Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive condition in chickens, caused by the infectious bursal disease virus (IBDV). The recent emergence of novel variant IBDV (nVarIBDV) poses a significant threat to the global poultry industry. However, currently available vaccines provide only limited protection against nVarIBDV strains. In this study, a recombinant avian metapneumovirus subtype B (aMPV/B) expressing the nVarIBDV VP2 gene (rLN16A-nVarVP2) was successfully rescued by inserting the gene between the G and L genes of the attenuated aMPV/B strain LN16-A. Immunofluorescence and Western blotting analyses confirmed stable VP2 expression in vitro. Further evaluation showed that VP2 insertion did not alter the growth kinetics of the parental virus, and the expression remained stable after 20 serial passages. A single immunization with rLN16A-nVarVP2 elicited robust humoral and cellular immune responses in specific pathogen-free chickens, inducing high levels of neutralizing antibodies against both nVarIBDV and aMPV/B, as well as Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-6) cytokines. Moreover, rLN16A-nVarVP2 conferred complete (100

Avian metapneumovirus subtype B (aMPV/B) infections significantly affect the global poultry industry. However, the virulence determinants and attenuation mechanism remain unknown. Here, a series of chimeric and mutant viruses was constructed, and their pathogenicity was evaluated in a specific-pathogen-free (SPF) chicken model. First, substitutions in different genes (N, P, F, SH, G, and L) and amino acid sites (323rd, 396th, and 522nd residues in F protein) in virulent (LN16-V) and attenuated (LN16-A) viruses revealed that the residue 396th in F protein is closely related to replication ability in vitro and in vivo and is a critical determinant of aMPV/B virulence in chickens. Further studies revealed that the K396R mutation in the F protein decreases the adsorption of aMPV/B to DF-1 cells, which reduces the binding of the F protein with αVβ1 integrin. Structural and surface plasmon resonance analysis indicated that the K396R mutation primarily reduced the electrostatic potential of the RDD motif of the F protein that binds with αVβ1 and decreased the binding between the F protein and αVβ1, which is a critical determinant of the replication ability of aMPV/B. Collectively, these findings not only contribute to a better understanding of the aMPV attenuation mechanism but also offer novel strategies for developing Metapneumovirus members live vaccines.IMPORTANCEBoth aMPV and hMPV belong to the Metapneumovirus family and cause the most acute respiratory diseases in poultry and humans, respectively. Recently, an outbreak of severe respiratory disease occurred on turkey and chicken farms across different states in the USA, largely attributed to aMPV/B infections. Live-attenuated vaccines developed by the blind passage of virulent strains in tissue culture have been widely used to prevent aMPV/B infection. However, the mechanism of aMPV/B attenuation remains unclear. Here, we identified the F gene as a key determinant of the virulence of aMPV/B and confirmed that residue 396 in the F protein plays an important role in attenuating the virulence of aMPV/B. Importantly, we found that the K396R mutation decreased the binding affinity between the F protein and αVβ1 and reduced the replication ability of aMPV/B. This is the first study to identify the key virulence genes and amino acid residues of aMPV/B and elucidate the molecular mechanisms underlying the attenuation of virulence. Our work provides fundamental insights into aMPV/B pathogenicity and offers direction for guiding the rational design of novel and more effective vaccines against aMPV/B and, by extension, related pathogens, such as hMPV.

Chicken infectious anemia (CIA) is a highly contagious disease caused by the chicken infectious anemia virus (CIAV), and it poses a serious threat to the poultry industry. However, effective control measures and strategies have not been identified. In this study, a recombinant Marek’s disease virus (rMDV) expressing the VP1 and VP2 proteins of CIAV was successfully constructed using CRISPR/Cas9, and a commercial Marek’s disease virus (MDV) vaccine strain was used as the vector. VP1 and VP2 expression by rMDV was confirmed by immunofluorescence assay and western blot analysis, which revealed robust in vitro expression. Further analysis showed that the VP1 and VP2 genes integrated into the MDV genome did not alter the growth kinetics of the virus and remained stable even after 20 passages, indicating the genetic stability of the recombinant virus. In animal studies, vaccination of one-day-old specific-pathogen-free chickens with rMDV induced high levels of CIAV-specific antibodies (1 × 105) and neutralizing antibodies (1:25) and a potent cellular immune response. Moreover, rMDV vaccination conferred an 85% protective index against challenge with a highly virulent strain of CIAV, significantly reducing the occurrence of anemia and thymic atrophy caused by CIAV infection and dramatically suppressing CIAV replication in the thymus. Collectively, these results highlight the potential of rMDV as a vaccine candidate for preventing and controlling CIAV infection, thus offering a new avenue for mitigating the impact of CIA on the poultry industry.

The influenza viral ribonucleoprotein (vRNP) serves as the machinery for viral RNA replication. It self - assembles from the RNA - dependent RNA polymerase (RdRp), viral RNA, and nucleoprotein (NP). During vRNP replication, NP proteins must rapidly switch between monomeric and polymeric states to sustain dynamic equilibrium, which relies on NP phosphorylation-dephosphorylation cycling and is crucial for viral nucleic acid replication. However, the regulatory mechanisms controlling this process remain poorly understood. In this study, we employed an RdRp/vRNP-targeted affinity mass spectrometry-based differential analysis method, discovered that the cellular protein phosphatase Mg2+/Mn2+-dependent 1G (PPM1G) is a phosphatase of NP and required for maintaining viral polymerase activity. Overexpression of PPM1G decreased viral replication. In ppm1g flox/flox-Sftpc Cre mice, lethal influenza infection led to survival and mild disease, as well as reduced lung viral loads and inflammation. These results suggest that PPM1G is a key host molecule for maintaining steady state of viral replication. PPM1G expression is upregulated upon influenza virus infection. This heightened expression occurs in response to increased viral protein production, simultaneously leading to elevated dephosphorylation of NP, which accelerates NP polymerization to promote viral replication. Meanwhile, surplus NP can be degraded through the ATG7 autophagylysosome pathway. Our research elucidates a mechanism that PPM1G maintains viral replication homeostasis by regulating NP status and fate. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, 32330103 National Natural Science Foundation of China, 32300126 China Postdoctoral Science Foundation, 2023M733820

Subgroup J avian leukosis virus (ALV-J), a retrovirus, elicits immunosuppression and persistent infections in chickens. Although it is widely acknowledged that ALV-J can evade the host's innate immune defenses, the mechanisms behind this immune evasion remain elusive. N6-methyladenosine (m6A), the most prevalent internal RNA modification, plays a role in innate immune evasion. Our research identified ALV-J as an inefficient stimulator of innate immunity in vitro and in vivo, with its genomic RNA featuring m6A modifications predominantly in the envelope protein (Env) region and 3' untranslated region (3'UTR). To elucidate the functional consequences of m6A modification, we subsequently generated m6A-deficient ALV-J through its culturing in the DF-1 overexpressing fat mass and obesity-associated protein (FTO) cells. The m6A-deficient ALV-J virus, or its RNAs significantly enhanced IFN-β production compared to the wild-type (wt) ALV-J, suggesting a pivotal regulatory function of m6A modifications in modulating innate immune response. Mechanistically, the m6A modification of the ALV-J genomic RNA directly impacted its recognition by MDA5, weakening its binding and ubiquitination and attenuating IFN-β activation. Moreover, m6A-deficient ALV-J, created by inducing mutations in m6A sites within Env and 3'UTR, exhibited reduced replication capacity and elevated IFN-β expression in host cells. Importantly, this phenomenon was abolished in MDA5-knockout DF-1 cells, further demonstrating the core role of MDA5. These data demonstrate that m6A modification of ALV-J genomic RNA dampens the host's innate immune response through MDA5 signaling pathway.

Avian metapneumovirus (aMPV), a paramyxovirus, causes acute respiratory diseases in turkeys and swollen head syndrome in chickens. This study established a reverse genetics system for aMPV subtype B LN16-A strain based on T7 RNA polymerase. Full-length cDNA of the LN16-A strain was constructed by assembling 5 cDNA fragments between the T7 promoter and hepatitis delta virus ribozyme. Transfection of this plasmid, along with the supporting plasmids encoding the N, P, M2-1, and L proteins of LN16-A into BSR-T7/5 cells, resulted in the recovery of aMPV subtype B. To identify an effective insertion site, the enhanced green fluorescent protein (EGFP) gene was inserted into different sites of the LN16-A genome to generate recombinant LN16-As. The results showed that the expression levels of EGFP at the site between the G and L genes of LN16-A were significantly higher than those at the other two sites (between the leader and N genes or replacing the SH gene). To verify the availability of the site between G and L for foreign gene expression, the VP2 gene of very virulent infectious bursal disease virus (vvIBDV) was inserted into this site, and recombinant LN16-A (rLN16A-vvVP2) was successfully rescued. Single immunization of specific-pathogen-free chickens with rLN16A-vvVP2 induced high levels of neutralizing antibodies and provided 100% protection against the virulent aMPV subtype B and vvIBDV. Establishing a reverse genetics system here provides an important foundation for understanding aMPV pathogenesis and developing novel vector vaccines.

Hemorrhagic fever with renal syndrome (HFRS), caused by Hantaan virus, poses a serious public health threat. Current diagnostic methods remain limited by low sensitivity, complex procedures, and high sample requirements. To address this, we developed a highly sensitive single-molecule biosensor using multi-fluorophore nucleic acid probes and STORM imaging for the detection of Hantaan virus RNA. The probe was synthesized via PCR incorporating EdUTP, enabling site-specific coupling of multiple Cy5 fluorophores through copper-catalyzed click chemistry. This multi-fluorophore probe, combined with magnetic beads and a capture sequence, specifically targeted viral RNA and enabled quantification by super-resolution imaging. Compared to conventional single-fluorophore probes, our system exhibited a significantly lower detection limit of 57.54 aM. Notably, this is the first application of STORM to a single-molecule viral RNA detection platform. The method offers a broadly applicable, ultrasensitive strategy for early clinical diagnostics of Hantaan virus and potentially other pathogens.

Host restriction of avian influenza virus (AIV) polymerase in human cells is driven by species-specific differences in ANP32A/B proteins. While AIV polymerase relies on ANP32A/B containing a 33-amino-acid insert unique to avian species, the structural and/or mechanistic basis for this requirement remains poorly characterized. Here, we demonstrate that chicken ANP32A (chANP32A) displays three functional determinants enabling its species-specific support of AIV polymerase: (1) a SUMO-interacting motif (SIM), (2) SUMOylation at residues K68/K153, and (3) a 28-amino-acid segment within the avian-specific insertion. These determinants function synergistically and redundantly, requiring at least two for optimal activity, to enhance interactions between AIV viral ribonucleoprotein (vRNP) and chANP32A, thereby promoting AIV vRNP assembly. In contrast, human ANP32A/B-which lack the other two determinants and rely solely on SUMOylation-exhibit a limited capacity to support AIV polymerase activity. Our findings unveil a cooperative mechanism where SUMO-dependent processes and structural motifs in chANP32A enforce species-specific adaptation of AIV polymerase, shedding light on how ANP32A/B governs host restriction of AIV polymerase. ### Competing Interest Statement The authors have declared no competing interest.

AbstractInfluenza viruses and thogotoviruses account for most recognized orthomyxoviruses. Thogotoviruses, exemplified by Thogoto virus (THOV), are capable of infecting humans using ticks as vectors. THOV transcribes mRNA without the extraneous 5′ end sequences derived from cap-snatching in influenza virus mRNA. Here, we report cryo-EM structures to characterize THOV polymerase RNA synthesis initiation and elongation. The structures demonstrate that THOV RNA transcription and replication are able to start with short dinucleotide primers and that the polymerase cap-snatching machinery is likely non-functional. Triggered by RNA synthesis, asymmetric THOV polymerase dimers can form without the involvement of host factors. We confirm that, distinctive from influenza viruses, THOV-polymerase RNA synthesis is weakly dependent of the host factors ANP32A/B/E in human cells. This study demonstrates varied mechanisms in RNA synthesis and host factor utilization among orthomyxoviruses, providing insights into the mechanisms behind thogotoviruses’ broad-infectivity range.

ABSTRACT Infectious bursal disease (IBD) is an acute and fatal immunosuppressive disease caused by infectious bursal disease virus (IBDV). As an obligate intracellular parasite, IBDV infection is strictly regulated by host factors. Knowledge on the antiviral activity and possible mechanism of host factors might provide the theoretical basis for the prevention and control of IBD. In this study, RNA-sequencing results indicated that many host factors were induced by IBDV infection, among which the expression levels of OASL (2´,5´-oligadenylate synthetase-like protein) was significantly upregulated. OASL overexpression significantly inhibited IBDV replication, whereas OASL knockdown promoted IBDV replication. Interestingly, the antiviral ability of OASL was independent of its canonical enzymatic activity, i.e., OASL targeted viral protein VP2 for degradation, depending on the autophagy receptor p62/SQSTM1 in the autophagy pathway. Additionally, the 316 lysine (K) of VP2 was the key site for autophagy degradation, and its replacement with arginine disrupted VP2 degradation induced by OASL and enhanced IBDV replication. Importantly, our results for the first time indicate a unique and potent defense mechanism of OASL against double-stranded RNA virus by interaction with viral proteins, which leads to their degradation. IMPORTANCE OASL (2´,5´-oligadenylate synthetase-like protein) exhibits broad-spectrum antiviral effects against single-stranded RNA viruses in mammals, potentially serving as a promising target for novel antiviral strategies. However, its role in inhibiting the replication of double-stranded RNA viruses (dsRNA viruses), such as infectious bursal disease virus (IBDV), in avian species remains unclear. Our findings indicated a unique and potent defense mechanism of OASL against dsRNA viruses. It has been previously shown in mammals that OASL inhibits virus replication through increasing interferon production. The groundbreaking aspect of our study is the finding that OASL has the ability to interact with IBDV viral protein VP2 and target it for degradation and thus exerts its antiviral effect. Our results reveal the interaction between avian natural antiviral immune response and IBDV infection. Our study not only enhances our understanding of bird defenses against viral infections but can also inform strategies for poultry disease management.

The subgroup J avian leukosis virus (ALV-J), a retrovirus, uses its gp85 protein to bind to the receptor, the chicken sodium hydrogen exchanger isoform 1 (chNHE1), facilitating viral invasion. ALV-J is the main epidemic subgroup and shows noteworthy mutations within the receptor-binding domain (RBD) region of gp85, especially in ALV-J layer strains in China. However, the implications of these mutations on viral replication and transmission remain elusive. In this study, the ALV-J layer strain JL08CH3-1 exhibited a more robust replication ability than the prototype strain HPRS103, which is related to variations in the gp85 protein. Notably, the gp85 of JL08CH3-1 demonstrated a heightened binding capacity to chNHE1 compared to HPRS103-gp85 binding. Furthermore, we showed that the specific N123I mutation within gp85 contributed to the enhanced binding capacity of the gp85 protein to chNHE1. Structural analysis indicated that the N123I mutation primarily enhanced the stability of gp85, expanded the interaction interface, and increased the number of hydrogen bonds at the interaction interface to increase the binding capacity between gp85 and chNHE1. We found that the N123I mutation not only improved the viral replication ability of ALV-J but also promoted viral shedding in vivo. These comprehensive data underscore the notion that the N123I mutation increases receptor binding and intensifies viral replication.

Human ANP32A/B (huANP32A/B) poorly support the polymerase activity of avian influenza viruses (AIVs), thereby limiting interspecies transmission of AIVs from birds to humans. The SUMO-interacting motif (SIM) within NS2 promotes the adaptation of AIV polymerase to huANP32A/B via a yet undisclosed mechanism. Here we show that huANP32A/B are SUMOylated by the E3 SUMO ligase PIAS2 alpha, and deSUMOylated by SENP1. SUMO modification of huANP32A/B results in the recruitment of NS2, thereby facilitating huANP32A/B-supported AIV polymerase activity. Such a SUMO-dependent recruitment of NS2 is mediated by its association with huANP32A/B via the SIM-SUMO interaction module, where K68/K153-SUMO in huANP32A or K68/K116-SUMO in huANP32B interacts with the NS2-SIM. The SIM-SUMO-mediated interactions between NS2 and huANP32A/B function to promote AIV polymerase activity by positively regulating AIV vRNP-huANP32A/B interactions and AIV vRNP assembly. Our study offers insights into the mechanism of NS2-SIM in facilitating AIVs adaptation to mammals.

Avian metapneumovirus (aMPV) is a highly contagious pathogen that causes acute upper respiratory tract diseases in chickens and turkeys, resulting in serious economic losses. Subtype B aMPV has recently become the dominant epidemic strain in China. We developed an attenuated aMPV subtype B strain by serial passaging in Vero cells and evaluated its safety and efficacy as a vaccine candidate. The safety test showed that after the 30th passage, the LN16-A strain was fully attenuated, as clinical signs of infection and histological lesions were absent after inoculation. The LN16-A strain did not revert to a virulent strain after five serial passages in chickens. The genomic sequence of LN16-A differed from that of the parent wide-type LN16 (wtLN16) strain and had nine amino acid mutations. In chickens, a single immunization with LN16-A induced robust humoral and cellular immune responses, including the abundant production of neutralizing antibodies, CD4+ T lymphocytes, and the Th1 (IFN-γ) and Th2 (IL-4 and IL-6) cytokines. We also confirmed that LN16-A provided 100% protection against subtype B aMPV and significantly reduced viral shedding and turbinate inflammation. Our findings suggest that the LN16-A strain is a promising live attenuated vaccine candidate that can prevent infection with subtype B aMPV.

Species-specific differences in acidic nuclear phosphoprotein 32 family member A (ANP32A) determine the restriction of avian-signature polymerase in mammalian cells. Mutations that evade this restriction, such as PB2-E627K, are frequently acquired when avian influenza A viruses jump from avian hosts to mammalian hosts. However, the mechanism underlying this adaptation process is still unclear. Here, we report that host factor ANP32 proteins can be incorporated into influenza viral particles through combination with the viral RNA polymerase (vPol) and then transferred into targeted cells where they support virus replication. The packaging of the ANP32 proteins into influenza viruses is dependent on their affinity with the vPol. Avian ANP32A (avANP32A) delivered by avian influenza A virions primes early viral replication in mammalian cells, thereby favoring the downstream interspecies transmission event by increasing the total amount of virus carrying adaptive mutations. Our study clarifies one role of avANP32A where it is used by avian influenza virus to help counteract the restriction barrier in mammals.

Avian leukosis is an important tumorigenic disease caused by the avian leukosis virus (ALV) in poultry. ALVs belong to the retroviral family and are classified into 11 subgroups (ALV-A to ALV-K). Among them, ALV-J was first introduced into China in 1999, spreading widely and evolving from infecting meat-type chickens to layer chickens and Chinese local chickens. ALV-J typically induces myeloid leukosis in infected chickens, but also induces a high proportion of hemangiomas in infected layer chickens, posing a serious threat to poultry breeds in China. As a retrovirus, the genome of ALV-J has undergone significant mutations, which may be related to the expansion of the infection host range and increased pathogenicity of ALV-J. Over the last two decades, the introduction and spread of ALV-J in China have caused substantial losses to the poultry industry. Specialized detection assays have been developed to combat ALV-J infections in China. Additionally, ongoing research aims to employ gene-editing technology as a novel antiviral strategy to control the spread of ALV infections. This review highlights the importance of understanding the impact of ALV-J on the Chinese poultry industry and emphasizes the need for ongoing research and innovation to safeguard poultry health and promote sustainable poultry farming practices in China.

Avian leukosis virus subgroup J (ALV-J), a member of the genus Alpharetrovirus, possesses a small genome and exploits a vast array of host factors during its replication cycle. To identify host factors required for ALV-J replication and potentially guide the development of key therapeutic targets for ALV-J prevention, we employed a chicken genome-wide CRISPR/Cas9 knockout library to screen host factors involved in ALV-J infection within DF-1 cells. This screening revealed 42 host factors critical for ALV-J infection. Subsequent knockout assays showed that the absence of the genes encoding cycle-regulatory proteins, namely Cables1, CDK1, and DHFR, significantly inhibited ALV-J replication. Notably, Cables1 knockout cell lines displayed the most pronounced inhibitory effect. Conversely, overexpression assays confirmed that Cables1 significantly promotes ALV-J replication. Immunoprecipitation assays further indicated that Cables1 specifically interacts with the viral protein p15 (viral protease) among all ALV-J proteins, enhancing ALV-J p15 polyubiquitination. Additionally, we identified 26 lysine residues of ALV-J p15 as key sites for ubiquitination, and their replacement with arginine attenuated the replication ability of ALV-J in both in vitro and in vivo assays. This study demonstrates that Cables1 is a critical replication-dependent host factor of ALV-J by enhancing p15 ubiquitination and thereby promoting viral replication. Overall, these findings contribute to a deeper understanding of the ALJ-V replication mechanism and offer a potential target for the prevention and control of ALV-J infection.

Species differences in the host factor ANP32A/B result in the restriction of avian influenza virus polymerase (vPol) in mammalian cells. Efficient replication of avian influenza viruses in mammalian cells often requires adaptive mutations, such as PB2-E627K, to enable the virus to use mammalian ANP32A/B. However, the molecular basis for the productive replication of avian influenza viruses without prior adaptation in mammals remains poorly understood. We show that avian influenza virus NS2 protein help to overcome mammalian ANP32A/B-mediated restriction to avian vPol activity by promoting avian vRNP assembly and enhancing mammalian ANP32A/B-vRNP interactions. A conserved SUMO-interacting motif (SIM) in NS2 is required for its avian polymerase-enhancing properties. We also demonstrate that disrupting SIM integrity in NS2 impairs avian influenza virus replication and pathogenicity in mammalian hosts, but not in avian hosts. Our results identify NS2 as a cofactor in the adaptation process of avian influenza virus to mammals.

Host ANP32 family proteins are crucial for maintaining the activity of influenza RNA polymerase and play an important role in the cross-species transmission of influenza viruses. To date, the molecular properties of equine ANP32 (eqANP32) protein are poorly understood, particularly the mechanisms that affect equine influenza virus (EIV) RNA polymerase activity. Here, we found that there are six alternative splicing variants of equine ANP32A (eqANP32A) with different levels of expression. Further studies showed that these six splicing variants of eqANP32A supported the activity of EIV RNA polymerase to varying degrees, with the variant eqANP32A_X2 having the highest expression abundance and exhibiting the highest support of polymerase activity. Sequence analysis demonstrated that the differences in the N-Cap regions of the six splicing variants significantly affected their N-terminal conformation, but did not affect their ability to bind RNA polymerase. We also demonstrated that there is only one transcript of eqANP32B, and that this transcript showed only very low support to the EIV RNA polymerase. This functional defect in eqANP32B is caused by the sequence of the 110-259 amino acids at its C -terminus. Our results indicated that it is the eqANP32A_X2 protein that mainly determines the efficiency of the EIV replication in horses. In conclusion, our study parsed the molecular properties of eqANP32 family proteins and revealed the sequence features of eqANP32A and eqANP32B, suggesting for the first time that the N-Cap region of ANP32A protein also plays an important role in supporting the activity of the influenza virus polymerase.

Subgroup K avian leukosis virus (ALV-K) is a novel subgroup of ALV isolated from Chinese native chickens. As for a retrovirus, the interaction between its envelope protein and cellular receptor is a crucial step in ALV-K infection. Tva, a protein previously determined to be associated with vitamin B12/cobalamin uptake, has been identified as the receptor of ALV-K. However, the molecular mechanism underlying the interaction between Tva and the envelope protein of ALV-K remains unclear. In this study, we identified the C-terminal loop of the LDL-A module of Tva as the minimal functional domain that directly interacts with gp85, the surface component of the ALV-K envelope protein. Further point-mutation analysis revealed that E53, L55, H59, and G70, which are exposed on the surface of Tva and are spatially adjacent, are key residues for the binding of Tva and gp85 and facilitate the entry of ALV-K. Homology modeling analysis indicated that the substitution of these four residues did not significantly impact the Tva structure but impaired the interaction between Tva and gp85 of ALV-K. Importantly, the gene-edited DF-1 cell line with precisely substituted E53, L55, H59, and G70 was completely resistant to ALV-K infection and did not affect vitamin B12/cobalamin uptake. Collectively, these findings not only contribute to a better understanding of the mechanism of ALV-K entry into host cells but also provide an ideal gene-editing target for antiviral study.

The acidic leucine-rich nuclear phosphoprotein 32 kDa (ANP32) family consists of evolutionarily conserved proteins of 220–291 amino acids characterized by an N-terminal leucine-rich repeat domain (LRR) and a C-terminal low-complexity acidic region (LCAR). ANP32 family proteins regulate a variety of physiological functions, including chromatin remodeling, apoptosis and nervous system development. Abnormal ANP32 expression is closely related to tumorigenesis. In recent years, the role of ANP32 family proteins in viral infections has received considerable attention due to their activity supporting influenza virus replication and restriction of virus cross-species transmission. Moreover, ANP32 proteins are closely related to the replication of HIV and nonsegmented negative-strand RNA viruses (NNSVs). In this review, the general physiological functions of ANP32 family proteins, as well as their roles in virus replication, are summarized in detail.