Recombinant Isolates Research Articles

Development of High-Throughput Methods for Nano- and Bulk Silver Toxicity Assays Using Bioluminescent Recombinant <i>Pseudomonas</i> Wastewater Isolates

Silver get washed into sewerage systems and eventually to wastewater treatment plant (WWTP) due to its utilization in industries. This poses concerns about the toxicity of these particles to microorganisms which are involved in biodegradation of organic wastes in biological WWTP. Pseudomonas species (Biosensor cell A, B, C, D and E) originally isolated from WWTP and modified by incorporating a stable chromosomal copy of the lux operon (lux CDABE) derived from <i>Escherichia coli</i> S17ƛ pir were sensitive immediately upon addition of silver nanoparticles (AgNPs) and bulk silver in short terms of incubation ranging from 0 to 300minutes. Microtitre plate luminometre was used to generate detailed luminescence reduction data for the silver particles tested against the bacterial cells in various concentrations ranging from 9µg/ml to 2500µg/ml. The EC₅₀ values generated at various time points showed that the highest toxicity was observed at time point, 0 of incubation for both AgNPs and bulk silver (158µg/ml and 618µg/ml EC₅₀ values respectively); these EC₅₀ values also indicate that AgNPs are much more toxic than bulk silver. Two putative biosensors, E and D showed proportional responses of bioluminescence reduction with increasing toxicant concentrations up to 2500µg/ml, hence displaying dose-dependent responses, superior operational range and sensing capabilities; good features for toxicity assay. Therefore, the recombinant isolate can be used to assay the toxicity of silver particles.

Virulence and Molecular Characterization of Experimental Isolates of the Stripe Rust Pathogen (Puccinia striiformis) Indicate Somatic Recombination.

Puccinia striiformis causes stripe rust on wheat, barley, and grasses. Natural population studies have indicated that somatic recombination plays a possible role in P. striiformis variation. To determine whether somatic recombination can occur, susceptible wheat or barley plants were inoculated with mixed urediniospores of paired isolates of P. striiformis. Progeny isolates were selected by passing through a series of inoculations of wheat or barley genotypes. Potential recombinant isolates were compared with the parental isolates on the set of 18 wheat or 12 barley genotypes that are used to differentiate races of P. striiformis f. sp. tritici (the wheat stripe rust pathogen) and P. striiformis f. sp. hordei (the barley stripe rust pathogen), respectively, for virulence changes. They were also tested with 51 simple-sequence repeat and 90 single-nucleotide polymorphism markers for genotype changes. From 68 possible recombinant isolates obtained from nine combinations of isolates based on virulence tests, 66 were proven to be recombinant isolates by molecular markers. Various types of recombinants were determined, including lost virulence from both virulent parental isolates, gained virulence from both avirulent isolates, combined virulences from both parents, and inherited virulence from one parent and avirulence from another. Marker data indicate that most of the recombinants were produced through chromosome reassortment and crossover after the hybridization of two parental isolates. The results demonstrate that somatic recombination is a mechanism by which new variants can be generated in P. striiformis.

Molecular genetic analysis and evolution of begomoviruses and betasatellites causing yellow mosaic disease of bhendi.

In India, Bhendi yellow vein mosaic disease (BYVMD) is one of the most economically important diseases of bhendi/okra and is caused by a complex of monopartite begomovirus (Bhendi yellow vein mosaic virus-BYVMV) and betasatellite (Bhendi yellow vein betasatellite-BYVB). In this study, we have analyzed the role of possible evolutionary factors involved in the evolution of BYVMV and BYVB isolates. Evidence of inter-species and inter-strain recombination events was detected among the viral isolates, and majority of these recombinant isolates possess microsatellites in their genome. Recombination analysis suggests that cotton-infecting and bhendi-infecting begomoviruses probably share a recent common ancestor. In addition to genetic differentiation and gene flow, high degree of genetic variability was detected among the viral population. A strong purifying selection seems to be acting on the viral coding regions. The nucleotide substitution rate of V1 gene (for BYVMV) and βC1 gene (for BYVB) was estimated to be 7.55×10-4 and 2.25×10-3 nucleotide substitutions/site/year, respectively. The present study underlines that the evolution of BYVMD-associated viral components is driven by selection acting on the genetic variation generated by recombination and mutation.

Mutations in VP1 and 3A proteins improve binding and replication of rhinovirus C15 in HeLa-E8 cells

Viruses in the rhinovirus C species (RV-C) can cause severe respiratory illnesses in children including pneumonia and asthma exacerbations. A transduced cell line (HeLa-E8) stably expressing the CDHR3-Y529 receptor variant, supports propagation of RV-C after infection. C15 clinical or recombinant isolates replicate in HeLa-E8, however progeny yields are lower than those of related strains of RV-A and RV-B. Serial passaging of C15 in HeLa-E8 resulted in stronger cytopathic effects and increased (≥10-fold) virus binding to cells and progeny yields. The adaptation was acquired by two mutations which increased binding (VP1 T125K) and replication (3A E41K), respectively. A similar 3A mutation engineered into C2 and C41 cDNAs also improved viral replication (2–8 fold) in HeLa but the heparan sulfate mediated cell-binding enhancement by the VP1 change was C15-specific. The findings now enable large-scale cost-effective C15 production by infection and the testing of RV-C infectivity by plaque assay.

Three variants of cotton leaf curl begomoviruses with their satellite molecules are associated with cotton leaf curl disease aggravation in New Delhi

Cotton leaf curl disease (CLCuD), caused by monopartite begomoviruses and its satellite molecules, is one of the serious constrains in cultivation of cotton in India. In the present study, five CLCuD-begomovirus and its associated satellite molecules were characterized based on rolling circle amplification and sequencing of complete genome. Sequence analysis showed 82–99 % nucleotide identity among them. The phylogenetic analysis and nt identity matrix determined that of the five CLCuD-begomovirus isolates, three IARI-34, IARI-42 and IARI-50 were members of Cotton leaf curl Multan virus (CLCuMuV)-Rajasthan isolates, designated as CLCuMuV-Rajasthan-34 and two, IARI-30 and IARI-45 of Cotton leaf curl Kokhran virus (CLCuKoV)-Burewala isolates, designated as CLCuKoV-Burewala-45. The present CLCuMuV-Rajasthan-34 is recombinant isolate showing recombination events in IR, C1 and C4 regions of its genome with high probality (P = 9.9 × 10−10–3.2 × 10−6). Same species of betasatellite (1371 nt) molecules obtained from both the present isolates was related with cotton leaf curl Multan betasatellite by 89–97 % nt identity. Three alphasatellites (1366–1396 nt) related to Cotton leaf curl Burewala alphasatellite and Gossypium darwinii symptomless alphasatellite by 86 % nt identity were also obtained. This is the first report of appearance of CLCuKoV-Burewala isolate and CLCuD associated alphasatellites in New Delhi. The present study demonstrated that CLCuD in New Delhi is caused by three kinds of variants, two are strains of CLCuMuV and one of CLCuKoV, either by single or mixed infection along with beta- and alpha-satellite molecues.

Near Full-Length Genome Identification of a Novel HIV-1 Unique (B/C) Recombinant Isolate in Sichuan, China.

A novel HIV-1 unique recombinant virus (XC2014EU20) was identified. It was isolated from an HIV-positive man who was infected through heterosexual sex in Sichuan, China. The near full-length genome analyses showed that the novel recombinant was composed of three subtype B regions in a subtype C backbone. Different from the other circulating recombinant forms and unique recombinant forms, six recombinant breakpoints of XC2014EU20 with seven fragments were observed in the pol, vpu, and nef genes, respectively. The emergence of B/C recombinant strain indicated the increasing complexity of the HIV-1 epidemic in Sichuan, China.

Occurrence and molecular analysis of quarantine virus in lily cultivation areas in Brazil

Abstract: The objective of this work was to describe the occurrence of quarantine Tulip breaking virus (TBV, synonym Lily mottle virus - LMoV) and Lily symptomless virus (LSV), and their respective molecular analyses, to provide data for supporting TBV removal from the Brazilian A1 quarantine pest list, since this virus has spread among the main commercial lily crops in Brazil. The occurrence of these viruses was detected in 12 cultivation areas through multiplex reverse transcription (RT-PCR), using specific primers to genes encoding the respective coat proteins (CP). Eight fragments of 800 nucleotides (nt) obtained from the LMoV-infected lilies and nine fragments of 600 nt from LSV-infected lilies were sequenced. Phylogenetic tree reconstruction showed a robust branch containing the LMoV Brazilian sequences, other LMoV isolates, TBV, and Tulip band breaking virus, suggesting that all are LMoV isolates, although they are clustered into two subgroups. Phylogenetic analysis also showed a robust branch supporting all Brazilian and other LSV sequences, except for an LSV Japanese isolate. Recombination analyses also showed an LMoV recombinant isolate, whereas no recombination events were found among LSV isolates. Lily mottle virus is the prevalent virus in lily crops in Brazil, in single and mixed infections with LSV or Cucumber mosaic virus (CMV).

Genome sequence and origin analyses of the recombinant novel IBV virulent isolate SAIBK2.

Recombination between infectious bronchitis viruses (IBVs), together with point mutations, insertions, and deletions, is thought to be responsible for the emergence of new IBV variants. SAIBK2 is a nephropathogenic strain isolated from layer flocks vaccinated with live attenuated H120 vaccine in Sichuan province, China in 2011. SAIBK2 causes severe kidney lesions and results in 50 % mortality in 30-day-old specific-pathogen-free chickens (with a dose of 105 EID50/0.1 mL SAIBK2 per chicken). The complete genome of SAIBK2 consists of 27669 nucleotides, excluding the poly-A tail at the 3′ end. SAIBK2 has the highest identity to YX10 in terms of complete genome. Phylogenetic analysis of complete sequence showed that SAIBK2 belongs to the most dominant genotype in China. Comparison and recombination analyses with other IBV strains revealed that SAIBK2 may originate from recombination events among a YX10-, a YN-, and a Mass-like strain. Furthermore, whole gene 5 and parts of nsp 3, nsp 4, nsp 16, and N genes are involved in the recombination events, and the uptake of these regions from YN and Mass strains by SAIBK2 may increase its replication efficiency and be responsible for its increased virulence in specific-pathogen-free chickens.

Near Full-Length Genome Sequences of Two Novel HIV-1 Recombinant Forms Detected in Henan Province, China.

In recent years, the population infected through sexual contact has seen the fastest growing prevalence of HIV transmission in Henan province, China. Here, we report two novel human immunodeficiency virus type 1 (HIV-1) recombinant form detected from a comprehensive HIV-1 molecular epidemiologic study among heterosexuals. Recombinant analyses of the near full-length genome of the two novel HIV-1 recombinant isolates: 01B.CN.2012.11092 was CRF01_AE that was partly replaced by a subtype B' fragment of 414 bp (from 4482-4896 according to the HXB2 calibrator). 01BC.CN.2011.11312 was composed of three segments (CRF01_AE/CRF_07BC/B') with breakpoints 4274 and 4833 according to the HXB2 calibrator. They are different from previously identified recombinant strains reported in China.

Identification of a naturally occurring Bean common mosaic virus recombinant isolate infecting azuki bean.

The near complete nucleotide sequence of Bean common mosaic virus (BCMV) infecting azuki bean from China was determined to be 10047 nucleotides in length excluding the 3′-terminal poly (A) sequence. The isolate was designated as BCMV-Az and caused stunting, mosaic and crumpling on leaves of azuki plants. It contains a large open reading frame (ORF) flanked by a 131 nt 5′-untranslated region (UTR) and a 253 nt 3′-UTR and the putative polyprotein encoded by this large ORF is comprised of 3220 amino acid residues. Phylogenetic analysis showed that BCMV-Az clustered with strains isolated from different hosts and geographic origins, suggesting that it is a novel strain. Recombination analyses indicated that it was a recombinant originating from isolates R (AJ312437) and US10 (KF919299).

Molecular characterization and detection of a recombinant isolate of bamboo mosaic virus from China.

The complete genome sequences of three isolates of bamboo mosaic virus (BaMV) from mainland China were determined and compared to those of BaMV isolates from Taiwan. Sequence analysis showed that isolate BaMV-JXYBZ1 from Fuzhou shares 98% nucleotide sequence identity with BaMV-YTHSL14 from nucleotides 2586 to 6306, and more than 94% nucleotide sequence identity with BaMV-MUZHUBZ2 in other regions. Recombination and phylogenetic analyses indicate that BaMV-JXYBZ1 is a recombinant with one recombination breakpoint. To our knowledge, this is the first report of a BaMV recombinant worldwide.

The complete genome sequences of two naturally occurring recombinant isolates of Sugarcane mosaic virus from Iran.

Sugarcane mosaic virus (SCMV) is the most prevalent virus causing sugarcane mosaic and maize dwarf mosaic diseases. Here, we presented the first two complete genomic sequences of Iranian SCMV isolates, NRA and ZRA from sugarcane and maize. The complete genome sequences of NRA and ZRA were, respectively, 9571 and 9572 nucleotides (nt) in length, excluding the 3'-terminal poly(A) tail. Both isolates contained a 5'-untranslated region (UTR) of 149 nt, an open reading frame of 9192 nt encoding a polyprotein of 3063 amino acids (aa), and 3'-UTR of 230 nt for NRA and 231 nt for ZRA. SCMV-NRA and -ZRA genome nucleotide sequences were 97.3 % identical and shared nt identities of 79.1-92 % with those of other 21 SCMV isolates available in the GenBank, highest with the isolate Bris-A (AJ278405) (92 and 91.7 %) from Australia. When compared for separate genes, most of their genes shared the highest identities with Australian and Argentinean isolates. Phylogenetic analysis of the complete genomic sequences reveals that SCMV can be clustered to three groups. Both NRA and ZRA were clustered with sugarcane isolates from Australia and Argentina in group III but formed a separate sublineage. Recombination analysis showed that both isolates were intraspecific recombinants, and represented two novel recombination patterns of SCMV (in the P1 coding region). NRA had six recombination sites within the P1, HC-Pro, CI, NIa-Vpg, and NIa-pro coding regions, while ZRA had four within the P1, HC-Pro, NIa-Pro, and NIb coding regions.

Population Analysis of Iranian Potato virus Y Isolates Using Complete Genome Sequence.

In this study, the full-length nucleotide sequences of four Iranian PVY isolates belonging to PVYN strain were determined. The genome of Iranian PVY isolates were 9,703–9,707 nucleotides long encoding all potyviral cistrons including P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP with coding regions of 825, 1,395, 1,095, 156, 1,902, 156, 564, 732, 1,557 and 801 nucleotides in length, respectively. The length of pipo, embedded in the P3 cistron, was 231 nucleotides. Phylogenetic analysis showed that the Iranian isolates clustered with European recombinant NTN isolates in the N lineage. Recombination analysis demonstrated that Iranian PVYN isolates had a typical European PVYNTN genome having three recombinant junctions while PVYN and PVYO were identified as the parents. We used dN/dS methods to detect candidate amino acid positions for positive selection in viral proteins. The mean ω ratio differed among different genes. Using model M0, ω values were 0.267 (P1), 0.085 (HC-Pro), 0.153 (P3), 0.050 (CI), 0.078 (VPg), 0.087 (NIa-pro), 0.079 (NIb) and 0.165 (CP). The analysis showed different sites within P1, P3 and CP were under positive selection pressure, however, the sites varied among PVY populations. To the best of our knowledge, our analysis provides the first demonstration of population structure of PVYN strain in mid-Eurasia Iran using complete genome sequences and highlights the importance of recombination and selection pressure in the evolution of PVY.

C-5-Modified Tetrahydropyrano-Tetrahydofuran-Derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs, including Darunavir.

We identified three nonpeptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097, containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C-5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1(WT)), with 50% effective concentrations (EC50s) of 3.0 to 49 nM, and exhibited minimal cytotoxicity, with 50% cytotoxic concentrations (CC50) for GRL-015, -085, and -097 of 80, >100, and >100 μM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multidrug-resistant HIV-1 variants (HIVMDR). Importantly, darunavir (DRV) was >1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1DRV(R) P51); the three compounds remained active against HIV-1DRV(R) P51 with only a 6.8- to 68-fold reduction. Moreover, the emergence of HIV-1 variants resistant to the three compounds was considerably delayed compared to the case of DRV. In particular, HIV-1 variants resistant to GRL-085 and -097 did not emerge even when two different highly DRV-resistant HIV-1 variants were used as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings indicate that the three compounds warrant further study as possible therapeutic agents for treating individuals harboring wild-type HIV and/or HIVMDR. Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1 strains and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1 strains (HIVDRV(R) ) has recently been observed in vivo and in vitro. Here, we identified three novel HIV-1 protease inhibitors (PIs) containing a tetrahydropyrano-tetrahydrofuran (Tp-THF) moiety with a C-5 hydroxyl (GRL-015, -085, and -097) which potently suppress the replication of HIVDRV(R) . Moreover, the emergence of HIV-1 strains resistant to the three compounds was considerably delayed compared to the case of DRV. The C-5 hydroxyl formed a strong hydrogen bonding interaction with the carbonyl oxygen atom of Gly48 of protease as examined in the structural analyses. Interestingly, a compound with Tp-THF lacking the hydroxyl moiety substantially decreased activity against HIVDRV(R) . The three novel compounds should be further developed as potential drugs for treating individuals harboring wild-type and multi-PI-resistant HIV variants as well as HIVDRV(R) .

Complete genome analysis of a novel recombinant isolate of pepper veinal mottle virus from mainland China.

BackgroundPepper veinal mottle virus (PVMV) was well established in Africa, and also reported infecting pepper (Capsicum annuum L) in Taiwan and India. However, there is not available of PVMV in mainland China. Here, the first complete genome sequence of PVMV isolated from pepper in mainland China was reported.FindingThe complete genomic sequence of isolate PVMV-HN isolated from pepper in mainland China is reported in this study. The genome of PVMV-HN is 9793 nucleotides (nt) excluding the poly (A) tail, shares 98-99 % nucleotide sequence identity with those two PVMV isolates from Ghana and Taiwan. Recombinant analysis showed that PVMV-HN probably represents a novel recombinant of PVMV. The phylogenetic relationship of PVMV-HN isolate to other PVMV isolates and other potyviruses based on genome or polyprotein sequence level and CP gene level, was also analyzed in this study.ConclusionThe current study will help to understand phylogenetic relationship of isolate PVMV-HN.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0419-9) contains supplementary material, which is available to authorized users.

Characterization of Phytophthora infestans populations in Canada during 2012

The late blight pathogen (Phytophthora infestans) continues to cause major losses on potato and tomato in Canada and worldwide. An increased diversity of P. infestans and dramatic shifts in pathogen populations have occurred in Canada in recent years. In 2011, a survey identified different genotypes of P. infestans in Canada, including the new US-22, US-23 and US-24 genotypes, which were dominant in various Canadian provinces. In 2012, analysis of samples collected from infected potato and tomato plants from different regions in Canada indicated a rapid change in P. infestans populations in most provinces within a single year. For example, in Prince Edward Island, the US-8 genotype that dominated the P. infestans landscape for many years was displaced by the US-23 genotype, a phenomenon similar to that which occurred in western Canada in prior years. In British Columbia, however, US-11 and the new CA-12 were the dominant genotypes while in Ontario the tested isolates were US-22. Evidence for recombination was found, and increasing insensitivity to mefenoxam was apparent among isolates of some populations. Independent segregation of either Gpi, mating type or RG57 loci occurred in a number of the recombinant isolates, resulting in increased diversity of P. infestans populations. The unexpected change in composition of P. infestans genotypes supports the need for continued monitoring of this pathogen.

Sexual recombination as a tool for engineering industrial Penicillium chrysogenum strains.

The recent discovery and functional characterization of opposite mating-type loci in the industrial penicillin producer Penicillium chrysogenum demonstrated their regulatory role in sexual as well as asexual development. Subsequent experiments further showed that a sexual life cycle can be induced in P. chrysogenum that was for long believed to reproduce exclusively by asexual propagation. Finally, crossing of wild type and production strains resulted in the generation of recombinant ascospore isolates. We predict from these recent findings that recombinant progeny for industrial applications can be obtained by sexual crossings and discuss experimental difficulties that occur when parental strains with karyotype heterogeneity are used for mating.

Incidence of Garlic common latent virus in Argentina, and phylogenetic and recombination analyses of isolates

The objective of this work was to estimate the incidence and prevalence of Garlic common latent virus (GarCLV) in the main production regions of garlic (Allium sativum) in Argentina, and to perform phylogenetic and recombination analyses in isolates from these regions. Leaf samples (3,050) were taken from four garlic commercial types, in 13 departments of the four main garlic-producing provinces of Argentina, in a 1,175-ha sampling area. Virus infection was evaluated with DAS-Elisa test using specific antiserum, and the phylogenetic and recombination analyses were done with capsid protein (CP) nucleotide sequence of seven GarCLV isolates from the provinces. The incidence of GarCLV in the evaluated provinces varied between 6.7 and 22% of the samples, whereas the prevalence varied between 52.6 and 70%. In the analysis of garlic commercial types, Morado showed the highest incidence of the virus, in the province of San Juan, whereas Rosado Paraguayo had the lowest incidence, in the province of Cordoba. Nucleotide identity in the CP sequences ranged between 80.3 and 97.6%. The phylogenetic analysis shows the presence of two main groups of GarCLV and of a possible third group that would include only a German isolate. The recombination analysis between isolates from different parts of the world evidences the presence of recombinant isolates from Poland and Australia.

Biological and molecular variation amongst Australian Turnip mosaic virus isolates

Turnip mosaic virus (TuMV) causes crop losses worldwide. Eight Australian TuMV isolates originally obtained from five different species in two plant families were inoculated to 14 plant species belonging to four families to compare their host reactions. They differed considerably in virulence in Brassicaceae crop species and virus indicator hosts belonging to three other families. The isolates infected most Brassica species inoculated, but not Raphanus sativus, usually causing systemic mosaic symptoms, so they resembled TuMV biological host type [B]. Whole genome sequences of seven of the Australian isolates were obtained and had lengths of 9834 nucleotides (nt). When they were compared with 37 non‐recombinant TuMV genomes from other continents and another whole genome from Australia, six of them formed an Australian group within the overall world‐B phylogenetic grouping, while the remaining new genome sequence and the additional whole genome from Australia were part of the basal‐B grouping. When the seven new Australian genomes and the additional whole genome from Australia were subjected to recombination analysis, six different recombination events were found. Six genomes contained one or two recombination events each, but one was non‐recombinant. The non‐recombinant isolate was in the Australian grouping within the overall world‐B group while the remaining recombinant isolates were in the basal‐B and world‐B phylogenetic groups.

Molecular and Biological Characterization of Potato virus Y Isolates from Vietnam

AbstractEight isolates from different potato growing regions in Vietnam were characterized. All were highly pathogenic in some potato cultivars, but did not overcome the extreme resistance of Solanum stoloniferum and Solanum demissum. RT‐PCR analysis revealed that all of these isolates are recombinants. Sequence data for 4 isolates were obtained, and their reaction in potato cultivars harbouring specific N genes was determined. Different phylogenetic analyses of viral sequences confirmed previous results that the recombinant isolates evolved from different parental sequences. One of the Vietnamese isolates investigated had a specific structure. The need for a clear classification of PVYNWi isolates is discussed.