BackgroundMosquito-borne infectious diseases pose a severe threat to public health in many areas of the world. Current methods for pathogen detection and surveillance are usually dependent on prior knowledge of the etiologic agents involved. Hence, efficient approaches are required for screening wild mosquito populations to detect known and unknown pathogens.Methodology/principal findingsIn this study, we explored the use of Next Generation Sequencing to identify viral agents in wild-caught mosquitoes. We extracted total RNA from different mosquito species from South China. Small 18–30 bp length RNA molecules were purified, reverse-transcribed into cDNA and sequenced using Illumina GAIIx instrumentation. Bioinformatic analyses to identify putative viral agents were conducted and the results confirmed by PCR. We identified a non-enveloped single-stranded DNA densovirus in the wild-caught Culex pipiens molestus mosquitoes. The majority of the viral transcripts (.>80% of the region) were covered by the small viral RNAs, with a few peaks of very high coverage obtained. The +/− strand sequence ratio of the small RNAs was approximately 7∶1, indicating that the molecules were mainly derived from the viral RNA transcripts. The small viral RNAs overlapped, enabling contig assembly of the viral genome sequence. We identified some small RNAs in the reverse repeat regions of the viral 5′- and 3′ -untranslated regions where no transcripts were expected.Conclusions/significanceOur results demonstrate for the first time that high throughput sequencing of small RNA is feasible for identifying viral agents in wild-caught mosquitoes. Our results show that it is possible to detect DNA viruses by sequencing the small RNAs obtained from insects, although the underlying mechanism of small viral RNA biogenesis is unclear. Our data and those of other researchers show that high throughput small RNA sequencing can be used for pathogen surveillance in wild mosquito vectors.

During an investigation of arboviruses in China, a novel densovirus (DNV) was isolated from the adult female Culex pipiens pallens. The virus, designated Culex pipiens pallens densovirus (CppDNV), caused cytopathic effect in C6/36 cells. The virus particles were icosahedral, non-enveloped and had a mean diameter of 24 nm. The complete coding region of CppDNV was found to be 3335 nt and it contained three open reading frames (ORFs). CppDNV shares 82-93 % identical nucleotides with isolates of the Aedes albopictus densovirus [isolates AalDNV-1, AalDNV-2 (C6/36 DNV) and , Aedes aegypti densovirus (AaeDNV) and Haemagogus equines densovirus (HeDNV). The nucleotide sequence identity among CppDNV isolates exceeds 98 %. Phylogenetic trees based on non-structural (NS1 and NS2) and capsid (VP) genes show that CppDNV clustered with the species AaeDNV and represents a novel variant of this species within the genus Brevidensovirus.Densoviruses (DNVs) are classified as members of the genera Densovirus, Iteravirus, Brevidensovirus and Pefudensovirus (subfamily Densovirinae) within the family Parvoviridae. They represent a group of non-enveloped viruses, with single-stranded DNA genomes, encapsidated within icosahedrally arranged viral particles. Their host range is limited to a few closely related invertebrates, particularly insects. However, some DNVs also infect and multiply in shrimps. Members of the genera Densovirus and Iteravirus infect lepidopterans (Tijssen & Arella., 1991). Members of the genus Brevidensovirus infect mosquitoes (Ward et al., 2001), while members of the genus Pefudensovirus infect cockroaches. Members of the genera Densovirus and Pefudensovirus have ambisense genomes that are 5.5-6 kb long. Their structural and non-structural proteins are encoded from separate strands, while those of the genera Iteravirus and Brevidensovirus are monosens (encoded from the same strand) and are~5 and 4 kb long, respectively (Jousset et al., 1993;O'Neill et al., 1995;Fauquet et al., 2005).The identification of DNVs in various mosquito cells and wild mosquitoes suggests that the brevidensoviruses have a widespread distribution (O'Neill et al., 1995). The type species of the genus Brevidensovirus is Aedes aegypti densovirus (AaeDNV) (Buchatsky., 1989). The genus contains several other isolates including Aedes albopictus densovirus (AalDNV) [this species contains to date three distinct viruses from C6/36 cells all identified as AalDNV, we shall refer to these in this paper as AalDNV-1 (identified by Jousset et al., 1993;Boublik et al., 1994), AalDNV-2 (identified by Chen et al., 2004) and AalDNV-3 (identified by Paterson et al., 2005)]. Culex pipiens densovirus (CpDNV) was isolated from Culex pipiens larvae; however, it is genetically related to the type speciesThe GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are EF579756-EF579771.Supplementary tables are available with the online version of this paper. (2008( ), 89, 195-199 DOI 10.1099 Here we report the isolation and characterization of a ...

From July to September in 2005 and 2006, a survey was conducted to identify mosquito species and mosquito-borne arboviruses at elevations ranging from 900-3280 m between 24 degrees 00' N and 29 degrees 00' N latitude in the northwestern part of Yunnan Province, China. A total of 54,879 mosquitoes representing 15 species and 4 genera was collected using UV light traps at 59 sites. Culex tritaeniorhynchus and Anopheles sinensis were the most abundant species. The density of mosquitoes as well as the diversity of species decreased with increasing altitude. A total of 21,008 mosquitoes in 281 pools representing all of the 15 species was tested for the presence of viruses using cell culture. Viruses identified included Japanese encephalitis virus (13 isolates), Getah virus (five isolates), Banna virus (three isolates), Kadipiro virus (five isolates), and Densovirus (seven isolates). These isolates were obtained from Culex tritaeniorhynchus (20 isolates), Anopheles sinensis (three isolates), Armigeres subalbatus (six isolates), Culex pipiens quinquefasciatus (two isolates), and from unidentified, mixed mosquitoes (two isolates). Most of the isolates were from collections made at elevations below 2,500 m. Vector Borne Zoonotic Dis. 0, 000-000.

Aims The response of fine roots to soil moisture is very sensitive. Climate change scenarios predict changes in precipitation which influence soil moisture directly. Plants optimize resource acquisition by fine root morphological plasticity and biomass redistribution when soil moisture changes. Therefore, it is important to study the effect of precipitation increase and decrease on fine roots and reveal the response of ecosystem carbon cycling to global climate change. Methods We collected 202 sets of data from 48 published domestic and foreign articles, and analized the responses of fine root biomass, production, turnover, root length density, specific root length and soil microbial biomass carbon which reflects fine root decomposition dynamic to precipitation change by the meta-analysis. RR++ (weighted response ratio) was used to quantify the effect size of the response of fine roots to precipitation change. Important findings (1) The significance and magnitude of the precipitation effects on fine roots varied among plant types. Shrub fine roots had stronger response than tree fine roots. (2) The response of fine roots differed across soil depth. Fine root had most significant responses when the precipitation increased or decreased 50%. A 50% increase in precipitation had a significant positive impact on both fine root biomass in 20-40 cm soil and specific root length in 0-10 cm soil depth. A 50% decreased in precipitation had a significant negative impact on fine root production in 20-40 cm soil but positive impact on root length density in 0-10 cm soil. (3) The duration of experiment affected the response of fine roots, fine roots responded to precipitation changes (increase and decrease) by morphological plasticity in short-term experiments, and by biomass redistribution in long-term experiments. (4) Increasing precipitation contributed to the nutrient release of fine roots, because soil microbes accelerated the decomposability of fine roots due to sufficient substrate resources stimulated their own activity.

No abstract