Avian Cell Line Research Articles

Cytotoxicity of the Fusarium mycotoxin deoxynivalenol on mammalian and avian cell lines

Trichothecenes are mycotoxins that occur in grains and can lead to acute and chronic poisoning in animals and humans. Deoxynivalenol (DON) is a type B trichothecene affecting protein synthesis, immune system, leading to brain, blood and kidney disorders. The aim of this work was to evaluate in vitro the cytotoxicity and the pathological effects of DON in short-term experiments on cells from non-tumour and tumour permanent cell lines and to compare their sensitivity. Cell cultivation of BALB/c 3T3, DEC 99, MDA-MB-231, MCF-7 and Hela cells was performed. Quantitative and qualitative methods evaluating cytotoxicity on the base of statistical and morphological analyses for determining the impact on the viability and proliferative activity were used: Neutral Red Uptake (NRU) cytotoxicity test, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and fluorescence microscopy. The cytotoxic effect of DON was assessed after an exposure period of 24 h. DON treatment induced significant alterations in the growth and morphology of the cells, involving early and late apoptosis and necrosis signs. Statistically significant decrease of the viability of all cell lines was established at concentrations of DON starting from 1.9 µg/mL to 3.7 µg/mL, the mean IC50 concentrations were calculated. According to the IC50 values the hierarchical order of cell lines’ sensitivity was determined.

Avian Cell Line DuckCelt®-T17 Is an Efficient Production System for Live-Attenuated Human Metapneumovirus Vaccine Candidate Metavac®.

The development of a live-attenuated vaccine (LAV) for the prevention of human metapneumovirus (HMPV) infection is often hampered by the lack of highly efficient and scalable cell-based production systems that support eventual global vaccine production. Avian cell lines cultivated in suspension compete with traditional cell platforms used for viral vaccine manufacture. We investigated whether the DuckCelt®-T17 avian cell line (Vaxxel), previously described as an efficient production system for several influenza strains, could also be used to produce a new HMPV LAV candidate (Metavac®, SH gene-deleted A1/C-85473 HMPV). To that end, we characterized the operational parameters of MOI, cell density, and trypsin addition to achieve the optimal production of Metavac®, and demonstrated that the DuckCelt®-T17 cell line is permissive and well-adapted to the production of the wild-type A1/C-85473 HMPV and the Metavac® vaccine candidate. Moreover, our results confirmed that the LAV candidate produced in DuckCelt®-T17 cells conserves its advantageous replication properties in LLC-MK2 and 3D-reconstituted human airway epithelium models, and its capacity to induce efficient neutralizing antibodies in a BALB/c mouse model. Our results suggest that the DuckCelt®-T17 avian cell line is a very promising platform for the scalable in-suspension serum-free production of the HMPV-based LAV candidate Metavac®.

Marker counter-selection via CRISPR/Cas9 co-targeting for efficient generation of genome edited avian cell lines and germ cells

Efficient isolation of genetically modified cells that are phenotypically indistinguishable from the unmodified cells remains a major technical barrier for the broader utilization of CRISPR/Cas9. Here, we report a novel enrichment approach to select the genome engineered cells by co-targeting a genomically integrated GFP gene along with the endogenous gene of interest (GOI). Using this co-targeting approach, multiple genomic loci were successfully targeted in chicken (DF1) and quail (CEC-32) fibroblast cell lines by transient transfection of Cas9 and guide RNAs (gRNAs). Clonal isolation of co-targeted DF1 cells showed 75% of cell clones had deletion of GFP and biallelic deletion of the GOI. To assess the utility of this approach to generate genome modified animals, we tested it on chicken primordial germ cells (PGCs) expressing GFP by co-targeting with gRNAs against GFP and endogenous ovomucoid (OVM) gene. PGCs enriched for loss of GFP and confirmed for OVM deletion, derived by co-targeting, were injected into Hamburger and Hamilton stage 14–15 chicken embryos, and their ability to migrate to the genital ridge was confirmed. This simple, efficient enrichment approach could easily be applied to the creation of knock-out or edited cell lines or animals.

Monitoring the replication of structured DNA through heritable epigenetic change.

DNA can adopt non-B form structures that create significant blocks to DNA synthesis and seeking understanding of the mechanisms cells use to resolve such impediments continues to be a very active area of research. However, the ability to monitor the stalling of DNA synthesis at specific sites in the genome in living cells, of central importance to elucidating these mechanisms, poses a significant technical challenge. Replisome stalling is often transient with only a small fraction of events leading to detectable genetic changes, making traditional reporter assays insensitive to the stalling event per se. On the other hand, the imprint stalling leaves on the epigenome can be exploited as a form of biological 'tape recorder' that captures episodes of fork stalling as heritable changes in histone modifications and in transcription. Here we describe a detailed protocol for monitoring DNA structure-dependent epigenetic instability of the BU-1 locus in the avian cell line DT40, which has proved a sensitive tool for understanding the mechanisms by which structured DNA is replicated in a vertebrate system.

Gga-miR-1603 and gga-miR-1794 directly target viral L gene and function as a broad-spectrum antiviral factor against NDV replication

ABSTRACT As the causative agent of Newcastle disease (ND), Newcastle disease virus (NDV) has seriously restricted the development of the poultry industry. Previous research has shown that miRNAs, members of the small noncoding RNA family, are implicated in the regulation NDV replication through extensive interactions with host mRNAs, but whether miRNAs affect NDV replication by directly binding to the NDV antigenome remains unclear. In this study, potential Gallus gallus miRNAs targeting the antigenome of NDV were bioinformatically predicted using the online software RegRNA 2.0, and gga-miR-1603 and gga-miR-1794 were identified as targeting the viral L gene directly through dual-luciferase reporter assays. Sequence alignment analysis demonstrated that multiple genotypes of NDVs harbored highly conserved binding sites for gga-miR-1603 and gga-miR-1794 in the viral antigenome located at 8611–8634 nt and 14,490–14,514 nt, respectively. Meanwhile, we found that gga-miR-1603 and gga-miR-1794 negatively regulated the expression of viral L gene at both the RNA and protein levels, as well as viral replication in vitro. Furthermore, NDV infection had no effect on endogenous gga-miR-1603 and gga-miR-1794 expression in various avian cell lines. Overall, our present study demonstrated that gga-miR-1603 and gga-miR-1794 directly bind to the viral L gene to facilitate ts degradation and inhibit the replication of multiple genotypes of NDVs in vitro. These findings will provide us with important clues for antiviral therapy against NDV infection.

Type I Interferon acts as a major barrier to the establishment of infectious bursal disease virus (IBDV) persistent infections.

Infectious bursal disease virus (IBDV), the best characterized member of the Birnaviridae family, is a highly relevant avian pathogen causing both acute and persistent infections in different avian hosts. Here, we describe the establishment of clonal, long-term, productive persistent IBDV infections in DF-1 chicken embryonic fibroblasts. Although virus yields in persistently-infected cells are exceedingly lower than those detected in acutely infected cells, the replication fitness of viruses isolated from persistently-infected cells is higher than that of the parental virus. Persistently-infected DF-1 and IBDV-cured cell lines derived from them do not respond to type I interferon (IFN). High-throughput genome sequencing revealed that this defect is due to mutations affecting the IFNα/β receptor subunit 2 (IFNAR2) gene resulting in the expression of IFNAR2 polypeptides harbouring large C-terminal deletions that abolish the signalling capacity of IFNα/β receptor complex. Ectopic expression of a recombinant chicken IFNAR2 gene efficiently rescues IFNα responsiveness. IBDV-cured cell lines derived from persistently infected cells exhibit a drastically enhanced susceptibility to establishing new persistent IBDV infections. Additionally, experiments carried out with human HeLa cells lacking the IFNAR2 gene fully recapitulate results obtained with DF-1 cells, exhibiting a highly enhanced capacity to both survive the acute IBDV infection phase and to support the establishment of persistent IBDV infections. Results presented here show that the inactivation of the JAK-STAT signalling pathway significantly reduces the apoptotic response induced by the infection, hence facilitating the establishment and maintenance of IBDV persistent infections.IMPORTANCE Members of the Birnaviridae family, including infectious bursal disease virus (IBDV), exhibit a dual behaviour, causing acute infections that are often followed by the establishment of life-long persistent asymptomatic infections. Indeed, persistently infected specimens might act as efficient virus reservoirs, hence potentially contributing to virus dissemination. Despite the key importance of this biological trait, information about mechanisms triggering IBDV persistency is negligible. Our report evidences the capacity of IBDV, a highly relevant avian pathogen, to establishing long-term, productive, persistent infections in both avian and human cell lines. Data presented here provide novel and direct evidence about the crucial role of type I IFNs on the fate of IBDV-infected cells and their contribution to controlling the establishment of IBDV persistent infections. The use of cell lines unable to respond to type I IFNs opens a promising venue to unveiling additional factors contributing to IBDV persistency.

A highly pathogenic recombinant infectious bronchitis virus with adaptability in cultured cells

Infectious bronchitis virus (IBV) of GI-19 (QX), GI-7 (TW), GI-13 (4/91) and GI-1 (Mass) lineages have been frequently detected in China in recent years. Here, An IBV strain, referred as GD17/04, was isolated from the dead yellow feather chicken vaccinated with H52 and 4/91 vaccines, whose genome sequence was obtained through high-throughput sequencing. Then it has been confirmed by the RDP and SimPlot analysis that GD17/04 is a recombinant strain deriving from YX10, 4/91, TW 2575/98 and H52 strains. Therein S1 gene of GD17/04 consists of sequences of TW2575/98 and 4/91, the former for the region of 20,371 to 21,072 nt and 21,847 to 21,975 nt, the latter for the sandwiched region of 21,073 to 21,846 nt. Moreover, as a nephropathogenic variant which caused high morbidity of 100 % and mortality of 60 %, unlike most other IBV strains, GD17/04 can cause obvious cell lesion in primary CEK cell, and even in DF-1 cells, without the process of continuous passage. As the few IBV strain can infect avian passage cell line, GD17/04 provides a material basis for further study of the interaction mechanism between IBV and avian host. Collectively, the findings highlight the significance that biological characteristics of novel strain should be studied, in addition to constant epidemiologic and molecular surveillance for IBV.

Discoveries of Exoribonuclease-Resistant Structures of Insect-Specific Flaviviruses Isolated in Zambia.

To monitor the arthropod-borne virus transmission in mosquitoes, we have attempted both to detect and isolate viruses from 3304 wild-caught female mosquitoes in the Livingstone (Southern Province) and Mongu (Western Province) regions in Zambia in 2017. A pan-flavivirus RT-PCR assay was performed to identify flavivirus genomes in total RNA extracted from mosquito lysates, followed by virus isolation and full genome sequence analysis using next-generation sequencing and rapid amplification of cDNA ends. We isolated a newly identified Barkedji virus (BJV Zambia) (10,899 nt) and a novel flavivirus, tentatively termed Barkedji-like virus (BJLV) (10,885 nt) from Culex spp. mosquitoes which shared 96% and 75% nucleotide identity with BJV which has been isolated in Israel, respectively. These viruses could replicate in C6/36 cells but not in mammalian and avian cell lines. In parallel, a comparative genomics screening was conducted to study evolutionary traits of the 5′- and 3′-untranslated regions (UTRs) of isolated viruses. Bioinformatic analyses of the secondary structures in the UTRs of both viruses revealed that the 5′-UTRs exhibit canonical stem-loop structures, while the 3′-UTRs contain structural homologs to exoribonuclease-resistant RNAs (xrRNAs), SL-III, dumbbell, and terminal stem-loop (3′SL) structures. The function of predicted xrRNA structures to stop RNA degradation by Xrn1 exoribonuclease was further proved by the in vitro Xrn1 resistance assay.

Production of Modified Vaccinia Ankara Virus by Intensified Cell Cultures: A Comparison of Platform Technologies for Viral Vector Production.

Modified Vaccinia Ankara (MVA) virus is a promising vector for vaccination against various challenging pathogens or the treatment of some types of cancers, requiring a high amount of virions per dose for vaccination and gene therapy. Upstream process intensification combining perfusion technologies, the avian suspension cell line AGE1.CR.pIX and the virus strain MVA‐CR19 is an option to obtain very high MVA yields. Here the authors compare different options for cell retention in perfusion mode using conventional stirred‐tank bioreactors. Furthermore, the authors study hollow‐fiber bioreactors and an orbital‐shaken bioreactor in perfusion mode, both available for single‐use. Productivity for the virus strain MVA‐CR19 is compared to results from batch and continuous production reported in literature. The results demonstrate that cell retention devices are only required to maximize cell concentration but not for continuous harvesting. Using a stirred‐tank bioreactor, a perfusion strategy with working volume expansion after virus infection results in the highest yields. Overall, infectious MVA virus titers of 2.1–16.5 × 109 virions/mL are achieved in these intensified processes. Taken together, the study shows a novel perspective on high‐yield MVA virus production in conventional bioreactor systems linked to various cell retention devices and addresses options for process intensification including fully single‐use perfusion platforms.

Mini-Review; Deriving Avian Stem Cells by Small Molecules.

Avian embryos and related cell lines have found wide applications in basic and applied sciences. The embryonated egg is a great host for monoclonal antibodies and recombinant proteins. Avian cell lines derived from embryonated eggs have been used for the production of transgenic birds and virus inoculation in vaccine preparation. Hitherto, many efforts have been invested to develop efficient avian stem cell culture. Under the conventional conditions, there are various challenges, such as the type of feeder layers, conditioned medium, serum, and growth factors. Researchers have investigated different conditions to solve these problems. Recent studies have shown that targeted strategies using small molecule inhibitors could be used as alternatives to multi-growth factor delivery approaches. Since small molecule inhibitors were used for mammalian pluripotent stem cells (PSC), several kinds of research have examined the effect of the small molecule on self- -renewal and maintenance of avian PSC. Avian PSC can be derived from early blastodermal cells (stage X), circular primoridial germ cells (PGC; stage HH17), gonadal PGC (stage HH28), and embryonic germ cells (EGC; HH28). Previous studies have shown that the use of small molecule drugs such as PD0325901, SB431542, SC1, IDE1, Z-VAD, Blebbistatin, H-1152, and IDE1 could be an efficient method for the derivation of avian stem cells. This mini-review covers the recent development of avian stem cell culture by small molecules.

Saccharomyces boulardii attenuates inflammatory response induced by Clostridium perfringens via TLR4/TLR15-MyD8 pathway in HD11 avian macrophages

Macrophages are professional phagocytic cells that play a critical role in initiating immune responses by presenting antigen and phagocytic clearance. The macrophages can be targeted for immunomodulation by beneficial microbes, such as probiotics. The aim of this study is to investigate the protective effect of Saccharomyces boulardii against Clostridium perfringens infection in avian macrophage cell line HD11. In this study, HD11 macrophages were prestimulated with S. boulardii for 6 h and then infected with C. perfringens for 3 h. Results showed that S. boulardii enhanced phagocytosis and bactericidal capacity against C. perfringens by HD11 cells. The S. boulardii effectively promoted the mRNA expression of CD80, CD83, and CD197 cell-surface molecules in C. perfringens-infected HD11 cells. Moreover, we found that prestimulation with S. boulardii reduced the mRNA expression of CD40, toll-like receptor [TLR] 4, and TLR15 induced by C. perfringens and thereby downregulated the mRNA expression of myeloid differentiation primary response 88, TNF receptor associated factor 6, nuclear factor kappa-B p65 subunit, and c-Jun N-terminal kinase genes in HD11 cells. The upregulation of cytokines (interleukin [IL]-6, tumor necrosis factor alpha, and IL-10) and inducible nitric oxide synthase mRNA expression in C. perfringens-infected HD11 cells were noticeably inhibited by S. boulardii pretreatment. Conclusively, these results might provide a new insight into the role of S. boulardii in regulating avian immune defense against C. perfringens invasion and immune escape.

Ex vivo rescue of recombinant very virulent IBDV using a RNA polymerase II driven system and primary chicken bursal cells

Infectious Bursal Disease Virus (IBDV), a member of the Birnaviridae family, causes an immunosuppressive disease in young chickens. Although several reverse genetics systems are available for IBDV, the isolation of most field-derived strains, such as very virulent IBDV (vvIBDV) and their subsequent rescue, has remained challenging due to the lack of replication of those viruses in vitro. Such rescue required either the inoculation of animals, embryonated eggs, or the introduction of mutations in the capsid protein (VP2) hypervariable region (HVR) to adapt the virus to cell culture, the latter option concomitantly altering its virulence in vivo. We describe an improved ex vivo IBDV rescue system based on the transfection of an avian cell line with RNA polymerase II-based expression vectors, combined with replication on primary chicken bursal cells, the main cell type targeted in vivo of IBDV. We validated this system by rescuing to high titers two recombinant IBDV strains: a cell-culture adapted attenuated strain and a vvIBDV. Sequencing of VP2 HVR confirmed the absence of unwanted mutations that may alter the biological properties of the recombinant viruses. Therefore, this approach is efficient, economical, time-saving, reduces animal suffering and can be used to rescue other non-cell culture adapted IBDV strains.

Application of an Inclined Settler for Cell Culture-Based Influenza A Virus Production in Perfusion Mode.

Influenza viruses have been successfully propagated using a variety of animal cell lines in batch, fed-batch, and perfusion culture. For suspension cells, most studies reported on membrane-based cell retention devices typically leading to an accumulation of viruses in the bioreactor in perfusion mode. Aiming at continuous virus harvesting for improved productivities, an inclined settler was evaluated for influenza A virus (IAV) production using the avian suspension cell line AGE1.CR.pIX. Inclined settlers present many advantages as they are scalable, robust, and comply with cGMP regulations, e.g., for recombinant protein manufacturing. Perfusion rates up to 3000 L/day have been reported. In our study, successful growth of AGE1.CR.pIX cells up to 50 × 106 cells/mL and a cell retention efficiency exceeding 96% were obtained with the settler cooled to room temperature. No virus retention was observed. A total of 5.4–6.5 × 1013 virions were produced while a control experiment with an ATF system equaled to 1.9 × 1013 virions. For infection at 25 × 106 cells/mL, cell-specific virus yields up to 3474 virions/cell were obtained, about 5-fold higher than for an ATF based cultivation performed as a control (723 virions/cell). Trypsin activity was shown to have a large impact on cell growth dynamics after infection following the cell retention device, especially at a cell concentration of 50 × 106 cells/mL. Further control experiments performed with an acoustic settler showed that virus production was improved with a heat exchanger of the inclined settler operated at 27°C. In summary, cell culture-based production of viruses in perfusion mode with an inclined settler and continuous harvesting can drastically increase IAV yields and possibly the yield of other viruses. To our knowledge, this is the first report to show the potential of this device for viral vaccine production.

Performance of an acoustic settler versus a hollow fiber\u2013based ATF technology for influenza virus production in perfusion

Process intensification and integration is crucial regarding an ever increasing pressure on manufacturing costs and capacities in biologics manufacturing. For virus production in perfusion mode, membrane-based alternating tangential flow filtration (ATF) and acoustic settler are the commonly described cell retention technologies. While acoustic settlers allow for continuous influenza virus harvesting, the use of commercially available membranes for ATF systems typically results in the accumulation of virus particles in the bioreactor vessel. Accordingly, with one single harvest at the end of a cultivation, this increases the risk of lowering the product quality. To assess which cell retention device would be most suitable for influenza A virus production, we compared various key performance figures using AGE1.CR.pIX cells at concentrations between 25 and 50 × 106 cells/mL at similar infection conditions using either an ATF system or an acoustic settler. Production yields, process-related impurities, and aggregation of viruses and other large molecules were evaluated. Taking into account the total number of virions from both the bioreactor and the harvest vessel, a 1.5–3.0-fold higher volumetric virus yield was obtained for the acoustic settler. In addition, fewer large-sized aggregates (virus particles and other molecules) were observed in the harvest taken directly from the bioreactor. In contrast, similar levels of process-related impurities (host cell dsDNA, total protein) were obtained in the harvest for both retention systems. Overall, a clear advantage was observed for continuous virus harvesting after the acoustic settler operation mode was optimized. This development may also allow direct integration of subsequent downstream processing steps.Key points• High suspension cell density, immortalized avian cell line, influenza vaccine.

Comparative Study between Avian Cell and Mammalian Cell in Production of Influenza Vaccine Shariah Compliance

Nowadays, the manufacturing industry of the influenza vaccine always make the improvement for their production. Most of them are looking for the new production cell line which are easily scalable, high yield production of viral and highly tolerant to multiple viruses. This situations happens as the high demand of the vaccine for every year from around the world. It can be seen that the capacity for the vaccines’ production are really critical in the pharmaceutical industry. However, certain Muslims’ community do not gain a trust from the vaccine’s ingredient itself. This is due to the production process involves blood consist of serum and cell components that may come from the prohibited animals such as pig and dog. Therefore, shariah compliance vaccine has been studied in this research. The avian cell lines are now competing with the mammalian cell currently at advance stage of commercial development for the manufacture of influenza vaccines. Avian cell line chosen are AGE1.CR.pIX cells and DuckCelt-T17 will be compared with the mammalian cells which is Vero and Madin Darby Canine Kidney (MDCK) cells in producing the shariah compliance vaccine. Different type of mediums also will be considered to produce shariah compliance vaccine. The artificial media will be compared with the natural media that contain the non-shariah components. Hence, the shariah status of the enzyme and media will be known in this research. In this study, the production of shariah compliance vaccine mainly focused on the bioreactor and simulated by using a software called SuperPro® Designer. Results gained from the simulation shows the highest yield at 72 hours which is 93.51 % conversion. These study will acknowledge the halal’s status of the vaccine and contribute the development of the halal vaccine processing industry.

Coinfection Mechanisms ofCampylobacter andEscherichia coli in Human and Chicken Epithelial cells

Campylobacter jejuniis the world’s most common food-borne pathogen. Campylobacterpathogenesis involves translocation across the intestinal epithelial cell barrier in both humans and chickens and previous work has suggested that strains of E. coli (ExPEC / APEC) may facilitate this process. This study aims to determine the effect that this relationship may have upon the invasive and adhesive potential of the two microbes using our recently published human and avian in vitromodel. Invasiveness of C. jejuni and E. coli strains were measured using human (Caco-2) and avian (8E-11) epithelial cell lines derived from the gastrointestinal tract and were characterised by epifluorescent and confocal microscopy. Adhesion and invasion assays were carried out to determine the pathogenicity of the different bacteria. Metabolic activity of Caco-2 and 8E-11 together with bacterial strains using alamar blue assay. Confocal and epifluorescence microscopy determined the strong presence of cytokeratin in Caco-2 cells whilst weak to medium signals were detected in 8E-11 cells. Optimal doses and times for of gentamicin across all strains was 0.02mg/ml for 90 minutes which did not affect metabolic activity of epithelial cells. Significant diversity was found in the adhesive/invasive potential of bacteria when exposed to human and avian cell types. The metabolic rate of C. jejuni (11168) and E. coli (K12) was investigated with the presence of K12 having negative impacts upon the activity of 11168. The model described here will provide opportunity to improve our understanding of Campylobacterinvasion mechanisms in human and chickens so that improved strategies to negate these consequences may be designed.

Isolation of Ontario aquatic bird bornavirus 1 and characterization of its replication in immortalized avian cell lines

BackgroundAquatic bird bornavirus 1 (ABBV-1) has been associated with neurological diseases in wild waterfowls. In Canada, presence of ABBV-1 was demonstrated by RT-qPCR and immunohistochemistry in tissues of waterfowls with history of neurological disease and inflammation of the central and peripheral nervous tissue, although causation has not been proven by pathogenesis experiments, yet. To date, in vitro characterization of ABBV-1 is limited to isolation in primary duck embryo fibroblasts. The objectives of this study were to describe isolation of ABBV-1 in primary duck embryonic fibroblasts (DEF), and characterize replication in DEF and three immortalized avian fibroblast cell lines (duck CCL-141, quail QT-35, chicken DF-1) in order to evaluate cellular permissivity and identify suitable cell lines for routine virus propagation.MethodsThe virus was sequenced, and phylogenetic analysis performed on a segment of the N gene coding region. Virus spread in cell cultures, viral RNA and protein production, and titres were evaluated at different passages using immunofluorescence, RT-qPCR, western blotting, and tissue culture dose 50% (TCID50) assay, respectively.ResultsThe isolated ABBV-1 showed 97 and 99% identity to European ABBV-1 isolate AF-168 and North American ABBV-1 isolates 062-CQ and CG-N1489, and could infect and replicate in DEF, CCL-141, QT-35 and DF-1 cultures. Viral RNA was detected in all four cultures with highest levels observed in DEF and CCL-141, moderate in QT-35, and lowest in DF-1. N protein was detected in western blots from infected DEF, CCL-141 and QT-35 at moderate to high levels, but minimally in infected DF-1. Infectious titre was highest in DEF (between approximately 105 to 106 FFU / 106 cells). Regarding immortalized cell lines, CCL-141 showed the highest titre between approximately 104 to 105 FFU / 106 cells. DF-1 produced minimal infectious titre.ConclusionsThis study confirms the presence of ABBV-1 among waterfowl in Canada and reported additional in vitro characterization of this virus in different avian cell lines. ABBV-1 replicated to highest titre in DEF, followed by CCL-141 and QT-35, and poorly in DF-1. Our results showed that CCL-141 can be used instead of DEF for routine ABBV-1 production, if a lower titre is an acceptable trade-off for the simplicity of using immortalized cell line over primary culture.

Orbitally Shaken Single-Use Bioreactor for Animal Cell Cultivation: Fed-Batch and Perfusion Mode.

Increasing the cultivation volume from small to large scale can be a rather complex and challenging process when the method of aeration and mixing is different between scales. Orbitally shaken bioreactors (OSBs) utilize the same hydrodynamic principles that define the success of smaller-scale cultures, which are developed on an orbitally shaken platform, and can simplify scale-up. Here we describe the basic working principles of scale-up in terms of the volumetric oxygen transfer coefficient (kLa) and mixing time and how to define these parameters experimentally. The scale-up process from an Erlenmeyer flask shaken on an orbital platform to an orbitally shaken single-use bioreactor (SB10-X, 12 L) is described in terms of both fed-batch and perfusion-based processes. The fed-batch process utilizes a recombinant variant of the mammalian cell line, Chinese hamster ovary (CHO), to express a biosimilar of a therapeutic monoclonal antibody. The perfusion-based process utilizes either an alternating tangential flow filtration (ATF) or a tangential flow filtration (TFF) system for cell retention to cultivate an avian cell line, AGE1.CR.pIX, for the propagation of influenza A virus, H1N1, in high cell density. Based on two example cell cultivations, processes outline the advantages that come with using an orbitally shaken bioreactor for scaling-up a process. The described methods are also applicable to other suspension cell lines.

Sarcocystis falcatula-like derived from opossum in Northeastern Brazil: In vitro propagation in avian cells, molecular characterization and bioassay in birds

Most reported isolates of Sarcocystis spp. derived from Brazilian opossums (Didelphis sp.) have genetic characteristics distinct from the known species of Sarcocystis, but behave similarly as Sarcocystis falcatula, as they are infective to budgerigars. In previous studies, these Brazilian isolates, classified as Sarcocystis falcatula-like, were originated from South and Southeast regions of Brazil. In the current work, we aimed to culture and to perform multilocus sequence analysis of Sarcocystis sp. derived from a Brazilian opossum (D. aurita/D. marsupialis) that inhabited the city of Salvador, Bahia, in the Northeast of Brazil. The parasite was isolated in Vero cells, referred here as Sarco-BA1, and propagated in avian cells (DF-1). Molecular analysis of Sarco-BA1 revealed that the nucleotide sequence of the internal transcribed spacer 1 (ITS1) of the rDNA was identical to all isolates (n = 19) of Sarcocystis spp. reported in two studies from South and Southeast regions of the country. Two budgerigars were inoculated with 10 and 1000 sporocysts of Sarco-BA1, respectively, and developed acute sarcocystosis, showing that the parasite behaves like S. falcatula. It was interesting to observe that Sarco-BA1 had almost identical ITS1 and SAG sequences to all 16 isolates of S. falcatula-like recently described in Magellanic penguins (Spheniscus magellanicus) rescued on the coast of Espírito Santo state, Brazil. Our results suggest that Sarco-BA1 and S. falcatula-like may represent a single species of Sarcocystis. Propagation of the parasite in a permanent avian cell line significantly improved the yield of merozoites in cell culture. To our knowledge, this is the first molecular study and in vitro isolation of S. falcatula-like derived from Northeastern Brazil. Studies are under way to determine the infectivity of Sarco-BA1 to other animal species, as well as to investigate serological cross-reactivity among Sarco-BA1, S. neurona and related species.

PFOS mediates immunomodulation in an avian cell line that can be mitigated via a virus infection

BackgroundPer- and polyfluoroalkyl substances (PFASs) are environmentally persistent and bioaccumulative chemicals. Immunomodulation is among the most concerning of toxic effects linked with PFAS exposure in mammalian models. However, no studies had yet shown this to be true in birds. Thus, we designed and conducted the first study to determine if PFASs could cause immunomodulation in birds. Secondly, we wanted to determine the effects on an avian host when exposed not only to immunomodulating chemicals, but also to a viral challenge. The aim, to determine if PFAS mediated immunmodulation functionally affects a pathogen challenge for a host. As innate immune system signalling pathways initiate crucial responses against a pathogen challenge, and are lesser studied than their adaptive counterparts, we focused on these pathways. To provide the first information on this, an in vitro experiment was designed and performed using chicken embryo fibroblasts exposed to perfluorooctane sulfonate (PFOS) (22 ppm) and immune markers characterised before and after being infected with gallid herpesvirus-2 (GaHV-2).ResultsThe expression of two pro-inflammatory cytokines, namely interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α), the nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB), and the anti-inflammatory cytokine interleukin 4 (IL-4) were investigated in various scenarios. These results showed that exposure to PFOS decreased immune gene expression in chicken fibroblasts from 36 h post-exposure. Next, it was shown that this decrease could be mitigated by infection with gallid herpesvirus-2, which increased gene expression back to the baseline/control levels.ConclusionsNot only is this the first study to provide the expected evidence that PFOS has immunomodulatory potential in birds, it also provides unexpected data that virus infections can mitigate this negative effect. Thereby, further research, including in vivo and in situ studies, on the impact of PFOS on host-virus interactions is now warranted, as it has been overlooked and might contribute to our understanding of recent disease outbreaks in wildlife. The mechanisms by which gallid herpesvirus mitigates immunomodulation were beyond the scope of this study, but are now of interest for future study.