Research Article
Identification of a novel totivirus from Culex tritaeniorhynchus in China
https://doi.org/10.21203/rs.3.rs-2713063/v1
This work is licensed under a CC BY 4.0 License
published 01 Jun, 2024
posted
You are reading this latest preprint version
After the first Totiviridae member IMNV was isolated from an arthropod penaeid shrimp, more toti-like viruses are being identified from arthropods. In this study, we isolated a novel totivirus-like particle from Culex tritaeniorhynchus designated NODE2. Sequence and phylogenetic analyses showed that NODE2 has a similar genome organization as some other members of Totiviridae family, and it may represent an entirely novel genus within the Totiviridae family.
Totiviridae is a family of non-enveloped, non-segmented, double-stranded RNA (dsRNA) viruses. Totiviridae genomes are 4.6-6.7 kb in size and contain two ORFs (RdRp) [1]. Totivirus, Victorivirus, Leishmaniavirus, Giardiavirus, and Trichomonasvirus are the five identified families. Totiviruses and Victoriviruses are fungi that attack yeast, smut fungi, and filamentous fungi, respectively, whereas Giardiaviruses and Leishmaniaviruses are parasitic protozoa [2]. Several totivirus-like genomes with comparable structures and morphology but limited similarity to members of other viruses have been discovered in recent years, for instance Totiviridae families have been found in non-fungal hosts such as shrimp, fish, and mosquitoes [3-8].
Mosquitoes carry parasites that cause major public health diseases.
Mosquito-borne viruses, in particular, are research hotspots as a result of new and re-emerging arboviral diseases, as well as recent global warming, which may assist in the spread of vector mosquitoes [9]. Deep sequencing from mosquitos identified some toti-like dsRNA viruses during an arbovirus study in China [10].
In this study, we isolated a novel virus of the Totiviridae family from Culex tritaeniorhynchus in Chuxiong, Yunnan, China, which we designated NODE2. The cell line used was C6/36 and inoculated with virus for 72 h until a cytopathic effect was observed. Viral RNA was extracted and used to conduct a whole-genome sequencing on an Illumina Hiseq platform. The DNA sequence data was uploaded on GenBank, with the entry code QQ443038. The NODE2 genome is 7714 bp long, with two ORFs, and a 13-nt-long untranslated region (UTR) at the 5' end and a 161-nt-long UTR at the 3' end. ORF1 encodes a protein of 1689 amino acids ranging from nt 14 to 5083. (5070 nt), which correlates with the capsid protein of other Totiviridae members. ORF2 spans nt 4789-7554 encoding 921 amino acids and corresponds to the RNA-dependent RNA polymerase. It has a putative RaRp motif (RDRP-4) from amino acid position 408 to 480.
Sequence analysis revealed an overlap between ORF1 and ORF2, implying a possible ribosomal-1 in NODE2. A heptamer UUUUUUA was identified in the overlap region at nt slippery position 4912, which corresponds to the earlier suggested XXXYYYZ (where X is A, C, G, or U, Y is A or U, and Z is A, C, or U) [11, 12]. This heptamer was followed by a pseudoknot at nt 4919-5053 (http://bibiserv.techfak.uni-bielefeld.de/knotinframe/). RNA pseudoknots are known to aid the ribosome in stopping translation, so an increase in frameshift and -1 frame-shifting would have to occur within the ORF1-ORF2 overlap region [13, 14]. The NODE2 gene contained a ribosomal-1 translational frameshift. The discovery of a 'slippery heptamer' followed almost immediately by a pseudoknot supports this hypothesis. Hence, a pseudosynaptic 'slippery heptamer' can be used to validate the existence or lack of ribosome-1 translational frameshift in the genome [8].
Some viruses with positive-stranded RNA and dsRNA have "2A" or "2A-like" nucleotide patterns, which induce ribosome "skipping" and apparent fragmentation of the viral polyprotein precursor [5]. Translation of the expected amino acid sequence from NODE2 ORF1, one 2A-like sequence motif (EGIEPNPGP) was discovered at amino acid location 504-512. The finding of such a structure in NODE2 suggests that the ORF1 product undergoes co-translational processing into two polypeptides. AsTV isolated from mosquitoes showed one 2A-like sequence in ORF1 (GDVESNPGP, amino acid positions 313-321), and DTV also showed one 2A-like sequence in ORF1 (EGVKPNPGP, amino acid positions 71-79). Other totiviruses like IMNV, OMRV, ToV-TJ and GSTV were found to have two conserved 2A-like sequence motifs in ORF1. IMNV showed at amino acid positions 86-94 (GDVESNPGP) and 370-378 (GDVEENPGP). OMRV-AK4 showed at amino acid positions 88-96 (EGVEPNPGP) and 500-508 (EGIEPNPGP), respectively. In the ToV-TJ genome sequence, at amino acid positions 88-96 and 500-508, there are two 2A-like sequence motifs (EGVEPNPGP), and the motifs for GSTV are EGVEKNPGP and GDIESNPGP at 305-313 and 466-474, respectively. Two conserved 2A-like sequence patterns are likely to split ORF1 polyprotein into three products [15].
The NODE2 CP showed maximum similarity to the strains Yuanmou totivirus and ToV-TJ (99% and 99.64%, respectively) based on amino acid sequence BLASTp analysis. The ORF2 amino acid sequence of NODE2 shared substantial similarity with RdRps from viruses in the Totiviridae family, especially ToV-TJ (99.73%) and OMRV-AK4 (98.4%). Genomic comparisons between mosquito strains (Yuanmou totivirus, OMRV-AK4, OMRV-Y61, CTV-NJ2 and CTV-NJ3) revealed that NODE2 shared maximum similarity with the strain Yuanmou totivirus (98%) but not the strains CTV-NJ2 or CTV-NJ3, although the strains of NODE2, CTV-NJ2 and CTV-NJ3 were isolated from the same place.
A phylogenetic analysis based on ORF1 CP and ORF2 RdRp sequences was performed to investigate the historical connection of NODE2 to other viruses in the Totiviridae family. NODE2, Yuanmou totivirus, TOV-TJ, OMRV-AK4, OMRV-Y61, DTV and IMNV are members of a distinct but closely related cluster, and in both the CP and RaRp trees (Fig. 1), showing the existence of a novel genus within the Totiviridae family. It suggests that these unidentified Totiviridae family members should be considered as members of a new species [8].
Conflict of interest: The authors declare no conflicts of interest.
published 01 Jun, 2024
posted
You are reading this latest preprint version