The PCV3 Cap Virus-like Particle Vaccine with the Chimeric PCV2-Neutralizing Epitope Gene Is Effective in Mice.

Porcine circovirus type 3 (PCV3) infection can cause symptoms similar to those of porcine circovirus type 2 (PCV2) infection, and coinfections with both PCV2 and PCV3 are observed in the swine industry. Consequently, developing chimeric vaccines is essential to prevent and control porcine circovirus infections. In this study, we used both E. coli and mammalian expression systems to express PCV3 Cap (Cap3) and a chimeric gene containing the PCV2-neutralizing epitope within the PCV3 Cap (Cap3-Cap2E), which were assembled into virus-like particle (VLP) vaccines. We found that Cap3 lacking nuclear localization signal (NLS) could not form VLPs, while Cap3 with a His-tag successfully assembled into VLPs. Additionally, the chimeric of PCV2-neutralizing epitopes did not interfere with the assembly process of VLPs. Various immunization approaches revealed that pCap3-Cap2E VLP vaccines were capable of activating high PCV3 Cap-specific antibody levels and effectively neutralizing both PCV3 and PCV2. Furthermore, pCap3-Cap2E VLPs demonstrated a potent ability to activate cellular immunity, protecting against PCV3 infection and preventing lung damage in mice. In conclusion, this study successfully developed a PCV3 Cap VLP vaccine incorporating chimeric PCV2-neutralizing epitope genes, providing new perspectives for PCV3 vaccine development.

Patient-specific temperature distribution prediction in laser interstitial thermal therapy: single-irradiation data-driven method.

Laser interstitial thermal therapy (LITT) is popular for treating brain tumours and epilepsy. The strict control of tissue thermal damage extent is crucial for LITT. Temperature prediction is useful for predicting thermal damage extent. Accurately predictingin vivobrain tissue temperature is challenging due to the temperature dependence and the individual variations in tissue properties. Considering these factors is essential for improving the temperature prediction accuracy.Objective. To present a method for predicting patient-specific tissue temperature distribution within a target lesion area in the brain during LITT.Approach. A magnetic resonance temperature imaging (MRTI) data-driven estimation model was constructed and combined with a modified Pennes bioheat transfer equation (PBHE) to predict patient-specific temperature distribution. In the PBHE for temperature prediction, the individual specificity and temperature dependence of thermal tissue properties and blood perfusion, as well as the individual specificity of optical tissue properties were considered. Only MRTI data during one laser irradiation were required in the method. This enables the prediction of patient-specific temperature distribution and the resulting thermal damage region for subsequent ablations.Main results. Patient-specific temperature prediction was evaluated based on clinical data acquired during LITT in the brain, using intraoperative MRTI data as the reference standard. Our method significantly improved the prediction performance of temperature distribution and thermal damage region. The average root mean square error was decreased by 69.54%, the average intraclass correlation coefficient was increased by 37.5%, the average Dice similarity coefficient was increased by 43.14% for thermal damage region prediction.Significance. The proposed method can predict temperature distribution and thermal damage region at an individual patient level during LITT, providing a promising approach to assist in patient-specific treatment planning for LITT in the brain.

Correction: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication.

[This corrects the article DOI: 10.1371/journal.ppat.1006851.].

Characterization of a novel recombinant NADC30­like porcine reproductive and respiratory syndrome virus in Shanxi Province, China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens affecting the swine industry. In this report, a novel PRRSV strain SXht2012 was isolated from Shanxi province in China. To identify genetic characteristics of SXht2012, we conducted phylogenetic and homology analyses after sequencing its complete genome. The results revealed that SXht2012 belonged to NADC30-like strain and shared 91.3% nucleotide (nt) identity with strain NADC30. Notably, sequence alignment showed that a distinctive feature in the NSP2 region, where a 131-amino acid (aa) deletion was found in the hypervariable region (HVR). Additionally, variations were also detected in the GP5 protein, specifically in the decoy peptide, T cell peptide, and a potential glycosylation site (aa 32). Furthermore, we also found that SXht2012 was likely a recombination virus originating from NADC30-like and JXA1-like strains, and three recombination breakpoints were identified in the genome at nt positions 1516, 5280 and 6851, which correspond to the NSP2, NSP3, and NSP7 regions. Overall, these findings have significant implications for understanding the genetic variation and evolutionary dynamics of PRRSV strains.

Case report: Treatment of Wilson's disease by human amniotic fluid administration.

Background: Wilson's disease (WD) is not an uncommon genetic disease in clinical practice. However, the current WD therapies have limitations. The effectiveness of stem cell therapy in treating WD has yet to be verified, although a few animal studies have shown that stem cell transplantation could partially correct the abnormal metabolic phenotype of WD. In this case report, we present the therapeutic effect of human amniotic fluid containing stem cells in one WD patient. Case presentation: A 22-year-old Chinese woman was diagnosed with WD 1 year ago in 2019. The available drugs were not effective in managing the progressive neuropsychiatric symptoms. We treated the patient with pre-cultured human amniotic fluid containing stem cells. Amniotic fluid was collected from pregnant women who underwent induced labor at a gestational age of 19-26 weeks, and then, the fluid was cultured for 2 h to allow stem cell expansion. Cultured amniotic fluid that contained amniotic fluid derived stem cells (AFSC) in the range of approximately 2.8-5.5 × 104/ml was administrated by IV infusion at a rate of 50-70 drops per minute after filtration with a 300-mu nylon mesh. Before the infusion of amniotic fluid, low-molecular-weight heparin and dexamethasone were successively administrated. The patient received a total of 12 applications of amniotic fluid from different pregnant women, and the treatment interval depended on the availability of amniotic fluid. The neuropsychiatric symptoms gradually improved after the stem cell treatment. Dystonia, which included tremor, chorea, dysphagia, dysarthria, and drooling, almost disappeared after 1.5 years of follow-up. The Unified Wilson's Disease Rating Scale score of the patient decreased from 72 to 10. Brain magnetic resonance imaging (MRI) showed a reduction in the lesion area and alleviation of damage in the central nervous system, along with a partial recovery of the lesion to the normal condition. The serum ceruloplasmin level was elevated from undetectable to 30.8 mg/L, and the 24-h urinary copper excretion decreased from 171 to 37 µg. In addition, amniotic fluid transplantation also alleviates hematopoietic disorders. There were no adverse reactions during or after amniotic fluid administration. Conclusion: Amniotic fluid administration, through which stem cells were infused, significantly improves the clinical outcomes in the WD patient, and the finding may provide a novel approach for managing WD effectively.

Genomic Characterization and gE/gI-Deleted Strain Construction of Novel PRV Variants Isolated in Central China.

Pseudorabies virus (PRV) variants have caused substantial economic losses in the swine industry in China since 2011. To surveil the genetic variation in PRV field strains, here, two novel variant strains of PRV were isolated from Shanxi Province in central China and were designated SX1910 and SX1911. To identify the genetic characteristics of the two isolates, their complete genomes were sequenced, and phylogenetic analysis and sequence alignment revealed that field PRV variants have undergone genetic variations; notably, the protein-coding sequences UL5, UL36, US1 and IE180 exhibited extensive variation and contained one or more hypervariable regions. Furthermore, we also found that the glycoproteins gB and gD of the two isolates had some novel amino acid (aa) mutations. Importantly, most of these mutations were located on the surface of the protein molecule, according to protein structure model analysis. We constructed a mutant virus of SX1911 with deletion of the gE and gI genes via CRISPR/Cas9. When tested in mice, SX1911-ΔgE/gI-vaccinated mice were protected within a comparable range to Bartha-K61-vaccinated mice. Additionally, a higher dose of inactivated Bartha-K61 protected the mice from lethal SX1911 challenge, while a lower neutralization titer, higher viral load and more severe microscopic lesions were displayed in Bartha-K61-vaccinated mice. These findings highlight the need for continuous monitoring of PRV and novel vaccine development or vaccination program design for PRV control in China.

High-dose insulin and dexamethasone combined with radiotherapy in endometrial stromal sarcoma recurring with multiple metastases: A case report.

RATIONALE: Endometrial stromal sarcoma (ESS) is a rare malignant tumor. There is insufficient data supporting the efficiency of current treatments in multiple metastatic settings, and novel therapeutic options for ESS are considered an area of high unmet clinical need. PATIENT CONCERNS: We report the case of a 28-year-old woman who was diagnosed with ESS after undergoing total hysterectomy and left adnexectomy at another hospital. Two years later, the disease recurred, with multiple abdominal cavities and lung metastases. The patient was treated with a variety of chemotherapeutic drugs, including tyrosine kinase inhibitors, at the same hospital; however, none of them inhibited disease progression. DIAGNOSES: Computed tomography (CT) revealed multiple masses in the abdominal and pelvic cavities and multiple pulmonary nodules. Ultrasound-guided biopsy was performed and the tumor tissue was histologically confirmed after treatment. INTERVENTIONS: Insulin 300-400 IU was administrated by intravenous infusion in 10% glucose (500 mL) with disodium adenosine triphosphate 60 mg, coenzyme A 100 units, 10% potassium chloride 5 mL and 25% magnesium sulfate 5 mL. Dexamethasone (20-25 mg/d) was diluted with 10 mL of 2% lidocaine and then intraperitoneally injected after ascites draw. After 9 months, the patient was referred to another center for radiotherapy. OUTCOMES: CT images tomography showed recurrent pelvic masses, and multiple abdominal cavity and lung metastases gradually shrunk with treatment. Histological biopsy revealed growth arrest of tumor cells. The patient experienced for 3-years survival. LESSONS: High-dose insulin and dexamethasone combined with radiotherapy provides a novel and promising option for patients with multiple ESS metastases.

RAP44 phage integrase-guided 50K genomic island integration in Riemerella anatipestifer.

Bacteriophages are viruses that infect bacteria. Bacteria and bacteriophages have been fighting for survival. Over time, the evolution of both populations has been affected. Pathogenic Flavobacteriaceae species including Riemerella anatipestifer mainly infects ducklings, geese, and turkeys. However, it does not infect humans, rats, or other mammals, and is a suitable and safe research object in the laboratory. Our previous study showed that there is a 10K genomic island in R. anatipestiferIn this study, we found another integrated 50K genomic islands and focused on the relationship between R. anatipestifer genomic islands and the RAP44 phage genome. The phage RAP44 genome was integrated into R. anatipestifer chromosome, and an evolutionary relationship was evident between them in our comparative analysis. Furthermore, the integrated defective RAP44 phage sequence had the function of integration, excision, and cyclization automatically. Integrases are important integration elements. The integrative function of integrase was verified in R. anatipestifer. The integrase with the attP site can be integrated stably at the attB locus of the R. anatipestifer genome. A recombinant strain can stably inherit and express the exogenous gene. By studying the integration between host bacterium and phage, we have provided evidence for the evolution of the genomes in R. anatipestifer.

PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence.

Host defense systems employ posttranslational modifications to protect against invading pathogens. Here, we found that protein inhibitor of activated STAT 1 (PIAS1) interacts with the nucleoprotein (NP), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) of influenza A virus (IAV). Lentiviral-mediated stable overexpression of PIAS1 dramatically suppressed the replication of IAV, whereas siRNA knockdown or CRISPR/Cas9 knockout of PIAS1 expression significantly increased virus growth. The expression of PIAS1 was significantly induced upon IAV infection in both cell culture and mice, and PIAS1 was involved in the overall increase in cellular SUMOylation induced by IAV infection. We found that PIAS1 inhibited the activity of the viral RNP complex, whereas the C351S or W372A mutant of PIAS1, which lacks the SUMO E3 ligase activity, lost the ability to suppress the activity of the viral RNP complex. Notably, the SUMO E3 ligase activity of PIAS1 catalyzed robust SUMOylation of PB2, but had no role in PB1 SUMOylation and a minimal role in NP SUMOylation. Moreover, PIAS1-mediated SUMOylation remarkably reduced the stability of IAV PB2. When tested in vivo, we found that the downregulation of Pias1 expression in mice enhanced the growth and virulence of IAV. Together, our findings define PIAS1 as a restriction factor for the replication and pathogenesis of IAV.

Effects of vitamin D with or without calcium on pathological ossification: A retrospective clinical study.

Vitamin D protects against the development and severity of several rheumatic diseases. However, the effect of vitamin D on the pathological ossification associated with rheumatic diseases remains unknown. The present retrospective study analyzed the clinical outcomes of vitamin D without calcium compared with vitamin D with calcium on pathological ossification in joints and ligaments. Data were collected from patients who were diagnosed with osteoarthritis, rheumatoid arthritis or spondylarthritis, and the presence of pathological ossification in joints or ligaments was confirmed by X-ray, computed tomography or magnetic resonance imaging examination. A total of 2,965 patients aged 18-75 years old were included, among who, 1,725 were included in the vitamin D alone group and 1,240 in the vitamin D with calcium group. Vitamin D was administered intramuscularly (300,000 IU) once every 7-10 days, 4-6 times in total. Patients who ingested an oral calcium supplement (1,000 mg/day; ≥5 days/week) were considered the vitamin D with calcium group. The clinical outcome was evaluated based on the imaging changes of pathological ossification, which were classified as alleviation, aggravation and unchanged. The bone mineral density (BMD) was determined, and the calcium concentration in the serum and urine was measured. The results revealed that vitamin D alone alleviated pathological ossification, while vitamin D combined with calcium aggravated pathological ossification in the majority of patients (P<0.0001) independent of disease type and patient age. BMD measurements demonstrated a decreasing trend in the vitamin D alone group, whereas they exhibited an increasing trend in the vitamin D combined with calcium group. The urine calcium concentration increased after vitamin D treatment alone. Therefore, it was concluded that vitamin D exerted both pro-resorptive and anti-resorptive actions on pathological ossification. The bidirectional action of vitamin D on bone metabolism may depend on exogenous calcium supplementation.

A convenient and stable vertebrae instance segmentation method for transforaminal endoscopic surgery planning.

PURPOSE: Transforaminal endoscopic surgery (TES) is effective for treatment of intervertebral disc-related diseases. To avoid injury to the critical structures, preoperative planning is required to find a safe working channel. Therefore, accurate patient-specific vertebral segmentation is important. The purpose of this work is to develop a convenient, stable and feasible lumbar vertebrae segmentation method for TES planning. METHODS: Based on the chain structure of the spine, an interactive dual-output vertebrae instance segmentation network was designed to segment the specific vertebrae in CT images. First, an initialization locator module was set up to provide an initial locating box. Then the dual-output network was designed to segment two adjacent vertebrae inside the locating box. Finally, iteration was performed until all the expected vertebrae were segmented. RESULTS: Verification on reconstructed public dataset showed that the vertebral segmentation Dice coefficient was 96.8 ± 1.2% and average surface distance (ASD) was 0.25 ± 0.10 mm. For intervertebral foramen (IVF) region, the Dice coefficient was 96.1 ± 1.5% and ASD was 0.29 ± 0.10 mm. For IVF forming region, the Dice coefficient was 93.4 ± 3.1% and ASD was 0.28 ± 0.13 mm. The evaluation on private dataset showed that more than 90% of the segmentation were suitable for TES planning. For IVF region, the Dice coefficient was 94.4 ± 1.8% and ASD was 0.71 ± 0.49 mm. CONCLUSION: This work provides a convenient, stable and feasible segmentation method for lumbar vertebrae, IVF region, and IVF forming region. The segmentation can meet the requirement for TES planning.

Viral RNA-binding ability conferred by SUMOylation at PB1 K612 of influenza A virus is essential for viral pathogenesis and transmission.

Posttranslational modifications, such as SUMOylation, play specific roles in the life cycle of invading pathogens. However, the effect of SUMOylation on the adaptation, pathogenesis, and transmission of influenza A virus (IAV) remains largely unknown. Here, we found that a conserved lysine residue at position 612 (K612) of the polymerase basic protein 1 (PB1) of IAV is a bona fide SUMOylation site. SUMOylation of PB1 at K612 had no effect on the stability or cellular localization of PB1, but was critical for viral ribonucleoprotein (vRNP) complex activity and virus replication in vitro. When tested in vivo, we found that the virulence of SUMOylation-defective PB1/K612R mutant IAVs was highly attenuated in mice. Moreover, the airborne transmission of a 2009 pandemic H1N1 PB1/K612R mutant virus was impaired in ferrets, resulting in reversion to wild-type PB1 K612. Mechanistically, SUMOylation at K612 was essential for PB1 to act as the enzymatic core of the viral polymerase by preserving its ability to bind viral RNA. Our study reveals an essential role for PB1 K612 SUMOylation in the pathogenesis and transmission of IAVs, which can be targeted for the design of anti-influenza therapies.

Amniotic Fluid Transplantation Alleviates Hematopoietic Deficits in Experimental Rat Aplastic Anemia.

BACKGROUND Hematopoietic stem cell (HSC) transplantation is the most effective therapy for hematopoietic impairment. However, maintenance and self-renewal of HSCs in culture is still a central focus of HSC research. It is known that amniotic fluid contains a heterologous population of stem cells (AFSCs) and nutrients as well as various types of growth factors. We hypothesize that AFSCs may be expanded in vitro in pure amniotic fluid. MATERIAL AND METHODS Amniotic fluid with transparent appearance was harvested at embryo age of 13.5-15.5 days in rats and was placed in a cell culture CO2 incubator. The cell number in the amniotic fluid was counted before and after culture of amniotic fluid. Then, the effect of amniotic fluid transplantation on 5-fluorouracil combined with busulfan induced-rat aplastic anemia was investigated. RESULTS We found that after a short time (about 30 min) culture, the number of AFSCs expanded more than 100-fold. Flow cytometry showed that there was a population of cells expressing hematopoietic markers CD45 and CD34 in addition to a higher proportional of mesenchymal stem cells (MSCs) markers CD29 and CD90. Transplantation of the expanded AFSCs possessed significant therapeutic effect in toxic chemicals induced-aplastic anemia rats, manifested by decreasing animal mortality and alleviating the reduction of the 3 lineages of hematopoietic cells in blood. CONCLUSIONS Our observation for the first time demonstrates that amniotic fluid is an excellent medium for stem cells to maintain "stemness" proliferation and provides novel evidence to support the potential use of in vitro-expanded AFSCs for the treatment of hematopoietic disorders.

Multiple objective planning for thermal ablation of liver tumors.

PURPOSE: Preoperative treatment planning is key to ensure successful thermal ablation of liver tumors. The planning aims to minimize the number of electrodes required for complete ablation and the damage to the surrounding tissues while satisfying multiple clinical constraints. This is a challenging multiple objective planning problem, in which the trade-off between different objectives must be considered. METHODS: We propose a novel method to solve the multiple objective planning problem, which combines the set cover-based model and Pareto optimization. The set cover-based model considers multiple clinical constraints and generates several clinically feasible treatment plans, among which the Pareto optimization is performed to find the trade-off between different objectives. RESULTS: We evaluated the proposed method on 20 tumors of 11 patients in two different situations used in common thermal ablation approaches: with and without the pull-back technique. Pareto optimal plans were found and verified to be clinically acceptable in all cases, which can find the trade-off between the number of electrodes and the damage to the surrounding tissues. CONCLUSION: The proposed method performs well in the two different situations we considered: with or without the pull-back technique. It can generate Pareto optimal plans satisfying multiple clinical constraints. These plans consider the trade-off between different planning objectives.

TRIM35 mediates protection against influenza infection by activating TRAF3 and degrading viral PB2.

Tripartite motif (TRIM) family proteins are important effectors of innate immunity against viral infections. Here we identified TRIM35 as a regulator of TRAF3 activation. Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon (IFN) in response to viral infection. Trim35-deficient mice were more susceptible to influenza A virus (IAV) infection than were wild-type mice. TRIM35 promoted the RIG-I-mediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1. IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3. TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2, thereby antagonizing its suppression of TRAF3 activation. Our in vitro and in vivo findings thus reveal novel roles of TRIM35, through catalyzing Lys63- or Lys48-linked polyubiquitination, in RIG-I antiviral immunity and mechanism of defense against IAV infection.

Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) causes the infectious disease COVID-19 (coronavirus disease 2019), which was first reported in Wuhan, China, in December 2019. Despite extensive efforts to control the disease, COVID-19 has now spread to more than 100 countries and caused a global pandemic. SARS-CoV-2 is thought to have originated in bats; however, the intermediate animal sources of the virus are unknown. In this study, we investigated the susceptibility of ferrets and animals in close contact with humans to SARS-CoV-2. We found that SARS-CoV-2 replicates poorly in dogs, pigs, chickens, and ducks, but ferrets and cats are permissive to infection. Additionally, cats are susceptible to airborne transmission. Our study provides insights into the animal models for SARS-CoV-2 and animal management for COVID-19 control.

Knockdown Rab11-FIP2 inhibits migration and invasion of nasopharyngeal carcinoma via suppressing Rho GTPase signaling.

Rab11 family interacting protein 2 (Rab11-FIP2) is a conserved protein and effector molecule for the small GTPase Rab11. By interacting with Rab11 and MYO5B, Rab11-FIP2 regulates endosome trafficking of plasma membrane proteins, promoting cellular motility. The endosomal trafficking system in nasopharyngeal carcinoma (NPC) remains unclear. Here, an outlier analysis using the Oncomine database suggested that Rab11-FIP2 but not Rab11 and MYO5B was overexpressed in NPC. We confirmed that the transcription of Rab11-FIP2 was upregulated in NPC cell lines and primary tumor tissues as compared with a normal nasopharyngeal epithelial cell line and normal nasopharynx tissues. We further confirmed the elevated protein expression level of Rab11-FIP2 in NPC biopsies. Instead of regulating the epithelial-mesenchymal transition or Akt signaling pathway, knockdown of Rab11-FIP2 inhibited the migration and invasion ability of NPC cell lines by decreasing the expression of Rac and Cdc42. In summary, Rab11-FIP2 could be an oncogene in NPC, mainly contributing to metastatic capacity by activating Rho GTPase signaling.

Automatic Radiofrequency Ablation Planning for Liver Tumors With Multiple Constraints Based on Set Covering.

Radiofrequency ablation (RFA) is now a widely used minimally invasive treatment method for hepatic tumors. Preoperative planning plays a vital role in RFA therapy. With increasing tumor size, multiple overlapping ablations are needed, which are challenging to optimize while considering clinical constraints. In this paper, we present a new automatic RFA planning method. First, a 2-steps set cover-based model is formulated, which can integrate multiple clinical constraints for optimization of overlapping ablations. To ensure that the planning model can be solved in a reasonable time, a search space reducing strategy is then proposed. We also developed an algorithm for automatic RFA electrode selection, which provides a proper electrode ablation zone for the planning model. The proposed method was evaluated with 20 tumors of varying sizes (0.92 cm3 to 28.4 cm3). Results showed that the proposed method can generate clinical feasible RFA plans with a minimum number of RFA electrodes and ablations, complete tumor coverage and minimized ablation of normal tissue.

The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry.

Influenza A virus (IAV) coopts numerous host factors to complete its replication cycle. Here, we identify free fatty acid receptor 2 (FFAR2) as a cofactor for IAV entry into host cells. We found that downregulation of FFAR2 or Ffar2 expression significantly reduced the replication of IAV in A549 or RAW 264.7 cells. The treatment of A549 cells with small interfering RNA (siRNA) targeting FFAR2 or the FFAR2 pathway agonists 2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide (4-CMTB) and compound 58 (Cmp58) [(S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide] dramatically inhibited the nuclear accumulation of viral nucleoprotein (NP) at early time points postinfection, indicating that FFAR2 functions in the early stage of the IAV replication cycle. FFAR2 downregulation had no effect on the expression of sialic acid (SA) receptors on the cell membrane, the attachment of IAV to the SA receptors, or the activity of the viral ribonucleoprotein (vRNP) complex. Rather, the amount of internalized IAVs was significantly reduced in FFAR2-knocked-down or 4-CMTB- or Cmp58-treated A549 cells. Further studies showed that FFAR2 associated with ß-arrestin1 and that ß-arrestin1 interacted with the ß2-subunit of the AP-2 complex (AP2B1), the essential adaptor of the clathrin-mediated endocytosis pathway. Notably, siRNA knockdown of either ß-arrestin1 or AP2B1 dramatically impaired IAV replication, and AP2B1 knockdown or treatment with Barbadin, an inhibitor targeting the ß-arrestin1/AP2B1 complex, remarkably decreased the amount of internalized IAVs. Moreover, we found that FFAR2 interacted with three G protein-coupled receptor (GPCR) kinases (i.e., GRK2, GRK5, and GRK6) whose downregulation inhibited IAV replication. Together, our findings demonstrate that the FFAR2 signaling cascade is important for the efficient endocytosis of IAV into host cells.IMPORTANCE To complete its replication cycle, IAV hijacks the host endocytosis machinery to invade cells. However, the underlying mechanisms of how IAV is internalized into host cells remain poorly understood, emphasizing the need to elucidate the role of host factors in IAV entry into cells. In this study, we identified FFAR2 as an important host factor for the efficient replication of both low-pathogenic and highly pathogenic IAV. We revealed that FFAR2 facilitates the internalization of IAV into target cells during the early stage of infection. Upon further characterization of the role of FFAR2-associated proteins in virus replication, we found that the FFAR2-ß-arrestin1-AP2B1 signaling cascade is important for the efficient endocytosis of IAV. Our findings thus further our understanding of the biological details of IAV entry into host cells and establish FFAR2 as a potential target for antiviral drug development.

Low Polymerase Activity Attributed to PA Drives the Acquisition of the PB2 E627K Mutation of H7N9 Avian Influenza Virus in Mammals.

Avian influenza viruses (AIVs) must acquire mammalian-adaptive mutations before they can efficiently replicate in and transmit among humans. The PB2 E627K mutation is known to play a prominent role in the mammalian adaptation of AIVs. The H7N9 AIVs that emerged in 2013 in China easily acquired the PB2 E627K mutation upon replication in humans. Here, we generate a series of reassortant or mutant H7N9 AIVs and test them in mice. We show that the low polymerase activity attributed to the viral PA protein is the intrinsic driving force behind the emergence of PB2 E627K during H7N9 AIV replication in mice. Four residues in the N-terminal region of PA are critical in mediating the PB2 E627K acquisition. Notably, due to the identity of viral PA protein, the polymerase activity and growth of H7N9 AIV are highly sensitive to changes in expression levels of human ANP32A protein. Furthermore, the impaired viral polymerase activity of H7N9 AIV caused by the depletion of ANP32A led to reduced virus replication in Anp32a-/- mice, abolishing the acquisition of the PB2 E627K mutation and instead driving the virus to acquire the alternative PB2 D701N mutation. Taken together, our findings show that the emergence of the PB2 E627K mutation of H7N9 AIV is driven by the intrinsic low polymerase activity conferred by the viral PA protein, which also involves the engagement of mammalian ANP32A.IMPORTANCE The emergence of the PB2 E627K substitution is critical in the mammalian adaptation and pathogenesis of AIV. H7N9 AIVs that emerged in 2013 possess a prominent ability in gaining the PB2 E627K mutation in humans. Here, we demonstrate that the acquisition of the H7N9 PB2 E627K mutation is driven by the low polymerase activity conferred by the viral PA protein in human cells, and four PA residues are collectively involved in this process. Notably, the H7N9 PA protein leads to significant dependence of viral polymerase function on human ANP32A protein, and Anp32a knockout abolishes PB2 E627K acquisition in mice. These findings reveal that viral PA and host ANP32A are crucial for the emergence of PB2 E627K during adaptation of H7N9 AIVs to humans.