HYDROPXRICAru)II'M SYI{DROME (ANGARA DISEASE) IN ...

"ENMTNT.It
PURIFICATION OF TIIE flPS VfRUS
Purificario. ol hydr op4iwdium syndrom. vitus (HpSV) ws c.died oul A ddcnbed
b, Cavanigh (1t83) pith somo hodi6c6ior! Bdcny, lh. cluificd ftsoc tuhurc pbpagded
virus and orhd honogo.rs in 500 ml quinliry w.r.6lrr.d rhrouai 03 and 045
micrometer fiheN (Millipor. Corpor.tion, B.dford, MA ) ARd filiralion, HISV ws pelt.red
by ce.trinrgaion at25,000x81o.2 t hn ar 4'C. Th. Np.m ttnras removed ddrhevirus
p.ller wd sspddcd lo l% ofitl orisin.l volunc in NET buff.r (loo rll,t N.Ct, I nM
EDTA l0 nM Tns HCI; pH 7 4). Th. virur srDonon w$ r[.n Sddy pdsd $roush a
slsilc 25€cuB. n*dl. to dispcrs. vid! chnp. ud ws l.i o!.rnishl 4qC x wG tho
sonidted al 20 pd cnt powa lor l0 s@ndr using . W-l?5 Soniotor (Hat Syed-
Ult@nic, 1.c., Flmingdal., Nr. Equd voluft! of FluorMbor ws nix.d uirh cqual
vdum of cdkitugcd natdial ud @ rifu8.d rr 30O r S for 20 nin. Two ro rhre '
q@ity of rhe aqumus ph& ofvitus $spdrion wa phc.d onlo ! disminuous sooe
gEdidr, which bmpnsd of.{u!! qu.nrity ofscoc 6ging fom 2l - 45% scms ( v)
DEp.r€d in NET bufl.r, A dinind ,l'.rp int.rf@ eB obeo.n betwen ech layd The
gddiot was c.dntuged ar 90,000 X 8 lor 2 3 hoB ar 4'C. Ai6 ccotritugotion, . vifls b.nd
Ms obseftd it the iot.rfe of thc 25% lid 35% suqos l.y.rs *ith rh. cid ol r.flecred
liBh rammnr.d lhrough rh. bouoh of rha tub.. Th. vids bmd wrs h@tcd by scrior
with a q/ring€ €quippd wilhr6inch.s lon!, 14 !,iug. n.edle th. rip ofwhich w.s bent ar a
nght ogle. Il ws gendy inen.d fton th. top of th. tub. and th. e.tire $cros Iays dd lhe
vitus blnd was h!ryosl.d h w.r mixcd wirh NET butrcr and vjrus was pelt.t.d 0r 2t,000 em
fot 2 h6. The pellet w8 again olpcndcd in NET butr r 6nd laved for cirhs pado8cn.sir
studies or tunher proc.$ed for puiicaion ihtouSh ccsium chloide (CaCl) eradicnt For ttis
purpose virus suspc.,ion wls pl.c.! oD two layets ol CaCl solution (0.3 nl of densiry 145
g/nl ind I t ml of donsity 133 dml in NET hF.r pH 74) in 5 nl ce.tritusc lub.s lnd