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infection but also cause a progressive, fatal immune-mediated disease, feline infectious peritonitis (FIP). The structural proteins of FCoVs include the spike (S), the membrane (M), and, the most representative, the nucleocapsid protein (N). Coronavirus N proteins vary from 377 to 455 amino acids in length, are highly basic, have a high serine content (7–11%), and are potential targets for phosphorylation. Antigenic studies have shown that the N protein is one of the immunodominant antigens in the CoV family. The cellular immune response against the N protein of some animal coronaviruses can enhance recovery from the virus infection. Immunization with a cell lysate using a recombinant baculovirus-expressing feline infectious peritonitis virus (FIPV) nucleocapsid protein was effective in preventing the progression of FIP. To investigate the antigenic role of the N protein, we carried out a computational analysis of the N protein of FCoV strains detected in healthy and diseased cats on the basis of the primary amino acid sequences.

The full-length F1L gene was amplified by PCR and cloned into pCR T7/CT-TOPO vector fused with a six-histidine tag at the C terminus. Full-length F1L protein was expressed in Escherichia coli BL21 and was purified using a metal-chelate affinity chromatography. Two rabbits were immunized with the purified recombinant full-length F1L protein in Gerbu adjuvant and the immune response of the animals was checked by Western immunoblot analysis and virus neutralization. The results showed that F1L protein is able to stimulate the production of neutralizing antibodies .

Betanodaviruses are small ssRNA viruses responsible of Viral Nervous Necrosis in marine fishes worldwide. Genetic analysis of a fragment of the coat protein gene sequences permit to divide Betanodavirus in four genotypes. Preview studies evidenced Betanodavirus infection in several wild asymptomatic fish species, also in the Mediterranean Sea (1) and in invertebrates from Korea, mostly crustaceans and in a mussel (2). Both fish and invertebrates could be a potential source of virus for farmed fish. In this study, we examined bivalve mollusks for the presence of Betanodavirus and genetically analyzed the detected viruses to find out whether these animals could be a source of genetically closely related Betanodaviruses transmissible horizontally to wild and farmed fish. A total of 46 samples were analyzed including species reared on the seabed (Tapes philippinarum; Crassostrea gigas) and in the water column (Mytilus galloprovincialis). Betanodavirus presence was investigated by RT-nested/PCR (3). Positive samples were sequenced to study the variable region of the viral RNA2 coding for the capside protein. No viruses were detected in mussels (M. galloprovincialis), whereas clams (T. philippinarum) and oysters (C. gigas), both species reared on the seabed, resulted highly positive (26.6%). Phylogenetic analysis showed the belonging of these viruses to the RGNNV genotype. Betanodaviruses from clams clustered with viruses detected in wild and farmed fish collected from the same region (Adriatic Sea). Oyster viruses clustered separately, however inside the same genotype; considering the foreign origin of oysters (France), they could act as vectors of the infection between different areas. This is the first study focusing on Betanodavirus in bivalve mollusks; it evidences important presence of Betanodaviruses in bivalve mollusks; in some cases viral strains are highly correlated to those found in fish. These results highlight the possible role of mollusks in the spread of Betanodavirus to wild and farmed fish.

Nervous Necrosis Virus (NNV) represents one of the most threatening pathogens for Mediterranean aquaculture. Several NNV strains are currently co-circulating in the Mediterranean Basin with a high prevalence of the RGNNV genotype and the RGNNV/SJNNV reassortant strain and a more limited diffusion of the SJNNV genotype and the SJNNV/RGNNV reassortant. In the present study, a one-step multiplex RT-PCR (mRT-PCR) assay was developed as an easy, cost-effective and rapid diagnostic technique to detect RGNNV and the reassortant RGNNV/SJNNV strain and to distinguish them from SJNNV and the reassortant SJNNV/RGNNV strain in a single RT-PCR reaction. A unique amplification profile was obtained for each genotype/reassortant enabling their rapid identification from cell culture lysates or directly from brain tissues of suspected fish. The method’s detection limit varied between 102.3 and 103.4 TCID50 ml-1 depending on viral strains. No cross-reacitivty with viruses and bacteria frequently associated with gilthead seabream, European seabass and marine environment was observed. The mRT-PCR was shown to be an accurate, rapid and affordable method to support traditional diagnostic techniques in the diagnosis of VNN, being able to reduce considerably the time to identify the viral genotype or the involvement of reassortant strains.

Lymphocystis is a cutaneous viral disease, caused by a virus (LCDV) of genus Lymphocystivirus, family Iridoviridae. The disease is frequently benign and in the Mediterranean area it affects mainly Gilthead seabream (Sparus aurata). This disease, in seabream farms, causes significant economic losses related to poor growth rate, non-marketability of fish with lesions and mortality due to secondary infections. LCDV infection causes single or clustered tumour-like cutaneous nodules. Histology, generally, does not evidence lesions in internal organs, however, recent studies have revealed the systemic spread of the virus. The in vitro isolation of LCDV is difficult, reducing the possibilities of pathogenetical investigations on lymphocystis. Recently, our research team has developed a real time PCR (qPCR) assay able to detect and quantify the LCDV DNA both in diseased and recovered fish. In this study, the qPCR assay has been applied to detect the LCDV DNA in target (pectoral and caudal fins) and not target (spleen, brain and eyes) organs of Gilthead seabream at different clinical stage (diseased/in regression) and asymptomatic. This study has revealed the viral DNA presence in both target and not target organs with statistically higher values in pectoral and caudal fins than in internal organs. Diseased, in regression and asyntomatic fish have shown similar viral DNA distribution.

Spring viremia of carp is a highly contagious viral disease caused by Rhabdovirus carpio which is a single strand RNA virus. A combination of one step RT-PCR (reverse transcription) and Semi-nested PCR was used to detect spring viremia of carp virus in infected cell cultures (EPC) and fish tissues. All the fish samples were collected from Lake Shkodra and from artificial ponds in different region of Albania. Total RNA was extracted from 500 μl of fish tissue extract or from 100 μl of supernatant from infected EPC cell cultures. One step RT-PCR is performed using a modification of the methods of Stone et al.(1) in a 15 μl reaction mix volume. We used SVCV F1 (primer forward) 5’- TCT-TGG-AGC-CAA-ATA-GCT-CAR*-R*TC-3’ and SVCV R2 ( primer reverse) 5’-AGA-TGG-TAT-GGACCC- CAA-TAC-ATH*-CAN*-CAY*-3 primers for the amplification of 714 bp fragment of SVCV cDNA (2). The reaction mix was subjected to thermal cycles (2). Amplified cDNA was analyzed in agarose gel electrophoresis. For the second round of amplification we used a semi-nested PCR with SVCV F1 5’-TCT-TGG-AGC-CAA-ATA-GCT-CAR*- R*TC-3’ and SVCV R4 5’-CTG-GGG-TTT-CCN*-CCT-CAA-AGY*-TGY*-3* primers (1). Amplified fragment cDNA (606 bp) is analyzed by agarose gel electrophoreses (2).The results of gel electrophoreses was positive for three fish samples (Cyprinus carpio) collected from Lake Shkodra. This is the first report of the detection of spring viremia of carp virus in Albania in a natural habitat like Lake Shkodra.

I virus sono parassiti endocellulari obbligati in grado di provocare patologie negli animali, nell’uomo e nelle piante. Queste forme di vita relativamente semplici hanno sviluppato strategie complesse per sopravvivere e diffondersi. I virus animali sono frequentemente causa di epidemie con grave impatto socioeconomico e rivestono una notevole importanza sia in campo veterinario che umano. Molti virus animali quali il virus della rabbia, Nipah, Hendra, Monkeypox sono trasmissibili all’uomo e come tali sono responsabili di importanti zoonosi. Inoltre, la recente epidemia di SARS nella popolazione umana, ha nuovamente sottolineato l’importanza degli animali come serbatoi di agenti virali patogeni per l’uomo. La ricerca sui virus animali ha da sempre contribuito ad accrescere le conoscenze sulle interazioni virus-ospite, la replicazione e l’evoluzione di agenti virali di interesse umano. Ancora oggi, i virus animali vengono largamente utilizzati come surrogati di virus umani nella ricerca su nuovi farmaci antivirali o per la messa a punto di vaccini innovativi. Si stima che circa il 20% delle forme tumorali sia provocato da agenti virali ed i virus vengono oggi utilizzati anche come vettori per il delivery genico nella terapia antitumorale e nella cura di malattie genetiche. L’obiettivo principale di questo volume è quello di fornire le nozioni di base sugli agenti virali responsabili delle più importanti malattie degli animali ai fini di comprenderne i meccanismi patogenetici e le strategie utilizzate per il loro controllo e la loro prevenzione.