Phylogenetic analysis of human metapneumovirus from New York State patients during February through April 2010

Background

Human metapneumovirus (hMPV) is the second leading cause of lower respiratory infection (LRI) in children around the world and has been linked to LRI in multiple studies. Currently, hMPV is classified into 2 major subtypes (A and B), each with 2 subgroups (1 and 2).

Objective

To determine which hMPV genotypes were present in NYS patients with influenza-like illness (ILI) from February through April 2010, during a period of unusually heightened activity.

Study design

Specimens were collected from February through April of 2010 from patients presenting with ILI who were previously confirmed as positive for hMPV by real-time RT-PCR. A 700 base pair region of the hMPV fusion (F) gene was amplified, sequenced and resulting sequences aligned. A phylogenic tree was constructed based on prototype strains, and the partial F gene sequences obtained in this study.

Results

Bi-directional sequence was obtained from 30 patient samples and included in the phylogenic analysis. Specimen sequences grouped into hMPV genotype A2a (16), A2b (9), B2 (4) and B1 (1). No A1 genotypes were found.

Conclusion

Previously, reports have demonstrated that genotypes A1, A2, B1 and B2 circulate every season, usually with one dominant strain. In contrast, late in the 2009-2010 respiratory season, 4 of the 5 recognized genotypes of hMPV were isolated from NYS ILI patients, and by sequencing a larger portion of the fusion gene, we were able to identify the A2a and A2b genotypes.     

A phylogenetic study of hepatitis B virus in chronically infected Brazilian patients of Western and Asian descent

Background  Hepatitis B virus (HBV) causes one of the most important chronic viral infections worldwide. HBV is classified into eight genotypes whose epidemiology varies geographically. In Brazil, genotypes A, D, and F are more frequent, while in East Asia, genotypes B and C predominate. Several studies showed that immigrants retain the HBV infection pattern of their ancestral country. Purpose  To identify HBV genotypes infecting chronic carriers in Brazilian families of Western and Asian descent by Hepatitis B surface antigen gene sequencing and analyze the route of viral transmission by phylogenetic analysis of viral sequences. Methods  Eighty-seven people chronically infected with HBV were separated into two groups: Western descent (27) and Asian descent (60). Surface and pre-core/core genes were amplified from serum HBV-DNA and sequences were subjected to phylogenetic analysis. Results  HBV genotype A was found in 74% of Western subjects, while genotype C was found in 94% of Asian patients. Thirty-eight percent of Western families were infected with HBV with similar pre-core/core sequences, while only 25% of Asian families showed similarity in these sequences. Conclusions  Phylogenetical analysis of pre-core/core HBV gene suggested intra-familial transmission of HBV in 38% of Western families and 25% of Asian families. Analysis of HBsAg gene sequences helped to define the HBV genotype but did not allow inferring route of transmission as its sequences showed a smaller phylogenetic signal than pre-core/core sequences. Chronic HBV carriers of Asian descent born in or living in Brazil were infected with the same HBV genotype predominant in their ancestral country.     

Characterization and Phylogenetic Analysis of Brazilian Chicken Anaemia Virus

Chicken anaemia virus (CAV) was detected by a Nested-PCR assay in field samples from different regions of Brazil. The 539 bp amplified fragments of vp1 gene from 44 field samples were sequenced and 10 new nucleotide sequences of CAV were observed. These sequences were phylogenetically analysed by Mega2 using neighbour joining distance methods with 1000 bootstrap replications. Phylogenetic analysis did not show correlation between CAV pathology pattern and genetic groups. The 10 nucleotide sequences of the Brazilian samples were also analysed together with 30 sequences of CAV strains previously described from other countries. The genetic variability observed was not related to the geographical distribution. Amino acid substitutions were detected at 9 positions of the Brazilian sequences and two of them had not been observed before, ⁶⁵R replacing the Q residue and ⁹⁸F replacing Y residue. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers AY855079 to AY855088.     

Molecular analyses of microbial diversity associated with the Lonar soda lake in India: an impact crater in a basalt area

The prokaryotic diversity associated with an Indian soda lake (Lonar Crater Lake) located in a basaltic soil area was investigated using a culture-independent approach. Community DNA was extracted directly from four sediment samples obtained by coring to depths of 10-20 cm. Small subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific to the domains Bacteria and Archaea. The PCR products were cloned and sequenced. For the bacterial rDNA clone library, 500 clones were randomly selected for further analysis. After restriction fragment length polymorphism (RFLP) analysis and subsequent sequencing, a total of 44 unique phylotypes were obtained. These phylotypes spanned a wide range within the domain Bacteria, occupying eight major lineages/phyla. 34% of the clones were classified as firmicutes. The other clones were grouped into proteobacteria (29.5%), actinobacteria (6.8%), deinococcus-thermus (4.5%), cytophages-flavobacterium-bacteroidetes (13.3%), planctomycetes (6.8%), cyanobacteria (4.5%) and spirochetes (2.27%). In the case of the archaeal 16S rDNA library, analysis of 250 randomly selected clones revealed the presence of 13 distinct phylotypes; 5 phylotypes were associated with Crenarchaeota and 8 with Euryarchaeota. Most of the euryarchaeota sequences were related to methanogens. Findings from this molecular study of a site investigated for the first time have revealed the presence of a highly diverse bacterial population and a comparatively less diverse archaeal population. The majority ( approximately 80%) of the cloned sequences show little affiliation with known taxa (<97% sequence similarity) and may represent novel taxa/sequences and organisms specifically adapted to this basaltic soda lake environment. Diversity analyses demonstrate greater diversity and evenness of bacterial species compared to a skewed representation of species for Archaea.     

Genetic encapsulation among Near Eastern populations

This report aims to genetically characterize the relationships between geographically targeted human populations covering an expanse from east sub-Saharan Africa northeastward into northern India with an emphasis on the Near East. A number of parameters of population genetics interest were examined based on allele frequencies from 15 forensic autosomal STR markers [D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA]. The phylogenetic analyses generated from genetic profiles of 885 individuals indicate that populations west of and including Iran have experienced substantial gene flow. Accordingly, our findings delineate a region of genetic homogeneity concentrated within the Near East with increasing genetic differentiation moving south into Africa and further east into Asia. We suggest that the Saharan desert, the Hindu Kush mountain range and perhaps to a lesser extent, the deserts of Iran may have acted as southern, eastern and northern geographical barriers, respectively, forming a genetic enclosure that allows limited gene flow outside the Near East. The biparental genetic landscape supports a picture of close contact between the Arab and Persian populations, perhaps beginning during the initial settlement of Asia from Africa extending to recent times.     

Identification of Tunisian Leishmania spp. by PCR amplification of cysteine proteinase B (cpb) genes and phylogenetic analysis

Discrimination of the Old World Leishmania parasites is important for diagnosis and epidemiological studies of leishmaniasis. We have developed PCR assays that allow the discrimination between Leishmania major, Leishmania tropica and Leishmania infantum Tunisian species. The identification was performed by a simple PCR targeting cysteine protease B (cpb) gene copies. These PCR can be a routine molecular biology tools for discrimination of Leishmania spp. from different geographical origins and different clinical forms. Our assays can be an informative source for cpb gene studying concerning drug, diagnostics and vaccine research.     

Lactovum miscens gen. nov., sp. nov., an aerotolerant, psychrotolerant, mixed-fermentative anaerobe from acidic forest soil

An aerotolerant, psychrotolerant anaerobe, anNAG3, was isolated from an acidic forest floor solution (in situ pH of 4.5). Cells of anNAG3 stained Gram-positive did not form spores, and were not motile. Cells were ovoid, approximately 1 microm long and 0.7 microm wide, mostly in pairs, and contained a multi-layered cell wall and intracytoplasmic membranes. Growth was observed at pH 3.5-7.5 and 0-35 degrees C. Glucose, galactose, fructose, mannitol, glucosamine, N-acetylglucosamine, cellobiose, and maltose supported growth. Lactate, ethanol, formate, and acetate were end products. H(2) and CH(4) were not detected, and only very minor amounts of CO(2) were produced. The relative amount of a particular product was dependent on the substrate utilized, and product profiles indicated that (i) sugars were initially metabolized to pyruvate via glycolysis, and (ii) lactate dehydrogenase and pyruvate-formate lyase were responsible for the subsequent metabolism of pyruvate. O(2) was not significantly utilized and was not toxic to growth. anNAG3 did not contain detectable membranous or cytoplasmic cytochromes. Nitrate, sulfate, and Fe(III) were not dissimilated. Thus, anNAG3 was characterized as an aerotolerant, non-acetogenic chemoorganotroph with a mixed-fermentative metabolism. The G + C content of the DNA was 37.6 mol%. The similarity of the 16S rRNA gene sequence of anNAG3 to that of its closest phylogenetic relatives (which were in the genera Lactococcus and Streptococcus) approximated 88-89%, indicating that anNAG3 constitutes the type species of a new genus. Based on the collective properties of anNAG3, it is proposed that anNAG3 be termed Lactovum miscens.     

Conservation of DNA photolyase genes in group II nucleopolyhedroviruses infecting plusiine insects

DNA photolyase genes (phr) encode photoreactive enzymes, which are involved in the repair of UV-damaged DNA. Cyclobutane pyrimidine dimer (CPD) specific photolyase genes are present in nucleopolyhedroviruses isolated from Chrysodeixis chalcites (ChchNPV) and Trichoplusia ni (TnSNPV), insects belonging to the Plusiinae (Noctuidae). To better understand the occurrence and evolution of these genes in baculoviruses, we investigated their possible conservation in other group II NPVs, which infect plusiine insects. A PCR based strategy using degenerate phr-specific primers was designed to detect and analyze possible photolyase genes. Six additional Plusiinae-infecting NPVs were analyzed and all, except Thysanoplusia oricalcea NPV A28-1, which is a group I NPV, contained one or more phr-like sequences. Phylogenetic analysis revealed that all photolyase genes of the tested Plusiinae-infecting baculoviruses group in a single clade, separated into three subgroups. The phylogeny of the polyhedrin sequences of these viruses confirmed that the analyzed viruses also formed a single clade in group II NPVs. We hypothesize that all plusiine group II NPVs contain one or more photolyase genes and that these have a common ancestor.     

Identification and molecular characterization of new STLV-1 and STLV-3 strains in wild-caught nonhuman primates in Cameroon

Humans and simian species are infected by deltaretroviruses (HTLV and STLV respectively), which are collectively called primate T-cell lymphotropic viruses (PTLVs). In humans, four types of HTLV have been described (HTLV-1 to -4) with three of them having closely related simian virus analogues named STLV-1, 2 and 3. In this study, our aim was to search for a simian HTLV-4-related virus and to document and characterize further the diversity of STLV infections in wild primate populations. We screened 1297 whole blood samples from 13 different primate species from southern Cameroon. Overall, 93 samples gave HTLV-1, HTLV-2 or dual HTLV-1/-2 INNOLIA profiles, 12 were HTLV positive but untypeable and 14 were indeterminate. Subsequently, we performed generic and specific (STLV-1 to -3) tax-rex PCRs to discriminate the different PTLV types, completed with phylogenetic analysis of 450-bp LTR sequences for STLV-1 and 900 bp pX-LTR sequences for STLV-3. We show for the first time that Lophocebus albigena and Cercopithecus cephus carry both STLV-1 and a divergent STLV-3. We also identified a new STLV-1 lineage in one C. cephus. Finally, we identify relative divergence levels in the tax/rex phylogeny suggesting that additional types of PTLV should be defined, particularly for the highly divergent STLV-1(MarB43) strain that we provisionally name STLV-5.