Spreading of canine parvovirus type 2c mutants of Asian origin in southern Italy.

Abstract Canine parvovirus type 2 (CPV‐2) emerged as dog pathogen in the late 1970s, causing severe and often fatal epizootics of gastroenteritis in the canine population worldwide. Although to date CPV‐2 is circulating in all continents, most of the current studies have analysed the amino acid changes accounted in the VP2 gene sequence, with limited information on virus introductions from other countries. The aim of this study was to analyse the genetic features of CPV‐2c strains currently spreading in Italy. Swabs and tissue samples were collected from dogs suspected of CPV infection. The nearly complete genome sequence from the CPV‐positive samples was obtained. The co‐circulation of two different but related CPV‐2c strains, with amino acid changes characteristic of CPV strains of Asian origin (NS1: 60V, 544F, 545F, 630P – NS2: 60V, 151N, 152V ‐ VP2: 5A/G, 267Y, 297A, 324I, 370R), were observed. The phylogenetic analyses inferred from the NS1 and VP2 gene sequences confirmed the relationship with Asian CPV‐2c strains. This study reports the spread of novel CPV‐2c mutants in Italy and supports further studies to evaluate the coexistence of genetically divergent CPV strains in the same geographical environment.

Previous studies provided information on the CPV strains spreading in Italy (Decaro, Desario, et al., 2007;Decaro et al., 2013Decaro et al., , 2006Dei Giudici et al., 2017;Mira, Dowgier, et al., 2018;Purpari et al., 2018;Tucciarone et al., 2018), suggesting the need of a continuous epidemiological survey to evaluate the CPV circulation and evolution, whereas limited data are available on the spread of novel strains imported from other continents (Mira, Purpari, Lorusso, et al., 2018). The aim of this study was the detection and molecular analysis of CPV strains displaying genetic features of Asian viruses spreading in southern Italy.

| MATERIAL S AND ME THODS
During an epidemiological survey, rectal swabs (n = 3) and tissue samples (n = 19) from seven dogs suspected of CPV infection (Table 1), collected in southern Italy (Sicily) from August 2018 to March 2019, were analysed at the Istituto Zooprofilattico Sperimentale della Sicilia "A. Mirri" (Palermo, Italy) for diagnostic purposes. DNA and RNA were extracted from swab/organ homogenates, obtained as previously described  Presence of CPV DNA was evaluated by a diagnostic PCR using a primer pair targeting the VP2 gene (Touihri et al., 2009), as previously described (Mira, Purpari, Lorusso, et al., 2018), and each amplicon was analysed by electrophoresis on a 3% agarose gel supplemented with ethidium bromide.
Sequencing encompassing both ORFs (NS and VP genes) was carried out using primer pairs developed by Pérez et al. (2014), as previously described . Sequences were assembled according to an overlapping strategy and analysed using BioEdit ver 7.0.5.3 software (Hall, 1999). Assembled nucleotide sequences were submitted to nBLAST program (Zhang, Schwartz, Wagner, & Miller, 2000) to search related sequences in public domain databases.
These sequence data have been submitted to the DDBJ/EMBL/ GenBank databases under accession numbers MK802679-85. The obtained sequences were aligned with reference sequences retrieved from the GenBank database, which included the sequence (accession number MF510157) of a CPV-2c strain collected from the same region and previously analysed (Mira, Purpari, Lorusso, et al., 2018 To elucidate the genetic relationships of the analysed CPV strains, two phylogenetic trees, based on the full-length VP2 and NS1 gene sequences, were constructed with the MEGA X software (Kumar, Stecher, Li, Knyaz, & Tamura, 2018), using the maximum-likelihood F I G U R E 1 Maximum-likelihood tree based on 50 full-length VP2 gene sequences of canine parvovirus type 2 strains (bootstrap 1,000 replicates; bootstrap values greater than 65 are shown). Black dots markings (•) indicate CPV strains analysed in this study. Each sequence is indicated with virus type (FPLV: feline panleukopenia virus-CPV: canine parvovirus) or variant (CPV-2, CPV-2a, CPV-2b, CPV-2c), country and year of collection, and accession number method according to the Tamura 3-parameter (T92) and Hasegawa-Kishino-Yano (HKY) models, with discrete Gamma distribution (five rate categories) (G) and invariant sites (I) (bootstrap analyses with 1,000 replicates). The models selection was performed using the best-fit model of nucleotide substitution with MEGA X software (VP2 gene: T92+G+I; NS1 gene: HKY+G).
CPV strain IZSSI_PA5632/19 evidenced an additional change at residue 492 of NS1 protein (Table S1). Only one mutation (A/G) was observed among the analysed strains at residue 5 of the VP2 protein, which suggests the circulation of two different but related CPV-2c strains in southern Italy.
Amino acid change I60V in NS1 also lies at the same residue in the NS2-encoding sequence, while change at codon 630 of NS1 sequences did not result in any changes in the encoded NS2 protein.
Additional two amino acid changes in the NS2-encoding sequences were observed among the analysed strains: D151N and M152V (Table S1). These changes resulted in silent mutations in the corresponding encoded NS1 protein.
Phylogenetic analysis inferred from VP2 sequences indicated that analysed strains are more related to Asian than to European CPV strains, clustering in a separate clade (Figure 1). Phylogenetic tree inferred from NS1 gene sequences shows that strains clustered within the phylogeny according to the geographical origin and the year of collection rather than to the CPV variant ( Figure 2).
The present molecular analysis of CPV strains detected in southern Italy provides new data about the viral spread and dynamics of CPV mutants circulating in Italy. In the last decades, several studies analysed the spread of CPV strains in Italy, and in 2001, the emergence of the CPV-2c variant was firstly reported (Buonavoglia et al., 2001).
In the following years, all three CPV variants were described in Italy, with a slightly higher prevalence of the CPV-2a and CPV-2c variants (Decaro, Desario, et al., 2007;Decaro et al., 2013Decaro et al., , 2006Tucciarone et al., 2018). More recently, a CPV-2c strain displaying genetic signatures typical of Asian viruses was detected in southern Italy (Mira, Purpari, Lorusso, et al., 2018), thus suggesting the introduction of the virus from other countries, as reported for other canine viruses (Decaro, Campolo, et al., 2007;Martella et al., 2006;. Therefore, a continuous molecular survey was assessed to point out eventual introduction and spread of CPV strains originated from other geographic areas in the Italian canine population. Since  (Hoelzer & Parrish, 2010). According to this study, the spread of Asian CPV strains in a separate geographical area different from Asian countries could be suggested, as previously described in South America (Grecco et al., 2018;Maya et al., 2013).
The classification system based on single amino acids (426 and 297) of the VP2 protein does not clearly reflect the phylogenetic relationships of the strains, better supported to proposed "clade" or "lineage/sub-lineage" new classification criteria (Grecco et al., 2018;Zhuang et al., 2019;Mira et al., 2019). As observed also in this study, phylogeny lacks any clustering based on the single VP2 aa residue 426 (CPV-2a/2b/2c), as well as on the geographic origin and period of sample collection. Therefore, a wider evolutionary analysis further supports the thesis to consider the CPV antigenic variants as variants of CPV-2a rather than distinct subtypes (Organtini, Allison, Lukk, Parrish, & Hafenstein, 2015) and could be considered as a more reliable tool in outbreak tracing.
This study reported the early evidence and spread of CPV-2c strains of Asian origin in the Italian canine population. As observed in South America (Grecco et al., 2018), studies based on the complete coding genome are useful to monitor the spread of CPV strains with Asian origin in a different continent and highlight the need of further studies to evaluate the CPV evolution due to the coexistence of genetically divergent strains in the same geographical environment.

ACK N OWLED G EM ENTS
The Authors would like to thank the Centro Veterinario Darwin