Growth of adeno-associated virus in embryonated eggs

Journal of Virological Methods, @ Elsevier/North-Holland

2 (1981)

Biomedical

287-292

GROWTH OF ADENO-ASSOCIATED

BETTY

(ALTENBURG)

Departments

of

PETRIE’

287

Press

VIRUS IN EMBRYONATED

and HEATHER

’ Virology and ‘Microbiology,

EGGS

D. MAYOR* * Baylor College of Medicine,

Houston,

TX 77030,

U.S.A. (Accepted

6 January

We describe chicken

1981)

a method

for preparing

eggs using an avian adenovirus

into mammalian

cultures

without

high-titered (CELO)

purification

adeno-associated

as helper.

Subsequently,

and elimination

virus (AVV)

in embryonated

the AAV may be inoculated

of the avian helper virus.

INTRODUCTION

defective members of the parvoAdeno -associated viruses (AAV) are unconditionally virus family. They are able to produce mature, infectious progeny only in the presence of replicating adenoviruses (Atchison et al., 1965; Hoggan et al., 1966; Parks et al,, 1967). Therefore, stock preparations of AAV invariably contain substantial quantities of adenovirus.. Separation of AAV from its helper virus can be accomplished by pelleting from infected tissue culture fluids followed by banding in cesium chloride density gradients (Mayor et al., 1965; Parks et al., 1967). However, this procedure is lengthy and results in considerable loss of infectious AAV. Alternatively, the adenovirus present in AAV stock.s can be inactivated by heating at 56°C for 30 min (Ito and Mayor, 1968). Although simple to perform,

heat inactivation

is often not 100% effective and must be carried out

just prior to use of the AAV stock, since some reactivation of virus occurs during subsequent storage at low temperatures. Therefore, it would be advantageous to grow stock.s of AAV using a helper virus which would not require inactivation. AAV type

1 (AAVl),

a virus of suspected

simian origin, can be grown in a wide

variety of cell types using appropriate helper adenoviruses. Blacklow et al. (1968) and Ishibashi and Ito (1971) have reported that chicken embryo lethal orphan (CELO) virus., an adenovirus isolated from chickens, can provide the necessary helper functions for AAVl replication in cultured chicken cells. Because CELO virus can be grown to a higher titer in the allantoic fluid of embryonated eggs than in cultured cells, stocks of CELO virus are usually prepared by this method. In this paper, we report that AAVl can also be grown to a high titer in eggs using C’ELO virus as the helper. * To whom

requests

for reprints

should

be addressed.

288

MATERIALS

AND METHODS

The AAVl stock used to infect embryonated eggs was produced in an established African green monkey kidney cell line, CV- I, using the simian adenovirus SV15 as a helper. This stock has a complement fixing (CF) titer of 1 : 32 determined by standard methods(Melnick,

1969).CELOvirus

lo’.’ egg ID,,/ml)

was obtained

from the American

Type Culture Collection. This virus was diluted 1 : 100 in serum-free MEM prior to use. 0.1 ml AAVl and 0.1 ml CELO virus were injected into the allantoic cavities of IO-dayold embryonated eggs. Control eggs were inoculated with 0.1 ml CELO virus alone. After incubation for 3 days at 37”C, the allantoic fluid (usually 338 ml) was harvested from each egg, clarified by low speed centrifugation and stored at -60°C. The infectivity of AAVl stocks was determined using a modification of the immunofluorescence (IF) assay described by Ito et al. (1967). Briefly, either CV-1 or HeLa cells were grown to confluency on 12 mm round glass coverslips. The monolayers were infected simultaneously with 0.5 ml each of various dilutions of AAVl and helper adenovirus. SV15 (lo7 p.f.u./ml) was used as the helper in CV-1 cells and human adenovirus type 2 (Ad2) (107.’ p.f.u./ml) was used with HeLa cells. The coverslips were harvested at 24 h after infection, air-dried, fixed in acetone at room temperature for 10 min and dried again. AAVl antigens were detected by indirect immunofluorescence procedures using a 1 : 640 dilution of hyperimmune anti-AAVl serum prepared in guinea pigs followed by fluorescein-conjugated rabbit anti-guinea pig globulin (Hyland) diluted 1 : 30. The number of fluorescent nuclei was counted in 30 ~-50 randomly selected fields using a Leitz microscope equipped with epifluorescence optics and a 40X objective lens. For electron microscopic examination, the allantoic fluids from infected eggs were used either undiluted or mixed with an equal volume of 1 : 20 dilution of the anti-AAVl serum. After adsorption onto carbon-coated copper grids, the virus particles were negatively stained with 2% phosphotungstate 1 A electron microscope at 80 kV.

(PTA), pH 7.0, and examined

using a Siemens

RESULTS

When examined by electron microscopy, the allantoic fluids of eggs infected with both AAVl and CELO virus were found to contain small groups of 20.-22 nm particles and occasional adenovirions (CELO) (Fig. la). Allantoic fluids from eggs infected with CELO virus alone appeared to contain only adenovirions (Fig. 1b). This suggested that the small particles seen in the fluids from eggs coinfected with AAVl and CELO resulted from the replication of AAVl and not from possible avian viruses (Tattersah and Ward, 1978) which might have been present in the eggs. The addition of specific antiserum against AAVl to the drops of allantoic fluid from co-infected eggs produces large clumps of AAV particles (Fig. lc), confirming that AAVl was able to replicate in embryonated eggs. No clumped particles were found in similarly treated allantoic fluids from eggs infected with CELO virus alone.

289

Fig. 1. Electron

micrographs

of virus particles

with AAVl

and CELO virus. Arrow

virus alone.

c) Same preparation

indicates

as (a) treated

seen in allantoic

fluids of infected

20 nm particles

of AAVl.

with antiserum

against

eggs. a) Eggs infected

b) Eggs infected

AAVl.

with CELO

X 61,000.

For comparative purposes, the yield of AAVl produced in embryonated eggs was determined by complement fixation (CF) (Melnick, 1969), using aIlantoic fluids pooled from several eggs co-infected with AAVl and CELO virus. A positive reaction was obtained when the virus stock was diluted up to 1 : 64. A CF titer of 1 : 16 or 1 : 32 was typical when AAVl was grown in monolayer cultures of CV-1 cells using SV15 as the helper adenovirus. To determine whether the egg-grown AAVl was able to replicate in mammalian cells, coverslip cultures of CV-1 cells were simultaneously infected with SV15 and a 1 : 10 dilut.ion of allantoic fluid containing AAVl . The cultures were incubated at 37°C for 24 h, then processed for AAVl antigen detection by immunofluorescence microscopy. As shown in Fig. 2a, many infected cell nuclei contained AAVl-specific antigens and appeared brightly fluorescent. Similarly stained CV-1 cells with AAVl only had no fluorescent nuclei (Fig. 2b). Since some CELO virus was known to be present in the egggrown AAVl stock (Fig. la), the data suggest that CELO is unable to support AAVl replication in W-1 cells. These experiments were repeated in HeLa cells using the egggrown AAVl stock with and without the helper virus Ad2. The results were similar to those obtained with CV-1 cells and SV1.5 adenovirus. In addition, HeLa cells were infec:ted with egg-grown AAVl plus additional CELO virus (lo6 p.f.u./ml). No nucleicontaining AAVl antigens were detected in this experiment, conlirming that CELO does not support AAVl replication in mammalian cells. To determine if CELO interferes with the helper functions of other adenoviruses, HeLa cells were infected with AAVl and Ad2 or with AAV, Ad2, and CELO. The numbers of AAV antigen-positive cells were counted and found to average 0.4 per field in the presence of CELO virus and 0.5 per field in the absence of CELO virus. Thus CELO does not seem to interfere significantly with the replication of AAVl in a mammalian cellhelper virus system. The infectivity of AAVl grown in allantoic fluid (CF titer 1 : 64) was compared to

Fig. 2. Indirect infected

immunofluorescence

with AAVl

grown

staining

of CV-1 cells using antiserum

in eggs and SV15 adenovirus.

b) Cells infected

against

with AAVl

AAVl.

a) Cells

alone.

that of AAVI grown in CV-1 cells with SV15 (CF titer 1 : 32). HeLa cell cultures were infected with Ad2 and with various dilutions of the AAVl stocks. The cells were processed as described above and the numbers of fluorescent nuclei were counted. The results are shown in Fig. 3. The number of AAVl-positive cells was proportional to the amount of input AAVl , except at the highest virus concentrations (undiluted AAVl) where other factors such as helper adenovirus concentration or cell susceptibility may have been limiting. The infectivity titer of the AAVl stock grown in eggs was 223-fold higher than that of the CV-l/SVlS -grown AAVI stock, in general agreement with the observed complement fixation titers. Infectious virus could readily be detected in the egg-grown AAVl at dilutions

of 1Oe5, the greatest dilution

tested.

DISCUSSION

The results presented here show that AAVl, a parvovirus of putative simian origin, can be grown in embryonated chicken eggs to produce an AAVl stock of sufficiently high titer for use in many experiments without additional concentration. The yield of 3-8 ml aIlantoic fluid per egg makes this a convenient and extremely economical method for the production of moderate quantities of AAVl at a time when tissue culture reagents such as fetal calf serum are exhorbitantly expensive. Although CELO is capable of supplying the helper functions for AAV replication in chicken cells, our data indicate that it cannot supply these functions in mammalian cells. It is not known whether or not CELO can infect CV-1 or HeLa ceil cultures specifically,

291

AAVl

DlLUflONl

Fig. 3. infectivity titrations of egg-grown AAVl (9) and tissue culture (CV-1 cells, SVlS helper) AAVl (0). The CF titer of undiluted egg-grown AAVl was 1 : 64, the CF titer of tissue culture grown AAVl was 1: 32. grown

but it has been shown to induce tumors in hamsters {Sarma et al., l%S),

to transform

hamster cells in cultures (Anderson et al., 1969) and to induce a specific T antigen in human fibroblast cultures (Potter et al., 1970). Since CELL neither supports AAVl replication in mammalian cells nor interferes with the helper function of mammalian adenoviruses, the egg-grown AAVl can be used for many types of experiments without further purification. If desired, however, AAVl could be purified from allantoic fluids by differential centrifugation. ACKNOWLEDGEMENT

This investigation National Institutes

was supported

by grant CA 14618 from the Cancer Institute

of Health and a grant from the Cockrell Foundation.

of the

292

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