The transcription factor TCF-1 initiates the differentiation of T(FH) cells during acute viral infection.

Induction of the transcriptional repressor Bcl-6 in CD4(+) T cells is critical for the differentiation of follicular helper T cells (T(FH) cells), which are essential for B cell-mediated immunity. In contrast, the transcription factor Blimp1 (encoded by Prdm1) inhibits T(FH) differentiation by antagonizing Bcl-6. Here we found that the transcription factor TCF-1 was essential for both the initiation of T(FH) differentiation and the effector function of differentiated T(FH) cells during acute viral infection. Mechanistically, TCF-1 bound directly to the Bcl6 promoter and Prdm1 5' regulatory regions, which promoted Bcl-6 expression but repressed Blimp1 expression. TCF-1-null T(FH) cells upregulated genes associated with non-T(FH) cell lineages. Thus, TCF-1 functions as an important hub upstream of the Bcl-6-Blimp1 axis to initiate and secure the differentiation of T(FH) cells during acute viral infection.

3'UTR of ALV-J can affect viral replication through promoting transcription and mRNA nuclear export.

IMPORTANCE: 3'UTRs can affect gene transcription and post-transcriptional regulation in multiple ways, further influencing the function of proteins in a unique manner. Recently, ALV-J has been mutating and evolving rapidly, especially the 3'UTR of viral genome. Meanwhile, clinical symptoms caused by ALV-J have changed significantly. In this study, we found that the ALV-J strains containing △-r-TM-type 3'UTR are the most abundant. By constructing ALV-J infectious clones and subgenomic vectors containing different 3'UTRs, we prove that 3'UTRs directly affect viral tissue preference and can promote virus replication as an enhancer. ALV-J strain containing 3'UTR of △-r-TM proliferated fastest in primary cells. All five forms of 3'UTRs can assist intron-containing viral mRNA nuclear export, with similar efficiency. ALV-J mRNA half-life is not influenced by different 3'UTRs. Our results dissect the roles of 3'UTR on regulating viral replication and pathogenicity, providing novel insights into potential anti-viral strategies.

Amino Acid Variation at Hemagglutinin Position 193 Impacts the Properties of H9N2 Avian Influenza Virus.

Despite active control strategies, including the vaccination program in poultry, H9N2 avian influenza viruses possessing mutations in hemagglutinin (HA) were frequently isolated. In this study, we analyzed the substitutions at HA residue 193 (H3 numbering) of H9N2 and investigated the impact of these mutations on viral properties. Our study indicated that H9N2 circulating in the Chinese poultry have experienced frequent mutations at HA residue 193 since 2013, with viruses that carried asparagine (N) being replaced by those with alanine (A), aspartic acid (D), glutamic acid (E), glycine (G), and serine (S), etc. Our results showed the N193G mutation impeded the multiple cycles of growth of H9N2, and although most of the variant HAs retained the preference for human-like receptors as did the wild-type N193 HA, the N193E mutation altered the preference for both human and avian-like receptors. Furthermore, these mutations substantially altered the antigenicity of H9N2 as measured by both monoclonal antibodies and antisera. In vivo studies further demonstrated that these mutations showed profound impact on viral replication and transmission of H9N2 in chicken. Viruses with D, E, or S at residue 193 acquired the ability to replicate in lungs of the infected chickens, whereas virus with G193 reduced its transmissibility in infected chickens to those in direct contact. Our findings demonstrated that variations at HA residue 193 altered various properties of H9N2, highlighting the significance of the continued surveillance of HA for better understanding of the etiology and effective control of H9N2 in poultry. IMPORTANCE H9N2 are widespread and have sporadically caused clinical diseases in humans. Extensive vaccinations in poultry helped constrain H9N2; however, they might have facilitated the evolution of the virus. It is therefore of importance to monitor the variation of the circulating H9N2 and evaluate its risk to both veterinary and public health. Here, we found substitutions at position 193 of HA from H9N2 circulated since 2013 and assessed the impact of several mutations on viral properties. Our data showed these mutations resulted in substantial antigenic change. N193E altered the binding preference of HA for human-like to both avian and human-like receptors. More importantly, N193G impaired the growth of H9N2 and its transmission in chickens, whereas mutations from N to D, E, and S enhanced the viral replication in lungs of chickens. Our study enriched the knowledge about H9N2 and may help implement an effective control strategy for H9N2.

10× single-cell sequencing revealed cellular composition heterogeneity in cardiac myxoma with malignant glandular properties.

Cardiac myxoma is the most common primary cardiac tumor in adults. The histogenesis and cellular composition of myxoma are still unclear. This study aims to reveal the role of myxoma cell components and their gene expression in tumor development. We obtained single living cells by enzymatic digestion of tissues from 4 cases of surgically resected cardiac myxoma. Of course, there was 1 case of glandular myxoma and 3 cases of nonglandular myxoma. Then, 10× single-cell sequencing was performed. We identified 12 types and 11 types of cell populations in glandular myxoma and nonglandular myxoma, respectively. Heterogeneous epithelial cells are the main components of glandular myxoma. The similarities and differences in T cells in both glandular and nonglandular myxoma were analyzed by KEGG and GO. The most important finding was that there was active communication between T cells and epithelial cells. These results clarify the possible tissue occurrence and heterogeneity of cardiac myxoma and provide a theoretical basis and guidance for clinical diagnosis and treatment.

Glycosylation of ALV-J Envelope Protein at Sites 17 and 193 Is Pivotal in the Virus Infection.

Glycans on envelope glycoprotein (Env) of the subgroup J avian leukosis virus (ALV-J) play an essential role in the virion integrity and infection process. In this study, we found that, among the 13 predicted N-linked glycosylation sites (NGSs) in gp85 of Tibetan chicken strain TBC-J6, N17, and N193/N191 are pivotal for virus replication. Further research illustrated that a mutation at N193 weakened Env-receptor binding in a blocking assay of the viral entrance, coimmunoprecipitation, and ELISA. Our studies also showed that N17 was involved in Env protein processing and later virion incorporation based on the detection of p27 and Env protein in the supernatant and gp37 in the cell culture. This report is systematic research on clarifying the biological function of NGSs on ALV-J gp85, which would provide valuable insight into the role of gp85 in the ALV life cycle and anti-ALV-J strategies. IMPORTANCE ALV-J is a retrovirus that can cause multiple types of tumors in chickens. Among all the viral proteins, the heavily glycosylated envelope protein is especially crucial. Glycosylation plays a major role in Env protein function, including protein processing, receptor attachment, and immune evasion. Notably, viruses isolated recently seem to lose their 6th and 11th NGS, which proved to be important in receptor binding. In our study, the 1st (N17) and 8th (N193) NGS of gp85 of the strain TBC-J6 can largely influence the titer of this virus. Deglycosylation at N193 weakened Env-receptor binding while mutation at N17 influenced Env protein processing. This study systemically analyzed the function of NGSs in ALV-J in different aspects, which may help us to understand the life cycle of ALV-J and provide antiviral targets for the control of ALV-J.

A novel mAb broadly neutralizes SARS-CoV-2 VOCs in vitro and in vivo, including the Omicron variants.

Novel immune escape variants have emerged as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide. Many of the variants cause breakthrough infections in vaccinated populations, posing great challenges to current antiviral strategies targeting the immunodominance of the receptor-binding domain within the spike protein. Here, we found that a novel broadly neutralizing monoclonal antibody (mAb), G5, provided efficient protection against SARS-CoV-2 variants of concern (VOCs) in vitro and in vivo. A single dose of mAb G5 could significantly inhibit the viral burden in mice challenged with the mouse-adapted SARS-CoV-2 or SARS-CoV-2 Omicron BA.1 variant, as well as the body weight loss and cytokine release induced by mouse-adapted SARS-CoV-2. The refined epitope recognized by mAb G5 was identified as 1148 FKEELDKYF1156 in the stem helix of subunit S2. In addition, a human-mouse chimeric mAb was generated based on the variable region of heavy chain and VL genes of mAb G5. Our study provides a broad antibody drug candidate against SARS-CoV-2 VOCs and reveals a novel target for developing pan-SARS-CoV-2 vaccines.

Investigation of Pre-service Teachers' Conceptions of the Nature of Science Based on the LDA Model.

This study used the Latent Dirichlet Allocation (LDA) topic model to analyze pre-service teachers' views on the nature of science (NOS). This approach can be used to automate the classification of documents, and at the same time, the researcher does not need to deduce with a NOS framework prior to evaluation. Participants were 155 pre-service teachers studying at the Shandong Normal University in China. To gather our data, we used an open questionnaire, namely, the Views of Nature of Science Questionnaire-Form C (VNOS-C). LDA topic modeling was used to classify the document, which was divided into 12 topics. By comparing the LDA topic modeling results with the theoretical framework behind the VNOS-C questionnaire, we categorized these 12 topics into eight descriptive aspects of the NOS: The Empirical Nature of Scientific Knowledge, Observation, Inference, and Theoretical Entities in Science, Scientific Theories and Laws, The Theory-Laden Nature of Scientific Knowledge, The Social and Cultural Embeddedness of Scientific Knowledge, The Myth of The Scientific Method, The Tentative Nature of Scientific Knowledge, and The Nature of Scientific Theory. The results show that pre-service teachers usually hold naive or mixed views of the NOS. In addition, each aspect of NOS is not independent of each other but interrelated and influencing each other. In the future, more consideration can be given to the relationship between each aspect of NOS.

LDHA mediated degradation of extracellular matrix is a potential target for the treatment of aortic dissection.

Aortic dissection (AD) is a disease with high mortality and lacks effective drug treatment. Recent studies have shown that the development of AD is closely related to glucose metabolism. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme and plays an important role in cardiovascular disease. However, the role of LDHA in the progression of AD remains to be elucidated. Here, we found that the level of LDHA was significantly elevated in AD patients and the mouse model established by BAPN combined with Ang II. In vitro, the knockdown of LDHA reduced the growth of human aortic vascular smooth muscle cells (HAVSMCs), glucose consumption, and lactate production induced by PDGF-BB. The overexpression of LDHA in HAVSMCs promoted the transformation of HAVSMCs from contractile phenotype to synthetic phenotype, and increased the expression of MMP2/9. Mechanistically, LDHA promoted MMP2/9 expression through the LDHA-NDRG3-ERK1/2-MMP2/9 pathway. In vivo, Oxamate, LDH and lactate inhibitor, reduced the degradation of elastic fibers and collagen deposition, inhibited the phenotypic transformation of HAVSMCs from contractile phenotype to synthetic phenotype, reduced the expression of NDRG3, p-ERK1/2, and MMP2/9, and delayed the progression of AD. To sum up, the increase of LDHA promotes the production of MMP2/9, stimulates the degradation of extracellular matrix (ECM), and promoted the transformation of HAVSMCs from contractile phenotype to synthetic phenotype. Oxamate reduced the progression of AD in mice. LDHA may be a therapeutic target for AD.

Identification of three novel B cell epitopes in ORF2 protein of the emerging goose astrovirus and their application.

The outbreak of goose gout disease caused by novel goose astrovirus type 1 (GAstV-1) has resulted in huge economic losses to the goose industry in China since 2017. However, little is known about the B cell epitopes in major antigen of GAstV-1 and the serological approach for detection of GAstV-1 is not available. In this study, three novel monoclonal antibodies (mAbs) against the ORF2 protein were first generated and designated as 3G6, 5H7, and 6C6, respectively. Epitope mapping revealed that mAb 3G6, 5H7, and 6C6 recognized 695AVRFEKGGHE704, 685EKALSAPQAG694, and 635DDDPLSDVTS644 in ORF2, respectively. Sequence alignments found that the three epitopes were highly conserved in GAstV-1 but not in other AAstV members. Moreover, a mAb-based sandwich ELISA for the detection of GAstV-1 was first developed using mAb 6C6. The sandwich ELISA only reacted with GAstV-1 but not with GAstV-2 and the other goose-associated viruses tested. The limit of the detection of the sandwich ELISA reaches 1.58 × 103 TCID50/mL of GAstV-1. Notably, mAb 6C6 could also efficiently react with the GAstV-1 in tissue frozen sections of the clinical infected goose through IFA. The mAbs generated in this study pave the way for further studying on the role of ORF2 in the infection and pathogenesis of GAstV, and the sandwich ELISA and the tissue frozen section-IFA approaches established here provide efficient and rapid serological diagnostic tools for detection of GAstV-1. KEY POINTS: • Three novel B cell epitopes were identified in ORF2 of GAstV-1. • mAb-based ELISA and IFA for detection of GAstV-1 were developed.

Chicken hepatomegaly and splenomegaly associated with novel subgroup J avian leukosis virus infection.

BACKGROUND: Subgroup J avian leukosis virus (ALV-J) is an oncovirus which can induce multiple types of tumors in chicken. In this report, we found novel ALV-J infection is closely associated with serious hepatomegaly and splenomegaly in chicken. CASE PRESENTATION: The layer chickens from six flocks in Jiangsu province, China, showed serious hemoperitoneum, hepatomegaly and splenomegaly. Histopathological results indicated focal lymphocytic infiltration, cell edema and congestion in the liver, atrophy and depletion of lymphocyte in the spleen. Tumor cells were not detected in all the organs. avian hepatitis E virus (aHEV), which is thought to be the cause of a very similar disease, big liver and spleen disease (BLS), was not detected. Other viruses causing tumors or liver damage including Marek's disease virus (MDV), reticuloendotheliosis virus (REV), fowl adenovirus (FAdV) and chicken infectious anemia virus (CIAV) were also proved negative by either PCR or RT-PCR. However, we did detect ALV-J in those chickens using PCR. Only novel ALV-J strains were efficiently isolated from these chicken livers. CONCLUSIONS: This is the first report that chicken hepatomegaly and splenomegaly disease was closely associated with novel ALV-J, highlighting the importance of ALV-J eradication program in China.

RETRACTED: The role of ATG-7 contributes to pulmonary hypertension by impacting vascular remodeling.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). At the request of the authors, this article has been retracted following questions of data ownership that were brought to the Editor's attention after publication. Concerns were raised over whether all data were generated in the authors' lab, and over ownership of the models used. The authors were unable to reach a resolution with other labs involved, and as a result all authors have agreed to a retraction of this article.

Fiber-1, Not Fiber-2, Directly Mediates the Infection of the Pathogenic Serotype 4 Fowl Adenovirus via Its Shaft and Knob Domains.

Recently, the disease of hepatitis-hydropericardium syndrome (HPS) caused by serotype 4 fowl adenovirus (FAdV-4) has spread widely and resulted in huge economic losses to the poultry industry. Although the genome of FAdV-4 has two fiber genes (fiber-1 and fiber-2), the exact role of the genes in the infection of FAdV-4 is barely known. In this study, through superinfection resistance analysis and an interfering assay, we found that fiber-1, but not fiber-2, was the key factor for directly triggering the infection of FAdV-4. The truncation analysis further revealed that both of the shaft and knob domains of fiber-1 were required for the infection. Moreover, the sera against the knob domain were able to block FAdV-4 infection, and the knob-containing fusion protein provided efficient protection against the lethal challenge of FAdV-4 in chickens. All the data demonstrated the significant roles of fiber-1 and its knob domain in directly mediating the infection of FAdV-4, which established a foundation for identifying the receptor of FAdV-4 and developing efficient vaccines against FAdV-4.IMPORTANCE Among 12 serotypes of fowl adenovirus (FAdV), FAdV-1, FAdV-4, and FAdV-10 all carry two fiber genes (i.e., fiber-1 and fiber-2), whereas other serotypes have only one. As important viral surface proteins, the fibers play vital roles in the infection and pathogenesis of FAdV. However, the importance of the fibers to the infection and pathogenesis of FAdV may be different from each other. Recent studies reveal that fiber-2 is identified as a determinant of virulence, but which fiber triggers the infection of FAdV-4 remains unknown. In this study, fiber-1 was identified as a key factor for directly mediating the infection of FAdV-4 through its shaft and knob domains, whereas fiber-2 did not play a role in triggering FAdV-4 infection. The results suggest that fiber-1 and its knob domain may serve as a target for identifying the receptor of FAdV-4 and developing efficient drugs or vaccines against FAdV-4.

OASL Triggered by Novel Goose Astrovirus via ORF2 Restricts Its Replication.

Although astroviruses causes enteric diseases and encephalitis in humans and nephritis and hepatitis in poultry, astrovirus infection is thought to be self-limiting. However, little is known about its molecular mechanism. In this study, we found that a novel goose astrovirus (GAstV), GAstV-GD, and its open reading frame 2 (ORF2) could efficiently activate the innate immune response and induce a high level of OASL in vitro and in vivo The truncation assay for ORF2 further revealed that the P2 domain of ORF2 contributed to stimulating OASL, whereas the acidic C terminus of ORF2 attenuated such activation. Moreover, the overexpression and knockdown of OASL could efficiently restrict and promote the viral replication of GAstV-GD, respectively. Our data not only give novel insights for elucidating self-limiting infection by astrovirus but also provide virus and host targets for fighting against astroviruses.IMPORTANCE Astroviruses cause gastroenteritis and encephalitis in human, and nephritis, hepatitis, and gout disease in poultry. However, the host immune response activated by astrovirus is mostly unknown. Here, we found that a novel goose astrovirus, GAstV-GD, and its ORF2 protein could efficiently induce a high level of OASL in vitro and in vivo, which could feed back to restrict the replication of GAstV-GD, revealing novel innate molecules triggered by astroviruses and highlighting that the ORF2 of GAstV-GD and OASL can be potential antiviral targets for astroviruses.

Gp37 Regulates the Pathogenesis of Avian Leukosis Virus Subgroup J via Its C Terminus.

Different from other subgroups of avian leukosis viruses (ALVs), ALV-J is highly pathogenic. It is the main culprit causing myeloid leukemia and hemangioma in chickens. The distinctiveness of the env gene of ALV-J, with low homology to those of other ALVs, is linked to its unique pathogenesis, but the underlying mechanism remains unclear. Previous studies show that env of ALV-J can be grouped into three species based on the tyrosine motifs in the cytoplasmic domain (CTD) of Gp37, i.e., the inhibitory, bifunctional, and active groups. To explore whether the C terminus or the tyrosine motifs in the CTD of Gp37 affect the pathogenicity of ALV-J, a set of ALV-J infectious clones containing different C termini of Gp37 or the mutants at the tyrosine sites were tested in vitro and in vivo Viral growth kinetics indicated not only that ALV-J with active env is the fastest in replication and ALV-J with inhibitory env is the lowest but also that the tyrosine sites essentially affected the replication of ALV-J. Moreover, in vivo studies demonstrated that chickens infected by ALV-J with active or bifunctional env showed higher viremia, cloacal viral shedding, and viral tissue load than those infected by ALV-J with inhibitory env Notably, the chickens infected by ALV-J with active or bifunctional env showed significant loss of body weight compared with the control chickens. Taken together, these findings reveal that the C terminus of Gp37 plays a vital role in ALV-J pathogenesis, and change from inhibitory env to bifunctional or active env increases the pathogenesis of ALV-J.IMPORTANCE ALV-J can cause severe immunosuppression and myeloid leukemia in infected chickens. However, no vaccine or antiviral drug is available against ALV-J, and the mechanism for ALV-J pathogenesis needs to be elucidated. It is generally believed that gp85 and LTR of ALV contribute to its pathogenesis. Here, we found that the C terminus and the tyrosine motifs (YxxM, ITIM, and ITAM-like) in the CTD of Gp37 of ALV-J could affect the pathogenicity of ALV-J in vitro and in vivo The pathogenicity of ALV-J with Gp37 containing ITIM only was significantly less than ALV-J with Gp37 containing both YxxM and ITIM and ALV-J with Gp37 containing both YxxM and ITAM-like. This study highlights the vital role of the C terminus of Gp37 in the pathogenesis of ALV-J and thus provides a new perspective to elucidate the interaction between ALV-J and its host and a molecular basis to develop efficient strategies against ALV-J.

A novel fiber-2-edited live attenuated vaccine candidate against the highly pathogenic serotype 4 fowl adenovirus.

Recently, the outbreaks of hydropericardium-hepatitis syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry globally. Although several inactivated or subunit vaccines have been developed against FAdV-4, live-attenuated vaccines for FAdV-4 are rarely reported. In this study, a recombinant virus FA4-EGFP expressing EGFP-Fiber-2 fusion protein was generated by the CRISPR/Cas9 technique. Although FA4-EGFP shows slightly lower replication ability than the wild type (WT) FAdV-4, FA4-EGFP was significantly attenuated in vivo compared with the WT FAdV-4. Chickens infected with FA4-EGFP did not show any clinical signs, and all survived to 14 day post-infection (dpi), whereas those infected with FAdV-4 showed severe clinical signs with HHS and all died at 4 dpi. Besides, the inoculation of FA4-EGFP in chickens provided efficient protection against lethal challenge with FAdV-4. Compared with an inactivated vaccine, FA4-EGFP induced neutralizing antibodies with higher titers earlier. All these data not only provide a live-attenuated vaccine candidate against the highly pathogenic FAdV-4 but also give a potential insertion site for developing FAdV-4-based vaccine vectors for delivering foreign antigens.

Peptide enzyme-linked immunosorbent assay (pELISA) as a possible alternative to the neutralization test for evaluating the immune response to IBV vaccine.

BACKGROUND: Infectious bronchitis virus (IBV), a coronavirus, is one of the most important poultry pathogens worldwide due to its multiple serotypes and poor cross-protection. Vaccination plays a vital role in controlling the disease. The efficacy of vaccination in chicken flocks can be evaluated by detecting neutralizing antibodies with the neutralization test. However there are no simple and rapid methods for detecting the neutralizing antibodies. RESULTS: In this study, a peptide enzyme-linked immunosorbent assay (pELISA) as a possible alternative to the neutralization test for evaluating the immune response to IBV vaccine was developed. The pELISA could indirect evaluate neutralizing antibody titers against different types of IBV in all tested sera. The titers measured with the pELISA had a coefficient of 0.83 for neutralizing antibody titers. CONCLUSIONS: The pELISA could detect antibodies against different types of IBV in all tested sera. The pELISA has the potential to evaluate samples for IBV-specific neutralizing antibodies and surveillance the infection of IBV.

A modified loop-mediated isothermal amplification method for detecting avian infectious laryngotracheitis virus.

This study was aimed to establish a highly specific and sensitive loop-mediated isothermal amplification (LAMP) method for diagnosing avian infectious laryngotracheitis (AILT). DNA was extracted from isolated infectious laryngotracheitis virus (ILTV) strains and control samples, followed by PCR using three sets of six specific primers. The detection efficiency of the LAMP assay was evaluated by the turbidity and calcein methods. The sensitivity of LAMP was then assessed using a concentration gradient followed by a specificity analysis. Furthermore, the detection efficiency of LAMP and PCR was compared. Finally, a clinical test was performed to evaluate the value of the LAMP assay. The optimal temperature for the LAMP reaction was 66 °C. Meanwhile, the primers selected for the LAMP assay were highly specific for the target virus. The sensitivity of the turbidity and calcein methods for LAMP was consistent. The minimum detection concentration of LAMP was 0.06 pg/µL, which was 100-fold higher than that of PCR. Furthermore, the results from clinical samples showed that the LAMP method could identify AILT from many samples. The newly designed LAMP assay was an effective method for AILT detection at an optimal temperature of 66 °C with a minimum detection concentration of 0.06 pg/µL.

HSC70 is required for infectious bursal disease virus (IBDV) infection in DF-1 cells.

BACKGROUND: Infectious bursal disease (IBD) is a highly contagious infectious disease that causes severe immunosuppression and damage to the bursa of Fabricius in chickens. Several proteins involved in IBD virus (IBDV) infection, such as surface immunoglobulin M, integrin, annexin A2 and chicken heat shock protein 90, have been identified. However, the main protein that plays key roles in virus infection has not yet been confirmed. METHODS: DF-1 cell line was transfected with the pcDNA-VP2 plasmid and analyzed by immunofluorescence assay. The proteins reacted with VP2 of IBDV in DF-1 cells were pulldown with the monoclonal antibody and identified by mass spectrometry. Heat shock cognate protein 70 (HSC70), one of these proteins, was selected to be investigated in the function in IBDV infection by specific antibody and its inhibitor. RESULTS: The DF-1 cell line was transfected with the pcDNA-VP2 plasmid, and expression of IBDV VP2 in DF-1 cells was confirmed by immunofluorescence assays. Heat shock cognate protein 70 (HSC70) was one of the proteins identified by coimmunoprecipitation using a monoclonal antibody (2H11) against VP2 and mass spectrometry analysis. IBDV infection in DF-1 cells was strongly inhibited by both an anti-HSC70 antibody and a HSC70 inhibitor (VER155008). CONCLUSION: These results suggest that HSC70 may be an essential factor for IBDV infection.

Generation and molecular characteristics of a highly attenuated GPV strain through adaptation in GEF cells.

BACKGROUND: Goose parvoviruses (GPVs) spread globally and cause a huge economic loss to the poultry industry. Although the attenuated GPV vaccines play a key role in preventing the disease caused by GPV, the molecular basis for the attenuation of GPV is barely known. RESULTS: A highly attenuated GPV strain, GPV-CZM-142, was generated through blindly passaging of the highly pathogenic strain, GPV-CZM, in goose embryonic fibroblasts (GEF) for 142 generations. The GEF-adapted GPV strain's virulence was 10,000 times weaker than its wild type counterpart, GPV-CZM, based on the ELD50 (50% Embryo Lethal Dose). By comparing with the wild type strain, genome sequencing analysis identified adapted mutations either in ITR or in NS and VP1 of GPV-CZM-142. CONCLUSIONS: The highly attenuated GPV strain, GPV-CZM-142, provides a GPV vaccine candidate, and the identified virulence-related mutations give a novel insight into the molecular determinants of GPV virulence.

A novel monoclonal antibodies-based sandwich ELISA for detection of serotype 4 fowl adenovirus.

As a major causative agent for hepatitis-hydropericardium syndrome (HPS) in chickens, serotype 4 fowl adenovirus (FAdV-4) has caused huge economic losses in the poultry industry globally. However, there is no commercial diagnostic test for FAdV-4 antigens. To generate a rapid approach for specific detection of FAdV-4, a monoclonal antibodies (mAbs)-based sandwich ELISA was developed. In this ELISA, a purified mAb 4A3 and a HRP-labelled mAb 3C2 specific to the fiber-2 of FAdV-4 were used as the capture antibody and detection antibody respectively. Specificity assay revealed the ELISA only reacted with FAdV-4, not with other avian viruses tested. Sensitivity assay showed the limit of detection of the ELISA was 1000 TCID50/ml and 12.5 ng/ml for the FAdV-4 and the purified GST-Fiber2 protein respectively. Moreover, the ELISA could be efficiently applied in detecting the FAdV-4 in tissue samples from a clinically-diseased chicken flock. All these data demonstrated that the ELISA developed here provides a promising tool for rapid and efficient diagnosis of clinical infection with FAdV-4.