Baculovirus expression of the glycoprotein gene of Lassa virus and characterization of the recombinant protein

virus Research, 25 (1992179-90 0 1992 Elsevier Science Publishers

VIRUS

79 B.V. All rights reserved

0168-1702/92/$05.00

00811

Baculovirus expression of the glycoprotein gene of Lassa virus and characterization of the recombinant protein Kimberly

B. Hummel,

Mary Lane Martin

and David D. Auperin

*

Special Pathogens Branch, DirQsion of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers For Disease Control, Public Health Sewice, US Department of Health and Human Sewices, Atlanta. GA 30333, USA (Received

16 March

1992: revision

received

18 May 1992; accepted

26 May 1992)

Summary

A recombinant baculovirus was constructed that expresses the glycoprotein gene of Lassa virus (Josiah strain) under the transcriptional control of the polyhedrin promoter. The expressed protein (B-LSGPC) comigrated with the authentic viral glycoprotein as observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), was reactive with monoclonal antibodies (MAbsl in Western blots, and was glycosylated. Although the recombinant protein was not processed into the mature glycoproteins, Gl and G2, it demonstrated reactivity with all known epitopes as measured by indirect immunofluorescence (IFA), and it was immunogenic, eliciting antisera in rabbits that recognized whole virus in IFAs. Regarding future applications to diagnostic assays, the recombinant glycoprotein proved to be an effective substitute for Lassa virus-infected mammalian cells in IFAs and it was able to distinguish sera from several human cases of Lassa fever, against a panel of known negative sera of African origin, in an enzyme immunoassay (EIA). Baculovirus; Glycoprotein

gene; Lassa virus: Recombinant

protein

Correspondence to: K.B. Hummel, Respiratory and Enterovirus Branch, National Center for Infections Diseases, Public Health Service. US Dept. of Health and Human Services, Atlanta, GA 30333, USA, Fax: (11 (4041 639-1307. * Present address: Department of Molecular Genetics, Central Research Division. Pfizer Incorporated, Eastern Point Road. Groton. CT 06340, USA.

Introduction Lassa fever. which is endemic to western sub-Saharan Africa, results in a spectrum of illnesses in humans ranging from asymptomatic infection to fatal hemorrhagic disease (McCormick et al., 1987a, b). The etiologic agent has been identified as Lassa virus, a member of the Arenaviridae (Frame et al., 19701, which is harbored in the natural host the multimammate rat, ~~~~~~~~ ~~~~~e~z~~~ (Monath et al., 1974; Murphy and Walker, 19781. Human infection occurs primarily by contact with urine-contaminated food, water, or soil, as a result of rodent infestation (Keenlyside et al., 19831, and overall estimates of antibody prevalence range from 4-6% in Guinea to as high as 1520% in Nigeria (McCormick, 1987; Tomori and McCormick, 1988). Although ribavirin appears to be an effective drug therapy (McCormick et al., 19851, diagnosis and treatment are often not available in many areas. Consequently, Lassa fever accounts for 10-l%% of all adult medical admissions in West Africa, resulting in over 100,000 infections and LOOO-3.000 deaths per year (McCormick, 1987). The extremely pathogenic nature of this virus has resulted in its classification as a biosafety IeveI 4 agent (P4), requiring maximum biocontainment facihties in which to culture live virus. Such precauti~)na~ measures, although required for human safety, are an impediment to conducting fundamental research on the molecular biology and pathogenesis of the virus. AccordingIy, headway in diagnostic development and implementation has also been limited. The most common method of serologic diagnosis of Lassa virus remains the IFA, in which infected cell monolayers must be grown under P4 conditions and subsequently inactivated with gamma irradiation. In an effort to produce a non-infectious source of antigen for both research and diagnostic applications, we constructed a recombinant Lassa virus glycoprotein by using a baculovirus expression system. Recombinant baculoviruses are well known for their ability to express large quantities of foreign protein under the transcriptional control of the polyhedrin gene promoter (Matsuura et al., 1987). The Lassa virus glycoprotein is post-translationally cleaved to yield the two envelope glycoproteins Gl and G2, which in addition to the internal nucleoprotein, comprise the structural components of the virus. The nucleoprotein gene of Lassa virus has recently been expressed in a baculovirus system and the resultant protein was indistinguishable from the authentic viral nucleoprotein as determined by SDSPAGE and immunoblotting using Lassa virus-specific antibodies (Barber et al., 1990). In this study, we characterize the antigenic properties of the expressed glycoprotein and evaluate its efficacy as an improved diagnostic antigen.

Materials and Methods Cells and I.GUSSpodoptera fmgiperda (Sf9) ceils and Autographa californica nuclear polyhedrosis virus (AcNPV) were obtained from Dr. M.D. Summers (Department of Ento-

81

mology, Texas A&M University, College Station, TX). Sf9 cells were cultured in TNM-FH medium (Hazelton, Lenexa, KS) supplemented with 10% fetal bovine serum (FBSI, 50 pg/ml gentamycin sulfate, and 2.5 pg/ml fungizone.

The transfer vector pAcYM1 (Matsuura et al., 19871, obtained from Dr. D.H.L. Bishop (NERC Institute of Virology, Oxford, UK), was modified by the insertion of a multipte cloning region at the original Barn HI site to produce pAcYMIB/S and was donated by Anthony Sanchez (Centers for Disease Control, Atlanta, GA), Plasmid manipulations to generate a recombinant baculovirus expressing the glycoprotein gene of Lassa virus (Josiah strain) were performed essentially as described by Maniatis et al. (1982). The plasmid LS1337, which contained the complete Lassa virus glycoprotein gene (Auperin et al., 1986), was digested with Apa I and Barn HI to liberate a fragment suitable for subcloning into pAcYM1B. Transfection of Sf9 cells with both transfer vector and AcNPV DNA was achieved through use of a calcium phosphate precipitation technique as described by Summers and Smith (1987). Dot blot and Southern blot analyses were performed on Genescreen hybridization transfer membranes (New England Nuclear, Boston, MA), using “2P-Iabeled nick-translated cDNA probes. A recombinant baculovirus expressing the nucleoprotein gene of Ebofa virus was used as a polyhedrin-minus negative control in IFAs and was donated by Anthony Sanchez (Centers for Disease Control, Atlanta, GA). Protein analyses Sf9-infected cell lysates prepared from wildtype or recombinant baculoviruses were resolved by SDS-PAGE as described by Laemmli (1970). Proteins were visualized by Coomassie brilliant blue stain or were transferred to nitrocellulose and probed with MAbs specific for the Lassa glycoprotein. In some instances, urea was added to the sample dissociation buffer (8 MI, to the stacking gel (5 Ml, and to the resolving gel (2.5 Mf to ensure complete dissociation of the polyhedrin protein. For pulse-labeling experiments, wildtype and recombinant baculovirus-infected Sf9 cells were starved in methionine-free medium prior to the addition of [35S]methionine (50 ~Ci/mI) for 6 h. Cell monolayers were harvested at 24, 48, 72, 96, and 120 h postinfection (pi.1 and lysates were prepared for resolution by SDS-PAGE. Infected cell cultures were also labeled continuously for 24 h with [3H]glucosamine or [“Hlmannose (250 pCi/ml) beginning at 72 h p.i., and Lassa virus-specific proteins were immunoprecipitated using MAbs specific for the glycoprotein as previously described (Auperin et al., 1988). Immunojluorescence Lassa virus protein expression was assayed in acetone-fixed and live Sf9 cells by IFA (Weller and Coons, 1954) using a panel of mouse MAbs specific for Lassa Gl

x2

and G2 proteins. A fluorescein used as a detector.

isothiocyanate-conjugated

secondary

antibody

was

Antibody generation in rabbits Monospecific antibodies to the recombinant glycoprotein were generated by injecting New Zealand White rabbits with crude freeze-thawed lysates from baculovirus-infected Sf9 sells. To prepare the lysates, infected cells (MO1 = 10) were harvested 72 h pi., diluted to 5.5 x lo6 cells/ml in phosphate-buffered saline (PBS), and lysed by 3 successive rounds of freeze-thawing. Aliquots (1.5 ml) of lysate were emulsified in an equal volume of Freund’s complete adjuvant and were injected subcutaneously. Two weeks later, a second injection of lysate, emulsified in Freund’s incomplete adjuvant, was similarly administered. At 3 and 5 weeks after the initial injection. 1.5 ml ~01s. of lysate and alum (2 mg/mll were administered by intraperitoneal injection. Serum specimens were drawn from rabbits before immunization and 10 days after the third and fourth rounds of injections. Lassa antibody titers were determined by IFA on Lassa virus-infected E6 Vero cells.

ElAS Cells infected with the recombinant baculovirus (MO1 = 101 were harvested 72 h pi. and lysates prepared as described above. A 1: 20 working dilution of the B-LSGPC protein was derived at by titration against a known positive human antiserum for Lassa virus, used at a dilution of 1 : 100. Lysate prepared from uninfected Sf9 cells was used as a negative control. Serum samples were collected as part of the Lassa Fever Research Project in Sierra Leone, West Africa from hospitalized patients infected with Lassa virus as confirmed by IFA using whole virus and in some cases, by virus isolation. Negative control sera were collected from hospitalized febrile patients who showed no seroconversion to Lassa virus as determined by IFA, or who had no IgM antibody to Lassa virus and unsuccessful virus isolation. Wells of Immulon 2 flat-bottom microtiter plates (Dynatech Laboratories. Inc., Alexandria, VA) were pretreated with 0.2% glutaraldehyde in PBS at 37°C before antigen was immobilized overnight at 4°C. Plates were blocked with 0.01 M PBS containing 0.05% Tween 20 and 10% FBS (PBST/FBS), and test serum was titrated in two-fold increments beginning at 1:200 with PBST/FBS as diluent. After incubation at 37°C with goat anti-human IgG horseradish peroxidase conjugate (Tago Inc., Burlingame, CA), plates were washed before the enzyme substrate TMB (3, 3’. 5, 5’ tetramethylbenzidine dihydrochloride, Sigma Chemical Co., St. Louis, MO) was added. Color development was assayed after 10 min by using a Perkin Elmer Lambda reader at 450 nm. Positive wells displayed an absorbance reading of 0.100 or greater and at least twice that of corresponding uninfected control wells.

83

Results Recombinant LG-usconstruction and purification The inverted complementary sequences present in the intergenic region of the arenavirus S RNA were removed from the 3’ end of the Lassa virus glycoprotein gene prior to insertion into the transfer vector pAcYM1B. This step was done to eliminate the possibility that the hairpin structure present in this region of DNA might interfere with gene expression in the recombinant baculovirus. All plasmid constructions were confirmed by restriction endonuclease and DNA sequence analyses over ligation junctions before transfection of AcNPV and vector DNA into cultured Sf9 cells. Limiting dilution cloning, using dot blot hybridization to cDNA probes of the Lassa glycoprotein, was performed to isolate a recombinant virus. Each round of successive screening was confirmed by IFA on infected Sf9 cells using MAbs to the glycoprotein. A plaque-purified recombinant baculovirus yielded a high-titer stock of 1.5 X 10” PFU/mi, and displayed no evidence of occluded virus in infected cell nuclei or of the polyhedrin protein by SDS-PAGE. Southern blot analyses (Fig. 1) of viral DNA were performed to confirm the recombination of the Lassa glycoprotein gene at the polyhedrin locus in the

7.30

kb

2.97

kb *

Probe:

AcNPV

DNA

Lasso

GPC cDNA

Fig. 1. Southern blot of viral DNA preparations digested to completion with EcoRI and resolved by electrophoresis in a 0.7% agarose gel. The filter was hybridized to “‘P-labeled nick-translated probes representative of AcNPV and Lassa GPC DNA. The 7.3 kb AcNPV EcoRi fragment is as indicated.

84

P

24

h,

48

h,

12

“r

56

h,

W”

h,

Fig. 2. Autoradiogram of recombinant CB-LSGPC) and wildtype CAcNPV) baculovirus-infected cell lysates labeled with [3’S]methionine, harvested at 24, 48, 72, 96, and 120 h pi., and resolved on 10% SDS-PAGE with fluorographic enhancement. Positions of the putative glycosylated CGPC) and unglycosylated (G’) forms of the recombinant glycoprotein, and the polyhedrin protein (P) are as indicated. A recombinant haculovirus (B-LSN) expressing the nucleoprotein WI is also shown for comparison.

wildtype AcNPV genome. The recombinant baculovirus lacked the 7.30 kb EcoRI fragment, which contained the intact polyhedrin gene, and hybridized as two smaller fragments due to an internal EcoRI site within the glycoprotein gene. Protein analyses

Cells infected with recombinant or wildtype baculoviruses were harvested 72, 96, and 120 h p.i. and protein lysates were resolved by SDS-PAGE (data not shown). The recombinant glycoprotein migrated as a broad band at approximately 72-75 kDa and reached maximum cellular levels by 72 h p.i. as detected by Coomassie blue stain. A 57 kDa protein was also evident in the recombinant baculovirus-infected cell lysate. The rate of recombinant glycoprotein synthesis was compared with that of polyhedrin protein synthesis via a series of pulse-labeling analyses (Fig. 2). Polyhedrin protein synthesis was clearly evident 48 h p.i. and maintained maximum levels throughout 120 h p.i. Synthesis of both species of the recombinant glycoprotein became apparent by 48 h p.i., reached maximum levels by 72 h, and declined thereafter. Radioimmune precipitations of [“SSlmethionine-, [“Hlglucosamine-, and [3H]mannose-labeled cell lysates using Lassa MAbs (Fig. 3) were performed to determine if the 72-75 kDa and 57 kDa proteins observed by SDS-PAGE represented glycosylated (GPC) and unglycosylated (G’) forms of the gtycoprotein, respectively. Both protein species iabeted well with [3sS]methionine and were reactive with MAbs, even in the absence of enzymatic cleavage to yield the mature

2 -4

04 ,

&

& co b7-,+* +

z

:

*

Jt

5

$

g

b

5

3H-Glucosamine

3H-Mannose

Total Proteins

a a+

s 91

+

9

1

Total Proteins 35 S-Met.

I- GPC

35S-Met.

Pulse

Label

Fig. 3. Autoradiogram of recombinant (B-LSGPC) bac~~ovirus-infected cell lysates labeled with [3*S]methionine (left panel) and with [3~]mannose or [3H]giucosamin~ (right panel) and reacted with MAbs. Immune complexes were precip~tat~d with ~tap~~foc~cu~ aureus protein A and resolved on 10% SDS-PAGE with fluorographic enhancement. Identified Gl and G2 monoclonal antibodies were pooled for immunoprecipitation of [“H]labeled proteins. Positions of the glycosylated (GPO and unglycosylated (G’) forms of the recombinant glycoprotein are as indicated. Total [35S]radiolabelrd proteins from a baculovirus-infected cell lysate are also shown for comparison.

glycoproteins GI (45 kDa) and G2 (38 kDa), lnt~rmed~at~ coprecipitated proteins also observed probably represented differentially glycosylated species of the expressed glycoprotein. In contrast, upon [3Hllabeling, only the 72-75 kDa protein incorporated mannose and glucosamine residues, thus suggesting that the 57 kDa form was unglycosylated. Furthermore, Western blot analyses, using iysates derived from recombinant baculovirus-infected Sf9 cells and Lassa virus-infected BHK-21 cells, confirmed that the glycosylated form of the expressed protein was comigrant with the authentic Lassa glycoprotein (data not shown).

The antigeni~i~ of the baculovirus-expressed glycoprotein was compared with that of Lassa virus by IFA using a pane1 of MAbs reactive with multiple distinct epitopes on Gl and G2 (Table 11. As non-specific background problems are

86 TABLE

1

Indirect immunofluorescent antibodies MAb 71-J 133-23

64-5 53-14 161-16 71-5 135-17 C 73-h 85-6 121-22 772-7 195-Z 165-4 216-7 138-33 154-18

Protein specificity Gl Cl Cl Gl Gl GI G’ G;! G7 G’ G2 G7 G7 G7 G2 G’7

reactivity

of a recombinant

glycoprotein

to Lassa virus-specific

Lassa I’

B-EBOLAN

1

+

_

1 I

++ ++

II II III I I II III III III IV IV V VI

+ + t t + t t+ t + + + +t ++

Epitopic

h

monoclonal

B-LSGPC

h

group

Antibody preparations diluted to 1 : 64 unless otherwise to +++. ’ Lassa virus-infected BHK-21 cells. h recombinant baculovirus-infected Sf9 cells. ‘ 1 :32 dilution of antibody.

t+t +t+ ttt t++

_ _ _ _ _ _ _

++t +t+

t t+t tt tt+ +++ +++ t tt ttt t++

_ _ _ indicated.

increasing

amount

of fluorescence

+

commonly observed with the polyhedrin protein, a polyhedrin-minus baculovirus expressing the nucleoprotein of Ebola virus was used as a negative control (Materials and Methods). For all MAbs tested, the fluorescence observed by using the B-LSGPC protein was equal to or greater in intensity than that obtained by using whole virus. Additionally, transport of the recombinant glycoprotein to the plasma membrane was detected in approximately 5% of infected Sf9 cells as assessed by IFA on unfixed cells. Monospecific antibodies generated in rabbits against the recombinant glycoprotein were also reactive (titer < = 1: 16) in IFAs using whole virus derived from Lassa virus-infected E6 Vero cells. Pre-bleed serum in all cases was negative. EIAS Preliminary studies were undertaken to determine the efficacy of the recombinant glycoprotein as a source of antigen in an EIA. Twenty-five serum samples from 9 patients infected with Lassa virus were tested and results were compared with those obtained by IFA using whole virus (Table 2). Twelve samples from 9 Lassa virus-negative patients served as control sera and were unreactive (data not shown). For whole virus IFA titers greater than or equal to 1 : 64, the recombinant glycoprotein was capable of detecting serum IgG antibodies against Lassa virus. In

87

TABLE 2 Comparison of serum IgG antibody titers to Lassa virus by whole virus 1FA and recombinant reactivity Outbreak serum no.

Days since onset

Lassa isolation (logs of virus)

83087486-l X3087486-6 X3087486-8 ~3OS74~~-1 83087488-5 83~~7~~~-1~ 83087489-J 83087489-3 83087489-8 83087492- 1 83U87492-3 X3087492-4 83087492-7 83087586-1 83087586-2 8308758f?-4 83MX93-2 83087620-3 83087620-5 83087640-J 83087640-8 83087865-3 83087865-4 83087865-6 83087RhS-7

4 8 12 10 I4 3.5 8 10 17 3 5 6 I# 8 9 17 75 9 12 10 18 9 11 15 29

0.0 ND ND 3.6 2.1 0.0 2.1 ND 0.0 1.6 3.1 ND 0.0 0.0 ND ND ND ND ND ND ND 7.1 0.0 0.0 0.0

IFA titer 2%

EIA titer B-LSGPC

> 1,024

800 1,600

> 1,024

1.600

> 1,024 > I.024 256

> 1.024 Ih 64 > 1.024 16 256 > 1,024 1,024 512 > 1,024 118 > 1.034 16 32 ‘56 > I,024

EIA

_ 3,200 3,200 1.600 1,fiOO 3,xX3

400 3.200 400 800 800 800 400 1,600 400 800

-

Negative value for IFA indicates titer of I : 4 or less. Negative value for EIA indicates titer less than 1:200. ND = not determined.

addition, a rise in IFA titer associated with viral clearance was also reflected in the EIA titer using the recombinant antigen. In three patients, ~3~8748~~ 83087492, and 83087865, IFA and EIA values were discordant until serum samples from later in the course of illness were analyzed. In the latter patient, however, the B-LSGPC protein remained unreactive.

Discussion A recombinant baculovirus containing the Lassa virus glycoprotein gene was shown to induce the synthesis of a glycosylated protein that was similar in electrophoretic mobihty to the authentic viral glycoprotein and that was reactive with MAbs specific for the glycoprotein in both immnno~uorescent and immunoprecipitation assays. Although the composition of the side chains on the expressed

protein was not determined, it has been previously reported that complex oiigosaccharides, which may influence many biological functions such as targeting to different cellular compartments and epitopic maintenance of glycoproteins, are often truncated in insect cells (Kuroda et al.. 1990). Thus, as the carbohydrate moieties added to the B-LSGPC protein were probably not of the complexity of the authentic Lassa glycoprotein, this may have interfered with effective transport to the cell surface. Such impaired glycosylation may also have contributed to the observation that the recombinant glycoprotein was not cleaved into the mature glycoproteins Gl and G2, as has been previously demonstrated in a vaccinia virus expression system (Auperin et al.. 1988). Cleavage of baculovirus-expressed glycoproteins has been documented, albeit at various rates, for several viruses (Kuroda et al., 1990, Schmaljohn et al.. 1989, Vialard et al., 19901, although other viral glycoproteins appear to have a host-mediated cleavage requirement (Madisen et al.. 1987). If indeed the Sf9 cell line lacks the protease necessary for cleavage into Gl and G2, the absence of such processing apparently was not critical for the antigenicity of the recombinant glycoprotein. The expressed glycoprotein was also evaluated as a potential source of antigen for diagnostic assays as 50% of patients by day 5 of illness, and virtually all by day 15, have detectable IgG antibody levels against the Lassa glycoprotein as measured by IFA (McCormick 1990). The recombinant glycoprotein proved to be an effective substitute for Lassa virus-infected BHK-21 cells in IFAs using a panel of mouse MAbs. Such findings corroborated the work of Barber et al. (19901, which demonstrated that a recombinant nucleoprotein was an adequate replacement for mammalian cells infected with whole virus in both 1FAs and EIAs. The B-LSGPC protein in EIAs, however. did not detect Lassa-specific IgG antibodies in all of the human outbreak serum samples, and in such a format may not be suitable as a single replacement source of antigen. As the broadest cross-reacting and most conserved antigens appear to be located on the G2 viral proteins of both African and South American arenaviruses (Rue et al., 19911, such a lack in reactivity may be in part due to ~onformational-dependent epitopes not presented on the recombinant glycoprotein. Additionally, there may be some degree of heterogeneity in the Lassa viruses of Africa, as a variable antigenic site has been identified on the Gl protein, such that a Gl MAb of the Josiah strain from Sierra Leone identified only local isolates or those from immediately adjacent countries (Rue et al., 19911. Nonetheless, these data suggest that using recombinant Lassa proteins in either an IFA or an EIA format for the detection of antibodies to Lassa virus would be efficacious in terms of safety. without a loss of sensitivity, when compared with current diagnostic assays. The generation of a recombinant glycoprotein may also provide a means by which to study viral-host interactions such as the cell-mediated immune response. as the Lassa virus glycoprotein may be an important target as has been demonstrated for Iymphocytic choriomeningitis virus (Oldstone et al., 1988, Whitton et al., 1988). Indeed, previous studies have demonstrated that a vaccinia recombinant expressing the Lassa glycoprotein, when administered as vaccine, prevented severe disease and death in challenged monkeys, although a recombinant expressing the

89

nucleoprotein was ineffective (Fisher-Hoch et al., 1989). Clearly, a better understanding of the immunogenic roles each structural protein plays in establishing lifelong immunity must be elucidated before a genetically engineered vaccine can be developed for the prophylaxis of Lassa fever in areas where the virus is endemic.

Acknowledgements

The authors respectfully acknowledge the advice given on baculovirus cloning by David O’Reilly and Lois K. Miller of the Department of Genetics and Entomology, University of Georgia, Athens, Georgia, and by Anthony Sanchez of the Special Pathogens Branch, Centers for Disease Control, Atlanta, GA. The authors also thank Paul A. Rota for assistance in manuscript preparation.

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90 McCormick, J.B. (1990) Arenaviruses. in: B.N. Fields and D.M. Knipe tEdsI, Virology. 2nd ed.. pp. 1245-1267. Raven Press, New York. McCormick. J.B.. King. I.J., Webb. P.A.. Scribner. C.L., Craven. R.B.. Johnson. K.M., Elliott. L.H. and Williams. B. (19861 Lassa fever. effective therapy with ribavirin. N. Engl. J. Med. 314. 3-2h. McCormick, J.B.. Webb. P.A., Krehs. J.W.. Johnson. K.M. and Smith. ES. tlYK7a) A prospective study of the epidemiology and ecology of Lassa fever. J. Infect. Dis. 155, 337-444. McCormick, J.B.. King, I.J., Wehh. P.A., Johnson. K.M.. O’Sullivan. R., Smith. ES. Trippel, S. and Tong, T.C. (1987bl A case-control study of the clinical di~~~nosis and course of Lassa fever. J. Infect. Dis. 155, 445-455. Monath. T.P.. Newhouse. V.F.. Kemp. G.E.. Setzer. H.W. and Cacciapuoti. A. (lY74) Lassa virus isolation from ~~~~ff~~z~~~rur~le~l~~j.~rodents during an epidemic in Sierra Leone. Science iii.;. ‘63-265. Murphy. F.A. and Walker, D.H. (lY7Xf Arenaviruses: persistent infection and viral survival in reservoir hosts. In: E. Kurstak and K. Maramor~)s~h tEds.1. Viruses and Environment. pp. 155-1X0. Academic Press, New York. Oldstone, M.B.A., Whitt~)n, J.L., Lewicki, H. and Tishon, A. (1YXKf Fine dissection of a nine amino acid giycoprotein epitope. a major determinant recognized by Iymphocytic chori~~menin~iti~ virus-specific class I restricted H-2Dh cytotoxic T lymphocytes. J. Exp. Med. 168, 559-570. Ruo. S.L.. Mitchell. S.W.. Kiley, M.P.. Rounlillat. L.F,. Fisher-Hoch, S.P.. and McCormick. J.B. (IYYI 1 Antigenie relatedness between arenavirns~s defined at the epitope level by monoclonal antihodirs. J. Gen. Virol. 72. 539-555. Schmaljohn. C.S.. Parker. M.D.. Ennis. W.H.. Dalrympie, J.M., Collett. MS., Suzich. J.A. and Schmaljohll. A.L. (19891 Baculovirus expression of the M genome segment of Rift Valley fever virus and examination of antigenic and immlln(~genic properties of the expressed proteins. Virology 1711, 184-191. Summers. M.D. and Smith, G.E. t1987f. A manual of methods for Bacul(~v~rus vectors and insect cell culture procedures. Texas AgriculttIral Experiment Station. Bulletin No. 1555. Tomori. O.W. and McCormick. J.B. (19881 Viral hemorrhagic fever. Antib(~dies in Nigerian populations. Am. J. Trop. Med. Hyg. 2, 407-41 I. T., Briedis. ct., Alkhatib, G.. Henning, D., Levin. D. and Vialard, J., Lalumiere. M., Vernet. Richardson, C. fl990) Synthesis of the membrane fusion and hemaggit~tirlin proteins of measles virus, using a novel haculovirus vector containing the B-galactosidase gene. J. Viral. 61. 37-50. Weller. T.H. and Coons. AH. (1954) Fluorescent antibody studies with antigens of varicella and herpes zoster propagated in vitro. Pro?. Sot. Exp. Biol. Med. 86, 789. Whitton. J.L.. Gebhard, J.R.. Lewicki. H., Tishnn, .A. and Ofdstone. M.B.A. (1988) Molecular definition of a major cytotoxic T-lymphocyte epitope in the glycoprotein of lymphocytic clloriomeningitis virus. J. Viral. 62. 687-695.