Massimo Giangaspero

A novel method for pestivirus genotyping based on palindromic nucleotide substitutions in the 5'-untranslated region.

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.

Giraffe Strain of Pestivirus: Its Taxonomic Status Based on the 5′‐Untranslated Region

The 5′‐untranslated region (5′‐UTR) of the ‘Giraffe’ strain of pestivirus was sequenced for comparison with those of other pestiviruses from cattle, sheep, goats, and swine. A phylogenetic tree constructed with these strains suggested that the ‘Giraffe’ strain was allocated to a new taxon. This observation was also confirmed by a newly proposed method based on palindromic nucleotide substitutions (PNS) at the three variable regions in the 5′‐UTR. Other reported pestivirus strains isolated from deer were assigned as bovine viral disease virus (BVDV)‐1 according to the PNS as well as phylogenetic analysis, suggesting that BVDV‐1 strains can cross‐infect deer as well as cattle, sheep, goats, and swine, and that wild deer may serve as a reservoir of BVDV‐1. We also identified the genovar of a deer isolate, SH9/11, as BVDV‐1c by the PNS method.

Genotypic Analysis of the 5′‐Untranslated Region of a Pestivirus Strain Isolated from Human Leucocytes

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%.

CHARACTERIZATION OF MYCOPLASMA ISOLATED FROM AN IBEX (CAPRA IBEX) SUFFERING FROM KERATOCONJUNCTIVITIS IN NORTHERN ITALY

In 2005 a Mycoplasma species was isolated from ocular-conjunctival swabs from an adult male Alpine ibex (Capra ibex) from the Valle dAosta Region, Northern Italy. The animal suffered from bilateral ocular discharge with diffuse inflammation, severe corneal involvement of the left eye and mild corneal opacity of the right eye. Histologic examination revealed a keratoconjunctivitis characterized by lymphocytic and plasmacellular infiltration. Laboratory investigations of the isolate included culture, transmission electron microscopy, PCR, and denaturing gradient gel electrophoresis, as well as DNA sequencing of the 16S rDNA gene. These tests identified the isolate as Mycoplasma mycoides subsp. capri large-colony serovar, an organism that has occasionally been associated with keratoconjunctivitis in goats. For a correct diagnosis, it is necessary to carry out laboratory investigations, as clinical cases of keratoconjunctivitis in wild ruminants are not always ascribable to Mycoplasma conjunctivae.

Genetic Variation in the 5′ End and NS5B Regions of Classical Swine Fever Virus Genome among Japanese Isolates

Sixteen clinical strains of classical swine fever virus (CSFV) isolated in Japan were subjected to analyses of nucleotide sequence variations in the 5′ end and NS5B regions of the genome. These isolates were divided into three genovars, CSFV‐1, CSFV‐2 and CSFV‐3, based on palindromic nucleotide substitutions at the three variable loci in the 5′ untranslated region (UTR). Phylogenetic trees constructed from nucleotide sequences in the 5′‐UTR and NS5B gene indicated that the CSFV strains were divided into three clusters, I, II and III. CSFV strains included in clusters I, II and III were identical to those in the CSFV‐1, CSFV‐2 and CSFV‐3 genovars, respectively.

Species characterization in the genus Pestivirus according to palindromic nucleotide substitutions in the 5′-untranslated region

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.

Ovine pestiviruses: their taxonomic status revealed by palindromic nucleotide substitutions

We examined previously identified border disease virus (BDV) strains by using a newly proposed genotyping procedure based on palindromic nucleotide substitutions (PNS) in the 5-untranslated region (UTR), and found 22 (41.5%) out of 53 strains of BDV in the nucleotide sequence databases are not of BDV. All the 22 ovine pestivirus strains were allocated to the BVDV species according to the PNS, and were compared with reference strains of pestivirus 1 (BVDV-Ia,-Ib, and-Ic genovars), pestivirus 2 (BVDV-II genovar), pestivirus 3 (BDV) and pestivirus 4 (CSFV), respectively. Ten strains (Weybridge, A553, B1056, D771/1, D861, D1120/1, D1432/P, Q1161/1, Q1161/2, 114817) showed a palindromic structure in the 5-UTR characteristic to the BVDV-Ia genovar, three strains (7535, 7546, 7548) were characteristic to the BVDV-Ib genovar, and nine strains (BD-78, 59386, SCP, Lees, C413, 167237, 168149, 173157, 175375) belonged to the BVDV-II genovar.

Molecular Evidence for Hemotropic Mycoplasma Infection in a Japanese Badger (Meles meles anakuma) and a Raccoon Dog (Nyctereutes procyonoides viverrinus )

Abstract We report detection of hemoplasma in wild Japanese badgers (Meles meles anakuma) and raccoon dogs (Nyctereutes procyonoides viverrinus). Sequence analysis of the entire 16S rRNA genes identified Mycoplasma haemocanis in the raccoon dog sample, and a potential novel Mycoplasma species in the Japanese badger.

Seroepidemiological survey of sheep flocks from Northern Japan for Mycoplasma ovipneumoniae and Mycoplasma agalactiae

Sheep flocks from Hokkaido, Iwate and Aomori, three northern prefectures of Japan, were screened for antibodies to Mycoplasma ovipneumoniae and Mycoplasma agalactiae by ELISA. Sixty four animals out of 246 (26%) were seropositive to M. ovipneumoniae, with positive results obtained from all three prefectures. None of the sera tested were serologically positive to M. agalactiae.

Characterization of genotypes among bovine viral diarrhea virus type 1 strains according to palindromic nucleotide substitutions in the genomic 5′-untranslated region

Two-hundred and eighty-one strains of bovine viral diarrhea virus type 1 (BVDV-1) were evaluated according to the palindromic nucleotide substitutions (PNS) method, a simple and practical genotyping procedure, at the three variable loci, V1, V2 and V3, in the 5-untranslated region of genomic RNA. Ten PNSs were characteristic to the Pestivirus genus, located in the V1 and V2 loci. The BVDV-1 species shared a U-A pairing, a species-characteristic PNS, in position fifteen common to the V1 locus, and a G-C pairing in position five common to the V2 and V3 loci. Within the BVDV-1 species, 15 genotypes, BVDV-1a, BVDV-1b, BVDV-1c, BVDV-1d, and the newly described from BVDV-1e to BVDV-1o were identified based on characteristic nucleotide base pairings. The nomenclature reflected the genotypes level of divergence within the species. Genotypes BVDV-1l, BVDV-1m and BVDV-1o were borderline in the species.