Pairwise Nucleotide Research Articles

Identification and estimation of sequence variation dynamics of Tomato Leaf curl Palampur virus and betasatellite complex infecting a new weed host

Leaf samples from the symptomatic Solanum nigrum plants showing curling on leaf margins, leaf chlorosis, leaf crumpling and stunting in growth were collected during spring 2018. Subsequently, the standard procedures of total genomic DNA isolation, enrichment via rolling circle amplification, polymerase chain reaction, cloning and complete genome sequencing were performed. Pairwise nucleotide (nt) sequence comparison and phylogenetic studies of the inferred genome sequences confirmed the identity of a bipartite begomovirus Tomato leaf curl Palampur virus (ToLCPalV) in the symptomatic plant samples sharing 97.6 and 95.5% nt identity with ToLCPalV isolates identified from Pakistan and India. In the phylogenetic analysis, ToLCPalV grouped with ToLCPalV isolates previously described from Pakistan and India. Additional attempts to identify any putative DNA-satellites in S. nigrum plant samples revealed an associated betasatellite component, cotton leaf curl Multan betasatellite (CLCuMuB). This is the first identification of cotton leaf curl disease-associated betasatellite with ToLCPalV in this region. The nt diversity analysis showed that ToLCPalV populations have a high mutation rate and evolve independently under strong purifying selection, but not via recombination.

1397. Modern Lineages of Mycobacterium tuberculosis Were Recently Introduced in Western India and Demonstrate Increased Transmissibility

Background Mycobacterium tuberculosis (Mtb) transmissibility may vary between lineages (or variants) and this may contribute to the slow decline of tuberculosis incidence. The objective of our study was to compare transmissibility across four major lineages (L1-4) of Mtb in Pune, India.MethodsWe performed whole-genome sequencing (WGS) of Mtb isolated from sputum culture of adult patients with pulmonary TB. We performed genotypic susceptibility testing for both first- and second-line drugs using a previously validated random forest predictor. We identified single nucleotide polymorphisms and generated a multiple sequence alignment excluding drug resistance conferring mutations to avoid skewing the phylogeny due to convergent evolution in these regions. We used Bayesian molecular dating to generate phylogenies and compared tree characteristics using a two-sample Kolmogorov-Smirnov (KS) test.ResultsOf the 642 isolates from distinct study participants that underwent WGS, 612 met quality criteria. The median age of participants was 31 years (range 18-74), the majority were male (64.7%) and sputum smear-positive (83.3%), and 6.7% had co-infection with HIV (Table 1). There was no significant difference in baseline characteristics between lineages. The majority of isolates belonged to L3 (44.6%). The majority (61.1%) of multidrug-resistant (MDR, resistant to isoniazid and rifampin) isolates belonged to L2. In phylogenetic analysis, we found evidence of higher transmissibility of L2 as indicated by shorter branch lengths (i.e., less time had elapsed between transmission and sampling) and more genetic similarity (smaller pairwise single nucleotide polymorphism [SNP] distances) among L2 isolates as compared to other lineages (Figure 1). Branching times for L2 and L4 were smaller than L1 and L3 indicating recent introduction into the region (p < 0.001 [KS test]). Figure 1: Lineage-wise distribution of A) phylogenetic tree branch lengths (log) and B) pairwise single nucleotide polymorphism (SNP) distance, using 612 tuberculosis isolates from Pune, India. P values calculated using two-sample Kolmogorov-Smirnov test. Table 1: Demographic characteristics of study participants included in the study, by lineage.ConclusionModern Mtb lineages (L2 and L4) were relatively recently introduced in western India, as compared to older lineages (L1 and L3), with the more drug-resistant L2 showing higher transmissibility. These findings highlight the need for early detection and treatment initiation to interrupt transmission with important implications for antimicrobial stewardship and heightened surveillance of TB resistance rates.Disclosures All Authors: No reported disclosures

Genetic diversity and population structure of three Hydroides species (Sedentaria, Serpulidae) in the Persian Gulf and Gulf of Oman, with the possible indication of heteroplasmy

The Persian Gulf, an enclosed sea connected to the Gulf of Oman through the narrow Strait of Hormuz, is known as the world’s hottest sea. Owing to its harsh environmental conditions, genetic isolation has been proposed for the Gulf’s biota. During the present study, population genetic and phylogeographic analyses were carried out focusing on potential barriers to dispersal in some polychaete populations between the Persian Gulf and the Gulf of Oman. Ninety-three specimens from three defined spatial populations of three Hydroides species, H. bulbosa, H. elegans and H. operculata, were sequenced for regions of two nuclear ribosomal (18S and ITS2) and two mitochondrial (COI and Cyt-b) genes to compare the genetic structure among the sampled populations. Haplotype networks of the three species showed no significant genetic differentiation and were consistent with their geographic distribution patterns. Population genetic analyses such as pairwise FST, nucleotide diversity and neutrality tests revealed some levels of intraspecific genetic diversities, suggesting different demographic histories. The genetic diversity observed in the more conservative genes (18S, Cyt-b, COI) in populations of H. elegans and H. operculata could reflect a more diverse genetic structure along their entire distribution range unlike the endemic species, H. bulbosa that presented a unique haplotype for each of those genes. Using Extended Bayesian Skyline plot analyses, populations of H. bulbosa and H. operculata showed signs of recent demographic expansion within the post-glacial transgression time frame of the Persian Gulf, while populations of H. elegans remained relatively constant. Another finding of the present study is the possible indication of heteroplasmy in H. elegans where two distinct mitochondrial lineages with about 11% of genetic divergence seem to coexist in the same individuals. We discuss also the alternative explanation of NUMTs (nuclear mitochondrial DNA), but regard it as substantially less likely.

Natural host range, incidence on overwintering cotton and diversity of cotton leafroll dwarf virus in Georgia USA

In 2018–19, cotton leafroll dwarf virus (CLRDV) was reported from several cotton-producing states in the southern United States. An extensive survey was conducted in the spring, summer, and fall of 2019 to identify weeds and overwintering cotton as sources for primary inoculum for the next season crop. Foliage samples of 57 different weed species were collected from fields formerly planted to cotton and analyzed using RT-PCR with CLRDV specific primers. CLRDV was detected from 23 weed species belonging to 16 different botanical families. Overwintering cotton stalks (48%) and regrowth leaves (75%) both harbored CLRDV. A phylogenetic analysis conducted using nucleotide and amino acid sequences of complete ORF 0, ORF 3, and ORF 4 determined that the CLRDV from all weeds and overwintered cotton stalks from Georgia clustered with CLRDV isolates collected in the USA but differed from isolates reported from South America and Asia. Pairwise nucleotide and amino acid identity showed 91–100% sequence similarity for complete ORF3. For ORF4, pairwise identity among the nucleotide ranged from 92-100%, while amino acids ranged from 90.2-100% with isolates reported from the USA and South America, with the exception of three Asian CLRDV isolates and two weed isolates from Georgia. Similarly, CLRDV isolates from Georgia weeds shared 91.6–93% nucleotide and 88–90.8% amino acid for silencing suppressor compared to most of the isolates from North America and South America. The role of alternative hosts on disease incidence and spread has not been studied in cotton-producing countries where the disease is prevalent. This is the first comprehensive study that identifies weeds and overwintering cotton as a potential green bridge for the year-round survival of CLRDV.

Detection and Phylogenetic Characterization of a Novel Herpesvirus in Sooty Terns Onychoprion fuscatus.

Since 2005, we have recorded annual episodes of alphaherpesvirus outbreaks in chicks of magnificent frigatebird Fregata magnificens on the Ile du Grand Connétable Nature Reserve in French Guiana. In 2009, we found sooty terns, Onychoprion fuscatus, that live sympatrically with frigatebirds, with visible clinical signs of a potential viral infection. To determine if the symptoms observed in sooty terns could be associated with an alphaherpesvirus previously identified in frigatebirds, we carried out molecular screening of samples collected from seven individuals. We identified and characterized a novel viral sequence from five birds. BLAST searches, pairwise nucleotide, and amino acid sequence comparisons, as well as phylogenetic analyses confirmed that the sequence belonged to the Herpesviridae family, of the Alphaherpesvirinae subfamily. We observed that it clustered with strains isolated from Podargidae (Caprimulgiformes), Columbiformes, and Falconiformes, but was distinct from the frigatebird herpesvirus. We have tentatively named it Onychoprion fuscatus alphaherpesvirus 1, (OfusAHV1). These two sequences, although found syntopic on the Ile du Grand Connétable, belong to two distinct alphaherpesvirus strains. Thus, the clinical symptoms showed by sooty terns do not likely result from a cross-species transmission event. Future work is needed to better characterize the virus and to investigate herpesvirus prevalence in healthy, free-ranging sooty terns, and to assess the impact of the virus on population viability.

Novel herpesviruses in neotropical bats and their relationship with other members of the Herpesviridae family

In the past decade, a large number of studies have detected herpesvirus sequences from many bat species around the world. Nevertheless, the discovery of bat herpesviruses is geographically uneven. Of the various bat species tested to date, only a few were from the New World. Seeking to investigate the distribution and diversity of herpesviruses circulating in neotropical bats, we carried out molecular screening of 195 blood DNA samples from 11 species of three bat families (Phyllostomidae, Mormoopidae, and Molossidae). Using polymerase chain reaction amplification, with degenerate consensus primers targeting highly conserved amino acid motifs of the herpesvirus DNA polymerase and Glycoprotein B genes, we characterized novel viral sequences from all tested species. BLAST searches, pairwise nucleotide and amino acid sequence comparisons, as well as phylogenetic analyses confirmed that they all belonged to the Herpesviridae family, of the Beta- and Gammaherpesvirinae subfamilies. Fourteen partial DNA polymerase gene sequences, of which three beta- and 11 gamma-herpesviruses, were detected. A total of 12 partial Glycoprotein B gene sequences, all gamma-herpesviruses, were characterized. Every sequence was specific to a bat species and in some species (Desmodus rotundus, Carollia perspicillata, and Pteronotus rubiginosus) multiple viruses were found. Phylogenetic analyses of beta- and gammaherpesvirus sequences led to the identification of bat-specific clades. Those composed of sequences obtained from different bat species belonging to distinct subfamilies follow the taxonomy of bats. This study confirms the astonishing diversity of bat herpesviruses and broadens our knowledge of their host range. Nevertheless, it also emphasizes the fact that, to better appreciate the evolutionary history of these viruses, much remains to be done at various taxonomic levels.

First Report of Papaya leaf curl China virus infecting Acalypha australis in China

Papaya leaf curl China virus (PaLCCNV), a monopartite begomovirus (genus Begomovirus, family Geminiviridae), was originally reported on Carica papaya in Guangxi province of China (Wang et al. 2004). The virus has been reported to infect C. papaya, Solanum lycopersicum, Nicotiana tabacum, Corchoropsis tomentosa, Siegesbeckia orientalis, and Ageratum conyzoides. In January 2015, plants of Acalypha australis, a common and widespread weed species, were found exhibiting yellow vein symptoms in fields of Nanning city, Guangxi province. Five symptomatic leaf samples were collected from each of five diseased plants. Total DNA was extracted from the five samples with the EasyPure Plant DNA kit (TransGen Biotech, Beijing, China) and was used as a template for polymerase chain reaction with the degenerate begomovirus primers AV494 (5′-gccyatrtayagraagccmag-3′) and CoPR (5′-gangsatghgtrcadgccatata-3′). The expected approximately 570-bp fragment was detected from all five samples, indicating that A. australis plants showing yellow vein symptoms were infected by a begomovirus. Begomoviral genomes were amplified from the total DNA using rolling circle amplification (RCA) (TempliPhi kit; GE Healthcare, Buckinghamshire, UK), followed by digestion with endonucleases BamH I, EcoR I, Hind III or Pst I, respectively. Amplicons corresponding to full-length begomoviral genomic components, obtained from RCA products digested with Hind III, were gel purified, ligated into the plasmid pGEM-3Z (previously digested with the corresponding restriction enzyme mentioned above), and transformed into Escherichia coli DH5α. Three clones from each sample were selected for sequencing (Invitrogen, Shanghai, China). Sequencing results were assembled and analyzed using DNAStar software version 5.0 (DNAStar, Madison, WI). A similarity search for each sequence was carried out using BLASTn to identify related sequences in the GenBank database (https://www.ncbi.nlm.nih.gov/). Pairwise nucleotide (nt) sequence identities were determined using the Species Demarcation Tool (SDT1.0). Full-length begomoviral genomic sequences were 2,735 nt long and encoded six putative open reading frames. The cloned genomic sequences shared 99.5 to 100% nt identities with each other. One representative sequence was deposited in GenBank as the accession number KX273343, which shared 91.8 to 99.6% nt identities with isolates of PaLCCNV in the database, with the highest nt identities to PaLCCNV-G111 [CN:Gx:Age:14] (HG003651). In accordance with the threshold of 91% for begomovirus species demarcation (Brown et al. 2015), the virus associated with A. australis yellow vein disease is an isolate of PaLCCNV. According to the obtained sequences, the specific primer pairs W-F2 (5′-GTATACACGCCACTCTCGCATTG-3′) and W-R2 (5′-CTGGACAATCAAAAATCCCCTAT-3′) were designed to amplify the full-length genome and detect the presence of PaLCCNV in A. australis samples. The expected approximately 2.7-kb full-length genomic fragment was amplified from all five samples, indicating that A. australis plants showing yellow vein symptoms were infected by PaLCCNV. To our knowledge, this is the first report of natural occurrence of PaLCCNV on A. australis in China. A. australis may play an important role as a reservoir of PaLCCNV, especially during the non-crop-planting period, but additional studies analyzing a large number of plant samples from different geographical locations within China are necessary to establish the role of this host as a virus reservoir.

First Report of Cucumber vein yellowing virus on Cucumber in Lebanon.

In a 2-year (2008 to 2009) wide-scale survey of viruses infecting cucurbits, a limited number of greenhouse-grown cucumber (Cucumis sativus) plants showed vein-yellowing symptoms. Greenhouses were infested with whiteflies and infection with Cucumber vein yellowing virus (CVYV) was suspected. CVYV is widely distributed in southern Europe in both open field and protected cucurbit crops (2). Total RNA was extracted from seven plants with vein yellowing symptoms using TRI Reagent (Sigma-Aldrich, St Louis, MO). RT-PCR tests using CVYV-specific primers (CV+/CV-) targeting the coat protein of CVYV (2) gave amplicons of the expected size from seven plants. The sequence of one representative isolate, CVYV-LB3 (GenBank Accession No. JF289167), showed 97, 95.6, and 95.2% pairwise nucleotide identity with isolates from Tunisia (EF538680), Israel (AF233429), and Jordan (JF460793), respectively. In 2012, CVYV like symptoms were not observed in greenhouses in the same areas. In early spring 2013, a total of 16 leaf samples with vein-yellowing symptoms were collected from the northern coastal areas (Jbeil, Amshit, Tabarja) and 11 samples showing only yellowing on older leaves from the southern coast (Jiyeh). CVYV was detected in all samples from the northern coast and in four samples from the southern coast. Four isolates from the North and two from the South were sequenced (KC990497 to KC990502) and showed high sequence variation. The pairwise nucleotide and amino acid identities between these isolates ranged from 95.1 to 100% and 98.5 to 100%, respectively. Pairwise nucleotide and amino acid identities of the isolates with CVYV-LB3 showed variable homology between 93.7 to 98.2% and 94.6 to 96.6%, respectively. The inter-population structure of CVYV in Lebanon showed high variability as compared to the homogenous Spanish population (4). For transmission tests, non-viruliferous whiteflies (Bemisia tabaci) were exposed for an 18-h acquisition access period on vein-yellowed leaves followed by a 24-h inoculation access period to healthy cucumber plants (5-leaf stage). In addition, leaves with vein-yellowing symptoms were ground in 0.1 M phosphate buffer (pH 7.0) and sap-inoculated on carborundum-dusted cucumber (cv. Delta) and squash (Cucurbita pepo cv. FarajF1) plants. Vein yellowing symptoms developed 9 to 11 days post-inoculation on all whitefly inoculated plants, while symptoms were delayed till 3 weeks post inoculation on seven out of eight sap-inoculated plants. All symptomatic plants were positive for CVYV by RT-PCR. Furthermore, surveyed plants were also tested for Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV), two criniviruses reported previously in Lebanon, by RT-PCR (1). Double or triple infection of CCYV and CYSDV occurred in 18 out of 20 of the CVYV-infected plants. During the past 5 years, a limited number of cucumber plants showed CVYV symptoms. This indicates that CVYV occurrence is sporadic. However, its occurrence in mixed infection with criniviruses may have damaging economic implications to cucurbit production (3). To our knowledge, this is the first report of CVYV on cucurbits in Lebanon and its occurrence in co-infection with CCYV.

Population genetic structure of northern Dolly Varden char Salvelinus malma malma in Asia and North America

The level of genetic differentiation of northern Dolly Varden char Salvelinus malma malma from Asia and North America was evaluated using the data on mtDNA variation (regions ND1/ND2, ND5/ND6, and Cytb/D loop) obtained by means of PCR-RFLP analysis. For S. m. malma, the mean values of haplotype and nucleotide diversity were 0.5261 +/- 0.00388 and 0.001558, respectively. The mean estimate of the population nucleotide divergence constituted 0.055%. It was demonstrated that S. m. malma on the most part of the species range examined (drainages of the Beaufort Sea, Chukotka Sea, Bering Sea, and the Sea of Okhotsk) was characterized by the population genetic structure with the low level of genetic differentiation and divergence. At the same time, populations from the Pacific Ocean Gulf of Alaska demonstrated marked genetic differentiation, supported by the high pairwise phi(ST) values (from 0.4198 to 0.5211) and nucleotide divergence estimates (mean divergence, 0.129%), from Asian and North American populations. Nested analysis of molecular variance (AMOVA) showed that most of the mtDNA variation in S. m. malma fell in the intrapopulation component (72.5%). At the same time, the differences between the populations (21.1%) and between the regions (6.4%) made lower contribution to the total variation.

Partial molecular characterisation of New World non-human primate lymphocryptoviruses

The description of numerous viruses belonging to the Lymphocryptovirus genus from different Old and New World non-human primate species during the past 10 years has led to developing and supporting co-speciational evolution hypotheses for these viruses and their hosts. Among the different primate species tested, only a few were from the New World. This study attempted to achieve a better understanding of the evolutionary processes within the Platyrrhini branch. Molecular screening of 253 blood DNA samples from 20 New World non-human primate species from Central and South America was carried out using polymerase chain reaction amplification with degenerate consensus primers targeting highly conserved amino acid motifs of the herpesvirus DNA polymerase gene. In addition to the 33 samples from which we have already described three lymphocryptoviruses, amplification products were detected in 17 other samples originating from 11 species (13 sub-species). BLAST searches, pairwise nucleotide and amino acid sequence comparisons, and phylogenetic analyses confirm that they all belong to the Lymphocryptovirus genus. Fourteen distinct Lymphocryptovirus sequences were detected, of which nine have never been reported. Phylogenetic analyses showed that, as expected, the New World virus lineage formed a sister clade to that of the Old World viruses. The parallel determination of the host taxa has demonstrated a good correlation between the distinct monophyletic clades of viruses and the infected primates at the sub-family level. In addition, these results further suggest the existence of two distinct groups within the Cebidae for Saimirinae and Cebinae primates. Nevertheless, based on the current genetic data, this study fell short of achieving a tree that was completely resolved within the lineage of Platyrrhini viruses. Further studies will be needed to better assess the evolutionary relationships between these viruses.

Nucleotide sequence analysis and detection of a Greek isolate of Tomato chlorosis virus

Tomato chlorosis virus (ToCV) is a whitefly‐transmitted crinivirus with a bipartite RNA genome inducing yellowing diseases in greenhouse and outdoor tomato plants. In this study, both genomic RNA components of a Greek isolate (Gr‐535) of ToCV were sequenced. They contained 8594 nucleotides (nt) and 8242 nt and shared 97% and 99% sequence identity with Spanish and American isolates, respectively. Phylogenetic analysis and pairwise nucleotide and amino acid sequence comparisons showed that the Greek isolate clustered together with the American ToCV isolate. Nevertheless, the Greek and Spanish isolates shared several common deletions and extra stretches of nucleotides in the untranslated regions of their genomes when compared to the American isolate, suggesting genetic recombination. Prediction of putative structures of the 3′‐terminus of ToCV RNA 1 showed the presence of four stem loops and a pseudo‐knot, while the putative structure of the 3′‐terminus of ToCV RNA 2 varied between the three sequenced isolates. Diagnostic dot‐blot hybridization and reverse transcription–polymerase chain reaction (RT‐PCR) assays indicated that ToCV could easily be detected in 20 ng of total RNA extracts from infected plants. Dot‐blot hybridization could also be performed for virus diagnosis using infected crude plant extracts.