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Effects of NV gene knock-out recombinant viral hemorrhagic septicemia virus (VHSV) on Mx gene expression in Epithelioma papulosum cyprini (EPC) cells and olive flounder (Paralichthys olivaceus)
Fish & Shellfish Immunology 32 (2012) 459e463
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Effects of NV gene knock-out recombinant viral hemorrhagic septicemia virus (VHSV) on Mx gene expression in Epithelioma papulosum cyprini (EPC) cells and olive flounder (Paralichthys olivaceus) Min Sun Kim, Ki Hong Kim* Department of Aquatic Life Medicine, Pukyong National University, Nam-gu 599-1, Busan 608-737, Republic of South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 July 2011 Received in revised form 28 December 2011 Accepted 28 December 2011 Available online 2 January 2012
To determine whether the NV gene of viral hemorrhagic septicemia virus (VHSV) is related to the type I interferon response of hosts, expression of Mx gene in Epithelioma papulosum cyprini (EPC) cells and in olive flounder (Paralichthys olivaceus) in response to infection with either wild-type VHSV or recombinant VHSVs (rVHSV-DNV-EGFP and rVHSV-wild) was investigated. A reporter vector was constructed for measuring Mx gene expression using olive flounder Mx promoter, in which the reporter Metridia luciferase was designed to be excreted to culture medium to facilitate measurement. The highest increase of luciferase activity was detected from supernatant of cells infected with rVHSV-DNV-EGFP. In contrast cells infected with wild-type VHSV showed a slight increase of the luciferase activity. Interestingly, cells infected with rVHSV-wild that has artificially changed nucleotides just before and after the NV gene ORF, also showed highly increased luciferase activity, but the increased amplitude was lower than that by rVHSV-DNV-EGFP. These results strongly suggest that the NV protein of VHSV plays an important role in suppressing interferon response in host cells, which provides a condition for the viruses to efficiently proliferate in host cells. In an in vivo experiment, the Mx gene expression in olive flounder challenged with the rVHSV-DNV-EGFP was clearly higher than fish challenged with rVHSV-wild or wild-type VHSV, suggesting that lacking of the NV gene in the genome of rVHSV-DNV-EGFP brought to strong interferon response that subsequently inhibit viral replication in fish. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Recombinant VHSV Role of NV gene Type I interferon response Mx gene expression
1. Introduction Viral hemorrhagic septicemia virus (VHSV), a member of the genus Novirhabdovirus in the family Rhabdoviridae, has the negative-sense, single-stranded RNA genome and is notorious for causing mass mortality in farm-reared freshwater and marine fish worldwide [1e5]. Four genotypes based on the sequence of glycoprotein (G) and nucleoprotein (N) genes have been reported in the world [6e8]. In Korea, only genotype IVa has been identified from marine fishes, and epizootics of VHS have severely affected on the productivity of olive flounder (Paralichthys olivaceus) that is the most important cultured fish in Korea [9,10]. The genome of Novirhabdovirus consists of five structural genes encoding a nucleoprotein (N), a polymerase-associated phosphoprotein (P), a matrix protein (M), a glycoprotein (G), an RNAdependent RNA polymerase (L), and one nonstructural gene
* Corresponding author. Tel.: þ82 51 629 5943; fax: þ82 51 629 5938. E-mail address: [email protected] (K.H. Kim). 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.12.014
encoding non-virion protein (NV) that is present only in species belonging to Novirhabdovirus [11,12]. The role of NV protein of Novirhabdovirus in viral replication and pathogenicity has been investigated using NV gene-knockout recombinant viruses generated by reverse genetics technology. However, the results were variable according to species. In snakehead rhabdovirus (SHRV), the NV protein was not necessary for virus replication in cell culture and did not affect on the in vivo pathogenicity [13]. In contrast, in infectious hematopoietic necrosis virus (IHNV) and VHSV, the NV protein played a significant role in viral replication efficiency and in vivo pathogenesis [14e16]. Similarly, we recently reported the generation of recombinant VHSV that has EGFP gene instead of NV gene (rVHSV-DNV-EGFP) using reverse genetics method, and demonstrated that the NV protein affected on viral replication rate and in vivo virulence [17,18]. Induction of type I interferon response is the first line defense in most vertebrates against viral infections [19]. As viruses are sensitive to interferon-induced antiviral proteins, mechanisms to evade or to suppress host’s interferon response have been adopted in many viruses that are pathogenic to vertebrates. In the present
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study, to determine whether the NV gene of VHSV is related to the interferon response of hosts, Epithelioma papulosum cyprini (EPC) cells and olive flounder were infected with either wild-type VHSV or recombinant VHSVs (rVHSV-DNV-EGFP and rVHSV-wild), and expression of Mx gene, a representative interferon-induced antiviral protein gene [20,21], was analyzed. 2. Materials and methods 2.1. Cells and viruses Epithelioma papulosum cyprini (EPC) cells were cultured in Leibovitz medium (L-15, Sigma) supplemented with penicillin (100 U/ ml), streptomycin (100 mg/ml) and 10% fetal bovine serum (FBS, Gibco). The wild-type VHSV KJ2008 and the recombinant viruses rVHSV-wild and rVHSV-DNV-EGFP [18] were inoculated in monolayer of EPC cells at 15 C in the presence of 2% FBS and antibiotics. Cultures displaying extensive cytopathic effect (CPE) were harvested and centrifuged 4000 g for 10 min at 4 C, and the supernatants were stored at 80 C. 2.2. Construction of a reporter vector to measure Mx gene expression The promoter fragment of olive flounder’s Mx gene was PCRamplified using a primer pair (OFMxF; 50 - AGCGCTGCTGGCAGCATGAATACCTT-30 with AfeI site and OFMxR 50 - ACCGGTCGGGACTCCTGGTCGGTGATGG-30 with AgeI site) and a genomic DNA isolated from olive flounder as a template. The promoter fragment was previously demonstrated as the region that can induce the strongest expression of a reporter gene by stimulation with polyinosinic:polycytidilic acid (poly I:C) [22]. The amplified product (848 bp) was cloned into the pGEM T-easy vector (Promega) and sequenced using an automatic sequencer (Applied biosystems). Then, the cloned fragment was digested with AfeI and AgeI, and subcloned into the pNFkB-MetLuc2-Reporter plasmid (Clontech) that was pre-digested with the same restriction enzymes (by which a region ranging from the NFkB enhancer element to the minimal TA promoter was eliminated), and named it as pOFMx-reporter. 2.3. Selection of EPC cells harboring pOFMx-reporter vector EPC cells were seeded in 35 mm-diameter dishes (3 105 cells/ dish) at 28 C in L-15 medium supplemented with 20 mM HEPES and 10% FBS. Cells were grown to about 80% confluence and transfected with the plasmids pOFMx-reporter using FuGENE6 (Roche) according to manufacturer’s instructions. At 3 days posttransfection, cells were trypsinized using TrypLE Express (Gibco), sub-cultured in culture medium containing G-418 (400 mg/ml, Sigma) for selection. 2.4. Quantitation of Mx1 gene expression in EPC cells using luciferase reporter system EPC cells harboring pOFMx-reporter vector were cultured in 6well plates (3 105 cells/well) at 28 C in L-15 medium containing 10% FBS and antibiotics. Cells were grown to 90% confluence and inoculated with either wild-type VHSV or recombinant VHSVs (rVHSV-wild and rVHSV-DNV-EGFP) at MOI 1.0. After 24, 48 and 72 h of the infection, 50 ml of each cell culture medium was collected, added into 96-well plates, and analyzed for luciferase activity using Ready-To-Glow secreted luciferase reporter assay kit (Clontech) according to the manufacturer’s instructions. The signals of luciferase activity were measured in a VICTOR3 multilabel plate reader (PerkinElmer).
2.5. Quantitation of NV gene expression in EPC cells using semiquantitative RT-PCR EPC cells were incubated in 35 mm-diameter dishes (3 105 cells/ dish) at 28 C with 2 ml of L-15 medium containing 10% FBS and antibiotics (penicillin-streptomycin). Next day, cell monolayers were washed and cultured with fresh medium containing 2% FBS and antibiotics. Cells were infected with either wild-type VHSV or rVHSVwild at MOI 1.0 and were incubated at 15 C. After 24, 48 and 72 h of the infection, total RNA was extracted using RNeasy Plus Mini-Kit (Qiagen) according to the manufacturer’s instruction, and 1 mg of total RNA was incubated with 0.5 ml of random primer (0.5 mg/ml, Promega) at 80 C for 5 min and further incubated at 42 C for 60 min in reaction mixture containing 2 ml of each 10 mM dNTP mix (TaKaRa), 0.5 ml of M-MLV reverse transcriptase (Promega) and 0.25 ml of RNase inhibitor (Promega) in a final reaction volume of 10 ml. PCR was performed with 2 Prime Taq Premix (Genet Bio) and 1 ml of 101 diluted cDNA template. The PCR primer pair used for amplification of the target NV gene was NV-F, 50 - ATGACGACCCAGTCGGCAC-30 , NV-R, 50 TCATGGGGGAGATTCGGAGC-30 ; and for a control P gene was P-F, 50 ATGACTGATATTGAGATGAG-30 , P-R, 50 - CTACTCCAACTTGTCCAAC-30 ; and for another control G gene was G-F, 50 - ATGGAATGGAATACTTTTTTCTTGG-30 , G-R, 50 - TCAGACCATCTGGCTTCTGGAG-30 . Thermal cycling condition was 1 cycle of 3 min at 95 C (initial denaturation) followed by 27 cycles (for NV) or 33 cycles (for P and G) of 30 s at 95 C, 30 s at 60 C, 30 s at 72 C, with a final extension step of 7 min at 72 C. PCR samples to be compared were electrophoresed on the 1% agarose gel, and stained with ethidium bromide (EtBr). 2.6. Quantitation of Mx1 gene expression in olive flounder using semi-quantitative RT-PCR To estimate in vivo Mx gene expression level by infection of the wild-type VHSV, rVHSV-wild, or rVHSV-DNV-EGFP, olive flounder fingerlings reared in 50 L tanks at 15 C were intramuscularly injected with wild-type VHSV, rVHSV-wild, or rVHSV-DNV-EGFP at a dose of 105 PFU per fish. At 24 and 48 h post-infection, the kidney and liver were sampled from 3 fish in each group, and total RNA was extracted using RNAiso plus from each excised organ. Before synthesis of first-strand cDNA, 1 mg of total RNA was treated with DNaseI (promega), and was performed as described above to synthesize cDNA. The PCR primer pair used for amplification of the target Mx gene was OFMx-F, 50 -AACAGCCAAGGCAAAGATTG-30 , and OFMx-R, 50 -AATGTCCAGCTCCTCCTTCA-30 ; and for a control 18S ribosomal RNA gene was 18S-F, 50 -CAAGACGGACGAAAGCGAAAGCAT-30 , and 18S-R, 50 - TGGCATCGTTTACGGTCGGAACTA-30 ; for the P gene was P-F & P-R; and for the NV gene was NV-F & NV-R. Thermal cycling condition was 1 cycle of 3 min at 95 C (initial denaturation) followed by 18 cycles (for 18S rRNA), 24 cycles (for Mx), 33 cycles (for P) or 27 cycles (for NV) of 30 s at 95 C, 30 s at 60 C, 30 s at 72 C, with a final extension step of 7 min at 72 C. PCR samples to be compared were electrophoresed on the 1% agarose gel, and stained with ethidium bromide (EtBr). 2.7. Statistical analyses Data were analyzed by the Student’s t-test. Significant differences were determined at P < 0.05. 3. Results 3.1. Analysis of Mx1 gene expression in EPC cells Type I interferon response of EPC cells against infection of either wild-type VHSV or recombinant VHSVs (rVHSV-wild and rVHSV-
M.S. Kim, K.H. Kim / Fish & Shellfish Immunology 32 (2012) 459e463
DNV-EGFP) was evaluated by analysis of Mx1 gene expression using luciferase reporter system. At 24 h post-infection, all cells infected with VHSVs showed statistically significant increase of luciferase activity (Fig. 1A). EPC cells infected with rVHSV-DNVEGFP showed the strongest luciferase activity, and cells infected with rVHSV-wild showed the second highest luciferase activity (Fig. 1A). Although cells infected with wild-type VHSV also showed significantly higher luciferase activities than mock-infected cells, the increased amplitude was not high. At 48 and 72 h postinfection, the pattern of luciferase activities in cells infected with VHSVs was similar to the pattern shown at 24 h post-infection
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(Fig. 1B,C). The luciferase activity in EPC cells was increased to 48 h post-infection, then was not further increased at 72 h postinfection. 3.2. Analysis of NV gene expression in cells by semi-quantitative RTPCR To know whether the insertion of restriction enzyme sites (ClaI and NarI) just before and after the NV gene ORF of rVHSV-wild affects on the NV gene expression, EPC cells were infected with either wild-type VHSV or rVHSV-wild, and expression of NV gene according to time lapse was compares with that of P and G genes. As a result, cells infected with rVHSV-wild showed markedly lower expression of NV gene and slightly lower expression of P and G genes than cells infected with wild-type VHSV at 24 h postinfection (Fig. 2). The expression level of NV gene in rVHSV-wild at 42 h post-infection was still lower than that in wild-type VHSV, whereas there were no differences in expression of NV, P and G genes expression between cells infected with wild-type VHSV and rVHSV-wild at 72 h post-infection (Fig. 2). 3.3. Analysis of Mx gene expression in olive flounder by semiquantitative RT-PCR To investigate the role of VHSV NV protein in inducing type I interferon responses in olive flounder, fish were challenged with wild-type VHSV, rVHSV-wild, and rVHSV-DNV-EGFP, and expression of Mx gene in the liver and the head-kidney was analyzed using semi-quantitative RT-PCR. As a result, fish infected with rVHSV-DNV-EGFP showed clearly higher Mx gene expression than fish infected with wild-type VHSV and rVHSV-wild at 24 and 48 h post-infection. Although expression of Mx gene was detected from the head kidney of fish infected with wild-type VHSV at 24 and 48 h p.i., the expression level was lower than that of fish infected with rVHSV-wild (Fig. 3A, B). The expression of viral NV and P genes from fish infected with rVHSV-wild was weaker than that from fish infected with wild-type VHSV. The fish infected with rVHSV-DNV-EGFP showed the lowest expression of P gene. 4. Discussion Recently, Ooi et al. [22] functionally characterized olive flounder Mx promoter by construction of reporter vector that had a cassette
Fig. 1. Analysis of Mx1 gene expression in Epithelioma papulosum cyprini (EPC) cells by measuring activity of luciferase that was used as the reporter protein. EPC cells were infected with wild-type VHSV, rVHSV-wild, or rVHSV-DNV-EGFP at MOI 1.0, and luciferase activity was analyzed at (A) 24 h, (B) 48 h, and (C) 72 h post-infection. Different letters on the bars represent significantly different at P < 0.05.
Fig. 2. Analysis of NV gene expression Epithelioma papulosum cyprini (EPC) cells by semi-quantitative RT-PCR. EPC cells were inoculated with wild-type VHSV (wV) and rVHSV-wild (rV) at MOI. 1.0, and relative expression of NV gene to structural viral protein P and G gene was analyzed at 24 h, 48 h and 72 h post-infection.
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Fig. 3. Analysis of Mx gene expression in olive flounder (Paralichthys olivaceus) by semi-quantitative RT-PCR. Olive flounder fingerlings were intramuscularly injected with wildtype VHSV (wtV), rVHSV-wild (rV-wild), and rVHSV-DNV-EGFP (rV-DNV) at a dose of 105 PFU per fish, and relative expression of Mx gene, viral NV gene and P gene to 18S ribosomal RNA gene in the liver and the kidney was analyzed at (A) 24 h and (B) 48 h post-infection.
of the olive flounder Mx promoter-driven luciferase reporter gene. In the present study, we generated a reporter vector for measuring Mx gene expression using olive flounder Mx promoter, in which the reporter luciferase was designed to be secreted to culture medium that confers convenience for measurement. Feasibility of the EPC cells harboring the reporter vector to measure Mx gene expression was preliminarily confirmed by comparison of the luciferase activity with the value of real time RT-PCR. Induction of Mx gene expression by VHSV infection has been demonstrated in vitro and in vivo experiments in rainbow trout [23,24] and in olive flounder [25,26]. Furthermore, antiviral activity of Mx proteins in fish has also been demonstrated from Atlantic salmon Mx1 protein that suppressed infectious pancreatic necrosis virus (IHNV) replication [27], and from olive flounder Mx protein that inhibit replication of two novirhabdoviruses, hirame rhabdovirus (HIRRV) and VHSV [28]. In mammalian rhabdoviruses, such as vesicular stomatitis virus and rabies virus, several mechanisms that can antagonize type I interferon response have been reported, which results in proliferation of the viruses in their hosts [29]. However, there has been no report in VHSV on the viral interfering mechanisms against host’s type I interferon response. In the present results, the highest increase of luciferase activity was detected from supernatant of cells infected with rVHSV-DNVEGFP. In contrast cells infected with wild-type VHSV showed a slight increase of the luciferase activity. These results suggest that the NV protein of VHSV might have a capability to suppress type I interferon response in EPC cells. Interestingly, cells infected with rVHSV-wild also showed highly increased luciferase activity compared to cells infected with wild-type VHSV, but the increased amplitude was lower than that by rVHSV-DNV-EGFP. Although the NV gene ORF of rVHSV-wild has the same nucleotides with wildtype VHSV, restriction enzyme sites (ClaI and NarI) were artificially made just before and after the NV gene ORF, respectively, by replacement of the original nucleotides [18]. This change might
affect on the NV gene expression, which would lead to the present intermediate expression of Mx gene in EPC cells infected with rVHSV-wild. In fact, previously, we had demonstrated that the replication efficiency and in vivo virulence of rVHSV-wild were lower than those of wild-type VHSV [17,18]. These results strongly suggest that the NV protein of VHSV plays an important role in suppressing type I interferon response in host cells, which provides a condition for the viruses to efficiently proliferate in host cells. In this study, the luciferase activities in cells infected with rVHSV-DNV-EGFP and rVHSV-wild were steeply increased until 48 h post-infection, whereas there were no further increase of the luciferase activities between 48 and 72 h post-infection, suggesting that EPC cells might respond to rVHSV-DNV-EGFP and rVHSV-wild by early interferon responses, and, then, the intensity of interferon responses might be diminished according to increase of the virus titer and host cell damage. The present in vivo experimental results also provide evidence for the effect of NV protein on fish type I interferon response. The Mx gene expression in olive flounder challenged with the rVHSVDNV-EGFP was clearly higher than fish challenged with rVHSVwild or wild-type VHSV, suggesting that a high interferon response can be elicited by lack of the NV gene of the challenged virus. In our previous study, it was demonstrated that pathogenicity of rVHSV-DNV-EGFP was so lowered that intramuscular (i.m.) injection of olive flounder with even 105 PFU of rVHSV-DNVEGFP induced no mortality, and the mutated VHSV showed limited persistence in internal organs of the fish [17]. Therefore, it might be conjectured that lacking of the NV gene in the genome of rVHSVDNV-EGFP brought to strong interferon response that subsequently inhibit viral replication in fish. In conclusion, we have firstly demonstrated the type I interferon-suppressive role of the NV protein of VHSV through in vitro EPC cells and in vivo olive flounder experiments. Further studies on the NV protein-mediated signal pathway are needed to uncover the action mechanism of the NV protein.
M.S. Kim, K.H. Kim / Fish & Shellfish Immunology 32 (2012) 459e463
Acknowledgment This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0011027). References [1] Schlotfeldt HJ, Ahne W. Epizootics in brown trout (Salmo trutta fario) caused by VHSV-F1. J App Ichthyol 1988;4:147e8. [2] Schlotfeldt HJ, Ahne W, Vestergard-Jorgensen PE, Glende W. Occurrence of viral haemorrhagic septicaemia in turbot (Scophthalmus maximus) - a natural outbreak. EAFP Bull 1991;11:105e7. [3] Mortensen HF, Heuer OE, Lorenzen N, Otte L, Olesen NJ. Isolation of viral haemorrhagic septicaemia virus (VHSV) from wild marine fish species in the Baltic Sea, Kattegat, Skagerrak and the North Sea. Virus Res 1999;63:95e106. [4] Isshiki T, Nagano T, Miyazaki T. Susceptibility of various marine fish species to viral hemorrhagic septicemia virus isolated from Japanese flounder. Fish Pathol 2003;38:113e5. [5] Skall HF, Olesen NJ, Mellergaard S. Viral hemorrhagic septicemia virus in marine fish and its implications for fish farming - a review. J Fish Dis 2005;28:509e29. [6] Einer-Jensen K, Ahrens P, Forsberg R, Lorenzen N. Evolution of the fish rhabdovirus viral haemorrhagic septicaemia virus. J Gen Virol 2004;85:1167e79. [7] Snow M, Cunningham CO, Melvin WT, Kurath G. Analysis of the nucleoprotein gene identifies distinct lineages of viral haemorrhagic septicaemia virus within the European marine environment. Virus Res 1999;63:35e44. [8] Snow M, Bain N, Black J, Taupin V, Cunningham CO, King JA, et al. Genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV). Dis Aquat Org 2004;61:11e21. [9] Kim SM, Lee JI, Hong MJ, Park HS, Park SI. Genetic relationship of the VHSV (viral hemorrhagic septicaemia virus) isolated from cultured olive flounder, Paralichthys olivaceus in Korea. J Fish Pathol 2003;16:1e12 [in Korean with English abstract]. [10] Kim WS, Kim SR, Kim D, Kim JO, Park MA, Kitamura SI, et al. An outbreak of VHSV (viral hemorrhagic septicemia virus) infection in farmed olive flounder Paralichthys olivaceus in Korea. Aquaculture 2009;296:165e8. [11] Schütze H, Mundt E, Mettenleiter TC. Complete genomic sequence of viral haemorrhagic septicemia virus, a fish rhabdovirus. Virus Genes 1999;19: 59e65. [12] Tordo N, Benmansour A, Calisher C, Dietzgen RG, Fang RX, Jackson AO, et al. Family Rhabdoviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, editors. Virus taxonomy. Eight report of the international committee on taxonomy of viruses. London: Elsevier Academic Press; 2005. p. 623e53. [13] Alonso M, Kim CH, Johnson MC, Pressley M, Leong JC. The NV gene of snakehead rhabdovirus (SHRV) is not required for pathogenesis, and a heterologous glycoprotein can be incorporated into the SHRV envelope. J Virol 2004;78:5875e82.
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[14] Biacchesi S, Thoulouze MI, Bearzotti M, Yu YX, Bremont M. Recovery of NV knockout infectious hematopoietic necrosis virus expressing foreign genes. J Virol 2000;74:11247e53. [15] Thoulouze MI, Bouguyon E, Carpentier C, Bremont M. Essential role of the NV protein of novirhabdovirus for pathogenicity in rainbow trout. J Virol 2004; 78:4098e107. [16] Ammayappan A, Kurath G, Thompson TM, Vakharia VN. A reverse genetics system for the Great Lakes strain of viral hemorrhagic septicemia virus: the NV gene is required for pathogenicity. Mar Biotechnol 2010;169:112e8. [17] Kim MS, Kim KH. Protection of olive flounder, Paralichthys olivaceus, against viral hemorrhagic septicemia virus (VHSV) by immunization with NV geneknockout recombinant VHSV. Aquaculture 2011;314:39e43. [18] Kim MS, Kim DS, Kim KH. Generation and characterization of NV geneknockout recombinant viral hemorrhagic septicemia virus (VHSV) genotype IVa. Dis Aquat Org 2011;97:25e35. [19] Levy DE, Garcia-Sastre A. The virus battles: IFN induction of the antiviral state and mechanisms of viral evasion. Cytokine Growth Factor Rev 2001;12: 143e56. [20] Haller O, Kochs G. Interferon-induced Mx proteins: dynamin-like GTPases with antiviral activity. Traffic 2002;3:710e7. [21] Haller O, Staeheli P, Kochs G. Interferon-induced Mx proteins in antiviral host defense. Biochimie 2007;89:812e8. [22] Ooi EL, Hirono I, Aoki T. Functional characterisation of the Japanese flounder, Paralichthys olivaceus, Mx promoter. Fish Shellfish Immunol 2006;21: 293e304. [23] O’Farrell C, Vaghefi N, Cantonnet M, Buteau B, Boudinot P, Benmansour A. Survey of transcript expression in rainbow trout leukocytes reveals a major contribution of interferon-responsive genes in the early response to a rhabdovirus infection. J Virol 2002;76:8040e9. [24] Tafalla C, Chico V, Pérez L, Coll JM, Estepa A. In vitro and in vivo differential expression of rainbow trout (Oncorhynchus mykiss) Mx isoforms in response to viral haemorrhagic septicaemia virus (VHSV) G gene; Poly I: C and VHSV. Fish Shellfish Immunol 2007;23:210e21. [25] Matsuyama T, Fujiwara A, Takano T, Nakayasu C. Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in Japanese flounder Paralichthys olivaceus leucocytes during Edwardsiella tarda and viral hemorrhagic septicemia virus infection. Fish Shellfish Immunol; 2011. doi:10.1016/j.fsi.2011.06.015. [26] Avunje S, Kim W-S, Park C-S, Oh M-J, Jung S- J. Toll-like receptors and interferon associated immune factors in viral haemorrhagic septicaemia virus-infected olive flounder (Paralichthys olivaceus). Fish Shellfish Immunol; 2011. doi:10.1016/j.fsi.2011.06.009. [27] Larsen R, Røkenes TP, Robertsen B. Inhibition of infectious pancreatic necrosis virus replication by Atlantic salmon Mx1 protein. J Virol 2004;78(15): 7938e44. [28] Caipang CM, Hirono I, Aoki T. In vitro inhibition of fish rhabdoviruses by Japanese flounder, Paralichthys olivaceus Mx. Virology 2003;317:373e82. [29] Faul EJ, Lyles DS, Schnell MJ. Interferon response and viral evasion by members of the family Rhabdoviridae. Viruses 2009;1:832e51.
Contents lists available at SciVerse ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Effects of NV gene knock-out recombinant viral hemorrhagic septicemia virus (VHSV) on Mx gene expression in Epithelioma papulosum cyprini (EPC) cells and olive flounder (Paralichthys olivaceus) Min Sun Kim, Ki Hong Kim* Department of Aquatic Life Medicine, Pukyong National University, Nam-gu 599-1, Busan 608-737, Republic of South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 July 2011 Received in revised form 28 December 2011 Accepted 28 December 2011 Available online 2 January 2012
To determine whether the NV gene of viral hemorrhagic septicemia virus (VHSV) is related to the type I interferon response of hosts, expression of Mx gene in Epithelioma papulosum cyprini (EPC) cells and in olive flounder (Paralichthys olivaceus) in response to infection with either wild-type VHSV or recombinant VHSVs (rVHSV-DNV-EGFP and rVHSV-wild) was investigated. A reporter vector was constructed for measuring Mx gene expression using olive flounder Mx promoter, in which the reporter Metridia luciferase was designed to be excreted to culture medium to facilitate measurement. The highest increase of luciferase activity was detected from supernatant of cells infected with rVHSV-DNV-EGFP. In contrast cells infected with wild-type VHSV showed a slight increase of the luciferase activity. Interestingly, cells infected with rVHSV-wild that has artificially changed nucleotides just before and after the NV gene ORF, also showed highly increased luciferase activity, but the increased amplitude was lower than that by rVHSV-DNV-EGFP. These results strongly suggest that the NV protein of VHSV plays an important role in suppressing interferon response in host cells, which provides a condition for the viruses to efficiently proliferate in host cells. In an in vivo experiment, the Mx gene expression in olive flounder challenged with the rVHSV-DNV-EGFP was clearly higher than fish challenged with rVHSV-wild or wild-type VHSV, suggesting that lacking of the NV gene in the genome of rVHSV-DNV-EGFP brought to strong interferon response that subsequently inhibit viral replication in fish. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Recombinant VHSV Role of NV gene Type I interferon response Mx gene expression
1. Introduction Viral hemorrhagic septicemia virus (VHSV), a member of the genus Novirhabdovirus in the family Rhabdoviridae, has the negative-sense, single-stranded RNA genome and is notorious for causing mass mortality in farm-reared freshwater and marine fish worldwide [1e5]. Four genotypes based on the sequence of glycoprotein (G) and nucleoprotein (N) genes have been reported in the world [6e8]. In Korea, only genotype IVa has been identified from marine fishes, and epizootics of VHS have severely affected on the productivity of olive flounder (Paralichthys olivaceus) that is the most important cultured fish in Korea [9,10]. The genome of Novirhabdovirus consists of five structural genes encoding a nucleoprotein (N), a polymerase-associated phosphoprotein (P), a matrix protein (M), a glycoprotein (G), an RNAdependent RNA polymerase (L), and one nonstructural gene
* Corresponding author. Tel.: þ82 51 629 5943; fax: þ82 51 629 5938. E-mail address: [email protected] (K.H. Kim). 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.12.014
encoding non-virion protein (NV) that is present only in species belonging to Novirhabdovirus [11,12]. The role of NV protein of Novirhabdovirus in viral replication and pathogenicity has been investigated using NV gene-knockout recombinant viruses generated by reverse genetics technology. However, the results were variable according to species. In snakehead rhabdovirus (SHRV), the NV protein was not necessary for virus replication in cell culture and did not affect on the in vivo pathogenicity [13]. In contrast, in infectious hematopoietic necrosis virus (IHNV) and VHSV, the NV protein played a significant role in viral replication efficiency and in vivo pathogenesis [14e16]. Similarly, we recently reported the generation of recombinant VHSV that has EGFP gene instead of NV gene (rVHSV-DNV-EGFP) using reverse genetics method, and demonstrated that the NV protein affected on viral replication rate and in vivo virulence [17,18]. Induction of type I interferon response is the first line defense in most vertebrates against viral infections [19]. As viruses are sensitive to interferon-induced antiviral proteins, mechanisms to evade or to suppress host’s interferon response have been adopted in many viruses that are pathogenic to vertebrates. In the present
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study, to determine whether the NV gene of VHSV is related to the interferon response of hosts, Epithelioma papulosum cyprini (EPC) cells and olive flounder were infected with either wild-type VHSV or recombinant VHSVs (rVHSV-DNV-EGFP and rVHSV-wild), and expression of Mx gene, a representative interferon-induced antiviral protein gene [20,21], was analyzed. 2. Materials and methods 2.1. Cells and viruses Epithelioma papulosum cyprini (EPC) cells were cultured in Leibovitz medium (L-15, Sigma) supplemented with penicillin (100 U/ ml), streptomycin (100 mg/ml) and 10% fetal bovine serum (FBS, Gibco). The wild-type VHSV KJ2008 and the recombinant viruses rVHSV-wild and rVHSV-DNV-EGFP [18] were inoculated in monolayer of EPC cells at 15 C in the presence of 2% FBS and antibiotics. Cultures displaying extensive cytopathic effect (CPE) were harvested and centrifuged 4000 g for 10 min at 4 C, and the supernatants were stored at 80 C. 2.2. Construction of a reporter vector to measure Mx gene expression The promoter fragment of olive flounder’s Mx gene was PCRamplified using a primer pair (OFMxF; 50 - AGCGCTGCTGGCAGCATGAATACCTT-30 with AfeI site and OFMxR 50 - ACCGGTCGGGACTCCTGGTCGGTGATGG-30 with AgeI site) and a genomic DNA isolated from olive flounder as a template. The promoter fragment was previously demonstrated as the region that can induce the strongest expression of a reporter gene by stimulation with polyinosinic:polycytidilic acid (poly I:C) [22]. The amplified product (848 bp) was cloned into the pGEM T-easy vector (Promega) and sequenced using an automatic sequencer (Applied biosystems). Then, the cloned fragment was digested with AfeI and AgeI, and subcloned into the pNFkB-MetLuc2-Reporter plasmid (Clontech) that was pre-digested with the same restriction enzymes (by which a region ranging from the NFkB enhancer element to the minimal TA promoter was eliminated), and named it as pOFMx-reporter. 2.3. Selection of EPC cells harboring pOFMx-reporter vector EPC cells were seeded in 35 mm-diameter dishes (3 105 cells/ dish) at 28 C in L-15 medium supplemented with 20 mM HEPES and 10% FBS. Cells were grown to about 80% confluence and transfected with the plasmids pOFMx-reporter using FuGENE6 (Roche) according to manufacturer’s instructions. At 3 days posttransfection, cells were trypsinized using TrypLE Express (Gibco), sub-cultured in culture medium containing G-418 (400 mg/ml, Sigma) for selection. 2.4. Quantitation of Mx1 gene expression in EPC cells using luciferase reporter system EPC cells harboring pOFMx-reporter vector were cultured in 6well plates (3 105 cells/well) at 28 C in L-15 medium containing 10% FBS and antibiotics. Cells were grown to 90% confluence and inoculated with either wild-type VHSV or recombinant VHSVs (rVHSV-wild and rVHSV-DNV-EGFP) at MOI 1.0. After 24, 48 and 72 h of the infection, 50 ml of each cell culture medium was collected, added into 96-well plates, and analyzed for luciferase activity using Ready-To-Glow secreted luciferase reporter assay kit (Clontech) according to the manufacturer’s instructions. The signals of luciferase activity were measured in a VICTOR3 multilabel plate reader (PerkinElmer).
2.5. Quantitation of NV gene expression in EPC cells using semiquantitative RT-PCR EPC cells were incubated in 35 mm-diameter dishes (3 105 cells/ dish) at 28 C with 2 ml of L-15 medium containing 10% FBS and antibiotics (penicillin-streptomycin). Next day, cell monolayers were washed and cultured with fresh medium containing 2% FBS and antibiotics. Cells were infected with either wild-type VHSV or rVHSVwild at MOI 1.0 and were incubated at 15 C. After 24, 48 and 72 h of the infection, total RNA was extracted using RNeasy Plus Mini-Kit (Qiagen) according to the manufacturer’s instruction, and 1 mg of total RNA was incubated with 0.5 ml of random primer (0.5 mg/ml, Promega) at 80 C for 5 min and further incubated at 42 C for 60 min in reaction mixture containing 2 ml of each 10 mM dNTP mix (TaKaRa), 0.5 ml of M-MLV reverse transcriptase (Promega) and 0.25 ml of RNase inhibitor (Promega) in a final reaction volume of 10 ml. PCR was performed with 2 Prime Taq Premix (Genet Bio) and 1 ml of 101 diluted cDNA template. The PCR primer pair used for amplification of the target NV gene was NV-F, 50 - ATGACGACCCAGTCGGCAC-30 , NV-R, 50 TCATGGGGGAGATTCGGAGC-30 ; and for a control P gene was P-F, 50 ATGACTGATATTGAGATGAG-30 , P-R, 50 - CTACTCCAACTTGTCCAAC-30 ; and for another control G gene was G-F, 50 - ATGGAATGGAATACTTTTTTCTTGG-30 , G-R, 50 - TCAGACCATCTGGCTTCTGGAG-30 . Thermal cycling condition was 1 cycle of 3 min at 95 C (initial denaturation) followed by 27 cycles (for NV) or 33 cycles (for P and G) of 30 s at 95 C, 30 s at 60 C, 30 s at 72 C, with a final extension step of 7 min at 72 C. PCR samples to be compared were electrophoresed on the 1% agarose gel, and stained with ethidium bromide (EtBr). 2.6. Quantitation of Mx1 gene expression in olive flounder using semi-quantitative RT-PCR To estimate in vivo Mx gene expression level by infection of the wild-type VHSV, rVHSV-wild, or rVHSV-DNV-EGFP, olive flounder fingerlings reared in 50 L tanks at 15 C were intramuscularly injected with wild-type VHSV, rVHSV-wild, or rVHSV-DNV-EGFP at a dose of 105 PFU per fish. At 24 and 48 h post-infection, the kidney and liver were sampled from 3 fish in each group, and total RNA was extracted using RNAiso plus from each excised organ. Before synthesis of first-strand cDNA, 1 mg of total RNA was treated with DNaseI (promega), and was performed as described above to synthesize cDNA. The PCR primer pair used for amplification of the target Mx gene was OFMx-F, 50 -AACAGCCAAGGCAAAGATTG-30 , and OFMx-R, 50 -AATGTCCAGCTCCTCCTTCA-30 ; and for a control 18S ribosomal RNA gene was 18S-F, 50 -CAAGACGGACGAAAGCGAAAGCAT-30 , and 18S-R, 50 - TGGCATCGTTTACGGTCGGAACTA-30 ; for the P gene was P-F & P-R; and for the NV gene was NV-F & NV-R. Thermal cycling condition was 1 cycle of 3 min at 95 C (initial denaturation) followed by 18 cycles (for 18S rRNA), 24 cycles (for Mx), 33 cycles (for P) or 27 cycles (for NV) of 30 s at 95 C, 30 s at 60 C, 30 s at 72 C, with a final extension step of 7 min at 72 C. PCR samples to be compared were electrophoresed on the 1% agarose gel, and stained with ethidium bromide (EtBr). 2.7. Statistical analyses Data were analyzed by the Student’s t-test. Significant differences were determined at P < 0.05. 3. Results 3.1. Analysis of Mx1 gene expression in EPC cells Type I interferon response of EPC cells against infection of either wild-type VHSV or recombinant VHSVs (rVHSV-wild and rVHSV-
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DNV-EGFP) was evaluated by analysis of Mx1 gene expression using luciferase reporter system. At 24 h post-infection, all cells infected with VHSVs showed statistically significant increase of luciferase activity (Fig. 1A). EPC cells infected with rVHSV-DNVEGFP showed the strongest luciferase activity, and cells infected with rVHSV-wild showed the second highest luciferase activity (Fig. 1A). Although cells infected with wild-type VHSV also showed significantly higher luciferase activities than mock-infected cells, the increased amplitude was not high. At 48 and 72 h postinfection, the pattern of luciferase activities in cells infected with VHSVs was similar to the pattern shown at 24 h post-infection
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(Fig. 1B,C). The luciferase activity in EPC cells was increased to 48 h post-infection, then was not further increased at 72 h postinfection. 3.2. Analysis of NV gene expression in cells by semi-quantitative RTPCR To know whether the insertion of restriction enzyme sites (ClaI and NarI) just before and after the NV gene ORF of rVHSV-wild affects on the NV gene expression, EPC cells were infected with either wild-type VHSV or rVHSV-wild, and expression of NV gene according to time lapse was compares with that of P and G genes. As a result, cells infected with rVHSV-wild showed markedly lower expression of NV gene and slightly lower expression of P and G genes than cells infected with wild-type VHSV at 24 h postinfection (Fig. 2). The expression level of NV gene in rVHSV-wild at 42 h post-infection was still lower than that in wild-type VHSV, whereas there were no differences in expression of NV, P and G genes expression between cells infected with wild-type VHSV and rVHSV-wild at 72 h post-infection (Fig. 2). 3.3. Analysis of Mx gene expression in olive flounder by semiquantitative RT-PCR To investigate the role of VHSV NV protein in inducing type I interferon responses in olive flounder, fish were challenged with wild-type VHSV, rVHSV-wild, and rVHSV-DNV-EGFP, and expression of Mx gene in the liver and the head-kidney was analyzed using semi-quantitative RT-PCR. As a result, fish infected with rVHSV-DNV-EGFP showed clearly higher Mx gene expression than fish infected with wild-type VHSV and rVHSV-wild at 24 and 48 h post-infection. Although expression of Mx gene was detected from the head kidney of fish infected with wild-type VHSV at 24 and 48 h p.i., the expression level was lower than that of fish infected with rVHSV-wild (Fig. 3A, B). The expression of viral NV and P genes from fish infected with rVHSV-wild was weaker than that from fish infected with wild-type VHSV. The fish infected with rVHSV-DNV-EGFP showed the lowest expression of P gene. 4. Discussion Recently, Ooi et al. [22] functionally characterized olive flounder Mx promoter by construction of reporter vector that had a cassette
Fig. 1. Analysis of Mx1 gene expression in Epithelioma papulosum cyprini (EPC) cells by measuring activity of luciferase that was used as the reporter protein. EPC cells were infected with wild-type VHSV, rVHSV-wild, or rVHSV-DNV-EGFP at MOI 1.0, and luciferase activity was analyzed at (A) 24 h, (B) 48 h, and (C) 72 h post-infection. Different letters on the bars represent significantly different at P < 0.05.
Fig. 2. Analysis of NV gene expression Epithelioma papulosum cyprini (EPC) cells by semi-quantitative RT-PCR. EPC cells were inoculated with wild-type VHSV (wV) and rVHSV-wild (rV) at MOI. 1.0, and relative expression of NV gene to structural viral protein P and G gene was analyzed at 24 h, 48 h and 72 h post-infection.
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Fig. 3. Analysis of Mx gene expression in olive flounder (Paralichthys olivaceus) by semi-quantitative RT-PCR. Olive flounder fingerlings were intramuscularly injected with wildtype VHSV (wtV), rVHSV-wild (rV-wild), and rVHSV-DNV-EGFP (rV-DNV) at a dose of 105 PFU per fish, and relative expression of Mx gene, viral NV gene and P gene to 18S ribosomal RNA gene in the liver and the kidney was analyzed at (A) 24 h and (B) 48 h post-infection.
of the olive flounder Mx promoter-driven luciferase reporter gene. In the present study, we generated a reporter vector for measuring Mx gene expression using olive flounder Mx promoter, in which the reporter luciferase was designed to be secreted to culture medium that confers convenience for measurement. Feasibility of the EPC cells harboring the reporter vector to measure Mx gene expression was preliminarily confirmed by comparison of the luciferase activity with the value of real time RT-PCR. Induction of Mx gene expression by VHSV infection has been demonstrated in vitro and in vivo experiments in rainbow trout [23,24] and in olive flounder [25,26]. Furthermore, antiviral activity of Mx proteins in fish has also been demonstrated from Atlantic salmon Mx1 protein that suppressed infectious pancreatic necrosis virus (IHNV) replication [27], and from olive flounder Mx protein that inhibit replication of two novirhabdoviruses, hirame rhabdovirus (HIRRV) and VHSV [28]. In mammalian rhabdoviruses, such as vesicular stomatitis virus and rabies virus, several mechanisms that can antagonize type I interferon response have been reported, which results in proliferation of the viruses in their hosts [29]. However, there has been no report in VHSV on the viral interfering mechanisms against host’s type I interferon response. In the present results, the highest increase of luciferase activity was detected from supernatant of cells infected with rVHSV-DNVEGFP. In contrast cells infected with wild-type VHSV showed a slight increase of the luciferase activity. These results suggest that the NV protein of VHSV might have a capability to suppress type I interferon response in EPC cells. Interestingly, cells infected with rVHSV-wild also showed highly increased luciferase activity compared to cells infected with wild-type VHSV, but the increased amplitude was lower than that by rVHSV-DNV-EGFP. Although the NV gene ORF of rVHSV-wild has the same nucleotides with wildtype VHSV, restriction enzyme sites (ClaI and NarI) were artificially made just before and after the NV gene ORF, respectively, by replacement of the original nucleotides [18]. This change might
affect on the NV gene expression, which would lead to the present intermediate expression of Mx gene in EPC cells infected with rVHSV-wild. In fact, previously, we had demonstrated that the replication efficiency and in vivo virulence of rVHSV-wild were lower than those of wild-type VHSV [17,18]. These results strongly suggest that the NV protein of VHSV plays an important role in suppressing type I interferon response in host cells, which provides a condition for the viruses to efficiently proliferate in host cells. In this study, the luciferase activities in cells infected with rVHSV-DNV-EGFP and rVHSV-wild were steeply increased until 48 h post-infection, whereas there were no further increase of the luciferase activities between 48 and 72 h post-infection, suggesting that EPC cells might respond to rVHSV-DNV-EGFP and rVHSV-wild by early interferon responses, and, then, the intensity of interferon responses might be diminished according to increase of the virus titer and host cell damage. The present in vivo experimental results also provide evidence for the effect of NV protein on fish type I interferon response. The Mx gene expression in olive flounder challenged with the rVHSVDNV-EGFP was clearly higher than fish challenged with rVHSVwild or wild-type VHSV, suggesting that a high interferon response can be elicited by lack of the NV gene of the challenged virus. In our previous study, it was demonstrated that pathogenicity of rVHSV-DNV-EGFP was so lowered that intramuscular (i.m.) injection of olive flounder with even 105 PFU of rVHSV-DNVEGFP induced no mortality, and the mutated VHSV showed limited persistence in internal organs of the fish [17]. Therefore, it might be conjectured that lacking of the NV gene in the genome of rVHSVDNV-EGFP brought to strong interferon response that subsequently inhibit viral replication in fish. In conclusion, we have firstly demonstrated the type I interferon-suppressive role of the NV protein of VHSV through in vitro EPC cells and in vivo olive flounder experiments. Further studies on the NV protein-mediated signal pathway are needed to uncover the action mechanism of the NV protein.
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