Transcriptome analysis reveals molecular mechanisms of lymphocystis formation caused by lymphocystis disease virus infection in flounder (Paralichthys olivaceus).

Lymphocystis disease is frequently prevalent and transmissible in various teleost species worldwide due to lymphocystis disease virus (LCDV) infection, causing unsightly growths of benign lymphocystis nodules in fish and resulting in huge economic losses to aquaculture industry. However, the molecular mechanism of lymphocystis formation is unclear. In this study, LCDV was firstly detected in naturally infected flounder (Paralichthys olivaceus) by PCR, histopathological, and immunological techniques. To further understand lymphocystis formation, transcriptome sequencing of skin nodule tissue was performed by using healthy flounder skin as a control. In total, RNA-seq produced 99.36%-99.71% clean reads of raw reads, of which 91.11%-92.89% reads were successfully matched to the flounder genome. The transcriptome data showed good reproducibility between samples, with 3781 up-regulated and 2280 down-regulated differentially expressed genes. GSEA analysis revealed activation of Wnt signaling pathway, Hedgehog signaling pathway, Cell cycle, and Basal cell carcinoma associated with nodule formation. These pathways were analyzed to interact with multiple viral infection and tumor formation pathways. Heat map and protein interaction analysis revealed that these pathways regulated the expression of cell cycle-related genes such as ccnd1 and ccnd2 through key genes including ctnnb1, lef1, tcf3, gli2, and gli3 to promote cell proliferation. Additionally, cGMP-PKG signaling pathway, Calcium signaling pathway, ECM-receptor interaction, and Cytokine-cytokine receptor interaction associated with nodule formation were significantly down-regulated. Among these pathways, tnfsf12, tnfrsf1a, and tnfrsf19, associated with pro-apoptosis, and vdac2, which promotes viral replication by inhibiting apoptosis, were significantly up-regulated. Visual analysis revealed significant down-regulation of cytc, which expresses the pro-apoptotic protein cytochrome C, as well as phb and phb2, which have anti-tumor activity, however, casp3 was significantly up-regulated. Moreover, bcl9, bcl11a, and bcl-xl, which promote cell proliferation and inhibit apoptosis, were significantly upregulated, as were fgfr1, fgfr2, and fgfr3, which are related to tumor formation. Furthermore, RNA-seq data were validated by qRT-PCR, and LCDV copy numbers and expression patterns of focused genes in various tissues were also investigated. These results clarified the pathways and differentially expressed genes associated with lymphocystis nodule development caused by LCDV infection in flounder for the first time, providing a new breakthrough in molecular mechanisms of lymphocystis formation in fish.

Peripheral Blood B-Lymphocytes Are Involved in Lymphocystis Disease Virus Infection in Flounder (Paralichthys olivaceus) via Cellular Receptor-Mediated Mechanism.

Previous studies imply that peripheral blood leukocytes (PBLs) may play an important role in systemic lymphocystis disease virus (LCDV) dissemination, but whether the PBLs are susceptible and permissive to LCDV infection and the dissemination mechanism need to be clarified. In this study, LCDV was firstly confirmed to infect the PBLs in flounder (Paralichthys olivaceus) in vivo, and to replicate in PBLs in vitro. Subsequently, the 27.8 kDa receptor protein (27.8R), a functional receptor mediating LCDV infection in flounder gill cells, was shown to locate on the cell membrane of PBLs and co-localize with LCDV in PBLs, while blocking of the 27.8R via pre-incubation of anti-27.8R MAb with the PBLs could obviously inhibit LCDV infection, revealing the 27.8R as a receptor for LCDV entry into PBLs. Multicolor fluorescence imaging studies verified that IgM+ and IgD+ B-lymphocyte were involved in LCDV infection. In the sorted IgM+ B-cells, 27.8R+ and LCDV+ signals were simultaneously observed, and LCDV copy numbers increased with time, indicating that IgM+ B-cells expressed the 27.8R and were permissive to LCDV infection. Furthermore, the dynamic changes of IgM+, 27.8R+, LCDV+ and LCDV+/IgM+ PBLs were monitored during the early phase of LCDV infection. It was found that the percentage of IgM+ B-cells in PBLs clearly declined first and then increased, suggesting LCDV infection facilitated damage to B-cells, whereas the amounts of 27.8R+ and LCDV+ PBLs, as well as LCDV-infected IgM+ B-cells, showed an opposite trend. These results proved that IgM+ B-lymphocytes could be infected by LCDV via a receptor-mediated mechanism and support viral replication, which provided novel insights for the first time into the role of B-lymphocytes in LCDV dissemination and pathogenesis in teleost fish.

Nuclear soluble cGAS senses double-stranded DNA virus infection.

The DNA sensor cGAS detects cytosolic DNA and instigates type I interferon (IFN) expression. Recent studies find that cGAS also localizes in the nucleus and binds the chromatin. Despite the mechanism controlling nuclear cGAS activation is well elucidated, whether nuclear cGAS participates in DNA sensing is unclear. Here, we report that herpes simplex virus 1 (HSV-1) infection caused the release of cGAS from the chromatin into the nuclear soluble fraction. Like its cytosolic counterpart, the leaked nuclear soluble cGAS also sensed viral DNA, produced cGAMP, and induced mRNA expression of type I IFN and interferon-stimulated genes. Consistently, the nuclear soluble cGAS limited HSV-1 infection. Furthermore, enzyme-deficient mutation (D307A) or cGAS inhibitor RU.251 abolished nuclear cGAS-mediated innate immune responses, suggesting that enzymatic activity is also required for nuclear soluble cGAS. Taken all together, our study demonstrates that nuclear soluble cGAS acts as a nuclear DNA sensor detecting nuclear-replicating DNA viruses.

Amphibian (Xenopus laevis) Tadpoles and Adult Frogs Differ in Their Antiviral Responses to Intestinal Frog Virus 3 Infections.

The global amphibian declines are compounded by ranavirus infections such as Frog Virus 3 (FV3), and amphibian tadpoles more frequently succumb to these pathogens than adult animals. Amphibian gastrointestinal tracts represent a major route of ranavirus entry, and viral pathogenesis often leads to hemorrhaging and necrosis within this tissue. Alas, the differences between tadpole and adult amphibian immune responses to intestinal ranavirus infections remain poorly defined. As interferon (IFN) cytokine responses represent a cornerstone of vertebrate antiviral immunity, it is pertinent that the tadpoles and adults of the anuran Xenopus laevis frog mount disparate IFN responses to FV3 infections. Presently, we compared the tadpole and adult X. laevis responses to intestinal FV3 infections. Our results indicate that FV3-challenged tadpoles mount more robust intestinal type I and III IFN responses than adult frogs. These tadpole antiviral responses appear to be mediated by myeloid cells, which are recruited into tadpole intestines in response to FV3 infections. Conversely, myeloid cells bearing similar cytology already reside within the intestines of healthy (uninfected) adult frogs, possibly accounting for some of the anti-FV3 resistance of these animals. Further insight into the differences between tadpole and adult frog responses to ranaviral infections is critical to understanding the facets of susceptibility and resistance to these pathogens.

PCV2 targets cGAS to inhibit type I interferon induction to promote other DNA virus infection.

Viruses use diverse strategies to impair the antiviral immunity of host in order to promote infection and pathogenesis. Herein, we found that PCV2 infection promotes the infection of DNA viruses through inhibiting IFN-ß induction in vivo and in vitro. In the early phase of infection, PCV2 promotes the phosphorylation of cGAS at S278 via activation of PI3K/Akt signaling, which directly silences the catalytic activity of cGAS. Subsequently, phosphorylation of cGAS at S278 can facilitate the K48-linked poly-ubiquitination of cGAS at K389, which can been served as a signal for recognizing by the ubiquitin-binding domain of histone deacetylase 6 (HDAC6), to promote the translocation of K48-ubiquitinated-cGAS from cytosol to autolysosome depending on the deacetylase activity of HDAC6, thereby eventually resulting in a markedly increased cGAS degradation in PCV2 infection-induced autophagic cells relative to Earle's Balanced Salt Solution (EBSS)-induced autophagic cells (a typical starving autophagy). Importantly, we found that PCV2 Cap and its binding protein gC1qR act as predominant regulators to promote porcine cGAS phosphorylation and HDAC6 activation through mediating PI3K/AKT signaling and PKCδ signaling activation. Based on this finding, gC1qR-binding activity deficient PCV2 mutant (PCV2RmA) indeed shows a weakened inhibitory effect on IFN-ß induction and a weaker boost effect for other DNA viruses infection compared to wild-type PCV2. Collectively, our findings illuminate a systematic regulation mechanism by which porcine circovirus counteracts the cGAS-STING signaling pathway to inhibit the type I interferon induction and promote DNA virus infection, and identify gC1qR as an important regulator for the immunosuppression induced by PCV2.

Phosphorylation of Shrimp Tcf by a Viral Protein Kinase WSV083 Suppresses Its Antiviral Effect.

Nuclear DNA-binding TCF proteins, which act as the main downstream effectors of Wnt signaling, are essential for the regulation of cell fate and innate immunity. However, their role during viral infection in shrimp remains unknown. Herein, we demonstrated that Litopenaeus vannamei TCF (LvTcf) acts independently of Lvß-catenin to promote interferon-like protein LvVago1 production, thus mounting the response to WSSV infection. Further, we observed that WSV083, a WSSV serine/threonine protein kinase, bound to LvTcf and phosphorylated it. Phosphorylated LvTcf was then recognized and degraded via the ubiquitin-proteasome pathway. Moreover, mass spectrometry analyses indicated that the T39 and T104 residues of LvTcf were target sites phosphorylated by WSV083. Point mutation analyses suggested that additional sites of LvTcf may undergo phosphorylation via WSV083. Taken together, the current work provides valuable insights into host immunity and viral pathogenesis. LvTcf is not only a modulator of shrimp innate immunity but is also an important target for WSSV immune evasion. Thus, the current findings will help improve disease control in shrimps.

LvCSN5 is involved in WSSV infection via interaction with wsv006.

As an extremely virulent pathogen, white spot syndrome virus (WSSV) greatly threatens shrimp aquaculture worldwide. The interaction between virus and host is important for viral infection. In the present study, a yeast two-hybrid (Y2H) library was constructed to clarify the functions of wsv006, and the interaction between wsv006 and shrimp Litopenaeus vannamei (L. vannamei) was analyzed. Furthermore, we explored the role of the wsv006-interacting molecule L. vannamei COP9 constitutive photomorphogenic-like protein subunit 5 (LvCSN5) in WSSV infection. Y2H assay showed that wsv006 interacted with LvCSN5, and co-immunoprecipitation (Co-IP) assay confirmed such interaction. Multiple alignments of amino acid sequences with other species revealed that the LvCSN5 had high identity with Penaeusmonodon CSN5 (PmCSN5). LvCSN5 was mainly expressed in intestine, eye and hepatopancreas. In addition, the relative expression of LvCSN5 was significantly up-regulated both in intestine and hepatopancreas following the WSSV challenge. Besides, the relative expressions of IE1 and VP28, as well as the viral copy numbers were significantly increased in the LvCSN5-silenced shrimp. Our findings suggested that LvCSN5 was involved in WSSV infection by interacting with wsv006.

Bid is involved in apoptosis induced by Chinese giant salamander iridovirus and contributes to the viral replication in an amphibian cell line.

Bid is a pro-apoptotic BH3-only member of the Bcl-2 superfamily that functions to link the extrinsic apoptotic pathway and the mitochondrial amplification loop of the intrinsic pathway. In this study, the expression and functions of Chinese giant salamander (Andrias davidianus) Bid (AdBid) were investigated. The AdBid cDNA sequence contains an open reading frame (ORF) of 576 nucleotides, encoding a putative protein of 191 aa. AdBid possesses the conserved BH3 interacting domain and shared 34-52% sequence identities with other amphibian Bid. mRNA expression of AdBid was most abundant in muscle. The expression level of AdBid in Chinese giant salamander muscle, kidney and spleen significantly increased after Chinese giant salamander iridovirus (GSIV) infection. Additionally, a plasmid expressing AdBid was constructed and transfected into the Chinese giant salamander muscle cell line (GSM cells). The morphology and cytopathic effect (CPE) and apoptotic process in AdBid over-expressed GSM cells was significantly enhanced during GSIV infection compared with that in control cells. Moreover, a higher level of the virus major capsid protein (MCP) gene copies and protein synthesis was confirmed in the AdBid over-expressed cells. These results indicated that AdBid played a positive role in GSIV induced apoptosis and the viral replication. This study may contribute to the better understanding on the infection mechanism of iridovirus-induced apoptosis.

Fish RIP1 Mediates Innate Antiviral Immune Responses Induced by SGIV and RGNNV Infection.

Receptor interacting protein 1 (RIP1) is an essential sensor of cellular stress, which may respond to apoptosis or cell survival and participate in antiviral pathways. To investigate the roles of fish RIP1 in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, a RIP1 homolog from orange-spotted grouper (Epinephelus coioides) (EcRIP1) was cloned and characterized. EcRIP1 encoded a 679 amino acid protein that shares 83.28% identity with that of Perca flavescens and contained a homologous N-terminal kinase (S-TKc) domain, a RIP isotype interaction motif (RHIM), and a C-terminal domain (DD). EcRIP1 was predominantly detected in immune tissues, and its expression was induced by RGNNV or SGIV infection in vitro. Subcellular localization showed that EcRIP1 was distributed in the cytoplasm with point-like uniform and dot-like aggregation forms. Overexpression of EcRIP1 inhibited SGIV and RGNNV replication and positively regulated the expression levels of interferon (IFN) and IFN-stimulated genes and pro-inflammatory factors. EcRIP1 may interact with grouper tumor necrosis factor receptor type 1-associated DEATH domain protein (EcTRADD) to promote SGIV-induced apoptosis, and interact with grouper Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-ß (EcTRIF) and participate in Myeloid Differentiation Factor 88 (MyD88)-independent toll-like receptor (TLR) signaling. EcRIP1 may also interact with grouper tumor necrosis factor receptor-associated factors (TRAFs) as intracellular linker proteins and mediate the signaling of various downstream signaling pathways, including NF-κB and IFN. These results suggest that EcRIP1 may inhibit SGIV and RGNNV infection by regulating apoptosis and various signaling molecules. Our study offers new insights into the regulatory mechanism of RIP1-related signaling, and provides a novel perspective on fish diseases mediated by RIP1.

Palmitic Acid Promotes Virus Replication in Fish Cell by Modulating Autophagy Flux and TBK1-IRF3/7 Pathway.

Palmitic acid is the most common saturated fatty acid in animals, plants, and microorganisms. Studies highlighted that palmitic acid plays a significant role in diverse cellular processes and viral infections. Accumulation of palmitic acid was observed in fish cells (grouper spleen, GS) infected with Singapore grouper iridovirus (SGIV). The fluctuated content levels after viral infection suggested that palmitic acid was functional in virus-cell interactions. In order to investigate the roles of palmitic acid in SGIV infection, the effects of palmitic acid on SGIV induced cytopathic effect, expression levels of viral genes, viral proteins, as well as virus production were evaluated. The infection and replication of SGIV were increased after exogenous addition of palmitic acid but suppressed after knockdown of fatty acid synthase (FASN), of which the primary function was to catalyze palmitate synthesis. Besides, the promotion of virus replication was associated with the down-regulating of interferon-related molecules, and the reduction of IFN1 and ISRE promotor activities by palmitic acid. We also discovered that palmitic acid restricted TBK1, but not MDA5-induced interferon immune responses. On the other hand, palmitic acid decreased autophagy flux in GS cells via suppressing autophagic degradation, and subsequently enhanced viral replication. Together, our findings indicate that palmitic acid is not only a negative regulator of TBK1-IRF3/7 pathway, but also a suppressor of autophagic flux. Finally, palmitic acid promotes the replication of SGIV in fish cells.

KAT5 acetylates cGAS to promote innate immune response to DNA virus.

The DNA sensor cGMP-AMP synthase (cGAS) senses cytosolic microbial or self DNA to initiate a MITA/STING-dependent innate immune response. cGAS is regulated by various posttranslational modifications at its C-terminal catalytic domain. Whether and how its N-terminal unstructured domain is regulated by posttranslational modifications remain unknown. We identified the acetyltransferase KAT5 as a positive regulator of cGAS-mediated innate immune signaling. Overexpression of KAT5 potentiated viral-DNA-triggered transcription of downstream antiviral genes, whereas a KAT5 deficiency had the opposite effects. Mice with inactivated Kat5 exhibited lower levels of serum cytokines in response to DNA virus infection, higher viral titers in the brains, and more susceptibility to DNA-virus-induced death. Mechanistically, KAT5 catalyzed acetylation of cGAS at multiple lysine residues in its N-terminal domain, which promoted its DNA-binding ability. Our findings suggest that KAT5-mediated cGAS acetylation at its N terminus is important for efficient innate immune response to DNA virus.

Adenosine Receptor Modulates Permissiveness of Baculovirus (Budded Virus) Infection via Regulation of Energy Metabolism in Bombyx mori.

Although the modulation of host physiology has been interpreted as an essential process supporting baculovirus propagation, the requirement of energy supply for host antivirus reactions could not be ruled out. Our present study showed that metabolic induction upon AcMNPV (budded virus) infection of Bombyx mori stimulated virus clearance and production of the antivirus protein, gloverin. In addition, we demonstrated that adenosine receptor signaling (AdoR) played an important role in regulating such metabolic reprogramming upon baculovirus infection. By using a second lepidopteran model, Spodoptera frugiperda Sf-21 cells, we demonstrated that the glycolytic induction regulated by adenosine signaling was a conservative mechanism modulating the permissiveness of baculovirus infection. Another interesting finding in our present study is that both BmNPV and AcMNPV infection cause metabolic activation, but it appears that BmNPV infection moderates the level of ATP production, which is in contrast to a dramatic increase upon AcMNPV infection. We identified potential AdoR miRNAs induced by BmNPV infection and concluded that BmNPV may attempt to minimize metabolic activation by suppressing adenosine signaling and further decreasing the host's anti-baculovirus response. Our present study shows that activation of energy synthesis by adenosine signaling upon baculovirus infection is a host physiological response that is essential for supporting the innate immune response against infection.

Ginkgolic acid inhibits fusion of enveloped viruses.

Ginkgolic acids (GA) are alkylphenol constituents of the leaves and fruits of Ginkgo biloba. GA has shown pleiotropic effects in vitro, including: antitumor effects through inhibition of lipogenesis; decreased expression of invasion associated proteins through AMPK activation; and potential rescue of amyloid-ß (Aß) induced synaptic impairment. GA was also reported to have activity against Escherichia coli and Staphylococcus aureus. Several mechanisms for this activity have been suggested including: SUMOylation inhibition; blocking formation of the E1-SUMO intermediate; inhibition of fatty acid synthase; non-specific SIRT inhibition; and activation of protein phosphatase type-2C. Here we report that GA inhibits Herpes simplex virus type 1 (HSV-1) by inhibition of both fusion and viral protein synthesis. Additionally, we report that GA inhibits human cytomegalovirus (HCMV) genome replication and Zika virus (ZIKV) infection of normal human astrocytes (NHA). We show a broad spectrum of fusion inhibition by GA of all three classes of fusion proteins including HIV, Ebola virus (EBOV), influenza A virus (IAV) and Epstein Barr virus (EBV). In addition, we show inhibition of a non-enveloped adenovirus. Our experiments suggest that GA inhibits virion entry by blocking the initial fusion event. Data showing inhibition of HSV-1 and CMV replication, when GA is administered post-infection, suggest a possible secondary mechanism targeting protein and DNA synthesis. Thus, in light of the strong effect of GA on viral infection, even after the infection begins, it may potentially be used to treat acute infections (e.g. Coronavirus, EBOV, ZIKV, IAV and measles), and also topically for the successful treatment of active lesions (e.g. HSV-1, HSV-2 and varicella-zoster virus (VZV)).

Sp-CBL inhibits white spot syndrome virus replication by enhancing apoptosis in mud crab (Scylla paramamosain).

In mammals, casitas B-lineage lymphoma (CBL) family proteins, a RING-type E3 ubiquitin ligase, are involved in many signal transduction pathways. However, the functions of CBL in invertebrates are not well elucidated. In this study, Sp-CBL containing CBL-N, CBL-2, CBL-3 and RING domains was identified in mud crab Scylla paramamosain. Sp-CBL was widely expressed in all tissues tested and found to be significantly up-regulated in the hemocytes of mud crab challenged by white spot syndrome virus (WSSV). The RNA interference of Sp-CBL increased the copy number of WSSV and declined the apoptosis rate of hemocytes. In addition, Sp-CBL could affect the activities of caspase 3 and the mitochondrial membrane potential. Taken together, the results of this study revealed that Sp-CBL could restrict WSSV proliferation through enhancing the apoptosis of the hemocytes, which would provide a novel insight into the anti-viral response in the innate immunity system of mud crab.

Chinese Giant Salamander (Andrias davidianus) Iridovirus Infection Leads to Apoptotic Cell Death through Mitochondrial Damage, Caspases Activation, and Expression of Apoptotic-Related Genes.

Chinese giant salamander iridovirus (GSIV) is the causative pathogen of Chinese giant salamander (Andrias davidianus) iridovirosis, leading to severe infectious disease and huge economic losses. However, the infection mechanism by GSIV is far from clear. In this study, a Chinese giant salamander muscle (GSM) cell line is used to investigate the mechanism of cell death during GSIV infection. Microscopy observation and DNA ladder analysis revealed that DNA fragmentation happens during GSIV infection. Flow cytometry analysis showed that apoptotic cells in GSIV-infected cells were significantly higher than that in control cells. Caspase 8, 9, and 3 were activated in GSIV-infected cells compared with the uninfected cells. Consistently, mitochondria membrane potential (MMP) was significantly reduced, and cytochrome c was released into cytosol during GSIV infection. p53 expression increased at an early stage of GSIV infection and then slightly decreased late in infection. Furthermore, mRNA expression levels of pro-apoptotic genes participating in the extrinsic and intrinsic pathway were significantly up-regulated during GSIV infection, while those of anti-apoptotic genes were restrained in early infection and then rose in late infection. These results collectively indicate that GSIV induces GSM apoptotic cell death involving mitochondrial damage, caspases activation, p53 expression, and pro-apoptotic molecules up-regulation.

Glutamine Metabolism in Both the Oxidative and Reductive Directions Is Triggered in Shrimp Immune Cells (Hemocytes) at the WSSV Genome Replication Stage to Benefit Virus Replication.

White spot syndrome virus (WSSV) is the causative agent of a shrimp disease that has caused huge global economic losses. Although its pathogenesis remains poorly understood, it has been reported that in the shrimp immune cells (hemocytes) targeted by WSSV, the virus triggers both the Warburg effect and glutamine metabolism at the WSSV genome replication stage (12 h post infection). Glutamine metabolism follows two pathways: an oxidative pathway mediated by α-KGDH (α-ketoglutarate dehydrogenase) and an alternative reductive pathway mediated by IDH1 and IDH2 (isocitrate dehydrogenase 1 and 2). Here we used isotopically labeled glutamine ([U-13C]glutamine and [1-13C]glutamine) as metabolic tracers to show that, at the replication stage, both the oxidative and reductive glutamine metabolic pathways were activated. We further show that the mRNA expression levels of α-KGDH and IDH1 were increased in WSSV-infected shrimps and that silencing of α-KGDH, IDH1, and IDH2 with their respective dsRNAs led to a decrease in WSSV gene expression and WSSV replication. Taken together, our findings provide new evidence for WSSV-induced metabolic reprogramming in hemocytes and demonstrate its importance in virus replication.

Know Thyself: RIG-I-Like Receptor Sensing of DNA Virus Infection.

The RIG-I-like receptors (RLRs) are double-stranded RNA-binding proteins that play a role in initiating and modulating cell intrinsic immunity through the recognition of RNA features typically absent from the host transcriptome. While they are initially characterized in the context of RNA virus infection, evidence has now accumulated establishing the role of RLRs in DNA virus infection. Here, we review recent advances in the RLR-mediated restriction of DNA virus infection with an emphasis on the RLR ligands sensed.

Accelerated Metabolite Levels of Aerobic Glycolysis and the Pentose Phosphate Pathway Are Required for Efficient Replication of Infectious Spleen and Kidney Necrosis Virus in Chinese Perch Brain Cells.

Glucose is a main carbon and energy source for virus proliferation and is usually involved in the glycolysis, pentose phosphate pathway (PPP), and tricarboxylic acid cycle (TCA cycle) pathways. In this study, we investigated the roles of glucose-related metabolic pathways during the replication of infectious spleen and kidney necrosis virus (ISKNV), which has caused serious economic losses in the cultured Chinese perch (Siniperca chuatsi) industry. We found that ISKNV infection enhanced the metabolic pathways of the PPP and the TCA cycle at the early stage of the ISKNV infection cycle and enhanced the glycolysis pathway at the late stage of the ISKNV infection cycle though the comprehensive analysis of transcriptomics, proteomics, and metabolomics. The advanced results proved that ISKNV replication induced upregulation of aerobic glycolysis at the late stage of ISKNV infection cycle and aerobic glycolysis were required for ISKNV multiplication. In addition, the PPP, providing nucleotide biosynthesis, was also required for ISKNV multiplication. However, the TCA cycle involving glucose was not important and necessary for ISKNV multiplication. The results reported here provide new insights into viral pathogenesis mechanism of metabolic shift, as well as antiviral treatment strategies.

Human DNA Virus Exploitation of the MAPK-ERK Cascade.

The extracellular signal-regulated kinases (ERKs) comprise a particular branch of the mitogen-activated protein kinase cascades (MAPK) that transmits extracellular signals into the intracellular environment to trigger cellular growth responses. Similar to other MAPK cascades, the MAPK-ERK pathway signals through three core kinases-Raf, MAPK/ERK kinase (MEK), and ERK-which drive the signaling mechanisms responsible for the induction of cellular responses from extracellular stimuli including differentiation, proliferation, and cellular survival. However, pathogens like DNA viruses alter MAPK-ERK signaling in order to access DNA replication machineries, induce a proliferative state in the cell, or even prevent cell death mechanisms in response to pathogen recognition. Differential utilization of this pathway by multiple DNA viruses highlights the dynamic nature of the MAPK-ERK pathway within the cell and the importance of its function in regulating a wide variety of cellular fates that ultimately influence viral infection and, in some cases, result in tumorigenesis.

Ranaviruses Bind Cells from Different Species through Interaction with Heparan Sulfate.

Ranavirus cross-species infections have been documented, but the viral proteins involved in the interaction with cell receptors have not yet been identified. Here, viral cell-binding proteins and their cognate cellular receptors were investigated using two ranaviruses, Andrias davidianus ranavirus (ADRV) and Rana grylio virus (RGV), and two different cell lines, Chinese giant salamander thymus cells (GSTC) and Epithelioma papulosum cyprinid (EPC) cells. The heparan sulfate (HS) analog heparin inhibited plaque formation of ADRV and RGV in the two cell lines by more than 80% at a concentration of 5 µg/mL. In addition, enzymatic removal of cell surface HS by heparinase I markedly reduced plaque formation by both viruses and competition with heparin reduced virus-cell binding. These results indicate that cell surface HS is involved in ADRV and RGV cell binding and infection. Furthermore, recombinant viral envelope proteins ADRV-58L and RGV-53R bound heparin-Sepharose beads implying the potential that cell surface HS is involved in the initial interaction between ranaviruses and susceptible host cells. To our knowledge, this is the first report identifying cell surface HS as ranavirus binding factor and furthers understanding of interactions between ranaviruses and host cells.