Plant or insect RNA viruses identified in whiteflies and amino acid (aa) identity ranges.

<p>Unassigned virus groups refer to contigs that had significant matches to more than one virus with BLASTn scores within 10% of each other. Multiple matches make it impossible to assign these partial sequences. Viruses found in hosts other than plants or insects are identified (**). Viruses highlighted in boldface are known to be whitefly – transmitted.</p><p>K = known virus, and known to occur in Florida at this site, N = known virus, but not previously reported from Florida, U = most likely a new, uncharacterized virus.</p

Responses observed in a range of selected host plants mechanically inoculated with the Cowpea mild mottle virus Florida isolate.

<p>¹ Number of infected plants/number of plants inoculated. The number of infected plants was determined by ELISA.</p><p>² Abbreviations used: NS - no symptoms, Mo – mottle, R – rugose, LD – leaf deformation, mMo – mild mottle, VC – veinal chlorosis, RV – red vein, RLL – red local lesions, CLL – chlorotic local lesions.</p><p>Infected species as determined by visible symptoms and/or ELISA are highlighted in boldface.</p

Schematic genome organization of Cowpea mild mottle virus (CpMMV) isolates identified in Ghana (top) and Florida (middle) as well as the general genome organization observed in carlavirus species (bottom; exact species shown in phylogenetic tree in Figure 2).

<p>Each block represents identified open reading frames (ORFs) in these genomes and the encoded protein names are given on top of the CpMMV Florida panel, including the replication polyprotein, triple gene block (TGB), capsid (CP), and nucleic acid binding (NB) protein. The four different domains within the replication polyprotein exhibiting viral methyltransferase, peptidase, superfamily one (SF1) helicase, and RNA-dependent RNA polymerase (polymerase) motifs are indicated. Percentage values in the CpMMV Ghana genome schematic represent amino acid pairwise identities among individual ORFs of CpMMV Ghana and CpMMV Florida. Percentages in the Carlaviruses genome schematic represent amino acid pairwise identity ranges observed between different carlavirus species and CpMMV Florida. ORFs highlighted in grey represent ORFs without a standard start codon (a lighter grey in the carlaviruses panel indicates that some carlavirus species have a standard start codon while others do not).</p

Maximum likelihood phylogenetic tree of capsid proteins (CP) found in different carlavirus species.

<p>Cowpea mild mottle virus (CpMMV) isolates identified in Florida, USA in this study are highlighted in grey. Branch support was assessed with the approximate likelihood ratio test and values >70% are shown.</p

Fecally Borne PMMV Is Infectious to a Pepper Plant

<div><p>(A) Fecal supernatant containing PMMV was inoculated on to a leaf of a <i>Capsicum</i> plant (Day 0). After 7 d of inoculation, this leaf developed typical symptoms of viral infection (Day 7).</p> <p>(B) RNA extracts from uninfected control leaves (lane 1) and PMMV-positive fecal supernatant challenged leaves (lane 2) were tested for PMMV by RT-PCR.</p></div

Alignment of Assembled PMMV-Like Sequences from Three Shotgun Libraries with the Reference PMMV Genome Sequence

<p>The PMMV-like viral genome sequence segments from Lib 1 (A), Lib 2 (B), and Lib 3 (C) were aligned with the reference PMMV genome sequence (6,357 bp). Colored bars (D) indicated the similarity level between library sequences with template sequences as measured by BLAST score.</p

Different PBV Strains Found in Two Fecal Samples from the Same Individual

<div><p>(A) The PBV-like sequence segments identified in Lib 1 were aligned to the partial genome sequence of PBV strain 4-GA-91 using BLASTn. The identities of nucleotide sequence between the contigs and the reference PBV sequence were 95%–99%.</p> <p>(B) The PBV-like sequence segments in Lib 2 were too remote to both known PBV strains (4-GA-91 and 1-CHN-97) at the nucleotide level, but could be aligned to the PBV strain 1-CHN-97 using tBLASTx. The identity of amino acid sequences between the PBV-like sequence segments in Lib 2 and the reference PBV genome sequence were 46%–69%.</p> <p>(C) Colored bars indicate the similarity level between library sequences with template sequences as measured by BLAST score.</p></div

Phylogenetic Tree of PMMV Sequences

<p>A region of 101 bp in the PMMV CP gene was chosen for sequence comparison and phylogenetic analysis. A total of 44 assembled sequences (>50 bp) from the three fecal virus libraries were located within this region. These sequences were aligned with 21 known PMMV CP genes from GenBank using ClustalW with default parameter settings. In this phylogenetic tree of the PMMV CP gene, sequences from Lib 1 are highlighted in pink, Lib 2 in blue, and Lib 3 in yellow. GenBank accession numbers of known CP genes are shown unshaded.</p

RT-PCR Detection of Fecally Borne RNA Viruses

<div><p>(A) PMMV (lane 1) was detected by RT-PCR using PMMV specific primers in a fecal RNA extract. This PMMV band is PMMV primer-specific (lane 2) and dependent on reverse transcription (lane 3).</p> <p>(B) The specificity of the RT-PCR reaction for detecting fecal PMMV was further assessed by the use of nonspecific PCR primers (lane 1 versus lane 2) and respiratory syncytial virus (RSV; American Type Culture Collection #VR-1401) as a nonspecific RNA template (lanes 1 and 2 versus lanes 3 and 4). The identities of RT-PCR products (PMMV in lane 1; RSV in lane 4) were confirmed by sequencing analysis.</p> <p>(C) RNA viruses were directly detected by RT-PCR from the total RNA of fecal sample 2: PMMV (lane 1), MCMV (lane 2), PBV, segment 2 (lane 3), OCSV (lane 4), and PanMV (lane 5).</p> <p>(D) Equal amounts of dry weight of food (meal samples for 2 d prior to fecal collection) and feces from three individuals were assayed by RT-PCR to compare the amounts of PMMV present. The estimated numbers of virions in 1 g of dry food and feces were 1.21 × 10⁶ (lane 1), 2.3 × 10⁷ (lane 2), 1.63 × 10⁷ (lane 3), 3.64 × 10⁹ (lane 4), 2.42 × 10⁷ (lane 5), and 1.95 × 10⁸ (lane 6) as determined by TaqMan RT-PCR.</p> <p>(E) Fecal samples from six additional individuals from San Diego were analyzed for detection of PMMV. The positive control is shown in lane 7.</p> <p>(F) Detection of PMMV in fecal samples of nine individuals from Singapore, including one infant (lane 9). Lane 10 is the positive control.</p> <p>(G) Detection of PMMV from seven chili sauces from Singapore.</p></div

Monte Carlo Simulation of Cross-Contigs between Metagenomic Samples

<p>(A) For the intersample analysis, the maximum likelihood occurred at 35% fraction permuted and 100% fraction shared. (B) The maximum likelihood was between 0% and 0.5% fraction permuted and 85% and 95 % fraction shared for the intrasample controls.</p