Nucleotide Sequence Differences Research Articles

Heteroallelic diploid pollen led to self-compatibility in tetraploid cultivar ‘Sha 01’ (Pyrus sinkiangensis Yü)

Pear cultivar ‘Sha 01’ is a sport from ‘Kuerlexiangli’. Field pollination data revealed that ‘Sha 01’ displayed self-compatibility (SC), whereas ‘Kuerlexiangli’ showed self-incompatibility upon self-pollination. Reciprocal pollinations between the two varieties showed that 76% of ‘Kuerlexiangli’ flowers pollinated with ‘Sha 01’ pollen set fruit, but only 7% of ‘Sha 01’ flowers set fruit when pollinated with ‘Kuerlexiangli’ pollen. The pollen performance was monitored with fluorescence microscopy, and it was observed that ‘Sha 01’ accepted self-pollen but rejected ‘Kuerlexiangli’ pollen, whereas ‘Kuerlexiangli’ rejected self-pollen but accepted ‘Sha 01’ pollen. Taken together, ‘Sha 01’ showed SC as a pollen-part mutant. Molecular S-genotyping of ‘Sha 01’, its selfed progeny, and wild-type ‘Kuerlexiangli’ showed that all contained S 22-RNase and S 28-RNase alleles, but showed no nucleotide sequence difference or changes in transcription. After flow cytometry and chromosome number analyses, ‘Sha 01’ was found to be a tetraploid (2n = 68) and ‘Kuerlexiangli’ a diploid (2n = 34). Thus, the S-genotype for ‘Sha 01’ was S 22 S 22 S 28 S 28 and for ‘Kuerlexiangli’ was S 22 S 28 . Ploidy level of ‘Sha 01’ selfed progeny were also determined from leaves using flow cytometry; all seedlings were tetraploids. The S-genotyping for the progeny was identified by S-RNase gene relative optical density for both alleles. Closer inspection of 52 progeny showed that they could be classified into three types S 22 S 22 S 28 S 28 : S 22 S 22 S 22 S 28 : S 22 S 28 S 28 S 28 with the distribution 28:10:14 ≈ 4:1:1; no individual homozygous for either allele was found. Therefore, it could be concluded that only heteroallelic diploid pollen S 22 S 28 could achieve ‘Sha 01’ fertilization through a so-called competitive interaction.

Phosphate transporter genes as reliable gene markers for the identification and discrimination of arbuscular mycorrhizal fungi in the genus glomus.

An inorganic phosphate transporter gene sequence (852-bp section) allowed discrimination between 10 Glomus fungal species represented by 25 strains. It was particularly valuable in differentiating between morphologically similar species with nucleotide and amino acid sequence differences higher than 3%. This gene is proposed as a reliable barcode for the Glomeromycetes.

A complex hepatitis B virus (X/C) recombinant is common in Long An county, Guangxi and may have originated in southern China.

Recently, a complex (X/C) hepatitis B virus (HBV) recombinant, first reported in 2000, was proposed as a new genotype; although this was refuted immediately because the strains differ by less than 8 % in nucleotide distance from genotype C. Over 13.5 % (38/281) of HBV isolates from the Long An cohort in China were not assigned to a specific genotype, using current genotyping tools to analyse surface ORF sequences, and these have about 98 % similarity to the X/C recombinants. To determine whether this close identity extends to the full-length sequences and to investigate the evolutionary history of the Long An X/C recombinants, 17 complete genome sequences were determined. They are highly similar (96–99 %) to the Vietnamese strains and, although some reach or exceed 8 % nucleotide sequence difference from all known genotypes, they cluster together in the same clade, separating in a phylogenetic tree from the genotype C branch. Analysis of recombination reveals that all but one of the Long An isolates resembles the Vietnamese isolates in that they result from apparent recombination between genotype C and a parent of unknown genotype (X), which shows similarity in part to genotype G. The exception, isolate QL523, has a greater proportion of genotype C parent. Phylogeographic analysis reveals that these recombinants probably arose in southern China and spread later to Vietnam and Laos.

Identification of Simian Agent 10 as Human Parainfluenza Virus Type 3 Suggests Transmission of a Human Virus to an African Monkey

The use of nonhuman primates in biomedical research has led to the isolation of many simian viruses (4, 5). These viruses were given either consecutive SV (simian virus; for isolates from Asian monkeys) or SA (simian agent; for isolates from African monkeys) numbers (7, 10). SA10 was first isolated from the mouth of a samango monkey (Cercopithecus mitis) in 1963 (10). Based on serological findings (10), SA10 has long been classified as a distinct simian virus in the genus Respirovirus within the family Paramyxoviridae (2, 7). We obtained SA10 from the ATCC and determined a complete consensus sequence for the genome using standard procedures (9). The genome of SA10 was determined to be 15,462 nucleotides (nt) in length (GenBank accession number {type:entrez-nucleotide,attrs:{text:HM583801,term_id:312618595,term_text:HM583801}}HM583801), which is identical to that of human parainfluenza virus type 3 (HPIV3). Comparison with full-length genome sequences of other members of the family Paramyxoviridae showed that SA10 clustered with HPIV3. The extent of nucleotide sequence difference between SA10 and the various strains of HPIV3 is the same as that between the HPIV3 strains (Table ​(Table11). TABLE 1. Nucleotide sequence identity among the complete genome sequences of the indicated viruses The general features of the genome of SA10 (Fig. ​(Fig.1a)1a) are identical to those of the genome of HPIV3. Like HPIV3, the P gene of SA10 contains an additional open reading frame (ORF) encoding the accessory C protein and a putative RNA editing site, 2498UUUUUUCCCCC2508, that is identical in position and sequence to that of HPIV3 (3). Like HPIV3, the insertion of two G residues at this site by the RNA editing mechanism would cause a frameshift and create an ORF encoding a D protein. FIG. 1. SA10 genome map and phylogeny. (a) The genome of SA10 is shown in 3′-to-5′ orientation. The six genes are identified by their encoded proteins. The nucleotide length of each gene is shown above the map, as well as the amino acid length(s) ... Furthermore, SA10 is identical to HPIV3 with regard to the nucleotide lengths of all of the genes, the exact spacing of each gene within the genome and within the respective hexamer subunits (8), and the lengths of the leader, trailer, and extragenic regions (Fig. ​(Fig.1a).1a). The predicted lengths of the unmodified encoded proteins also are identical to those of various strains of HPIV3, with some heterogeneity in the predicted P and HN protein lengths, whereas the lengths of the other proteins are invariant among the HPIV3 strains and SA10. Analysis using 17 HPIV3 HN protein sequences showed that the SA10 HN protein sequence clustered with those of the HPIV3 strains (Fig. ​(Fig.1b).1b). The cleavage site of SA10 is 104DPRTKR↓F110, which conforms to the furin cleavage site (underlining indicates the basic amino acids in the cleavage site). The veritable identity of SA10 with HPIV3 suggests that SA10, rather than being a simian virus, is a strain of HPIV3. In particular, the few differences that occur between SA10 and various HPIV3 strains are of the same frequency as those occurring among the HPIV3 strains. A more likely possibility is that HPIV3 had been transmitted to the monkey from human handlers. A serologic survey of Indonesian macaques showed that nearly half of the sampled wild adult animals were seropositive for HPIV3, whereas no seropositive animals among the sampled wild infant, juvenile, and subadult animals were observed, implying that transmission can occur frequently, even to wild animals (6). In particular, human respiratory syncytial virus was originally isolated from captive chimpanzees and initially was called “chimpanzee coryza agent” (1) but was quickly recognized as a human pathogen and not a natural pathogen of chimpanzees. The present findings on SA10 clarify the taxonomy of Paramyxoviridae, illustrate that host range identification can be ambiguous, and provide a cautionary tale for hasty virus classification.

Remarkable Polymorphism of HLA-B27: An Ongoing Saga

Histocompatibility antigen HLA-B27 is a normal gene that is distributed worldwide with variable prevalence and shows a remarkable association with ankylosing spondylitis and related spondyloarthropathies. The precise biological explanation for this remarkable association remains elusive. HLA-B27 represents a family of closely related proteins encoded by an ever-increasing number of alleles; there are 75 alleles of HLA-B27 known thus far, based on nucleotide sequence differences, but at the translated protein level, there are 62 known subtypes of HLA-B27. Not all subtypes are disease associated. Moreover, existence of a possible hierarchical ranking among some of the subtypes for their disease association has been observed.

Methicillin Resistance Transfer from Staphylocccus epidermidis to Methicillin-Susceptible Staphylococcus aureus in a Patient during Antibiotic Therapy

BackgroundThe mecA gene, encoding methicillin resistance in staphylococci, is located on a mobile genetic element called Staphylococcal Cassette Chromosome mec (SCCmec). Horizontal, interspecies transfer of this element could be an important factor in the dissemination of methicillin-resistant S. aureus (MRSA). Previously, we reported the isolation of a closely related methicillin-susceptible Staphylococcus aureus (MSSA), MRSA and potential SCCmec donor Staphylococcus epidermidis isolate from the same patient. Based on fingerprint techniques we hypothesized that the S. epidermidis had transferred SCCmec to the MSSA to become MRSA. The aim of this study was to show that these isolates form an isogenic pair and that interspecies horizontal SCCmec transfer occurred.Methodology/ResultsWhole genome sequencing of both isolates was performed and for the MSSA gaps were closed by conventional sequencing. The SCCmec of the S. epidermidis was also sequenced by conventional methods. The results show no difference in nucleotide sequence between the two isolates except for the presence of SCCmec in the MRSA. The SCCmec of the S. epidermidis and the MRSA are identical except for a single nucleotide in the ccrB gene, which results in a valine to alanine substitution. The main difference with the closely related EMRSA-16 is the presence of SaPI2 encoding toxic shock syndrome toxin and exfoliative toxin A in the MSSA-MRSA pair. No transfer of SCCmec from the S. epidermidis to the MSSA could be demonstrated in vitro.ConclusionThe MSSA and MRSA form an isogenic pair except for SCCmec. This strongly supports our hypothesis that the MRSA was derived from the MSSA by interspecies horizontal transfer of SCCmec from S. epidermidis O7.1.

Two Pione species (Hadromerida, Clionaidae) from the Red Sea: a taxonomical challenge

Boring sponges of the genus Pione (Hadromerida, Clionaidae) are easily recognizable due to their spiculation. However, species identification is challenging, as the potentially diagnostic morphological character states of different species often overlap. For this reason, this group of species is frequently referred to as the ‘Pione vastifica complex’, after the most well-studied species of the genus. Boring-sponge samples were collected in the Red Sea and identified as P. cf. lampa and P. cf. vastifica, respectively. So far, these two species names have usually been considered as valid, although some authors suggested them to be synonymous. Morphological analyses were performed on spicules and micro-erosion patterns by means of both light and scanning electron microscopy. Two apparent morphotypes can be distinguished, mainly by the growth form, but statistical analysis does not support a clear separation in two species. In addition, a DNA barcoding approach using sequences of CO1 has not identified any nucleotide sequence differences. These data support the hypothesis that P. cf. lampa and P. cf. vastifica from the Red Sea are conspecific.

Prolactin Family of the Guinea Pig, Cavia porcellus

Prolactin (PRL) is a multifunctional hormone with prominent roles in regulating growth and reproduction. The guinea pig (Cavia porcellus) has been extensively used in endocrine and reproduction research. Thus far, the PRL cDNA and protein have not been isolated from the guinea pig. In the present study, we used information derived from the public guinea pig genome database as a tool for identifying guinea pig PRL and PRL-related proteins. Guinea pig PRL exhibits prominent nucleotide and amino acid sequence differences when compared with PRLs of other eutherian mammals. In contrast, guinea pig GH is highly conserved. Expression of PRL and GH in the guinea pig is prominent in the anterior pituitary, similar to known expression patterns of PRL and GH for other species. Two additional guinea pig cDNAs were identified and termed PRL-related proteins (PRLRP1, PRLRP2). They exhibited a more distant relationship to PRL and their expression was restricted to the placenta. Recombinant guinea pig PRL protein was generated and shown to be biologically active in the PRL-responsive Nb2 lymphoma cell bioassay. In contrast, recombinant guinea pig PRLRP1 protein did not exhibit PRL-like bioactivity. In summary, we have developed a new set of research tools for investigating the biology of the PRL family in an important animal model, the guinea pig.

Identification of SNAPc subunit domains that interact with specific nucleotide positions in the U1 and U6 gene promoters.

The small nuclear RNA (snRNA)-activating protein complex (SNAPc) is essential for transcription of genes coding for the snRNAs (U1, U2, etc.). In Drosophila melanogaster, the heterotrimeric DmSNAPc recognizes a 21-bp DNA sequence, the proximal sequence element A (PSEA), located approximately 40 to 60 bp upstream of the transcription start site. Upon binding the PSEA, DmSNAPc establishes RNA polymerase II preinitiation complexes on U1 to U5 promoters but RNA polymerase III preinitiation complexes on U6 promoters. Minor differences in nucleotide sequence of the U1 and U6 PSEAs determine RNA polymerase specificity; moreover, DmSNAPc adopts different conformations on these different PSEAs. We have proposed that such conformational differences in DmSNAPc play a key role in determining the different polymerase specificities of the U1 and U6 promoters. To better understand the structure of DmSNAPc-PSEA complexes, we have developed a novel protocol that combines site-specific protein-DNA photo-cross-linking with site-specific chemical cleavage of the protein. This protocol has allowed us to map regions within each of the three DmSNAPc subunits that contact specific nucleotide positions within the U1 and U6 PSEAs. These data help to establish the orientation of each DmSNAPc subunit on the DNA and have revealed cases in which different domains of the subunits differentially contact the U1 versus U6 PSEAs.

Mapping SNAPc subunit domains that interact with specific nucleotide positions in the U1 and U6 snRNA gene promoters

The small nuclear RNA activating protein complex (SNAPc) is essential for transcription of genes coding for the snRNAs (U1, U2, etc.). In D. melanogaster, the heterotrimeric DmSNAPc recognizes a 21 base‐pair DNA sequence, the PSEA, located approximately 40–60 base pairs upstream of the transcription start site. Upon binding the PSEA, DmSNAPc establishes RNA polymerase II pre‐initiation complexes on U1‐U5 promoters but RNA polymerase III pre‐initiation complexes on U6 promoters. Minor differences in nucleotide sequence of the U1 and U6 PSEAs determine RNA polymerase specificity; moreover, DmSNAPc adopts different conformations on these different PSEAs. We have proposed that such conformational differences in DmSNAPc play a key role in determining the different polymerase specificities of the U1 and U6 promoters. To better understand the structure of DmSNAPc‐PSEA complexes, we have developed a novel protocol that combines site‐specific protein‐DNA photo‐cross‐linking with chemical cleavage of the protein. This has allowed us to map regions of the three subunits that contact specific nucleotide positions within the U1 and U6 PSEAs. These data help to establish the orientation of each DmSNAPc subunit on the DNA and have revealed cases in which different domains of the subunits differentially contact the U1 versus U6 PSEAs. (Supported by NSF and in part by the California Metabolic Research Foundation.)

Comparison of mitochondrial and chloroplast genome segments from three onion (Allium cepa L.) cytoplasm types and identification of a trans-splicing intron of cox2

To study genetic relatedness of two male sterility-inducing cytotypes, the phylogenetic relationship among three cytotypes of onions (Allium cepa L.) was assessed by analyzing polymorphisms of the mitochondrial DNA organization and chloroplast sequences. The atp6 gene and a small open reading frame, orf22, did not differ between the normal and CMS-T cytotypes, but two SNPs and one 4-bp insertion were identified in CMS-S cytotype. Partial sequences of the chloroplast ycf2 gene were integrated in the upstream sequence of the cob gene via short repeat sequence-mediated recombination. However, this chloroplast DNA-integrated organization was detected only in CMS-S. Interestingly, disruption of a group II intron of cox2 was identified for the first time in this study. Like other trans-splicing group II introns in mitochondrial genomes, fragmentation of the intron occurred in domain IV. Two variants of each exon1 and exon2 flanking sequences were identified. The predominant types of four variants were identical in both the normal and the CMS-T cytotypes. These predominant types existed as sublimons in CMS-S cytotypes. Altogether, no differences were identified between normal and CMS-T, but significant differences in gene organization and nucleotide sequences were identified in CMS-S, suggesting recent origin of CMS-T male-sterility from the normal cytotype.

Pepino mosaic virus: a successful pathogen that rapidly evolved from emerging to endemic in tomato crops

Pepino mosaic virus (PepMV) belongs to the Potexvirus genus of the Flexiviridae family. PepMV virions are nonenveloped flexuous rods that contain a monopartite, positive-sense, single-stranded RNA genome of 6.4 kb with a 3' poly-A tail. The genome contains five major open reading frames (ORFs) encoding a 164-kDa RNA-dependent RNA polymerase (RdRp), three triple gene block proteins of 26, 14 and 9 kDa, and a 25-kDa coat protein. Four PepMV genotypes, with an intergenotype RNA sequence identity ranging from 78% to 95%, can be distinguished: the original Peruvian genotype (LP); the European (tomato) genotype (EU); the American genotype US1; and the Chilean genotype CH2. PepMV is very efficiently transmitted mechanically, and a low seed transmission rate has been demonstrated. In addition, bumblebees have been associated with viral transmission. Similar to other Potexviruses, PepMV has a rather narrow host range that is thought to be largely restricted to species of the Solanaceae family. After originally being isolated from pepino (Solanum muricatum), PepMV has been identified in natural infections of the wild tomato species S. chilense, S. chmielewskii, S. parviflorum and S. peruvianum. PepMV is causing significant problems in the cultivation of the glasshouse tomato Solanum lycopersicum, and has been identified in weeds belonging to various plant families in the vicinity of tomato glasshouses. PepMV symptoms can be very diverse. Fruit marbling is the most typical and economically devastating symptom. In addition, fruit discoloration, open fruit, nettle-heads, leaf blistering or bubbling, leaf chlorosis and yellow angular leaf spots, leaf mosaic and leaf or stem necrosis have been associated with PepMV. The severity of PepMV symptoms is thought to be dependent on environmental conditions, as well as on the properties of the viral isolate. Minor nucleotide sequence differences between isolates from the same genotype have been shown to lead to enhanced aggressiveness and symptomatology. Prevention of infection through strict hygiene measures is currently the major strategy for the control of PepMV in tomato production. Cross-protection can be effective, but only under well-defined and well-controlled conditions, and the effectiveness depends strongly on the PepMV genotype.

Molecular Variation and Distribution ofAnopheles fluviatilis(Diptera: Culicidae) Complex in Iran

Anopheles fluviatilis James (Diptera: Culicidae) is one of the known malaria vectors in south and southeastern Iran. Earlier ITS2 sequences analysis of specimens from Iran demonstrated only a single genotype that was identical to species Y in India, which is also the same as species T. We identified 2 haplotypes in the An. fluviatilis populations of Iran based on differences in nucleotide sequences of D3 domain of the 28S locus of ribosomal DNA (rDNA). Comparison of sequence data from 44 Iranian specimens with those publicly available in the Genbank database showed that all of the 28S-D3 sequences from Kazeroun and Khesht regions in Fars Province were identical to the database entry representing species U in India. In other regions, all the individuals showed heterozygosity at the single nucleotide position, which identifies species U and T. It is argued that the 2 species may co-occur in some regions and hybridize; however, the heterozygosity in the 28S-D3 locus was not reflected in ITS2 sequences and this locus for all individuals was identical to species T. This study shows that in a newly diverged species, like members of An. fluviatilis complex, a single molecular marker may not be sufficiently discriminatory to identify all the taxa over a vast geographical area. In addition, other molecular markers may provide more reliable information for species discrimination.

Molecular characterization through IGS sequencing of formae speciales of Fusarium oxysporum pathogenic on lamb's lettuce.

Twenty-nine strains of Fusarium oxysporum isolated from wilted lamb’s lettuce plants ( Valerianella olitoria ) along with eight ATCC reference strains were examined for differences in the nucleotide sequences of the ribosomal DNA (rDNA) intergenic spacer (IGS) region, which is about 2.5 kb long in these strains. A phylogenetic (neighbour-joining) analysis was performed on the strains and identified four phylogenetic groups, I, II, III, and IV. Most F. oxysporum isolates recovered from wilted lamb’s lettuce plants cultivated in northern Italy clustered in group I and were very similar to F. oxysporum f. sp. raphani . All the isolates, including the 8 control strains, were tested for pathogenicity on lamb’s lettuce cv. Trophy cultivated in the glasshouse. Most isolates from northern Italy were pathogenic on lamb’s lettuce, displaying varying degrees of virulence, and their IGS sequence were similar to the forma specialis raphani . However, four isolates were not pathogenic on lamb’s lettuce. Among the control strains, six showed moderate virulence; these belonged to the formae speciales raphani and conglutinans ; and two, belonging to the forma specialis matthioli (ATCC16602 and ATCC16603) were not pathogenic on lamb’s lettuce. In conclusion, analysis of the IGS sequences indicated that the isolates studied had different origins, and that phylogeny and pathogenicity were related; non-pathogenic isolates differed genetically from isolates that were poorly, moderately or highly virulent. To the best of our knowledge, this is the first report on the differences between formae speciales of F. oxysporum on lamb’s lettuce plants as determined by IGS sequence analysis.

Frequent gene conversion events between the X and Y homologous chromosomal regions in primates

BackgroundMammalian sex-chromosomes originated from a pair of autosomes. A step-wise cessation of recombination is necessary for the proper maintenance of sex-determination and, consequently, generates a four strata structure on the X chromosome. Each stratum shows a specific per-site nucleotide sequence difference (p-distance) between the X and Y chromosomes, depending on the time of recombination arrest. Stratum 4 covers the distal half of the human X chromosome short arm and the p-distance of the stratum is ~10%, on average. However, a 100-kb region, which includes KALX and VCX, in the middle of stratum 4 shows a significantly lower p-distance (1-5%), suggesting frequent sequence exchanges or gene conversions between the X and Y chromosomes in humans. To examine the evolutionary mechanism for this low p-distance region, sequences of a corresponding region including KALX/Y from seven species of non-human primates were analyzed.ResultsPhylogenetic analysis of this low p-distance region in humans and non-human primate species revealed that gene conversion like events have taken place at least ten times after the divergence of New World monkeys and Catarrhini (i.e., Old World monkeys and hominoids). A KALY-converted KALX allele in white-handed gibbons also suggests a possible recent gene conversion between the X and Y chromosomes. In these primate sequences, the proximal boundary of this low p-distance region is located in a LINE element shared between the X and Y chromosomes, suggesting the involvement of this element in frequent gene conversions. Together with a palindrome on the Y chromosome, a segmental palindrome structure on the X chromosome at the distal boundary near VCX, in humans and chimpanzees, may mediate frequent sequence exchanges between X and Y chromosomes.ConclusionGene conversion events between the X and Y homologous regions have been suggested, mainly in humans. Here, we found frequent gene conversions in the evolutionary course of primates. An insertion of a LINE element at the proximal end of the region may be a cause for these frequent conversions. This gene conversion in humans may also be one of the genetic causes of Kallmann syndrome.

Clonal fingerprinting in the genus Populus L. by nuclear microsatellite loci regarding differences between sections, species and hybrids

Reliable methods for clone identification are desired to characterise and distinguish breeding products within the genus Populus L. (Salicaceae). Ten nuclear microsatellite loci (PMGC14, PMGC456, PMGC2163, PTR2, PTR7, WPMS05, WPMS09, WPMS14, WPMS15 and WPMS20) were applied on a clone collection with several species and hybrids belonging to the sections Tacamahaca (balsam poplars), Aigeiros (black poplars, cottonwoods) and Populus (white poplars and aspens) and intersectional hybrids between black and balsam poplars. The members of the different sections and species do not always share their allelic ladders. Some shifts of one or two nucleotides in allele length were observed for several loci. This could be explained by nucleotide sequence differences in the flanking regions of loci in diverse taxonomic groups. Such shifts of allelic ladders result in irregular patterns in hybrid genotypes. The set of loci should have a sufficient amount of variation for a differentiation between clones, even if they are full siblings originating from crossing experiments.

CHARACTERIZATION OF LEPTOSPIRES USING V3 REGION OF 16S rDNA BY DENATURING GRADIENT GEL ELECTROPHORESIS: A CASE STUDY

Serological and molecular characterization of Leptospiral isolates helps us to identify serovar, which is useful, for epidemiological study. Serological characterization is tedious and requires a panel of monoclonal antibodies and expertise to read the results. This study is a preliminary work to evaluate the usefulness of Denaturing Gradient Gel Electrophoresis (DGGE) to identify serovars of leptospira. The V3 region of most conserved 16S rDNA of five pathogenic leptospiral serovars and one saprophytic serovar was characterized. DGGE method was employed to separate the amplified V3 region based on the nucleotide sequence. On DGGE, amplified V3 region of leptospiral serovars, under study, showed bands at different positions indicating DGGE as the effective method of characterization in the future. DNA sequencing of V3 region of the three serovars showed great difference in nucleotide sequence supporting the results of DGGE.

Genotyping of hepatitis A virus prevalent strains in Xinjiang Hetian of China in 2006

To analysis the genotypes of wild type hepatitis A virus circulated in Xinjiang Hetian of China in 2006. The Vp1-2A region of HAV genome was amplified and sequenced from serum samples collected in Xinjiang Hetian of China in 2006, and subjected to phylogenetic analysis by Neighbor Joining (NJ) method. The nucleotide sequence differences in the VP1-2A region among Xinjiang Hetian HAV strains ranged from 0%-3.9%, all belonged to sub-genotype 1A. Genetically similar strains were identified among Xinjiang Hetian 2006 and Xinjiang Yili 2005 of China isolates. Only 0-2 amino acid differences were found among the Xinjiang Hetian HAV isolates in the VP1-2A region. There were different HAV strains existing in the investigated areas, these strains may have different transmission pathways for the spread of the disease. The results indicate the usefulness of molecular epidemiological methods in studying changes in the circulating HAV strains and in tracing transmission routes, and also for effectively control measures to prevent the spread of the disease.

Differentiation of field isolates and vaccine strains of infectious laryngotracheitis virus by DNA sequencing

Two different regions of the infected cell protein 4 (ICP4) gene of infectious laryngotracheitis virus (ILTV) were amplified and sequenced for characterization of field isolates and tissue culture-origin (TCO) and chicken embryo-origin (CEO) vaccine strains. Phylogenetic analysis of the two regions showed differences in nucleotide and amino acid sequences between field isolates and attenuated vaccines. The PCR-RFLP results were identical to those obtained by DNA sequencing and validated their use to differentiate ILTV strains. The approach using the sequencing of the two fragments of the ICP4 gene showed to be an efficient and practical procedure to differentiate between field isolates and vaccine strains of ILTV.

Rapid ABO Genotyping Using Whole Blood without DNA Purification

ABO genotyping is commonly used in cases of an ABO discrepancy between cell typing and serum typing, as well as in forensic practice for personal identification and paternity testing. We evaluated ABO genotyping via multiplex allele-specific PCR (ASPCR) amplification using whole blood samples without DNA purification. A four-reaction multiplex ASPCR genotyping assay was designed to detect specific nucleotide sequence differences between the six ABO alleles A101, A102, B101, O01, O02, and cis-AB01. The ABO genotypes of 127 randomly chosen samples were determined using the new multiplex ASPCR method. The genotypes of the 127 samples were found to be A101/A102 (n=1), A102/A102 (n=9), A101/O01 (n=3), A102/O01 (n=12), A102/O02 (n=14), B101/B101 (n=5), B101/O01 (n=18), B101/O02 (n=15), O01/O01 (n=14), O02/O02 (n=8), O01/O02 (n=14) and A102/B101 (n=14), from which phenotypes were calculated to be A (n=39), B (n=38), O (n=36) and AB (n=14). The multiplex ASPCR assay results were compared with the serologically determined blood group phenotypes and genotypes determined by DNA sequencing, and there were no discrepancies. This convenient multiplex ASPCR assay, performed using whole blood samples, provides a supplement to routine serological ABO typing and might also be useful in other genotyping applications.