Many RNA viruses exhibit error-prone replication. Continuous generation of erroneous copies accelerates evolution. Avian leukosis virus subgroup J (ALV-J), an avian oncogenic virus, is a classical model virus for studying retroviruses. ALV-J's high mutation rates drive continuous evolution of its envelope and pathogenicity, posing significant challenges to the poultry industry. Here we employed deep mutational scanning to systematically assess envelope-wide mutation effects on ALV-J replication, integrating high-throughput sequencing with mutant libraries to identify critical envelope residues impacting viral fitness. Following 10 passages, the library virus exhibited enhanced replication capacity. Moreover, the library virus derived from SPF chickens displays screening results similar to those of the DF-1 cell-passaged virus. Most mutations were progressively eliminated during viral passaging, especially the first 80 amino acids of ALV-J envelope. Critical amino acid mutations, preferential deletion/insertion mutations and glycosylation patterns recapitulate evolutionary patterns observed in natural ALV-J isolates. Incorporation of all identified mutations into ALV-J J1 significantly increased in vivo replication efficiency and viral shedding of the recombinant virus. Functional study demonstrated that two key mutations independently promote viral replication: A64T enhancing entry via receptor-binding optimization, H304R promoting maturation through envelope cleavage efficiency. These insights enable targeted antiviral design by predicting evolutionary paths.

Infectious bursal disease virus (IBDV) primarily triggers severe immunosuppression in birds and has resulted in substantial economic losses to the poultry industry globally. Here, a novel naturally reassortant IBDV strain, designated IBDV_G3, was isolated from quails during a disease outbreak in Jiangsu Province, China. Genome sequencing revealed that IBDV_G3 carried segment A derived from the serotype-2-related A0 lineage, while segment B clustered within the B1a classical-like lineage, and this genomic constellation was further confirmed in the original uncultured quail tissues. IBDV_G3 replicated efficiently in LMH cells without prior adaptation, and its VP2 was recognized by the serotype 1 VP2-specific monoclonal antibody 5G10. These findings suggest that quails may harbor reassortant IBDV strains and that the quail–chicken interface warrants continuous surveillance for IBDV reassortment and evolutionary variation.

Marek's disease virus (MDV), an avian α-herpesvirus, heavily relies on host metabolic reprogramming during infection. However, the precise regulatory mechanisms governing MDV-induced nucleotide metabolic remodeling remain poorly characterized. The study explores how MDV induces changes in nucleotide metabolism during infection. Our results demonstrated that MDV infection significantly upregulates nucleotide synthesis metabolism, particularly purine de novo synthesis in chicken embryonic fibroblast (CEF) cells. Metabolomic analysis identified 19 upregulated metabolites related to nucleotide metabolism post-infection. Functional assays revealed that adenine and guanine supplementation enhanced MDV replication, while the purine inhibitor 6-mercaptopurine (6MP) suppressed it. The transcription factor c-Myc was found to activate purine synthesis enzymes during MDV infection, with c-Myc knocked down reducing viral replication and overexpression increasing it. Additionally, MDV thymidine kinase UL23 was identified as crucial in reprogramming nucleotide metabolism, promoting c-Myc-mediated nucleotide anabolism and viral replication. This research highlights the potential of targeting nucleotide metabolism as an antiviral strategy.

Upon infection, viruses reprogram the host metabolic system to hijack metabolic resources for proliferation. Avian leukosis virus subgroup J (ALV-J), an avian oncogenic virus, poses significant challenges to the poultry industry. ALV-J infection upgrades monosaccharide N-acetylgalactosamine (GalNAc) and galactosyltransferase (core 1 β3-Gal-T) in DF-1 cells, both of which are crucial for O-linked glycosylation. Addition of GalNAc or overexpression of core 1 β3-Gal-T in DF-1 cells can promote ALV-J replication. ALV-J envelope protein (Env) undergoes complex post-translational modifications. Two O-linked glycosylation sites (T32 and T271) located in the head region of the ALV-J Env have been identified for the first time using liquid chromatography–mass spectrometry (LC–MS). The results of coimmunoprecipitation and flow cytometry indicate that mutations in T32 or T271 diminish ALV-J infection by affecting viral internalization, rather than attachment. The viral internalization efficiency was partially restored under a low pH environment. Incorporation of T32A and T271A into ALV-J led to significantly reduced replication capacity in vivo and viral shedding of the recombinant virus. These findings are valuable for our understanding of the roles of glycans in the ALV-J infection cycle, as well as for providing potential anti-ALV-J strategies.

Goose astrovirus (GAstV) is a newly emerged viral pathogen in goose, characterized by a high incidence and significant mortality rates. This etiology has repeatedly occurred in coastal areas of China, rapidly spreading to inland provinces in recent decade, and thus imposed huge economic losses on China’s goose industry. Therefore, it is essential to establish a rapid, accurate and sensitive method for GAstV diagnosis. In this study, we employed reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay combined with a Pyrococcus furiosus Argonaute (PfAgo) system, providing a simple and precise approach for GAstV. Specific primers and guide DNA (gDNA) were designed to target conserved regions of the GAstV genome. The assay achieved a detection limit of 10 copies/μL in preliminary validation assays when targeting conserved regions of the viral genome with optimized reaction conditions. Importantly, the assay exhibited no cross-reactivity with other viruses, including Goose parvovirus (GPV), Goose circovirus (GoCV), Tembusu Virus (TMUV), Muscovy Duck Reovirus (MDRV), and Escherichia coli (E. coli). Detection results from 59 clinical samples demonstrated complete concordance in positive rates between the LAMP-PfAgo and qPCR methods.

Some subtypes of avian influenza A viruses (IAVs) have been associated with human infections (e.g. H3N8, H5Ny, H7Ny, H9N2, and H10Ny), and the ability of these viruses to cause zoonotic infections further increases the public health risk of avian IAVs. Among them, H9N2 virus is one of particular importance, both in its own right and as a contributor of internal gene segments to other emerging zoonotic avian IAVs. In recent years, an increasing number of H9N2 strains have been observed to be highly pathogenic in mice without prior adaptation. In this study, we found that a naturally occurring H9N2 isolate, A/chicken/Jiangxi/198/2019 (JX198), is lethal in mice. To investigate the molecular basis for the high virulence of JX198 in mice, a series of reassortants and mutants were generated and tested. We found that the PA, especially D347G mutation in PA, is responsible for the increased pathogenicity of JX198 in mice. Notably, D347G in PA of JX198 significantly alters the polymerase activity, vRNA production, and plaque-formation. Thus, our data demonstrate that a novel molecular marker, D347G in PA, determines the virulence of H9N2 in mice, posing a potential risk of H9N2 with D347G in PA for public health and highlighting the significance of continuing surveillance of H9N2 field strains.

Endogenous retroviruses (ERVs), the genomic remnants of ancient retroviral infections, constitute a significant and dynamic component of livestock and poultry genomes. This review synthesizes current knowledge on the dual roles of ERVs, from drivers of genomic instability and disease to essential agents of evolution and innovation in domesticated species. We detail the classification, structure, and life cycle of ERVs, highlighting the distinct genomic landscapes and host-silencing strategies that differentiate avian and mammalian lineages, with poultry genomes maintaining lower ERV loads through persistent repression compared to livestock. A critical examination reveals the "dark side" of ERVs, including their capacity for insertional mutagenesis, contribution to diseases like avian leukosis, and zoonotic risks, exemplified by porcine endogenous retroviruses (PERVs). Conversely, we talk over how ERVs have been harnessed as allies, where their insertions create structural variants that shape economically vital traits—such as metabolism, growth, and unique phenotypes—and where domesticated viral genes support crucial functions like placental development and innate immunity. Finally, we discuss the transformative potential of CRISPR-Cas9 genome editing as a precise tool for silencing hazardous ERVs and exploiting their beneficial aspects. A comprehensive understanding of ERV biology is thus pivotal for advancing genetic improvement, disease resistance, and the sustainability of livestock and poultry production systems.

Goose astrovirus (GAstV) is a newly emerged viral pathogen in goose, characterized by a high incidence and significant mortality rates. This etiology has repeatedly occurred in coastal areas of China, rapidly spreading to inland provinces in recent decade, and thus imposed huge economic losses on China's goose industry. Therefore, it is essential to establish a rapid, accurate and sensitive method for GAstV diagnosis. In this study, we employed reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay combined with a Pyrococcus furiosus Argonaute (PfAgo) system, providing a simple and precise approach for GAstV. Specific primers and guide DNA (gDNA) were designed to target conserved regions of the GAstV genome. The assay achieved a detection limit of 10 copies/mu L in preliminary validation assays when targeting conserved regions of the viral genome with optimized reaction conditions. Importantly, the assay exhibited no cross-reactivity with other viruses, including Goose parvovirus (GPV), Goose circovirus (GoCV), Tembusu Virus (TMUV), Muscovy Duck Reovirus (MDRV), and Escherichia coli (E. coli). Detection results from 59 clinical samples demonstrated complete concordance in positive rates between the LAMP-PfAgo and qPCR methods.

Recently, chicken infectious bronchitis (IB), caused by the infectious bronchitis virus (IBV), has emerged as one of the top diseases in the poultry industry in China. Site mutations and recombination events contribute to the high genetic diversity of IBV. In this study we considered the S1 sequences of 30 GI-22 lineage strains, including 24 isolates previously identified, isolated from flocks with the clinical signs of nephritis, egg production drop or oviduct lesions, which shared common clinical signs with the classical GI-22 lineage IBV. The S1 amino acid sequences of all 30 isolates were highly diverse from those of classical GI-22 viruses and these 30 isolates could be grouped into five clusters with high diversity of S1 protein with each other, the S1 amino acid sequence homology ranging from 83.48% to 100%. Cross-neutralization assays revealed that these 30 novel GI-22 lineage isolates exhibited at least three serotypes. Furthermore, infection studies showed that these novel IBV isolates could cause lesions of glandular stomach, kidney and reproductive system in chickens, and showed potential efficient transmission ability with high mortality. Analysis of the receptor binding domain (RBD) revealed that these novel IBV strains of GI-22 lineage also carried 14-amino-acid insertion in HVR1, which was distinctly different from the current vaccine strains. Overall, our data provided insights into the emergence and highly variant antigenicity of novel GI-22 lineage IBVs, highlighting the need for development of IB vaccines against novel and pathogenic GI-22 lineage IBVs.

Gosling gout disease is an infectious disease caused by goose astrovirus (GAstV), which can result in urate deposition in the internal organs and joints of goslings. Since 2015, outbreaks of gosling gout disease have occurred in several goose-producing areas in China. Subsequently, the disease spread to the vast majority of eastern China, becoming a major threat to goose farms and causing huge economic losses to the goose industry. Meanwhile, GAstV can infect species of birds other than geese. It is worth noting that, as an emerging virus, the research on GAstV is still in the early stages. Therefore, the investigation of GAstV has become an urgent issue, which can improve understanding of GAstV and develop effective measures to control its threat to poultry. The purpose of this review is to summarize the latest research progress on GAstV in recent years, mainly focusing on the genetic evolution, pathogenesis, diagnostic detection, and control strategies of GAstV, aiming to provide a reference for scientific prevention and control of GAstV infection.

Chicken Infectious Anemia Virus (CIAV) infection causes severe anemia, hematopoietic disorders, and immunosuppression in poultry. However, the mechanisms underlying its pathogenicity remain poorly understood. In this study, RNA sequencing (RNA-seq) was employed to investigate the transcriptional response of MSB1 cells to CIAV infection at 12, 24, and 48 hours post-infection. The results revealed differential gene expression associated with immune response, inflammatory response, apoptosis, and metabolic pathways. Several immune-related factors, including IL18, TRAF1, MYD88, IRF2, and CD28, were found to be downregulated. VP1, the capsid protein of CIAV, plays a significant role in viral pathogenicity. To explore how VP1 affects CIAV replication and pathogenicity, co-immunoprecipitation (Co-IP) was used to identify host proteins interacting with VP1. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that VP1 interacts with a range of host proteins involved in metabolic pathways, the PI3K-AKT signaling pathway, and ribosomal functions. Based on RNA-seq and LC-MS findings, ANXA6 and HSP90aa1 were identified as key interacting partners, which co-localize in the cytoplasm of infected cells. Both proteins are involved in immune responses and metabolic regulation and were found to be downregulated in CIAV-infected cells. Notably, overexpression of ANXA6 or HSP90aa1 inhibited CIAV infection during the early stages of the viral lifecycle. These findings enhance our understanding of the host factors influencing CIAV infection and provide new insights into potential therapeutic targets for managing CIAV-induced diseases.

Since 2015, hepatitis-hydropericardium syndrome (HHS) induced by the infection of highly pathogenic serotype 4 fowl adenovirus (HP-FAdV-4) has caused severe economic losses to the poultry industry. Although Hexon protein is closely associated with the pathogenicity of HP-FAdV-4, its antigenic epitopes remain poorly elucidated. In this study, two monoclonal antibodies (mAb) against Hexon were generated, designated as 2C5 and 4H7, respectively. Cross-reactivity with different FAdVs showed that mAb 2C5 only reacted with HP-FAdV-4 but not with low pathogenic FAdV-4 (LP-FAdV-4), whereas mAb 4H7 reacted with both HP-FAdV-4 and LP-FAdV-4. Epitope mapping revealed that the mAb 2C5 and 4H7 recognized 185GPGRNP190 and 161TSTSKDT167 in Hexon, respectively. Moreover, 188R in Hexon was identified as the key site recognized by mAb 2C5, and 162S and 166D in Hexon were identified as the critical sites recognized by mAb 4H7. Using mAb 2C5 as a capture antibody and HRP-conjugated mAb 4H7 as a detection antibody, we developed a novel sandwich ELISA to efficiently differentiate HP-FAdV-4 from LP-FAdV-4 and other serotypes of FAdV. All these data give novel insights into the epitopes and key antigenic sites in Hexon of FAdV-4 and provide efficient differentiating diagnostics approaches for HP-FAdV-4 endemic in China.

The emergence of novel H3N8 and H10N3 avian influenza A viruses (IAVs) circulating in chicken flocks in China has raised significant concerns to their spillovers in humans and associated economic losses. In this study, we isolated a novel H3N3 strain from chickens exhibiting respiratory symptoms, designated as A/chicken/Shandong/118/2023(H3N3) (SD118). Genetic analyses revealed that SD118 was a triple-reassortment virus, possessing hemagglutinin (HA) gene originating from H3N8, neuraminidase (NA) gene originating from H10N3, and other six internal genes were from H9N2, each of which is currently circulating in chicken flocks in China. In vitro studies confirmed that SD118 could effectively replicate in mammalian cells (MDCK and A549 cells), and exhibited dual receptor-binding affinity for both avian-like (α-2, 3 sialic acid) and human-like (α-2, 6 sialic acid) receptors. Moreover, SD118 could replicated effectively not only in the lungs and nasal turbinates of the infected mouse with more than 10 % bodyweight loss, but also in the upper respiratory tract and intestines of the infected chickens with efficient transmission ability. Our findings highlight the ongoing evolution of H3 avian IAVs through reassortment with other subtypes in poultry and the enhanced surveillance is critical to monitor the genetic diversity and zoonotic potential of H3 viruses in chickens.

Subgroup J avian leukosis virus (ALV-J) has significantly affected the global poultry industry, with rapid mutations and frequent outbreaks in the poultry industry of China, challenging the current eradication strategies. In this study, virus isolation, identification, and complete genome sequencing of suspected ALV-J-infected samples collected from Guangxi and Jiangsu provinces during 2022-2023 were performed. Four ALV-J strains (designated as, GXZM01, GXZM02, JSZM02, and JSZM963) were successfully isolated and sequenced, with genome lengths ranging from 7487 to 7636 bp and a homology of 82.8 %-96.5 % with the reference ALV-J strains. The amino acid sequences of the gag and pol genes of the isolated strains showed relatively high conservation, with homologies of 95-100 % and 97.1-99.5 % compared with the reference strains, respectively. Analysis of the 3'UTR revealed that the 4 ALV-J isolates show different type of mutations compared to the reference strains, with all the isolates containing a 175 bp deletion at the r-TM, while the JSZM02 strain uniquely showed a 127 bp deletion in the E element. The homology of Env protein and Gp85 protein between the 4 isolates and the reference strains ranged from 86.5 to 95.6 % and 83.7 to 93.8 %, respectively. Notably, in comparison with ALV-J prototype strain HPRS-103, we found that a distinct deletion of 11 aa located at 209-219 aa of Gp85 protein of GXZM02 strain which located at the receptor-binding domain (RBD) and partially overlapped with hypervariable region2 (hr2). Structural prediction analysis revealed that the Gp85 proteins of these ALV-J strains exhibited certain differences in the number of α-helices and β-sheets. Importantly, viral growth kinetic showed that the replication ability of GXZM02 was the weakest one among the 4 isolates, which is potentially related to the deletion in the RBD. Our findings contribute to enrich the epidemiological data on ALV-J and reveal the evolution of prevalent ALV-J strains in clinic, which will provide scientific basis for ALV prevention and control.

The national breeding and disease eradication programs in China are crucial for the healthy and sustainable development of the poultry industry. Among them, avian leukosis virus, the causative agent of avian leukosis (AL), is one of the important pathogens that must be strictly controlled due to its effects on immune suppression, tumorigenicity, and decreased production performance on the chicken flocks. Although scientists have made many efforts in various directions, currently, there are still no effective prevention and control strategies available except for eradication which has been implemented and confirmed by many countries, including China. However, due to the low efficacy, time-consuming, and high cost, current eradication programs struggle to keep up with the rapid development of the poultry industry in China. Thus, enhancing eradication efficiency and reducing operational cost are critical determinants for holistically expediting China's AL eradication program. This review aims to supply several potential strategies for accelerating the eradication of AL in China, including: improving the efficiency of virus isolation and the sensitivity of current detection methods; vaccines, therapeutic antibodies, and small molecule antiviral drugs; AL resistance breeding; comprehensive prevention and control of AL and the construction of biosecurity system; other significant measures, including epidemiological investigations, talents cultivation, top-down design, publicity and education, etc.

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections that have shaped vertebrate genomes through evolution. In poultry, ERVs remain understudied despite their potential roles in genome plasticity, gene regulation, and disease resistance. Here, we presented a comprehensive analysis of ERVs across 8 poultry species from three families (Anatidae, Phasianidae, and Numidae), combining de novo mining, phylogenetic classification, and functional characterization. Our study revealed substantial variation in ERV abundance, ranging from 46,326 (Cairina moschata) to 79,018 (Meleagris gallopavo) elements per genome. We discovered 23 distinct ERV groups, including 20 novel groups, with dominance of Alpharetroviruses (10 groups), suggesting lineage-specific expansion. Several ERVs grouped closely with known poultry ERVs, indicating common evolutionary origins, they have been distributed across different families, highlighting lineage-specific expansion patterns and suggesting that they may play conserved roles in host genome regulation. Notably, ERV-derived sequences contribute significantly to both protein-coding (29.4-44.8 %) and long non-coding RNA (22.5-61.2 %) genes, with a pronounced depletion in coding regions (CDS: 63.2-98.3 %) but enrichment in regulatory regions. We further identified polymorphic ERV insertions in key developmental genes (e.g., CLC2DL4/5, TYR, CNTNAP2, CNTN5, and LUZP2), implying roles in post-transcriptional regulation. However, these polymorphic insertions were specifically observed in 2 species, Gallus gallus (4 insertions) and Numida meleagris (1 insertion). PCR genotyping confirmed active ERV polymorphisms in a small population of chickens (n = 24) and ducks (n = 24), indicating ongoing genomic dynamism. These findings underscore ERVs as dual agents of genetic innovation and structural variation, with implications for avian genome evolution, host-pathogen interactions, and poultry breeding strategies.

Waterfowl play a pivotal role in the evolution of the influenza A virus (IAV), which harbor an extensive repertoire of IAV and act as long-term reservoirs in which low-pathogenic strains can reassort and acquire new genomic segments. In this study, three H1N1 IAVs, named as A/duck/Jiangxi/DP771/2021(H1N1) (DP771), A/duck/DP1433/2021(H1N1) (DP1433), and A/duck/Jiangxi/DP1467/2021(H1N1) (DP1467), were isolated from wild ducks and characterized. Phylogenetic analysis revealed that all these three viruses belonged to Eurasian lineages. In vitro, all these three isolates replicated efficiently in both A549 and MDCK cells, and demonstrated dual receptor binding properties (α2,3- and α2,6-linked sialic acids). Most strikingly, the mouse study showed that two of these three H1N1 viruses, DP1433 and DP1467, replicated efficiently in lungs without pre-adaptation, and DP1433 caused about 20 % bodyweight loss in mice. All these data provide valuable insight into the molecular epidemiology and pathogenicity of duck-original H1N1 viruses circulating in China, and continued surveillance to monitor the diversity of IAVs in ducks is critical to understand the natural history of IAVs and develop efficient strategies against IAVs.

Since 2016, the novel variant infectious bursal disease virus (nVarIBDV) has emerged as the predominant strain in China, which characterized by causing subclinical but persistent immunosuppression and substantial economic losses in chickens. However, the global transcriptomic changes and regulatory mechanisms in the bursa of Fabricius (BF) during nVarIBDV infection remain unclear. In this study, transcriptome sequencing of BF from infected chickens identified 5,775 differentially expressed mRNAs (DEmRNAs), 535 lncRNAs (DElncRNAs), and 1,072 circRNAs (DEcircRNAs) at 7 days post-infection (dpi), along with 5,275 DEmRNAs at 14 dpi. Functional enrichment analysis showed these genes were involved in immune and inflammatory responses, such as NF-kappa B signaling, cytokine-cytokine receptor interaction, T cell activation, and extracellular matrix remodeling. Protein-protein interaction networks highlighted hub genes related to antiviral response (e.g., CD4, IFN-γ, GZMA), T cell exhaustion (e.g., CTLA-4, LAG3, PD-L1), and fibrosis (e.g., COL1A1, MMP9). Notably, key B-cell-related genes (BTK, RAG2, SYK) were significantly down-regulated, indicating impaired humoral immunity. Competing endogenous RNA (ceRNA) network analysis suggested that DElncRNAs and DEcircRNAs may act as miRNA sponges, regulating genes in Hippo, Ras, and cAMP pathways. RT-qPCR validation of 20 differentially expressed genes (DEGs) confirmed the RNA-seq reliability. This study reveals a dynamic host response involving immune activation, T cell exhaustion, B-cell depletion, and maladaptive tissue repair, offering the first comprehensive transcriptomic landscape of chicken BF during nVarIBDV infection and insights into its immunosuppressive mechanisms.