Santino Prosperi

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

The VP2 genes of Italian canine parvovirus (CPV) type 2 strains isolated from dogs and wolves were sequenced and a three-dimensional model of the VP2 capsid protein was constructed. Two mutations were detected in the VP2 sequences of the Italian strains: one at residue 297 and one at residue 265. Variant 297 is the predominant CPV isolate in Europe, whereas variant 265 has never been detected before. The mutation at residue 265 causes a disruption in a G strand of the beta-barrel in the VP2 protein. Data on strains isolated from wolves demonstrated that the same strain of CPV can circulate among domestic and wild canids; therefore, this result leads us to exclude the possibility that a separate parvovirus pool exists in wild populations.

Genetic analysis of canine parvovirus isolates (CPV-2) from dogs in Italy.

Genetic and antigenic properties of 62 field isolates of canine parvovirus (CPV-2) collected from 1994 to 2001 in Italy were investigated. Antigenic characterisation was conducted using specific monoclonal antibodies (Mabs). The VP1\VP2 gene was amplified by PCR and characterised with restriction endonucleases to detect the 297 and 265 variant. The VP2 gene of 16 isolates was sequenced and molecular genetic analysis was conducted. The antigenic type prevalent among our isolates is type 2a as well as the 297 variant, which is also prevalent in the rest of Europe. Only the 9.7% of the isolates have the T265P mutation. The VP2 sequences of CPV-2 isolates were very similar to recent Asian isolates. In the threefold spike of CPV-699 a coding change was detected in the 440 residue where threonine was substituted by alanine: the same mutation has been found in two Asian CPV-2 isolates from leopard cats [Virology 278 (2000) 13]. Phylogenetic analysis revealed that the Italian CPV-2 strains followed the same evolution as observed in other countries and they gave no indication of a separate lineage.

Genetic complexity and multiple infections with more Parvovirus species in naturally infected cats

Parvoviruses of carnivores include three closely related autonomous parvoviruses: canine parvovirus (CPV), feline panleukopenia virus (FPV) and mink enteritis virus (MEV). These viruses cause a variety of serious diseases, especially in young patients, since they have a remarkable predilection for replication in rapidly dividing cells. FPV is not the only parvovirus species which infects cats; in addition to MEV, the new variants of canine parvovirus, CPV-2a, 2b and 2c have also penetrated the feline host-range, and they are able to infect and replicate in cats, causing diseases indistinguishable from feline panleukopenia. Furthermore, as cats are susceptible to both CPV-2 and FPV viruses, superinfection and co-infection with multiple parvovirus strains may occur, potentially facilitating recombination and high genetic heterogeneity. In the light of the importance of cats as a potential source of genetic diversity for parvoviruses and, since feline panleukopenia virus has re-emerged as a major cause of mortality in felines, the present study has explored the molecular characteristics of parvovirus strains circulating in cat populations. The most significant findings reported in this study were (a) the detection of mixed infection FPV/CPV with the presence of one parvovirus variant which is a true intermediate between FPV/CPV and (b) the quasispecies cloud size of one CPV sample variant 2c. In conclusion, this study provides new important results about the evolutionary dynamics of CPV infections in cats, showing that CPV has presumably started a new process of readaptation in feline hosts.

Quasispecies composition and phylogenetic analysis of feline coronaviruses (FCoVs) in naturally infected cats

Abstract Quasispecies composition and tissue distribution of feline coronaviruses (FCoVs) were studied in naturally infected cats. The genomic complexity of FCoVs was investigated using single-strand conformational polymorphism (SSCP) analysis of N and ORF7b amplicons, and the evolutionary process was investigated by sequence-based phylogenetic analysis. SSCP analysis showed high heterogeneity of the FCoV genome which was correlated with the seriousness of the clinical form. The two genomic regions analysed showed different levels of variation; the N region demonstrated significant heterogeneity as compared to ORF7b. Phylogenetic analysis of the nucleotide sequences showed the clear separation of sequences analysed on the basis of virulence and geographical origin. A maximum likelihood analysis of N and ORF7b data sets showed a situation of strong heterogeneity for the N region.

Molecular Analysis of the NP Gene of Italian CDV Isolates

Canine distemper virus (CDV) is a highly contagious pathogen that occurs worldwide, causing a fatal disease in young carnivores. This virus is a member of the genus Morbillivirus in the family Paramixoviridae. The disease has been controlled throughout the world using live attenuated vaccines, but in the last few years an increasing number of distemper cases in vaccinated dogs has been recorded in Italy as well as in other European countries. (Blixenkrone-Moller et al., 1993). Epidemiological, serological and histopatological research suggested that the ‘new CDVs’ had altered antigenicities and/or different pathogenicities from the old strains (Yoshida et al. 1998). Infections caused by these new viruses may explain the increase of distemper cases in Italy. Analysis of the nucleocapsid protein (NP) gene of wildtype strains indicated a separate cluster from the vaccine strain (Yoshida et al., 1998). The NP protein is the most abundant viral structural protein and has been was demonstrated to influence viral persistence as well as regulatory functions such as transcription and replication (Stettler and Zurbriggen, 1995). In this study we analysed a partial nucleotide sequence of the NP gene of four wildtype CDV strains isolated in Italy.

Isolation and Genetic Characterization of Betanodavirus from Wild Marine Fish from the Adriatic Sea

Ciulli, S., Galletti, E., Grodzki, M., Alessi, A., Battilani, M. and Prosperi, S., 2007. Isolation and genetic characterization of Betanodavirus from wild marine fish from the Adriatic Sea. Veterinary Research Communications, 31(Suppl. 1), 221–224

Characterisation of immunodominant protein encoded by the F1L gene of orf virus strains isolated in Italy.

Summary. We analysed the molecular properties of the immunodominant protein of different orf virus strains isolated in Italy. The F1L encoding genes and the deduced amino acid sequences of all strains were determined and compared, and they showed several mutations. Structural analysis was carried out in order to assess the influence of amino acid variations on protein structure demonstrating a conservation of the secondary structure. Western blot analysis and immunogold electron microscopy showed that all orf virus strains were antigenically identical. The results of our study confirmed the immunogenicity of the F1L protein; furthermore, our data suggest a possible involvement of the protein in the virus cycle.

Development and application of a real-time PCR assay for the detection and quantitation of lymphocystis disease virus

Lymphocystis disease virus (LCDV) is responsible for a chronic self-limiting disease that affects more than 125 teleosts. Viral isolation of LCDV is difficult, time-consuming and often ineffective; the development of a rapid and specific tool to detect and quantify LCDV is desirable for both diagnosis and pathogenic studies. In this study, a quantitative real-time PCR (qPCR) assay was developed using a Sybr-Green-based assay targeting a highly conserved region of the MCP gene. Primers were designed on a multiple alignment that included all known LCDV genotypes. The viral DNA segment was cloned within a plasmid to generate a standard curve. The limit of detection was as low as 2.6DNA copies/μl of plasmid and the qPCR was able to detect viral DNA from cell culture lysates and tissues at levels ten-times lower than conventional PCR. Both gilthead seabream and olive flounder LCDV has been amplified, and an in silico assay showed that LCDV of all genotypes can be amplified. LCDV was detected in target and non-target tissues of both diseased and asymptomatic fish. The LCDV qPCR assay developed in this study is highly sensitive, specific, reproducible and versatile for the detection and quantitation of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains.

In Vitro Evaluation of Antiviral Activity of Ribavirin Against Canine Distemper Virus

Canine distemper virus (CDV) a member of the Morbillivirus genus of the family Paramyxoviridae, is a highly infectious and an acknowledged lethal pathogen of many carnivores. The disease is controlled by vaccination but an increasing number of distemper cases has been recorded even in vaccinated dogs all over the world and the affected animals can display catharral and or neurological symptoms. At present, there is no specific therapy, even if there is an increasing demand for one from the dog owners. For this reason the research on antiviral compounds against distemper is aimed at finding active molecules to treat the infection. Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carbox-amide, RBV) is a broad spectrum antiviral agent (Garcia et al., 2001; Crance et al., 2003; Poynard et al., 1998) that has been licensed for clinical use (as an aerosol) in the treatment of respiratory syncytial virus and for the therapy of measles pneumonia (Forni et al., 1994) and subacute slerosing panencephalitis (Hara et.al, 2003; Hosoya et al., 2004). The aim of the present study was to evaluate the in vitro antiviral activity of ribavirin against CDV.

Development of a SYBR Green real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of canine adenovirus type 1 and type 2.

Canine adenovirus type 1 (CAdV-1) and canine adenovirus type 2 (CAdV-2) cause infectious canine hepatitis (ICH) and infectious tracheobronchitis (ITB) in dogs, respectively. Cases of ICH have been documented in recent years and recent surveys have demonstrated a wide percentage of asymptomatic CAdV-1 infection in the canine population. Since both CAdV types are detectable in the same biological matrices, and viral coinfection with CAdV-1 and CAdV-2 are reported with high frequency, it is urgent to have available a rapid, highly sensitive and specific assay for the diagnosis of CAdV infection and distinction between CAdV-1 and CAdV-2. In order to detect canine adenovirus in biological samples and to rapidly distinguish the two viral types, a SYBR Green real-time PCR assay was optimized to discriminate CAdV-1 and CAdV-2 via a melting curve analysis. The developed assay showed high sensitivity and reproducibility and was highly efficient and specific in discriminating the two CAdV types. This reliable and rapid technique may represent a simple, useful and economic option for simultaneous CAdV types detection, which would be feasible and attractive for all diagnostic laboratories, both for clinical purposes and for epidemiological investigations.