Acessibilidade / Reportar erro

Time course of virus-like particles (VLPs) double-stranded rna accumulation in toxigenic and non-toxigenic strains of Aspergillus flavus

Distribuição de vírus RNA de fita dupla em cepas de Aspergillus flavus toxigênica e não toxigênica

Abstracts

Two strains of Aspergillus flavus, non-toxigenic NRRL 6550 and toxigenic NRRL 5940, were studied over a period of 44 days, in order to detect the presence of virus-like particles (VLPs) by means of electron microscopy (EM) and nucleic acids electrophoresis. Only the toxigenic strain contained VLPs, presenting three-segmented dsRNA. An increase in VLPs number was observed during the exponential phase of fungal growth, up to day 12 of culture; after this, higher levels of aflatoxin production in toxigenic NRRL 5940 mycelia occurred in parallel with decreased VLPs replication.

mycovirus; double-stranded RNA; virus-like particles; Aspergillus flavus


Neste trabalho, foram estudadas duas cepas de Aspergillus flavus não toxigênica (NRRL 6550) e toxigênica (NRRL 5940). As cepas foram cultivadas durante 44 dias objetivando a pesquisa de partículas semelhantes a vírus (VLPs), por meio de microscopia eletrônica e pesquisa de ácidos nucléicos, através de eletroforese. Somente as cepas toxigênicas continham "VLPs", as quais apresentaram 3 segmentos de RNA de fita dupla. Um aumento no número de "VLPs" foi observado com 12 dias de cultivo, período correspondente a fase exponencial de crescimento de A. flavus.

micovírus; RNA de fita dupla; partículas semelhantes a vírus; Aspergillus flavus


TIME COURSE OF VIRUS-LIKE PARTICLES (VLPS) DOUBLE-STRANDED RNA ACCUMULATION IN TOXIGENIC AND NON-TOXIGENIC STRAINS OF ASPERGILLUS FLAVUS

1Valéria N. Silva; 1Edison Luiz Durigon; 2Maria de Fátima Costa Pires; 1Alexandre Lourenço; 1Maria Jacinta de Faria; 1Benedito Corrêa* * Corresponding author. Mailing address: Av. Prof. Lineu Prestes 1374, Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, CEP 05508-900, São Paulo, SP, Brasil, Tel.: (+5511) 3818-7295; FAX: (+5511) 3818-7354; E-mail: correabe@usp.br

1Departamento de Microbiologia, Instituto de Ciências Biomédias, Universidade de São Paulo, São Paulo, SP, Brasil.

2Seção de Microscopia Eletrônica, Divisão de Virologia, Instituto Adolfo Lutz, São Paulo, SP, Brasil

Submitted: February 10, 2000; Returned to authors for corrections: April 13, 2000; Approved: January 18, 2001

ABSTRACT

Two strains of Aspergillus flavus, non-toxigenic NRRL 6550 and toxigenic NRRL 5940, were studied over a period of 44 days, in order to detect the presence of virus-like particles (VLPs) by means of electron microscopy (EM) and nucleic acids electrophoresis. Only the toxigenic strain contained VLPs, presenting three-segmented dsRNA. An increase in VLPs number was observed during the exponential phase of fungal growth, up to day 12 of culture; after this, higher levels of aflatoxin production in toxigenic NRRL 5940 mycelia occurred in parallel with decreased VLPs replication.

Key words: mycovirus, double-stranded RNA, virus-like particles, Aspergillus flavus

INTRODUCTION

Virus-like particles (VLPs) were discovered in 1962 by Hollings, who extracted three types of particles from the fruit bodies of diseased mushrooms in England. Differents VLPs may be present in a single fungal strain (9) and most of them are found to posses segmented, double-stranded RNA genomes (13). They are widespread among fungi and, despite their presence being typically symptomless (3), some exceptionally cause adverse effects on their hosts. Examples of these effects are 'La France' disease of the white-button mushroom Agaricus bisporus (11, 12), expression of lethal toxins encoded by viral dsRNA in certain strains of Saccharomyces cerevisiae and Ustilago maydis (1, 2), and induction of hypovirulence in the chestnut blight fungus Cryphonectria parasitica associated with dsRNA (7).

Several Aspergillus species have been found to be infected with mycoviruses, including A. foetidus (4, 17), Aspergillus section Circundati and Fumigati (23), Aspergillus section Nigri and section Flavi (8, 22).

Certain strains of Aspergillus flavus and all strains of A. parasiticus have been reported to synthesize aflatoxins, which are highly toxic and potent carcinogenic secondary metabolites (14). The absence of aflatoxin production in NRRL A - 12268, a strain of Aspergillus flavus, originally correlated with the occurrence of nucleic acid-free VLPs (16). However, a more extensive investigation failed to establish a relationship between absence of VLPs and aflatoxin production in other fungal strains (10, 24). Double stranded RNA particles in non-toxigenic strain of A. flavus (NRRL 5565 = NRRL A -12268) as shown by Mackenzie and Adler (16) had a genome identical in size to a dsRNA found in Penicillium chrysogenum (20). Strain NRRL 5565 cured of dsRNA infection by exposing it to an RNA synthesis inhibitor (cicloheximide) developed the ability to produce aflatoxins (18); furthermore, after artificial infection with virus isolated from P. chrysogenum, the production of aflatoxins ceased. However, Elias and Cotti (8) detected dsRNA genomic elements in 10 out of 92 isolates of Aspergillus section Flavi yet the aflatoxin-producing ability of the isolates was not affected by dsRNA infection (16, 24). Strains that produced high levels of aflatoxins were as likely to be infected by dsRNA as strains that produced no aflatoxins. Moreover, curing strains infected with dsRNA did not result in altered aflatoxin-producing ability, as previously suggested (10, 18, 19, 20). The high frequency of A. flavus in the ecosystem, its serious implications to human and animal health due to ingestion of food contaminated with aflatoxins, and the limited data on fungi/VLPs interactions, motivated the present investigation. We therefore evaluated the presence of VLPs in toxigenic and non-toxigenic strains of A. flavus over a total growth period of 44 days, by electron microscopy and electrophoresis techniques.

MATERIALS AND METHODS

Strains of Aspergillus flavus

The two strains of A. flavus used, namely: toxigenic NRRL 5940 (producer of aflatoxin B1 and B2), and non-toxigenic NRRL 6550, were provided by Northern Regional Research Laboratory (NRRL), Department of Agriculture - Illinois - USA.

Spore suspension

A. flavus was grown on potato dextrose agar at 25ºC for 7 days. Spores were harvested by rinsing the culture tube with sterile 0.1M sodium phosphate buffer (PBS) pH 7.2 and then transferred to a sterile tube containing a drop (0.05 mL) of Tween 80, which was added to disperse the spores and to homogenize the suspension. Spore counts were done in a Neubauer chamber; the final suspension was adjusted to approximately 105 spores per 0.3 mL with sterile 0.1M PBS, pH 7.2.57

Growth Curves

Growth curves were established on the basis of three determinations of fungal dry weight values, recorded every 4 days over a period of 44 days; the first measure was taken after 2 days of cultivation. Spore suspensions were inoculated into 12 Roux bottles containing yeast extract sucrose (YES) semi-synthetic medium, normally employed in B1 Aflatoxin (AFB1) production tests. To avoid light degradation of AFB1, incubation was carried out at 25ºC in total darkness. At the indicated time intervals, the Roux bottles content was filtered through Whatman paper number 1. For quantitation of AFB1 levels, broth and mycelium were used for the toxigenic samples and only the mycelium for the non-toxigenic samples. Mycelia were washed with sterile sodium 0.1M PBS, pH 7.2, and incubated at 80ºC for 4 days, in order to achieve a constant weight of the dried fungal mass.

Quantification of aflatoxin

The extraction of aflatoxin was carried out as previously described (5) and quantitated in a xenon bulb photodensitometer (Shimadzu CS9000).

Preparation of samples for EM and Electrophoresis

According to the growth curves and AFB1 production, the following time schedule was established to search for VLPs: 2, 4, 12, 20, 24, 28 and 40 days for NRRL 6550 and 2, 4, 12, 28, 32, 36 and 40 days for NRRL 5940. At the indicated time intervals, Roux bottles contents were filtered, as previously described, and the mycelia were washed 3 times with sterile distilled water; the recovered broth and mycelia were conditioned and stored at -20ºC until use.

Electron microscopy

Sampled mycelia were freeze-thawed 4 times in order to break the cell wall and release VLPs. The material was centrifuged at 353xg for 20 minutes and the supernatant ultracentrifuged at 153 610xg for 4 hours. Using the "reverse drop" technique, the ultracentrifuged sediment was allowed to adhere to a formvar-carbon coated grid (300 mesh) for 30 minutes. The grid was then stained with 2% potassium phosphotungstate (PTK), pH 6.4, for 5 minutes and examined for VLPs in a Phillips EM 400 T microscope. Estimates of VLPs number at each time point studied were done by counting particles in only one grade per period. These estimates were done so as to evaluate the amount of particles present in mycelia and to compare it with the concentration of dsRNA.

Extraction of nucleic acids

The mycelium (0.2 g) of each strain was ground under liquid nitrogen and the frozen powder was suspended in 0.1M TRIS/HCl pH 7.3 containing 10% SDS and Proteinase K (PK). The sample was incubated for 30 minutes at 37ºC, and the nucleic acids were extracted with phenol:chloroform:isoamyl alcohol (25:24:1). Cellular debris was removed by centrifugation (3920xg, 5 min.); nucleic acids were precipitated with ethanol and 20% NaCl (-20ºC overnight), centrifuged (6625xg, 30 minutes, 4ºC), and dried at room temperature.

VLPs from the filtrate were concentrated by PEG 6000 precipitation (15). Briefly, the filtrate was diluted in 50 mM glycin, pH 9.0 added of 10% tryptose phosphate buffer (TPB) and after 30 sec the pH was adjusted to 9.0 with 1N NaOH. This material was agitated (30 sec) and centrifuged (10 000xg, 30 min., 4ºC); pH of the supernatant was adjusted to 7.5 with 1N HCl. To each 10 mL of supernatant, 0.8g of PEG 6000 was added and the pH adjusted to 7.5; the sample was then incubated in a shaker (1h and 30 min, 4ºC) and centrifuged (10 000xg, 20 min, 4ºC). For the RNA extraction, the pellet obtained was resuspended in 10 mL of 150 mM NaHPO4 9.5, and the pH was adjusted to 9.0; the sample was then sonicated twice (30 sec, 50 mA, on ice), agitated (30 sec) at room temperature and centrifuged (10 000xg, 30 min, 4ºC). The pH of the supernatant was adjusted to 7.2 and 7.5 and the material was stored at -20ºC until use for electrophoresis of nucleic acids.

The extracted nucleic acids were separated on 7.5% polyacrylamide gel (5h, 40mA) as whole samples and after digestion with DNase I and RNase I "A" in DNase buffer (50 mM TRIS, pH 7.5 added of 10 mM MgCl2) using NCDV (Nebrask Calf Diarrhea Virus) dsRNA as standard (silver nitrate staining). Agarose gel electrophoresis of the material was also performed (0.8% agarose, 4h, 40V) using the 1 Kb DNA ladder molecular markers (ethidium bromide staining). Band concentration and molecular weight markers determinations were made with the aid of the computer program "DNAstar-Computer Systems for Molecular Biology and Genetics" (1990).

RESULTS

The exponential phase of growth started on day 2; maximum biomass accumulation was recorded on day 24 for the non-toxigenic strain NRRL 6550 and on day 36 for the toxigenic strain NRRL 5940. The production of aflatoxin B1 in mycelia of the toxigenic strain (NRRL 5940) began on day 4 and peaked at day 32 of culture. No production of aflatoxin B1 was noted in the non- toxigenic strain.

VLPs were detected in mycelia of the toxigenic strain at all time points studied and measured approximately 33nm in diameter (Fig. 1). The number of particles increased from day 2 to day 12 and decreased thereafter, dropping to very small numbers on days 28, 32, 36 and 40 (Table 1). No VLPs were observed in the mycelia of non-toxigenic strain, or in the culture filtrates of both strains.


Agarose and polyacrilamyde gel electrophoresis of nucleic acids from mycelia of the toxigenic strain revealed the presence of 3 bands at all the time points studied. The intensity of these bands increased from day 2 till day 12 of culture and tended to decrease thereafter (Table 1). The bands were unaffected by digestion with Proteinase K and DNase I but were sensitive to RNase I "A", indicating that they were dsRNAs. No bands were detected in nucleic acid extracts from mycelia of the non-toxigenic strain.

The A. flavus dsRNA migrated at positions equivalent to 3.7, 3.4 and 2.9 Kb of the DNA ladder, and those from other time points with the 3.5, 3.0 and 2.8 Kb positions (Fig. 2). The corrected size of the A. flavus dsRNA using RNA standards has not been determined yet. The estimated concentrations of dsRNA correlated with the data on number of VLPs visualized by electron microscopy (Table 1). The fungal biomass, concentration of aflatoxin B1 and estimates of dsRNA of the toxigenic strain are shown in Fig. 3.



DISCUSSION

Our comparison of growth curve, estimated VLPs number and concentration of dsRNA revealed a correlation between fungal growth, increase in VLPs count and concentration of dsRNA at day 12 of culture - a time when the fungus was undergoing a transition from trophophase (characterized by its primary metabolism) to idiophase (characterized by its secondary metabolism). Also, day 12 of culture was part of the exponential phase of growth, when cells are metabolically active. This, in fact, may have provided ideal conditions for viral replication and could explain the occurrence of greater number of VLPs at such time period. Some authors, on the other hand, have reported maximum viral replication associated with the stationary growth phase of fungi (6, 21, 22). In this study, we observed that, during the period when AFB1 concentrations were low (up to day 28 of culture), VLPs counts and viral dsRNA concentrations fluctuated and peaked on day 12. Conversely, when the AFB1 levels reached higher values (after day 28), the VLPs counts and dsRNA concentrations were low.

The results obtained in this study apparently showed a lack of correlation between production of aflatoxins and presence of VLPs in the A. flavus strain NRRL 5940. This agree with the findings of Elias and Cotti (8), who detected dsRNA in 10 out of 92 strains of Aspergillus seção Flavi and found no correlation between production of aflatoxins and presence dsRNA, as previously suggested (16, 24). According to these authors, the molecular size estimates of the dsRNA components ranged from 0.4 to greater than 10.0 Kb, relative to the migration of dsRNA fragments of phage submitted to digestion with hindIII. The number of dsRNA components infecting an individual isolate ranged from one to nine, and apparently no isolate contained the same molecular size as estimated by eletrophoretic migration in 1.0% agarose.

It is quite possible that different types of VLPs may influence positively or negatively the production of aflatoxins by activating or inhibiting aflatoxin gene expression. The results obtained in this study can be used in experimental transmission to other fungal strains.

RESUMO

Distribuição de vírus RNA de fita dupla em cepas de Aspergillus flavus toxigênica e não toxigênica

Neste trabalho, foram estudadas duas cepas de Aspergillus flavus não toxigênica (NRRL 6550) e toxigênica (NRRL 5940). As cepas foram cultivadas durante 44 dias objetivando a pesquisa de partículas semelhantes a vírus (VLPs), por meio de microscopia eletrônica e pesquisa de ácidos nucléicos, através de eletroforese. Somente as cepas toxigênicas continham "VLPs", as quais apresentaram 3 segmentos de RNA de fita dupla. Um aumento no número de "VLPs" foi observado com 12 dias de cultivo, período correspondente a fase exponencial de crescimento de A. flavus.

Palavras-chave: micovírus, RNA de fita dupla, partículas semelhantes a vírus, Aspergillus flavus

  • 1. Adler, J.; Wood, H.A.; Bozarth, R.F. Virus-like particles from killer, neutral and sensitive strains of Saccharomyces cerevisiae J.Virol., 17: 472-476, 1976.
  • 2. Bozarth, R.F.; Koltin, Y.; Weissman, M.B.; Parker, R.L.; Dalton, R.E.; Steinlauc, R. The molecular weight and packaging of dsRNAs in the mycovirus from Ustilago maydis killer strains. Virology, 113: 492-502, 1981.
  • 3. Buck, K.W. Fungal virology: an overview. In: Buck, K.W. (Editor), Fungal Virology. CRC Press, Boca Raton, 1-84, 1986.
  • 4. Buck, K.W.; Ratti, G. Biophysical and Biochemical properties of two viruses isolated from Aspergillus foetidus J. Gen. Virol., 27: 211-224, 1975.
  • 5. Davis N.D.; Diener, U.L.; Eldridge, D.W. Production of aflatoxin B1, G1 by Aspergillus flavus in a semisynthetic medium. Appl. Microbiol., 14: 378-380, 1969.
  • 6. Detroy, R.W.; Still, P.E. Patulin inhibition of mycovirus replication in Penicillium stoloniferun J. Gen. Microbiol., 92: 167-174, 1975.
  • 7. Dodds, J.A. Association of type 1 virus-like dsRNA with club-shaped particles in hypovirulent strains of Endothia parasitica Virology, 107: 1-12, 1980.
  • 8. Elias, K.S.; Cotti, P.J. Incidence and stability of infection by double-stranded RNA genetic elements in Aspergillus section Flavi and effects on aflatoxigenicity. Can. J. Bot, 74: 716-725, 1996.
  • 9. Ghabrial, S.A. Effects of fungal viruses on their hosts. Ann. Rev. Phytopathol., 18: 441-461, 1980.
  • 10. Gussack, G.; Bennet, J.W.; Cavalier, S.; Yatsu, L. Evidence for the parassexualcycle in a strain of Aspergillus flavus containing virus-like particles. Mycopathologia, 61(3): 159-165, 1977.
  • 11. Hollings, M. Viruses associated with a Die-Back disease of cultivated mushroom. Nature, 196: 962-965, 1962.
  • 12. Hollings, M.; Gandy, D.G.; Last, F.T. A virus disease of a fungus: die-back of a cultivated mushroom. Endeavour, 22: 112-117, 1963.
  • 13. Inglis, P.W.; Valadares-Inglis, M.C. Rapid isolation of double-stranded RNAs from entomopathogenic species of the fungus Paecilomyces using a commercial minicolumn system. J. Virol. Meth., 67: 113-116, 1997.
  • 14. Krishna, S.B.; Kaushal, K.S. Aflatoxins: their biological effects and ecological significance. In: Bhatnagar D.; Lillehoj E.B.; Arora D.K. (Editors), Handbook of Applied Mycology Mycotoxins in Ecological Systems. Marcel Dekker, New York, 59, 1992.
  • 15. Lewis, G.D.; Metcalf, T.G. Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus from oystes, water, and sediment samples. Appl. Environm. Microbiol., 54: 1983-1988, 1989.
  • 16. Mackenzie, D.E.; Adler, J.P. Virus-like particles in toxigenic Aspergilli. Abstr. Annu. Meet. Am. Soc. Microbiol., American Society for Microbiology, Washington, D.C., 68, 1972.
  • 17. Ratti, G.; Buck, K.W. Virus particles in Aspergillus foetidus: a multicomponent system. J. Gen. Virol., 14: 165-175, 1972.
  • 18. Schmidt, F.R.; Davis, N.D.; Diener, U.L.; Lemke, P.A. Cyclohexamide induction of aflatoxin synthesis in a non-toxigenic strain of Aspergillus flavus Bio/Technology, 1: 794-795, 1983.
  • 19. Schmidt, F.R.; Esser, K. Aflatoxins: Medical, Economic impact, and prospects for control. Proc. Biochem, 167-174, 1985.
  • 20. Schmidt, F.R.; Lemke, P.A.; Esser, K. Viral influences on aflatoxin formation by Aspergillus flavus Appl. Microbiol. Biotechnol, 24: 248-252, 1986.
  • 21. Still, P.E.; Detroy, R.W.; Hesseltine, C.W. Penicillium stoloniferum virus altered replication in ultraviolet derived mutants. J. Gen. Virol., 27: 275, 1975.
  • 22. Varga, J.; Kevey, F.; Vágvölgyi, C. Double-stranded RNA mycoviruses in section Nigri of the Aspergillus genus. Can. J. Microbiol., 40: 325-329, 1994.
  • 23. Varga, J.; Rinyu, E.; Kevei, E.; Tóth, B.; Kozakiewicz, Z. Double-stranded RNA mycoviruses in species of Aspergillus sections Circundati and Fumigati Can. J. Microbiol., 44: 569-574, 1998.
  • 24. Wood, H.A.; Bozarth, R.F.; Adler, J.; Mackenzie, W. Proteinaceus virus-like particles from an isolated of Aspergillus flavus J. Virol., 13: 532-534, 1974.
  • *
    Corresponding author. Mailing address: Av. Prof. Lineu Prestes 1374, Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, CEP 05508-900, São Paulo, SP, Brasil, Tel.: (+5511) 3818-7295; FAX: (+5511) 3818-7354; E-mail:
  • Publication Dates

    • Publication in this collection
      02 Aug 2001
    • Date of issue
      Mar 2001

    History

    • Accepted
      18 Jan 2001
    • Reviewed
      13 Apr 2000
    • Received
      10 Feb 2000
    Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
    E-mail: bjm@sbmicrobiologia.org.br