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Phylogenetic analysis of the genus Aquaspirillum based on 16S rRNA gene sequences.

Phylogenetic analysis of 15 species of the genus Aquaspirillum based on 16S rRNA gene (rDNA) sequences indicated that the genus Aquaspirillum is phylogenetically heterogeneous and the species could be divided into four groups as follows: Aquaspirillum serpens, the type species of this genus, A. dispar and A. putridiconchylium are situated in the family Neisseriaceae; members of the second group, A. gracile, A. delicatum, A. anulus, A. giesbergeri, A. sinuosum, A. metamorphum and A. psychrophilum, are included in the family Comamonadaceae; the two members of the third group, A. arcticum and A. autotrophicum, are included in the family Oxalobacteriaceae; and members of the fourth group, A. polymorphum, A. peregrinum, and A. itersonii, are included in the alpha-subdivision of Proteobacteria. Thus, phylogenetic studies indicated that all the species excepting A. serpens, the type species, should be transferred to distinct genera.

Fresh Water↗

Phylogenetic analysis of GB virus C: comparison of isolates from Africa, North America, and Taiwan.

A group of flavi-like GB viruses (GBV-A, -B, and -C) has been discovered and partially characterized. Only GBV-C is found in humans. Nucleotide sequences of the putative helicase gene of the GBV-C genome were determined in 21 Taiwanese patients and compared with isolates from Africa and North America by phylogenetic analysis. The average similarities of Taiwanese isolates to those from West Africa, East Africa, Canada, and United States were 83%, 85%, 78%, and 82%, respectively. Phylogenetic analysis of the NS3 region showed that 81% of the Taiwanese isolates were more closely related to the East African isolate. GBV-C isolates from Taiwan could be classified into at least 3 groups. These data suggest that GBV-C isolates cloned from different geographic areas have genetic heterogeneity.

Africa↗

Use of tuf sequences for genus-specific PCR detection and phylogenetic analysis of 28 streptococcal species.

A 761-bp portion of the tuf gene (encoding the elongation factor Tu) from 28 clinically relevant streptococcal species was obtained by sequencing amplicons generated using broad-range PCR primers. These tuf sequences were used to select Streptococcus-specific PCR primers and to perform phylogenetic analysis. The specificity of the PCR assay was verified using 102 different bacterial species, including the 28 streptococcal species. Genomic DNA purified from all streptococcal species was efficiently detected, whereas there was no amplification with DNA from 72 of the 74 nonstreptococcal bacterial species tested. There was cross-amplification with DNAs from Enterococcus durans and Lactococcus lactis. However, the 15 to 31% nucleotide sequence divergence in the 761-bp tuf portion of these two species compared to any streptococcal tuf sequence provides ample sequence divergence to allow the development of internal probes specific to streptococci. The Streptococcus-specific assay was highly sensitive for all 28 streptococcal species tested (i.e., detection limit of 1 to 10 genome copies per PCR). The tuf sequence data was also used to perform extensive phylogenetic analysis, which was generally in agreement with phylogeny determined on the basis of 16S rRNA gene data. However, the tuf gene provided a better discrimination at the streptococcal species level that should be particularly useful for the identification of very closely related species. In conclusion, tuf appears more suitable than the 16S ribosomal RNA gene for the development of diagnostic assays for the detection and identification of streptococcal species because of its higher level of species-specific genetic divergence.

DNA Primers↗

A phylogenetic analysis of microbial communities associated with methane hydrate containing marine fluids and sediments in the Cascadia margin (ODP site 892B).

Methane hydrates represent an enormous carbon and energy source in many low temperature deep marine sediments. However, little information is available concerning the nature of the microbial communities associated with these structures. Here, we describe a phylogenetic analysis based on ribosomal DNA (rDNA) sequences obtained from sediment and fluid samples present in a region of gas hydrate formation in shallow sediments within the Cascadia margin in and around Ocean Drilling Program (ODP) Site 892B. Our studies detected diverse sulfur-utilizing microbes, methanogens, methanotrophs, and non-thermophilic members of the kingdom Crenarchaeota. This is the first culture-independent phylogenetic analysis of a gas hydrate habitat.

Archaea↗

Sequencing and phylogenetic analysis of human genotype P[6] rotavirus strains detected in Hungary provides evidence for genetic heterogeneity within the P[6] VP4 gene.

Although rotavirus genotype P[6] is one of the three most common VP4 specificities associated with human infection, the relatively few sequence data available in public databases suggest that the genetic variability within P[6] might be presently unexplored. Thus far, two human P[6] lineages (M37-like and AU19-like) and a single porcine P[6] lineage (Gottfried-like) have been identified by phylogenetic analysis. Serologic studies demonstrated that these three lineages are antigenically distinct from each other, a finding based on which they were classified into three subtypes, P2A[6] (M37-like), P2B[6] (Gottfried-like), and P2C[6] (AU19-like). To study heterogeneity within this genotype, we selected for molecular characterization a total of six P[6] strains detected during an ongoing surveillance in Hungary. The variable region of the VP4 gene was subjected to sequencing and phylogenetic analysis. Our data indicated that these six strains fell into two phylogenetic lineages distinguishable from the human lineages M37-like and AU19-like and from the porcine lineage Gottfried-like. Further studies are needed to understand whether these two novel lineages are genuine human strains or might have originated from animal strains and to evaluate the antigenic relationship of the novel Hungarian P[6] strains to the three established subtypes.

Amino Acid Sequence↗

Tracing the origins of louping ill virus by molecular phylogenetic analysis.

The nucleotide and deduced amino acid sequences of louping ill (LI) virus isolates, collected from representative regions of the British Isles and Norway, were determined for either the entire envelope gene (20 isolates) or for a portion of the envelope gene that spans a hypervariable region and includes an LI virus specific marker sequence (53 isolates). Phylogenetic analysis reveals the presence of three major geographical populations of LI virus in the British Isles, viz. Irish, Welsh and British LI viruses, which all cause encephalomyelitis in animals, predominantly sheep, and co-habit the same tick population. British LI virus occurs throughout Scotland, England, Ireland and Norway. Irish and Welsh LI viruses occur only in Ireland and Wales, respectively. Phylogenetic analysis also predicts that LI virus initially emerged in Ireland and that a descendant was introduced into Great Britain via Wales and was subsequently transported to the borders of Scotland, from where it was dispersed throughout Scotland, northern England and Norway. More recently, the British LI virus was reintroduced into Ireland and also into south-west England. Dates of lineage divergence, calculated from the synonymous substitution rate, indicate that LI virus emerged in the British Isles less than 800 years ago and most LI virus dispersal occurred during the last 300 years. By combining these data with historical records it appears that livestock movement can be implicated in the dispersal of LI virus.

Animals↗

[Phylogenetic analysis of the surface glycoprotein genes of an aquatic bird origin influenza virus isolate A/Duck/Yangzhou/233/2002 (H6N2)].

Several H6 subtype avian A influenza viruses were isolated from aquatic birds in some live bird markets when we surveyed the ecology of the influenza in East China for more than two years and identified by specific RT-PCR. In this paper, the hemagglutinin (HA) and neuraminidase (NA) gene of one representative virus named A/Duck/Yangzhou/233/ 2002 (H6N2) (Dk/YZ/233/02) had been sequenced. Phylogenetic analysis of the H6 gene sequences available in the Influenza Sequence Database showed that the Dk/YZ/233/02 viruse cluster together with Dk/HK/3461/99 (H6N1) and Ck/Taiwan/na3/98 (H6N1). Phylogenetic analysis of the N2 NA genes of Dk/YZ/233/02 revealed that the NA gene of Dk/YZ/233/02 had genetically close relationships with that of H9N2 viruses isolated from duck in Japan and from chickens in South Korea, which were distinct from those of Ck/Beijing/94 (H9N2). The sequence of cleavage site between HA1 and HA2 of Dk/YZ/233/02 is P-Q-I-E-T-R-D, which was the typical characterization of the LPAIV.

Animals↗

Genetic diversity and phylogenetic analysis of human immunodeficiency virus type 1 subtypes circulating in French Guiana.

We investigated the characterization of different HIV-1 subtypes present in French Guiana by use of three different methods. Serological methods were used for the initial screening, which were then confirmed by the heteroduplex mobility assay (HMA). The V3 env region was subsequently sequenced for phylogenetic analysis, to confirm the subtype of the samples, and to assign a subtype to samples that gave results that were difficult to interpret or discordant by serology or HMA. A total of 221 HIV-1 seropositive samples were typed; 110 of them were confirmed by HMA and 16 were sequenced. Of the 221 samples tested 210 patients (95%) were found to be infected with subtype B, 10 (4.5%) were infected with subtype A, and one patient was infected with subtype F. Phylogenetic analysis demonstrated that the strains from French Guiana were closely related to the subtype A and B subtypes, and that one strain was closely related to an F subtype (100% bootstrap value). Four strains from French Guiana clustered in the subtype A (99% bootstrap value) and the other strains were associated with subtype B (100% bootstrap value). The geographic position of French Guiana suggested that HIV-1 was probably introduced into the country via several routes, and thus the pattern of the HIV-1 epidemic might evolve in the near future.

AIDS Serodiagnosis↗

Phylogenetic analysis of the major capsid protein gene of iridovirus isolates from cultured flounders Paralichthys olivaceus in Korea.

In 2003, 13 isolates of iridovirus were obtained from cultured flounders Paralichthys olivaceus during epizootics in Korea. The full open reading frames (ORFs) encoding the major capsid protein (MCP) (1362 bp) from the 13 flounder iridoviruses (FLIVs) were sequenced and the deduced amino acid sequences were phylogenetically analyzed. Phylogenetic analysis of the MCP revealed that all 13 FLIVs were the same species as rock bream iridovirus (RBIV), red sea bream iridovirus (RSIV), and infectious spleen and kidney necrosis virus (ISKNV), and were grouped into an unknown genus which was different from the 2 genera known to infect fish, Ranavirus and Lymphocystivirus. This is the first report on the isolation and phylogenetic analysis of the iridovirus of unknown genus from flounders during epizootics.

Amino Acid Sequence↗

Phylogenetic analysis of bacterial communities associated with ectoparasitic chewing lice of pocket gophers: a culture-independent approach.

This study identifies the bacteria associated with ectoparasitic chewing lice that live in the fur of pocket gophers. Samples of chewing lice were collected from pocket gopher hosts in Florida, Missouri, New Mexico, and Costa Rica. We used a molecular sampling method whereby total community DNA was extracted from samples of chewing lice, and PCR was used to selectively amplify small-subunit rRNA genes from bacteria. This culture-independent method yielded ca. 35 distinct lineages representing eight widely divergent groups within the domain Bacteria. Phylogenetic analysis of two lineages (Acinetobacter and Staphylococcus) provides evidence that multiple species of each group are found in chewing lice. Phylogenetic analysis also demonstrated that diversification within chewing lice may be evident in both Acinetobacter and Staphylococcus. Some clones amplified from chewing louse hosts appeared to be distinct from known species of Acinetobacter and Staphylococcus. This diversification may be the result of the extreme isolation of populations of both chewing lice and their pocket gopher hosts.

Animals↗

Classification of hepatitis C viruses based on phylogenetic analysis of the envelope 1 and nonstructural 5B regions and identification of five additional subtypes.

Genotyping of hepatitis C virus-positive sera by means of a line probe assay indicated that < 3% of European samples, but up to 30% of Gabonese sera, could not be classified as either 1a, 1b, 2a, 2b, 3a, 3b, 4c, 5a, or 6a. Such samples were analyzed in the 5' untranslated region and in the nonstructural 5 (NS5) region. Classification based on phylogenetic analysis of the commonly used 222-bp-long NS5B region was possible for most but not all of the selected sera. Therefore, the core/envelope 1 region (579 bp) and a larger NS5B (340 bp) region were also analyzed. Only the phylogenetic analysis of the 340-bp NS5B region of these newly identified and published isolates provided unambiguous classification into types and subtypes. Furthermore, unequivocal evidence for four subtypes in type 2 and eight subtypes in type 4 was provided. A specific recognition sequence in the 5' untranslated region was observed for every newly identified subtype. Based on 1830 pair-wise comparisons in NS5B, isolates belonging to the same subtype showed evolutionary distances of < 0.127 and isolates of the same type exhibited evolutionary distances of < 0.328. These phylogenetic border distances can be conveniently used for classification of hepatitis C virus isolates into types and subtypes.

Amino Acid Sequence↗

Genotyping of Giardia in Dutch patients and animals: a phylogenetic analysis of human and animal isolates.

Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is a protozoan organism that can infect the intestinal tract of many animal species including mammals. Genetic heterogeneity of G. duodenalis is well described but the zoonotic potential is still not clear. In this study, we analysed 100 Giardia DNA samples directly isolated from human stool specimens, to get more insight in the different G. duodenalis assemblages present in the Dutch human population. Results showed that these human isolates could be divided into two main Assemblages A and B within the G. duodenalis group on the basis of PCR assays specific for the Assemblages A and B and the DNA sequences of 18S ribosomal RNA and the glutamate dehydrogenase (gdh) genes. Genotyping results showed that G. duodenalis isolates originating from Dutch human patients belonged in 35% of the cases to Assemblage A (34/98) and in 65% of the cases to Assemblage B (64/98) whereas two human cases remained negative in all assays tested. In addition, we compared these human samples with animal samples from the Netherlands and human and animal samples from other countries. A phylogenetic analysis was carried out on the DNA sequences obtained from these Giardia and those available in GenBank. Using gdh DNA sequence analysis, human and animal Assemblage A and B Giardia isolates could be identified. However, phylogenetic analysis revealed different sub-clustering for human and animal isolates where host-species-specific assemblages (C, D, E, F and G) could be identified. The geographic origin of the human and animal samples was not a discriminating factor.

Animals↗

Application of polymerase chain reaction and subsequent phylogenetic analysis to the diagnosis of enteroviral infection in the central nervous system.

BACKGROUND: Enteroviral infections of the central nervous system (CNS) are often difficult to diagnose, even if consistent conventional laboratory methodologies are used. OBJECTIVES: To clarify the efficiency of two polymerase chain reaction (PCR) methods for the sensitive detection of enteroviruses and for the identification of enteroviral genotypes based on phylogenetic analysis of the amplified genome sequences, and to facilitate the diagnosis of enteroviral infection in CNS. STUDY DESIGN: Cerebrospinal fluid (CSF), throat swab, rectal swab, and/or serum samples were collected from 171 patients with aseptic meningitis and 67 patients with febrile seizures. The samples were tested for the presence of enteroviruses by cell culture and PCR methods for the detection and identification of enteroviruses. RESULTS: In 111 (64.9%) of 171 patients with aseptic meningitis, enteroviruses were isolated by cell cultures from any site. In 143 (83.6%) patients, including 110 of 111 patients with aseptic meningitis, the enteroviral genome was detected in CSF by PCR. No enterovirus was isolated from any site for the 67 patients with febrile seizures. PCR detected the enteroviral genome in CSF samples from 13 (61.9%) of 21 patients who developed febrile seizures in the summer (June-August). Phylogenetic analysis of amplified genome sequences showed that the major pathogens of febrile seizures in summer were group A coxsackieviruses, which are usually difficult to isolate by cell culture. CONCLUSION: PCR methods for the detection and identification of enteroviruses were useful for the diagnosis of enteroviral infection in CNS.

Central Nervous System Viral Diseases↗

Ultrastructure and phylogenetic analysis of 'Candidatus Neoehrlichia mikurensis' in the family Anaplasmataceae, isolated from wild rats and found in Ixodes ovatus ticks.

A novel bacterium that infects laboratory rats was isolated from wild Rattus norvegicus rats in Japan. Transmission electron microscopy of the spleen tissue revealed small cocci surrounded by an inner membrane and a thin, rippled outer membrane in a membrane-bound inclusion within the cytoplasm of endothelial cells. Phylogenetic analysis of the 16S rRNA gene sequence of the bacterium found in R. norvegicus rats and Ixodes ovatus ticks in Japan revealed that the organism represents a novel clade in the family Anaplasmataceae, which includes the Schotti variant found in Ixodes ricinus ticks in the Netherlands and the Ehrlichia-like Rattus strain found in R. norvegicus rats from China. The novel clade was confirmed by phylogenetic analysis of groESL sequences found in R. norvegicus rats and Ixodes ovatus ticks in Japan. No serological cross-reactivity was detected between this bacterium and members of the genera Anaplasma, Ehrlichia or Neorickettsia in the family Anaplasmataceae. It is proposed that this new cluster of bacteria should be designated 'Candidatus Neoehrlichia mikurensis'.

Anaplasmataceae↗

Phylogenetic analysis of hepatitis E virus isolates from Egypt.

Hepatitis E virus (HEV) genome was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in fecal samples of two sporadic cases of hepatitis E in Cairo Egypt. Sequence of the complete putative structural region [open reading frame (ORF)-2] and complete region of unknown function (ORF-3) was determined for the two HEV isolates. Phylogenetic analysis of the nucleotide sequences was performed using neighbor joining or maximum parsimony methods of tree reconstruction. Direct correspondence between the HEV evolutionary trees and geographic origin of the HEV isolates was observed. Three genotypes of HEV were identified: genotype I (Asia-Africa), genotype II (US), and genotype III (Mexico). Genotype I was further divided into two subgenotypes (Asia and Africa). In the Asian subgenotype, three smaller genetic clusters were observed (China-like sequences, Burma-like sequences, and sequence from a fulminant case of HEV). The segregation of all these genetic clusters was supported by the high level of bootstrap probabilities. Four regions of the HEV genome were used for phylogenetic analysis. In all four regions, Egyptian HEV isolates were grouped in a separate African clade.

Adult↗

Application of hepatitis B virus genotyping and phylogenetic analysis in intrafamilial transmission of hepatitis B virus.

BACKGROUND: Infection with hepatitis B virus (HBV) in early life frequently results in persistent infection, and clustering of the chronic infection within a family is common. However, the relative contribution of perinatal mother-to-infant transmission or early horizontal transmission to the intrafamilial clustering of HBV infection remains unclear. Therefore, we used HBV genotyping and phylogenetic analysis to elucidate the modes of intrafamilial HBV transmission in Taiwan. METHODS: HBV genotypes and serological markers were determined for 103 individuals from 20 families with evidence of clustering HBV infection. RESULTS: Three patterns of intrafamilial clustering of HBV infection were identified. Among the 20 families, 8 included a hepatitis B surface antigen (HBsAg)-positive mother (pattern I), 7 included an HBsAg-positive father (pattern II), and in the remaining 5, both parents were positive for HBsAg (pattern III). The rates of HBsAg positivity for children of the 3 representative groups of families were 85.7%, 65.4%, and 87.5%, respectively (P = .16). The identical genotyping results between index parent and carrier children indicated that pattern I clustering was caused by maternal transmission, whereas pattern II clustering was caused by paternal transmission. In pattern III clustering, a concordant HBV genotype between carrier children and mother or father was found in 3 and 2 families, respectively. The modes of transmission were confirmed by phylogenetic analysis in 1 family of each pattern. CONCLUSIONS: In Taiwan, maternal and paternal transmissions are both important in the intrafamilial spread of HBV infection.

Adult↗

galaxieEST: addressing EST identity through automated phylogenetic analysis.

BACKGROUND: Research involving expressed sequence tags (ESTs) is intricately coupled to the existence of large, well-annotated sequence repositories. Comparatively complete and satisfactory annotated public sequence libraries are, however, available only for a limited range of organisms, rendering the absence of sequences and gene structure information a tangible problem for those working with taxa lacking an EST or genome sequencing project. Paralogous genes belonging to the same gene family but distinguished by derived characteristics are particularly prone to misidentification and erroneous annotation; high but incomplete levels of sequence similarity are typically difficult to interpret and have formed the basis of many unsubstantiated assumptions of orthology. In these cases, a phylogenetic study of the query sequence together with the most similar sequences in the database may be of great value to the identification process. In order to facilitate this laborious procedure, a project to employ automated phylogenetic analysis in the identification of ESTs was initiated. RESULTS: galaxieEST is an open source Perl-CGI script package designed to complement traditional similarity-based identification of EST sequences through employment of automated phylogenetic analysis. It uses a series of BLAST runs as a sieve to retrieve nucleotide and protein sequences for inclusion in neighbour joining and parsimony analyses; the output includes the BLAST output, the results of the phylogenetic analyses, and the corresponding multiple alignments. galaxieEST is available as an on-line web service for identification of fungal ESTs and for download / local installation for use with any organism group at http://galaxie.cgb.ki.se/galaxieEST.html. CONCLUSIONS: By addressing sequence relatedness in addition to similarity, galaxieEST provides an integrative view on EST origin and identity, which may prove particularly useful in cases where similarity searches return one or more pertinent, but not full, matches and additional information on the query EST is needed.

Computational Biology↗

Characterization of agglutinin-like sequence genes from non-albicans Candida and phylogenetic analysis of the ALS family.

The ALS (agglutinin-like sequence) gene family of Candida albicans encodes cell-surface glycoproteins implicated in adhesion of the organism to host surfaces. Southern blot analysis with ALS-specific probes suggested the presence of ALS gene families in C. dubliniensis and C. tropicalis; three partial ALS genes were isolated from each organism. Northern blot analysis demonstrated that mechanisms governing expression of ALS genes in C. albicans and C. dubliniensis are different. Western blots with an anti-Als serum showed that cross-reactive proteins are linked by beta 1,6-glucan in the cell wall of each non-albicans Candida, suggesting similar cell wall architecture and conserved processing of Als proteins in these organisms. Although an ALS family is present in each organism, phylogenetic analysis of the C. albicans, C. dubliniensis, and C. tropicalis ALS genes indicated that, within each species, sequence diversification is extensive and unique ALS sequences have arisen. Phylogenetic analysis of the ALS and SAP (secreted aspartyl proteinase) families show that the ALS family is younger than the SAP family. ALS genes in C. albicans, C. dubliniensis, and C. tropicalis tend to be located on chromosomes that also encode genes from the SAP family, yet the two families have unexpectedly different evolutionary histories. Homologous recombination between the tandem repeat sequences present in ALS genes could explain the different histories for co-localized genes in a predominantly clonal organism like C. albicans.

Agglutinins↗