A 6K-Deletion Variant of Salmonid Alphavirus Is Non-Viable but Can Be Rescued through RNA Recombination

Guo, Tz Chun; Johansson, Daniel X.; Haugland, Øyvind; Liljeström, Peter; Evensen, Øystein

doi:10.1371/journal.pone.0100184

Cited by 15 publications

(31 citation statements)

References 47 publications

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“…Notably, in contrast to RRV-(⌬6K), the SFV 6k deletion mutant demonstrated a dramatic decrease in the rate of viral replication (5,11). In addition, a 6k deletion mutant of salmonid alphavirus (SAV) was found to be nonviable (34), outlining the functional divergence of the 6k proteins in different alphaviruses. In animal studies, RRV-(⌬6K)-infected mice did not show disease of the same severity as that in WT RRV-infected mice.…”

Section: Discussionmentioning

confidence: 99%

Effects of an In-Frame Deletion of the 6k Gene Locus from the Genome of Ross River Virus

Taylor

Melton

Herrero

et al. 2016

J Virol

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The alphaviral 6k gene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the 6k proteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel 6k in-frame deletion mutant. Comprehensive microscopic analysis revealed that the 6k proteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the 6k proteins 6K and TF [RRV-(⌬6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the 6k deletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequent in vivo studies demonstrated that RRV-(⌬6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(⌬6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the 6k proteins may contribute to alphaviral disease manifestations and suggest that manipulation of the 6k gene may be a potential strategy to facilitate viral vaccine development. IMPORTANCE Arthritogenic alphaviruses, such as chikungunya virus (CHIKV) and Ross River virus (RRV), cause epidemics of debilitating rheumatic disease in areas where they are endemic and can emerge in new regions worldwide. RRV is of considerable medical significance in Australia, where it is the leading cause of arboviral disease. The mechanisms by which alphaviruses persist and cause disease in the host are ill defined. This paper describes the phenotypic properties of an RRV 6k deletion mutant. The absence of the 6k gene reduced virion release from infected cells and also reduced the severity of disease and viral titers in infected mice. Immunization with the mutant virus protected mice against viremia not only upon exposure to RRV but also upon challenge with CHIKV. These findings could lead to the development of safer and more immunogenic alphavirus vectors for vaccine delivery.

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Section: Discussionmentioning

confidence: 99%

Effects of an In-Frame Deletion of the 6k Gene Locus from the Genome of Ross River Virus

Taylor

Melton

Herrero

et al. 2016

J Virol

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“…Virus-inoculated cells were incubated at 15 uC in L-15 medium supplemented with 2% FBS and 50 mg gentamicin ml 21 for both cell lines and these conditions were also used in the following studies unless otherwise stated. The SAV3 isolate used in this study originates from heart tissue from a fish diagnosed with PD as described by Guo et al (2014) and Xu et al (2010). Homogenate supernatant was used for initial inoculation and further propagated by serial passages until the 11th passage in the CHSE cell line.…”

Section: Methodsmentioning

confidence: 99%

“…H10 P11 has previously been subjected to full-length genome sequencing as described in an earlier study where an infectious SAV3 cDNA clone was constructed using H10 P11 genome sequence as the cDNA template (Guo et al, 2014; SAV3-H10 GenBank accession no. JQ799139.1) and H10 P3 and H10 P14 were full-length genome sequenced using the same procedures.…”

Section: Virus Replication Kinetics In Cells Measured By Real-time Pcrmentioning

confidence: 99%

In vitro adaptation of SAV3 in cell culture correlates with reduced in vivo replication capacity and virulence to Atlantic salmon (Salmo salar L.) parr

Petterson

Guo

Evensen

et al. 2015

Journal of General Virology

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In vitro adaptation of SAV3 in cell culture correlates with reduced in vivo replication capacity and virulence to Atlantic salmon (Salmo salar L.) parr Salmonid alphavirus (SAV) is the causative agent of pancreas disease affecting Atlantic salmon and rainbow trout and causes a major burden to the aquaculture industry. This study describes a Norwegian subtype SAV3 virus isolate (SAV3-H10) subjected to serial passages in Chinook salmon embryo cells (CHSE-214) followed by Asian Grouper skin cells (AGK). Two passages from CHSE and one after transfer to AGK cells were chosen for further investigation, based on variation in degree and development of cytopathic effect (CPE). After plaque purification, several in vitro studies were performed. Cell viability after infection, viral replication and ability to cause morphological changes in CHSE and AGK cells was studied for the three isolates. The AGK-transferred isolate was identified with the strongest abilities to reduce cell viability, replicate more and cause more CPE in cell culture when compared with the early and late CHSE-grown isolates. Subsequently, the isolates were tested in an experimental fish challenge, showing higher viral load and higher pathological score for the least cell-cultured isolate. Fulllength sequencing of the viral genome of the three isolates revealed divergence in four amino acid positions and the AGK-grown isolate also had a 3 nt deletion in the 39UTR. In conclusion, we show that cell culture of SAV3-H10 selects for strains inducing earlier CPE in vitro with increased viral replication. In vivo, the effect is reversed, with lower replication levels and lower pathology scores in target organs. This study outlines a path to identify potential virulence motifs of SAV3.

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“…Modifications to the construct for improved levels of protein expression would therefore be valuable, as it is well known that antigen dose in fish vaccines is well correlated with protection (Munang'andu et al, 2013). The objective of the present study was to develop a SAV3 replicon vaccine vector, based on a previously constructed infectious full-length cDNA clone (Guo et al, 2014), and to evaluate different modifications to the constructs for enhanced expression of heterologous protein.…”

mentioning

confidence: 99%

“…Initially, the effect of inserting a hammerhead (HH) ribozyme sequence, as described previously (Guo et al, 2014), to ensure precise cleavage at the 59 end was evaluated. Chinook salmon embryonic (CHSE-214) cells were transfected (Fugene HD transfection reagent, Roche) with an infectious full-length SAV3 clone, with or without the HH ribozyme sequence fused at the 59 end (pSAV3-HHFL and pSAV3-FL, respectively), and incubated at 15 u C. Cell supernatant was collected at 2, 4, 8, 9, 11 and 13 days post-transfection (dpt), followed by titration of rescued virus on CHSE-214 cells by the method of Spearman and Kärber (Miller & Ulrich, 2001).…”

mentioning

confidence: 99%

Modification of a salmonid alphavirus replicon vector for enhanced expression of heterologous antigens

Guo

Johansson

Liljeström

et al. 2015

Journal of General Virology

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A salmonid alphavirus (SAV) replicon has been developed to express heterologous antigens but protein production was low to modest compared with terrestrial alphavirus replicons. In this study, we have compared several modifications to a SAV replicon construct and analysed their influence on foreign gene expression. We found that an insertion of a translational enhancer consisting of the N-terminal 102 nt of the capsid gene, together with a nucleotide sequence encoding the footand-mouth disease virus (FMDV) 2A peptide, caused a significant increase in EGFP reporter gene expression. The importance of fusing a hammerhead (HH) ribozyme sequence at the 59 end of the viral genome was also demonstrated. In contrast, a hepatitis D virus ribozyme (HDV-RZ) sequence placed at the 39 end did not augment expression of inserted genes. Taken together, we have developed a platform for optimized antigen production, which can be applied for immunization of salmonid fish in the future.

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A 6K-Deletion Variant of Salmonid Alphavirus Is Non-Viable but Can Be Rescued through RNA Recombination

Cited by 15 publications

References 47 publications

Effects of an In-Frame Deletion of the 6k Gene Locus from the Genome of Ross River Virus

Effects of an In-Frame Deletion of the 6k Gene Locus from the Genome of Ross River Virus

In vitro adaptation of SAV3 in cell culture correlates with reduced in vivo replication capacity and virulence to Atlantic salmon (Salmo salar L.) parr

Modification of a salmonid alphavirus replicon vector for enhanced expression of heterologous antigens

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