Gabriele Chiaramonte

Canine coronavirus (CCoV) is a common agent of gastroenteritis in dogs, although some variants have been found associated with systemic and often fatal diseases. Distinct genotypes (CCoV‐I and CCoV‐II) and subgenotypes (CCoV‐IIa and CCoV‐IIb) are worldwide distributed. In Italy, CCoV infections have been occasionally evaluated, but information about the molecular epidemiology and the genomic features of currently circulating strains is limited. This study reports the detection and molecular characterization of CCoV strains from samples collected from 284 dogs in Italy between 2019 and 2021. CCoV RNA was detected in 39 (13.7%) dogs, as a single viral agent (5 animals, 12.8%) or with other viral pathogens (canine parvovirus types 2a/2b/2c; canine adenovirus type 1; norovirus GIV.2) (34 animals, 87.2%). A total of 48 CCoV strains were detected either alone (CCoV‐I: 51.3%, CCoV‐IIa: 20.5%) or in copresence (CCoV‐I and CCoV‐IIa, 23.1%); surprisingly, CCoV‐IIb was not identified in this study. Five clusters of CCoV‐I were detected, and their spike gene sequences showed the highest nucleotide identities with CCoV‐I strains collected from Greece in 2008/2009 and from China in 2021. CCoV‐IIa spike gene sequences (three variants) had the highest nucleotide identities with CCoV‐IIa strains collected in Greece in 2008/2009 and in Italy in 2009/2011. Given the high CCoV diversity and the variable pathogenicity potential, we underline the need of further surveillance studies to increase our understanding of the epidemiology and evolution of these viruses.

Canine parvovirus (CPV-2) modified-live virus vaccine strain can replicate in lymphoid tissues and intestinal mucosa after administration, being shed through canine faeces. Detection of vaccine strains has been reported in the bloodstream and faeces, potentially interfering with molecular diagnostic tests. The persistence of these strains in canine tissues has not yet been described. With this aim, canine tissues were tested during a molecular survey to screen for the presence of canine enteric viruses. Tissue samples from 165 dead dogs were tested by a conventional PCR assay. Positive samples and five commercial vaccines were subjected to sequence analysis. Vaccinal strains were detected and virus load was measured by using a set of real-time PCR assays using minor-groove binder (MGB) probes. Seventy-five dogs (45.4%) tested positive for CPV-2. Strains from 70 dogs were characterised as field variants. The presence of CPV sequences of vaccine origin was observed in the spleen, intestine, and mesenteric lymph nodes of five young dogs. Vaccinal strains were detected from 12 to 24 days after the last vaccine administration. Viral loads comprised between 6.3 × 10² and 9.95 × 10⁴ DNA copies/10 µl of template. This study confirms that CPV vaccinal strains can be detected in canine tissues after vaccination, so post-mortem diagnosis of CPV infection needs further molecular analyses to assess the viral type (vaccine or field strains). The present study updates the current information on the persistence of CPV vaccine strains in canine tissues and their possible interference with molecular assays.

Canine adenovirus type 1 (CAdV-1) is the causative agent of a systemic and potentially fatal viral disease of domestic and wild canids. In Italy, CAdV-1 infection has also been occasionally described in dogs, but information on the epidemiology and its genomic features is still limited. A study was conducted on 291 dogs suspected of infectious gastrointestinal disease. Samples collected from dogs in southern Italy between 2017 and 2020 were analyzed. Virological and histopathological assays were carried out. The presence of CAdVs and other canine viral enteropathogens was investigated, and sequence and phylogenetic analyses were performed. CAdV-1 was detected in six (2.1%) dead stray dogs alone or in mixed infections with other viruses. Gross lesions and histopathological findings referred to CAdV infection were observed, also involving the central nervous system tissues. All inoculated samples were successfully isolated. Sequence analysis evidenced divergences with the circulating strains previously described in Italy and a closer relation with older CAdV-1 strains collected from other countries, suggesting a genetic heterogeneity of CAdV-1 in Italy. The evidence of the circulation of CAdV-1 and its genomic features allows us to have more in-depth knowledge of the epidemiology and evolution of the CAdV-1 genomic variants.

Canine parvovirus type 2 (CPV-2) is an infectious agent relevant to domestic and wild carnivorans. Recent studies documented the introduction and spread of CPV-2c strains of Asian origin in the Italian canine population. We investigated tissue samples from a puppy collected during necropsy for the presence of viral enteropathogens and all samples tested positive only for CPV-2. The full coding sequence of a CPV-2b (VP2 426Asp) strain was obtained. This virus was related to CPV-2c strains of Asian origin and unrelated to European CPV-2b strains. The sequence had genetic signatures typical of Asian strains (NS1: 60Val, 545Val, 630Pro; VP2: 5Gly, 267Tyr, 324Ile) and mutations rarely reported in Asian CPV-2b strains (NS1: 588N; VP2: 370Arg). Phylogenetic analyses placed this strain in well-supported clades, including Asian CPV-2c-like strains, but always as a basal, single-sequence long branch. No recombination was observed for this strain, and we speculate that it could have originated from an Asian CPV-2c-like strain that acquired the 426Asp mutation. This study reports the first evidence of an Asian-like CPV-2b strain in Italy, confirming the occurrence of continuous changes in the global CPV-2 spread. Since positive convergent mutations complicate data interpretation, a combination of phylogenetic and mutation pattern analyses is crucial in studying the origin and evolution of CPV-2 strains.

Canine parvovirus type 2 (CPV-2) represents a major viral threat to dogs. Considering the potential effects of pets on antimicrobial resistance, information on the CPV and associated bacterial co-infections is limited. The aim of this study was to analyze the antimicrobial susceptibility and multidrug-resistance profiles of bacterial species from tissue samples of dogs with canine parvovirus infection. A set of PCR assays and sequence analyses was used for the detection and the molecular characterization of the CPV strains and other enteric viruses. Bacterial isolation, the determination of antimicrobial susceptibility via the disk diffusion method, and the determination of the minimum inhibitory concentration were performed. The detection of β-lactamase genes and toxin genes for specific bacteria was also carried out. CPV infection was confirmed in 23 dogs. Forty-three bacterial strains were isolated and all showed phenotypic resistance. Seventeen multidrug-resistant bacteria and bacteria with high resistance to third- and fourth-generation cephalosporins and metronidazole were detected. Almost 50% of the isolated Enterobacteriaceae were positive for at least one β-lactamase gene, with the majority carrying more genes as well. The evidence for multi-resistant bacteria with the potential for intra- or cross-species transmission should be further considered in a One Health approach.

Equid and asinine gammaherpesviruses (GHVs; genus Percavirus) are members of the Herpesviridae family. Though GHVs have been reported in horse populations, less studies are available on gammaherpesviral infections in donkeys. This study reports the co-infection with two GHVs in Pantesco breed donkeys, an endangered Italian donkey breed. Samples (n = 124) were collected on a breeding farm in Southern Italy from 40 donkeys, some of which were healthy or presented erosive tongue lesions and/or mild respiratory signs. Samples were analysed by using a set of nested PCRs targeting the DNA polymerase, glycoprotein B, and DNA-packaging protein genes, and sequence and phylogenetic analyses were performed. Twenty-nine donkeys (72.5%) tested positive, and the presence of Equid gammaherpesvirus 7 and asinine herpesvirus 5 was evidenced. In 11 animals, we found evidence for co-infection with viruses from the two species. Virions with herpesvirus-like morphology were observed by electron microscopic examination, and viruses were successfully isolated in RK-13-KY cell monolayers. The histological evaluation of tongue lesions revealed moderate lympho-granulocytic infiltrates and rare eosinophilic inclusions. The detection of GHVs in this endangered asinine breed suggests the need long-life monitoring within conservation programs and reinforces the need for further investigations of GHV’s pathogenetic role in asinine species.

Canine parvovirus type 2 (CPV‐2) emerged suddenly in the late 1970s as pathogen of dogs, causing a severe and often fatal gastroenteric disease. The original CPV‐2 was replaced by three antigenic variants, CPV‐2a, CPV‐2b and CPV‐2c, which to date have gained a worldwide distribution with different relative proportions. All previous studies conducted in Africa were based on partial VP2 gene sequences. The aim of this study was to provide a genome analysis to characterize the CPV strains collected in Nigeria, Africa. Rectal swab samples (n = 320) were collected in 2018 and tested by means of an immunochromatographic assay. Among the 144 positive samples, 59 were selected for further analyses using different molecular assays. The results revealed a high prevalence of CPV‐2c (91.5%) compared to the CPV‐2a variant (8.5%). The VP2 gene sequences showed a divergence from the strains analysed in 2010 in Nigeria and a closer connection with CPV strains of Asian origin. The non‐structural genes analysis evidenced amino acid changes never previously reported. The molecular analysis based on genomic sequences evidenced a geographical pattern of distribution of the analysed strains, suggesting a potential common evolutionary origin with CPV of Asian origin. This study represents the first CPV molecular characterization including all the encoding gene sequences conducted in the African continent and contributes to define the current geographical spread of the CPV variants worldwide.

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.

Carnivore protoparvovirus 1 is the etiological agent of a severe disease of terrestrial carnivores. This unique specie encompasses canine parvovirus type 2 (CPV-2) and feline panleukopenia virus (FPLV). Studies widely analyzed the main capsid protein (VP2), but limited information is available on the nonstructural genes (NS1/NS2). This paper analyzed the NS1 gene sequence of FPLV and CPV strains collected in Italy in 2009–2017, along with worldwide related sequences. Differently from VP2, only one NS1 amino-acid residue (248) clearly and constantly distinguished FPLV from CPV-2, while five possible convergent amino-acid changes were observed that may affect the functional domains of the NS1. Some synonymous mutation in NS1 were non-synonymous in NS2 and vice versa. No evidence for recombination between the two lineages was found, and the predominance of negative selection pressure on NS1 proteins was observed, with low and no overlap between the two lineages in negatively and positively selected codons, respectively. More sites were under selection in the CPV-2 lineage. NS1 phylogenetic analysis showed divergent evolution between FPLV and CPV, and strains were clustered mostly by country and year of detection. We highlight the importance of obtaining the NS1/NS2 coding sequence in molecular epidemiology investigations.