Identification and functional analysis of salmon annexin 1 induced by a virus infection in a fish cell line

In this study, we investigated changes in protein expression of fish cells induced by infection of infectious pancreatic necrosis virus (IPNV) using two-dimensional electrophoresis and matrix-assisted laser desorption-time of flight proton motive force analysis and identified a novel type of salmon annexin 1 that is induced in fish cells by infection with IPNV. Northern blotting showed that this annexin is overexpressed in IPNV-infected cells compared to control cells, and further analysis revealed that it has a 1,509-bp full-length cDNA sequence with an open reading frame encoding 339 amino acids (GenBank accession no. AY944135). Amino acid sequence analysis revealed that this protein belongs to the annexin 1 subfamily. By applying RNA interference, the mRNA levels of salmon annexin 1 were suppressed and, under these conditions, apoptosis of IPNV-infected cells was significantly increased. While small interfering RNA (siRNA) treatment did not affect the levels of the viral proteins significantly until 10 h postinfection, it reduced the titer of extracellular virus to 25% of that of a scrambled siRNA-treated control. These data provide evidence of an antiapoptotic function for salmon annexin 1 that is important for IPNV growth in cultured cells.     

Induction of apoptotic death in cells via Bad gene expression by infectious pancreatic necrosis virus infection

A Bcl-2 related family member, Bad, promotes cell death, and its function is regulated by phosphorylation. In this study, we show how the IPNV elicits the induction of Bad gene expression and promotes host apoptotic death. Anti-IPNV-E1S polyclonal and anti-VP3 monoclonal antibodies are used to neutralize the virus that blocks the prime death signal via the virus receptor. In the viability assay, each antibody could also enhance cell viability during IPNV infection. We tested tyrosine kinase inhibitors on IPNV-infected cells in order to assess their effect on blocking the death signal. With 100 microg/ml genistein treatment, Bad-like gene expression was blocked, either by rescuing the IPNV-infected CHSE-214 cells or by blocking internucleosomal DNA cleavage; but the tyrphostin group did not block Bad expression. For CHSE-214 cells, treatment with the protein synthesis-inhibitor, cycloheximide (1microg/ml), blocked new protein synthesis via activated tyrosine kinase during IPNV infection. We found that Bad protein expression could be blocked, and apoptotic death prevented. Together, these results demonstrate that the IPNV exerts up-regulation of a pro-apoptotic death gene (Bad), the expression of which serves to trigger apoptotic cell death. Our data also suggests that the IPNV induces apoptotic death via a viral receptor which triggers death effector Bad gene expression, possibly through a tyrosine kinase-dependent pathway.     

Stage-specific expression of TNFα regulates bad/bid-mediated apoptosis and RIP1/ROS-mediated secondary necrosis in Birnavirus-infected fish cells

Infectious pancreatic necrosis virus (IPNV) can induce Bad-mediated apoptosis followed by secondary necrosis in fish cells, but it is not known how these two types of cell death are regulated by IPNV. We found that IPNV infection can regulate Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic death pathways via the up-regulation of TNFα in zebrafish ZF4 cells. Using a DNA microarray and quantitative RT-PCR analyses, two major subsets of differentially expressed genes were characterized, including the innate immune response gene TNFα and the pro-apoptotic genes Bad and Bid. In the early replication stage (0-6 h post-infection, or p.i.), we observed that the pro-inflammatory cytokine TNFα underwent a rapid six-fold induction. Then, during the early-middle replication stages (6-12 h p.i.), TNFα level was eight-fold induction and the pro-apoptotic Bcl-2 family members Bad and Bid were up-regulated. Furthermore, specific inhibitors of TNFα expression (AG-126 or TNFα-specific siRNA) were used to block apoptotic and necrotic death signaling during the early or early-middle stages of IPNV infection. Inhibition of TNFα expression dramatically reduced the Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic cell death pathways and rescued host cell viability. Moreover, we used Rip1-specific inhibitors (Nec-1 and Rip1-specific siRNA) to block Rip1 expression. The Rip1/ROS-mediated secondary necrotic pathway appeared to be reduced in IPNV-infected fish cells during the middle-late stage of infection (12-18 h p.i.). Taken together, our results indicate that IPNV triggers two death pathways via up-stream induction of the pro-inflammatory cytokine TNFα, and these results may provide new insights into the pathogenesis of RNA viruses.     

Dynamics of nontypical apoptotic morphological changes visualized by green fluorescent protein in living cells with infectious pancreatic necrosis virus infection

Morphologically, apoptotic cells are characterized by highly condensed membrane blebbing and formation of apoptotic bodies. Recently, we reported that apoptosis precedes necrosis in a fish cell line infected with infectious pancreatic necrosis virus (IPNV). In the present study, we tested the possibility that nontypical apoptosis is a component of IPNV-induced fish cell death. A variant type of green fluorescent protein (EGFP) was expressed in a fish cell line such that EGFP served as a protein marker for visualizing dynamic apoptotic cell morphological changes and for tracing membrane integrity changes during IPNV infection. Direct morphological changes were visualized by fluorescence microscopy by EGFP in living cells infected with IPNV. The nontypical apoptotic morphological change stage occurred during the pre-late stage (6 to 7 h postinfection). Nontypical apoptotic features, including highly condensed membrane blebbing, occurred during the middle apoptotic stage. At the pre-late apoptotic stage, membrane vesicles quickly formed, blebbed, and were finally pinched off from the cell membrane. At the same time, at this pre-late apoptotic stage, apoptotic cells formed unique small holes in their membranes that ranged from 0.39 to 0.78 micrometer according to examination by scanning electron microscopy and immunoelectron microscopy. Quantitation of the intra- and extracellular release of EGFP by CHSE-214-EGFP cells after IPNV infection was done by Western blotting and fluorometry. Membrane integrity was quickly lost during the late apoptotic stage (after 8 h postinfection), and morphological change and membrane integrity loss could be prevented and blocked by treatment with apoptosis inhibitors such as cycloheximide, genistein, and EDTA before IPNV infection. Together, these findings show the apoptotic features at the onset of pathology in host cells (early and middle apoptotic stages), followed secondarily by nontypical apoptosis (pre-late apoptotic stage) and then by postapoptotic necrosis (late apoptotic stage), of a fish cell line. Our results demonstrate that nontypical apoptosis is a component of IPNV-induced fish cell death.     

Aquatic birnavirus induces necrotic cell death via the mitochondria-mediated caspase pathway

Aquatic birnavirus induces necrotic cell death by an ill-understood process. Presently, we demonstrate that infectious pancreatic necrosis virus (IPNV) induces post-apoptotic necrotic cell death through loss of mitochondrial membrane potential (MMP) followed by caspase-3 activation in CHSE-214 cells. Progressive phosphatidylserine externalization was observed at 6 h post-infection (p.i.). This was followed by the development of bulb-like vesicles (bleb formation) at 8 h p.i. Progressive loss of MMP was also observed in IPNV-infected CHSE-214 cells beginning at 6 h p.i. At 8 h and 12 h p.i., IPNV-infected cells demonstrated a dramatic increase in MMP loss, rapid entry into necrotic cell death, and activation of caspase-9 and -3. Additionally, treatment with an inhibitor of MMP loss, bongkrekic acid, an adenine nucleotide translocase inhibitor, blocked IPNV-induced PS exposure and MMP loss, as well as reduced the activation of caspase-3. Taken together, our results suggest that IPNV induces apoptotic cell death via loss of MMP, thereby triggering secondary necrosis and caspases-3 activation. Furthermore, this death-signaling pathway is disrupted by bongkrekic acid in fish cells, indicating that this drug may serve to modulate IPNV-induced pathogenesis.     

Establishment and characterization of the Epithelioma Papulosum Cyprini (EPC) cell line persistently infected with Infectious Pancreatic Necrosis Virus (IPNV), an aquabirnavirus

Infectious pancreatic necrosis virus (IPNV), a type species of aquabirnaviruses in the family Birnaviridae, is an etiological agent of infectious pancreatic necrosis and has been isolated from epizootics of cultured salmonids. In the present study, an epithelioma papulosum cyprini (EPC) cell line persistently infected with IPNV (PI-EPC) was experimentally established by subculturing EPC cells surviving IPNV infection, and was characterized. PI-EPC cells were morphologically indistinguishable from EPC, but continued to grow and yield IPNV. PI-EPC cells showed no cytopathic effect due to IPNV inoculation, and susceptibility of PI-EPC cells against heterologous viruses was not different from that of EPC cells. Only one cell of 10^3.5 PI-EPC cells produced IPNV at approximately 10^0.5 50% tissue culture infectious dose (TCID50)/cell/day, which was approximately 1,000 times lower than that of normal EPC cells. PI-EPC cells that did not yield IPNV (N-PI-EPC) were screened. The IPNV genome was detected from both PI-EPC and N-PI-EPC cells, and the IPNV VP2 structural protein was detected from both cell lines, but no other IPNV proteins were observed by Western blot analysis with anti-IPNV serum. Thus, multiplication of IPNV in PI-EPC cells was regulated by some host cell factors, except interferon.     

Flow cytometry detection of infectious pancreatic necrosis virus (IPNV) within subpopulations of Atlantic salmon (Salmo salar L.) leucocytes after vaccination and during the time course of experimental infection

In the present study, intracellular infectious pancreatic necrosis virus (IPNV) in salmon leucocytes was detected by flow cytometry after experimental cohabitant challenge. IPNV vaccinated, non-vaccinated and intraperitoneally (i.p.) infected salmon (virus shedders) were analysed at different times throughout the period when mortality occurred. Fish that had survived 61 days post challenge (carriers) were also analysed. In particular, we analysed the presence of IPNV in B-cells (C7G7+cells) and in neutrophils (E3D9+ cells) in head kidney leucocytes (HKL) and in peripheral blood leucocytes (PBL).IPNV was present in HKL and PBL from all challenged fish groups at all samplings, including carriers. IPNV was also found intracellular in other leucocytes than B-cells and neutrophils. During the time course of infection there were changes in proportion of B-cells and neutrophils and in proportions of IPNV+ cells. In vaccinated fish, a delay in the changes observed in the proportion of IPNV+ cells and in the proportions of the two subpopulations was identified. The vaccinated fish were protected against disease as no fish died compared to 30.8% of non-vaccinated cohabitant fish. All i.p. infected fish, except one, survived the challenge. This is consistent with previous studies and confirmed that the routes of infection can influence mortality. The analyses in this study could not identify any factors enlightening this absence of mortality in i.p. infected fish, but both flow cytometry and qRT-PCR showed that i.p. infected fish were carriers of IPNV. The present study also found that IPNV was present in both B-cells and neutrophils as well as in other leucocytes in all carriers after cohabitant challenge. These fish had survived 9 weeks post challenge and 4 weeks after mortality has ceased. The fish harbouring virus within their leucocytes might become life long carriers and represent a risk for disease outbreaks, being virus shedders. Such fish are protected from later infections if the virus exposure has resulted in protective immunity. Flow cytometry was found to be very suitable for detection of intracellular virus after in vivo challenge and the sensitivity was demonstrated by the detection of virus in carriers.     

Alphavirus-induced apoptosis in mouse brains correlates with neurovirulence.

Sindbis virus induces apoptotic cell death in cultured cell lines, raising the possibility that apoptosis of infected neurons and other target cells in vivo may contribute to the resulting disease and mortality. To investigate the role of apoptosis in Sindbis virus pathogenesis, infected mouse brains were assayed by the in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique and for DNA ladder formation. Infection with recombinant Sindbis virus strain 633 resulted in widespread apoptosis in newborn mouse brains and spinal cords, but few apoptotic cells were observed following infection of 2-week-old animals. This finding correlates with the age-dependent mortality observed in mice. The more neurovirulent virus TE, which differs from 633 by a single amino acid in the E2 glycoprotein, induced significant apoptosis in brains and spinal cords of 2-week-old animals, consistent with its ability to cause fatal disease in older animals. Double-labeling experiments demonstrated that the apoptotic cells were also infected with Sindbis virus. Thus, Sindbis virus-induced apoptosis appears to be a result of virus infection and is likely to reflect pathogenic mechanisms for other viruses.     

Varicella-zoster virus-infected human sensory neurons are resistant to apoptosis, yet human foreskin fibroblasts are susceptible: evidence for a cell-type-specific apoptotic response

The induction of apoptosis or programmed cell death in virus-infected cells is an important antiviral defense mechanism of the host, and some herpesviruses have evolved strategies to modulate apoptosis in order to enhance their survival and spread. In this study, we examined the ability of varicella-zoster virus (VZV) to induce apoptosis in primary human dorsal root ganglion neurons and primary human foreskin fibroblasts (HFFs). Three independent methods (annexin V, TUNEL [terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling] staining, and electron microscopy) were used to assess apoptosis in these cells on days 1, 2, and 4 postinoculation. By all three methods, apoptosis was readily detected in VZV-infected HFFs. In stark contrast, apoptosis was not detected during productive VZV infection of neurons. The low-passage clinical isolate Schenke and the tissue culture-adapted ROka strain both induced apoptosis in HFFs but not in neurons, suggesting that this cell-type-specific apoptotic phenotype was not VZV strain specific. These data show that the regulation of apoptosis differs markedly between HFFs and neurons during productive VZV infection. Inhibition of apoptosis during infection of neurons may play a significant role in the establishment, maintenance, and reactivation of latent infection by promoting survival of these postmitotic cells.     

Comparison of two birnavirus-rhabdovirus coinfections in fish cell lines

Aquabirnaviruses, such as the infectious pancreatic necrosis virus (IPNV), Novirhabdoviruses, such as the infectious hematopoiteic necrosis virus (IHNV) and the viral hemorrhagic septicemia virus (VHSV), cause considerable losses to the salmonid industry worldwide. Coinfections of 2 viruses have been described, but the interactions between rhabdoviruses and birnaviruses have not been examined closely. Using virus titration, flow cytometry and RT-PCR assays, we compared the effect of IPNV on the replication of IHNV and VHSV in tissue culture cells. RT-PCR assays indicated that simultaneous infection of IPNV with VHSV does not affect the replication of the rhabdovirus either in the first or successive passages; the infective titers were similar in single and double infections. In contrast, coinfection of IPNV with IHNV induced a fall in infectivity, with reduced expression of IHNV viral antigens in BF-2 cells from Lepomis macrochirus and a loss of 4.5 log10 units of the infective titer after 3 successive passages. It was possible to stimulate BF-2 cells to produce significant interferon-like activity against IHNV but not against VHSV.     

中华鳖病毒感染宿主的细胞病理学研究

中华鳖病毒经人工感染可引起健康中华鳖发病和死亡。对病鳖组织细胞瓣超微结构观察,同时见有以染色质高度凝集和边缘化,核裂并形成“凋亡小体”等表现有典型的类似于细胞凋记形态变化的细胞,和以核膜崩解、线粒体膜膨大、细胞膜破裂、细胞质严重液泡化等表现为坏死性变化的细胞。ＤＮＡ片段化分析表明,感病鳖组织细胞的ＤＮＡ发生了严重的片段化,且以肝组织细胞的ＤＮＡ片段化最为严重。研究提示,中华鳖病毒导致宿主死亡同时存     

汉滩病毒诱导小鼠骨髓瘤SP2／0细胞凋亡的初步研究

为研究汉滩病毒对肿瘤细胞的诱导凋亡作用,以一定量病毒悬液感染体外培养的SP2／0细胞,接种后一定时间将细胞消化甩片行Gimsa染色观察凋亡细胞核的变化,制细胞悬液以流式细胞仪测细胞周期,并用免疫组化的方法检测凋亡分子Fas和FasL的表达。结果示经病毒诱导后细胞出现生长特性及形态学变化,Giemsa染色观察到典型凋亡细胞：流式细胞仪显示有凋亡峰出现;免疫组化检测出感染后SP2／0细胞中Fas和FasL表达明显升高。该结果表明汉滩病毒可诱导体外培养SP2／0细胞凋亡,其发生可能与凋亡分子Fas和FasL有关。     

ORF390 of white spot syndrome virus genome is identified as a novel anti-apoptosis gene

Apoptosis serves as an important defense strategy employed by host cells against viral invasion. Many viruses contain the anti-apoptotic genes to block the defense-by-death response of host cells. In this study, we tried to identify the putative anti-apoptotic genes in white spot syndrome virus (WSSV) genome. We confirmed that actinomycin D could induce apoptosis of shrimp primary cells. However, the apoptosis triggered by actinomycin D was inhibited by WSSV infection. As mutants of Autographa californica nucleopolyhedrovirus (AcMNPV), AcMNPVDelta35k/pol+ lacks a functional P35 gene undergoing apoptosis and its infection could induce Sf9 cell apoptosis. To identify the putative apoptotic suppressor gene of WSSV, overlapping cosmid clones representing the entire WSSV genome were individually cotransfected along with genome DNA of AcMNPVDeltaP35k/pol+. Using this marker rescue assay, a WSSV DNA fragment that was able to rescue AcMNPVDeltaP35k/pol+ infection in Sf9 cells was isolated. By further sequence analysis and rescue assay, the ORF390 was identified as a novel anti-apoptotic gene. The ORF displays two putative caspase9 cleavage sites LLVETDGPS, VKLEHDGSK, and a caspase3 cleavage site EEDEVDGVP. The ORF was cloned into the pIE1 vector and then the recombinant vector was transfected into Sf9 cells. The Sf9 cells did not show obvious characteristics of apoptosis when infected with AcMNPVDeltaP35k/pol+. And the transient expression of ORF390 allowed AcMNPVDeltaP35k/pol+ replication in Sf9 cells and resulted in the formation of polyhedra successfully. The results indicate that function of ORF390 in WSSV is a kind of apoptotic suppressor like P35 in AcMNPV.     

Study of the viral interference between infectious pancreatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss)

The resistance of rainbow trout (Oncorhynchus mykiss) to an infectious haematopoietic necrosis virus (IHNV) challenge following a preceding non-lethal infection with infectious pancreatic necrosis virus (IPNV) was investigated through experimental dual infections. Trout initially infected with IPNV were inoculated 14 days later with IHNV. Single infections of trout with 1 of the 2 viruses or with cell culture supernatant were also carried out and constituted control groups. No mortality was noted in fish after a single infection with IPNV. This virus had no influence on the head kidney leucocyte phagocytic activity and plasma haemolytic complement activity. IHNV induced a high mortality (72%) and reduced the macrophage phagocytic activity and complement haemolytic activity. It also induced a late production of anti-IHNV antibodies which occurred after clearance of the virus in the fish. In trout co-infected with both viruses, a mortality rate of 2% occurred and the immune parameters were similar to those observed in the fish infected with IPNV only, demonstrating that in co-infected trout IPNV inhibits the effects of IHNV. The studied parameters did not allow us to define the mechanism of interference occurring between these 2 viruses, but some hypothesis are put forward to explain the interference between the 2 viruses.     

VP3, a structural protein of infectious pancreatic necrosis virus, interacts with RNA-dependent RNA polymerase VP1 and with double-stranded RNA

Infectious pancreatic necrosis virus (IPNV) is a bisegmented, double-stranded RNA (dsRNA) virus of the Birnaviridae family that causes widespread disease in salmonids. Its two genomic segments are encapsulated together with the viral RNA-dependent RNA polymerase, VP1, and the assumed internal protein, VP3, in a single-shell capsid composed of VP2. Major aspects of the molecular biology of IPNV, such as particle assembly and interference with host macromolecules, are as yet poorly understood. To understand the infection process, analysis of viral protein interactions is of crucial importance. In this study, we focus on the interaction properties of VP3, the suggested key organizer of particle assembly in birnaviruses. By applying the yeast two-hybrid system in combination with coimmunoprecipitation, VP3 was proven to bind to VP1 and to self-associate strongly. In addition, VP3 was shown to specifically bind to dsRNA in a sequence-independent manner by in vitro pull-down experiments. The binding between VP3 and VP1 was not dependent on the presence of dsRNA. Deletion analyses mapped the VP3 self-interaction domain within the 101 N-terminal amino acids and the VP1 interaction domain within the 62 C-terminal amino acids of VP3. The C-terminal end was also crucial but not sufficient for the dsRNA binding capacity of VP3. For VP1, the 90 C-terminal amino acids constituted the only dispensable part for maintaining VP3-binding ability. Kinetic analysis revealed the presence of VP1-VP3 complexes prior to the formation of mature virions in IPNV-infected CHSE-214 cells, which indicates a role in promoting the assembly process.