Chicken Fibroblasts Research Articles

DF-1 cells prevent MG-HS infection through gga-miR-24-3p/RAP1B mediated decreased proliferation and increased apoptosis

Mycoplasma gallisepticum (MG) is a major poultry pathogen that can induce Chronic Respiratory Disease (CRD) in chickens, causing serious economic losses in the poultry industry worldwide. Increasing evidence suggests that microRNAs (miRNAs) act as a vital role in resisting microbial pathogenesis and maintaining cellular mechanism. Our previous miRNAs sequencing data showed gga-miR-24-3p expression level was significantly increased in MG-infected chicken lungs. The aim of this study is to reveal the cellular mechanism behind the MG-HS infection. We found that gga-miR-24-3p was significantly upregulated and Ras-related protein-B (RAP1B) was downregulated in chicken fibroblast cells (DF-1) with MG infection. Dual luciferase reporting assay and rescue assay confirmed that RAP1B was the target gene of gga-miR-24-3p. Meanwhile, overexpressed gga-miR-24-3p increased the levels of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), and significantly inhibited cell proliferation as well as promoted MG-infected DF-1 cell apoptosis, whereas inhibition of gga-miR-24-3p had the opposite effect. More importantly, the results of overexpression and knockdown of target gene RAP1B demonstrated that the presence of RAP1B promoted cell proliferation and it saved the reduced or increased cell proliferation caused by overexpression or inhibition of gga-miR-24-3p. Furthermore, the overexpression of gga-miR-24-3p could significantly inhibit the expression of MG-HS adhesion protein. Taken together, these findings demonstrate that DF-1 cells can resist MG-HS infection through gga-miR-24-3p/RAP1B mediated decreased proliferation and increased apoptosis, which provides a new mechanism of resistance to MG infection in vitro.

COMPARATIVE STUDY BETWEEN ROTA VIRUSES ISOLATED FROM DIARRHEATIC INFANT BABIES AND NEWLY BORN CALVES

Rota viruses were isolated in vero continuous cell line from infant babies and newly borne calves affected by diarrhea after treatment in every passage with 10 mg/ml of trypsin and adding 0.5 mg/ml of trypsin in the maintenance media. The isolated viruses induced distinctive and progressive type of cytopatheic effect in infected cells and highest titer was 2x10 6. TCID,50/0.1 for bovine viral isolate. The isolated 50/0.1 for human viral isolate and 2x10 » viruses were identified by indirect fluorescent antibody technique by using reference calf rotavirus antisera. Comparative study were conducted on both human and bovine viral isolate including growth in different cell culture, vero cell line was very sensitive to support growth of both viruses than secondary embryonic calf kidney cell culture, lamb testes and primary embryonic chicken fibroblast cell culture. Both viruses induced morphologically similar kind or plaques in vero cell line but plaqes formed by human isolate were larger in size about 1.5 - 2.5 mm in diameter than those of bovine isolate 1.5 - 2 mm. Cross reactive viral antigens were detected between the isolated viruses in indirect fluorescent antibody technique but not in serum neutralization test by using reference bovine rotavirus antisera.

THE RESULTS OF THE USE OF BIOTECHNOLOGICAL DRUGS IN VETERINARY MEDICINE, PROMISING FOR MEDICINE (PART 1)

Cell and serum nucleases are considered as a natural biological barrier, one of the first links in the body’s antiviral defense: nucleases act on viruses that the usual immunological barrier cannot resist. Inactivation of the enzymatic properties of nucleases also leads to the loss of their antiviral activity. It was found that the nucleases do not inactivate the native virus outside the cell and act mainly on the virus that multiplies in the cells. There is a direct dependence of the antiviral activity of endonuclease on the concentration of the enzyme in the medium. The first antiviral drug created on the basis of the Serratia marcescens endonuclease was called bacterial endonuclease. Its technology was developed by joint research of NIKTI BAS of the Ministry of Medical Industry (Berdsk, Novosibirsk region) and ICIG SB of the USSR Academy of Sciences in 1973–1984. Serratia marcescens endonuclease is capable of cleaving the nucleic acids of both RNA and DNA-containing viruses. The enzyme inhibits the reproduction of vesicular stomatitis and smallpox vaccine viruses in chicken fibroblast cell culture, respectively. It was the first antiviral drug in the history of beekeeping, designed to prevent acute and chronic paralysis and other viral diseases of bees. The drug was also intended as an antiviral agent for a wide range of diseases in various organisms. The effectiveness of bacterial endonuclease as a means of prevention and treatment of respiratory viral diseases in calves was studied. The research was carried out at the industrial complex for growing heifers of the Gosnensky state farm in the Leningrad region on calves of a black-and-white breed of 20–60 days old. For the experiments, we used bacterial endonuclease produced by the Vyshnevolotsky Plant of Enzyme Preparations or NICTI BAS of the Ministry of Medical Industry. In the future, NIKTI BAV together with Diafarm LLC developed a second – generation antiviral drug, endoglucin, based on bacterial endonucleosis. Production experiments to study its effect as a therapeutic and prophylactic agent for respiratory diseases of calves were conducted during 2007– 2011. in the agricultural complex «Prichulymsky» of the Achinsky district of the Krasnoyarsk Territory with the participation of the Department of Epizootology and Parasitology of the Faculty of Veterinary Medicine of KrasGAU and in CJSC «Suzdalskoye» of the Dovolensky district of the Novosibirsk region. Prophylactic use of endoglucin in calves with bronchopneumonia can reduce the incidence from 2.2 to 3.1 times. The drug endoglucin in combination with drugs indicated for the treatment of pulmonary pathology, has therapeutic effectiveness in bronchopneumonia of calves.

BIOTECHNOLOGICAL RESEARCH IN THE CREATION AND PRODUCTION OF ANTIRABIC VACCINES

Rabies is a neurological disease of a viral nature, leading to death. Rabies virus is an RNA virus that invades the central nervous system, leading to neuronal dysfunction. Timely vaccination can prevent the diseases development. Aim. The article is devoted to immunobiotechnological research aimed at creating antirabic vaccines. Results. The history of the antirabic vaccines creation from the first inactivated vaccines obtained from nervous tissue to the cultivation of the virus on animal cell cultures is considered. The article presents commercially available anti-rabies vaccines: their composition, the used rabies virus strains, cell cultures, the methods of inactivation and purification. The technology of producing an anti-rabies vaccine based on a Pitman Moore virus strain and a chicken fibroblast cell culture is presented. The advantages of different vaccine types are considered: live attenuated, peptide, liposomal, RNA vaccines, vaccines based on viral vectors, transgenic plants and reverse genetics methods. Conclusions. The development of biotechnology, immunology and virology makes it possible to improve constantly vaccine preparations, including those against rabies, increasing their effectiveness and safety.

Molecular and Cultures based Diagnosis of Infectious Bursal Disease Virus (IBDV) in Vaccinated and Non Vaccinated Poultry Flocks

Background: In recent years, Infectious bursal disease is continuously occurring even after vaccination in India and requires an inclusive diagnosis. Therefore, the present study was undertaken to diagnose IBD through molecular and culture methods. Methods: One pooled sample, from each of 54 flocks having birds with IBD like symptoms, was collected. History of bird type, age and vaccination was recorded. Samples were subjected to RT-PCR, egg embryo culture and chicken fibroblast cells culture. Result: A total of 49669 out of 517900 (9.59 %) of birds, aging 3-6 weeks, were displaying the signs similar to IBD.In RT-PCR, 21 (38.88%) samples were found positive which belonged to11 (52.38%) vaccinated and 10 (47.62%) unvaccinated flocks.The RT-PCR positive samples were successfully cultivated for the virus through egg embryo and cell culture. The CEF culture was found least sensitive compared to egg embryo culture and RT-PCR.

In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells.

Advances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the Cas13a-msGFP-NLS (pT-Cas13a) transgene, followed by a stable insertion of the Cas13a transgene into the genome of DF1 cells to generate stable DF1-Cas13a cells. To assess the Cas13a-mediated functional knockdown, DF1-Cas13a cells were transfected with the combination of a plasmid encoding DsRed coding sequence (pDsRed) and DsRed-specific crRNA (crRNA-DsRed) or non-specific crRNA (crRNA-NS). Fluorescence-activated cell sorting (FACS) and a microscopy analysis showed reduced levels of DsRed expression in cells transfected with crRNA-DsRed but not in crRNA-NS, confirming a sequence-specific Cas13a mediated mRNA knockdown. Next, we designed four crRNAs (crRNA-IAV) against the PB1, NP and M genes of influenza A virus (IAV) and cloned in tandem to express from a single vector. DF1-Cas13a cells were transfected with plasmids encoding the crRNA-IAV or crRNA-NS, followed by infection with WSN or PR8 IAV. DF1 cells transfected with crRNA-IAV showed reduced levels of viral titers compared to cells transfected with crRNA-NS. These results demonstrate the potential of Cas13a as an antiviral strategy against highly pathogenic strains of IAV in chickens.

Interferonogenic activity of the strain BH-3 duck hepatitis virus type I (Picornaviridae: Avihepatovirus: Avihepatovirus A)

Duck viral hepatitis type I (DVH-I) is a poorly studied contagious disease caused by RNA-containing duck (Anatinae) hepatitis virus type I (Picornaviridae: Avihepatovirus: Avihepatovirus A). This infection is widespread in many countries, including Russia, and causes significant damage to industrial duck breeding. The study of interferonogenic activity of its etiologic agent strains is of great importance in solving the problem of developing effective means to control the disease. Strain BH-3 of duck hepatitis virus type I isolated from the liver of sick ducklings was used in the study. The strain was adapted to developing 10-12 day old duck embryos, to the cell culture of chicken and duck fibroblasts and deposited in the State Collection of Viruses of the D.I. Ivanovsky Institute of Virology of FSBI «National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya» of the Ministry of Health of Russia. Experiments were performed using the standard tissue culture method. Data on the ability of the viral strain BH-3 to induce interferon (IFN) and its sensitivity to the action of exogenous interferon in the culture of duck fibroblasts are presented. It has been shown that the interferonogenic activity of this strain of the hepatitis virus is in direct proportion to the multiplicity of infection. The maximum induction of IFN (1 : 256 CEPD50) was observed at a dose of 1.0 TCD50/cell in 72-96 hrs after inoculation of the cell culture. Exogenous IFN at a dose of 1 : 128 completely suppressed the cytopathic effect and death of duck embryos infected with hepatitis virus at a dose of 100 TCD50/cell. The data obtained allow us to state that the vaccine strain BH-3 of duck hepatitis virus type I has a pronounced interferonogenic activity and sensitivity to the action of exogenous IFN. This may have implications for the development of effective therapeutic agents against DVH-I.

Thymic transcriptome analysis after Newcastle disease virus inoculation in chickens and the influence of host small RNAs on NDV replication

Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV), is a contagious viral disease affecting various birds and poultry worldwide. In this project, differentially expressed (DE) circRNAs, miRNAs and mRNAs were identified by high-throughput RNA sequencing (RNA-Seq) in chicken thymus at 24, 48, 72 or 96 h post LaSota NDV vaccine injection versus pre-inoculation group. The vital terms or pathways enriched by vaccine-influenced genes were tested through KEGG and GO analysis. DE genes implicated in innate immunity were preliminarily screened out through GO, InnateDB and Reactome Pathway databases. The interaction networks of DE innate immune genes were established by STRING website. Considering the high expression of gga-miR-6631-5p across all the four time points, DE circRNAs or mRNAs with the possibility to bind to gga-miR-6631-5p were screened out. Among DE genes that had the probability to interact with gga-miR-6631-5p, 7 genes were found to be related to innate immunity. Furthermore, gga-miR-6631-5p promoted LaSota NDV replication by targeting insulin induced gene 1 (INSIG1) in DF-1 chicken fibroblast cells. Taken together, our data provided the comprehensive information about molecular responses to NDV LaSota vaccine in Chinese Partridge Shank Chickens and elucidated the vital roles of gga-miR-6631-5p/INSIG1 axis in LaSota NDV replication.

The SOX11 Transcription Factor Upregulates Growth Differentiation Factor‐5 During Joint Formation Through a Downstream Enhancer Element

The direct molecular mechanism underlying joint formation and remodeling has been studied for decades yet still is not fully understood. Growth differentiation factor 5 (GDF5), a critical joint-associated transcription factor linked to accelerated osteoarthritis, has been shown to be one of the intermediate markers for precursors of joint associated cells. Understanding the mechanisms regulating GDF5 expression will further illuminate some of the key steps of joint development. Previously, we identified a GDF5 Associated Regulatory Region (GARR) with putative Sox binding sites; the SOX11 transcription factor has been shown to upregulate GDF5. Hence, we hypothesize that SOX11 (SOXC family) is necessary for GARR-mediated expression of GDF5 during joint development and maintenance. We previously demonstrated that site-directed mutagenesis of the putative Sox binding sites in a GARR reporter construct markedly reduced activity. To confirm the role of SOX11 we performed over expression experiments in transformed chicken fibroblast (DF1) cells, a primary limb bud micromass culture, and in ovo in the developing chicken wing. Co-transfection studies demonstrated increased GARR activity both in vivo and in vitro with over expression of SOX11. Taken together, these data suggest that SOX11 may play a role in the regulation of GDF5 through GARR.

Cytokine inducible SH2-containing protein potentiate J subgroup avian leukosis virus replication and suppress antiviral responses in DF-1 chicken fibroblast cells

Cytokine-inducible Srchomology2 (SH2)-containing protein (CIS) belongs to the suppressors of cytokine signaling (SOCS) protein family function as a negative feedback loop inhibiting cytokine signal transduction. J subgroup avian leukosis virus (ALV-J), a commonly-seen avian virus with a feature of immunosuppression, poses an unmeasurable threat to the poultry industry across the world. However, commercial medicines or vaccines are still no available for this virus. This study aims to evaluate the potential effect of chicken CIS in antiviral response and its role on ALV-J replication. The results showed that ALV-J strain SCAU-HN06 infection induced CIS expression in DF-1 cells, which was derived from chicken embryo free of endogenous avian sarcoma-leukosis virus (ASLV) like sequences. By overexpressing CIS, the expression of chicken type I interferon (IFN-I) and interferon-stimulated genes (ISGs; PKR, ZAP, CH25H, CCL4, IFIT5, and ISG12) were both suppressed. Meanwhile, data showed that CIS overexpression also increased viral yield. Interestingly, knockdown of CIS enhanced induction of IFN-I and ISGs and inhibited viral replication. Collectively, we proved that modulation of CIS expression not only affected SCAU-HN06 replication in vitro but also altered the expression of IFN-I and ISGs that act as an essential part of antiviral innate immune system. Our data provide a potential target for developing antiviral agents for ALV-J.

Role of the chicken oligoadenylate synthase-like gene during in vitro Newcastle disease virus infection

The enzyme 2′-5′ oligoadenylate synthase (OAS) is one of the key interferon-induced antiviral factors that act through inhibition of viral replication. In chickens, there is a single well-characterized OAS gene, oligoadenylate synthase-like (OASL) that has been shown to be upregulated after infection with various viruses. However, a deeper understanding of how chicken OASL acts against viral infection is still necessary. In this study, we tested the hypothesis that OASL short interfering RNA (siRNA)–mediated knockdown would decrease the host gene expression response to the Newcastle disease virus (NDV) by impacting antiviral pathways. To assess our hypothesis, a chicken fibroblast cell line (DF-1) was infected with the NDV (LaSota strain) and OASL expression was knocked down using a specific siRNA. The level of NDV viral RNA in the cells and the expression of interferon response- and apoptosis-related genes were evaluated by quantitative PCR at 4, 8, and 24 h postinfection (hpi). Knockdown of OASL increased the level of NDV viral RNA at 4, 8, and 24 hpi (P < 0.05) and eliminated the difference between NDV-infected and noninfected cells for expression of interferon response- and apoptosis-related genes (P > 0.05). The lack of differential expression suggests that knockdown of OASL resulted in a decreased response to NDV infection. Within NDV-infected cells, OASL knockdown reduced expression of signal transducer and activator of transcription 1, interferon alfa receptor subunit 1, eukaryotic translation initiation factor 2 alpha kinase 2, ribonuclease L, caspase 8 (CASP8) and caspase 9 (CASP9) at 4 hpi, CASP9 at 8 hpi, and caspase 3, CASP8, and CASP9 at 24 hpi (P < 0.05). We suggest that the increased NDV viral load in DF-1 cells after OASL knockdown was the result of a complex interaction between OASL and interferon response- and apoptosis-related genes that decreased host response to the NDV. Our results provide comprehensive information on the role played by OASL during NDV infection in vitro. Targeting this mechanism could aid in future prophylactic and therapeutic treatments for Newcastle disease in poultry.

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.

Cellular CARD11 Inhibits the Fusogenic Activity of Newcastle Disease Virus via CBM Signalosome-Mediated Furin Reduction in Chicken Fibroblasts.

Newcastle disease virus (NDV) causes an infectious disease that poses a major threat to poultry health. Our previous study identified a chicken brain-specific caspase recruitment domain-containing protein 11 (CARD11) that was upregulated in chicken neurons and inhibited NDV replication. This raises the question of whether CARD11 plays a role in inhibiting viruses in non-neural cells. Here, chicken fibroblasts were used as a non-neural cell model to investigate the role. CARD11 expression was not significantly upregulated by either velogenic or lentogenic NDV infection in chicken fibroblasts. Viral replication was decreased in DF-1 cells stably overexpressing CARD11, while viral growth was significantly increased in the CARD11-knockdown DF-1 cell line. Moreover, CARD11 colocalized with the viral P protein and aggregated around the fibroblast nucleus, suggesting that an interaction existed between CARD11 and the viral P protein; this interaction was further examined by suppressing viral RNA polymerase activity by using a minigenome assay. Viral replication was inhibited by CARD11 in fibroblasts, and this result was consistent with our previous report in chicken neurons. Importantly, CARD11 was observed to reduce the syncytia induced by either velogenic virus infection or viral haemagglutinin-neuraminidase (HN) and F cotransfection in fibroblasts. We found that CARD11 inhibited the expression of the host protease furin, which is essential for cleavage of the viral F protein to trigger fusogenic activity. Furthermore, the CARD11-Bcl10-MALT1 (CBM) signalosome was found to suppress furin expression, which resulted in a reduction in the cleavage efficiency of the viral F protein to further inhibit viral syncytia. Taken together, our findings mainly demonstrated a novel CARD11 inhibitory mechanism for viral fusogenic activity in chicken fibroblasts, and this mechanism explains the antiviral roles of this molecule in NDV pathogenesis.

ChGRP78 is Required for the Activation and Phosphorylation of AKT1 at Serine 478 in Chicken Sells

Background: Chicken serum-mediated proliferation regulates chGRP78 to prevent apoptosis in chicken cells via chGRP78-mediated anti-apoptosis. However, the precise molecular mechanisms underlying the chGRP78-mediated protection against apoptosis remain undefined. In an earlier study, we have shown that chGRP78 is critical for chicken embryo fibroblast (CEF) and DF-1 cell proliferation.Methods: In this experiment, we highlight AKT1 as a key target of GRP78 during apoptosis. We used 2D gel-based proteomics and bioinformatics prediction analysis for our studies. Result: Here, we detected chGRP78 binding sites in AKT1-rgulated proteins. chGRP78 promoted AKT1 activation and chGRP78 silencing decreased AKT1 levels. Taken together, we suggest that the AKT1-mediated signaling pathway plays a critical role in GRP78-stimulated fibroblast survival and anti-apoptosis. Our findings have important implications for the maintenance of chicken fibroblast cells via the inhibition of apoptosis.

KAv-1 is Better Suited to Chick Fibroblast Culture than DMEM or 199 Media.

Cultured cells are a useful resource for poultry scientists, since these cells allow scientists to evaluate biological responses to conditions such as infectious diseases in vitro while mimicking the whole-body response in birds. However avian cell culture requires an optimized basal medium, and there are currently relatively few options for this basal medium (medium 199 and KAv-1). This means that there is still room for the development of an optimal basal medium for avian cell culture. Here we compare KAv-1 medium, Dulbecco's modified Eagle medium (DMEM) and medium 199 during the culture of chick fibroblasts and determine that KAv-1 remains the optimal medium for these assays. Our results show that DNA damage is reduced in fibroblasts cultured in the KAv-1 medium, when compared to both DMEM and Medium 199 and that these cells also display improved growth dynamics in KAv-1 medium when compared to both DMEM and medium 199. To the best of our knowledge, this is the first study to describe a comparative analysis of culture media for avian cells, which would provide useful information for poultry scientists.

The expression of Lin28B was co-regulated by H3K4me2 and Wnt5a/β-catenin/TCF7L2

Lin28A and Lin28B are homologous RNA-binding proteins that participate in the development of primordial germ cells. The mechanisms underlying expression and regulation of Lin28A have been well documented, but such information for Lin28B is limited. In this study, a fragment of the Lin28B promoter was cloned, the pEGFP-pLin28B vector was constructed. DF-1 chicken fibroblasts were transfected and the expression of green fluorescent protein (GFP) was measured. Furtherly, Lin28B promoter of different lengths fragments was cloned using the chromosome-walking method and the fragments were ligated into the PGL3-Basic vector, and transfected into DF-1 cells. Results of dual-luciferase reporter assay showed that the core of the Lin28B promoter was included in the sequence from –1 431 to –1 034 bp. The binding sites of the transcription factor TCF7L2 was showed within this sequence by bioinformatics analysis. The promoter activity of Lin28B was downregulated (P<0.05) when the TCF7L2 binding site was mutated. Further experiments suggested that Lin28B promoter activity responded to the activation or inhibition of Wnt signaling. Results of chromatin immunoprecipitation and quantitative PCR showed that β-catenin-TCF7L2 may be enriched in the Lin28B promoter core area. In vivo and in vitro activation or inhibition of Wnt signaling significantly up- or down-regulated (P<0.05) Lin28B expression. H3K4me2 enriched in the promoter of Lin28B, which affected the regulation of Wnt signaling to Lin28B. In conclusion, our results showed that H3K4me2 and Wnt5a/β-catenin/TCF7L2 were the positive regulators of Lin28B expression. Findings of this study may lay a theoretical foundation for illuminating the mechanism underlying Lin28B expression.

Isolation and evolutionary analyses of gout-associated goose astrovirus causing disease in experimentally infected chickens

Astroviruses are a common cause of gastroenteritis in humans and animals. They are also associated with extraintestinal infections, including hepatitis in ducklings, nephritis in chickens, as well as fatal meningitis and encephalitis in humans and other mammals. Since 2014, outbreaks of disease characterized by visceral gout and swelling of kidneys have been reported in goslings and ducklings in China, with the causative agent revealed to be a novel avian astrovirus designated goose astrovirus (GoAstV). In the present study, this novel gout-associated GoAstV was identified in diseased goslings from 2 farms in Hunan province, China. Three genomes were successfully sequenced and analyzed and were shown to have high identities of 99.7 to 99.8% between each other, with some specific amino acid alterations revealed in open reading frame 2 when compared with other gout-associated GoAstVs. Two strains were further efficiently isolated in the DF-1 chicken fibroblast cell line with high virus titers of 1011 viral genomic copies per mL of culture media. A pilot virus challenge study using GoAstV in chickens demonstrated that this virus can cause clinical visceral gout in chickens, indicating its ability to cross the species barrier. Based on the phylogenetic analyses of capsid sequences, the identified GoAstVs were proposed to be classified into 2 genotypes, GoAstV1 and GoAstV2, and the novel gout-associated GoAstVs were all clustered in GoAstV2. Further Bayesian inference analyses indicated a nucleotide substitution rate of 1.46 × 10−3 substitutions/site/year for avian astrovirus based on open reading frame 2 sequences, and the time to the most recent common ancestor of GoAstVs was estimated to be around 2011. This is the first report to confirm GoAstV can infect chickens while also providing an estimation of the evolutionary rates of Avastroviruses.

In Vitro and In Ovo Host Restriction of Aquatic Bird Bornavirus 1 in Different Avian Hosts.

Aquatic bird bornavirus 1 (ABBV-1) is associated with chronic meningoencephalitis and ganglioneuritis. Although waterfowl species act as the natural host of ABBV-1, the virus has been sporadically isolated from other avian species, showing the potential for a broad host range. To evaluate the host restriction of ABBV-1, and its potential to infect commercial poultry species, we assessed the ability of ABBV-1 to replicate in cells and embryos of different avian species. ABBV-1 replication was measured using multi- and single-step growth curves in primary embryo fibroblasts of chicken, duck, and goose. Embryonated chicken and duck eggs were infected through either the yolk sac or chorioallantoic cavity, and virus replication was assessed by immunohistochemistry and RT-qPCR in embryonic tissues harvested at two time points after infection. Multi-step growth curves showed that ABBV-1 replicated and spread in goose and duck embryo fibroblasts, establishing a population of persistently infected cells, while it was unable to do so in chicken fibroblasts. Single-step growth curves showed that cells from all three species could be infected; however, persistence was only established in goose and duck fibroblasts. In ovo inoculation yielded no detectable viral replication or lesion in tissues. Data indicate that although chicken, duck, and goose embryo fibroblasts can be infected with ABBV-1, a persistent infection is more easily established in duck and goose cells. Therefore, ABBV-1 may be able to infect chickens in vivo, albeit inefficiently. Additionally, our data indicate that an in ovo model is inadequate to investigating ABBV-1 host restriction and pathogenesis.

Highly elevated base excision repair pathway in primordial germ cells causes low base editing activity in chickens.

Base editing technology enables the generation of precisely genome-modified animal models. In this study, we applied base editing to chicken, an important livestock animal in the fields of agriculture, nutrition, and research through primordial germ cell (PGC)-mediated germline transmission. Using this approach, we successfully produced two genome-modified chicken lines harboring mutations in the genes encoding ovotransferrin (TF) and myostatin (MSTN); however, only 55.5% and 35.7% of genome-modified chickens had the desired base substitutions in TF and MSTN, respectively. To explain the low base-editing activity, we performed molecular analysis to compare DNA repair pathways between PGCs and the chicken fibroblast cell line DF-1. The results revealed that base excision repair (BER)-related genes were significantly elevated in PGCs relative to DF-1 cells. Subsequent functional studies confirmed that the editing activity could be regulated by modulating the expression of uracil N-glycosylase (UNG), an upstream gene of the BER pathway. Collectively, our findings indicate that the distinct DNA repair property of chicken PGCs causes low editing activity during genome modification, however, modulation of BER functions could promote the production of genome-modified organisms with the desired genotypes.

Electrochemiluminescence immunosensor for 5-hydroxymethylcytosine detection based on PAMAM-nanosilver‑nitrogen doped graphene nanocomposite

As a significant potential biomarker for early disease diagnostics, the detection of 5-hydroxymethylcytosine (5hmC) has garnered widespread attention. In this work, a simple and sensitive electrochemiluminescence (ECL) was developed for 5hmC detection. After substrate electrode preparation, anti-5hmC antibody was captured on electrode surface by the formation of amide bond. Then, through immunoreaction, 5hmC was specifically recognized and captured. Utilizing the crosslink effect of Zr 4+ with phosphoric acid recognition, aminomethyl phosphate was modified on electrode to further capture PAMAM-silver nanoclusters/Nitrogen-doped graphene nanocomposite (PAMAM-Ag-NG), where this nanocomposite was employed as ECL signal probe. The designed immunosensor showed a linear concentration range from 10 pM to 30 nM with a low detection limit of 2.47 pM (S/N = 3). This method presented good selectivity and reproducibility. To prove the applicability, this method was further used to study the influence of phytohormone and avian leukemia virus on 5hmC content in maize seedling leaves and chicken fibroblast cells, respectively. • A novel ECL strategy was developed for sensitive detection of 5hmC. • PAMAM-silver nanoclusters/Nitrogen-doped graphene nanocomposite was employed as ECL probe. • Avian leukemia virus decreased 5hmC content in chicken fibroblast cells. • Abscisic acid and gibberellinon decreased 5hmC content in maize seedling leaves. • The low detection limits of 2.47 pM was achieved.