Genome sequencing and phylogenetic analyses as a basis for molecular subtyping of male-specific (FRNA) coliphages

An international collection of male-specific ssRNA (FRNA) coliphages comprising the Leviviridae family exists but the genetic diversity of these strains is poorly characterized. FRNA coliphages belonging to the family Leviviridae are genetically divided into two genera (Levivirus and Allolevivirus) which can be further divided into four different genogroups (I, II, III and IV). To better characterize this virus family and its genetic genogroups, strains were collected from the United States, Japan, Europe, Brazil, Taiwan and/or Mexico. The complete genomic sequences of 19 new FRNA strains (10 Levivirus and 9 Allolevivirus) from diverse sources were determined and compared to the eleven known genome sequences available in GenBank, for a total of thirty FRNA genomes. Phylogenetic analyses demonstrated all strains clustered into two genera, Levivirus and Allolevivirus, and in four distinct genogroups, I, II, III and IV. Out of ten genogroup I strains, nine strains shared nucleotide sequence similarities ranging from 91.68% 99% whereas genogroup I strain fr shared a 75.27-77.65% sequence identity with the other genogroup I phages. Genogroup II FRNA strains shared 83.30-93.84% nucleotide similarity. Allolevivirus strains shared a nucleotide similarity range of 69.77-95.69% and 74.90-95.03% for genogroups III and IV, respectively. An approximate 50% nucleotide sequence identity was shared between Levivirus groups I and II and between Allolevivirus groups III and IV. For all strains 57 genomic full-length nucleotide and individual protein phylogenetic trees were compared. Genogroup II lysis protein tree formed a unique branch that was not observed in the fulllength nucleotide tree. Thus, both full-length nucleotide and individual proteins need to be evaluated when genotyping or phylogenetically clustering these FRNA coliphages. Data for amino acid composition, nucleotide similarities and replicase catalytic domain location contributed to phylogenetic branches or strain subclusters. Eight nucleotides at the 3' termini clearly distinguished between the Levivirus, 5' ACCACCCA 3' from the Allolevivirus 5' TCCTCCCA 3' genera. This evidence suggests that the sequence data is valid. Introduction Male-specific RNA (FRNA) coliphages are single-strand RNA (ssRNA) viruses possessing a positive sense genome ranging from 3.8 to 4.2 kb in size enclosed by a nonenveloped 26 nm icosahedral-shaped capsid (Buchen-Osmond, 2003). The bacterial host is restricted to the gram-negative bacteria of the genera Escherichia, Pseudomonas, Caulobacter, Salmonella, and Vibrio (Loeb and Zinder, 1961). For successful infection, the host must possess a fertility (F) sex-pilus, coded on the F-plasmid of E. coli (Paranchych, 1975), as infection occurs by attachment to this receptor site (Crawford and Gesteland, 1964). As the sex-pili are only expressed at temperatures greater than 30 o C (Grabow, 2001), phage replication is restricted to environments such as the intestinal tract of warmblooded animals. The inability to replicate outside the gut, environmental stability, high numbers in sewage and a strong correlation with water-borne pathogens are some of the properties which have