Incomplete bunyavirus particles can cooperatively support virus infection and spread

Bunyaviruses lack a specific mechanism to ensure the incorporation of a complete set of genome segments into each virion, explaining the generation of incomplete virus particles lacking one or more genome segments. Such incomplete virus particles, which may represent the majority of particles produced, are generally considered to interfere with virus infection and spread. Using the three-segmented arthropod-borne Rift Valley fever virus as a model bunyavirus, we here show that two distinct incomplete virus particle populations unable to spread autonomously are able to efficiently complement each other in both mammalian and insect cells following co-infection. We further show that complementing incomplete virus particles can co-infect mosquitoes, resulting in the reconstitution of infectious virus that is able to disseminate to the mosquito salivary glands. Computational models of infection dynamics predict that incomplete virus particles can positively impact virus spread over a wide range of conditions, with the strongest effect at intermediate multiplicities of infection. Our findings suggest that incomplete particles may play a significant role in within-host spread and between-host transmission, reminiscent of the infection cycle of multipartite viruses

Primers for cDNA synthesis of viral genome segments.

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Visualization of viral genome segments in RVFV-infected cells and in immobilized RVFV virions from virus stocks and co-infection supernatants.

(A) Schematic representation of the single-molecule viral RNA FISH-immunofluorescence method used to visualize the intracellular viral ribonucleoprotein (vRNP) composition of infected cells and the intravirion genomic composition of virus stocks and culture supernatants (illustration based on [22]). (B) BSR-T7/5 cells were mock infected, infected exclusively with one population of incomplete particles (MOI 1), or simultaneously infected with iRVFV-SL-eGFP and iRVFV-ML (MOI 1 for each virus), and fixed at 16 h post-infection. Cells infected with a three-segmented RVFV-Clone 13 (MOI 1) were used as positive control. (C) RVFV virions from incomplete particle stocks and supernatants of co-infected BSR-T7/5 and C6/36 cells were immobilized on coverglass by incubation for 5 h at 28°C. The S segment (N gene, red), M segment (polyprotein gene, blue), and L segment (RdRp gene, yellow) were hybridized using probe sets labeled with CAL Fluor Red 610, Quasar 670, and Quasar 570, respectively. RVFV particles (green) were detected with antibody 4-D4 [47] targeting the Gn glycoprotein in combination with Alexa Fluor 488–conjugated secondary antibodies. Cell nuclei (cyan) were visualized with DAPI. Merge images show the overlay of five (for cells) or four (for virions) individual channels. Individual spots, each representing either a single vRNP or a virus particle, were detected and assessed for co-localization in ImageJ with the plugin ComDet. Colored circles display the spots detected in each channel and their co-localization in the merge image. Scale bars, 10 μm (B), 5 μm (C). Illustration in Fig 5A was created with BioRender.com.</p

Co-infection of mammalian and insect cells with recombinant three-segmented RVFV reporter viruses.

(A) Schematic representation of the T7 polymerase-based reverse genetics system. Fluorescently marked variants of RVFV were generated by simultaneous transfection of BSR-T7/5 cells with the transcription plasmids pUC57_S, pUC57_M, and pUC57_L encoding the RVFV-35/74 S, M and L genome segments, respectively, in antigenomic-sense orientation. The pUC57_S plasmid additionally encoded for either eGFP or mCherry2 in place of the NSs gene. (B) Direct fluorescence detection of BSR-T7/5 cells infected with either RVFV-eGFP (green) or RVFV-mCherry2 (red) (MOI 0.5) at 24 h post-infection. (C) Growth kinetics of RVFV-eGFP and RVFV-mCherry2 after infection of BSR-T7/5 cells at an MOI of 0.01. Virus titers were determined with an end-point dilution assay (fluorescence microscopy readout). Dots represent means of biological replicates (n = 3) at each time point, and the shaded area represents the standard deviation. The dashed line indicates the limit of detection (101.8 TCID50/mL). Source data are provided in S1 Data. (D) Schematic representation of the simultaneous infection of mammalian (BSR-T7/5) or insect (C6/36) cells with RVFV-eGFP and RVFV-mCherry2. (E) Direct fluorescence detection of BSR-T7/5 and C6/36 cells co-infected with RVFV-eGFP and RVFV-mCherry2 (MOI 0.5 for each virus) at 24 h (BSR-T7/5) or 72 h (C6/36) post-infection. Inset images are magnifications of a region of interest (indicated as a dashed box). Co-infected cells co-express eGFP (green) and mCherry2 (red) and thus appear yellow. Scale bars, 200 μm (inset images 100 μm). Illustrations in Fig 1A and 1D were created with BioRender.com.</p

Quantitative assessment of mammalian cells co-infected with non-spreading two-segmented RVFV reporter viruses.

(A) Schematic representation of the reverse genetics system used to create two-segmented RVFV reporter viruses. Incomplete RVFV-SL particles were generated by co-transfection of BSR-T7/5 cells with the transcription plasmids pUC57_S, encoding either eGFP or mCherry2 in place of the NSs gene, pUC57_L, and the protein expression plasmid pCAGGS_NSmGnGc. (B) Immunofluorescence assay for detection of BSR-T7/5 cells infected with iRVFV-SL-eGFP or iRVFV-SL-mCherry2 (MOI 0.5) at 24 h post-infection. (C) Supernatants from cells primarily infected with the incomplete RVFV particles in (B) were passaged onto naive BSR-T7/5 cells. Cells were examined at 24 h post-infection for expression of eGFP (green) and mCherry2 (red) by fluorescence microscopy. Expression of the Gn glycoprotein (green or red, depending on the virus) was detected with rabbit polyclonal anti-Gn serum in combination with FITC-conjugated (green) or Alexa Fluor 568–conjugated (red) secondary antibodies. Cell nuclei (cyan) were visualized with DAPI. Scale bars, 50 μm. (D) Schematic representation of the simultaneous infection of mammalian (BSR-T7/5) cells with non-spreading iRVFV-SL-eGFP and iRVFV-SL-mCherry2 particles. Co-infections were done at increasing MOI (ranging from 0.1 to 2.5 for each virus). After 48 h, cells were examined by fluorescence microscopy and fixed for flow cytometry analysis. (E) Direct fluorescence detection of BSR-T7/5 cells co-infected (MOI of 0.5 for each virus) with the two-segmented incomplete particle populations. The inset image is a magnification of a region of interest (indicated as a dashed box). Co-infected cells co-express eGFP (green) and mCherry2 (red) and thus appear yellow. Scale bars, 200 μm (inset images 100 μm). (F) Cells expressing eGFP, mCherry2, or both were quantified by flow cytometry. Mock-infected cells and cells infected exclusively with only one population of incomplete particles (MOI 0.5) were used as controls. (G) Relationship between the fraction of infected (left) and co-infected (right) cells as a function of the MOI. Dots represent experimental data points. Dashed lines represent the predictions of a model based on the assumptions that genome segments are randomly packaged into virus particles and that host susceptibility is heterogeneous. The code required to reproduce the model predictions is provided as S1 File. Source data are provided in S1 Data. Illustrations in Fig 2A and 2D were created with BioRender.com.</p

Sensitivity analysis of the infection model parameters.

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RVFV incomplete particles complement upon co-infection and allow virus replication and spread in mosquitoes.

(A) Schematic representation of the experimental design. Five groups of Aedes aegypti mosquitoes (n = 15–62 per group) were fed a blood meal spiked with different RVFV preparations and housed at 28°C. After 12–15 d, mosquitoes were sedated with CO2 and body and saliva samples were collected. RVFV infection of mosquito bodies and transmission to mosquito saliva were assessed via virus isolation with a fluorescence microscopy readout. (B) RVFV infectious titers in mosquito bodies. Dots represent individual mosquitoes and are color-coded gray for negative bodies, light red (group #4) or light green (group #5) for positive bodies, and solid red (group #4) or solid green (group #5) for both body and saliva virus-positive mosquitoes. The dashed line indicates the limit of detection (101.5 TCID50/mL). The incidence (in absolute numbers and percentages) of RVFV infection (bodies) and transmission (saliva) is indicated at the bottom for each group. Data corresponding to mosquitoes from groups #4 and #5 derive from two independent experiments. Source data are provided in S1 Data. (C) Representative fluorescence microscopy images of BSR-T7/5 cells inoculated with virus-positive body and virus-positive saliva samples from groups #4 (RVFV-mCherry2) and #5 (iRVFV: SL-eGFP + ML) at 48 h post-infection. Merge images show the overlay of two individual channels (eGFP and mCherry2). Scale bars, 200 μm. Illustration in Fig 6A was created with BioRender.com.</p

Oligonucleotide sequences of RNA FISH probe sets.

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Antibodies used in immunostaining assays.

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