Identification of multiple novel viruses, including a parvovirus and a hepevirus, in feces of red foxes

Abstract

Red foxes (Vulpes vulpes) are the most widespread members of the order of Carnivora. Since they often live in (peri)urban areas, they are a potential reservoir of viruses that transmit from wildlife to humans or domestic animals. Here we evaluated the fecal viral microbiome of 13 red foxes by random PCR in combination with next-generation sequencing. Various novel viruses, including a parvovirus, bocavirus, adeno-associated virus, hepevirus, astroviruses, and picobirnaviruses, were identified.

Fig 1

Genome organization and phylogenetic analysis of fox parvovirus. (A) Genome organization of the fox parvovirus. Indicated are the locations of the major ORFs (gray) and the locations of the start and stop codons on the nucleotide level, starting from the known sequence of fox parvovirus at the 5′ end. The locations of the theoretical minor ORFs are indicated by white areas. In addition, the predicted coding fragment of the VP1 is indicated based on a spliced transcript (white bar) with predicted splicing donor AAGAG|GTCAG and acceptor (TTTAG|GTTGG) sites. (B) Phylogenetic neighbor-joining tree with p-distance and 1,000 bootstrap replicates of the deduced amino acid sequences of VP2 genes of various viruses of the subfamily Parvovirinae. Significant bootstrap values are shown. GenBank accession no.: feline parvovirus V142, AB054225; mink enteritis virus, AY665656; blue fox parvovirus, EU698028; canine parvovirus 2b, JQ335978; canine parvovirus 2a, JQ996152; canine parvovirus (raccoon), JN867616; porcine parvovirus Tai'an, FJ853421; porcine parvovirus WB-639, JQ249918; rat minute virus 2a, EF029111; mouse parvovirus 2, NC_008186; mouse parvovirus UT, AB234204; fox parvovirus, KC692368; Aleutian mink disease (AMD) parvovirus, GU183264; gray fox amdovirus, JN202450.1; bufavirus 2 BF 39, JX027297; swine parvovirus H-1, AB076669; human parvovirus 4, AY622943; simian parvovirus, U26342; B19 parvovirus, NC_000883; duck parvovirus, NC_006147; adeno-associated virus 2, NC_001401; porcine bocavirus 5, JN831651; canine minute virus SH1, FJ899734; human bocavirus 3, HM132056.

Fig 2

Phylogenetic analysis of fox hepevirus. (A) Schematic representation of sequences of fox hepevirus detected by random PCR (RaPCR) in combination with next-generation sequencing (NGS) and specific PCR using the complete genome of rat hepatitis E virus (R63/DEU/2009) as a reference. The PCR fragment that was used to prepare the phylogenetic tree is shown in gray. (B) A short fragment of the polyprotein (RdRp) gene of 360 nt corresponding to nt 4015 to 4377 of the complete genome of rat hepatitis E virus (GU345042) was aligned using ClustalW with MEGA5 to the corresponding genome fragments of the indicated viruses. A phylogenetic maximum likelihood (NNI) tree was prepared using the Tamura-Nei model with 500 bootstrap replicates. Bootstrap values are shown. For each sequence, the GenBank accession number, the species from which it was isolated, and the country are indicated.

Fig 3

Genome organization and phylogenetic analysis of fox astrovirus F5. (A) Genome organization of the near complete fox astrovirus F5 genome. Indicated are the locations of three major ORFs (1a, 1b, and 2), the locations of the start and stop codons on the nucleotide level starting from the known sequence of Fox astrovirus at the 5′ end (the unknown sequence is indicated by the dotted line), the poly(A) tail, and a ribosomal frameshift (A)6C sequence. (B) Phylogenetic neighbor-joining tree with p-distance and 1,000 bootstrap replicates based on alignment of ORF2 nucleotide sequences of various astroviruses, including fox astrovirus F5. Bootstrap values are shown. GenBank accession no.: California sea lion astrovirus, FJ890351; mink astrovirus, NC_004579; swine astrovirus PoAstV16-2, HM756261; fox astrovirus F5, KC692365; HMO astrovirus A, NC_013443.1; HMO astrovirus B, GQ415661; ovine astrovirus, NC_002469; bat astrovirus 1, EU847155; bottlenose dolphin astrovirus, FJ890355; minke whale astrovirus, HQ668143; bovine astrovirus B76-2, HQ916317; deer astrovirus, HM447045; porcine astrovirus, Y15938; feline astrovirus, AF056197; human astrovirus 4, Z33883; human astrovirus 1, Z25771; human astrovirus 5, U15136; turkey astrovirus TAstV/TX/00, EU143850; duck astrovirus 1, NC_012437.