Genome Sequences of Anelloviruses, Genomovirus, and Papillomavirus Isolated from Nasal Pharyngeal Swabs.

The genome sequences of three anelloviruses (genus Alphatorquevirus), a genomovirus (genus Gemykolovirus), and an unclassified papillomavirus were identified in four human nasopharyngeal swabs, and one was positive for influenza A and one for influenza B virus. The influenza B virus-positive sample had a coinfection with an anellovirus and a papillomavirus.

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Nasal pharyngeal swabs were taken from four patients with influenza-like illness as part of a routine clinical testing for influenza virus on a university campus in Arizona between February and March 2020. Two of these swabs tested positive for seasonal influenza viruses, namely, one influenza A virus (IAV) and the other influenza B virus (IBV), via rapid lateral flow immunoassay (Abbott BinaxNow). To identify associated DNA viruses in these samples, 200 μL of the buffer from the lateral flow assay was used to extract viral DNA with the high pure viral nucleic acid kit (Roche Diagnostics, USA). Circular DNA was amplified by rolling circle amplification (RCA) using an Illustra TempliPhi kit (GE Healthcare, USA). The RCA products were used to prepare libraries using a TruSeq Nano DNA kit (Illumina, USA). The 2× 150-bp libraries were sequenced on a NovaSeq6000 sequencer at Psomagen Inc. (USA). The reads were trimmed using Trimmomatic v0.39 (1) and de novo assembled with metaSPAdes 3.14.0 (2). We identified complete genome sequences (based on terminal redundancy) of anelloviruses (n = 3), genomovirus (n = 1), and a papillomavirus (n = 1) using the BLASTn (3) nonredundant (nr) database online on May 2021. Reads were mapped to the viral genome sequences using BBMap (4) and showed depth coverage of 24× to 715× and mapped reads per genome of 365 to 17,483 (Fig. 1A). In sample S1 (IBV positive), we found a coinfection with an anellovirus and a papillomavirus (Fig. 1).

FIG 1

(A) Summary of the genomes determined from the four nasopharyngeal swabs with accession number, genome length, GC content, read depth, and coverage. (B) Genome organization of the anelloviruses, genomovirus, and papillomavirus identified from the swab samples. (C) Taxonomic classification of the anelloviruses, genomovirus, and papillomavirus and the summary of the BLASTn analysis.

Anelloviruses are circular single-stranded DNA (ssDNA) viruses (2.0 to 3.9 kb) (5). They are diverse and highly prevalent but are not associated with disease (6). Anellovirus genomes have three large open reading frames (ORFs). The three anelloviruses identified here are ~3.7 kb (with GC content of 52% to 53%) and share >83% nucleotide identity with those previously identified in humans (Fig. 1B and C). Based on the guidelines for the classification of mammal-infecting members of the Anelloviridae family (7), torque teno virus AZ1_2 (MW679003), torque teno virus AZ6_5 (MW679005), and torque teno virus AZ7_4 (MW679006) identified in the nasal pharyngeal swabs all belong to the genus Alphatorquevirus and species Torque teno virus 24, Torque teno virus 29, and Torque teno virus 19, respectively (Fig. 1C).

Genomoviruses are circular ssDNA viruses (2 to 2.4 kb) that encode bidirectionally transcribed ORFs, capsid protein (CP), and a replication associated protein (Rep), with the exception of Fusarium graminearum gemytripvirus 1 which is a tripartite virus (8–10). Although ubiquitous in nature, only two genomoviruses have confirmed hosts, namely, Sclerotinia sclerotiorum (11) and Fusarium graminearum (12). Genomovirus AZ4_17 (GenBank accession number MW679004; 2.28 kb with GC content of 53.3%) shares ~89.6% genome-wide nucleotide identity with tortoise genomovirus 13 (MK570213) (Fig. 1C) (13) and is part of the genus Gemykolovirus and species Gemykolovirus gopha2 based on the classification guideline for Genomoviridae (10). Genomoviruses have been identified previously in human blood, cerebrospinal fluid, and pericardial fluid (14–18), and we speculate that the one identified here may be those that infect oral fungi (yeast).

Papillomaviruses are circular double-stranded DNA viruses (~5.7 to 8.6 kb) and are classified within two subfamilies (Firstpapillomavirinae and Secondpapillomavirinae) (19). Members of the Firstpapillomavirinae encode at least seven genes (19). Human papillomavirus AZ1_1 (MW679002; 7.3 kb with GC content of 36.1%) identified in the swab shares ~97% nucleotide identity with HPV-mSK_175 (MH777317) from a human skin swab (20) (Fig. 1) and HPV_SD2R (KC113191) from an oral swab (21); neither have been assigned a type or classified.

Sample collection was part of routine clinical care which is approved by Arizona State University Institutional Review under study identifier (ID) number STUDY00008985.

Data availability.

The sequences of the viruses identified in this study have been deposited in GenBank with accession numbers MW679002, MW679003, MW679004, MW679005, and MW679006. Raw reads have been deposited in SRA project number PRJNA701833 with SRA accessions SRR13720055, SRR13720056, SRR13720057, and SRR13720061.