Development of TaqMan RT-qPCR for the detection of regulated citrus viruses and viroids in Aotearoa New Zealand.

The major citrus species include several economically important fruits, such as orange, mandarin, lemon, limes, grapefruit and pomelos. Since the 1980 s, total production and consumption of citrus has grown strongly with the current annual worldwide production at over 105 million tonnes. New Zealand's citrus exports, for instance, had an estimated worth of NZ$ 11.6 million (approx. US$ 7 million) in 2020. Citrus plants are prone to viral diseases, which can lead to substantial economic losses. In New Zealand, the citrus Import Health Standard (IHS) has identified 22 viruses and viroids that are subject to regulation and requires citrus nursery stock to be free of these pathogens. As such, there is a need for reliable, sensitive, and rapid detection methods to screen for these viruses and viroids during post entry quarantine. In this study, we developed TaqMan RT-qPCR assays for the detection of nine of these regulated viruses and viroids, namely citrus leaf rugose virus (CiLRV), citrus leprosis virus C (CiLV-C), citrus leprosis virus C2 (CiLV-C2), citrus leprosis virus N (CiLV-N), citrus psorosis virus (CPsV), citrus yellow mosaic virus (CYMV), citrus bent leaf viroid (CBLVd), citrus viroid V (CVd-V), and citrus viroid VI (CVd-VI). These assays have been validated and found to be highly sensitive, specific, and reliable. The implementation of these assays will facilitate the safe importation of citrus nursery stock, thus safeguarding the country's horticultural and economic interests.

Lavender Harbors More Viruses than Previously Thought: First Report of Raspberry Ringspot Virus and Phlox Virus M in Lavandula × intermedia.

Plants of the genus Lavandula are thought to be rarely infected by viruses. To date, only alfalfa mosaic virus, cucumber mosaic virus, tobacco mosaic virus, and tomato spotted wilt virus have been reported in this host. In this study, we identified for the first time raspberry ringspot virus (RpRSV) and phlox virus M (PhlVM) in lavender using herbaceous indexing, enzyme-linked immunosorbent assay, and high-throughput sequencing. Nearly complete genome sequences for both viruses were determined. Phylogenetic and serological characterizations suggest that the obtained RpRSV isolate is a raspberry strain. A preliminary survey of 166 samples indicated RpRSV was spread only in the lavender cultivar 'Grosso', while PhlVM was detected in multiple lavender cultivars. Although RpRSV raspberry strain may have spread throughout Auckland and nearby areas in New Zealand, it is very likely restricted to the genus Lavandula or even to the cultivar 'Grosso' due to the absence or limited occurrence of the nematode vector. Interestingly, all infected lavender plants, regardless of their infection status (by RpRSV, PhlVM, or both) were asymptomatic. RpRSV is an important virus that infects horticultural crops including grapevine, cherry, berry fruits, and rose. It remains on the list of regulated pests in New Zealand. RpRSV testing is mandatory for imported Fragaria, Prunus, Ribes, Rosa, Rubus, and Vitis nursery stock and seeds for sowing, while this is not required for Lavandula importation. Our study revealed that lavender could play a role not only as a reservoir but also as an uncontrolled import pathway of viruses that pose a threat to New Zealand's primary industries.

Detection, Characterization, and Distribution of the First Case of Pepino Mosaic Virus in Aotearoa New Zealand.

Tomato (Solanum lycopersicum L., family Solanaceae) represents one of the most economically valuable horticultural crops worldwide. Tomato production is affected by numerous emerging plant viruses. We identified, for the first time in New Zealand (NZ), Pepino mosaic virus (PepMV) in greenhouse grown tomato crops using a combination of methods from electron microscopy and herbaceous indexing to RT-qPCR and high-throughput sequencing. Phylogenetic and genomic analysis of a near-complete PepMV genome determined that the detected strain belonged to the mild form of the CH2 lineage of the virus. Subsequently, a delimiting survey of PepMV was conducted, and PepMV was detected at four additional locations. PCR-derived sequences obtained from samples collected from different greenhouses and from herbaceous indicator plants were identical to the original sequence. Since PepMV has never been reported in NZ before, seed pathways are speculated to be the most likely source of entry into the country.

First report of Streptocarpus flower break virus in Streptocarpus hybrids in Aotearoa New Zealand.

Streptocarpus (Cape primrose, family Gesneriaceae) is a genus of plants native to Southern Africa commonly grown indoors for their foliage and trumpet-shaped flowers. In Aoteroa New Zealand (NZ) to date, no viruses have been reported to infect plants of the Gesneriaceae (Veerakone et al. 2015). In September 2022, a plant of Streptocarpus hybrid exhibiting necrotic rings was observed in a hobbyist's greenhouse in Auckland, NZ. High-Throughput Sequencing (HTS) using MinIONTM (Oxford Nanopore Technologies), was applied as a first screen (Liefting et al. 2021). Phylogenetic analysis was performed using Geneious Prime 2021 (Biomatters Ltd, NZ). A BLASTn search with 622,847 obtained reads resulted in 3,260 and 4,340 matches to the sequences of Streptocarpus flower break tobamovirus (SFBV) and Impatiens necrotic spot orthotospovirus (INSV), respectively. A near-complete (98.5%) genome sequence of SFBV was obtained (GenBank accession No. OQ970154), which shared 99.52% nucleotide identity to a SFBV type isolate from Germany (GenBank accession No. NC_008365). A phylogenetic tree was also generated (e-Xtra). To confirm the presence of both viruses, leaf tissue was rub inoculated onto herbaceous indicator plants as described by Tang et al. (2013). Chenopodium amaranticolor and C. quinoa plants developed local lesions while Nicotiana occidentalis plants showed local necrosis followed by systemic leaf puckering by 14 days post inoculation (dpi). Nicotiana benthamiana and N. clevelandii plants showed systemic chlorosis but N. tabacum plants did not exhibit any symptoms by 28 dpi. Samples from indicators and Streptocarpus were tested by RT-PCR (SFBV) or RT-qPCR (INSV), using in-house designed primers: SFBV-forward (5'-GTCATCAGCCGGAGAGGTTC-3'), SFBV-reverse (5'-AGGGCGAGTCTCTTCCTCTG-3'), INSV-forward (5'-CAATCAGAGGGTGACTTGGAA-3'), INSV-reverse (5'-GACTTTCCGAAGACTTGATGC-3') and INSV-probe (5'-CCATTGTCCTTTATCATTCCAACAAG-3'). RT-PCR products (across MP and CP regions) of the expected size (357 bp) were amplified from the Streptocarpus sample and symptomatic indicators. All amplicons were sequenced in both directions and found to be identical to the obtained HTS sequence. The presence of INSV was confirmed in all Streptocarpus and inoculated indicators except N. tabacum by INSV-specific RT-qPCR. A further 77 Streptocarpus plants were collected from a greenhouse in Auckland that holds a collection of multiple Streptocarpus cultivars from across NZ and overseas. Twenty-five plants, either displaying flower colour-break (only one plant) or asymptomatic (24 plants), tested positive for SFBV by RT-PCR. All amplicons were sequenced and found to be identical. SFBV was first described from naturally infected Streptocarpus plants in 1995 in the Netherlands (Verhoeven et al. 1995), and then in Germany (Heinze et al. 2006) and the United States (Pappu & Druffel 2007). While INSV has been found in NZ in several plant genera (Veerakone et al., 2015), to our knowledge, this is the first report of SFBV in NZ. SFBV was thought to be associated with colour breaking of Streptocarpus flowers (Verhoeven et al. 1995) but the virus was detected in asymptomatic Streptocarpus plants in this study and in California (Pappu & Druffel 2007). Given SFBV-infected plants were purchased from several sources, and leaf cuttings for propagation are shared among hobbyists, SFBV is likely to have spread throughout NZ. How this will affect production is unclear at this stage.

Eradication of Potato Virus S, Potato Virus A, and Potato Virus M From Infected in vitro-Grown Potato Shoots Using in vitro Therapies.

Certain viruses dramatically affect yield and quality of potatoes and have proved difficult to eradicate with current approaches. Here, we describe a reliable and efficient virus eradication method that is high throughput and more efficacious at producing virus-free potato plants than current reported methods. Thermotherapy, chemotherapy, and cryotherapy treatments were tested alone and in combination for ability to eradicate single and mixed Potato virus S (PVS), Potato virus A (PVA), and Potato virus M (PVM) infections from three potato cultivars. Chemotherapy treatments were undertaken on in vitro shoot segments for four weeks in culture medium supplemented with 100 mg L-1 ribavirin. Thermotherapy on in vitro shoot segments was applied for two weeks at 40°C (day) and 28°C (night) with a 16 h photoperiod. Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture followed by exposure to PVS2 either without or with liquid nitrogen (LN, cryotherapy) treatment. The virus status of control and recovered plants following therapies was assessed in post-regeneration culture after 3 months and then retested in plants after they had been growing in a greenhouse for a further 3 months. Microtuber production was investigated using in vitro virus-free and virus-infected segments. We found that thermotherapy and cryotherapy (60 min PVS2 + LN) used alone were not effective in virus eradication, while chemotherapy was better but with variable efficacy (20-100%). The most effective result (70-100% virus eradication) was obtained by combining chemotherapy with cryotherapy, or by consecutive chemotherapy, combined chemotherapy and thermotherapy, then cryotherapy treatments irrespective of cultivar. Regrowth following the two best virus eradication treatments was similar ranging from 8.6 to 29% across the three cultivars. The importance of virus removal on yield was reflected in "Dunluce" free of PVS having higher numbers of microtubers and in "V500' free of PVS and PVA having a greater proportion of microtubers > 5 mm. Our improved procedure has potential for producing virus-free planting material for the potato industry. It could also underpin the global exchange of virus-free germplasm for conservation and breeding programs.

First report of Grapevine Algerian latent virus in carnation in New Zealand.

Carnation (Dianthus caryophyllus) is a popular ornamental plant widely used as a cut flower and in landscaping. In New Zealand, several viruses are known to infect plants of the genus Dianthus: arabis mosaic virus, carnation etched ring virus (CERV), carnation latent virus, carnation mottle virus, carnation necrotic fleck virus, carnation ringspot virus, carnation vein mottle virus and cucumber mosaic virus (Veerakone et al. 2015). In October 2020, a carnation sample with leaf chlorotic spots and distortion from a home garden in Auckland, New Zealand was submitted to the Plant Health and Environment Laboratory (PHEL) for virus testing. Leaf tissue of the sample was mechanically inoculated onto a range of herbaceous species using the method described in Tang et al. (2013). Chenopodium amaranticolor and C. quinoa plants developed local necrotic pinpoint spots while Nicotiana benthamiana, N. clevelandii, and N. occidentalis plants exhibited systemic leaf mosaic symptoms 7 days post-inoculation. The carnation plant and all five symptomatic indicator species tested positive for tombusviruses using an in-house designed generic RT-qPCR (available on request). Direct sequencing of the ~140 bp PCR product revealed the presence of grapevine Algerian latent virus (GALV). To further characterise the detected sequence, forward (5'-GTAGCGATGTATTGGGATAAGGA-3') and reverse (5'-TGCCGACACCCCGAAAGGT-3') primers were designed based on an alignment of the conserved region in the coat protein (CP) of 19 GALV isolates deposited in GenBank. Products of the expected size of 406 bp were amplified from all infected plants and their sequences found to be identical (GenBank accession No. OM891837). BLAST searches showed that the CP region of the sequence shared 97.0% (nucleotide) and 97.8% (amino acid) identity to the type isolate of GALV (GenBank accession no. NC_011535). GALV was first reported in Italy from a symptomless Algerian grapevine (Vitis vinifera) (Gallitelli et al., 1989), and is the only report of GALV in Vitis in the world. Since then, GALV has been reported in Germany, the Netherlands and Japan in several ornamental plant species including Alstroemeria sp. (Tomitaka et al., 2016), Gypsophila paniculata, Limonium sinuatum (Koenig et al., 2004, Fujinaga et al., 2009) and Solanum mammosum (Ohki et al., 2006). These infected ornamental host plants were reported to show various types of foliar symptoms, including chlorotic leaf spots. The GALV-infected carnation plant in this study was tested by PCR for all viruses that are known to infect D. caryophyllus in New Zealand, and CERV was identified. It is therefore unclear if the observed symptoms were induced by either GALV or CERV, or if they were the results of a synergistic interaction between GALV and CERV. Samples from a further 11 plants, comprised of nine symptomatic Dianthus spp. and two asymptomatic Alstroemeria spp. were collected from the same address and tested individually using the GALV-specific RT-PCR. GALV (along with CERV) was detected from all Dianthus plants while the Alstroemeria samples were negative. To our knowledge, this is the first report of GALV in New Zealand, and the first report in the host Dianthus in the world. Given the GALV-infected carnation plants were purchased from a local garden centre between 2007-2020, and plants from this garden centre have been widely distributed over this period of time to various customers, the virus is very likely to have spread throughout the country.

Complete nucleotide sequence of sweetbriar rose curly-top associated virus, a tentative member of the genus Waikavirus.

A novel virus, tentatively named "sweetbriar rose curly-top associated virus" (SRCTaV), was identified in sweetbriar rose (Rosa rubiginosa) using high-throughput sequencing. The complete genome sequence of SRCTaV was determined and characterized. Phylogenetic analysis revealed that SRCTaV is closely related to members of the genus Waikavirus.

Partial biological and molecular characterization of a novel citrivirus from Nandina domestica.

We report the complete genome sequence of a novel virus isolated from Nandina domestica 'Firepower' in Auckland, New Zealand. It was mechanically transmitted to Nicotiana species, although all of these infections were symptomless. The complete genome of the new virus is 8892 nucleotides (nt) long, excluding the 3' poly(A) tail, contains three open reading frames (ORF), and is most closely related to citrus leaf blotch virus (CLBV) Actinidia isolate (CLBV-Act; 72% nt sequence identity), a member of the genus Citrivirus. Replicase and coat proteins, encoded by genome ORFs 1 and 3 respectively, shared 81-83% and 76-79% amino acid (aa) sequence identity, respectively, with CLBV-Act. Computer-based analysis suggests that this novel virus is the result of recombination between CLBV-Act and an unknown virus, highlighting the importance of this phenomenon for betaflexivirus evolution.

Development of TaqMan real-time RT-PCR for sensitive detection of diverse Raspberry ringspot virus isolates.

Raspberry ringspot virus (RpRSV) is an important virus that infects horticultural crops including grapevine, cherry, berry fruit and rose. The genome sequences of RpRSV are highly diverse between isolates and this makes the design of a PCR-based detection method difficult. In this study, a TaqMan real-time RT-PCR assay was developed for the rapid and sensitive detection of RpRSV. Primers and probes targeting the most conserved region of the movement protein gene were designed to amplify a 229 bp fragment of RpRSV RNA-2. The assay was able to amplify all RpRSV isolates tested. The detection limit of the RpRSV target region was estimated to be 61-98 copies, depending on the RpRSV strain. The sensitivity was about 100 times greater than the conventional RT-PCR assay using the same primers as the real-time RT-PCR assay. A comparison with published conventional RT-PCR assays indicated that both published assays lacked reliability and sensitivity, as neither were able to amplify all RpRSV isolates tested, and both were at least 1000 times less sensitive than the novel TaqMan real-time RT-PCR assay. The assay can also be run as a duplex reaction with the nad5 plant internal control primers and probe to simultaneously verify the PCR competency of the samples. The amplicon obtained with the real-time RT-PCR assay is suitable for direct sequencing if it is necessary to further confirm the RpRSV identity or determine the RpRSV strain.

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 3' 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.

Diversity and evolutionary history of lettuce necrotic yellows virus in Australia and New Zealand.

Lettuce necrotic yellows virus (LNYV) is the type member of the genus Cytorhabdovirus, family Rhabdoviridae, and causes a severe disease of lettuce (Lactuca sativa L.). This virus has been described as endemic to Australia and New Zealand, with sporadic reports of a similar virus in Europe. Genetic variability studies of plant-infecting rhabdoviruses are scarce. We have extended a previous study on the variability of the LNYV nucleocapsid gene, comparing sequences from isolates sampled from both Australia and New Zealand, as well as analysing symptom expression on Nicotiana glutinosa. Phylogenetic and BEAST analyses confirm separation of LNYV isolates into two subgroups (I and II) and suggest that subgroup I is slightly older than subgroup II. No correlation was observed between isolate subgroup and disease symptoms on N. glutinosa. The origin of LNYV remains unclear; LNYV may have moved between native and weed hosts within Australia or New Zealand before infecting lettuce or may have appeared as a result of at least two incursions, with the first coinciding with the beginning of European agriculture in the region. The apparent extinction of subgroup I in Australia may have been due to less-efficient dispersal than that which has occurred for subgroup II - possibly a consequence of suboptimal interactions with plant and/or insect hosts. Introduction of subgroup II to New Zealand appears to be more recent. More-detailed epidemiological studies using molecular tools are needed to fully understand how LNYV interacts with its hosts and to determine where the virus originated.

Opium poppy mosaic virus, a new umbravirus isolated from Papaver somniferum in New Zealand.

A novel virus, tentatively named "opium poppy mosaic virus" (OPMV), was isolated from Papaver somniferum (opium poppy) with leaf mosaic and mottling symptoms in Auckland, New Zealand, in 2006. The virus was mechanically transmitted to herbaceous plants of several species, in which it induced local and/or systemic symptoms. No virus particles were observed by electron microscopy in the diseased P. somniferum or any of the symptomatic herbaceous plants. The complete genomic sequence of 4230 nucleotides contains four open reading frames (ORF) and is most closely related (59.3 %) to tobacco bushy top virus, a member of the genus Umbravirus. These data suggest that OPMV is a new umbravirus.

Red clover vein mosaic virus-A Novel Virus to New Zealand that is Widespread in Legumes.

Red clover vein mosaic virus (RCVMV) is an important virus of leguminous crops that can cause devastating losses. During a routine survey of legumes conducted on the South Island of New Zealand, RCVMV was found in mixed infections in clover plants with Alfalfa mosaic virus and White clover mosaic virus. The full-length sequence of the New Zealand isolate RCVMV-NZ from clover shared 96% nucleotide sequence identity with a chickpea isolate previously described from Washington (United States). Targeted surveys of pea, faba bean, and pasture crops showed that RCVMV-NZ is widespread on the South Island in New Zealand. This isolate is causing mild if any symptoms on experimental hosts and naturally infected plants.

Novel genus-specific broad range primers for the detection of furoviruses, hordeiviruses and rymoviruses and their application in field surveys in South-East Australia.

A number of viruses from the genera Furovirus, Hordeivirus and Rymovirus are known to infect and damage the four major temperate cereal crops, wheat, barley, sorghum and oats. Currently, there is no active testing in Australia for any of these viruses, which pose a significant biosecurity threat to the phytosanitary status of Australia's grains industry. To address this, broad spectrum PCR assays were developed to target virus species within the genera Furovirus, Hordeivirus and Rymovirus. Five sets of novel genus-specific primers were designed and tested in reverse-transcription polymerase chain reaction assays against a range of virus isolates in plant virus diagnostic laboratories in both Australia and New Zealand. Three of these assays were then chosen to screen samples in a three-year survey of cereal crops in western Victoria, Australia. Of the 8900 cereal plants screened in the survey, all were tested free of furoviruses, hordeiviruses and rymoviruses. To date, there were no published genus-specific primers available for the detection of furoviruses, hordeiviruses and rymoviruses. This study shows for the first time a broad-spectrum molecular test being used in a survey for exotic grain viruses in Australia. Results from this survey provide important evidence of the use of this method to demonstrate the absence of these viruses in Victoria, Australia. The primer pairs reported here are expected to detect a wide range of virus species within the three genera.

A Preliminary Study of an Integrated and Culturally Attuned Cognitive Behavioral Group Treatment for Chinese Problem Gamblers in Hong Kong.

Chinese people may have a higher rate of gambling problems than other cultural groups. However, there are very few clinical outcome studies that have demonstrated the effectiveness of clinical interventions for helping Chinese gamblers. Cognitive behavioural therapy (CBT) has been found to be effective for helping problem gamblers to significantly reduce their gambling problems in western countries. Very few CBT clinical trials have been conducted with the Chinese populations, and the results were masked by methodological limitations. This preliminary study attempted to test the effectiveness of an integrated and culturally attuned CBT group treatment for Chinese problem gamblers in Hong Kong. This study adopted a randomized control design and 38 participants were allocated randomly to the experimental condition (n = 18) and control condition (n = 20). The experimental group received 10 weekly CBT group sessions and individual counseling services while control group only received the individual counseling services. Significant decreases in gambling severity and frequencies of gambling were found in the experimental group. The findings also showed that a change in gambling cognitions predicted the changes in gambling severity and gambling urge while a change in gambling severity was also linked to a change in depression. Preliminary evidence highlights the potential benefits of an integrated and culturally attuned CBT group treatment for Chinese problem gamblers in Hong Kong. However, a more vigorous research design with a larger sample is needed to provide solid evidence of the effectiveness of the model for Chinese problem gamblers.

Suicidal ideation and familicidal-suicidal ideation among individuals presenting to problem gambling services: a retrospective data analysis.

BACKGROUND: Studies have consistently reported high rates of suicidal ideation (SI) among individuals with disordered gambling. None have explored gambling-related familicidal-suicidal ideation (FSI). AIMS: This study examined the (1) prevalence of SI and FSI among treatment-seeking gamblers in Hong Kong, (2) characteristic profile of factors associated with SI and FSI, and (3) factors that predict SI and FSI. METHOD: This is a retrospective analysis of data collected at initial clinical assessments from a specialized gambling counseling centre in Hong Kong. Participants were gamblers (N = 3,686) who sought treatment at the centre between 2003 and 2012. Information about socio-gambling demographics, physical and mental health status, current presenting problems, self-rated South Oaks Gambling Screen (SOGS, Chinese version), and occurrence of SI or FSI were examined. Descriptive analysis and ordinal regression analysis were used to investigate the characteristics of the gamblers and the association of variables. RESULTS: In our sample, 720 (20.0%) individuals reported SI, and 22 (0.6%) individuals reported FSI at the initial assessment. Individuals with SI and FSI differed from the nonsuicidal individuals in terms of their demographics, gambling experiences and severity, mental and physical wellbeing, and types of gambling-related problems. The adjusted ordinal regression model shows that participating in table games in casinos and having familial and financial problems seem to enhance the likelihood of having SI and FSI. CONCLUSION: While mental health issues are significantly related to SI and FSI among gambling treatment seekers, the impacts of physical, family, and financial strains should not be underestimated.

Sensitive detection of Tomato ringspot virus by real-time TaqMan RT-PCR targeting the highly conserved 3'-UTR region.

A real-time TaqMan RT-PCR assay was developed for the rapid and sensitive detection of Tomato ringspot virus (ToRSV), an important plant virus which infects a wide range of fruit and ornamental crops. Primers and a probe were designed based on the highly conserved 3'-untranslated region (UTR) sequences of ToRSV, to amplify a 182bp fragment of this region of RNA-1 and RNA-2. The assay was demonstrated to reliably amplify all ToRSV isolates tested. The detection limit was estimated to be about 12 copies of the ToRSV target region. No amplification was observed from the RNA of other nepoviruses or healthy host species. A comparison with a published conventional RT-PCR and a SYBR-based qRT-PCR indicated that both of the published assays lacked reliability and sensitivity, as neither were able to amplify all ToRSV isolates tested, and both were approximately 1000 times less sensitive than the novel TaqMan real-time assay. This TaqMan real-time assay was tested using four different reagent kits and was shown to be robust and stable, with no significant differences in sensitivity between kits. It is expected that the implementation of this TaqMan real-time RT-PCR assay will facilitate efficient phytosanitary certification of nursery stock requiring testing for ToRSV by regulatory agencies, and will also have wider uses for the general detection of ToRSV in a range of hosts.

Phylogenetic analysis of New Zealand tomato spotted wilt virus isolates suggests likely incursion history scenarios and mechanisms for population evolution.

Tomato spotted wilt virus (TSWV) is an internationally significant pathogen with a wide host range, vectored by thrips. We have studied the sequence variation and evolutionary mechanisms at play in parts of the L, M and S subgenomes of 23 New Zealand TSWV isolates collected between 1992 and 2009, aiming to identify the possible geographic origins of isolates. Maximum-likelihood-based phylogenetic analyses of New Zealand and overseas TSWV isolates placed the L and M subgenome sequences of two isolates (MAF04 and PFR04) in distinct clades composed primarily of Korean, Japanese and Chinese isolates, in contrast to the remaining 21 isolates, which clustered with a cosmopolitan group of isolates. The nucleocapsid (N) gene sequences of MAF04 and PFR04 plus MAF02 clustered with Japanese isolates. Consequently, we postulate that these isolates may represent a distinct incursion into New Zealand, but we do not have enough evidence to indicate an incursion pathway. Alternately, these isolates may have arrived with an incursion that included a mixture of TSWV isolates of diverse international origins. The sequences of four of the TSWV isolates contained a number of sites with a mixture of nucleotides, suggesting that these isolates either consisted of several sequence variants or were from plants with mixed infections. One isolate (MAF02) was shown to be a either a reassortant or an S subgenome recombinant. Large amounts of low-level polymorphism were detected with low amino acid change fixation rates (purifying selection). Negative selection was indicated at four amino acid sites in the New Zealand TSWV N gene sequences.