Interaction of Mesonivirus and Negevirus with arboviruses and the RNAi response in Culex tarsalis-derived cells

Abstract

Background: Mosquito-specific viruses (MSVs) comprise a variety of different virus families, some of which are known to interfere with infections of medically important arboviruses. Viruses belonging to the family Mesoniviridae or taxon Negevirus harbor several insect-specific viruses, including MSVs, which are known for their wide geographical distribution and extensive host ranges. Although these viruses are regularly identified in mosquitoes all over the world, their presence in mosquitoes in Germany had not yet been reported.

Methods: A mix of three MSVs (Yichang virus [Mesoniviridae] and two negeviruses [Daeseongdong virus and Dezidougou virus]) in a sample that contained a pool of Coquillettidia richiardii mosquitoes collected in Germany was used to investigate the interaction of these viruses with different arboviruses in Culex-derived cells. In addition, small RNA sequencing and analysis of different mosquito-derived cells infected with this MSV mix were performed.

Results: A strain of Yichang virus (Mesoniviridae) and two negeviruses (Daeseongdong virus and Dezidougou virus) were identified in the Cq. richiardii mosquitoes sampled in Germany, expanding current knowledge of their circulation in central Europe. Infection of mosquito-derived cells with these three viruses revealed that they are targeted by the small interfering RNA (siRNA) pathway. In Culex-derived cells, co-infection by these three viruses had varying effects on the representative arboviruses from different virus families (Togaviridae: Semliki forest virus [SFV]; Bunyavirales: Bunyamwera orthobunyavirus [BUNV]; or Flaviviridae: Usutu virus [USUV]). Specifically, persistent MSV co-infection inhibited BUNV infection, as well as USUV infection (but the latter only at specific time points). However, the impact on SFV infection was only noticeable at low multiplicity of infection (MOI 0.1) and at specific time points in combination with the infection status.

Conclusions: Taken together, these results are important findings that will lead to a better understanding of the complex interactions of MSVs, mosquitoes and arboviruses.

Keywords: Aedes; Arbovirus; Culex; Interference; Mesonivirus; Mosquito-specific viruses; Negevirus; RNAi.

Fig. 1

Maximum likelihood phylogeny of MeSV and NeV samples. The phylogenetic inference of MeSV was based on a concatenated amino acid alignment of the conserved regions of the 3CLpro, RdRp and helicase domains (a). The phylogenetic inference of NeV was based on a concatenated amino acid alignment of the conserved regions of the methyltransferase, helicase and RdRp domains (b). The virus species identified in this study are marked with a red star. The bootstrap values represent 1000 replicates. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. MeSV, Mesoniviridae; NeV, taxon Negevirus

Fig. 2

Mosquito-derived and mammalian cell lines co-infected with YicV/DeziV/DaesV. a Hsu (Culex quinquefasciatus), CT (Culex tarsalis), Aag2 (Aedes aegypti), Vero (Chlorocebus sp.) and BHK (Mesocricetus auratus) cells infected with the YicV/DeziV/DaesV mix (representative data of 2 independent repeats; except for Hsu cells). Right panel shows two additional repeats for Hsu-infected cells (Hsu-1 and Hsu-2, respectively). a, b The presence of YicV, DeziV and DaesV was detected by reverse transcription and virus-specific primers at 72 hpi (a) or at several passages for persistently infected CT and Hsu cells (b). The asterisk indicated an unspecific PCR band (verified by Sanger sequencing) in Hsu cells. DaesV, Daeseongdong virus (taxon Negevirus); DeziV, Dezidougou virus (taxon Negevirus); hpi, hours post infection; P, passage; YicV, Yichang virus (family Mesoniviridae)

Fig.3

Effect of YicV/DeziV/DaesV infection status on Semliki Forest or Bunyamwera orthobunya arbovirus infections in CT (Culex tarsalis)-derived cells. CT cells were infected with BUNV-NLuc (BUNV expressing NLuc; MOI 0.1) (a) or SFV6-2SG-Nluc (SFV-Nluc, SFV expressing Nluc; MOI 1 or MOI 0.1) (b, c), either single, acutely co-infected with YicV/DeziV/DaesV (YicV/DeziV/DaesV + SFV-Nluc; MOI 1, MOI 0.1 or YicV/DeziV/DaesV + BUNV-NLuc; MOI 0.1) or using cells persistently infected with YicV/DeziV/DaesV (pYicV/DeziV/DaesV + SFV-Nluc; MOI 1, MOI 0.1 or pYicV/DeziV/DaesV + BUNV-NLuc; MOI 0.1). Cells were lysed at the indicated hours post infection (hpi) and luciferase activity was measured. Results of three independent experiments performed in technical triplicates are presented. Mean values with standard error of the mean are shown. Significance was tested via unpaired t-test. BUNV infection: acute infection (24 hpi: t = 2.448, *P = 0.0263; 72 hpi: t = 2.389, *P = 0.0296); persistent infection (24 hpi: t = 5.180, ***P = 0.0002; 48 hpi: t = 9.981, ****P < 0.0001; 72 hpi: t = 11.69, ****P < 0.0001). SFV infection MOI 0.1: acute infection (24 hpi: t = 3.143, **P = 0.0063; 72 hpi: t = 10.59, ****P < 0.0001); persistent infection (18 hpi: t = 5.962, ****p < 0.0001). BUNV, Bunyamwera orthobunya virus; DaesV, Daeseongdong virus; DeziV, Dezidougou virus; MOI, multiplicity of infection; Nluc, Nano luciferase; ns, not significant; YicV, Yichang virus; SFV Semliki forest virus

Fig. 4

Effect of YicV/DeziV/DaesV infection status on Usutu arbovirus infections in CT (Culex tarsalis)-derived cells. CT cells singly infected with Usutu virus (USUV, MOI 10) were compared to cells either acutely co-infected with YicV/DeziV/DaesV and USUV (YicV/DeziV/DaesV + USUV; MOI 10) or to USUV infection in cells persistently infected with YicV/DeziV/DaesV (pYicV/DeziV/DaesV + USUV; MOI 10). Supernatants were collected for USUV titration via TCID₅₀ at 0, 6, 10, 24, 48 and 72 hpi for 3 independent experiments in technical triplicates. Mean values with standard error of the mean are shown. Significance was tested via the unpaired t-test. Acute infection (48 hpi: t = 4.507, ***p = 0.0004; 72 hpi: t = 4.706, ***p = 0.0002); persistent infection (24 hpi = t = 9.487, ****p < 0.0001; 48 hpi: t = 8.959, ****p < 0.0001). DaesV, Daeseongdong virus; DeziV, Dezidougou virus; MOI, multiplicity of infection; TCID₅₀, median tissue culture infectious dose assay; USUV Usutu virus; YicV, Yichang virus

Fig. 5

Production of DaesV-specific sRNAs in CT (Culex tarsalis) and Aag2 (Aedes aegypti) cells acutely infected with YicV/DeziV/DaesV or CT cells persistently infected with YicV/DeziV/DaesV. For acute infection, cell were infected with the DaesV/DeziV/YicV mix, and total RNA was isolated at 24 h post-infection. RNA of persistently infected CT cells (passage 3) were isolated, followed by β-elimination treatment (Additional file 8: Figure S4) and control (CT persistent). a The absolute frequency of sRNAs ranging in length from 18 to 31 nt that were mapped to the virus genome/antigenome. b The distribution of 21-nt-long sRNA to the indicated virus genome/antigenome. c The the distribution of 26- to 30-nt-long sRNAs (piRNA-sized) to the virus genome/antigenome is shown. Positive values (green) represent sense reads, negative values (purple) represent antisense reads. Y-scale values give the read counts, with the scale values mentioned above the graph. Representative results of the acute infection of a duplicate (Additional file 7: Figure S3). DaesV, Daeseongdong virus; DeziV, Dezidougou virus; nt, nucleotide; sRNAs, small RNAs; YicV, Yichang virus

Fig. 6

Production of DeziV-specific sRNAs in CT (Culex tarsalis) and Aag2 (Aedes aegypti) cells acutely infected with YicV/DeziV/DaesV or CT cells persistently infected ith YicV/DeziV/DaesV. For acute infection, cells were infected with the DaesV/DeziV/YicV mix and total RNA was isolated at 24 h post-infection. RNA of persistently infected CT cells (passage 3) were isolated, followed by β-elimination treatment (Additional file 8: Figure S4) and control (CT persistent). a The absolute frequency of sRNAs ranging in length from 18 to 31 nt that were mapped to the virus genome/antigenome. b The distribution of 21-nt-long sRNA to the indicated virus genome/antigenome. c The distribution of 26- to 30-nt-long sRNAs (piRNA-sized) to the virus genome/antigenome. Positive values (green) represent sense reads, negative values (purple) represent antisense reads. Y-scale values give the read counts, with the scale values mentioned above the graph. Representative results of the acute infection of a duplicate (Additional file 7: Figure S3). DaesV, Daeseongdong virus; DeziV, Dezidougou virus; nt, nucleotide; sRNAs, small RNAs; YicV, Yichang virus

Fig. 7

Production of YicV-specific small RNAs in YicV/DeziV/DaesV acutely infected Aag2 (Aedes aegypti) cells. Cells were infected with the DaesV/DeziV/YicV mix and total RNA was isolated at 24 h post infection. a The absolute frequency of sRNAs ranging in length from 18 to 31 nt that were mapped to the virus genome/antigenome. b The distribution of 21-nt-long sRNA to the indicated virus genome/antigenome. c The distribution of 26- to 30-nt-long sRNAs (piRNA-sized) to the virus genome/antigenome. Positive values (green) represent sense reads, negative values (purple) represent antisense reads. Y-scale values give the read counts, with the scale values mentioned above the graph. Results of two independent experiments are shown (I and II). DaesV, Daeseongdong virus; DeziV, Dezidougou virus; nt, nucleotide; sRNAs, small RNAs; YicV, Yichang virus