Development and evaluation of a chicken embryo fibroblast cell culture based live attenuated Indian strain duck plague vaccine.

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 107.5 TCID50/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.

In Ovo Models to Predict Virulence of Highly Pathogenic Avian Influenza H5-Viruses for Chickens and Ducks.

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.

Cross-species spill-over potential of the H9N2 bat influenza A virus.

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.

Complex N-glycans are important for interspecies transmission of H7 influenza A viruses.

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.

Genome-wide characterization and comparison of endogenous retroviruses among 3 duck reference genomes.

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_wild_1.0), and Shaoxing duck (CAU_laying_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.

Impact of duck astrovirus on susceptibility to infection across duck ages.

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.

Duck Tembusu virus NS3 protein induces apoptosis by activating the PERK/PKR pathway and mitochondrial pathway.

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.

Placenta-specific 8 facilitates the infection of duck hepatitis A virus type 1 by inhibiting the TLR7 MyD88-dependent signaling pathway.

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.

Duck Enteritis Virus Protein Kinase US3 Inhibits DNA Sensing Signaling by Phosphorylating Interferon Regulatory Factor 7.

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-ß and expression of interferon-stimulated genes induced by interferon-stimulatory DNA and poly(dA-dT). US3 also inhibited viral DNA-triggered IFN-ß activation and promoted DEV replication in duck embryo fibroblasts, while knockdown of US3 during DEV infection enhances the IFN-ß 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-ß 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.

The Sialyl Lewis X Glycan Receptor Facilitates Infection of Subtype H7 Avian Influenza A Viruses.

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 [SLeX]) in chickens and five common dabbling duck species and the association between SLeX 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 SLeX; H7 IAVs replicated more efficiently in SLeX-overexpressed than SLeX-deficient MDCK cells. While chickens and all tested dabbling ducks expressed abundant SA2,3Gal and SA2,6Gal, SLeX 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 SLeX but fewer bind to mallard colon crypt cells, which do not express SLeX; 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 SLeX 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 SLeX 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 SLeX expression varies among wild dabbling ducks. Although it facilitated virus binding and affected infection of H7 IAV in cells, SLeX 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.

Differential expression profile and in-silico functional analysis of long noncoding RNA and mRNA in duck embryo fibroblasts infected with duck plague virus.

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.

An Antibody Neutralization Determinant on Domain III and the First α-Helical Domain in the Stem-Anchor Region of Tembusu Virus Envelope Protein.

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.

A new clade 2.3.4.4b H5N1 highly pathogenic avian influenza genotype detected in Europe in 2021.

Despite their widespread distribution, only a single genotype variant of clade 2.3.4.4b H5N1 influenza viruses has been found so far in Europe. Here, we report the detection of a new highly pathogenic avian influenza H5N1 genotype in geese and ducks from a backyard farm in the Czech Republic. Phylogenetic analysis revealed that the Czech H5N1 virus retained the A/Eurasian_Wigeon/Netherlands/1/2020-like backbone with an altered PB2 segment obtained from co-circulating low-pathogenic avian influenza viruses.

Effects of duck circovirus on immune function and secondary infection of Avian Pathogenic Escherichia coli.

Duck circovirus (DuCV) infection occurs frequently in ducks in China and is generally believed to lead to immunosuppression and secondary infection, though there has been a lack of detailed research and direct evidence. In this study, one-day-old Cherry Valley ducklings were artificially infected with DuCV alone and co-infected with DuCV and Avian Pathogenic Escherichia coli (APEC). The immune indexes at 32 d old were systematically monitored, including immune organ weight, lymphocyte transformation rate, IL-10, IL-12, soluble CD4 (sCD4), soluble CD8 (sCD8), IFN-γ, viral loads in each organ, APEC colonization, and so on. The results showed the development of immune organs in ducklings was affected, resulting in a decrease in the lymphocyte transformation rate (LTR), IL-12, sCD4, sCD8, IFN-γ and an increase in IL-10 content at 8 to 32 d postinfection (dpi). In the detection of virus loads in some organs, it was found that 8 dpi, DuCV existed stably in various organs, suggesting the importance of preventing and controlling the virus in the early stage of culture. The results of exploring the DuCV infection that shows some influence on secondary infection by APEC. The results showed that DuCV infection could significantly enhance the pathogenicity of APEC and the colonization ability of APEC in vivo. DuCV can induce more serious APEC infection in 24 dpi than in 14 dpi. Based on the above results, it can be concluded that DuCV infection will affect the immune system, cause immunosuppression, and lead to more serious secondary infection.

Non-Structural Protein 3 of Duck Tembusu Virus Induces Autophagy via the ERK and PI3K-AKT-mTOR Signaling Pathways.

Despite autophagy's pivotal role in the replication of viruses such as duck Tembusu virus (DTMUV), which has caused massive economic losses to the poultry industry in the world, the specific relationships between DTMUV and cellular autophagy remain largely unknown. In response, we investigated the interactions between autophagy and DTMUV, the effects of the structural and non-structural proteins of DTMUV on autophagy, and the autophagy-related signaling pathways induced by DTMUV. Among the results, DTMUV increased the autophagy flux in duck embryo fibroblasts (DEF) and BHK-21 cells, while autophagy facilitated viral replication. After we pharmacologically induced autophagy with rapamycin (RAPA), the replication of DTMUV increased by 15.23-fold compared with the control group of DEF cells. To identify which DTMUV protein primarily induced autophagy, all three structural proteins and seven non-structural proteins of DTMUV were transfected into cells, and the results showed that non-structural protein 3 (NS3) induced significant autophagy in DEF cells. By means of Western blot, immunofluorescence, and transmission electron microscopy, we confirmed that NS3 protein could significantly induce autophagy and autophagy flux. Furthermore, we showed that NS3 induced autophagy in DEF cells through extracellular signal-regulated kinase 2 (ERK2) and phosphatidylinositol-3-kinase (PI3K)/AKT and the mammalian target of rapamycin (mTOR) signaling pathways using specific inhibitors and RNA interference assays. Finally, autophagy induced by NS3 promoted DTMUV replication. These results provide novel insight into the relationship between DTMUV and autophagy, broadening the current understanding of the molecular pathogenesis of DTMUV.

Surveillance and genetic diversity analysis of avian astrovirus in China.

Avian astroviruses (AAstVs) have caused major problem for poultry breeding industries in China in recent years, and the goose gout caused by goose astrovirus has produced particularly great economic losses. To better understand the prevalence and genetic diversity of AAstVs in China, 1210 poultry samples collected from eight provinces were tested with reverse transcription-polymerase chain reaction (RT-PCR) to detect AAstV infections in different poultry populations. Then, Open reading frames 2 (ORF2) was amplified by specific primers, and the genetic evolution was analyzed. Our surveillance data demonstrate the diversity of AAstVs in China insofar as we detected 17 AAstVs, including seven chicken astroviruses (CAstVs), five avian nephritis viruses (ANVs), two goose astroviruses (GoAstVs), two duck astrovirus (DAstVs), and one new AAstV belonging to Avastrovirus Group 3. The positive rate of AAstV infection was 1.40%. Host analysis showed that CAstVs and ANVs were isolated from chickens, DAstVs and GoAstVs were isolated from ducks. Host-species-specific AAstVs infections were also identified in numerous samples collected at each stage of production. This study provides further evidence to better understand the epidemiology of AAstVs in different species of poultry in China.

Tropism of Highly Pathogenic Avian Influenza H5 Viruses from the 2020/2021 Epizootic in Wild Ducks and Geese.

Highly pathogenic avian influenza (HPAI) outbreaks have become increasingly frequent in wild bird populations and have caused mass mortality in many wild bird species. The 2020/2021 epizootic was the largest and most deadly ever reported in Europe, and many new bird species tested positive for HPAI virus for the first time. This study investigated the tropism of HPAI virus in wild birds. We tested the pattern of virus attachment of 2020 H5N8 virus to intestinal and respiratory tissues of key bird species; and characterized pathology of naturally infected Eurasian wigeons (Mareca penelope) and barnacle geese (Branta leucopsis). This study determined that 2020 H5N8 virus had a high level of attachment to the intestinal epithelium (enterotropism) of dabbling ducks and geese and retained attachment to airway epithelium (respirotropism). Natural HPAI 2020 H5 virus infection in Eurasian wigeons and barnacle geese also showed a high level of neurotropism, as both species presented with brain lesions that co-localized with virus antigen expression. We concluded that the combination of respirotropism, neurotropism, and possibly enterotropism, contributed to the successful adaptation of 2020/2021 HPAI H5 viruses to wild waterbird populations.

A Highly Divergent Hepacivirus Identified in Domestic Ducks Further Reveals the Genetic Diversity of Hepaciviruses.

Hepaciviruses represent a group of viruses that pose a significant threat to the health of humans and animals. During the last decade, new members of the genus Hepacivirus have been identified in various host species worldwide, indicating the widespread distribution of genetically diversified hepaciviruses among animals. By applying unbiased high-throughput sequencing, a novel hepacivirus, provisionally designated Hepacivirus Q, was discovered in duck liver samples collected in Guangdong province of China. Genetic analysis revealed that the complete polyprotein of Hepacivirus Q shares 23.9-46.6% amino acid identity with other representatives of the genus Hepacivirus. Considering the species demarcation criteria for hepaciviruses, Hepacivirus Q should be regarded as a novel hepacivirus species of the genus Hepacivirus within the family Flaviviridae. Phylogenetic analyses also indicate the large genetic distance between Hepacivirus Q and other known hepaciviruses. Molecular detection of this novel hepacivirus showed an overall prevalence of 15.9% in duck populations in partial areas of Guangdong province. These results expand knowledge about the genetic diversity and evolution of hepaciviruses and indicate that genetically divergent hepaciviruses are circulating in duck populations in China.

Reduced Replication of Highly Pathogenic Avian Influenza Virus in Duck Endothelial Cells Compared to Chicken Endothelial Cells Is Associated with Stronger Antiviral Responses.

Highly pathogenic avian influenza viruses (HPAIVs) cause fatal systemic infections in chickens, which are associated with endotheliotropism. HPAIV infections in wild birds are generally milder and not endotheliotropic. Here, we aimed to elucidate the species-specific endotheliotropism of HPAIVs using primary chicken and duck aortic endothelial cells (chAEC and dAEC respectively). Viral replication kinetics and host responses were assessed in chAEC and dAEC upon inoculation with HPAIV H5N1 and compared to embryonic fibroblasts. Although dAEC were susceptible to HPAIV upon inoculation at high multiplicity of infection, HPAIV replicated to lower levels in dAEC than chAEC during multi-cycle replication. The susceptibility of duck embryonic endothelial cells to HPAIV was confirmed in embryos. Innate immune responses upon HPAIV inoculation differed between chAEC, dAEC, and embryonic fibroblasts. Expression of the pro-inflammatory cytokine IL8 increased in chicken cells but decreased in dAEC. Contrastingly, the induction of antiviral responses was stronger in dAEC than in chAEC, and chicken and duck fibroblasts. Taken together, these data demonstrate that although duck endothelial cells are permissive to HPAIV infection, they display markedly different innate immune responses than chAEC and embryonic fibroblasts. These differences may contribute to the species-dependent differences in endotheliotropism and consequently HPAIV pathogenesis.

Detection of a Novel Reassortant H9N9 Avian Influenza Virus in Free-Range Ducks in Bangladesh.

Wild aquatic birds are the primary natural reservoir for influenza A viruses (IAVs). In this study, an A(H9N9) influenza A virus (A/duck/Bangladesh/44493/2020) was identified via routine surveillance in free-range domestic ducks in Bangladesh. Phylogenetic analysis of hemagglutinin showed that the H9N9 virus belonged to the Y439-like lineage. The HA gene had the highest nucleotide identity to A/Bean Goose (Anser fabalis)/South Korea/KNU 2019-16/2019 (H9N2). The other seven gene segments clustered within the Eurasian lineage.