Case studies of the spatial heterogeneity of DNA viruses in the cystic fibrosis lung.

Microbial communities in the lungs of patients with cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) have been shown to be spatially heterogeneous. Viral communities may also vary spatially, leading to localized viral populations and infections. Here, we characterized viral communities from multiple areas of the lungs of two patients with late-stage CF using metagenomics, that is, the explanted lungs from a transplant patient and lungs acquired postmortem. All regions harbored eukaryotic viruses that may infect the human host, notably herpesviruses, anelloviruses, and papillomaviruses. In the highly diseased apical lobes of explant lungs, viral diversity was extremely low, and only eukaryotic viruses were present. The absence of phage suggests that CF-associated microbial biofilms may escape top-down controls by phage predation. The phages present in other lobes of explant lungs and in all lobes of postmortem lungs comprised distinct communities, and encoded genes for clinically important microbial phenotypes, including small colony variants and antibiotic resistance. Based on the these observations, we postulate that viral communities in CF lungs are spatially distinct and contribute to CF pathology by augmenting the metabolic potential of resident microbes, as well as by directly damaging lung tissue via carcinomas and herpesviral outbreaks.

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Identification of a novel human papillomavirus by metagenomic analysis of samples from patients with febrile respiratory illness.

As part of a virus discovery investigation using a metagenomic approach, a highly divergent novel Human papillomavirus type was identified in pooled convenience nasal/oropharyngeal swab samples collected from patients with febrile respiratory illness. Phylogenetic analysis of the whole genome and the L1 gene reveals that the new HPV identified in this study clusters with previously described gamma papillomaviruses, sharing only 61.1% (whole genome) and 63.1% (L1) sequence identity with its closest relative in the Papillomavirus episteme (PAVE) database. This new virus was named HPV_SD2 pending official classification. The complete genome of HPV-SD2 is 7,299 bp long (36.3% G/C) and contains 7 open reading frames (L2, L1, E6, E7, E1, E2 and E4) and a non-coding long control region (LCR) between L1 and E6. The metagenomic procedures, coupled with the bioinformatic methods described herein are well suited to detect small circular genomes such as those of human papillomaviruses.

Spatial distribution of microbial communities in the cystic fibrosis lung.

Cystic fibrosis (CF) is a common fatal genetic disorder with mortality most often resulting from microbial infections of the lungs. Culture-independent studies of CF-associated microbial communities have indicated that microbial diversity in the CF airways is much higher than suggested by culturing alone. However, these studies have relied on indirect methods to sample the CF lung such as expectorated sputum and bronchoalveolar lavage (BAL). Here, we characterize the diversity of microbial communities in tissue sections from anatomically distinct regions of the CF lung using barcoded 16S amplicon pyrosequencing. Microbial communities differed significantly between different areas of the lungs, and few taxa were common to microbial communities in all anatomical regions surveyed. Our results indicate that CF lung infections are not only polymicrobial, but also spatially heterogeneous suggesting that treatment regimes tailored to dominant populations in sputum or BAL samples may be ineffective against infections in some areas of the lung.

Molecular characterization and expression of vitellogenin receptor from white perch (Morone americana).

A full-length (4021 base pair [bp]) cDNA encoding a polypeptide (844 amino acids) with a predicted mass of 93 kDa and other characteristic structural features of a vertebrate vitellogenin receptor (VgR) was isolated from a white perch (Morone americana) ovarian cDNA library. Northern blotting performed using a specific digoxygenin-labeled VgR cDNA probe revealed a distinct approximately 4.1 kilobase (kb) hybridization signal in an mRNA preparation obtained from previtellogenic perch ovaries. The deduced amino acid sequence of the perch VgR was 89% and 82% identical, respectively, to that of the tilapia and rainbow trout. Because it possessed an eight-repeat ligand-binding domain (LR8) but lacked an O-linked sugar domain (-), the perch VgR was identified as a non-O-linked form of VgR (LR8-). Unlike the case in other vertebrates investigated, including tilapia and trout, no species of mRNA encoding an O-linked form of VgR (LR8+) could be detected when perch ovarian or liver mRNA reverse transcripts or cDNA libraries were screened by PCR using primer sets flanking the putative O-linked sugar domain. These novel findings call into question the assumptions that an LR8+ splice variant of the VgR always is dominantly present in somatic tissues and exists at lower levels in ovarian tissues to sequester lipoproteins distinct from Vg. A SYBR-green-based real-time reverse transcription-polymerase chain reaction assay was developed and used to quantitatively measure VgR expression in gonadal and somatic tissues, for the first time in any vertebrate. The main site of perch VgR mRNA expression was the ovary and the highest level of VgR mRNA expression was in ovaries whose largest follicles contained previtellogenic oocytes. Expression of VgR mRNA decreased with oocyte growth during vitellogenesis and was very limited in ovulated eggs. These quantitative results verify the concept that growing oocytes must extensively recycle LR8- forms of the VgR.