The gray fox studied here was identified during the summer of 2009 in Sonoma County, California. It had severe gait abnormalities, lymphadenopathy, and acute muscle inflammation, and was euthanized at a wildlife rehabilitation center. Using the generic viral particle enrichment method previously described for tissues (13
), we generated ≈14,000 sequence reads from spleen and lung samples. We found 136 sequence reads in spleen tissue that were related to AMDV by using BLASTx (E score <10–5
); these could be assembled into 24 contigs covering ≈60% of the viral genome. By connecting gaps between sequenced viral fragments and amplifying the genome extremities by using PCR primers based on AMDV sequences, the nearly complete genome of the new amdovirus (GenBank accession no. JN202450) was acquired. We temporarily named it gray fox amdovirus (GFADV).
The partial GFADV genome was 4,441 nt in length with a low guanine–cytosine content of 37%. Similar to that of AMDV, the GFADV genome contained 2 major ORFs. The left ORF (LORF) contains the bulk of the sequences for the putative NS1, and the right ORF (RORF) codes for VP2 (). Two small middle ORFs (67 and 75 aa long) with putative alternative start codons were detected in the 448-bp region between the ORFs. The theoretical proteins showed 55% and 59% aa identity with the 2 similarly located middle ORFs reported in AMDV. The partial 5′ untranslated region (UTR) was 109 nt and the partial 3′ UTR was 191 nt. Potential RNA splicing signals in AMDV were present on the GFADV genome (Technical Appendix Figure 1
). The predicted spliced transcripts encode hypothetical NS1, NS2, and NS3 of 635 aa, 115 aa, and 80 aa, respectively, and a capsid protein VP1 of 674 aa. The putative VP2 was predicted to arise from the intact transcript of RORF encoding a 630-aa protein.
Figure 1 Open reading frames (ORFs) in gray fox amdovirus genome. Three possible reading frames of the plus-strand sequence with the stop codons indicated by red lines and ATG codons by green flags. Two major ORFs, left (LORF) and right (RORF), are indicated by (more ...)
Sequence analyses confirmed that GFADV was a divergent amdovirus with 76% nt identity with the genome of AMDV. Conserved protein domains typical of parvoviruses were identified in GFADV. In the LORF, the GKRN domain was found (Technical Appendix Figure 2
), which may act as the nuclear transport signal of NS1 protein. In the RORF, 3 conserved domains (TPW, YNN, and PIW) of unknown biologic significance were detected (6,14
) (Technical Appendix Figure 3
). The phosholipase 2 motif in the N terminal VP1 region, generally conserved in parvoviruses, was not found in either GFADV not AMDV (Technical Appendix Figure 3
), which suggests that this parvovirus genus uses a different mechanism to escape the endosome during infection (15
Comparison of NS1 regions showed GFADV shared ≈74% nt and 67% aa similarities with AMDV strains, whereas different strains of AMDV shared >87% nt and 82% aa similarities. Alignments of the VP2 region showed GFADV shared ≈78% nt and 80% aa similarities with AMDV strains, whereas strains of AMDV had >92% nt and 91% aa similarities (Technical Appendix Figure 4
, Technical Appendix Figure 5
). To determine the relationship between GFADV and AMDV strains, phylogenetic analyses of the NS1 and VP2 proteins were performed, which showed that in both genome regions GFADV was more closely related to AMDV strains than to those of minute virus of mice or other parvoviruses analyzed (data not shown), but was distinct from the 3 AMDV groups (). Pending review by the International Committee on Taxonomy of Viruses, GFADV thus appears to be the second reported parvovirus species in the genus Amdovirus
Figure 2 Phylogenetic analyses of gray fox amdovirus (GFADV) (red dots) and Aleutian mink disease virus (AMDV) based on the complete amino acid sequence of nonstructural protein 1 region (A) and viral protein 1 region (B). The neighbor-joining method was used (more ...)
GFADV sequences were also detected in the lung and heart tissues of the same animal by using a GFADV-specific nested PCR targeting a ≈400-bp segment of the VP2 gene, as well as in the heart tissue of another gray fox, which had the same signs, collected in Sonoma County in summer 2009. Further PCR screening of 19 tissue samples, including spleen, lung, liver, lymph node, and muscle from 9 other gray foxes with similar gait abnormalities and chronic muscle lesions, collected in 2008 (n = 2) and 2010 (n = 7) were negative by the same GFADV PCR.