Nucleotide Substitution Model Research Articles

Phylodynamics and Phylogenetic Analysis of Recent SARS CoV 2 Viral Strains from Pune, Maharashtra

In December 2019, medical practitioners from China identified a novel strain of severe acute respiratory syndrome-CoV (SARS-CoV). The disease was allotted a zoonotic origin and the spillover event is associated with Huanan Wholesale Seafood Market in Wuhan City, Hubei Province, China. On 30 January 2020, the World Health Organization (WHO) declared COVID- 19 as an international public-health emergency concern. In India, the index case was reported on 30th January in the district of Thrissur, state of Kerala and by 25th March 2020, the country was lockdown. This research aims to analyze the viral phylogenetic and phylodynamics of strains from Pune, Maharashtra, followed by a comparison against real time data and an analysis of the potency of government prevention strategies. Publicly available SARS-CoV-2 sequences, specific to Pune, Maharashtra, were downloaded from GISAID during the time frame of the epidemics. Phylogenetic analysis of the sequences, including Maximum Likelihood trees and the nucleotide substitution model, were done using IQ-TREE software. Phylodynamic tools available as part of the BEAST2 software was used to estimate the evolution of R number through time. GTR+F+I was found to be the best-fit nucleotide substitution model for the data acquired. ML trees constructed reported a log likelihood of 44842.9931. Analysis of trace estimates reported R number with an average of 1, indicating transmission of virus. The data contains controversial elements which can only be clarified upon further sequencing of the SARS- CoV-2 strains, however, the results obtained are parallel to real time statistics.

The emergence and circulation of human immunodeficiency virus (HIV)-1 subtype C.

Introduction. Human immunodeficiency virus (HIV)-1 subtype C is the most prevalent globally and is thought to have originated in non-human primates in the Democratic Republic of Congo.Hypothesis/Gap Statement. Although the global dominance of HIV-1 subtype C is well established, a thorough understanding of its evolutionary history and transmission dynamics across various risk populations remains elusive. The current knowledge is insufficient to fully capture the global diversification and dissemination of this subtype.Aim. We for the first time sought to investigate the global evolutionary history and spatiotemporal dynamics of HIV-1 subtype C using a selection of maximum-likelihood-based phylodynamic approaches on a total of 1210 near full-length genomic sequences sampled from 32 countries, collected in 4 continents, with sampling dates between 1986-2019 among various risk groups were analysed.Methodology. We subsampled the HIV-1 subtype C genomic datasets based on continent and risk group traits, and performed nucleotide substitution model selection analysis, maximum likelihood (ML) phylogenetic reconstruction, phylogenetic tree topology similarity analysis, temporal signal analysis and traced the timings of viral spread both geographically and by risk group.Results. Based on the phylodynamic analyses of four datasets (full1210, locrisk626, loc562 and risk393), we inferred the time to the most recent common ancestor (TMRCA) in the 1930s and an evolutionary rate of 0.0023 substitutions per site per year. The total number of introduction events of HIV-1 subtype C between continents and between risk groups is estimated to be 71 and 115, respectively. The largest number of introductions occurred from Africa to Europe (n=32), from not-recorded to heterosexual (n=40) and from heterosexual to not-recorded (n=51) risk groups.Conclusion. Our results emphasize that HIV subtype C has mainly spread from Africa to Europe, likely through heterosexual transmission.

Phylogenetic position and genetic features of HIV-1 in CNS

Background. Due to the wide coverage with antiretroviral therapy, the life expectancy of HIV infected people has significantly increased. Against the background of a decrease in mortality from HIV infection, HIV-associated neurocognitive disorders, which develop even during effective treatment, are of high importance. The overall prevalence of this pathology among HIV-infected people reaches 42.6%.
 The objective of the study was to research the genetic features and phylogenetic position of HIV-1 persisting in the central nervous system.
 Materials and methods. The clinical study group consisted of 38 patients with severe neurocognitive disorders against the background of HIV infection in stage 4B. The viral load of HIV-1 in blood plasma and cerebrospinal fluid (CSF) was measured using the "AmpliSens HIV Monitor-FRT" reagents kit. Sanger sequencing was performed using the AmpliSens HIV-Resist-Seq assay kit on an Applied Biosystems 3500 analyzer. Phylogenetic analysis of the pol gene fragments of HIV-1 strains (the site encoding the viral protease and part of the reverse transcriptase) was carried out using maximum likelihood method with the GTR+G nucleotide substitution model. Comparisons of the tertiary structure of viral proteins were performed according to three-dimensional models of the protease and p51 and p66 reverse transcriptase subunits obtained by homologous reconstruction using the SWISS-MODEL tools.
 Results. The viral load in the sample of patients with severe CNS lesions in blood plasma was 6.27 times higher than in CSF and amounted to 4.67 and 3.87 lg copies/ml respectively by median (p = 0,004).
 Phylogenetic analysis with the use of all available HIV-1 genomes from GenBank, which differed from the studied ones by less than 5% showed close genetic relations of viruses circulating in Chelyabinsk region, apart from strains circulating in Russian Federation, with viruses circulating in neighboring countries, in most abundance — from Ukraine and Kyrgyzstan, slightly less — from Belarus, Tajikistan, Kazakhstan and Armenia and also with strains from certain foreign countries: Poland and Germany. Phylogenetic analysis of 38 HIV-1 genomes revealed significant genetic distances between HIV isolates from blood plasma and CSF in 5 patients, 4 of whom were PWID, which may indicate an event of superinfection.
 The amount of independent amino acid substitutions in protease in isolates from blood plasma ranged from 1 to 3, in isolates from CSF — from 1 to 2. An amount of such substitutions in a fragment of reverse transcriptase in isolates from blood plasma ranged from 1 to 6, while in isolates from CSF, it ranged from 1 to 7. HIV isolates from blood plasma and CSF from 5 patients had differences in the tertiary structure of HIV-1 reverse transcriptase p51 subunit in amino acid positions 16–20 and 210–235. Isolates from 3 other patients differed in the tertiary structure only in amino acid positons 210–235. Isolates from 3 patients differed in the structure of HIV-1 RT p66 subunit in a non-nucleoside reverse transcriptase inhibitor binding pocket (NNRTI) region. Fixed differences in the tertiary structure of p51 subunit required at minimum only 1 amino acid substitution to emerge. Alterations in the tertiary structure of p66 subunit required at least 3 amino acid substitutions.
 Conclusion. Microevolution of HIV-1 proceeds in parallel within the same patient, in different compartments, which is reflected in the accumulation of amino acid substitutions different from another compartment in the conserved pol gene. There is a weak correlation between the viral load level in plasma and in CSF. The genetic heterogeneity of HIV strains from patients of the Chelyabinsk region indicates a high frequency of reintroduction of HIV infection in the region from other countries. Differences in the tertiary structure of HIV-1 reverse transcriptase between blood plasma and CSF isolates are regularly fixed in certain domens, which also confirms the presence of parallel HIV microevolution during virus persistence in tissues separated by the blood-brain barrier which allows a better understanding of the fixation trends of individual amino acid substitutions during HIV-induced damage to central nervous system.

First Report of Vascular Wilt on Peanut in Mexico Caused by Fusarium incarnatum.

During the fall of 2020, wilt symptoms were presented in a commercial peanut field (Arachis hypogaea L.), variety 'CHAMPS' in Buenavista de Benito Juárez, México (18.460501 N, 98.627100 W). A peanut field was scout 80 days after planting, and plants presenting symptoms of root and crown rot, vascular chlorosis, and tissue death, were sampled. Disease incidence was estimated at 55% of the field. Isolations of the pathogen were made from stem and root tissues. These samples were disinfected by immersion in 1% sodium hypochlorite (NaClO) for 3 min and immersion in 70% ethanol for 1 min with 3 rinses with sterile distilled water. Subsequently, 0.5 cm fragments were removed and placed on media of potato dextrose agar (PDA) and Rose Bengal. Petri dishes were incubated in complete darkness at 26 °C for 7 days. Abundant aerial white mycelium was observed, which turned tan to brown and showed a slightly orange color on the back of the plate. Finally, pure cultures were obtained by single sporing (Aslam et al. 2020). Colonies identified as Fusarium spp. (Leslie and Summerell, 2006) were sub cultured on PDA agar media and Spezieller Nährstoffarmer (Pérez-Vázquez et al. 2022) to observe microscopic characteristics of ten isolates. Colonies of a representative strain (MA-PET-03) produced hyaline septate hyphae, macroconidia dorsoventrally curved, tapering towards both ends of 51-57 × 4.6-5.4 μm (n = 80) with most having 7 septa. Microconidia were unicellular, nonseptate, hyaline, and ovoid, 12.4-20.6 × 3.6-4.1 μm (n = 80). Chlamydospores were abundant and globose 5-11 μm diam (n = 80), intercalary, and solitary in short chains (Figure 1). The observed microscopic characteristics correspond to the description of Fusarium incarnatum (Khoa et al. 2006; Leslie and Summerell, 2006; Xia et al. 2019). The molecular analyses were done with genomic DNA extracted as previously reported by Pérez et al. (2022). A region from the translation elongation factor gene was PCR amplified using EF688/EF1251 primers (Alves et al. 2008) and from the calmodulin (CMDA) gene, using CALDF1 and CALDR1 primers (Noel et al. 2022). The corresponding PCR products were purified with the Gen Elute™ PCR Clean-Up Kit from Sigma-Aldrich Co. (St. Louis Mo. USA) and sequenced at Macrogen Inc. (Seoul, South Korea). The phylogenetic analysis was inferred using the Bayesian Inference method with 1 million generations, final standard deviation was 0.008516. The nucleotide substitution model for Calmodulin (CMDA) was GTR + G and for TEF1 GTR + I + G. This analysis showed that strain MA-PET-03 shared 100% identity (Figure 2) with F. incarnatum ex-type strain CBS 132.73 (CMDA: MN170342; TEF1: JMN1704761) from Pointed gourd (Trichosanthes dioica) in Malawi Africa. The sequences of strain MA-PET-03 were deposited in GenBank (CMDA: OQ679820; TEF1: OQ679821). The pathogenicity tests were carried out with a total of 20 peanut plants, variety 'CHAMPS', 18 days after having been sown in groups of five seeds in 250 g plastic pots, containing a sterilized mixture of Peatmoss and Agrellite (1:1 v./v), with four repetitions. The seeds were inoculated by immersion in 20 mL of spore suspension (106 conidia/mL) isolated from F. incarnatum for 10 min. The plants were maintained in a greenhouse (70% relative humidity and 28 °C) until the appearance of disease symptoms of. Likewise, 10 control plants were inoculated with sterile water. The experiment was repeated twice. The symptoms developed 15 days after inoculation, the plants presented symptoms of chlorosis, wilting of leaves, stems, and roots, a manifestation similar to that observed in the field, while the control plants remained healthy. F. incarnatum was consistently reisolated from inoculated stems and roots and identified by the microscopic characteristics described above. Peanut leaf blight and wilt disease caused by F. incarnatum has been reported in India (Thirumalaisamy et al. 2019). This first report emphasizes that this phytopathogen is a new threat for peanut producers in Mexico, which is why our finding suggests the need to seek new strategies for its control.

The complete mitochondrial genome of Semiothisa cinerearia Bremer & Grey 1853 (Lepidoptera: Geometridae: Ennominae) and its phylogenetic analysis

Semiothisa cinerearia Bremer & Grey, 1853 belongs to the lepidopteran family Geometridae, subfamily Ennominae. We sequenced the complete mitochondrial genome of S. cinerearia by PCR and Sanger sequencing method. The mitochondrial genome of S. cinerearia is 15,523 bp in length and contains a typical set of 37 genes with ‘MIQ’ type gene arrangement and a 394 bp AT-rich regions. Except for cox1 using CGA as initiation codon, all protein-coding genes (PCGs) start with ATN codons and except for nad2 and nad4l using TAG as termination codon, all PCGs terminated with TAA codon. A phylogenetic tree including 39 genus of subfamily Ennominae was first reconstructed based on the mitochondrial genome sequences with nucleotide substitution model GTR + G + I, which showed that the genera Amraica, Jankowskia, and Ectropis are not monophyletic and S. cinerearia and Macaria notata are classified together.

Deep Sequencing to Reveal Phylo-Geographic Relationships of Juquitiba Virus in Paraguay.

Several hantaviruses result in zoonotic infections of significant public health concern, causing hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in the Old and New World, respectively. Given a 35% case fatality rate, disease-causing New World hantaviruses require a greater understanding of their biology, genetic diversity, and geographical distribution. Juquitiba hantaviruses have been identified in Oligoryzomys nigripes in Brazil, Paraguay, and Uruguay. Brazil has reported the most HCPS cases associated with this virus. We used a multiplexed, amplicon-based PCR strategy to screen and deep-sequence the virus harbored within lung tissues collected from Oligoryzomys species during rodent field collections in southern (Itapúa) and western (Boquerón) Paraguay. No Juquitiba-like hantaviruses were identified in Boquerón. Herein, we report the full-length S and M segments of the Juquitiba hantaviruses identified in Paraguay from O. nigripes. We also report the phylogenetic relationships of the Juquitiba hantaviruses in rodents collected from Itapúa with those previously collected in Canindeyú. We showed, using the TN93 nucleotide substitution model, the coalescent (constant-size) population tree model, and Bayesian inference implemented in the Bayesian evolutionary analysis by sampling trees (BEAST) framework, that the Juquitiba virus lineage in Itapúa is distinct from that in Canindeyú. Our spatiotemporal analysis showed significantly different time to the most recent ancestor (TMRA) estimates between the M and S segments, but a common geographic origin. Our estimates suggest the additional geographic diversity of the Juquitiba virus within the Interior Atlantic Forest and highlight the need for more extensive sampling across this biome.

Mutational and Phylogenetic Analyses of SARS-Cov-2: Bangladesh Perspective

Severe acute respiratory syndrome or SARS-Cov-2 is a global pandemic causing over 6.5 million deaths worldwide. As a part of the global village, Bangladesh is also heavily affected with more than 29,000 deaths in those two years of pandemic. Though the severity and mortality rate are not so high compared to the other countries, Bangladesh also faced many difficulties combating the mutated and newer types of the Covid-19. The evolution and phylogenetic study of the virus is one of those key areas to focus in order to understand the nature of virus, its mutation pattern and gene prediction. This study investigates the evolutionary links among the older and newly emerged SARS‐Cov‐2 variants. In this study, the entire genome sequences of SARS‐Cov‐2 variants were obtained, aligned using muscle alignment, pairwise comparison was computed, differences, gaps and mutations were noted. The phylogenetics of different types of covid-19 variants were determined using a variety of evolutionary substitution models. The ultrametric and metric clustering methods, such as UPGMA and neighbor‐joining (NJ), using nucleotide substitution models allowed the inclusion of nucleotide transitions and transversions as Kimura 80 models. The findings revealed that Omicron variant forms a new monophyletic clade that is distant from other SARS‐Cov‐2 variants but surprisingly in proximity to the alpha variant, which was actually dominant in the early stage of pandemic. This finding also indicates that the omicron variant might have been there for a while, hiding the virulence and later becomes the variant of concern in the latest wave of the pandemic. This may pave the way for new researchers to find proper insight of different variants of Bangladesh in treatment plan and vaccine designing. Bangladesh Pharmaceutical Journal 26(2): 204-211, 2023 (July)

Nucleotide Substitution Model Selection Is Not Necessary for Bayesian Inference of Phylogeny With Well-Behaved Priors.

Model selection aims to choose the most adequate model for the statistical analysis at hand. The model must be complex enough to capture the complexity of the data but should be simple enough not to overfit. In phylogenetics, the most common model selection scenario concerns selecting an adequate substitution and partition model for sequence evolution to infer a phylogenetic tree. Previously, several studies showed that substitution model under-parameterization can bias phylogenetic studies. Here, we explored the impact of substitution model over-parameterization in a Bayesian statistical framework. We performed simulations under the simplest substitution model, the Jukes-Cantor model, and compare posterior estimates of phylogenetic tree topologies and tree length under the true model to the most complex model, the $\text{GTR}+\Gamma+\text{I}$ substitution model, including over-splitting the data into additional subsets (i.e., applying partitioned models). We explored 4 choices of prior distributions: the default substitution model priors of MrBayes, BEAST2, and RevBayes and a newly devised prior choice (Tame). Our results show that Bayesian inference of phylogeny is robust to substitution model over-parameterization and over-partitioning but only under our new prior settings. All 3 current default priors introduced biases for the estimated tree length. We conclude that substitution and partition model selection are superfluous steps in Bayesian phylogenetic inference pipelines if well-behaved prior distributions are applied and more effort should focus on more complex and biologically realistic substitution models.

DNA Sequences Are as Useful as Protein Sequences for Inferring Deep Phylogenies.

Inference of deep phylogenies has almost exclusively used protein rather than DNA sequences based on the perception that protein sequences are less prone to homoplasy and saturation or to issues of compositional heterogeneity than DNA sequences. Here, we analyze a model of codon evolution under an idealized genetic code and demonstrate that those perceptions may be misconceptions. We conduct a simulation study to assess the utility of protein versus DNA sequences for inferring deep phylogenies, with protein-coding data generated under models of heterogeneous substitution processes across sites in the sequence and among lineages on the tree, and then analyzed using nucleotide, amino acid, and codon models. Analysis of DNA sequences under nucleotide-substitution models (possibly with the third codon positions excluded) recovered the correct tree at least as often as analysis of the corresponding protein sequences under modern amino acid models. We also applied the different data-analysis strategies to an empirical dataset to infer the metazoan phylogeny. Our results from both simulated and real data suggest that DNA sequences may be as useful as proteins for inferring deep phylogenies and should not be excluded from such analyses. Analysis of DNA data under nucleotide models has a major computational advantage over protein-data analysis, potentially making it feasible to use advanced models that account for among-site and among-lineage heterogeneity in the nucleotide-substitution process in inference of deep phylogenies.

Variability of <i>pgm</i>‑Region Genes in <i>Yersinia pestis</i> Strains from the Caspian Sandy and Adjacent Plague Foci

The aim of the study was to compare the nucleotide sequences of pgm‑region genes in Yersinia pestis strains isolated on the territory of the Caspian sandy and adjacent plague foci in 1925–2015. Materials and methods. 65 Y. pestis strains from the Caspian sandy and adjacent plague foci were used in the work. DNA isolation was performed using the PureLink Genomic DNA Mini Kit. Whole genome sequencing was conducted in Ion S5 XL System (Thermo Fischer Scientific). Data processing was carried out using Ion Torrent Suite software package 3.4.2 and NewblerGS Assembler 2.6. To compare the obtained sequences with the NCBI GenBank database, the Blast algorithm was used. The phylogenetic analysis was performed according to the data of whole genome SNP analysis based on 1183 identified SNPs. The search for marker SNPs was performed using the Snippy 4.6 program. The phylogenetic tree was constructed using the Maximum Likelihood algorithm, the GTR nucleotide substitution model. Results and discussion. The nucleotide sequences of pgm‑region genes of 65 Y. pestis strains from the Caspian sandy and adjacent plague foci have been assessed. Single nucleotide substitutions have been identified in Y. pestis strains from the Caspian sandy and Kobystan plain-foothill foci in the hmsR, astB, ybtS, ypo1944, ypo1943, ypo1936 genes, as well as a deletion of 5 bp in the ypo1945 gene, which is characteristic of strains of one of the phylogenetic lines of Y. pestis from the foci of Caucasus and Transcaucasia, isolated in 1968–2001. The data obtained can be used to differentiate Y. pestis strains from the Caspian sandy focus, as well as to establish the directions of microevolution of the plague pathogen in this region and adjacent foci.

Next-generation development and application of codon model in evolution.

To date, numerous nucleotide, amino acid, and codon substitution models have been developed to estimate the evolutionary history of any sequence/organism in a more comprehensive way. Out of these three, the codon substitution model is the most powerful. These models have been utilized extensively to detect selective pressure on a protein, codon usage bias, ancestral reconstruction and phylogenetic reconstruction. However, due to more computational demanding, in comparison to nucleotide and amino acid substitution models, only a few studies have employed the codon substitution model to understand the heterogeneity of the evolutionary process in a genome-scale analysis. Hence, there is always a question of how to develop more robust but less computationally demanding codon substitution models to get more accurate results. In this review article, the authors attempted to understand the basis of the development of different types of codon-substitution models and how this information can be utilized to develop more robust but less computationally demanding codon substitution models. The codon substitution model enables to detect selection regime under which any gene or gene region is evolving, codon usage bias in any organism or tissue-specific region and phylogenetic relationship between different lineages more accurately than nucleotide and amino acid substitution models. Thus, in the near future, these codon models can be utilized in the field of conservation, breeding and medicine.

Measuring the relative contribution to predictive power of modern nucleotide substitution modeling approaches.

Traditional approaches to probabilistic phylogenetic inference have relied on information-theoretic criteria to select among a relatively small set of substitution models. These model selection criteria have recently been called into question when applied to richer models, including models that invoke mixtures of nucleotide frequency profiles. At the nucleotide level, we are therefore left without a clear picture of mixture models' contribution to overall predictive power relative to other modeling approaches. Here, we utilize a Bayesian cross-validation method to directly measure the predictive performance of a wide range of nucleotide substitution models. We compare the relative contributions of free nucleotide exchangeability parameters, gamma-distributed rates across sites, and mixtures of nucleotide frequencies with both finite and infinite mixture frameworks. We find that the most important contributor to a model's predictive power is the use of a sufficiently rich mixture of nucleotide frequencies. These results suggest that mixture models should be given greater consideration in nucleotide-level phylogenetic inference.

Phylogeography of hepatitis B virus: The role of Portugal in the early dissemination of HBV worldwide.

In Portugal, the genetic diversity, origin of HBV and the Portuguese role in the dissemination of HBV worldwide were never investigated. In this work, we studied the epidemic history and transmission dynamics of HBV genotypes that are endemic in Portugal. HBV pol gene was sequenced from 130 patients followed in Lisbon. HBV genotype A was the most prevalent (n = 54, 41.5%), followed by D (n = 44, 33.8%), and E (n = 32, 24.6%). Spatio-temporal evolutionary dynamics was reconstructed in BEAST using a Bayesian Markov Chain Monte Carlo method, with a GTR nucleotide substitution model, an uncorrelated lognormal relaxed molecular clock model, a Bayesian skyline plot, and a continuous diffusion model. HBV subgenotype D4 was the first to be introduced in Portugal around 1857 (HPD 95% 1699-1931) followed by D3 and A2 a few decades later. HBV genotype E and subgenotype A1 were introduced in Portugal later, almost simultaneously. Our results indicate a very important role of Portugal in the exportation of subgenotypes D4 and A2 to Brazil and Cape Verde, respectively, in the beginning of the XX century. This work clarifies the epidemiological history of HBV in Portugal and provides new insights in the early and global epidemic history of this virus.

1374. Utilizing Genomic Epidemiology to Explore SARS CoV-2 Transmission Patterns and Support Outbreak Investigations in Long Term Care Facilities, Washington State, April-October 2021

Abstract Background In Washington State, COVID-19 cases in long-term care facilities (LTCF) have accounted for less than 3% of all cases, yet 30% of all COVID-19 deaths. Understanding transmission patterns and outbreak epidemiology informs outbreak management. From April to October 2021, two large LTCFs experienced COVID-19 outbreaks. Whole genome sequencing and phylogenetic analysis were leveraged to explore transmission patterns and complement outbreak epidemiology. Methods Epidemiologic data was exported from the Washington Disease Reporting System. Sequences, retrieved from GISAID, were aligned to the Wuhan-1 reference genome using Nextalign version 1.11.0. Pairwise single nucleotide polymorphism (SNP) distance matrices were calculated using SNP-Dists version 0.8.2. Phylogenetic trees for each outbreak were generated using IQ-Tree multicore version 2.2.0-beta COVID-edition using the GTR+F+G4 nucleotide substitution model with 1000 bootstrap replicates. MicrobeTrace was used to visualize the phylogeny, SNP heatmap, and identify clusters among sequences. Results Weekly, LTCF A tested 162 residents and 800 staff, and LTCF B tested 60 residents and 144 staff. Of all cases in LTCF A (n= 119), 23% (n =27) were residents and 77% (n = 92) were staff, compared to 78% (n =28) residents and 22% (n =7) staff among total LTCF B cases (n=36). In LTCF A, 34% (n=40) of the cases had high-quality sequences available. Seven clusters of two or more genetically related sequences and thirteen genetically unrelated sequences were identified. Five of the clusters involved resident and staff cases, linked by unit. Two clusters and remaining unrelated sequences were among staff. In LTCF B, 40% (n=14) of the cases had high-quality sequences available. One cluster of genetically related sequences was identified, all from residents of two floors. The SNP differences between sequences from LTCF A ranged from 0 to 70, whereas SNP differences between LTCF B sequences ranged from 0 to 6. Conclusion Phylogenetic analysis of the two outbreaks confirms differences in disease transmission patterns. Multiple independent introductions of SARS-CoV-2 were identified in LTCF A, compared to a single introduction in LTCF B. Genomic epidemiology is a valuable resource for outbreak investigation and management. Disclosures All Authors: No reported disclosures.

Phylogenetic Tree Selection by Testing Substitution Number in Clade

The phylogenetic tree is a widely-used tool to show the evolutionary relationship between taxa. There are many types of phylogenetic trees proposed in the literature such as maximum likelihood, neighbor-joining, and UPGMA trees. The topologies of different types of trees are not the same. Even for the same type of tree, the topologies are different when they embed different nucleotide substitution models, such as the JC69 model, K80 model, TN93 model, and so on. Although each type of tree has its advantages, selecting a suitable tree among these choices becomes a challenging problem. In this study, we propose a method based on testing the nucleotide substitution number between sequences to select trees. An Ebolavirus example is used to illustrate the method. In addition, this approach can select a suitable nucleotide substitution model for a particular type of tree. For this ebolavirus example, the JC69 model is the selected substitution model for the maximum likelihood tree.

First Report of Cucurbit Chlorotic Yellows Virus Infecting Watermelon and Zucchini in the Canary Islands, Spain.

In July 2019, eleven watermelon (Citrullus lanatus) plants, six 'Augusta negra' and five 'Kasmira', and seven zucchini (Cucurbita pepo) 'Marcado' plants showing yellowing and vein clearing on the leaves were collected in Arico, in Tenerife Island of the Canary Islands. Analysis with enzyme-linked immunosorbent assay (ELISA) with polyclonal antibodies using kits from (LOEWE® Biochemica GmbH, Sauerlach, Germany) detected cucurbit chlorotic yellows virus (CCYV) in four watermelon (two 'Augusta negra' and two 'Kasmira') plants and one zucchini plant. Reverse transcription-polymerase chain reaction (RT-PCR) with primers CCYV-CPs (5'-ATGGAGAAGACTGACAATAAACAA-3') and CCYV-CPas (5'-TTTACTACAACCTCCCGGTG-3') (Hamed et al. 2011) yielded 750 bp DNA fragments, which were purified, and directly sequenced by Sanger sequencing. The four CCYV sequences obtained from watermelon plants were identical and showed a 99.6% nucleotide identity with the CCYV isolate from Zucchini. The nucleotide sequences of CCYV isolates 351/19 from watermelon and 361/19 from zucchini were deposited in GenBank under accessions OK562588 and OK562589, respectively. BLASTn analysis showed high nucleotide identity, greater than 99 % with most worldwide CCYV isolates (AB523789, HM581658, JF502222, JF807053, JN126045-6, JQ904629, JX014262, KC990503-5, KC990507, KJ735450, KJ149806, KT946809, KT946816, KU507602, KX118632, KY400632-4, KY618799, LT716000, LT716003-4, MH477612, MH806868, MH819191, MN529560, MN815012-3, MT396249 MT048669, MW033301, MW584337, MW251342, MW521380, MW521381, MW584335-6, MW629380, MZ325848-9 and MZ405664), about 98% with two Saudi Arabian isolates (KT946810 and KT946815) and about 95 % with three Iranian isolates (KC577201-3). These genetic relationships were confirmed with a phylogenetic tree (Supplementary Fig. 1) inferred with the maximum likelihood method with 500 bootstrap replicates and the nucleotide substitution model HKY+G implemented in the program MEGA-X (Kumar et al., 2018). There is no correlation between geographic distance and genetic distance. CCYV is related to viruses in the genus Crinivirus, family Closteroviridae (Okuda et al., 2010). It is widespread and causes high economic losses (yield reduction and unmarketable fruits) in cucurbit crops from East Asia: Japan, China, Taiwan and South Korea; North America (the USA states of California, Texas, Georgia and Alabama); Middle East: Sudan, Saudi Arabia and Iran; and the Mediterranean basin: Egypt, Israel, Lebanon, Turkey, Greece, Algeria and peninsular Spain (Kheireddine et al., 2020; Chynoweth et al., 2021; Jailani, et al., 2021; Kwak et al., 2021). This is the first report of CCYV infecting cucurbits in the Canary Islands. The emergence of this virus poses a threat to the cucurbit crops in the Canary Islands and requires control measures to prevent its spread in Tenerife and the other Canary Islands.

Genetic diversity of the cultivated Salvia miltiorrhiza populations revealed by four intergenic spacers.

For better understanding the genetic diversity and phylogeny of the cultivated Salvia miltiorrhiza populations, four intergenic spacer sequences, ETS, psbA-trnH, trnL-trnF, and ycf1-rps15 of the 40 populations collected from China were Polymerase Chain Reaction (PCR) amplified, analyzed both individually and in combination. Haplotype diversity analysis showed that the cultivated S. miltiorrhiza populations had a very rich genetic diversity and an excellent capacity to resist environmental pressure. The best-fit nucleotide substitution models for ETS, psbA-trnH, trnL-trnF, ycf1-rps15, and their combined sequences were HKY+I, T92, T92, T92+G, and T92+G, respectively; the nucleotide conversion frequency in the combined sequences was lower than the transversion, and the relatively high nucleotide substitution frequencies suggests its high genetic variability. Neutral tests showed that the spacer sequences of the populations conform with the neutral evolution model, and there has been no current expansion events occurred. Phylogeny analyses based on both the individual and the combined sequences showed that the 40 populations were clustered in two clades with a very similar topological structure. The discrimination rate of the combined sequence marker is significantly increased to 52.5% (21 populations) over the highest 35% (13 populations) by the single marker of ETS, though still inadequate but a big step forward. Further exploration of more DNA markers is needed. This study for the first time revealed the rich genetic diversity and phylogeny of the currently cultivated S. miltiorrhiza populations in China and provides novel alternative molecular markers for the genetic identification and resources evaluation of the cultivated S. miltiorrhiza populations.

Molecular characterization of sapovirus from children with gastroenteritis in Botswana

Sapoviruses are an important cause of acute viral gastroenteritis in children. The objective of this study was to determine the burden and genetic diversity of sapoviruses in children hospitalized with acute gastroenteritis in Botswana between 2016 and 2018. Samples were screened using the BioFire® Filmarray® GI Panel assay and for positive sapovirus samples, conventional RT-PCR was used to amplify the partial VP1 gene. Strain characterization was performed using both the Human Calicivirus Typing tool and the NCBI GenBank BLAST. Phylogenetic and evolutionary analysis was inferred using BEAST V1.10.4. General time-reversible was used as a nucleotide substitution model. Sapovirus was detected in 6.5% of participants and two different sapovirus genotypes GI.1 (5/7) and GI.6 (2/7) were identified from the seven samples. This study provides the first data on sapovirus infections and their genetic diversity in a clinical setting in Botswana, contributing to the global knowledge of this virus.

Integrative taxonomy peeps a new torrent catfish species of genus Amblyceps (Siluriformes: Amblycipitidae) in Kaladan River of Mizoram, India.

Explorations of the Kaladan River of Mizoram, India during the last decade have given a large number of new species. Integrative taxonomy, when used at the time of discovery of new species, can correlate the morphological characters with the species-specific DNA signatures and ameliorate the process of species identification. Based on this approach, Amblyceps hmolaii sp. nov., a new torrent catfish species is described from the Kaladan River drainage. The new species is distinguished from all twenty-two congeners by morphometric measurements and meristic counts. Sequence analysis of mitochondrial gene cytochrome c oxidase subunit I (COI), generated in this study and those available in NCBI, separated A. hmolaii sp. nov. from seven species of Amblyceps. Analysis of COI sequences, with ABGD software to delimit the species, also provided eight stable groups corresponding to the eight species of Amblyceps. The new species was separated from its congeners with an average genetic distance of 11.77%. Maximum-likelihood (ML) phylogenetic tree was constructed using the best fit nucleotide substitution model HKY + G + I. This study used all the twenty-two congeners in morphomeristic analysis and seven congeners in molecular analysis, for comparison with the new species. This approach unambiguously resolved the new species from other species of Amblyceps and created its species-specific DNA signatures. The discovery of new species even marked the first record of genus Amblyceps from the Kaladan drainage.

Omicron variant genome evolution and phylogenetics.

Following the discovery of the SARS‐CoV‐2 Omicron variant (B.1.1.529), the global COVID‐19 outbreak has resurfaced after appearing to be relentlessly spreading over the past 2 years. This new variant showed marked degree of mutation, compared with the previous SARS‐CoV‐2 variants. This study investigates the evolutionary links between Omicron variant and recently emerged SARS‐CoV‐2 variants. The entire genome sequences of SARS‐CoV‐2 variants were obtained, aligned using Clustal Omega, pairwise comparison was computed, differences, identity percent, gaps, and mutations were noted, and the identity matrix was generated. The phylogenetics of Omicron variants were determined using a variety of evolutionary substitution models. The ultrametric and metric clustering methods, such as UPGMA and neighbor‐joining (NJ), using nucleotide substitution models that allowed the inclusion of nucleotide transitions and transversions as Kimura 80 models, revealed that the Omicron variant forms a new monophyletic clade that is distant from other SARS‐CoV‐2 variants. In contrast, the NJ method using a basic nucleotide substitution model such as Jukes–Cantor revealed a close relationship between the Omicron variant and the recently evolved Alpha variant. Based on the percentage of sequence identity, the closest variants were in the following order: Omicron, Alpha, Gamma, Delta, Beta, Mu, and then the SARS‐CoV‐2 USA isolate. A genome alignment with other variants indicated the greatest number of gaps in the Omicron variant's genome ranging from 43 to 63 gaps. It is possible, given their close relationship to the Alpha variety, that Omicron has been around for much longer than predicted, even though they created a separate monophyletic group. Sequencing initiatives in a systematic and comprehensive manner is highly recommended to study the evolution and mutations of the virus.