Identification and Genome Characterization of Novel Feline Parvovirus Strains Isolated in Shanghai, China.

Feline panleukopenia virus (FPV) is the causative agent of hemorrhagic gastroenteritis in feline animals. FPV has been evolving over time, and there have been several different strains of the virus identified. Some of these strains may be more virulent or more resistant to current vaccines than others, which highlights the importance of ongoing research and monitoring of FPV evolution. For FPV genetic evolution analysis, many studies focus on the main capsid protein (VP2), but limited information is available on the nonstructural gene NS1 and structural gene VP1. In the present study, we firstly isolated two novel FPV strains circulating in Shanghai, China, and performed full-length genome sequencing for the desired strains. Subsequently, we focused on analyzing the NS1, VP1 gene, and the encoding protein, and conducted a comparative analysis among the worldwide circulating FPV and Canine parvovirus Type 2 (CPV-2) strains, which included the strains isolated in this study. We found that the 2 structural viral proteins, VP1 and VP2, are splice variants, and VP1 has a 143 amino-acid-long N-terminal compared to VP2. Furthermore, phylogenetic analysis showed that divergent evolution between FPV and CPV-2 virus strains were clustered mostly by country and year of detection. In addition, much more continuous antigenic type changes happened in the process of CPV-2 circulating and evolution compared to FPV. These results stress the importance of the continuous study of viral evolution and provide a comprehensive perspective of the association between viral epidemiology and genetic evolution.

Hepatitis E as a Zoonosis.

Hepatitis E viruses in the family of Hepeviridae have been classified into 2 genus, 5 species, and 13 genotypes, involving different animal hosts of different habitats. Among all these genotypes, four (genotypes 3, 4, 7, and C1) of them are confirmed zoonotic causing sporadic human diseases, two (genotypes 5 and 8) were likely zoonotic showing experimental animal infections, and the other seven were not zoonotic or unconfirmed. These zoonotic HEV carrying hosts include pig, boar, deer, rabbit, camel, and rat. Taxonomically, all the zoonotic HEVs belong to the genus Orthohepevirus, which include genotypes 3, 4, 5, 7, 8 HEV in the species A and genotype C1 HEV in the species C. In the chapter, information of zoonotic HEV such as swine HEV (genotype 3 and 4), wild boar HEV (genotypes 3-6), rabbit HEV (genotype 3), camel HEV (genotype 7 and 8), and rat HEV (HEV-C1) was provided in detail. At the same time, their prevalence characteristics, transmission route, phylogenetic relationship, and detection technology were discussed. Other animal hosts of HEVs were introduced briefly in the chapter. All these information help peer researchers have basic understanding of zoonotic HEV and adopt reasonable strategy of surveillance and prevention.

Porcine Epidemic Diarrhea Virus ORF3 Protein Is Transported through the Exocytic Pathway.

Accessory genes occurring between the S and E genes of coronaviruses have been studied quite intensively during the last decades. In porcine epidemic diarrhea virus (PEDV), the only gene at this location, ORF3, encodes a 224-residue membrane protein shown to exhibit ion channel activity and to enhance virus production. However, little is known about its intracellular trafficking or about its function during PEDV infection. In this study, two recombinant PEDVs were rescued by targeted RNA recombination, one carrying the full-length ORF3 gene and one from which the gene had been deleted entirely. These viruses as well as a PEDV encoding a naturally truncated ORF3 protein were employed to study the ORF3 protein's subcellular trafficking. In addition, ORF3 expression vectors were constructed to study the protein's independent transport. Our results show that the ORF3 protein uses the exocytic pathway to move to and accumulate in the Golgi area of the cell similarly in infected and transfected cells. Like the S protein, but unlike the other structural proteins M and N, the ORF3 protein was additionally observed at the surface of PEDV-infected cells. In addition, the C-terminally truncated ORF3 protein entered the exocytic pathway but it was unable to leave the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartment (ERGIC). Consistently, a YxxØ motif essential for ER exit was identified in the C-terminal domain. Finally, despite the use of sensitive antibodies and assays no ORF3 protein could be detected in highly purified PEDV particles, indicating that the protein is not a structural virion component.IMPORTANCE Coronaviruses typically express several accessory proteins. They vary in number and nature, and only one is conserved among most of the coronaviruses, pointing at an important biological function for this protein. PEDV is peculiar in that it expresses just this one accessory protein, termed the ORF3 protein. While its analogs in other coronaviruses have been studied to different extents, and these studies have indicated that they share an ion channel property, little is still known about the features and functions of the PEDV ORF3 protein except for its association with virulence. In this investigation, we studied the intracellular trafficking of the ORF3 protein both in infected cells and when expressed independently. In addition, we analyzed the effects of mutations in five sorting motifs in its C-terminal domain and investigated whether the protein, found to follow the same exocytic route by which the viral structural membrane proteins travel, is also incorporated into virions.

SALL1 functions as a tumor suppressor in breast cancer by regulating cancer cell senescence and metastasis through the NuRD complex.

BACKGROUND: SALL1 is a multi-zinc finger transcription factor that regulates organogenesis and stem cell development, but the role of SALL1 in tumor biology and tumorigenesis remains largely unknown. METHODS: We analyzed SALL1 expression levels in human and murine breast cancer cells as well as cancer tissues from different types of breast cancer patients. Using both in vitro co-culture system and in vivo breast tumor models, we investigated how SALL1 expression in breast cancer cells affects tumor cell growth and proliferation, metastasis, and cell fate. Using the gain-of function and loss-of-function strategies, we dissected the molecular mechanism responsible for SALL1 tumor suppressor functions. RESULTS: We demonstrated that SALL1 functions as a tumor suppressor in breast cancer, which is significantly down-regulated in the basal like breast cancer and in estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2) triple negative breast cancer patients. SALL1 expression in human and murine breast cancer cells inhibited cancer cell growth and proliferation, metastasis, and promoted cell cycle arrest. Knockdown of SALL1 in breast cancer cells promoted cancer cell growth, proliferation, and colony formation. Our studies revealed that tumor suppression was mediated by recruitment of the Nucleosome Remodeling and Deacetylase (NuRD) complex by SALL1, which promoted cancer cell senescence. We further demonstrated that the mechanism of inhibition of breast cancer cell growth and invasion by SALL1-NuRD depends on the p38 MAPK, ERK1/2, and mTOR signaling pathways. CONCLUSION: Our studies indicate that the developmental control gene SALL1 plays a critical role in tumor suppression by recruiting the NuRD complex and thereby inducing cell senescence in breast cancer cells.

Molecular detection of hepatitis E virus in sheep from southern Xinjiang, China.

Hepatitis E virus (HEV) is a causative agent of infectious hepatitis in animals and humans both in developing and developed countries. Here, we collected 500 sheep sera and 75 raw sheep liver samples from a slaughterhouse in the southern part of the Xinjiang region, China, along with 26 sera of butchers from the same slaughterhouse. All serum samples were tested for anti-HEV antibody by enzyme-linked immunosorbent assay. Both serum and liver samples were evaluated for the presence of HEV RNA by nested polymerase chain reaction targeting partial nucleotide sequences of open reading frame 2 (ORF2). The results indicate that sheep seroprevalence was 35.20 % (176/500) and that four of the 75 (5.3 %) sheep livers showed detectable amounts of HEV RNA. The seroprevalence of the butchers was 57.7 % (15/26). The four amplicons shared 97.8-100 % nucleotide sequence identity and had pairwise sequence identities of 81.6-85.3 %, 84.2-85.3 %, 82.1-85.3 % and 84.7-97.9 % with the corresponding regions of genotypes 1, 2, 3 and 4 of HEV, respectively. A phylogenetic tree was constructed based on alignments of an amplified 186-bp ORF2 sequence and corresponding reference strains. The analysis showed that the four sheep strains detected in our study formed a lineage within a genotype 4 cluster that contains hb-3, bjsw1, T1, swCH189 and swCH25, all of which belong to genotype 4, subtype 4d. The results indicated a high level of seroconversion in sheep and suggested that sheep liver may be a source of foodborne HEV infection in humans.

Construction of an infectious cDNA clone of a swine genotype 3 HEV strain isolated in Shanghai, China.

OBJECTIVES: Infectious cDNA clones are important tools for studying molecular mechanisms in RNA viruses. The aim of this study was to construct an infectious cDNA clone for SAAS-JDY5, which is a genotype 3 HEV strain of swine origin. METHODS: Construction employed overlapping PCR and restriction analysis to ligate nine cDNA fragments into a full-length cDNA clone containing 14 mutations compared to the consensus HEV genome sequence. Megaprimer PCR-directed mutagenesis restored nine non-silent mutations back to the consensus sequence while the other five silent mutations were maintained as genetic markers. RESULTS: HEV proteins were identified by an immunofluorescence assay in Huh7 cells infected with capped RNA transcripts of the full-length cDNA clone, while HEV viremia, fecal HEV RNA and seroconversion were recorded in inoculated Sprague-Dawley rats. CONCLUSIONS: Our data confirmed the successful construction of an infectious cDNA clone of swine HEV strain pGEM4z-SAAS-JDY5, and support the use of rats as an HEV infectious model.

Towards a Safer Future: Enhancing Vaccine Development to Combat Animal Coronaviruses.

Coronaviruses (CoVs) are a large class of positively stranded RNA viruses that pose a significant threat to public health, livestock farming, and wild animals. These viruses have the ability to cross species barriers and cause devastating epidemics. Animals are considered to be intermediate hosts for many coronaviruses, and many animal coronaviruses also have the potential for cross-species transmission to humans. Therefore, controlling the epidemic transmission of animal coronaviruses is of great importance to human health. Vaccination programs have proven to be effective in controlling coronaviruses infections, offering a cost-effective approach to reducing morbidity and mortality, so the re-emergence of lethal coronaviruses emphasizes the urgent need for the development of effective vaccines. In this regard, we explore the progress in animal coronavirus vaccine development, covering the latest taxonomy of the main animal coronaviruses, spillover events, diverse vaccine development platforms, potential main targets for animal coronavirus vaccine development, and primary challenges facing animal coronavirus vaccines. We emphasize the urgent need to create a "dual-effect" vaccine capable of eliciting both cellular and humoral immune responses. The goal is to highlight the contributions of veterinary scientists in this field and emphasize the importance of interdisciplinary collaboration between the veterinary and medical communities. By promoting communication and cooperation, we can enhance the development of novel and super vaccines to combat human and animal coronavirus infections in the future.

Developing Next-Generation Live Attenuated Vaccines for Porcine Epidemic Diarrhea Using Reverse Genetic Techniques.

Porcine epidemic diarrhea virus (PEDV) is the etiology of porcine epidemic diarrhea (PED), a highly contagious digestive disease in pigs and especially in neonatal piglets, in which a mortality rate of up to 100% will be induced. Immunizing pregnant sows remains the most promising and effective strategy for protecting their neonatal offspring from PEDV. Although half a century has passed since its first report in Europe and several prophylactic vaccines (inactivated or live attenuated) have been developed, PED still poses a significant economic concern to the swine industry worldwide. Hence, there is an urgent need for novel vaccines in clinical practice, especially live attenuated vaccines (LAVs) that can induce a strong protective lactogenic immune response in pregnant sows. Reverse genetic techniques provide a robust tool for virological research from the function of viral proteins to the generation of rationally designed vaccines. In this review, after systematically summarizing the research progress on virulence-related viral proteins, we reviewed reverse genetics techniques for PEDV and their application in the development of PED LAVs. Then, we probed into the potential methods for generating safe, effective, and genetically stable PED LAV candidates, aiming to provide new ideas for the rational design of PED LAVs.

Genetic distribution, characterization, and function of Escherichia coli type III secretion system 2 (ETT2).

Many Gram-negative bacteria use type Ⅲ secretion system (T3SS) to inject effector proteins and subvert host signaling pathways, facilitating the growth, survival, and virulence. Notably, some bacteria harbor multiple distinct T3SSs with different functions. An extraordinary T3SS, the Escherichia coli Type III Secretion System 2 (ETT2), is widespread among Escherichia coli (E. coli) strains. Since many ETT2 carry genetic mutations or deletions, it is thought to be nonfunctional. However, increasing studies highlight ETT2 contributes to E. coli pathogenesis. Here, we present a comprehensive overview of genetic distribution and characterization of ETT2. Subsequently, we outline its functional potential, contending that an intact ETT2 may retain the capacity to translocate effector proteins and manipulate the host's innate immune response. Given the potential zoonotic implications associated with ETT2-carrying bacteria, further investigations into the structure, function and regulation of ETT2 are imperative for comprehensive understanding of E. coli pathogenicity and the development of effective control strategies.

Blocking senescence and tolerogenic function of dendritic cells induced by γδ Treg cells enhances tumor-specific immunity for cancer immunotherapy.

BACKGROUND: Regulatory T (Treg) cells are a key component in maintaining the suppressive tumor microenvironment and immune suppression in different types of cancers. A precise understanding of the molecular mechanisms used by Treg cells for immune suppression is critical for the development of effective strategies for cancer immunotherapy. METHODS: Senescence development and tolerogenic functions of dendritic cells (DCs) induced by breast cancer tumor-derived γδ Treg cells were fully characterized using real-time PCR, flow cytometry, western blot, and functional assays. Loss-of-function strategies with pharmacological inhibitor and/or neutralizing antibody were used to identify the potential molecule(s) and pathway(s) involved in DC senescence and dysfunction induced by Treg cells. Impaired tumor antigen HER2-specific recognition and immune response of senescent DCs induced by γδ Treg cells were explored in vitro and in vivo in humanized mouse models. In addition, the DC-based HER2 tumor vaccine immunotherapy in breast cancer models was performed to explore the enhanced antitumor immunity via prevention of DC senescence through blockages of STAT3 and programmed death-ligand 1 (PD-L1) signaling. RESULTS: We showed that tumor-derived γδ Treg cells promote the development of senescence in DCs with tolerogenic functions in breast cancer. Senescent DCs induced by γδ Treg cells suppress Th1 and Th17 cell differentiation but promote the development of Treg cells. In addition, we demonstrated that PD-L1 and STAT3 signaling pathways are critical and involved in senescence induction in DCs mediated by tumor-derived γδ Treg cells. Importantly, our complementary in vivo studies further demonstrated that blockages of PD-L1 and/or STAT3 signaling can prevent γδ Treg-induced senescence and reverse tolerogenic functions in DCs, resulting in enhanced HER2 tumor-specific immune responses and immunotherapy efficacy in human breast cancer models. CONCLUSIONS: These studies not only dissect the suppressive mechanism mediated by tumor-derived γδ Treg cells on DCs in the tumor microenvironment but also provide novel strategies to prevent senescence and dysfunction in DCs and enhance antitumor efficacy mediated by tumor-specific T cells for cancer immunotherapy.

Coronavirus accessory protein ORF3 biology and its contribution to viral behavior and pathogenesis.

Coronavirus porcine epidemic diarrhea virus (PEDV) is classified in the genus Alphacoronavirus, family Coronaviridae that encodes the only accessory protein, ORF3 protein. However, how ORF3 contributes to viral pathogenicity, adaptability, and replication is obscure. In this review, we summarize current knowledge and identify gaps in many aspects of ORF3 protein in PEDV, with emphasis on its unique biological features, including membrane topology, Golgi retention mechanism, potential intrinsic disordered property, functional motifs, protein glycosylation, and codon usage phenotypes related to genetic evolution and gene expression. In addition, we propose intriguing questions related to ORF3 protein that we hope to stimulate further studies and encourage collaboration among virologists worldwide to provide constructive knowledge about the unique characteristics and biological functions of ORF3 protein, by which their potential role in clarifying viral behavior and pathogenesis can be possible.

A roadmap for developing Venezuelan equine encephalitis virus (VEEV) vaccines: Lessons from the past, strategies for the future.

Venezuelan equine encephalitis (VEE) is a zoonotic infectious disease caused by the Venezuelan equine encephalitis virus (VEEV), which can lead to severe central nervous system infections in both humans and animals. At present, the medical community does not possess a viable means of addressing VEE, rendering the prevention of the virus a matter of paramount importance. Regarding the prevention and control of VEEV, the implementation of a vaccination program has been recognized as the most efficient strategy. Nevertheless, there are currently no licensed vaccines or drugs available for human use against VEEV. This imperative has led to a surge of interest in vaccine research, with VEEV being a prime focus for researchers in the field. In this paper, we initially present a comprehensive overview of the current taxonomic classification of VEEV and the cellular infection mechanism of the virus. Subsequently, we provide a detailed introduction of the prominent VEEV vaccine types presently available, including inactivated vaccines, live attenuated vaccines, nucleic acid, and virus-like particle vaccines. Moreover, we emphasize the challenges that current VEEV vaccine development faces and suggest urgent measures that must be taken to overcome these obstacles. Notably, based on our latest research, we propose the feasibility of incorporation codon usage bias strategies to create the novel VEEV vaccine. Finally, we prose several areas that future VEEV vaccine development should focus on. Our objective is to encourage collaboration between the medical and veterinary communities, expedite the translation of existing vaccines from laboratory to clinical applications, while also preparing for future outbreaks of new VEEV variants.

Three Amino Acid Substitutions in the Spike Protein Enable the Coronavirus Porcine Epidemic Diarrhea Virus To Infect Vero Cells.

Porcine epidemic diarrhea virus (PEDV), a continuously evolving pathogen, causes severe diarrhea in piglets, with high mortality rates. To prevent or mitigate the disease, it is common practice to develop live or inactivated PEDV vaccines based on cell-adapted viral variants. Propagating wild-type PEDV in cultured cells is, however, often challenging due to the lack of knowledge about the requirements for the cell adaptation of PEDV. In the present study, by using the RNA-targeted reverse genetic system for PEDV to apply S protein swapping followed by the rescue of the recombinant viruses, three key amino acid mutations in the S protein, A605E, E633Q, and R891G, were identified, which enable attenuated PEDV strain DR13 (DR13att) to efficiently and productively infect Vero cells, in contrast to the parental DR13 strain (DR13par). The former two key mutations reside inside and in the vicinity of the receptor binding domain (RBD), respectively, while the latter occurs at the N-terminal end of the fusion peptide (FP). Besides the three key mutations, other mutations in the S protein further enhanced the infection efficiency of the recombinant viruses. We hypothesize that the three mutations changed PEDV tropism by altering the S2' cleavage site and the RBD structure. This study provides basic molecular insight into cell adaptation by PEDV, which is also relevant for vaccine design. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) is a lethal pathogen for newborn piglets, and an efficient vaccine is needed urgently. However, propagating wild-type PEDV in cultured cells for vaccine development is still challenging due to the lack of knowledge about the mechanism of the cell adaptation of PEDV. In this study, we found that three amino acid mutations, A605E, E633Q, and R891G, in the spike protein of the Vero cell-adapted PEDV strain DR13att were critical for its cell adaptation. After analyzing the mutation sites in the spike protein, we hypothesize that the cell adaptation of DR13att was achieved by altering the S2' cleavage site and the RBD structure. This study provides new molecular insight into the mechanism of PEDV culture adaptation and new strategies for PEDV vaccine design.

Magnolol, a Neolignan-like Drug, Inhibits Porcine Epidemic Diarrhea Virus Replication in Cultured Cells.

Porcine epidemic diarrhea virus (PEDV) is a destructive pathogen that continues to adversely affect the swine industry worldwide due to a current lack of vaccines and drugs capable of effective disease control. In the present study, the neolignan-like drug, magnolol (MAG), was tested for its ability to inhibit a Vero-cell adapted PEDV strain DR13att. Our data revealed that MAG exhibited anti-PEDV activity in vitro, with IC50 and CC50 values of 28.21 µM and 57.28 µM, respectively. MAG was an efficient inhibitor of viral replication, and repression of viral proliferation was strongest when the host cells were exposed to MAG and the virus at the same time. Although our data indicate that MAG has the potential to be a useful PEDV control agent, in vivo testing of the drug, using animal hosts, is required.

Design and Identification of a Novel Antiviral Affinity Peptide against Fowl Adenovirus Serotype 4 (FAdV-4) by Targeting Fiber2 Protein.

Outbreaks of hydropericardium hepatitis syndrome caused by fowl adenovirus serotype 4 (FAdV-4) with a novel genotype have been reported in China since 2015, with significant economic losses to the poultry industry. Fiber2 is one of the important structural proteins on FAdV-4 virions. In this study, the C-terminal knob domain of the FAdV-4 Fiber2 protein was expressed and purified, and its trimer structure (PDB ID: 7W83) was determined for the first time. A series of affinity peptides targeting the knob domain of the Fiber2 protein were designed and synthesized on the basis of the crystal structure using computer virtual screening technology. A total of eight peptides were screened using an immunoperoxidase monolayer assay and RT-qPCR, and they exhibited strong binding affinities to the knob domain of the FAdV-4 Fiber2 protein in a surface plasmon resonance assay. Treatment with peptide number 15 (P15; WWHEKE) at different concentrations (10, 25, and 50 µM) significantly reduced the expression level of the Fiber2 protein and the viral titer during FAdV-4 infection. P15 was found to be an optimal peptide with antiviral activity against FAdV-4 in vitro with no cytotoxic effect on LMH cells up to 200 µM. This study led to the identification of a class of affinity peptides designed using computer virtual screening technology that targeted the knob domain of the FAdV-4 Fiber2 protein and may be developed as a novel potential and effective antiviral strategy in the prevention and control of FAdV-4.

Determination of the full-genome sequence of hepatitis E virus (HEV) SAAS-FX17 and use as a reference to identify putative HEV genotype 4 virulence determinants.

BACKGROUND: Four major genotypes of hepatitis E virus (HEV), the causative agent of hepatitis E, have so far been recognized. While genotypes 3 and 4 are both zoonotic, the disease symptoms caused by the latter tend to be more severe. To examine if specific nucleotide/amino acid variations between genotypes 3 and 4 play a role in determining the severity of hepatitis E disease, the complete genome of one swine HEV genotype 4 isolate, SAAS-FX17, was determined and compared with other genotype 4 and genotype 3 genomes to identify putative HEV genotype 4 virulence determinants. RESULTS: A total of 42 conformable nt/aa variations between genotype 3 and 4 HEVs were detected, of which 19 were proposed to be potential disease severity determinants for genotype 4 strains. CONCLUSIONS: One potential determinant was located in each of the 5'-UTR and 3'-UTR, 3 and 12 within ORF1 and ORF2 respectively, and 2 in the junction region.

Adaptation of genotype 3 hepatitis E virus in Eastern China and inverse correlation with genotype 4 hepatitis E virus.

We studied the distribution and development of hepatitis E virus (HEV) genotypes 3 and 4 in pig farms located within the Shanghai metropolitan area. A total of 1,487 swine fecal samples were collected from 39 pig farms during 2009-2010. The average incidence rates for genotype 3 and genotype 4 HEV were 10.6 and 9.3%, respectively. The frequencies of genotype 3 and genotype 4 HEV among the farms were inversely related (r = -0.7423), suggesting that the two genotypes competed within the environmental niche provided by the porcine host. In addition, all of the farms tested positive for both genotype 3 and genotype 4 HEV, indicating that the former is becoming more prevalent in the Shanghai area. The overall HEV incidence rate during 2009-2010 was 20%, which supports the existence of homeostasis within the porcine HEV reservoir.

Deep decoding of codon usage strategies and host adaption preferences of soybean mosaic virus.

Soybean mosaic virus (SMV) has threatened the global yield of Leguminosae crops, but the mechanism of its infection, spread, and evolution remains unknown. A systemic analysis of 107 SMV strains was performed to explore the genome-wide codon usage profile and the various factors influencing the codon usage patterns of SMV, which provides insight into its molecular evolution and elucidates its unknown host adaptation pattern. The overall nucleotide composition and correlation analysis revealed that the preferred synonymous codons mostly end with A/U. Clustering by RSCU value of each strain and phylogenetic tree analysis showed that the SMV isolates studied were divided into four clades, with a low overall extent of codon usage bias (CUB) in SMV. According to the ENC, PR2, neutrality plot, and correspondence analysis, natural selection of geographical diversity may play a critical role in the CUB. Higher adaptability was shown in Glycine with SMV and more pressure was received by clade III. These findings could not only provide valuable information about the overall codon usage pattern of the SMV genome, but could also aid in the clarification of the involved mechanisms that dominate the codon usage patterns and genetic evolution of the SMV genome.

When liquid-liquid phase separation meets viral infections.

Eukaryotic cells have both membranous and membraneless organelles. While the formation mechanism of membranous organelles is well understood, the formation mechanism of membraneless organelles remains unknown. Many biomolecules in the cytoplasm transition from the liquid phase to the agglutinated phase are known as liquid-liquid phase separation (LLPS). The biomolecular agglomerates' physical properties enable them to function as dynamic compartments that respond to external pressures and stimuli. Scientists have gradually recognized the importance of phase separation during viral infections. LLPS provides a powerful new framework for understanding the viral life cycle from viral replication to evasion of host immune surveillance. As a result, this review focuses on the progress of LLPS research in viral infection and immune regulation to provide clues for antiviral therapeutic strategies.