An iridescent virus from Simulium vittatum (Diptera: Simuliidae) in Saskatchewan

JOURNAL

OF INVERTEBRATE

An Iridescent

PATHOLOGY

56, 8-14 (1990)

Virus from Simulium vittafum in Saskatchewan’ MARTIN A. ERLANDSON AND PETERG.

Agriculture

(Diptera: Simuliidae) MASON

Canada Research Station, 107 Science Crescent, Saskatoon, Saskatchewan, Canada S7N OX2 Received January 27, 1989; accepted October 4, 1989

An iridescent virus was isolated from larvae of the black fly, Sirnulium vittatum, collected from the South Saskatchewan River. Infected larvae displayed a brilliant blue-green iridescence. The virions occurred in paracrystalline arrays in the cytoplasm of fat body and epithelial cells. The apex-apex diameter of the icosahedral virions measured 127.7 * 0.9 nm in neg@ively stained preparations. Characterization of virion structural polypeptides on SDS-polyacrylamide gels resolved 26 polypeptides ranging from 12.7 to 205.8 kDa. Analysis of EcoRI, HindUI, and PsrI restriction endonuclease digests of purified viral DNA indicated that the genome had an approximate size of 153 kbp. On the basis of the restriction endonuclease profiles, this iridescent virus is a new isolate from black flies. KEY WORDS: Iridovirus; black fly; Simulium vitfatum; characterization.

INTRODUCTION

1981). Its replication, in vitro, has been studied in Aedes albopictus cell cultures (Kelly, 1976). During a survey of black fly larval populations in Saskatchewan, a variety of pathogens were observed including several species of microsporida and an iridescent virus. This paper reports the occurrence of an IV in S. vittatum for the first time and includes a biochemical characterization of the virus.

Iridescent viruses (IVs) are large icosahedral cytoplasmic DNA viruses which have been isolated from a variety of insects (Kelly, 1985) including a number of black fly species (Simuliidae) (Avery and Bauer, 1984; Batson et al., 1976; Batson, 1986; Weiser, 1968). Iridovirus infections of simuliids are characterized by a bright blue iridescence caused by quasi-crystalline arrays of vu-ions in the cytoplasm of infected cells. The fat body is the primary site of infection but hemocytes and epidermal and tracheal cells may also be infected (Avery and Bauer, 1984; Batson, 1986). All IV isolates from black flies have been classified in the small-sized invertebrate iridovirus group ranging in diameter from 130 to 140 nm. The iridescent virus isolated from Simulium sp. in Wales, classed as IV type 22 (Batson et al., 1976), has been characterized biochemically in terms of structural polypeptides and its serological relationship to other insect IVs (Elliott et al., 1977; Kelly et al., 1979). Its genome consists of a linear, double-stranded DNA molecule of MW 128 to 138 kDa (Hibbin and Kelly, ’ Contribution

MATERIALS

Collections. Black fly larvae were collected at 22 sites in 13 rivers and creeks in the northeastern agricultural area of Saskatchewan. One representative larval sample (n 5 100) was collected at each site per sampling date and larvae were examined in the field for signs of disease or parasitism. The total number of larvae in samples containing-IV-infected individuals was noted to determine incidence of infection. In the laboratory, larvae with IV infections were rinsed with ethanol, blotted dry on filter paper, and frozen at - 20°C until processing. Electron microscopy. Immediately upon arrival at the laboratory, several patently infected larvae were transected into l- to 2-mm-long cross-sectional pieces in 2%

No. 996, from the Research Station. 8

0022-2011190 $1.50

AND METHODS

: Simulium

vittatum

9

for 15 min. Proteinase K was added to a final concentration of 0.5 mg/ml and the incubation continued for a further 3 hr at 37°C. The preparation was extracted twice with phenol:chloroform:isoamyl alcohol (25:24: 1) and once with chlorofornuisoamyl alcohol (24: 1). The final aqueous phase was dialyzed extensively against TE, pH 7.5. Purified DNA was digested with EcoRI, HindIII, or PstI restriction endonucleases (GIBCOBRL) following the method suggested by the supplier. The DNA restriction fragments were separated by electrophoresis on 0.5 or 1% agarose gels at 60 V (50 mA) for approximately 18 hr. Gels were stained with ethidium bromide (0.5 l&nl), exposed to a UV light source, and photographed. The sizes of the DNA restriction fragments were estimated by comparison of their mobilities to that of A-phage DNA digested with HindIII. RESULTS AND DISCUSSION Characteristic iridescent blue coloration, car a=an indication of IV infection, was observed at 260 nm. The fractions containing virus in black fly larvae at 2 of the 22 collection were pooled, diluted with TE, pH 7.5, and sites. At one of these sites a single Simucentrifuged in a Beckman SW28 rotor at fium luggeri larva was found to be patently 26K for 45 min. The resulting pellet, which infected with IV. At the second site IVhad a bright blue iridescence, was resus- infected S. vittatum larvae were found on pended in TE, pH 7.5, at approximately 1 .O two sampling dates and the incidence of inmg/ml of protein. fection was 16% (10/61) and 17% (41/238), Analysis of viral proteins. Virion struc- respectively. tural polypeptides were analyzed by SDSThe IV from S. vittatum produced a discrete band (1.275 g/cm3) in a 40-66% w/w PAGE (4% stacking gel and 10% separating gel) using the discontinuous buffer system sucrose gradient centrifuged for 1 hr at in negativeof Laemmli (1970). Purified virus was pel- 100,OOOg. When examined leted, resuspended in sample buffer (0.06 M stained preparations the banded material particles Tris-HCl, pH 6.8, 10% glycerol, 1% w/v consisted of icosahedral-shaped with an apex-apex diameter of 127.7 + 0.9 SDS, 1% v/v 2-mercaptoethanol), and nm (114.3-132.7 nm, n = 75) (Fig. 1). heated to 100°C for 5 min prior to electroThese virions are in the size range of the phoresis. The gels were stained with 0.1% small iridescent virus group (Kelly, 1981) Coomassie blue R250 or a Kodak Kodavue and similar in size and form to IVs previelectrophoresis visualization kit. ously described from simuliid larvae Nucleic acid analysis. Purified virus (1.0 (Avery and Bauer, 1984; Batson, 1986; Batmg/ml in TE, pH 7.5) was incubated at 60°C son et al., 1976). for 15 min prior to DNA extraction to inacElectron microscope examination of thin tivate nucleases. The virion suspension was sections of tissues from infected larvae adjusted to 1% SDS and incubated at 37°C

_. FIG. 1. Electron micrograph of a negative-stained preparation of Simulium purified on a sucrose gradient. Bar = 100 nm.

virtarum

FIG. 2. Electron micrograph of a section through a fat body cell of a Simulium infected with iridescent virus (icosahedral particles). Bar = 500 nm. 10

iridescent virus

vittarum

larva

IRIDOVIRUS

11

FROM Simulium vittatum

showed numerous virions, which were hexagonal in cross section, packed in paracrystalline arrays in the cytoplasm of cells in both fat body and epidermal tissues (Fig. 2). The mean apex-apex diameter of the virions was 129.5 + 2.4 nm (115-140 nm, n = 50). Analysis of the structural polypeptides of purified virions on SDS-polyacrylamide gels resolved up to 26 bands (Fig. 3). The polypeptide profiles in lanes 1 and 2 were of viruses isolated from black fly larvae collected on two different sampling dates and are essentially similar in form. Major polypeptides with molecular weights of 170.6, 48.7, and 12.7 kDa were detected. In addition, other less prominent polypeptides ranging from 205.8 to 14.4 kDa were detected in Kodavue-stained gels. The polypeptide profile of S. vittatum IV resembled that of IV type 22, isolated from Simulium sp., (Elliott et al., 1977; Kelly et al., 1979), but there were considerable differences between the two for estimated molecular weights of many of the polypeptides, particularly the high-molecular-weight polypeptides. This may reflect differences in the SDS-PAGE conditions and the different standard markers used in our analysis or it may be attributed to unique differences in the virus isolates. Both isolates have a major structural polypeptide of approximately 50 kDa, similar to that of many other IVs. However, neither the lower-molecularweight polypeptide of 12.7 kDa nor the 34.6-kDa polypeptide, which may correspond to VP39 in type 22, appeared to be as predominant in the S. vittatum IV as in the type 22 virus (Elliott et al., 1977). The DNA fragment patterns of S. vittaturn IV DNA are shown in Figure 4. The EcoRI, HindIII, and PstI fragment patterns were different than those published for type 22 virus DNA (Hibbin and Kelly, 1981). Each of the restriction endonucleases generated fewer DNA fragments from S. vittaturn IV DNA (Table 1) than from type 22 virus DNA (Tables 3a and 3b in Hibbin and Kelly, 1981). The relative size of the genome, 153.4 kbp (151.37-156.04 kbp), was

12 205.0

170.6

142.3

L

03.5 76.0

60.9

79.47 64.1 56.3

40.7 46.0 45.0 42.4 -

37.35 34.6 33.4 31.4

-

29.75

-

21.2

-

16.7 16.6 15.9 14.4

12.7

FIG. 3. SDS-polyacrylamide gel of the structural polypeptides of Simulium vittatum iridescent virus. The estimated molecular weights (kDa) of the polypeptides are given on the right side of the panel. Lanes 1 and 2 represent viruses isolated from larvae from two different sampling dates.

estimated from the totals of the mean molecular weights of the DNA fragments from four different gels (Table 1). The total molecular weight of the S. vittatum IV genome was considerably smaller than the estimates for type 22 virus DNA which range from 189.4 to 209.1 kbpI (Hibbin and Kellv,_ I ~

12

ERLANDSON

AND

MASON

Kbp 2313 9.42 6.56 2313 4.36

6 56

2.32

4 36

2.03

232

FIG. 4. Restriction endonuclease fragment profiles of Simulium vittatum iridescent virus DNA digested with EcoRI, HindIII, or PstI and electrophoresed on a 0.5% (A) or 1% (B) agarose gel. The migration of h-phage DNA Hind111 restriction fragments and their size in kbp is indicated to the left of each panel. The faint bands (arrows) in the EcoRI digest (A) probably represent restriction fragments present in submolar ratios.

181). Although most of the small insect IVs, Tar characterized, have DNA genomes in the size range of 180-215 kbp (Kelly, 1981; Wa rd and Kalmakoff, 1987); Boucias et al. SO1

(1987) isolated an IV from the south em mole cricket, Scapteriscus vicinus, wklich has an estimated genome size of 144 kt ‘PThere appeared to be DNA fragme :nts

IRIDOVIRUS

TABLE 1 Simulium vittatum IRIDESCENT RESTRICTION

END~NUCLEASE IN kpb

EcoRI Band A B C D E F G H I J K L M N 0 t R s T U V W X Y

FRAGMENT

Hind111 12.44 11.00 9.50 1.53 7.13 6.90 6.90 6.90 5.91 5.80 5.10 4.88 4.88 4.88 4.44

2.36 2.65 2.17 1.98

4.09 3.68 3.31 3.36 3.04 3.04 3.00 2.88 2.88 2.76

SIZES

PstI 25.00 25.00 21.00 18.50 18.50 13.75 12.00 5.59 5.59 4.93 3.02

156.04

polypeptides were similar in form to those published for type 22 IV from Simulium sp. (Elliott et al., 1977; Kelly et al., 1979) but differences in the size and relative abundance of specific polypeptides were evident. The results suggest that the virus isolated from S. vittatum was related to IVs isolated from other black fly species; however, the estimates of genome size and the restriction endonuclease patterns of the S. vittatum IV DNA clearly indicated that it was a different virus isolate than IV type 22 from a Simulium sp. in Wales (Hibbin and Kelly, 1981). ACKNOWLEDGMENTS

REFERENCES S. W., AND BAUER, L. 1984. Iridescent virus from Prosimulium collected in Maine. .I. Znvertebr. Pathol., 43, 43w31. BATSON, B. S. 1986. A small iridescent virus (Iridovirus) from Simulium neornatipes Dumbleton (Diptera: Simuliidae) in the South Pacific island of New Caledonia. J. Znvertebr. Pathol., 48, 384-381. AVERY,

BATSON, B. S., JOHNSTON, M. K., AND KELLY, D. C.

2.10 2.23 1.99 1.84 1.53 151.37

13

Simuiium vittatum

We thank Keith Moore, Peter Kusters, and Peter Burgess for their technical assistance. We also thank Chris Hinks and Lome Duczek for reviewing the manuscript.

2.36 2.70

: 4 g Total

VIRUS DNA

21.96 19.19 19.19 14.35 9.27 9.27 8.28 8.15 1.90 1.48 5.20 4.99 4.38 3.86 3.41

t

FROM

152.88

Note. Mean = 153.4 * 2.38 kbp.

present in submolar quantities in the EcoRI digest of S. vittatum IV DNA (arrows in Fig. 4A) suggesting that there was a mixture of genotypes in the virus population isolated from the black fly larvae. Hibbin and Kelly (1981) also observed submolar bands in digests of type 22 virus DNA. The gross pathology of infected black fly larvae and the size and morphology of the virus particles described in this report indicated that an IV had been isolated. The SDS-PAGE profiles of virion structural

M.

R. L.,

ARNOLD,

1976. An iridescent virus from Simulium sp. (Diptera: Simuliidae) in Wales. .Z. Znvertebr. Pathol., 27, 133-135. BOUCIAS, D. G., MARUNIAK, J. E., AND PENDLAND, J. C. 1987. Characterization of an iridovirus isolated from the Southern Mole Cricket, Scapteriscus vicinus. J. Znvertebr. Pathol., 50, 238-245. ELLIOTT, R. M., LESCOTT, T., AND KELLY, D. C. 1977. Serological relationships of an iridescent virus (Type 25) recently isolated from Tipula sp. with two other iridescent viruses (Types 2 and 22). Virology, 81, 309-316. HIBBIN, J. A., AND KELLY, D. C. 1981. Iridescent virus type 22 DNA. Arch. Viral., 68, 9-18. KELLY, D. C. 1976. Iridescent virus Type 22 replication in Aedes albopictus cells in culture. J. Znvertebr. Pathol., 27, 415-418. KELLY, D. C. 1981. Non-occluded viruses. In “Pathogenesis of Invertebrate Microbial Diseases” (E. W. Davidson, Ed.), pp. 34-60. Allanhead Osmun, Totowa, New Jersey. KELLY, D. C. 1985. Insect iridescent viruses. Curr. Top. Microbial. Zmmunol., 116, 23-35. KELLY, D. C., AYRES, M. D., LESCOTT, T., ROBERT-

14

ERLANDSON

SON, J. S., AND HAPP, G. M. 1979. A small iridescent virus (Type 29) isolated from Tenebrio molitor: A comparison of its proteins and antigens with six other iridescent viruses. J. Gen. Virol., 42, 95-105.

U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227, 680-685.

LAEMMLI,

REYNOLDS,

E. S. 1963. The use of lead citrate at high

AND

MASON

pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17, 208-212. WARD, V. K. AND KALMAKOFF, J. 1987. Physical mapping of the DNA genome of insect iridescent virus type 9 from Wiseana spp. larvae. Virology, 160, 507-510. WEISER, J. 1968. Iridescent virus from the black fly Simulium ornatum in Czechoslovakia. J. Invertebr. Parho/.. 12, 36-39.