Search

Hypomyelination is a rare consequence of in utero bovine viral diarrhoea virus (BVDV) infection. We describe a BVDV outbreak in a naïve Holstein dairy herd in northern Italy, with an unusually high prevalence of calves with neurological signs, generalised tremors and ataxia. Histological analysis showed that hypomyelination was the predominant lesion and that the most typical BVDV neuropathological findings (e.g. cerebellar hypoplasia) were absent. Virological and molecular analyses showed that non-cytopathic BVDV genotype 1b was associated with the calves’ neurological signs and excluded other viruses responsible for congenital infection or neurological disorders. Whole-genome sequencing of BVDVs from the brain of a calf with neurological signs and the whole blood of a persistently infected herd-mate with no such sign showed >99.7 % sequence identity. Analysis of the quasispecies distribution revealed the greatest variation rates in regions coding for the structural proteins E1 and E2. Variation was slightly greater in the brain- than in the blood-derived sequence and occurred at different sites, suggesting the occurrence of distinct evolutionary processes in the two persistently infected calves. Molecular characterisation of BVDV genomes from five other calves with neurological signs from the same farm confirmed that the E1 and E2 regions were the most variable. Several factors, including genetic variability and host factors, appear to have contributed to the observed unique BVDV disease phenotype, characterised by hypomyelination and neurological signs.

Apart from the established pestivirus species Pestivirus A to Pestivirus K novel species emerged. Pigs represent not only hosts for porcine pestiviruses, but are also susceptible to bovine viral diarrhea virus, border disease virus (BDV) and other ruminant pestiviruses. The present study focused on the characterization of the ovine Tunisian sheep-like virus (TSV) as well as Bungowannah virus (BuPV) and BDV strain Frijters, which were isolated from pigs. For this purpose, we performed genetic characterization based on complete coding sequences, studies on virus replication in cell culture and in domestic pigs, and cross-neutralization assays using experimentally derived sera. TSV forms a distinct phylogenetic group more closely related to Pestivirus C (classical swine fever virus, CSFV) than to Pestivirus D (BDV). In contrast to BDV and BuPV, TSV replicates by far more efficiently on ovine than on porcine cells. Nevertheless, pigs were susceptible to TSV. As a consequence of close antigenic relatedness of TSV to CSFV, cross-reactivity was detected in CSFV-specific antibody assays. In conclusion, TSV is genetically closely related to CSFV and can replicate in domestic pigs. Due to close antigenic relatedness, field infections of pigs with TSV and other ruminant pestiviruses can interfere with serological diagnosis of classical swine fever.

Redspotted grouper nervous necrosis virus (RGNNV), genus Betanodavirus, family Nodaviridae, is the causative agent of viral encephalopathy and retinopathy (otherwise known as viral nervous necrosis) and can infect several fish species worldwide. Betanodaviruses, including RGNNV, are very resilient in the aquatic environment, and their presence has already been reported in several wild marine species including invertebrates. In order to investigate the interaction between a bivalve mollusc (Manila clam Ruditapes philippinarum) and RGNNV, we optimised a culture-based method. The bioaccumulation of the pathogenic RGNNV by R. philippinarum and the potential shedding of viable RGNNV from RGNNV-exposed clams were evaluated through a culture-based method. R. philippinarum clearly accumulated viable RGNNV in their hepatopancreatic tissue and were able to release viable RGNNV via faecal matter and filtered water into the surrounding environment. The role of clams as bioaccumulators and shedders of viable RGGNV could put susceptible cohabiting cultured fish at risk. RGNNV-contaminated molluscs could behave as reservoirs for this virus and may modify the virus epidemiology.

Background Cutaneous squamous cell carcinoma (cSCC) is an uncommon disease affecting koi carp (Cyprinus carpio). Cutaneous papilloma (carp pox) is a benign epidermal proliferation reported in koi and has been shown to be caused by Cyprinid Herpesvirus 1 (CyHV1). Hypothesis/Objectives Histological, ultrastructural and molecular investigations were carried out aiming to investigate the aetiology of cSCC within archived tissue samples. Animals Surgical samples of masses located on the integument, fins and lips of 13 koi carp belonging to different private owners were included in this retrospective study. Methods CyHV1 DNA and RNA presence were investigated in five cSCC formalin-fixed paraffin-embedded tissue samples to recognize CyHV1 presence and its replication activity. Results All cases were histologically diagnosed as cSCC. The ultrastructural observations confirmed the squamous differentiation of neoplastic epithelial cells, which showed abundant tonofilament bundles and desmosomes. Although no virus particles were revealed ultrastructurally, the molecular investigation detected viral DNA in five epidemiologically unrelated cSCC. Viral transcript analysis revealed no evidence for viral replication in the tested cSCC, which could be consistent with latent infection. Conclusions and clinical importance These findings illustrate the frequent association of carp cSCC with CyHV1, although a direct cause–effect relationship cannot be established at this time. Therefore, surveillance programmes should take into account the suspected viral origin of cSCC to better inform prevention and control of CyHV1 in the future.

The genus Pestivirus, which belongs to the Flaviviridae family, includes ssRNA+ viruses responsible for infectious diseases in pigs, cattle, sheep, goats and other domestic and wild ruminants. Like most of the RNA viruses, pestivirus has high genome variability with practical consequences on disease epidemiology, diagnosis and control. In addition to the officially recognized species in the genus Pestivirus, such as BVDV-1, BVDV-2, BDV and CSFV, other pestiviruses have been detected. Furthermore, most of the ruminant pestiviruses show low or absent species specificity observed in serological tests and are able to infect multiple species. Particularly, small ruminants are receptive hosts of the most heterogeneous group of pestiviruses. The aim of this study was to carry out the molecular characterization of pestiviruses isolated from sheep and goats in Sicily, Italy. Phylogenetic analysis of two viral genomic regions (a fragment of 5'-UTR and the whole Npro regions) revealed the presence of different pestivirus genotypes in the analysed goat and sheep herds. Two of five viral isolates were clustered with BVDV-1d viruses, a strain widespread in Italy, but never reported in Sicily. The other three isolates formed a distinct cluster with high similarity to Tunisian isolates, recently proposed as a new pestivirus species. This represents the first evidence for Tunisian-like pestivirus presence in small ruminants in Italy. Furthermore, one of the isolates was collected from a goat, representing the first isolation of Tunisian-like pestivirus from this species.

The major viral diseases that affect rainbow trout (Oncorhynchus mykiss) are viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and sleeping disease (SD). In the presented study, we developed a multiplex RT-PCR (mRT-PCR) assay for the simultaneous detection of these four rainbow trout viruses in a single assay. The choice of primers was carried out based on the expected size of the fragments, the temperature and time required for the amplification, and the specificity for the target sequence. Firstly, the method was optimised using reference strains of viral haemorrhagic septicaemia virus (VHSV), infectious haematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV) and sleeping disease virus (SDV) cultivated with permissive cell culture lines; subsequently, the method was used for the identification of these viral infections in rainbow trout samples. Twenty-two samples of rainbow trout, clinically suspected of having viruses were analysed by the developed method to detect the presence of the four viruses, by directly analysing the animal tissues. The mRT-PCR method was able to efficiently detect the viral RNA in infected cell culture supernatants and in tissue samples, highlighting the presence of single infections as well as co-infections in rainbow trout samples. VHSV/SDV and IHNV/SDV co-infections were demonstrated for the first time in rainbow trout. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.

This study reports the detection of co-infection by multiple CPV variants and the high genetic complexity of a CPV-2 strain detected in a domestic cat. The CPV variants selected by cloning the VP2 gene were sequenced, and genetic diversity and selection pressure were investigated. Comparison of the nucleotide sequences has evidenced 10 different viral populations, and, in the same animal, more CPV variants coexist. Our analysis excludes the possibility that the recombination events took place during infection and that negative selection acted on the VP2 gene. These findings confirm that CPV-2 shows high genetic heterogeneity resembling the quasispecies found in RNA viruses.

The orf virus is the type species of the Parapoxvirus genus and is the causative agent of contagious echtyma, a debilitating skin disease of sheep and goats, which can also affect man. The virus exhibits a restricted host range, even if it has been shown to bind to a wide range of tissues of non-permissive species. This ability is an argument for its potential use as an expression vector. Since most mammalian cell types express heparan sulfate (HS) surface receptors, we assumed that HS could serve as receptors to mediate orf virus binding. In this study, we showed that orf virus is inhibited by the addition of soluble heparin in cell cultures. Affinity chomatography using heparin agarose demonstrated that orf virus F1L is the major heparin binding protein. Furthermore, the recombinant F1L protein was visualised on the cell surface by confocal microscopy, and rabbits immunised with recombinant F1L protein produced virus neutralising antibodies. These results confirm that the F1L immunodominant protein is also involved in virus binding to cells as for the vaccinia homologue H3L protein. Heparin also inhibited the binding of the F1L protein to cells showing that this protein has a role in the early stages of infection.

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.

Proliferative orf virus infections in adult sheep have increased in Italy in the past few years: these extreme cases are frequently fatal and difficult to differentiate from other infectious diseases of sheep such as blue tongue. A probable explanation for the proliferative and highly vascularized nature of the lesions was found in the expression of the VEGF-E gene encoded by the orf virus. To investigate a possible role of the viral VEGF in the pathogenesis of severe persistent orf virus lesions, the activity of four VEGF-E variants was compared by an angiogenesis in vitro model. Similar angiogenic activity was found between strains isolated from the classical and the proliferative forms of the disease, even if the latter was able to develop a higher number of vessels during the first 24 h of infection. Our in vitro findings seems to exclude that the VEGF variants encoded by the strain isolated from the atypical form of the disease could be the responsible for the histopathological aspect of the proliferative lesions.

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.

This study examined the in vitro effects of temperature on Betanodavirus infection in the SSN-1 cell line. A Betanodavirus isolated from moribund sea bass fry (Dicentrarchus labrax) farmed in the Adriatic Sea and characterised as a RGNNV (Redspotted grouper nervous necrosis virus) genotype was used in our study. Virus infected SSN-1 cells were incubated at temperatures between 10° and 30°C and observed for cytopathic effects daily for 15 days. Cell-free and cell-associated viral growth were evaluated by 50% tissue culture infectious dose (TCID50) titration at 0, 24, 48, 72, 96, 144, 192, 240, 312 and 360 hours post-infection. Virus replication was observed at all temperatures from 15°C to 30°C. The optimal temperature for virus growth was 25°C. A temperature of 10°C was detrimental to the growth of the SSN-1 cells and cell death interfered with interpretations of viral growth. The isolate of Betanodavirus from Italian sea bass in this study demonstrates a different temperature range for growth compared to previous reports for related Betanodavirus strains and most likely this is due to an adaptation to the normal environmental temperatures of the host fish species of origin.

A rotor-gene SYBR Green base real time system was developed for the detection and quantification of Betanodavirus. Two primers set were designed to target conserved sequence in the nodavirus capsid protein gene, yielding an amplification product of 122 and 116 nucleotides. A plasmid was cloned and quantify for producing standard curve. The method was then apply to cDNA retrotranscripted from RNA extracted from cell-supernatants and directly from tissues of infected animals. The assay allowed analitycal enumeration of 300 copies of plasmid and 500 target cDNA copies for primer set 1 and 10 copies of plasmid and 20 target cDNA copies for primer set 2. The reproducibility of the assay was evaluated by intra-assay variability of standard control that showed a coefficient of variation (CV) ranging from 1.5 to 4.6 and from 1.5 to 4.9 respectively for the two assaysas. Intra-assay and inter-assay variability were further assessed using high and low amounts of cDNA showing a high reproducibility. The method revealed to be very sensitive and quick, reducing post-PCR processing and suitable for both diagnostic and pathogenesis studies.

A real time quantitative PCR assay based on TaqMan technology was developed for orf virus (ORFV) DNA quantification in clinical samples, infected cells and organotypic cultures. This method was based on the amplification of a 70 bp fragment from the ORFV B2L gene (orthologue of the Vaccinia virus Copenhagen F13L gene) that encodes the major envelope protein. Both intra- and inter-assay variability were well within +/-0.25 log(10) S.D. showing the high efficiency and reproducibility of the assay. The TaqMan PCR was subsequently used to determine the titre of several batches of the ORFV strain NZ-2, with it being possible to quantify virus solutions in the range of 1 x 10(1) to 1 x 10(6) TCID(50)/ml. A good correlation between the titre determined by the TaqMan PCR and by conventional endpoint dilution was found. The PCR assay is reproducible and can be used for a rapid quantification of ORFV in vitro and ex vivo, being readily achievable within 1h.

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 .