A new species of mesonivirus from the Northern Territory, Australia

Abstract

Here we describe Casuarina virus (CASV), a new virus in the family Mesoniviridae. This is the first report of a mesonivirus in Australia, which extends the geographical range of this virus family to 3 continents. The virus was isolated in 2010 from Coquillettidia xanthogaster mosquitoes during surveillance in the suburbs of Darwin, the capital of the Northern Territory. Cryo-electron microscopy of the CASV virions revealed spherical particles of 65 nm in size with large club-shaped projections of approximately 15 nm in length. The new virus was most closely related to Alphamesonivirus 1, the only currently recognized species in the family. In 2013 a further 5 putative new mesonivirus species were described: Hana, Méno, Nsé, Moumo and Dak Nong viruses. The evolutionary distance between CASV and two of its closest relatives, Cavally and Hana viruses (Jones-Taylor-Thornton distance of 0.151 and 0.224, respectively), along with its isolation from a different genus of mosquitoes captured on a separate continent indicate that CASV is a new species.

Figure 1. CASV morphology and structural proteins.

(A,B) Negative-staining electron micrograph of CASV particles following potassium tartrate gradient purification and staining with 1% uranyl acetate. Visible spike protrusions are marked with arrows. Each scale bar represents 100 nm. (C) SDS-PAGE analysis of gradient-purified CASV virions. The virion proteins were assessed for the presence of N-linked glycans by treatment with PNGAse F (+) or left untreated (−). Putative protein identity is shown on the left of the image.

Figure 2. Cryo-electron imaging and in silico virion surface reconstruction.

(A) Cryo-electron micrograph of purified CASV particles imbedded in vitrious ice layer within a holey carbon support. Scale bar in 100 nm (white). Representative virions shown for cryo-projection (B) and tomographic reconstruction (inverted contrast - C). Stratified surface densities at the virion surface due to M protein layer imbedded in virion envelope can be seen clearly together with an electron dense core containing the ribonucleoprotein (RNP) complex. (D) Representative surface projections extracted from a 10Å filtered tomogram show 15 nm spikes at the virion surface, comparable in dimensions to that observed for SARS coronavirus spike (shown in lower right inset – scale bars 15 nm. Refer to EMBD-1423 [28]). (E) Proposed virion architecture equivalent to that observed within the Coronaviridae family, with trimeric spike proteins projecting from an ordered M protein/lipid bilayer support. The RNP layer lies below a low density region but is tethered to the envelope through interactions with M protein projections.

Figure 3. Analysis for mesonivirus core using NP-40 treatment.

Gradient-purified CASV particles were left untreated (A) or treated with 0.05% NP-40 for 6 min (B) or 0.25% NP-40 for 30 min (C) to disrupt the lipid envelope. No proteainaceous internal component was released suggesting the absence of a core. An uncoating virion is highlighted in panel B with an arrow. Scale bars: A = 500 nm, B and C = 200 nm.

Figure 4. Phylogenetic analysis of the replicase domains of pp1ab.

Unrooted phylogenies were generated from the protein sequences of the indicated replicase domains using the maximum likelihood method. Bootstrapping replicates (1000) are rounded to a value out of 100. The branch length is proportional to the evolutionary distance as shown by the scale bar (substitutions per amino acid site).