key: cord-0778212-c4ut7vn7
authors: Palombieri, Andrea; Di Profio, Federica; Lanave, Gianvito; Capozza, Paolo; Marsilio, Fulvio; Martella, Vito; Di Martino, Barbara
title: Molecular detection and characterization of Carnivore chaphamaparvovirus 1 in dogs
date: 2020-10-01
journal: Vet Microbiol
DOI: 10.1016/j.vetmic.2020.108878
sha: 64ac184c71a1ddcf6767aeb616a6905fc0785890
doc_id: 778212
cord_uid: c4ut7vn7

Canine chaphamaparvovirus (CaChPV) is a newly recognised parvovirus discovered by metagenomic analysis during an outbreak of diarrhoea in dogs in Colorado, USA, in 2017 and more recently detected in diarrhoeic dogs in China. Whether the virus plays a role as canine pathogen and whether it is distributed elsewhere, in other geographical areas, is not known. We performed a case-control study to investigate the possible association of CaChPV with enteritis in dogs. CaChPV DNA was detected both in the stools of diarrhoeic dogs (1.9%, 3/155) and of healthy animals (1.6%, 2/120). All the CaChPV-infected dogs with diarrhea were mixed infected with other enteric viruses such as canine parvovirus (formerly CPV-2), canine bufavirus (CBuV) and canine coronavirus (CCoV), whilst none of the asymptomatic CaChPV positive animals resulted co-infected. The nearly full-length genome and the partial capsid protein (VP) gene of three canine strains, Te/36OVUD/19/ITA, Te/37OVUD/19/ITA and Te/70OVUD/19/ITA, were reconstructed. Upon phylogenetic analyses based on the NS1 and VP aa sequences, the Italian CaChPV strains tightly clustered with the American reference viruses. Distinctive residues could be mapped to the deduced variable regions of the VP of canine and feline chaphamaparvoviruses, considered as important markers of host range and pathogenicity for parvoviruses.

Parvoviridae are small (~25 nm diameter), non-enveloped, single-stranded and negative-sense DNA viruses of 3.9-6.3 kb in length, with the coding region bracketed by terminal repeats that can fold into hairpin-like structures (Berns and Parrish, 2013) . They have a large host spectrum, spanning from invertebrates to mammals (Pénzes et al., 2019) . Parvoviruses have long been known in dogs, since the identification of canine minute virus, or canine parvovirus (CPV) type 1 (CPV-1; genus Bocaparvovirus), in 1967 from the faecal samples of healthy dogs (Binn et al., 1970) . CPV-1 infection is responsible for reproductive disorders and occasionally for respiratory and gastrointestinal signs in young dogs (Decaro et al., 2012) . A second CPV (CPV-2; genus Protoparvovirus) was reported in the 1970s in Europe and North America in puppies with signs of haemorrhagic gastroenteritis and myocarditis (Appel et al., 1979) . CPV-2 is currently regarded as the major causative agent of severe gastroenteritis in puppies (Decaro and Buonavoglia, 2012) . Recent advances in molecular technologies have been allowed the discovery of novel parvoviruses in dogs, including two additional bocaparvoviruses species, CBoV-2 and CBoV-3 identified respectively from healthy and sick dogs respiratory samples (Kapoor et al., 2012) and from the liver of a dog with multiorgan failure (Li et al., 2013) , and the still unclassified carnivore protoparvoviruses likebufaviruses (CBuVs) detected in stool samples of dogs with or without enteric disease and in the nasal and oropharyngeal swabs of animals with respiratory signs. More recently, assessing faecal samples from an unexplained outbreak of diarrhoea in Colorado (USA), a novel canine virus has been discovered by metagenomic approach (Fahsbender et al., 2019) . Upon sequence analysis of the nearly complete genome of the two strains identified, Cachavirus-1A strain IDEXX1 (GenBank accession number MH893826) and Cachavirus-1B strain IDEXX2 (MK448316), the highest genetic relatedness (65.4-74.4% nt identity) was found to members of the genus Chaphamaparvovirus, previously described under an unofficial umbrella term "Chapparvovirus" (Pénzes et al., 2019) . On the basis of the International Committee on Taxonomy of Viruses (ICTV) J o u r n a l P r e -p r o o f classification criteria for Parvoviridae species demarcation, both the canine strains were classified within the species Carnivore chaphamaparvovirus 1 (Pénzes et al., 2019) .

The genus Chaphamaparvovirus includes viruses infecting vertebrate hosts that are more closely related to invertebrate-infecting parvoviruses than to members of the Parvovirinae. The discovery of ChPVs has compelled a radical rethink on the evolution and genetic relationships among Parvoviridae, leading to a recent taxonomical re-classification characterised by the introduction of the novel subfamily Hamaparvovirinae that encompasses divergent densoviruses and vertebrateinfecting parvoviruses (Pénzes et al., 2019) . In the last few years, ChPVs have been identified in several animal species including bats, rodents, birds, pigs and domestic cats (Berker et al., 2013; Reuter et al., 2014; Yang et al., 2016; Palinski et al., 2016; Lima et al., 2017; de Souza et al., 2017; Yinda et al., 2018; Roediger et al., 2018; William et al., 2018; . However, information on their epidemiology and/or ability to cause disease in their natural hosts are still limited. Preliminary epidemiological data collected by Fahsbender et al. (2019) address the question on the possible role of canine ChPVs (CaChPVs) in the aetiology of dog enteritis. Molecular analysis by quantitative PCR (q-PCR) of a large set of enteric samples revealed the presence of CaChPVs DNA in the faeces of dogs with (4.35%) or without (1.47%) diarrhoea, although a clear association with enteric signs was not demonstrated. In a more recent study performed in China , the detection rate of CaChPVs DNA was 0% and 1.55% in healthy or diarrhoeic dogs, respectively. However, association between CaChPVs and gastrointestinal disease was not supported by statistical analysis.

In order to gather additional information on the distribution of this novel parvovirus in dogs and to investigate its possible association with enteritis, during the year 2019 a surveillance study was initiated by implementing with CaChPV-specific assays the diagnostic algorithms of cases of acute gastro-enteritis admitted to the veterinary hospital of the Faculty of Veterinary Medicine, University of Teramo (Italy).

J o u r n a l P r e -p r o o f

A case-control study was conducted using two subsets of dogs selected on the basis of the presence of acute gastroenteric signs for clinical cases and the absence of enteritis for controls. A total of 155 rectal swab samples (subset A) was collected from household dogs suffering from gastro-enteric disorders. The inclusion criteria were the presence of mild to severe diarrhoea and age comprised between 2 and 6 months old. Rectal specimens (n. 120) randomly recruited among healthy dogs during routine visits in 2019 and matching with cases for living condition and age were included in the study as control group (subset B). Informed consent was obtained from all animal owners.

The collected swabs were immersed in 1 ml of viral transport medium consisting of Dulbecco's modified Eagle's medium (D-MEM), and subsequently clarified by centrifuging at 2500g for 10 min.

DNA and RNA were extracted from 200 μl of viral suspension by using the QIAamp Cador Pathogen Mini Kit (Qiagen S.p.A., Milan, Italy), following the manufacturer's instructions. All faecal samples were screened for CPV-2, CBuV, canine coronavirus (CCoV), canine kobuvirus (CaKoV) and norovirus (NoV) by conventional PCR and reverse transcription (RT)-PCR (Pratelli, et al., 1999; Buonavoglia et al., 2001; Vennema et al., 2002; Di Martino et al., 2013; Martella et al., 2018) . The presence of CaChPV DNA was assessed by nested-PCR using diagnostic primer sets CPV_625F/CPV_948R and CPV18_687FN/CPV_911RN, following chemical and thermal conditions previously described (Fahsbender et al., 2019) A standard curve was generated using 10 0 to 10 9 copies per reaction of CaChPV plasmid DNA containing the synthetised fragment. Viral DNA quantification was performed using TaqMan and type 2). The qPCR assay was able to detect >10 1 DNA copies/5 μL of standard DNA and 4.0 × 10 1 DNA copies/5 μL of DNA template extracted from clinical samples.

In order to gather sequence information from the CaChPVs detected in our study, attempts were made J o u r n a l P r e -p r o o f

By using diagnostic primer sets (Fahsbender et al., 2019) , CaChPV DNA was detected in a total of 5 faecal samples, with an overall prevalence of 1.9% (5/275 On phylogenetic analysis based on the complete NS1 protein (Fig. 1A) (Yinda et al., 2018) and the ChPVs of feline origin . The aa identities within this group was 58.5-66.3%. The high genetic conservation J o u r n a l P r e -p r o o f among all the CaChPVs identified to date (with an overall aa identity of 99.0-99.1%) was also confirmed in the VP capsid based tree (Fig. 1B) . Identities to the feline and bat ChPVs were 74.5-74.7% and 71.1-71.2%, respectively. In order to further investigate the relationship between the canine and the feline ChPVs, the variable regions (VRs) of the CaChPV strains 1B-IDEXX2 and Te/36OVUD/19/ITA and those of the feline ChPVs 1/VRI/849 and IDEXX-1 were mapped as described elsewhere (Pénzes et al., 2019 ) and compared to each other. Out of ten VRs (VR-I to VR-X), VR-II resulted more conserved, whilst the largest differences were located in VR-III and VR-VI to VR-X (Fig. 2) .

The findings of this study, while providing firm evidence that CaChPV is a common component of canine faecal virome, do not allow to make any solid conclusions on the potential role of this novel parvovirus as a primary causative agent of gastrointestinal disease. In our analysis, CaChPV DNA was detected in animals with enteric signs only in co-infection with other canine viral pathogens.

Similar results were also obtained in the Chinese study, in which two out of the five diarrhoeic Although most ChPV strains identified to date in the various animal species have been discovered serendipitously in metagenomic studies of enteric virome, evidence for the pathogenic potential of these viruses have been reported. Horizontal transmission of the mouse kidney parvovirus (MKPV) (Rodent chaphamaparvoviruses 1 species) has been proved to induce inclusion body nephropathy (IBN) and kidney fibrosis in aged immunodeficient mice and, to lesser extent, in immunocompetent mice (Roediger et al., 2018; Ge et al., 2020; Lee et al., 2020) . More recently, a newly identified ChPV was identified by qPCR and immunohistochemistry in the heart, intestine, liver, and lung of a dead peafowl suffering of enteritis and pneumonia .

Sequence analysis of the nearly complete genome of the strains Te/36OVUD/19/ITA and Te/37OVUD/19/ITA and of ~ 1.1-kb long partial VP of the CaChPV strain Te/70OVUD/19/ITA revealed a strong sequence conservation with the viruses detected in diarrhoeic dogs in USA (Fahsbender et al., 2019) , either in the NS1 (98.6-99.8%) or in the VP (99.0-99.1%) encoding genes.

Upon phylogenetic analysis based on the NS1 aa sequence, all the strains of canine origin grouped tightly within the species Carnivore chaphamaparvovirus 1. Interestingly, this group segregated apart from but close to a ChPV identified in stools of a Cameroonian fruit bat (Yinda et al., 2018) and to

ChPVs recently identified in cats with gastroenteritis . The genetic relatedness among J o u r n a l P r e -p r o o f the CaChPVs and the strains detected in cats and bats (58.5-66.7%) was consistent with the ranges established by ICTV for classification of parvoviruses into the same genus (cut-off > 35.0% aa identity with a NS1 coverage > 80.0%), whilst it did not match the criteria required to demark the same species (cut-off > 85.0% aa identity) (Pénzes et al., 2019) . The correlation between the canine and feline viruses (74.5-74.7% nt identities in the nearly complete genome) was also confirmed in the VP-based tree. For parvoviruses, the capsid is the major determinant of host range (Hueffer et al., 2003) and subject to antibody-mediated selection (Nelson et al., 2007) . Minor genetic changes in these proteins are known to alter the host range and pathogenic potential of parvoviruses (Parrish and Kawaoka, 2005) . When comparing the VP sequences of the canine strains 1B-IDEXX2 and Te/36OVUD/19/ITA with those of the feline ChPVs 1/VRI/849 and IDEXX-1, the highest divergence was observed in the predicted VRs of the capsid (Fig. 3) , with several aa changes, including residue insertions and deletions, located at VR-III and VR-VII that have been involved in control of tissue tropism, antibody recognition and receptor attachment of several parvoviruses (Halder et al., 2012; Kailasan et al., 2015) . The marked genetic heterogeneity observed between the CaChPVs and the feline strains indicates that at least two distinct groups of ChPVs, each characterised by peculiar genetic signatures, circulate in dogs and cats. Since some canine viruses can infect cats and vice versa (Martella et al., 2002; Matthijnssens et al., 2011; Di Martino et al., 2016; Di Martino et al., 2018; Diakoudy et al., 2019) , it will be important to explore whether cross-species transmission of ChPVs might occur between the two carnivore species.

We gathered epidemiological information of CaChPV in a large dog population demonstrating that the circulation of this novel canine parvovirus is not geographically restricted to the American and Asiatic continents where the virus was so far detected (Fahsbender et al., 2019; Hu et al., 2020) . This firmly demonstrates that CaChPV is a common component of canine enteric virome. The impact of these viruses on canine health remains to establish. In our study, we did not find any possible J o u r n a l P r e -p r o o f association between CaChPV and gastro-enteric disease in young dogs. Animal experiments or detailed observational studies would be required to address this thoroughly and to investigate whether there may be other possible implications for canine health.

All Authors declare that there are no financial or other relationships that might lead to a conflict of interest. All authors have seen and approved the manuscript and have contributed significantly to the work.

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Funding: this work was financed by grants from Università degli Studi di Teramo.J o u r n a l P r e -p r o o f