isolation of a reovirus from the snake,python regius

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Page 1: Isolation of a reovirus from the snake,Python regius

Arch Virol (1987) 94:135-139 Archives of Virology

© by Springer-Verlag 1987

Isolation of a Reovirus from the Snake, Python reffius

Brief Report

By

W. AHNE I, I. TIIOMSEN I, and J. WINTON 2

i Institute of Zoology and Hydrobiology, University of Munich, Munich, Federal Republic of Germany

2 Department of Microbiology, Oregon State University, Corvallis, Oregon, U.S.A.

With 3 Figures

Accepted December 12, 1986

Summary

A reovirus isolated from a moribund snake was not neutralized by anti- sera against mammalian serotypes, did not agglutinate human 0 erythro- eytes, and produced fusion of IgH 2 cells. The genome contained 10 seg- ments of dst~NA.

Members of the Reoviridae have been isolated from plants, insects, fish, birds, and mammals (6). The existence of viruses in the herpetofauna is of interest since reptiles (e.g. snakes) are known reservoirs for certain viruses transmitted by- mosquitoes (3, 11). RAY~'AVD and AY)R.IA~¢ (12) observed reo- virus-like particles by electron microscopy in cells from the skin of a green lizard (Lacerta viridis). In this paper we describe a reovirus isolated from a python (Python regius). Although members of other virus families have been isolated from reptiles (1, 10, 11), we believe this is the first reovirus recovered from a snake.

In April of 1982, kidney, liver and spleen samples were collected from a moribund adult python showing slight hemorrhagic lesions in the kidney. The samples were homogenized, passed through a 300 nm pore size mem- brane filter, and dilutions were inoculated onto monolayer cultures of IgH 2 cells (2). Infected cells incubated at 2 8 - 3 0 ° C for 24 hours showed eyto- pathogenic effects consisting of granulation, rounding, and lysis of the celts. In fixed and Giemsa-stained cultures, focal cytoplasmic fusion of cells resul-

Page 2: Isolation of a reovirus from the snake,Python regius

136 W. AHNE, I. THOMSEN, and J. WINTON:

Fig. 1. Syneytia formed in a monolayer eulture o f IgH 2 cells after infection with the python reovirus. Cells were incubated at 28 ° C for 24 hours

ted in extensive syncytia (Fig. 1). Treatment of the inoeulum with chloroform and exposure of the cells to 5-iodo-2'-deoxyuridine did not, affect viral repli- cation indicating the absence of a lipid containing envelope and suggesting the virus had an t~NA genome. Staining of infected cells with acridine orange showed green cytoplasmic inclusions characteristic of those produced by reoviruses (4).

Electron microscopy of negatively stained virions revealed spherical to iscosahedral particles 70-75 nm in diameter (Fig. 2 a) composed of a double capsid layer (Fig. 2 b). Follm~dng t rea tment with a-ehymotrypsin (7), the outer capsid was removed leaving 50-55 nm subviral particles (Fig. 2 c). Partially purified virions were treated with freon and the dst~NA was extrac- ted with phenol and precipitated with ethanol. Polyaerylamide gel electro- phoresis using 9 per cent acrylamide SDS-PAGE vertical slab gels and the buffer system of LAE~I~LI (9) showed the viral genome contained ten seg- ments of dsRNA. The relative migration of the three large, three medium and fbur small segments was similar to that of reovirus 3 run in the same gel (Fig. 3). The greatest differences were observed in the migration rates of the large and medium segments of dsRNA. Antisera against mammalian reo- virus serotypes 1, 2, and 3 did not neutralize infectivity of the python isolate. The virus was not able to hemagglutinate human type 0 erythrocytes at either 4 ° C or room temperature.

Page 3: Isolation of a reovirus from the snake,Python regius

l~eovirus from a Snake 137

Our resul ts showed t ha t while the reovirus f rom py thon was similar to the mammMian r eov imses in size, morphology , eapsid s t ructure , and orga- nizat ion of the dsRNA genome (6), the virus differed f rom them by being able to cause cell fusion in vitro and by the lack of hemagglut ina t ing act ivi ty for h u m a n e ry th roey tes . The py thon isolate was no t neut ra l ized by ant i sera against any of the th ree mammal i an sero types . The absence of hemaggluti- na t ion and the ability to cause cell fusion are typical character is t ics of avian reovi ruses (8); however , the p y t h o n reovirus had an e l ee t rophe ro type differ- en t f rom the p a t t e r n gene ra t ed by avian isolates (5), par t icu lar ly in the migra t ion ra te of the S 1 segment . W I L c o x and Co?aPANs (13) descr ibed a reovirus r ecove red f rom the flying fox (Pter(ypu8 poliocephalus) t h a t showed character is t ics in t e rmed ia te be tween the reoviruses of mammal i an and avian origin. SerologieM and biochemical compar i son of the py thon reovirus with avian isolates and the Nelson Bay virus will be neces sa ry before taxo-

Fig. 2. Electron micrographs of negatively stained virions and cores of the python reo- virus. A a complete particle with typical reovirus-like morphology; B a typical empty shell. The double eapsid structure becomes evident by the particle in C where virions in various stages of digestion following treatment with a-ehymotrypsin are seen. Intact. virions were 70-75 nm in diameter and cores were 50--55 nm. All mierographs are the same magnifica-

tion. Bar equals 75 nm

Page 4: Isolation of a reovirus from the snake,Python regius

138 W. A ~ E , I. TtIOMSEN, and J. WINTON:

Fig. 3. Comparison of the genomes of mammalian reovirus type 3 and the reovirus recove- red from python. Ten segments of dsRNA were observed in the genome of the new isolate using 9 per cent polyaerylamide gels. Reovirus type 3 (Dearing strain) segments are in A

and B; C blank space; python reovirus segments arc in D and E

nomic placement, of this new virus can be made. The python isolate also differed from reoviruses recovered from other poikilothermie vertebrates (e.g. fish) that have eleven dsRNA segments (14).

Acknowledgements The authors with to thank Ms. Cathy Lannan for her excellent technical assistance and

Mr. A1 Soeldncr for performing the electron microscopy. This work was supported by the Deutsche ForschungsgemeinschM't, Bonn, Federal Republic of Germany.

References

1. AgNE W (1977) Bei Reptilien vorkommende Viren. In: REICHENBAcH-KLINKE HH (ed) Krankheiten der Reptilien, 2nd edn. Gustav Fischer, Stuttgart New York, pp 13- 3O

Page 5: Isolation of a reovirus from the snake,Python regius

Reovirus from a Snake 139

2. CLARK HF, COttEN MM, KARYON DT (1970) Characterization of reptilian cell lines established at incubation temperatures of 23 to 36 ° C. Proc Soc Exp Biol Med 133: 1039-1040

3. GERHARDT LD, SWANTON GJ, HILL DW, COLLETT GC (1964) Natural overwintering hosts of the western equine encephalitis. N Engl J Med 217:172-177

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6. JOKLIK V~'K (1983) The reovinls particle. In: JoKLIK WK (ed) The reoviridae. Plenum, NewYork London, pp 9-78

7. JOKLIK W ~ (1972) Studies of the effect of chymotrypsin on reovirions. Virology 49: 700-715

8. KAWAMURA H, SHIMIZU F, MAEDA M, TSURAItARA H (t965) Avian reovirus: its pro- perties and serological classification. Nail Inst Anim Health Q 5:115-124

9. LAEMMLI UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227:680-685

10. LUNaER PD, CLARK HF (1978) Reptilia related viruses. Adv Virus Res 23:159-204 11. MARCUS LC (1981) Veterinary biology and medicine of captive amphibians and repti-

les. Lea& Febiger, Philadelphia, p l l0 12. RAYNAUD A, ADRIAN M (1976) Lesions cutanee a structure papillomateuse associees a

des virus chez le lezard vert (Lacerta viridis). C R Acad Sci [III] 283:845-847 13. WILOX GE, Co~]'A~s RW (1983) Characterization of Nelson Bay virus and virus-indu-

ced cell fusion. In: COMFA~'S RW, BISROP DHL (eds) Double-stranded RNA viruses. Else~der, NewYork, pp 391-403

14. WINTON JR, LANNAN CN, FRYER JL (1983) Further characterization of a new reo- virus of poikilothermic vertebrates. In: COMI~ANS RW, BlSttOP DHL (eds) Double- stranded I~NA viruses. Elsevier, NewYork, pp 231-236

Authors' address: Dr. Vg. AHNE, Institute of Zoology and Hydrobiology, University of Munich, Kaulbachstrasse 37, D-8000 Munich 22, Federal l~epublie of Germany.

Received November 25, 1986