IHNV persistently infects chinook salmon embryo cells

VIROLOGY

109,

37-58

(1981)

IHNV Persistently

Infects

H. M. ENGELKING

Chinook AND

Salmon

Embryo

Cells

J. C. LEONG’

Chinook salmon embryo cells have been infected with infectious hematopoietic necrosis virus (IHNV) to form virus-producing and nonproducing cell lines. In the producing persistently infected cells, high levels of virus were released into the medium. Yet. these cells c’on tinued to grow \vell in culture. Persistence is apparently mediated by several factors uhich include development of temperature-sensitive mutants, small plaque mutants, and defective interfering particles. Interferon did not play a significant role in the maintenance of virus persistence since these cells were susceptible to heterologous virus infection. This laborator) model of the virus-carrier state should be valuable in defining those factor.:: \vhich infuenw the outcome of IHNV infection of salmon.

to be resistant to the disease. In the hatchery environment where fish population Infectious hematopoietic necrosis virus density is high, an IHNV epizootie can re(IHNV) is a rhabdovirus that causes an sult in complete destruction of the fish proacute disease with a high mortality rate in duced for that year. Thus, IHNV can have young trout and salmon (Amend et al., 1969). a tremendous economic impact on the aquaIn surviving fish, virus-carrier states may culture industry. Any procedures instituted become established. Estimates of the carrier to prevent this disease must rely on a firm rate are based on the isolation of virus from understanding of the formation and maintespawning adult fish and range from 10% in nance of the IHN virus-carrier state in fish. sockeye salmon (Amend and Wood, 1972) to This study was instigated to develop a model 26.2% in rainbow trout (Amend, 1975). The of the virus-carrier state i?/ /litro and to carrier fish are apparently normal and have identify the host and viral components that no detectable virus in their tissues during may contribute to virus persistence. adult life (Amend, 1975). However, high The mechanismsdetermining the outcome concentrations of virus do appear in their of an IHNV infection have been examined ovarian or seminal fluid during spawning. using persistent infections ivith IHNV in Virus has been isolated from brain tissue cells obtained from the normal host, chinook and fecal matter from adult, nonspawning salmon (O~zco~hynchus fshawytscha). The kokanee salmon (Hedrick, personal com- virus is highly cytolytic in these cells, but a munication). Thus, IHNV infection can re- small number do survive virus replication sult either in death or in the establishment and become persistently infected (PI) cell of a persistent virus infection uith no appar- lines. We have investigated the role of deent disease symptoms. fective interfering (DI) virus particles and The disease is endemic in the sockeye mutant viruses in the maintenance of virus salmon population in Alaska and is found persistence. among feral fish in coastal streams in Oregon and Washington. The virus is MATERIALS AND METHODS thought to be transmitted by water (horizontal transmission) or by virus-contamiCells. Chinook salmon (Oncorhynchus nated ovarian or seminal fluid to newly tchawytscha) embryo cells (CHSE-214) and hatched or juvenile fish. Adult fish appear epithelioma papillosum cyprini (EPC) cells were grown in minimal essential medium ’ To whom all irquiries should be addressed. INTRODUCTION

48

ENGELKINGANDLEONG

(MEM) (AutopoLv MEM, Flovv Lab.) supplemented with 10% fetal calf serum, 10 mM glutamine, jO0 units/ml penicillin, and 500 Fgiml streptomycin. The salmon cells were obtained from J. L. Fryer, Dept. Microbiology, Oregon State University, Corvallis, Oregon. The EPC cells were obtained from D. Mulcahy, National Marine Fisheries Research Center, Seattle, Washington. Both cell lines were passaged weekly in 75 cm’ plastic tissue culture flasks (Corning). V~TUS. Infectious hematopoietic necrosis virus (IHNV) was isolated from kokanee salmon (0~~corh~]?lchzc,sxwkn) obtained from the Metolius River in Central Oregon. Stock virus was propagated on CHSE-214 cells. Virus \vas adsorbed to cell monolayer at a multiplicity of infection (m.0.i.) of lOmA for 30 min at 16” and then media vvas added. The cells \vere incubated for seven days at 16”. At that time, the supernatant fluid \vas harvested and centrifuged for 10min at 2000 rpm in a Sorvall HS-4 rotor at 4”. The clarified supernatant fluid contained 0.5- 1 x IOx TCID,,, units of IHNV per ml. Stock virus was stored by freezing at -73” in l-ml aliquots. Vir7~s nssays. TCID,,, assay: Virus titers were determined by tissue culture infective close 50% (TCID,,,) assays on EPC or CHSE-214 cells. Titrations were done in 96 well plastic tissue culture plates (Linbro) sealed with adhesive mylar sheets. In a series of lo-fold dilutions, 0.1 ml of viral dilution in MEM bvith 2% dialyzed fetal calf serum (MEM-Zd) ancl 0.01 mgiml polybrene (Calbiochem) was added to each of 12 wells for each lo-fold dilution. Then, 0.1 ml of cell suspension containing approximately 5 x lOAcells was added to each well. The cells \vere incubated at 16 or 22” as indicated for 7 clays, fixed with neutral buffered formalin, and stained with 1% crystal violet. The 50%’ endpoint tissue culture infective dose was calculated according to Reed and Muench (1938). Plaque assay: Virus titer was also determined by plaque assay on CHSE-214 cells. Tissue culture fluid was diluted with MEM2d with 0.005 mg/ml polybrene. Confluent monolayers of cells on 35mm plastic petri dishes (Corning) were infected with 0.1 ml of the appropriate viral dilution. After adsorp-

tion for 30 min with intermittent rotation of the plates at 16”, 2.0 ml of MEM media containing 5% fetal calf serum and 0.8% gum tragacanth (Fisher) vvas added. The cells were incubated for 7 days at 16 or 22” and fixed and stained by the method of Dobos (1976). Virxlewce nssnys it{ ,fish. Rainbo\v trout (Snlwo gnirdl~eri) weighing from 0.5 to 1.0 g \vere ‘infected with wild-type IHNV or \vith virus obtained from persistently infected cells. Groups of 12-15 fish were exposed to varying dilutions of virus in nO0ml of water at 10”. After 24 hr the fish were placed in 10 liters of dechlorinated and aerated tap water and held for 21 days at 10”. Mortalities were recorded twice a day. The dead fish were stored at -85” and examined for IHN virus at the end of the experiment. Arrti-ZHNV mbbit SPI’IL))I.CHSE-214 cell monolayers \vere infected with IHNV at an m.o.i. of 1OY and incubated at IR”. The supernatant fluid was harvested when the cell monolayer was destroyed completely. Cell clebris was removed by centrifugation at 1000 g for 15 min and the supernatant collected. The supernatant was layered onto a 2 ml pad of 50% glycerol in STE (0.15 M NaCl, 0.01 M Tris, pH 7.5, 0.01 M EDTA, pH 7.0) and centrifuged in a Beckman SW27 rotor, 90 min, 80,000 g. The virus pellet was resuspended in STE and homogenized with an equal volume of Freund’s complete ac!juvant (Difco). Ne\v Zealand white rabbits ivere injected intramuscularly with 1 ml of the virus antigen suspension. After an interval of 2 weeks another 1 ml inoculum of virus suspension was again administered intramuscularly. T\\-o weeks later the rabbit was bled from the ear vein and the serum was isolated. This serum \vas adsorbecl 5 times with CHSE-214 cells before use in the fluorescent antibody staining procedure. All serum was stored at -75” in O.&ml aliquots. Fluorescein-conjugated goat anti-rabbit y-globulin Lvasobtained from Grand Island Biologicals Company. i,1tliwct

,fl7mesce,rt

CWtibOd~J stni?rinq.

Normal, IHNV-infected, or persistently infected cells were grown on glass coverslips. Tlventy-four to 48 hr later, the cells were

IHNVINF'ECTSSALMONEMBRYOCELLS Six

Lines-

/ CHSE-214-H%-214 Cells MO1 = 1

Cell

7-10 16C

Death

passages

(IHK) 16 c

16 C 34 days

\

One

Line

-. 3 passages 16 C

NP cells 10

/

(3bb)

passages

\ 22 c 50 passages

16 LIISE-214 C?llS

CPE

___ !I01 = 20 extensive 16 c 3 days

/

(11-16)

\

\

c ___ Nip cells 50 passages

(11-22)

of IHN\' of IHNI'

FIG.

1. Development

of persistently

infected

carefully washed in phosphate-buffered saline (PBS), pH ‘7.0, and fixed in cold acetone. After drying the coverslips were stored at -20” or stained immediately. In the staining procedure, a solution of antiIHNV rabbit serum (1:lO and 1:5 dilutions in PBS) was applied to the cells for 1 hr at 37”. After extensive washing with PBS, the cells \vere covered for 30 min with a solution (1:ZO dilution in PBS) of fluorescein-conjugated goat anti-rabbit 7 S globulin (Grand Island Biologicals Co.). After another careful wash with PBS, the cells were dried and mounted on glass slides with Elvanol (DuPont). The stained cells were observed and photographed through a Nikon fluorescence microscope. RESULTS Establishment

(IHF)-3c)

60 days 22

:ii' = Nonproducer P = Producer

C ___ NP cells 50 passages

r cells (CHSE-214

?fPersistently

Infected Cells

The IHN virus-carrier state irr vitro was initiated by infection of chinook salmon embryo cells at different multiplicities of infection. CHSE-214 cells infected at an m.o.i. of 1 TCID,,,/per cell exhibited a delayed virusinduced cytopathic effect (CPE); complete CPE was only observed 21 days after infection. Under usual conditions at low m.o.i. (lo-’ to lo-” TCID,,/per cell), complete CPE is achieved in 7 to 9 days. The cells

(PI)

cell lines

after

IHNV

infection.

that survived the virus infection regrew to confluent monolayers in 34 days. A diagrammatic description of the development of the PI cell lines is shown in Fig. 1. Seven different cell lines were established in this manner and each initially released large quantities of IHN virus (lo4 to 10” TCID,,/ml) into the culture medium. The cell monolayers in all cases showed microscopic evidence of viral infection with large, rounded cells containing sharply marginated nuclei floating in the culture medium (see Fig. 2). The persistently infected cells were subcultured at 3 to 4 day intervals at 75-100% confluency. In six of these lines, an increasing length of time was required before confluent monolayers were obtained. Successive subcultures became more difficult to maintain as the development of virus-induced CPE was increasingly rapid. These cells did not grow after 10 cell passages. One line, CHSE-214-(IHNV)-3c, was established as a persistently infected cell line which continually produces large quantities of virus and grows well in culture. This line is maintained by growth at 22” rather than 16”, the optimal temperature for IHN virus growth. At 22”, these cells continue to grow well and produce virus. This paper describes the properties of these persistently infected cells and the virus

ENGELKING

AND

LEONG

FIG. 2. Photomicrographs of (A) normal, uninfected CHSE-214 cells, (B) WT-IHNV infected CHSF:214 cells at 48 hr postinfection, (C) nonproducing CHSE;-214 cell line selected after IHNV infected, and (D) virus-producing CHSE-214 (IHNV)-3c cell line. Arrows indicate rounded, “balloon-shaped” cells with marginated nuclei that are characteristic of IHNV-infection in these salmon cells. Magnification x250.

selected by growth in CHSE-214-(IHN)-3c cells. In a second isolation of IHNV persistently infected cells, CHSE-214 cells were infected at an m.o.i. of 20. The development of extensive CPE was rapid and by 3 days postinfection, only a few cells remained attached to the flask. With weekly medium change, these cells grew to confluent monolayers by 60 days postinfection. Two lines were then established, one at 16” and one at 22” (II-16 and 11-22). Both lines did not produce IHN virus and were desig-

nated nonproducer or NP cells. By microscopic examination these cells are mdistinguishable from normal CHSE-214 cells (Fig. 2). ch aracteristics Persistently

of the Virus-Producing I?lfecfed Ceil Livp

The CHSE-214-(IHN)-3c line (designated 3c line also in the text) has been maintained in culture for more than 2 years and has been passaged more than 50 times at 22”. This persistently infected cell line produces

IHNV

INFECTS

SALMON

EMBRYO

CELLS

.j 1

107 2? 5

106

z

IO5 0 !n

g t-

IO4 IO3

0

106 1 CELL

PASSAGE

NUMBER

3. IHNV production by CHSE-214 (IHNV)-3c cells as a function of passage level. Each point represents the concentration of infectious virus (TCID,,/ml) in the tissue culture fluid harvested from a confluent monolayer of the PI cells in a %-cm” plastic flask. EPC cells were infected and maintained at Iti” for infectivity assays. FIG.

virus at concentrations ranging from 10z to 10’ TCID,,,/ml (Fig. 3). Cell growth is not affected by virus production although virusinfected cells are evident (Fig. 2). These cells will form confluent monolayers and reach saturation densities of 1.3-2.7 x 10” cells/cm’. Uninfected CHSE-214 cells will grolv to saturation densities of 2-3 x 10; cells/cm”. Maintenance of both cell growth and virus production in CHSE-214-(IHNV)-3c at 22” is thought to result from a balance between viral and cellular replication. At 22” virus replication is presumably held in check while cellular replication proceeds at a normal or even faster rate (Phillipon-Fried, 1979). In fact \vhen the 3c cells were shifted back dovvn to 16”, a temperature that is optimal for virus growth, a “crisis” was observed. Greater numbers of rounded, virus-infected cells appeared floating in the tissue culture fluid. Subsequent efforts to passage these cells Lvere unsuccessful. of Homologous and Heterologem Virus i?l Persistently Injected Gel Is

Replication

Both homologous virus, IHNV, and heterologous virus, infectious pancreatic necrosis virus (IPNV), were tested for growth on the persistently infected cells. In the virus-producing cell line, CHSE-214(IHNV)-3c. neither virus was able to form

visible plaques. In contrast, control uninfected CHSE-214 cells exhibited more than 100 plaques per dish with the same virus dilution (Fig. 4). A similar experiment measuring tissue culture infectivity with assays of IHNV in the 3c line confirmed this observation. IPNV did produce CPE in a TCID,,, assay on 3c cells (Table 1). The nonvirus producing cell lines allou replication of both viruses. However. IHNV exhibited consistently a lower titer in these cells than in control CHSE-214 cells (Table 1). The decrease in titer ranged from 4- to 150-fold by plaque and TCID,,, assay. Differences in virus titer were more pronounced when assays were conducted at 22”. Thus, although the nonproducing lines appear normal upon microscopic examination some change(s) occurred that made them resistant to IHNV infection. IPN virus, a piscine diplorna virus, will produce large plaques on CHSE-214 cells (Hedrick et al., 1978). The titer of IPNV was not reduced on the nonproducing cells at 16 or 22” (Table 1) by plaque or TCID,,, assay. In fact, the titer was increased by lofold m the nonproducing lines. Preserlce of ViTal Antigeus irl PI Cell Lirles

Both producing and nonproducing cell lines were checked for the presence of viral antigen by indirect fluorescent antibody

ENGELKINGANDLEONG

IHNV

INFECTS

SALMON

EMBRYO

TABLE GROWTH

Cell line

Control cells

OF WT.IHNV

Difference in virus titer

AND

.j:i

CELLS

1 IPNV

IS NP AND

Xc CELLS"

Average difference --

1. WT-IHNV

Plaque assay 11-16” 5.14 5.45 7.83 11-p 3.90 3.20 3bb16 4.53 4.90 7.10 dc-22 0 0 ‘KID,,, assay II-16 4.43 4.36 5.32 11-22” 2.00 3.64 4.11

infection of NP, 3c, and control CHSE-214 cells”

214-16 5.24 6.54 x.39 214-22” 4.84 5.30 214-16 j.24 6.54 8.39 214-22 7.40 5.30

214-16 4.90 4.67 5.48 214-22 3.17 4.67 5.48

0.10 1.09 0.56

O..i8

3bb16 3.43 3.67 4.16 3c--22” 0 II.

0.94 2.10

IPNV

1.32

0.61 1.64 1.29

1.18

7.40 5.30

6.35

0.47 0.11 0.16

0.2.5

1.17 1.03 1.35

1.18

214-16 4.90 4.67 .i. 50 214-92 4.67

1.47 1.00 1.34

1.27

4.67

4.67

infection of NP, 3c, and control CHSE-214 cells

Plaque assay 11-22” 7.20

214-22 7.10

0.10

0. 1

TCID;,, assa) II-22 6.16 K.56

214-92 Y 5.33 7.44

PO.83 -1.12

~ 0.98

3c-22 7.86 8.16 7.75

214-22 7.90 7.94 8.00

~ 0.04 0.22 -0.23

0.03

8.10

7.90

0.20

” mT:T-IHSV was titeretl hg pl;cque or T(‘Il);,, assay on NP cells on wntrol (‘HSF:-21,1 cells at l(i 01’ L’2 as intlicatetl. The difference in IHNV titer was obtaine(l on SF’ cells from that obtainetl in the control cells. ” Titer is c~xprrssetl as the logarithmic exponent to the h;i,qr trn.

staining. In the case of the virus-producing line, CHSE-214-(IHN)-3c, nearly all of the cells showed a positive reaction, as illustrated in Fig. 5A. The antigen was distributed throughout the cytoplasm; in some cells, antigen xas detected as brightly fluorescing patches in the cytoplasm. The specificity of the immunofluorescent stain is evidenced by the lack of staining in control cells (Fig. 5A) and the specific staining of a focus of IHNV-infected cells (Fig. 5B). The nonproducing cells were also found to contain viral antigen. Approximately; 5- 10% of the cells exhibited diffuse staining ovel the cytoplasm of the cell.

Plccque .fbrmntiorl. The virus produced after contmuous growth in the long-term carrier cells, CHSE-214-(IHN)-3c, Leas esamined for alterations in its plaque morphology or in the rate of increase in its plaque size. Ten days after infection, wild-type IHNV (WT-IHNV) forms uniform plaques that are 3 mm in diameter. Virus produced in the persistently infected cells at passage level 45 (3c-IHNV) forms t\vo different types of plaques at 16”. Minute plaques 0.5-l mm in diameter, accounted for ZO60% of the total number of plaques. The

ENGE:LKINGAK;D

(FIG.

percentage of minute plaques remained constant for lo-11 days. This result suggests that small plaque do not become large plaques. The larger plaques ranged in size from 1 mm to 2 mm in diameter. The rate of increase in plaque diameter was measured for WT-IHNV and 3c-IHNV on CHSE-214 cells at 16”. As shown in Fig. 6, the size of WT-IHNV plaques increased with respect to time in a linear relationship for lo-11 days after infection, whereas the 3c-IHNV plaque size remained constant during this time period. This result suggests that the minute plaques do not progressively enlarge during the 10 to 11 day interval.

LEONG

5)

Tempemture Sensitivity

of SC-IHNV

Since the virus-producing cell line was maintained at 22”, the growth properties of the 3c virus was examined at 16 and 22”. Wild-type IHNV did not show any difference in titer at 22 or 16“. However, 3c virus did show a marked difference in growth at the two temperatures, as illustrated in Fig. 7A. At 16”, the virus titer, as determined by the number of wells in which evidence of CPE was observed microscopically, is approximately 3.5 x 10: TCID,,/ml. In contrast (Fig. 7B) at 22”, the number of viruspositive wells is less clearly defined. In fact, with undiluted and 1:lO dilution of virus

IHNV

INFECTS

SALMON

EMBRYO

CELLS

FIG. j. Presence of viral antigen in PI cells. (A) normal CHSE-214 cells. (B) CHSE-214 cells infected with IVT-IHNV at a m.o.i. of 0.1 at 48 hr postinfection, and (C) CHSE-214-(IHNV)-3c virus-producing PI cells \vere stained with rabbit anti-IHNV and goat anti-rabbit y-globulin as described in Materials and Methods. In 5B, a focus of IHNV-infected cells is shown. Magnification x400.

sample the cell monolayer is not completely removed by virus-induced cytolysis. “Interference” Lvlth viral replication is apparent in these wells receiving undiluted virus. Individual plaques are actually seen in wells receiving 1OF, lo-“, and lo-’ dilutions of the PI virus. If the virus titer is computed by the number of Lvells containing any evidence of virus infection, there is a significant difference between the 3c virus titer at 16 and 22”. Virus obtained from the 3c line at different passage levels exhibited greater differences in infectivitv at the two temperatures as demonstrated in Table 2. These results suggest that 3c virus is less infectious or replicates at a slower rate at 22” and that there are fluctuations in the apparent temperature sensitivity of the 3c virus.

Sedimentation analysis of [13H]uridinelabeled virus produced by the 3c cell line does show that these cells produce at least two types of particles. [“H]Uridine was added to 3c cells (20 pCi/ml) for 24 hr at 22”. The virus containing culture fluid was harvested and freed from cell debris by centrifugation. The virus was concentrated as de-

scribed in Materials and IMethods. The virus pellet was resuspended in STE, layered onto a 5 to 30% (w/v) linear gradient of sucrose in STE, and centrifuged at 40,000 rpm for 40 min in an SW41 rotor. The faster seclimenting peak contained IHNV infectivity and a 42 S viral RNA. The more slowly sedimenting particle is associated with interfering activity and contains an 8-10 S RNA.

The virulence of the 3c virus was determined by exposing young rainbow trout to the virus in water. At 10” TCID,,, units of virus per ml of water, a 6’% mortality was observed in the group receiving 3c virus. A 21% mortality was observed in the control group receiving WT-IHNV at 10” TCID,,, units per ml of water. The virus produced by persistently infected cells is apparently less virulent than WT-IHNV. DISCUSSION

Virus-carrier states in cell culture have been described for a variety of virus-cell systems and it has become obvious that a number of factors (DI particles, interferon,

56

ENGELKING

I

I

I

I

I

I

4

6

8

IO

12

DAYS

14

POSTINFECTION

FIG. 6. Increase in plaque diameter as a function of time after infection of CHSE-214 cells by (0) WTIHNV and (0) PI-IHNV-SC. Insert contains data relating to the percentage of plaques appearing as minute plaques (5 1 mm diameter) at different days after infection.

AND

LEONG

tenance of virus persistence since CHSE214 cells do not produce interferon (MacDonald and Kennedy, 1979) and the 3c cell line was susceptible to IPNV infection. The virus-producing cell line has been maintained in culture for more than 50 passages. In this time the temperature sensitivity of the 3c virus was assessed by measuring its culture infectivity at 16 and 22”. At the onset of these studies it seemed probable that continued growth of the 3c line at 22” would lead to the selection of temperature-resistant mutants. We have shown that temperature-sensitive (ts) mutants are produced instead. The 3c-IHN virus exhibited a lower titer at 22 than 16”. However, although ts mutants appear in the extracellular fluid, continuous production of large numbers of ts mutants was not found. Great fluctuations in the frequency of ts mutants were observed (Table 2). There was a correlation between the degree of temperature sensitivity and the appearance of the 3c line in the culture. At passages 31 to 35, the growth of the 3c virus TABLE EFFECT

and temperature-sensitive mutants) are involved in the formation of persistent viral infections (Nishiyama, 1977; Friedman and Ramseur, 1979; Holland and Villareal, 1977; Preble and Youngner, 1975). We have established a chronic infection of salmon cells in vitro with a piscine rhabdovirus, IHNV, that grows optimally in salmon cells at 1618” (McAllister et al.). The cell line is maintained at 22” so that virus production and cell growth can proceed concomitantly. The IHNV-producing cell line had the following characteristics: (1) virus production fluctuated with passage level and a range in virus concentration of 10’ to 1Oi TCID,,,/ml was observed; (2) these cells were resistant to superinfection by homologous virus (Fig. 4) but sensitive to infection by heterologous virus (Table 1); (3) nearly 100% of the cells were stained with anti-IHNV in an indirect immunofluorescence test for viral antigen; (4) no addition of antiviral antibody was required for the maintenance of the carrier culture; (5) endogenous interferon production was probably not involved in the main-

OF TEMPERATURE OK PI VIRUS DIFFERENT PASSAGE LEVELS Titer

Passage level 24 41 32 34 35 36 37 38 39 40 41 43 44 4c5 46 47 48 49 .iO

2 AT

(TCID,,/ml)”

16

22”

Difference (16” ~ 22”)

3.63 5.67 5.91 5.84 6.87 3.75 4.86 7.00 6.40 5.75 4.75 3..;0 6.00 5.50 5.54 6.38 4.5 .5.33 .I. -0

.5.63 4.20 4.67 4.75 1.00 .5.67 3.60 6.43 4.60 4.00 4.30 .i.25 5.67 5.43 4.65 6.6 4..i 4.86 4.86

0 1.57 1.24 1.09 5.00 0.08 1.26 0.37 1.80 1.75 0.45 0.2, 0.33 0.07 0.89 -.z 0.00 0.47 0.14

” Titer is expressed to the base ten.

as the

logarithmic

exponent

IHNV

INFECTS

SALMON

EMBRYO

CELLS

FIG. 7. Growth of PI-IHNV-3c virus at 16 and 22”. Virus obtained from the persistently infected cell line, CHSE-214-(IHNV)-3c at passage level 46, was assayed by endpoint dilution in EPC cells at (A) 16 and (B) 22”. The virus titer was determined by both macroscopic and microscopic observation of the cells in each well. The virus titer was 3.5 x 10” TCID,,,/ml at 16 C and 4.5 x IO4 TCID,,,/ml at 22”.

at 22” was very temperature sensitive and the 3c cells appeared more like normal CHSE-214 cells. In contrast more CPE was observed in the cell monolayer when the virus infectivity differed slightly or not at all at the two temperatures. These results suggest that ts mutants ameliorate the effects of virus replication in cells. In fact when the 3c line was shifted to 16”, virus-

induced CPE and rising titers of extracelMar virus developed within 48 hr of the temperature shift. Increasing temperature sensitivity of the vesicular stomatitis virus (VSV) produced in persistently infected mouse L cells has been reported previously (Youngner et al., 1976). When VSV infection was initiated in the presence of large numbers of DI parti-

58

ENGELKING

cles, there was a rapid selection of ts mutants. A steady increase in the frequency of ts clones in the virus population occurred to cell passage level 6. Characterization of the virus at later cell passages was not reported. Thus, no fluctuations in the frequency of ts clones were observed in this study. Nishiyama (1977) reported for VSV chronic infections in L cells that by passage 19, 100% of the plaques were small at the nonpermissive temperature. The small plaque phenotype was associated with temperature-sensitive virus replication since 95% of these plaques were large at the permissive temperature. In the IHNV study, the frequency of small plaque mutants in the 3c virus stock was determined at 16”, the permissive temperature. At passage 45, 50-60% of the plaques were small and remained small for 11 days (Fig. 6). There was very little difference in virus titer at 16 or 22” at passage 45 by TCID,,, assay (Table 2). Thus, these studies suggest that a substantial portion of small plaque mutants do not exhibit the small plaque phenotype because of temperature-sensitive virus replication. An important factor in the establishment and maintenance of virus persistence is the DI particle (Huang and Baltimore, 1970; Holland and Villarreal, 1974). In the case of VSV, the DI particle is smaller in size than normal virions and there are deletions in the viral genome (Holland et al., 1976). We have detected interference in assays of stock preparations of 3c virus (Fig. 7B). Interference is observed only at 22” when standard virus particle growth is slower. A preliminary characterization of defective interfering particles is not reported here. The data indicate that the 3c line does produce virus particles that are smaller than the standard IHNV. Electron microscopic evidence for DI particle production in IHNV infection has been reported previously (McAllister and Pilcher, 1974). ACKNOWLEDGMENTS The authors thank Drs. S. Turner and J. L. Fryer for their help. This work was supported by a grant from the Office of Water Research and Technology, 17. S. Department of Interior.

AND

LEONG

AMEND, D. F., and WOOD, infectious hematopoietic Washington salmon. Bog.

AMEND, D. F.. YASUTAKE. W. T., and MEAD, R. W. (1969). A hematopoietic virus disease of rainboLt trout and sockeye salmon. Trans. Amer. Fish. Sot. 98, 7964304. DOBOS, P. (1976). Use of gum tragacanth overlay, applied at room temperature, in the plaque assay of fish and other animal viruses. J. Clin. ,%ficro. 3, 373-37;5. FRIEDMAN, R. W., and RAMSEUR, J. (1979). nisms of persistent infections by cytopathic in tissue culture. Arch. Viral. 84, 83-103.

D. F. (197.5). Detection and transmission of infectious hematopoietic necrosis virus in rainbow trout. b. Wild/ifr Dis. 11, 471-480.

AMEND,

Mechaviruses

HOLLAND. .J. J., and VILLARREAL, L. P. (1974). Persistent non-cytocidal vesicular stomatitis virus infections mediated by T particles that suppress virion transcriptase. Proc. Nat. Acad. Sci. USA 71, 2956-2960. HOLLAND. .J. J., VILLARREAL, L. P., and BRENDL, 11. (1976). Factors involved in generation and replication of rhabdovirus defective T particles. d. Viral. 17, 805-815. HUANG. A. S., and BALTIMORE, D. (1970). Defective viral particles and viral disease processes. Nnfllrr flondorr) 226, 325-327. HEDRICK, R. P., LEONG, J., and FRYER, J. L. (1978). Persistent infection in salmonid fish cells with infectious pancreatic necrosis virus (IPNV). d. Fish ,%a. 1, 297-308. MACDONALD, R. I).. and KENNEDY, virus persistently infects Chinook cells independently of interferon.

C. (1979). IPN Salmon Embryo Virolog:y 95,

260-264.

MCALLISTER, P. E., and PILCHER, K. S. (1974). Autointerference in infectious hematopoietic necrosis virus of salmonid fish. Proc. Sot. Exp. Biol. Med. 145,

840-846.

NISHIYAMA, Y. (1977). Studies of L cells persistently infected with VSV: Factors involved in regulation of persistent infection. .I. Gen. Viral. 35, 265-279. PHILLIPON-FRIED, M. (1980). of Six Established Salmonid University, p. dissertation.

Partial Characterization Cell Lines. Oregon State

PREBLE, 0. T., and YOUNGNER, J. S. (1973). Selection of temperature-sensitive mutants during persistent infection: Role in maintenance of persistent Newcastle disease virus infections of I, cells. d. Viral. 12, 481-491. REED, L. J., and MUENCH, H. (1938). A simple of estimating fifty percent endpoints. Amer.

method J. Hyg.

27.493-497.

J. S.. DUBOVI, E. J., QUAGLIANA, D. O., KELLY, M., and PREBLE, 0. (1976). Role oftemperature-sensitive mutants in persistent infections initiated with vesicular stomatitis virus. d. Viral. 19, 90-101.

YOUNGNER.

REFERENCES

J. W. (1972). Survey for necrosis (IHN) virus in Fish. Cult. 34, 143-147.