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. 2018 Oct 29;12(10):e0006884.
doi: 10.1371/journal.pntd.0006884. eCollection 2018 Oct.

Development of Reverse Genetics Systems and Investigation of Host Response Antagonism and Reassortment Potential for Cache Valley and Kairi Viruses, Two Emerging Orthobunyaviruses of the Americas

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Free PMC article

Development of Reverse Genetics Systems and Investigation of Host Response Antagonism and Reassortment Potential for Cache Valley and Kairi Viruses, Two Emerging Orthobunyaviruses of the Americas

James I Dunlop et al. PLoS Negl Trop Dis. .
Free PMC article

Abstract

Orthobunyaviruses such as Cache Valley virus (CVV) and Kairi virus (KRIV) are important animal pathogens. Periodic outbreaks of CVV have resulted in the significant loss of lambs on North American farms, whilst KRIV has mainly been detected in South and Central America with little overlap in geographical range. Vaccines or treatments for these viruses are unavailable. One approach to develop novel vaccine candidates is based on the use of reverse genetics to produce attenuated viruses that elicit immune responses but cannot revert to full virulence. The full genomes of both viruses were sequenced to obtain up to date genome sequence information. Following sequencing, minigenome systems and reverse genetics systems for both CVV and KRIV were developed. Both CVV and KRIV showed a wide in vitro cell host range, with BHK-21 cells a suitable host cell line for virus propagation and titration. To develop attenuated viruses, the open reading frames of the NSs proteins were disrupted. The recombinant viruses with no NSs protein expression induced the production of type I interferon (IFN), indicating that for both viruses NSs functions as an IFN antagonist and that such attenuated viruses could form the basis for attenuated viral vaccines. To assess the potential for reassortment between CVV and KRIV, which could be relevant during vaccination campaigns in areas of overlap, we attempted to produce M segment reassortants by reverse genetics. We were unable to obtain such viruses, suggesting that it is an unlikely event.

Conflict of interest statement

I have read the journal’s policy and the authors of this manuscript have the following competing interests: Anti CVV and KRIV N antibodies described in this study were produced by the MRC-University of Glasgow Centre for Virus Research in collaboration with Cambridge Research Biochemicals (CRB). Antibodies are distributed by CRB under license from the University of Glasgow.

Figures

Fig 1
Fig 1. Growth of CVV and KRIV.
(a) Plaque phenotype of authentic wt CVV and KRIV denoted as wt CVV and wt KRIV; recombinant wt CVV and KRIV, denoted as rCVV and rKRIV and NSs-deletant CVV and KRIV, denoted as rCVVdelNSs and rKRIVdelNSs. BHK-21 cells were used in all plaque assays. The plaque pictures shown are representative of several plaque assays. (b) Western blot analysis showing reactivity of infected BHK-21 cell lysate with anti-KRIV N and anti-CVV N antibodies. Molecular weight markers (M) in kD. The blot shown is a representative of 3 experiments with identical results; U, uninfected cells.
Fig 2
Fig 2. Design of CVV and KRIV that do not express NSs.
Shown are sections of S segment for the N-termini of the N and overlapping NSs proteins. Mutations were added to disrupt the reading frames of the NSs proteins for both viruses, without changing the amino acid sequence of the overlapping N protein. For CVV, two methionines were changed to threonine (denoted in bold) and two stop codons introduced (denoted with an asterisk). For KRIV, mutational sites were used where three methionines were changed to threonines and two stop codons introduced. Protein representation is not to scale.
Fig 3
Fig 3. CVV growth in cell culture.
Cell lines BHK-21 (a), Vero-E6 (b), A549 (c), A549 NPro (d), Aag2 (e) and SFT-R (f) were infected at a MOI of 0.1. The curves show the titre of CVV accumulated at 12 or 24 hr intervals. The titre of the virus at each time point was determined by plaque assay; virus titres are indicated in plaque forming units per ml (PFU/ml). Representative experiments are shown (n = 2). Error bars indicate standard deviation (SD).
Fig 4
Fig 4. KRIV growth in cell culture.
Cell lines BHK-21 (a), Vero-E6 (b) A549 (c), A549 Npro (d) Aag-2 (e) and the equine cell line, E-Derm (f) were infected at a MOI of 0.1 or 3 as indicated. The curves show the titre of KRIV accumulated after 12 or 24 hr intervals. The titre of virus at each time point was determined by a plaque assay; virus titres are indicated in PFU/ml. Representative experiments are shown (n = 2). Error bars indicate SD.
Fig 5
Fig 5. Type I IFN protection assay for orthobunyaviruses.
A549 cells were infected with rBUNV, rCVV, rKRIV or rCVVdelNSs, rBUNVdelNSs (clone rBUNVdelNSs2) or rKRIVdelNSs at a MOI of 1 and incubated at 37 °C for 48 hrs. Twofold dilutions of the clarified and UV-treated supernatant were used to treat fresh A549 NPro cells in a 96-well plate for 24 hrs. The cells were then infected with EMCV and the development of CPE was monitored at 96 hrs post-infection by staining with crystal violet. The production of IFN was calculated according to the highest dilution of supernatant giving protection against EMCV infection and is expressed as relative IFN units. The experiment was conducted three times and gave identical results.
Fig 6
Fig 6. Phylogenetic analysis of selected viruses from the Bunyamwera serogroup with the newly acquired sequences from CVV (6V633) and KRIV (TR8900).
The bootstrap supports shown at the nodes on the phylogeny (* represents 100 percent bootstrap support) and La Crosse virus (California serogroup) was used as an outgroup: (A) nucleoprotein N (from S segment) using the JTT+G substitution model, (B) RNA dependent RNA polymerase (from L segment) using the LG protein substitution model and (C) the M segment open reading frame (encoding glycoproteins and NSm) using the FLU+I+G+F substitution model. The arrows on the phylogeny indicate the phylogenetic position of the newly sequenced full genomes.
Fig 7
Fig 7. Cross-recognition of UTRs between CVV and KRIV in minigenome activity assay.
BSR-T7/5 CL21 cells were transfected with pTM1-N and/or pTM1-L and M-derived minigenome plasmids (as described in Methods) pUC57-T7-KRIVMRen(-) (a), or pUC57-T7-CVVMRen(-) (b). Firefly luciferase (FF)-expressing pTM1-FF-Luc was used as a transfection control. At 24 hrs post-transfection, cells were lysed and Ren and Firefly luciferase activities were measured using Dual-Luciferase Reporter Assay kit (Promega). Luciferase values were normalised and minigenome activity is expressed as relative light units (RLU). Error bars indicate SD (n = 3). This is one representative experiment of three repeats with very similar results. pTM1-CVV or pTM1-KRIV refers to the viral origin of L or N proteins.
Fig 8
Fig 8. Attempted rescues of CVV and KRIV M segment reassortants.
Western blot analysis showing the lack of N protein produced in 3 independent rescues for the hybrid viruses CKC and KCK (with segment order S-M-L). Supernatant from rescue experiment was passaged (1 ml of rescue supernatant added to BHK-21 cells for 5–7 days) and cell lysates used for western blotting using an anti-CVV N antibody for the hybrid CKC and an anti-KRIV N antibody for KCK. +ve 1, rCVV-infected cell lysate; +ve 2, rKRIV-infected cell lysate; U, uninfected cells.

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