Duck Cells Research Articles

Influenza viruses production: Evaluation of a novel avian cell line DuckCelt®-T17

The influenza vaccine manufacturing industry is looking for production cell lines that are easily scalable, highly permissive to multiple viruses, and more effective in term of viral productivity. One critical characteristic of such cell lines is their ability to grow in suspension, in serum free conditions and at high cell densities. Influenza virus causing severe epidemics both in human and animals is an important threat to world healthcare. The repetitive apparition of influenza pandemic outbreaks in the last 20years explains that manufacturing sector is still looking for more effective production processes to replace/supplement embryonated egg-based process. Cell-based production strategy, with a focus on avian cell lines, is one of the promising solutions. Three avian cell lines, namely duck EB66®cells (Valneva), duck AGE.CR® cells (Probiogen) and quail QOR/2E11 cells (Baxter), are now competing with traditional mammalian cell platforms (Vero and MDCK cells) used for influenza vaccine productions and are currently at advance stage of commercial development for the manufacture of influenza vaccines.The DuckCelt®-T17 cell line presented in this work is a novel avian cell line developed by Transgene. This cell line was generated from primary embryo duck cells with the constitutive expression of the duck telomerase reverse transcriptase (dTERT). The DuckCelt®-T17 cells were able to grow in batch suspension cultures and serum-free conditions up to 6.5×106cell/ml and were easily scaled from 10ml up to 3l bioreactor. In the present study, DuckCelt®-T17 cell line was tested for its abilities to produce various human, avian and porcine influenza strains. Most of the viral strains were produced at significant infectious titers (>5.8 log TCID50/ml) with optimization of the infection conditions. Human strains H1N1 and H3N2, as well as all the avian strains tested (H5N2, H7N1, H3N8, H11N9, H12N5) were the most efficiently produced with highest titre reached of 9.05 log TCID50/ml for A/Panama/2007/99 influenza H3N2. Porcine strains were also greatly rescued with titres from 4 to 7 log TCID50/ml depending of the subtypes. Interestingly, viral kinetics showed maximal titers reached at 24h post-infection for most of the strains, allowing early harvest time (Time Of Harvest: TOH). The B strains present specific production kinetics with a delay of 24h before reaching the maximal viral particle release. Process optimization on H1N1 2009 human pandemic strain allowed identifying best operating conditions for production (MOI, trypsin concentration, cell density at infection) allowing improving the production level by 2 log. Our results suggest that the DuckCelt®-T17 cell line is a very promising platform for industrial production of influenza viruses and particularly for avian viral strains.

Cross-Species Antiviral Activity of Goose Interferons against Duck Plague Virus Is Related to Its Positive Self-Feedback Regulation and Subsequent Interferon Stimulated Genes Induction.

Interferons are a group of antiviral cytokines acting as the first line of defense in the antiviral immunity. Here, we describe the antiviral activity of goose type I interferon (IFNα) and type II interferon (IFNγ) against duck plague virus (DPV). Recombinant goose IFNα and IFNγ proteins of approximately 20 kDa and 18 kDa, respectively, were expressed. Following DPV-enhanced green fluorescent protein (EGFP) infection of duck embryo fibroblast cells (DEFs) with IFNα and IFNγ pre-treatment, the number of viral gene copies decreased more than 100-fold, with viral titers dropping approximately 100-fold. Compared to the control, DPV-EGFP cell positivity was decreased by goose IFNα and IFNγ at 36 hpi (3.89%; 0.79%) and 48 hpi (17.05%; 5.58%). In accordance with interferon-stimulated genes being the “workhorse” of IFN activity, the expression of duck myxovirus resistance (Mx) and oligoadenylate synthetases-like (OASL) was significantly upregulated (p < 0.001) by IFN treatment for 24 h. Interestingly, duck cells and goose cells showed a similar trend of increased ISG expression after goose IFNα and IFNγ pretreatment. Another interesting observation is that the positive feedback regulation of type I IFN and type II IFN by goose IFNα and IFNγ was confirmed in waterfowl for the first time. These results suggest that the antiviral activities of goose IFNα and IFNγ can likely be attributed to the potency with which downstream genes are induced by interferon. These findings will contribute to our understanding of the functional significance of the interferon antiviral system in aquatic birds and to the development of interferon-based prophylactic and therapeutic approaches against viral disease.

Fiber knob domain lacking the shaft sequence but fused to a coiled coil is a candidate subunit vaccine against egg-drop syndrome

Egg-drop syndrome (EDS) virus is an avian adenovirus that causes a sudden drop in egg production and in the quality of the eggs when it infects chickens, leading to substantial economic losses in the poultry industry. Inactivated EDS vaccines produced in embryonated duck eggs or cell culture systems are available for the prophylaxis of EDS. However, recombinant subunit vaccines that are efficacious and inexpensive are a desirable alternative. In this study, we engineered chimeric fusion proteins in which the trimeric fiber knob domain lacking the triple β-spiral motif in the fiber shaft region was genetically fused to trimeric coiled coils, such as those of the engineered form of the GCN4 leucine zipper peptide or chicken cartilage matrix protein (CMP). The fusion proteins were expressed predominantly as soluble trimeric proteins in Escherichia coli at levels of 15–80mg/L of bacterial culture. The single immunization of chickens with the purified fusion proteins, at a dose equivalent to 10μg of the knob moiety, elicited serum antibodies with high hemagglutination inhibition (HI) activities, similar to those induced by an inactivated EDS vaccine. A dose–response analysis indicated that a single immunization with as little as 1μg of the knob moiety of the CMP–knob fusion protein was as effective as the inactivated vaccine in inducing antibodies with HI activity. The immunization of laying hens had no apparent adverse effects on egg production and effectively prevented clinical symptoms of EDS when the chickens were challenged with pathogenic EDS virus. This study demonstrates that the knob domain lacking the shaft sequence but fused to a trimeric coiled coil is a promising candidate subunit vaccine for the prophylaxis of EDS in chickens.

Hydropericardium Hepatitis Syndrome Emerged in Cherry Valley Ducks in China

Since June 2015, a highly pathogenic disease occurred in duck flocks in China, causing pericardial effusion, enlarged discoloured liver, renal enlargement and haemorrhagic lung with a mortality ranging from 5% to 20%. Previous study confirmed that Fowl adenovirus group C (FAdV-C) and some field FAdVs isolates had been identified as causative agents of hydropericardium hepatitis syndrome (HHS) in chickens and geese world widely. In this study, we firstly report the isolation of FAdV-C from ducks with HHS. The two isolates, designated as SDSX and SDJX, were separated from liver samples using 9-day-old SPF chicken embryos and could cause severe cytopathic effects in duck and chicken embryonic kidney cells. The entire ORF sequences of hexon gene of the two isolates were amplified, sequenced and analysed by restriction fragment length polymorphism. Phylogenetic analysis of loop 1 sequences of hexon gene of FAdVs revealed that the two isolates were closely related to FAdV-C isolates, which could cause HHS in chickens. Experimental infection indicated that the isolate was high pathogenicity to 20-day-old ducks. Our study shows that the recently emerged HHS in ducks was caused by FAdV-C and may possess a potential risk to other poultry flocks.

Genetic Diversity of NHE1, Receptor for Subgroup J Avian Leukosis Virus, in Domestic Chicken and Wild Anseriform Species.

J subgroup avian leukosis virus (ALV-J) infects domestic chicken, jungle fowl, and turkey and enters the host cell through a receptor encoded by tvj locus and identified as Na+/H+ exchanger 1 (NHE1). The resistance to ALV-J in a great majority of examined galliform species was explained by deletions or substitutions of the critical tryptophan 38 in the first extracellular loop of NHE1, and genetic polymorphisms around this site predict the susceptibility or resistance of a given species or individual. In this study, we examined the NHE1 polymorphism in domestic chicken breeds and documented quantitative differences in their susceptibility to ALV-J in vitro. In a panel of chicken breeds assembled with the aim to cover the maximum variability encountered in domestic chickens, we found a completely uniform sequence of NHE1 extracellular loop 1 (ECL1) without any source of genetic variation for the selection of ALV-J-resistant poultry. In parallel, we studied the natural polymorphisms of NHE1 in wild ducks and geese because of recent reports on ALV-J positivity in feral Asian species. In anseriform species, we demonstrate a specific and highly conserved critical ECL1 sequence without any homologue of tryptophan 38 in accordance with the resistance of duck cells to prototype ALV-J. Last, we demonstrated that the new Asian strains of ALV-J have not evolved their envelope glycoprotein to the entry the duck cells. Our results contribute substantially to the current discussion of possible heterotransmission of ALV-J and its spill-over into the wild ducks and geese.

Duck tembusu virus and its envelope protein induce programmed cell death.

The cytopathic effect produced in cells infected with duck tembusu virus (DTMUV) suggests that this emerging virus may induce apoptosis in primary cultures of duck embryo fibroblasts (DEF). Here, we present evidence that DTMUV infection of cultured cells activates apoptosis and that the ability of DTMUV to induce apoptosis is not restricted to cell type because DTMUV-induced apoptosis in duck and mammalian host cells. We further investigated which viral components induce apoptosis in DTMUV-infected host cells. The major envelope glycoprotein (E) was investigated for its apoptotic activities in expressed cells. Transient expression of the E protein alone triggered apoptosis in DEF, Vero, and BHK cells. Expression of the E protein resulted in activation of caspase-3-like proteases in cultured cells. These results indicate that infection of cells with DTMUV or expression of DTMUV E protein alone induces apoptosis, providing the basis for future to define the molecules that play key roles in the fate of DTMUV-infected cells.

Two myeloid differentiation factor 88 (MyD88) isoforms identified in ducks

MyD88 is an adaptor protein involved in the interleukin-1 receptor-induced and Toll-like receptor (TLR)-induced activation of nuclear factor-κB (NF-κB). In this study, we identified two isoforms of MyD88 gene, designated DuMyD88-X1 and DuMyD88-X2, from duck cells. Both variants were determined to have a death domain at the N-terminal and a Toll/IL-1R (TIR) domain at the C-terminal; however, the TIR domain of DuMyD88-X2 was incomplete and was 81 amino acids shorter than DuMyD88-X1. Quantitative real-time reverse transcription PCR revealed broad expression of both MyD88s. During Newcastle disease virus (NDV) challenge experiments, expression of the two genes increased significantly, with DuMyD88-X1 having a larger amplitude and longer duration. Overexpression of DuMyD88-X1 and DuMyD88-X2 induced the activation of NF-κB and IL-6 in vitro, suggesting that DuMyD88-X1 and DuMyD88-X2 may be important in the innate immune response. The results verify the existence of a MyD88-dependent signaling pathway in ducks and contribute to understanding the potential role of MyD88s in the innate immune response.

DNA microarray global gene expression analysis of influenza virus-infected chicken and duck cells.

The data described in this article pertain to the article by Kuchipudi et al. (2014) titled “Highly Pathogenic Avian Influenza Virus Infection in Chickens But Not Ducks Is Associated with Elevated Host Immune and Pro-inflammatory Responses” [1]. While infection of chickens with highly pathogenic avian influenza (HPAI) H5N1 virus subtypes often leads to 100% mortality within 1 to 2 days, infection of ducks in contrast causes mild or no clinical signs. The rapid onset of fatal disease in chickens, but with no evidence of severe clinical symptoms in ducks, suggests underlying differences in their innate immune mechanisms. We used Chicken Genechip microarrays (Affymetrix) to analyse the gene expression profiles of primary chicken and duck lung cells infected with a low pathogenic avian influenza (LPAI) H2N3 virus and two HPAI H5N1 virus subtypes to understand the molecular basis of host susceptibility and resistance in chickens and ducks. Here, we described the experimental design, quality control and analysis that were performed on the data set. The data are publicly available through the Gene Expression Omnibus (GEO)database with accession number GSE33389, and the analysis and interpretation of these data are included in Kuchipudi et al. (2014) [1].

Identification of morphological differences between avian influenza A viruses grown in chicken and duck cells

Although wild ducks are considered to be the major reservoirs for most influenza A virus subtypes, they are typically resistant to the effects of the infection. In contrast, certain influenza viruses may be highly pathogenic in other avian hosts such as chickens and turkeys, causing severe illness and death. Following in vitro infection of chicken and duck embryo fibroblasts (CEF and DEF) with low pathogenic avian influenza (LPAI) viruses, duck cells die more rapidly and produce fewer infectious virions than chicken cells. In the current study, the morphology of viruses produced from CEF and DEF cells infected with low pathogenic avian H2N3 was examined. Transmission electron microscopy showed that viruses budding from duck cells were elongated, while chicken cells produced mostly spherical virions; similar differences were observed in viral supernatants. Sequencing of the influenza genome of chicken- and duck-derived H2N3 LPAI revealed no differences, implicating host cell determinants as responsible for differences in virus morphology. Both DEF and CEF cells produced filamentous virions of equine H3N8 (where virus morphology is determined by the matrix gene). DEF cells produced filamentous or short filament virions of equine H3N8 and avian H2N3, respectively, even after actin disruption with cytochalasin D. These findings suggest that cellular factors other than actin are responsible for the formation of filamentous virions in DEF cells. The formation of elongated virions in duck cells may account for the reduced number of infectious virions produced and could have implications for virus transmission or maintenance in the reservoir host.

Chicken and duck myotubes are highly susceptible and permissive to influenza virus infection.

ABSTRACTSkeletal muscle, at 30 to 40% of body mass, is the most abundant soft tissue in the body. Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines and respond to proinflammatory cytokines. Little is known about the role of skeletal muscle during systemic influenza A virus infection in any host and particularly avian species. Here we used primary chicken and duck multinucleated myotubes to examine their susceptibility and innate immune response to influenza virus infections. Both chicken and duck myotubes expressed avian and human sialic acid receptors and were readily susceptible to low-pathogenicity (H2N3 A/mallard duck/England/7277/06) and high-pathogenicity (H5N1 A/turkey/England/50-92/91 and H5N1 A/turkey/Turkey/1/05) avian and human H1N1 (A/USSR/77) influenza viruses. Both avian host species produced comparable levels of progeny H5N1 A/turkey/Turkey/1/05 virus. Notably, the rapid accumulation of viral nucleoprotein and matrix (M) gene RNA in chicken and duck myotubes was accompanied by extensive cytopathic damage with marked myotube apoptosis (widespread microscopic blebs, caspase 3/7 activation, and annexin V binding at the plasma membrane). Infected chicken myotubes produced significantly higher levels of proinflammatory cytokines than did the corresponding duck cells. Additionally, in chicken myotubes infected with H5N1 viruses, the induction of interferon beta (IFN-β) and IFN-inducible genes, including the melanoma differentiation-associated protein 5 (MDA-5) gene, was relatively weak compared to infection with the corresponding H2N3 virus. Our findings highlight that avian skeletal muscle fibers are capable of productive influenza virus replication and are a potential tissue source of infection.IMPORTANCE Infection with high-pathogenicity H5N1 viruses in ducks is often asymptomatic, and skeletal muscle from such birds could be a source of infection of humans and animals. Little is known about the ability of influenza A viruses to replicate in avian skeletal muscle fibers. We show here that cultured chicken and duck myotubes were highly susceptible to infection with both low- and high-pathogenicity avian influenza viruses. Infected myotubes of both avian species displayed rapid virus accumulation, apoptosis, and extensive cellular damage. Our results indicate that avian skeletal muscle fibers of chicken and duck could be significant contributors to progeny production of highly pathogenic H5N1 viruses.

Highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses.

Highly pathogenic avian influenza (HPAI) H5N1 viruses cause severe infection in chickens at near complete mortality, but corresponding infection in ducks is typically mild or asymptomatic. To understand the underlying molecular differences in host response, primary chicken and duck lung cells, infected with two HPAI H5N1 viruses and a low pathogenicity avian influenza (LPAI) H2N3 virus, were subjected to RNA expression profiling. Chicken cells but not duck cells showed highly elevated immune and pro-inflammatory responses following HPAI virus infection. HPAI H5N1 virus challenge studies in chickens and ducks corroborated the in vitro findings. To try to determine the underlying mechanisms, we investigated the role of signal transducer and activator of transcription-3 (STAT-3) in mediating pro-inflammatory response to HPAIV infection in chicken and duck cells. We found that STAT-3 expression was down-regulated in chickens but was up-regulated or unaffected in ducks in vitro and in vivo following H5N1 virus infection. Low basal STAT-3 expression in chicken cells was completely inhibited by H5N1 virus infection. By contrast, constitutively active STAT-3 detected in duck cells was unaffected by H5N1 virus infection. Transient constitutively-active STAT-3 transfection in chicken cells significantly reduced pro-inflammatory response to H5N1 virus infection; on the other hand, chemical inhibition of STAT-3 activation in duck cells increased pro-inflammatory gene expression following H5N1 virus infection. Collectively, we propose that elevated pro-inflammatory response in chickens is a major pathogenicity factor of HPAI H5N1 virus infection, mediated in part by the inhibition of STAT-3.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-014-0118-3) contains supplementary material, which is available to authorized users.

Duck enteritis virus glycoprotein D and B DNA vaccines induce immune responses and immunoprotection in Pekin ducks.

DNA vaccine is a promising strategy for protection against virus infection. However, little is known on the efficacy of vaccination with two plasmids for expressing the glycoprotein D (gD) and glycoprotein B (gB) of duck enteritis virus (DEV) in inducing immune response and immunoprotection against virulent virus infection in Pekin ducks. In this study, two eukaryotic expressing plasmids of pcDNA3.1-gB and pcDNA3.1-gD were constructed. Following transfection, the gB and gD expressions in DF1 cells were detected. Groups of ducks were vaccinated with pcDNA3.1-gB and/or pcDNA3.1-gD, and boosted with the same vaccine on day 14 post primary vaccination. We found that intramuscular vaccinations with pcDNA3.1-gB and/or pcDNA3.1-gD, but not control plasmid, stimulated a high frequency of CD4+ and CD8+ T cells in Pekin ducks, particularly with both plasmids. Similarly, vaccination with these plasmids, particularly with both plasmids, promoted higher levels of neutralization antibodies against DEV in Pekin ducks. More importantly, vaccination with both plasmids significantly reduced the virulent DEV-induced mortality in Pekin ducks. Our data indicated that vaccination with plasmids for expressing both gB and gD induced potent cellular and humoral immunity against DEV in Pekin ducks. Therefore, this vaccination strategy may be used for the prevention of DEV infection in Pekin ducks.

The effect of avian influenza virus NS1 allele on virus replication and innate gene expression in avian cells

The NS1 gene encoded by Type A influenza virus circulates as two alleles, the A and B allele. The immunomodulatory properties of the NS1 A allele have been thoroughly examined; however, comparisons of allele function have been predominantly made in mammalian systems. Here we show that counter to the current understanding of allele function in mammals, the two alleles similarly regulate elements of the type I interferon (IFN) signaling pathway, including the interferon-inducible genes Mx and 2′–5′ oligoadenylate synthase (2′–5′ OAS), and IL-6, which share the same induction pathway as the interferons in embryo fibroblasts from chickens, turkeys or ducks. Replication of two reassortant viruses demonstrated that the B allele virus replicates more and to higher titers than the A allele virus in duck cells; however, the A allele virus replicates more in the cells from chickens and turkeys. Finally, chimeric constructs were used to identify a region of the NS1 gene that conferred the statistically significant differences in expression and replication observed between the alleles.

Deletion of Marek's Disease Virus Large Subunit of Ribonucleotide Reductase Impairs Virus Growth In Vitro and In Vivo

Marek's disease virus (MDV), a highly cell-associated lymphotropic alphaherpesvirus, is the causative agent of a neoplastic disease in domestic chickens called Marek's disease (MD). In the unique long (UL) region of the MDV genome, open reading frames UL39 and UL40 encode the large and small subunits of the ribonucleotide reductase (RR) enzyme, named RR1 and RR2, respectively. MDV RR is distinguishable from that present in chicken and duck cells by monoclonal antibody T81. Using recombinant DNA technology we have generated a mutant MDV (Md5deltaRR1) in which RR1 was deleted. PCR amplification of the RR gene in Md5deltaRR1-infected duck embryo fibroblasts (DEF) confirmed the deletion of the 2.4 kb RR1 gene with a resultant amplicon of a 640-bp fragment. Restriction enzyme digests with SalI confirmed a UL39 deletion and the absence of gross rearrangement. The biologic characteristics of Md5deltaRR1 virus were studied in vitro and in vivo. The Md5deltaRR1 replicated in DEF, but significantly slower than parental Md5-BAC, suggesting that RR is important but not essential for replication in fibroblasts. In vivo studies, however, showed that the RR1 deletion virus was impaired for its ability to replicate in chickens. Inoculation of specific-pathogen-free (SPF) chickens with Md5deltaRR1 showed the mutant virus is nonpathogenic and does not induce MD in birds. A revertant virus, Md5deltaRR1/R, was generated with the restored phenotype of the parental Md5-BAC in vivo, indicating that RR is essential for replication of the virus in chickens. Protection studies in SPF chickens indicated that the Md5deltaRR1 virus is not a candidate vaccine against MD.

The Turkey Ig-Like Receptor Family: Identification, Expression and Function

The chicken leukocyte receptor complex located on microchromosome 31 encodes the chicken Ig-like receptors (CHIR), a vastly expanded gene family which can be further divided into three subgroups: activating CHIR-A, bifunctional CHIR-AB and inhibitory CHIR-B. Here, we investigated the presence of CHIR homologues in other bird species. The available genome databases of turkey, duck and zebra finch were screened with different strategies including BLAST searches employing various CHIR sequences, and keyword searches. We could not identify CHIR homologues in the distantly related zebra finch and duck, however, several partial and complete sequences of CHIR homologues were identified on chromosome 3 of the turkey genome. They were designated as turkey Ig-like receptors (TILR). Using cDNA derived from turkey blood and spleen RNA, six full length TILR could be amplified and further divided according to the typical sequence features into one activating TILR-A, one inhibitory TILR-B and four bifunctional TILR-AB. Since the TILR-AB sequences all displayed the critical residues shown to be involved in binding to IgY, we next confirmed the IgY binding using a soluble TILR-AB1-huIg fusion protein. This fusion protein reacted with IgY derived from various gallinaceous birds, but not with IgY from other bird species. Finally, we tested various mab directed against CHIR for their crossreactivity with either turkey or duck leukocytes. Whereas no staining was detectable with duck cells, the CHIR-AB1 specific mab 8D12 and the CHIR-A2 specific mab 13E2 both reacted with a leukocyte subpopulation that was further identified as thrombocytes by double immunofluorescence employing B-cell, T-cell and thrombocyte specific reagents. In summary, although the turkey harbors similar LRC genes as the chicken, their distribution seems to be distinct with predominance on thrombocytes rather than lymphocytes.

18S rRNAis a reliable normalisation gene for real time PCR based on influenza virus infected cells

BackgroundOne requisite of quantitative reverse transcription PCR (qRT-PCR) is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), under different experimental settings. However, ACTB and GAPDH remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ACTB, GAPDH, 18S ribosomal RNA (18S rRNA), ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9) (ATP5G1)] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes.ResultsThe relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs), pig tracheal epithelial cells (PTECs), and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. Analysis of qRT-PCR data from virus and mock infected cells using NormFinder and BestKeeper software programmes found that 18S rRNA was the most stable gene in HBECs, PTECs and avian lung cells.ConclusionsBased on the presented data from cell culture models (HBECs, PTECs, chicken and duck lung cells) infected with a range of influenza viruses, we found that 18S rRNA is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including ACTB and GPADH) were highly affected by influenza virus infection and hence are not reliable as reference genes for RNA normalisation.

Regulatory T cell properties of thymic CD4+CD25+ cells in ducks

Thymic CD4+CD25+ cells from ducks were characterized for mammalian T regulatory cells’ suppressive and cytokine production properties. The cross reactivity of anti-chicken CD25 monoclonal antibody with duck CD25 was confirmed by evaluating Concanavalin-A-stimulated CD25 upregulation in splenocytes. CD4+CD25+ cells were detectable in the thymus, spleen, cecal tonsil, and lung (airsacs), but not in the bursa. Duck CD4+CD25+ cells had approximately nine-fold higher IL-10 mRNA, 12-fold higher TGF-β, 16-fold higher CTLA-4, and nine-fold higher LAG-3 mRNA amounts than thymic CD4+CD25− cells. Thymic CD4+CD25+ cells had no detectable levels of IL-2 mRNA. Duck CD4+CD25+ cells had a three-fold higher IL-10 mRNA amount than chicken CD4+CD25+ cells. Duck CD4+CD25+ cells were anergic in vitro. Duck CD4+CD25+ cells suppressed naive cell proliferation at effector: responder cell ratios above 0.5:1 in both contact-dependent and -independent pathways. It could be concluded that thymic CD4+CD25+ cells in ducks are most likely the counterpart of mammalian T regulatory cells.

Establishment of a duck cell line susceptible to duck hepatitis virus type 1

Until recently, there was no cell line that could produce continuously high-titer duck hepatitis virus type 1 (DHV-1). In this study, a duck embryo fibroblast (DEF) cell line was established, and the susceptibility of this cell line to DHV-1 was determined. The primary culture of DEF cells was from a duck embryo that was partially digested with trypsin. Digested tissue pieces were cultured at 37°C in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum. The cultured DEF cells, which had the morphology of fibroblast, proliferated to 100% confluence four days later. An immortalized DEF cell line, named DEF-TA, was established and subcultured to passage 33, and the susceptibility of that cell line to DHV-1 was determined. In the DHV-1 susceptibility tests, cytopathic effects and the propagation of virus were observed in DEF-TA cells after DHV-1 infection. This continuous DHV-1-susceptible DEF cell line may serve as a valuable cell line for studies of cell-virus interactions and the pathogenesis of DHV-1 and may be useful for the development of an inactivated vaccine.

Adaptation of a Duck Influenza A Virus in Quail

Quail are thought to serve as intermediate hosts of influenza A viruses between aquatic birds and terrestrial birds, such as chickens, due to their high susceptibility to aquatic-bird viruses, which then adapt to replicate efficiently in their new hosts. However, does replication of aquatic-bird influenza viruses in quail similarly result in their efficient replication in humans? Using sialic acid-galactose linkage-specific lectins, we found both avian (sialic acid-α2-3-galactose [Siaα2-3Gal] linkages on sialyloligosaccharides)--and human (Siaα2-6Gal)-type receptors on the tracheal cells of quail, consistent with previous reports. We also passaged a duck H3N2 virus in quail 19 times. Sequence analysis revealed that eight mutations accumulated in hemagglutinin (HA) during these passages. Interestingly, many of the altered HA amino acids found in the adapted virus are present in human seasonal viruses, but not in duck viruses. We also found that stepwise stalk deletion of neuraminidase occurred during passages, resulting in reduced neuraminidase function. Despite some hemagglutinin mutations near the receptor binding pocket, appreciable changes in receptor specificity were not detected. However, reverse-genetics-generated viruses that possessed the hemagglutinin and neuraminidase of the quail-passaged virus replicated significantly better than the virus possessing the parent HA and neuraminidase in normal human bronchial epithelial cells, whereas no significant difference in replication between the two viruses was observed in duck cells. Further, the quail-passaged but not the original duck virus replicated in human bronchial epithelial cells. These data indicate that quail can serve as intermediate hosts for aquatic-bird influenza viruses to be transmitted to humans.

Chicken interferon alpha pretreatment reduces virus replication of pandemic H1N1 and H5N9 avian influenza viruses in lung cell cultures from different avian species

BackgroundType I interferons, including interferon alpha (IFN-α), represent one of the first lines of innate immune defense against influenza virus infection. Following natural infection of chickens with avian influenza virus (AIV), transcription of IFN-α is quickly up regulated along with multiple other immune-related genes. Chicken IFN-α up regulates a number of important anti-viral response genes and has been demonstrated to be an important cytokine to establish anti-viral immunity. However, the mechanisms by which interferon inhibit virus replication in avian species remains unknown as does the biological activity of chicken interferon in other avian species.MethodsIn these studies, we assessed the protective potential of exogenous chicken IFN-α applied to chicken, duck, and turkey primary lung cell cultures prior to infection with the pandemic H1N1 virus (A/turkey/Virginia/SEP-4/2009) and an established avian H5N9 virus (A/turkey/Wisconsin/1968). Growth kinetics and induction of select immune response genes, including IFN-α and myxovirus-resistance gene I (Mx), as well as proinflammatory cytokines (IL-1β and IL-6), were measured in response to chicken IFN-α and viral infection over time.ResultsResults demonstrate that pretreatment with chicken IFN-α before AIV infection significantly reduced virus replication in both chicken-and turkey-origin lung cells and to a lesser degree the duck-origin cells. Virus growth was reduced by approximately 200-fold in chicken and turkey cells and 30-fold in duck cells after 48 hours of incubation. Interferon treatment also significantly decreased the interferon and proinflammatory response during viral infection. In general, infection with the H1N1 virus resulted in an attenuated interferon and proinflammatory response in these cell lines, compared to the H5N9 virus.ConclusionsTaken together, these studies show that chicken IFN-α reduces virus replication, lower host innate immune response following infection, and is biologically active in other avian species.