L. I. Ward

Occurrence of two different types of RNA-5-containing beet necrotic yellow vein virus in the UK

Summary.Two types of RNA-5-containing beet necrotic yellow vein virus (BNYVV) have been detected in the UK at different sites in Norfolk. On the basis of nucleotide (nt) sequence comparisons, one virus source (UK-MH) was clearly identified as P type BNYVV, a virus type that had previously only been detected in two widely separated parts of the world, France and Kazakhstan. The other virus source (UK-FF) has a complex genome composition. The analysed portions of its RNAs 2 and 4 are closely related to the corresponding portions in the RNAs of the East Asian A type isolate S, whereas those of its RNAs 1 and 3 resemble P type RNA 1 from Kazakhstan and European A type RNA 3, respectively. Interestingly, the P25 encoded on its RNA 3 has an unique TYHG tetrad in the highly variable amino acid positions 67–70. RNA 5 of the UK-FF BNYVV source shares properties with P type RNA 5, but also with East Asian types of RNA 5. The possible origin and epidemiology of BNYVV types is discussed.

First detection of Kashmir bee virus in the UK using real-time PCR

Kashmir bee virus (KBV) often persists in bees as a covert infection with no apparent symptoms. The virus can switch to become an overt lethal infection, especially in the presence of Varroa mites. Although the virus is distributed worldwide, it was thought to be absent from the UK. A real-time PCR assay was developed for specific detection of KBV. No cross-reaction was observed with other bee viruses. KBV was successfully amplified from different life stages of honey bees and from a wasp and bumble bee. Using the real-time PCR assay, a survey of hives was conducted in England and Wales to investigate the presence and geographical distribution of the virus. KBV was detected within three colonies at two locations. The virus titre in the positive samples was quantified and found to contain similar levels to other bees with covert KBV infection. We conclude that KBV is present in the UK and cannot now be considered an exotic disease. The discovery of KBV in the UK has major significance for import policies.ZusammenfassungWie viele andere Bienenviren ist auch das Kaschmir-Bienenvirus (KBV) in vielen Bienen als latente Infektion ohne offensichtliche Symptome präsent. In der Anwesenheit von Varroamilben kann der Virusbefall sich jedoch zu einer lethalen Infektion entwickeln. Das KBV ist zwar weltweit verbreitet, für Grossbritannien wurde jedoch bislang kein Vorkommen gemeldet.Wir entwickelten ein Protokoll zur spezifischen Detektion von KBV mittels Real-Time-quantitativer PCR. Dieses zeigte keine Kreuzreaktionen mit RNA anderer Bienenviren, wie ABPV, BQCV, SBV, CWV, SPV, DWV, CPV und AIV, und ermöglichte die Ampflifikation von PCR-Produkten aus virenbefallenen Bienenproben der verschiedenen Stadien des Lebenszyklus, ebenso wie von KBV-infizierten Bienen aus verschiedenen geographischen Regionen. PCR-Fragmente konnten auch aus RNA-Proben einer Wespe (Vespula germanica) aus Australien und aus einer aus Hummeln aufgereinigten KBV16 Virusprobe amplifiziert werden. Alle PCR-Produkte wurden kloniert und mittels Sequenzierung als KBV-RNA identifiziert.Mittels dieses Protokolls einer Real-Time-quantitativer PCR analysierten wir Proben aus 458 Völkern aus ganz England und Wales, um die eventuelle Anwesenheit und die geographische Verbreitung des KBV-Virus zu erfassen. Diese Übersichtsstudie zeigte die Präsenz des Virus in drei Völkern von zwei Ständen an. Mittels Real-Time-quantitativer PCR konnten wir zeigen, dass der Virentiter in diesen positiven Proben ähnliche KBV-Werte aufwies wie in Bienenproben aus Australien, die einen klaren Befall gezeigt hatten. Wir schliessen daraus, dass das KBV in Grossbritannien vorkommt, und dass es jetzt nicht länger als eine exotische Krankheit betrachtet werden kann. Die Klärung der Auswirkung des KBV-Befalls auf Völker in Grossbritannien erfordert weitere Untersuchungen, aber es scheint nicht die grosse Bedrohung zu sein, für die es bislang gehalten wurde, und es kann durchaus auch sein, dass das KBV keine grössere Bedrohung darstellt als andere, bereits in Grossbritannien vorkommende Viren. Die Entdeckung des KBV in Grossbritannien ist jedoch von Bedeutung in Hinsicht auf Importregelungen und belegt die Notwendigkeit von genauen Übersichtsdaten über das Vorkommen von pathogenen Agentien für die Erstellung formaler Importrisikoanalysen und für Entscheidungsprozesse innerhalb der Vereinbarungen des Internationalen Büros für Epizootien (OIE) und der WTO.

Two generic PCR primer sets for the detection of members of the genus Torradovirus.

Two degenerate primer pairs were designed for the universal detection of members of the genus Torradovirus. Primer pair Torrado-1F/Torrado-1R was designed based on the RNA-dependent RNA polymerase region located in RNA1 and primer pair Torrado-2F/Torrado-2R based on a region overlapping the two first coat proteins Vp35 and Vp26 in RNA2. The primers were used in two-step and one-step RT-PCR protocols. Both primer pairs were able to detect 14 out of 15 isolates belonging to the two torradovirus species Tomato torrado virus (ToTV) and Tomato marchitez virus (ToMarV) and the two tentative species Tomato chocolate spot virus (ToChSV) and Tomato chocolàte virus (ToChV). Due to poor sample quality, one isolate of ToTV was detected with primer pair Torrado-2F/Torrado-2R and not with primer pair Torrado-1F/Torrado-1R, suggesting that the latter primer pair was less sensitive. Nevertheless, both primer pairs proved to be suitable for the universal RT-PCR detection of torradoviruses and can be deployed for the detection of all currently known torradoviruses and possibly for the detection of new members of this group.

Detection of Tomato black ring virus by real-time one-step RT-PCR.

A TaqMan-based real-time one-step RT-PCR assay was developed for the rapid detection of Tomato black ring virus (TBRV), a significant plant pathogen which infects a wide range of economically important crops. Primers and a probe were designed against existing genomic sequences to amplify a 72 bp fragment from RNA-2. The assay amplified all isolates of TBRV tested, but no amplification was observed from the RNA of other nepovirus species or healthy host plants. The detection limit of the assay was estimated to be around nine copies of the TBRV target region in total RNA. A comparison with conventional RT-PCR and ELISA, indicated that ELISA, the current standard test method, lacked specificity and reacted to all nepovirus species tested, while conventional RT-PCR was approximately ten-fold less sensitive than the real-time RT-PCR assay. Finally, the real-time RT-PCR assay was tested using five different RT-PCR reagent kits and was found to be robust and reliable, with no significant differences in sensitivity being found. The development of this rapid assay should aid in quarantine and post-border surveys for regulatory agencies.

A DNA method for screening hive debris for the presence of small hive beetle (Aethina tumida)

The small hive beetle (SHB) is a parasite and scavenger of honey bee colonies. It has recently become an invasive species creating the need for an efficient and reliable detection method. We present a method to screen hive debris for the presence of SHB using real-time PCR in conjunction with an automated DNA extraction protocol. The method was able to detect DNA from SHB eggs, larvae and adult specimens collected from Africa, Australia and North America. The method was used to successfully detect SHB DNA extracted from spiked and naturally infested debris. An Apis mellifera 18S rRNA real-time PCR assay was used as an internal positive control (IPC). The IPC showed that the method was reliable for detection as extraction efficiency was consistent between hive debris samples. If the SHB were to establish at new locations, the availability of such a method would be a valuable support tool to enable species identification and rapid screening of hive debris for delimiting surveys.ZusammenfassungZiel der gegenwärtigen Studie war die Entwicklung und Validierung eines schnellen Extraktionsprotokolls, das in Verbindung mit einem quantitativen PCR-Ansatz die artspezifische Erkennung des Kleinen Beutenkäfers (KBK) und seine Detektion im Stockmüll erlauben sollte. Wir gingen davon aus, dass angesichts der raschen Verbreitung des KBK ein schnelles und zuverlässiges Detektionsverfahren ein wertvolles Werkzeug in der KBK-Kontrolle darstellen könnte.Das quantitative PCR-Verfahren wurde auf die Detektion des Cytochrom c-Oxidase I Gens von A. tumida angelegt. Zur Validierung wurden KBK-Eier, Larven und Imagines aus Afrika, Australien und Nordamerika eingesetzt. Das Verfahren wurde auch auf Kreuzreaktionen gegen die Milben Varroa destructor and Tropilaelaps clareae, sowie gegen eine Reihe an gängig in Stockmüll vorkommenden Insektenarten getestet. Es erwies sich als spezifisch für KBK und erlaubte die Detektion aller Stadien des Lebenszyklus und auch von KBK-Proben verschiedener geographischer Herkunft.Anschliessend wurde das Verfahren auf die Erkennung von KBK in Stockmüllproben optimiert. Dazu wurde Stockmüll mit unterschiedlichen Mengen an Käfern und Larven versetzt und in Lysepuffer zermahlen. DNA wurde aus diesen Proben mittels eines automatisierten Verfahrens extrahiert, bevor die KBK-DNA-Menge dieser Proben im zuvor etablierten quantitativen PCR-Protokoll bestimmt wurde. Weitere Methodentests wurden mit natürlichen Stockmüllproben durchgeführt und mit Proben, denen KBK zugesetzt worden war. Die Ergebnisse zeigten, dass es möglich war, eine KBK-Menge von 17 mg in einer Gesamtmenge von 30 g Stockmüll zu detektieren. Die Zuverlässigkeit der Extraktionsmethode wurde ebenfalls mittels quantitativer PCR getestet, und zwar für das 18S rRNA Gen der Honigbiene, das ebenfalls aus Stockmüllproben amplifiziert werden konnte. Die CT-Werte jedes Experiments lagen im Schwankungsbereich von je einem Zyklus, was darauf hinweist, dass die Effizienz des Extraktionsverfahrens für alle Proben ähnlich war. Die Amplifizierung des 18S rRNA-Gens stellt somit eine geeignete interne positive Kontrolle für die Stockmüllextraktion dar. Eine Verringerung der Sensitivität der PCR-Methode wurde in DNA-Proben registiert, denen Stockmüll zugesetzt worden war, was auf die Anwesenheit von PCR-Inhibitoren in den DNA-Extrakten hinweist.Wir stellen in dieser Arbeit ein Hochdurchsatzverfahren zur Detektion von KBK vor, das besonders dann von Nutzen sein kann, wenn ein KBK-Ausbruch an neuen Standorten zu verzeichnen ist. Das Verfahren kann noch weiter verfeinert werden, so dass noch grössere Probenvolumina gleichzeitig getestet werden können. Dies setzt allerdings eine weiter Validierung voraus. Die gegenwärtige Methode ist jedoch direkt einsetzbar und transferierbar für Forschungs- und Inspektions- und Überwachungsprogramme und stellt ein Mittel dar, KBK-verdächtige Stockproben zu testen.

Loop-Mediated Isothermal Amplification for the Detection of Plant Pathogens

Loop-mediated isothermal amplification (LAMP) is a technique involving the use of four to six primers (two inner primers, two outer primers, and two loop primers) and the strand displacement activity of Bacillus subtilis-derived (Bst) DNA polymerase. The end result of strand displacement and loop formation and synthesis is the single-temperature amplification of a highly specific fragment from a DNA template at a much greater titre than that obtained with polymerase chain reaction. With LAMP, there are several methods to determine a positive reaction. Presented here are three alternative methods: gel electrophoresis, hydroxynaphthol blue colorimetric dye, and the fluorescent intercalating PicoGreen(®) reagent.

First report of Narcissus degeneration virus, Narcissus late season yellows virus, and Narcissus symptomless virus on Narcissus in New Zealand.

In September 2008, Narcissus plants originating from commercial nurseries in Taranaki (TK) in New Zealands North Island and Canterbury (CB) in the South Island were received showing leaf mottling, flower distortion, and color break. The CB plant also showed stunting. Filamentous virus particles (700 to 900 nm long) were seen in crude sap of both plants with a transmission electron microscope. Total RNA was isolated from the leaves of both plants with an RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA), and cDNA was synthesized by Superscript III (Invitrogen, Carlsbad, CA). cDNA was used in PCR to test for viruses in the following genera: Allexivirus, Carlavirus, Cucumovirus, Nepovirus A and B, Potyvirus, Potexvirus, Tospovirus, and Tobravirus. Both plants tested positive for potyvirus using generic potyvirus primers (3). Amplicons from both plants were directly sequenced. The forward and reverse sequence from the CB plant matched sequences in the GenBank database for Narcissus late season yellows virus (NLSYV) and Narcissus degeneration virus (NDV), respectively. The potyvirus amplicon from the CB plant was cloned and sequenced. Sequence from independent clones was obtained for NLYSV only (No. FJ546721), and this sequence showed 97% nucleotide identity to NLYSV No. EU887015. The CB plant was tested with a second set of generic potyvirus primers using forward (PV1SP6) (2) and reverse primers (U335) (1). BLASTN analysis of the sequence obtained from independent clones (No. FJ543718) matched sequence for NDV only (97% nucleotide identity to No. AM182028). BLASTN analysis of the potyvirus obtained for the TK plant (No. FJ546720) showed 97% nucleotide identity to NLSYV (No. EU887015). The TK plant also tested positive for a carlavirus using commercial primers (Agdia, Elkhart, IN) and unpublished generic carlavirus primers (A. Blowers, personal communication). Amplicons from both PCRs were cloned and sequenced. BLASTN analysis of both sequences (Nos. FJ546719 and GQ205442) showed 94% nucleotide identity to Narcissus symptomless virus (NSV) No. AM182569. Both plants were also tested for NLSYV, Narcissus virus Q, Narcissus latent virus, and Narcissus yellow stripe virus by indirect ELISA (Neogen, Lansing, MI). Results confirmed the presence of NLSYV in both plants but the plants were negative for the other viruses. NLSYV has been detected previously from Narcissus pseudonarcissus L. (daffodil) (D. Hunter, personal communication); however, to our knowledge, this is the first official report of NDV, NLSYV, and NSV in New Zealand. Since both plants tested negative for several other viruses by PCR and ELISA, this would suggest that the symptoms observed may have been caused by NSV, NLSYV, NDV, or as a result of a mixed infection. However, symptoms were not confirmed using Kochs postulate. NSV has been reported in the literature as symptomless. NLYSV has been reported to be a possible cause of leaf chlorosis and striping and NDV has been associated with chlorotic leaf striping in N. tazetta plants (4). Since Narcissus is an important flower crop for domestic production in New Zealand, the reduction in flower quality observed when these viruses are present may be of economic significance in commercial nurseries. References: (1) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991. (2) A. M. Mackenzie et al. Arch Virol. 143:903, 1998. (3) V. Marie-Jeanne et al. J. Phytopathol. 148:141, 2000. (4) W. P. Mowat et al. Ann. Appl. Biol. 113:531, 1988.

A review of the plant virus, viroid, liberibacter and phytoplasma records for New Zealand

A complete review of the records of plant virus, viroid, liberibacter and phytoplasma in New Zealand has found evidence for 220 viruses, seven viroids, two liberibacters and two phytoplasmas. Of these, 80 viruses, one viroid and two species of liberibacter have been reported as new to New Zealand since the last review in 2006. Ten viruses and two viroids, which were previously placed in the unconfirmed category, have now been confirmed. Based on insufficient evidence, 25 virus, three viroid, three mollicute and 36 disease records are considered unconfirmed.

A survey of viral diseases of Allium crops in New Zealand

Targeted surveillance was done in 2004/2005 and 2007 to determine the presence of viruses in ornamental and vegetable Allium crops in New Zealand. Several samples were tested for regulated viruses and viruses that commonly infect New Zealand’s Allium crops using enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. Most viruses were found to occur in mixed infections in garlic, onion, shallot, and in the ornamentals collected from the North and South Islands. New host associations were observed for some viruses previously recorded in New Zealand. Three viruses, Garlic virus A, Iris yellow spot virus and Shallot virus (syn. Shallot mite-borne latent virus), were detected for the first time in New Zealand. However, Impatiens necrotic spot virus, Shallot yellow stripe virus, Sint-Jan’s onion latent virus, Tobacco rattle virus and Tomato black ring virus were not detected in the Allium plants tested. Australasian Plant Pathology Society 2009

The complete nucleotide sequence and genome organisation of a novel member of the family Betaflexiviridae from Actinidia chinensis

We report the complete genome sequence of a novel virus, tentatively named “actinidia seed-borne latent virus” (ASbLV), isolated from Actinidia chinensis in Auckland, New Zealand. The complete genome of ASbLV is 8,192 nucleotides long, excluding the 3ʹ poly(A) tail, contains four open reading frames, and is most closely related to Caucasus prunus virus (56% nucleotide sequence identity), a member of the genus Prunevirus. Based on the demarcation criteria of the family Betaflexiviridae, ASbLV is a new member of the genus Prunevirus.