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.     

Evidence for recombination among isolates of <Emphasis Type="Italic">Tobacco leaf curl Japan virus</Emphasis> and <Emphasis Type="Italic">Honeysuckle yellow vein mosaic virus</Emphasis>

Summary Complete nucleotide sequences of Tobacco leaf curl Japan virus (TbLCJV) isolates from infected tomato (Lycopersicon esculentum) plants in Nara (-[Jp2], 2764nt; -[Jp3], 2761nt), Kochi (-[Koc], 2760nt) and Yamaguchi (-[Yam], 2758nt) Prefectures, of Japan were determined. These sequences were compared with each other and the sequences of further begomoviruses from Japan. TbLCJV, TbLCJV-[Jp2], TbLCJV-[Jp3], TbLCJV-[Koc], TbLCJV-[Yam], Honeysuckle yellow vein mosaic virus (HYVMV), Eupatorium yellow vein virus (EpYVV), EpYVV-[MNS2], EpYVV-[SOJ3], EpYVV-[Yam] and EpYVV-[Tob] are monophyletic. The intergenic region (IR) of TbLCJV has highest nucleotide sequence identity with that of HYVMV (93%) whereas the rest of the genomic DNA had higher identity with that of TbLCJV-[Jp2] or -[Jp3] (91100%) than with that of HYVMV. In conclusion, TbLCJV has a chimeric genome which may have arisen by recombination between TbLCV-[Jp2] or -[Jp3]-like and HYVMV-like ancestors. Similarly, TbLCJV-[Yam] DNA has a hybrid genome, with a major parent HYVMV and minor parent TbLCJV-[Koc].     

The complete sequence of <Emphasis Type="Italic">Cymbidium mosaic virus</Emphasis> from <Emphasis Type="Italic">Vanilla fragrans</Emphasis> in Hainan,China

The complete nucleotide sequence of Cymbidium mosaic virus (CymMV) isolated from vanilla in Hainan province, China was determined for the first time. It comprised 6,224 nucleotides; sequence analysis suggested that the isolate we obtained was a member of the genus Potexvirus, and its sequence shared 86.67–96.61% identities with previously reported sequences. Phylogenetic analysis suggested that CymMV from vanilla fragrans was clustered into subgroup A and the isolates in this subgroup displayed little regional difference.     

Breakage of resistance to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> by co-infection with <Emphasis Type="Italic">Zucchini yellow mosaic virus</Emphasis>: enhancement of CMV accumulation independent of symptom expression

Summary. Resistance to the cucumovirus Cucumber mosaic virus (CMV) in cucumber cv. Delila was manifested as a very low level of accumulation of viral RNA and capsid protein, and an absence of CMV-induced symptoms. In addition, resistance was observed at the single cell level, with a reduction in accumulation of CMV RNAs, compared to accumulation in cells of the susceptible cucumber cv. Bet Alpha. Resistance to CMV in cv. Delila was broken by co-infection with the potyvirus Zucchini yellow mosaic virus (ZYMV). Resistance breakage in cv. Delila plants was manifested by an increase in the accumulation of (+) and (–) CMV RNA as well as CMV capsid protein, with no increase in the level of accumulation of ZYMV. Resistance breakage in the resistant cultivar by ZYMV also occurred at the single cell level. Thus, synergistic interactions known to occur between a potyvirus and a cucumovirus led to resistance breakage during a double infection. However, resistance breakage was not accompanied by an increase in disease symptoms beyond those induced by ZYMV itself. On co-inoculation with an asymptomatic variant of ZYMV-AG an enhancement of CMV infection occurred without disease manifestation. Consequently, intensification of viral RNA and capsid protein accumulation can occur without a corresponding increase in disease development, suggesting that different host genes regulate viral accumulation and disease development in the CMV-resistant cucumber plants.     

Molecular phylogenetic analysis of <Emphasis Type="Italic">Barley yellow mosaic virus</Emphasis>

Complete nucleotide sequences of three strains (I, III, and IV) of Barley yellow mosaic virus (BaYMV) isolated in Japan were determined. The length of the genome was the same among the three strains; RNA1 was 7,642 nt and RNA2 was 3,585 nt. The molecular phylogenetic analysis showed that strain I was most closely related to the Chinese isolate, and these two strains formed one cluster with European isolates. Strains II, III, and IV, and the Korean isolate formed another cluster. Amino acid sequences of each viral gene product were compared among strains. The sequences of the VPg protein showed less identity among almost strains (less than 92%) than the sequences of other proteins (more than 93%). VPg is thought to be involved in interactions with host factors, especially initiation factor 4E (eIF4E) or eIF(iso)4E, and infection. Therefore, the relationship between amino acid substitutions and infection of host plants is analyzed.     

Characterisation of a satellite RNA of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> that induces chlorosis in <Emphasis Type="Italic">Capsicum annuum</Emphasis>

The presence of Cucumber mosaic virus (CMV) satellite RNA dramatically changes symptoms on some hosts. A satellite RNA present in a strain of CMV (PepY-CMV) that induced chlorosis in pepper (Capsicum annuum) was shown to induce chlorosis in pepper in combination with another strain (Fny-CMV) that by itself induced a green mosaic symptom. The location of sequences within the PepY satellite RNA (PepY-satRNA) of CMV that conferred the ability to induce chlorosis on pepper plants were analyzed by exchanging sequence domains between cDNA clones of PepY-satRNA and an attenuated mosaic satellite RNA (Paf-satRNA), as well as site-directed mutagenesis of various clusters of the 22-nt sequence differences between the two satellite RNAs in the delimited central domain. The symptoms induced by site-directed mutants of PepY-satRNA and Paf-satRNA in the presence of Fny-CMV demonstrated an insertion within PepY-satRNA of 11 nt at positions 86–96 relative to Paf-satRNA determined the chlorosis-inducing phenotype. Within the chlorosis-inducing domain, deletion of nucleotides did not affect the satRNA replication but abolished the ability of PepY-satRNA to elicit chlorosis symptom. Conversely, a mutant satellite RNA derived from Paf-satRNA in which eleven nucleotides were inserted indicated that sequences of 11 nucleotides were found to be sufficient for chlorosis induction in pepper.     

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.     

A naturally occurring recombinant isolate of <Emphasis Type="Italic">Lettuce mosaic virus</Emphasis>

Summary. LMV-Common and LMV-Most are two seed-borne types of Lettuce mosaic virus (LMV), genus Potyvirus. LMV-Most, but not LMV-Common, overcomes the resistance afforded to lettuce by two recessive genes, mo1 ¹ and mo1 ². An RT-PCR-based assay thought to be specific for LMV-Most also amplified LMV-Tn2, previously typified as LMV-Common. The sequence of selected regions along the genome indicated that LMV-Tn2 is a natural recombinant between LMV-Most and LMV-Common isolates, with a putative recombination site located within the P3 coding region. This is the first evidence of a naturally occurring LMV recombinant isolate.Present address: Departamento de Produção Vegetal, UNESP/FCA, 18603-000, Botucatu, SP, Brazil     

Genetic diversity of Hungarian <Emphasis Type="Italic">Maize dwarf mosaic virus</Emphasis> isolates

The genetic diversity of the coat-protein (CP) region and the untranslated C-terminal region (3′UTR) of Maize dwarf mosaic virus (MDMV) was analyzed to evaluate the variability between isolates (inter-isolate sequence diversity). The results of inter-isolate sequence diversity analysis showed that the diversity of the MDMV CP gene is fairly high (p-distance: up to 0.136). During sequence analysis, a 13 amino-acid residue insertion and an 8 amino-acid residue deletion were found within the N-terminal region of the CP gene. The phylogenetic analysis showed that—unlike other potyvirus species in this subgroup—the MDMV isolates could not be distinguished on the basis of their host plants or geographic origins.     

<Emphasis Type="Italic">Pepo aphid</Emphasis>-<Emphasis Type="Italic">borne yellows virus</Emphasis>: a new species in the genus <Emphasis Type="Italic">Polerovirus</Emphasis>

Pepo aphid-borne yellows virus (PABYV) has been proposed as a putative representative of a new species in the genus Polerovirus in the family Luteoviridae. The genomes of two South African (SA) isolates of cucurbit-infecting PABYV were described in this record. Total RNA, extracted from a pattypan (Cucurbita pepo L.) and a baby marrow (C. pepo L.) leaf samples, was subjected to next-generation sequencing (NGS) on the HiSeq Illumina platform. Sanger sequencing was subsequently used to authenticate the integrity of PABYV’s genome generated from de novo assembly of the NGS data. PABYV genome of SA isolates consists of 5813 nucleotides and displays an organisation typical of poleroviruses. Genome sequence comparisons of the SA PABYV isolates to other poleroviruses support the classification of PABYV as a new species in the genus Polerovirus. Recombination analyses showed that PABYV and Cucurbit aphid-borne yellows virus (CABYV) shared the same ancestor for the genome part situated between breaking points. Phylogenetic analyses of the RNA-dependent RNA polymerase and the coat protein genes showed that SA PABYV isolates shared distant relationship with CABYV and Suakwa aphid-borne yellows virus. Based on our results, we propose that PABYV is a distinct species in the genus Polerovirus.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Complete nucleotide sequence of the genomic RNA of <Emphasis Type="Italic">Poplar mosaic virus</Emphasis> (genus <Emphasis Type="Italic">Carlavirus</Emphasis>)

Summary. The complete nucleotide sequence of the Poplar mosaic virus (PopMV) genome was determined. The genomic RNA of PopMV is 8,742 nucleotides in length. Comparative sequence analysis supports earlier research suggesting that this virus is a member of the genus Carlavirus. For example, as is the case for all carlaviruses, there are 6 predicted ORFs in the PopMV genome. The first ORF, ORF1, encodes a predicted helicase/replicase, which corresponds to ORF1 from other carlaviruses, while ORF2, ORF3, ORF4, ORF5 and ORF6 encode the three triple block proteins, the coat protein, and a putative nucleic acid-binding protein respectively.     

The complete nucleotide sequence of <Emphasis Type="Italic">Alternanthera mosaic virus</Emphasis> infecting <Emphasis Type="Italic">Portulaca grandiflora</Emphasis> represents a new strain distinct from phlox isolates

A southeastern European isolate of Alternanthera mosaic virus (AltMV-MU) of the genus Potexvirus (family Flexiviridae) was purified from the ornamental plant Portulaca grandiflora. The complete nucleotide sequence (6606 nucleotides) of AltMV-MU genomic RNA was defined. The AltMV-MU genome is different from those of all isolates described earlier and is most closely related to genomes of partly sequenced portulaca isolates AltMV-Po (America) and AltMV-It (Italy). Phylogenetic analysis supports the view that AltMV-MU belongs to a new “portulaca” genotype distinguishable from the “phlox” genotype.     

Variability and transmission by <Emphasis Type="Italic">Aphis glycines</Emphasis> of North American and Asian <Emphasis Type="Italic">Soybean mosaic virus</Emphasis> isolates

Summary. The variability of North American and Asian strains and isolates of Soybean mosaic virus was investigated. First, polymerase chain reaction (PCR) products representing the coat protein (CP)-coding regions of 38 SMVs were analyzed for restriction fragment length polymorphisms (RFLP). Second, the nucleotide and predicted amino acid sequence variability of the P1-coding region of 18 SMVs and the helper component/protease (HC/Pro) and CP-coding regions of 25 SMVs were assessed. The CP nucleotide and predicted amino acid sequences were the most similar and predicted phylogenetic relationships similar to those obtained from RFLP analysis. Neither RFLP nor sequence analyses of the CP-coding regions grouped the SMVs by geographical origin. The P1 and HC/Pro sequences were more variable and separated the North American and Asian SMV isolates into two groups similar to previously reported differences in pathogenic diversity of the two sets of SMV isolates. The P1 region was the most informative of the three regions analyzed. To assess the biological relevance of the sequence differences in the HC/Pro and CP coding regions, the transmissibility of 14 SMV isolates by Aphis glycines was tested. All field isolates of SMV were transmitted efficiently by A. glycines, but the laboratory isolates analyzed were transmitted poorly. The amino acid sequences from most, but not all, of the poorly transmitted isolates contained mutations in the aphid transmission-associated DAG and/or KLSC amino acid sequence motifs of CP and HC/Pro, respectively.Received March 20, 2002; accepted May 14, 2003     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

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.     

Transmission of <Emphasis Type="Italic">Yersinia pestis</Emphasis> cultures with different plasmid content from <Emphasis Type="Italic">Xenopsylla cheopis</Emphasis> to <Emphasis Type="Italic">Calomys callosus</Emphasis>

Most Brazilian Yersinia pestis isolates display a typical plasmid profile composed of the three classical plasmids: pYV, pPst and pFra. However, some cultures lack at least one of these plasmids, while a few of them harbour atypical DNA bands of molecular weight ranging from 147 to 11.5 kb. To investigate whether Y. pestis displaying atypical plasmid content could be propagated among rodents in nature through flea bites, we carried out studies with fleas ( Xenopsylla cheopis) and rodents ( Calomys callosus) reared in the laboratory and five Y. pestis cultures differing in plasmid content. The results suggest that: (1) the single presence of pYV is not sufficient for the transmission of Y. pestis by fleas, (2) pPst is not essential for transmission, (3) two atypical DNA bands of molecular weight of 30 kb and >90 kb have no biological role, and (4) pFra is required for the transmission of Y. pestis by flea bites. Other studies are needed to determine whether this plasmid alone is sufficient for transmission.