Synthetic recovery of Yada Yada virus expands insect-specific alphavirus knowledge and facilitates production of chimeric viruses

Abstract

Few insect-specific alphaviruses (ISA) have been discovered, with even fewer culturable to facilitate full characterisation. Here, we report the recovery of an infectious clone of Yada Yada virus (YYV)-a virus previously only detected by metagenomic sequencing of mosquito homogenates. Using the infectious clone, we confirmed the inability of YYV to replicate in vertebrate cells in vitro, with replication limited to only Aedes mosquito-derived cell lines. We further produced and characterised the first monoclonal antibodies (mAbs) to ISAs. Through successful replacement of the structural proteins of YYV with those of other ISAs, Eilat virus, Agua Salud (ASALV), Taï Forest (TALV) and Mwinilunga alphaviruses (MWAV), we established that a replication block for in vitro culture of TALV and MWAV in mosquito cells does not exist at virus entry. Unexpectedly, ASALV structural proteins were recognised by cross-reactive mAbs made to chikungunya (CHIKV) and Ross River viruses (RRV), suggesting a potential antigenic link between ASALV and pathogenic alphaviruses. The YYV genetic backbone was also investigated to generate chimeras displaying the structural proteins of various pathogenic vertebrate-infecting alphaviruses including CHIKV, RRV, Barmah Forest, Sindbis and Mayaro viruses. These chimeras retained the antigenic properties of the parental viruses and did not replicate in vertebrate cells, demonstrating the potential of the YYV platform for vaccine and diagnostic antigen production.

Fig. 1. Recovery of YYV and EILV infectious clones by CPER.

A Schematic of the YYV genome, overlapping fragments and linker fragment used for CPER assembly. B Schematics of alphavirus genomes showing virus and chimera construction. Construct 1 (C1): EILV (blue) infectious clone; construct 2 (C2), EILV/YYV-sP chimera – EILV backbone (blue), YYV structural proteins (green); construct 3 (C3), EILV/RRV-sP/YYV-3’UTR: EILV 5’ UTR and non-structural proteins (blue), RRV structural proteins (red), YYV 3’UTR (green), with addition of UC nucleotides prior to the poly(A) tail; Construct 4 (C4): final YYV infectious clone (YYV_EILV; green) including the UC nucleotides. C IFA staining of C6/36 cells infected constructs of panel B. Mock and infectious clone/chimera infected and fixed cell monolayers were probed with anti-dsRNA (see also Fig. S4A), anti-YYV E2 (6C12), anti-EILV E2 (1E1) or cross-reactive anti-CHIKV capsid (5.5G9) mAbs (green). Nuclei were stained with Hoechst 33342 (blue). Images taken at 40× magnification. D Comparative growth kinetics of EILV, YYV_EILV (YYV infectious clone including 5’ terminal 11 nt of EILV genome) and YYV_TALV (YYV infectious clone including 5’ terminal 11 nt of TALV genome) in C6/36 cells, infected at an MOI of 0.1 (n = 3). Statistical analyses: two-way ANOVA, and comparisons between individual time-points with Tukey’s multiple comparisons test. *P ≤ 0.05. E Representative image of gradient-purified YYV. The blue band is concentrated virus, and the white band is cell debris from C6/36 cells. F SDS-PAGE analysis of gradient-purified infectious clones and chimeric viruses. G TEM of purified YYV imaged at ×100,000 magnification. H IFA analysis of insect and mammalian cells infected with YYV, YYV/RRV-sP, and RRV_WT or West Nile virus (WNV_WT) virus at an MOI of 1. YYV-infected cell lines probed with anti-YYV mAb 6C12, YYV/RRV-sP and RRV with cross-reactive anti-CHIKV mAb 5.5G9, and WNV with anti-flavivirus envelope mAb 4G2. Nuclei were stained with Hoechst 33342. Images taken at 63× magnification.

Fig. 2. Recovery of chimeric insect-specific alphaviruses.

A Schematics of alphavirus genomes showing ASALV, MWAV, and TALV structural protein chimeric virus construction with YYV backbone. For each construct, the YYV structural protein cassette has been replaced with that of ASALV (pink), MWAV (orange) or TALV (yellow), whilst retaining the YYV non-structural protein genes and UTRs (green). B IFA staining of C6/36 cells infected with supernatant post-CPER transfection of constructs listed in panel B. Mock and chimera-infected and fixed cell monolayers were probed with anti-dsRNA mAbs (see also Fig. S4A), anti-YYV mAb 6C12, anti-EILV mAb 1E1, or cross-reactive anti-CHIKV capsid mAb 5.5G9 (green). Nuclei were stained with Hoechst 33342 (blue). Images taken at 40 × magnification. C SDS-PAGE of YYV and EILV infectious clone and YYV/ISA-sP chimeric virus virions treated with (+) or without (–) PNGase F enzyme. The alphavirus structural proteins (E1/E2 and C) are indicated. D Western blot analysis of purified EILV, YYV, YYV/ASALV-sP and YYV/TALV-sP antigen probed with hyperimmune mouse sera derived from mice injected with antigens as labelled. Visual reactivity of each sera to viral E1/E2 or C proteins is indicated below the Western blot panels: –, non-reactive; +, reactive. E TEM of purified YYV/ASALV-sP, Y/MWAV-sP, and Y/TALV-sP stained with 2% uranyl acetate and imaged at ×100,000 magnification.

Fig. 3. Characterisation of YYV chimeras for vertebrate-infecting alphaviruses.

A Schematic of YYV chimeras displaying VIA structural proteins genes (YYV/VIA-sP). B IFA staining of C6/36 cells infected with supernatant post CPER transfection of virus constructs. Mock and infectious clone/chimera-infected and fixed cell monolayers were probed with cross-reactive anti-RRV E2 mAb B10 (YYV/BFV-sP only) or cross-reactive anti-CHIKV capsid mAb 5.5G9 (other viruses), and anti-YYV E1/E2 mAb 6C12 (green). Nuclei were stained with Hoechst 33342 (blue). Images taken at ×40 magnification. C Coomassie-stained SDS-PAGE of gradient-purified YYV/CHIKV-sP and YYV/RRV-sP. D TEM of purified YYV/CHIKV-sP and YYV/RRV-sP stained with 2% uranyl acetate and imaged at ×100,000 magnification. E Analysis of virus titre of various infectious clones, chimeras and wild type viruses 4 dpi after infection of C6/36 cells at MOI of 0.01 (n = 3). 1: YYV infectious clone, 2: YYV/RRV-sP, 3: YYV/CHIKV-sP, 4: EILV infectious clone, 5: EILV/RRV-sP, 6: YYV/RRV-sP/EILV-3’UTR, 7: EILV/RRV-sP/YYV-3’UTR, 8: RRV_WT (GenBank KY302801). Statistical analyses to compare viruses performed with two-way ANOVA, and comparisons between individual time-points with Tukey’s multiple comparisons test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.