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.

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.

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.

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.

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.

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.

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.

Survey of low pathogenic avian influenza viruses in live poultry markets in Guangxi Province, Southern China, 2016-2019.

Low pathogenic avian influenza viruses (LPAIVs) have been widespread in poultry and wild birds throughout the world for many decades. LPAIV infections are usually asymptomatic or cause subclinical symptoms. However, the genetic reassortment of LPAIVs may generate novel viruses with increased virulence and cross-species transmission, posing potential risks to public health. To evaluate the epidemic potential and infection landscape of LPAIVs in Guangxi Province, China, we collected and analyzed throat and cloacal swab samples from chickens, ducks and geese from the live poultry markets on a regular basis from 2016 to 2019. Among the 7,567 samples, 974 (12.87%) were LPAIVs-positive, with 890 single and 84 mixed infections. Higher yearly isolation rates were observed in 2017 and 2018. Additionally, geese had the highest isolation rate, followed by ducks and chickens. Seasonally, spring had the highest isolation rate. Subtype H3, H4, H6 and H9 viruses were detected over prolonged periods, while H1 and H11 viruses were detected transiently. The predominant subtypes in chickens, ducks and geese were H9, H3, and H6, respectively. The 84 mixed infection samples contained 22 combinations. Most mixed infections involved two subtypes, with H3 + H4 as the most common combination. Our study provides important epidemiological data regarding the isolation rates, distributions of prevalent subtypes and mixed infections of LPAIVs. These results will improve our knowledge and ability to control epidemics, guide disease management strategies and provide early awareness of newly emerged AIV reassortants with pandemic potential.

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.

NOD1 Is Associated With the Susceptibility of Pekin Duck Flock to Duck Hepatitis A Virus Genotype 3.

Duck viral hepatitis (DVH) is an acute, highly lethal infectious disease of ducklings that causes huge losses in the duck industry. Duck hepatitis A virus genotype 3 (DHAV-3) has been one of the most prevalent DVH pathogen in the Asian duck industry in recent years. Here, we investigated the genetic basis of the resistance and susceptibility of ducks to DVH by comparing the genomes and transcriptomes of a resistant Pekin duck flock (Z8) and a susceptible Pekin duck flock (SZ7). Our comparative genomic and transcriptomic analyses suggested that NOD1 showed a strong signal of association with DVH susceptibility in ducks. Then, we found that NOD1 showed a significant expression difference between the livers of susceptible and resistant individuals after infection with DHAV-3, with higher expression in the SZ7 flock. Furthermore, suppression and overexpression experiments showed that the number of DHAV-3 genomic copies in primary duck hepatocytes was influenced by the expression level of NOD1. In addition, in situ RNAscope analysis showed that the localization of NOD1 and DHAV-3 in liver cells was consistent. Altogether, our data suggested that NOD1 was likely associated with DHAV-3 susceptibility in ducks, which provides a target for future investigations of the pathogenesis of DVH.

Comparative susceptibility of the common teal (Anas crecca) to infection with high pathogenic avian influenza virus strains isolated in Japan in 2004-2017.

High pathogenic avian influenza viruses (HPAIVs) of the H5 subtype have spread in poultry and wild birds worldwide. Current studies have highlighted the association between the migration of wild birds and the spread of HPAIVs. However, virological studies examining responsible species of migratory birds to spread HPAIVs are limited. In Japan, the common teal (Anas crecca) arrives in great numbers for overwintering every autumn-spring season; therefore, we performed experimental infection using six H5 HPAIVs isolated in past outbreaks in Japan (A/chicken/Yamaguchi/7/2004 (H5N1), A/whooper swan/Akita/1/2008 (H5N1), A/mandarin duck/Miyazaki/22M-765/2011 (H5N1), A/duck/Chiba/26-372-48/2014 (H5N8), A/duck/Hyogo/1/2016 (H5N6) and A/mute swan/Shimane/3211A002/2017 (H5N6)) to evaluate the susceptibility of the species to HPAIV infection. The results illustrated that most birds in all experimental groups were infected by the strains, and they shed viruses for a prolonged period, in trachea than cloaca, without displaying distinctive clinical signs. In addition, comparative analysis using calculation value of total viral shedding during the experiment revealed that the birds shed viruses at above a certain level regardless of the differences of strains. These results suggested that the common teal could be a migratory bird species that disseminates viruses in the environment, thereby influencing HPAI outbreaks in wild birds in Japan.

Genetic Characterization and Pathogenesis of Three Novel Reassortant H5N2 Viruses in South Korea, 2018.

The outbreaks of H5N2 avian influenza viruses have occasionally caused the death of thousands of birds in poultry farms. Surveillance during the 2018 winter season in South Korea revealed three H5N2 isolates in feces samples collected from wild birds (KNU18-28: A/Wild duck/South Korea/KNU18-28/2018, KNU18-86: A/Bean Goose/South Korea/KNU18-86/2018, and KNU18-93: A/Wild duck/South Korea/KNU18-93/2018). Phylogenetic tree analysis revealed that these viruses arose from reassortment events among various virus subtypes circulating in South Korea and other countries in the East Asia-Australasian Flyway. The NS gene of the KNU18-28 and KNU18-86 isolates was closely related to that of China's H10N3 strain, whereas the KNU18-93 strain originated from the H12N2 strain in Japan, showing two different reassortment events and different from a low pathogenic H5N3 (KNU18-91) virus which was isolated at the same day and same place with KNU18-86 and KNU18-93. These H5N2 isolates were characterized as low pathogenic avian influenza viruses. However, many amino acid changes in eight gene segments were identified to enhance polymerase activity and increase adaptation and virulence in mice and mammals. Experiments reveal that viral replication in MDCK cells was quite high after 12 hpi, showing the ability to replicate in mouse lungs. The hematoxylin and eosin-stained (H&E) lung sections indicated different degrees of pathogenicity of the three H5N2 isolates in mice compared with that of the control H1N1 strain. The continuing circulation of these H5N2 viruses may represent a potential threat to mammals and humans. Our findings highlight the need for intensive surveillance of avian influenza virus circulation in South Korea to prevent the risks posed by these reassortment viruses to animal and public health.

Tissue Specific Transcriptome Changes Upon Influenza A Virus Replication in the Duck.

Ducks are the natural host and reservoir of influenza A virus (IAV), and as such are permissive to viral replication while being unharmed by most strains. It is not known which mechanisms of viral control are globally regulated during infection, and which are specific to tissues during infection. Here we compare transcript expression from tissues from Pekin ducks infected with a recombinant H5N1 strain A/Vietnam 1203/04 (VN1203) or an H5N2 strain A/British Columbia 500/05 using RNA-sequencing analysis and aligning reads to the NCBI assembly ZJU1.0 of the domestic duck (Anas platyrhynchos) genome. Highly pathogenic VN1203 replicated in lungs and showed systemic dissemination, while BC500, like most low pathogenic strains, replicated in the intestines. VN1203 infection induced robust differential expression of genes all three days post infection, while BC500 induced the greatest number of differentially expressed genes on day 2 post infection. While there were many genes globally upregulated in response to either VN1203 or BC500, tissue specific gene expression differences were observed. Lungs of ducks infected with VN1203 and intestines of birds infected with BC500, tissues important in influenza replication, showed highest upregulation of pattern recognition receptors and interferon stimulated genes early in the response. These tissues also appear to have specific downregulation of inflammatory components, with downregulation of distinct sets of proinflammatory cytokines in lung, and downregulation of key components of leukocyte recruitment and complement pathways in intestine. Our results suggest that global and tissue specific regulation patterns help the duck control viral replication as well as limit some inflammatory responses in tissues involved in replication to avoid damage.

Single-Cell Analysis of the In Vivo Dynamics of Host Circulating Immune Cells Highlights the Importance of Myeloid Cells in Avian Flaviviral Infection.

Ducks are an economically important waterfowl but a natural reservoir for some zoonotic pathogens, such as influenza virus and flaviviruses. Our understanding of the duck immune system and its interaction with viruses remains incomplete. In this study, we constructed the transcriptomic landscape of duck circulating immune cells, the first line of defense in the arthropod-borne transmission of arboviruses, using high-throughput single-cell transcriptome sequencing, which defined 14 populations of peripheral blood leukocytes (PBLks) based on distinct molecular signatures and revealed differences in the clustering of PBLks between ducks and humans. Taking advantage of in vivo sex differences in the susceptibility of duck PBLks to avian tembusu virus (TMUV) infection, a mosquito-borne flavivirus newly emerged from ducks with a broad host range from mosquitos to mammals, a comprehensive comparison of the in vivo dynamics of duck PBLks upon TMUV infection between sexes was performed at the single-cell level. Using this in vivo model, we discovered that TMUV infection reprogrammed duck PBLks differently between sexes, driving the expansion of granulocytes and priming granulocytes and monocytes for antiviral immune activation in males but decreasing the antiviral immune activity of granulocytes and monocytes by restricting their dynamic transitions from steady states to antiviral states with a decrease in the abundance of circulating monocytes in females. This study provides insights into the initial immune responses of ducks to arthropod-borne flaviviral infection and provides a framework for studying duck antiviral immunity.