key: cord-288948-89cdfhi0
authors: Campalto, M.; Carrino, M.; Tassoni, L.; Rizzo, G.; Rossmann, M. C.; Cocchi, M.; De Benedictis, P.; Beato, Maria Serena
title: Divergent minute virus of canines strains identified in illegally imported puppies in Italy
date: 2020-10-08
journal: Arch Virol
DOI: 10.1007/s00705-020-04800-6
sha: 
doc_id: 288948
cord_uid: 89cdfhi0

Minute virus of canines (MVC) belongs to the family Parvoviridae, genus Bocaparvovirus, and has been mainly described during enteritis episodes in young dogs. This study reports the characterization of four divergent MVC strains detected between 2012 and 2018, three of which were from dogs illegally imported into Italy, most probably from Eastern Europe, that cluster together phylogenetically but share low genetic similarity with the fourth MVC from an autochthonous dog and other available MVC sequences. Our data indicate that the introduction of genetically distinct MVC strains occurred through the illegal movement of dogs from a geographic area where a distinct MVC lineage was most likely circulating. Enforced surveillance of MVC in the dog population of Eastern Europe and its neighboring countries may shed light on, and eventually trace back to, illegal animal movements.

Minute virus of canines (MVC) belongs to the family Parvoviridae, genus Bocaparvovirus, and is antigenically distinct from canine parvovirus 2 (CPV2), which belongs to the genus Protoparvovirus and is a major causative agent of gastroenteritis [1] . Canine bocaparvoviruses (CBoVs) are non-enveloped single-stranded linear DNA viruses with a genome size of about 5.4 kb with three open reading frames (ORFs) coding for two non-structural proteins, NS1 and NP1, and two overlapping capsid proteins, VP1 and VP2 [1] . ORF1, located at the 5′ end, is 2,325 bp long and encodes NS1 (774 amino acids [aa]) [2] , which is involved in replication, regulation of viral expression, and cytotoxicity [3] . ORF2, located at the 3′ end, is 2,112 bp long and encodes VP1 (703 aa) and VP2 (571 aa) [2] . The VP1 protein is critical for MVC infection, while VP2 mediates receptor recognition and nuclear translocation [3] . ORF3 (561 bp long) partially overlaps with ORF1 (191 bp) and ORF2 (17 bp) and encodes the NP1 protein (186 aa) [2] , which plays an essential role in accumulating capsid mRNAs and proteins [3] . Genetic differences in the coding genes for NS1, NP1 and VP1/VP2 allow three genotypes to be distinguished: CBoV1, CBoV2, CBoV3. MVC corresponds to CBoV1 [1] and was first isolated in 1967 from the feces of a clinically healthy military dog [7] . MVC is distributed worldwide in domestic dogs of different ages. Its clinical significance and virulence are uncertain. It produces mild to unapparent infections, mainly enteritis, in puppies and is weakly pathogenic in adults [8] . Pneumonitis, myocarditis, lymphadenitis and hepatitis have been reported in dogs with MVC infection [9] [10] [11] . MVC may cross the placenta, causing early fetal death and birth defects [9, 10] . The undefined pathogenic role of MVC compared to CPV2 may account for the limited information available on MVC. In Italy, only one report is available describing a high-mortality episode in puppies of a shelter in Southern Italy in 2011 [9] . Novel cases of MVC infection have been detected in Italy at the Istituto , and sterile ultrapure water to volume. The following thermal profile was used: Taq polymerase activation at 95 °C for 5 min followed by 40 cycles of denaturation at 95 °C for 15 s and annealing at 53 °C or 59 °C for 30 s for MVC and CPV2, respectively. All of the archived MVC-positive diagnostic samples were re-analysed by real-time PCR, and those with cycle threshold (Ct) values below 27 were sequenced by the Sanger method. Positive MVC samples identified during the diagnostic activity were subjected to virus isolation attempts. Five hundred µL of tissue homogenate was passed through a 0.45µm filter and used to infect a confluent Madin-Darby canine kidney (MDCK, NBL-2, ATCC CLL-34) cell monolayer, maintained at 37 °C in a 5% CO 2 atmosphere, and monitored daily. After three blind passages, MVC real-time PCR was carried out on cell supernatants to confirm the isolation of the virus. Tissues and cell cultures that were confirmed to be positive for MVC were used to determine the full MVC genome sequence. Six different endpoint PCRs were performed using primer pairs described by Shan et al. [11] covering the entire MVC genome with the exception of a small gap of 32 nucleotides (nt) in the NS1 gene (from nt 996 to 1,028). To cover this gap, an additional primer pair was designed ad hoc (MVC 550F, 5′-GGA TGC CTG GTC CCG ATA G-3′; MVC 1450R, 5′-ATA AGT TTG TTC CCG CCC GA-3′). The endpoint PCRs were conducted with 5 µl of PrimeSTAR GXL Buffer (5x) (Takara, Japan), 0.75 µl of each primer (10 µM), 2 µl of dNTPs (2.5 mM), 0.5 µl of PrimeSTAR GXL Taq polymerase (1.25 U/µl) (Takara, Japan), and 11 µl of sterile ultrapure water to 25 µl. The conditions for PCR were as follows: denaturation at 98 °C for 10 s, annealing at the primer annealing temperature for 15 s, and extension at 68 °C for 1 min, for 40 cycles. PCR products were subjected to electrophoresis in 7% acrylamide gels. Positive samples were subjected to Sanger sequencing using the same primers used for amplification. Complete sequences of CBoV1 available in the GenBank database were aligned, and a phylogenetic tree was constructed by the maximum-likelihood (ML) method, using the software IqTree-1.6.1, with the TMP3u + F + R2 nucleotide substitution model and of 100 replicates of bootstrap analysis. Further phylogenetic analysis was conducted for the individual ORFs in the MVC genome. For each analysis, the ORF sequences of the MVC strains identified in the present study (completely or partially sequenced) were aligned with those of the twelve complete genome sequences available in the GenBank database. Additional partial sequences were also included in the analysis ORF2. The nt substitution models for ORF1 (NS1), ORF2 (VP1/VP2), and ORF3 (NP1) were HKY + F + I, TVM + F + R5, and HKY + F + G4, respectively. The matrices of nt distances for the whole genome sequences and the VP1/ VP2 sequences were calculated using MEGA software 6.06. (Fig. 1a) . In detail: the complete ORF1, encoding the NS1 protein, of three of the four MVC isolates (Italy/4033/2012, BIO-CRIME/2018 and Italy/59116/2017, which had a gap of 17 nt) was sequenced and was 2325 bases in length, corresponding to 774 aa (Fig. 1a) . The complete ORF2, 2112 bp long, encoding the VP1/VP2 proteins (703-aa VP1 and 571-aa VP2), was fully sequenced for two out of four MVC isolates: Italy/4033/2012 (with a small gap of 17 nt) and Italy/59116/2017. Partial ORF2 sequences were obtained for Italy/50120/2018 and BIO-CRIME/2018: missing 322 nt at 3′ end of VP2 and 26 nt at the 5′end and 326 nt at the 3′ end of VP2 (Fig. 1a) . A full ORF3 sequence of 561 nt, encoding the NP1 protein (186 aa), was obtained for three out of the four MVC isolates: Italy/4033/2012, Italy/59116/2017 and BIO-CRIME/2018, but not for Italy/50120/2018 (Fig. 1a) . The nearly complete genome sequences of Italy/4033/2012, Italy/59116/2017 and BIO-CRIME/2018, the two partial sequences of strain Italy/50120/2018, and the 12 whole MVC genome sequences available in the GenBank database were used for phylogenetic analysis. BIO-CRIME/2018 and Italy/59116/2017 were found to cluster together in a distinct monophyletic branch; by contrast, Italy/50120/2018 and Italy/4033/2012 clustered independently (Fig. 1b) . To test the robustness of our analysis, we also generated phylogenetic trees using whole genome sequences trimmed to the shortest sequence length possible and including or excluding the region corresponding to the gap in the Italy/50120/2018 sequence. Although the topologies of the phylogenetic trees generated were slightly different, BIO-CRIME/2018 and Italy/59116/2017 strains always clustered together, and the corresponding nodes were supported by high bootstrap values (data not shown). The matrix of nt distances (Table 1 ) revealed a high similarity between BIO-CRIME/2018 and Italy/59116/2017 (99.21%) and showed less than 98% sequence identity to all of the full MVC sequences available in GenBank (Table 1) . Conversely, Italy/50120/2018 and Table 1 ). The topologies of the phylogenetic trees based on each individual ORF (Fig. 2) was similar to that observed for the whole genome (Fig. 1b) , with Italy/59116/2017 and BIO-CRIME/2018, both of which were obtained from smuggled puppies, clustering together in a separate new branch and the other two MVC strains falling in different branches (Fig. 2) . The phylogenetic tree based on the ORF2 nt sequences encoding the VP1/VP2 proteins (Fig. 2c) , was constructed using all 124 MVC sequences available in the GenBank database and included the only Italian MVC sequence available, Italy/285_11/2011 (JQ612703_Canine_ minute_virus_strain_285_11_Italy_2011), for which only the partial VP1/VP2 sequence is present [9] . Interestingly, the phylogenetic analysis based on ORF2 sequences, highlighted that, among the MVC strains identified in the present study, the Italy/50120/2018 strain, from the autochthonous dog, is the only one that shares the highest nt sequence identity with Italy/285_11/2011 (99.07%) and is 100% identical to two Chinese strains identified in 2016 (MH051146 and MH051145) ( Table 1) . Moreover, the aa sequences encoded by the ORF1, ORF2 and ORF3 regions of the four MVCs identified were compared with those of the twelve complete MVC genome sequences available in the GenBank database. The aa sequence of the Italy/50120/2018 strain was analyzed for ORF1 and ORF2, but the ORF3 was not available. Several aa mutations Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.

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Acknowledgements This study was partially funded by the European Union through the BIO-CRIME Project-Animal diseases (zoonoses) and illegal trade of young animals in the Alps-Adriatic region (animal welfare)-(ITAT3002), Interreg Italia-Osterreich. The authors thank Francesca Ellero for English revisions and Anna Toffan for providing strain MN947833_Minute_virus_of_canines_4033_Italy_2012 (Italy/4033/2012).