Molecular characterization of two Chinese isolates of <Emphasis Type="Italic">Beet mosaic virus</Emphasis>

The complete genomic sequences of Beet mosaic virus Xinjiang (BtMV-XJ) and Inner Mongolia (BtMV-IM) isolates from China were determined and compared with US and German isolates, reported previously. Results showed that viral genome of the two isolates both comprise 9,591 nucleotides, and contain the large single open reading frame (ORF) encoding a single polyprotein of 3,085 amino acid residues, from which ten putative functional proteins may be produced by autolytic cleavage processing as the US (BtMV-Wa) and German (BtMV-G) isolates. Sequence comparisons showed that BtMV-XJ shared 89.8% and 98.3% overall nucleotide identity with BtMV-Wa and BtMV-G isolates, and BtMV-IM exhibited the overall identities of 91.6% and 93.8% with BtMV-Wa and BtMV-G, respectively. Further, analyses revealed that BtMV-XJ shared higher identities in almost every region to BtMV-G than to BtMV-Wa both at the nucleotide and the amino acid levels. While BtMV-IM in the regions (6,666–7,671 and 7,672–9,591) showed highest homology with BtMV-XJ and BtMV-G, especially, after nt 7,672 with similarity up to 99.2% with BtMV-G; the region (2,331–4,083) showed highest identity (98.0% nt identity) with BtMV-Wa. That suggested BtMV-XJ had a more close relationship to BtMV-G, while BtMV-IM was more likely to be a natural recombination virus. In addition, phylogenetic analysis of the available BtMV CP sequences showed that BtMV isolates fell into two distinct groups: Euroasia group (Europe and China) and America group (USA). To the best of our knowledge, this study reported the complete sequences of two BtMV isolates from Asia for the first time. H. Xiang and Y.-H. Han contributed equally to this paper.     

Sequencing and computational analysis of complete genome sequences of <Emphasis Type="Italic">Citrus yellow mosaic badna virus</Emphasis> from acid lime and pummelo

Citrus yellow mosaic badna virus (CMBV), a member of the Family Caulimoviridae, Genus Badnavirus, is the causative agent of Citrus mosaic disease in India. Although the virus has been detected in several citrus species, only two full-length genomes, one each from Sweet orange and Rangpur lime, are available in publicly accessible databases. In order to obtain a better understanding of the genetic variability of the virus in other citrus mosaic-affected citrus species, we performed the cloning and sequence analysis of complete genomes of CMBV from two additional citrus species, Acid lime and Pummelo. We show that CMBV genomes from the two hosts share high homology with previously reported CMBV sequences and hence conclude that the new isolates represent variants of the virus present in these species. Based on in silico sequence analysis, we predict the possible function of the protein encoded by one of the five ORFs. The nucleotide sequences reported in this paper have been submitted to the NCBI GenBank nucleotide sequence database and have been assigned the accession numbers EU489744 and EU489745.     

Molecular characterization and evolutionary analysis of <Emphasis Type="Italic">soybean mosaic virus</Emphasis> infecting <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Twenty-nine Pinellia ternata specimens were collected from representative areas in China, including the major production provinces of Zhejiang, Henan, Shanxi, Hunan, Shandong and Hubei. Seven isolates related to soybean mosaic virus (SMV), which could be pathogenic on P. ternata and some soybean [Glycine max (L.) Merr.] cultivars, were detected using double antibody sandwich immunosorbent assay (DAS-ELISA) and RT-PCR amplification performed with degenerate primer of potyviruses. It is revealed that the common potyvirus infecting P. ternata is, indeed, only SMVs rather than Dasheen mosaic virus (DsMV) as previously reported. Further molecular phylogenetic analysis of the coat protein (CP) genes of these SMV isolates from P. ternata and G. max, along with some other potyvirus members, such as DsMV and Watermelon mosaic virus (WMV) reconstructed the evolutionary route on both nucleotide and amino acid levels. Similarity and homology of nucleotide sequences for SMV CP genes demonstrated high host correlation and low partial habitat correlation, while those of amino acid sequences also showed that the host correlation was more notable than the habitat correlation. The amino acid sequence of conserved region within CP determines the main function, which shows high homology between species. This study outspreaded from the viruses themselves and their relationship to the infected hosts and revealed the evolutionary strategies, especially the rapid variation or recombination of SMV of P. ternata, in order to adapt itself naturally to the special host. The GenBank Accession numbers of the sequences reported in this article are DQ360817-DQ360823.     

Complete genomic RNA sequence of the Polish <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> isolate belonging to the US2 strain

We have determined the complete nucleotide and amino acid sequences of the Polish Pepino mosaic virus (PepMV) isolate marked as PepMV-PK. The PepMV-PK genome consists of a single positive-sense RNA strand of 6412-nucleotide-long that contains five open reading frames (ORFs). ORF1 encodes the putative viral polymerase (RdRp), ORFs 2–4 the triple gene block (TGB 1–3), and ORF5-coat protein CP. Two short untranslated regions flank the coding ones and there is a poly (A) tail at the 3′ end of the genomic RNA. Thus, the genome organization of PepMV-PK is that of a typical member of the genus Potexvirus. Phylogenetic analysis based on full-length genomes of PepMV sequences showed that PepMV-PK was most closely related to the Ch2 isolate from Chile. Comparison of PepMV-PK and Ch2 showed the following nucleotide identities: 98% for the RdRp, 99% for the CP genes, and 98, 99, and 98% for the TGB1, TGB2, and TBG3, respectively. This high level of nucleotide sequence identity between the Chilean and Polish PepMV-PK isolates suggest their common origin.     

Molecular evidence and sequence analysis of a natural reassortant between <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> subgroup IA and II strains

Cucumber mosaic virus (CMV) is a tripartite RNA virus and has been divided into three subgroups, named IA, IB, and II. Some studies have found a few natural reassortants between CMV subgroups, although reassortment between CMV subgroups is infrequent. In our present work, a CMV reassortant, named CMV-Tsh, was obtained from a tomato plant. The complete sequence of CMV-Tsh genomic RNAs has been determined and analyzed. The results of sequence comparisons and phylogenetic analyses revealed that CMV-Tsh RNAs 1 and 3 are derived from one or two CMV subgroup II strain(s), while RNA2 is derived from a CMV subgroup IA strain. A PCR and restriction enzyme analysis-based method was developed to analyze the possibility of mixed infection by CMV strains of different subgroup in the CMV-Tsh-infected tomato plant. The results of the restriction enzyme analysis proved that CMV-Tsh is the unique strain in the tomato plant. Taken together, CMV-Tsh is a natural reassortant having CMV subgroup IA RNA2 and subgroup II RNAs 1 and 3. The GenBank Accession numbers of the sequences reported in this paper are EF202595- EF202597.     

Population structure of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> from field Argentinean isolates

We studied the genetic variability of three genomic regions (p23, p25 and p27 genes) from 11 field Citrus tristeza virus isolates from the two main citrus growing areas of Argentina, a country where the most efficient vector of the virus, Toxoptera citricida, is present for decades. The pathogenicity of the isolates was determinated by biological indexing, single-strand conformation polymorphism analysis showed that most isolates contained high intra-isolate variability. Divergent sequence variants were detected in some isolates, suggesting re-infections of the field plants. Phylogenetic analysis of the predominant sequence variants of each isolate revealed similar grouping of isolates for genes p25 and p27. The analysis of p23 showed two groups contained the severe isolates. Our results showed a high intra-isolate sequence variability suggesting that re-infections could contribute to the observed variability and that the host can play an important role in the selection of the sequence variants present in these isolates. N. G. Iglesias and S. P. Gago-Zachert contributed equally to this study.     

Association analysis of <Emphasis Type="Italic">SLC22A4</Emphasis>, <Emphasis Type="Italic">SLC22A5</Emphasis> and <Emphasis Type="Italic">DLG5</Emphasis> in Japanese patients with Crohn disease

Crohn disease (CD) is an inflammatory bowel disease characterized by chronic transmural, segmental, and typically granulomatous inflammation of the gut. Recently, two novel candidate gene loci associated with CD, SLC22A4 and SLC22A5 on chromosome 5 known as IBD5 and DLG5 on chromosome 10, were identified through association analysis of Caucasian CD patients. We validated these candidate genes in Japanese patients with CD and found a weak but possible association with both SLC22A4 (P=0.028) and DLG5 (P=0.023). However, the reported genetic variants that were indicated to be causative in the Caucasian population were completely absent in or were not associated with Japanese CD patients. These findings imply significant differences in genetic background with CD susceptibility among different ethnic groups and further indicate some difficulty of population-based studies.     

Complete genomic sequence analyses of <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> basal-BR isolates from China

Isolates of Turnip mosaic virus (TuMV) are divided into four molecular lineages based on host range and geographical origins. Basal-BR is one of the four lineages and represented a new emergent lineage in East Asia. In one previous paper, we report the occurrence of basal-BR isolates in China. Here, we presented the first two complete genomic sequences of Chinese TuMV basal-BR isolates, WFLB06 and TANX2. The genomes of both isolates were 9833 nucleotides excluding the poly(A) tail, and had identical genomic structure. Most of their genes shared the highest identities with Japanese isolates. Recombination analysis showed that WFLB06 was an interlineage recombinant of basal-BR and Asian-BR parents, while TANX2 was an intralineage recombinant of basal-BR parents, and these two isolates represented two novel recombination patterns of TuMV. The ratio of nonsynonymous and synonymous substitution for the P1 gene of Chinese TuMV population was the highest and amounted to 12 times higher than that for the NIa-Pro gene, which implies that the selection pressure on the P1 gene was the highest among the genes present in the genome. Hong-Yan Wang and Jin-Liang Liu contributed equally.     

Multiple interspecies recombination events within RNA2 of <Emphasis Type="Italic">Grapevine fanleaf virus</Emphasis> and <Emphasis Type="Italic">Arabis mosaic virus</Emphasis>

Sequence alignments and SISCAN analyses inferred multiple interspecies recombination events within RNA2 of strains GHu of Grapevine fanleaf virus (GFLV) and Ta of Arabis mosaic virus (ArMV), two closely related subgroup A nepoviruses in the family Comoviridae. Interspecies recombination events were identified in the 5' untranslated region, the putative homing protein and movement protein genes but not in the coat protein gene and 3' untranslated region. These findings suggest a dynamic relationship between GFLV and ArMV, and a differential selection pressure on RNA2-encoded proteins with constraints in terms of function and co-adaptation that limit interspecies recombination to certain gene segments.     

Detection and Localization of <Emphasis Type="Italic">Rice stripe virus</Emphasis> Gene Products <Emphasis Type="Italic">In Vivo</Emphasis>

The genome of the Tenuivirus, Rice stripe virus (RSV) comprises four RNAs, the smallest three of which each contain two open reading frames (ORFs) arranged in an ambisense manner. The expression of the ORFs from RNAs 2–4 in plants and the insect vector, Laodelphax striatellus, was studied using antisera raised against the gene products. In Western blotting of the proteins from infected plants, the molecular masses of p2, p3, pc3 (nucleocapsid protein, N) and p4 (major non-structural protein, NCP) were as expected; that of pc4 appeared larger than expected. Antisera to the N- and C-terminal parts of the complementary ORF on RNA 2, analogous to that encoding glycoproteins on genomes of bunyaviruses and tospoviruses, revealed banding patterns suggestive of processing of the product; the possible processing is discussed. Four types of inclusion bodies were identified by immunofluorescent and immunogold microscopy of thin sections of infected leaves. Most electron-dense amorphous semi-electron-opaque inclusion bodies (dASO) contained only p4 while some contained at least p2, pc2-N, p3, pc3 as well as p4. A ring-like structure containing at least pc2-N, p4 and pc4 was also identified in infected plant cells. Fibrillar amorphous semi-electron-opaque inclusion bodies (fASO) contained only p4. Filamentous electron-opaque inclusion bodies (FEO), which consist of pc2-N^.and p4, were found both in infected plant cells and in the mid-gut lumen and mid-gut epithelial cells of L. striatellus. This suggests an interaction between p4 and pc2-N and a function of pc2-N distinct from that of its-homologue in Bunyaviridae. Our results confirm the in vivo ambisense coding strategy of Tenuivirus RNA 2 and provide further evidence that RSV does not produce enveloped virions in infected rice plants.     

Functional analysis of <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> carboxylic acids permeases: heterologous expression of <Emphasis Type="Italic">KlJEN1</Emphasis> and <Emphasis Type="Italic">KlJEN2</Emphasis> genes

The present work describes a detailed physiological and molecular characterization of the mechanisms of transport of carboxylic acids in Kluyveromyces lactis. This yeast species presents two homologue genes to JEN1 of Saccharomyces cerevisiae: KlJEN1 encodes a monocarboxylate permease and KlJEN2 encodes a dicarboxylic acid permease. In the strain K. lactis GG1888, expression of these genes does not require an inducer and activity for both transport systems was observed in glucose-grown cells. To confirm their key role for carboxylic acids transport in K. lactis, null mutants were analyzed. Heterologous expression in S. cerevisiae has been performed and chimeric fusions with GFP showed their proper localization in the plasma membrane. S. cerevisiae jen1Δ cells transformed with KlJEN1 recovered the capacity to use lactic acid, as well as to transport labeled lactic acid by a mediated mechanism. When KlJEN2 was heterologously expressed, S. cerevisiae transformants gained the ability to transport labeled succinic and malic acids by a mediated mechanism, exhibiting, however, a poor growth in malic acid containing media. The results confirmed the role of KlJen1p and KlJen2p as mono and dicarboxylic acids permeases, respectively, not subjected to glucose repression, being fully functional in S. cerevisiae. O. Queirós and L. Pereira contributed equally to this work.     

Association of positional and functional candidate genes<Emphasis Type="Italic"> FGF1</Emphasis>,<Emphasis Type="Italic"> FBN2</Emphasis>, and<Emphasis Type="Italic"> LOX</Emphasis> on 5q31 with intracranial aneurysm

We previously performed a genome-wide linkage study of intracranial aneurysm (IA) and found positive evidence of linkage at chromosomes 5q22–31, 7q11, and 14q22. In the present study, we focus on 5q31, where three candidate genes, fibroblast growth factor 1 (FGF1), fibrillin 2 (FBN2), and lysyl oxidase gene (LOX) lie, and evaluate associations with IA. Genomic DNAs were obtained from 172 IA patients and 192 controls. Association analysis was performed with ten, five, and four single-nucleotide polymorphisms (SNPs) identified in FGF1, FBN2, and LOX, respectively. A difference in allelic frequency was observed for only the SNP at intron 4 in FGF1 (χ²=4.44, df=1, P=0.035). Although a haplotype association was observed with the combination of ten SNPs in FGF1 (χ²=16.04, df=1, P=0.00006), significant haplotype associations were not observed when haplotypes were constructed with the three, two, and four SNPs in FGF1 according to the linkage disequilibrium structure. No associations of FBN2 and LOX with IA were detected in the present study.     

<Emphasis Type="Italic">MECP2 </Emphasis>and <Emphasis Type="Italic">CDKL5</Emphasis> gene mutation analysis in Chinese patients with Rett syndrome

Rett syndrome (RTT) is a progressive neurodevelopmental disorder that is caused by mutations in the X-linked methyl-CpG-binding protein2 (MECP2) gene. In this study, the MECP2 sequences in 121 unrelated Chinese patients with classical or atypical RTT were screened for deletions and mutations. In all, we identified 45 different MECP2 mutations in 102 of these RTT patients. The p. T158M mutation (15.7%) was the most common, followed in order of frequency by p. R168X (11.8%), p. R133C (6.9%), p. R270X (6.9%), p. G269fs (6.9%), p. R255X (4.9%), and p. R306C (3.9%). In addition, we identified five novel MECP2 mutations: three missense (p. K305E, p. V122M, p. A358T), one insertion (c.45-46insGGAGGA), and one 22 bp deletion (c.881-902del22). Large deletions represented 10.5% of all identified MECP2 mutations. Conversely, mutations in exon 1 appeared to be rare (0.9%). The remaining cases without MECP2 mutations were screened for the cyclin-dependent kinase-like 5 (CDKL5) gene using denaturing high-performance liquid chromatography (DHPLC). One synonymous mutation (p. I72I) was found in exon 5, suggesting that CDKL5 is a rare cause of RTT. The overall MECP2 mutation detection rate for this patient series was 84.3:87.9% in 107 classical RTT cases and 57.1% in 14 atypical RTT cases. Moreover, there were two patients with homozygous mutations and normal female karyotypes. However, we did not pinpoint a significant relationship between genotype and phenotype in these cases.     

Functional and structural characterisation of <Emphasis Type="Italic">Ag</Emphasis>MNPV <Emphasis Type="Italic">ie1</Emphasis>

We have located and cloned the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genomic DNA fragment containing the immediate early 1 ORF and its flanking regions. Computer assisted analysis of the complete ie1 locus nucleotide sequence information was used to locate regulatory signals in the upstream region and conserved nucleotide and amino acid sequences. Comparative studies led to the identification of several characteristic protein motifs and to the conclusion that AgMNPV-2D is more closely related to Choristoneura fumiferana defective NPV than to other Group I nucleopolyhedrovirus. We have also shown that the AgMNPV IE1 protein was able to transactivate an early Autographa californica MNPV promoter and its own promoter in transient expression assays. In order to investigate the biological functionality of the ie1 promoter, the ie1 upstream activating region (UAR) was molecularly dissected and cloned upstream of the E. coli lacZ ORF. The results obtained, after transfection of UFL-AG-286 insect cells, leading us to find that the −492 and −357 versions contains sequence motifs important for the level of the lacZ reporter gene expression. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. The GenBank accession number of the sequence reported in this paper is AF368905.     

Repeat induced point mutation in two asexual fungi, <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Penicillium </Emphasis><Emphasis Type="Italic">chrysogenum</Emphasis>

Repeat induced point mutation (RIP) is a gene silencing mechanism present in fungal genomes. During RIP, duplicated sequences are efficiently and irreversibly mutated by transitions from C:G to T:A. For the first time, we have identified traces of RIP in transposable elements of Aspergillus niger and Penicillium chrysogenum, two biotechnologically relevant fungi. We found that RIP in P. chrysogenum has affected a large set of sequences, which also contain other mutations. On the other hand, RIP in A. niger is limited to only few sequences, but literally all mutations are RIP-like. Surprisingly, RIP occurred only in transposon sequences that have disrupted open reading frames in A. niger, a phenomenon not yet reported for other fungi. In both fungal species, we identified two sequences with strong sequence similarity to Neurospora crassa RID. RID is a putative DNA methyltransferase and the only known enzyme involved in the RIP process. Our findings suggest that both A. niger and P. chrysogenum either had a sexual past or have a sexual potential. These findings have important implications for future strain development of these fungi.