ICTV Virus Taxonomy Profile: Inoviridae.

Members of the family Inoviridae are non-enveloped flexible filamentous bacteriophages (600-2500×6-10 nm) with supercoiled, circular, positive-sense, single-stranded DNA genomes of 5.5-10.6 kb, encoding 7-15 proteins. They absorb to the pili of Gram-negative bacteria and replicate their DNA by a rolling-circle mechanism with progeny released from cells by extrusion without killing the host. Phage DNA can persist extra-chromosomally or integrate into the bacterial genome. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Inoviridae, which is available at ictv.global/report/inoviridae.

Unravelling the consequences of the bacteriophages in human samples.

Bacteriophages are abundant in human biomes and therefore in human clinical samples. Although this is usually not considered, they might interfere with the recovery of bacterial pathogens at two levels: 1) by propagating in the enrichment cultures used to isolate the infectious agent, causing the lysis of the bacterial host and 2) by the detection of bacterial genes inside the phage capsids that mislead the presence of the bacterial pathogen. To unravel these interferences, human samples (n = 271) were analyzed and infectious phages were observed in 11% of blood culture, 28% of serum, 45% of ascitic fluid, 14% of cerebrospinal fluid and 23% of urine samples. The genetic content of phage particles from a pool of urine and ascitic fluid samples corresponded to bacteriophages infecting different bacterial genera. In addition, many bacterial genes packaged in the phage capsids, including antibiotic resistance genes and 16S rRNA genes, were detected in the viromes. Phage interference can be minimized applying a simple procedure that reduced the content of phages up to 3 logs while maintaining the bacterial load. This method reduced the detection of phage genes avoiding the interference with molecular detection of bacteria and reduced the phage propagation in the cultures, enhancing the recovery of bacteria up to 6 logs.

Cryptic inoviruses revealed as pervasive in bacteria and archaea across Earth's biomes.

Bacteriophages from the Inoviridae family (inoviruses) are characterized by their unique morphology, genome content and infection cycle. One of the most striking features of inoviruses is their ability to establish a chronic infection whereby the viral genome resides within the cell in either an exclusively episomal state or integrated into the host chromosome and virions are continuously released without killing the host. To date, a relatively small number of inovirus isolates have been extensively studied, either for biotechnological applications, such as phage display, or because of their effect on the toxicity of known bacterial pathogens including Vibrio cholerae and Neisseria meningitidis. Here, we show that the current 56 members of the Inoviridae family represent a minute fraction of a highly diverse group of inoviruses. Using a machine learning approach leveraging a combination of marker gene and genome features, we identified 10,295 inovirus-like sequences from microbial genomes and metagenomes. Collectively, our results call for reclassification of the current Inoviridae family into a viral order including six distinct proposed families associated with nearly all bacterial phyla across virtually every ecosystem. Putative inoviruses were also detected in several archaeal genomes, suggesting that, collectively, members of this supergroup infect hosts across the domains Bacteria and Archaea. Finally, we identified an expansive diversity of inovirus-encoded toxin-antitoxin and gene expression modulation systems, alongside evidence of both synergistic (CRISPR evasion) and antagonistic (superinfection exclusion) interactions with co-infecting viruses, which we experimentally validated in a Pseudomonas model. Capturing this previously obscured component of the global virosphere may spark new avenues for microbial manipulation approaches and innovative biotechnological applications.

Complete genome sequence of a filamentous bacteriophage, RS611, that infects the phytopathogen Ralstonia solanacearum.

Filamentous bacteriophage RS611 (ϕRS611), which infects the phytopathogen Ralstonia solanacearum, had a circular single-stranded DNA genome that was characterized as an Ff-type phage belonging to the family Inoviridae. The ϕRS611 genome was composed of 6386 bases with a G + C content of 62.1 % and contained 11 putative open reading frames. The ϕRS611 genome showed high similarity to those of Ralstonia phages RSS0 and RSS1. However, approximately 900-nucleotide deletions were found in the region corresponding to open reading frames 10 and 11 of ϕRSS0 and ϕRSS1.

Isolation and characterization of a novel filamentous phage from Stenotrophomonas maltophilia.

In this study, a novel filamentous phage, φSHP1, of the environmental Stenotrophomonas maltophilia strain P2 was isolated and characterized. Electron microscopy showed that φSHP1 resembled members of the family Inoviridae and was about 2.1 µm long. The 6,867-nucleotide genome of φSHP1 was a circular single-stranded DNA and had a replication form designated pSH1. Ten putative open reading frames (ORFs) were found in the φSHP1 genome, and six predicted proteins showed similarity to proteins in databases. Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis of φSHP1 displayed one major structural polypeptide of approximately 4.0 kDa. N-terminal sequencing showed that it was the mature product of ORF5 and that its N-terminal 27 amino acid residues had been cleaved off from the predicted nascent protein. Finally, phylogenetic trees were constructed to analyze the phylogenetic relationship of φSHP1 to other known filamentous phages. φSHP1 appears to be the first reported Stenotrophomonas filamentous phage.

Application of real-time PCR assays to genotyping of F-specific phages in river water and sediments in Japan.

Genotyping of F-specific RNA phages is currently one of the most promising approaches to differentiate between human and animal fecal contamination in aquatic environments. In this study, a total of 18 river water and sediment samples were collected from the Tonegawa River basin, Japan, in order to describe the genogroup distribution of F-specific RNA and DNA phages using genogroup-specific real-time PCR assays. F-specific phages were detected in nine (100%) river water and six (67%) sediment samples. Eighty-five phage plaques were isolated from these samples and subjected to real-time PCR assays specific for the phages. F-specific RNA phages of human genogroups (II and III) were detected in 32 (38%) plaques, whereas those of animal genogroups (I and IV) were detected in 17 (20%) plaques. No correlation was observed between the genogroup distribution of F-specific RNA phages and the occurrence of human adenovirus genomes, suggesting that genotyping of the phages alone is inadequate for the evaluation of the occurrence of viruses in aquatic environments. SYBR Green-based real-time PCR assay revealed the presence of F-specific DNA phages in four (5%) plaques, which were further classified into two genogroups (fd- and f1-like phages) by sequence analysis. Thirty-two (38%) plaques were not classified as the F-specific phage genogroups, indicating the limited applicability of these real-time PCR assays to a wide range of aquatic environmental samples worldwide.

Salmonella phages examined in the electron microscope.

Out of 177 surveyed bacteriophages, 161 (91%) are tailed and belong to the Myoviridae, Siphoviridae, and Podoviridae families (43, 55, and 59 viruses, respectively). Sixteen filamentous or isometric phages are members of the Inoviridae, Leviviridae, Microviridae, and Tectiviridae families (9%). Many tailed phages belong to established phage genera (P22, T1, T5, and T7), which are widespread in enterobacteria and other Gram-negatives of the Proteobacteria phylum.

Isolation and characterization of Thermus bacteriophages.

One-hundred-fifteen bacteriophage strains were isolated from alkaline hot springs in Iceland, New Zealand, Russia (Kamchatka), and the U.S.A. The phages belonged to the Myoviridae, Siphoviridae, Tectiviridae, and Inoviridae families. Over 50% of isolates were isometric or filamentous. One type of siphovirus had giant tails of over 800 nm in length. Phages were further characterized by host range, genome size, DNA restriction endonuclease digestion patterns, and temperature and pH sensitivity. Myoviruses and tectiviruses had a worldwide distribution. Most phages were narrowly host-specific and all were highly resistant against heating and alkaline and acidic pH. This is the first time that tectiviruses and filamentous phages are reported for bacteria of the Thermus-Deinococcus phylum. The presence of tectiviruses, inoviruses, and myoviruses is attributed to acquisition from ancestral gamma-proteobacteria by horizontal gene transfer.

Molecular detection and genotyping of male-specific coliphages by reverse transcription-PCR and reverse line blot hybridization.

In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Q beta, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies.

Bacteriophages that infect the cellulolytic ruminal bacterium Ruminococcus albus AR67.

AIM: To isolate bacterial viruses that infect the ruminal cellulolytic bacterium Ruminococcus albus. METHODS: Four phages infecting R. albus AR67 were isolated under anaerobic conditions using the soft-agar overlay technique. The phages were characterized on morphology, solvent stability, nucleic acid type and digestion characteristics. Two phages, phiRa02 and phiRa04 comprised icosahedral virions with linear double-stranded DNA and appeared to belong to the family Podoviridae [corrected] The other two phages are most likely filamentous phages with circular single-stranded DNA of the family Inoviridae. SIGNIFICANCE OF THE STUDY: Viruses of the family Inoviridae [corrected] have not previously been isolated from rumen bacteria. The phages isolated in this study are the first phages shown to infect the cellulolytic bacteria of the rumen. This suggests that the cellulolytic populations of the rumen are subject to lytic events that may impact on the ability of these bacteria to degrade plant fibre and on the nutrition of the animal.