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.
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Introduction
Alphaviruses are small, enveloped, positive-sense RNA viruses in the Togaviridae family, with the Alphavirus genus comprising over 30 distinct members that can be further separated into seven antigenic complexes plus unclassified alphaviruses1,2. Most alphaviruses are transmitted by haematophagous vectors, typically mosquitoes, and can cause severe disease in humans, livestock and wildlife, courtesy of the host range extending from mammals, birds and reptiles, through to amphibians and fish for some species3,4. The arthritogenic alphaviruses, such as chikungunya (CHIKV), Mayaro (MAYV), Sindbis (SINV), Ross River (RRV) and Barmah Forest viruses (BFV) cause a variety of disease manifestations in humans, some severe, and often including chronic polyarthritis. In comparison, the new world alphaviruses such as the closely related equine encephalitis viruses, Eastern, Western and Venezuelan equine encephalitis viruses (EEEV, WEEV, and VEEV, respectively) can cause neurological disease and encephalitis in horses, humans and some bird species2,5. Despite the continued worldwide burden of these viruses, only a single vaccine for CHIKV has been recently licensed and there are no specific antiviral drugs6,7. Hence, treatment is restricted to symptomatic relief.
The recent surge in virus discovery by metagenomic studies has facilitated the detection of previously unknown viruses, drastically expanding viral families and the identification of viruses with attributes that may be exploited to combat pathogens8,9. Indeed, the surveillance of mosquito populations worldwide revealed the existence of viruses that do not infect or replicate in vertebrates, despite their close ecological and evolutionary relationships to pathogenic arthropod-borne viruses10. Termed insect-specific viruses (ISVs), these viruses have provided clues to arbovirus evolution, vector-borne pathogen transmission and, through engineering, some insect-specific flaviviruses (ISFs) have been used in recombinant vaccine and diagnostic antigen development based on their favourable safety profile11,12,13,14. Eilat virus (EILV), the first insect-specific alphavirus (ISA) discovered, has been investigated in this context and has been engineered to produce a chimeric viral platform whereby the insect-specific virus genetic backbone is used to support the expression of viral particles displaying pathogenic alphavirus structural proteins15,16,17,18. In both systems, the ISV backbone renders the replication of the chimera restricted to mosquito cells – a key advantage to support the handling under lower biocontainment conditions.
Surprisingly, in comparison to the numerous isolations of ISF species, only five ISA species have been detected globally to date (Fig. S1). Further investigations into the evolutionary relationships of ISAs with their pathogenic alphavirus relatives are complicated by a lack of immunological tools for their detection and characterisation. Another limitation into the research of ISAs is that only two (EILV and Agua Salud alphavirus, ASALV) replicate efficiently in mosquito cells in vitro19,20. No viral isolates for the remaining three ISAs (Taï Forest alphavirus, TALV; Mwinilunga alphavirus, MWAV; and Yada Yada virus, YYV) exist and attempts to derive the Culex spp. mosquito associated MWAV and TALV through Aedes-derived C6/36 mosquito cultures were unsuccessful, perhaps due to a narrow mosquito host range21,22.
Within the Asia-Pacific region, YYV remains the only ISA to be detected, and was found via RNA sequencing performed on pooled mosquitoes from three traps set in Victoria, Australia23. The host mosquito species of YYV is unknown; however, two possible vectors are Anopheles annulipes or Culex australicus as these were the only mosquito species present in all three pools. A sequence fragment deposited in GenBank with high identity to YYV was also detected in a single Aedes notoscriptus mosquito collected in Melbourne, Australia, in 201124, incriminating this mosquito species also as a potential vector. Assembly of high throughput RNA sequencing data contigs elucidated the near-complete YYV genome, including complete coding sequence for the two open-read frames (ORFs), and partial sequences for the untranslated regions (UTRs; 33 nt of 5’UTR, and 470 nt of 3’UTR, respectively)23. The assembled genome was consistent with that typical of an alphavirus. Alphaviral genomes are typically 11 to 12 kb, featuring a 5’ cap and 5’UTR, followed by the non-structural protein gene sequences (nsPs; nsP1, nsP2, nsP3, and nsP4), and then the structural protein gene sequences (sPs; Capsid, E3, E2, 6 K, and E1), which are transcribed via a 26 S subgenomic promoter overlapping the 3’-end of nsP4. At the terminus of the genome is a 3’UTR, and a poly(A) tail25,26. Phylogenetic comparisons grouped YYV within the putative mosquito-restricted ISA clade, however, no attempts were made to recover an isolate to confirm this phenotype23.
Here, we describe the recovery and characterisation of an infectious clone of YYV using the circular polymerase extension reaction (CPER) reverse genetics method27. We also report the development of the first monoclonal antibodies (mAbs) directed to ISAs and the use of the YYV backbone to rapidly produce chimeric viruses displaying the structural proteins of various ISAs and the alphaviral pathogens, CHIKV, RRV, MAYV, SINV and BFV. Together, these facilitated investigation of antigenic relationships and possible host restriction factors of ISAs.
Results
Elucidation of the YYV genome untranslated regions
To date, YYV RNA detection has been exclusively via metagenomic sequencing of mosquito homogenates23. Attempts herein at recovering the virus by the transfection of C6/36 cells with RNA extracted from the YYV-positive homogenates were not successful. Instead, the +ssRNA virus, Yongsan picorna-like virus 2, the sequence for which was detected in the homogenate RNA raw sequencing data and has been identified previously in mosquitoes, was recovered28,29 (Fig. S2). Therefore, we aimed to design and recover an infectious clone of YYV using the reverse genetics CPER technique27. The coding sequence of the YYV genome was previously assembled via metagenomic sequencing of RNA extracted from mosquito homogenates, however, the 5’ and 3’ UTR termini were not complete23. To complete the UTR sequences, further rounds of de novo assembly were performed on the available sequencing data sets. These assemblies identified an additional 171 nucleotides of the 3’UTR sequence and a partial poly(A) tail (Fig. S3A), while the 5’UTR terminus was extended by 13 nt. However, multiple sequence alignment of YYV to the other four ISAs revealed a predicted 11 nt segment was still missing from the extreme 5’ end of the YYV virus genome (Fig. S3B). Therefore, for the purposes of this study, the terminal 5’ 11 nt from either EILV or TALV were substituted into the infectious clone design using overhang primers, denoted as YYVEILV or YYVTALV, respectively, to complete the 5’UTR and create functional infectious clones of YYV and subsequent chimeras as detailed in the following sections (Fig. S3C).
De novo generation of YYV and EILV infectious clones via CPER
Fragments covering the full-length YYV genome, including the extreme 5’ 11 nt from EILV or TALV (as detailed above) were amplified from cDNA generated from the YYV-positive mosquito homogenate RNA to create seven dsDNA fragments with overlapping ends of 21–40 nt. These fragments were combined with a CPER linker fragment, which was generated by amplifying from a previously established plasmid encoding the OpIE2 promoter and HDVr, for precise 3’ trimming of the transcript27,30, and incorporating YYV 5’ and 3’UTR-overhangs via the primers. The CPER was transfected into C6/36 cells, but as initial attempts to recover a YYV infectious clone via this CPER technique (Fig. 1A) were unsuccessful, the YYV infectious clone recovery was performed in a stepwise manner using CPER27. The available YYV genome fragments were tested systematically by creating several chimeric viruses to confirm the accuracy of the elucidated sequences (Fig. 1B). The structural proteins of RRV were routinely used in selected chimera designs due to the ease of detecting replicating virus with specific anti-RRV sP mAbs, prior to the availability of mAbs to EILV and YYV.
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 (YYVEILV; 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, YYVEILV (YYV infectious clone including 5’ terminal 11 nt of EILV genome) and YYVTALV (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 RRVWT or West Nile virus (WNVWT) 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.
Given the prior research on EILV and its application as a chimeric viral platform15,17,18,31, we chose EILV as a comparator for this study and as an aid to identify the cause for the lack of the YYV infectious clone recovery. An EILV infectious clone was constructed via CPER using synthetic dsDNA gBlocks. As with the YYV infectious clone design (Fig. 1A), the full-length EILV genome, excluding the 3’ UTR, was encoded by six overlapping gBlocks (Fig. 1B, construct 1). Due to multiple repeat sequences in the EILV 3’UTR, synthesis as one discrete gBlock was not possible. Therefore, a single PCR fragment encompassing the 3’UTR region was generated using three smaller dsDNA gBlocks of 129 to 200 bp. This PCR product was subsequently joined to a 200 nt synthetic ultramer containing the terminal 156 nt of the EILV 3’UTR, the poly(A) tail with 26 adenines, and an 18 nt overlap with the CPER linker fragment (Fig. 1B). The fragments covering the viral genome and the linker fragment were assembled by CPER and an infectious clone of EILV was successfully recovered by transfecting into C6/36 cells (Fig. 1C, Fig. S4A). Replication of EILV was initially detected via staining in immunofluorescence assays (IFA) with anti-dsRNA mAbs32 and subsequently verified using the first EILV-specific mAbs generated later in the project.
To confirm the ability of the structural proteins of YYV to facilitate binding and entry of C6/36 mosquito cells, a chimera was produced by exchanging the EILV structural protein genes with those of YYV (Fig. 1B, construct 2; EILV/YYV-sP). Successful recovery of the chimera via CPER was confirmed by IFA and suggested that the elucidated genetic sequence of the YYV sP genes was sound (Fig. 1C). A chimera with the YYV 5’UTR and nsPs, RRV sPs, and the EILV 3’UTR (YYV/RRV-sP/EILV-3’UTR) was also recovered successfully (Fig. S4B), suggesting the YYV 5’ UTR and nsPs gene sequences were also correct. However, a reverse chimera containing the EILV 5’UTR and nsPs, RRV-sPs and the YYV 3’UTR (EILV/RRV-sP/YYV-3’UTR) failed to recover, implicating the newly elucidated YYV 3’UTR as a potential issue.
Examination of the elucidated YYV 3’UTR sequence in comparison to other alphaviruses identified that a pair of conserved nucleotides, ‘UC’ were missing from the 3’ terminus; a deletion previously shown to be lethal for alphaviruses33. Further investigation found that the raw sequencing reads for the YYV isolate did possess these UC nucleotides but were not included in the final consensus sequence when the de novo genome was constructed. Confirmation that the YYV 3’UTR was functional when UC nucleotides were present was achieved by recovery of construct 3 (Fig. 1B, C construct 3; YYV 3’UTR independently assessed away from the full YYV genome using EILV 5’UTR and nsPs; RRV sP’s); a chimera which had previously failed to recover when the UC nucleotides were not present at the YYV 3’UTR terminus. Subsequently, infectious clones of YYV were successfully launched with the two alternative 5’UTR substitutions described above: YYVEILV and YYVTALV (Fig. 1B construct 4, Figs. 1D, S3).
Infectious clones of both EILV, YYVEILV, and YYVTALV displayed typical alphavirus replication in C6/36 cells, reaching peak titres of 108.24 TCID50/mL, 106.91 TCID50/mL, and 106.25 TCID50/mL by 96 h, respectively, with a significant difference in replication between EILV and YYV viruses (Fig. 1D, P < 0.05). There was no statistical difference between replication of YYVEILV and YYVTALV, but the YYVTALV 5’UTR was chosen for all subsequent experiments in this paper, hereafter referred to as YYV. Both YYV and EILV caused little overt cytopathic effect (CPE), consistent with reports describing the replication of wild-type EILV20. While no cell death was observed, some visible morphological changes were observed in the C6/36 cell monolayer upon infection with either virus, such as vacuolisation, filopodial extensions, and slowing of cell growth compared to uninfected cells (Fig. S5). Virus supernatant for YYV, EILV, and EILV/YYV-sP were concentrated and purified via potassium tartrate gradient ultracentrifugation (Fig. 1E) and analysed by SDS-PAGE (Fig. 1F). Distinct protein bands were visible at expected sizes of E1/E2 and capsid viral proteins. The apparent molecular weight of the EILV and YYV E1/E2 proteins of ~53 kDa and ~49 kDa, respectively, as determined by SDS-PAGE, were larger than the in silico predicted E1/E2 of 47.0/46.0 kDa and 47.3/45.9 kDa, respectively (Table S2). The decreased mobility may be due to differences in E1/E2 glycosylation, which was further analysed later in this study. The apparent molecular weight of YYV capsid protein was larger than that of EILV, consistent with the predicted sizes (Table S2) and the YYV structural protein band sizes were indistinguishable regardless of expression via the infectious clone, or via use of an EILV backbone (Fig. 1F). Transmission electron microscopy (TEM) analysis of the YYV virions showed spherical virions approximately 70 nm in diameter (69.67 nm ± 4.29 nm, n = 50), which is characteristic of an alphavirus (Fig. 1G).
YYV displays a mosquito-restricted tropism
Initial phylogenetic analyses of YYV suggested that it was an insect-specific alphavirus23, however, this required verification. To investigate YYV host tropism, a YYV-backboned chimera was constructed by replacing the YYV structural proteins with the structural proteins of RRV (YYV/RRV-sP). This chimera would assumably be capable of binding and entering mammalian cells such that the ability of the YYV nsPs to launch replication could be independently investigated. Our experiments showed that YYV, as the infectious clone, was unable to replicate in an extensive panel of mammalian cell lines after inoculation with virus at an MOI of 1 (Fig. 1H, Table S1). Cell lines tested included Vero cells, which lack a functional interferon response to viral infection34, BSR cells, which are partially interferon response deficient35, Ju56 (wallaby) cells, as wallabies and other macropods are likely host reservoirs for pathogenic alphaviruses36, and SW-13 (human) cells, to demonstrate the safety of YYV and YYV chimeras in the context of human cells. Similarly, YYV did not replicate in mosquito cell lines derived from Culex and Anopheles mosquitoes, nor in the Drosophila S2 line. Parallel inoculation of the cell lines with the YYV/RRV-sP chimera revealed a similar inability of this chimera to replicate in any of the assessed cell lines, apart from those derived from Aedes albopictus mosquitoes. Together, these data suggested that YYV has a mosquito-specific host tropism and that a block to replication in mammalian cells and non-Aedes mosquito cells likely occurs post cell entry (Fig. 1H).
YYV is antigenically distinct from EILV and other alphaviruses
Antigenic analysis of insect-specific alphaviruses is limited to date due to the lack of suitable antibodies. To assess the antigenic relatedness and evolutionary relationships between ISAs, crucial research tools were developed. A panel of 18 hybridomas secreting mAbs reactive to YYV or EILV proteins were generated through hybridoma fusion technology and characterised (Table 1; Fig. S6). Of the 18 hybridomas recovered, six reacted specifically to YYV, while 12 reacted to EILV and did not react to a panel of other ISA structural proteins in fixed-cell ELISA (Table 1). All the newly created anti-YYV and anti-EILV mAbs were determined to target an envelope glycoprotein by Western blot, however, it was not possible to differentiate between those that bound E1 or E2 due to the similar protein size. Most mAbs were IgG isotypes except for six that were typed as IgM. Ten mAbs (three YYV, seven EILV) neutralised virus to low levels, with titres ranging between 1 and 8 (Table 1). These mAbs represent the first reported mAbs that are directed to insect-specific alphaviruses.
YYV chimeras facilitate the study of the structural proteins of other ISAs
In contrast to the successful replication of ASALV and EILV in C6/36 cells19,20, cell culture isolation of TALV and MWAV has not been successful21,22 (Fig. S1B). To determine if the block to replication of TALV and MWAV occurs at the initial stages of virus binding and entry, chimeric viruses using the YYV backbone and expressing the structural protein genes of ASALV, MWAV, or TALV (YYV/ISA-sP: YYV/ASALV-sP, YYV/MWAV-sP, YYV/TALV-sP) were generated by CPER, transfected into C6/36 cells and detected by IFA using anti-dsRNA mAbs (Fig. 2A, Fig. S4A). All three chimeras were successfully recovered, as indicated by extensive cytoplasmic signal in IFA (Fig. 2B). Sanger sequencing over the junction between YYV nsP4 and the ISA structural proteins confirmed the identify of each chimera. The successful recovery of the YYV/ISA-sP chimeras indicated that the inability of MWAV and TALV to replicate in C6/36 cells likely occurs post-entry and is linked to the MWAV and TALV non-structural proteins or UTRs, rather than a failure of the structural proteins to mediate binding and entry.
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.
To extend the ISA antigenic analysis, YYV/ASALV-sP, YYV/MWAV-sP and YYV/TALV-sP chimeras were assessed in ELISA with the newly created panels of anti-YYV and EILV mAbs, as well as those directed to CHIKV and RRV (Table 1). While there was no cross-reactivity of the majority of the mAbs assessed against the structural proteins of ASALV, MWAV, and TALV in ELISA, anti-EILV mAb 1E1 weakly bound MWAV structural proteins and this reactivity was more pronounced in IFA (Fig. 2B, Table 1). Interestingly, the cross-reactive anti-CHIKV capsid mAbs 5.5G9 and 5.5D11 and cross-reactive anti-RRV mAb B10 bound the capsid and E2 proteins of ASALV (Table 1).
YYV/ISA-sP chimeras were concentrated, purified, and analysed via SDS-PAGE alongside YYV and EILV (Fig. 2C) where distinct protein bands were visible, presumably E1/E2 and capsid. This revealed some differences in structural protein sizes that diverged from the in silico predictions, likely due to glycosylation in the context of E1/E2 (Table S2). PNGase F digest of the purified virions resulted in an increased protein mobility for E1/E2 of ~5–7 kDa for YYV, EILV, YYV/MWAV-sP, and YYV/TALV-sP, consistent with the removal of 2–3 glycans for both E1 and E2 (Fig. 2C). Post-PNGase F digest, the YYV/ASALV-sP E1/E2 proteins were clearly separated, suggesting that one glycoprotein possessed fewer N-linked glycans than the other. This was in contrast to the pattern of other ISAs but consistent with the fewer predicted glycosylation motifs for ASALV (Table S2, Fig. S7). As expected, none of the capsid proteins were glycosylated. However, the ASALV capsid protein had distinctly lower motility than that of other the ISAs and was ~4–5 kDa larger than the predicted 29.3 kDa. TEM analysis of purified ISA chimeras (Fig. 2E) showed typical alphavirus virions (YYV/ASALV-sP virion diameter: 69.17 nm ± 5.02 nm, n = 50), similar to YYV.
To further examine the antigenic similarity between the ISA structural proteins, the chimeric viruses were assessed with hyperimmune anti-YYV and -EILV polyclonal anti-serum (Fig. 2D). Although none of the anti-YYV and anti-EILV mAbs displayed any cross-reactivity to other ISAs (except for mAb E-1E1 in IFA), immunoblots of the ISAs with anti-YYV and anti-EILV hyperimmune anti-sera showed evidence for the presence of cross-reactive epitopes between the ISA E1/E2 and capsid proteins. The immune antiserum from the mouse injected with a combination of EILV and YYV resulted in weak capsid detection for EILV, YYV and TALV.
YYV as a platform to display the structural proteins of pathogenic vertebrate-infecting alphaviruses
To further assess the versatility of the YYV backbone to be used as a recombinant system to produce chimeric viruses for pathogenic vertebrate-infecting alphaviruses (VIAs), chimeras were constructed displaying the structural proteins of the arthritogenic alphaviruses BFV, CHIKV, MAYV, SINV and RRV (Fig. 3A). Each of the YYV-backboned chimeras were successfully recovered via CPER in C6/36 cells, as indicated by staining of viral proteins in IFA (Fig. 3B). Antigenic similarity to wild-type VIAs was confirmed through analysis with mAbs specific for pathogenic alphaviruses (Fig. 3B, Table 2). Purification of YYV/CHIKV-sP and YYV/RRV-sP via high-speed centrifugation and subsequent analysis by SDS-PAGE showed E1/E2 and capsid proteins at the predicted sizes (Fig. 3C). Further visualisation of the purified YYV/CHIKV-sP (67.70 nm ± 4.09 nm, n = 50) and YYV/RRV-sP virions (68.55 nm ± 9.76 nm, n = 30) by negative-stained TEM revealed spherical virions typical of alphaviruses within the expected size of ~60–70 nM (Fig. 3D). A small number of larger virions (~90 nm) were observed for YYV/RRV-sP, likely multi-cored alphavirus virions reported in previous cryo-EM studies37,38.
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: RRVWT (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.
The YYV/CHIKV-sP and YYV/RRV-sP chimeras replicated efficiently, reaching titres of 108.4 and 107.6 TCID50/mL, respectively, over 4 days, with yields similar to that of the YYV infectious clone (Fig. 3E, constructs 1-3). Simultaneous comparison with an EILV-backboned construct displaying the RRV structural proteins (EILV/RRV-sP, Fig. 3E, construct 5) revealed significantly higher replication of EILV/RRV-sP in comparison to YYV/RRV-sP (P < 0.01), consistent with the higher replication capacity of the EILV infectious clone when compared with YYV. The plasticity and effect of 3’ UTR exchanges for these two chimeras, YYV/RRV-sP with EILV 3’UTR and EILV/RRV-sP with YYV 3’UTR (Fig. 3E, constructs 6,7), was further examined. In both cases, a non-significant difference in titre was observed (P > 0.1). Comparatively, there was a significant difference between EILV/RRV-sP and YYV/RRV-sP/EILV-3’UTR chimeras (P < 0.001). Future studies will ascertain whether the nsPs or 5’UTR of YYV can be manipulated to increase replication characteristics further.
Discussion
The study of ISVs has rapidly increased in recent years as they provide crucial insights into the evolution and emergence of mosquito-borne viral pathogens and represent novel options for the development of new biocontrol agents and recombinant antigens for vaccine and diagnostic applications39,40,41,42. Despite these advances, much is yet to be understood regarding the evolutionary history, antigenic properties, host tropism, and virion structure of many ISVs. In this study, we report the first use of CPER to construct infectious clones and chimeras of ISAs, and generated the first mAbs against ISAs to further explore their antigenic profiles and host characterisation.
Within the Asia-Pacific region, more than ten ISFs – the most studied mosquito-associated ISV group, have been detected10,43. In sharp contrast, YYV remains the only ISA detected in this region23, despite extensive screening of mosquitoes and virome analyses42,44,45,46,47,48,49. As no attempts had been made to isolate YYV, RNA extracted from a YYV-positive mosquito homogenate was transfected into C6/36 cells. While Yongsan picorna-like virus 2, another +ssRNA virus, was successfully recovered, suggesting that the approach was sound and the RNA of sufficient quality, replicating YYV was not detected, potentially through replication competition. As we have demonstrated the use of CPER for launching alphaviruses previously27, this approach was used to successfully recover an infectious clone of YYV. De novo genome assembly extended the YYV 5’UTR and completed the 3’UTR missing from the published genome23. Initial attempts to recover a YYV infectious clone were unsuccessful due to a 2 nt deletion of ‘UC’ nucleotides at the end of the newly-elucidated 3’UTR. Indeed, previous studies investigating mutagenesis of the SINV 3’UTR found the deletion of these two highly conserved nucleotides to be lethal33. Restoration of these two nucleotides enabled successful recovery of a YYV infectious clone and indicated that this critical terminal ‘UC’ conservation extends to ISAs. While the YYV 5’UTR was extended from the published sequence, sequence alignments with other ISAs revealed that approximately 11 nt remained unknown from the extreme 5’ end. Through incorporating the predicted 11 missing nt into the YYV infectious clone design from either EILV or TALV, the virus was successfully launched. Given that within the terminal 11 nt sequence used of EILV or TALV, that the sequences differ by only 2 nt, it was unsurprising that the resulting infectious clone replication for each was similar. As new detections of YYV are made within Australian mosquitoes, further attempts to elucidate the complete YYV 5’UTR will be made.
EILV, a virus that had previously been used in successful reverse genetics systems15,17,18,40, represented an ideal tool to assist with the recovery strategies for a YYV infectious clone. Herein, we reported the first use of CPER to recover an alphavirus (EILV) entirely from synthesised dsDNA fragments. The many repeat sequence elements (RSEs) in the 3’UTR of EILV prompted the 3’UTR to be synthesised as four smaller dsDNA fragments, requiring subsequent annealing via PCR. As many alphavirus 3’UTRs contain RSEs of varying number and complexity, similar difficulties in CPER design may be encountered for the synthetic generation of other alphaviruses.
Peak replication of YYV via growth kinetics was achieved by 48 h and reached titres up to 108.1 TCID50/ml in some assays, albeit at consistently lower titres than those achieved by EILV. Through the systematic exchange of genes between YYV, EILV and RRV to validate the accuracy of the elucidated YYV sequences and arrive at the final infectious clone of YYV, we established amenable exchange of 3’UTRs and structural proteins between these viruses. The alphavirus 3’UTR is linked to virus replication in insect hosts, and mutations or deletions in this region can attenuate virus replication in mosquito cells33,50,51. In this study, we saw only a moderate attenuation in replication when the 3’UTR of EILV was exchanged for YYV in the EILV/RRV-sP chimera and vice versa. This is consistent with analysis of an EILV reporter chimeric virus with the SINV 3’UTR, whereby no decrease in virus titre in C7/10 cells compared to wild-type EILV reporter virus was found16. Comparatively, there was a significant decrease in viral titre observed when comparing EILV/RRV-sP (entire EILV backbone) to YYV/RRV/EILV-3’UTR, suggesting a sequence factor in the 5’UTR or nsPs of YYV may be contributing to lower levels of replication. Until additional isolates of YYV are identified, it is unknown whether the 11 nt of EILV or TALV used in the YYV infectious clone strategy recapitulates the wild type virus. From other alphaviral studies, it is known that the 5’UTR contains important promoter elements, and interactions between the 5’UTR and 3’UTR are required to initiate negative-strand synthesis in viral replication52. Serial passaging of YYV in C6/36 cells may also assist in increasing viral titres, as observed for a chimera of the ISF Aripo virus and pathogenic Zika virus (ARPV/ZIKV), whereby 14 serial passages increased genome copies (GC) by approximately 104 GC/mL compared to early passage ARPV/ZIKV and wild-type ARPV13.
Assessment of the YYV infectious clone in a variety of mammalian and insect cell lines revealed a narrow host range, with replication supported by only Aedes albopictus cells. While initial reports implicated An. annulipes and Cx. australicus as the potential host species23, the detection of a partial YYV nsP4 sequence in Ae. notoscriptus supports a potential Aedes host species for YYV24. Such mosquito genus restrictions have similarly been observed for ISFs, with Parramatta River virus isolated only from Aedes vigilax mosquitoes and in vitro tropism confined to Aedes-derived cell line53. In contrast, previous studies with EILV and ASALV show that replication of other ISAs is supported by multiple mosquito genera. EILV replicated in vitro in a wide range of invertebrate cell lines derived from insects outside of the natural vectors from which it has been isolated (Anopheles coustani20 and Culex pipiens54) including, Ae. albopictus, Cx. tarsalis and Phlebotomus papatasi, while recent in vivo studies revealed that EILV could replicate in mosquito tissues of Ae. albopictus, Aedes aegypti, Anopheles gambiae, Culex quinquefasciatus and Cx. tarsalis55,56. ASALV, initially isolated from Culex declarator mosquitoes, can also replicate in Aedes albopictus cell lines and in vivo in Ae. aegypti mosquitoes19,57.
Wild-type RRV (RRVWT) entered and replicated in many of the insect and mammalian cell lines tested in this study. However, the Aedes-restricted tropism of the YYV/RRV-sP chimera was identical to that of YYV, despite displaying the RRV structural proteins to facilitate cell binding and entry. These data suggest a replication block post-entry, conferred by the non-structural genes or UTRs of YYV. EILV host restriction is multigenic and occurs at both the cell entry and replication levels16, and EILV/CHIKV-sP is able to enter cells (facilitated by the CHIKV structural proteins) yet not replicate17. The host restriction of YYV may be similarly multigenic, and this will be investigated in future work through cell transfection studies, reverse chimeras (e.g. VIA/YYV-sP), and cell binding assays. While YYV and YYV/RRV-sP viruses were unable to replicate in several mammalian cell lines assessed herein, potential cryptic vertebrate hosts for YYV may exist, warranting future assessment of the viruses and chimeras in avian and reptilian cell lines. Future experiments may also investigate temperature-associated host restriction, as demonstrated for ASALV, which did not replicate above 32 °C in C6/36 cells19.
To date, isolation of infectious MWAV and TALV has been unsuccessful21,22. While RT-PCR confirmed a lack of viral replication in MWAV experiments22 following serial passaging, the recovery of TALV relied solely on CPE presence21, which may have caused potential TALV replication to be overlooked should it induce minimal or no CPE. Indeed, the YYV infectious clone generated in this study displayed no overt CPE, and YYV chimeras of pathogenic alphaviruses (e.g. YYV/RRV-sP) caused markedly less CPE than their EILV counterparts (EILV/RRV-sP; Fig. S5), though this may be due to lower overall virus titre. Through the generation of chimeras displaying the structural protein genes of MWAV and TALV, we demonstrated that the structural genes of these viruses could facilitate binding and entry into the C6/36 cell. These data indicated that a blockage to replication may exist post-entry and is linked to the MWAV and TALV non-structural genes or UTRs. Alternatively, TALV and MWAV may exhibit a narrow in vitro host range, as demonstrated for YYV, or high host-specificity. Indeed, some Anopheles-associated ISFs can only replicate in their mosquito vector, and not in cell culture in vitro, or other mosquito species in vivo58. Currently, no wild-type isolates of MWAV, TALV, or YYV have been successfully recovered from mosquitoes. Small RNA sequencing of mosquito homogenate would verify these viruses are actively infecting their mosquito hosts, as performed for ASALV in vitro and in vivo19,57,59.
N-linked glycosylation of alphaviral glycoproteins (E1, E2, and E3) is known to be associated with multiple viral factors, including replication and virulence, in both mammalian and insect systems60,61. Whilst extensively researched for VIAs, potential glycosylation sites of ISAs and implications on viral replication or host tropism has yet to be investigated. N-linked glycan prediction for all ISAs identified multiple sites on E1 and E2, several of which aligned with analogous regions in the VIA sequences (Fig. S7). YYV, EILV, MWAV and TALV shared numerous predicted glycosylation motifs at the same location and indeed PNGase F digests indicated that identical numbers of these motifs were utilised. Upon comparison with VIA sequences, only two predicted N-linked glycosylation motifs of ISAs, excluding ASALV, aligned with VIA glycosylation motifs - amino acids E1-139, predicted in EILV, MWAV, and TALV, and E1-270, predicted in YYV, EILV, MWAV, and TALV. In studies with SINV, removal of the N-linked glycosylation at E1-139 decreased replication in BHK cells61, whereas E1-270 in Getah virus (GETV) is possibly associated with viral immune evasion or host cell attachment as it is surface-exposed62. In contrast, fewer N-linked glycosylation motifs were predicted for the ASALV E1/E2 proteins and were more likely to align with the N-linked glycosylation sites of VIAs. For example, E1-245 glycosylation also occurs in SINV, the mutation of which decreased replication of SINV in both BHK and C7/10 cells61. Glycosylation of E2-196, also occurs in SINV; and E2-318, in SINV, VEEV, and EEEV61,63,64. Both N-linked glycans are associated with heparan sulphate binding, and mutation of either in SINV resulted in increased replication in BHK cells and increased virulence in mice61.
The commonality of N-linked glycosylation sites within the E1/E2 proteins of ASALV with VIAs rather than the other ISAs is consistent with previous phylogenetic evidence showing that ASALV is placed in a solitary deep-rooting lineage, basal to the WEE serocomplex and ISA clade19. The presence of three predicted glycosylation sites in ASALV with links to replication and virulence in vertebrates is also possible evidence for ASALV descending from an ancestral alphavirus with the ability to infect vertebrates, or, is evolving traits conducive to vertebrate cell infection19. Further evolutionary links to the VIAs was seen in antigenic analysis whereby ASALV structural proteins were successfully detected by several anti-VIA cross-reactive mAbs that bind to E2 (mAb B10; reactive to viruses of the Semliki Forest virus complex)65 and capsid (mAbs 5.5D11, 5.5G9; reactive to CHIKV, RRV, MAYV, SINV), although it should be highlighted that mAbs 5.5D11 and 5.5G9 have previously been shown to bind an overlapping epitope66. The structural proteins of none of the other ISAs were bound by these mAbs. Viruses of different antigenic complexes usually diverge >38% in nucleotide sequence and >40% in amino acid sequence, and ASALV has 44–45% nucleotide divergence and 43–45% amino acid divergence to other ISAs67. Together, these data provide extra evidence to suggest that ASALV represents a separate antigenic complex from other ISAs and supports the theory that ASALV may be an evolutionary intermediate between the vertebrate-infecting alphaviruses and other ISAs.
Prior to this report, there have been minimal antigenic analyses of ISAs, and no ISA-reactive monoclonal antibodies available for use as research tools. The only previous antigenic analyses of EILV in serological assays found evidence of cross-reactivity, although minimal, with Aura virus (AURAV), Trocara virus (TROV), SINV, WEEV, VEEV, and EEEV using hyperimmune mouse fluids; no other ISAs had been discovered at this time20. Through our production of anti-YYV and -EILV mAbs, pAbs and the construction of a variety of ISA chimeras, we demonstrated that cross-reactive epitopes do exist between the ISAs, but that the mAbs generated indicate that a virus-specific antibody response may have dominated in the immunised mice .
Alphaviruses commonly elicit a high prevalence of cross-reactive antibodies within phylogenetic or antigenic clades, thus the generation of only a single cross-reactive mAb made to EILV (E-1E1, anti-E1/E2) that also detects MWAV in IFA was unexpected. Phylogenetic analysis of the structural polyprotein shows MWAV is most closely related to TALV (76.1% identity), followed by EILV (74.5% identity), hence there are likely many conserved epitopes between them. Some of the generated mAbs may share a common epitope, although the varying neutralising properties suggests this is not the case for all. While it was not possible to determine whether the mAbs were reactive to E1 or E2, as neutralising epitopes are frequently directed to E2 of other alphaviruses68,69, it is likely that a number of the mAbs are E2-reactive but will require confirmation. Producing anti-E1 mAbs targeting conserved epitopes may be the best strategy for generating ISA cross-reactive mAbs3,70,71,72.
In addition to YYV/ISA-sP chimeras, YYV was also amenable to exchange of structural proteins with vertebrate-infecting alphaviruses, as has been demonstrated with EILV using CHIKV, SINV, WEEV, VEEV, EEEV, ONNV, and MAYV15,16,17,18. Previously, EILV was the only ISA that had been used as a chimeric platform, compared to the multiple ISFs investigated in the same manner11,13,14. In addition to YYV/CHIKV-sP and YYV/MAYV-sP, this study reports the first insect-specific virus-backboned chimeras for the arthritogenic alphaviruses RRV and BFV. These YYV chimeras were readily recognised by mAbs specific for the parental VIA in ELISA and comparing YYV/CHIKV-sP and YYV/RRV-sP to their wild-type counterparts found identical antibody binding profiles. YYV/RRV-sP was only able to infect Ae. albopictus cell lines, indicating these chimeras retain the host-specific replication restriction of the YYV backbone. The chimeras generated in this study were able to be concentrated and readily purified as authentic alphavirus virions (as seen in TEM) with distinct viral proteins visible via protein separation. Further experiments will investigate the use of these chimeras as potential diagnostic and vaccine antigens, as a safe alternative to handling wild-type isolates of these pathogenic alphaviruses.
In conclusion, we have recovered an infectious form of the first ISA in the Asia-Pacific region, YYV, and generated the first mAbs specific for ISAs, which are valuable research tools to investigate antigenic relationships and host restriction. This study demonstrates the versatility of the CPER method, as YYV was recovered by amplifying from mosquito homogenate RNA that contained other +ssRNA viruses, and EILV was constructed entirely from synthesised DNA. Furthermore, the first chimeras of other ISAs were created – including MWAV and TALV, of which no viral isolate exists – and used to investigate this largely uncharacterised group of insect-specific viruses. However, more research is required to understand the underlying causes of the mosquito-specific host tropism of ISAs and what structural and antigenic relationships these viruses have to the broader group of alphaviruses.
Materials and methods
Ethics statement and approvals
All mouse work was conducted in accordance with the “Australian code for the care and use of animals for scientific purposes” as defined by the National Health and Medical Research Council of Australia. The animal experiments were approved by the University of Queensland Animal Ethics Committee (AEC permit number 2021/AE000626). All wild-type CHIKV work was conducted in a biosafety level-3 (BSL3) facility at the QIMR Berghofer MRI (Australian Department of Agriculture, Fisheries and Forestry certification Q2326). All work with infectious and CPER-generated viruses was approved by the UQ Institutional Biosafety Committee (UQ IBC, approvals IBC/1246/SCMB/2019, IBC/1389/SCMB/2021, and IBC/1402/SCMB/2022).
Cell culture
C6/36 (Aedes albopictus, RNAi-deficient) and MOS. 55 (Anopheles gambiae) cell lines were maintained in RPMI1640 medium supplemented with 5% heat-inactivated (HI) fetal bovine serum (FBS, Bovogen) at 28 °C. Chao ball (Culex tarsalis) and RML-12 [Aedes albopictus, RNAi-intact53] cell lines were maintained in L-15 medium supplemented with 10% FBS and 10% tryptose phosphate broth at 28 °C. S2 (Drosophilia melanogaster) cell line was maintained in Schneider’s Drosophila medium (Gibco) supplemented with 10% FBS. All insect cells were maintained at 28 oC. Vero (Cercopithecus aethiops, African green monkey kidney), BSR (Mesocricetus auratus, baby hamster kidney), JU56 (Protemnodon bicolour, black-tailed wallaby buccal mucosa fibroblasts), and SW-13 (Homo sapiens, human adrenal gland/cortex) cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% FBS at 37 °C. All media were supplemented with 50 U/mL penicillin, 50 μg/mL streptomycin, and 2 mmol/L L-glutamine (PSG).
Virus culture
Wild-type viruses used herein were: RRV-TT (GenBank KY302801), CHIKV Mauritius strain (GenBank MH229986), and WNV Kunjin NSW2011 strain (GenBank JN887352). WNV was used as a positive control in Fig. 1H as RRV did not infect S2 or Ju56 cells. Virus stocks were generated by infecting C6/36 cell monolayers with virus diluted to a multiplicity of infection (MOI) of 0.1 or a dilution of 1/100 (if the virus titre was unknown, in the case of ISA stocks grown prior to generation of specific mAbs) in 2% HI-FBS RPMI media and incubating the monolayers on a rocking platform for 1 h at room temperature (RT). Following this, the virus inoculum was removed and replaced with fresh 2% HI-FBS RPMI media and incubated at 28 °C. The supernatant was harvested for virus stocks 5 days post-infection (dpi), or earlier if extensive CPE developed, by centrifuging at 3000 rpm for 10 min at 4 °C. Clarified supernatant was supplemented with additional HI-FBS to increase the total concentration to 10%, aliquoted and stored at −80 °C until required. Virus titres were determined with a 50% tissue culture infectious dose (TCID50) assay73 using methods previously described74. The stocks were titrated on C6/36 cells and stained by fixed-cell ELISA with virus-specific mAbs.
For analysis by SDS-PAGE and electron microscopy, sub-confluent monolayers of C6/36 cells were infected with the infectious clones and chimeras at an MOI of 0.1. The supernatant was collected at 3-, 5- and 7-dpi. The virus culture supernatant was clarified by centrifugation at 3000 rpm for 30 min at 4 °C and filtered through a 0.22 μM filter. After each collection, cells were replenished with fresh RPMI containing 2% FBS. The virions were precipitated via the addition of polyethylene glycol 8000 to a final concentration of 8% and slow stirring overnight at 4 °C. The virus was pelleted at 8000 rpm (Beckman Coulter JLA 10.500 rotor) for 1.5 h at 4 °C, before ultracentrifugation through a sucrose cushion and a potassium tartrate gradient as described previously11,75. However, YYV/ASALV-sP, YYV/MWAV-sP, YYV/TALV-sP, and YYV/RRV-sP were not taken through the potassium gradient, as sucrose cushion resulted in sufficient sample purity, due to YYV chimeras causing minimal CPE. Purified virus was collected, and buffer exchanged into sterile PBS using a 30 kDa molecular weight cut-off Amicon filter and stored at 4 °C. Purified virion proteins were resolved by SDS-PAGE (NuPAGE 4-12% Bis-Tris gels; Invitrogen, ThermoFisher Scientific) and staining with Coomassie blue stain (Bio-Rad) following the manufacturer’s guidelines.
Method on UTR elucidation for YYV
Additional 5’ and 3’ UTR sequences of YYV were recovered from deposited high throughput sequencing libraries containing k-mers of YYV identified on the NCBI SRA database. This identification was performed using Google BigQuery with “Yada yada virus” as a query, following a previously established pipeline76. This analysis revealed three accessions from metagenomics datasets from mosquito pools in Mildura, Victoria, Australia from November 201645. The accessions were SRR12113258 (YYV k-mers: 1145, Sample name: M450R), SRR12113256, (YYV k-mers: 850, Sample name: M4602), and SRR12113252 (YYV k-mers: 144, Sample name: M4702). Raw data from these libraries and the original Mildura sample (SRA accessions: SRR10607433 and SRR1211325623) was downloaded. Fastp (v0.23.2)77 was used to trim adapters and low-quality reads under default settings. De novo assembly of clean reads was undertaken using Trinity (v2.9.1)78 under default settings. BLASTn was used to identify contigs in the assembly bearing high similarity to the YYV genome (GenBank MN733821). The largest YYV contig bearing the complete coding sequence was identified and used for remapping and polishing using Bowtie2 (v2.4.5)79. Mapped reads were extracted and reassembled iteratively until no further 5’ and 3’ UTR sequences were recovered. The final binary alignment file of the assembly was visualised using Integrative Genomics Viewer (v2.6.2)80. Terminal assembled 5’ and 3’ UTR sequences were subsequently queried against deposited sequencing data using Geneious Prime 2022.1 (https://www.geneious.com). This was to identify reads that were not assembled but shared high similarity to the UTRs. To identify viruses that might have been recovered in the M4602 transfected sample in C6/36 cells, M4602 libraries SRR10607433 and SRR12113256 were assembled using Trinity as above. Other putative positive-sense RNA viruses were identified using a virus discovery pipeline81. RT-PCR primers for potential viruses were designed based on viral contigs.
Infectious clone and chimeric virus generation
Infectious DNA constructs were created using the circular polymerase extension reaction (CPER) method27. Briefly, cDNA was synthesised from extracted viral RNA using Superscript IV Reverse Transcriptase (Invitrogen) using an Oligo(dT) primer, random hexamers or a virus-specific reverse primer as per manufacturer’s instructions. The cDNA was then used as a template to generate overlapping dsRNA PCR fragments containing 15-30 nt overhangs at both 5’ and 3’ ends using Q5 High-Fidelity DNA Polymerase [New England Biolabs (NEB)] as per the manufacturer’s instructions. For the generation of the EILV infectious clone, synthetic dsDNA gBlocks (IDT) were designed using the published genome (GenBank JX67873020). Likewise, the structural proteins of ASALV (GenBank MK959115), MWAV (GenBank LC361437), and TALV (GenBank KY303625), were derived from synthesised dsRNA gBlocks (IDT). The structural protein cassettes as dsDNA were generated from the following viruses using viral RNA: BFV (GenBank JX855115), CHIKV (GenBank MH229986), MAYV (GenBank MK573238), RRV (GenBank KY302801), and SINV (GenBank AF429428). A complete list of primers used to generate the infectious clones and chimeras can be found in Tables S3–S5.
For each CPER assembly, equimolar amounts (0.1 pmol) of each viral cDNA fragment, together with a linker region (amplified with virus-specific overhangs from plasmid DNA) containing a modified insect promoter and hepatitis delta virus ribozyme as previously described27,30, were added to a Q5 PCR reaction as per the manufacturer’s instructions and cycled under the following conditions: 98 °C for 2 min (1 cycle), 98 °C for 30 s, 55 °C for 30 s, 72 °C for 6 min (2 cycles), 98 °C for 30 s, 55 °C for 30 s, 72 °C for 8 min (10 cycles). The entire reaction was transfected using TransIT-LT1 Transfection Reagent (Mirus) as per the manufacturer’s instructions and incubated for 5–7 dpi. Cells and virus supernatant (P0) were passaged onto freshly seeded C6/36 cell monolayers for IFA analysis as described below to confirm the presence of successful CPER virus recovery. Coverslips were stained with virus-specific E1/E2 mAbs, or anti-dsRNA mAbs (3G1/2G4)32 if no mAbs were available for the target virus. Virus identity was confirmed by RT-PCR of RNA extracted from cell culture supernatant and Sanger sequencing performed at the Australian Genome Research Facility (AGRF, Brisbane, Australia).
Immunofluorescence Assay (IFA)
Monolayers of virus-infected or mock-infected cells grown on glass coverslips were fixed in 100% ice-cold acetone. IFA analysis was performed as previously described32 with all steps performed at room temperature with rocking. Briefly, fixed coverslips were submerged in blocking buffer for 1 h, followed by incubation of primary antibodies (1/10 dilution of hybridoma supernatant) either generated in this study or previously published. All previously published mAbs used in this study are referenced in Table 1 and Table 2, except for anti-dsRNA mAbs, 2G4 and 3G1, which were generated by O’Brien, et al.32. Coverslips were washed 3 times with PBS-T and incubated for 1 h with secondary antibody; goat anti-mouse IgG H + L AlexaFluor 488 (Invitrogen) at 1/500 dilution. After washing, the coverslips were drained, mounted in ProLong Gold antifade reagent (Invitrogen) and viewed using Zeiss LSM 900 confocal microscope.
Fixed-cell ELISA
Fixed-cell ELISA82 was used for viral titre determination and antigenic characterisation. C6/36 cell monolayers were infected with virus and incubated at 28 °C for 5 dpi prior to fixation in 20% acetone, 0.02% bovine serum albumin (BSA) in PBS overnight at 4 °C and air-dried. After blocking with TENTC blocking buffer (0.5 M Tris-Cl, 1 mM EDTA, 0.15 M NaCl, 0.05% Tween-20 (v/v), 0.2% casein w/v)) at RT for 1 h, diluted mAbs in TENTC blocking buffer were added for 1 h. Plates were washed 4 times with PBS containing 0.05% Tween-20 (PBS-T) and horse-radish peroxidase (HRP)-conjugated goat anti-mouse Ig (DAKO), diluted 1/3000 added for 1 h. After washing 6 times with PBS-T, substrate solution comprising 1 mM 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 3 mM H2O2 in a buffer prepared by mixing 0.1 M citric acid with 0.2 M Na2HPO4 was added to each well and incubated in the dark at RT for 1 h. Absorbance was measured at 405 nm and a positive well calculated by the average of mock-infected wells plus three standard deviations.
PNGase F digestion
Purified virions were digested with PNGase F (NEB) with modification to previously described protocol83. Briefly, purified virions were combined with GlycoBuffer 2 and H2O to 20 µL total volume. PNGase F was added (or H2O for mock samples) and samples incubated at 37 °C overnight. The Western blot was immunostained with anti-YYV or anti-EILV mAbs as required following resolving of the proteins using SDS-PAGE as described above.
Transmission electronic microscopy
Purified virions were diluted in PBS, before adsorbing onto carbon-coated grids (ProSciTech, Thuringowa, QLD, Australia) for 2 min and glow-discharged for 5 sec in 25 mA. The grids were blotted and washed three times in water and stained twice with a 2% uranyl acetate solution, with blotting in between. The grids were air-dried and imaged using a JEOL JEM-1011 microscope operated at 80 kV.
MAb production
To generate mAbs reactive to YYV and EILV, BALB/c mice (Animal Resources Centre, Murdoch, Western Australia) were immunised with purified YYV, EILV or EILV/YYV-sP adjuvanted with AdvaxTM (Vaxine Pty Ltd., Adelaide, Australia), as previously described11. Prior to splenectomy, mice were euthanised by cervical dislocation and hyperimmune polyclonal sera were obtained from cardiac bleeds. Fusion of the spleen cells with NS0 (European Collection of Cell Cultures) myeloma cells was performed as previously described84. Hybridomas secreting YYV or EILV-reactive mAbs were identified by fixed-cell ELISA as previously described82,69. The mAbs were further analysed for cross-reactivity against other alphaviruses by Western blot and fixed-cell ELISA using methods previously detailed82. The isotype of each mAb was determined using mouse monoclonal antibody isotyping reagents (Sigma-Aldrich, St Louis, Missouri, United States) as per the manufacturer’s instructions. The neutralisation capabilities of each mAb were assessed using methods previously detailed84.
In vitro host range assessment
To assess the in vitro host range of YYV, vertebrate and insect cell infection assays were performed as previously detailed11,53,85,86,87,88. Briefly, monolayers of selected insect and vertebrate cells were grown on glass coverslips before inoculation with YYV, YYV/RRV-sP, RRV, or WNV at an MOI of 1. WNV was used as a positive control for cell lines that RRV did not replicate in. Cells and virus were incubated at room temperature with rocking for 15 min, then at the optimal growth temperature for each cell line (28 °C or 37 °C) for a further 45 min with rocking every 15 min. Afterwards, the inoculum was removed, and the coverslips were washed three times with sterile PBS before adding appropriate growth media back onto the coverslips. The coverslips were cultured for 5 dpi; the supernatant was then harvested and stored at –80°C, and the coverslips were fixed in 100% ice-cold acetone. IFA analysis to assess virus replication was then performed using virus-specific mAbs, as detailed in above methods.
Viral growth kinetics
Replication kinetics were used to compare the growth of the different infectious clones and chimeras generated here. In a 24-well plate, C6/36 cells were pre-seeded in triplicate at a density of 1 × 105 cells/mL, before incubating overnight at 28 °C. The following day, the cell monolayers were infected with virus diluted to an MOI 0.1 (except for Fig. 3E, which used an MOI of 0.01) or mock-infected and incubated for 1 h at RT with rocking before washing with sterile PBS to remove residual virus, then replenishing with fresh 2% HI-FBS RPMI and incubating at 28 °C. Supernatant was harvested at 0, 6, 24, 48, 72, and 96 h (except for experiment in Fig. 3E, where supernatant was harvested at only 96 h), and stored at −80 °C prior to titration. Viral titres were determined on C6/36 cells using TCID50 method, as described above, except four wells were used for each dilution instead of the standard ten wells73,88. Statistical analyses to compare viruses performed with two-way ANOVA and comparisons between individual time-points with Tukey’s multiple comparisons test.
Data availability
Accessions numbers for raw sequencing data analysed in this study are described in Methods. All data generated in this study are available within this paper and its supplementary files.
Change history
05 March 2025
A Correction to this paper has been published: https://doi.org/10.1038/s44298-025-00094-0
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Acknowledgements
The authors acknowledge the facilities and the scientific and technical assistance of the Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, University of Queensland. We thank N. Petrovsky (Vaxine, Australia) for provision of Advax adjuvant used for the mAb production. We thank Daniel Rawle, Wilson Nguyen, and Andreas Suhrbier (QIMR Berghofer MRI) for providing Mayaro virus RNA and CHIKV fixed plates. We thank Alberto Amarilla Ortiz for assistance in CPER construct design and productive discussions. We thank Humda Zainab (UQ) for excellent technical assistance. Computer resources for viral sequencing analysis were provided by the Australian Galaxy service (https://usegalaxy.org.au). This project was funded by an Advance Queensland Industry Research Fellowship awarded to J.H-P. (AQIRF067-2020-CV). J.J.H. and N.M. were supported by Australian Research Council Discovery Early Career Researcher Awards, D.W. was supported by a CSL Centenary Fellowship, M.G.B. was supported by a Research Training Program Stipend from the University of Queensland.
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Experimental design: M.G.B., J.J.H., N.M., R.H.P., R.A.H., J.H-P. Experiments: M.G.B., J.J.H, R.H.P., T.S.E.L., G.H., I.E.M., M.A.T., J.H-P. Provision of reagents: J.B., S.E.L., D.W. Funding: D.W., J.H-P., J.J.H. Writing, original draft preparation: M.G.B, J.J.H., J.H-P., N.M., R.H.P. Writing, review, and editing: all authors.
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Bell, M.G., Parry, R.H., Lee, T.S.E. et al. Synthetic recovery of Yada Yada virus expands insect-specific alphavirus knowledge and facilitates production of chimeric viruses. npj Viruses 2, 45 (2024). https://doi.org/10.1038/s44298-024-00052-2
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DOI: https://doi.org/10.1038/s44298-024-00052-2
