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.

TEXT

Recent outbreaks of disease caused by infection with viruses of animal origin, like the human coronavirus HCoV-EMC (human coronavirus—Erasmus Medical Center), and recurring infections with Ebola virus and Hendra virus have highlighted the importance of wild and domestic animals as a reservoir of viruses that are harmful to humans and other animals (1–3). Knowledge of viruses present in wildlife may be used to predict future transmission risks or eventually outbreaks of viral disease among humans and domestic animals (4, 5). In addition, the investigation of viruses present in healthy wildlife can provide a baseline level of viruses that are present in healthy hosts, which might help in understanding the role of certain pathogens in case of an outbreak of infectious disease in wildlife.

In a number of studies, viral metagenomics has been performed in healthy wildlife using random PCR and next-generation sequencing (6–9). Especially carnivores and omnivores are of interest, since they can provide information about the presence of known and unknown viruses specific for the host, and in fecal material of these animals, viruses can be detected that are derived from their prey. For example, viral sequences of nodaviruses (which infect fish and insects) have been detected in feces of California sea lions, and insect viruses have been detected in insectivorous bats (9, 10).

The most widespread member of the order Carnivora is the red fox (Vulpes vulpes) (Global Invasive Species Database; http://www.issg.org/database/welcome/). These animals are distributed across the complete Northern Hemisphere and have a broad habitat, ranging from urban areas and farmlands to remote forests (11, 12). In addition, red foxes are known carriers of a number of pathogens that are harmful to humans, including Echinococcus multilocularis (13) and, in certain parts of the world, rabies virus (14, 15). However, there has been no thorough analysis of fecal material of these animals. In the present study, we evaluated the fecal viral flora of 13 red foxes using random PCR in combination with next-generation sequencing. Foxes were provided by hunters according to Dutch wildlife regulations in The Netherlands in 2012, and samples of foxes were collected in a monitoring program for parasites in foxes (13). All foxes were collected in southern Flevoland, The Netherlands (perirural/periurban area) (see Fig. S1 in the supplemental material).

Collected fox carcasses (see Table S1 in the supplemental material) were frozen at −80°C for 1 week, and after defrosting, fecal materials were collected from the rectum of the foxes and frozen in vials at −80°C until processing. Subsequently, molecular virus screening was performed as described previously (7, 16). In brief, a 10% (wt/vol) mixture of feces and phosphate-buffered saline (PBS) was prepared, and after centrifugation and filtration through a 0.45-μm-pore filter, samples were treated with Omnicleave endonuclease (Epicentre, Illumina). RNA and DNA were extracted using the Nucleospin RNA XS kit (Macherey-Nagel) and the High Pure viral nucleic acids kit (Roche). After first- and second-strand syntheses, random PCR amplification was performed and PCR products were purified using the MinElute PCR purification kit (Qiagen). Subsequently, unique sequence tags were added to the PCR products of each sample using the GS FLX Titanium rapid library MID adaptor kit, and a library of DNA fragments was prepared using a GS FLX Titanium library preparation kit (454 Life Sciences, Roche). The library of DNA fragments was sequenced on a 454 GS Junior instrument (454 Life Science, Roche). Obtained sequence reads of all individual fecal samples were analyzed by removing adaptor and primer sequences and assembled into contigs using CLC Genomics Workbench 5.5.1 (CLC Bio). Contigs and single reads were analyzed according to nucleotide (BLASTN) and translated nucleotide (BLASTX) BLAST. As cutoff values for significant virus hits for BLASTN and BLASTX, E values of 1.0 × 10⁻³ and 1.0 × 10⁻¹⁰, respectively, were used. Classification of sequences based on the taxonomic origin of the best-hit sequence was performed using MEGAN 4.70.4 (17).

Overview of detected viral sequences.

Approximately 29% of the detected sequences were related to bacteria, and 3% were related to eukaryota. In addition, in all samples sequences of the order Caudovirales were detected, and in all samples, sequences were detected that were related to viruses known to infect eukaryotes. Sequences related to known viruses of the Picobirnaviridae family were found in 11 foxes, while sequences related to viruses of the Astroviridae and Picornaviridae families were detected in 5 out of 13 samples. Sequences with homology to viruses of the families Circoviridae and Parvoviridae (genera Parvovirus and Bocavirus) were found in 3 out of 13 samples. In addition, sequences related to viruses of the genus Dependovirus (Parvoviridae) and the Hepeviridae family were found in 2 out of 13 samples. Some of the detected sequences had identities of more than 90% on the nucleotide level with known sequences of the genera Parvovirus genus (97% identity with Kilham rat virus) and Bocavirus (porcine bocavirus; 91%) and the families Picobirnaviridae (Microtus picobirnavirus; 92%) and Astroviridae (rat astrovirus; 93%). However, the majority of the sequences had relatively low homology to known viruses and were detected by BLASTX analysis (see Table S1 in the supplemental material). A subset of these novel viruses were further characterized.

Novel viruses belonging to the subfamily Parvovirinae.

Parvoviruses are small single-stranded DNA viruses. At present, the genera Parvovirus, Erythrovirus, Dependovirus, Amdovirus, and Bocavirus have been recognized by the International Committee on Taxonomy of Viruses (ICTV) in the family of Parvoviridae subfamily Parvovirinae (http://www.ictvonline.org/virusTaxonomy.asp). In addition, new genera (Bufavirus and Partetravirus) within this subfamily have been proposed recently (18, 19). Dependent on the species, clinical signs following infection with a parvovirus can be absent or severe, including death (20). In general, parvoviruses are species specific, but cross-species transmission of parvoviruses has been reported (for a review, see reference 21). In the analyzed fecal samples of foxes, sequences were detected with homology to viruses belonging to the genera Bocavirus, Dependovirus, and Parvovirus.

Sequences with the best hit with viruses of the genus Parvovirus of fox 6 were further analyzed, and the complete coding sequence of this novel virus, tentatively named fox parvovirus (GenBank accession no. KC692368), was obtained by Sanger sequencing based on overlapping sequences using specific primers and a degenerate primer at the 5′ end based on an alignment of multiple parvovirus sequences. In foxes 9 and 11, reads were detected that showed >95% identity to the sequence of fox parvovirus, indicating that this virus is present in feces of multiple foxes included in this study.

Two major open reading frames (ORFs) were identified, with one major ORF coding for the putative nonstructural (NS) protein (nucleotides [nt] 137 to 2191) and a second ORF coding for the structural protein VP2 (nt 2674 to 4425). In addition, five other ORFs of more than 100 nt were detected. One of these ORFs was situated between the two major ORFs with a predicted spliced transcript coding for the VP1 of a total of 716 amino acids (aa) (Fig. 1A). In the deduced amino acid sequence of the NS protein, the conserved GKRN domain was present, while in the deduced amino acid sequence of the VP1 gene, the phospholipase 2 motif and three other conserved domains (TPW, YNN, and QIW) were recognized. The genomic organization of this virus indicates that it is an exogenous parvovirus and not an endogenous parvovirus as has been demonstrated recently for the endogenous rat parvovirus (22).

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.

Nucleotide and deduced amino acid sequences of the NS1 and VP2 gene of the fox parvovirus were aligned with various other parvoviruses using ClustalW in MEGA5 (23). In addition, a neighbor-joining phylogenetic tree was generated using the alignment of the VP2 and the NS1 sequences (Fig. 1B; see Fig. S2 in the supplemental material). In contrast to parvoviruses recently detected in raccoons and other carnivore hosts (24, 25), the VP2 sequence of fox parvovirus was not similar to sequences of canine and feline parvoviruses, nor was it similar to the blue fox parvovirus and gray fox amdovirus. Since the pairwise sequence identities of the nucleotide sequence of the NS1 and VP2 genes of fox parvovirus with viruses of the genus Parvovirus were, respectively, <66% and <60% and <65% and <50% on the deduced amino acid sequence and even larger with viruses of other genera of the subfamily Parvovirinae, fox parvovirus might belong to either the genus Parvovirus or to a new genus (see Tables S2 and S3 in the supplemental material).

In addition to fox parvovirus, reads were detected that were related to bocaviruses detected in swine and pine martens in the fecal material of fox 6 (7, 26, 27). A fragment of 810 nt, covered by at least three reads for each nucleotide, corresponding to nt 3944 to 4753 of porcine bocavirus SX/China/2010 (27), was aligned with various partial VP1 genes of similar lengths of viruses of the genus Bocavirus. A neighbor-joining phylogenetic tree prepared on the basis of this alignment confirmed that this fragment of this novel virus, tentatively called fox bocavirus (GenBank accession no. KC878870), is most closely related to the pine marten bocavirus and bocavirus detected in swine (7, 26, 27) (see Fig. S3 in the supplemental material). In feces of fox 4, reads were detected related to the VP1 gene of porcine adeno-associated virus Po3 (28). A fragment of 1,680 nt, covered by at least three reads for each nucleotide, was aligned with fragments of the VP1 gene of various other adeno-associated viruses. Based on this alignment, a neighbor-joining phylogenetic tree was prepared, which showed that this fragment of this novel virus, tentatively called fox adeno-associated virus (GenBank accession no. KC878874), is most closely related to porcine adeno-associated virus Po3 and bat adeno-associated virus strain YNM (28, 29) (see Fig. S4 in the supplemental material).

Fox hepevirus.

Hepatitis E viruses (family Hepeviridae) are single-stranded positive-sense RNA viruses. Hepatitis E viruses are an important cause of hepatitis of humans in both developing and industrialized countries (30). Since hepatitis E viruses have also been detected in domestic animals, such as rabbits and swine, it is thought that these animals can play a role as a reservoir for hepatitis E viruses that circulate among humans (31). Members of the family of Hepeviridae that are more distantly related to human hepatitis E viruses have been detected in rats, birds, trout, and, recently, in bats and ferrets (32, 33).

In feces of foxes 3 and 5, sequences were detected with the highest homology to hepatitis E viruses detected in rats (Fig. 2A). Using specific primers, one fragment of the polyprotein gene of 362 nt (GenBank accession no. KC692370) and one fragment of the capsid protein of 295 nt (GenBank accession no. KC692369) were amplified and 454 sequences were confirmed by Sanger sequencing on PCR amplicons obtained using specific primers (Fig. 2A). Sequences of both PCR fragments were identical in feces of foxes 3 and 5. Both fragments were most closely related to hepatitis E viruses detected in rats, with identities on the deduced amino acid level of 73% (fragment polyprotein gene) and 85% (fragment capsid protein gene). The fragment of the polyprotein of the novel hepevirus, tentatively named fox hepevirus, was aligned with the same fragment of various other hepatitis E viruses using ClustalW, and a phylogenetic maximum likelihood tree was prepared based on this alignment (Fig. 2B). This phylogenetic tree showed that the fox hepevirus belongs most likely to the group of ferret and rodent hepeviruses. All samples of foxes from this study were screened for the presence of fox hepevirus sequences using the polyprotein-specific PCR, but no additional positive samples were identified. In addition, more sequences could not be obtained by Sanger sequencing, probably due to fragmentation of the RNA present in the samples. The detection of a novel hepevirus in red foxes indicates that also in fecal material of foxes, viruses of the family of Hepeviridae are present. It is unknown, however, whether this virus is circulating among foxes or is derived from their prey (e.g., rats) (34–36). Additional screening of samples from foxes is necessary to demonstrate if foxes are widespread carriers of this virus. In addition, serology could be used to measure infection of foxes with this virus.

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.

Fox astroviruses.

Astroviruses are small nonenveloped, unsegmented RNA viruses. Viruses of the family Astroviridae are divided into two genera, Aviastrovirus and Mamastrovirus, which is based on the host from which they were isolated (37). In a number of recent studies in wildlife, multiple new astroviruses have been described (for a review, see reference 38). Astroviruses are found often in fecal samples from humans and animals with and without gastroenteritis (8, 9, 37, 39–41). The genetic relatedness between viruses detected in various animal species and humans suggests that cross-species transmission has occurred and could occur in the future (38).

In fecal material from 5 out of 13 foxes in this study, sequences related to astroviruses were identified. The highest number of reads obtained by 454 sequencing related to astroviruses was detected in fecal material of fox 5. The partial genome of a novel astrovirus was identified and confirmed by Sanger sequencing on overlapping PCR amplicons using specific primers. The 3′ end was obtained using the First Choice RLM-RACE (rapid amplification of cDNA ends) kit (Ambion, TX) according to the instructions of the manufacturer. The complete coding sequences of the genes coding for the RdRp and the capsid protein and the partial coding sequence of the nonstructural gene of this novel virus, tentatively called fox astrovirus F5 (GenBank accession no. KC692365), were obtained. Three major ORFs were identified (ORF1a, ORF1b, and ORF2) with a similar genome organization to other astroviruses, including a ribosomal frameshift sequence (37, 42) (Fig. 3A). Alignment of the sequence of ORF2 with those of various other astroviruses, subsequent phylogenetic analysis using the neighbor-joining method, and analysis of the pairwise identities on the nucleotide and deduced amino acid level showed that fox astrovirus F5 is most closely related to the human astrovirus HMO and astroviruses detected in mink, swine, and California sea lions (9, 43–45) (Fig. 3B; see Table S4 in the supplemental material).

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.

Astrovirus-related reads detected in fecal material of other foxes were compared with the sequence of fox astrovirus F5, and in foxes 2, 7, and 8, reads were present with >95% homology on the nucleotide level with the fox astrovirus F5. In fecal material of fox 4, a contig of 332 nt was detected that had the highest homology to astroviruses other than fox astrovirus F5. The sequence of this second novel astrovirus, tentatively called fox astrovirus F4 (GenBank accession no. KC878875), corresponded to nt 2608 to 2939 of the complete genome of porcine astrovirus 4 strain 35/USA (ORF1b) (46). Alignment of the sequence of this novel virus with the same fragment of various other astroviruses and subsequent phylogenetic analysis using the neighbor-joining method shows that this virus was most closely related to astroviruses detected in mice, pigs, and wild boars (47–49) (see Fig. S5 in the supplemental material).

Fox picobirnavirus.

Picobirnaviruses are small, nonenveloped, bisegmented double-stranded RNA viruses. We detected sequences with homology to picobirnaviruses in 11 out of 13 samples of this study, which is a higher prevalence than that identified in other host species previously (7, 9, 16, 46). The complete coding sequence of the RNA-dependent RNA polymerase (RdRp) gene of segment 2 of a picobirnavirus from fox 5, tentatively called fox picobirnavirus F5-1, was obtained, which consists of 1,620 nt (GenBank accession no. KC692366). In addition, a near complete segment 1 of this novel picobirnavirus was obtained (GenBank accession no. KC692367) in which two major ORFs were identified coding for a hypothetical protein and the (partial) capsid precursor protein. The genome of this novel picobirnavirus was partially confirmed by Sanger sequencing using specific primers based on 454 sequencing reads.

Alignment and phylogenetic analysis of the sequences obtained by Sanger sequencing using the neighbor-joining method with the p-distance method in the MEGA5 program with other RdRp gene sequences of picobirnaviruses of similar length showed that obtained sequences of the RdRp gene were most closely related to the human picobirnavirus VS-10 and human picobirnavirus 1 (see Fig. S6A in the supplemental material) (17, 50), which was confirmed by calculation of the pairwise identities on the nucleotide and deduced amino acid levels (see Table S5 in the supplemental material). Sequences of the deduced hypothetical protein and capsid precursor of fox picobirnavirus F5-1 were more divergent (see Fig S6B and C). Of interest, in the deduced amino acid sequence of ORF1, multiple repeats of the ExxRxNxxxE motif were present, which has been described previously in known sequences of this ORF (51).

Picobirnavirus reads were detected in some of the other foxes with higher sequence homology to picobirnaviruses other than fox picobirnavirus F5-1. In samples from foxes 5, 7, and 9, three or more reads were detected that had the best hit with a fragment of the RdRp gene (corresponding to nt 1071 to 1465 of human picobirnavirus VS-10). Alignment of the consensus sequence of these reads using the neighbor-joining method with p-distance showed that in foxes 5 and 9, picobirnaviruses (fox picobirnavirus F5-2 and F9; GenBank accession no. KC878872 and KC878871, respectively) were detected that are divergent from fox picobirnavirus F5-1, while the picobirnavirus detected in fox 7, fox picobirnavirus F7 (GenBank accession no. KC878873), had 99% homology to fox picobirnavirus F5-1 on the nucleotide level (see Fig S6D in the supplemental material).

In the present study, we have identified a number of previously unknown viruses in feces of red foxes, including a new parvovirus, bocavirus, adeno-associated virus, hepevirus, astroviruses, and picobirnaviruses. It is unknown whether these viruses can cause disease in foxes, and no conclusions can be drawn about the prevalence of these viruses in the total population due to the small size of this study. Further research is necessary to elucidate the biological relevance of these viruses and to understand if these viruses can transmit to and cause disease in other animal species or humans. In addition to the viruses described in the present study, few sequences were detected related to other viruses, like Circoviridae and Picornaviridae, which are often detected in fecal samples (8, 46, 52). Most of the sequences detected in the present study were related to viruses detected in fecal samples or nasopharyngeal swabs previously (see Table S1 in the supplemental material) (8, 52–55). Future research will focus on the characterization of these viruses.

Known zoonotic viruses were not identified in fecal material of foxes. However, there is serological evidence that rat hepatitis E viruses are able to transmit to humans based on a study among forestry workers (56). As rat hepatitis E viruses are close relatives of the fox hepevirus, transmission of fox hepevirus to humans may occur as well.

In addition, various viruses were identified that are close relatives to human viruses based on phylogenetic analysis, suggesting that these viruses may have been transmitted from foxes to humans or vice versa in the past. The newly discovered viruses of the present study are examples of the diversity of viruses that are present in wildlife. Since foxes often live close to humans, the opportunities exist for cross-species transmission to humans or their pets. Since multiple recent outbreaks of viral disease among humans are caused by viruses that originate from (wild) animals, expansion of the current knowledge of viruses that circulate in these animals is needed. A timely identification of reservoir animal hosts in case of novel outbreaks of disease in humans is crucial in mitigating infectious threats (5).

Nucleotide sequence accession number.

All reads were deposited at the Sequence Read Archive (SRA) under archive no. ERP002446.