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

Extensive attempts at virus isolation from adult female mosquitoes pooled in small numbers (up to 23 mosquitoes per pool) yielded virus in 40 (9.3%) of the 432 pools tested. As shown in Table 1 , virus was most frequently isolated from Culex mosquitoes, especially Culex nebulosus. Mosquitoes of the genera Aedes, Anopheles, and Uranotaenia were also found to be infected but at lower rates. CAVV was present in all sampled habitat types but with the highest prevalence in human settlements (Table 1) . Analysis of variance showed the observed virus isolation rate in human settlements (and only there) to be significantly different from the mean isolation rates (F test, P Ͻ 0.00001) and from all isolation rates in any other habitat. In order to gain insight into the genetic diversity of CAVV, a 603-nucleotide (nt) genome fragment representing the 1b polymerase gene region was amplified and sequenced from all positive pools. The pairwise genetic distance between the isolates was up to 15% at the nucleotide level and up to 9.9% at the amino acid level. In particular, the nucleotide distance of isolate CAVV/A4/CI/2004 from all other isolates ranged between 13.5 and 15%, whereas the range of distances was below 1.8% for all other CAVV isolates. This suggested the presence of a diversified virus population comprising two different clusters whose distance is compatible with different species. CAVV is an insect-associated virus whose hosts were encountered along a gradient of anthropogenic habitat modifications. To examine possible influences of habitat modification on virus diversity, average nucleotide distances over sequence pairs were determined within the amplified 603-nt fragments from all pools and were assigned to one of the habitat types from where the respective isolates originated, i.e., (i) primary forest or research camps within a primary forest (9 isolates), (ii) secondary forest (5 isolates), (iii) plantations (6 isolates), and (iv) villages (19 isolates). The mean numbers of nucleotide exchanges in pairwise sequence comparisons were determined within the samples pertaining to each habitat type. CAVV/A4/CI/2004 was excluded to prevent bias from a virus that may be evolutionarily disconnected from the main clade (putative second species). In habitat types i to iv, the mean pairwise exchange rates within the 603-nt fragment were 4.35, 2.8, 0.6, and 2, respectively. Even though isolates from villages predominated numerically in the data set, the level of diversification was highest in primary forest (chi-square test on exchange rates, P Ͻ 0.01).