Immunocapture PCR Research Articles

The emergence of begomovirus-induced yellow leaf curl disease of tomato ( Lycopersicon esculentum mill.) in Ethiopia

A systematic survey was carried out to determine the incidence and distribution of tomato virus diseases in Ethiopia. Fifty seven tomato fields were inspected, 570 plant samples were collected and tested by DAS-ELISA against eight tomato infecting viruses. The presence of a Tomato yellow leaf curl virus (TYLCV) in selected symptomatic samples was confirmed by immunocapture-polymerase chain reaction (IC-PCR). The data showed that TYLCV and Tomato mosaic virus (ToMV) were the two widespread viruses detected in 24.6% and 47.37% of the fields, respectively; whereas Tomato mild mottle virus (TMMV) followed by P otato virus Y (PVY) were detected only in few samples or completely absent in most fields in the other parts of southern and eastern parts of the country. All tomato samples were negative for Tomato spotted wilt virus (TSWV), Tomato ring spot virus (ToRSV), Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV). High incidence (up to 95%) and prevalence of leaf curl disease was observed in fields in the Rift Valley areas (Upper Awash, Merti, Melkasa and fields along the road Mojo to Zwai). The result indicated that tomato yellow leaf curl has emerged as a major tomato virus disease causing serious losses in the fields of Rift Valley areas. Key words/phrases : Begomoviruses, DAS-ELISA, IC-PCR, Tomato.

Anthrax Outbreak Among Cattle and its Detection by Extractable Antigen 1 (EA1) Based Sandwich ELISA and Immuno PCR

Anthrax, a fatal zoonotic disease caused by spore forming bacteria Bacillus anthracis is predominantly witnessed among herbivorous animals that are highly sus- ceptible to the pathogen. Typically B. anthracis spores existing in soil, initiate the infection during grazing and animal to animal spread has been rarely reported. A sus- pected outbreak of anthrax was observed among cattle in Gundlupet taluk neighboring Bandipur national park and tiger reserve during January 2013. Blood samples collected from domestic cattle in Gundlupet region were examined by EA1 sandwich ELISA and immunocapture PCR. Of the 64 samples examined, 12 recovered during the course of outbreak were found positive by the diagnostic methods and further resulted in recovery of B. anthracis isolates that were confirmed by standard methods indicating prevalence of the pathogen among diseased cattle. The outbreak could be contained by the administration of oxytetracycline and live spore vaccine. The isolates recovered from the cattle were susceptible to the major antibiotics penicillin, ampi- cillin, ciprofloxacin, nalidixic acid, tetracycline, erythro- mycin and chloramphenicol whereas resistance could be seen for the antibiotic neomycin (25 %). The ambiguity for initial symptomatic identification due to the absence of bleeding from natural orifices could be overcome by the EA1 immunoassay kits employed in this study.

Immunodiagnosis of episomal Banana streak MY virus using polyclonal antibodies to an expressed putative coat protein

A cryptic Badnavirus species complex, known as banana streak viruses (BSV) poses a serious threat to banana production and genetic improvement worldwide. Due to the presence of integrated BSV sequences in the banana genome, routine detection is largely based on serological and nucleo-serological diagnostic methods which require high titre specific polyclonal antiserum. Viral structural proteins like coat protein (CP) are the best target for in vitro expression, to be used as antigen for antiserum production. However, in badnaviruses precise CP sequences are not known. In this study, two putative CP coding regions (p48 and p37) of Banana streak MY virus (BSMYV) were identified in silico by comparison with caulimoviruses, retroviruses and Rice tungro bacilliform virus. The putative CP coding region (p37) was in vitro expressed in pMAL system and affinity purified. The purified fusion protein was used as antigen for raising polyclonal antiserum in rabbit. The specificity of antiserum was confirmed in Western blots, immunosorbent electron microscopy (ISEM) and antigen coated plate–enzyme linked immunosorbent assay (ACP–ELISA). The antiserum (1:2000) was successfully used in ACP–ELISA for specific detection of BSMYV infection in field and tissue culture raised banana plants. The antiserum was also utilized in immuno-capture PCR (IC-PCR) based indexing of episomal BSMYV infection. This is the first report of in silico identification of putative CP region of BSMYV, production of polyclonal antiserum against recombinant p37 and its successful use in immunodetection.

Development and evaluation of IgY ImmunoCapture PCR ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference

In the present study, a sensitive and specific IgY mediated ImmunoCapture-PCR-ELISA (IC-PCR-ELISA) was developed for the detection of staphylococcal enterotoxin A (SEA) from culture supernatants and suspected contaminated samples. Due to the virtue of avian immunoglobulins (IgY) to have the least affinity towards staphylococcal protein A (SpA) responsible for false positives, we employed anti-SEA IgY for capture of SEA toxin and revealed with SEA specific rabbit antibodies conjugated to a 524bp DNA marker. Biotin-11-dUTP was incorporated during PCR amplification and post PCR analysis was performed by PCR-ELISA. Unlike IgG immunocapture, IgY mediated immunocapture of SEA was free from false positives due to protein A. The developed assay was specific to SEA except for minor cross reactivity with staphylococcal enterotoxin E (SEE). Several raw milk samples were evaluated for the presence of SEA with and without enrichment. Three samples were found to be positive for SEA after enrichment for 8h. Though IC-PCR-ELISA for SEA showed 100% correlation with PCR analysis for sea gene, the assay was unique in terms of sensitivity of detecting ~10pg/ml of SEA toxin from spiked milk samples. Result of IC-PCR-ELISA was further confirmed by conventional methods of isolation and characterization. The presented method can be very useful for rapid analysis of milk samples for SEA contamination and can be further extended for detection of multiple SE's in different wells of same PCR plate using common DNA substrate.

Development of a quantitative immunocapture real-time PCR assay for detecting structurally intact adenoviral particles in water

Development of rapid, sensitive and specific methods for detection of infectious enteric viruses in water is challenging but crucial for gaining reliable information for risk assessment. An immunocapture real-time PCR (IC-qPCR) was designed to detect jointly the two major viral particle components, i.e. the capsid protein and the viral genome. Targeting both constituents helps circumventing the technical limits of cell culture approaches and the inability of PCR based methods to predict the infectious status. Two waterborne pathogenic virus models, human adenovirus types 2 and 41, were chosen for this study. IC-qPCR showed a detection limit of 10MPNCU/reaction with a dynamic range from 102 to 106MPNCU/reaction. Sensitivity was thus 100-fold higher compared to ELISA-based capture employing the same anti-hexon antibodies. After optimisation, application on environmental water samples was validated, and specificity towards the targeted virus types was obtained through the qPCR step. Heat-treated pure samples as well as surface water samples brought evidence that this method achieves detection of encapsidated viral genomes while excluding free viral genome amplification. As a consequence, adenovirus concentrations estimated by IC-qPCR were below those calculated by direct qPCR. The results demonstrate that the IC-qPCR method is a sensitive and rapid tool for detecting, in a single-tube assay, structurally intact and thus potentially infectious viral particles in environmental samples.

Lack of evidence for an interaction between Buchnera GroEL and Banana bunchy top virus (Nanoviridae)

Circulative plant viruses such as luteovirids and geminiviruses have been shown to bind to GroEL proteins produced by endosymbiotic bacteria harboured within hemipteran vectors. These interactions seem to prevent the degradation of the viral particles in the aphid's haemocoel. Similarly to luteovirids and geminiviruses, Banana bunchy top virus (BBTV), a member of the Nanoviridae family, is transmitted in a persistent, circulative manner and can be detected in the haemolymph of the aphid vector, Pentalonia nigronervosa. To date, it is not known if BBTV can interact with GroEL. In this study, we localised and inferred the phylogeny of a Buchnera aphidicola endosymbiont inhabiting P. nigronervosa. Furthermore, we predicted the 3D structure of Buchnera GroEL and detected the protein in the haemolymph of P. nigronervosa. Interactions were tested using 3 different assays: immunocapture PCR, dot blot, and far-western blot assays; however, none of them showed evidence of a BBTV–GroEL interaction. We concluded that it was unlikely that BBTV interacted with Buchnera GroEL either in vitro or in vivo and we discuss possible alternatives by which BBTV viral particles are able to avoid the process of degradation in the aphid haemocoel.

Immuno capture PCR for rapid and sensitive identification of pathogenic Bacillus anthracis

Immuno capture PCR (IPCR) is a technique capable of detecting the pathogens with high specificity and sensitivity. Rapid and accurate detection of Bacillus anthracis was achieved using anti-EA1 antibodies to capture the cells and two primer sets targeting the virulence factors of the pathogen i.e., protective antigen (pag) and capsule (cap) in an IPCR format. Monoclonal antibodies specific to B. anthracis were generated against extractable antigen 1 protein and used as capture antibody onto 96 well polystyrene plates. Following the binding of the pathogen, the DNA extraction was carried out in the well itself and further processed for PCR assay. We compared IPCR described here with conventional duplex PCR using the same primers and sandwich ELISA using the monoclonal antibodies developed in the present study. IPCR was capable of detecting as few as 10 and 100 cfu ml⁻¹ of bacterial cells and spores, respectively. IPCR was found to be 2-3 logs more sensitive than conventional duplex PCR and the sandwich ELISA. The effect of other bacteria and any organic materials on IPCR was also analyzed and found that this method was robust with little change in the sensitivity in the presence of interfering agents. Moreover, we could demonstrate a simple process of microwave treatment for spore disruption which otherwise are resistant to chemical treatments. Also, the IPCR could clearly distinguish the pathogenic and nonpathogenic strains of B. anthracis in the same assay. This can help in saving resources on unnecessary decontamination procedures during false alarms.

Immunocapture Loop Mediated Isothermal Amplification for Rapid Detection of Tomato Yellow Leaf curl Virus (TYLCV) without DNA Extraction

To diminish the time required for some diagnostic assays including polymerase chain reaction (PCR), loopmediated isothermal amplification (LAMP; due to mainly DNA extraction step) and also DAS-ELISA into a minimum level, an innovative immunocapture LAMP (IC–LAMP) and immunocapture PCR (IC-PCR) protocol on the basis of Tomato Yellow Leaf curl Virus (TYLCV) genome were used and optimized. Even though DAS-ELISA, IC-PCR and IC–LAMP assays could successfully detect positive infected plant samples, considering the time, safety, sensitivity, cost, no need of DNA extraction and simplicity the last one was overall superior. The hydroxynaphthol blue could produce long stable colour change and brightness in a close tube-based approach to prevent cross-contamination risk. Altogether, as IC–LAMP is sensitive, cost effective, fairly user friendly and also can generate more accurate results than previous diagnostic procedures, we accordingly propose this assay as a highly reliable alternative viral recognition system regarding TYLCV recognition and probably other viral-based diseases.

Progressive aggregation of Tomato yellow leaf curl virus coat protein in systemically infected tomato plants, susceptible and resistant to the virus

Tomato yellow leaf curl virus (TYLCV) coat protein (CP) accumulated in tomato leaves during infection. The CP was immuno-detected in the phloem associated cells. At the early stages of infection, punctate signals were detected in the cytoplasm, while in the later stages aggregates of increasing size were localized in cytoplasm and nuclei. Sedimentation of protein extracts through sucrose gradients confirmed that progress of infection was accompanied by the formation of CP aggregates of increasing size. Genomic ssDNA was found in the cytoplasm and in the nucleus, while the dsDNA replicative form was exclusively associated with the nucleus. CP–DNA complexes were detected by immuno-capture PCR in nuclear and cytoplasmic large aggregates. Nuclear aggregates contained infectious particles transmissible to test plants by whiteflies. In contrast to susceptible tomatoes, the formation of large CP aggregates in resistant plants was delayed. By experimentally changing the level of resistance/susceptibility of plants, we showed that maintenance of midsized CP aggregates was associated with resistance, while large aggregates where characteristic of susceptibility. We propose that sequestering of virus CP into midsized aggregates and retarding the formation of large insoluble aggregates containing infectious particles is part of the response of resistant plants to TYLCV.

Leptospirosis-persistence of a Dilemma: An Overview with Particular Emphasis on Trends and Recent Advances in Vaccines and Vaccination Strategies

Leptospirosis, caused by pathogenic spirochetes of the genus Leptospira, affects both humans and animals and is among the most common but neglected direct zoonotic disease in the world, particularly in untreated or undiagnosed animals as well as humans. Now, it has been considered as a re-emerging disease causing global health problem due to its increasing incidences in developing as well as developed nations. It is a multisystemic disease leading to death. Diagnostic tests of importance are Latex Agglutination Test (LAT), lateral flow and immunoglobulin M (IgM) based ELISA, PCR based assays, Multiple-microscopic Agglutination Test (MAT), Loop-mediated Isothermal Amplification (LAMP) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Molecular tools like PCR-RFLP, real-time PCR, multiplex PCR, qPCR and immunocapture PCR have all been found useful for rapid and confirmatory detection and differentiation of pathogenic and non-pathogenic leptospires. Inactivated/killed and attenuated vaccines are always attempted, since the beginning of vaccine and vaccination story, against all emerging pathogens with mixed success stories. The advanced tools and techniques like recombinant DNA technology, reverse genetics, DNA vaccination, molecular genetics and proteomics approaches are being explored for search of novel antigens, proteins and genes as potential candidates to discover safer, efficient and better vaccines for leptospirosis. The present review highlights the leptospirosis, susceptible population, disease transmission and epidemiology, treatment, trends and advances in diagnosis, vaccines and vaccination strategies in humans and animals with a view to combat this organism having public health significance.

Implication of Bemisia tabaci heat shock protein 70 in Begomovirus-whitefly interactions.

The whitefly Bemisia tabaci (Gennadius) is a major cosmopolitan pest capable of feeding on hundreds of plant species and transmits several major plant viruses. The most important and widespread viruses vectored by B. tabaci are in the genus Begomovirus, an unusual group of plant viruses owing to their small, single-stranded DNA genome and geminate particle morphology. B. tabaci transmits begomoviruses in a persistent circulative nonpropagative manner. Evidence suggests that the whitefly vector encounters deleterious effects following Tomato yellow leaf curl virus (TYLCV) ingestion and retention. However, little is known about the molecular and cellular basis underlying these coevolved begomovirus-whitefly interactions. To elucidate these interactions, we undertook a study using B. tabaci microarrays to specifically describe the responses of the transcriptomes of whole insects and dissected midguts following TYLCV acquisition and retention. Microarray, real-time PCR, and Western blot analyses indicated that B. tabaci heat shock protein 70 (HSP70) specifically responded to the presence of the monopartite TYLCV and the bipartite Squash leaf curl virus. Immunocapture PCR, protein coimmunoprecipitation, and virus overlay protein binding assays showed in vitro interaction between TYLCV and HSP70. Fluorescence in situ hybridization and immunolocalization showed colocalization of TYLCV and the bipartite Watermelon chlorotic stunt virus virions and HSP70 within midgut epithelial cells. Finally, membrane feeding of whiteflies with anti-HSP70 antibodies and TYLCV virions showed an increase in TYLCV transmission, suggesting an inhibitory role for HSP70 in virus transmission, a role that might be related to protection against begomoviruses while translocating in the whitefly.

Whitefly-mediated transmission of cotton leaf curl Multan betasatellite: evidence for betasatellite encapsidation in coat protein of helper begomoviruses

Cotton leaf curl Multan betasatellite (CLCuMB) is responsible for symptom expression of a devastating disease of cotton in the Indian subcontinent. CLCuMB depends on helper virus replication-associated protein for its replication and on viral coat protein (CP) for its encapsidation. However, no direct evidence of encapsidation of CLCuMB in viral CP has been available. In the present study, non-viruliferous whiteflies were placed on tomato plants that had been agroinoculated with infectious clones of an Iranian isolate of tomato yellow leaf curl virus (TYLCV-[Ab]) and CLCuMB for an acquisition access period of 72 h and then transferred to healthy tomato seedlings at the 3- to 4-leaf stage. Typical symptoms of TYLCV-[Ab] appeared on inoculated seedlings 30-45 days post-inoculation. The presence of TYLCV-[Ab] and CLCuMB DNAs in symptomatic test plants and viruliferous whiteflies was confirmed by PCR analysis using specific primers and DIG Southern blotting. Furthermore, the possibility of CLCuMB DNA encapsidation in TYLCV-[Ab] CP within infected plants was examined by immunocapture PCR. The results showed that CLCuMB DNA was encapsidated in TYLCV-[Ab] CP. Whitefly-mediated transmission of CLCuMB in the presence of helper virus is additional evidence for encapsidation of CLCuMB by TYLCV-[Ab] CP.

Leptospirosis diagnosis by immunocapture polymerase chain reaction: a new tool for early diagnosis and epidemiologic surveillance

The aim of this study was to develop an immunocapture polymerase chain reaction (IC-PCR) protocol for leptospirosis. For the standardization of IC-PCR, polyclonal (AS) and monoclonal (MAb) antibodies against different serogroups and serovars of Leptospira were coupled to polystyrene plates. Human sera were artificially contaminated with leptospires and incubated on plates. The bacterial DNA was obtained and used in a multiplex PCR. Sensitivity was tested using sera contaminated with crescent concentrations of leptospires, while specificity was established using sera contaminated with different bacterial genera and sera obtained from patients positive for viral infections. IC-PCR using AS was able to recognize specific serogroups, although some cross-reactions have been observed. No cross-reactions were observed when MAbs were used; however, the sensitivity in this case was lower than that of IC-PCR using AS. IC-PCR proved to be specific to Leptospira and is a promising tool for early diagnosis of leptospirosis, providing additional information about the infecting serovar or serogroup.

Detection and Differentiation of Potato Virus Y Strains from Potato Using Immunocapture Multiplex RT-PCR

Potato virus Y (PVY) is a serious problem for the seed potato industry in the United States. The maximum allowable infection level of PVY in certified seed potatoes is 2 % and is substantially lower in early generations of seed production. Moreover, recent emergence of genetically recombinant and serologically different strains of Potato virus Y has led to the development of diagnostic procedures to determine strain identity and to detect mixed strain infections more easily, sensitively and accurately. In the studies reported here a protocol for the detection of single or mixed PVY infections in potato incorporates the advantages of enzyme-linked immunoassay (ELISA) and multiplex reverse transcriptase PCR (RT-PCR). The viral particles from plant sap were enriched by ELISA and then lysed by heating to release the viral RNA for the reverse transcriptase. The cDNA product was used as template for the detection of infection by multiplex PCR eliminating the need for RNA extraction and handling. The sensitivity tests conducted indicate that the immunocapture reverse transcriptase PCR was more sensitive in detecting PVY in infected plant sap than multiplex RT-PCR or ELISA alone while retaining the ability to differentiate strains of PVY that can infect potato in the United States. The immunocapture multiplex RT-PCR described will be particularly useful for seed certification and diagnostic laboratories as a confirmatory test in conjunction with ELISA.

Rolling Circle Amplification Is More Sensitive than PCR and Serology-Based Methods in Detection of <i>Banana streak virus</i> in <i>Musa</i> Germplasm

Banana (Musa sp.) is a popular and important crop among many communities in East Africa. Banana production is however threatened by the wide-spread banana streak disease (BSD), caused by Banana streak virus (BSV). The success of BSV management is inherently coupled to the availability of a sensitive indexing method. In this study, the sensitivity of three BSV detection techniques: rolling circle amplification (RCA), immunocapture PCR (with degenerate and Gold finger primers) and standard PCR was compared. A set of 32 BSD-asymptomatic samples were used to compare the techniques. Analysis of variance (ANOVA) for comparison of the four techniques showed that there were significant differences (P Musa tissues for BSV. This study unveils a more reliable BSV detection method, a need that has remained unaddressed for a long while.

High molecular variability of sugarcane bacilliform viruses in Guadeloupe implying the existence of at least three new species

Thirty-five unique partial sugarcane bacilliform virus (SCBV) sequences extending over 529bp were identified in sugarcane samples from Guadeloupe diagnosed by Immunocapture-PCR (IC-PCR) using specific badnavirus primers. Phylogenetic analysis of these sequences along with the two known genome sequences of Sugarcane bacilliform Mor virus (SCBMV) and Sugarcane bacilliform IM virus (SCBIMV) revealed high molecular variability in the SCBV genome. Seven phylogenetic groups, named A to G, were characterized: virus isolates from groups A-B, C and D are proposed to be members of three additional SCBV species.The two (7446 and 7444bp) and one (7317bp) complete sequences of SCBV isolates from groups A and D, respectively, likely represented the genome of two new species. Phylogenetic analysis of the complete genome and RT/RNase H sequences confirmed the polyphyletic structure of SCBV isolates and the absence of a clear separation between SCBV and Banana streak virus (BSV) isolates within badnavirus group 1. These results showed that reconsideration of taxonomy and classification of SCBV and BSV are necessary.

Spiroplasma citri: A Wide Host Range Phytopathogen

Spiroplasmas are helical motile filamentous, wall-less and culturable mollicutes. Thirty six spiroplasma species have been identified. Only S. citri, S. kunkelii and S. phoeniceum have been identified as plant pathogens. Spiroplasma citri, the causal agent of citrus stubborn disease, have a wide host range. S. citri infects most citrus species and cultivars and a wide range of non-rutaceous plant species. Citrus srubborn disease widely distributed in the southwestern united states of America, northern Africa and Mediterranean countries. It is naturally transmitted by phloem-feeding leafhopper vectors. S. citri can be detected by grafting to citrus indicators, culturing on artificial media, serological, DNA probes, dot-immunobinding assay, Immunocapture Polymerase Chain Reaction (I C- PCR), Polymerase Chain Reaction (PCR) and real-time PCR. There is genetic variability among isolates of S. citri. © 2011 Asian Network for Scientific Information.

DEVELOPMENT OF PCR-BASED TECHNIQUES FOR THE DETECTION OF IMMOBILISED POTATO VIRUS Y VIRIONS

Detection of Potato virus Y (PVY), one of the important potato viruses, was undertaken using various PCRbased techniques, i.e immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR), direct binding RT-PCR (DB-RT-PCR) and print capture RTPCR (PC-RT-PCR). These techniques were evaluated for detection of PVY virions immobilized on solid supports. The 5’UTR and P1 region (969 bp) of the PVY genome were amplified using these techniques, which were compared with DAS-ELISA and nucleic acid spot hybridization (NASH) using fluorescein labeled cDNA probes. The detection limit of purified PVY in serially diluted healthy potato leaf sap was 10 ng for DAS-ELISA, 1 ng for NASH and DB-RT-PCR, 1pg for PC-RT-PCR, ICRT- PCR and standard RT-PCR. Of all techniques, PCRT- PCR proved to be the simplest, economical and reliable. It does not require antibodies for the capture of virions nor lengthy RNA extraction processes.

Consistent detection of bovine papillomavirus in lesions, intact skin and peripheral blood mononuclear cells of horses affected by hoof canker

Equine hoof canker is a chronic proliferative pododermatitis of as yet unknown aetiology. Like equine sarcoid disease, canker is a therapy-resistant disorder characterised by hyperkeratosis, acanthosis and a marked tendency to recur. There is an association of sarcoid-inducing bovine papillomaviruses of types 1 and 2 (BPV-1, BPV-2) with hoof canker disease. Using PCR-based techniques, we assessed canker tissue, intact skin and/or peripheral blood mononuclear cells (PBMCs) of 25 canker-affected horses for the presence of sarcoid-associated BPV-1 and -2. Conventional PCR revealed BPV-1/-2 DNA in 24/24 canker, 12/13 skin and 10/11 PBMC DNA isolates. Using inverse PCR, full-length BPV episomes were detected in 1/5 canker specimens. Sequencing of viral early and late genes amplified from canker, intact skin and PBMC DNA of 2 cases revealed an overall identity of 98% to BPV-1. Viral DNA loads amounted to ≤16 copies per cell in canker tissue and intact skin, and to ≤0.35 copies per PBMC, as determined by quantitative PCR. Using RT-PCR, the viral major oncogene E5 was shown to be transcribed in 2/4 canker tissue specimens and 5/7 PBMC isolates. Immunocapture PCR from 7 canker and 6 skin extract supernatants revealed capsomere-associated viral DNA in one canker and one skin sample. Hoof tissue, skin and PBMCs collected from 13 individuals with no signs of canker or BPV-related malignancies scored negative throughout the experiments. These findings suggest that the observed presence of BPV-1/-2 in canker-affected horses is not coincidental but indicative of an active contribution to hoof canker disease. The use of antivirals and/or immune modulators may help improving canker therapy.

Immunofluorescence localisation of Banana bunchy top virus (family Nanoviridae) within the aphid vector, Pentalonia nigronervosa, suggests a virus tropism distinct from aphid-transmitted luteoviruses

We have applied immunocapture PCR and developed an immunofluorescence assay to specifically detect Banana bunchy top virus (BBTV; family Nanoviridae, genus Babuvirus) within its aphid vector, Pentalonia nigronervosa (Hemiptera, Aphididae). BBTV was localised using either monoclonal or polyclonal antibodies into the anterior midgut (stomach) and into specific cells forming the principal salivary glands. These results suggest a distinct path of virus translocation that likely differs from the one described for aphid-transmitted luteovirus, which enter hemocoels through the hindguts and posterior midguts and that penetrate the accessory salivary glands of their competent vectors. To our understanding, this is the first work analysing the localisation of a virus member of the family Nanoviridae within an aphid vector.