*Author for correspondence: Yasushi Okinaka. Telephone: Fax:

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1 Hata et al., 00 Identification of RNA regions that determine temperature sensitivities in betanodaviruses 0 Naomi Hata, Yasushi Okinaka,, * Tokinori Iwamoto, Yasuhiko Kawato, Koh-Ichiro Mori, and Toshihiro Nakai Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima -, Japan Atlantic Veterinary College, University of Prince Edward Island, PE Canada CA P, Canada National Research Institute of Aquaculture, Fisheries Research Agency, Saiki -0, Japan *Author for correspondence: Yasushi Okinaka. Telephone: Fax: okinaka@hiroshima-u.ac.jp Running title: RNA regions controlling temperature sensitivity in betanodaviruses Key words: betanodavirus, temperature sensitivity, reassortant, chimeric virus, viral growth, RNA replication 0

2 Hata et al., Summary Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The larger genomic segment RNA (~. kb) encodes an RNA-dependent RNA polymerase (protein A) and the smaller genomic segment RNA (~. kb) codes for the coat protein. The viruses can be classified basically into four genotypes, designated striped jack nervous necrosis virus (SJNNV), redspotted grouper nervous necrosis virus (RGNNV), tiger puffer nervous necrosis virus (TPNNV), and barfin flounder nervous necrosis virus (BFNNV), based on similarities in the partial RNA sequences. The optimal temperatures for the growth of these viruses are 0 C (SJNNV), 0 C (RGNNV), 0 C (TPNNV), and 0 C (BFNNV). However, little is known about the mechanisms underlying temperature sensitivities of these viruses. We first constructed two reassortants between SJNNV and RGNNV to test their temperature sensitivity. The levels of viral growth and RNA replication of these reassortants and parental viruses in cultured fish cells were similar at C. However, the levels of all the viruses but RGNNV were markedly reduced at 0 C. These results indicate that both RNA and RNA control temperature sensitivities of betanodaviruses via modulating RNA replication or earlier viral growth processes. We then constructed 0 mutated RGNNV, the RNA segment of which was chimeric between SJNNV and RGNNV, and showed that only chimeric viruses bearing the RGNNV RNA region, encoding th amino acid residues, grew similarly to the parental RGNNV at 0 C. The portion of protein A is known to serve a mitochondrial-targeting signal rather than functions as an enzymatic domain.

3 Hata et al., Introduction Betanodaviruses cause a highly destructive disease of hatchery-reared larvae and juveniles of a variety of marine fish. Adult and mature fish have also sufferred from the disease in some species in aquaculture facilities. The disease, designated as viral nervous necrosis (VNN) or viral encephalopathy and retinopathy, has spread worldwide to a large munber of marine fish species [, 0]. The viruses multiply in the brains, spinal cords, and retinas of the affected fish. Recently, the viruses were detected in the organs of apparently healthy wild marine fish [, ]. Betanodavirus is comprised in the family Nodaviridae along with the other member alphanodavirus that infects insects []. Betanodaviruses are nonenveloped, spherical viruses (~ nm in diameter) with a bipartite positive-sense RNA genome RNA (~. kb) and RNA (~. kb), which encode an RNA-dependent RNA polymerase (protein A) and the coat protein (CP), respectively [, ]. Recently, we characterized a subgenomic RNA (0. kb), which encodes protein B being a suppressor for post-transcriptional gene silencing []. The viruses can be classified basically into four genotypes, designated striped jack nervous necrosis virus (SJNNV), barfin flounder nervous necrosis virus (BFNNV), tiger puffer nervous necrosis virus (TPNNV), and redspotted grouper nervous necrosis virus (RGNNV), based on similarities in the partial RNA sequences [, ]. Recently, a betanodavirus isolate from turbot Scophthalmus maximus (TNV) was suggested to belong to a fifth genotype []. The host ranges of TPNNV are limited to tiger puffer (Takifugu rubripes). SJNNV was isolated from only striped jack (Pseudocaranx dentex) with some exceptions [, ]. BFNNV has been isolated from some coldwater species, such as barfin flounder (Verasper moseri), Pacific cod (Gadus macrocephalus) and Atlantic halibut (Hippoglossus hippoglossus).

4 Hata et al., 00 0 In contrast, RGNNV has a broad host range and causes the disease among a variety of warm water fish species, particularly groupers and sea bass []. RNA is known to be a determinant for host-specificity of the viruses []. Recently, the partial RNA region, used to classify betanodaviruses [], was shown to be suffucuent to control host-specificity []. The optimal temperatures for the growth of RGNNV are 0 C which are relatively higher than those of SJNNV (0 C), TPNNV (0 C), and BFNNV ( 0 C) []. Some RGNNV isolates were shown to multiply at up to C []. However, little is known about the mechanisms underlying temperature sensitivities of these viruses [,, 0, ]. In this study, we constructed reassortants and chimeric viruses between SJNNV and RGNNV to investigate their temperature sensitivities in some viral growth steps. Base on the obtained data, we also refer to possible temperature sensitivity mechanisms in betanodaviruses. 0 Materials and methods Cells. The E- cell line [], cloned from the striped snakehead (Ophicephalus striatus) cell line (SSN-) [], was grown at C in Leibovit s L- medium (Invitrogen) supplemented with % foetal bovine serum. Plasmids. The plasmids used or constructed in this study are listed in Table. These plasmids contain the cdnas of SJNNV (SJNag strain), RGNNV (SGWak strain) [], and the chimeric viruses constructed from SJNNV and RGNNV. Identities of RNA and RNA sequences between SJNagA and SGWak are % and 0%, respectively []. A series of chimeric viral cdnas was constructed by replacing the original cdna sequences with corresponding heterologous viral cdnas synthesized using a polymerase chain reaction (PCR)-based method (Table ). All the recombinant

5 Hata et al., DNA techniques used in this study are described in Sambrook et al. []. The nucleotide sequences of the constructed viral cdnas were verified by DNA sequencing. Preparation of chimeric viruses. The synthesis of infectious transcripts from the viral cdnas (Table ) and the transfection of E- cells with these transcripts were performed to produce mature viruses (Table ), as described previously []. Viral growth then was confirmed by the appearance of cytopathic effects. The culture supernatants containing infectious virus particles were stored at 0 C before use. Viral growth test. E- cells grown in a -well plate (Iwaki) were inoculated with each of the viruses (Table ) at a multiplicity of infection (MOI) of.0. After incubation at, 0,, or 0 C for h, the inoculum was replaced by Leibovitz s L- medium (Invitrogen) supplemented with % fetal bovine serum ( ml per well) and the cells were cultured at the same temperatures. Fifty µl culture supernatant was collected periodically and its viral titer was measured by determining the 0 % tissue culture infectious dose (TCID 0 )/ml as described in Reed and Muench []. RNA replication and encapsidation tests. E- cells were inoculated with the viruses (Table ) and cultured under the same condition as used for the viral growth test. Periodically, the inoculated cells were washed once with Hanks balanced salt solution (HBSS) (Nissui) and were homogenized with 0 µl ISOGEN (Nippon gene) to prepare total RNA according to the manufacturer s instruction. Obtained RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water and used for Northern hybridization experiments. Northern hybridization was performed as described previously [, ] using DIG-Labelling and Detection Kit (Roche) according to the supplier s instructions. Briefly, to prepare probes for positive-sense RNA and RNA, psjbs and prgbs were linearized with SalI, followed by transcription with T RNA polymerase (Takara).

6 Hata et al., Similarly, to prepare probes for positive-sense RNA, psjbs and prgbs were linearized with EcoRI before transcription with the polymerase. Hybridization signals were detected by exposing hybridization blots to X-ray films (RX-U; Fuji film). For the encapsidation competence test, virion fractions were prepared periodically from the inoculated cells as described previously []. Virion RNA was prepared from the virion fractions using ISOGEN, dissolved in DEPC-treated water, and used for Northern hybridization experiments as described avobe. RNA transfection. Viral RNA was prepared from SJNNV or RGNNV virions as described previously [] and separated on a % agarose gel (SeqPlaque; FMC). RNA was recovered from the gel, purified using RNeasy Mini Kit (Qiagen), and dissolved in DEPC-treated water. E- cells grown in a -well plate (Iwaki) were transfected with the purified RNA (0 ng/well) as described previously [] and cultured at or 0 C. Total RNA was prepared periodically from the transfected cells and used for Northern hybridization experiments as described above. Binding assay. E- cells grown in a -well plate (Iwaki) were washed once with ml HBSS and inoculated with the reassortant or parental virus resuspended in ml HBSS. The inoculated cells were incubated at or 0 C for 0 min, washed four times with ml HBSS, and sacrificed to prepared total RNA from them using RNeasy Mini Kit. First strand cdnas were synthesized from the SJRG- primer ( -CGCCGAAGCGTAGGACAGCA- ) using the SuperScript Chioce System for cdna Synthesis kit (Invitrogen) and used as the templates for PCR. PCR was performed using the SJRG-F ( -AGTTCCACTGAGCTGGTTGA- ) and SJRG-R ( -AGCTCGACGAGGTGCTGATC- ) primers with cycles of denaturation at C for 0 sec, annealing at C for 0 sec, and extension at C

7 Hata et al., 00 for 0 sec. SJNNV and RGNNV RNAs shared these three primers sequences and gave approximately -kb PCR products. 0 0 Results Effects of temperatures on the growth of reassortant and parental viruses. To identify viral segments that control temperature sensitivities of betanodaviruses, the growth of SJ/SJ, RG/RG, SJ/RG, and RG/SJ were examined at different temperatures. At C, all the four viruses grew well, showing similar growth rates throughout the experimental periods (Fig. ). Similarly, no significant difference in viral growth rates was observed among the four viruses at or 0 C though the rates at and 0 C were lower and slightly lower than those at C, respectively. In contrast, at 0 C, RG/RG exclusively grew to the similar levels occurred at C (Fig. ). RG/SJ slightly produced progeny viruses of up to ca. 0. TCID 0 /ml at 0 C. While SJ/SJ and SJ/RG barely grew at this temperature from one to three days after inoculations, their titers finally reduced to the background levels. These data indicate that both RNA and RNA control temperature sensitivity of these viruses. Effects of temperatures on the RNA replication, encapsidation competence, and CP-expression. Involvement of both RNA and RNA in the determination of temperature sensitivity encouraged us to identify the mechanism underlying it. RNA replication of the reassortants and parental viruses was first examined by detecting their progeny viral RNA in the inoculated cultured cells at or 0 C. At C, all the four viruses showed similar RNA replication levels at five days after inoculations. However, at 0 C, only RG/RG exhibited RNA replication similar to the levels at C (Fig. ). No RNA replication was detected at 0 C for SJ/SJ or SJ/RG. RG/SJ gave a

8 Hata et al., small amount of progeny viral RNA at 0 C (Fig. ). These RNA replication data of the four viruses well correlated with the viral growth shown in Fig.. The kinetics of virion RNA production was closely similar to that of total viral RNA production (Fig. ). Moreover, the levels of CP expression for the viruses were also correlated to those of RNA replication and viral growth (Fig. ). At C, the signals of SJNNV CP were slightly stronger than those of RGNNV despite the similar levels of RNA replication between SJNNV and RGNNV because the anti-sjnnv antibody used in this study cross-reacts SJNNV CP higher than RGNNV CP (). Collectively, temperature seemed to affect RNA replication or earlier viral growth processes. Virion stability of reassortant and parental viruses. Possible differences in the stability of the inocula and progeny viruses at 0 C might have affected the viral growth and RNA replication shown in Figs. -. Hence, the reassortant and parental viruses were exposed at 0 C for days to examine their stability at the temperature. The viral titers of the incubated viral solutions showed that the survival rates of SJ/SJ, SJ/RG, RG/SJ, and RG/RG were.,., 0., and. %, respectively. These small differences in virion stability do not seem to explain the drastic differences in the viral growth or RNA replication in Figs. -. Nevertheless, the higher virion stability of RG/RG may contribute to some extent to its adaptation at 0 C. Effects of temperatures on the bindings of reassortant and parental viruses to E- cells. One of the viral growth steps that could be affected by temperature is virion attachment to the host cell surface. Binding of SJ/SJ, RG/RG, SJ/RG, and RG/SJ virions to E- cells at or 0 C were evaluated using an RT-PCR based method. At C, SJ/RG bound the most abundantly to E- cells among the four viruses (Fig. ). Binding was slightly reduced for RG/RG and was further reduced for

9 Hata et al., RG/SJ and SJSJ. This tendency was true of their bindings with E- cells at 0 C (Fig. ). These results imply that elevating the incubation temperature from to 0 C does not affect markedly the levels of binding between the four viruses and E- cells. Effects of temperatures on RNA replication. One plausible scenario of the RNA-dependent control of temperature sensitivity (Figs. and ) is that an enzyme activity of protein A, encoded by RNA, is regulated by temperatures. To test this possibility, RNA purified from the virions of SJNNV or RGNNV was transfected into E- cells and their replication were examined by Northern hybridization. At C, both the RNA replicated and produced RNA similarly (Fig. ). However, at 0 C, only RGNNV RNA replicated and produced RNA as occurred at C. No RNA replication and RNA production was obtained for SJNNV RNA at 0 C throughout the experiment periods (Fig. ). These results indicate that RNA can control temperature sensitivity of betanodaviruses, at least in part, independent of RNA. RNA regions that control temperature sensitivity. Chimeric RGNNV viruses, parts of RGNNV RNA of which were replaced by the corresponding parts of SJNNV RNA, were prepared to identify the regions that determine temperature sensitivity (Fig. A). RG/RG, SJ/RG, and all the nine chimeric viruses grew similarly at C, showing cytopathic effects (CPE). The viruses gave viral titers from 0. to 0. TCID 0 /ml at this temperature (Fig. A). At 0 C, RGSJ-Cla/RG, RGSJ-Hpa/RG, RGSJ-Eco/RG, RGSJ-Sty/RG, and SJRGSJ-UTR/RG grew, giving CPE and similar levels of viral titers to those obtained at C. In contrast, RGSJ-Nhe/RG, RGSJ-Bst/RG, SJRGSJ-Nde/RG, SJRGSJ-Eco/RG, SJ/RG grew less efficiently, showing the viral titers from less than 0. to 0. TCID 0 /ml (Fig. A). No CPE was observed in these infections during the experimental periods. These results

10 Hata et al., 00 0 indicate that the RGNNV RNA region from to nt is important for viral growth at 0 C. This region encodes th amino acid residues of RGNNV protein A. RNA regions that control temperature sensitivity. To identify an RNA region that controls temperature sensitivity, chimeric RGNNV viruses, parts of RGNNV RNA of which were replaced by the corresponding parts of SJNNV RNA, were prepared. Seven chimeric viruses were designed to investigate systematically the RNA segment. However, unfortunately, of the seven chimeric viruses, only the four chimeric viruses shown in Fig. were grew well at C (data not shown). RG/RG, RG/SJ, and the four chimeric viruses grew similarly at C showing CPE (data not shown). The titers of their progeny viruses at this temperature were from 0. to 0. TCID 0 /ml (Fig. ). In contrast, at C, RG/RGSJAfl gave a low level of titer, 0. TCID 0 /ml and RG/RGSJORF showed a further reduced titer of 0.0 TCID 0 /ml. Replacing the 0 entire UTR or a part of the UTR gave no significant effect on temperature sensitivity, which was evidenced by the inoculations of RG/RGSJUTR-ORF and RG/RGSJORF-UTR0. Nevertheless, all the chimeric viruses produced more progeny viruses at C than RG/SJ. These results indicate that temperature sensitivity determinants are dispersed throughout the RNA segment. In this RNA investigation, the viruses were cultured at C instead of 0 C because the low level of RG/SJ growth at 0 C (Fig. ) was completely suppressed by elevating the culture temperature to C, which produced more differences in the titers of the six viruses tested. Discussion Effects of temperatures on viral growth, RNA replication, virion stability, and 0

11 Hata et al., virion binding to host cells were examined using SJNNV, RGNNV, and their reassortant viruses. Obtained data demonstrate that both RNA and RNA play a role in determining temperature sensitivity, and that temperatures affected viral RNA replication but had no significant influence on virion stability or virion attachment to host cells. Further experiments by transfecting E- cells with SJNNV RNA or RGNNV RNA revealed that RNA can control temperature sensitivity without interacting with RNA, which confirmed the vulnerability of viral RNA replication processes to temperature changes. It remains to be addressed whether RNA itself and/or encoded protein A confer the temperature sensitivity. In many plant and animal viruses, temperature sensitivities are controlled by viral RNA replication processes. In those cases, RNA replication of wild type [,, ] or mutant [,,,, ] viruses, the latters of which carry amino acid substitutions in the replicases, were examined at different temperatures. However, direct evidence for the effects of temperatures on viral replicase activities has not yet been studied extensively in those viruses [e. g., ]. One possible reason is a difficulty to express or purify an active viral replicase [0, 0,, 0]. Our attempts to evaluate SJNNV and RGNNV protein A activities at and 0 C have never been succeeded due to the poor expression of protein A in cultured fish cells or the insoluble nature of the protein A expressed in Escherichia coli cells (authors unpublished data). We need an improved experimental method to evaluate the enzyme activities of SJNNV and RGNNV protein As, which will help us to confirm the temperature sensitivity mechanisms of betanodaviruses. Our temperature sensitivity assay using the RNA-chimeric viruses revealed that the RNA region, which encoded the th amino acid residues of protein A, seemed sufficient to control temperature sensitivity in those viruses (Fig. A). These

12 Hata et al., amino acid residues contain the putative signal to target protein A onto mitochondrial membranes (Fig. B) [, ]. The remaining C-terminal region of protein A harbors a putative RdRp catalytic domain. No other apparent functional domain was predicted in either of SJNNV or RGNNV protein A (authors unpublished data). These results suggest that temperatures affect transferring protein A to mitochondrial membranes or the protein A conformation, rather than modulate substrate bindings to the putative RdRp domain. Alternatively, the RNA sequence itself may be important to confer the temperature sensitivity [, ]. RNA-RNA interactions within or between viral segments are important to control RNA replication and basically occur between loop and/or bulge structures of RNA [,,, ]. Such the secondary structures of RNA are known to change depending on the temperature conditions []. RGSJ-Cla/RG, in which the entire RGNNV RNA sequence was replaced by that of SJNNV, still retained strong viral growth at 0 C. This result demonstrates that either RNA or encoded protein B does not confer temperature sensitivity in these two viruses used in this study. Our experiments using the RNA-chimeric viruses did not identify a distinct RNA region that controls temperature sensitivity though RNA as well as RNA served as a factor affected by temperature. Conversely, such regions were revealed to be dispersed throughout the. kb RNA segment. A mechanism of the RNA-dependent control is still not clear since temperatures gave no significant influence on virion stability or attachment of the parental and reassortant viruses onto host cells (Fig. ). Nevertheless, the low levels of viral growth and RNA replication of RG/SJ at 0 C (Figs. and ), which was not correlated with the profile of RGNNV RNA and RNA production at the same temperature (Fig. ), indicates that SJNNV RNA inhibits the production of

13 Hata et al., RGNNV RNA and RNA at 0 C. That is, direct or indirect interactions between RNA and RNA (and/or RNA and RNA) may occur during RNA replication and RNA production in betanodavirus. In the insect alphanodavirus, Flock house virus (FHV), RNA produced from RNA interacts physically with RNA and triggers the replication of RNA. Increased RNA then interacts physically with RNA again and inhibits the production of RNA, which finally terminates the replication of RNA []. If a similar RNA replication system is recruited in betanodavirus, one of the possible temperature-sensitive events for this virus is the interaction between RNA and RNA since a direct interaction between RNA and RNA do not occur during the regulation process in FHV. Unfortunately, temperature sensitivity of RNA replication has not been well studied in alphanodavirus []. In this report, we thus far propose that RNA replication is a plausible temperature-sensitive processe in betanodavirus growth, which is evidenced by our experiments using chimeric and reassortant viruses. Nonetheless, we do not deny the existence of other temperature-sensitive processes for this virus. In some animal viruses, temperature sensitivity is controlled by capsid proteins; the temperature sensitive mutant of reovirus (tsa) is blocked in transmembrane transport of virions, the characteristics of which is determined by its M capsid gene []. In herpes simplex virus, the VP capsid protein fails to localize to nucleus under nonpermissive temperature [] and uncoating is temperature dependent []. In tobamovirus that infects capsicum plants, L-gene-mediated defense systems are elicited by coat protein in temperature dependent manner []. Finally, some temperature sensitive phage P variants, the coat proteins of which bear amino acid substitutions, were incompetent for assembly at the nonpermissive temperature [, ]. In our study, an effect of

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21 Hata et al., 00 Figure legends Fig.. Growth of the reassortant and parental viruses at different temperatures. E- cells were inoculated with SJ/SJ, SJ/RG, RG/SJ, or RG/RG, and cultured at the indicated temperatures. Culture supernatants then were collected periodically and their viral titers were examined. Data are represented as means ± standard deviations from two ( and 0 C) or three ( and 0 C) independent experiments. 0 Fig.. RNA replication, encapsidation, and CP expression of the reassortant and parental viruses at different temperatures. E- cells were inoculated with each of the viruses and cultured at or 0 C. Total RNA, virion RNA, and total protein fractions were prepared from the inoculated cells days after inoculation. The RNA samples were used for Northern hybridization experiments to detect positive-sense viral RNAs. Five µg total RNA and 0 ng virion RNA were run on the each lane. Total protein fractions were analysis by Western blotting using anti-sjnnv rabbit serum. Representative data were shown from three independent experiments. 0 Fig.. Semi-quantitative RT-PCR for the detection of cell-bound virions. E- cells were incubated with SJ/SJ, SJ/RG, RG/SJ, or RG/RG virions for 0 min at the indicated temperatures, followed by the extraction of total RNA from the cells. The extracted samples were used for the detection of RNA by semi-quantitative RT-PCR, which was evidenced by the amplification of approximately -kb fragments. Fig.. Replication of SJNNV RNA and RGNNV RNA at different temperatures. E- cells were transfected with SJNNV RNA or RGNNV RNA and cultured at or

22 Hata et al., 00 0 C. Total RNA was isolated from the cells at 0 0 h post transfections and used for Northern hybridization experiments to detect positive-sense RNA and RNA. Five µg total RNA was run on the each lane. Representative data were shown from three independent experiments. C; No RNA was transfected. P; 0 ng of virion RNA from SJNNV or RGNNV were loaded per lane as the positive controls. 0 Fig.. RNA regions which control temperature sensitivities of SJNNV and RGNNV. Blue and red bars show SJNNV RNA and RGNNV RNA segments, respectively. Thin and thick bars indicate the untranslated regions and the ORF for protein A, respectively. Numbers on the RNA segments indicate the nucleotide positions of chimeric junctions. (A) E- cells were inoculated with the viruses, each of which had the chimeric or parental RNA along with RGNNV RNA, and were cultured at or 0 C. Five days after inoculations, culture supernatants were collected and their viral titers were examined. Data are represented as means ± standard deviations from three independent experiments. (B) The putative transmenbrane (TM) and RdRp domains are denoted on the protein A map. 0 Fig.. RNA regions which control temperature sensitivities of SJNNV and RGNNV. Blue and red bars show SJNNV RNA and RGNNV RNA segments, respectively. Thin and thick bars indicate the untranslated regions and the ORF for CP, respectively. Numbers on the RNA segments indicate the nucleotide positions of chimeric junctions. E- cells were inoculated with the viruses, each of which had RGNNV RNA and the chimeric or parental RNA, and were cultured at or C. Five days after inoculations, culture supernatants were collected and their viral titers

23 Hata et al., 00 were examined. Data are represented as means ± standard deviations from three independent experiments.

24 Hata et al., Table. Plasmids used in this study Plasmid Characteristics Reference Wild type viral cdna psjtk full-length cdna of SJNNV RNA Iwamoto et al. [] psjtk0 full-length cdna of SJNNV RNA Iwamoto et al. [] prgtk a full-length cdna of RGNNV RNA Iwamoto et al. [] prgtk a full-length cdna of RGNNV RNA Iwamoto et al. [] prgtkb a 0 th T in the prgtk cdna is replaced by A Ito et al. [] Chimeric viral cdna prgsj-cla ca. 0 bp Cla I EcoR I fragment in prgtk is replaced by that from psjtk This study prgsj-hpa ca. 0 bp Hpa I EcoR I fragment in prgtk is replaced by that from psjtk This study prgsj-eco ca. 0 bp EcoR V EcoR I fragment in prgtk is replaced by that from psjtk This study prgsj-sty ca. 0 bp Sty I EcoR I fragment in prgtk is replaced by that from psjtk This study prgsj-nhe ca. 0 bp Hind III-Nhe I fragment in psjtk is replaced by that from prgtk This study prgsj-bst ca. 00 bp Hind III-BstBI fragment in psjtk is replaced by that from prgtk This study psjrgsj-utr ca. 0 bp Hind III-BstE II fragment in prgsj-rv is replaced by that from psjtk This study psjrgsj-nde ca. 0 bp Hind III-Nde I fragment in prgsj-rv is replaced by that from psjtk This study psjrgsj-eco ca. 0 bp Hind III-EcoT I fragment in prgsj-rv is replaced by that from psjtk This study prgsjafl ca. 0 bp Afl III BspE I fragment in prgtkb is replaced by that from psjtk0 Ito et al. [] prgsjorf the ORF in prgtk is replaced by that from psjtk0 This study prgsjutr-orf the UTR and ORF in prgtk is replaced by that from psjtk0 This study prgsjorf-utr0 the UTR terminus ( bp) in prgsjorf was replaced by that from psjtk0 This study Viral cdna for probe preparation psjbs b ca. 0 bp Cla I EcoR I fragment of SJNNV RNA cdna in pbluescript SK(-) c Iwamoto et al. [] psjbs ca. 0 bp BamH I EcoR I fragment of SJNNV RNA cdna in pbluescript SK(-) Iwamoto et al. [] prgbs b ca. 0 bp Cla I EcoR I fragment of RGNNV RNA cdna in pbluescript SK(-) Hata et al. [] prgbs ca. 0 bp BamH I EcoR I fragment of RGNNV RNA cdna in pbluescript SK(-) Hata et al. [] a prgtk, prgtk, and prgtkb were described previously as psgtk, psgtk, and psgtkb, respecvively [, ]. b Riboprobes transcribed from these cdna detected both RNA and RNA. c pbluescript SK(-) was purchased from Stratagene (La Jolla, CA, USA).

25 Hata et al., Table. Viruses used in this study Viral cdna clone b Virus RNA RNA Reference Parental virus SJ/SJ psjtk psjtk0 Iwamoto et al. [] RG/RG a prgtk prgtk Iwamoto et al. [] Reassortant virus SJ/RG a psjtk prgtk Iwamoto et al. [] RG/SJ a prgtk psjtk0 Iwamoto et al. [] Chimeric virus RGSJ-Cla/RG prgsj-cla prgtk This study RGSJ-Hpa/RG prgsj-hpa prgtk This study RGSJ-Eco/RG prgsj-eco prgtk This study RGSJ-Sty/RG prgsj-sty prgtk This study RGSJ-Nhe/RG prgsj-nhe prgtk This study RGSJ-Bst/RG prgsj-bst prgtk This study SJRGSJ-UTR/RG psjrgsj-utr prgtk This study SJRGSJ-Nde/RG psjrgsj-nde prgtk This study SJRGSJ-Eco/RG psjrgsj-eco prgtk This study RG/RGSJAfl prgtk prgsjafl Ito et al. [] RG/RGSJORF prgtk prgsjorf This study RG/RGSJUTR-ORF prgtk prgsjutr-orf This study RG/RGSJORF-UTR0 prgtk prgsjorf-utr0 This study a RG/RG, SJ/RG, and RG/SJ are described previously as SG/SG, SJ/SG, and SG/SJ, respectively []. b In vitro transcripts from the indicated viral cdnas were co-transfected into E- cells to obtain the viruses.

26 Hata et al., 00 Fig.

27 Hata et al., 00 Fig.

28 Hata et al., 00 Fig. SJ/SJ SJ/RG RG/SJ RG/RG Mock 0

29 Hata et al., 00 Fig.

30 Hata et al., 00 Fig. A Viral titer ( log 0 TCID 0 /ml) 0 h 0 h RGSJ-Cla/RG RGSJ-Hpa/RG RGSJ-Eco/RG RGSJ-Sty/RG RGSJ-Nhe/RG RGSJ-Bst/RG SJRGSJ-UTR/RG SJRGSJ-Nde/RG SJRGSJ-Eco/RG < <.... RG/RG RG <... B SJ/RG SJ + RG... RNA Protein A TM RdRp 0

31 Hata et al., 00 Fig. Viral titer ( log 0 TCID 0 /ml) 0 h 0 h RG/RGSJAfl 0... RG/RGSJORF..0.0 RG/RGSJUTR-ORF... RG/RGSJORF-UTR RG/RG RG <... RG/SJ SJ + RG.. <.