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ATTEMPTS TO CLASSIFY UNGROUPED ARBOVIRUSES BY ELECTRON MICROSCOPY

A. EL MEKKI (1), G. VAN DER GROEN (1), S.R. PATTYN (1,2)
1. Instituut voor Tropische Geneeskunde, Laboratory of Bacteriology and Virology, Nationalestraat 155, 2000 Antwerpen, Belgium.
2. University of Antwerp, Wilrijk, Belgium.

It is a general biological rule that each morphological virus group contains numerous serotypes. Therefore the Marburg virus group for which at present only two different viruses are known: Marburg and Ebola, may be expected to contain more viruses with the same morphology.

There is a possibility that such viruses may be present among the "ungrouped" arboviruses. Indeed numerous viruses are isolated in arbovirus laboratories all over the world. The general procedure is isolation of virus by intra-cerebral inoculation of newborn mice, after which the isolate is tested for ether and/or desoxycholate sensitivity and a haemagglutinin prepared. If haemagglutinin is not present or if antigenic relationship with known arboviruses is not detected, the new virus is classified as "ungrouped". The morphology of such isolates is generally not studied. There is possibility that other members of the MarburgEbola virus group might be present among ungrouped arboviruses. We therefore started a study of the morphology of some of these viruses. Because there is a definite advantage to work with tissue culture harvests, all viruses were also inoculated into Vero cells. Preliminary results are given in the present paper.

MATERIAL AND METHODS

Viruses. Ungrouped arboviruses listed in table 1 not producing a haemagglutinin were investigated.

All viruses were received in the freeze dried state, except Tettnang which was a frozen mouse brain suspension. Upon receipt the viruses were rehydrated and inoculated intra-cerebrally into newborn mice and in Vero cells.

ELECTRON MICROSCOPY

a. Floating method. A carbon coated grid was deposited on a drop of virus sus pension for one minute, the grid was then blotted with paper, floated on a drop of stain for 3 to 5 seconds and blotted again. The following stains have been used: phosphotungstic acid at 0.5, 1 and 2%, pH ranging from 6.4 to 7.1 and uranyl-acetate 0.5, 1 and 2%.

An alternative technique is to put a drop of virus suspension on a grid, let it dry and float the grid on a drop of stain for 30 seconds and finally blot it with filter paper.

b. Immune-electron microscopy. The method used is a modification of the one described by Anderson and Doane I for identification of rotaviruses. Serum-agar mixtures containing noble agar 1% in phosphate buffered saline and antisera diluted 1/50 or 1/100 depending upon their neutralization titers are pipetted into disposable microtitre plates, (sealed with sterile adhesive tape, these can be stored at 4º until use). A drop of the virus suspension to be examined is put on the agar containing homologous antiserum. A grid is floated on it and incubated at 37ºC for 80 minutes. Grids are then removed and floated for 3 seconds on 2% P.T.A. pH 6.8. Both pialoform and carbon coated grids were used.

c. Pseudo-replica technique. The method used is a modification from Scharp (2) Agarose 2% is prepared in petridishes. A block of about one square cm is cut and mounted on the edge of a glass slide. A drop of virus suspension is put on the surface of the agarose and allowed to dialize and evaporate until the surface is completely dry, this usually takes about 10 minutes. Two or three drops may be applied. Two drops of formvar are then added and left for a few seconds and excess formvar is removed by keeping the slide vertically against blotting paper. When the film is dry, the edges of the block are cut with a sharp knife. By dipping the block gently into stain, a film is released. A grid mounted on a metal rod is pushed onto the floating film, through the stain and turned out of it. Excess film and stain are removed by rotating the edge of the rod against a blotting paper. Uranylacetate in 0.5 and 2% concentrations and P.T.A. in concentrations of 2 to 4% pH 6.8 were used. Electron microscopic observations were done at an accelerating voltage of 80v.

TABLE 1 UNGROUPED ARBOVIRUSES INVESTIGATED


Name


Abrv.


Isolation


Origin


Mice


Vero


EM


Bangoran


BGN


Culex


Dakar


+


-


-


Bangui


BGI


Man


Dakar


+


-

 

Bobaya


BOB


Trudus


Dakar


+


+


+


Bimbo


BBO


Euplectus


Dakar


+


-


-


Gomoka


GOM


Anopheles


Dakar


+


-


-


Gossas


GOS


Tadaria


Dakar


+


-

 

Kannamangalam


KAN


Bird


Yaru


+


-


-


Keuraliba


KEU


Tetara Kempi


Yaru


+


-


-


Kolongo


KOL


Euplectus


Yaru


+


-

 

Kowanyama


KOX


Anopheles


Yaru


+


-


-


Landjia


LJA


Bird


Dakar


+


-


-


Le Dantec


LD


Man


Yaru


+


-


-


Minnal


MIN


Culex


Yaru


+


-

 

Ouango


OUA


Bird


Yaru


+


-


-


Oubangui


OUB


Culex


Dakar


+


-


-


Salanga


SAL


Athomys M.


Dakar


+


-


-


Sandjimba


SJA


Bird


Dakar


+


-


-


Sebokele


SEB


Tick


Dakar


+


-

 

Tanga


TAN


Anopheles


Yaru


+


-

 

Tataguine


TAT


Culex


Yaru


+


-

 

Tembe


TME


Anopheles


Yaru


+


-


-


Thottapalayam


TPM


Shrew


Yaru


+


-


-


Toure


TOU


Tetara Kempi


Yaru


+


-


-


Trinity


TNT


Mosquito


Yaru


+


+


+


Upolu


UPO


Bird


Yaru


+


-


-


Wongorr


WGR


Aedes sp.


Yaru


+


-

 

Yata


YATA


Mansonia


Yaru


+


-


+


Yogue


YOG


Bat


Yaru


+


-


-


Zinga


ZGA


Mansonia


Dakar


+


+


+


An B 4268

   

Dakar


+


+


-


An Br 1398d

   

Dakar


+


-


-


An B 4289

   

Dakar


+


-


-


An Y 1444

   

Dakar


+


-


-

TABLE 2 COMPARISON OF EM TECHNIQUES ON A SELECTED SAMPLE OF VIRUSES


Virus Tested


Titre TCD 50ml


Floating Method


Serum-In-Agar


Pseudo-replication


Bobaya (TC)


10^6


-


-


+


Zinga (TC)


10^5.7


-

 

+


Trinity (TC)


10^7


-

 

+


Yata (MB) -

 

+

   

Keuraliba (MB)

 

+

 

+


VSV (TC)


10^6.7


+

 

+


MB (TC)


10^3.5


-


+


+


Chik. (TC)


10^6


-


+


+


SF. (TC)


10^6.2


-


+


+


SI. (TC)


10^6.7


-


+


+


YF. (MB)


10^5.2


-


+


+


WN. (MB)


10^5


-


+


+


BUN. (TC)


10^7.2


-


+


+


Polio 1 (TC)


10^4.7


-


+


+


Polio 3 (TC)


10^5.7


-


+


+


TC = tissue culture harvest

MB = mouse brain harvest

2. Results of Electron Microscopy.

1. Ungrouped Arboviruses producing CPE in Vero cells. Table 2 shows the viruses producing CPE in Vero cells. One virus, Trinity, did not produce CPE when the hydrated freeze dried virus was inoculated. A newborn mouse brain harvest however, inoculated into Vero cells produced CPE and the virus could be repeatedly passed in Vero cells.

2. Results of Electron Microscopy

a. We first investigated those viruses producing CPE in Vero cells. Although VSV, used as a model and harvested from Vero cells (titer 10^6.7 TCD 50/ml) could be readily detected by the floating techniques described, none of the following viruses could be detected in the electron microscope, by this technique : (Vero titer: 10^7 TCD 50/ml), Trinity. (Vero titer: 10^6 TCD 50/ml), Bobaya. Vero titer: 10^5.7 TCD 50/ml), Zinga. Vero titer: 10^4.5 TCD 50/ml), AbB 4268.

b. Immune electron microscopy. To test this method known arboviruses were investigated. Virus-antibody aggregates were detected as indicated in table 2. Aggregates of virus particles were surrounded by a diffuse electron-transparent halo of antibody. These complexes were sometimes mixed populations of empty and full particles. Similar complexes were identifiable when antisera and viruses were added on 1% noble agar, but not when the viruses were inactivated by 2.5% glutaraldehyde at 37oC for 30 minutes. Although the titer of the virus used (MIB) was very low (10^3.5 TCD 50/ml), we were able to detect virus-antibody aggregates but the empty particles outnumbered the full ones. When viruses and antisera were mixed in a parafilm, incubated at 37ºC for 30 minutes, no complexes were identifiable. Since antisera against ungrouped viruses were not available, this technique could not be applied to them.

c. Pseudoreplica technique. By this method all the viruses indicated in table 2 were detected: enteroviruses, a rhabdovirus (VSV), Bunyamwera, flaviviruses and alphaviruses. Three ungrouped viruses harvested from Vero cells could be visualized : Bobaya, Zinga and Trinity. They are spherical viruses with an average diameter of 90-100 mm resembling Bunyamwera virus. Two viruses, Yata and Keuraliba that do not propagate in Vero cells have been visualized in a membrane filtered mouse brain suspension and identified as rhabdoviridae, average dimensions of 65 x 180 nm. Several attempts to visualize other ungrouped arboviruses prepared from mouse brain remained negative.

d. Comparison of the sensitivity of the antiserum-in-agar and the pseudo replica techniques. A suspension of Sindbis virus (10^6 TCD 50/ml) was diluted 1/4, 1/16, 1/164, 1/256 and examined by both techniques. Results were as follows :


VIRUS VISUALIZED BY

 

Serum-in-agar


Pseudo replication


pure (10^6)


+


+


1/4


-


+


1/16


-


+


1/64


-


+


1/256


-


-


The pseudoreplica technique thus seems to be more sensitive. It also allows an easier and more rapid screening of the grids and produces much clearer pictures for morphologic study.


DISCUSSION

It is not surprising that the floating technique did not allow to detect any viruses except VSV because of the relatively low titers of the suspensions that could be tested. It is often stated in the literature that to detect virus particles by this technique, these should be present in concentrations of more than 10^7 or 10^8 particles/ml.

The serum-in-agar technique appears to be more sensitive than the floating method and viruses of titers 10^5 TCD 50/ml could be visualized. In fact, this method could serve as a rapid typing method as well provided the corresponding antisera are available. Besides, it offers the advantage that several viruses can be screened in one microtiter plate and that these plates can be stored at 4ºC for several months without deterioration.

It seems however that the most sensitive method is pseudoreplication. We have been able to detect the viruses shown in table 2 by applying both one and three drops on the agarose surface, despite the relatively low titers of some of them. However, AnB 4268 virus, producing a low titer in tissue culture cells (10^4.5 TCD 50/ml) was not visualized. Pseudoreplication of ungrouped arboviruses harvested from mouse brain was unsuccessful except for two rhabdoviruses.

Among ungrouped arboviruses, Trinity, Bobaya, Zinga and AnB 4268 multiply in Vero cells producing CPE.Bobaya, Trinity and Zinga have been classified as Bunyawera viridae and Yata and Keuraliba as rhabdoviridae.

ACKNOWLEDGMENTS

Viruses were kindly provided by Dr. Yves Robin, Director of Pasteur Institute of Dakar, Robert E. Shope, Director of Yale Arbovirus Research Unit, James D. Converse, U.S. Naval Medical Research Unit, Dr. Brunhilde, KUpper University of Cologne, Dr. Pierre Sureau, Pasteur Institute, Paris.

The electron microscopy work was performed in the University of Antwerp, Laboratory of Electron Microscopy (Director W. Jacob).

REFERENCES
1. Anderson and Doane, F.W. (1973), Can. J. Microbiol. 19, 585-589.
2. Scharp, D.G. (1960), in Proc. 4th Intern. Conf. Electron Microscopy, Springer, Berlin, 2, 542-548.


DISCUSSION
P. Brès : Dr. El Mekki, has Le Dantec virus been examined ?
A. El Mekki : Not yet, it is on our programme. But there is another interesting virus : Wanowrie, isolated in India from the brain of a patient who died in Sri Lanka from a disease which, as far as the clinical history goes, resembles very much a hemorrhagic syndrome.
J. Casals : Dr. van der Groen, I wonder whether you tried the CER cell line. It proved the most satisfactory cell line to work with a number of viruses and particularly Congo-Krimean hemorrhagic fever virus which gives no CPE and no plaques or with great difficulty, however the virus replicates extensively without visible CPE but it is really very good for immunofluorescence.
G. van der Groen : We tried different cell lines not so much to find a more sensitive one, but also to obtain an indication concerning a possible virus reservoir or transmitter. These are continuous cell lines and I know one cannot conclude from the sensitivity of cell lines that the corresponding animal would be the reservoir, but it gives some indication.

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