ABSTRACT
The generation of recombinant group A rotaviruses (RVAs) entirely from cloned cDNAs has been described only for a single animal RVA strain, simian SA11-L2. We recently developed an optimized RVA reverse genetics system based on only RVA cDNAs (11-plasmid system), in which the concentration of cDNA plasmids containing the NSP2 and NSP5 genes is 3- or 5-fold increased in relation to that of the other plasmids. Based on this approach, we generated a recombinant human RVA (HuRVA)-based monoreassortant virus containing the VP4 gene of the simian SA11-L2 virus using the 11-plasmid system. In addition to this monoreassortant virus, authentic HuRVA (strain KU) was also generated with the 11-plasmid system with some modifications. Our results demonstrate that the 11-plasmid system involving just RVA cDNAs can be used for the generation of recombinant HuRVA and recombinant HuRVA-based reassortant viruses.
IMPORTANCE Human group A rotavirus (HuRVA) is a leading pathogen causing severe diarrhea in young children worldwide. In this paper, we describe the generation of recombinant HuRVA (strain KU) from only 11 cloned cDNAs encoding the HuRVA genome by reverse genetics. The growth properties of the recombinant HuRVA were similar to those of the parental RVA, providing a powerful tool for better understanding of HuRVA replication and pathogenesis. Furthermore, the ability to manipulate the genome of HuRVAs “to order” will be useful for next-generation vaccine production for this medically important virus and for the engineering of clinical vectors expressing any foreign genes.
INTRODUCTION
Group A rotavirus (RVA), a member of the genus Rotavirus and family Reoviridae, is a leading cause of severe gastroenteritis in the young of humans and many animal species worldwide. Each host species is known to have its own particular RVAs. Human RVA (HuRVA) is estimated to cause 215,000 deaths each year (1). The RVA virion encapsidates an 11-segment genome of double-stranded RNA (dsRNA) (2). Although several animal RVAs have been shown to grow to high titers in cell cultures, the cultivation of HuRVAs has been generally found to be more difficult (3–7). Of note, this fastidious nature was overcome by replacing the VP4 gene segment with that of animal RVA, which is associated with a restriction of growth in cell cultures (8).
Reverse genetics is a powerful tool for studying virus replication and pathogenicity and also for vaccine development. For RVA, partial plasmid-based reverse genetics systems that require a helper virus have been exploited to engineer novel replicative recombinant RVAs having a cDNA-derived gene segment (9–13). In the initial reverse genetics system for RVA described, a VP4 gene segment expressed from cDNA could be rescued in an infectious virus (helper virus) using strong selection conditions (10). Subsequently, other helper virus-dependent systems were developed through modification of the first system. However, because these systems require strong selection conditions to separate and rescue the recombinant RVA from a helper virus, their use is restricted to only a few gene segments. Alternatively, a versatile entirely plasmid-based reverse genetics system was developed recently (14). Entirely plasmid-based reverse genetics systems have been developed only for a single animal RVA strain, simian SA11-L2, which grows to high titers in cell cultures (14–16). Three strategies for entirely plasmid-based reverse genetics have been developed for this animal virus. The primary method was based on the transfection of 14 plasmids; BHK-T7 cells, stably expressing T7 RNA polymerase, were transfected with 14 plasmids (11 RVA cDNA plasmids for 11 gene segments, flanked by the T7 RNA polymerase promoter and hepatitis delta virus [HDV] ribozyme sequences, in combination with three expression plasmids encoding the Nelson Bay orthoreovirus fusion-associated small transmembrane protein and the two subunits of the vaccinia virus [VV] capping enzyme) (14). The second strategy was based on the transfection of 12 plasmids; cotransfection of two expression plasmids encoding the VV capping enzyme was shown not to be essential for virus rescue (15). Finally, the third most recently reported strategy is based on the transfection of just 11 RVA plasmids for its 11 gene segments with an increased proportion (3- or 5-fold) of plasmids for the NSP2 and NSP5 genes (11-plasmid system); this system is highly efficient and does not require the use of any plasmids other than the 11 RVA cDNAs (16). By using the above-mentioned three methods, several recombinant SA11-L2 viruses carrying foreign genes and monoreassortants with 10 segments from SA11-L2 virus and one segment (VP6 or NSP5) from HuRVA strain KU were generated (14–16).
Compared to animal RVAs, HuRVAs have distinct properties, as follows. (i) HuRVAs have distinct antigenic structures such as neutralization epitopes on both outer capsid proteins, VP7 and VP4 (17–20). (ii) HuRVAs have different receptor aspects: HuRVAs bind to internal sialic acid moieties on glycans or to modified sialic acid moieties in oligosaccharide structures, such as those present in the GM1 ganglioside, and histo-blood group antigens, whereas most animal RVAs bind to external sialic acid residues on glycans (21–28). (iii) HuRVAs exhibit a higher dependency for trypsin-like proteases for infectivity activation than animal RVAs (29, 30). (iv) HuRVAs grow less efficiently than most animal RVAs in cultured cells and in suckling mice as an infection model (31). Thus, for studying the molecular biology of HuRVA infection in humans, for developing new-generation vaccines for humans, and for applying recombinant HuRVAs as viral vectors in humans, we need to apply the 11-plasmid system to the rescue of HuRVAs. A HuRVA strain with high-yield growth properties in cell cultures, analogous to simian SA11-L2 virus, has not been reported; thus, the establishment of an entirely plasmid-based reverse genetics system for HuRVAs has been more difficult than for animal RVA strains. To date, despite extensive efforts in many laboratories, no entirely plasmid-based reverse genetics system for HuRVA rescue has been established.
In this paper, we report a reverse genetics system for HuRVAs based on the 11-plasmid system that enables the generation of recombinant HuRVAs entirely from cloned cDNAs encoding the HuRVA genome. Utilizing the 11-plasmid reverse genetics system, we first generated a HuRVA-based monoreassortant virus containing the RNA segment encoding VP4 of the SA11-L2 virus. Then, by making minor modifications to the 11-plasmid system, we also generated authentic HuRVA (strain KU) from just 11 HuRVA plasmids containing its 11 gene cDNAs as inserts. The availability of recombinant HuRVAs and their recombinant reassortants will provide a genetic platform for a better understanding of the replication, pathogenicity, and other biological characteristics of HuRVAs and enable the rational development of next-generation HuRVA vaccines.
RESULTS AND DISCUSSION
Construction of rescue T7 plasmids for HuRVA strain KU.As a first step in developing a reverse genetics system for HuRVAs, we tried to determine the complete sequences of all 11 gene segments of HuRVA strain KU (G1P[8]), which is genetically and serologically related to representative reference HuRVA strain Wa (G1P[8]) (7), by performing deep sequencing using Illumina MiSeq five times. This approach allowed determination of the complete nucleotide sequences of all 11 gene segments of the KU virus. To generate recombinant HuRVA entirely from cloned cDNAs, we newly constructed nine T7 rescue plasmids expressing the individual mRNAs for nine gene segments of strain KU, the exceptions being the VP6 and NSP5 genes, based on the determined sequences. We had previously constructed T7 plasmids carrying the full-length VP6 and NSP5 genes of KU virus (14, 15). The full-length cDNA fragments of the remaining nine gene segments were biochemically synthesized (VP1 and VP3 genes) or amplified by reverse transcription-PCR (RT-PCR) from viral genomic dsRNAs of KU (VP2, VP4, VP7, and NSP1 to NSP4 genes). The cDNA of each segment was inserted into a T7-driven plasmid. Each plasmid contains a full-length gene segment cDNA of KU virus flanked by the T7 RNA polymerase promoter and HDV ribozyme sequences.
Generation of recombinant HuRVA-based monoreassortant virus possessing a SA11-L2-derived VP4 gene segment.To generate recombinant authentic KU virus from the constructed 11 rescue T7 plasmids encoding the KU genome, BHK/T7-9 cells stably expressing T7 RNA polymerase were transfected with the 11 HuRVA plasmids according to the 11-plasmid reverse genetics system we developed recently for simian SA11-L2 virus (16). Following many unsuccessful attempts to rescue replicative recombinant KU virus, we then tried to generate HuRVA-based monoreassortant virus containing an SA11-L2-derived VP4 segment to evaluate the functionalities of the newly constructed rescue T7 plasmids for the KU genome except for the VP4 gene, because the growth of HuRVAs with the SA11-L2 VP4 gene had been found to be much better than that of the original HuRVA in reassortment experiments (10, 32). To generate HuRVA-based monoreassortant virus containing an SA11-L2-derived VP4 segment, the VP4 rescue T7 plasmid, which encodes the full-length VP4 gene of simian SA11-L2 virus, was cotransfected into BHK/T7-9 cells with the other 10 plasmids from KU virus, and the recombinant virus was successfully rescued using the 11-plasmid reverse genetics system (Fig. 1A). Polyacrylamide gel electrophoresis (PAGE) analysis of viral double-stranded RNAs (dsRNAs) showed that the VP4 gene of the rescued monoreassortant virus, named rKU-VP4SA11, migrated to the same position as the corresponding VP4 gene of the parental SA11-L2 with a KU backbone (Fig. 1B). To confirm that the rescued monoreassortant virus was generated from the cloned cDNAs, pT7/VP4SA11-ΔPstI (10, 15) was used as the VP4 rescue T7 plasmid, in which a unique PstI site was destroyed in the VP4 gene of SA11-L2 by the introduction of two silent mutations (T to C and A to C at nucleotide positions 1365 and 1368, respectively) (Fig. 1C). The full-length VP4 genes (2,362 bp) of parental SA11-L2 and rKU-VP4SA11 were amplified by RT-PCR. The amplified VP4 gene derived from the parental SA11-L2 was digested to produce 1,368- and 994-bp fragments, whereas the VP4 segment from rKU-VP4SA11 was not digested (Fig. 1D). This demonstrated that the rKU-VP4SA11 originated from the cloned cDNAs and that recombinant HuRVA-based monoreassortant virus possessing a SA11-L2-derived VP4 segment could be generated by means of reverse genetics, indicating the functionalities of the newly constructed rescue T7 plasmids for KU virus except for the VP4 gene for the generation of recombinant HuRVA.
Generation of recombinant HuRVA-based monoreassortant rKU-VP4SA11 virus. (A) Schematic presentation of an 11-plasmid reverse genetics system to generate HuRVA-based monoreassortant. The 11 rescue T7 plasmids encode the full-length segment cDNA of an individual gene of RVA, flanked by the T7 RNA polymerase promoter (PT7) and the HDV ribozyme (Rib). To generate rKU-VP4SA11, BHK/T7-9 cells were cotransfected with the 11 rescue T7 plasmids (10 plasmids for the 10 genes, the exception being the VP4 gene of KU, in combination with the plasmid for the VP4 gene of SA11-L2) with 3-fold increased amounts of the two plasmids carrying the NSP2 and NSP5 genes. rKU-VP4SA11 was rescued from the cultures of the transfected BHK/T7-9 cells, followed by amplification on CV-1 cells with 0.3 μg/ml trypsin. (B) PAGE of viral dsRNAs extracted from KU, rKU-VP4SA11, and SA11-L2. Lanes 1 and 3, dsRNAs from KU (lane 1) and SA11-L2 (lane 3); lane 2, dsRNA from the rescued rKU-VP4SA11. The numbers on the left indicate the order of the genomic dsRNA segments of KU. (C) Rescued rKU-VP4SA11 virus contains a signature mutation in its VP4 gene as a gene marker. Nucleotide substitutions (T to C and A to C at nucleotide positions 1365 and 1368, respectively) were introduced to eliminate a unique PstI site in the VP4 gene (10). (D) The VP4 gene RT-PCR products were digested with PstI, followed by separation in a 1.2% agarose gel. SA11-L2 (lanes 1 and 2) and rKU-VP4SA11 (lanes 3 and 4) are shown. The 2,362-bp fragments (lanes 1 and 3) were digested with PstI (lanes 2 and 4). M, 1-kb DNA ladder marker (TaKaRa Bio).
Characterization of recombinant HuRVA-based monoreassortant virus with a SA11-L2-derived VP4 gene segment.To assess the replication kinetics of the rescued monoreassortant virus, multiple-step growth curves for KU, rKU-VP4SA11, and SA11-L2 were determined after infection of MA104 cells at a low multiplicity of infection (MOI) of 0.01 PFU/cell. As observed in previous studies with the KU-based monoreassortant virus carrying the authentic SA11-L2 VP4 gene (10, 33), viral titers similar to those of the parental SA11-L2 were attained for the generated rKU-VP4SA11 (Fig. 2A). We then examined the plaque sizes in CV-1 cells for these viruses by measuring the mean diameters of 25 plaques each in two different assays (Fig. 2B). The virus growth titers correlated well with the sizes of the plaques formed: viruses with high infectivity (SA11-L2 and rKU-VP4SA11) formed large plaques (diameters, 3.34 ± 0.64 and 2.53 ± 0.44 mm, respectively), as has been demonstrated for SA11-L2 VP4-carrying viruses (32, 33), while the authentic human virus KU exhibiting low infectivity formed small plaques (1.09 ± 0.27 mm). These results suggested that the 11-plasmid reverse genetics system allows the generation of HuRVA-based monoreassortant viruses possessing an animal RVA-derived VP4 gene with higher infectivity than the original HuRVAs.
Growth properties of the rKU-VP4SA11 virus. (A) Multiple-step growth curves for KU, rKU-VP4SA11, and SA11-L2. MA104 cells were infected with KU, rKU-VP4SA11, or SA11-L2 at an MOI of 0.01 and then incubated for various numbers of hours. The virus titers in the cultures were determined by plaque assay. The data shown are the mean viral titers and standard deviations (SDs) from three independent cell cultures. *, P < 0.05 (as determined by t test). (B) Plaque formation by KU, rKU-VP4SA11, and SA11-L2. KU, rKU-VP4SA11, or SA11-L2 was directly plated onto CV-1 cells for plaque formation. The experiment was repeated three times with similar results, and representative results are shown.
Generation of recombinant authentic HuRVA from just 11 T7 rescue plasmids.Since we confirmed the consistency and feasibility of at least 10 rescue T7 plasmids for KU virus, we reattempted to rescue recombinant authentic HuRVA virus. To rescue replicative recombinant KU virus from the 11 rescue T7 plasmids encoding the KU genome, BHK/T7-9 cells were transfected with the 11 HuRVA plasmids according to the 11-plasmid reverse genetics system. As a modification, the trypsin concentration for coculture of transfected BHK/T7-9 cells and overlaid CV-1 cells was 3-fold increased to 0.9 μg/ml due to the high dependency for infectivity activation of HuRVAs. In addition, the roller-tube culture technique with MA104 cells was applied to amplify recombinant HuRVAs within the coculture of transfected BHK/T7-9 cells with overlaid CV-1 cells. Although a significant cytopathic effect (CPE) could not be observed in the first passage in MA104 cells of a roller-tube culture with the lysates of the cocultures of transfected BHK/T7-9 cells and overlaid CV-1 cells, MA104 cells of a roller-tube culture in the second passage showed characteristic RVA-induced CPE. These results suggested that recombinant authentic HuRVA, named rKU virus, was generated with the 11-plasmid reverse genetics system entirely from cloned cDNAs with some modifications.
PAGE analysis of viral genomic dsRNAs extracted from the rescued viruses showed that rKU exhibited an RNA migration pattern that was indistinguishable from that of native KU virus (Fig. 3A). To confirm that the rescued virus was generated from the cloned cDNAs, unique HindIII and EcoRI sites were destroyed in the VP1 and NSP4 genes, respectively, by the introduction of one silent mutation (G to A) each at nucleotide position 3066 in the pT7/VP1KU plasmid (Fig. 3B) and A to G at nucleotide position 482 in the pT7/NSP4KU plasmid (Fig. 3C). The full-length VP1 gene (3,302 bp) and NSP4 gene (750 bp) of native KU and rKU were amplified by RT-PCR. The amplified VP1 and NSP4 genes derived from native KU were digested to produce 3,064- and 238-bp fragments and 480- and 270-bp fragments, respectively, whereas the corresponding genes from rKU were, as expected, not digested (Fig. 3D and E). Whole-genomic sequencing using Illumina MiSeq confirmed the presence of the expected G-to-A and A-to-G mutations in the VP1 gene and NSP4 gene, respectively, and the absence of additional mutations on all 11 gene segments of rescued rKU (data not shown). Thus, these results demonstrated that recombinant HuRVA originating from the cloned cDNAs could be generated with the 11-plasmid reverse genetics system with minor modifications.
Generation of recombinant HuRVA rKU virus. (A) PAGE of viral genomic dsRNAs extracted from the native KU and recombinant rKU viruses. Lane 1, dsRNA from KU; lane 2, dsRNA from rescued rKU. The numbers on the left indicate the order of the genomic dsRNA segments of KU. (B) The rescued rKU virus contains a signature mutation in its VP1 gene as a gene marker. A nucleotide substitution (G to A at nucleotide position 3066) was introduced to eliminate a unique HindIII site in the VP1 gene. (C) The rescued rKU contains a signature mutation in its NSP4 gene as a gene marker. A nucleotide substitution (A to G at nucleotide position 482) was introduced to eliminate a unique EcoRI site in the NSP4 gene. (D) The VP1 gene RT-PCR products were digested with HindIII, followed by separation in a 2% agarose gel. KU (lanes 1 and 2) and rKU (lanes 3 and 4) are shown. The 3,302-bp fragments (lanes 1 and 3) were digested with HindIII (lanes 2 and 4). M, 1-kb (left) and 100-bp (right) DNA ladder markers (TaKaRa Bio). (E) The NSP4 gene RT-PCR products were digested with EcoRI, followed by separation in a 2% agarose gel. KU (lanes 1 and 2) and rKU (lanes 3 and 4) are shown. The 750-bp fragments (lanes 1 and 3) were digested with EcoRI (lanes 2 and 4). M, 100-bp ladder DNA marker.
Characterization of recombinant HuRVA rKU virus.To confirm that the rescued rKU has the characteristics of the native KU, multiple-step growth curves for KU and rKU were obtained after infection of MA104 cells at an MOI of 0.01 PFU/cell. The growth curves demonstrated that the replication of rKU was virtually identical to that of the native KU (Fig. 4A). We then examined the plaque sizes in CV-1 cells for these viruses by measuring the mean diameters of 25 plaques each in two different assays (Fig. 4B). rKU formed plaques of virtually the same size (diameter, 1.21 ± 0.36 mm) as those of the native KU (diameter, 1.32 ± 0.35 mm), which is indistinguishable from that shown in Fig. 2B (diameter, 1.09 ± 0.27 mm). These results showed that the replication characteristics of rKU are indistinguishable from those of the native KU.
Growth properties of the rKU virus. (A) Multiple-step growth curves for KU and rKU. MA104 cells were infected with KU or rKU at an MOI of 0.01 and then incubated for various numbers of hours. The virus titers in the cultures were determined by plaque assay. The data shown are the mean viral titers and SDs from three independent cell cultures. NS, P > 0.05 (as determined by t test). (B) Plaque formation by KU and rKU. KU or rKU was directly plated onto CV-1 cells for plaque formation. The experiment was repeated three times with similar results, and representative results are shown.
The fact that we are able to rescue HuRVAs after transfection of just 11 HuRVA rescue T7 plasmids into BHK/T7-9 cells proves that the 11-plasmid reverse genetics system with some modifications is robust enough for the recovery of recombinant HuRVAs. In addition, the ability to generate and manipulate infectious HuRVAs with a given desired gene segment combination by reverse genetics will provide a genetic platform for the rational design of live HuRVA vaccines and vaccine vectors. We will soon be able to prepare recombinant HuRVAs, such as wild-type circulating strains, expressing reporters that encode fluorescent proteins, having mosaic antigenic structures, and so on.
Although the modifications of the 11-plasmid system described in this study appear to be minor and simple, rotavirologists understand that the important point for the generation of recombinant HuRVAs is the cultivation of very few infectious viruses produced within the transfected cells. This process is like the propagation of fastidious wild-type circulating HuRVAs from stool specimens.
The establishment of an entirely plasmid-based reverse genetics system for HuRVAs, which exhibit a much lower replication efficiency than the simian SA11-L2 virus, is an important advance that provides a new tool for the better understanding of different aspects of HuRVA replication, pathogenesis, and other complex biological activities. Furthermore, this HuRVA reverse genetics system may be useful for next-generation vaccine production and clinical vector engineering.
MATERIALS AND METHODS
Cells and viruses.A baby hamster kidney cell line, BHK/T7-9 (34), constitutively expressing T7 RNA polymerase, was cultured in Dulbecco’s modified Eagle medium (DMEM; Nissui) supplemented with 5% fetal calf serum (FCS; Gibco) (complete medium) in the presence of 600 ng/ml hygromycin (Invitrogen). Monkey kidney cell lines MA104 and CV-1 were cultured in complete medium. HuRVA strain KU (G1P[8]) (35) and simian RVA strain SA11-L2 (G3P[2]) (32) were propagated as described previously (10). Briefly, KU and SA11-L2 were pretreated with trypsin (type IX, from porcine pancreas; 10 μg/ml; Sigma-Aldrich) and then propagated in MA104 cells in Eagle’s minimum essential medium (MEM; Nissui) without FCS (incomplete medium) but containing trypsin (1 μg/ml).
cDNA library building, Illumina MiSeq sequencing, and sequence analysis.Preparation of a cDNA library and Illumina MiSeq sequencing were performed as described previously (36, 37). Briefly, a 200-bp fragment library ligated with barcoded adapters was built using an NEBNext Ultra RNA Library Prep kit for Illumina v1.2 (New England Biolabs) according to the manufacturer’s instructions. The cDNA library was purified using Agencourt AMPure XP magnetic beads (Beckman Coulter). After assessing the quality and quantity of the purified cDNA library, nucleotide sequencing was carried out on an Illumina MiSeq sequencer (Illumina) using a MiSeq Reagent kit v2 (Illumina) to generate 151 paired-end reads. Data analysis was performed using a CLC Genomics Workbench v8.0.1 (CLC Bio). Contigs were assembled from the obtained sequence reads (trimmed) by de novo assembly. Using the assembled contigs as query sequences, the Basic Local Alignment Search Tool (BLAST) nonredundant nucleotide database was searched to determine which contig represents the full-length nucleotide sequence for each segment of KU and rKU viruses.
Construction of rescue T7 plasmids carrying the VP1 to VP3, VP4, VP7, and NSP1 to NSP4 gene segments of HuRVA KU virus.To establish a HuRVA reverse genetics system, we newly constructed nine T7 plasmids for transcription of the individual mRNAs of nine gene segments, the exceptions being the VP6 and NSP5 gene segments, of KU virus. As vector backbones, we used a pUC57R vector (VP1 and VP3 genes) and a traditional pX8dT vector (38) (VP2, VP4, VP7, and NSP1 to NSP4 genes). To create T7-driven plasmids pT7/VP1KU and pT7/VP3KU, the full-length cDNA fragments of the VP1 and VP3 gene segments of KU virus were artificially synthesized by GENEWIZ. To construct T7-driven plasmids pT7/VP2KU, pT7/VP4KU, pT7/VP7KU, pT7/NSP1KU, pT7/NSP2KU, pT7/NSP3KU, and pT7/NSP4KU, cDNAs, each of which encodes the full-length nucleotide sequence of the corresponding gene segment of KU virus, were amplified by RT-PCR from the genomic dsRNAs with ReverTra Ace reverse transcriptase (Toyobo), PrimeStar HS DNA polymerase (TaKaRa Bio), and specific primers. As described previously (10, 15, 16), the forward primers contain the T7 RNA polymerase promoter sequence and a sequence corresponding to the 5′ terminus of each viral segment. After digestion with restriction enzymes, the prepared cDNAs were ligated into the corresponding restriction enzyme sites of T7 expression plasmids. As T7 expression plasmids, we employed a pUC57-derived pUC57R vector that carries the HDV ribozyme and T7 RNA polymerase terminator sequences (artificially synthesized by GENEWIZ) (for VP1 and VP3 genes) and a modified pX8dT vector (16, 38) (for VP2, VP4, VP7, and NSP1 to NSP4 genes). The constructed T7 plasmids contain the full-length segment cDNAs of individual genes of KU virus (GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences of the VP1 to VP4, VP6, VP7, and NSP1 to NSP5 genes are LC438379 to LC438388 and LC309019, respectively), flanked by the T7 RNA polymerase promoter and HDV ribozyme sequences (9), and followed by the T7 RNA polymerase terminator sequence. The rescue T7 plasmids containing signature mutations to destroy unique restriction enzyme sites (HindIII in the VP1 gene [position 3066], pT7/VP1KU-ΔHindIII; and EcoRI in the NSP4 gene [position 482], pT7/NSP4KU-ΔEcoRI) were generated using a QuikChange II site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer’s instructions. The nucleotide sequences of all the constructed plasmids were confirmed by direct sequencing. The primer sequences used for plasmid construction will be provided on request.
An 11-plasmid reverse genetics system for HuRVAs.The protocol was basically as described by Komoto et al. (16), with some modifications. Eleven T7 plasmids encoding the genome of KU virus, pT7/VP1KU-ΔHindIII, pT7/VP2KU, pT7/VP3KU, pT7/VP4KU, pT7/VP6KU (14), pT7/VP7KU, pT7/NSP1KU, pT7/NSP2KU, pT7/NSP3KU, pT7/NSP4KU-ΔEcoRI, and/or pT7/NSP5KU (15), were employed. Briefly, the protocol was as follows. Monolayers of BHK/T7-9 cells in 6-well plates (Falcon) were cotransfected with 11 T7 plasmids representing the cloned cDNAs of 11 RVA gene segments, pT7/VP1KU-ΔHindIII (0.75 μg), pT7/VP2KU (0.75 μg), pT7/VP3KU (0.75 μg), pT7/VP6KU (0.75 μg), pT7/VP7KU (0.75 μg), pT7/NSP1KU (0.75 μg), pT7/NSP2KU (2.25 μg), pT7/NSP3KU (0.75 μg), pT7/NSP4KU-ΔEcoRI (0.75 μg), and pT7/NSP5KU (2.25 μg), in combination with pT7/VP4KU (0.75 μg) or pT7/VP4SA11-ΔPstI (0.75 μg). Following a 1-day incubation, the transfected BHK/T7-9 cells were washed with incomplete medium and then cocultured with overlaid CV-1 cells (5 × 104/well) for 3 days in incomplete medium containing trypsin (0.3 or 0.9 μg/ml). After incubation, the cultures were subjected to two cycles of freezing and thawing and then treated with trypsin (10 μg/ml) for RVA activation, followed by inoculation onto MA104 cells in a roller-tube culture (5). The cultures were passaged once in MA104 cells in a roller-tube culture. After a 1-day incubation, recombinant HuRVAs were rescued and then plaque purified in CV-1 cells as described previously (20).
PAGE analysis of viral genomic dsRNAs.Viral genomic dsRNAs were extracted from cell cultures using a QIAamp Viral RNA Mini kit (Qiagen). The extracted viral dsRNAs were subjected to PAGE analysis. The dsRNAs were electrophoresed in 10% polyacrylamide gels for 16 h at 20 mA at room temperature, followed by silver staining (10) to determine the genomic dsRNA migration profiles.
Multiple-step virus replication.Monolayers of MA104 cells in 12-well plates (Falcon) were infected in triplicate with trypsin-pretreated RVAs at an MOI of 0.01, washed twice with incomplete medium, and then incubated in incomplete medium with trypsin (1 μg/ml) for various numbers of hours. The infected cells were frozen and thawed twice before the determination of virus titers by plaque assay.
Plaque assay.The plaque assays were carried out as described previously (39). Briefly, monolayers of CV-1 cells in 6-well plates (Thermo Fisher Scientific) were infected with trypsin-pretreated RVAs, washed twice with incomplete medium, and then cultured with trypsin (1 μg/ml) in primary overlay medium (0.7% agarose). After 2 days, the cells were stained with secondary overlay medium containing 0.005% neutral red (Sigma-Aldrich) and 0.7% agarose. Plaque sizes were determined by measuring the mean diameters of 25 plaques in two independent assays.
Statistics.Virus titers were evaluated by means of a t test. Statistical analyses were carried out using GraphPad Prism 7 (GraphPad Software). P values of <0.05 were considered statistically significant.
ACKNOWLEDGMENTS
We thank Chihiro Yamashiro (Fujita Health University, Aichi) for technical assistance.
This study was supported in part by the MEXT-supported program for the Research Program on Emerging and Re-emerging Infectious Diseases of the Japan Agency for Medical Research and Development, AMED (18fk0108018h0403 and 18fk0108034h1102 to S.K.), JSPS KAKENHI (15K08505 and 18K07150 to S.K.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (S.K.), and the Takeda Science Foundation (S.K.).
We have no conflicts of interest to declare.
FOOTNOTES
- Received 15 December 2018.
- Accepted 28 January 2019.
- Accepted manuscript posted online 6 February 2019.
- Copyright © 2019 American Society for Microbiology.
