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Construction of the full-length cDNA clone of IPNV segment A for the generation of plus-sense RNA transcript with T7 RNA polymerase. The gene structure of IPNV segment A and its encoded proteins are shown at the top. Overlapping cDNA segments of IPNV were generated by RT-PCR and cloned into a pCR2.1 vector to obtain various pCR clones, as indicated. These plasmids were digested with appropriate restriction enzymes, and the resulting segments were cloned into a pUC19 vector to obtain plasmid pUC19WBA. This plasmid contains a T7 RNA polymerase promoter sequence at its 5 end. Restriction enzymes used for the construction or linearization of the full-length clone are indicated. Abbreviations: A, ApaI; E, EcoRI; K, Asp718; S, SalI.
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We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of the Birnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segm...
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... were cloned into the EcoRI site of the pCR2.1 vector (Invitrogen Corp.) to obtain plasmids pCR#8, pCR#11, and pCR#23 ( Fig. 1). The insert DNA in all these plasmids was sequenced by the dideoxy chain termination method with an Applied Biosystem automated DNA sequencer, and the se- quence data were analyzed by using PC/GENE (Intelligenetics) software. To construct a full-length cDNA clone of segment A, plasmids pCR#8, pCR#11, and pCR#23 were double digested ...
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... enzyme pairs Asp718 plus ApaI, ApaI plus SalI, and SalI plus EcoRI to release 670-, 1,520-, and 904-bp fragments, respectively. These fragments were then cloned between the EcoRI and Asp718 sites of pUC19 vector to obtain plasmid pUC19WBA. This plasmid contains a full-length copy of segment A, which encodes all of the structural and NS proteins ( Fig. 1). Similarly, to prepare cDNA clones of segment B, three primer pairs (B-B5NC plus B-HindR, B-HindF plus B-PstR, and B-PstF plus B-Bgl3NC) were used to generate overlapping cDNA fragments of segment B by RT-PCR amplification (Table 1). Amplified fragments were cloned into pCR2.1 vector as described above to obtain plasmids pCR#4.1, ...
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... and transfection of synthetic RNAs. Plasmid pUC19WBA and plasmids pUC19WBB and pUC19WBB-Sma were digested with SmaI and with both BglII and SmaI, respectively ( Fig. 1 and 2), and used as templates for in vitro transcription with T7 RNA polymerase (Promega). Similarly, plasmids pUC19FLAD78 and pUCD78B were linearized with BsrGI and PstI enzymes, respectively, as described earlier (24,32). ...
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... analysis of IPNV genome. We determined the complete nucleotide sequences of IPNV genome segments A and B, including the precise 5-and 3-terminal sequences. Seg- ment A is 3,097 bp long and contains two overlapping ORFs. The major ORF encodes the structural VP2 and VP3 proteins and the NS protease, whereas the minor ORF codes for the NS protein (Fig. 1). Segment B is 2,783 bp long and encodes VP1, which is the RNA-dependent RNA polymerase (Fig. 2). Com- parison of the 5-and 3-terminal sequences of segments A and B of WB strain with those of the Jasper strain (GenBank ac- cession no. M18049 and M58756) showed some minor differ- ences. For example, in segment A, a deletion of a T ...
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... clones of segments A and B of IPNV strain WB. Plas- mid pUC19WBA, upon digestion with SmaI and transcription in vitro by T7 RNA polymerase, yielded RNA with precise 5 and 3 ends, and it encoded all of the structural and NS pro- teins (Fig. 1). However, after linearization with BglII and tran- scription, plasmid pUC19WBB yielded RNA with the correct 5 end but with an additional five nucleotides at the 3 end, and it encoded VP1 protein (Fig. 2). We also constructed plasmid pUC19WBB-Sma with a genetic tag (the elimination of an in- ternal SmaI site) to identify the virus as ...
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... 954- bp fragment was obtained from both rWB and rWB-Sma vi- ruses (lanes 5 and 6), but not from the CHSE cells (lane 4). Moreover, no PCR product was detected in mock-infected or IPNV-infected cells if the RT was omitted from the reaction before PCR (lanes 1 to 3). This indicates that the PCR product was derived from RNA and not from contaminating DNA. ...
Similar publications
The nucleotide sequence of genome segment A cDNA of the STC strain of infectious bursal disease virus (IBDV) was determined and compared with sequences of the homologous genome segment of the 002-73 strain of IBDV and the Jasper strain of infectious pancreatic necrosis virus (IPNV). The STC-IBDV genome segment A was determined to be 3262 base pairs...
Citations
... *Modified initiators, modified nucleotides were highlighted in bold. a Initiators previously reported byYao and Vakharia (1998); b Initiators designed to amplify a 1259 Pb PCR product of the 3' RNC of the polyprotein; c Primers previously reported byBlake et al. (2001). BAUTISTA-GÓMEZ ET AL. ...
El virus de la necrosis pancreática infecciosa es uno de los agentes infecciosos más importantes de la industria trutícola y del salmón, dado que causa alta mortalidad en crías de primera alimentación. En México, es la única enfermedad viral que afecta a la trucha arcoíris, por lo tanto, información sobre la caracterización y origen genético de los asilados virales que circulan en nuestro país, ya han sido descritos, sin embargo, un análisis con el genoma completo no, motivo por el cual, el presente trabajo de investigación tiene por objetivo analizar el genoma completo del virus de la necrosis pancreática infecciosas que comúnmente circula en el país. A partir de las relaciones genéticas de un prototipo de este virus aislado en México en relación con otros aislados que circulan a nivel mundial, se encontró que el aislado mexicano del virus de la necrosis pancreática infecciosas pertenece al genogrupo I y es originado a partir de la cepa Buhl.
... With this aim, recombinant viruses displaying mutations in the mentioned positions were obtained by reverse genetics. This technique has been successfully used to study both, negativeand positive-stranded RNA fish viruses 11,[19][20][21][22][23][24][25] . ...
European sea bass (Dicentrarchus labrax) is severely affected by nervous necrosis disease, caused by nervous necrosis virus (NNV). Two out of the four genotypes of this virus (red-spotted grouper nervous necrosis virus, RGNNV; and striped jack nervous necrosis virus, SJNNV) have been detected in sea bass, although showing different levels of virulence to this fish species. Thus, sea bass is highly susceptible to RGNNV, whereas outbreaks caused by SJNNV have not been reported in this fish species. The role of the capsid protein (Cp) amino acids 247 and 270 in the virulence of a RGNNV isolate to sea bass has been evaluated by the generation of recombinant RGNNV viruses harbouring SJNNV-type amino acids in the above mentioned positions (Mut247Dl965, Mut270Dl965 and Mut247 + 270Dl965). Viral in vitro and in vivo replication, virus virulence and fish immune response triggered by these viruses have been analysed. Mutated viruses replicated on E-11 cells, although showing some differences compared to the wild type virus, suggesting that the mutations can affect the viral cell recognition and entry. In vivo, fish mortality caused by mutated viruses was 75% lower, and viral replication in sea bass brain was altered compared to non-mutated virus. Regarding sea bass immune response, mutated viruses triggered a lower induction of IFN I system and inflammatory response-related genes. Furthermore, mutations caused changes in viral serological properties (especially the mutation in amino acid 270), inducing higher seroconversion and changing antigen recognition.
... Viral genome sequencing was performed on RNA isolated by PatoGen and transcribed to cDNA using Superscript ® III First-Strand Synthesis from Invitrogen by suppliers instruction. The PCR for sequencing was performed on segment A using primers described previously (Santi et al., 2004;Yao & Vakharia, 1998). The sequencing was performed by the dideoxy chain termination method in an automated DNA sequencer (Applied Biosystems) using sequence-specific primers. ...
Infectious pancreatic necrosis (IPN) is an important restraint to production of salmonids in aquaculture globally. In order to implement efficacious mitigation strategies for control of this disease, it is important to understand infection routes under current production systems. IPN virus has been shown to be transmitted vertically in Rainbow trout, from broodstock to fingerlings in hatcheries, and there is circumstantial evidence suggesting that vertical transmission can also occur in Atlantic salmon, in addition to horizontal transmission between grow‐out fish in farms. In this study, we show that the smolt carries infection with IPN from hatchery to the marine farm. We do this by comparing sequences from fish groups taken both in hatcheries and on corresponding marine grow‐out farms. We use statistical analysis to prove that sequences obtained from the same fish group in both hatchery and marine farm are more similar than sequences obtained from random fish groups on hatcheries and marine farms.
... Reverse genetics (RG) has been developed for viruses of many families including those with double stranded RNA (dsRNA) segmented genomes. The latter includes members of Birnaviridae [52,53], and different genera within the Reoviridae family [25]. RG for BTV, the prototype member of the Orbivirus genus has also been developed. ...
Background:
Bluetongue virus (BTV) and African horse sickness virus (AHSV) are distinct arthropod borne virus species in the genus Orbivirus (Reoviridae family), causing the notifiable diseases Bluetongue and African horse sickness of ruminants and equids, respectively. Reverse genetics systems for these orbiviruses with their ten-segmented genome of double stranded RNA have been developed. Initially, two subsequent transfections of in vitro synthesized capped run-off RNA transcripts resulted in the recovery of BTV. Reverse genetics has been improved by transfection of expression plasmids followed by transfection of ten RNA transcripts. Recovery of AHSV was further improved by use of expression plasmids containing optimized open reading frames.
Results:
Plasmids containing full length cDNA of the 10 genome segments for T7 promoter-driven production of full length run-off RNA transcripts and expression plasmids with optimized open reading frames (ORFs) were used. BTV and AHSV were rescued using reverse genetics. The requirement of each expression plasmid and capping of RNA transcripts for reverse genetics were studied and compared for BTV and AHSV. BTV was recovered by transfection of VP1 and NS2 expression plasmids followed by transfection of a set of ten capped RNAs. VP3 expression plasmid was also required if uncapped RNAs were transfected. Recovery of AHSV required transfection of VP1, VP3 and NS2 expression plasmids followed by transfection of capped RNA transcripts. Plasmid-driven expression of VP4, 6 and 7 was also needed when uncapped RNA transcripts were used. Irrespective of capping of RNA transcripts, NS1 expression plasmid was not needed for recovery, although NS1 protein is essential for virus propagation. Improvement of reverse genetics for AHSV was clearly demonstrated by rescue of several mutants and reassortants that were not rescued with previous methods.
Conclusions:
A limited number of expression plasmids is required for rescue of BTV or AHSV using reverse genetics, making the system much more versatile and generally applicable. Optimization of reverse genetics enlarge the possibilities to rescue virus mutants and reassortants, and will greatly benefit the control of these important diseases of livestock and companion animals.
... Similarly, segment B forward primer was based on Yao and Vakharia's work from 1998[15], and the corresponding reverse primer designed in accordance to IPNV sequences available at GenBank,For both, A and B segments amplification, 3.0 µL of viral RNA were mixed with primers A-A5EJ'NC and A-IPNVEJ R for segment A and B-B5EJ'NC and B-IPNVEJ R for segment B at a final concentration of 0.5 µM in 15 µL of 2X DreamTaqTM Green PCR Master Mix (Fermentas, Vilnius, Lithuania), 0.8 µL of MMLV Reverse Transcriptase and RNase Block (Stratagene, La Jolla, CA, USA) and 8.2 µL of RNase free water in 30 µL reaction volume. The primers used are shown in table 1. ...
Background: The genomes of several infectious pancreatic necrosis viruses (IPNVs) isolated in Chile were
sequenced with a single amplification approach for both segments A and B. The resulting sequences were then used to determine the conservation of the primer-binding regions used in polymerase chain reaction
(PCR)-based diagnostic methods proposed in the literature. Thus, the robustness of each technique was
studied, particularly the eventual effect of further mutations within the primer-binding sites.
Results: On analysis, most methods currently used to detect Chilean IPNV varieties were deemed adequate.
However, the primers were designed to be genogroup specific, implying that most detection methods pose
some risk of detecting all strains prevalent in the country, due to the coexistence of genogroups 1 and 5.
Conclusions: Negative results must be interpreted carefully given the high genomic variability of IPNVs. Detection techniques (quantitative reverse transcription (qRT)-PCR) based on degenerate primers can be used to minimize the possibilities of false-negative detections.
... At various intervals postinfection, cells were trypsinized, transferred to microcentrifuge tubes, washed twice with PBS, and fixed in 70% ethanol at 4°C overnight. Cells were washed twice with PBS and stained with Krishan's stain, containing 3.8 mM trisodium citrate (Sigma), 70 M propidium iodide (Sigma), 0.01% Nonidet P-40 (Sigma), and 0.01 mg of RNase A (Boehringer Mannheim) per ml (43). Cellular DNA content was quantified using a Coulter Epics XL flow cytometer (Beckman-Coulter). ...
Reovirus nonstructural protein σ1s is implicated in cell cycle arrest at the G2/M boundary and induction of apoptosis. However, the contribution of σ1s to these effects in an otherwise isogenic viral background
has not been defined. To evaluate the role of σ1s in cell cycle arrest and apoptosis, we used reverse genetics to generate
a σ1s-null reovirus. Following infection with wild-type virus, we observed an increase in the percentage of cells in G2/M, whereas the proportion of cells in G2/M following infection with the σ1s-null mutant was unaffected. Similarly, we found that the wild-type virus induced substantially
greater levels of apoptosis than the σ1s-null mutant. These data indicate that σ1s is required for both reovirus-induced cell
cycle arrest and apoptosis. To define sequences in σ1s that mediate these effects, we engineered viruses encoding C-terminal
σ1s truncations by introducing stop codons in the σ1s open reading frame. We also generated viruses in which charged residues
near the σ1s amino terminus were replaced individually or as a cluster with nonpolar residues. Analysis of these mutants revealed
that amino acids 1 to 59 and the amino-terminal basic cluster are required for induction of both cell cycle arrest and apoptosis.
Remarkably, viruses that fail to induce cell cycle arrest and apoptosis also are attenuated in vivo. Thus, identical sequences in σ1s are required for reovirus-induced cell cycle arrest, apoptosis, and pathogenesis. Collectively,
these findings provide evidence that the σ1s-mediated properties are genetically linked and suggest that these effects are
mechanistically related.
... Generation of full-length cDNA clones of the entire coding and non-coding regions of NVI-015 RNA segment A and B was performed according to procedures described by Yao and Vakharia [16]. The recombinant IPNV Sp strains rNVI-15TA was generated as described in previous studies [10,11]. ...
We have studied stress-induced reversion to virulence of infectious pancreatic necrosis virus (IPNV) in persistently infected Atlantic salmon (Salmo salar L.) fry. Naïve fry were persistently infected with a virulent strain (T(217)A(221) of major structural virus protein 2, VP2) or a low virulent (T(217)T(221)) variant of IPNV. The fry were infected prior to immunocompetence as documented by lack of recombination activating gene-1, T-cell receptor and B-cell receptor mRNA expression at time of challenge. The fish were followed over 6 months and monitored monthly for presence of virus and viral genome mutations. No mutation was identified in the TA or TT group over the 6 months period post infection. Six months post infection TA and TT infected groups were subject to daily stress for 7 days and then sampled weekly for an additional period of 28 days post stress. Stress-responses were documented by down-regulation of mRNA expression of IFN-α1 and concomitant increase of replication levels of T(217)T(221) infected fish at day 1 post stress. By 28 days post stress a T221A reversion was found in 3 of 6 fish in the T(217)T(221) infected group. Sequencing of reverted isolates showed single nucleotide peaks on chromatograms for residue 221 for all three isolates and no mix of TA and TT strains. Replication fitness of reverted (TA) and non-reverted (TT) variants was studied in vitro under an antiviral state induced by recombinant IFN-α1. The T(217)A(221) reverted variant replicated to levels 23-fold higher than the T(217)T(221) strain in IFN-α1 treated cells. Finally, reverted TA strains were virulent when tested in an in vivo trial in susceptible salmon fry. In conclusion, these results indicate that stress plays a key role in viral replication in vivo and can facilitate conditions that will allow reversion from attenuated virus variants of IPNV.
... A recombinant virus strain made by reverse genetics from the highly virulent Norwegian Sp strain rNVI015 (Genbank: AY379740) [26] was used for production of vaccines, challenge and as an antigen for ELISA. The virus was maintained in Rainbow trout gonad cells (RTG-2) grown in Leibowitz L-15 media (Gibco ® Life Technologies) supplemented with 10% fetal bovine serum (FBS) and gentamycin 500 l. ...
... Generation of full-length cDNA clones of the entire coding and non-coding regions of NVI-015 RNA segment A and B was performed according to procedures described by Yao and Vakharia [26] . The construction of recombinant viruses rNVI- 15TAT and rNVI-15PAA has been described elsewhere [27]. ...
Infectious pancreatic necrosis virus (IPNV) is a member of the family Birnaviridae that has been linked to high mortalities in juvenile salmonids and postsmolt stages of Atlantic salmon (Salmo salar L.) after transfer to seawater. IPN vaccines have been available for a long time but their efficacy has been variable. The reason for the varying immune response to these vaccines has not well defined and studies on the importance of using vaccine trains homologous to the virulent field strain has not been conclusive. In this study we prepared one vaccine identical to the virulent Norwegian Sp strain NVI-015 (NCBI: 379740) (T(217)A(221)T(247) of VP2) and three other vaccine strains developed using the same genomic backbone altered by reverse genetics at three residues yielding variants, T(217)T(221)T(247), P(217)A(221)A(247), P(217)T(221)A(247). These 4 strains, differing in these three positions only, were used as inactivated, oil-adjuvanted vaccines while two strains, T(217)A(221)T(247) and P(217)T(221)A(247), were used as live vaccines. The results show that these three residues of the VP2 capsid play a key role for immunogenicity of IPNV vaccines. The virulent strain for inactivated vaccines elicited the highest level of virus neutralization (VN) titers and ELISA antibodies. Interestingly, differences in immunogenicity were not reflected in differences in post challenge survival percentages (PCSP) for oil-adjuvanted, inactivated vaccines but clearly so for live vaccines (TAT and PTA). Further post challenge viral carrier state correlated inversely with VN titers at challenge for inactivated vaccines and prevalence of pathology in target organs inversely correlated with protection for live vaccines. Overall, our findings show that a few residues localized on the VP2-capsid are important for immunogenicity of IPNV vaccines.
... Development of a plasmid-based reverse genetics system for members of the Birnaviridae family, which contain two genomic dsRNA segments, suggested that delivery of viral positive-strand RNA alone could launch successful viral progeny production for a dsRNA virus (28). Furthermore, introduction of message-sense RNA isolated from virions of Reoviridae family members leads to the production of infectious progeny virions in some cases (6,29,30). ...
Mammalian orthoreoviruses (reoviruses) are highly tractable models for studies of viral replication and pathogenesis. The versatility of reovirus as an experimental model has been enhanced by development of a plasmid-based reverse genetics system. Infectious reovirus can be recovered from cells transfected with plasmids encoding cDNAs of each reovirus gene segment using a strategy that does not require helper virus and is independent of selection. In this system, transcription of each gene segment is driven by bacteriophage T7 RNA polymerase, which can be supplied transiently by recombinant vaccinia virus (rDIs-T7pol) or by cells that constitutively express the enzyme. Reverse genetics systems have been developed for two prototype reovirus strains, type 1 Lang (T1L) and type 3 Dearing (T3D). Each reovirus cDNA was encoded on an independent plasmid for the first-generation rescue system. The efficiency of virus recovery was enhanced in a second-generation system by combining the cDNAs for multiple reovirus gene segments onto single plasmids to reduce the number of plasmids from 10 to 4. The reduction in plasmid number and the use of baby hamster kidney cells that express T7 RNA polymerase increased the efficiency of viral rescue, reduced the incubation time required to recover infectious virus, and eliminated potential biosafety concerns associated with the use of recombinant vaccinia virus. Reovirus reverse genetics has been used to introduce mutations into viral capsid and nonstructural components to study viral protein-structure activity relationships and can be exploited to engineer recombinant reoviruses for vaccine and oncolytic applications.
