Metagenomic Analysis of RNA Viruses in a Fresh Water Lake

Freshwater lakes and ponds present an ecological interface between humans and a variety of host organisms. They are a habitat for the larval stage of many insects and may serve as a medium for intraspecies and interspecies transmission of viruses such as avian influenza A virus. Furthermore, freshwater bodies are already known repositories for disease-causing viruses such as Norwalk Virus, Coxsackievirus, Echovirus, and Adenovirus. While RNA virus populations have been studied in marine environments, to this date there has been very limited analysis of the viral community in freshwater. Here we present a survey of RNA viruses in Lake Needwood, a freshwater lake in Maryland, USA. Our results indicate that just as in studies of other aquatic environments, the majority of nucleic acid sequences recovered did not show any significant similarity to known sequences. The remaining sequences are mainly from viral types with significant similarity to approximately 30 viral families. We speculate that these novel viruses may infect a variety of hosts including plants, insects, fish, domestic animals and humans. Among these viruses we have discovered a previously unknown dsRNA virus closely related to Banna Virus which is responsible for a febrile illness and is endemic to Southeast Asia. Moreover we found multiple viral sequences distantly related to Israeli Acute Paralysis virus which has been implicated in honeybee colony collapse disorder. Our data suggests that due to their direct contact with humans, domestic and wild animals, freshwater ecosystems might serve as repositories of a wide range of viruses (both pathogenic and non-pathogenic) and possibly be involved in the spread of emerging and pandemic diseases

Dispatched and Scube Mediate the Efficient Secretion of the Cholesterol-Modified Hedgehog Ligand

The Hedgehog (Hh) signaling pathway plays critical roles in metazoan development and in cancer. How the Hh ligand is secreted and spreads to distant cells is unclear, given its covalent modification with a hydrophobic cholesterol molecule, which makes it stick to membranes. We demonstrate that Hh ligand secretion from vertebrate cells is accomplished via two distinct and synergistic cholesterol-dependent binding events, mediated by two proteins that are essential for vertebrate Hh signaling: the membrane protein Dispatched (Disp) and a member of the Scube family of secreted proteins. Cholesterol modification is sufficient for a heterologous protein to interact with Scube and to be secreted in a Scube-dependent manner. Disp and Scube recognize different structural aspects of cholesterol similarly to how Niemann-Pick disease proteins 1 and 2 interact with cholesterol, suggesting a hand-off mechanism for transferring Hh from Disp to Scube. Thus, Disp and Scube cooperate to dramatically enhance the secretion and solubility of the cholesterol-modified Hh ligand

RNA Viruses Families in Lake Needwood.

<p>Classified RNA viral families are listed along with seasonal and host distribution. Families were assigned to sequences based on best BLASTX matches (e-value<1e-5 or lower). Host type was assigned using viral species (not viral family) data, and is therefore not reflective of the full host range for a given viral family.</p

Phylogenetic tree of circoviruses.

<p>A region of 90 amino at the 3′ end of the circoviruses polymerase gene was selected for sequence comparison analysis. Other sequences were downloaded from the GOS and the NCBI databases. Selected sequences were analyzed using ClustalX with default parameter settings as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007264#s4" target="_blank">Materials and Methods</a>. Consensus tree bootstrapping was performed with Geneious 4.0.4 using the neighbor-joining method and 1,000 samples.</p

Summary of candidates disease causing and novel viruses identified in Lake Needwood.

<p>Summary of candidates disease causing and novel viruses identified in Lake Needwood.</p

Distribution of viral reads by viral types.

<p>Names were assigned to assemblies based on the best BLASTX match. Assemblies with the same virus name were grouped together, and the numbers of reads comprising these assemblies were added to generate a reads per unique types value. Although RNA viruses were the target of this study and the best-represented in the data, DNA viruses have been included as well for comparison.</p

Taxonomic classification of assemblies.

<p>Assemblies were classified based on comparison to the CAMERA database using the BLASTX algorithm and an e-value of 1×10e-5 or lower. Sequences in assemblies without significant matches to existing protein sequences (e-value>1E-5) were classified as “Unknown”. The remaining sequences were classified based on best BLASTX hits for their assemblies. Of the “known” sequences, 67% of the November sample and 70% of the June sample had homology to published viral sequences.</p

Comparison with other viral metagenomics data sets.

<p>Comparison with other viral metagenomics data sets.</p

Total sequences metrics and preliminary classification.

<p>Total sequences metrics and preliminary classification.</p