A GH19 lysozyme and peptidase from Myoviridae cyanophages lacking the typical holin-endolysin system exhibit lytic activity.

Most of the dsDNA cyanophages employ holin-endolysin lysis systems to damage the host cells. This study aimed to elucidate the lytic activity of ORF91 and ORF117 in the cyanophage MaMV-DH01, which lacked a conventional cholinesterase system. These two proteins contained Lyz-like superfamily domains and were annotated as a member of GH family 19 (named DHGH19) and peptidase (named DHpeptidase), respectively. Overexpression of DHGH19 in E. coli over a 5 h course demonstrated potent bactericidal activity, evident from significant growth inhibition, membrane damage, and leakage of intracellular enzymes of E. coli cells. However, the lytic activity of DHpeptidase was relatively weaker, exhibiting a bacteriostatic effect. It was important to highlight that the specific mutation of enzyme-catalyzed residues in DHGH19 (E122 and E131) showed that these were the essential amino acids for DHGH19 to exert its bactericidal activity. Furthermore, the lytic function of DHGH19 and DHpeptidase on cyanobacteria cells was confirmed by their overexpression in the cyanobacterium Synechocystis sp. PCC6803. Overall, this study provides novel insights into the lytic mechanism of Myoviridae cyanophage, offering potential alternatives for the development of GH19 and peptidase as new antibacterial agents in the future.

Eukaryotic translation elongation factor 1 alpha (eEF1A) inhibits Siniperca chuatsi rhabdovirus (SCRV) infection through two distinct mechanisms.

IMPORTANCE: Although a virus can regulate many cellular responses to facilitate its replication by interacting with host proteins, the host can also restrict virus infection through these interactions. In the present study, we showed that the host eukaryotic translation elongation factor 1 alpha (eEF1A), an essential protein in the translation machinery, interacted with two proteins of a fish rhabdovirus, Siniperca chuatsi rhabdovirus (SCRV), and inhibited virus infection via two different mechanisms: (i) inhibiting the formation of crucial viral protein complexes required for virus transcription and replication and (ii) promoting the ubiquitin-proteasome degradation of viral protein. We also revealed the functional regions of eEF1A that are involved in the two processes. Such a host protein inhibiting a rhabdovirus infection in two ways is rarely reported. These findings provided new information for the interactions between host and fish rhabdovirus.

Advances on genomes studies of large DNA viruses in aquaculture: A minireview.

Genomic studies of viral diseases in aquaculture have received more and more attention with the growth of the aquaculture industry, especially the emerging and re-emerging viruses whose genome could contain recombination, mutation, insertion, and so on, and may lead to more severe diseases and more widespread infections in aquaculture animals. The present review is focused on aquaculture viruses, which is belonged to two clades, Varidnaviria and Duplodnaviria, and one class Naldaviricetes, and respectively three families: Iridoviridae (ranaviruses), Alloherpesviridae (fish herpesviruses), and Nimaviridae (whispoviruses). The viruses possessed DNA genomes nearly or larger than 100 kbp with gene numbers more than 100 and were considered large DNA viruses. Genome analysis and experimental investigation have identified several genes involved in genome replication, transcription, and virus-host interactions. In addition, some genes involved in virus genetic variation or specificity were also discussed. A summary of these advances would provide reference to future discovery and research on emerging or re-emerging aquaculture viruses.

Genome Characteristics of Two Ranavirus Isolates from Mandarin Fish and Largemouth Bass.

Ranaviruses are promiscuous pathogens that threaten lower vertebrates globally. In the present study, two ranaviruses (SCRaV and MSRaV) were isolated from two fishes of the order Perciformes: mandarin fish (Siniperca chuatsi) and largemouth bass (Micropterus salmoides). The two ranaviruses both induced cytopathic effects in cultured cells from fish and amphibians and have the typical morphologic characteristics of ranaviruses. Complete genomes of the two ranaviruses were then sequenced and analyzed. Genomes of SCRaV and MSRaV have a length of 99, 405, and 99, 171 bp, respectively, and both contain 105 predicted open reading frames (ORFs). Eleven of the predicted proteins have differences between SCRaV and MSRaV, in which only one (79L) possessed a relatively large difference. A comparison of the sequenced six ranaviruses from the two fish species worldwide revealed that sequence identities of the six proteins (11R, 19R, 34L, 68L, 77L, and 103R) were related to the place where the virus was isolated. However, there were obvious differences in protein sequence identities between the two viruses and iridoviruses from other hosts, with more than half lower than 55%. Especially, 12 proteins of the two isolates had no homologs in viruses from other hosts. Phylogenetic analysis revealed that ranaviruses from the two fishes clustered in one clade. Further genome alignment showed five groups of genome arrangements of ranaviruses based on the locally collinear blocks, in which the ranaviruses, including SCRaV and MSRaV, constitute the fifth group. These results provide new information on the ranaviruses infecting fishes of Perciformes and also are useful for further research of functional genomics of the type of ranaviruses.

Biological and Genomic Characteristics of MaMV-DH01, a Novel Freshwater Myoviridae Cyanophage Strain.

The genomic traits of cyanophages and their potential for metabolic reprogramming of the host cell remain unknown due to the limited number of studies on cyanophage isolates. In the present study, a lytic Microcystis cyanophage, MaMV-DH01, was isolated and identified. MaMV-DH01 has an icosahedral head approximately 100 nm in diameter and a tail 260 nm in length. Its burst size is large, with approximately 145 phage particles/infected cell; it has a latent period of 2 days, and it shows high stability under pH and temperature stresses. Multiple infection (multiplicity of infection [MOI] 0.0001 to 100) results showed that when the MOI was 0.0001, MaMV-DH01 needed a longer time to lyse host cells. Cyanophage MaMV-DH01 has a double-stranded DNA genome of 182,372 bp, with a GC content of 45.35% and 210 predicted open reading frames (ORFs). These ORFs are related to DNA metabolism, structural proteins, lysis, host-derived metabolic genes, and DNA packaging. Phylogenetic trees based on the whole genome and two conserved genes (TerL and capsid) indicate that MaMV-DH01 is clustered with Ma-LMM01 and MaMV-DC, which are independent of other cyanophages. Collinearity analysis showed that the complete genome of MaMV-DH01 was longer than those of Ma-LMM01 and MaMV-DC, with lengths of 20,263 bp and 13,139 bp, respectively. We verified the authenticity of these excess DNA fragments and found that they are involved to various degrees in the MaMV-DH01 transcription process. Map overlays of environmental virus macrogenomic reads onto the MaMV-DH01 genome revealed that viral sequences similar to that of MaMV-DH01 are widespread in the environment. IMPORTANCE A novel freshwater Myoviridae cyanophage strain, MaMV-DH01, was isolated; this strain infects Microcystis aeruginosa FACHB-524, and the biological and genomic characteristics of MaMV-DH01 provide new insights for understanding the mechanism by which cyanophages infect cyanobacterial blooms.

Siniperca chuatsi Rhabdovirus (SCRV)-Induced Key Pathways and Major Antiviral Genes in Fish Cells.

Fish rhabdoviruses, including Siniperca chuatsi rhabdovirus (SCRV), are epidemic pathogens that harm fish aquaculture. To clarify the interactions between SCRV and its host and explore antiviral targets, the present study performed transcriptome analysis in a cultured S. chuatsi skin cell line (SCSC) after SCRV infection at 3, 12, 24, and 36 h post-infection (hpi). Comparison with control obtained 38, 353, 896, and 1452 differentially expressed genes (DEGs) in the detected time points, respectively. Further analysis of the Go terms and KEGG pathways revealed the key pathways "Cytokine-cytokine receptor interaction" and "interferon related pathways" in SCSC cells responding to SCRV infection. The significantly up-regulated genes in the pathways were also verified by qPCR. Furthermore, gene cloning and overexpression revealed that five interferon-stimulated genes (ISGs) IFI4407, IFI35, Viperin, IFIT1, and IFIT5 had the ability to inhibit SCRV replication in FHM (Fathead minnow) cells, especially an inhibition efficiency more than 50% was observed in IFI35 overexpressed cells. In summary, current study revealed the main innate immune pathways in S. chuatsi cells induced by SCRV infection and the major ISGs of S. chuatsi in controlling SCRV replication.

Environmental Factors and Their Threshold Affecting the Survival of Five Aquatic Animal Viruses in Different Animal Cells.

Aquatic animal viruses infect and transmit in aquatic environments, causing serious harm to the aquaculture industry and a variety of wild aquatic animals. How are they affected by environmental factors and do they represent potential threat to mammalian heath or not? Here, the effects of environmental factors (ultraviolet radiation (UV), temperature, pH, and drying) and their threshold on five epidemic aquatic animal viruses infecting amphibians and bony fish, including Rana grylio virus (RGV), Andrias davidianus ranavirus (ADRV), Grass carp reovirus (GCRV), Paralichthys olivaceus rhabdovirus (PORV), and Scophthalmus maximus rhabdovirus (SMRV), were measured and compared in a fish cell line. The examination of virus titers after different treatment in fish cells showed that the two iridoviruses, RGV and ADRV, had a higher tolerance to all of the environmental factors, such as they only had a decay rate of 22-36% when incubated at 37 °C for 7 days. However, the rhabdovirus SMRV was sensitive to all of the factors, with a decay rate of more than 80% in most of the treatments; even a complete inactivation (100%) can be observed after drying treatment. To address the potential threat to mammals, infectivity and limitation factors of the five viruses in Baby hamster kidney fibroblast cells (BHK-21) were tested, which showed that three of the five viruses can replicate at a low temperature, but a high temperature strongly inhibited their infection and none of them could replicate at 37 °C. This study clarified the sensitivity or tolerance of several different types of aquatic animal viruses to the main environmental factors in the aquatic environment and proved that the viruses cannot replicate in mammalian cells at normal physiological temperature.

Development and characterization of a skin cell line from Chinese perch (Siniperca chuatsi) and its application in aquatic animal viruses.

Chinese perch (Siniperca chuatsi), an important fish for the aquaculture industry of China, is often affected by viral diseases. A stable and sensitive cell line can play an important role in virus identification and isolation, functional gene identification, virus pathogenic mechanism and antiviral immunity study. In the present study, a new cell line (S. chuatsi skin cell, SCSC) derived from the skin of S. chuatsi was established. The SCSC mainly consisted of fibroblastic-like cells, which grew well in M199 medium supplemented with 10% foetal bovine serum at 25°C. Chromosome analysis revealed that the SCSC (44%) has a diploid chromosome number of 2n = 48. The SCSC can be transfected and expressed exogenous gene efficiently. It also showed high sensitivity to several aquatic animal viruses from different families including Rhabdoviridae, Iridoviridae and Reoviridae. In addition, RT-PCR showed that S. chuatsi rhabdovirus (SCRV) started genome replication as early as 3 h post infection in the cells, which also induced the up-regulation of a variety of immune-related genes including these related to interleukin family, pattern recognition receptors, JAK-STAT pathway and interferon regulatory factors. In summary, current study provided a new tool in research of fish viruses and its interaction with host.

Functional Analysis of the Endopeptidase and Holin From Planktothrix agardhii Cyanophage PaV-LD.

A cyanophage PaV-LD, previously isolated from harmful filamentous cyanobacterium Planktothrix agardhii, was sequenced, and co-expression of its two ORFs in tandem, ORF123 and ORF124, inhibited growth on the model cyanobacterium Synechocystis sp. PCC6803 cells. However, the mechanism of action of ORF123 and ORF124 alone remains to be elucidated. In this study, we aimed to study the individual function of ORF123 or ORF124 from PaV-LD. Our data showed that the ORF123 encoded an endopeptidase, which harbored an M23 family peptidase domain and a transmembrane region. The expression of the endopeptidase in Escherichia coli alone revealed that the protein exhibited remarkable bacteriostatic activity, as evidenced by observation of growth inhibition, membrane damage, and leakage of the intracellular enzyme. Similarly, the holin, a membrane-associated protein encoded by the ORF124, showed weak bacteriostatic activity on E. coli. Moreover, deletion mutations indicated that the transmembrane domains of endopeptidase and holin were indispensable for their bacteriostatic activity. Meanwhile, the bacteriostatic functions of endopeptidase and holin on cyanobacteria cells were confirmed by expressing them in the cyanobacterium Synechocystis sp. PCC6803. Collectively, our study revealed the individual role of endopeptidase or holin and their synergistic bacteriolytic effect, which would contribute to a better understanding of the lytic mechanism of cyanophage PaV-LD.

ADRV 12L: A Ranaviral Putative Rad2 Family Protein Involved in DNA Recombination and Repair.

The Andrias davidianus ranavirus (ADRV) is a member of the family Iridoviridae and belongs to the nucleocytoplasmic large DNA viruses. Based on genomic analysis, an ADRV-encoding protein, ADRV 12L, and its homologs from other iridoviruses were predicted as Rad2 family proteins based on the conserved amino acids, domains, and secondary structures. Expression analysis showed that the transcription of ADRV 12L started at 4 h post infection, and its expression was not inhibited by a DNA-replication inhibitor. Meanwhile, immunofluorescence localization showed that ADRV 12L mainly localized in viral factories and colocalized with the viral nascent DNA, which hinted at a possible role in DNA replication. Furthermore, a mutant ADRV lacking 12L (ADRV-Δ12L) was constructed. In both luciferase assays based on homologous recombination (HR) and double-strand break repair (DSBR) that followed, ADRV-Δ12L induced less luciferase activity than the wild-type ADRV, indicating that HR and DSBR were impaired in ADRV-Δ12L infected cells. In addition, infection with ADRV-Δ12L resulted in smaller plaque sizes and lower viral titers than that with wild-type ADRV, indicating an important role for 12L in efficient virus infection. Therefore, the results suggest that Rad2 homologs encoded by iridovirus have important roles in HR- and DSBR-process of the viral DNA and, thus, affect virus replication and the production of progeny virions.

Replication and transcription machinery for ranaviruses: components, correlation, and functional architecture.

BACKGROUND: Ranaviruses (family Iridoviridae) are promiscuous pathogens that can infect across species barriers in poikilotherms and can replicate in amphibian and fish cells and even in cultured mammalian cells. However, as nucleocytoplasmic large DNA viruses (NCLDVs), their replication and transcription mechanisms remain largely unknown. Here, we screened and uncovered the replication and transcription machinery of two ranaviruses, Andrias davidianus ranavirus (ADRV) and Rana grylio virus (RGV), by a combination of methods, including the isolation of proteins on nascent DNA, recombinant virus-based affinity, and NanoLuc complementation assay. RESULTS: The ranavirus replication and transcription machinery was deeply dissected and identified as a complicated apparatus containing at least 30 viral and 6 host proteins. The viral proteins ADRV-47L/RGV-63R (DNA polymerase, vDPOL), ADRV-23L/RGV-91R (proliferating cell nuclear antigen, vPCNA), ADRV-85L/RGV-27R (single-stranded DNA binding protein, vSSB), ADRV-88L/RGV-24R (vhelicase/primase), etc., constitute the core replisome. Specifically, the core of the transcription complex, the viral RNA polymerase, contain the host RNAPII subunits Rpb3, Rpb6, and Rpb11, which was a first report in NCLDVs. Furthermore, correlations and interactions among these factors in the machinery were described. Significantly, the replisome core protein vDPOL (ADRV-47L) can interact with numerous viral and host proteins and could act as a linker and regulation center in viral DNA replication and transcription. Thus, these results depicted an architecture for ranavirus replication and transcription. CONCLUSIONS: Up to 36 components from ranavirus and their host were found to form viral replisomes and transcription complexes using a series of precise methods, which further constructed an architecture for ranavirus replication and transcription in which vDPOL was a key central factor and various components correlated and cooperated. Therefore, it provides a cornerstone for further understanding the mechanisms of the replication and transcription of ranaviruses which can ensure the efficient production of progeny virus and adaptation to cross-species infection.

Two Duplicated Ptpn6 Homeologs Cooperatively and Negatively Regulate RLR-Mediated IFN Response in Hexaploid Gibel Carp.

Src homology region 2 domain-containing phosphatase 1 (SHP1), encoded by the protein tyrosine phosphatase nonreceptor type 6 (ptpn6) gene, belongs to the family of protein tyrosine phosphatases (PTPs) and participates in multiple signaling pathways of immune cells. However, the mechanism of SHP1 in regulating fish immunity is largely unknown. In this study, we first identified two gibel carp (Carassius gibelio) ptpn6 homeologs (Cgptpn6-A and Cgptpn6-B), each of which had three alleles with high identities. Then, relative to Cgptpn6-B, dominant expression in adult tissues and higher upregulated expression of Cgptpn6-A induced by polyinosinic-polycytidylic acid (poly I:C), poly deoxyadenylic-deoxythymidylic (dA:dT) acid and spring viremia of carp virus (SVCV) were uncovered. Finally, we demonstrated that CgSHP1-A (encoded by the Cgptpn6-A gene) and CgSHP1-B (encoded by the Cgptpn6-B gene) act as negative regulators of the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via two mechanisms: the inhibition of CaTBK1-induced phosphorylation of CaMITA shared by CgSHP1-A and CgSHP1-B, and the autophagic degradation of CaMITA exclusively by CgSHP1-A. Meanwhile, the data support that CgSHP1-A and CgSHP1-B have sub-functionalized and that CgSHP1-A overwhelmingly dominates CgSHP1-B in the process of RLR-mediated IFN response. The current study not only sheds light on the regulative mechanism of SHP1 in fish immunity, but also provides a typical case of duplicated gene evolutionary fates.

Divergent Antiviral Mechanisms of Two Viperin Homeologs in a Recurrent Polyploid Fish.

Polyploidy and subsequent diploidization provide genomic opportunities for evolutionary innovations and adaptation. The researches on duplicated gene evolutionary fates in recurrent polyploids have seriously lagged behind that in paleopolyploids with diploidized genomes. Moreover, the antiviral mechanisms of Viperin remain largely unclear in fish. Here, we elaborate the distinct antiviral mechanisms of two viperin homeologs (Cgviperin-A and Cgviperin-B) in auto-allo-hexaploid gibel carp (Carassius gibelio). First, Cgviperin-A and Cgviperin-B showed differential and biased expression patterns in gibel carp adult tissues. Subsequently, using co-immunoprecipitation (Co-IP) screening analysis, both CgViperin-A and CgViperin-B were found to interact with crucian carp (C. auratus) herpesvirus (CaHV) open reading frame 46 right (ORF46R) protein, a negative herpesvirus regulator of host interferon (IFN) production, and to promote the proteasomal degradation of ORF46R via decreasing K63-linked ubiquitination. Additionally, CgViperin-B also mediated ORF46R degradation through autophagosome pathway, which was absent in CgViperin-A. Moreover, we found that the N-terminal α-helix domain was necessary for the localization of CgViperin-A and CgViperin-B at the endoplasmic reticulum (ER), and the C-terminal domain of CgViperin-A and CgViperin-B was indispensable for the interaction with degradation of ORF46R. Therefore, the current findings clarify the divergent antiviral mechanisms of the duplicated viperin homeologs in a recurrent polyploid fish, which will shed light on the evolution of teleost duplicated genes.

Structural and Functional Diversity among Five RING Finger Proteins from Carassius Auratus Herpesvirus (CaHV).

Carassius auratus herpesvirus (CaHV) has been identified as a high-virulence pathogenic virus that infects aquatic animals, but the key factor for virus-host interaction is still unclear. Five Really interesting new genes (RING) finger proteins (39L, 52L, 131R, 136L, and 143R) of CaHV were screened to determine structural diversity. RING finger proteins were also predicted in other known fish herpesviruses, with an arrangement and number similar to CaHV. We performed multifaceted analyses of the proteins, including protein sizes, skeleton structures, subcellular localizations, and ubiquitination activities, to determine their precise roles in virus-host interactions. The five proteins were overexpressed and detected different levels of ubiquitination activities, and 143R showed the highest activity. Then, the prokaryotic expressed and purified full-length proteins (131R and 136L), RING domain isolates (131R12-43 and 136L45-87), and RING domain-deleted mutants (131RΔ12-43 and 136LΔ45-87) were prepared to detect their activities through ubiquitination assays. The results indicate that both full-length proteins and their isolates have activities that catalyze ubiquitination, and the full-length proteins possess higher activity than the isolates, but RING domain-deleted mutants lose their activities. Furthermore, the activities of the five proteins were verified as E3 ubiquitin ligase activity, showing that the RING domains determine the ubiquitination activity. These proteins present different subcellular localization. RING domain-deleted mutants showed similar subcellular localization with their full-length proteins, and all the isolates diffused in whole cells. The current results indicate that the sequence outside the RING domain determines subcellular localization and the level of ubiquitination activity, suggesting that the RING finger proteins of fish herpesviruses might have diverse functions in virus-host interaction.

Infectious pancreatic necrosis virus inhibits infectious hematopoietic necrosis virus at the early stage of infection in a time dependent manner during Co-infection in Chinook salmon embryo cell lines.

Salmonids can be co-infected by infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) under natural or experimental conditions. To reveal the influence of IPNV on IHNV in co-infections, CHSE-214 cells were inoculated with IPNV at different time intervals prior to or after IHNV infection. Propagation of IHNV was determined by an immunofluorescence antibody test, real-time quantitative polymerase chain reaction, flow cytometry, and virus titration. The results showed that when cells were inoculated with IPNV prior to IHNV, IHNV multiplication was inhibited. This inhibitory effect became stronger with increasing time intervals (P < 0.05). When cells were inoculated with IPNV after IHNV, the inhibitory effect became weaker with increasing time intervals (P < 0.05), and no significant inhibition was observed at 12 h (P > 0.05) compared with the single IHNV infection group. The findings suggest that IHNV is inhibited at the early stage of infection by IPNV and in a time dependent manner during co-infection. Furthermore, the effect of IPNV on IHNV entry and expression of IHNV entry-related genes clathrin, dynamin-2, adaptor protein 2, and vacuolar protein sorting 35 were also determined. The results showed that IPNV did not affect the amount of IHNV entering the cells. However, the expression levels of clathrin and dynamin-2 were significantly lower in co-infection than those in single IHNV infection, which suggests that IPNV likely inhibits IHNV by affecting IHNV invasion via downregulating IHNV entry-related genes clathrin and dynamin-2.

Function characterization and expression regulation of two different-sized 3' untranslated region-containing interferon genes from clone F of gibel carp Carassius auratus gibelio.

Fish interferon (IFN)-mediated antiviral innate immunity is the first line of defense against virus invasion. In the present study, we identify two fish IFN genes (here tentatively named IFNa and IFNc) with different-sized 3' UTRs from clone F strain of gibel carp Carassius auratus gibelio. Carp IFNa has a relatively short 3'UTR without AU-rich elements (AREs) but IFNc has a long one with 9 AREs. Functionally, carp IFNa and IFNc display significantly antiviral potential to viral infection, likely through induction of downstream IFN-stimulated genes (ISGs). Both carp IFN genes are induced by viral infection, poly(I:C) treatment and IRF3/7, which are ascribed to the IFN-sensitive response elements (ISRE) within their promoters. Carp IFN genes are also induced by each other and by themselves, indicating existence of a positive feedback loop in fish IFN-mediated antiviral immune response. Comparative analyses of 3'UTR-mediated expression regulation at mRNA and protein levels show that the ARE-containing 3'UTR of carp IFNc rather than the short 3'UTR of carp IFNa promotes mRNA decay but instead results in high-level protein expression, indicating that 3'UTR of fish IFN mRNAs might be a potential factor for regulation of IFN-mediated antiviral immune response. Considering a fact that a given protein function is largely related to its protein level, these results suggest that both promoter and 3'UTR contribute to the transcription and translation of fish IFN genes, thus shaping their eventually antiviral potential.

Fish herpesvirus protein (CaHV-138L) can target to mitochondrial protein FoF1 ATPase.

Herpesvirus infection usually relies on the interaction between viral protein and host protein to enhance replication of the enveloped virus. Fish Carassius auratus herpesvirus (CaHV) is highly pathogenic pathogen causing gill acute hemorrhages of crucian carp (Carassius auratus) and high moritality rates among those infected fish. The protein of CaHV (CaHV-138 L) containing two transmembrane (TM) domains and an immunoglobulin C-2 Type (IGc2) domain was predicted as a viral membrane protein. In this investigation, fluorescence observation showed that full-length CaHV-138 L mainly localized on the plasma membrane or around nuclear membrane of fish fathead minnow (FHM) cells in a punctate pattern. The TM domain deletion mutants of CaHV-138 L (ΔTM1, ΔTM2, and ΔTM1&ΔTM2) diffusely distributed in both the cytoplasm and the nucleus, mainly presented patchy fashion in the cytoplasm, and mainly presented both in the nucleus and in the cytoplasm, respectively. Obviously, the TM domain deletion mutants significantly affected CaHV-138 L subcellular localization. Meanwhile, colocalization assay showed that the full-length viral protein colocalized with mitochondria. Furthermore, the interaction between CaHV-138 L and host protein was identified by yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) assays. The host mitochondrial protein FoF1 ATP synthase (FoF1-ATPase) that interacts with this viral protein was screened. The data indicated that CaHV-138 L can target to mitochondrial protein FoF1-ATPase, which might provide energy for virus replication through mediating mitochondrial ATP synthesis. This study has provided valuable information for better understanding of the links of herpesvirus proteins with aquaculture animal proteins.

A transmembrane domain of Andrias davidianus ranavirus 13R is crucial for co-localization to endoplasmic reticulum and viromatrix.

13R, a core gene of Andrias davidianus ranavirus (ADRV), encoded a protein containing a transmembrane domain (TMD) and a restriction endonuclease-like domain. However, the characterization and function of 13R and the protein it encodes remain unclear. In this study, Chinese giant salamander thymus cell (GSTC) was used to investigate the function of 13R. The results showed that the 13R transcripts were detected first at 8 h post-infection (hpi) by RT-PCR and the protein was detected first at 24 hpi by western blot, but the transcription was inhibited by cycloheximide and cytosine arabinofuranoside, indicating that 13R is a viral late gene. Subcellular localization showed that the 13R was co-localized with endoplasmic reticulum (ER) in the cytoplasm, while 13R deleting TMD (13RΔTM) was distributed in cytoplasm and nucleus. During ADRV infection, 13R was observed first in the cytoplasm and nucleus, and later aggregated into the viromatrix, whereas 13RΔTM remain dispersed in cytoplasm and nucleus. Western blot analysis suggested that 13R was a viral non-structural protein and its overexpression did not affect the viral titer in GSTC. All these indicated that the TMD of 13R is crucial for the co-localization into the ER and the viromatrix.

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.