Removal of Leafroll Viruses from Infected Grapevine Plants by Droplet Vitrification

Transcription

1 Removal of Leafroll Viruses from Infected Grapevine Plants by Droplet Vitrification R. Pathirana 1, A. McLachlan 1, D. Hedderley 1, A. Carra 2, F. Carimi 2 and B. Panis 3 1 The New Zealand Institute for Plant & Food Research Ltd., Private Bag 11600, Palmerston North 4442, New Zealand 2 Consiglio Nazionale delle Ricerche, Istituto di Genetica Vegetale, Corso Calatafimi 414, I Palermo, Italy 3 Bioversity International, c/o KU Leuven, Willem de Croylaan 42 bus 2455, 3001 Leuven, Belgium Keywords: Closteroviridae, cryopreservation, cryotherapy, germplasm, salicylic acid, sucrose, Vitis vinifera Abstract A robust method for removing all microorganisms from infected tissue is important for cultivar imports, germplasm maintenance and to produce healthy grafted material for the grapevine industry. In the droplet vitrification method of cryopreservation, plant tissue pre-treated with a vitrification solution is placed on aluminium foil in a droplet of vitrification solution and directly immersed in liquid nitrogen. Only highly cytoplasmic, non-vacuolar meristematic cells survive freezing. Therefore, cryopreservation can be considered a precise method of meristem culture and is developing into a new method for virus eradication in horticultural species called cryotherapy. To test the suitability of cryotherapy for virus eradication, we used Chardonnay and Lakemont Seedless infected with Grapevine leafroll associated virus-3 (GLRaV-3, an Ampelovirus), Pinot gris and Sauvignon blanc 316 infected with Grapevine leafroll associated virus-2 (a Closterovirus), and another clone of Sauvignon blanc infected with both Grapevine leafroll associated virus-1 (an Ampelovirus) and GLRaV-3. Plants regenerated after cryo-treatment tested negative (DAS-ELISA) for all three viruses, whereas untreated control plants tested positive. Droplet vitrification has the potential to be a novel and precise tool for virus eradication and establishment of high-health grapevine germplasm collections. In addition to removing virus infections, the method provides a cost-effective way of maintaining clonal plant material. INTRODUCTION As an ancient crop, 10,000-14,000 different grapevine cultivars are thought to be held in germplasm collections around the world (Alleweldt and Dettweiler, 1994). Maintenance of field collections is expensive, which has led to the erosion of valuable germplasm resources and carries the risk of pathogen transmission through vegetative multiplication and insects (Barba et al., 2008; Carimi et al., 2011). Virus infections in clonally propagated grapevine material are a serious problem (Pathirana and McKenzie, 2007; Carimi et al., 2011). For example, 45-85% of plants tested were infected with leafroll virus in six wine grape cultivars and in American rootstocks in Turkey (Azeri, 1990). Autochthonous germplasm in Sicily and Sardinia has a high level of virus infection, and sanitary improvement is considered important (Garau et al., 2003; Barba et al., 2008). In the Czech Republic, 14-25% vines tested were positive for Grapevine leafroll associated virus-1 (GLRaV-1), Grapevine fleck virus or Grapevine virus A (GVA) (Kominek, 2008) while in Hungary 16 virus or virus-like diseases have been reported (Lazar, 2003). Some viruses significantly reduce berry weight and fruit quality (Lazar, 2003; Akbas et al., 2009). In French-American hybrid cultivars, Grapevine leafroll associated virus-3 (GLRaV-3) is latent but is linked to reduced berry weight and higher titratable acidity (Kovacs, 2001). Cryopreservation is the storage of viable cells, tissues, organs and organisms at ultra-low temperatures, usually in liquid nitrogen (LN) and/or its vapour phase, at Proc. VIII th IS on In Vitro Culture and Horticultural Breeding Eds.: J.M. Canhoto and S.I. Correia Acta Hort. 1083, ISHS

2 temperatures of c to -140 C (Benson, 2008). It is generally accepted that cryopreservation is the best technique currently available to ensure the safe and costefficient long-term conservation of germplasm of vegetatively propagated species (Benson, 2008; Keller et al., 2008). Despite its value as a crop, there is no collection of grapevine germplasm in cryostorage, partly because of recalcitrance of some genotypes to cryopreservation as demonstrated by Ganino et al. (2012). In addition to the storage of germplasm resources by cryopreservation, recent studies have shown that viruses can be eradicated by cryopreservation (Helliot et al., 2002) and these methods are now developing into a new technology called cryotherapy (Nukari et al., 2009; Wang and Valkonen, 2009) and are being incorporated into highhealth conservation programmes. This work was aimed at applying a droplet vitrification protocol to Vitis vinifera and testing the feasibility of using cryotherapy to remove viruses of the family Closteroviridae. MATERIALS AND METHODS Plant Material Virus infected plant material used in the experiments included Pinot gris and Sauvignon blanc 316 sourced from Marlborough, New Zealand infected with Grapevine leafroll associated virus-2 (GLRaV-2), a Closterovirus from family Closteroviridae. Chardonnay sourced from Hawke s Bay, New Zealand and Lakemont Seedless, a table grape cultivar sourced from Marlborough were infected with GLRaV-3, an Ampelovirus of the family Closteroviridae. Another Sauvignon blanc clone from Marlborough was infected with both GLRaV-3 and GLRaV-1, an Ampelovirus from Closteroviridae. The virus infections in these clones were tested and certified by the Linnaeus Laboratories, Gisborne, New Zealand a NZS ISO/IEC 17025:2005 accredited laboratory. Initiation of Axenic Cultures and Tissue Culture Conditions The buds of woody shoots collected as cuttings were induced to produce new green shoots under greenhouse conditions by holding them in coarse sand in a mist bed with bottom heating to 28 C. When the green shoots were cm long, they were harvested for initiation of in vitro cultures as described by Pathirana and McKenzie (2005). The basal medium (BM) comprised half-strength Murashige and Skoog (1962) (MS) macronutrients, MS micronutrients, B5 vitamins (Gamborg et al., 1968) and 58.5 mm sucrose solidified by addition of 3 g L -1 Gelrite. BM supplemented with 2.22 µm N 6 -benzylaminopurine (BA) was used to promote shoot growth from axillary buds. For all experiments, the ph of culture media was adjusted to ph 5.8 using either NaOH or HCl before autoclaving the medium for 20 min at 121 C. In vitro growing plants were multiplied by shoot tip and nodal cuttings comprising segments with two nodes at 4-week intervals in BM. Cultures were initiated in 9-cm petri plates holding 20 ml of medium and plantlets were multiplied in 290 ml clear wide-mouth disposable polystyrene tissue culture tubs. Culture rooms and growth chambers were maintained at 24±1 C with a 16-h photoperiod and a photosynthetic photon flux of 30 µmol m -2 s -1 at the top of the culture vessels provided by Phillips cool-white 18 W fluorescent lamps. Following initiation of in vitro cultures, work was carried out under aseptic conditions, until plants were deflasked. Droplet Vitrification In vitro multiplied grapevine material was transferred to BM supplemented with 0.1 mm salicylic acid (SA) and maintained for two weeks. Explants comprising apical and axillary buds were harvested at the end of two weeks and held on sterile filter paper (Whatman Qualitative Grade 2) on BM with increasing sucrose concentrations of 0.25 to 0.5 to 0.75 then 1 M sequentially over 4 days. The explants were prepared by dissecting most of the protective scale leaves from the bud. Once all the explant material was 492

3 prepared, it was immersed in Loading Solution (LS) for 20 min at room temperature; LS comprised a half-strength MS (macro and micro-nutrients) medium supplemented with 2 M glycerol and 0.4 M sucrose. The explants were then immersed in PVS2 solution (15% w/v ethylene glycol, 15% w/v DMSO, 30% w/v glycerol, and 13.7% w/v sucrose) in MS macro and micro salts (Sakai, 2004) on ice for 36 (apical buds) or 41.5 min (axillary buds). After PVS2 treatment the explants were transferred to a drop of PVS2 solution on sterile aluminium foil (8 25 mm) and the foil was plunged into LN and transferred to 1.8-ml cryo tubes (Nunc, Roskilde, Denmark) filled with LN. About four explants were used per foil. After a minimum of 1 h in LN, the aluminium foils with explants were transferred to Recovery Solution (RS) (comprising 1.2 M sucrose in MS macro- and micro-salts) at room temperature for 20 min before transfer to Recovery Medium (RM) (0.6 M sucrose in agar-solidified MS macro- and micro-salts) on sterile filter paper in petri plates. The cultures were maintained in the dark for 24 h at 24±1 C. After incubation on RM, the filter papers with explants were transferred to regeneration medium (REM) that comprised BM supplemented with 3 µm BA and 0.05 µm 1-naphthaleneacetic acid. REM plates were changed every 4 weeks. The cultures were maintained in darkness for 1 week before transfer to light. Deflasking and Acclimation of Plants to the Greenhouse Conditions Plantlets regenerated from the PVS2 control and after cryopreservation were transferred to BM in tissue culture tubs. The plantlets produced roots in 3-4 weeks. After about six weeks of growth, the rooted plants were deflasked, maintained in a fog tent inside a greenhouse for 1 week before transfer to a mist bench for another week. The plants were then transferred to a greenhouse bench with capillary watering for two weeks, before transfer to a greenhouse bench with overhead watering. The greenhouse was heated from 15 C and vented from 25 C with bottom heating of 28 C for the first two weeks of acclimation. Only natural lighting in the greenhouse was used. Virus Indexing The effectiveness of droplet vitrification as a method for removing Closteroviridae viruses was assessed. Plants regenerated after PVS2 treatment but without immersion in LN (PVS2 control) and those regenerated in REM from shoot apices with no other treatments provided positive controls for assessing the role of cryopreservation in virus elimination. Fully expanded leaves and their petioles harvested from regenerated plants growing in tissue culture and the greenhouse three and six months after acclimation were sampled for virus indexing. Virus indexing was conducted for GLRaV-1, GLRaV-2 and GLRaV-3 in all samples, using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). This assay uses antibodies bound to the surface of a microplate to capture the antigens. The presence of the antigen is detected using specific antibodies coupled with alkaline phosphatase. The addition of the enzyme substrate (pnpp) produces a yellow product, detectable at 405 nm, when the antigen is present. The DAS-ELISA kit for detecting GLRaV-1 was supplied by Sediag (Colmer, France), for GLRaV-2 by Agritest (Valenzano, Italy) and for GLRaV-3 by Bioreba (Reinach, Switzerland) and the assays were conducted according to the manufacturer s instructions at a NZS ISO/IEC 17025:2005 accredited independent laboratory (Linnaeus Laboratories, Gisborne, New Zealand). A minimum of six independently regenerated plants per infected clone after PVS2 treatment (Control) and LN treatment were used in the assays. RESULTS AND DISCUSSION All the five virus-infected accessions regenerated after cryopreservation, but at different rates (Table 1). Our preliminary experiments showed that SA and sucrose pretreatments are essential for recovery after cryopreservation of grapevine buds. We optimised the PVS2 treatment in preliminary experiments and used treatment times that gave 50% regeneration for apical (36 min) and axillary buds (41.5 min) in 493

4 cryopreservation experiment. Virus-infected material regenerated at low rates and we applied probabilistic tools to ensure post-storage regeneration to be accurately predicted. We predict that even for the most difficult genotype Chardonnay, storage of 22 explants cryopreserved by droplet vitrification will ensure regeneration of at least one plant at 95% probability, and 33 explants would increase that to 99% (Table 1). Cryopreservation is an ideal method for long-term preservation of clonally propagated fruit and berry crops (Benson, 2008; Keller et al., 2008; Benelli et al., 2013). Cryopreservation techniques have been successfully applied to several vegetatively propagated species. Thus, for example, the Malus germplasm collection is held in the form of vegetative buds cryopreserved by a slow freezing method (Towill et al., 2004; Lambardi et al., 2011; Benelli et al., 2013), whereas banana and raspberry collections are cryopreserved by droplet vitrification (Panis et al., 2010; Benelli et al., 2013). A potato gene bank has been established using droplet freezing (Keller et al., 2008). The virus-infected material was difficult to propagate in tissue culture and therefore lower regeneration of this material after cryopreservation was expected. This confirms previous observations by Guta et al. (2008), who reported poor regeneration of shoot tips in tissue cultures of leafroll virus-infected V. vinifera Feteasca neagra. Plant regeneration of healthy clones that were used to develop the protocol had much higher regeneration ( %; data not shown). The physiological condition of the explants at the time of cryopreservation is critical for survival in LN and regeneration after cryopreservation (Johnston et al., 2007; Benson, 2008; Ganino et al., 2012). Some Vitis genotypes have proven recalcitrant to cryopreservation (Ganino et al., 2012), and even to in vitro multiplication if infected with virus (Guta et al., 2008). The introduction of SA pre-treatment of plantlets and high sucrose prior to PVS2 treatment was designed to increase freezing tolerance and tolerance to desiccation imparted by PVS2 solution. High sucrose concentration increases the osmotic potential in media and imparts desiccation tolerance to tissue (Gonzalez-Benito et al., 2009). It adjusts the osmotic potential in the cytoplasm, minimising ice crystal formation (Ganino et al., 2012). High sucrose concentrations in media have proven useful when explants have been sensitive to PVS2 solution or to physical methods of desiccation before cryopreservation (Johnston et al., 2007; Halmagyi et al., 2010; Ganino et al., 2012). Wang et al. (2003) acclimated encapsulated V. vinifera Bruti shoot tips in media enriched with 0.25, 0.5, 0.75 and 1.0 M sucrose concentrations before cryopreservation by vitrification. The difference in our experiments was that we used explants from plantlets growing in SA-supplemented media for sucrose pre-treatment followed by droplet vitrification, without encapsulating the explants. Droplet vitrification was investigated because it is proving applicable to many species (Gonzalez-Arnao et al., 2008; Condello et al., 2009; Nukari et al., 2009; Halmagyi et al., 2010; Kim et al., 2012). Additionally, it allows exposure of unprotected meristematic tissue to LN, making droplet vitrification more suitable than encapsulation-based methods for virus eradication. Lakemont Seedless and Chardonnay cultivars were infected with GLRaV-3, Sauvignon blanc 316 and Pinot gris with GLRaV-2, and another Sauvignon blanc accession was infected with both GLRaV-3 and GLRaV-1 (Table 2). PVS2 treatment alone was not sufficient to remove virus from infected plants (Table 2). Virus was not detected in tissue-cultured or greenhouse-grown plants following cryopreservation by droplet vitrification (Table 2). We tested a minimum of six independently regenerated plants per clone in tissue culture (after 3 months of growth) as well as 3 and 6 months after greenhouse acclimation. None of the tested plants showed presence of virus. Mean absorbance values recorded for LN-treated material after regeneration ranged from , whereas to consider the virus status to be inconclusive, an absorbance value of 0.03 needs to be reached and this value has to be above 0.1 for the material to be regarded as infected. The observation that droplet vitrification appears to eliminate virus from infected material is of particular interest. Droplet vitrification offers a viable method for improving 494

5 the virus status of Vitis. No virus was detected in any of the plants recovered from infected clones following cryopreservation when screened using DAS-ELISA for both greenhouse and in vitro growing plants (Table 2). The LN treatment step was confirmed as the important step for virus elimination as the PVS2 treated control plants were tested positive for virus (Table 2). More effective removal of vacuolated cells during droplet vitrification may be the reason for high effectiveness of our cryotherapy protocol. Recent studies have shown that viruses, including GVA, can be eradicated by cryopreserving plant material (Helliot et al., 2002; Wang et al., 2003; Wang and Valkonen, 2009). This result was extended in our research by the recovery of plants free of several viruses of the family Closteroviridae, which are some of the most damaging viruses in Vitis spp. (Kovacs, 2001; Pathirana and McKenzie, 2005; Akbas et al., 2009). Among the opportunities that open up through this research, the ability to develop, maintain and deliver high-health plant material appears the most obvious. It also offers an opportunity to speed the introduction of new germplasm from overseas, especially important for countries lacking autochthonous germplasm. These technologies also offer an opportunity to establish a repository of verified high-health (free of known virus) germplasm from which high-health plant material can be generated for use in the industry. In conclusion this work describes a method for cryotherapy of Vitis spp. using droplet vitrification of shoot tips and axillary buds, which was capable of removing three Closteroviridae viruses at 100% success rate. Viruses were successfully removed from a grapevine clone co-infected with two viruses. The method opens up opportunities for speedier introduction of cultivars with less rigorous quarantine procedures and delivery of high-health planting material to industry. High recovery rates in virus-free V. vinifera make the method suitable for cryobanking of grapevine. Supplementation of culture media with salicylic acid and pre-treatment of explants with sucrose were of critical importance for the success of the protocol. ACKNOWLEDGEMENTS This work was funded by NZ Winegrowers (NZW Cryopreserved grapevine: a new way to maintain high-health germplasm and cultivar imports with less rigorous quarantine ). Virus testing was conducted by Ilze Greyling at Linnaeus Laboratories, Gisborne, New Zealand. The authors thank Tony Baker, Vaughn Bell and Dion Mundy for the supply of grapevine material infected with virus. Thanks to Ed Morgan and Marian McKenzie for helpful comments on the manuscript. Literature Cited Akbas, B., Kunter, B. and Ilhan, D Influence of leafroll on local grapevine cultivars in agroecological conditions of Central Anatolia region. Hortic. Sci. 36: Alleweldt, G. and Dettweiler, E The Genetic Resources of Vitis: World List of Grapevine Collections. 2 nd ed. Geilweilerhof, Siebeldingen. Azeri, T Detection of Grapevine leafroll virus in different grapevine varieties by indexing. Journal of Turkish Phytopathology 19: Barba, M., Lernia, G.d., Carimi, F., Carra, A., Abbate, L. and Chiota, G Rescuing autochthonous grape vines thanks to virus elimination. Informatore Agrario Supplemento 64: Benelli, C., De Carlo, A. and Engelmann, F Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis. Biotechnology Advances 31: Benson, E.E Cryopreservation of phytodiversity: a critical appraisal of theory & practice. Critical Reviews in Plant Sciences 27: Carimi, F., Pathirana, R. and Carra, A Biotechnologies for germplasm management and improvement. p In: P.V. Szabo and J. Shojania (eds.), Grapevines 495

6 Varieties, Cultivation and Management Nova Science Publishers, New York. Condello, E., Andre, E., Piette, B., Druart, P., Caboni, E. and Panis, B Improvement of the cryopreservation of apple through the droplet vitrification method. Cryoletters 30: Gamborg, O.L., Miller, R.A. and Ojima, K Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research 50: Ganino, T., Silvanini, A., Beghe, D., Benelli, C., Lambardi, M. and Fabbri, A Anatomy and osmotic potential of the Vitis rootstock shoot tips recalcitrant to cryopreservation. Biologia Plantarum 56: Garau, R., Prota, V.A., Tolu, G., Mungianu, M.P.M., Sechi, A. and Prota, U On the sanitary improvement of grapevine in Sardinia. Informatore Fitopatologico 53: Gonzalez-Arnao, M.T., Panta, A., Roca, W.M., Escobar, R.H. and Engelmann, F Development of large scale application of cryopreservation techniques for shoot and somatic embryo cultures of tropical crops. Plant Cell Tissue and Organ Culture 92:1-13. Gonzalez-Benito, M.E., Martin, C. and Vidal, J.R Cryopreservation of embryogenic cell suspensions of the Spanish grapevine cultivars Albarino and Tempranillo. Vitis 48: Guta, I.C., Visoiu, E. and Buciumeanu, E.C In vitro investigation of grapevine in the presence of virus infection (V. vinifera L., Feteasca neagra cv.). Lucrari Stiintifice 51: Halmagyi, A., Deliu, C. and Isac, V Cryopreservation of Malus cultivars: comparison of two droplet protocols. Scientia Horticulturae 124: Helliot, B., Panis, B., Poumay, Y., Swennen, R., Lepoivre, P. and Frison, E Cryopreservation for the elimination of Cucumber mosaic and Banana streak viruses from banana (Musa spp.). Plant Cell Reports 20: Johnston, J.W., Harding, K. and Benson, E.E Antioxidant status and genotypic tolerance of Ribes in vitro cultures to cryopreservation. Plant Science 172: Keller, E.R.J., Kaczmarczyk, A. and Senula, A Cryopreservation for plant genebanks - a matter between high expectations and cautious reservation. Cryoletters 29: Kim, H.-H., Popova, E., Shin, D.-J., Yi, J.-Y., Kim, C.H., Lee, J.-S., Yoon, M.-K. and Engelmann, F Cryobanking of Korean Allium germplasm collections: results from a 10 year experience. Cryoletters 33: Kominek, P Distribution of grapevine viruses in vineyards of the Czech Republic. Journal of Plant Pathology 90: Kovacs, L.G., Hanami, H., Fortenberry, M. and Kaps, M.L Latent infection by leafroll agent GLRaV-3 is linked to lower fruit quality in French-American hybrid grapevines Vidal blanc and St. Vincent. American Journal of Enology and Viticulture 52: Lambardi, M., Benelli, C., Carlo, A., Ozudogru, E.A. and Previati, A Cryopreservation of ancient apple cultivars of Veneto: a comparison between PVS2- vitrification and dormant-bud techniques. Acta Hort. 908: Lazar, J Sanitary aspects and results of the Hungarian grape breeding. Acta Hort. 603: Murashige, T. and Skoog, F A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: Nukari, A., Uosukainen, M., Rokka, V.-M., Antonius, K., Wang, Q. and Valkonen, J.P.T Cryopreservation techniques and their application in vegetatively propagated crop plants in Finland. Agricultural and Food Science 18: Panis, B., Garming, H., Piette, B., Roux, N., Swennen, R. and Van den houve, I Banana conservation activities in the Bioversity International Transit Centre (ITC), Belgium. Cryoletters 31: Pathirana, R. and McKenzie, M.J Early detection of Grapevine leafroll virus in Vitis vinifera using in vitro micrografting. Plant Cell Tissue and Organ Culture 81:11-496

7 18. Pathirana, R. and McKenzie, M Micrografting grapevine for virus indexing. p In: S.M. Jain and H. Häggman (eds.), Protocols for Micropropagation of Woody Trees and Fruits Springer. Sakai, A Plant Cryopreservation. p In: B. Fuller, N. Lane and E.E. Benson (eds.), Life in the Frozen State CRC Press, London, U.K. Towill, L.E., Forsline, P.L., Walters, C., Waddell, J.W. and Laufmann, J Cryopreservation of Malus germplasm using a winter vegetative bud method: results from 1915 accessions. Cryoletters 25: Wang, Q. and Valkonen, J.P.T Cryotherapy of shoot tips: novel pathogen eradication method. Trends in Plant Science 14: Wang, Q.C., Mawassi, M., Li, P., Gafny, R., Sela, I. and Tanne, E Elimination of Grapevine virus A (GVA) by cryopreservation of in vitro-grown shoot tips of Vitis vinifera L. Plant Science 165: Tables Table 1. Mean regeneration percentages of explants of virus-infected grapevine accessions after cryopreservation by droplet vitrification and the predicted numbers of explants to be cryopreserved to ensure a 95 or 99% reliability of recovering at least one plant. Genotype Regeneration Probability (%) 95% 99% Chardonnay 13.0 (3.8) Pinot gris 13.6 (4.0) Sauvignon blanc (4.5) Lakemont Seedless 16.2 (4.1) Sauvignon blanc 30.0 (5.3)

8 Table 2. Results of virus testing by DAS-ELISA of control (PVS2-treated) plants and plants regenerated after cryopreservation by droplet vitrification. Plants from tissue cultures and greenhouse after three and six months from deflasking were sampled for the analyses. Cultivars Treatment a Mean absorbance at 405 nm b GLRaV-1 c GLRaV-2 c GLRaV-3 c Lakemont Seedless Control (-) (-) (+) Cryo (-) (-) (-) Sauvignon blanc 316 Control (-) (+) (-) Cryo (-) (-) (-) Pinot gris Control (-) (+) (-) Cryo (-) (-) (-) Sauvignon blanc Control (+) (-) (+) Cryo (-) (-) (-) Chardonnay Control (-) (-) (+) Cryo (-) (-) (-) Negative control Positive control a Control = plants from PVS2 treatment without freezing; Cryo = Cryopreserved by droplet vitrification and maintained in liquid nitrogen for a minimum of 1 h. b Mean values from a minimum of six independently regenerated plants at three stages of growth. None of the plants regenerated after cryopreservation showed an absorbance value above 0.03 considered inconclusive ( inconclusive, >0.1 considered infected). c ( ) = not detected, (+) = positive for virus. 498