Goose Embryo Fibroblasts Research Articles

First Report of a Novel Goose Adenovirus Outbreak in Lion Head Gooses in China

In April 2022, a novel Goose adenovirus (GoAdV) isolated from diseased Lion head gooses exhibiting swelling and hemorrhage of liver and kidney, accumulation of fluid in pericardial, in Fujian province of China. The GoAdV was propagated in goose embryo fibroblasts (GEFs), the morphological properties of the virions were studied by electron microscopy, and the full genome sequence was determined and analyzed. The results revealed that the infected cells became round and clustered like grapes, virions accumulated and were arranged in crystal lattice formations in the nucleus of GEFs with a diameter of ∼80 nm. The new isolate (named CH-FJZZ-202201) has a viral genome size of 43,480 bp and shared 96.69% sequence identity with GoAdV-4 (P29), representing the species Goose aviadenovirus A. Phylogenetic analysis showed that CH-FJZZ-202201 was in the same genetic evolutionary branch with the viruses of Aviadenovirus and was the closest relative to GoAdV-4 P29/Hungary. This is the first report of the GoAdV-4 outside of Hungary, indicating the reemergence of new AdV strains in China.

Goose STING mediates IFN signaling activation against RNA viruses.

Stimulator of the interferon gene (STING) is involved in mammalian antiviral innate immunity as an interferon (IFN) activator. However, there is still a lack of clarity regarding the molecular characterization of goose STING (GoSTING) and its role in the innate immune response. In the present study, we cloned GoSTING and performed a series of bioinformatics analyses. GoSTING was grouped into avian clades and showed the highest sequence similarity to duck STING. The in vitro experiments showed that the mRNA levels of GoSTING, IFNs, IFN-stimulated genes (ISGs), and proinflammatory cytokines were significantly upregulated in goose embryo fibroblast cells (GEFs) infected with Newcastle disease virus (NDV). Overexpression of GoSTING in DF-1 cells and GEFs strongly activated the IFN-β promoter as detected by a dual-luciferase reporter assay. Furthermore, overexpression of GoSTING induced the expression of other types of IFN, ISGs, and proinflammatory cytokines and inhibited green fluorescent protein (GFP)-tagged NDV (NDV-GFP) and GFP-tagged vesicular stomatitis virus (VSV) (VSV-GFP) replication in vitro. In conclusion, these data suggest that GoSTING is an important regulator of the type I IFN pathway and is critical in geese’s innate immune host defense against RNA viruses.

Molecular characterisation and function analysis of NOD1 gene from Yangzhou goose (Anser cygnoides domesticus)

ABSTRACT 1. NOD1 is a significant member of the NOD-like receptor (NLR) family. Its main role is to identify microorganisms that invade the body, transmit immune signals and regulate innate immune responses. However, the expression and role of the NOD1 in immune defence against infection in geese remain unknown. 2. The RT-PCR method and rapid amplification of cDNA ends (RACE) was used to obtain the full-length goose NOD1 (gNOD1) cDNA series. The cDNA for gNOD1 contains 2856-bp nucleotides, i.e. 47-bp 5ʹ UTR, 135-bp 3ʹ UTR, and 1275-bp ORF region, and encodes a 951-amino-acids (AAs) polypeptide chain. The nucleotide sequence of gNOD1 was found more than 90% similar to its homologs from other avian organisms. 3. The qRT-PCR results showed that gNOD1 mRNA was widely distributed in different tissues, but highly expressed in liver, spleen, lung and caecum tissues. 4. Following stimulation of goose embryo fibroblasts (GEFs) with lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (poly(I:C)), the expression of gNOD1 and cytokines, such as IL-1β, IL-6, IL-18, and TNF-α, changed with the response-efficacy correlation at 24 and 48 h post-infection (hpi). 5. When the goslings were challenged with Salmonella entertidis (SE) and LPS, the expression of gNOD1 was up-regulated at 3 and 6 hpi in the spleen and caecum tissues, respectively. However, after SE infection, the expression level of gNOD1 fluctuated, while in the LPS group, gNOD1 mRNA increased immediately at a peak time of 6 hpi and then steadily declined. These results indicated that NOD1 was associated with the potency to resist bacterial and viral infections in the goose, both in vivo and in vitro.

Goose parvovirus and the protein NS1 induce apoptosis through the AIF-mitochondrial pathway in goose embryo fibroblasts

In this study, the effects of Goose parvovirus (GPV) infection as well as the possible role of NS1 protein on apoptosis induction in goose embryo fibroblast (GEF) cells were examined. Flow cytometry analysis and TUNEL assays revealed that GPV infection and NS1 transfection induced significant apoptosis in GEF cells compared to what was observed in mock-infected cells. Interestingly, the increase in the rate of apoptosis detected in GPV-infected GEFs was accompanied by an increased viral load in the cells. In addition, the apoptotic pathway was mediated by apoptosis-inducing factors (AIFs) and internal factors that influence the release of AIFs. The results indicated that the mitochondrial membrane potential was decreased, and AIF expression was increased in the nucleus (P < 0.01). Reactive oxygen species (ROS) increased gradually within 48 h (P < 0.001). Cathepsin D activities were also increased (P < 0.05). The results demonstrated that the AIF-mediated pathway is a new mitochondrial apoptotic pathway and that mitochondrial depolarization, ROS content, and cathepsin D activities are the key factors influencing apoptosis in GEF cells.

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.

The 164\u2009K, 165\u2009K, and 167\u2009K residues of VP1 are vital for goose parvovirus proliferation in GEFs based on PCR-based reverse genetics system

BackgroundGoose parvovirus (GPV) is the etiological agent of Derzsy’s disease and is fatal for gosling. Research on the molecular basis of GPV pathogenicity has been hampered by the lack of a reliable reverse genetics system. At present, the GPV infectious clone has been rescued by transfection in the goose embryo, but the growth character of it is unclear in vitro.MethodsIn this study, we identified the full-length genome of GPV RC16 from the clinical sample, which was cloned into the pACYC177, generating the pIRC16. The recombinant virus (rGPV RC16) was rescued by the transfection of pIRC16 into goose embryo fibroblasts (GEFs). The rescued virus was characterized by whole genome sequencing, indirect immunofluorescence assays (IFA) and western blot (WB) using rabbit anti-GPV Rep polyclonal antibody as the primary antibody. Previously, we found the 164 K, 165 K, and 167 K residues in the 160YPVVKKPKLTEE171 are required for the nuclear import of VP1 (Chen S, Liu P, He Y, et al. Virology 519:17–22). According to that, the GPV infectious clones with mutated K164A, K165A, or K167A in VP1 were constructed, rescued and passaged.ResultsThe rGPV RC16 has been successfully rescued by transfection of pIRC16 into the GEFs and can proliferate in vitro. Furthermore, the progeny virus produced by pIRC16 transfected cells was infectious in GEFs. Moreover, mutagenesis experiments showed that the rGPV RC16 with mutated 164 K, 165 K and 167 K in VP1 could not proliferate in GEFs based on the data of IFA and WB in parental virus and progeny virus.ConclusionsThe rGPV RC16 containing genetic maker and the progeny virus are infectious in GEFs. The 164 K, 165 K, and 167 K of VP1 are vital for the proliferation of rGPV RC16 in vitro.

Geese not susceptible to virulent subgroup J avian leukosis virus isolated from chickens

ABSTRACT To determine whether geese are susceptible to infection by avian leukosis virus (ALV), 702 serum samples from domestic and foreign goose breeds were screened for p27 antigen as well as being inoculated into DF-1 cell cultures to isolate ALV. Although 5.7% of samples were positive for p27 antigen, reactivity appeared to be non-specific because no ALV was detected in the corresponding DF-1 cultures. To further determine whether geese are susceptible to ALV-J isolated from chickens, ALV-J strain JS09GY7 was artificially inoculated into 10-day-old goose embryos, with one-day-old hatched goslings then screened for p27 antigen and the presence of ALV. In all cases, the results of both tests were negative. Liver tissues from the 1-day-old goslings were screened using a polymerase chain reaction-based assay, which failed to amplify ALV-J gene fragments from any of the samples. Further, no histopathological damage was observed in the liver tissues. ALV-J was further inoculated intraperitoneally into one-day-old goslings, with cloacal swabs samples and plasma samples then collected every 5 days for 30 days. All samples were again negative for the presence of p27 antigen and ALV, and liver tissues from the challenged geese showed no histopathological damage and were negative for the presence of ALV-J gene fragments. Furthermore, p27 antigen detection, PCR-based screening, and indirect immunofluorescence assays were all negative following the infection of goose embryo fibroblasts with ALV-J. Together, these results confirm that virulent chicken-derived ALV-J strains cannot infect geese, and that p27 antigen detection in goose serum is susceptible to non-specific interference.

Growth characteristics of the novel goose parvovirus SD15 strain in vitro

BackgroundShort beak and dwarfism syndrome (SBDS) was caused by novel goose parvovirus (NGPV)--a variant of goose parvovirus (GPV). Ducks infected with NGPV shows clinical signs including growth retardation and protrusion of the tongue from an atrophied beak. SBDS outbreak was first reported at the northern coastal provinces of China during 2015 and it was again reported in Sichuan, an inland province of China in 2016. The disease caused a huge economic loss in Chinese duck feeding industry.ResultsThe SD15 strain of NGPV was isolated from liver and intestinal tract tissue samples of infected ducks. Real-time quantitative PCR (qPCR) was used to estimate viral load in embryonated eggs and cells infected with adapted virus. The data showed that duck embryo fibroblasts (DEFs) were permissive to NGPV, while goose embryo fibroblasts (GEFs) cells were not, and the copy numbers of SD15 in the allantoic fluid of infected eggs remained at 105.0–106.5 copies/ml. The adaption procession of the virus was determined via qPCR, and viral proliferation was detected through indirect fluorescent antibody assay (IFA) in DEFs. It was further determined that viral copy numbers peaked at 96 h post-inoculation (hpi), which is the best time to harvest the virus in DEFs. Cytotoxic effects and cell death were observed at 72 hpi in SD15 infected DEFs, yet SD15 did not induce apoptosis.ConclusionsThe growth characteristics of SD15 strain of NGPV determined would be beneficial for further molecular characterization of these viruses and develop potential vaccines if required.

Goose MAVS functions in RIG-I-mediated IFN-β signaling activation

Mitochondrial antiviral-signaling protein (MAVS) is an essential adaptor protein in retinoic acid-inducible gene I (RIG-I)-mediated antiviral innate immunity in mammals. In this study, the goose MAVS gene (goMAVS) was identified. The 2019 bp-long goMAVS exhibits 96.2% amino acid similarity compared to the predicted goMAVS. Quantitative real-time polymerase chain reactions showed that goMAVS mRNA was widely expressed in different tissues. The overexpression of goMAVS in goose embryo fibroblast cells up-regulated the mRNA levels of goose interferon-stimulated genes. We concluded that MAVS mediates the activation of type I interferon (IFN) pathway in a species-specific manner. We further demonstrated that a CARD-like domain, transmembrane domain and two previously unidentified domains of goMAVS were essential for the activation of type I IFN pathway. GoMAVS inhibited Newcastle disease virus replication by activating type I IFN pathways, especially at the early stages of infection. Finally, the interaction between goMAVS and goose RIG-I was confirmed. The CARD domain of goMAVS plays a vital role in the interaction. Together, we identified goMAVS as a goRIG-I interactive protein and concluded that goMAVS is involved in the activation of type I IFN pathways in goose cells.

Identification of Goose PKR Gene: Structure, Expression Profiling, and Antiviral Activity Against Newcastle Disease Virus.

Double-stranded RNA-dependent protein kinase (PKR) is an important antiviral IFN-stimulated gene (ISGs) that recognizes double-stranded RNA (dsRNA) and mediates inhibition of translation initiation and protein synthesis in various types of viral infection. In this study, the complete coding sequence (CDS) of goose PKR (goPKR) is identified and characterized. The open reading frame (ORF) of goPKR is 1668 bp, which encodes a polypeptide of 555 amino acids. The sequence identity results demonstrate that the goose PKR is most closely related to duck PKR gene, with nucleotide identities of 91.6%, whereas nucleotide identity of the goose PKR to chicken, human, and mouse PKR is 76.4%, 51.9%, and 52.0%, respectively. Interestingly, the deduced amino acid sequence of goose PKR contains 3 main structure domains, including 2 double-strand RNA-binding motif (dsRBM) domains and one serine/threonine protein kinase domain. This is similar to the chicken and mammals, whereas it is different from duck PKR protein, which contains only one dsRBM1 domain and one serine/threonine protein kinase domain. Quantitative real-time PCR analysis indicates that goose PKR mRNA is widely expressed in all sampled tissues. It is highly expressed in the blood, spleen, lung, and bursa of Fabricius and jejunum and is slightly expressed in heart, muscle, trachea, and brain. The results of confocal microscopy suggest that PKR-EGFP is mainly localized in the cytoplasm, and overexpression of goPKR protein significantly reduces Newcastle disease virus (NDV) replication (viral copies and viral titer) in goose embryo fibroblasts. These findings show that goose PKR is an important antiviral ISG, involved in the antiviral innate immune defense to NDV in geese.

The 125th Lys and 145th Thr Amino Acids in the GTPase Domain of Goose Mx Confer Its Antiviral Activity against the Tembusu Virus.

The Tembusu virus (TMUV) is an avian pathogenic flavivirus that causes a highly contagious disease and catastrophic losses to the poultry industry. The myxovirus resistance protein (Mx) of innate immune effectors is a key antiviral “workhorse” of the interferon (IFN) system. Although mammalian Mx resistance against myxovirus and retrovirus was witnessed for decades, whether or not bird Mx has anti-flavivirus activity remains unknown. In this study, we found that the transcription of goose Mx (goMx) was obviously driven by TMUV infection, both in vivo and in vitro, and that the titers and copies of TMUV were significantly reduced by goMx overexpression. In both primary (goose embryo fibroblasts, GEFs) and passaged cells (baby hamster kidney cells, BHK21, and human fetal kidney cells, HEK 293T), it was shown that goMx was mainly located in the cytoplasm, and sporadically distributed in the nucleus. The intracellular localization of this protein is attributed to the predicted bipartite nuclear localization signal (NLS; 30 residues: the 441st–471st amino acids of goMx). Intuitively, it seems that the cells with a higher level of goMx expression tend to have lower TMUV loads in the cytoplasm, as determined by an immunofluorescence assay. To further explore the antiviral determinants, a panel of variants was constructed. Two amino acids at the 125th (Lys) and 145th (Thr) positions in GTP-binding elements, not in the L4 loop (40 residues: the 532nd–572nd amino acids of goMx), were vital for the antiviral function of goMx against TMUV in vitro. These findings will contribute to our understanding of the functional significance of the antiviral system in aquatic birds, and the development of goMx could be a valuable therapeutic agent against TMUV.

Molecular identification of goose (Anser cygnoide) suppressor ubiquitin-specific protease 18 (USP18) and the effects of goose IFN and TMUV on its comparative transcripts

Ubiquitin-specific protease 18 (USP18) is known as an inhibition factor and has been associated with the innate immune response to pathogens. USP18 is the only deconjugating protease with specificity for interferon-stimulated gene 15 (ISG15), which is supposed to be missing in birds. To analyze the efficacy of goose USP18 (goUSP18) against Tembusu virus (TMUV) infection, we first cloned USP18 homologous cDNA from TMUV infected geese. The coding sequence was 1131bp, and the deduced amino acid sequence shared conserved motifs with its homologues. Tissue-specific expression has shown that goUSP18 transcripts are strongly expressed in the spleen and liver of adult geese, as well as in the pancreas of goslings. Moreover, the goUSP18 transcripts were induced by goose interferons (goIFN) in goose embryo fibroblasts (GEF) and by TLR ligands in peripheral blood mononuclear cells (PBMC). Notably, goUSP18 transcripts were highly up-regulated by TMUV infection compared to the basal level in uninfected birds. Taken together, these results suggested that goUSP18 was involved in host innate immunity against TMUV infection.

Construction of expression vectors of capsid proteins from goose parvovirus and investigation of the immunogenicity

Goose parvovirus (GPV) is a highly contagious and lethal disease in goslings and Muscovy ducklings, and is of concern to the waterfowl industry. With the aim of comparing the cellular immunogenicity of three capsid proteins of GPV, plasmids of pcDNA3.1(+)-VP1, pcDNA3.1(+)-VP2, and pcDNA3.1(+)-VP3 were constructed, and the recombinant protein VPs were expressed using an eukaryotic expression system. We detected the levels of immune-related genes (CD4, CD8α, IL-1β, IL-6, IFNα, IFNγ, and IFNλ) in both goose embryo fibroblasts (GEF) and goose peripheral blood mononuclear cells (PBMCs) cellular models. The immune response conferred by a VP2 DNA vaccine in vivo was observed in a time course. Our data suggested that the cellular immune response to VP2 and VP3 was stronger than that to VP1, while VP2 and VP3 shared similar cellularimmunereactivity. In addition, vaccination with VP2 plasmid can induce high level of IgY antibody that continued to increase through 28 days post vaccination. Therefore, our findings shed light on the host cellular immune response against GPV capsid proteins. Keywords: GPV; capsid proteins; cellular immune response; humoral immunity.

Cross-species antiviral activity of goose interferon lambda against duck plague virus is related to its positive self-regulatory feedback loop.

Duck plague virus (DPV) is a virus of the Herpesviridae family that leads to acute disease with a high mortality rate in ducks. Control of the disease contributes to the development of poultry breeding. Type III IFN family (IFN-λs) is a novel member of the IFN family, and goose IFN-λ (goIFN-λ) is a newly identified gene whose antiviral function has only been investigated to a limited extent. Here, the cross-species antiviral activity of goIFN-λ against DPV in duck embryo fibroblasts (DEFs) was studied. We found that pre-treatment with goIFN-λ greatly increased the expression of IFN-λ in both heterologous DEFs and homologous goose embryo fibroblasts (GEFs), while differentially inducing IFNα- and IFN-stimulated genes. Additionally, a positive self-regulatory feedback loop of goIFN-λ was blocked by a mouse anti-goIFN-λ polyclonal antibody, which was confirmed in both homologous GEFs and goose peripheral blood mononuclear cells (PBMCs). The suppression of the BAC-DPV-EGFP by goIFN-λ in DEFs was confirmed by fluorescence microscopy, flow cytometry (FCM) analysis, viral copies and titre detection, which can be rescued by mouse anti-goIFN-λ polyclonal antibody incubation. Finally, reporter gene assays indicated that the cross-species antiviral activity of goIFN-λ against BAC-DPV-EGFP is related to its positive self-regulatory feedback loop and subsequent ISG induction. Our data shed light on the fundamental mechanisms of goIFN-λ antiviral function in vitro and extend the considerable range of therapeutic applications in multiple-poultry disease.

Identification of 2'-5'-Oligoadenylate Synthetase-Like Gene in Goose: Gene Structure, Expression Patterns, and Antiviral Activity Against Newcastle Disease Virus.

2'-5'-oligoadenylate synthetase-like (OASL) is a kind of antiviral protein induced by interferons (IFNs), which plays an important role in the IFNs-mediated antiviral signaling pathway. In this study, we cloned and identified OASL in the Chinese goose for the first time. Goose 2'-5'-oligoadenylate synthetase-like (goOASL), including an ORF of 1527bp, encoding a protein of 508 amino acids. GoOASL protein contains 3 conserved motifs: nucleotidyltransferase (NTase) domain, 2'-5'-oligoadenylate synthetase (OAS) domain, and 2 ubiquitin-like (UBL) repeats. The tissue distribution profile of goOASL in 2-week-old gosling and adult goose were identified by Real-Time quantitative PCR, which revealed that the highest level of goOASL mRNA transcription was detected in the blood of adult goose and gosling. The mRNA transcription level of goOASL was upregulated in all tested tissues of duck Tembusu virus (DTMUV)-infected 3-day-old goslings, compared with control groups. Furthermore, using the stimulus Poly(I: C), ODN2006, R848, and lipopolysaccharide (LPS) as well as the viral pathogens DTMUV, H9N2 avian influenza virus (AIV), and gosling plague virus (GPV) to treat goose peripheral blood mononuclear cells (PBMCs) for 6 h, goOASL transcripts level was significantly upregulated in all treated groups. To further investigate the antiviral activity of goOASL, pcDNA3.1(+)-goOASL-His plasmid was constructed, and goOASL was expressed by the goose embryo fibroblast cells (GEFs) transfected with pcDNA3.1(+)-goOASL-His. Our research data suggested that Newcastle disease virus (NDV) replication (viral copies and viral titer) in GEFs was significantly reduced by the overexpression of goOASL protein. These data were meaningful for the antiviral immunity research of goose and shed light on the future prevention of NDV in fowl.

Characterization of monoclonal antibodies against waterfowl parvoviruses VP3 protein

BackgroundThe VP3 protein of goose parvovirus (GPV) or Muscovy duck parvovirus (MDPV), a major structural protein, can induce neutralizing antibodies in geese and ducks, but monoclonal antibodies (MAbs) against VP3 protein has never been characterized.ResultsThree hybridoma cell lines secreting anti-GPV VP3 MAbs were obtained and designated 4A8, 4E2, and 2D5. Immunoglobulin subclass tests differentiated them as IgG2b (4A8 and 4E2) and IgG2a (2D5). Dot blotting assays showed that three MAbs reacted with His-VP3 protein in a conformation-independent manner. A competitive binding assay indicated that the MAbs delineated two epitopes, A and B of VP3. Immunofluorescence assay showed that MAbs 4A8, 4E2, and 2D5 could specifically bind to goose embryo fibroblast cells (GEF) or duck fibroblast cells (DEF) infected with GPV and MDPV. Dot blotting also showed that the MAbs recognized both nature GPV and MDPV antigen. Western blotting confirmed that the MAbs recognized VP3 proteins derived from purified GPV and MDPV particles. The MAbs 4A8 and 2D5 had universal reactivity to heterologous GPV and MDPV tested in an antigen-capture enzyme-linked immunosorbent assay.ConclusionsPreparation and characterization of these the MAbs suggests that they may be useful for the development of a MAb-capture ELISA for rapid detection of both GPV and MDPV. Virus isolation and PCR are reliable for detecting GPV and MDPV infection, but these procedures are laborious, time-consuming, and requiring instruments. These diagnosis problems highlight the ongoing demand for rapid, reproducible, and automatic methods for the sensitive detection of both GPV and MDPV infection.

Goose RIG-I functions in innate immunity against Newcastle disease virus infections

Mammalian retinoic acid-inducible gene I (RIG-I) is a chief antiviral gene sensing viral RNA molecules including Newcastle disease virus (NDV). In this study, goose RIG-I gene (gRIG-I) was identified. The 2805bp-long gene encodes a gRIG-I protein that exhibits 93.8% amino acid identity to duck RIG-I. DF-1 chicken fibroblast cells transfected with full-length of gRIG-I or CARD domain of gRIG-I plasmids respond significantly to the agonist of 21-mer 5′ppp RNA, evident through enhancement of IFN-β promoter activity. Goose RIG-I transfected 293T/17 cells were then tested for the response to NDV infection, resulting in up-regulated activity of IFN-β promoter, and mRNA levels of IRF-3 and IFIT1, but decreased virus titer. Similar results were obtained in transfected DF-1 chicken fibroblast cells and goose embryo fibroblast cells in response to NDV infections Animal experiments further support a role of gRIG-I in goose innate immunity against NDV infections by showing increased gRIG-I mRNA levels and decreased virus titer in geese lung and air sac post-infection.

Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy’s disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy’s disease and vehicles for the delivery of drugs.

In Vitro Host-Cell Susceptibility to Usutu Virus

We investigated the susceptibility to Usutu virus (Flavivirus) of 13 permanent cell lines, 3 primary cell cultures, and chicken embryos. Vero, PK-15, and goose embryo fibroblast cells developed cytopathic effects; however, viral multiplication was detected in all mammalian cell types by immunohistochemical tests. Chicken embryo fibroblast cells and chicken embryos were resistant.

Comparative studies on duck viral enteritis (DVE) virus strains in geese

DVE virus strains isolated from ducks (KPV strain), geese (KLM strain) and an attenuated strain designated DP were compared. Geese vaccinated with the avirulent strain showed 100% protection against the KPV strain, but no or only partial protection against strain KLM, depending on age and sex. Protection against the KLM strain was achieved by using a vaccine containing an avirulent strain and a strain of medium virulence. In goose embryo fibroblast (GEF) cell cultures the virulent strains caused small plaques with sharp margins, whereas the avirulent strain produced large plaques of uneven outline. In the cross-neutralisation tests all strains studied proved to be antigenically identical. The three strains showed similar sensitivity to heat-treatment (at 50 degrees C) and phosphonoacetic acid (PAA), while the KLM strain was slightly more resistant to trypsin-treatment.