








key: cord-318237-22s13v2y
authors: Mira, Francesco; Purpari, Giuseppa; Di Bella, Santina; Colaianni, Maria Loredana; Schirò, Giorgia; Chiaramonte, Gabriele; Gucciardi, Francesca; Pisano, Patrizia; Lastra, Antonio; Decaro, Nicola; Guercio, Annalisa
title: Spreading of canine parvovirus type 2c mutants of Asian origin in southern Italy
date: 2019-07-14
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.13283
sha: 
doc_id: 318237
cord_uid: 22s13v2y

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.

. After its emergence, the original type CPV-2 was replaced by three antigenic variants termed CPV-2a, and CPV-2c (Buonavoglia et al., 2001; Parrish et al., 1991; Parrish, O'Connell, Evermann, & Carmichael, 1985) . During the years, several amino acid (aa) changes were accounted in the VP2 gene sequence (Battilani et al., 2001; Geng et al., 2015; Jeoung, Ahn, & Kim, 2008; Nakamura et al., 2004; Truyen, 1999) and, only recently, the analysis of the NS1 gene sequence was included in the CPV phylogenies (Canuti, Rodrigues, Whitney, & Lang, 2017; Grecco et al., 2018; Han et al., 2015; Li et al., 2018; Mira et al., 2019; Pérez et al., 2014; Zhuang et al., 2019) .

Previous studies provided information on the CPV strains spreading in Italy (Decaro, Desario, et al., 2007; Decaro et al., 2013 Decaro et al., , 2006 Dei 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.

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).

Extracted DNA/RNA were also amplified using a set of PCR assays for the detection of canine distemper virus (CDV) , canine adenovirus (CAdV) type 1 and type 2 (Dowgier et al., 2016) , canine herpesvirus (CaHV-1) (Decaro et al., 2010) , canine coronavirus (CCoV) (Decaro et al., 2004) and canine rotavirus (CRoV) (Freeman, Kerin, Hull, McCaustland, & Gentsch, 2008) . Sequence analysis revealed amino acid changes previously described in Asian CPV-2c strains (NS1: 60V, 544F, 545F, 630P-NS2:

60V, 151N, 152V-VP2: 5A/G, 267Y, 297A, 324I, 370R) (Table S1 ).

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., 2013 Decaro et al., , 2006 Tucciarone 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) .

Whereas CPV-2a and CPV-2b are the prevalent variants circulating in Asia (Yi, Tong, Cheng, Song, & Cheng, 2016) , and more recently, CPV-2c has been described in the same continent (Chiang, Wu, Chiou, Chang, & Lin, 2016; Geng et al., 2015; Nakamura et al., 2004; Wang et al., 2016; Zhao et al., 2017; Zhou, Zeng, Zhang, & Li, 2017; Zhuang et al., 2019) , showing molecular signatures different from those of other continents. Indeed, the Asian CPV-2c variant shows specific amino acids in NS1 (60V, 544F, 545V, 630P) and VP2 (5A/G, 267Y, 297A, 324I, 370R) gene sequences. Most of these amino acids have been described in the VP2 of CPV-2a/2b/2c strains collected in China, Vietnam, India, Taiwan, South Korea, Thailand and Japan (Chiang et al., 2016; Geng et al., 2015; Han et al., 2015; Jeoung et al., 2008; Lin et al., 2014; Mukhopadhyay et al., 2014; Nakamura et al., 2004; Phromnoi, Sirinarumitr, & Sirinarumitr, 2010; Soma, Taharaguchi, Ohinata, Ishii, & Hara, 2013; Xu et al., 2015; Yi et al., 2016; Zhang et al., 2010) . In particular, CPV-2c strains displaying the amino acid glycine (G) instead of the highly conserved alanine (A) at residue 5 of the VP2 have been previously detected in China (Wang et al., 2016) and Italy (Mira, Purpari, Lorusso, et al., 2018) . More recently, molecular analyses including the NS1 gene sequence showed the presence of molecular signatures of the Asian CPV strains even in this region (Han et al., 2015; Mira, Purpari, Lorusso, et al., 2018; Zhuang et al., 2019) . Indeed, it results critical to extend the analysis to other genomic regions to properly infer the spread of the genetic CPV variants (Grecco et al., 2018) . (NS1: 60V, 544F, 545F, 630P -NS2: 60V, 151N, 152V) could contribute to further elucidate its evolution ).

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.

The Authors would like to thank the Centro Veterinario Darwin 

Analysis of canine parvovirus sequences from wolves and dogs isolated in Italy

Evidence for evolution of canine parvovirus type 2 in Italy

Introduction of canine parvovirus 2 into wildlife on the Island of Newfoundland

Identification of a novel canine parvovirus type 2c in Taiwan

ICTV virus taxonomy profile: parvoviridae

Development and validation of a real-time PCR assay for specific and sensitive detection of canid herpesvirus 1

Canine parvovirus-A review of epidemiological and diagnostic aspects, with emphasis on type 2c

Infectious canine hepatitis: An "old" disease reemerging in Italy

The study molecular epidemiology of canine parvovirus

Detection of a canine parvovirus type 2c with a non-coding mutation and its implications for molecular characterisation

Genetic analysis of canine parvovirus type 2c

Characterisation of the canine parvovirus type 2 variants using minor groove binder probe technology

Quantitation of canine coronavirus RNA in the faeces of dogs by TaqMan RT-PCR

First molecular characterization of canine parvovirus strains in Sardinia

A duplex real-time PCR assay based on TaqMan technology for simultaneous detection and differentiation of canine adenovirus types 1 and 2

Detection of canine distemper virus in dogs by real-time RT-PCR

Enhancement of detection and quantification of rotavirus in stool using a modified real-time RT-PCR assay

Co-Circulation of the Rare CPV-2c with Unique Gln370Arg Substitution, New CPV-2b with Unique Thr440Ala Substitution, and New CPV-2a with High Prevalence and Variation in Heilongjiang Province, Northeast China

Inter-and intracontinental migrations and local differentiation have shaped the contemporary epidemiological landscape of canine parvovirus in South America

BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT

Fulllength genomic characterizations of two canine parvoviruses prevalent in Northwest China

The emergence of parvoviruses of carnivores

Genetic analysis of VP2 gene of canine parvovirus isolates in Korea

MEGA X: Molecular evolutionary genetics analysis across computing platforms

Genetic characterization of the complete genome of a mutant canine parvovirus isolated in China

Genetic characterization of type 2a canine parvoviruses from Taiwan reveals the emergence of an Ile324 mutation in VP2

Heterogeneity within the hemagglutinin genes of canine distemper virus (CDV) strains detected in Italy

Phylodynamics analysis of canine parvovirus in Uruguay: Evidence of two successive invasions by different variants

Molecular characterization and evolutionary analyses of carnivore protoparvovirus 1 NS1 gene. Viruses, 11

Molecular typing of a novel canine parvovirus type 2a mutant circulating in Italy. Infection

Update on canine distemper virus (CDV) strains of Arctic-like lineage detected in dogs in Italy

Introduction of Asian canine parvovirus in Europe through dog importation

Phylogenetic analysis of canine parvovirus partial VP2 gene in India

A novel antigenic variant of Canine parvovirus from a Vietnamese dog

Global displacement of canine parvovirus by a host-adapted variant: Structural comparison between pandemic viruses with distinct host ranges

Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus

Natural variation of canine parvovirus

Selective regimen shift and demographic growth increase associated with the emergence of high-fitness variants of canine parvovirus

Phylogenetic and genome-wide deep-sequencing analyses of canine parvovirus reveal co-infection with field variants and emergence of a recent recombinant strain

Sequence analysis of VP2 gene of canine parvovirus isolates in Thailand

Investigation on canine parvovirus circulation in dogs from Sicily (Italy) by biomolecular assay

Nucleotide sequence and genome organization of canine parvovirus

High rate of viral evolution associated with the emergence of carnivore parvovirus

Analysis of the VP2 protein gene of canine parvovirus strains from affected dogs in Japan

Molecular characterization of canine parvovirus-2 variants circulating in Tunisia

Emergence and recent evolution of canine parvovirus

Evolution of canine parvovirus-a need for new vaccines? Veterinary Microbiology

Molecular insight into Italian canine parvovirus heterogeneity and comparison with the worldwide scenario

Continuing evolution of canine parvovirus in China: Isolation of novel variants with an Ala5Gly mutation in the VP2 protein

Phylogenetic analysis of canine parvovirus isolates from Sichuan and Gansu Provinces of China in 2011

Phylogenetic analysis of canine parvovirus VP2 gene in China

Phylogenetic analysis of the VP2 gene of canine parvoviruses circulating in China

A greedy algorithm for aligning DNA sequences

Typing of canine parvovirus strains circulating in North-East China

The genetic evolution of canine parvovirus -A new perspective

Genome sequence characterization of canine parvoviruses prevalent in the Sichuan province of China

Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article



