Palindromic Nucleotide Substitutions Research Articles

Pestivirus A Bovine viral diarrhea virus type 1 species genotypes circulating in China and Turkey.

Pestivirus A Bovine viral diarrhea virus type 1 (BVDV-1) is a heterogeneous species within the genus, affecting cattle and other ruminants, with economic impact on livestock production. The study aimed to update the taxonomy of the Pestivirus A, BVDV-1 species and to verify the clustering of the strains reported as genotype 1v, originating from different countries. Recently deposited strains from China, Turkey, and Iran have been evaluated by the palindromic nucleotide substitutions (PNS) genotyping method. Based on secondary structure analysis of the 5'-UTR sequences, strains reported as 1v from China were clustered as sub genotype 1.7.3 (1o). Genotype 1.19 (1w) was restricted to China and genotype 1.21 (1v) was present only in Turkey and Iran. The application of the PNS method clarified the taxonomical status of strains, revealing the homonymy of genetically different clusters. Furthermore, these observations indicated geographic segregation in the Pestivirus A species, and confirmed the occurrence of new atypical genetic variants, with potential implications on control and prophylaxis.

Retracted: Artifactual Palindrome and Mutations Resulting from the Stable Secondary Structure of Templates in Sanger Sequencing

AbstractDue to its advantages of high precision and low cost, automated Sanger sequencing technology remains useful for many applications despite the emergence of next‐generation sequencing (NGS) platforms. Few studies revealed the sequence errors in sanger sequencing because the instrumentation and chemistry currently used for Sanger sequencing is highly reliable and reproducible. Unexpectedly, in our study, the sequencing artifacts were identified in sequencing the PCR product of rAAV‐ITR (recombination Adeno‐Associated Virus‐inverted terminal repeat) which formed highly stable T‐shaped hairpin and played critical role in virus production. The artifactual sequences were characterized by the 3’ end palindromic sequence and nucleotide substitutions. We confirmed that the presence of stable hairpin in template results in sequencing artifacts by employing disruptor to eliminate the structure, sequencing the PCR product and the synthezized strands containing stable or weak hairpin respectively for comparation. Furthermore, we proposed that endonuclease activity of Taq polymerase cleaves the templates containing stable structure to generate annealed 3’end which acts as primer to synthesize the artifactual palindrome.

Characterization of internal ribosome entry sites according to secondary structure analysis to classify border disease virus strains

Applying palindromic nucleotide substitutions (PNS) method, variable loci of the internal ribosome entry site (IRES) secondary structure in the 5’ untranslated region (UTR) of Border disease virus sequences were analysed allowing their allocation into ten IRES classes within the species. Sequence characteristics of Turkish and Chinese strains were highly divergent from other genogroups, indicating geographic segregation and micro-evolutive steps within the species. Observed heterogeneity in the BDV species has to be considered for potential implications on diagnostic tests, control and preventive measures.

Pathogen spread and globalization: The case of Pestivirus heterogeneity in southern Italy

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.

Heterogeneity of <i>Pestivirus</i> Species in Asia

Pestivirus are responsible for cosmopolitan diseases affecting cattle, pigs and other ruminants, presenting a wide range of clinical manifestations, with relevant impact on zootechnic production. Understanding genomic characteristic and virus taxonomy is fundamental in order to sustain control and prophylactic programs. Given the recent various studies reporting a relatively high number of new strains, in particular from Asian countries, in the present study, six hundred-fifty-one genomic sequences have been considered applying the palindromic nucleotide substitutions method for genotyping. Based on the secondary structure analysis of the 5’ untranslated region of RNA, sequence characteristics among Asian genomic clusters within the different Pestivirus species suggested geographic segregation and occurrence of micro-evolutive steps in the genus evolutionary history. This aspect was particularly evident in atypical sequences originated from China or Turkey, indicating risk of diffusion by animals and products trade or contamination of biological products as bovine calf serum, with potential diagnostic and control difficulties.

Bovine viral diarrhea virus type 1 current taxonomy according to palindromic nucleotide substitutions method

Pestivirus bovine viral diarrhea virus type 1 species is responsible for cosmopolitan diseases affecting cattle and other ruminants, presenting a wide range of clinical manifestations, with relevant impact on zootechnic production. Understanding genomic characteristic and virus taxonomy is fundamental in order to sustain control and prophylactic programs. Given the recent various studies reporting a relatively high number of new strains, in particular from Asian countries, in the present study, four hundred-eighty-two genomic sequences have been evaluated applying the palindromic nucleotide substitutions method for genotyping. Based on the secondary structure alignment and computing genetic distance among strains in the 5′ untranslated region of Pestivirus RNA, the current taxonomy of the species was reviewed. Twenty-two genotypes have been identified, applying a nomenclature based on divergence in the genus.

Improved Palindromic Nucleotide Substitutions Software Version 2.0. Genotyping Based on the Secondary Structure Alignment in the 5 Untranslated Region of Pestivirus RNA

Improved Palindromic Nucleotide Substitutions Software Version 2.0. Genotyping Based on the Secondary Structure Alignment in the 5 Untranslated Region of <I>Pestivirus</I> RNA

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.

Numerical taxonomy of the genus Pestivirus: New software for genotyping based on the palindromic nucleotide substitutions method

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.

Palindromic Nucleotide Substitutions Software Version 2.0. Genotyping Based on the Secondary Structure Alignment in the 5' Untranslated Region of &lt;I&gt;Pestivirus&lt;/I&gt; RNA

Palindromic Nucleotide Substitutions Software Version 2.0. Genotyping Based on the Secondary Structure Alignment in the 5' Untranslated Region of &lt;I&gt;Pestivirus&lt;/I&gt; RNA

Palindromic-nucleotide substitutions (PNS) of hepatitis C virus genotypes 1 and 5a from South Africa

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.

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.

Rapid Identification of Hemoplasma Species by Palindromic Nucleotide Substitutions at the GAAA Tetraloop Helix in the Specificity Domain of Ribonuclease P RNA

We examined secondary structures of the ribonuclease P RNA sequences obtained from DNA databases, and identified a determinative prototype of the P12 helix peculiar to each species of hemoplasmas. This key structure will provide a rapid means for species identification of these uncultivable pathogens without making a phylogenetic tree based on alignments of nucleotide sequences. This procedure based on palindromic nucleotide substitutions at the stem portion of the P12 helix provide clear information such as the level of heterogeneity within a species, the relatedness between species, or facilitating the characterization and clustering of specific strains. In conclusion, the PNS analysis is based on the evaluation of only the strategic and highly conserved genomic region in the specificity domain of RNase P RNA.

Taxonomy of genus Hepacivirus. Application of palindromic nucleotide substitutions for the determination of genotypes of human hepatitis C virus species

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.

Genoepidemiological Evaluation of Bovine viral diarrhea virus 2 Species Based on Secondary Structures in the 5' Untranslated Region

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.

Numerical taxonomy of the genus Pestivirus based on 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 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.

Evidence for Pestivirus Infection in Free‐Living Japanese Serows,Capricornis crispus

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.

Genotypic characteristics of bovine viral diarrhea virus 2 strains isolated in northern Italy.

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.

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.

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.