Either cDNA derived from RNA enriched for non-polyadenylated bacterial mRNA (MD 257) or cDNA sequencing library samples derived from reverse transcribed poly(A) + RNA (for MD OCT-pool and MD 80228) were used as templates for the generation of 16S rRNA V6 hypervariable region-specific amplicons using the FastStart High Fidelity PCR System (Roche, Switzerland). PCR conditions were 50 cycles of 94uC for 30 sec, 55uC for 45 sec and 72uC for 45 sec. The yielded amplicon products were purified using AMPure, and the resulting individual amplicon DNA libraries were clonally amplified by multiplex emulsion PCR followed by sequencing using the GS FLX pyrosequencing platform. The sequencing output data were computationally divided into subsets according to the barcodes (and the corresponding mule deer sample) and the primers A or B. Figure S1 Comparative MEGAN analysis of (A) MD 80228 and (B) MD OCT-pool transcript-tags analyzed by comparison to the protein database (red) and the ribosomal database (blue), and of amplicon 16S rRNA-tags compared to the ribosomal database (green). Bit score cutoff for the protein database comparison was set at 50, and confidence cutoffs for the ribosomal database comparisons were set at 80% and 80%, respectively.  In Vitro and In Vivo Studies Identify Important Features of Dengue Virus pr-E Protein Interactions Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV) pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors. The emergence and resurgence of human viral pathogens can be traced to a complex variety of causes including increased urbanization, human contact with animal reservoirs, a decrease in effective public health systems, and the spread of insect vectors that disseminate some viral infections [1, 2, 3] . Flaviviruses are a genus in the Flaviviridae family and include important emerging and resurgent human pathogens such as dengue virus (DENV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV) and yellow fever virus [2, 4] . Flaviviruses are transmitted by insects such as mosquitoes and ticks, and can cause severe human diseases characterized by encephalitis, meningitis, and hemorrhages [2, 3] . More than one third of the world's population lives in dengue fever endemic areas, and there are an estimated 50-100 million cases of dengue infection and 500,000 cases of the more lethal complication, dengue hemorrhagic fever, per year [5, 6, 7, 8] . There are currently no antiviral therapies for flaviviruses. DENV vaccine development is underway but is problematic due to the presence of four DENV serotypes and the potential for antibody-dependent enhancement of infection [2, 6, 9, 10] . Antiviral therapies could thus be an important alternative for DENV and for viruses such as WNV in which the cost and potential side effects of vaccination must be weighed against the relatively low number of human cases [2] .