Characterization of a Novel Tanay Virus Isolated From Anopheles sinensis Mosquitoes in Yunnan, China

Abstract

Globally, mosquitoes are known to be competent vectors to various arboviruses that cause serious and debilitating diseases to humans and animals. Conversely, mosquitoes harbor a wide array of insect specific viruses (ISVs) that are generally neglected. Extensive characterization of these ISVs is important in understanding their persistence infection effect on host behavior and arbovirus transmission. Herein, we report first time isolation of Tanay virus (TANAV) isolate YN15_103_01 in Anopheles sinensis mosquitoes from Yunnan Province, China. Phylogenetically, the isolate's nucleotide identity had more than 14.47% variance compared to previous TANAV isolates, and it clustered into an independent branch within the genus Sandewavirus in the newly proposed taxon Negevirus. TANAV growth and high titers was attained in Aag2 cells (10⁷ PFU/mL) but with no CPE observed up to 7 days.p.i. compared to C6/36 cells that exhibited extensive CPE at 48 h.p.i. with titers of 10⁷ PFU/mL. Contrarywise, the viral isolate did not replicate in vertebrate cell lines. Electron microscopy analyses showed that its final maturation process takes place in the cell cytoplasm. Notably, the predicted viral proteins were verified to be corresponding to the obtained SDS-PAGE protein bands. Our findings advance forth new and vital knowledge important in understanding insect specific viruses, especially TANAV.

Keywords: Anopheles sinensis; Negevirus; Tanay virus; insect specific viruses; yunnan.

FIGURE 1

(A) Morphology of purified viral particles visualized by negative staining. (B) representative plaques of TANAV- isolate YN15_103_01 infected and uninfected (mock) C6/36 cells 48 h.p.i. Cells were fixed with 10% formalin and stained with crystal violet.

FIGURE 2

Transmission electron microscopy analysis of infected cells. Ultrastructure of mock-infected (A) and TANAV isolate YN15_103_01 infected (B-G) C6/36 cells at 48 h.p.i. (B) Para crystalline arrays could be observed surrounded by the ER (endoplasmic reticulum) (red arrows). (C) Expanded perinuclear space filled with vesicular (yellow arrows indicate the points of perinuclear membrane expansion). (D) Expanded ER stretch from the nuclear membrane to the cell membrane (blue arrows) and filled with microtubules (E) (brown dashed arrows), a few Mit (Mitochondria) were observed. (F) Cytoplasmic cytopathic vacuoles (CPVs), containing a lot of spherules (purple arrows) inside the vacuoles. (G) Several V (viroplasm-like particles) measuring approximately 40–50 × 60–70 nm (orange dashed arrows) and autolysosomes (pink arrows) were observed.

FIGURE 3

Replication of TANAV isolate YN15_103_01 in cells. (A–C) The growth curve of TANV isolate YN15_103_01 (MOI = 0.1–0.0001) was measured by Plaque assay at 6–168 h post infection.

FIGURE 4

Bioinformatic and physical analysis of TANAV isolate YN15_103_01 structural proteins. (A) Three open reading frames (ORFs) were predicted in full genome, ORF1 encoded viral methyltransferase (vMet), RNA ribosomal methyltransferase (FtsJ), Helicase Helicase (Hel), RNA dependent RNA polymerase (RdRp) ORF2 contained transmembrane regions, ORF3 encoded membrane protein (M). (B) N-terminal signal peptide (S) and a C-terminal transmembrane domain (TM) of ORF2 predicted by protter. This protein contains 4 transmembrane (TM) regions. (C) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) analysis performed on purified virions shows 6 abundant proteins corresponding to the predicted ORF2 (594 aa), RdRp (440 aa), Hel (418 aa), vMet (349 aa), FtsJ (200 aa), M (124 aa), respectively, ^∗ means no predicted functional protein was corresponding.

FIGURE 5

Analysis of a gap-free concatenated alignment of fused methyltransferase, viral helicase and RdRp domains of members of negeviruses, cileviruses, higreviruses and blunerviruses, as well as representative members of each genus of the Virgaviridae family. A: ML-Tree; B: SDT: TANAV isolate YN15_103_01 (red font); negeviruses (green font). Data shown on Supplementary File S1.

FIGURE 6

World map showing the global distribution of negeviruses. Data shown on Supplementary File S1. (A–C) indicates a partially enlarged area and numbers and sorts them from left to right.