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In order to assess the molecular epidemiology of African swine fever (ASF) in Sardinia, we analyzed a wide range of isolates from wild and domestic pigs over a 31-year period (1978–2009) by genotyping sequence data from the genes encoding the p54 and the p72 proteins and the CVR. On this basis, the analysis of the B602L gene revealed a minor difference, placing the Sardinian isolates into two clusters according to their temporal distribution. As an extension of this study, in order to achieve a higher level of discrimination, three further variable genome regions, namely p30, CD2v, and I73R/I329L, of a large number of isolates collected from outbreaks in the years 2002–14 have been investigated. Sequence analysis of the CD2v region revealed a temporal subdivision of the viruses into two subgroups. These data, together with those from the B602L gene analysis, demonstrated that the viruses circulating in Sardinia belong to p72/genotype I, but since 1990 have undergone minor genetic variations in respect to its ancestor, thus making it impossible to trace isolates, enabling a more accurate assessment of the origin of outbreaks, and extending knowledge of virus evolution. To solve this problem, we have sequenced and annotated the complete genome of nine ASF isolates collected in Sardinia between 1978 and 2012. This was achieved using sequence data determined by next-generation sequencing. The results showed a very high identity with range of nucleotide similarity among isolates of 99.5 per cent to 99.9 per cent. The ASF virus (ASFV) genomes were composed of terminal inverted repeats and conserved and non-conserved ORFs. Among the conserved ORFs, B385R, H339R, and O61R-p12 showed 100 per cent amino acid identity. The same was true for the hypervariable ORFs, with regard to X69R, DP96R, DP60R, EP153R, B407L, I10L, and L60L genes. The EP402R and B602L genes showed, as expected, an amino acid identity range of 98.5 per cent to 100 per cent and 91 per cent to 100 per cent, respectively. In addition, all of the isolates displayed variable intergenic sequences. As a whole, the results from our studies confirmed a remarkable genetic stability of the ASFV/p72 genotype I viruses circulating in Sardinia.

Bovine viral diarrhea viruses (BVDV) are members of the Pestivirus genus within the family Flaviviridae. Based on antigenic and nucleotide differences, BVDV are classified into two recognized species, BVDV-1 and BVDV-2. More recently, a new putative pestivirus species, tentatively called "HoBi-like", has been associated with bovine viral diarrhea. HoBi-like viruses were first identified in fetal bovine serum (FBS) imported from Brazil. Subsequently, a number of HoBi-like viruses have been detected as contaminants in FBS or cell culture and in live ruminants. To further investigate the possible pestivirus contamination in commercially available FBS batches, 26 batches of FBS with various countries of origin, were tested in this study for the presence of bovine pestiviruses. All the 26 batches were positive by RT-PCR for at least one species of bovine pestiviruses. HoBi-like viruses were detected in 15 batches. Analysis of the 5'UTR and N(pro) sequences of 15 newly identified HoBi-like viruses combined with analysis of additional sequences from GenBank, identified 4 genetic groups tentatively named 3a-3d. The current study confirmed the presence of the emerging HoBi-like viruses in FBS products labeled with different geographic origins. This finding has obvious implications for the safety of biological products, such cell lines and vaccines.

14 páginas, 4 figuras, African swine fever (ASF), one of the most important diseases of swine, has been endemic in the Italian island of Sardinia for more than 35 years. During these decades, several strategies and eradication efforts have been implemented in the island with limited success. Strong climatic and ecological similarities exist between Sardinia and one area of the Iberian Peninsula where Ornithodoros erraticus ticks were involved in the persistence of ASF from 1960 to 1995. This fact leads to the hypothesis that, potentially, Ornithodoros ticks could be also involved in the ASF cycle in Sardinia, thus accounting for some of the reoccurring ASF outbreaks in this island. Initial efforts aimed at detection of Ornithodoros ticks in Sardinia were performed during the 1970s/80s with no positive results. Accordingly, the absence of Ornithodoros ticks in Sardinia has been generally accepted. However, since a new and reinforced ASF eradication program has been recently launched in Sardinia, it is essential to clarify the presence and role of these soft ticks in the epizootiology of ASF in this island. For that purpose, 1767 porcine serum samples collected from all around the island (1261 from domestic and 506 from wild boar) were analyzed by ELISA for antibodies to salivary antigens of Ornithodoros erraticus. In addition, Ornithodoros ticks were directly searched in a number of pig premises that have suitable habitats for these ticks and were located in areas repeatedly affected by ASF. Only one serum sample resulted positive in the serological assay, and no Ornithodoros ticks were collected in none of the premises. These results indicate that these soft tick species are not involved in the epizootic cycle of ASF in Sardinia, and highlight the importance of controlling other risk factors still present in the island for effectively eradicate the disease., This study has been partially funded by the European Research Project ASFORCE (‘Targeted research effort on African swine fever’, grant agreement 311931). Lina Mur had a scholarship (FPU programme) from the Spanish Ministry of Education and received a scholarship for a short stay in Sardinia, which partially allowed the development of this study

SummaryTwo European laboratories independently detected atypical bovine pestiviral nucleic acids in three commercial batches of foetal bovine serum (FBS) that was claimed by the producers to be of Australian origin. Additional batches of FBS were obtained directly from Australia to exclude possible

African swine fever virus (ASFV) is the aetiological agent of a highly lethal haemorrhagic disease affecting pigs that inflicts significant economic damage on the swine industry. ASF is present in many African countries, in several eastern and central European countries and in Sardinia (Italy). Sequence analyses of variable genomic regions have been extensively used for molecular epidemiological studies of ASFV isolates. Previous sequencing data of genes that codify for viral protein p54, p72 and the central variable region (CVR) within the B602L gene revealed that Sardinian isolates show a very low level of variability. To achieve a finer level of discrimination among such closely related viruses, in this study, we have chosen three different genome regions to investigate the within-genotype relationships and to provide a more accurate assessment of the origin of outbreaks. The analysis of p30 and I73R/I329L sequences obtained from ASFV collected in Sardinia over a 13-years period confirms a remarkable genetic stability in these regions. The sequence comparison of the protein encoded by the EP402R gene (CD2v), carried out on various strains from 1978 to 2014, revealed a temporal subdivision of Sardinian viruses into two subgroups: one group includes the historical isolates from 1978 to 1990, and the second one is comprised of the viruses collected from 1990 until 2014. These data, together with those obtained from CVR within the B602L gene analysis, demonstrated that the viruses circulating in Sardinia belong to p72 genotype I, but have undergone genetic variations in two different regions of the genome since 1990. We proposed the cytoplasmic region of CD2v protein as a new genetic marker that could be use to analyse ASFVs from different locations to track virus spread. Our study reaffirms the need to analyse other genome regions in order to improve the molecular characterization of ASFV.

Currently, the genus Pestivirus comprises four approved species, namely bovine viral diarrhoea viruses 1 and 2 (BVDV-1, BVDV-2), classical swine fever virus and border disease virus (BDV). Recently, three major genotypes have been identified within the species BDV and termed as subgroups BDV-1, BDV-2 and BDV-3. Here, an isolate from animals in a herd showing BD-like syndromes, which occurred in central Italy was analysed. A reverse transcriptase polymerase chain reaction was performed using primers that specifically amplify a fragment of the 5'-non-coding region (5'-NCR) from BDV. Both the 5'-NCR fragment and the entire Npro gene were sequenced and used for genetic typing. The 5'-NCR sequence revealed that the newly isolated Pestivirus could be allocated to the BDV species. Interestingly, the Npro sequence of this virus isolate significantly differed from all the ovine pestiviruses previously described, providing evidence for the presence of an additional subgroup within the species BDV.

A variable region of the gene encoding the major glycoprotein (E2) of Classical Swine Fever Virus (CSFV) was sequenced from 12 Sardinian isolates which had been obtained from three geographically distinct regions of the Island. Phylogenetic analysis of these viruses and others characterized in previous studies [1, 2] indicated that (a) the Sardinian viruses were all members of the common European subgroup 2·3 and were clearly distinct from live vaccines recently used in this area; (b) they could be resolved into four distinct groups in accordance with the region or date of isolation; (c) in at least two regions wild boar/domestic swine contact was implicated in virus spread; (d) the oldest isolate (1983) and some of the recent isolates were possibly introduced from mainland Italy. In addition, this study has wider implications for the interpretation of CSFV variation. We have been able to demonstrate that small variations within this region of the virus genome (possibly less than 2·7% or five nucleotide substitutions) can be used to separate isolates into groups that precisely fit their geographical distribution. This finding is especially important for deducing the epidemiological relationships between multiple outbreaks caused by similar viruses that occur in close proximity.

The presence of antibodies against African swine fever (ASF), a complex fatal notifiable OIE disease of swine, is always indicative of previous infection, since there is no vaccine that is currently used in the field. The early appearance and subsequent long-term persistence of antibodies combined with cost-effectiveness make antibody detection techniques essential in control programmes. Recent reports appear to indicate that the serological tests recommended by the OIE for ASF monitoring are much less effective in East and Southern Africa where viral genetic and antigenic diversity is the greatest. We report herein an extensive analysis including more than 1000 field and experimental infection sera, in which the OIE recommended tests are compared with antigen-specific ELISAs and immuno-peroxidase staining of cells (IPT). The antibody detection results generated using new antigen-specific tests, developed in this study, which are based on production of antigen fractions generated by infection and virus purification from COS-1 cells, showed strong concordance with the OIE tests. We therefore conclude that the lack of success is not attributable to antigenic polymorphism and may be related to the specific characteristics of the local breeds African pigs. © 2012 Elsevier B.V.

Currently, few information is available on the stimulation of neutralizing antibodies (NA) to porcine circovirus type 2 (PCV2). The aim of the present study was to characterize the humoral response, particularly the production of NA in pigs inoculated with an inactivated vaccine against PCV2. Twenty-five conventional pigs, seronegative to PCV2, were divided into two groups. Group 1 was composed of 14 animals that were injected by intramuscular route with 1 ml of inactivated vaccine. Group 2 was composed of 11 pigs and was used as a negative control. ELISA antibodies and NA were evaluated and a virological investigation was performed. The vaccine did not induce any clinical signs in immunized pigs. The animals in Group 1 developed IgG antibodies, detected by ELISA tests, between 14 and 42 post-vaccination days (PVD). In the same group, NA were produced between 35 and 42 PVD. In Group 2, neither ELISA antibodies nor NA were produced. No virus was isolated from any of the pigs during the experimental period.

Two European laboratories independently detected atypical bovine pestiviral nucleic acids in three commercial batches of foetal bovine serum (FBS) that was claimed by the producers to be of Australian origin. Additional batches of FBS were obtained directly from Australia to exclude possible contamination of the Australian FBS with that of South American origin during manufacturing/packaging in European countries. RT-PCR amplification of partial 5'untranslated region and the complete N(pro) gene yielded a specific band with expected size, which was confirmed by DNA sequencing. Bayesian analysis of sequence data demonstrated a closer phylogenetic relation of the newly detected atypical bovine pestiviruses to those of South American origin, which were related to the recognized bovine pestivirus species, bovine viral diarrhoea virus. Taken together, the results indicated the presence of atypical bovine pestiviruses in the Australian FBS, and most likely in Australian Continent.

Classical swine fever (CSF) is responsible of devastating outbreaks with high socio-economic impacts. Since vaccination has been banned in Europe, the current control systems are still based on massive culling of infected and suspect infected animals. Stamping out is considered a cost effective strategy, but it negatively impacts on community acceptance and animal welfare. For this reason use of antiviral drugs has been suggested as additional tool for controlling the spread of CSF as a good alternative to vaccination in offering an immediate protection. In the last few years the efficacy of 5-[(4-Bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP), a representative of a class of imidazopyridines, has been demonstrated in vitro and in vivo. We and others have already demonstrated the efficacy of Proanthocyanidin A2 (PA2) against RNA viruses. In particular, preliminary results showed that this natural compound possesses an extracellular antiviral activity against animal pestiviruses. The aim of this study was to assess in vivo the antiviral activity of Proanthocyanidin A2 in the phytosome form, against CSFV. This formulation demonstrated greater affinity for the epithelial stratum corneum compared to the pure molecule itself, with the potential to enhance PA2 absorption through digestive mucosa. Specific-pathogen-free pigs (n=4) received a PA2 daily dose of 100 mg/kg (dry powder directly mixed in feed) for 15 consecutive days, starting 1 day before infection with the CSFV Alfort/187 strain. Pigs receiving PA2-free feed (n=4) were infected and housed in a separate isolation pen as a positive-control group. Treated pigs showed significantly lower clinical signs and a lower viral genome load compared to the controls. Such results seem to indicate that PA2 phytosome is able to reduce CSFV spread and suggest that it might be simply added to feed, thus being suitable to treat large numbers of animals in case of outbreak to exert either prophylactic and therapeutic effects.

In order to investigate the genetic heterogeneity of small ruminant lentivirus (SRLV) isolates in Italy, 55 clinical samples collected between 1998 and 2010 were analysed. The phylogenetic study was based on analysis of gag–pol sequences. Our findings revealed that the SRLVs belonged to the subtype A9 (n = 3, sheep), B1 (n = 5, goat), B2 (n = 3, sheep) and E2 (n = 5, goat). Interestingly, 39 isolates from both sheep and goat, significantly differed from all the other SRLVs previously described and formed two separate clusters within genotypes A and B tentatively named A11 (n = 27, goat and sheep) and B3 (n = 12, goat and sheep), which have never been shown before. These results revealed a marked diversity among Italian field SRLV strains which might reflect the absence of any systematic control measures.

During 1992 and 1993, outbreaks of an acute, highly fatal disease mainly affecting adult rabbits were observed in Tunisia. The clinical and pathological findings were consistent with rabbit haemorrhagic disease. A monoclonal antibody designated PG4G3 specific for surface determinants of the rabbit haemorrhagic disease virus was used to identify the aetiological agent by ELISA and by colloidal gold immunoelectron microscopy; a haemagglutination test and conventional immunoelectron microscopy were also used. The results confirmed the first recorded cases of the disease in Tunisia.

Two commercial marker vaccines against classical swine fever virus (CSFV) and companion diagnostic tests were examined in 160 conventional pigs. To test the vaccines in a "worst case scenario", group of 10 weaners were vaccinated using a single dose of an E2 (gp55) based vaccine at days -21, -14, -10 or -7, and subsequently challenged at day 0. The challenge virus was CSFV 277, originating from a recent outbreak of classical swine fever (CSF) in Germany. In all groups, only 5 out of 10 pigs were challenged; the remaining 5 pigs served as vaccinated contact controls. Also, three control groups, each consisting of 10 non-vaccinated pigs, were challenged in parallel to the vaccinated animals. CSFV could be isolated from all non-vaccinated pigs. Among these pigs 40% displayed a chronic course of the infection (virus positive for more than 10 days). Pigs vaccinated 21 or 14 days before challenge displayed no clinical signs of CSFV after challenge. However, they were still able to replicate CSFV when challenged, as measured by reisolation of CSFV from leukocytes of the directly challenged pigs. CSFV could be isolated from the leucocytes of 25% of the pigs vaccinated 21 days before challenge and 50% of the pigs vaccinated 14 days before challenge. Chronic infection was not observed, but transmission to one vaccinated contact pig occurred. From all pigs vaccinated 10 or 7 days before challenge, CSFV could be reisolated. We observed a chronic course of infection in 5% of pigs vaccinated 10 days before challenge and in 30% of pigs vaccinated 7 days before challenge. The mortality rate was 20% in the pigs vaccinated 10 days before challenge, and varied between 20 and 80% in pigs vaccinated 7 days prior to challenge. The contact animals had lower mortality (0-20%) than directly challenged pigs, probably mirroring the delayed time point of infection. There was thus some protection against clinical illness by both marker vaccines, but not a solid protection against infection and virus shedding. The efficacy of the vaccine was best if used 3 weeks before challenge and a clear correlation between time interval from vaccination to challenge and the level of virus shedding was observed. Each vaccine had its own accompanying discriminatory ELISA, but 18% of the virus positive pigs never seroconverted in these tests.

Six laboratories participated in a study to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 25 samples of random primed cDNA, synthesised from viral RNA representative of different pestiviruses. The other set comprised samples of blood and serum obtained from virus-free or CSFV-infected pigs. Each laboratory tested the samples using PCR/RT-PCR according to a set of standardised protocols that specified the exact conditions and requirements for inclusion of control samples. Two types of test were evaluated. One amplified a part of the 5'-non coding region of the pestivirus genome by means of a closed, one-tube RT-nested PCR. The other amplified a part of the NS5B gene using non-nested RT-PCR. The results of the laboratories were compared with one another, and with those obtained earlier when similar samples were tested by the same laboratories using non-standardised methods [Paton et al., Classical swine fever virus: a ring test to evaluate RT-PCR detection methods, Vet. Microbiol., in press]. Standardisation of the protocols resulted in a more consistent test sensitivity. Three laboratories avoided significant false positive results. Others that did not, could nevertheless recognise that test specificity was inadequate from the results obtained with the control samples. Minimum requirements for the inclusion of adequate controls and periodic proficiency testing are proposed.

A big epidemic of classical swine fever (CSF) occurred in the European Community in 1997. The first case was reported at the beginning of January 1997 from Germany. The disease presumably spread to the Netherlands, and from there to Italy, Spain and eventually to Belgium. About 30 isolates from these outbreaks were analysed by comparison of the nucleotide sequence data generated from fragments of both the E2 glycoprotein gene (190 nucleotides) and from the 5'-nontranslated region (5'-NTR; 150 nucleotides). By combining epidemiological data with genetic typing, it was found that the outbreaks were related and caused by a virus belonging to the genetic subgroup 2.1. As this type of virus had been reported infrequently in Europe and not at all since 1993, we postulate that it was newly introduced into the European Union (EU). (C) 2000 Elsevier Science B.V.

A workshop was convened, at which seven enzyme-linked immunosorbent assays (ELISAs) were compared with virus isolation for the detection of viraemia in serial blood samples collected from six pigs at up to fourteen days after inoculation with classical swine fever virus. All ELISAs were of the double antibody sandwich type, using monoclonal and/or polyclonal antibodies to detect a variety of viral proteins in leukocytes, or in anti-coagulated blood or serum. Compared to virus isolation, specificity of the ELISA was good: only one sample found negative by virus isolation yielded a positive result in a single ELISA. Some false-negative results occurred with samples collected at up to eight days after inoculation, but all tests found samples collected between nine and fourteen days post-inoculation to be positive. The ELISAs require less-specialised facilities and can be performed much more rapidly than virus isolation. They are therefore extremely promising tools for screening large numbers of live pigs.

Two pairs of oligonucleotide primers were designed that specifically amplified regions of the classical swine fever virus genome. These products, corresponding to a 671 bp portion of the genes encoding the E1 and E2 (gp33 and gp55) proteins and a 1090 bp portion of the putative polymerase gene, were amplified from eight virus isolates which had been responsible for a series of classical swine fever outbreaks in Italy involving both domestic pigs and wild boar. For each virus the fragments were partially sequenced to give 475 bp of the E1/E2 glycoprotein and 212 bp of the putative polymerase gene sequences. The data from each set of fragments were compared with one another and with reference strains. This allowed us confidently to assign most of the viruses to one of three subgroups. An analysis of the same viruses with a panel of monoclonal antibodies was much less informative. The subgrouping of the isolates suggested that, in this region of Italy, there had been at least two separate introductions of classical swine fever over a 7 year period and that virus had been transmitted between domestic pigs and wild boar. A consensus nucleotide sequence derived from the glycoprotein fragments of all the viruses examined revealed conservation at the wobble position of some codons.