Increased Host Range Research Articles

Comparing Methods to Genetically Engineer Bacteriophage and Increase Host Range.

Antibacterial resistance is an emerging problem in military medicine. Disruptions to the health care systems in war-torn countries that result from ongoing conflict can potentially exacerbate this problem and increase the risk to U.S. forces in the deployed environment. Therefore, novel therapies are needed to mitigate the impact of these potentially devastating infections on military operations. Bacteriophages are viruses that infect and kill bacteria. They can be delivered as therapeutic agents and offer a promising alternative to traditional antibiotic chemotherapy. There are several potential benefits to their use, including high specificity and comparative ease of use in the field setting. However, the process of engineering phages for military medical applications can be a laborious and time-consuming endeavor. This review examines available techniques and compares their efficacy. This review evaluates the scientific literature on the development and application of four methods of bacteriophage genome engineering and their consideration in the context of military applications. Preffered Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed for a systematic review of available literature that met criteria for analysis and inclusion. The research completed for this review article originated from the United States Military Academy's library "Scout" search engine, which compiles results from 254 available databases (including PubMed, Google Scholar, and SciFinder). Particular attention was focused on identifying useful mechanistic insight into the nature of the engineering technique, the ease of use, and the applicability of the technique to countering the problem of antimicrobial resistance in the military setting. A total of 52 studies were identified that met inclusion criteria following PRISMA guidelines. The bioengineering techniques analyzed included homologous recombination (12 articles), in vivo recombineering (9 articles), bacteriophage recombineering of electroporated DNA (7 articles), and the CRISPR-Cas system (10 articles). Rates of success and fidelity varied across each platform, and comparative benefits and drawbacks are considered. Each of the phage engineering techniques addressed herein varies in amount of effort and overall success rate. CRISPR-Cas-facilitated modification of phage genomes presents a highly efficient method that does not require a lengthy purification and screening process. It therefore appears to be the method best suited for military medical applications.

Distinct phenotype of SARS-CoV-2 Omicron BA.1 in human primary cells but no increased host range in cell lines of putative mammalian reservoir species

SARS-CoV-2′s genetic plasticity has led to several variants of concern (VOCs). Here we studied replicative capacity for seven SARS-CoV-2 isolates (B.1, Alpha, Beta, Gamma, Delta, Zeta, and Omicron BA.1) in primary reconstituted airway epithelia (HAE) and lung-derived cell lines. Furthermore, to investigate the host range of Delta and Omicron compared to ancestral SARS-CoV-2, we assessed replication in 17 cell lines from 11 non-primate mammalian species, including bats, rodents, insectivores and carnivores. Only Omicron's phenotype differed in vitro, with rapid but short replication and efficient production of infectious virus in nasal HAEs, in contrast to other VOCs, but not in lung cell lines. No increased infection efficiency for other species was observed, but Delta and Omicron infection efficiency was increased in A549 cells. Notably replication in A549 and Calu3 cells was lower than in nasal HAE. Our results suggest better adaptation of VOCs towards humans, without an extended host range, and may be relevant to the search for the putative intermediate host and reservoirs prior to the pandemic.

Dual labelled probe assays for differentiation of Botryosphaeria dothidea, Neofusicoccum mediterraneum and Neofusicoccum parvum, based on polymorphisms in the MAT1-2-1 gene

Botryosphaeriaceous fungi are widespread, and cause serious diseases in many economically important crops. Botryosphaeria dothidea, Neofusicoccum mediterraneum and N. parvum are the most important members of this family in the Mediterranean region. These fungi are frequently isolated from the same host, which together with their extensive and increasing host range necessitates development of rapid and reliable diagnostic tools. Species boundaries within the Botryosphaeriaceae have been defined based on phylogenetic analyses of multiple gene sequences, including those of mating type genes. The MAT1-2-1 gene displayed high sequence variability between Botryosphaeriaceous species, so was selected as the target for development of a definitive diagnostic tool. This paper outlines a new and robust molecular tool, composed of three TaqMan assays based on polymorphisms located in the MAT1-2-1 gene of B. dothidea, N. mediterraneum and N. parvum. Each assay differentiated the target species from other Botryosphaeriaceae, and from non-target fungi.

Distribution of Scirtothrips dorsalis (Thysanoptera: Thripidae) cryptic species complex in the United States and reproductive host assessment of its dominant member.

Chilli thrips, Scirtothrips dorsalis Hood, is a cryptic species complex of at least 9 species, 2 of which (South Asia 1 and East Asia 1) have been confirmed damaging >50 plant taxa in the United States. To develop a knowledge-based management program for this pest in the United States, the objectives of the study were to (i) survey for S. dorsalis species complex in the suspected regions and (ii) assess the reproductive hosts of the predominant member available. Thrips samples received from collaborators were subjected to morpholo-molecular characterization. Of the 101 thrips populations received across the United States between 2015 and 2021, 71 populations were confirmed as S. dorsalis, with ~25% further identified as East Asia 1, compared with ~41% identified as South Asia 1, suggesting the latter species is more prevalent in the US. East Asia 1 was primarily found in samples collected from Hydrangea sp. (95%) in Massachusetts, New York, and Pennsylvania, indicating geographic range expansion in the Northeast and successful overwintering in areas with hard freezes. While assessing the host range of South Asia 1 (dominant species), 62 plant taxa were evaluated under greenhouse conditions. Among these, 40 feeding and reproductive hosts were confirmed in Florida. We also found 18 new reproductive hosts and 3 feeding hosts of S. dorsalis, which were not previously reported in the literature. The results provide insight into the increasing host range and expanded geographical distribution of S. dorsalis species and will enable the development of a species-specific monitoring and management program.

Hybridization in heliothine moths: impacts on reproduction, pheromone communication, and pest management

Heliothine moths (Family Noctuidae : Subfamily Heliothinae) are ubiquitous crop pests with three documented species combinations known to hybridize: Helicoverpa zea x Helicoverpa armigera, H. armigera x Helicoverpa assulta, and Heliothis virescens x Heliothis subflexa. Hybrids can have advantageous traits, such as increased host range, resistance to insecticides, and increased mating success, posing serious threats to agriculture. However, deleterious traits of hybrids, such as developmental abnormalities or sterility, can be exploited for pest management. In this review, the characteristics of F1 hybrids and backcrosses are examined through a historical lens. Topics reviewed include developmental characteristics, sex pheromone synthesis and perception, mating and calling behavior, sperm morphology, gene expression, electrophysiological responses, structures of the central and peripheral nervous systems, hybrid sterility, and applications in pest control. Recommendations for future studies based on existing gaps of knowledge are given, as are proposed pest management strategies.

Cryptosporidium equi n. sp. (Apicomplexa: Cryptosporidiidae): Biological and genetic characterisations

The horse genotype is one of three common Cryptosporidium spp. in equine animals and has been identified in some human cases. The species status of Cryptosporidium horse genotype remains unclear due to the lack of extensive morphological, biological, and genetic data. In the present study, we have conducted biological and whole genome sequence analyses of an isolate of the genotype from hedgehogs and proposed to name it Cryptosporidium equi n. sp. to reflect its common occurrence in equine animals. Oocysts of C. equi measured 5.12 ± 0.36 μm × 4.46 ± 0.21 μm with a shape index of 1.15 ± 0.08 (n = 50). Cryptosporidium equi was infectious to 3-week-old four-toed hedgehogs (Atelerix albiventris) and mice, with a prepatent period of 2–9 days and a patent period of 30–40 days in hedgehogs. It was not infectious to rats and rabbits. Phylogenetic analyses of small subunit rRNA, 70 kDa heat shock protein, actin, 60 kDa glycoprotein and 100 other orthologous genes revealed that C. equi is genetically distinct from other known Cryptosporidium species and genotypes. The sequence identity between C. equi and Cryptosporidium parvum genomes is 97.9%. Compared with C. parvum, C. equi has lost two MEDLE genes and one insulinase-like protease gene and gained one SKSR gene. In addition, 60 genes have highly divergent sequences (sequence differences ≥ 5.0%), including those encoding mucin-like glycoproteins, insulinase-like peptidases, and MEDLE and SKSR proteins. The genetic uniqueness of C. equi supports its increasing host range and the naming of it as a valid Cryptosporidium species. This is the first known use of whole genome sequence data in delineating new Cryptosporidium species.

Potential Hybridization of Fasciola hepatica and F. gigantica in Africa-A Scoping Review.

The occurrence of Fasciola gigantica and F. hepatica in Africa is well documented; however, unlike in Asia, there is a paucity of information on the existence of hybrids or parthenogenetic species on the continent. Nonetheless, these hybrid species may have beneficial characteristics, such as increased host range and pathogenicity. This study provides evidence of the potential existence of Fasciola hybrids in Africa. A literature search of articles published between 1980 and 2022 was conducted in PubMed, Google Scholar, and Science Direct using a combination of search terms and Boolean operators. Fasciola species were documented in 26 African countries with F. hepatica being restricted to 12 countries, whilst F. gigantica occurred in 24 countries, identified based on morphological features of adult Fasciola specimens or eggs and molecular techniques. The co-occurrence of both species was reported in 11 countries. However, the occurrence of potential Fasciola hybrids was only confirmed in Egypt and Chad but is suspected in South Africa and Zimbabwe. These were identified based on liver fluke morphometrics, assessment of the sperms in the seminal vesicle, and molecular techniques. The occurrence of intermediate host snails Galba truncatula and Radix natalensis was reported in Ethiopia, Egypt, South Africa, Tanzania, and Uganda, where F. hepatica and F. gigantica co-occurrences were reported. The invasive Pseudosuccinea columella snails naturally infected with F. gigantica were documented in South Africa and Egypt. In Zimbabwe, P. columella was infected with a presumed parthenogenetic Fasciola. This suggests that the invasive species might also be contributing to the overlapping distributions of the two Fasciola species since it can transmit both species. Notwithstanding the limited studies in Africa, the potential existence of Fasciola hybrids in Africa is real and might mimic scenarios in Asia, where parthenogenetic Fasciola exist in most Asian countries. In South Africa, aspermic F. hepatica and Fasciola sp. have been reported already, and Fasciola hybrids have been reported? in Chad and Egypt. Thus, the authors recommend future surveys using molecular markers recommended to identify Fasciola spp. and their snail intermediate hosts to demarcate areas of overlapping distribution where Fasciola hybrids and/or parthenogenetic Fasciola may occur. Further studies should also be conducted to determine the presence and role of P. columella in the transmission of Fasciola spp. in these geographical overlaps to help prevent parasite spillbacks.

Identification of a Novel Solinvivirus with Nuclear Localization Associated with Mass Mortalities in Cultured Whiteleg Shrimp (Penaeus vannamei).

The emergence and spread of disease-causing viruses in shrimp aquaculture is not uncommon. Since 2016, unusual mortalities have been affecting the Brazilian shrimp industry and we have associated these unusual mortalities with a novel variant of infectious myonecrosis virus (IMNV). The transcriptome analysis of these diseased shrimp showed an additional divergent viral sequence that we have assigned to the family Solinviviridae. The novel virus has been tentatively termed Penaeus vannamei solinvivirus (PvSV) (GenBank accession: OP265432). The full-length genome of the PvSV is 10.44 kb (excluding the poly A tail) and codes for a polyprotein of 3326 aa. Five conserved domains coding for a helicase, RdRp, calicivirus coat protein, G-patch and tegument protein were identified. The genome organization of the PvSV is similar to other (Nylan deria fulva virus 1) solinvivirus. A unique feature of this virus that differs from other members of the Solinviviridae is the presence of putative nuclear localization signals. The tissue tropism of this virus is wide, infecting cells of the hepatopancreas, gastrointestinal tract, lymphoid organ and muscle tissue. Another unique feature is that it is the only RNA virus of penaeid shrimp that shows a nuclear localization by in situ hybridization. The PvSV has a wide distribution in Brazil and has been found in the states of Maranhão State (Perizes de Baixo), Piaui State (Mexeriqueira), Ceará State (Camocim, Jaguaruana, Aracati and Alto Santo) and Pará State where it has been detected in coinfections with IMNV. The diagnostic methods developed here (real-time RT-PCR and in situ hybridization) are effective for the detection of the pathogen and should be employed to limit its spread. Furthermore, the identification of the PvSV shows the increasing host range of the relatively new family Solinviviridae.

Emergence of Asian endemic begomoviruses as a pandemic threat.

Plant viruses are responsible for the most devastating and commercially significant plant diseases, especially in tropical and subtropical regions. The genus begomovirus is the largest one in the family Geminiviridae, with a single-stranded DNA genome, either monopartite or bipartite. Begomoviruses are transmitted by insect vectors, such as Bemisia tabaci. Begomoviruses are the major causative agents of diseases in agriculture globally. Because of their diversity and mode of evolution, they are thought to be geographic specific. The emerging begomoviruses are of serious concern due to their increasing host range and geographical expansion. Several begomoviruses of Asiatic origin have been reported in Europe, causing massive economic losses; insect-borne transmission of viruses is a critical factor in virus outbreaks in new geographical regions. This review highlights crucial information regarding Asia’s four emerging and highly destructive begomoviruses. We also provided information regarding several less common but still potentially important pathogens of different crops. This information will aid possible direction of future studies in adopting preventive measures to combat these emerging viruses.

Canine morbillivirus (CDV): a review on current status, emergence and the diagnostics.

The increasing host range of canine morbillivirus (CDV) affecting important wildlife species such as Lions, Leopard, and Red Pandas has raised the concern. Canine distemper is a pathogen of dogs affecting the respiratory, gastrointestinal, and nervous systems. Seventeen lineages of CDV are reported, and the eighteenth lineage was proposed in 2019 from India. Marked genomic differences in the genome of wild-type virus and vaccine strain are also reported.The variations at the epitope level can be differentiated using specific monoclonal antibodies in neutralization tests. Keeping in mind the current status of the emergence of CDV, genetic and molecular study of circulating strains of the specific geographical region are the essential components of the disease control strategy. New target-based diagnostics and vaccines are in need to counter the effects of the emerging virus population. Control of CDV is necessary to save the endangered, vulnerable, and many other wildlife species to maintain balance in the ecological system. This review provides an overview on emergence reported in CDV, diagnostics developed till today, and a perspective on the disease control strategy, keeping wildlife in consideration.

Whole-Genome Comparisons of Ergot Fungi Reveals the Divergence and Evolution of Species within the Genus Claviceps Are the Result of Varying Mechanisms Driving Genome Evolution and Host Range Expansion.

The genus Claviceps has been known for centuries as an economically important fungal genus for pharmacology and agricultural research. Only recently have researchers begun to unravel the evolutionary history of the genus, with origins in South America and classification of four distinct sections through ecological, morphological, and metabolic features (Claviceps sects. Citrinae, Paspalorum, Pusillae, and Claviceps). The first three sections are additionally characterized by narrow host range, whereas section Claviceps is considered evolutionarily more successful and adaptable as it has the largest host range and biogeographical distribution. However, the reasons for this success and adaptability remain unclear. Our study elucidates factors influencing adaptability by sequencing and annotating 50 Claviceps genomes, representing 21 species, for a comprehensive comparison of genome architecture and plasticity in relation to host range potential. Our results show the trajectory from specialized genomes (sects. Citrinae and Paspalorum) toward adaptive genomes (sects. Pusillae and Claviceps) through colocalization of transposable elements around predicted effectors and a putative loss of repeat-induced point mutation resulting in unconstrained tandem gene duplication coinciding with increased host range potential and speciation. Alterations of genomic architecture and plasticity can substantially influence and shape the evolutionary trajectory of fungal pathogens and their adaptability. Furthermore, our study provides a large increase in available genomic resources to propel future studies of Claviceps in pharmacology and agricultural research, as well as, research into deeper understanding of the evolution of adaptable plant pathogens.

Genetic diversity and expanded host range of astroviruses detected in small mammals in Singapore

Astroviruses are a genetically diverse group of viruses that infect a wide range of hosts, including small mammals. Small mammals were trapped at 19 sites across Singapore from November 2011 to May 2014. Pooled oropharyngeal and rectal swabs (n = 518) and large intestine tissue (n = 107) were screened using a PCR to detect the presence of Astrovirus RNA-dependent RNA polymerase (RdRp) gene. Astroviruses were detected in 93 of 625 (14.9%) of samples tested, with eight of 11 species of rats, shrews, and squirrels testing positive. This is the first detection of astroviruses in seven species (Callosciurus notatus, Mus castaneus, Rattus tanezumi, Rattus tiomanicus, Sundamys annandalei, Suncus murinus and Tupaia glis). Phylogenetic analysis of 10 RdRp gene sequences revealed that astroviruses from Singapore small mammals fall in three distinct clades, one that is specific to the common treeshrew (Tupaia glis), and two comprised of multiple species. One of these includes viruses from the cave nectar bat (Eonycteris spelaea), two rodent species, and a squirrel, suggesting that virus spillover from bats to small mammals may have occurred. Our results show an increased host range for astroviruses and highlight their potential for intra- and inter-species transmission.

Betanodavirus and VER Disease: A 30-year Research Review.

The outbreaks of viral encephalopathy and retinopathy (VER), caused by nervous necrosis virus (NNV), represent one of the main infectious threats for marine aquaculture worldwide. Since the first description of the disease at the end of the 1980s, a considerable amount of research has gone into understanding the mechanisms involved in fish infection, developing reliable diagnostic methods, and control measures, and several comprehensive reviews have been published to date. This review focuses on host–virus interaction and epidemiological aspects, comprising viral distribution and transmission as well as the continuously increasing host range (177 susceptible marine species and epizootic outbreaks reported in 62 of them), with special emphasis on genotypes and the effect of global warming on NNV infection, but also including the latest findings in the NNV life cycle and virulence as well as diagnostic methods and VER disease control.

Directed in Vitro Evolution of Therapeutic Bacteriophages: The Appelmans Protocol.

The ‘Appelmans protocol’ is used by Eastern European researchers to generate therapeutic phages with novel lytic host ranges. Phage cocktails are iteratively grown on a suite of mostly refractory bacterial isolates until the evolved cocktail can lyse the phage-resistant strains. To study this process, we developed a modified protocol using a cocktail of three Pseudomonas phages and a suite of eight phage-resistant (including a common laboratory strain) and two phage-sensitive Pseudomona aeruginosa strains. After 30 rounds of selection, phages were isolated from the evolved cocktail with greatly increased host range. Control experiments with individual phages showed little host-range expansion, and genomic analysis of one of the broad-host-range output phages showed its recombinatorial origin, suggesting that the protocol works predominantly via recombination between phages. The Appelmans protocol may be useful for evolving therapeutic phage cocktails as required from well-defined precursor phages.

Identifying mutation positions in all segments of influenza genome enables better differentiation between pandemic and seasonal strains

Influenza has a negative sense, single-stranded, and segmented RNA. In the context of pandemic influenza research, most studies have focused on variations in the surface proteins (Hemagglutinin and Neuraminidase). However, new findings suggest that all internal and external proteins of influenza viruses can contribute in pandemic emergence, pathogenicity and increasing host range. The occurrence of the 2009 influenza pandemic and the availability of many external and internal segments of pandemic and non-pandemic sequences offer a unique opportunity to evaluate the performance of machine learning models in discrimination of pandemic from seasonal sequences using mutation positions in all segments. In this study, we hypothesized that identifying mutation positions in all segments (proteins) encoded by the influenza genome would enable pandemic and seasonal strains to be more reliably distinguished. In a large scale study, we applied a range of data mining techniques to all segments of influenza for rule discovery and discrimination of pandemic from seasonal strains. CBA (classification based on association rule mining), Ripper and Decision tree algorithms were utilized to extract association rules among mutations. CBA outperformed the other models. Our approach could discriminate pandemic sequences from seasonal ones with more than 95% accuracy for PA and NP, 99.33% accuracy for NA and 100% accuracy, precision, specificity and sensitivity (recall) for M1, M2, PB1, NS1, and NS2. The values of precision, specificity, and sensitivity were more than 90% for other segments except PB2. If sequences of all segments of one strain were available, the accuracy of discrimination of pandemic strains was 100%. General rules extracted by rule base classification approaches, such as M1-V147I, NP-N334H, NS1-V112I, and PB1-L364I, were able to detect pandemic sequences with high accuracy. We observed that mutations on internal proteins of influenza can contribute in distinguishing the pandemic viruses, similar to the external ones.

Differential Effect of Newly Isolated Phages Belonging to PB1-Like, phiKZ-Like and LUZ24-Like Viruses against Multi-Drug Resistant Pseudomonas aeruginosa under Varying Growth Conditions.

In this study, we characterize three phages (SL1 SL2, and SL4), isolated from hospital sewage with lytic activity against clinical isolates of multi-drug resistant Pseudomonas aeruginosa (MDR-PA). The host spectrum ranged from 41% to 54%, with all three phages together covering 79% of all tested clinical isolates. Genome analysis revealed that SL1 (65,849 bp, 91 open reading frames ORFs) belongs to PB1-like viruses, SL2 (279,696 bp, 354 ORFs) to phiKZ-like viruses and SL4 (44,194 bp, 65 ORFs) to LUZ24-like viruses. Planktonic cells of four of five selected MDR-PA strains were suppressed by at least one phage with multiplicities of infection (MOIs) ranging from 1 to 10−6 for 16 h without apparent regrowth of bacterial populations. While SL2 was most potent in suppressing planktonic cultures the strongest anti-biofilm activity was observed with SL4. Phages were able to rescue bacteria-infected wax moth larvae (Galleria melonella) for 24 h, whereby highest survival rates (90%) were observed with SL1. Except for the biofilm experiments, the effect of a cocktail with all three phages was comparable to the action of the best phage alone; hence, there are no synergistic but also no antagonistic effects among phages. The use of a cocktail with these phages is therefore expedient for increasing host range and minimizing the development of phage resistance.

First Report of Tomato leaf curl New Delhi virus on Calotropis procera, a Weed as Potential Reservoir Begomovirus Host in Pakistan

HomePlant DiseaseVol. 101, No. 6First Report of Tomato leaf curl New Delhi virus on Calotropis procera, a Weed as Potential Reservoir Begomovirus Host in Pakistan Previous DISEASE NOTES OPENOpen Access licenseFirst Report of Tomato leaf curl New Delhi virus on Calotropis procera, a Weed as Potential Reservoir Begomovirus Host in PakistanS. S. Zaidi, S. Shakir, H. J. Malik, M. Farooq, I. Amin, and S. MansoorS. S. Zaidihttp://orcid.org/0000-0002-2976-4624Search for more papers by this author, S. ShakirSearch for more papers by this author, H. J. MalikSearch for more papers by this author, M. FarooqSearch for more papers by this author, I. AminSearch for more papers by this author, and S. MansoorSearch for more papers by this authorAffiliationsAuthors and Affiliations S. S. Zaidi , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan S. Shakir , Virology Laboratory, Center for Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad, Pakistan, and Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan H. J. Malik M. Farooq I. Amin S. Mansoor , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. Published Online:19 Apr 2017https://doi.org/10.1094/PDIS-10-16-1539-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In 2015–16, during field surveys of Sindh and Punjab provinces of Pakistan to observe virus symptoms on cultivated crops and nearby weeds, symptoms of leaf yellowing and yellow mosaic were observed on leaves of Calotropis procera growing around the cultivated fields. C. procera, also known as rubber bush or aak, is a common weed in Pakistan. To detect the possible presence of begomoviruses, five symptomatic plants and one asymptomatic plant were collected from Punjab and Sindh, and total DNA was extracted. Southern blot hybridization was performed using a DIG labeled probe to the Tomato leaf curl New Delhi virus (ToLCNDV) C1 gene. A strong hybridization signal was observed in symptomatic plants, while there was no signal in asymptomatic plants collected from the same fields. To further confirm the association of ToLCNDV, rolling circle amplification (RCA) was performed to enrich for the circular DNA molecules of the begomovirus genome, using phi29 DNA polymerase (Thermo Fisher Scientific, Waltham, MA). The RCA product was purified and sequenced by the Illumina MiSeq sequencing system. The Illumina NeoPrep automation system (Illumina, San Diego, CA) was used with the Illumina TruSeq Nano DNA Library Prep Kit (NP-101-1001). After sequencing, adapters were trimmed by the MiSeq Reporter Software and sequencing data were analyzed by the CLC Genomics Workbench 7.5 (https://www.qiagenbioinformatics.com). De novo assemblies were prepared following the standard quality parameters (quality score: 0.001 and Phred score: 30). The NCBI BLASTn results of assembled de novo reads confirmed the presence of ToLCNDV DNA-A and ToLCNDV DNA-B (sequence available in the database with the accession number KX710158 and KX710159, respectively). The species demarcation of ToLCNDV was also confirmed using the Sequence Demarcation Tool (SDT) program (http://web.cbio.uct.ac.za/∼brejnev/), following the revised taxonomic criteria of begomoviruses (Brown et al. 2015). The nBLAST analysis of ToLCNDV DNA-A reported in this study indicated that it was 98% identical to the ToLCNDV DNA-A reported previously in chili from Khanewal, Pakistan (DQ116880); while the nBLAST analysis of ToLCNDV DNA-B reported in this study indicated that it was 95% identical to the ToLCNDV DNA-B reported previously in Cucurbita pepo from Rawalpindi, Pakistan (KT948073). No evidence for the presence of alphasatellites or betasatellite either in the Illumina data or by PCR using universal primers for amplification of alphasatellites or betasatellites was found. Whitefly transmitted ToLCNDV (Gilbertson et al. 2015) infects several important crops like tomato, potato, cucurbits (Zaidi et al. 2016a), and cotton (Zaidi et al. 2016b). The occurrence of ToLCNDV on C. procera indicates the increasing host range of ToLCNDV, which is serious threat for other economically important crops. C. procera has been reported as a potential host for Papaya leaf curl virus (JQ407224; formerly known as Croton yellow vein mosaic virus) and croton yellow vein mosaic betasatellite (HQ631430) in India (Prajapat et al. 2012). C. procera is a common weed and may therefore serve as an alternative virus reservoir, with an important role in the epidemiology of the disease. To our knowledge, this is the first report of ToLCNDV infecting C. procera in Pakistan.

First Report of Tomato leaf curl Albatinah virus (ToLCABV) and its Associated Betasatellite Infecting Papaya in Oman.

Leaf curl disease with severe curling, vein darkening, and vein thickening was observed on papaya plants in a field in Qurayat district of Oman during December 2013. Disease incidence ranged from 50 to 70%, particularly in young papaya plants. The presence of a large population of whiteflies and symptoms observed on papaya plants suggested that the causal agent could be begomoviruses (family Geminiviridae) and associated satellites. Four leaf samples with mild and severe leaf curling were collected from the field. Total nucleic acid extracted from symptomatic and healthy plants using the CTAB method were used as a template to amplify circular DNAs using Φ29 DNA polymerase, and products were digested with restriction enzymes to identify fragments of 2.6 to 2.8 kb typical of geminiviruses. BamHI yielded fragments of ~2.8 and 1.4 kb when the digested products were resolved by electrophoresis on a 1% agarose gel. These fragments were cloned and sequenced using a primer walking strategy in both directions. Sequencing results confirmed the exact sizes of 1,303, 1,358, and 2,765 bp; the sequences were deposited in GenBank under the accession numbers HG969296, HG969297, and HG969260, respectively. BLAST results showed that the first two sequences are Tomato leaf curl betasatellite (ToLCB; isolates Pap-2 and Pap-3) showing 97% sequence identity with a previously reported ToLCB sequence (Accession No. KF229728). Both satellites encode a single gene in the complementary sense strand referred to as βC1, which showed 97% sequence identity to ToLCB (HE800551). The viral sequence (isolate Pap-6) showed four genes in the complementary sense (the replication-associated protein [Rep] gene, the transcription-activator protein [TrAP] gene, the replication-enhancer protein [REn] gene, and the C4 gene) and two genes (pre-coat protein [V2] and coat protein [CP]) in virion-sense (2). BLAST analysis showed 95.2% sequence identity to Tomato leaf curl Albatinah virus (ToLCABV; FJ956700), reported earlier to infect tomato in Oman (3). Amino acid sequence comparison of the four predicted proteins (Rep, TrAP, Ren, and C4) encoded by Pap-6 shared 95, 96, 100, and 100% sequence identity, whereas virion-sense proteins (V1 and V2) shared 99% sequence identity with ToLCABV (FJ956700). According to the recommendations of the International Committee on Taxonomy of Viruses, these results indicate that the virus identified in association with papaya leaf curl disease in Oman is a variant of ToLCABV (1). All infected samples showed the presence of ToLCABV, while no hybridization was observed in healthy control DNA using ToLCABV probe. These findings are indicative of the rapid spread of diseases involving Begomovirus and betasatellites, which often result in increased host range, as is evident from this study.

Modeling the decline and potential recovery of a native butterfly following serial invasions by exotic species

Population sizes and range of the native butterfly Pieris oleracea declined after habitat loss and parasitism by an exotic braconid wasp (Cotesia glomerata) introduced to control the exotic invasive butterfly Pieris rapae. Further declines are attributed to the invasive exotic weed garlic mustard (Alliaria petiolata), an oviposition sensory trap on which P. oleracea larval survival and growth are very poor. But a population of P. oleracea has adapted to garlic mustard over the past several decades, coincident with the introduction of a second parasitoid, C. rubecula, a specialist on P. rapae that is competitively dominant to C. glomerata. We used stochastic simulation models to assess the plausibility of a hypothesis that reduced parasitoid pressure over this time period enabled P. oleracea to adapt to A. petiolata. We simulated scenarios of trait proliferation via spontaneous mutation or immigration of the trait, and residual variation in the trait following the butterfly’s isolation in North America. Results indicate that the most likely scenario for the population that has adapted to garlic mustard includes (1) a change in selection following garlic mustard invasion to favor previously neutral residual variation in the population, (2) release from parasitism, and (3) evolution of improved larval survival on garlic mustard, which allowed an increased host range, and potentially, population size.

Understanding the undelaying mechanism of HA-subtyping in the level of physic-chemical characteristics of protein.

The evolution of the influenza A virus to increase its host range is a major concern worldwide. Molecular mechanisms of increasing host range are largely unknown. Influenza surface proteins play determining roles in reorganization of host-sialic acid receptors and host range. In an attempt to uncover the physic-chemical attributes which govern HA subtyping, we performed a large scale functional analysis of over 7000 sequences of 16 different HA subtypes. Large number (896) of physic-chemical protein characteristics were calculated for each HA sequence. Then, 10 different attribute weighting algorithms were used to find the key characteristics distinguishing HA subtypes. Furthermore, to discover machine leaning models which can predict HA subtypes, various Decision Tree, Support Vector Machine, Naïve Bayes, and Neural Network models were trained on calculated protein characteristics dataset as well as 10 trimmed datasets generated by attribute weighting algorithms. The prediction accuracies of the machine learning methods were evaluated by 10-fold cross validation. The results highlighted the frequency of Gln (selected by 80% of attribute weighting algorithms), percentage/frequency of Tyr, percentage of Cys, and frequencies of Try and Glu (selected by 70% of attribute weighting algorithms) as the key features that are associated with HA subtyping. Random Forest tree induction algorithm and RBF kernel function of SVM (scaled by grid search) showed high accuracy of 98% in clustering and predicting HA subtypes based on protein attributes. Decision tree models were successful in monitoring the short mutation/reassortment paths by which influenza virus can gain the key protein structure of another HA subtype and increase its host range in a short period of time with less energy consumption. Extracting and mining a large number of amino acid attributes of HA subtypes of influenza A virus through supervised algorithms represent a new avenue for understanding and predicting possible future structure of influenza pandemics.