Download One Defective Interfering Particle per Cell Prevents Influenza Virus

J. gen. Virol. (1988), 69, 1415-1419.
Printed in Great Britain
1415
Key words: defective interfering~influenza A virus/eytopathology
One Defective Interfering Particle per Cell Prevents Influenza Virusmediated Cytopathology: an Efficient Assay System
By L. M c L A I N , S. J. A R M S T R O N G AYD N. J. D I M M O C K *
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K.
(Accepted 12 February 1988)
SUMMARY
The titre of defective interfering (DI) influenza virus measured by an assay based on
the inhibition of cytopathology caused by A/WSN (H 1N l) influenza virus in MDCK
cells was 320000-fold greater than titres measured by inhibition of infectious centre
formation. Interference was less in other types of cell. By electron microscopy, we have
shown that the ratio between physical particles and DI units in preparations of the DI
virus was approximately unity, which suggested that one or few DI particles is/are
required to confer resistance of a MDCK cell to viral cytopathology. This human DI
virus interfered heterotypically with an avian H7N1 influenza virus.
The observation that the particle :infectivity ratio of influenza virus increases on passage at
high m.o.i., by several orders of magnitude, while the overall production of virus particles
remains constant (von Magnus, 1951) led to the recognition of what is now called defective
interfering (DI) virus. Physical studies demonstrated that populations of such particles had a
reduced content of RNA (Ada & Perry, 1955) and it was later shown that there was a reduction
in the three largest genomic RNA segments with the generation of several presumptive DI
RNAs, usually smaller than the segments that constitute the genome of the standard (infectious)
virus (Crumpton et al., 1978 ; Nayak et al., 1978 ; Janda et al., 1979). More recently, presumptive
DI RNAs have been sequenced and found to be highly variable in sequence and origin
(Jennings et al., 1983; Nayak & Sivasubramanian, 1983). By comparison, understanding of the
biology of DI influenza virus is incomplete, although it has been demonstrated that DI virus can
protect mice from lethal infection (Bernkopf, 1950; yon Magnus, 1951 ; Dimmock et al., 1986)
and prevent plaque formation in cell culture (Janda et al., 1979). Interference is associated with
small viral ribonucleoproteins (Janda & Nayak, 1979), and DI influenza virus can give rise to
persistent infections in vitro (De & Nayak, 1980). Furthermore, interference is stable in the
absence of virus multiplication in dividing cells; in MDCK cells its minimum half-life is 25 days
(Cane et al., 1987). We report on the number of aspects relating to the biology of DI influenza
virus and describe a simple, highly efficient assay based on the ability of DI virus to inhibit the
cytopathology produced by standard virus.
The original seed stock of influenza virus A/WSN (H1N1) was cloned for the experiments
described herein by picking plaques on chicken embryo fibroblast (CEF) cells (Morser et al.,
1973) and re-plaquing consecutively ten times, during which time the p.f.u. : haemagglutination
(HA) ratio increased to 106"4. Stocks of standard infectious virus were prepared by inoculating
approx. 103 p.f.u, into the allantoic cavity of fertile 10 day old chicken eggs, and harvesting after
a 2 day incubation at 33 °C. DI virus was prepared in the same way except that about 109 p.f.u.
was inoculated per egg and allantoic fluids were harvested after a 16 h incubation. After three
serial high multiplicity passages the p.f.u. :HA ratio fell to 102.6. DI viruses were stored in liquid
nitrogen and standard virus at - 70 °C. Viruses were assayed by their ability to agglutinate red
blood ceils, using the conventional assay with doubling dilutions in phosphate-buffered saline
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(PBS). Infectivity was estimated by plaque assay in CEF cells. Neuraminidase activity was
determined spectrophotometrically by the release of N-acetylneuraminic acid from fetuin
(Webster & Laver, 1967).
Interference by DI virus was measured by a new assay which detects its ability to prevent the
c.p.e, caused by standard virus in MDCK cells. In its optimized form, we use 96-well flat-bottom
plastic trays (Sterilin) seeded with 2 x 104 cells/well in 200 ~i DMEM containing I 0 ~ newborn
calf serum (Flow Laboratories). After rinsing twice with PBS, monolayers were infected with 10
p.f.u./cell standard virus in 20 p.1 for 30 rain at 37 °C. Monolayers were rinsed again and
inoculated with 20 ~tl of 0-5 log10 dilutions of DI virus, as before. The inoculum was removed and
monolayers were incubated in DMEM at 37 °C for 60 min, to allow attached, non-internalized
virus to elute. Monolayers were rinsed and then incubated in 200 ~tl DMEM containing 0.1
(w/v) bovine serum albumin for 30 h. Cells were fixed in formol-saline and stained with 1
(w/v) crystal violet (Fig. 1). The dilution of DI virus that gave 63 ~ protection of the monolayer
was determined by interpolation.
This assay system was used to investigate the effect of different types of cell upon interference.
We found that the ability of WSN to multiply in CEF, L929,BHK-21 and MDCK cells varied
greatly, but all gave sufficient progeny to allow measurement of interference which varied
considerably with cell type (Table l). There was no detectable inhibition in CEF and BHK-21
cells; interference was increased nearly 100-fold in L929 and over 105-fold in MDCK cells.
Table 2 shows that the inhibition of cytopathology assay (ICA) for DI WSN is 320000-fold
more sensitive than the infectious centre reduction assay (ICRA) of Janda et al. (1979) (as well as
being less labour-intensive and quicker to perform). Tables 1 and 2 show that sister preparations
of DI virus can vary 10-fold in the amount of DI virus present.
The assay was used to demonstrate that the interfering activity of DI WSN was sensitive to
inactivation by ~-propiolactone (BPL). BPL is an alkylating and acylating agent which
inactivates virus infectivity by reacting primarily with nucleic acid (Fraenkel-Conrat, 1981) and
would thus be expected to inactivate DI virus-mediated interference which operates through the
activity of the DI virus genome (Perrault, 1981). Incubation of DI WSN with BPL (Sigma),
under conditions described previously (Barrett et al., 1984), completely inactivated interference,
even though neither the HA titre nor the neuraminidase enzyme activity was affected by BPL
(data not shown).
In general, DI viruses only interfere with the strain of standard virus from which they
originated (homotypic interference). However, exceptions include the interference between DI
particles of the Indiana strain of vesicular stomatitis virus and the New Jersey serotype (Prevec
& Kang, 1978; Schnitzlein & Reichmann, 1976), between DI Semliki Forest virus and Sindbis
virus (Barrett & Dimmock, 1984) and between subtypes of human influenza virus (De & Nayak,
1980). We examined interference between human DI WSN (H1N1) and the avian strain
A/FPV/Rostock/34 (H7N 1). DI WSN interfered with the production ofc.p.e, by FPV, although
the extent of interference was reduced by about 100-fold from the homologous titre of 107.2 to
105.2 DI units (DIU)/ml.
Since WSN-induced cytopathology in MDCK cells is so sensitive to DI virus, we were
interested to know how many physical particles (PP) were required to protect a cell. This was
determined by comparing the concentration of PP with a known concentration of latex beads
(Agar Aids, Stansted, U.K.) using a JEOL JEM-1005 transmission electron microscope (Table
3). Infectious or DI virus was purified by velocity centrifugation through a gradient of 15700to
45 ~ sucrose and the peak of virus, as determined by HA, was pooled and pelleted. Both viruses
peaked at approximately the same position in the gradient. Resuspended virus was stained with
1 ~ sodium silicotungstate (Agar Aids) pH 6.5. The mean PP : DI particle (DIP) ratio, estimated
using the interference titre measured by the ICA, was 1-15. Thus, by inference, approximately 1
DIP/cell protects against infection with 10 p.f.u. (equivalent to about 1000 PP) of standard
virus. We do not understand the mechanism of what appears to be a very efficient process of
interference.
Although highly efficient, this assay for DI influenza virus will detect a minimum of 6.3 × l0 s
DIP/ml, which compares unfavourably with the highly sensitive 'negative plaquing' techniques
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Reciprocal
l o g Io
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dilution of DI virus
5
4
3
2
1
10
9
8
7
6
cc
%1
• ",
VC
Fig. 1. Assay for the inhibition of cytopathogenicity by DI influenza virus. MDCK cells were
inoculated with WSN (m.o.i. 10) followed by 0-5 log~o dilutions of DI WSN. After 30 h at 37 °C,
monolayers were fixed and stained with crystal violet. CC, mock-infected cells; VC, ceils inoculated
with standard WSN virus only.
Interference by DI WSN in various types of cell as measured by interference with the
development of cytopathology
T a b l e 1.
Cell type
CEF
L929
BHK-21
MDCK
No. cells/monolayer*
1.0 x 105
2'0 x 10a
5"0 x 104
1-0 x 104
Interference titre (loglo DIU/ml)')"
<0.7
2'52
<0'7
6'2
* In 96-well plates. Monolayers were inoculated with 10 p.f.u. WSN/cell and subsequently with 0.5 log~0
dilutions of DI virus (allantoic fluid) after incubating for 30 rain at 37 °C. Plates were fixed and stained 30 h later.
t DIU were calculated by interpolation from the dilution of DI virus that gave 63 ~ inhibition of cytopathology.
T a b l e 2.
Relative efficiencies of bioassays for interference by DI WSN influenza virus in MDCK
cells
DIU/ml
~,~
107.2
ICRA
ICA
DIP/mI*
107.s
1013"°
Efficiency of assayt
1
3"2 x l0 s
* The number of DI particles was calculated according to the Poisson distribution and the number of
ceUs/monolayer in each assay.
t Relative to the ICRA.
NA, Not applicable.
T a b l e 3.
Ratios of physical to biologically active particles in purified defective interfering and
standard A/WSN influenza virus preparations
Virus
Standard
DI
PP/ml*
4.59 x 101°
8.56 × 1010
HAU~'/ml
2.0 x 103
2.5 x 103
PP :HAU
2.3 x 107
3-4 × 107
DIP/mI~
~<6.30 × 105
9.95 × 101°
PP :DIP
/>7.29 x 104
0.86§
* The PP concentration was estimated by electron microscopy. At least 100 particles were counted.
t HA units.
:~ DIP were estimated by the ICA in MDCK cells.
§ With different DI and standard virus preparations this value was 1.45, giving a mean of 1-15 PP :DIP.
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used with DI lymphocytic choriomeningitis virus (Popescu et al., 1976), DI vesicular stomatitis
virus (Winship & Thacore, 1980) and DI rabies virus (Kawai & Matsumoto, 1982), all of which
detect around 10 DIU/ml. All available evidence indicates that the interfering activity was
mediated by the DI virus nucleic acid, because interference was sensitive to BPL and to u.v.
irradiation and has a genome about 20-fold smaller than that of infectious virus (data not
shown). These observations are consistent with known attributes of DI viruses (Perrault, 1981).
The ability of DI influenza virus to interfere heterotypically in vitro, not only between human
subtypes (De & Nayak, 1980) but between human and avian subtypes as shown above, makes it
essential to examine whether this property extends to the situation in vivo. As DI virus-mediated
interference in vitro operates at the level of macromolecular synthesis (Perrault, 1981),
heterotypic interference in vivo should not be limited by the antigenic variations of influenza
virus. However, protection of mice from lethal WSN pneumonia by DI WSN is not
accompanied by any demonstrable alteration in virus multiplication and may be mediated by an
entirely different mechanism (Dimmock et al., 1986).
We gratefully acknowledge the help of Janis Wignall with some of the early exploratory work and thank the
Agricultural Research Council, the Cancer Research Campaign, the Ministry of Defence and the Research and
Innovations Fund of the University of Warwick for financial support.
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