Detection at high prevalence of newlavirus (protoparvovirus) in the carcasses of red foxes

Highlights • Several novel parvoviruses have been identified in carnivores by deep sequencing.• Newlaviruses (NLV) are a candidate protoparvovirus species found in foxes in 2021.• Carcasses of red foxes were screened for NLV revealing a high (71%) prevalence.• Marked genetic diversity was found in the VP1/VP2 gene among NLV strains.• The evolutionary dynamics of NLV in fox and in other carnivores should be assessed.


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Parvoviruses, family Parvoviridae, comprise small, non-enveloped icosahedral viruses, approximately 20-30 nm in diameter, with a single-stranded linear DNA genome, 4-6 kb in length. Parvovirus genome possesses at both ends palindromic sequences that form hairpin structures, used for genomic replication, and two adjacent open reading frames (ORFs) which encode non-structural (NS) and structural (VP) proteins, respectively (Cotmore et al., 2019).
In 2021 a new candidate protoparvovirus species, provisionally designated as Newlavirus (NLV), was identified with a high prevalence (up to 44%) in foxes from Canada. The virus displayed elevated genetic diversity at the VP1 gene level (70.5-87.8%) and was mainly identified in the feces and spleen. NLV was not detected in other animal species, and it was assumed that foxes could be the natural reservoir of the virus (Canuti et al., 2021).
The interest in wildlife research has been increasing in recent years as the result of the awareness that the health of human, animals and the environment are tightly connected, as postulated in the One Health envision. The study of wildlife viral diseases mostly relies on opportunistic sampling of carcasses and this constitutes an important resource for passive disease surveillance although potentially introducing biases into epidemiological studies (Nusser et al., 2008).
In the present study we performed a molecular screening for NLV on tissues collected from necropsied fox. A total of 100 retropharyngeal lymph node samples were collected from foxes (Vulpes vulpes), found dead by hunters, to perform routine necropsy procedures for diagnostic purposes. Sampling was carried out in different regions of Southern Italy (Calabria and Campania) from 2013 to 2016 (Ndiana et al., 2021). Samples were homogenized by a Tissue Lyser (Qiagen GmbH, Hilden, Germany) in 2 mL Eppendorf safe-lock tubes containing 1 mL of Dulbecco Minimum Essential Medium and a 4.8 mm stainless-steel bead (30 Hz for 5 min). Homogenized samples were subsequently centrifuged at 13,000 rpm for 3 min. DNA extraction was performed using QIAampDNA Blood & Tissue kit (Qiagen, Hilden, Germania). DNA extracts were subjected to a real-time PCR assay (qPCR) for the detection and quantitation of NLV DNA (Table 1). To confirm the presence of NLV, samples tested positive by qPCR were re-tested using a nested-PCR protocol for the amplification of a partial portion (328 bp) of the VP1 gene (Canuti et al., 2021) (Table 1). NLV copy numbers were calculated on the basis of standard curves generated by 10-fold dilutions of two pEX-A128 standard plasmids containing 500 bp of ORF2 region of NLV strain FX74 (GenBank accession no. MZ813280). The inserted gene was synthetized and cloned by Eurofins Genomics (Ebersberg, Germany). Log10 dilutions of standard DNA were evaluated simultaneously in order to obtain a standard curve for absolute quantification. All standard dilutions and unknown samples were tested in triplicate.
In order to acquire the complete viral genome sequences of NLV strains, two multiplex PCR protocols amplifying sixteen PCR-tiling amplicons with a 400-475 bp size were designed, following an ARTIClike strategy (Quick et al., 2017). Primer pairs were designed on the consensus sequence of NLV genomes recovered from the NCBI database (Table 1). The PCR assays were performed with TaKaRa La Taq polymerase (Takara Bio Europe S.A.S. Saint-Germain-en-Laye, France). All PCR products were pooled in equimolar ratios, quantified by Qubit dsDNA HS assay (Thermo Fisher Scientific, Waltham, MA) and used for library preparation and adapter-ligation with Genomic DNA by Ligation kit SQK-LSK110 (Oxford Nanopore Technologies, ONT, Oxford UK) following manufacturer's guidelines. Libraries were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter™) and sequenced using flowcell flongle FLO-FLG001, R9.4.1 version adapted in a MinION Mk1C (ONT, Oxford UK) platform for 24 h. FastQ MinION files were subjected to quality control, trimming and reference assembly by Minimap2 and open reading frame (ORF) predictions, and annotations were performed in Geneious Prime software v. 2021.2.2 (Biomatters Ltd., Auckland, New Zealand). Sequence alignment was performed by MAFFT (Katoh et al., 2002). The phylogenetic analyses were performed using the maximum likelihood method implemented in MEGAX version 10.0.5 software (Kumar et al., 2018). Similarity plots were obtained using Simplot 3.5 (Lole et al., 1999).
NLV DNA was detected by qPCR with a 71% (71/100) prevalence. Viral load of NLV ranged from 2.3 × 10 1 to 4.0 × 10 6 DNA copies / ml (mean = 2.2 × 10 5 DNA copies / ml, median = 4.9 × 10 3 DNA copies / ml). A total of 21 positive samples, selected on the basis of viral DNA copies (≥ 10 3 DNA copies/ml), were amplified by nested PCR and sequenced. Partial VP1 sequences (328 bp) obtained exhibited a nt identity ranging from 95.3 to 100.0 to cognate NLV strains previously identified (Canuti et al., 2021). The nearly complete genome sequences of 7 NLV strains (GenBank accession nos. ON959793-ON959799) and the partial VP1/VP2 gene sequences (918 bp) of other 14 NLV strains (ON959800-ON959813) were obtained. The genome size of the 7 Italian NLV strains identified in this study ranged between 4709 and 4772 nt and displayed an overall nucleotide (nt) identity ranging from 88.5% to 99.7% to other NLV strains retrieved from the GenBank database. The genome features of the identified NLVs comprised two major open reading frames (ORFs): ORF1 (1854 nt) encoding for NS1 protein (617 aa), and the ORF2 (2262-2268 nt), encoding for the VP1 (753-755 aa) and VP2 (588-590 aa) proteins, respectively ( Table 2). The nucleotide  nt, nucleotides; aa, amminoacids * partial genome sequence ** partial VP1 sequence; # sequence with the highest identity on interrogation of GenBank database with BLAST. alignment of the complete NS1 and the partial VP1/VP2 (663 bp) sequences of NLV strains identified in this report and cognate reference strains recovered in the GenBank database displayed an overall nucleotide (nt) identity ranging from 93.8% to 100.0% and 70.2% to 100.0%, respectively. Upon phylogenetic analysis, in the NS1 region the 7 Italian NLV strains clearly diverged from the Canadian viruses (Fig. 1A), whilst in the partial VP1/VP2 gene (663 bp) the 21 Italian NLV strains were intermingled with the Canadian NLVs in three distinct genetic clusters (Fig. 1B), a pattern which may imply recombination, a phenomenon not uncommon in parvoviruses (Shackelton et al., 2007). By SimPlot analysis, a highly conserved region was mapped in the middle portion of the NS1 throughout the alternative splicing site of the N terminal domain of the VP1 region (from nt 1100 to nt 1900), using as reference the NLV strain FX25 (MZ813278) (Fig. 2). Another highly conserved region spanned the VP1 unique region throughout the initial part of the VP2 (from nt 2500 to 3120), where the qPCR was targeted. Genome variation was higher in the spliced region and, in particular, in the VP2 coding region.
Overall, the prevalence of NLV in foxes in Italy was higher than previously observed in Canada. In particular, considering only the lymph nodes collected from the head, in our study we observed a nearly 15-fold difference (71.0% versus 5.4%) (Canuti et al., 2021). This high detection rate of NLV in foxes could be accounted for by a pattern of persistent infection or, eventually, by reactivation of the virus in stressed/weakened animals, rather than being directly related to the death of the animals.
Finally, the high genetic diversity observed in the VP2 gene of NLV contrasts with the high genetic conservation of other protoparvoviruses, such as CPPV-1, where a few punctate mutations result in major biological changes (Ndiana et al., 2021;Tuteja et al., 2022), suggesting a long-term adaptation of NLV in foxes or intricated dynamics of circulation of virus variants among multiple susceptible hosts. Epidemiological studies in wild animals might help understand more in depth the pathogenic role, if any, of NLVs, and the evolutionary dynamics of these viruses in foxes and, eventually, in other animal hosts.

Ethical statement
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as this study was conducted on carcasses of animals found dead and submitted to routine necropsy procedures for diagnostic purposes. All experiments were performed in accordance with relevant guidelines and regulations.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability
Data will be made available on request.