Autophagy induced by Cyprinid herpesvirus 3 (CyHV-3) facilitated intracellular viral replication and extracellular viral yields in common carp brain cells.

Autophagy is a conservative and important process that exists in all eukaryotic cells in nature. Cyprinid herpesvirus 3 (CyHV-3), also known as KHV (Koi Herpesvirus), is a pathogen that mainly infecting common carp and koi. In the present study, we identified the CcLC3B gene, with a length of 379 bp and displaying a close evolutionary relationship with other sixteen different species, the tissue distribution and expression pattern of CcLC3 were also identified. We found that CyHV-3 infection could promote autophagy in CCB cells at the early stage but inhibit autophagy at the late stage by using confocal fluorescence microscopy, transmission electron microscopy and western blotting. And we measured the protein levels associated with the Akt/mTOR signalling pathway, intracellular replication of CyHV-3 at the mRNA and protein levels as well as viral titters. Collectively, the results taken together suggested that CyHV-3 infection could promote autophagy in CCB cells at the early stage but inhibit autophagy at the late stage via mTOR and that promoting autophagy could facilitate CyHV-3 intracellular replication and extracellular viral yields in CCB cells. These findings revealed the relationship between CyHV-3 and autophagy and provided a novel treatment strategy targeting the autophagy signalling pathway against CyHV-3 infection.

A 16-color full spectrum flow cytometric analysis for comprehensive evaluation of T-cell reconstitution in SIV-infected rhesus macaques.

T-cell reconstitution is central in human immunodeficiency virus (HIV) infection/disease progression. Simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) have been the most widely used animal model for HIV research so far. An effective flow cytometry panel is crucial for monitoring the T cell reconstitution in SIV infection progression. We developed this sixteen-color flow cytometry-based panel for a T cell subsets analysis by manual gating and, once successfully gated, to characterize T cells function in-depth in rhesus macaques. This panel included markers to characterize CD4+ T cells and CD8+ T cells, T regulatory cells (Tregs), and T cell differentiation status (CD45RA and CCR7). Additionally, we included antibodies that measure T cell activation and proliferation molecules (CD69, HLA-DR, CD38 and Ki67), antibodies that examine the expressions of key PD-1 pathway molecule (PD-1), SIV potential target (CD32) and the primary SIV co-receptor CCR5 (CD195). High-dimensional single cell analysis was also performed to identify CD3+ T cells immunophenotypes of SIV-infected rhesus macaques. We designed this panel to evaluate the responses of different T cell subsets to SIV in whole blood from SIV-infected rhesus macaques.

Deleting ORF71L of infectious spleen and kidney necrosis virus (ISKNV) resulted in virulence attenuation in Mandarin fish.

Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus, infects a variety of teleost fish species and causes substantial losses in the aquaculture industry worldwide. ISKNV ORF71L is 1611 bp in length, encodes a 537-amino-acid peptide and was previously identified as a viral structural protein in the ISKNV virion. In this study, the ORF71L deletion mutant virus strain ISKNV-Δ71 was obtained through a homologous recombination approach. The multistep growth curves showed that ISKNV-Δ71 replication was faster than ISKNV-WT replication in mandarin fish fry cells (MFF-1 cells) before 48 h post-infection (hpi). The cumulative mortality of ISKNV-Δ71-infected mandarin fish (Siniperca chuatsi) was lower than that of fish infected with ISKNV-WT. The copy numbers of viral genome equivalents (GEs) in ISKNV-Δ71-infected mandarin fish spleens were also lower than those in ISKNV-WT-infected spleens. Deletion of ORF71L resulted in ISKNV virulence attenuation in mandarin fish. Furthermore, we found that the number of melanomacrophage centers (MMCs) in ISKNV-Δ71-infected mandarin fish spleens was higher than that in ISKNV-WT-infected mandarin fish spleens. Transcriptomic analysis showed that the cytokine-cytokine receptor interaction pathway had the most significant change between ISKNV-Δ71- and ISKNV-WT-infected MFF-1 cells. These results indicated ORF71L is a virulence-related gene of ISKNV. ORF71L could be considered as a potential target for the development of engineered attenuated live vaccines via multigene deletion or as a potential insertion site for exogenous protein expression.

Differential autophagic effects triggered by five different vertebrate iridoviruses in a common, highly permissive mandarinfish fry (MFF-1) cell model.

Autophagy of five vertebrate iridoviruses, including one megalocytivirus (infectious spleen and kidney necrosis virus, ISKNV) and four ranaviruses (Chinese giant salamander iridovirus, CGSIV; Tiger frog virus, TFV; Grouper iridovirus, GIV; and Largemouth bass virus, LMBV) were investigated in a common, highly permissive mandarinfish fry (MFF-1) cell model. The results showed marked autophagosome formation in GIV- and LMBV-infected cells but not in ISKNV-, CGSIV- and TFV-infected MFF-1 cells. Strong evidence for the autophagosomes was provided by transmission electron microscopy, the detection of mandarinfish microtubule-associated protein 1 light chain 3B (mLC3)-based fluorescent dot formation and mLC3-I/mLC3-II conversion was provided by Western blotting. Pharmacological tests indicated that autophagy plays an antiviral role during GIV or LMBV infection. Collectively, our data are the first to show that antiviral autophagic effects can be triggered by GIV and LMBV but not by ISKNV, TFV and CGSIV in a common susceptible cell model. These results suggest that differential host-virus interaction strategies may be utilized against different vertebrate iridoviruses; they also indicate the potential effectiveness of an antiviral treatment that modulates autophagy to control iridoviral infections, such as GIV and LMBV.

Functional characterization of viral tumor necrosis factor receptors encoded by cyprinid herpesvirus 3 (CyHV3) genome.

Cyprinid herpesvirus 3 (CyHV3) is a large double-stranded DNA virus of Alloherpesviridae family in the order Herpesvirales. It causes significant morbidity and mortality in common carp and its ornamental koi variety, and threatens the aquaculture industries worldwide. Mimicry of cytokines and cytokine receptors is a particular strategy for large DNA viruses in modulating the host immune response. Here, we report the identification and characterization of two novel viral homologues of tumor necrosis factor receptor (TNFR) encoded by CyHV3-ORF4 and -ORF12, respectively. CyHV3-ORF4 was identified as a homologue of HVEM and CyHV3-ORF12 as a homologue of TNFRSF1. Overexpression of ORF4 and ORF12 in zebrafish embryos results in embryonic lethality, morphological defects and increased apoptosis. Although we failed to identify any interaction between the two vTNFRs and their potential ligands in zebrafish TNF superfamily by yeast two-hybrid system, the expression of some genes in TNF superfamily or TNFR superfamily were mis-regulated in ORF4 or ORF12-overexpressing embryos, especially the death receptor zHDR and its cognate ligand DL1b. Further studies showed that the apoptosis induced by the both CyHV3 vTNFRs is mainly activated through the intrinsic apoptotic pathway and requires the crosstalk between the intrinsic and extrinsic apoptotic pathway. Additionally, using RT-qPCR and Western blot assays, the expression patterns of the both vTNFRs were also analyzed during CyHV3 productive infection. Collectively, this is the first functional study of two unique vTNFRs encoded by a herpesvirus infecting non-mammalian vertebrates, which may provide novel insights into viral immune regulation mechanism and the pathogenesis of CyHV3 infection.

Identification and characterization of a novel FstK-like protein from spotted knifejaw iridovirus (genus Megalocytivirus).

Prokaryotes contain many DNA binding proteins with large molecular weights and multiple domains. DNA binding proteins are involved in DNA replication, transcription, and other physiological processes. In this study, a DNA binding protein, containing an Ftsk-like protein (FLP) domain, was cloned and characterized from SKIV-ZJ07, a member of the RSIV-type megalocytivirus, using bioinformatics and molecular biology approaches. SKIV-FLP is 3,762 base pairs long, encodes a viral protein of 1253 amino acid residuals, and contains an Ftsk (or EBV-NA3) and a Grx-2 domain. Virion localization indicated that SKIV-FLP is a major viral structural protein located below the major capsid protein. Laser confocal microscopy showed that SKIV-FLP is a cytoplasm-/nuclear-localized protein. However, the reconstruction experiments demonstrated that SKIV-FLP may contain three nuclear localization signals, each present in FLP-NT (1-380 aa), FtsK domain (380-880 aa), and Grx-2 domain (880-1253 aa). When SKIV-FLP was fused to the Gal-4 DNA-binding domain and co-transfected with L8G5-Luc, SKIV-FLP suppressed L8G5-Luc transcription. As a transcription inhibitor, SKIV-FLP also inhibited the transcription of NF-κB and IFN-γ (a type II IFN) promoter in HEK293T cells, suggesting that SKIV-FLP has a role in evading host immunity.