Discussion of whether virus have been found to return to heat treated plants.
In the following quotes, when something appears in bold print, it does not mean that it appeared that way in the original; the bold print was added by me (Henry Kuska) for emphasis.
On Paul Barden's web site a recent statement by Tom Liggett concerning the possibility of virus returning to heat treated roses is given, see http://www.rdrop.com/~paul/tom_virus.html
This statement is consistent with one Tom Liggett made in a 1998 posting: (click on "show quoted text")
My comment: That link states that the information was presented at a meeting and that others heard it also.
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A comment concerning Tom Liggett's statement has recently appeared (click on this link):
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The following link contains a statement that I feel supports Tom Liggett's statement about statements being made at a meeting concerning virus reappearing in "cleaned stock".
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The following quote from a UCDAVIS link concerning their general virus testing program may help show that a "virus free" by a seller may too optimistic:
"While the testing and disease elimination programs are conducted under
very exacting conditions, there is no ironclad guarantee that materials released
from the program are 100 percent clean, Golino says. "In a simpler world,
it would be wonderful if we could say this grape is disease or virus free, or we
know it's healthy," Golino says. "But the truth is, especially as the
technology becomes more complicated, we are finding that there is a lot we don't
know about diseases." While the goal of a perfectly healthy plant may be a
purely "academic" exercise, FPMS can assure its customers that the
material has undergone an extensive testing program, Golino says.
"We can describe a series of protocols to which our materials have been
subjected," Golino says. "We can guarantee that those tests have been
conducted, that the results have been negative and that we are selling something
that is certified." "
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The above should be sufficient for the general reader. Those with science backgrounds may want to continue reading to see if the above is supported by scientific literature.
The following 8 sections may relate to this question:
1) I was able to find a certification document of the European and Mediterranean Plant Protection Organization which covers rose virus certification, see: http://home.neo.rr.com/kuska/virus-testing.htm
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2) Another reference that may be very appropriate concerning this point is:
Title: Recent Issues raised in the Evaluation of Virus Removal and Inactivation
Authors: White, E.M., and Woodward, R.S.,
Published in: Genetic Engineering News, volumn 15, page 6, (1995).
(Note (H. Kuska comment), the page number is ambiguous in the source that I found this in.) Unfortunately no abstract was given. Can anyone provide the abstract for this article?
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3) A number of Austrian papers (by the same laboratory plus other laboratory co-authors) appear to say something which is related to this issue. I purchased one of the most recent of their papers.
Title: Phytosanitary Improvement of Fruit Tree Species: Diagnostic Strategies in Virus-Indexing of In Vitro Plants
Authors: A. da Camara Machado, D. MendonCa, M.S. Lopes, E. Knapp, V. Hanzer, W. Arthofer, H. Katinger, M. Laimer da Camara Machado
Authors affiliation: this was a combined publication from a laboratory in Portugal and a laboratory in Austria.
Published in: Acta Hort., volumn 472, pages 511-516, (1998).
H. Kuska comment: In the actual paper they say that one of the points of the investigation was: "2) Are virus elimination treatments merely reducing the virus titer, but are they failing to eliminate some viruses entirely. If so, how long does it take for those viruses to reaccumulate to detectable levels?"
My comment (H. Kuska) The above sounds like exactly what we are looking for (but not on roses).
In their Results section, they say:
"Furthermore, we tried to understand whether, in some cases, virus elimination treatments merely reduced the virus titer or failed to eliminate some viruses entirely. If so, how long does it take for those viruses to reaccumulate to detectable levels? Depending on this situation, how many tests are necessary to confirm initial negative test results? It is known that elimination treatments may depress the pathogen titer below the threshold level of detection (Fridlund, 1989). In this respect the effects of thermotherapy on the titer of ASGV and ACLSV were investigated in in-vitro-shoots of several apple cultivars. Irrespective of the duration of thermotherapy treatment (depending on heat sensitivity of the plant material), the virus titer of both ASGV and ACLSV initially was decreased dramatically. Shoots that were multiplied in vitro for over 2 years and used as starting material. Before undergoing thermotherapy, they tested 100 % positive for ACLSV, 100 % negative for ApMV and showed values around the threshold level of 36% for ASGV, as shown in Fig. 1 for the Austrian cultivar Maschanzker. After thermotherapy meristems were excised and regenerating shoots submitted to a multiplication step to increase the number of shoots. After 7 months, plantlets were tested again and showed mainly negative values or values around the threshold for ApMVand ACLSV, indicating that the elimination success was satisfactory. On the other hand, after 7 months 7% of the samples were positive when tested for ASGV, indicating a different reaction pattern (Fig. 1)...................................... It is a definite fact that plants with a double infection of ASGV and ACLSV are more difficult to- treat. However, we do not know, so far, on which mechanism this synergistic effect may rely. Samples of the different groups were compared (Fig. 2). From the nontreated positive control plants, only ApMV was not detected after this period in vitro. Values around the threshold were obtained from the originally negative plantlets. In other cultivars, from originally negative clones multiplied in vitro, a high number of samples showed ASGV positive results after more than one year of in vitro culture. ------------------------------------- The detection of ASGV by ITP in shoot tips from several potted plants of Maschanzker grown for 2 years in the greenhouse (data not shown) was, however, a concern. Therefore, the need for a more reliable detection system seems evident."
The DISCUSSION section contained the following: "As it is still common practice in sanitation programs to carry out in-vitro treatment and ex-vitro re-testing for selection of plant material. The result is a lack of knowledge of the speed of recovery of low pathogen levels under in-vitro conditions (IPGRI/FAO, 1994).......................... Elimination treatments depressed virus titer, as could be shown for a wide range of cultivars. Thermotherapy, however, alters or destroys viral proteins, therefore, serodiagnostics are of little value for reliable early screening. Furthermore, there remain the limitations of current ELISA-based serological tests which might be not sensitive enough to detect very low levels of virus. Also, the time required by the different viruses to recover up to a level of detection from low levels of infection will again be dependent on the pathogen host combination. ACLSV was readily detected after re-accumulation above the threshold level whereas the well-known problems in reliable diagnosis of ASGV were further encountered even after several tests of in-vitro cultures (Fuchs et at, 1988, Gilles and Verhoyen, 1992). We assume that molecular diagnostics will improve the aforementioned problems. Other methods which, so far, are not used for routine diagnostics, like PCR or immuno-capture-PCR, will be introduced into the sanitation program to improve the system of diagnosis for in-vitro plants."
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4) There were several Japanese papers which also appeared to be related to our topic. The following is one:
Title: Evaluation of virus-free bulblet production by antiviral and or heat treatment in in vitro scale cultures of Lilium longiflorum 'Georgia' and L-X 'Casablanca'
Authors: Xu PS, Niimi Y Authors affiliation: Xu PS,
Authors affiliation: Niigata Univ, Fac Agr, Ikarashi 2-8050, Niigata 9502181, Japan
Published in: JOURNAL OF THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE, volumn 68, pages 640-647, (1999).
Abstract: "This study evaluated the effects of chemotherapy (addition of ribavirin or 2-thiouracil to a medium) and/or heat treatment at 35 degrees C on the Production of virus-free bulblets in the scales culture of Lilium longtiflorum 'Georgia' and L. x'Casablanca', infected with lily symptomless virus (LSV), tulip breaking virus-lily (TBV-L), and cucumber mosaic virus (CMV).
1. When scales were cultured on a medium with ribavirin at 50 mu M, ELISA-absorbance values at 405 nm for LSV in the incubated scales decreased as the incubation time lengthened, whereas the TBV-L, values of the scales decreased at almost the same rate as those of the control.
2. When scales of L. longiflorum 'Georgia' were cultured on medium with antiviral chemicals, the number of bulblets formed decreased as concentrations of antiviral chemicals increased. Viruses were detected in about 20% of the bulblets at the end of culture when the scales were cultured on medium with ribavirin and thiouracil at 50 mu M; however, more: than 44% of the bulblets, which were transplanted into soil and cultivated in the greenhouse for 6 months, showed the positive reaction in viruses.
3. Scales excised from bulblets heat-treated at 35 degrees C for 4 weeks formed fewer bulblets than those of control, especially the scales of 'Georgia'.
4. Chemotherapy in combination with thermotherapy was more effective in decreasing the number of virus infected bulblets than was the single treatment. When scales, kept at 35 degrees C for 4 weeks, were cultured on medium with 5 mu M ribavirin, viruses were detected in 30% of the bulblets of 'Georgia' and 6% of those in 'Casablanca' at the end of in vitro culture. However, viruses were detected in 100% of the bulblets in 'Georgia' and 44% of those in 'Casablanca' which were transplanted into soil and cultivated in the greenhouse for 6 months."
My comment (H. Kuska) The Ohio State Research Laboratory at Wooster does subscribe to this journal. However, the article is written in Japanese so I cannot add to the abstract.
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My comment (Henry Kuska): it appears that, at least in some plant systems, heat treated material may have a virus level below the detection limit of ELISA, and that small amount of undetected virus may then grow to detectable levels.
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5) The following is a paper which compares the sensitivity of ELISA detection of PNRSV to other methods:
Title: Comparative analysis of ELISA, nonradioactive molecular hybridization and PCR for the detection of prunus necrotic ringspot virus in herbaceous and Prunus hosts
Authors: Sanchez-Navarro, Apancio, Rowhani & Pallas
Published in: Plant Pathology, volumn 47, page 780, (1998).
Abstract: "Comparative analysis of ELISA, nonradioactive molecular hybridization and PCR for the detection of prunus necrotic ringspot virus in herbaceous and Prunus hosts Three methods were compared for the detection of prunus necrotic ringspot virus in herbaceous and woody plants: DAS-ELISA, nonisotopic dot-blot hybridization and reverse transcriptional polymerase chain reaction (RT-PCR). When purified virus preparations were used, the detection limit of the RT-PCR technique was 1.28 pg mL-1 whereas nonisotopic molecular hybridization and DAS-ELISA allowed detection of 0.8 ng mL-1 and 4 ng mL-1, respectively. Several sample processing procedures were evaluated for virus detection by the nonisotopic molecular hybridization technique. When a very short and simple sample processing method was used, the detection limit of the nonisotopic molecular hybridization technique was 25 times higher than that of DAS-ELISA and 625 times lower than that of RT-PCR. A comparison of the level of virus accumulation in mature fruits and in leaf tissue showed that, on average, 125 times more virus was found in the fruits."
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6) The following paper reports that virus heat inactivation is a
second order (or higher) reaction process.
Many recent plant virus treatment papers use both heat treatment and tissue
culture growth plus often an antiviral drug in the tissue culture. Why do
they go through these extra steps? It is my understanding that the thermal and
chemical treatments reduce the concentration of the virus and then the tissue
culture is used in an attempt to
"outrun" the virus. i.e. the fast growing tissue culture growth may
produce some virus free cells.
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Title: Therapy of virus-infected plants by heat treatment. I. Properties of
tomato aspermy virus and its inactivation at 36.deg.
Authors: Johnstone, G. R.; Wade, G. C.
Authors affiliation: Dep. Agric. Sci., Univ. Tasmania, Hobart, Australia.
Published in: Australian Journal of Botany, volumn 22, pages 437-450, (1974).
Abstract: "Heat treatment therapy of tobacco plants infected with tomato
aspermy virus (TAV) could not be accounted for solely on the basis of a direct
thermal effect since there was no correlation either between the time of
treatment and the percentage of cures or between the time of treatment and the
rate at which TAV multiplied in plants not freed from infection. Inactivation of
TAV at 36° both in vivo and in vitro was a second or higher order reaction. The
infectivity of TAV inocula was increased when leaf tissue was extd. in the
presence of cysteine, and decreased when plants were sprayed with chlorogenic
acid or mushroom tyrosinase. The concn. of polyphenol oxidases increased greatly
and the chlorogenic acid content decreased in tobacco plants grown at 36° as
compared with plants grown at
20-3°. The effect of heat treatment in reducing the level of infective TAV was
partially reversed when the plants were sprayed with chlororesorcinol. The
infectivity of TAV was greatest at pH 7.5 and a buffer molarity of 0.3, and was
destroyed by incubation in vitro with RNase. In tobacco plants grown at 36°,
the pH and ionic strength of the sap decreased and the concn. of RNases
increased."
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7) Title: Characterization of F VIII concentrates produced by two
methods incorporating double virus inactivation.
Authors: Branovic, Karmen; Gebauer, Branka; Trescec, Anda; Benko, Bojan.
Authors affiliation: Institute of Immunology, Zagreb, Croatia.
Published in: Applied Biochemistry and Biotechnology, volumn 69, pages 99-111,
(1998).
Abstract: "The trend toward the prodn. of high purity factor VIII concs.
for clin. use is still in progress. Although all plasma derivs. must undergo
viral inactivation procedures, the possibility of transmission of viral
diseases is not completely eliminated. In order to reduce such risk, we have
included double virus inactivation in the procedure of factor VIII conc. prodn.
In a scale-up procedure for isolation of factor VIII from cryoppt., two methods
were used. The first is based on the chromatog. purifn. of factor VIII after
pasteurization of cryoppt. soln. and solvent/detergent (S/D) inactivation of
viruses. The second is based on multistep pptn. of factor VIII by sodium
chloride and glycine. Viral inactivation was performed by combination of S/D
treatment and heating of final freeze-dried product 30 min at 100°C. The
typical yield of factor VIII activity in the freeze-dried product was about 20%
for the first method, and 25-30% for the second. Electrophoretic analyses of
both factor VIII prepns. by SDS-PAGE and IEF show very low content of
contaminant proteins, in accordance with obsd. 400-650-fold increase of their
specific activity over plasma. Both factor VIII products were stable in the liq.
state for more than 24 h at room temp. The final products, after double viral
inactivation, are considered to be suitable for clin. evaluations."
Note (H. Kuska comment): "the possibility of transmission of
viral diseases is not completely eliminated."
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8) Also note, (H. Kuska comment) I have seen references using the term
"half-life" when describing thermal virus inactivation ( http://www.pp.nchu.edu.tw/cpps/%E6%A4%8D%E7%97%85%E8%A9%9E%E5%85%B8/H.htm
). Concentrations that can be described by "half-lives" never go to zero
(half of half of half, etc.). I wonder if we are looking at
something similar to the distinction between the term "disinfection"
and the term "sterilization" concerning
bacteria. "Disinfection" means to reduce the
bacterial numbers to some arbitrary acceptable number while
"sterilization" means to make completely free from live
bacteria. It appears (to me) that heat treatment, by itself, is similar
to a bacterial disinfection process.
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