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].     

HC-Pro,a potyvirus RNA silencing suppressor,cancels cycling of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> plants

The mixed infection of Cucumber mosaic virus (CMV) and a potyvirus has been known to increase CMV titer in Nicotiana benthamiana plants, resulting in synergistic viral symptoms. We found that among three potyviruses—Potato virus Y (PVY), Turnip mosaic virus (TuMV), and Clover yellow vein virus (C1YVV)—synergistic effects on CMV (or a recombinant CMV vector) titers were most efficiently induced by a co-infection with PVY in N. benthamiana plants. In addition, the helper component-proteinase (HC-Pro) gene of PVY expressed by transgenic plants, which is a viral RNA silencing suppressor, was sufficient to cancel the cycling pattern of CMV titer, resulting in increased levels of overall CMV accumulation. Surprisingly, we found that the levels of CMV and the foreign protein expressed from the CMV vector were much higher in the HC-Pro-transgenic plants than the levels detected in the plants mixed-infected with CMV and PVY. The mechanism for canceling the cyclic infection of CMV by the HC-Pro protein alone is discussed in view of the interaction between RNA silencing and HC-Pro, as well as the possible involvement of the 3a protein.     

Sequence characterization of Tomato leaf curl Sinaloa virus and <Emphasis Type="Italic">Tomato severe leaf curl virus</Emphasis>: Phylogeny of New World begomoviruses and detection of recombination

Summary. Diseases caused by begomoviruses (family Geminiviridae, genus Begomovirus) constitute a serious constraint to tomato production in Nicaragua. In this study, the complete nucleotide (nt) sequences of the DNA-A and DNA-B components were determined for the first time for Tomato leaf curl Sinaloa virus (ToLCSinV). In addition, the complete nt sequence was determined for the DNA-A component of two isolates of Tomato severe leaf curl virus (ToSLCV). The genome organization of ToLCSinV and ToSLCV was identical to the bipartite genomes of other begomoviruses described from the Americas. A phylogenetic analysis of DNA-A including 45 begomovirus species showed that the indigenous begomoviruses of the New World can be divided into three major clades and an intermediate group: AbMV clade, SLCV clade, “Brazil clade”, and BGYMV group. Phylogenetic analyses of the DNA-A and DNA-B components and their open reading frames indicated that ToLCSinV and ToSLCV belong to different clades: ToLCSinV to the AbMV clade, and ToSLCV to the SLCV clade. The two Nicaraguan isolates of ToSLCV showed a close relationship with ToSLCV from Guatemala (ToSLCV-[GT96-1]) and Tomato chino La Paz virus (ToChLPV), but differed significantly in the AV1 and AC1 regions, respectively. Computer-based predictions indicated that recombination with another begomovirus had taken place within AV1 of ToSLCV dividing this species into two strains. A high probability was also found that ToChLPV is involved in the evolution of ToSLCV.     

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.     

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.     

<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.     

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.     

Effect of bioactive fractions of <Emphasis Type="Italic">Citrullus vulgaris</Emphasis> Schrad. leaf extract against <Emphasis Type="Italic">Anopheles stephensi</Emphasis> and <Emphasis Type="Italic">Aedes aegypti</Emphasis>

The benzene extract of Citrullus vulgaris was tested against Anopheles stephensi and Aedes aegypti for the larvicidal activity and ovicidal properties. The crude benzene extract was found to be more effective against A. stephensi than A. aegypti. The LC₅₀ values were 18.56 and 42.76 ppm respectively. The LC₅₀ values for silica gel fractions (bioactive fractions I, II, III and IV) were 11.32, 14.12, 14.53 and 16.02 ppm respectively. The mean per cent hatchability of the egg rafts were observed after 48 h post treatment. The crude extract of benzene exerted 100% mortality at 250 ppm against A. stephensi and at 300 ppm against A. aegypti. The silica gel fractions I and II afforded 100% mortality at 100 ppm and III and IV exerted the hatchability rate of 4.9 and 5.3% at the same concentration against A. stephensi.     

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.     

Sub-genomic replicons of <Emphasis Type="Italic">Tick-borne encephalitis virus</Emphasis>

Summary We constructed three sub-genomic replicons of Tick-borne encephalitis virus (TBEV) (Oshima REP, Oshima REP-GFP and Oshima REP-Neo) by deleting genes coding for structural proteins without or with insertion of green fluorescent protein (GFP) or Neo genes, respectively. BHK cells transfected with Oshima REP expressed the viral non-structural antigens in immunofluorescent and western blot analyses. GFP and viral antigens were co-expressed in the transfected cells with Oshima REP-GFP. G418-resistant cells harboring Oshima REP-Neo consistently expressed the antigens without showing any apparent CPE. These replicons constructed in this study will be useful in studies on the replication, assembly and packaging of TBEV, and to develop vaccines and gene-delivering systems.     

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.     

<Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">shawi</Emphasis> purified antigens confer protection against murine cutaneous leishmaniasis

Objective  

Leishmania (Viannia) shawi was characterized only recently, and few studies concerning the immunogenic and protective properties of its antigens have been performed. The present study aimed to evaluate the protective potential of the five antigenic fractions isolated from L. (V.) shawi promastigotes in experimental cutaneous leishmaniasis.     

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.     

A conserved zinc finger motif in the coat protein of <Emphasis Type="Italic">Tomato leaf curl Bangalore virus</Emphasis> is responsible for binding to ssDNA

Summary. Tomato leaf curl Bangalore virus (ToLCBV-[Ban5]) from south India belongs to the genus Begomovirus of the family Geminiviridae. So far, only the DNA A component has been identified in this virus. In this paper we report the overexpression of ToLCBV-[Ban5] coat protein (CP) in E. coli and its characterization. The purified recombinant CP bound preferentially to ssDNA in a sequence non-specific manner. Deletion of 50 amino acids from the N-terminus, including a putative N-terminal helix, did not result in the loss of binding to DNA. A search for motifs responsible for DNA binding indicated a conserved putative zinc finger motif in the CPs (corresponding to residues 65–85 in ToLCBV-[Ban 5] CP) of begomoviruses. The role of this motif in DNA binding was evaluated through site-directed mutagenesis of the conserved cysteines (C68, C72) and histidines (H81, H85) present within this motif to alanine. The results indicate that the zinc finger motif is indeed involved in binding to zinc and DNA.     

Phylogenetic analysis of <Emphasis Type="Italic">Beet necrotic yellow vein virus</Emphasis> isolates from China

A survey detected Beet necrotic yellow vein virus (BNYVV) infection in six Chinese sugar-beet-growing regions. To study the diversity of virus isolates among the regions, nucleotide sequences of four proteins, namely CP, p25, p31, and p26, were determined and the amino acid sequences thus deduced were analyzed using sequence alignments and the phylogenetic method, respectively. Amino acid sequence analysis of CP revealed A-type isolates in Harbin, Hohhot, Baotou, Wuwei, and Jiuquan and B-type isolates in Hohhot and Changji; no Chinese isolate was found to cluster with European P-type isolates. Chinese p25 proteins clustered into three groups with seven tetrad motifs (positions 67–70). Of the seven, the tetrad ASHG has not been reported previously. Most Chinese p31 proteins clustered in p31-2 group, diverging from European p31 proteins. Isolates containing RNA 5 occurred in most of the investigated regions and were associated with both A-and B-types. Phylogenetic analyses of these four proteins showed complex patterns of genetic diversity among these Chinese isolates and indicated that the isolates of China and Japan were more closely related and may have a common origin. The nucleotide sequence data reported in this article have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers EF473089–EF473126.     

Genetic reassortment between high-virulent and low-virulent <Emphasis Type="Italic">Dobrava</Emphasis>-<Emphasis Type="Italic">Belgrade virus</Emphasis> strains

The tri-segmented RNA genome of hantaviruses facilitates genetic reassortment by segment swapping when cells are co-infected with different virus strains. We found efficient in vitro reassortment between members of two different genetic lineages of the Dobrava-Belgrade virus species, the weakly virulent DOBV-Aa and highly virulent DOBV-Af. In all reassortants, S and L segments originated from the same parental strain, and only the M segment was exchanged. To identify functional differences between the parental strains DOBV-Aa and DOBV-Af in cell culture and to compare them with the reassortants, we studied elements of the innate immunity in virus-infected cells. The contrasting phenotypes of the parental viruses were maintained by the reassortants carrying the respective S and L segments of the parental virus and were not influenced by the origin of the M segment.     

A simple and efficient method for agroinfection of <Emphasis Type="Italic">Vernonia cinerea</Emphasis> with infectious clones of <Emphasis Type="Italic">Vernonia yellow vein virus</Emphasis>

Vernonia yellow vein virus (VeYVV) is a distinct monopartite begomovirus associated with a satellite DNA β. After constructing dimers of both DNA A and DNA β in binary vectors, a number of infection methods were attempted. However, only a modified stem-prick method produced up to 83% infection in the natural host Vernonia cinerea, thus, fulfilling the Koch’s postulate. The presence of the viral DNA in the agroinfected plants was confirmed by rolling circle amplification (RCA), followed by Southern hybridization. DNA β induces typical symptoms of Vernonia yellow vein disease (VeYVD) when co-agroinoculated with the begomovirus to Vernonia and also leads to the accumulation of DNA A systemically. VeYVV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom induction.     

Characterisation of an isolate of <Emphasis Type="Italic">Narcissus degeneration virus</Emphasis> from Chinese narcissus (<Emphasis Type="Italic">Narcissus tazetta</Emphasis> var. <Emphasis Type="Italic">chinensis</Emphasis>)

Summary. A potyvirus from Chinese narcissus was transmitted mechanically to three species of Narcissus and to Lycoris radiata but not to 22 other test species. In western blot, the coat protein reacted strongly with Narcissus degeneration virus (UK isolate) antiserum. Antiserum raised to the Chinese virus did not react with eighteen other potyviruses. The complete nucleotide sequence (9816 nt) had the typical genome organisation for a member of the genus Potyvirus. Sequence comparisons and phylogenetic analysis showed that the Chinese virus was different from all previously sequenced potyviruses but distantly related to onion yellow dwarf and shallot yellow stripe viruses.