Multiple Lineages of Hantaviruses Harbored by the Iberian Mole (Talpa occidentalis) in Spain

The recent detection of both Nova virus (NVAV) and Bruges virus (BRGV) in European moles (Talpa europaea) in Belgium and Germany prompted a search for related hantaviruses in the Iberian mole (Talpa occidentalis). RNAlater®-preserved lung tissue from 106 Iberian moles, collected during January 2011 to June 2014 in Asturias, Spain, were analyzed for hantavirus RNA by nested/hemi-nested RT-PCR. Pairwise alignment and comparison of partial L-segment sequences, detected in 11 Iberian moles from four parishes, indicated the circulation of genetically distinct hantaviruses. Phylogenetic analyses, using maximum-likelihood and Bayesian methods, demonstrated three distinct hantaviruses in Iberian moles: NVAV, BRGV, and a new hantavirus, designated Asturias virus (ASTV). Of the cDNA from seven infected moles processed for next generation sequencing using Illumina HiSeq1500, one produced viable contigs, spanning the S, M and L segments of ASTV. The original view that each hantavirus species is harbored by a single small-mammal host species is now known to be invalid. Host-switching or cross-species transmission events, as well as reassortment, have shaped the complex evolutionary history and phylogeography of hantaviruses such that some hantavirus species are hosted by multiple reservoir species, and conversely, some host species harbor more than one hantavirus species.


Introduction
Hantavirology dates to the landmark discovery of Hantaan virus as the prototype virus of hemorrhagic fever with renal syndrome (HFRS) in the striped field mouse (Apodemus agrarius coreae) [1]. This was followed by the detection of other Hantaan-like viruses in the bank vole (Myodes glareolus) [2] and in the brown rat (Rattus norvegicus) and black rat (Rattus rattus) [3]. In the Americas, hantaviruses burst into the public consciousness when an outbreak of a rapidly progressive, frequently fatal cardiorespiratory disease, now known as hantavirus cardiopulmonary syndrome (HCPS), occurred in the southwestern United States [4,5]. Cases of HCPS, with case-fatality rates exceeding 20%, have since been diagnosed throughout North and South America [6,7].
Hantaviruses possess a single-stranded, negative-sense RNA genome consisting of three segments designated large (L), medium (M) and small (S), which encode a nucleocapsid (N) protein, two envelope glycoproteins (Gn and Gc), and an RNA-dependent 2.3. Next-Generation Sequencing cDNA samples were processed and run on the HiSeq 1500 System (Illumina, Inc., San Diego, CA, USA) at the University of Texas at Galveston Sequencing Core Facility, using the methods described previously [28,29]. Seven hantavirus-positive samples were mulitplexed on a single lane. Reads were sorted and barcodes removed, quality control was applied and the samples were assembled into contigs. Contigs were subjected to basic local alignment search tool (BLAST) analysis to determine their origins.

Genetic and Phylogenetic Analyses
Pairwise alignment and comparison of nucleotide sequences were performed using Clustal W [30]. Unrooted phylogenetic trees were generated by maximum-likelihood and Bayesian methods, implemented in RAxML Blackbox webserver [31] and MrBayes 3.1 [32], under the best-fit GTR + I + Г model of evolution selected by hierarchical likelihoodratio test in MrModeltest v2.3 [33] and jModelTest version 0.1 [34]. Two replicate Bayesian Metropolis-Hastings Markov Chain Monte Carlo runs, each comprising six chains of 10 million generations sampled every 100 generations with a burn-in of 25,000 (25%), resulted in 150,000 trees overall. Each genomic segment (S, M and L) was treated separately in phylogenetic analyses. The posterior node probabilities were based on 2 million generations and estimated sample sizes over 100 (implemented in MrBayes).

Host Identification and Phylogeny
To molecularly verify the species of the hantavirus-infected mole hosts, the 1140nucleotide cytochrome b gene of mitochondrial DNA (mtDNA) was amplified by PCR using universal primers (forward, 5 -CGAAGCTTGATATGAAAAACCAT-CGTTG-3 ; and reverse, 5 -CTGGTTTACAAGACCAGAGTAAT-3 ) [35]. PCR was performed in 50-µL reaction mixtures containing 200 mM dNTP and 1.25 U of LA Taq polymerase (Takara). Cycling conditions consisted of an initial denaturation at 95 • C for 4 min, followed by 40 cycles with denaturation at 94 • C for 1 min, annealing at 57 • C for 1 min, and elongation at 72 • C for 1 min in a GeneAmp PCR9700 thermal cycler. Phylogenetic analysis was performed using the maximum-likelihood method [36], and evolutionary analyses were conducted in MEGA 7 [37].

Hantavirus RNA Detection
Hantavirus RNA was detected by nested RT-PCR, using L-segment oligonucleotide primers, in 11 of 56 Iberian moles captured in Coceña, Fresnadiellu, Oles and Priesca, four parishes in the principality of Asturias, in northwestern Spain ( Figure 1 and Table 1). Additionally, 2 of the 15 crowned shrews, one each in VEV and Fresnu, were positive (Table 1).

Sequence Analysis
Based on the analysis of the partial L-segment sequences, BRGV, NVAV and ASTV were identified in six, four and one Iberian moles, respectively ( Table 2). Despite multiple attempts, we were unable to obtain S-and M-segment sequences of NVAV and BRGV from Iberian moles. Interestingly. two infected Iberian moles, captured in the same gallery on two consecutive days (TO11.06.14.02 and TO11.06.15.01) in Coceña, harbored different hantaviruses: NVAV 3873 and BRGV 3879. significant sequence dissimilarity with representative rodent-, shrew-, mole-and bat-borne hantaviruses, ranging from 30.2-80.5% and 40.2-82.1% at the nucleotide (nt) and amino acid (aa) levels, respectively ( Table 3). The novel hantavirus, which was named ASTV 3877 after the location where the Iberian mole was trapped, showed low sequence similarity with two recently described mole-borne hantaviruses: namely, ACDV Academ-Ta450 (S, 54.4% nt/52.2% aa; M, 60.2% nt/52.1% aa; L, 69.1% nt/71.3% aa) and LDRV MNHN-ZM-2017-2257 (S, 50.1% nt/50.8% aa) ( Table 3 and Supplemental Table S1). Table 3. Nucleotide and amino acid sequence similarities (%) between ASTV 3877 and other representative mole-, shrew-, bat-and rodent-borne hantaviruses.  The full-length 1979-nucleotide S-segment of ASTV 3877 encoded an N protein of 429 amino acids in length. As was in other mole-borne hantaviruses, an additional open reading frame for a nonstructural NSs protein was not present. Our analysis of ASTV also included 1971 and 1369 nt of the M and L segments, respectively, or approximately 50% and 20% of the M and L segments of ASTV.

S Segment
Of the seven hantavirus samples analyzed by next-generation sequencing, only one (ASTV 3877) had sufficient viral reads to produce viable contigs: 22 contigs from ASTV 3877 exhibited sequence similarity to known hantaviruses. These contigs were translated and aligned to a reference hantavirus (NVAV) to determine which segment they were from and where on the segment they corresponded to. Sequences of ASTV 3877 from next-generation sequences corresponded to that derived from Sanger sequencing.
Compared with other hantaviruses, sequence similarity of a 346-nt region of the L segment amplified from two crowned shrews (SWSV 4050 and SWSV 4056) was 80-85% and 96% at the nucleotide and amino acid levels, respectively, with SWSV.

Phylogenetic Analysis
Phylogenetic analyses based on the full-length S-segment and partial M-and Lsegment sequences, using maximum-likelihood and Bayesian methods, indicated that ASTV 3877 represented a distinct hantavirus ( Figure 2).
Analysis of the partial L-segment sequences showed the simultaneous circulation of three distinct hantavirus lineages, which did not segregate according to geography. That is, Iberian moles collected at the same sites showed NVAV mobatvirus (3873, 3931, 3943, 3945) and BRGV orthohantavirus (3879, 3884, 3890, 3914, 3930, 3947) ( Figure 2). Posterior node probabilities between BRGV strains from Belgium and Spain and between NVAV strains from Poland and Spain were 1.00 and 1.00, respectively ( Figure 2). In addition, phylogenetic analysis of SWSV 4050 and SWSV 4056 from crowned shrews shared a common ancestry with prototype SWSV mp70 and other SWSV strains harbored by soricine shrews (Supplemental Figure S1).
Phylogenetic analysis of the cytochrome b mtDNA sequences from the 11 hantavirus RNA-positive moles confirmed the host identity as Talpa occidentalis (GenBank accession numbers OQ915046-OQ915056) (Figure 3). Analysis of the partial L-segment sequences showed the simultaneous circulation of three distinct hantavirus lineages, which did not segregate according to geography. That is, Iberian moles collected at the same sites showed NVAV mobatvirus (3873, 3931, 3943, 3945) and BRGV orthohantavirus (3879, 3884, 3890, 3914, 3930, 3947) (Figure 2). Posterior node probabilities between BRGV strains from Belgium and Spain and between NVAV strains from Poland and Spain were 1.00 and 1.00, respectively (Figure 2). In addition, phylogenetic analysis of SWSV 4050 and SWSV 4056 from crowned shrews shared a common ancestry with prototype SWSV mp70 and other SWSV strains harbored by soricine shrews (Supplemental Figure S1).
Phylogenetic analysis of the cytochrome b mtDNA sequences from the 11 hantavirus RNA-positive moles confirmed the host identity as Talpa occidentalis (GenBank accession numbers OQ915046-OQ915056) (Figure 3). Phylogenetic tree based on full-length sequences of the cytochrome b mtDNA using the maximum-likelihood method. The percentage of trees in which the associated taxa clustered together is shown at the respective node. Initial trees for the heuristic search were obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Talpa occidentalis from this study are shown in red lettering.

Discussion
A genetically distinct hantavirus, designated ASTV, was detected in an Iberian mole captured in northwestern Spain. Iberian moles from Asturias were also shown to harbor BRGV and NVAV. Previously, European moles, the reservoir host of NVAV, was found to Figure 3. Phylogenetic tree based on full-length sequences of the cytochrome b mtDNA using the maximum-likelihood method. The percentage of trees in which the associated taxa clustered together is shown at the respective node. Initial trees for the heuristic search were obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Talpa occidentalis from this study are shown in red lettering.

Discussion
A genetically distinct hantavirus, designated ASTV, was detected in an Iberian mole captured in northwestern Spain. Iberian moles from Asturias were also shown to harbor BRGV and NVAV. Previously, European moles, the reservoir host of NVAV, was found to harbor BRGV in Belgium, Germany and the United Kingdom [23]. Thus, the co-circulation of ASTV, BRGV and NVAV in Iberian moles was not totally unexpected. With the recent discovery of ACDV [24] and LDRV [25] in the Siberian mole and Aquitania mole, respectively, the number of mole-borne hantaviruses is now nine: four in Europe (ASTV, BRGV, LDRV, NVAV), three in Asia (ACDV, ASAV, DHCV), and two in North America (OXBV, RKPV). Presumably, other hantaviruses are hosted by other mole species or the same mole species. In this regard, the eastern mole in the United States harbors a hantavirus that is distinct from RKPV (H.J. Kang and R. Yanagihara, unpublished observations).
The European mole, which harbors NVAV and BRGV, has an exceptionally vast geographic range across Europe and western Asia, extending northward to the United Kingdom and southern Scandinavia (Sweden and Finland), southward to northern Greece, and eastward to Poland, Ukraine and western Siberia. By contrast, the Iberian mole has a very limited geographic distribution, and is confined to only Spain and Portugal. Although the European mole is the sole mole species throughout most of its range, there is geographic overlap with the Aquitania mole. Although virus isolation is the gold standard to definitively demonstrate that virus gene amplification findings do not merely represent "spillover" events, the absence of the European mole in Asturias, Spain, would argue strongly against "spillover" and instead would support true infection and co-circulation of NVAV, BRGV and ASTV in the Iberian mole. Curiously, NVAV and BRGV were found more commonly than ASTV among Iberian moles in this study. Only 1 of the 11 hantavirusinfected Iberian moles had ASTV. Nevertheless, a limitation of our study was the failure to include European moles captured west of Asturias and/or elsewhere in northern Spain.
Interestingly, while analyzing cytochrome b mtDNA sequences of 85 European moles from 46 localities across nearly all of its geographic range, Feuda and colleagues found three differentiated mtDNA lineages, of which two were restricted to Spain and Italy and a third that was widespread across Europe [55]. Phylogenetic inferences and molecular clock analysis suggested that the European moles from Spain represented a highly divergent and ancient lineage.
Recently, in a reanalysis of cytochrome b mtDNA sequences from European moles from west and south of the Loire River in France and from northern Spain, a new mole species, tentatively named the Aquitanian mole (Talpa aquitania), was reported [56]. The Aquitanian mole is the reservoir host of a newly described hantavirus (LDRV) [25]. Unfortunately, our study did not include Aquitanian moles, as evidenced by mtDNA sequence analysis, which confirmed that all ASTV-, NVAV-and BRGV-infected moles were, in fact, Iberian moles. Nevertheless, future studies are warranted to analyze tissues from European moles and Aquitanian moles in northern Spain for LDRV and other hantaviruses.
A recently published proposal would require full coding sequences for classification of hantaviruses [57]. If approved by the International Committee on Taxonomy of Viruses, this will present obvious challenges for multiple well-known "classical" rodent-borne orthohantaviruses that have yet to be fully sequenced, as well as for many of the newfound hantaviruses detected in shrews, moles and bats. To date, non-rodent-borne hantavirus isolates exist only for Thottapalayam thottimvirus, Imjin thottimvirus and NVAV. Of the more than 30 recently described shrew-, mole-and bat-borne hantaviruses, none have been isolated in cell culture and most exist only as partial sequences.

Conclusions
The view that each hantavirus species is harbored by a single small-mammal host species now appears overly simplistic and invalid [58,59]. Host sharing, host-switching and spill-over events have occurred frequently in the evolutionary history of hantaviruses such that certain hantavirus species are carried by multiple sympatric small mammal hosts, and conversely, certain reservoir species may host more than one hantavirus species. Like the European mole, which hosts NVAV and BRGV, Iberian moles appear to harbor more than one hantavirus species. The definitive identification of hantaviruses in the Iberian mole must await whole-genome sequence analysis and virus isolation. Concurrently, sympatric soricine and crocidurine shrews need to be investigated to determine if they may represent the sources of hantavirus diversity in Iberian moles. Overall, this report supports the growing understanding of hantavirus and host ecology as an evolutionarily complex history that merits continued research into the patterns and mechanisms involved.

Institutional Review Board Statement:
The study was conducted in compliance with existing laws. Permission was not required to study unintentionally trapped moles, shrews and rodents. The study did not involve endangered or protected species.

Informed Consent Statement: Not applicable.
Data Availability Statement: GenBank accession numbers for phylogenetic analyses are available in Table 2 and in the legend of Figure 2. Other presented data are available on request from the corresponding author.