Evolutionary Distance Analysis Research Articles

Genetic evolution of Newcastle Disease Virus sub-genotype VII.2 isolates, diagnosed from vaccinated poultry farms of Gujarat, India

Newcastle disease was suspected in 37 commercial poultry farms, including 12 layer and 25 broiler farms in four districts of Gujarat, India. Vaccination had been done in 32 (20 broilers and 12 layers) farms. Tissue samples from each farm were pooled as one sample. In egg embryo inoculation, HA-HI and PCR, respectively, 32/37, 29/37, and 24/37 samples were found positive. Pathotyping by mean death time calculation and primer combination PCR revealed velogenic NDV, which was later confirmed with the presence of the 112-RRQKR*F-117 sequence at the F protein cleavage site. Phylogenetic analysis of full F gene sequences (N=10) confirmed the presence of sub-genotype VII.2 in 9/10 sequences, and genotype II in one sample. These 9 sequences were only 0.7 to 2.6 % divergent with two VII.2 (=VIIi) sequences (HQ697254.1 chicken/Banjarmas/Indonesia and KU862293.1 Parakeet/Karachi/Pakistan) but had 2.2 to 3.6 % diversion from two VII.2 sequences (OR185447 and MZ546197) from India. Then branching was found from sequences of VIIh, VIIk (VII.2), and VIIa (VII.1.2), and then from sub-genotypes VII.1.1 and VII.1.2. Due to less than 5 % diversion, these sequences could not be qualified as new sub-genotype in evolutionary distance analysis. At the amino acid level, our sequences had aa N-T-I-A-L-T at 24–79-125–385-445–482. Whereas at the same positions, in most of the retrieved VII.2 sequences and vaccines, the sequence was S-A-V-T-Q/I- E/A. Two sequences revealed additional six and four amino acid differences,respectively.This indicates rapid continuous genetic evolution of sub-genotype VII.2 and partially explains vaccinal immunity escape.

Phylotaxonomic assessment based on four core gene sets and proposal of a genus definition among the families Paracoccaceae and Roseobacteraceae

The families Paracoccaceae and Roseobacteraceae comprise metabolically, phenotypically and genotypically diverse members, and their descriptions rely heavily on 16S rRNA gene analysis. Hundreds of genera have been reported within the two families and misclassifications have been a reoccurring problem, even when the taxonomies have been established based on genome-scale phylogenetic reconstructions. In this study, we conducted a comprehensive phylotaxonomic assessment of the families Paracoccaceae and Roseobacteraceae based on four ubiquitous gene sets, bac120 (120 genes in Bacteria), rhodo268 (268 genes in ‘ Rhodobacteraceae ’, defined in this study), rp1 (16 ribosomal protein genes in Prokaryote) and rp2 (23 ribosomal protein genes in Prokaryote), using two tree-inferring applications and two approaches (supermatrix and consensus). The results suggested that the four supermatrix trees based on bac120 and rhodo268 shared a high proportion of common nodes (>88.4 %) and the topology was reproducible among all the trees within most of the genera. The evolutionary distance (ED) analysis showed significant overlapping between the intergeneric and intrageneric comparisons, implying that the proposal of some genera seemed to be unnecessary. In addition, the bac120 gene set and the FastTree program were found to be the most cost-effective way to conduct phylogenomic analysis of the families Paracoccaceae and Roseobacteraceae . An ED threshold of 0.21–0.23 based on either bac120 or rhodo268 was proposed as one standard for later genus delimitation in these families. A comprehensive phylogenetic framework is presented in this study and the proposed genus definition will help to establish a more reasonable taxonomy in the families Paracoccaceae and Roseobacteraceae .

Introduction of a Newcastle disease virus challenge strain (sub-genotype VII.1.1) isolated in Iran.

Newcastle disease virus (NDV) sub-genotype VII.1.1 is the most common circulating NDV in Iran. In this study, a velogenic NDV isolate was plaque purified and then characterized according to Office International des Epizooties (OIE) standard protocols. The biological properties of the purified isolate named CH/RT40/IR/2011 were characterized using sequencing and phylogenetic analysis, measurement of pathogenicity indexes and challenge studies. The isolate was plaque purified on chicken embryo fibroblast cells for three rounds and then characterized using molecular and biological approaches. Phylogenetic and evolutionary distance analysis of fusion and hemagglutinin-neuraminidase genes classified the virus in sub-genotype VII.1.1. No mutation was observed in the glycosylation and neutralizing epitope sites of the fusion and hemagglutinin-neuraminidase proteins compared to other reported Iranian NDV VII.1.1 isolates. The presence of the 112RRQKRF117 motif in the fusion protein cleavage site together with mean death time, intracerebral pathogenicity index and intravenous pathogenicity index of 57 hr, 1.80 and 2.50 respectively, revealed that the RT40 isolate was a velogenic NDV. In the challenge study, all chickens were inoculated via eye drop, and intranasal route with RT40 isolate died within a week. While all chickens in the vaccinated and challenged group survived and showed no clinical signs. In conclusion, according to genetic analysis, pathotyping and challenge study, the RT40 isolate was similar to virulent NDVs in Iran and was a suitable candidate for a national standard challenge strain, vaccine trials and vaccine production in commercial levers.

The monitoring of plum pox virus in Bursa, Bilecik, and Bolu provinces of Turkey refined MIs status as a variant of strain M

A total of 486 symptomatic and asymptomatic peach leaf samples were collected from Bursa, Bilecik, and Bolu provinces and tested for plum pox virus (PPV, Potyvirus) in response to recent reports on the new divergent subgroup MIs in the nearby Istanbul province of Turkey. The phylogenetic analysis of newly sequenced isolates indicated that two isolates from Bolu belong to subgroup MIs, confirming the existence of its isolates beyond Istanbul. Both Bolu MIs isolates cross-reacted with a mixture of polyclonal antibodies and monoclonal 5B-IVIA in a commercial DAS-ELISA kit for universal detection of PPV strains, elucidating some of MIs serological characters. The identity percentage, and also the evolutionary distance and diversity analyses showed that M isolates shared high genetic identities among them, and that the diversity among its subgroups was also low. Therefore, the results showed that MIs is not a new distinct strain but a variant of strain M. Supplemental data for this article is available online at https://doi.org/10.1080/03235408.2022.2052523 .

Genetic diversity of avian leukosis virus subgroup J (ALV-J): toward a unified phylogenetic classification and nomenclature system

Avian leukosis virus subgroup J (ALV-J) has infected a variety of birds, causing major economic losses in China. Understanding the comprehensive criteria of classification and nomenclature of ALV-J would be useful for the investigation of the viral evolution and also for the prevention and control of this infection. An in-depth analysis of the genetic diversity of ALV-J was performed in the present study. Four hundred and seventy-five sequences of the gp85 gene, including thirteen of avian endogenous retrovirus designated ev/J and 462 of ALV-J, were used in the phylogenetic and the evolutionary distance analysis for this classification. The study identified that the current ALV-J strains were divided into two first-order clades (Clades 1 and 2) and three second-order clades (Clades 1.1, 1.2 and 1.3). The current Chinese ALV-J strains are predominantly in Clade 1.3, and the Chinese and Egyptian chicken flocks have been facing the emerging Clade 2 viruses. This system pioneers the classification efforts for ALV-J, which uses Pilot tree for rapid classification of the new isolates and also the addition of possible new clades. The proposed unified classification system will facilitate future studies of ALV-J epidemiology and genetic evolution and of the comparison of sequences obtained across the world.

Identification of a novel hantavirus strain in the root vole (Microtus oeconomus) in Lithuania, Eastern Europe.

Hantaviruses are zoonotic pathogens that can cause subclinical to lethal infections in humans. In Europe, five orthohantaviruses are present in rodents: Myodes-associated Puumala orthohantavirus (PUUV), Microtus-associated Tula orthohantavirus, Traemmersee hantavirus (TRAV)/ Tatenale hantavirus (TATV)/ Kielder hantavirus, rat-borne Seoul orthohantavirus, and Apodemus-associated Dobrava-Belgrade orthohantavirus (DOBV). Human PUUV and DOBV infections were detected previously in Lithuania, but the presence of Microtus-associated hantaviruses is not known. For this study we screened 234 Microtus voles, including root voles (Microtus oeconomus), field voles (Microtus agrestis) and common voles (Microtus arvalis) from Lithuania for hantavirus infections. This initial screening was based on reverse transcription-polymerase chain reaction (RT-PCR) targeting the S segment and serological analysis. A novel hantavirus was detected in eight of 79 root voles tentatively named "Rusne virus" according to the capture location and complete genome sequences were determined. In the coding regions of all three genome segments, Rusne virus showed high sequence similarity to TRAV and TATV and clustered with Kielder hantavirus in phylogenetic analyses of partial S and L segment sequences. Pairwise evolutionary distance analysis confirmed Rusne virus as a strain of the species TRAV/TATV. Moreover, we synthesized the entire nucleocapsid (N) protein of Rusne virus in Saccharomyces cerevisiae. We observed cross-reactivity of antibodies raised against other hantaviruses, including PUUV, with this new N protein. ELISA investigation of all 234 voles detected Rusne virus-reactive antibodies exclusively in four of 79 root voles, all being also RNA positive, but not in any other vole species. In conclusion, the detection of Rusne virus RNA in multiple root voles at the same trapping site during three years and its absence in sympatric field voles suggests root voles as the reservoir host of this novel virus. Future investigations should evaluate host association of TRAV, TATV, Kielder virus and the novel Rusne virus and their evolutionary relationships.

Molecular phylogenetics of Newcastle disease viruses isolated from vaccinated flocks during outbreaks in Southern India reveals circulation of a novel sub-genotype.

Newcastle disease (ND) is an economically important, contagious poultry viral disease reported across the globe. In India, ND is endemic and episodes of ND outbreaks despite strict vaccinations are not uncommon. We isolated and characterized seven ND viruses from vaccinated commercial poultry farms during severe disease outbreaks in Tamil Nadu, in Southern India, between April 2015 and June 2016. All the seven isolates were categorized as virulent by mean death time (48-54hr) in embryonated chicken eggs. Also, their sequences carried the virulence signature of multi-basic amino acid residues in their fusion protein cleavage site (RRQ/RR/KRF). Phylogenetic and evolutionary distance analyses revealed circulation of a novel sub-genotype of genotype XIII, class II ND viruses, herein proposed as sub-genotype XIIIe. The genetic divergence between the circulating virulent strains and the vaccine strains could possibly explain the disease outbreak in the vaccinated flocks. Further, our study signifies the need to implement routine epidemiological surveillance and to revisit the current vaccination program.

Complete genome sequence of sacbrood virus isolated from Asiatic honey bee Apis cerana indica in India.

We determined the complete genome sequence of a sacbrood virus (SBV) infecting Indian honey bee (Apis cerana indica) from Tamil Nadu, India named as AcSBV-IndTN1. The genome of AcSBV-IndTN1 comprised of 8740 nucleotides, encoding a single large ORF containing 2849 amino acids flanked by 5' and 3' untranslated regions. Results of phylogenetic tree analysis based on complete genomes of SBV isolates indicated that the virus isolates from India isolated from the Asiatic honey bee A. cerana (AcSBVs) formed a separate group along with six Vietnam isolates and three Chinese isolates. The AcSBV-IndTN1 isolate showed closer genetic relationship with other isolates from India. The second major group had both AcSBVs and AmSBVs (virus isolated from European honey bee, Apis mellifera SBV) of Korea, China and Vietnam. The third and a distantly related group had AmSBVs of Australia, UK, USA and Korea. The results obtained from phylogenetic analysis were further supported with evolutionary distance analysis. AcSBV-IndTN1 isolate open reading frame had 95-99% amino acid sequence similarity with other Indian isolates and 92-96% with AcSBVs and AmSBVs of other geographical locations. In addition, sequence difference count matrix ranged from 154 to 907nt among all the SBV isolates. This suggests that the virus isolates have evolved significantly in different geographical locations but isolates on different hosts in a given location/country are closely related. The high similarity in the genome among the AcSBV and AmSBV isolates indicate possible cross-infections and recombination of SBV isolates in Asian continent where both the honey bee species are reared in close proximity. Gene flow between SBV population indicating that an infrequent gene flow occur between them. The pattern of molecular diversity in SBV population revealed that the occurrence of recent population expansion of SBV. To the best of our knowledge this is the first report of the complete nucleotide sequence of AcSBV from Tamil Nadu, India. This study provided an opportunity to establish the molecular evolution of SBV isolates and shall be useful in the development of diagnostics and effective disease control strategies.

Genome-wide Estimation of Evolutionary Distance and Phylogenetic Analysis of Homologous Genes.

Homologous genes, including paralogs and orthologs, are genes that share sequence homologies within or between different species. Homologous genes originate from a common origin through speciation, genetic duplication or horizontal gene transfer. Estimation of the sequence divergence of homologous genes help us to understand divergence time, which makes it possible to understand the evolutionary patterns of speciation, gene duplication and gene transfer events. This protocol will provide a detailed bioinformatics pipeline on how to identify the homologous genes, compare their sequence divergence and phylogenetic relationships, focusing on homologous genes that show syntenic relationships using soybean (Glycine max) and common bean (Phaseolus vulgaris) as example species.

Isolation and characterization of a novel mesonivirus from Culex mosquitoes in China

A new insect nidovirus (named Yichang virus) from the family Mesoniviridae was isolated, identified, and characterized from Culex mosquitoes in Hubei, China. Results showed a high number of viral RNA copies (up to 1011 copies/ml) within 48h in C6/36 cells. In addition, the titers of the Yichang virus reached maximal levels of 107 PFU/mL at 6 d post-infection (dpi). The virus produced moderate cytopathic effects when the multiplicity of infection ranged from 0.001–0.1 at 6 dpi, but did not replicate in mammalian cells. Under electron microscopy, the virion of the Yichang virus appeared as spherical particles with diameters of ∼80nm and large club-shaped projections. Although subsequent genomic sequence analysis revealed that the Yichang virus had similar protein patterns as those of other mesoniviruses, the nucleotide acids shared less than 20% BLAST query coverage with known viruses in the family Mesoniviridae, and showed a maximum sequence identity of 67% for RNA-dependent RNA polymerase (RdRp). The putative protein sequences showed slightly higher identity (28%–68%), and the most conserved domain was RdRp. Based on the phylogenetic and pairwise evolutionary distance analyses, the Yichang virus should be considered a new species belonging to a currently unassigned genus within the family Mesoniviridae.

Comparison of the genetic relationship between nine Cephalopod species based on cluster analysis of karyotype evolutionary distance.

Karyotype analysis was carried out on gill cells of three species of octopods using a conventional air-drying method. The karyotype results showed that all the three species have the same diploid chromosome number, 2n=60, but with different karyograms as 2n=38M+6SM+8ST+8T, FN (fundamental number)=104 (Cistopus chinensis Zheng et al., 2012), 2n=42M+6SM+4ST+8T, FN=108 (Octopus minor (Sasaki, 1920)) and 2n=32M+16SM+12T, FN=108 (Amphioctopus fangsiao (d’Orbigny, 1839–1841)). These findings were combined with data from earlier studies to infer the genetic relationships between nine species via cluster analysis using the karyotype evolutionary distance (De) and resemblance-near coefficient (λ). The resulting tree revealed a clear distinction between different families and orders which was substantially consistent with molecular phylogenies. The smallest intraspecific evolutionary distance (De=0.2013, 0.2399) and largest resemblance-near coefficient (λ=0.8184, 0.7871) appeared between O. minor and C. chinensis, and Sepia esculenta Hoyle, 1885 and S. lycidas Gray, 1849, respectively, indicating that these species have the closest relationship. The largest evolutionary gap appeared between species with complicated karyotypes and species with simple karyotypes. Cluster analysis of De and λ provides information to supplement traditional taxonomy and molecular systematics, and it would serve as an important auxiliary for routine phylogenetic study.

Emerging variant of genotype XIII Newcastle disease virus from Northeast India

Northeast India with its rich and diverse avifauna acts as a hotbed for emerging virulent Newcastle disease virus (NDV) strains. The present work describes the molecular and pathogenic characterization of NDV strain isolated from Pandu, Assam, India. Clinicopathological and genetic analysis showed the virulent nature of NDV strain Pandu. On molecular phylogenetic and evolutionary distance analysis, the NDV strain Pandu formed a distinct clade within the genotype XIII of class II NDV, suggesting a new sub-genotype XIIIc. The accumulation of mutations in the NDV strain Pandu makes it divergent enough to be considered as a new sub-genotype. The proposed NDV sub-genotype XIIIc consists of strains recently reported from eastern and northeastern India.

Genomic characterization of a wild-bird-origin pigeon paramyxovirus type 1 (PPMV-1) first isolated in the northwest region of China.

Pigeon paramyxovirus type-1(PPMV-1) is an enzootic in pigeon flocks and causes severe economic losses in the poultry industry in many countries. A PPMV-1 isolate, abbreviated as PPMV-1/QL-01/CH/15, was isolated from a great spotted woodpecker in the northwest region of China in 2015. The complete genome was sequenced, and the results showed that the virus genome was 15,192 nt in length, in the gene arrangement 3'-NP-P-M-F-HN-L-5'. Several amino acid mutations were identified in the functional domains of the F and HN proteins. The pathogenicity index of the isolate was evaluated, and the mean death time (MDT) was 72 h and the intracerebral pathogenicity index (ICPI) was 0.925, indicating that this isolate was mesogenic. Sequencing results showed that it had a virulent Newcastle disease virus cleavage motif 112R-R-Q-K-R-F117 at the fusion protein cleavage site. Morbidity and mortality were 70% and 50%, after inoculation of pigeons, whereas this virus was nonpathogenic in chickens. Different immune responses of pigeons and chickens were induced in vivo, which led to different HI serum antibody titers. The results of phylogenetic and evolutionary distance analysis showed that this PPMV-1 strain belonged to sub-genotype VIa in class II. To our knowledge, this is the first identification and analysis of PPMV-1 co-circulation among wild birds and domestic pigeon flocks in China. The data from this study highlight the important role of wild birds in the dissemination of PPMV-1 and provide useful references for improving our understanding of the distribution and evolution of PPMV-1 in China.

Characterization of an intracellular bacterium infecting the digestive gland of the South African abalone Haliotis midae

A Rickettsiales-like prokaryote was observed in digestive gland epithelium of farmed South African abalone Haliotis midae sampled for routine surveillance. Histological examination showed basophilic inclusions typical of rickettsial infections in shellfish (Mouton, 2000). This study aimed to identify the Rickettsiales-like prokaryote (RLP) colonising H. midae. Total genomic DNA was extracted from infected digestive gland sampled from cultured H. midae. PCR performed with “Candidatus Xenohaliotis californiensis” specific primers RA 5-1 and RA 3-6 did not yield positive results, indicating that the H. midae RLP is not the withering syndrome agent. The 16S rRNA gene sequence of the intracellular bacterium was determined following nested PCR using universal eubacterial primers fD1/Rp2 and the broad spectrum primers EHR16SD/EHR16SR which are specific for members of the Anaplasmataceae family within the Rickettsiales order. Phylogenetic and evolutionary distance analysis showed that the H. midae RLP is a unique member of the α-proteobacteria. The identity and location of the histologically observed intracellular bacterium was confirmed by in situ hybridization performed on sequential sections of infected H. midae digestive gland using a combination of three digoxigenin-labelled probes specific to various regions of the 16S rRNA gene of the RLP.Statement of relevanceThe manuscript shows that farmed abalone, showing no disease symptoms, can remain healthy despite the presence of intracellular bacteria colonising their digestive glands. The study shows that Rickettsiales-like prokaryotes detected in farmed abalone are not necessarily “Candidatus Xenohaliotis californiensis”, the causative agent of withering syndrome.

Evolutionary analysis of the ubiquitin gene of baculovirus and insect hosts.

Baculovirus is the only virus that has been found to encode the ubiquitin protein. In this study, ubiquitin sequences from 16 insects and 49 viruses were collected and compared. The resulting sequences were aligned with virus genomes. Then MAGE 5.0, k-estimated software, as well as other software programs were used for systemic evolutionary, selection pressure, and evolutionary distance analysis. The results of the pairwise ratio of non-synonymous to synonymous substitution values and evolutionary distances showed that ubiquitin from baculovirus and insect hosts have been under purifying selection during evolution and are thus evolutionarily conserved. Moreover, genes from insect hosts were more conserved than those in baculovirus. Analysis of the non-synonymous to synonymous substitution rates at each site and entropy calculations revealed the evolutionary status of every site in the ubiquitin genes of baculovirus and their hosts. Genome locations and phylogenetic trees indicated that granuloviruses and non-photosynthetic vegetation evolved, and granulovirus evolution was more similar to that of insect hosts. Our results suggest that the ubiquitin gene in baculovirus may have been acquired through horizontal transfer from the host.

Molecular characterization of Indian Sugarcane streak mosaic virus isolates reveals recombination and negative selection in the P1 gene

Sugarcane streak mosaic virus (SCSMV), a member of the genus Poacevirus is an important viral pathogen affecting sugarcane production in India. The P1 gene of ten Indian isolates was sequenced and compared with previously reported SCSMV isolates. Comparative sequence analysis revealed a high level of diversity in the P1 gene (83–98% nucleotide sequence identity; 87–100% amino acid sequence identity), and the Indian SCSMV isolates were found to be the most variable (up to 9% diversity at the amino acid level). Phylogenetic tree analysis showed clustering of 17 SCSMV isolates into two groups: group I included isolates from India (except SCSMV-TPT) and Pakistan, and group II consisted of isolates from Japan, Indonesia, Thailand and SCSMV-TPT. The results obtained from phylogenetic study were further supported by the different in silico analysis viz. SNPs (single nucleotide polymorphism), INDELs (insertion and deletion) and evolutionary distance analysis. A significant proportion of recombination sites were observed at the N terminal region of P1 gene. Analysis of selection pressure indicated that the P1 gene of the Indian SCSMV isolates is under strong negative or purifying selection. It is likely that recombination identified in Indian SCSMV isolates, along with strong purifying selection, enhances the speed of elimination of deleterious mutations in the P1 gene. The evolutionary processes (recombination and selection pressure) together contributed to the observed genetic diversity and population structure of Indian SCSMV isolates.

The molecular diversity and evolution of Rice tungro bacilliform virus from Indian perspective

Rice tungro disease is caused by a combination of two viruses: Rice tungro spherical virus and Rice tungro bacilliform virus (RTBV). This study was performed with the objective to decipher the molecular variability and evolution of RTBV isolates present in the tungro-affected states of Indian subcontinent. Phylogenetic analysis based on ORF-I, ORF-II, and ORF-IV sequences showed distinct divergence of Indian RTBV isolates into two groups; one consisted isolates from Hyderabad (Andhra Pradesh), Cuttack (Orissa), and Puducherry and another from West Bengal, Chinsura West Bengal, and Kanyakumari (Tamil Nadu). The results obtained from phylogenetic analysis were further supported with the single nucleotide polymorphisms (SNPs), insertion and deletion (INDELs) and evolutionary distance analysis. In addition, sequence difference count matrix revealed a maximum of 56 (ORF-I), 13 (ORF-II) and 73 (ORF-IV) nucleotides differences among all the Indian RTBV isolates taken in this study. However, at the protein level these differences were not significant as revealed by K (a)/K (s) ratio calculation. Sequence identity at nucleotide and amino acid level was 92-100 % (ORF-I), 96-100 % (ORF-II), 94-100 % (ORF-IV) and 86-100 % (ORF-I), 98-100 % (ORF-II) and 95-100 % (ORF-IV), respectively, among Indian isolates of RTBV. The divergence of RTBV isolates into two independent clusters of Indian and non-Indian was shown with the help of the data obtained from phylogeny, SNPs, and INDELs, evolutionary distance analysis, and conserved motifs analysis. The important role of ORF-I and ORF-IV in RTBV diversification and adaptation to different rice growing regions is also discussed.

Clustering analysis of karyotype resemblance-near coefficient for 6 Bupleurum species

To explore the genetic evolutionary distance between plants by using karyotype parameters identification of medicinal plants. The cluster analysis of karyotype resemblance-near coefficient and evolutionary distance was used for 6 Bupleurum species. The results showed that there were the biggest karyotype resemblance-near coefficient (0.9920) and the smallest evolutionary distance (D(e) = 0.0080) between B. scorzonerifolium and B. chinense, indicating the closest relationship, and the minimum karyotype resemblance-near coefficient (0.4794) and the maximum evolutionary distance (D(e) = 0.7352) between B. smityii and B. falcatum, indicating the most distant relationship. Karyotype was an important parameter for identification of medicinal plants because karyotype was stabilized for species. The genetic distance between in 6 species of Bupleurum species was obtained by karyotype clustering analysis of karyotype resemblance-near coefficient. There was the bigger evolutionary distance between the species which had different chromosome number.

Genetic variation of coat protein gene among the isolates of Rice tungro spherical virus from tungro-endemic states of the India

Rice tungro disease, one of the major constraints to rice production in South and Southeast Asia, is caused by a combination of two viruses: Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus (RTBV). The present study was undertaken to determine the genetic variation of RTSV population present in tungro endemic states of Indian subcontinent. Phylogenetic analysis based on coat protein sequences showed distinct divergence of Indian RTSV isolates into two groups; one consisted isolates from Hyderabad (Andhra Pradesh), Cuttack (Orissa), and Puducherry and another from West Bengal, Coimbatore (Tamil Nadu), and Kanyakumari (Tamil Nadu). The results obtained from phylogenetic study were further supported with the SNPs (single nucleotide polymorphism), INDELs (insertion and deletion) and evolutionary distance analysis. In addition, sequence difference count matrix revealed 2-68 nucleotides differences among all the Indian RTSV isolates taken in this study. However, at the protein level these differences were not significant as revealed by Ka/Ks ratio calculation. Sequence identity at nucleotide and amino acid level was 92-100% and 97-100%, respectively, among Indian isolates of RTSV. Understanding of the population structure of RTSV from tungro endemic regions of India would potentially provide insights into the molecular diversification of this virus.

Variation of Anaeromyxobacter community structure and abundance in paddy soil slurry over flooding time

Cultivation-independent techniques like PCR-amplified restriction fragment length polymorphism (PCR-RFLP) of 16S rRNA genes and real-time PCR were applied to assess the abundance, diversity and phylogenetic composition of Anaeromyxobacter communities over time in flooded, unplanted paddy soil slurries. Six Anaeromyxobacter communities were sampled from anoxic slurries at 1 h, and 1, 5, 10, 20 and 30 days while the Fe (II) concentrations were measured also. Bacterial Genomic DNA was extracted and PCR-amplified to obtain 16S rDNA fragments of Anaeromyxobacter which were cloned to construct 6 16s rDNA libraries. Eventually 10 major Anaeromyxobacter types were identified by RFLP fingerprintings. Results showed that the optimal increasing phase of Fe (II) was from 1h to nearly 10 days, being correspond with the growth phase of the abundance of Anaeromyxobacter. The highest diversity appeared in slurry at 30 days and the lowest was found at 30 days. Jackknife Environment Clusters by UniFrac showed that phylogenetic compositions of Anaeromyxobacter communities in slurries at 10 and 20 days were the most similar. By evolutionary distance analysis, our 10 major Anaeromyxobacter types were diverged into Group 1 and 2 in phylogenetic tree, while Group 1 was the exclusive collection of clones from our experiment. Major type P1 was present in all slurries abundantly and P9 only existed in slurry at 5 days. The abundance of Anaeromyxobacter spp., calculated as its proportion of 16S rDNA copies to the value of total Bacteria, was from 0.242% at 1 h to 5.135% at 10 days. We demonstrated that flooding time led to successional dynamics of major types and variable abundance of Anaeromyxobacter community. Flooding time also influenced the diversity of Anaeromyxobacter community on some extent. Canonical correspondence analysis (CCA) revealed that Anaeromyxobacteria spp. abundance had interrelation with Fe (II) content and the influenced the distribution of the slurries in the Biplot. Moreover, our study provides valuable information for the further isolation of Anaeromyxobacter strains from paddies. Key words: Anaeromyxobacter; paddy soil, community structure, abundance, PCR-RFLP, flooding time.