Nucleotide Sequence Assessment of Four ORFs of Citrus Tristeza Virus: Evidence of Recombination

Citrus Tristeza Virus (CTV), usually occurs in nature as a mixture of genotypes. Six naturally infected citrus (Citrus sinensis) trees grafted on sour orange rootstock were collected from three citrus growing governorates in Egypt (Sharqia, Qalyubia and Garbia). In this study, RT-PCR, Single-Strand Conformation Polymorphism (SSCP) and nucleotide sequence analysis were used for four independent CTV genomic regions (p65, p18, p20, and p23) to detect and assess the sequence and genetic variabilities among CTV Egyptian isolates. RTPCR products (650 bp) for the CTV p23 gene obtained from the selected isolates were used for the SSCP analysis and DNA sequencing. SSCP patterns of p23 gene for individual isolates yielded different complex haplotype patterns. Nucleotide sequence analysis of p23 region amplified from six isolates under study revealed that p23 shared high nucleotide identity 98.7% with T36 isolate from USA, Florida. Phylogenetic analysis of p23 gene indicated a close evolutionary relationship between all examined isolates and Qaha isolate (T36 isolate group), suggesting that they may have originated from closely related ancestors. Nucleotide sequence analysis of the three genes located on CTV 3′-coterminal overhang, p18, p20 and p65, amplified from isolate A3, Sharqia governorate, revealed that the p18, p65, and p20 genes were related to the T3-KB isolate from South Africa with 99%–100% sequence homology. Phylogenetic relationship analysis for p65, p18 and p20 ORFs clustered the current A3 isolate with T3 genotype group. The recombination analysis identified three of six isolates from Sharqia, and Garbia as potential recombinant for p23 gene. The isolates T36 and T3 were identified as major donors for recombination events in isolate A3. Our results concluded that p23 ORF likely to be as a hotspot region for recombination and originated through recombination event. The current study indicated that recombination is an important factor for the origin of CTV strains in Egypt.     

Identification,genotyping, and molecular evolution analysis of duck circovirus

Duck circovirus (DuCV) is a contagious immunosuppressive virus affecting many duck species, which is responsible for multiple outbreaks in poultry industries worldwide. In this study, the first DuCV isolate GH01 was identified in Sichuan by PCR, which shared a high level of nucleotide identity (81.8–99.4%) with sequences of other DuCV isolates available in GenBank. Comparative phylogenetic and pairwise sequence comparison analyses indicated that DuCV could be divided into two genotypes (DuCV-1 and DuCV-2) and six subtypes (1a, 1b, 1c, 2a, 2b and 2c) based on the complete genome sequence. The results revealed that both DuCV-1 and DuCV-2 had evolved from the same ancestor but undergone divergent evolution. Interestingly, phylogenetic analyses indicated that three isolates were classified into a cluster DuCV-2a using complete DuCV genome sequence and cap gene, except rep gene. Recombination analyses revealed that DuCV-2a arose from recombination between DuCV-1a and DuCV-2b isolates within the rep genes, and the recombination events mainly occur both in non-structural protein coding region and structural protein coding region. In addition, the mechanisms of recombination supporting the genetic variability in DuCV isolates were investigated. Likewise, selective pressure indicated that purifying selection had been a major driving force in maintaining diversity among the DuCV isolates. Because eradicating the virus from commercial ducks is impossible, it is necessary to take effective control measures and implement them throughout the world.     

Occurrence of grapevine leafroll-associated virus complex in Napa Valley

Grapevine leafroll disease (GLD) is caused by a complex of several virus species (grapevine leafroll-associated viruses, GLRaV) in the family Closteroviridae. Because of its increasing importance, it is critical to determine which species of GLRaV is predominant in each region where this disease is occurring. A structured sampling design, utilizing a combination of RT-PCR based testing and sequencing methods, was used to survey GLRaVs in Napa Valley (California, USA) vineyards (n = 36). Of the 216 samples tested for GLRaV-1, -2, -3, -4, -5, and -9, 62% (n = 134) were GLRaV positive. Of the positives, 81% (n = 109) were single infections with GLRaV-3, followed by GLRaV-2 (4%, n = 5), while the remaining samples (15%, n = 20) were mixed infections of GLRaV-3 with GLRaV-1, 2, 4, or 9. Additionally, 468 samples were tested for genetic variants of GLRaV-3, and of the 65% (n = 306) of samples positive for GLRaV-3, 22% were infected with multiple GLRaV-3 variants. Phylogenetic analysis utilizing sequence data from the single infection GLRaV-3 samples produced seven well-supported GLRaV-3 variants, of which three represented 71% of all GLRaV-3 positive samples in Napa Valley. Furthermore, two novel variants, which grouped with a divergent isolate from New Zealand (NZ-1), were identified, and these variants comprised 6% of all positive GLRaV-3 samples. Spatial analyses showed that GLRaV-3a, 3b, and 3c were not homogeneously distributed across Napa Valley. Overall, 86% of all blocks (n = 31) were positive for GLRaVs and 90% of positive blocks (n = 28) had two or more GLRaV-3 variants, suggesting complex disease dynamics that might include multiple insect-mediated introduction events.     

Occurrence of grapevine leafroll-associated viruses-1 and -3 in Crimea

Grapevine leafroll is one of the most widespread and harmful grapevine diseases. To investigate the occurrence of grapevine leafroll-associated viruses-1 and 3, the survey of vineyards has been conducted in 2015 in six regions of the Crimea. A total of 689 leaf samples with virus symptoms were collected. GLRaV-1 and GLRaV-3 were analyzed by RT-PCR with the specific primers, followed by sequencing of the PCR products. GLRaV-1 and GLRaV-3 were detected in 34 (4.9%) and 37 (5.4%) of the samples, respectively. Vineyards in Simferopol, Bakhchisarai, and Sevastopol regions were found to be free of GLRaV-1 and GLRaV-3.     

Genetic Diversity and Molecular Evolution of Plum bark necrosis stem pitting-associated virus from China

Plum bark necrosis stem pitting-associated virus (PBNSPaV), a member of the genus Ampelovirus in the family Closteroviridae, infects different Prunus species and has a worldwide distribution. Yet the population structure and genetic diversity of the virus is still unclear. In this study, sequence analyses of a partial heat shock protein 70 homolog (HSP70h) gene and coat protein (CP) gene of PBNSPaV isolates from seven Prunus species grown in China revealed a highly divergent Chinese PBNSPaV population, sharing nucleotide similarities of 73.1–100% with HSP70h gene, and 83.9–98.6% with CP gene. Phylogenetic analysis of HSP70h and CP sequences revealed segregation of global PBNSPaV isolates into four phylo-groups (I–IV), of which two newly identified groups, II and IV, solely comprised Chinese isolates. Complete genome sequences of three PBNSPaV isolates, Pch-WH-1 and Pch-GS-3 from peaches, and Plm-WH-3 from a plum tree, were determined. The three isolates showed overall nucleotide identities of 90.0% (Pch-GS-3) and 96.4% (Pch-WH-1) with the type isolate PL186, and the lowest identity of 70.2–71.2% with isolate Nanjing. For the first time, to the best of our knowledge, we report evidence of significant recombination in the HSP70h gene of PBNSPaV variant Pch2 by using five programs implemented in RDP3; in addition, five codon positions in its CP gene (3, 8, 44, 57, and 88) were identified that appeared to be under positive selection. Collectively, these results indicate a divergent Chinese PBNSPaV population. In addition, our findings provide a foundation for elucidating the epidemiological characteristics of virus population.     

Development of immunodiagnostics for the detection of <Emphasis Type="Italic">Grapevine leafroll</Emphasis>-<Emphasis Type="Italic">associated virus</Emphasis> 3 (GLRaV-3) in grapevine using in vitro expression and purification of its coat protein gene

A previously cloned coat protein (CP) gene of Grapevine leafroll-associated virus 3 (GLRaV-3) from cultivar Cabernet Souvignon was over-expressed in Escherichia coli strain BL21 expression system as ~ 43 kDa fusion protein containing polyhistidine tag (6His) at its N terminal. The protein was purified from insoluble fraction and reacted positively in western blotting with commercial anti GLRaV-3 polyclonal antiserum (Bioreba, Switzerland) and hence, used as immunogen for the production of polyclonal antisera in New Zealand white rabbits. Polyclonal antiserum specific to GLRaV-3 detected the virus by double antibody sandwich enzyme linked immunosorbent assay using commercial alkaline phosphatase (ALP) conjugated globulin fraction (Bioreba, Switzerland) in GLRaV-3 positive grapevine samples. The immunoreactivity of the antiserum was confirmed through western blotting. The purified antiserum was conjugated with ALP. The primary antiserum along with ALP conjugate successfully detected the GLRaV-3 from the infected sample at 1:8000 and 1:10,000 dilutions, respectively. To the best of our knowledge, it is the first global study wherein the CP of GLRaV-3 was cloned in pET28a(+) expression vector having many advantages over the earlier used expression vectors. The cloned CP gene was expressed, purified and subjected to the production of immunoreagents. The developed immunoreagents will be useful for certification programmes as well as for large scale virus screening to produce GLRaV-3 free grapevines. The indigenously developed immunereagents will provide a cost-effective way of managing grapevine leafroll disease in Indian sub-continent.     

Genomic Relatedness of Clostridium difficile strains from different toxinotypes and serogroups

Clostridium difficile strains can be divided into sixteen toxinotypes (0 and I to XV) according to changes in their toxin genes. To determine the genomic similarity between toxinotypes, two molecular typing techniques were used, AP-PCR and PFGE. Strains were selected from five serogroups (A1, A15, E, F, X) and represented non-toxinogenic isolates, strains with toxin genes identical to the reference C. difficile strain, VPI 10463 (toxinotype 0), and strains with variant toxin genes from toxinotypes III, IV, V, VI, VII, VIII, IX, and XI. The strains studied formed three main clusters, which correlated well with serogroups: in the first were strains from serogroup A15 and E; in the second, serogroup A1 strains; and in the third, strains from serogroups F and X. Within these three clusters strains of a single toxinotype were grouped together. Toxinotypes III, IV and VIII were more similar to strains with ordinary toxin genes or non-toxinogenic isolates within the same serogroup than to other toxinotypes. Toxinotypes V, VI, VII, and XI, which exhibit similar changes in their toxin genes, seem to be more closely related one to another than to other toxinotypes. It can be concluded that variant Clostridium difficile strains do not have a common ancestor and that groups of different toxinotypes arose independently from strains with ordinary toxin genes.     

The Internal Transcribed Spacer Region of Belonolaimus (Nemata: Belonolaimidae)

Belonolaimus isolates from six U.S. states were compared by restriction endonuclease digestion of amplified first internal transcribed spacer region (ITS1) of the nuclear ribosomal genes. Seven restriction enzymes were selected for evaluation based on restriction sites inferred from the nucleotide sequence of a South Carolina Belonolaimus isolate. Amplified product size from individuals of each isolate was approximately 700 bp. All Midwestern isolates gave distinct restriction digestion patterns. Isolates identified morphologically as Belonolaimus longicaudatus from Florida, South Carolina, and Palm Springs, California, were identical for ITS1 restriction patterns. The correlation between ITS1 restriction patterns and the distribution of B. longicaudatus isolates suggest that the California isolate is a relatively recent introduction into the state.     

Clonal Evolution of Enterocytozoon bieneusi Populations in Swine and Genetic Differentiation in Subpopulations between Isolates from Swine and Humans

Enterocytozoon bieneusi is a widespread parasite with high genetic diversity among hosts. Its natural reservoir remains elusive and data on population structure are available only in isolates from primates. Here we describe a population genetic study of 101 E. bieneusi isolates from pigs using sequence analysis of the ribosomal internal transcribed spacer (ITS) and four mini- and microsatellite markers. The presence of strong linkage disequilibrium (LD) and limited genetic recombination indicated a clonal structure for the population. Bayesian inference of phylogeny, structural analysis, and principal coordinates analysis separated the overall population into three subpopulations (SP3 to SP5) with genetic segregation of the isolates at some geographic level. Comparative analysis showed the differentiation of SP3 to SP5 from the two known E. bieneusi subpopulations (SP1 and SP2) from primates. The placement of a human E. bieneusi isolate in pig subpopulation SP4 supported the zoonotic potential of some E. bieneusi isolates. Network analysis showed directed evolution of SP5 to SP3/SP4 and SP1 to SP2. The high LD and low number of inferred recombination events are consistent with the possibility of host adaptation in SP2, SP3, and SP4. In contrast, the reduced LD and high genetic diversity in SP1 and SP5 might be results of broad host range and adaptation to new host environment. The data provide evidence of the potential occurrence of host adaptation in some of E. bieneusi isolates that belong to the zoonotic ITS Group 1.     

Molecular Characterization and Recombination Analysis of the Complete Genome of Apple Chlorotic Leaf Spot Virus

The complete nucleotide sequence of an Indian isolate of Apple chlorotic leaf spot virus (ACLSV) was determined and found to be 7,525 nt in length. The genome organization was similar to known isolates of ACLSV, encoding three ORFs. Comparisons indicated high sequence variability among known isolates with overall nucleotide sequence identities of 80 to 84%. A striking variable region was identified among the replicase protein upstream of the RNA‐dependent RNA polymerase (aa 1510–1590), which showed a 41–43% match with the corresponding region in other isolates. Phylogenetic analysis at the nucleotide level clustered the isolates into three groups, without any relation to geographical origin. Recombination analysis showed that the isolate is a recombinant with recombination sites spread throughout the genome, especially in the polymerase gene region (nt 4700–5400). Most recombination sites were bordered by an upstream region (5′) of GC‐rich and downstream region (3′) of AU‐rich sequences of similar length. Correlation of recombination site with host type is discussed, and it was found that there were more interlineage recombinations in the apple host compared with intralineage recombinations.     

Genetic Relatedness of Clinical and Environmental Vibrio cholerae Isolates Based on Triple Housekeeping Gene Analysis

Sequence analysis of dnaE, hlyA, and asd housekeeping genes were used to determine the genetic relatedness of our collection of Vibrio cholerae isolated from patients and surface waters over a 5-year period in Iran. The results showed 41, 17, and 9 variable sites throughout the sequenced fragments of dnaE (837 bp), hlyA (495 bp), and asd (295 bp), respectively. The results from sequence typing showed that all our clinical isolates were grouped in the same cluster. Eleven genotypes were identified among the environmental isolates. One environmental isolate was found to be in close genetic relatedness with our clinical isolates. One V. cholerae isolate showed a single-locus variant in the dnaE. For each of the studied genetic loci 10, 7, and 7 sequence types were observed for dnaE, hlyA, and asd, respectively. Only asd sequence analysis could make the distinction between the classical and El Tor isolates which emphasizes on selection of housekeeping locus with better discrimination power for analysis of different groups of isolates. Overall, the results indicated that surface waters in Tehran are a pool of non-toxigenic V. cholerae strains which are rarely related to clinical toxigenic isolates. In addition, our results verified that housekeeping gene sequence analysis could be a suitable approach for determination of the relatedness between clinical and environmental V. cholerae isolates.     

Independent Isolates of the Emerging Subgroup J Avian Leukosis Virus Derive from a Common Ancestor

A new subgroup of avian leukosis virus (ALV) that includes a unique env gene, designated J, was identified recently in England. Sequence analysis of prototype English isolate HPRS-103 revealed several other unique genetic characteristics of this strain and provided information that it arose by recombination between exogenous and endogenous virus sequences. In the past several years, ALV J type viruses (ALV-J) have been isolated from broiler breeder flocks in the United States. We were interested in determining the relationship between the U.S. and English isolates of ALV-J. Based on sequence data from two independently derived U.S. field isolates, we conclude that the U.S. and English isolates of ALV-J derive from a common ancestor and are not the result of independent recombination events.     

Molecular and evolutionary analyses of a variable series of genes in Borrelia burgdorferi that are related to ospE and ospF, constitute a gene family, and share a common upstream homology box.

In this study we report on the molecular characterization of a series of genes that constitute a gene family related to ospE and ospF. Some members of this family appear to represent recombined or variant forms of ospE and ospF. Variant ospE and ospF genes were found in several Borrelia burgdorferi isolates, demonstrating that their occurrence is not a phenomenon relevant to only a single isolate. Hybridization analyses revealed that the upstream sequence originally identified 5' of the full-length ospEF operon exists in multiple copies ranging in number from two to six depending on the isolate. This repeated sequence, which we refer to as the upstream homology box (UHB), carries a putative promoter element. In some isolates, UHB elements were found to flank copies of ospE and ospF that exist independently of each other. We refer to this group of UHB-flanked genes collectively as the UHB gene family. The evolutionary relationships among UHB gene family members were assessed through DNA sequence analysis and gene tree construction. These analyses suggest that some UHB-flanked genes might actually represent divergent forms of other previously described genes. Analysis of the restriction fragment length polymorphism patterns of the UHB-flanked genes among B. burgdorferi isolates demonstrated that these patterns are highly variable among isolates, suggesting that these genes are not phylogenetically conserved. The variable restriction fragment length polymorphism patterns could indicate recombinational activity in these sequences. The presence of numerous copies of the UHB elements and the high degree of homology among UHB-flanked genes could provide the necessary elements to allow for homologous recombination, leading to the generation of recombination variants of UHB gene family members.     

In vitro introduction of healthy and virus-infected genotypes of native Croatian grapevine cultivars

We evaluated the response of eight economically important Croatian grapevine cultivars and studied the impact of their sanitary status on in vitro introduction, by comparing the response of healthy and virus-infected genotypes of one cultivar. Nodal explant survival on three media, M1 (half-strength MS), M2 (full-strength MS) or M3 (full-strength MS with 4.4 µM L^?1 benzylaminopurine) was measured after 2 weeks and regrowth after 8 weeks. After 8 weeks, average shoot length and node number were significantly higher on M2 compared to M1 and M3. M3 induced significantly shorter average internode length, compared to M1 and M2. Survival of one healthy and of five cultivar Plavac mali genotypes infected with GFLV, GLRaV-1, GLRaV-3, GLRaV-3+GVA and GLRaV-1+GLRaV-3 was 97.5 and 82.8–87.5%, respectively. Regrowth of the healthy genotype reached 95.5%, but dropped to 5.5–31.4% in infected ones. The healthy genotype showed significantly higher shoot length (6.3 cm) and node number (7.3) compared to infected genotypes, with shoot length between 1.2–2.6 cm and node number between 1.2–3.0. By contrast, internode length was not significantly different between the healthy and the infected genotypes. The present work represents the first successful in vitro introduction for three of the eight native Croatian cultivars studied.