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Background: Classical swine fever virus (CSFV), species member of the family Flaviviridae, is generally considered restricted to domestic and wild suids. The circulation of CFSV has been detected in cattle herds in China and India. Natural infection appeared associated with clinical signs in some cases.Aim: The secondary structures of the internal ribosome entry site in the 5ʹ untranslated region (UTR) were used for the genomic characterization of bovine strains.Methods: Sequences have been compared to the representative CSFV strains isolated from pigs, vaccines, and contaminants from porcine cell lines and an ovine strain isolated in Spain.Results: The observed sequences from cattle showed a genetic relatedness with live-attenuated vaccine strains used in pigs. Sequence characteristics of the Chinese strain S171 are genetically distant from the previously reported CSFV genotypes, suggesting a new outgroup in the species, described for the first time, and named CSFV-d. Other Chinese strains were genetically closely related to CSFV genotype a2 (Alfort type) pig strains. Indian strains, reported from the states of Tamil Nadu and Meghalaya, were genetically closely related to CSFV genotype a1 (Brescia type) and a5 pig strains, respectively.Conclusion: These preliminary observations are new and relevant in countries, where CSFV control and eradication strategies are applied. Keywords: Cattle, Classical swine fever virus, Pestivirus, Secondary structure.

Pestiviruses are responsible for widespread diseases affecting cattle, pigs and other ruminants, presenting a wide range of clinical manifestations, with significant impact on animal production. Given the recent various reports of a relatively high number of new strains and atypical genomic variants, in the present study, ninety-seven genomic sequences from southern Italy have been evaluated applying the palindromic nucleotide substitutions method, based on 5’-UTR secondary structure alignment and computing genetic distance among strains in the internal ribosome entry site. Sequence analysis revealed a highly heterogeneous virus population, indicating the introduction of virus variants of Bovine viral diarrhea virus and Border disease virus species from foreign countries. The application of different analytical procedures was useful to avoid interpretation difficulties. Circulation of heterogeneous virus populations showed the need for more accurate epidemiological investigations and stringent veterinary controls to protect animal health and welfare.

The genus Pestivirus from the family Flaviviridae is represented by four established species; Bovine viral diarrhea virus 1 (BVDV-1); Bovine viral diarrhea virus 2 (BVDV-2); Border disease virus (BDV); and Classical swine fever virus (CSFV); as well a tentative species from a Giraffe. The palindromic nucleotide substitutions (PNS) in the 5′ untranslated region (UTR) of Pestivirus RNA has been described as a new, simple and practical method for genotyping. New software is described, also named PNS, that was prepared specifically for this PNS genotyping procedure. Pestivirus identification using PNS was evaluated on five hundred and forty-three sequences at genus, species and genotype level using this software. The software is freely available at www.pns-software.com .

Ovine herpesvirus 2 (OvHV-2) causes sheep-associated malignant catarrhal fever (SA-MCF), and is responsible for economic losses in cattle and other susceptible species around the world. A survey of 154 serum samples from 14 flocks in 3 Japanese prefectures (Hokkaido, Aomori and Iwate) was undertaken between 2007 and 2008 to test for antibodies to OvHV-2. OvHV-2 was present in 56 sheep and 2 goats, with 37.66% of samples having a positive reaction using a serum neutralization test. The immune reaction reported in goats could result from Caprine herpesvirus-2. These results indicate that sheep are reservoirs for OvHV-2 in the field in Japan, and they might transmit the virus to susceptible cattle and wild fauna.

The palindromic nucleotide substitutions (PNS) at the three variable loci (V1, V2 and V3) in the 5′-untranslated region (UTR) of the Pestivirus genome have been considered for taxonomical segregation of the species, through the evaluation of 534 strains. On the basis of qualitative and quantitative secondary structure characteristics, species have been identified within the genus, determining genetic distances between species isolates, clarifying borderline and multirelated sequences, and characterizing and clustering the Pestivirus strains showing unexpected genomic sequences. Nine genomic groups have been identified: the species Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Border disease virus (BDV) and Classical swine fever virus (CSFV) and the tentative species Pronghorn, Giraffe, Bovine viral diarrhea virus 3 (BVDV-3) (HoBi group), Border disease virus 2 (BDV-2) (Italian small ruminant isolates) and Bungowannah. Palindromic positions have been characterized according to changes in nucleotide base-pairs identifying low variable positions (LVP) including base-pairs present in less than 80% of the genus. The determination of divergence between single strain sequences or genetic groups was obtained easily by comparing base-pairing combinations from aligned secondary structures. This provided clear information such as the level of heterogeneity within a species, the relatedness between species, or facilitating the characterization and clustering of specific strains. The BVDV-1 and BDV species resulted heterogeneous, showing isolates located on a borderline in the species. Within the BVDV-2 species, two main genogroups were identified, with strains showing common sequence characteristics to both groups (multirelated strains). They could be allocated correctly by quantitative analysis. Similarly, the relation between CSFV and BDV species appeared very clearly. Also in this case, ambiguous strain sequences could be clustered in the species showing the lowest divergence values. In conclusion, the proposed taxonomical procedure is based on the evaluation of only the strategic and highly conserved genome regions in the 5′-UTR. Furthermore, the application of quantitative analytical procedure allowed for a better determination of relation among species.

The first epidemiological survey of Border disease virus (BDV) was undertaken in small ruminants in Japan. Ovine sera, collected from the northern prefectures of Hokkaido, Aomori and Iwate, were examined for the presence of antibodies against BDV using the neutralization peroxidase-linked antibody test. Twenty-nine (17.6%) of one hundred and sixty-five samples were seropositive for BDV. Results were specific, excluding cross-reactions with bovine viral diarrhea virus (BVDV). Only one sample (0.6%) was positive for BVDV, and was negative for BDV. Despite serological evidence of virus circulation, there have been no clinical cases of border disease in sheep in Japan. Although no diagnostic measures were performed, the infection did not appear to be associated with a reduction in ewe fertility nor with lamb mortality.

Bovine viral diarrhea virus 2 (BVDV-2) strains demonstrated in cattle, sheep, and adventitious contaminants of biological products have been evaluated by the palindromic nucleotide substitutions (PNS) method at the three variable loci (V1, V2 and V3) in the 5' untranslated region (UTR), to determine their taxonomical status. Variation in conserved genomic sequences was used as parameter for epidemiological evaluation of the species in relation with geographical distribution, animal host and virulence. Four genotypes, BVDV-2a, BVDV-2b, BVDV-2c, and BVDV-2d have been identified within the species. Taxonomical segregation corresponded to geographical distribution of genotype variants. Genotype 2a was present worldwide, and was the only circulating also in sheep, in addition to cattle. Genotypes 2b, 2c and 2d were restricted to South America. Contamination of biological products was related to genotypes 2a and 2d. Genetic variation could be related with chronological diffusion of the BVDV-2 species variants in different geographic areas. Chronologically, the species emerged in North America in 1978, spreading in UK and Japan, continental Europe, South America and New Zealand. Correlation between clinical features related with isolation of BVDV-2 strains and genetic variation indicated that subgenotype 1, variant 4 of genotype 2a was related with hemorrhagic syndrome. These observations suggest that evaluation of genomic secondary structure, by identifying markers for expression of virus biological activities and species evolutionary history, may be applied as useful tool for epidemiological evaluation of the BVDV-2 species, and possibly for other species of the genus Pestivirus.

Sixteen serum samples collected from free-living Japanese serows, Capricornis crispus, between 2001 and 2004 in Morioka and its vicinity were examined for the presence of pestivirus by reverse transcription-nested PCR procedure. Three out of the 16 samples produced a visible band in electrophoresed agarose gels. The nucleotide sequences of the three PCR products were found to be identical. The pestivirus found in the serow was identified as Bovine viral diarrhea virus 1 (BVDV-1) based on nucleotide sequence analyses by phylogeny as well as palindromic nucleotide substitutions at the 5' untranslated regions. Our data first indicated that BVDV-1 infection occurred continuously among the free-living serow populations though the role of BVDV-1 in wild ungulates is currently unknown.

Two strains of Bovine viral diarrhea virus 2 (BVDV-2) were isolated from calves in northern Italy. Variations in the 5'-untranslated region (UTR) of the genome were studied by primary structure alignment and neighbor-joining method based phylogenetic tree analyses and by palindromic nucleotide substitutions at the three variable loci in the 5'-UTR. Genetic analysis indicated their appurtenance to genovar BVDV-2a. Nucleotide sequence at the 5'-UTR of strain BS-95-II, one of the Italian isolates from healthy calves, showed 98% homology to that of the Japanese isolate OY89, a cytopathic strain derived from cattle with mucosal disease.

Bovine Viral Diarrhea virus species of the genus Pestivirus, important pathogens affecting zootechnics worldwide, have been reported as adventitious contaminants of biological products for veterinary and human use. The Bovine Viral Diarrhea virus 1 species showed potential for an emerging zoonosis. According to World Organization for Animal Health (Office International des Epizooties: OIE), Bovine Viral Diarrhea is a notifiable disease of importance to international trade. Recently, Bovine Viral Diarrhea virus 3 tentative species have been isolated from Brazil and Thailand. The virus has been diffused from South America to other countries probably through the commercialization of contaminated fetal bovine serum.

A seroepidemiologic survey was conducted in all provinces of the Syrian Arab Republic to determine prevalence of antibodies against Border Disease (BD) virus in Awassi sheep flocks. About 45% of the animals were positive (IIF method). Northwest provinces showed highest percentage of seropositive animals (up to 94%), while BD was almost absent in the southern provinces. Distribution of anti-BD antibodies appeared to be related to two aspects of flock management, distance of transhumance and the use and quality of pens. The higher prevalence in long distance transhumant flocks is possibly related to the higher risk of contacts with persistently infected and virus excreting animals. Similarly, stress due to sheltering in poorly ventilated and humid folds may facilitate occurrence of the infection. Sheep from the wettest regions, with more than 600 mm of rainfall per year (IA zone), showed a higher prevalence to BD, when compared to those from dry areas. This may be related to the rapid inactivation of the virus by desiccation. Occurrence of BD infection was related to a mortality rate up to 20% of lambs.

The HCV stem–loop subdomains III-a, -b and -c have been shown to reflect the characteristics of the virus and identify isolates by genus, genotype and subtype. The aim of this study was to investigate the genotype-specific PNS within the 5′UTR of prevalent HCV genotypes (1 and 5a) found in South Africa. The genotype 5a (N = 35) and genotype 1 sequences (N = 20) were from patients presenting with liver disease or haemophilia, respectively. PNS HCV typing characteristics, defined previously, were observed. The PNS method differentiated subtypes 1a and 1c from subtype 1b by the base change at nucleotide position 243. A lack of structural data from the variable loci V1 of the 5′UTR did not allow us to further differentiate the subtypes of 1. A nucleotide change from a thymine (T) to a cytosine (C) at position 183 was found among genotype 5a sequences. This mutation changed the stable U–AA bond to a Y AA bulge at base-pair position 32. There was an insertion of a single adenine (A) at position 207. At present PNS analysis is labour intensive but, with development of further software to aid the computer analysis, it has the potential to provide a rapid, reliable alternative to phylogenetic analysis.

Des sérums d'ovins Awassi de toutes les provinces syriennes ont été testés pour rechercher les anticorps du virus Maedi Visna (MVv) par le test d'immunodiffusion en gélose. Quatre vingt-sept animaux sur 1445 testés ont montré une positivité à la glycoprotéine 135 du MVv soit 6 %. L'infection a été décelée dans la plupart des provinces sauf dans celles, méridionales, de Damas, Konaitra et Sweida. Le mode d'utilisation et la qualité des bergeries ainsi que la pluviosité ont été considérés comme des facteurs influençant la distribution du MVv. L'infection pourrait être en relation avec les pertes de production ovine, et plus spécialement avec la diminution du pourcentage net annuel de survie des moutons (pourcentage de moutons qui ne meurent pas et ne sont pas réformés).

Bovine viral diarrhoea virus 2 (BVDV-2) strains demonstrated in cattle, sheep and adventitious contaminants of biological products were evaluated by the palindromic nucleotide substitutions (PNS) method at the three variable loci (V1, V2 and V3) in the 5’ untranslated region (UTR), to determine their taxonomic status. Variation in conserved genomic sequences was used as a parameter for the epidemiological evaluation of the species in relation to geographic distribution, animal host and virulence. Four genotypes were identified within the species. Taxonomic segregation corresponded to geographic distribution of genotype variants. Genotype 2a was distributed worldwide and was also the only genotype that was circulating in sheep and cattle. Genotypes 2b, 2c and 2d were restricted to South America. Genotypes 2a and 2d were related to the contamination of biological products. Genetic variation could be related to the spread of BVDV-2 species variants in different geographic areas. Chronologically, the species emerged in North America in 1978 and spread to the United Kingdom and Japan, continental Europe, South America and New Zealand. Correlation between clinical features related with isolation of BVDV-2 strains and genetic variation indicated that subgenotype 1, variant 4 of genotype 2a, was related to a haemorrhagic syndrome. These observations suggest that the evaluation of genomic secondary structures, by identifying markers for expression of virus biological activities and species evolutionary history, may be a useful tool for the epidemiological evaluation of BVDV-2 species and possibly of other species of the genus Pestivirus.

The palindromic nucleotide substitutions (PNS) in the 5′-untranslated region (UTR) of Pestivirus RNA have been described as a new, simple and practical method for genotyping. Given the genetic relatedness between Pestivirus and hepatitis C virus species, the application of the method was investigated preliminarily on 180 isolates, including reference strains. The keys for hepatitis C virus identification have been determined at the genus, species, genotype and subtype levels. Secondary structure nucleotide substitutions were characteristics to the genus included in a complex stem-loop structure composed of 112–115 nucleotides. Due to the worldwide importance of hepatitis C virus, and the difficulties encountered in the control of the disease, it is, therefore, important to understand the genetic aspects of the virus. The application of the PNS method might represent an additional useful tool for determining the genetic variations among hepatitis C virus strains. The identification of viral types or subtypes based on genetic changes should improve our understanding of hepatitis C virus and might provide markers for biological differences, such as virulence, and improve understanding of the evolution of the virus.

The palindromic nucleotide substitutions (PNS) at the three variable loci (V1, V2 and V3) in the 5' untranslated region (UTR) of Pestivirus RNA have been considered for taxonomical segregation of species, through the evaluation of 430 genomic sequences. On the basis of qualitative and quantitative secondary structure characteristics, six species have been identified: Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Classical swine fever virus (CSFV), Border disease virus (BDV), the tentative species Giraffe and a new proposed taxon named Pronghorn. The first step was qualitative and consisted in the characterization of the different positions of the three stems and loops in the 5' UTR sequences of all the strains under consideration belonging to the genus. Secondary structure sequences showing divergent base-pair combinations have been aligned for comparison. Palindromic positions have been characterized according to changes in nucleotide base-pairs identifying low-variable positions (LVP) including base-pairs present in less than 80% of the genus. The second step was quantitative, allowing the identification of genomic groups by clustering the base-pair combinations according to LVP. Relatedness among types was evaluated to identify homogeneous groups. Cross comparisons between types within the genus have been evaluated by computing the divergence percentage thus clarifying borderline and multirelated sequences.

Bovine viral diarrhea virus 2 (BVDV-2) strains, isolated from sheep showing clinical symptoms of border disease, have been evaluated by the palindromic nucleotide substitution (PNS) method at the three variable loci (V1, V2 and V3) in the 5'-untranslated region (UTR) of genomic RNA. The characteristic two base-pairings common to the BVDV-2 species, a C-G pairing which was common to the V1 locus, and a G*U pairing common to the V2 locus, were observed in all tested strains. Strains BD-78 and C413 were identified by a unique C-G pairing at position 4 from the bottom of the V2 stem region, which is characteristic to BVDV-2b. BVDV-2d characteristic U-A pairing at position 18 of the V1 stem region was observed in five strains, Lees, 167 237, 168 149, 173 157 and 175 375. No strains have been assigned to the genotypes BVDV-2a or BVDV-2c. Furthermore, the investigation at the level of the 5'-UTR excluded the application in sheep of the proposed BVDV-2 genetic virulence markers described in cattle. The two specific positions of uracil and cytosine nucleotides related to low or high virulence where indifferently present in the ovine BVDV-2 strains responsible of border disease.

Live virus vaccines for human use, 29 monovalent vaccines against measles, mumps, rubella or polio, eight polyvalent vaccines against measles-mumps-rubella and one bacterial polyvalent vaccine against Streptococcus pneumoniae, were tested by reverse transcriptase-nested PCR for the presence of petivirus or pestivirus RNA. Twenty-four samples were selected from European manufacturers, ten were from U.S.A. and four from Japan. Five (13.1%) out of 38 tested samples were positive for pestivirus RNA. Three vaccines (rubella and two measles) were from Europe and two (mumps and rubella) from Japan. The 5'-untranslated genomic region of the contaminant pestivirus RNA were amplified by reverse transcription-PCR and sequenced. Analyses based on primary nucleotide sequence homology and on secondary structures, characteristic to genotypes, revealed that the cDNA sequences belonged to bovine viral diarrhea virus (BVDV). A cDNA sequence, detected from one measles sample, belonged to BVDV-1b genotype. Pestiviral cDNA detected from the Japanese mumps and rubella vaccine samples, belonged to the BVDV genotypes 1a and 1c, respectively. Analysis on two cDNA sequences detected from measles and rubella vaccine samples from Europe showed their appurtenance to a new genotype, BVDV-1d. These findings indicate that contamination by animal pestivirus may occur in biological products for human use.

A simple and practical method was developed for pestivirus genotyping based on analysis of the secondary structures in the 5'-untranslated region (UTR). Three stable stem-loop structures, V1, V2 and V3, predicted by computer in the 5'-UTR, included strictly conserved consensus base-pairings which are shared by all the genotypes of pestivirus or are characteristic to each genotype of pestivirus. On the basis of the palindromic nucleotide substitution at the secondary structural level, six genotypes have been identified among pestivirus strains, irrespective of the cytopathic and non-cytopathic biotypes. They are genotypes Ia, Ib, Ic and II in bovine viral diarrhea virus, genotype III in border disease virus, and genotype IV in classical swine fever virus. The stable stem-loop structures, which were maintained by palindromic nucleotide substitutions in the stem region, may represent references for the classification and identification of pestivirus species and/or genotypes.

Awassi sheep sera from all the provinces of the Syrian Arab Republic were tested for RSV, P13, REO and Adeno viruses (IIF and AGID tests). RSV was the most prevalent infection with 63.6% of samples seropositive, followed by REO, P13 and Adenovirus with seroprevalences of 27.3%, 24% and 8.1% respectively. Animals were more frequently infected by RSV alone. Mixed infections were also identified but the occurrence was not high. The RSV and REO virus infections occurred more frequently when transhumant flocks travelled for long distances (P0.001). The prevalence of RSV, P13, REO and Adenovirus infections was higher in animals sheltered in poor conditions compared to those in good shelter or with no shelter (P0.001; P0.001; n.s.; P0.05, respectively). RSV and P13 infections were related to an adult mortality rate of 10 to 20% (P0.001); REO virus was also proportionally related to mortality from low to high rates (P0.05). Concerning mortality of lambs, only Adenovirus infection was related to losses (20%) (P0.001). REO virus was related to low milk yield (P0.05).

The 5'-untranslated genomic region of the pestivirus strain Europa, originated in human leucocytes and previously identified as bovine diarrhea virus (BVDV), was amplified by reverse transcription-PCR and sequenced. Analyses based on primary nucleotide sequence homology and on secondary palindromic sequence structures characteristic to genotypes revealed that this human isolate should be assigned to a novel genotype of pestivirus, type Ic. This newly emerged genotype was related to, but distinguishable from the three known BVDV genotypes, Ia, Ib and II. Three other bovine field isolates of BVDV originated from Germany were also found to belong to this new genotype Ic. Within pestivirus genotype Ic strains, the overall nucleotide sequence homology was 95-96%, and 88-92%, 88-90% and 77-79% with the other BVDV genotypes Ia, Ib and II, respectively. With the strains from border disease virus (genotype III) and hog cholera virus (genotype IV), homologies were less than 75%.

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Un dépistage des anticorps contre le virus de l'arthrite-encéphalite des caprins a été effectué au nord-ouest de la Syrie en utilisant le test d'immunodiffusion en gélose. Sur 72 sérums testés, 12,5 % montraient une réaction positive. Aucune évidence clinique de la maladie n'a été trouvée chez les animaux testés. Ces résultats indiquent que l'infection est endémique dans la région étudiée.