Sara Ciulli

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.

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.

Detection and quantification of Betanodavirus by real time PCR

Betanodavirus infection is now widespread in several fish species worldwide and it causes severe economic losses in European sea bass (Dicentrarchus labrax) farming in Italy. Betanodavirus are small ssRNA non-envelope viruses. Based on phylogenetic analysis of the coat protein gene, Betanodavirus can be classified into four genotypes: SJNNV, TPNNV, BFNNV, RGNNV (Nishizawa et al., 1997). The virus is widespread both in farmed and wild animals (Gagné et al., 2004; Gomez et al., 2004) and it has high resistance to environmental and chemico-physical disinfectant treatment, so control by direct prophylaxis is particularly difficult. Furthermore, fattening-farm is often conducted in sea cages or in extensive brackish ponds with no clear-cut separation between wild and farmed fish (Munday et al., 2002). Despite a recent work (Thiéry et al., 2004) reporting the presence of a SJNNV-like virus in a sole farmed in the Mediterranean, the main genotype widespread in this region and responsible for the outbreaks on sea bass farms is the RGNNV. Unfortunately, there is no vaccine available for this disease, due to a limited knowledge about the pathogenesis of Betanodavirus infection, so control is based on early diagnosis and broodstock control. In this study we set up a new Real Time PCR diagnostic method. This technique was found to be rapid, practical and sensitive. Several Real Time PCR methods have recently been set up to detect numerous pathogens significantly contributing to studies on pathogenesis (Gilad et al., 2004), diagnosis and antiviral activity (Yun et al., 2003).

Defensive response of European sea bass (Dicentrarchus labrax) against Listonella anguillarum or Photobacterium damselae subsp. piscicida experimental infection.

Sea bass were experimentally infected with Listonella anguillarum or Photobacterium damselae subsp. piscicida (Phdp). At 24 and 72h post-infection, the expression analysis of immune-relevant genes (IL-1β, IL-6, IL-8, Hepcidin), the transcriptional level and detection of HSP70, and the quantification of serum iron were investigated in association with the histological analysis and the bacterial recognition in tissues by immunohistochemistry. At 15 days post-infection, the specific antibody response was detected in surviving fish, as well as the transcriptional levels of TcR and BcR sequences. Both experimental infections were characterized by a similar acute response, whereas different histological and immunohistochemistry evidences were observed. In particular, the early reaction appeared suitable for the clearance of L. anguillarum, thus limiting the histological lesions, the bacterial dissemination and the further development of acquired immunity in surviving fish. On the contrary, the innate response appeared not enough to resolve the Phdp infection, which was characterized by tissue damage, bacterial widespread and substantial detection of specific humoral immunity in surviving fish, also associated to lymphocytes clonal expansion. Besides the opportunistic conditions involved in fish vibriosis and pasteurellosis, the comparison between these experimental infection models seems to suggest that the rate of development of the acquired immunity is strictly linked to the activation of the host innate response combined to the degree of bacterial virulence.

Genotyping of bovine viral diarrhea viruses (BVDV) isolated from cattle in Sicily

Bovine Viral Diarrhea–Mucosal Disease (BVD–MD) is a widely spread infectious disease that causes important economic losses in farms. Several epidemiological studies indicate a high genetic heterogeneity among Bovine Viral Diarrhea Virus (BVDV) strains circulating in Italy. The aim of this study was to investigate the genotypes of BVDV in Sicily, a region in the South of Italy. For this purpose, 17 BVDV strains collected from cattle breed in Sicily between 2005 and 2008 were genetically typed by sequencing of the 5′-untraslated region (5′-UTR) of the viral genome. In this study, phylogenetic analysis showed that all 17 examined strains were clustered within the BVDV genotype 1. Particularly, 14 of them were clustered with the BVDV-1b subgroup, while the remaining three strains were clustered with the BVDV-1e. Moreover, the restriction analysis indicated a bovine origin for all of the 17 strains typed in this study. These results could be useful to carry out an epidemiological survey and to create vaccines that protect cattle against BVDV different subgroups.

The use of sscp analisys to evidence genetic variability in the gene coding for immunodominant protein e2 of the BVDV

The use of sscp analisys to evidence genetic variability in the gene coding for immunodominant protein e2 of the BVDV S. Ciulli & E. Galletti & F. De Giorgi & M. Battilani & S. Prosperi Published online: 6 August 2008 # Springer Science + Business Media B.V. 2008