Platform establishment of the Cre-loxP recombination system for genetic manipulation of the Lumpy skin disease virus.

Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in Asia, including China. Genetic manipulation of the LSDV is essential for the elucidation of the pathogenic mechanism and biological function of the LSDV-encoded protein. In this study, we established a platform for the Cre-loxP recombination system under a modified early-late H5 promoter of the VACV for quick construction of the recombinant LSDV virus. The recombinant virus, LSDV-EGFP-ΔTK, was purified and obtained using serial limited dilution and picking the single cells methods. Using the lentiviral package system, a Cre recombinase enzyme stable expression MDBK cell line was established to supply the Cre recombinase for the reporter gene excision. A genetically stable, safe TK gene-deleted LSDV (LSDV-ΔTK) was constructed using homologous recombination and the Cre-loxP system. It was purified using limited dilution in the MDBK-Cre cell line. Establishing the Cre-loxP recombination system will enable sequential deletion of the interested genes from the LSDV genome and genetic manipulation of the LSDV genome, providing technical support and a platform for developing the attenuated LSDV vaccine.

Identification of the murine osteoblastic cell MC3T3-E1 as a permissive cell line in response to lumpy skin disease virus.

Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in China. Screening suitable cells for LSDV replication is vital for future research on pathogenic mechanisms and vaccine development. Previous comparative studies have identified that the rodent-derived BHK21 is a highly susceptible cell model to LSDV infection. Using western blot, indirect immune-fluorescence assay, flow cytometry, and transmission electron microscopy methods, this study is the first to identify the murine osteoblastic cell line MC3T3-E1 as a novel permissive cell model for LSDV infection. The establishment of MC3T3-E1 as a suitable infectious cell model enhances our understanding of the species range and cell types of the permissive cells and nonpermissive that support LSDV replication. It is helpful to accelerate future research on the pathogenesis, clinical application, and vaccine development of LSDV.

Foot-and-Mouth Disease Virus Capsid Protein VP1 Antagonizes Type I Interferon Signaling via Degradation of Histone Deacetylase 5.

Foot-and-mouth disease (FMD) is a highly contagious and economically important disease of cloven-hoofed animals that hampers trade and production. To ensure effective infection, the foot-and-mouth disease virus (FMDV) evades host antiviral pathways in different ways. Although the effect of histone deacetylase 5 (HDAC5) on the innate immune response has previously been documented, the precise molecular mechanism underlying HDAC5-mediated FMDV infection is not yet clearly understood. In this study, we found that silencing or knockout of HDAC5 promoted FMDV replication, whereas HDAC5 overexpression significantly inhibited FMDV propagation. IFN-ß and IFN-stimulated response element (ISRE) activity was strongly activated through the overexpression of HDAC5. The silencing and knockout of HDAC5 led to an increase in viral replication, which was evident by decreased IFN-ß, ISG15, and ISG56 production, as well as a noticeable reduction in IRF3 phosphorylation. Moreover, the results showed that the FMDV capsid protein VP1 targets HDAC5 and facilitates its degradation via the proteasomal pathway. In conclusion, this study highlights that HDAC5 acts as a positive modulator of IFN-ß production during viral infection, while FMDV capsid protein VP1 antagonizes the HDAC5-mediated antiviral immune response by degrading HDAC5 to facilitate viral replication.

Lumpy skin disease virus ORF127 protein suppresses type I interferon responses by inhibiting K63-linked ubiquitination of tank binding kinase 1.

Lumpy skin disease (LSD) is a severe animal infectious disease caused by lumpy skin disease virus (LSDV), inducing extensive nodules on the cattle mucosa or the scarfskin. LSDV genome encodes multiple proteins to evade host innate immune response. However, the underlying molecular mechanisms are poorly understood. In this study, we found that LSDV could suppress the expression of IFN-ß and interferon-stimulated genes (ISGs) in MDBK cells during the early stage of infection. Subsequently, an unbiased screen was performed to screen the LSDV genes with inhibitory effects on the type I interferon (IFN-I) production. ORF127 protein was identified as one of the strongest inhibitory effectors on the expression of IFN-ß and ISGs, meanwhile, the 1-43 aa of N-terminal of ORF127 played a vital role in suppressing the expression of IFN-ß. Overexpression of ORF127 could significantly promote LSDV replication through inhibiting the production of IFN-ß and ISGs in MDBK cells. Mechanism study showed that ORF127 specifically interacted with TBK1 and decreased the K63-linked polyubiquitination of TBK1 which suppressed the phosphorylation of TBK1 and ultimately decreased the production of IFN-ß. In addition, truncation mutation analysis indicated that the 1-43 aa of N-terminal of ORF127 protein was the key structural domain for its interaction with TBK1. In short, these results validated that ORF127 played a negative role in regulating IFN-ß expression through cGAS-STING signaling pathway. Taken together, this study clarified the molecular mechanism of ORF127 gene antagonizing IFN-I-mediated antiviral, which will helpfully provide new strategies for the treatment and prevention of LSD.

TMP269, a small molecule inhibitor of class IIa HDAC, suppresses RABV replication in vitro.

TMP269, a small molecular inhibitor of IIa histone deacetylase, plays a vital role in cancer therapeutic. However, the effect of TMP269 on the regulation of viral replication has not been studied. In the present study, we found that TMP269 treatment significantly inhibited RABV replication at concentrations without significant cytotoxicity in a dose-dependent manner. In addition, TMP269 can reduce the viral titers and protein levels of RABV at an early stage in the viral life cycle. RNA sequencing data revealed that immune-related pathways and autophagy-related genes were significantly downregulated after RABV infection treated with TMP269. Further exploration shows that autophagy enhances RABV replication in HEK-293T cells, while TMP269 can inhibit autophagy to decrease RABV replication. Together, these results provide a novel treatment strategy for rabies.

Phylogenetic and pathogenic characterization of lumpy skin disease virus circulating in China.

The genomic characterization of emerging pathogens is critical for unraveling their origin and tracking their dissemination. Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in Asia including China. Although the first Lumpy skin disease (LSD) outbreak was reported in 2019, the origin, transmission, and evolutionary trajectory of LSDV in China have remained obscure. The viral genome of a circulating LSDV strain in China, abbreviated LSDV/FJ/CHA/2021, was sequenced using the next-generation sequencing technique. The morphology and cytoplasmic viral factory of these LSDV isolates were observed using transmission electron microscopy. Subsequently, the genomic characterization of this LSDV isolate was systematically analyzed for the first time using the bioinformatics software. The current study revealed that several mutations in the genome of LSDV isolates circulating in China were identified using single nucleotide polymorphisms (SNPs) analysis, an instrument to evaluate for continuous adaptive evaluation of a virus. Furthermore, phylogenomic analysis was used to identify the lineage using the whole genome sequences of 44 LSDV isolates. The result revealed that the isolates from China were closely similar to that of the LSDV isolates from Vietnam, which are divided into a monophyletic lineage sub-group I. The SNPs and Simplot analysis indicate no significant occurrence of the recombinant event on the genome of LSDV isolates in China. Notably, the live virus challenge experiment demonstrated that the pathogenic characterization of this LSDV isolate belongs to a virulent strain. Collectively, we gain the first insight into the evolutionary trajectory, spatiotemporal transmission, and pathogenic characterization of circulating LSDV in China. This study provides a unique reference for risk assessment, guiding diagnostics, and prevention in epizootic and non-epizootic areas.

The comparative study revealed that the hTERT-CSF cell line was the most susceptible cell to the Lumpy skin disease virus infection among eleven cells.

Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in Asia, including China. Improving the propagation of LSDV is important for diagnostics and vaccine production. Our study identified and compared the LSDV susceptibility of eleven standard cells using western blot, indirect immune-fluorescence assay, quantitative PCR, and 50 % tissue culture infectious dose. Our finding revealed that the LSDV strain could infect five cell lines and show a cytopathic effect. Furthermore, the hTERT-CSF cell line had the highest level of virus in the five cell models, followed by BHK-21, MDBK, Vero, and hTERT-ST. Hence, hTERT-CSF could be used as a candidate cell line for basic and applied research, clinical application, and LSDV vaccine development, providing a vital reference in LSDV and other viruses.

9-Butyl-Harmol Exerts Antiviral Activity against Newcastle Disease Virus through Targeting GSK-3ß and HSP90ß.

The paramyxoviruses represent a large family of human and animal pathogens that cause significant health and economic burdens worldwide. However, there are no available drugs against the virus. ß-carboline alkaloids are a family of naturally occurring and synthetic products with outstanding antiviral activities. Here, we examined the antiviral effect of a series of ß-carboline derivatives against several paramyxoviruses, including Newcastle disease virus (NDV), peste des petits ruminants virus (PPRV), and canine distemper virus (CDV). Among these derivatives, 9-butyl-harmol was identified as an effective antiviral agent against these paramyxoviruses. Further, a genome-wide transcriptome analysis in combination with target validation strategies reveals a unique antiviral mechanism of 9-butyl-harmol through the targeting of GSK-3ß and HSP90ß. On one hand, NDV infection blocks the Wnt/ß-catenin pathway to suppress the host immune response. 9-butyl-harmol targeting GSK-3ß dramatically activates the Wnt/ß-catenin pathway, which results in the boosting of a robust immune response. On the other hand, NDV proliferation depends on the activity of HSP90. The L protein, but not the NP protein or the P protein, is proven to be a client protein of HSP90ß, rather than HSP90α. 9-butyl-harmol targeting HSP90ß decreases the stability of the NDV L protein. Our findings identify 9-butyl-harmol as a potential antiviral agent, provide mechanistic insights into the antiviral mechanism of 9-butyl-harmol, and illustrate the role of ß-catenin and HSP90 during NDV infection. IMPORTANCE Paramyxoviruses cause devastating impacts on health and the economy worldwide. However, there are no suitable drugs with which to counteract the viruses. We determined that 9-butyl-harmol could serve as a potential antiviral agent against paramyxoviruses. Until now, the antiviral mechanism of ß-carboline derivatives against RNA viruses has rarely been studied. Here, we found that 9-butyl-harmol exerts dual mechanisms of antiviral action, with its antiviral activities being mediated by two targets: GSK-3ß and HSP90ß. Correspondingly, the interaction between NDV infection and the Wnt/ß-catenin pathway or HSP90 is demonstrated in this study. Taken together, our findings shed light on the development of antiviral agents against paramyxoviruses, based on the ß-carboline scaffold. These results present mechanistic insights into the polypharmacology of 9-butyl-harmol. Understanding this mechanism also deepens the host-virus interaction and reveals new drug targets for anti-paramyxoviruses.

The 16S rRNA Gene Sequencing of Gut Microbiota in Chickens Infected with Different Virulent Newcastle Disease Virus Strains.

Newcastle disease virus (NDV) which is pathogenic to chickens is characterized by dyspnea, diarrhea, nervous disorder and hemorrhages. However, the influence of different virulent NDV strain infection on the host gut microbiota composition is still poorly understood. In this study, twenty 21-day-old specific pathogen free (SFP) chickens were inoculated with either the velogenic Herts33 NDV strain, lentogenic La Sota NDV strain or sterile phosphate buffer solution (PBS). Subsequently, the fecal samples of each group were collected for 16S rRNA sequencing. The results showed that the gut microbiota were mainly dominated by Firmicutes, Bacteroidetes and Proteobacteria in both healthy and NDV infected chickens. NDV infection altered the structure and composition of gut microbiota. As compared to the PBS group, phylum Firmicutes were remarkably reduced, whereas Proteobacteria was significantly increased in the velogenic NDV infected group; the gut community structure had no significant differences between the lentogenic NDV infected group and the PBS group at phylum level. At genus level, Escherichia-Shigella was significantly increased in both the velogenic and lentogenic NDV infected groups, but the lactobacillus was only remarkably decreased in the velogenic NDV infected group. Collectively, different virulent strain NDV infection resulted in a different alteration of the gut microbiota in chickens, including a loss of probiotic bacteria and an expansion of some pathogenic bacteria. These results indicated that NDV strains with different virulence have different impacts on chicken gut microbiota and may provide new insights into the intestinal pathogenesis of NDV.

Establishment of a fetal cow (Bos Borus) skin fibroblasts cell line with immortalized characterization through human telomerase reverse transcriptase (hTERT) ectopic expression.

The ectopic introduction of the human telomerase reverse transcriptase (hTERT) is an effective way to establish an immortalized cell line. Here, hTERT was obtained by RT-PCR, and the eukaryotic expression plasmid and lentivirus shuttle plasmid of hTERT was successfully constructed by the homologous recombination method. The stable expression of hTERT in fetal cow skin fibroblasts (CSF) was established using the lentivirus package system. The hTERT-CSF proliferate and have immortalized characteristics. Meanwhile, the chromosome analysis identified that the number and structure of the hTERT-CSF genome maintain stable. The indirect immunofluorescence, western blot, and flow cytometry showed that the hTERT gene had been successfully integrated into the primary genome of bovine skin and stably expressed. The viral infection experiment first identifies the hTERT-CSF as a vulnerable cell model responding to the Lumpy skin disease virus (LSDV). Establishing hTERT-CSF provides an important cell model for basic and applied research, clinical application, and vaccine development. It provides an essential reference for the future's rapid establishment of other immortalized cell lines.

Molecular characterization, expression, and functional identification of TANK-binding kinase 1 (TBK1) of the cow (Bos taurus) and goat (Capra hircus).

The role of TANK-binding kinase 1 (TBK1) of humans and mice in innate immunity is well elucidated. Still, the molecular characterization and biological function of the TBK1 gene in herbivorous animals are less studied. Here, the open reading frame (ORF) of TBK1 of the cow and goat was firstly cloned and successfully expressed. The Phylogenetic tree analysis reveals that the TBK1 gene of goats and cows is similar to chicken and mute swans, respectively. Some evolutionary distances of the TBK1 gene were still present among different species. A slightly subcellular distribution difference was observed among full-length and truncated TBK1 of goats and cows. Dual-luciferase reporter assay has shown that the full-length TBK1 of goats and cows plays a vital role in the induction of IFN-ß production. The viral infection experiment showed that the over-expression of the full-length TBK1 gene of the cow and goat significantly suppresses intracellular viral replication of the Lumpy skin disease virus (LSDV) in infected cells. Our study showed that TBK1 in the cows and goats is a crucial immunoregulatory for IFN-ß production during viral infection, contributing to a better understanding of innate immunity in the herbivorous animal.

Knockout of HDAC9 Gene Enhances Foot-and-Mouth Disease Virus Replication.

Foot-and-mouth disease virus (FMDV) is a highly contagious viral disease that mainly infects cloven-hoofed animals. Propagation of FMDV by cell culture is an important method to preserve viral biological and antigenic characteristics, which is crucial in FMD monitoring and vaccine production. However, only a few cell lines are sensitive to FMDV, and there is still a lot of room for improvement. Acetylation is an important post-translational modification, which is dynamically regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). However, the study of the relationship between FMDV and HDACs is still unclear. HDAC9 belongs to the class II of HDACs family; in this study, HDAC9 knockout (KO) BHK-21 cells were successfully established using CRISPR/cas9 technology. The results of karyotype analysis, growth curve analysis, and morphological observation showed that the HDAC9 knockout cell line was stable in growth and morphological characteristics. After infection with FMDV, the expression of viral RNA and protein, viral titers, and the copies of viral RNA in HDAC9-KO cells were significantly higher than those in NC cells. Meanwhile, RNA-seq technology was used to sequence HDAC9-KO cells and NC cells infected and uninfected with FMDV. It was found that the differentially expressed innate immune factors containing NFKBIA, SOD2, IL2RG, BCL2L1, CXCL1/2/3, and IL1RAP have significantly enriched in the Jak-STAT, NOD-like receptor, Toll-like receptor, NF-κB, and MAPK signaling pathway. RT-qPCR was performed to detect the expression level of differentially expressed genes and showed consistency with the RNA-seq data. These results preliminarily reveal the role of HDAC9 in host antiviral innate immune response, and the HDAC9-KO cell line could also serve as a useful tool for FMDV research.

ORF3a-Mediated Incomplete Autophagy Facilitates Severe Acute Respiratory Syndrome Coronavirus-2 Replication.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent for the coronavirus disease 2019 (COVID-19) pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin 1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 26 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate PI3KC3-C1 (Beclin-1-Vps34-Atg14) but selectively inhibit PI3KC3-C2 (Beclin-1-Vps34-UVRAG). Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raise the possibility of targeting the autophagic pathway for the treatment of COVID-19.

Newcastle Disease Virus Induced Pathologies Severely Affect the Exocrine and Endocrine Functions of the Pancreas in Chickens.

Newcastle disease virus (NDV) causes a highly contagious and devastating disease in poultry. ND causes heavy economic losses to the global poultry industry by decreasing the growth rate, decrease in egg production high morbidity and mortality. Although significant advances have been made in the vaccine development, outbreaks are reported in vaccinated birds. In this study, we report the damage caused by NDV infection in the pancreatic tissues of vaccinated and specific-pathogen-free chickens. The histopathological examination of the pancreas showed severe damage in the form of partial depletion of zymogen granules, acinar cell vacuolization, necrosis, apoptosis, congestion in the large and small vessels, sloughing of epithelial cells of the pancreatic duct, and mild perivascular edema. Increased plasma levels of corticosterone and somatostatin were observed in NDV-infected chicken at three- and five- days post infection (DPI). A slight decrease in the plasma concentrations of insulin was noticed at 5 DPI. Significant changes were not observed in the plasma levels of glucagon. Furthermore, NDV infection decreased the activity and mRNA expression of amylase, lipase, and trypsin from the pancreas. Taken together, our findings highlight that NDV induces extensive tissue damage in the pancreas, decreases the activity and expression of pancreatic enzymes, and increases plasma corticosterone and somatostatin. These findings provide new insights that a defective pancreas may be one of the reasons for decreased growth performance after NDV infection in chickens.

Identification of Newcastle disease virus P-gene editing using next-generation sequencing.

Avian paramyxoviruses 1 has the ability to edit its P gene to generate three amino-coterminal proteins (P, V and W), but its kinetic change is unclear. In this study, next-generation sequencing (NGS) was used to analyze the P-gene editing of Newcastle disease virus (NDV). Transcriptome analysis of chicken embryonic tissues and bursa of fabricius showed the P-gene editing frequencies were 45.46-52.70%. To investigate the rules of P-gene editing along time, the ratio of PVW was determined by PCR based deep sequencing at multiple time points in cells infected with velogenic and lentogenic strain respectively. The results confirmed similar editing frequencies with transcriptome data and the PVW ratios were stable along time among different NDVs, but had a greater V-gene transcript on velogenic strain infection (P<0.001), which were different from previous reports. Also, it was shown that the number of inserted G residues in P-derived transcripts was not limited to +9G, and +10G transcripts were identified. These results confirmed the NDV P-gene editing frequencies and provided a novel point of view on NDV P-gene editing with NDV virulence.

Newcastle disease virus induces testicular damage and disrupts steroidogenesis in specific pathogen free roosters.

Newcastle disease (ND), which is caused by Newcastle disease virus (NDV), can cause heavy economic losses to the poultry industry worldwide. It is characterised by extensive pathologies of the digestive, respiratory, and nervous systems and can cause severe damage to the reproductive system of egg-laying hens. However, it is unknown whether NDV replicates in the male reproductive system of chickens and induces any pathologies. In this study, we selected a representative strain (i.e. ZJ1) of the most common genotype (i.e. VII) of NDV to investigate whether NDV can induce histological, hormonal, and inflammatory responses in the testes of specific pathogen free (SPF) roosters. NDV infection increased the expression of toll like receptor TLR3, TLR7, MDA5, IFN-α, IFN-ß, IFN-γ, IL-8, and CXCLi1 in the testes of NDV-infected roosters at 5 days post-infection (dpi). Severe histological changes, including decrease in the number of Sertoli cells and individualized, shrunken spermatogonia with pyknotic nuclei, were observed at 3 dpi. At 5 dpi, the spermatogenic columns were disorganized, and there were fewer cells, which were replaced by necrotic cells, lipid vacuoles, and proteinaceous homogenous material. A significant decrease in the plasma concentrations of testosterone and luteinizing hormone (LH) and the mRNA expression of their receptors in the testes, steroidogenic acute regulatory protein, cytochrome P450 side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase in the NDV-infected group was observed relative to those in the control group (P < 0.05). Collectively, these results indicate that NDV infection induces a severe inflammatory response and histological changes, which decrease the steroidogenesis.

ATM-mediated DNA double-strand break response facilitated oncolytic Newcastle disease virus replication and promoted syncytium formation in tumor cells.

Deoxyribonucleic acid (DNA) damage response (DDR) is the fundamental cellular response for maintaining genomic integrity and suppressing tumorigenesis. The activation of ataxia telangiectasia-mutated (ATM) kinase is central to DNA double-strand break (DSB) for maintaining host-genome integrity in mammalian cells. Oncolytic Newcastle disease virus (NDV) can selectively replicate in tumor cells; however, its influence on the genome integrity of tumor cells is not well-elucidated. Here, we found that membrane fusion and NDV infection triggered DSBs in tumor cells. The late replication and membrane fusion of NDV mechanistically activated the ATM-mediated DSB pathway via the ATM-Chk2 axis, as evidenced by the hallmarks of DSBs, i.e., auto-phosphorylated ATM and phosphorylated H2AX and Chk2. Immunofluorescence data showed that multifaceted ATM-controlled phosphorylation markedly induced the formation of pan-nuclear punctum foci in response to NDV infection and F-HN co-expression. Specific drug-inhibitory experiments on ATM kinase activity further suggested that ATM-mediated DSBs facilitated NDV replication and membrane fusion. We confirmed that the Mre11-RAD50-NBS1 (MRN) complex sensed the DSB signal activation triggered by NDV infection and membrane fusion. The pharmacological inhibition of MRN activity also significantly inhibited intracellular and extracellular NDV replication and syncytia formation. Collectively, these data identified for the first time a direct link between the membrane fusion induced by virus infection and DDR pathways, thereby providing new insights into the efficient replication of oncolytic NDV in tumor cells.

Morphology Remodeling and Selective Autophagy of Intracellular Organelles during Viral Infections.

Viruses have evolved different strategies to hijack subcellular organelles during their life cycle to produce robust infectious progeny. Successful viral reproduction requires the precise assembly of progeny virions from viral genomes, structural proteins, and membrane components. Such spatial and temporal separation of assembly reactions depends on accurate coordination among intracellular compartmentalization in multiple organelles. Here, we overview the rearrangement and morphology remodeling of virus-triggered intracellular organelles. Focus is given to the quality control of intracellular organelles, the hijacking of the modified organelle membranes by viruses, morphology remodeling for viral replication, and degradation of intracellular organelles by virus-triggered selective autophagy. Understanding the functional reprogram and morphological remodeling in the virus-organelle interplay can provide new insights into the development of broad-spectrum antiviral strategies.

Hemagglutinin-neuraminidase and fusion proteins of virulent Newcastle disease virus cooperatively disturb fusion-fission homeostasis to enhance mitochondrial function by activating the unfolded protein response of endoplasmic reticulum and mitochondrial stress.

The fusogenically activated F and HN proteins of virulent NDV induce complete autophagic flux in DF-1 and A549 cells. However, the effect of both glycoproteins on mitochondria remains elusive. Here, we found that F and HN cooperation increases mitochondrial biogenesis but does not cause the mitochondria damage. We observed that both glycoproteins change the morphological characteristics and spatial distribution of intracellular mitochondria. F and HN cooperate cooperatively to induce ER stress and UPRmt. Our preliminary data suggested that F and HN cooperatively disturb mitochondrial fusion-fission homeostasis to enhance mitochondrial biogenesis, and eventually meet the energy demand of syncytium formation.