Molecular Survey on Kobuviruses in Domestic and Wild Ungulates From Northwestern Italian Alps

Since the first identification in 1989 in humans, kobuviruses (KoVs) have been identified from a wide range of animal species including carnivores, rodents, birds, ungulates, rabbits, and bats. Several studies have described the identification of genetically related KoVs in the fecal virome of domestic and wild animals suggesting a mutual exchange of viruses. By screening a total of 231 fecal samples from wild and domestic ungulates, KoVs RNA was detected in wild boars (3.2%; 2/63), chamois (4.6%; 2/43), and goats (2.6%; 2/77). On phylogenetic analysis of the partial RdRp sequence, the wild boar strains clustered within the species Aichivirus C whilst the strains identified in domestic and wild ruminants grouped into the species Aichivirus B. The complete VP1 gene was obtained for chamois and goat KoVs. Interestingly, upon phylogenetic analysis the strains grouped together with a KoV of ovine origin within a distinct genetic type (B3) of the species Aichivirus B.


INTRODUCTION
Kobuviruses (KoVs) are small (∼30-32 nm), icosahedral, non-enveloped viruses with a single stranded positive sense RNA genome of 8. 2-8.4 kb in length, classified in the genus Kobuvirus within the family Picornaviridae (1). The viral RNA, polyadenylated at the 3 ′ -end and covalently linked to a virus-encoded protein (VPg) at its 5 ′ -end, consists of a 5 ′ untranslated region (UTR) of 646-717 nucleotides (nt), an open reading frame (ORF) of 7,308-7,467 nt and a 3 ′ UTR of 241-244 nt. The unique ORF encodes a single large polyprotein that is post-translationally cleaved into three distinct functional P regions (P1-P3) with P1 encoding the viral capsid proteins (VP0, VP3, and VP1) and P2 and P3 encoding proteins involved in protease processing and genome replication (2).

Study Area and Sampling
Sampling covered the Region of Valle d'Aosta, in Northwestern Italy, with an overall geographic area of 3,261 km 2 . It is an alpine area characterized by the concomitant presence of domestic ruminant semi-rural farms and abundant ungulates wildlife. In this setting, the population of wild ungulates, composed primarily by wild boar (Sus scrofa), red deer (Cervus elaphus), and chamois (Rupicapra rupicapra), may occasionally enter in contact with domestic small ruminants through the use of pastures during seasonal transhumance or in the surrounding of backyard farms. Between September 2017 and December 2019, using a convenience sampling strategy, a total of 231 fecal samples was obtained from domestic and wild ungulates. Briefly, 95 fecal specimens were collected from 77 goats and 18 sheep, respectively, in 14 caprine and 3 ovine small farms from 16 municipalities in the Valle d'Aosta region ( Figure 1A), consisting of 4-10 animals. All animals were female and clinically healthy at the time of sampling. On the basis of the age, 25 goats and 6 sheep were from the defined age group <3 years, 22 goats and 3 sheep from 3 to 4 years group, 16 goats and 9 sheep were from the age group >4 years. Age was unknown for 15 goats. Among wild animals, a total of 136 stool specimens was collected from 63 wild boars, 30 red deer, and 43 chamois sampled during the regular hunting season in Valle d'Aosta Region and submitted to the National Reference Center for Wild Animal Diseases (Italy). Age data were not available for many of the wild species investigated.

Molecular Analyses
Total RNA was extracted from 200 µl of 10% (wt/vol) fecal suspension using TRIzol LS (Invitrogen, Ltd, Paisley, UK) procedure. KoVs RNA was detected by RT-PCR using broadly reactive primer pair, UNIV-kobu-F/UNIV-kobu-R (28) designed to amplify all members of the genus Kobuvirus and targeting a region of 217-bp of the viral RNA-dependent RNA polymerase complex (RdRp). In order to further investigate the genetic heterogeneity of the strains detected, all the samples yielding amplicons of the expected sizes were screened using specific primer sets ( Table 1) designed by multiple alignment of sequences currently available in GenBank, able to amplify the complete VP1 encoding gene. All the positive amplicons were purified with the QIAquick Gel Extraction Kit (Qiagen, Milan, Italy) and sequenced by using BigDye Terminator Cycle chemistry (Applied Biosystems, Foster City, California, US). Basic Local Alignment Search Tool (BLAST; http://www.ncbi. nlm.nih.gov) and FASTA (http://www.ebi.ac.uk/fasta33) with default values were used to find homologous hits. Multiple alignments were performed using the commercially available Geneious software package version 9.1.6 (Geneious software package vers. 9, Biomatters, New Zealand, https://www.geneious. com/ Biomatters). Phylogenetic tree (neighbor joining and Kimura 2-parameter model) with bootstrap analysis (1,000 replicates) were constructed by using the MEGA software package, version X (29).
However, none significant association was found between the detection of KoVs RNA and age. Also, on the basis of the positivities obtained, a geographical clustering between wild and domestic animals was not observed.
Partial RdRp sequences were determined from the KoV positive samples. By sequence analysis, two wild boar strains, WB-15/ITA and WB-28/ITA (GenBank accession numbers: MW307937-8), shared 98.6% nt identity to each other and displayed a closed relatedness (94.0-96.1% nt identity) to porcine KoV sequences detected from swine enteric samples with or without diarrhea (30)(31)(32)(33)(34)(35), whilst identities to KoVs identified in wild boar fecal samples (25,26) ranged from 91.7 to   (Figure 2). Out of the four KoV strains detected in ruminants, the complete VP1 encoding gene was obtained from three samples collected, respectively, from the two goats (Goat-23/ITA and Goat-24/ITA, MW307943-4) and a chamois (Chamois-8/ITA, MW307945). A selection of VP1 encoding gene representative of the Aichivirus B species was retrieved from GenBank. Based on the inspection of tree and according to the distance matrix, three genetic type groups could be distinguished (Figure 3). A large group included only viruses of bovine origin (Aichivirus B-1). The mean nt identity within this group was 88.7%. A second group, sharing more than 85.5% nt identity (mean identity 86.4%) comprised KoVs detected from ferrets (Aichivirus B-2). Group 3 included a strain of ovine origin, TB3/HUN/2009 (6), and the three KoVs found in goats and chamois in this study (Aichivirus B-3). Identity among these VP1 sequences was higher than 90% nt (mean identity 93.2%).  Tree was generated using the neighbor-joining method with kimura 2-parameter model and supplying statistical support with bootstrapping of 1,000 replicates. The arrows indicate the KoV strains detected in this study. Human aichivirus A strain A8461 (GenBank accession no. AB010145) was used as outgroup.

DISCUSSION
In this study we monitored circulation of KoVs among wild boars, goats and chamois in a restricted Italian geographical area. Identification of these viruses in wild boars and goats has been already described elsewhere (7,8,25,26,38,39), whilst KoV infection has never been documented thus far in chamois. Viruses virtually identical to porcine KoVs (species Aichivirus C-1) were first identified in wild boars in Hungary (25) with a prevalence of 100% (10/10) and subsequently in Serbia with a rate of 6.0% (6/100) (26). Although the detection rate obtained in our survey was lower (3.2%, 2/63), our results indicate that circulation of porcine KoVs among wild boars is not uncommon and it is not limited to some settings (25,26 larger animal populations in other geographic areas where wild ungulates are in contact with domestics could be useful to obtain a more complete picture of the ecology of these viruses. In our analysis, all of the tested animals were apparently healthy at time of sampling. The role of KoVs in the etiology of enteritis in animals is still controversial (36,40,41). In previous studies (36), Aichivirus B-3 has been found at higher positivity rates in goats with enteritis (6.5%, 3/46) than in goats without enteritis (5.4%, 5/93), although the difference was not statistically significant. The possible role of Aichivirus B-1 as enteric pathogen involved in neonatal calf diarrhea has been hypothesed (41). Furthermore, in many cases, KoVs have been reported as the sole enteric pathogen detected in diarrheic pigs (42,43), dogs (44), and cats (45,46). Structured surveillance studies could help understand the overall impact of KoVs on livestock and wild animals in terms of health and production.

DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are openly available in the GenBank database at https://www.ncbi.nlm. nih.gov/nucleotide/ under accession numbers MW307937-42 (BankIt2404995).