An Insect Nidovirus Emerging from a Primary Tropical Rainforest

Zirkel, Florian; Kurth, Andreas; Quan, Phenix-Lan; Briese, Thomas; Ellerbrok, Heinz; Pauli, Georg; Leendertz, Fabian H.; Lipkin, W. Ian; Ziebuhr, John; Drosten, Christian; Junglen, Sandra

We tested 269 pools of 1,716 adult male mosquitoes collected during this survey for CAVV infection by specific real-time reverse transcription (RT)-PCR (14) . CAVV was not detected in male mosquitoes, suggesting that CAVV is likely dependent on amplifying vertebrate hosts. Viral growth and morphology. Virus growth kinetics and morphological presentation in insect cells were studied. Cells showed strong cytopathic effects (CPE) manifesting in aggregation of cells at 48 h postinfection (hpi) ( Fig. 2A and B) . Virus replication was measured by real-time RT-PCR every 3 h for 2 days. Maximal RNA concentrations were reached at 15 to 18 hpi, indicating a fast replication cycle (Fig. 2C) . Enveloped, spherical CoV-like virions with a mean diameter of 120 nm and large, clubshaped surface projections were detected in cell culture supernatants at 48 hpi by negative staining electron microscopy (Fig. 3) . In ultrathin sections of fixed insect cells analyzed by transmission electron microscopy at 48 hpi, vesicles containing spherical, potentially enveloped particles were observed in the cytoplasm of infected cells. These were 50 to 60 nm in diameter and lacked surface projections ( Fig. 3A and B) . A role for these vesicles filled with virions resembling steps during virus maturation, as shown for other plus-strand RNA viruses, remains to be investigated (28) (29) (30) . Furthermore, tubular structures likely of viral origin were detected in the cytoplasm of infected cells (Fig. 3A) . Separation or adsorption of putative virions on cell membranes is shown in Fig. 3C . These particles were morphologically indistinguishable from typical particles encountered in cleared cell culture supernatant (Fig. 3D) .