191
J. gen. ViroL 0977), 37, 191-194
Printed in Great Britain
Separation and Infectivity of Two Particle Types of Human Rotavirus
(Accepted 25 February I977)
SUMMARY
Two morphological types of human rotavirus particles were separated in caesium
chloride density gradients. The particles of higher density ('incomplete' particles
without an outer capsid layer) banded at a density of 1-38 g/ml, while the less dense
('complete' particles with an outer as well as an inner capsid layer) banded at
a density of 1.36 g/ml. Some particles were found with an incomplete outer layer
of capsomeres. The particle/infectivity ratio for tissue cultures of the fraction containing complete particles was more than 3 logx0 higher than that of the fraction
containing incomplete particles. But, as there was a small number of complete
particles in the fraction containing mostly incomplete particles, it was impossible
to determine whether the incomplete particles had a low infectivity or whether they
had none at all.
Rotaviruses are found in association with diarrhoea of children, calves and other animals
in two different forms, complete particles possessing a double layer of capsomeres and
presenting a smooth rim-like outline when negatively stained and incomplete particles
lacking the outer layer of capsomeres (Flewett et aL I974 a, b). Bridger & Woode 0976)
showed that the complete and incomplete forms of calf rotavirus could be separated by
isopycnic centrifugation in a CsCI density gradient. The complete forms banded at
a buoyant density of 1.36 g/ml, and the incomplete forms at a density of 1.38 g/ml. The
particle/infectivity ratio for the complete particles, assayed by inoculation of tissue cultures,
was about Iooo-fold higher than that of the incomplete particles. A method described by
Banatvala et al. (I975) and Bryden et al. (I977) for detecting human virus in tissue cultures
and for titrating its infectivity has made it possible to determine whether complete and
incomplete human rotaviruses can similarly be separated and to titrate the infectivities of
the two forms of the virus. This paper describes the separation of the two kinds of human
rotavirus and their infectivity for tissue culture.
Virus suspensions were prepared from rotavirus-containing faeces collected from
children suffering from acute infectious diarrhoea. Ten to twenty per cent (v/v) faecal
suspensions were made in phosphate buffered saline (PBS), pH 7"3, and centrifuged at
I53 o g for I5 min; the supernatants were re-centrifuged at 7000 g for 30 min in an MSE
'8 x 35' angle-head rotor. Supernatants were then filtered, using negative pressure, through
a series of membrane filters (Sartorius membranfilters, West Germany) starting with one of
average pore diam. (a.p.d.) of 3"0 #m and ending with one of 0"45/zm a.p.d. The filtrate was
ultracentrifuged at I55OOO g for 3o min at 4 °C in a Spinco SW5o rotor. Pellets were re-suspended in sterile PBS and the complete and incomplete particles counted in the electron
microscope against a preparation of latex spheres of known concentration.
Thirty-six g, analytical grade CsCI were dissolved in 64 ml distilled water, giving a solution
of refractive index I "369 with a density of 1.366 g/ml. Two ml of the filtered and centrifuged
virus preparation was layered upon 8 ml of the CsC1 solution. Centrifugation was carried out
in an MSE Io x Io AR65 titanium rotor at I55ooo g at 4 °C for 8 h; the time did not include
I3
V I R 37
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192
1.44
1"42
1 "40
-
-
R
S
~
4-÷
xN,,
10lo
"5
1"38
1 "36
10 9
1"34
fll
1"32
1"30
16 x 103
II rl
1'28
108
L
o
8 X 10~ A ~ A
1 "26
4 x 103
1.24
2 x lOs
107
~A
~A
~A-~,,
10 6
A
1 "22
.ND
I
I
3
6
I
9
I
12
I
15 18
I
I
I
21 24 27 30
Fractions
I
I
I
I
I
|
33 36 39 42 45 48
Fig. t. Distribution of infectivity and virus particles in a caesium chloride gradient. A - A ,
Density of fraction (g/ml); O - - O , particle count/ml; • -- - 0 , number of fluorescing foci/ml
undiluted fraction; ND, not detected by E.M.
T a b l e I. Distribution of infectivity and virus-specific particles after centrifuging
to equilibrium in a CsCI gradient
Fraction
5-8
9
io
II
12
13
I4
15
Total particle
count/ml
1-8×
lO 9
0"9 X 108
Complete
Incomplete
(S)
(R)
particles/ml
particles/ml
Not detected by E.M.
I'4×
IO 7
I'8X
109
Approximate
R:S
ratio
-128:1
Not detected by E.M.
Not detected by E.M.
0' 9 X IOs
1"3 × IO8
Not detected by E.M.
Not detected by E.M.
Not detected by E.M.
* Calculated
No. of
foci/ml*
-240
--
40
20
16600
40
2.0
--
--
---
I : 69
--
acceleration a n d deceleration. E i g h t - d r o p (o.2 ml) fractions were collected by p u n c t u r i n g
the base o f the tubes. The refractive index of each fraction was m e a s u r e d a n d its virus
c o n t e n t d e t e r m i n e d by electron microscopy using negative staining. Particles in fractions
c o n t a i n i n g virus were mixed with a suspension o f latex spheres (3o0 n m in diam.) a n d
counted. A t least 5o0 particles in each fraction were examined to determine the ratio o f
complete to incomplete forms.
Tissue cultures of a c o n t i n u o u s line of m o n k e y kidney (LLC-MK~) cells were used for
infectivity titration of all fractions from 5 to 15 by inoculating, in triplicate, serial twofold
dilutions in PBS of each fraction into f l a t - b o t t o m e d wells of microtitre plates c o n t a i n i n g
cell monolayers. The plates were sealed with Sell•tape a n d centrifuged, after a d d i t i o n of
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193
the inoculum, at 12oo g for I h in an M.S.E. ' m a g n u m ' centrifuge, fitted with swing-out
platforms to hold the plates (Bryden et al. 1977). The plates were then incubated at 37 °C
for I8 to 20 h.
After fixation in chilled methanol, infected cells were 'stained' by an indirect immunofluorescent technique (Bryden et al. 1977) using fluorescein conjugated rabbit antibovine
globulin (Nordic Lab.) and counted. A serum from a gnotobiotic calf convalescent from
experimental rotavirus infection was used as the intermediate layer in the 'sandwich'.
Infectivity was expressed as the calculated number of fluorescing cells produced by I ml of
the undiluted fraction.
The starting virus preparation contained 4-4 x IOl°]ml, of which 3-2× Iol°]ml were
'incomplete' or 'rough' (R) particles and t-2 × Lo~°/ml complete or ' s m o o t h ' (S) particles;
the R : S ratio was approx. 3: I.
After isopycnic gradient centrifugation, 48 fractions were collected from the base o f each
tube. Fraction 9 with a refractive index of i "3700 (density 1"38 g/ml) contained mostly
R particles and fraction 12, with a refractive index of 1.3685 (density 1.36 g/ml) contained
mostly S particles. A certain degree of admixture existed in either fraction (Table I).
Small numbers of damaged particles were seen, especially at the top of the gradients.
The results of infectivity assays in the !'38 g/ml and I "36 g/ml fractions showed in three
separate experiments- all giving similar results- that the greatest infectivity coincided
with the complete particles in the 1.36 g/ml fraction, although the largest number of virus
particles was found in the I "38 g/ml fraction (Fig. I). The results of one experiment are shown
in Table I.
I'8 X IO9
Particle]infectivity (P:I) ratio for the 1.38 g/ml fraction - - 24o
- 7"5 × Io6:1
P : I ratio for the 1"36 g/ml fraction = o 166oo
9 x io s - 5.4 x io8:I
P: I ratio for the smooth particles included in the 1"38 g/ml fraction
=
I-4 x io 7
_
_
24o
_
5.8x
io4:i
Estimation of the P: I ratio for the two fractions showed that infectivity resided mainly in
the complete particle population. As about o.8 to 1"3 9/00(in repeated experiments) of particles
in the 1.38 g/ml fraction were complete, it is possible that infectivity in this fraction was
entirely due to the complete particles, but the possibility that incomplete particles also
contributed to the infectivity could not be excluded.
I thank Dr T. H. Flewett and Dr M. E. Thouless for their help in preparing this paper for
publication.
Regional Virus Laboratory
East Birmingham Hospital
Birmingham, B 9 5ST
England
M . M . ELIAS
13-2
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REFERENCES
BANATVALA, J. E., TOTTERDELL, B., CHRYSTIE, I. L. & WOODE, O. N. 0 9 7 5 ) . In vitro d e t e c t i o n o f h u m a n r o t a -
viruses. Lancet ii, 82 I.
BRIDGER, J. C. & WOODE, G. N. (I976). Characterization o f two particle types o f calf rotavirus. Journal of
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BRYDEN, A. S., DAVIES, H. A., THOULESS, M. E. & FLEWETT, T.H. 0 9 7 7 ) . D i a g n o s i s o f r o t a v i r u s i n f e c t i o n b y
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FLEWETT, T. H., BRYDEN, A. S., DAVIES, H., WOODE, G. N., BRIDGER, J. C. & DERRICK, J. M. ( I 9 7 4 a ) . R e l a t i o n
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FLEWETT, T. H., DAVIES, H., BRYDEN, A. S. & ROBERTSON, M. J. ( I 9 7 4 b ) . Acute gastroenteritis associated with
reovirus-like particles. Journal of Clinical Pathology 27, 6o8-6t4.
(Received 6 January I 9 7 7 )
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