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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still adapting to its new human host. Attention has focussed on the viral spike protein, but substantial variation has been seen in the ORF8 gene. Here, we show that SARS-CoV-2 ORF8 protein undergoes signal peptide-mediated processing through the endoplasmic reticulum and is secreted as a glycosylated, disulphide-linked dimer. The secreted protein from the prototype SARS-CoV-2 virus had no major effect on viability of a variety of cell types, or on IFN or NF-κB signalling. However, it modulated cytokine expression from primary CSF1-derived human macrophages, most notably by decreasing IL-6 and IL-8 secretion. Furthermore, a sequence polymorphism L84S that appeared early in the pandemic associated with the Clade S lineage of virus, showed a markedly different effect, of increasing IL-6 production. We conclude that ORF8 sequence polymorphisms can potentially affect SARS-CoV-2 virulence and should therefore be monitored in sequencing-based surveillance.

Additional file 2: Table S2. SNPs used in this study with their position on GalGal6. SNPs were derived from dbEST, sequencing and dbSNP.

Additional file 1: Table S1. Genetic map for the resolution of the QTL for bone quality in the F2 population. Marker names and distance between markers in cM.

Additional file 4: Mass Spectrophotometer transitions. The eluent from LC was passed onto the electrospray source of an amaZon ETD Ion Trap operated in MRM mode with the following transitions. The concentrations of components in samples were calculated by comparison with external calibration curves of authentic compounds.

Background The global market for protein drugs has the highest compound annual growth rate of any pharmaceutical class but their availability, especially outside of the US market, is compromised by the high cost of manufacture and validation compared to traditional chemical drugs. Improvements in transgenic technologies allow valuable proteins to be produced by genetically-modified animals; several therapeutic proteins from such animal bioreactors are already on the market after successful clinical trials and regulatory approval. Chickens have lagged behind mammals in bioreactor development, despite a number of potential advantages, due to the historic difficulty in producing transgenic birds, but the production of therapeutic proteins in egg white of transgenic chickens would substantially lower costs across the entire production cycle compared to traditional cell culture-based production systems. This could lead to more affordable treatments and wider markets, including in developing countries and for animal health applications. Results Here we report the efficient generation of new transgenic chicken lines to optimize protein production in eggs. As proof-of-concept, we describe the expression, purification and functional characterization of three pharmaceutical proteins, the human cytokine interferon α2a and two species-specific Fc fusions of the cytokine CSF1. Conclusion Our work optimizes and validates a transgenic chicken system for the cost-effective production of pure, high quality, biologically active protein for therapeutics and other applications. Electronic supplementary material The online version of this article (10.1186/s12896-018-0495-1) contains supplementary material, which is available to authorized users.

Objective The multifunctional NS1 protein of influenza A virus has roles in antagonising cellular innate immune responses and promoting viral gene expression. To better understand the interplay between these functions, we tested the effects of NS1 effector domain mutations known to affect homo-dimerisation or interactions with cellular PI3 kinase or Trim25 on NS1 ability to promote nuclear export of viral mRNAs. Results The NS1 dimerisation mutant W187R retained the functions of binding cellular NXF1 as well as stabilising NXF1 interaction with viral segment 7 mRNAs and promoting their nuclear export. Two PI3K-binding mutants, NS1 Y89F and Y89A still bound NXF1 but no longer promoted NXF1 interactions with segment 7 mRNA or its nuclear export. The Trim25-binding mutant NS1 E96A/E97A bound NXF1 and supported NXF1 interactions with segment 7 mRNA but no longer supported mRNA nuclear export. Analysis of WT and mutant NS1 interaction partners identified hsp70 as specifically binding to NS1 E96A/E97A. Whilst these data suggest the possibility of functional links between NS1’s effects on intracellular signalling and its role in viral mRNA nuclear export, they also indicate potential pleiotropic effects of the NS1 mutations; in the case of E96A/E97A possibly via disrupted protein folding leading to chaperone recruitment.

ObjectiveThe multifunctional NS1 protein of influenza A virus has roles in antagonising cellular innate immune responses and promoting viral gene expression. To better understand the interplay between these functions, we tested the effects of NS1 effector domain mutations known to affect homo-dimerisation or interactions with cellular PI3 kinase or Trim25 on NS1 ability to promote nuclear export of viral mRNAs.ResultsThe NS1 dimerisation mutant W187R retained the functions of binding cellular NXF1 as well as stabilising NXF1 interaction with viral segment 7 mRNAs and promoting their nuclear export. Two PI3K-binding mutants, NS1 Y89F and Y89A still bound NXF1 but no longer promoted NXF1 interactions with segment 7 mRNA or its nuclear export. The Trim25-binding mutant NS1 E96A/E97A bound NXF1 and supported NXF1 interactions with segment 7 mRNA but no longer supported mRNA nuclear export. Analysis of WT and mutant NS1 interaction partners identified hsp70 as specifically binding to NS1 E96A/E97A. Whilst these data suggest the possibility of functional links between NS1’s effects on intracellular signalling and its role in viral mRNA nuclear export, they also indicate potential pleiotropic effects of the NS1 mutations; in the case of E96A/E97A possibly via disrupted protein folding leading to chaperone recruitment.

Table S1. pCSF1-Fc purity assessed by LC-MS/MS analysis. The purified protein sample was digested in-solution using trypsin and analyzed on a micrOTOF-QII (Bruker) mass spectrometer. The spectral data was searched against Uniprot porcine protein sequence database to confirm the identity of pCSF1-Fc and against Uniprot chicken database to check the purity from chicken egg proteins using Mascot server. (A composite file of results from separate Mascot searches of LC-MS/MS data.) (DOCX 332 kb)

Figure S5. pCSF1-Fc mono-dispersity, size and purity analysis. A) Size-exclusion chromatography multi-angled light scattering (SEC-MALS) was used to determine the molecular and mono-dispersity of pCSF1-Fc in solution. pCSF1-Fc eluted as a single peak (≥ 97% purity; As = 0.91) from a Superdex-200 Increase 10/300 GL size exclusion column with apparent molecular mass of ~ 110 ± 5 kDa and an Rs of 4.03 ± 0.4 nm (mean ± SD, n = 3). B) Elution profile of pCSF1-Fc from an RPC 5 4.6/150 ST column run. Chromatogram shows single sharp peak, with ≥98% purity. (DOCX 188 kb)

Figure S3. Confirmation of pCSF1-Fc transgene and copy number in G1 birds by Southern transfer analysis, Southern transfer analysis of genomic DNA from individual G1 birds. A) Samples from 6 birds positive by PCR for HIV sequence were digested with BamHI (located at the 5â ˛ end of the promoter) and AvrII (located at the 3â ˛ end of the oPRE) to generate a 6.6â kb fragment spanning most of the ovalbumin promoter and the pCSF1-Fc coding sequence. Any smaller bands were considered to be truncated versions of the transgene.B) Samples from the three birds containing intact transgenes were digested with BamHI to detect insertion events, with each event expected to show a distinct band. (DOCX 7376 kb)

Figure S4. Comparison of expression between two generations and different individuals of pCSF1-Fc birds. 5â ÎźL egg white diluted 1:50 from a second generation pen (lane 1) and three third generation pens (lanes 3â 5) of pCSF1-Fc, compared by non-reducing western blot against each other and 500â ng of purified pCSF1-Fc from second generation egg white. (DOCX 267 kb)

Figure S6. Confirmation of hCSF1-Fc transgene and copy number in G1 birds by Southern transfer analysis. Southern transfer analysis of genomic DNA from individual G1 birds. A) Samples from 7 birds positive by PCR for HIV sequence were digested with BamHI (located at the 5â ˛ end of the promoter) and AgeI (located at the 3â ˛ end of the oPRE) to generate a 6.3â kb fragment spanning most of the ovalbumin promoter and the hCSF1-Fc coding sequence. B) Samples from the seven birds were digested with BamHI to detect insertion events, with each event expected to show a distinct band. (DOCX 15849 kb)

Figure S1. EREOVA2 sequence. DNA sequence of EREOVA2 promoter. (DOCX 16 kb)

Figure S2. Interferon α2A mono-dispersity, size and purity analysis. A) Size-exclusion chromatography multi-angled light scattering (SEC-MALS) was used to determine the molecular and mono-dispersity of Interferon α2A in solution. Interferon α2A eluted from a Superdex-200 Increase 10/300 GL size exclusion column as a single peak with slight trailing edge (≥ 96% purity; As > 2) with apparent molecular mass of ~ 33.2 ± 2 kDa and an Rs of 2.43 ± 0.2 nm (mean ± SD, n = 3). The molecular mass average across the elution profile is 18.7 kDa with average mono-dispersity (Mw/Mn = 1.038) and no significant aggregation. B) Elution profile of Interferon α2A from an RPC 5 4.6/150 ST column run. Chromatogram shows single sharp peak, with ≥98% purity. (DOCX 486 kb)

Ocular coloboma (OC) is a defect in optic fissure closure and is a common causeof severe congenital visual impairment. Bilateral OC is primarily genetically determined and shows marked locus heterogeneity. Whole exome sequencing was used to analyse twelve trios (child affected with OC and both unaffected parents), This identified de novo mutations in ten different genes in eight probands. Three of these genes encoded proteins associated with actin cytoskeleton dynamics: ACTG1, TWF1 and LCP1. Proband-only whole exome sequencing identified a second unrelated individual with isolated OC carrying the same ACTG1 allele, encoding p.(Pro70Leu). Both individuals have normal neurodevelopment with no extra-ocular signs of Baraitser Winter syndrome. We found this mutant protein to be incapable of incorporation into F-actin. The LCP1 and TWF1 variants each resulted in only minor disturbance of actin-interactions and no further plausibly causative variants were identified in these genes on re-sequencing 380 unrelated individuals with OC.

This study reports a proteomic analyses on porcine follicular fluid (FF) obtained from a previous nutritional trial, where oocytes from gilts fed a high-fibre (HF) diet for the first 19 days of their third oestrous cycle produced blastocysts with more cells following in vitro maturation (IVM) and IVF compared with oocytes from control-fed (CON) pigs. Oocytes were matured in TCM-199 supplemented with LH and FSH at 0.5 μg mL–1 and 10% of the animals’ own pooled FF. Following IVF, resultant embryos were cultured in NCSU-23 medium for 6 to 7 days. We hypothesize that FF protein composition is altered by the HF diet and that this confers the reproductive benefits previously observed. The FF had previously been stored at –80°C after the IVF trials and was thawed for the current study, which compared the protein composition of pooled Day 19 FF from 12 CON pigs and 12 HF pigs. These gilts were a subset of the pigs described above with the largest FF volumes. The protein composition of pooled FF from 6 CON pigs whose oocytes produced blastocysts was compared with FF from 6 CON pigs whose oocytes did not produce blastocysts. The same analysis was carried out with the 6 HF pigs that produced blastocysts and the 6 HF pigs that did not produce blastocysts. Equal numbers of samples from animals were selected for experimental balance. The proteomic study was carried out in duplicate. Abundant proteins were depleted from FF by Proteominer enrichment. Samples were labelled by isotopic di-methylation, where in each analysis, one sample was labelled with a heavy methyl group, the other with a light methyl group. Proteins were detected by liquid chromatography tandem mass spectrometry. Protein identifications were filtered using a 1% false discovery threshold and a requirement for two or more peptides detected for each protein. Differentially expressed proteins (DEPs) were identified as having heavy/light ratios greater than 1.2 or less than 0.8, which are recognised cut-off points for differential expression in proteomics. Over 140 DEPs were detected between CON and HF samples, indicating a nutritional influence on FF protein composition. Over one-third (37%) of these DEPs were also differentially expressed in the blastocyst versus no blastocyst analyses, suggesting that the altered FF protein composition may affect IVF outcome. DEPs were submitted into Ingenuity Pathway Analysis to highlight associated canonical pathways and upstream regulators. Top ranking canonical pathways detected included coagulation system, acute phase response, and LXR/RXR activation pathways. Potential upstream regulators detected by IPA included transforming growth factor beta, tumour protein P53, and beta-oestradiol. These pathways and upstream regulators could serve as potential avenues for elucidating the mechanism(s) by which the HF diet results in the reproductive benefits and could lead to the refinement of IVM and IVF culture conditions.