MS Spectrums of the Three Distinct Genotypes at SNV Position 1,727

<p>The T example is from the cultured viral isolate from patient Sin849, the T/C example is from the uncultured lung tissue sample from patient Sin849, and the C example is from the cultured viral isolate from patient Sin852.</p

The Molecular Relationship among 13 Singapore SARS-CoV Isolates Based on the Genotype Sharing Pattern of the Viral Isolates

<p>The Molecular Relationship among 13 Singapore SARS-CoV Isolates Based on the Genotype Sharing Pattern of the Viral Isolates</p

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

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