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Plant viruses of the families Luteoviridae and Geminiviridae rely on hemipteran vectors for the infection of their hosts. Several lines of evidence have revealed that these viruses are transmitted by competent vectors in a circulative manner, involving entry into the vector's body and the crossing of epithelial tissues forming the alimentary tract and the salivary glands. Similar to luteovirids and geminiviruses, a third family of plant viruses, the family Nanoviridae, have also been reported to be transmitted by aphids in a circulative manner. However, there is limited direct evidence of a possible path of translocation through the aphid vectors. Here, we used time-course experiments and transmission assays coupled with real-time PCR and immunofluorescence assays on dissected tissues to examine the translocation, compartmentalization and retention of banana bunchy top virus (BBTV) into the aphid vector Pentalonia nigronervosa. Our results indicate that BBTV translocates rapidly through the aphid vector; it is internalized into the anterior midgut in which it accumulates and is retained at concentrations higher than either the haemolymph or the principal salivary glands. Despite the large increase in viral concentration, we have failed to detect BBTV transcripts with RT-PCR. When tissues were not permeabilized, BBTV localized as distinct puncta in the proximity of the basal surface of the cells forming the anterior midgut and principal salivary glands, suggesting an on-going process of virion escape and internalization, respectively. Interestingly, we document that those organs can have direct contact within the aphid body, suggesting a possible haemolymph-independent translocation path.

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Banana bunchy top virus (BBTV) is the most destructive pathogenic virus of banana plants worldwide. The virus is transmitted in a circulative non-propagative manner by the banana aphid, Pentalonia nigronervosa Coquerel. In this work, we examined the localization, accumulation, and transmission efficiency of BBTV in four laboratory-established lineages of Pentalonia aphids derived from four different host plants: taro (Colocasia esculenta), heliconia (Heliconia spp.), red ginger (Alpinia purpurata), and banana (Musa sp.). Mitochondrial sequencing identified three and one lineages as Pentalonia caladii van der Goot, a recently proposed species, and P. nigronervosa, respectively. Microsatellite analysis separated the aphid lineages into four distinct genotypes. The transmission of BBTV was tested using leaf disk and whole-plant assays, both of which showed that all four lineages are competent vectors of BBTV, although the P. caladii from heliconia transmitted BBTV to the leaf disks at a significantly lower rate than did P. nigronervosa. The concentration of BBTV in dissected guts, haemolymph, and salivary glands was quantified by real-time PCR. The BBTV titer reached similar concentrations in the guts, haemolymph, and salivary glands of aphids from all four lineages tested. Furthermore, immunofluorescence assays showed that BBTV antigens localized to the anterior midguts and the principal salivary glands, demonstrating a similar pattern of translocations across the four lineages. The results reported in this study showed for the first time that P. caladii is a competent vector of BBTV.

BackgroundThe non-translated regions at the genome ends of RNA viruses serve diverse functions and can exhibit various levels of nucleotide (nt) heterogeneity. However, the extent of nt heterogeneity at the extreme termini of Citrus tristeza virus (CTV) genomes has not been comprehensively documented. This study aimed to characterize two widely prevalent CTV genotypes, T36-CA and T30-CA, from California that have not been sequenced or analyzed substantially. The information obtained will be used in our ongoing effort to construct the infectious complementary (c) DNA clones of these viruses.MethodsThe terminal nts of the viral genomes were identified by sequencing cDNA clones of the plus- and/or minus-strand of the viral double-stranded (ds) RNAs generated using 5′ and 3′ rapid amplification of cDNA ends. Cloned cDNAs corresponding to the complete genome sequences of both viruses were generated using reverse transcription-polymerase chain reactions, sequenced, and subjected to phylogenetic analysis.ResultsAmong the predominant terminal nts identified, some were identical to the consensus sequences in GenBank, while others were different or unique. Remarkably, one of the predominant 5′ nt variants of T36-CA contained the consensus nts “AATTTCAAA” in which a highly conserved cytidylate, seen in all other full-length T36 sequences, was absent. As expected, but never systematically verified before, unique variants with additional nt (s) incorporated upstream of the 5′ terminal consensus nts of T36-CA and T30-CA were also identified. In contrast to the extreme 5′ terminal nts, those at the extreme 3′ termini of T36-CA and T30-CA were more conserved compared to the reference sequences, although nt variants were also found. Notably, an additional thymidylate at the extreme 3′ end was identified in many T36-CA sequences. Finally, based on pairwise comparisons and phylogenetic analysis with multiple reference sequences, the complete sequences of both viruses were found to be highly conserved with those of the respective genotypes.ConclusionsThe extreme terminal nts in the T36-CA and T30-CA genomes were identified, revealing new insights on the heterogeneity of these CTV genomic regions. T36-CA and T30-CA were the first and the second genotypes, respectively, of CTV originating from California to be completely sequenced and analyzed.Electronic supplementary materialThe online version of this article (10.1186/s12985-018-1041-4) contains supplementary material, which is available to authorized users.

Banana bunchy top virus (BBTV) (Nanoviridae: Babuvirus) is transmitted by aphids of the genus Pentalonia in a circulative manner. The cellular mechanisms by which BBTV translocates from the anterior midgut to the salivary gland epithelial tissues are not understood. Here, we used multiple fluorescent markers to study the distribution and the cellular localization of early and late endosomes, macropinosomes, lysosomes, microtubules, actin filaments, and lipid raft subdomains in the gut and principal salivary glands of Pentalonia nigronervosa. We applied colabeling assays, to colocalize BBTV viral particles with these cellular compartments and structures. Our results suggest that multiple potential cellular processes, including clathrin- and caveolae-mediated endocytosis and lipid rafts, may not be involved in BBTV internalization.

In August 2011, tomato (Solanum lycopersicum L.) fruit from a University of Hawaii field trial displayed mottling symptoms similar to that caused by Tomato spotted wilt virus (TSWV) or other tospoviruses. The foliage from affected plants, however, appeared symptomless. Fruit and leaf tissue from affected plants were negative for TSWV analyzed by double antibody sandwich (DAS)-ELISA and/or TSWV ImmunoStrips (Agdia, Elkhart, IN) when performed following the manufacturer's instructions. Total RNA from a symptomatic and an asymptomatic plant was isolated using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and reverse transcribed using Invitrogen SuperScript III reverse transcriptase (Life Technologies, Grand Island, NY) and primer 900 (5′- CACTCCCTATTATCCAGG(T)16-3′) following the enzyme manufacturer's instructions. The cDNA was then used as template in a universal potyvirus PCR assay using primers 900 and Sprimer, which amplify sequences encoding the partial inclusion body protein (NIb), coat protein, and 3′ untranslated region of potyviruses (1). A ~1,700-bp product was amplified from the cDNA of the symptomatic plant but not the asymptomatic plant. This product was cloned using pGEM-T Easy (Promega, Madison, WI) and three clones were sequenced at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. The 1,747-bp consensus sequence of the three clones was deposited in GenBank (Accession No. JQ429788) and, following primer sequence trimming, found to be 97% identical to positions 7,934 through 9,640 of Pepper mottle virus (PepMoV; family Potyviridae, genus Potyvirus) accessions from Korea (isolate ‘217’ from tomato; EU586126) and California (isolate ‘C’ from pepper; M96425). To determine the incidence of PepMoV in the field trial, all 292 plants representing 14 tomato cultivars were assayed for the virus 17 weeks after planting using a PepMoV-specific DAS-ELISA (Agdia) following the manufacturer's directions. Plants were considered positive if their mean absorbance at 405 nm was greater than the mean absorbance + 3 standard deviations + 10% of the negative control samples. The virus incidence ranged from 4.8 to 47.6% for the different varieties, with an overall incidence of 19.9%. Although plant growth was not noticeably impaired by PepMoV infection, the majority of fruit from infected plants was unsaleable, making PepMoV a considerable threat to tomato production in Hawaii. PepMoV has been reported to naturally infect tomato in Guatemala (3) and South Korea (2). To our knowledge, this is the first report of this virus in Hawaii and the first report of this virus naturally infecting tomato in the United States. References: (1) J. Chen et al. Arch. Virol. 146:757, 2001. (2) M.-K. Kim et al. Plant Pathol. J. 24:152, 2008. (3) J. Th. J. Verhoeven et al. Plant Dis. 86:186, 2002.

Coconut rhinoceros beetle, Oryctes rhinoceros (L., 1758), is a large scarab beetle native to Southeast Asia and a major pest of coconut (Cocos nucifera) and oil (Elaeis guineensis) palms in its invaded range. Few tools are available for coconut rhinoceros beetle management, particularly for an emerging haplotype with resistance to known strains of Oryctes rhinoceros nudivirus, the traditional biological control agent used in coconut rhinoceros beetle management programs. RNA interference (RNAi) represents an emerging tool for insect pest control that exploits an existing pathway for gene regulation in the target organism. In this study, we evaluated RNAi as a potential tool for coconut rhinoceros beetle management. Using transcriptome data generated from gut tissue of early instar larvae, we identified 24 RNAi target sequences that were either highly expressed or had demonstrated efficacy in other insect systems. Double-stranded (ds)RNAs ranging from 249 to 297 bp in length were generated for 23 of these target sequences and 150 ng were microinjected into coconut rhinoceros beetle 1st, 2nd, and 3rd instar larvae and adults. Five of these dsRNAs that targeted genes putatively encoding V-type ATPase, polyadenylate binding protein, and three forms of actin induced 30.8–100% mortality within 14 days post injection (dpi). Microinjection of 2nd instars with 10 and 100 ng of these same five dsRNAs induced 20–100% and 80–100% mortality at 7 and 14 dpi, respectively. These results indicate RNAi should be explored as a possible management option for coconut rhinoceros beetle. Coconut rhinoceros beetle may also represent a model species for using RNAi in the management of large invasive insect species.