RNA viral community in human feces: prevalence of plant pathogenic viruses

Abstract

The human gut is known to be a reservoir of a wide variety of microbes, including viruses. Many RNA viruses are known to be associated with gastroenteritis; however, the enteric RNA viral community present in healthy humans has not been described. Here, we present a comparative metagenomic analysis of the RNA viruses found in three fecal samples from two healthy human individuals. For this study, uncultured viruses were concentrated by tangential flow filtration, and viral RNA was extracted and cloned into shotgun viral cDNA libraries for sequencing analysis. The vast majority of the 36,769 viral sequences obtained were similar to plant pathogenic RNA viruses. The most abundant fecal virus in this study was pepper mild mottle virus (PMMV), which was found in high concentrations--up to 10(9) virions per gram of dry weight fecal matter. PMMV was also detected in 12 (66.7%) of 18 fecal samples collected from healthy individuals on two continents, indicating that this plant virus is prevalent in the human population. A number of pepper-based foods tested positive for PMMV, suggesting dietary origins for this virus. Intriguingly, the fecal PMMV was infectious to host plants, suggesting that humans might act as a vehicle for the dissemination of certain plant viruses.

Figure 1. Different PBV Strains Found in Two Fecal Samples from the Same Individual

(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%. (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%. (C) Colored bars indicate the similarity level between library sequences with template sequences as measured by BLAST score.

Figure 2. Alignment of Assembled PMMV-Like Sequences from Three Shotgun Libraries with the Reference PMMV Genome Sequence

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.

Figure 3. Phylogenetic Tree of PMMV Sequences

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.

Figure 4. RT-PCR Detection of Fecally Borne RNA Viruses

(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). (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. (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). (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. (E) Fecal samples from six additional individuals from San Diego were analyzed for detection of PMMV. The positive control is shown in lane 7. (F) Detection of PMMV in fecal samples of nine individuals from Singapore, including one infant (lane 9). Lane 10 is the positive control. (G) Detection of PMMV from seven chili sauces from Singapore.

Figure 5. Fecally Borne PMMV Is Infectious to a Pepper Plant

(A) Fecal supernatant containing PMMV was inoculated on to a leaf of a Capsicum plant (Day 0). After 7 d of inoculation, this leaf developed typical symptoms of viral infection (Day 7). (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.