sensing pathway-mediated antiviral innate immunity is essential for the persistent infection and replication for many DNA viruses. However, the strategies used by DEV to block the DNA-sensing pathway are not well understood. In this study, DEV US3 protein kinase was demonstrated to inhibit the DNA-sensing signaling via binding to the activation domain of interferon regulatory factor 7 (IRF7), which induced the hyperphosphorylation of IRF7 and abolished IRF7 dimerization and nuclear translocation. Our findings provide insights into how duck herpesviral kinase counteracts host antiviral innate immunity to ensure viral replication and spread.

Subtype H7 avian influenza A viruses (IAVs) are enzootic in wild aquatic birds and have caused sporadic spillovers into domestic poultry and humans. Here, we determined the distribution of fucosylated α2,3 sialoglycan (i.e., sialyl Lewis X [SLe X ]) in chickens and five common dabbling duck species and the association between SLe X and cell/tissue/host tropisms of H7 IAVs. Receptor binding analyses showed that H7 IAVs bind to both α2,3-linked (SA2,3Gal) and α2,6-linked sialic acids (SA2,6Gal), but with a higher preference for SLe X ; H7 IAVs replicated more efficiently in SLe X -overexpressed than SLe X -deficient MDCK cells. While chickens and all tested dabbling ducks expressed abundant SA2,3Gal and SA2,6Gal, SLe X was detected in both respiratory and gastrointestinal tissues of chickens and mallard ducks and in only the respiratory tissues of gadwall, green-wing teal, and northern shoveler but not in wood ducks. Viral-tissue binding assays showed that H7 IAVs bind to chicken colon crypt cells that express SLe X but fewer bind to mallard colon crypt cells, which do not express SLe X ; H7 IAVs bind efficiently to epithelial cells of all tissues expressing SA2,3Gal. High viral replication was identified in both chickens and mallards infected with an H7 virus, regardless of SLe X expression, and viruses were detected in all cells to the same degree as viruses detected in the viral-tissue binding assays. In summary, this study suggests that SLe X facilitates infection of H7 viruses, but other types of SA2,3Gal glycan receptors shape the tissue/host tropisms of H7 IAVs. IMPORTANCE In addition to causing outbreaks in domestic poultry, subtype H7 IAVs can cause sporadic spillover infections in lower mammals and humans. In this study, we showed that SLe X expression varies among wild dabbling ducks. Although it facilitated virus binding and affected infection of H7 IAV in cells, SLe X expression is not the only determinant of viral replication at either the tissue or host level. This study suggested that access to heterologous SA2,3Gal glycan receptors, including fucosylated α2,3-linked sialoglycans, shape tissue and host tropism of H7 IAVs in aquatic wild birds.

Previous studies identified three neutralizing epitopes on domains I, II, and III of the Tembusu virus (TMUV) envelope (E). More evidence is needed to understand the molecular basis of Ab-mediated neutralization and protection against TMUV. In this study, we observed a neutralizing mAb, 6C8, that neutralized TMUV infection primarily by inhibiting cell attachment. In immunofluorescence assays, 6C8 recognized the premembrane and E proteins coexpressed in HEK-293T cells, but failed to react with premembrane or E expressed individually. Epitope mapping identified nine E protein residues positioned on BC/EF loops and F/G strands in domain III and the first α-helical domain in the stem region. Further investigation with mutant viruses showed that 6C8 pressure resulted in mutations at residues 330 of BC loop and 409 of the first α-helical domain, although 6C8 only exhibited a moderate neutralizing activity in BHK-21 cells and a weak protective activity in BALB/c mice and Shaoxing duck models. Mutations A330S and T409M conferred high- and low-level 6C8 resistance, respectively, whereas the combination of A330S and T409M mutations conferred moderate-level 6C8 resistance. As a result, a quasispecies comprising three groups of antigenic variants appeared in BHK-21 cell-derived viral stocks after repeated passages of TMUV strain Y in the presence of 6C8 treatment. Taken together, these findings have raised a concern about Ab-induced antigenic variations in vivo, and they have revealed information concerning the conformational structure of the 6C8 epitope and its role in constraint on antigenic variations. The present work contributes to a better understanding of the complexity of the TMUV immunogen., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Background: Duck plague virus (DPV), belonging to herpesviruses, is a linear double-stranded DNA virus. There are many reports about the outbreak of the duck plague in a variety of countries, which caused huge economic losses. Recently, increasing reports revealed that multiple long non-coding RNAs (lncRNAs) can possess great potential in the regulation of host antiviral immune response. Furthermore, it remains to be determined which specific molecular mechanisms are responsible for the DPV-host interaction in host immunity. Here, lncRNAs and mRNAs in DPV infected duck embryonic fibroblast (DEF) cells were identified by high-throughput RNA-sequencing (RNA-seq). And we predicted target genes of differentially expressed genes (DEGs) and formed a complex regulatory network depending on in-silico analysis and prediction., Result: RNA-seq analysis results showed that 2921 lncRNAs were found at 30 h post-infection (hpi). In our study, 218 DE lncRNAs and 2840 DE mRNAs were obtained in DEF after DPV infection. Among these DEGs and target genes, some have been authenticated as immune-related molecules, such as a Macrophage mannose receptor (MR), Anas platyrhynchos toll-like receptor 2 (TLR2), leukocyte differentiation antigen, interleukin family, and their related regulatory factors. Furthermore, according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis, we found that the target genes may have important effects on biological development, biosynthesis, signal transduction, cell biological regulation, and cell process. Also, we obtained, the potential targeting relationship existing in DEF cells between host lncRNAs and DPV-encoded miRNAs by software., Conclusions: This study revealed not only expression changes, but also the possible biological regulatory relationship of lncRNAs and mRNAs in DPV infected DEF cells. Together, these data and analyses provide additional insight into the role of lncRNAs and mRNAs in the host's immune response to DPV infection., (© 2022. The Author(s).)