Diversity and generation of defective interfering influenza virus particles

VIROLOGY

95,

48-58

(1979)

Diversity

and Generation of Defective Influenza Virus Particles

Interfering

J. MICHAEL JANDA, ALAN R. DAVIS, DEB1 P. NAYAK,’ AND BARUN K. DE Department

of Microbiology & immunology, Los Angeles, California Accepted

January

UCLA 90024

School

of Medicine,

3, 1979

A number of individually isolated, plaque-purified clones of ts-52 WSN influenza virus were analyzed for their ability to generate defective interfering (DI) virus particles. Clonal stocks, when passaged four or five times serially in MDBK cells at high multiplicity produced DI viruses each with a characteristic set of DI RNA segments. Aliquots of different viral passages from a given clone (VP1 through VP3), when passaged independently produced the same DI virus suggesting that DI viruses are already present in VPl. Furthermore, additional experiments suggested that DI viruses characteristic of a given clone were even present in the clonal stock virus and became amplified during undiluted passages. When aliquots of a virus clone (directly isolated from a plaque) were passaged independently some clones produced identical DI viruses at VP4 suggesting that DI viruses were already present in the plaque whereas aliquots of another clone produced different DI viruses suggesting that DI viruses were absent in the clone and were generated subsequently during passages. Thus DI virus can be generated even in the plaque produced by a single infectious virus. Large quantities of a particular DI virus could be produced by coinfection with a helper infectious virus. However, continued amplification of DI viruses for a large number of passages by a helper infectious virus resulted in disappearance of some DI RNA segments with the appearance of possible new DI RNA segments. Mixed infections of two different DI viruses in the presence of helper virus resulted in the production of both types of major DI viruses. Furthermore, we have developed an assay for quantitating DI virus directly using an infectious center reduction method. Our data suggest that a single DI virus particle can inhibit infectious center formation by standard virus and that standard virus is replaced almost entirely by DI virus after four undiluted passages. INTRODUCTION

the absence of helper virus, and their ability to interfere with infectious virus replication while being selectively amplified. Several well-characterized DI-RNA viruses have been investigated including vesicular stomatitis virus (VSV), Semliki Forest Virus (SFV), Sindbis virus and Sendai virus (Huang, 1977; Schlesinger et al., 1973; Stark and Kennedy, 1978; Kolakofsky, 1977). Factors involved in the appearance and generation of VSV-DI viruses have been studied by several groups. DI virus has been shown to be generated from cloned B virions during undiluted serial passages (Schincariol and Howatson, 1970; Reichmann et al., 1971; Holland et d., 1976). Holland et al. (1976) have shown that B

The formation of noninfectious virus that interferes with the replication of standard virus has now been described in a number of animal virus systems (Huang, 19’77; Huang and Baltimore, 1977). These defective interfering (DI) particles, a term first coined by Baltimore and Huang in 19’70, describe a unique group of noninfectious viruses that may play critical roles in the maintenance and persistance of viral infections in vivo (Doyle and Holland, 1974; Welsh and Oldstone, 1977; Holland and Levine, 1978). DI viruses have been characterized by their abbreviated genomes, inability to replicate in 1 To whom requests for reprints should be addressed. 0042~6322/79/079943-11$02.00/0 Copyright All rights

Q 1979 by Academic Press, Inc. of reproduction in any form reserved.

48

DIVERSITY

AND

GENERATION

virions are not genetically predisposed to generate a particular type of T particle but instead T particles are randomly generated during the preparation of fist-passage pools of cloned B virions. Kang et al. (19’78) have shown that the same clonal isolate of VSV in a given cell type produces identical patterns of DI particles, though different clonal isolates produce different DI. With influenza virus von Magnus (1954) described the formation of noninfectious virus particles upon passaging influenza virus at high multiplicities in eggs. Subsequent findings demonstrated a similar multiplicity dependent formation of noninfectious virus (Choppin, 1969; Choppin and Pons, 19’70; Nayak, 1972; Lenard and Compans, 1975). Yet the exact nature of these noninfectious particles produced at high multiplicity remained undetermined. Recently we have shown that these noninfectious particles are truly defective as well as interfering virus particles. They require the helper function of infectious virus for replication and in turn, interfere with the replication of homologous infectious virus (Nayak et al., 1978). In addition, these DI viruses also possess small subgenomic vRNA segments absent in infectious virus preparations. Our results, furthermore, have suggested that these small RNA segments, as with other DI viruses, may be responsible for their interfering activity. In this report we describe an infectious center reduction assay for quantitating DI virus. Additionally, we have studied the generation and diversity of DI influenza viruses. We 6nd that different clonal isolates of ts-52 give rise to different DI viruses. Each DI virus possesses a characteristic RNA pattern indicating the formation of different types of DI viruses. However, aliquots of clonal stock virus or of subsequent passages invariably give rise to the same DI virus upon independent passages suggesting DI virus is present in clonal stock virus and becomes amplified during undiluted passages. When aliquots of a virus clone are passaged independently some virus clones produce the same DI virus while others produce different DI viruses. This observation suggests that in some clones DI viruses may be generated

OF DI FLU

VIRUS

PARTICLES

49

even during the formation of a plaque while in others they are generated during subsequent passages. Furthermore, our observation also supports that the formation of DI virus is not predetermined. However, once formed these DI viruses maintain their characteristic RNA pattern. Finally, we show that a given DI or mixtures of DIs may be maintained in substantial quantities by amplification using helper virus. Coinfection for a large number of passages with helper virus may, however, result in a selection process whereby initial DI RNA segments may disappear with the appearance of new DI RNA segments. MATERIALS

AND

METHODS

Virus and cells. MDBK (bovine kidney) cells were grown and maintained as described by Sugiura et al. (1975). Wild-type (ts+> and ts-52 (a group II temperaturesensitive mutant) influenza virus of the WSN strain have been described (Sugiura et al., 1975). They were plaque cloned (Suguira et al., 1975) in MDBK cells monolayers by isolation of individual, well-circumscribed clones. Individual clones were transferred into sterile tubes containing 1 ml of phosphate-buffered saline containing 0.2% bovine albumin (PBS-BA). Each isolated clone was subjected to three consecutive freezethawings in dry ice-ethanol baths. This preparation is called virus clone. Subsequently one 150~cm2 flask of MDBK cells was infected with a single virus clone and infection was allowed to continue for 2-3 days until a 70% cytopathic effect was observed. The medium (20 ml) was clarified, aliquoted, and assayed for hemagglutination activity and infectivity at both the permissive (34”) and nonpermissive (39.5”) temperature. This viral preparation is called clonal stock virus. Production and propagation of DI virus. DI viruses were produced after repeated undiluted passages as reported previously (Nayak et al., 1978). Briefly a 150 cm2 flask of MDBK cells was inoculated with clonal stock virus at 20 PFU/cell; after 16-18 hr virus (20 ml) was harvested and subjected to two cycles of rapid freezing and thawing. This is designated Passage 1 (VPl). In sub-

50

JANDA

sequent passages flasks (150 cmz) of MDBK cells were infected with 10 ml of the undiluted virus preparations obtained from previous passages. Usually four undiluted passages (VP4) were used. Maintenance and propagation of DI virus was carried on by coinfection of DI virus with infectious ts-52 virus (or ts’) (1 PFU/cell) in MDBK cells as described previously (Nayaket ~2.) 1978). Polyacrylamide gel electrophoresis of viral RNA. Flasks (150 cm2) of cells were

infected with either DI virus alone or with 1 PFU/cell of ts-52 virus and 2 DIU/cell DI virus. After 1 hr at 37” inoculum was removed and phosphate-free maintenance medium containing 5.0 mCi 32Pi added. Viral RNA was extracted from a suspension of purified virus (Palese and Schulman, 1976). The RNA of virus particles was analyzed on 2.2% polyacrylamide, 0.6% agarose gels containing 6 M urea, 0.036 M Tris base, 0.03 M NaH,PO,, and 0.01 M EDTA as described by Floyd et al. (1974). The apparatus (20 x 40 x 0.15 cm) described by DeWachter and Fiers, 1971 was used. Electrophoresis was at room temperature and top and bottom reservoir buffers were recirculated. Other conditions are described in the text. Direct assay of DI virus using infectious center inhibition. A direct assay for DI

virus using infectious center reduction was used. Cells (6 x 106) were infected with different amounts of DI virus (0.1 to 2 ml). The inoculum was adjusted to 2 ml with maintenance medium and adsorbed at 37 for 60 min. Unadsorbed virus was removed and cells were washed once with PBS-BA and superinfected with standard ts-52 virus (4 PFU/cell) for 60 min at 37”. At the end of the adsorption with standard virus cells were trypsinized and counted. These infected cells (1000, 500, 250, and 125) in 0.1 ml were plated on monolayers of cells in duplicate. Cells were allowed to attach for 1 hr and subsequently overlaid with agar medium and incubated at 34”. Both controls, i.e., ts-52 alone and DI virus alone were similarly assayed. Plaques were counted on the fourth day and the total number of plaques and percentage reduction due to interference with DI were calculated. No plaques were present in infectious center

ET AL.

assay of cells infected with DI alone as the m.o.i. of infectious virus present in DI preparation was too low to yield any infectious centers per 1000 cells (maximum number of cells plated). The fraction of cells not receiving DI virus was determined directly from the yield of infectious centers at different DI virus concentrations compared to the infectious center yield of ts-52 alone. The. assumption is that infected cells not receiving DI virus will produce infectious centers whereas cells receiving one or more DI will not produce infectious centers. We can therefore calculate the multiplicity of DI using the formula for Poisson distribution or m = -In P(O),

P(0) = e-m

where m = multiplicity P(0) is the fraction

DI as determined ter yield.

of DI per cell and of cells not receiving from the infectious cen-

RESULTS

Direct Assay of DI Virus

We have previously shown that DI influenza virus can cause an interference of infectious virus replication by determining the yield of progeny virus upon coinfection. In the present method we determined directly the defective interfering unit (DIU) by the direct inhibition of infectious center production. Cells were infected with varying amounts of VP4 DI virus and superinfected with ts-52 infectious virus as described under Materials and Methods. The multiplicity of DI virus was determined from the number of infectious centers at different DI concentrations (see Materials and Methods). Figure la shows the value of m for the different amounts of DI inoculum. Several important conclusions can be drawn from these results. (a) DI can inhibit infectious centers produced by homologous infectious virus. (b) m (multiplicity of DI virus/cell) is directly proportional to the amount of DI virus suggesting that a single DI particle can inhibit infectious center formation by ts-52. (c) We can calculate from the graph DI units per milliliter. It can be seen that m is 1 when 1 ml of DI preparation is used.

DIVERSITY

a

Mlume

of DI virus

AND GENERATION

OF DI FLU VIRUS PARTICLES

Volume

(ml)

51

of DI virus(AJl)

FIG. 1. (A) Quantitation of DI virus by inhibition of infectious center assay. 6 x lo6 cells were infected with different amounts of DI virus and subsequently superinfected with infectious ts-52 virus (4 PFU/cell). Cells were trypsinized and plated for infectious centers. Multiplicity of DI virus in each plate was calculated using the formula for Possion distribution as stated under Materials and Methods. (B) A comparative analysis of a defective interfering virus preparation using the infectious center assay and inhibition assay upon coinfection of cells with infectious virus and defective virus. For infectious center inhibition assay (*), MDBK cells (5 x 10’7plate) were absorbed with different amounts of DI virus (VP4) and subsequently super-infected with infectious ts-52 (4 PFU/cell). Cells were trypsinized and plated for infectious center assay. For yield inhibition assay (a) various amounts of DI virus were incubated with MBDK cells (5 x 106/plate) followed by superinfection with ts-52 (4 PFU/cell). Excess virus was neutralized with WSN antiserum and then incubated for 14 hr at 34” using maintenance medium (Nayak et al., 19’78). The supernatant was used for the direct infectious center assay.

This DI preparation therefore, contains 6 x lo6 DIU (defective interfering units/ml). (d) The DI preparation contains 3 x lo4 PFU and 6 x lo6 DIU/ml, i.e., for every infectious virus in the DI preparation there are 200 DI viruses. (e) In the ts-52 stock virus PFUEIA was 1.5 x lo4 compared to DIU/HA in undiluted Passage 4 virus of 1.2 x 104. This suggests that in the passage 4 infectious particles have been almost entirely replaced by DI particles and that the entire loss of infectivity in the DI preparation can be accounted for by DI virus. Therefore, very little, if any, additional randomly incomplete noninfectious virus is formed during undiluted passages. In the next experiment we compared the sensitivity of the direct infectious

center inhibition assay to the reduction of yield of progeny following coinfection of DI virus and infectious virus (Nayak et al., 1978). As shown in Fig. lb both assays essentially produce similar results. We have therefore used the direct infectious center inhibition assay to quantitate DI virus in all subsequent experiments. Diversity

of DI Viruses

To determine the diversity among DI viruses a number of clones were passaged separately in MDBK cells and VP3, VP4, or VP5 viruses were analyzed for HA, PFU, and DIU per milliliter and for RNA patterns by gel electrophoresis. The properties of some of these DI viruses produced from

52

JANDA ET AL.

different clones are shown in Table 1. The PFU/HA ratio varied from 6-3000 in the DI virus preparation which contained 301500 DI units for every contaminating plaque-forming virus. When PFU/HA decreased DIU/PFU generally increased. However, the absolute amount of DIU per milliliter (which varied from 2 x 106-4.6 x 10’) was not affected by the amount of contaminating virus. In fact, the higher yield of DIU per milliliter was observed when the contaminating plaque-forming virus (PFU/HA) as well as the absolute amount of HA per milliliter were also higher. This was expected because limited replication of infectious virus is required for the amplification of DI virus. A high multiplicity of DI virus tends to suppress the total yield of virus particle production and thus reduce the yield of DIU per milliliter, PFU per milliliter, and HA per milliliter. RNA patterns of a number of DI viruses obtained from individual clones are shown in Figs. 2a and b. As shown earlier (Nayak et al., 19’78) eight bands (Vl-V8) migrated in seven major (Fig. 2a) or sometimes eight discrete bands (Fig. 2b). As reported by others (Palese and Ritchey, 1977) V4 of WSN virus appears diffuse under these conditions of electrophoresis, whereas V4 appears relatively sharp when electrophoresis is run at 4” (Nayak et al., 1978).

Smaller RNA molecules characteristic of DI viruses are absent in infectious ts-52 virus. A band migrating slightly faster than V6 is occasionally present in ts-52 as well as DI virus preparations (Fig. 2b) and is due to 18 S ribosomal RNA contamination from host cells. These DI RNA patterns (DI-c, DI-L, DI-e, DI-d, DI-ts+, in Fig. 2a and DI-b and DI-f in Fig. 2b) and others (not shown) indicate that each DI virus preparation obtained from individual viral clones possesses unique DI RNA segments and more than one DI RNA segment are often present in these DI virus populations. It also appears that some of the DI RNA segments migrate faster than the V8 segment. Clearly all these DI RNA segments do not appear in equimolar ratio. Also, some of the DI RNA segments of DI-e, DI-b, DI-L, and ts+ viruses are present in larger quantity than other DI RNA segments in the same preparation. DI virus containing pronounced DI RNA segments can be isolated from ts+ virus by passaging at high multiplicity (Fig. 2a). DI-d contains a strong V8 band which is a mixture of V8 and a DI band. These two segments became separated when the gel was run at 4” (not shown). Larger RNA segments, particularly Vl-V3, appeared in reduced amounts in many DI RNA preparations, except in DI-d where Vl and V2 RNA segments appear to be

TABLE

1

CHARACTERISTICS OF DEFECTIVE INTERFERING VIRUSES PREPARED FROM DIFFERENT CLONAL STOCKS OF WSN VIRUS Clone designation’

PFU/HA (clonal stock)*

PFU/HA

(DI virus)

DIU/mld

DIU/PFIJ’

b

2.8 x 104 9.1 x 105

300 33

3.2 x 10’ 3.0 x 100

27 350

ii

2.8 1.2 x lo5 105

94 66

3.1 2.0 x 10” 106

180 660

E

3.1 1.5 x 104 106

29296

4.6 x lo6 10’

1500 30

ts+lcic

cLb, c, d, e, and L were clones of ts-52 and ts+lc/c from wild-type WSN. DI virus used for analysis were prepared as follows: ts+/c/c (VP2), b (VP4), c (VP4), d (VP4), e (VP5), L (VP4). Initial multiplicity of infection (passage 1) was ts+/clc (2 PFU/cell), b and c (20 PFUlcell), d and e (1 PFU/cell), and L (5 PFU/cell). Subsequent passages were undiluted as described under Materials and Methods. b PFU/HA ratio of individually prepared clonal stocks from different clones before passage to DI. c PFU/HA ratio of different DI viruses used for labeling in Figs. 2a and 2b. d DIU (defective interfering units) of DI virus as determined by infectious center inhibition assay. e DIU/PFU in the DI virus preparation.

DIVERSITY

AND GENERATION

OF DI FLU VIRUS PARTICLES

53

FIG. 2. Analysis of [32P]RNA of different clones of ts-52 virus. (A) Cells were infected with ts-52 virus at 1 PFU/cell or were coinfected with 1 PFU/cell k-52 and 2 DIUlcell of ts-52 clone c(DI-c) or L(DI-L) that had been serially passaged four times without dilution. Clones e and d ofts-52 virus were passaged three and four times respectively without dilution and a clone of ts+ virus was passaged once without dilution. Clones e and d were labeled during undiluted passage 4 and 5 respectively. The clone oft& was labeled during undiluted passage 2. (B) Cells were either infected with k-52 virus at 1 PFU/cell or were infected and labeled during undiluted passage 4 of m-52 clones L and b. Clone f of m-52 virus after four serial undiluted passages was coinfected at 2 DIUkell with 1 PFUlcell of m-52 virus. Electrophoresis was for 20 hr at 140 V.

present in normal amounts. The other observation which is of interest is that V7 segment is present in increased amount (molar ratio 2 or more) in most DI RNA preparations (except in DI-e). Generation of DI Virus It appears from the above experiment that the formation of DI is a random event and that each clone may produce a DI virus with a characteristic set of DI RNA segments. Since DI virus was produced during passaging at high multiplicity we undertook experiments to determine at what passage level DI virus characteristic to that clone was being generated. Accordingly, aliquots

of virus from VPl, VP2, and VP3 were saved and passaged independently to VP4 (Fig. 3) when DI viral RNA was labeled and analyzed. The results show (Fig. 3) that major DI RNA segments in these preparations were essentially the same, i.e., DI RNA segments migrating faster than V7 and V8 RNA segments were identical although some additional minor DI RNA segments migrating between V6 and V7 were observed in DI viruses generated from VP1 and VP2 virus. It appears therefore that the majority of DI viruses are already generated at VP1 and subsequent passages are causing amplification of these DI RNA segments.

54

JANDA ET AL.

Since it appears that DI influenza virus is already present at the passage 1 level we decided to see if DI virus is already present in clonal stock virus prepared from a single virus clone. Accordingly, 20 ml clonal stock virus was prepared by infecting one flask (150 cm2) of MDBK cells and five aliquots of this clonal stock virus were passaged four times independently at high multiplicity. The passage 4 DI virus preparations were labeled and RNA was isolated and analyzed. As shown in Fig. 4, the RNA pattern of all five DI preparations were identical. This suggests that the preexist-

FIG. 4. Analysis of [9’]RNA of DI viruses prepared using aliquoted clonal stock virus. Clonal stock virus (20 ml) of clone p of ts-52 was prepared as described in the text and divided into five aliquots. Each was passaged independently three times without dilution. DI p-l to p-5 show the RNA patterns obtained when each was labeled during undiluted passage 4. Electrophoresis was for 21 hr at 140 V. Arrows indicate the position of DI RNA segments.

ing DI viruses were already present in the clonal stock virus prepared at a very low multiplicity from single clone. Clonal Origin of DI Virus

FIG. 3. Analysis of [32P]RNA of DI virus produced during independent passages of stock virus. Cells were infected with 20 PFU/cell of clone b of ts-52 virus. This virus was aliquoted and was independently passaged four times without dilution (a and d). Subsequently VP2 and VP3 were aliquoted and passaged two and one times respectively without dilution (b and c). a and b were labeled during undiluted passage 4 and c and d were labeled by coinfection with 1 PFU/cell ts-52 and 2 DIU/cell VP4 virus. Electrophoresis was for 20 hr at 140 V.

To determine whether a characteristic DI virus was present in the virus clone or generated during production of clonal stock virus (Holland et al., 1978), two separate virus clones (clone q and R) were resuspended directly in 1 ml medium each and divided into multiple aliquots. Each aliquot was inoculated into confluent 75cm* flasks for the production of separate stock viruses from a single clone. Each stock sample was then independently passaged and VP4 was labeled. The results are shown in Figs. 5a and 5b. Two aliquots of one of the clones (q) (DI-ql and DI-q2) produced identical RNA patterns. However, each aliquot of

DIVERSITY

AND GENERATION

clone R used produced different RNA patterns. This suggests in the case of clone q DI viruses were generated in the plaque whereas in clone R they were generated subsequent to plaque isolation. Amplification

of DI Virus

OF DI FLU VIRUS PARTICLES

55

However, in passages 6 and 10 there was a gradual decrease with eventual loss of large characteristic DI RNA segments with increasing passages. In addition, as these DI RNA segments decreased, DI viruses containing apparently new low molecular weight segments appeared (passage 6 and 10). These viruses were still noninfectious and retained the interfering property. Passage 10 DI-L contained 2.1 x lo7 DIU/ml which is essentially similar to that contained in starting DI-L (Table 1).

For production of large quantities of individual DI viruses, amplification of a DI clone upon coinfection was carried out. Accordingly, DI-L was coinfected with 1 PFUkell of ts-52 virus and passaged successively by coinfection of 2 ml of the pre- Ampli$cation of DI Virus in Mixedly vious passage with ts-52 virus (1 PFUkell). Infected Cells After 10 passages selected samples were analyzed. Figure 6 shows the results of When cells were infected with two DI this amplification process in passages 2, viruses (DI-L and DI-b) along with standard 6, and 10 of DI-L. Passage 2 was identical ts-52 virus, the progeny virus contained to the original DI-L (see Figs. la and lb). major DI viruses from both preparations

FIG. 5. Analysis of [“*P]RNA of DI virus prepared using aliquots of a virus clone. (A) A single plaque of ts-52 virus (clone q) that had previously been plaque cloned was picked. This plaque was frozen and thawed three times, diluted with 1 ml of PBS-BA, and separate aliquots were grown to stock virus and then passaged independently three times. Two fourth passage isolates (DI-ql and DI-q2) were labeled with 32P. Electrophoresis was for 19 hr at 140 V. (B) Clone R was treated as above and independent aliquots were passaged and the fourth passage preparations labeled and RNA patterns analyzed. Arrows indicate the position of some of the DI RNA segments.

56

JANDA ET AL.

(Fig. 7). This shows that both DI viruses can replicate in mixedly infected cells. These results were not unexpected since multiple DI RNA segments are found in the same DI virus preparation obtained from a single virus clone (Fig. 2). DISCUSSION

The data presented here show that defective interfering units (DIU) can be accurately determined using an infectious center reduction assay and that a single DI particle can prevent infectious center formation. Similar results and conclusions have been drawn in assaying DI virus from Sindbis virus (Johnston et al., 1975). However, lack of visible infectious center does not indicate complete inhibition of virus production in coinfected cells. Since multiple cycles of infection are required for the formation of a plaque and since DI virus tends to prevent the cytopathic effect in coinfected cells (Nayak, unpublished data), coinfected cells

FIG. ‘7. Analysis of DI viral RNA produced from cells mixedly infected with VP4 DI viruses obtained from two clones. Cells were mixedly coinfected with DI-L, DI-b (4 DIUkell) and standard ts-52 (1 PFUkell). This was then coinfected four times with ts-52 virus (1 PFUkell). The final progeny virus was isolated and RNA analyzed. Electrophoresis was for 20 hr at 140 V.

FIG. 6. DI viral RNA upon continued amplification with infectious ts-52 virus. DI clone L (VP4) was amplified continuously for 10 additional passages using helper infectious ts-52 virus. RNA patterns of labeled virus produced during passages 2, 6, and 10 were analyzed. Electrophoresis was for 20 hr at 140 V.

may produce both DI and infectious virus without producing a visible plaque. Our data show that different clonal stocks of ts-52 give rise to different DI viruses which are unique for a specific clone. Similar observation has been reported by Holland et al. (1976) who suggested that DI virus is formed in the clonal stock and becomes amplified later during high multiplicity passages. Using virus clones we fmd that some clones upon independent passages generate the same DI virus whereas others do not. This would suggest that DI virus may be generated even in some clones during the formation of plaque from a single infectious virus. Since WSN virus require multiple cycles of infection for plaque formation and since the multiplicity of infection in neighboring cells during plaque

DIVERSITY

AND

GENERATION

formation is likely to be high, DI virus may be generated and partially amplified during the formation of a plaque. Furthermore, since the formation of DI virus is not a predetermined event, formation can occur either early or late during plaque formation; therefore, the amount of DI virus present in a plaque may vary. Additionally, the formation of DI at different stages of plaque formation may explain why a variety of plaque sizes are observed even using cloned WSN virus. The production of different DI virus by each plaque suggests a random event leading to the formation of DI RNA either during plaque formation or during the preparation of clonal stock and subsequent amplification. Holland et al. (19’76) have reported similar observation for VSV-DI virus. It is unlikely that each clone is genetically predisposed for the formation of a specific set of DI viruses since all clones were isolated from a single clone. Since different clones give rise to different DI virus the possibility that the DI virus in the clone was due to coinfection of a preexisting DI virus rather than generation either during or subsequent to plaque formation is excluded. Additionally, such a cell coinfected with DI virus will not form a visible plaque. Furthermore, the m.o.i. during plaque isolation is very low (10-j to lop6 PFU/cell). A number of investigators have observed the diversity in DI virus preparation with different clones of vesicular stomatitis virus (Kang et al., 1978; Holland et al., 1976; Schincariol and Howatson, 1970). Our present data and earlier results (Nayak et al., 1978) clearly show that multiple DI RNA segments can be present in the DI virus preparation produced from a single clone. Fingerprint analysis of vRNA and DI RNA segments to determine if these multiple DI RNA segments from single clone arise from one or multiple vRNA segments are currently underway. RNA analysis of different DI viruses from specific clones also shows that the pattern of interference of RNA segments may differ (Figs. 2a and 2b). For example, in DI-e segment VI and V2 appear greatly reduced, in DI-d segment V3 is almost lacking al-

OF DI FLU

VIRUS

PARTICLES

57

though segment Vl is present in normal amount. These results suggest that the reduction of vRNA segments may vary in DI RNA preparations obtained from one clone to another. This may explain diverse results observed by different investigators (Duesberg, 1968; Choppin and Pons, 1970; Bean and Simpson, 1976; Nayak et al., 1978; Hay et al., 1977; Palese and Schulman, 1976; Crumpton et al., 1978). However, the mechanism of this differential reduction of different vRNA segments in different DI preparations is unknown. It would be tempting to speculate DI RNA would interfere most with the vRNA segment it came from. Some preliminary evidence from oligonucleotide mapping suggests that this may be the case (Davis and Nayak, unpublished data). Sugiura and his colleagues have obtained similar data (personal communication). Further experiments are necessary to confirm this preliminary observation. We proposed earlier (Nayak et al., 1978) that the loss of infectivity observed among the DI virus preparation may be due to a random loss of vRNA segments resulting from the DI virus mediated interference. Our present data show two other factors may contribute to the noninfectious nature of DI virus; (1) specific loss of some RNA segments and (2) presence of DI RNA segment(s) in the virion. Our data from infectious center reduction assay suggest that the DI RNA may render a virus particle both noninfectious and interfering. Our data show that upon coinfection for a limited number of passages the same DI virus is generated. Thus it is possible to generate large amounts of DI for biochemical studies. However, upon continued passages with coinfection DI viruses with smaller DI RNA segments are generated. Similar observations have been reported by Stark and Kennedy (1978) with Semliki Forest Virus. They further suggested that the larger DI RNA segments were intermediates in the formation of ultimate DI RNA. With influenza virus whether the formation of smaller DI RNA is due to the selection of preexisting DI RNA segments or via intermediary larger DI RNA segments is unknown. Furthermore, we do

58

JANDA ET AL

not know if there is an ultimate DI virus. Experiments are currently underway to answer these questions. ACKNOWLEDGMENTS The authors gratefully acknowledge the expert technical assistance of J. Lofgren. These studies were partly supported by Public Health Service Grant AI-12749 from the National Institutes of Allergy and Infectious Diseases and by a training grant from the National Cancer Institute (J.M.J.). REFERENCES BEAN, W. J., JR., and SIMPSON, R. W. (1976). Transcriptase activity and genome composition of defective influenza virus. J. Viral. 18, 365-369. CHOPPIN, P. W. (1969). Replication of influenza virus in a continuous cell line: High yield of infective virus from cells inoculated at high multiplicity. Virology 39, 130-134. CRUMPTON, W. M., DIMMOCK, N. J., MINOR, P. D., and AVERY, R. J. (1978). The RNAs of defectiveinterfering influenza virus. Virology 90, 370-373. DEWACHTER, R., and FIERS, W. (1971). Fractionation of RNA by electrophoresis on polyacrylamide slab gels. In “Methods in Enzymology” (L. Grossman and K. Moldave, eds.), Vol. 21, pp. 167-178. Academic Press, New York. DOYLE, M., and HOLLAND, J. J. (1973). Prophylaxsis and immunization in mice by use of virus-free defective T particles to protect against intracerebral infection by vesicular stomatitis virus. Proc. Nat. Acad. Sci. USA 70, 2105-2108. DUESBERG, P. H. (1968). The RNAs of influenza virus. Proc. Nat. Acad. Sci. USA 59, 930-937. FLOYD, R. W., STONE, M. P., and JOKLIK, W. K. (1974). Separation of single-stranded ribonucleic acid by acrylamide-agarose-urea electrophoresis. Anal.

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