Metagenomic Analysis of the Human Mouth Virus Population and Characterisation of Two Lytic Viruses

Viruses are biological agents that infect cellular organisms. Most viruses are bacteriophages, these are the most abundant biological entities on earth. Not much is known about virus diversity in the human mouth, including dental plaque, compared to other environments. A culture-independent based approach was tried using metagenomic analysis to characterize uncultured virus gene fragments in human dental plaque. The isolated viral genomes were amplified using a multiple displacement amplification method. Eighty, eleven and ten clones were sequenced from three volunteers, respectively. TBLASTX analysis showed that 44% of the sequences had significant identities to the GenBank databases. Of these 66% were viral; 12% human; 10% bacterial; 6% mobile and 6% eukarya. These sequences were sorted into six contigs and forty five single sequences. Four contigs and one single sequence were found to have a significant identity to a small region of a putative prophage in the Corynebcterium diphtheria genome. The gaps between these were filled by primer walking and PCR to give a continuous contig of 11554 bp. Two viruses A1 and A2 and their bacterial host were isolated from the human mouth. The 16S rRNA gene sequence of the host had a 99% identity to several Neisseria sp. The A1 virus was found to appear spontaneously on soft top agar plates, and might be a lysogenic virus. The A2 virus was a lytic virus. The two viruses have different morphological shapes. A1 has a varied isometric head size that ranges from 32 to 58 nm and no tail; it may belong to the Tectiviridae family. It has a linear dsDNA genome with a size between 12 kb and 23kb. A limited amount of the genome of the A1 virus was sequenced. The A2 virus has an icosohedral head with size of 60±3 nm and a sheathed rigid tail about 175 nm long with no detectable base plate or tail fibres. It can be classified into the order Caudovirales family Siphoviridae. The size of the A2 virus genome is estimated to be 35 to 40 kb. 31703 bp of unique sequence has been determined and sorted into three contigs and 14 single sequences. Further attempts at gap filling using primer walking and PCR were unsuccessful. It has a linear dsDNA genome, with a GC content of 49 mol%. A latent period of 25 min and a burst size of 25±2 particles were determined by a single step growth curve. Bioinformatic approaches were used to identify ORFs in the genome. A2 virion associated proteins were analysed by SDS–PAGE gel electrophoresis, and some proteins sequences were directly related to the translated genomic sequence