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[(TissueLyzer.)-343.7(The)-339.7(solid)-339.6(material)-342.2(was)-339.2(removed)-339.5(through)-336.1(cen-)]TJ
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[(trifugation)-296(\(20,000)]TJ
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7.6143 0 TD
(g)Tj
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[(,)-291.4(5)-162.4(min\))-293.7(and)-292.4(the)-286.8(cleared)-294.7(material)-296.1(was)-293.1(ti-)]TJ
-8.1154 -1.1116 TD
[(tered.)-195.7(Vero)-196.5(cells)-198(were)-197.6(infected)-196.8(with)-195.6(10-fold)-197.8(serial)-199(dilutions)-194.5(of)]TJ
T*
[(sample)-263.5(for)-256.7(12)-164.6(h.)-258(The)-259.1(inoculum)-259.1(was)-264.3(then)-253.4(removed,)-262.3(replaced)]TJ
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()Tj
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(-)Tj
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[(.)-481.5(Chicken)-489.3(embryos)-482.2(were)-485.6(scanned)]TJ
15.3611 67.9184 TD
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()Tj
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T*
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T*
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1.0137 -1.1174 TD
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(1)Tj
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[(-weighted)-531.7(and)-522.8(T)]TJ
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(1)Tj
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(2)Tj
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[(1)-168.1(mm\))-529.9(and)-528.6(T)]TJ
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()Tj
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0 Tc
(2)Tj
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[(,)-537.3(i).5(m)7.5(a)-2.9(ge)-536.2(matr)7.5(ix)]TJ
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[(256,)-537.7(1)7.1(6)-.9(22)-535.4(slice)7.1(s)-537.5(de-)]TJ
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1.0137 -1.1116 TD
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(R)Tj
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[(\(A)-291(Language)]TJ
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[(and)-373.1(Environment)-303.9(for)-297(Statistical)-304(Computing\).)-299.2(MRI)-304.7(volumet-)]TJ
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(P)Tj
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[(-value)-256.7(of)]TJ
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(<)Tj
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[(0.05)-352.3(indicated)-347.9(statistically)-352.4(signicant)-353.2(differences)-350.7(between)]TJ
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(groups.)Tj
1.0137 -1.1116 TD
[(BiosafetyAll)-295.3(studies)-285.2(were)-289.8(conducted)-290(in)-284.5(accordance)-294.4(with)]TJ
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[(NIH)-241.2(and)-240.6(UGA)-236.3(guidelines)-238.2(for)-239.4(embryonated)-241.6(chicken)-241.7(eggs.)-239.2(All)]TJ
0 -1.1116 TD
[(virus)-431.6(work)-431.5(was)-431.4(approved)-431.6(by)-428.4(the)-430.8(UGAs)-434.1(institutional)-436.7(bio-)]TJ
T*
[(safety)-337.1(committee.)]TJ
/F1 1 Tf
9.9626 0 0 9.9626 313.0582 376.044 Tm
[(Resu)-10.9(lts)]TJ
/F9 1 Tf
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/F4 1 Tf
9.843 0 0 9.843 323.0361 341.518 Tm
[(Embryonated)-673.6(broiler)-671.4(eggs)-667.7(were)-669.9(infected)-674.9(with)-667.9(ZIKV)]TJ
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[(MEX1-44,)-613.1(a)-612.8(strain)-612.6(isolated)-616.1(from)-610.3(a)-612.8(eld)-610.3(population)-611.6(of)]TJ
T*
[(Mexican)-426.9(mosquitoes)-416.5(in)-417(January)-424.5(2016)-419.1([10].)-416.7(Eggs)-420.1(were)-416.5(in-)]TJ
0 -1.1174 TD
[(ocula)-8(ted)-321.4(with)-328.1(an)-325.9(amniotic)-329.5(injection)-329.5(of)-324.7(ZIKV)-329.9(on)-324.7(embryonic)]TJ
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[(day)-298.2(2.5)-293.6(\(E2.5\))-298.1(or)-295.9(embryonic)-295.8(day)-298.2(5)-294.8(\(E5\),)-297(accomplished)-301.4(with)]TJ
T*
[(an)-395(established)-395.7(windowing)-392.1(procedure)-399.2(to)-388.2(access)-396.8(the)-390.5(embryo)]TJ
0 -1.1174 TD
[([12].)-462.8(Daily)-462.8(monitoring)-461.9(of)-457.2(embryo)-464(viability)-461(showed)-463.5(dose-)]TJ
0 -1.1116 TD
[(depen)-7.8(dent)-397.4(morality)-403.2(three)-405.3(days)-397(following)-402.8(infection.)-407.7(To)-400.8(de-)]TJ
T*
[(termine)-547.1(if)-543.7(ZIKV)-543(replication)-546(was)-540.8(occurring)-548(in)-538(the)-546(chick)]TJ
0 -1.1174 TD
[(embryos,)-301.3(a)-301.8(proportion)-299.2(of)-295.9(the)-298.3(embryos)-297.9(was)-298.9(harvested)-301.3(3)-300.6(days)]TJ
0 -1.1116 TD
[(postinfectio)-10.1(n)-531(\(dpi\))-535.5(and)-534.4(the)-534.5(presence)-537.4(of)-537.8(infectious)-537.6(ZIKV)]TJ
T*
[(particles)-295.8(was)-293.1(monitored)-294.8(with)-287.8(plaque)-291.3(assays.)-294(The)-293.6(percentage)]TJ
T*
[(of)-543.6(the)-546(population)-542.5(exhibiting)-542.6(ZIKV)-543(replication)-546(was)-546.6(dose)]TJ
0 -1.1174 TD
[(depen)-7.8(dent)-334(after)-336.2(both)-332.8(E2.5)-330.5(and)-338.5(E5)-331.7(infections)-336(\(Fig.)-336.1(1A\).)]TJ
1.0137 -1.1116 TD
[(The)-368.5(number)-371.8(of)-365(infectious)-370.6(viral)-369.8(particles,)-374.1(harvested)-370.4(from)]TJ
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[(each)-365(embryo,)-363.7(was)-362.2(at)-366.4(least)-359.2(100-fold)-366(greater)-362.6(than)-362.8(the)-361.7(inocu-)]TJ
0 -1.1174 TD
[(lum,)-353.7(conrming)-352.1(active)-355.9(virus)-356.8(replication)-355.9(\(Fig.)-347.6(1B\).)-353.3(In)-353.5(addi-)]TJ
0 -1.1116 TD
[(tion,)-399.8(chick)-399.6(embryos)-401.5(that)-403.2(were)-399.2(conrmed)-403.8(to)-394(contain)-402(active)]TJ
T*
[(ZIKV)-283.8(infections)-278.5(\(ZIKV)]TJ
/F7 1 Tf
7.3823 0 0 6.5614 407.7353 148.6488 Tm
(+)Tj
/F4 1 Tf
9.843 0 0 9.843 411.8172 144.2834 Tm
[(\))-277.5(showed)-279.2(dramatic)-278.7(mortality)-277.7(within)]TJ
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[(3)-168.1(dpi,)-248.8(and)-240.6(only)-246.4(embryos)-246(exposed)-251.8(to)-244.2(the)-246.5(three)-244.1(lowest)-250.6(doses)-242.4(of)]TJ
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[(ZIKV)-433.5(survived)-424.6(to)-428.5(embryonic)-428.3(day)-424.9(20)-428.4(\(E20\))-427.1(of)-428.4(in)-422.8(ovo)-423.8(de-)]TJ
T*
[(velopment)-399.6(\(Fig.)-393.7(1C\).)-399.4(In)-393.8(data)-398.5(not)-395.1(shown,)-395.5(a)-393.9(small)-399.6(group)-391.4(of)]TJ
T*
[(embryos)-246(was)-247.1(infected)-242.9(on)-244.1(embryonic)-244(day)-240.6(13)-244.1(\(E13\))-242.8(and)-240.6(active)]TJ
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[(ZIKV)-468.1(infection)-461.9(was)-465.9(identied)-467.5(7)-162.4(dpi)-464.2(on)-463(E20,)-463(yet)-459.6(no)-463(fetal)]TJ
0 -1.1116 TD
[(mortality)-341.1(was)-333.5(observed.)]TJ
1.0137 -1.1116 TD
[(The)-362.8(presence)-364.6(of)-359.3(virus)-362.5(observed)-362.3(at)-360.6(the)-361.7(end)-355.8(of)-365(in)-359.4(ovo)-354.6(de-)]TJ
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[(velopment)-296(was)-293.1(not)-291.4(signicantly)-297.9(altered)-293.6(by)-295.9(the)-292.6(day)-292.5(of)-290.2(ZIKV)]TJ
/F1 1 Tf
8.9663 0 0 8.9663 62.022 749.6502 Tm
(1692)Tj
43.8051 0 TD
[(GOODFELLOW)-334.8(ET)-338.7(AL.)]TJ
ET
endstream
endobj
51 0 obj
<>stream
/GS1 gs
BT
/F4 1 Tf
9.843 0 0 9.843 59.7543 254.0975 Tm
0 0 0 rg
0 Tc
0 Tw
[(infection)-329.4(or)-324.7(the)-327.1(narrow)-331.1(dose)-322.2(level,)-328.3(and)-327(therefore)-330.2(results)-325.5(for)]TJ
0 -1.1116 TD
[(these)-635.6(treatment)-631.3(groups)-628.4(were)-635.4(pooled)-629.9(for)-631(all)-632.5(subsequent)]TJ
0 -1.1174 TD
[(endpoints.)-613.6(High)-610.2(viral)-611.7(loads)-611.4(were)-612.3(consistently)-608.9(identied)]TJ
0 -1.1116 TD
[(within)-347.7(the)-338.7(brain)-346.6(and)-338.5(eyes)-346.4(of)-342(E20)-344.3(chick)-342(embryos)-343.9(\(Fig.)-341.8(1D\).)]TJ
T*
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0 -1.1174 TD
[(heart,)-362.7(intestine,)-355.8(and)-355.8(liver)-358.2(\(Fig.)-353.4(1D\).)-358.9(The)-357(widespread)-360.9(distri-)]TJ
0 -1.1116 TD
[(bution)-571.3(of)-572.4(replicating)-574.8(ZIKV)-577.5(particles)-572.3(in)-572.5(multiple)-576.2(organs)]TJ
T*
[(suggests)-291.7(that)-293.8(initial)-291.7(low-level)-289.9(infection)-294.8(of)-290.1(ZIKV)-289.5(can)-293.6(lead)-289.1(to)]TJ
0 -1.1174 TD
[(persistent)-364.7(viral)-364(growth)-358.9(in)-359.4(the)-361.7(chick)-359.3(embryos.)-364.7(The)-357(embryos)]TJ
0 -1.1116 TD
[(were)-295.6(not)-291.4(perfused)-293.1(before)-291.1(organ)-294.6(harvest)-292.2(and)-292.4(at)-291.5(least)-295.9(some)-288.8(of)]TJ
T*
[(the)-482.6(virus)-477.7(observed)-483.2(in)-474.6(the)-482.6(various)-475.3(tissues)-480.8(may)-483.7(have)-479(been)]TJ
0 -1.1174 TD
[(circulating)-338.6(in)-336.4(the)-332.9(blood.)]TJ
/F9 1 Tf
9.9626 0 0 9.9626 59.7543 107.5464 Tm
[(In)-339.2(vivo)-335.5(imag)-7.4(ing)-333.9(of)-333.6(Zika-)-8(induce)-10.3(d)-330(brain)-342.3(damage)]TJ
/F4 1 Tf
9.843 0 0 9.843 69.7323 89.6882 Tm
[(ZIKV-induced)-346.9(brain)-340.8(neuropathology)-340.5(was)-339.2(observed)-345(using)]TJ
-1.0137 -1.1116 TD
[(magnetic)-445.8(resonance)-440.6(imaging)-436.6(\()-87.5(MRI\).)-435.9(Chick)-439.8(embryos)-441.9(were)]TJ
T*
[(imaged)-436.5(on)-434.2(embryonic)-439.8(day)-436.4(15)-434.2(\(E15\))-432.9(and)-436.4(E20)-430.7(of)-434.1(develop-)]TJ
0 -1.1174 TD
[(ment.)-396.3(Eight)-398.6(sham-injected)-394.7(embryos)-401.5(and)-390.4(12)-393.9(ZIKV-infected)]TJ
25.5097 20.0438 TD
[(embryos)-326.7(were)-324.3(imaged.)-330.5(MR)-320.9(imaging)-327.1(slices)-325.6(were)-330.1(oriented)-328.2(to)]TJ
0 -1.1116 TD
[(keep)-398.4(bilateral)-399.8(symmetry)-398.2(on)-399.6(the)-396.3(head,)-401.8(by)-393.9(checking)-398.3(the)-402(ap-)]TJ
0 -1.1174 TD
[(pearance)-308.3(of)-307.4(the)-309.9(two)-309.6(eyeballs.)-309.5(Two)-304.9(of)-307.4(the)-309.9(12)-307.5(ZIKV-infected)]TJ
0 -1.1116 TD
[(embryos)-274.8(displayed)-271.5(gross)-275.8(defects)-276.1(on)-272.9(anatomical)-276.5(T)]TJ
7.3823 0 0 6.5614 506.7778 219.5716 Tm
(2)Tj
9.843 0 0 9.843 510.4628 221.2157 Tm
(-weighted)Tj
-20.2799 -1.1116 TD
[(MR)-580.1(images)-586.1(at)-579.5(E20)-580.5(\(Fig.)-583.7(2A\).)-577.8(Hyperintensity)-589.1(or)-578.1(bright)]TJ
0 -1.1174 TD
[(contrast)-518(shown)-518.8(in)-514.9(the)-517.2(inset)-519.3(box)-521.7(of)-514.8(ZIKV-infected)-522(chick)]TJ
0 -1.1116 TD
[(embryos)-332.4(indicates)-341.9(higher)-330.4(content)-338.6(of)-330.5(uid)-332.7(in)-336.4(the)-332.9(malformed)]TJ
T*
[(brain)-335.1(area.)]TJ
1.0137 -1.1174 TD
[(Segmentation)-697.8(of)-606.9(an)19.7(at)19.5(om)19.6(ic)19.5(al)-608.3(MR)18.9(I)-611.6(a)0(f)19.7(f)0(o)20.9(r)0(d)15.1(e)0(d)-602.4(v)0(o)20.8(l)0(u)20.7(m)0(e)18.4(t)0(r)20.8(i)0(c)]TJ
-1.0137 -1.1116 TD
[(qu)20.8(an)19.7(ti)20.6(c)19.8(at)19.5(io)15(n)-289.1(o)0(f)-278.6(s)0(p)21(e)0(c)18.5(i)0()20.9(c)-290.2(br)20.9(ai)13.8(n)-289.1(r)0(e)19.7(g)0(i)20.7(o)0(n)20.8(s)0(.)-280.8(W)0(h)18.6(e)0(n)-279.8(c)0(o)19.7(m)0(p)19.6(a)0(r)19.7(i)0(n)20.7(g)-289.1(un)20.8(-)]TJ
T*
[(in)20.7(fe)19.7(ct)19.5(ed)-636.9(an)19.7(d)-646.2(i)0(n)15(f)0(e)19.7(c)0(t)19.5(e)0(d)-636.9(e)0(m)18.5(b)0(r)20.9(y)0(o)20.8(s)0(,)-637.9(v)0(o)20.8(l)0(u)20.7(m)0(e)18.4(t)0(r)15(i)0(c)-637.1(q)0(u)20.8(a)0(n)19.7(t)0(i)20.6()0(c)19.8(a)0(t)19.5(i)0(o)20.7(n)]TJ
0 -1.1174 TD
[(re)19.7(ve)19.7(al)19.5(ed)-222.2(st)20.9(un)20.8(te)19.5(d)-231.5(C)0(N)18.9(S)-231.3(gr)20.9(ow)20(th)-221.2(be)19.7(tw)19.8(ee)18.5(n)-231.5(E)0(1)19.6(5)-231.5(an)19.7(d)-231.5(E)0(2)19.6(0)-225.7(in)-221.2(ZI)19.7(KV)19.1(-)]TJ
0 -1.1116 TD
[(in)20.7(fe)19.7(ct)19.5(ed)-302.9(em)24.2(br)15.1(yo)20.8(s)-306.2(\()0(F)21(i)0(g)14.9(.)-302.9(2B)14(F)21(\).)-298.2(No)20(ni)20.7(nf)20.9(ec)18.5(te)19.6(d)-312.1(e)0(m)18.5(b)0(r)20.9(y)0(o)20.8(s)-306.2(de)19.7(mo)19.6(n-)]TJ
T*
[(st)20.9(ra)19.7(te)19.5(d)-202.7(a)-203.8(si)20.9(gn)20.8(i)15.1(ca)18.5(nt)-192.4(in)20.7(cr)19.7(ea)18.5(se)-193.2(in)-192.4(wh)20(ol)20.7(e)-203.8(b)0(r)20.9(a)0(i)19.5(n)-202.7(vo)20.8(lu)20.7(me)-194.7(an)19.7(d)-202.7(w)0(i)19.8(t)0(h)20.7(i)0(n)]TJ
T*
[(al)19.5(l)-277.7(c)0(o)19.7(r)0(t)15(i)0(c)19.6(a)0(l)-268.4(b)0(r)20.9(a)0(i)19.5(n)-283.3(re)19.7(gi)20.7(on)20.8(s)-283.1(f)0(r)20.9(o)0(m)-268.4(E)0(1)19.6(5)-283.3(to)-267.3(E2)19.6(0)-283.3(\()0(F)21(i)0(g)20.7(.)-279.9(2B)19.8(D)20(\).)-269.4(Co)19.8(n-)]TJ
0 -1.1174 TD
[(ve)19.7(rs)21.1(el)19.5(y,)-263.7(ZI)19.7(KV)-263.1(in)20.7(fe)14(ct)19.6(io)20.7(ns)-261.2(st)20.9(un)15.1(te)19.5(d)-271.8(t)0(h)20.7(e)-273(gr)20.9(ow)20(th)-267.3(of)-261.4(th)20.7(e)-273(c)0(h)19.7(i)0(c)19.5(k)-271.8(em)18.4(-)]TJ
0 -1.1116 TD
[(br)20.9(yo)-480.3(CN)18.9(S)-490.5(a)0(s)-475.5(e)0(v)13.9(i)0(d)20.7(e)0(n)19.7(t)-490.8(by)-474.5(no)20.8(ns)15.3(ig)20.7(ni)20.7(c)19.8(an)19.7(t)-490.8(i)0(n)20.7(c)0(r)19.7(e)0(a)18.5(s)0(e)19.9(s)-490.5(in)-480.4(br)20.9(ai)19.5(n)]TJ
T*
[(vo)20.8(lu)20.7(me)-154.3(wi)19.8(th)-157.8(on)20.8(ly)-152.1(th)20.7(e)-169.3(c)0(e)18.5(r)0(e)19.7(b)0(e)19.7(l)0(l)20.6(u)0(m)-153.2(e)0(x)13.9(h)0(i)20.7(b)0(i)20.7(t)0(i)20.6(n)0(g)-152(s)0(i)15.1(g)0(n)20.8(i)0()20.9(c)0(a)18.5(n)0(t)-152.1(g)0(r)15.1(o)0(w)20(t)0(h)20.7(,)]TJ
0 -1.1174 TD
[(wh)20(il)20.6(e)-163.5(o)0(t)20.7(h)0(e)13.9(r)-162.3(co)19.7(rt)20.7(ic)19.6(al)-159(re)19.7(gi)20.7(on)20.8(s)-167.9(r)0(e)19.7(m)0(a)18.4(i)0(n)20.7(e)0(d)-153.1(u)0(n)20.8(d)0(e)19.7(r)0(s)15.3(i)0(z)19.6(e)0(d)19.7(.)-164.7(We)-155.4(ob)20.8(se)19.9(rv)20.9(ed)]TJ
ET
q
1 i
59.981 732.245 m
549.751 732.245 l
549.751 360.624 l
59.981 360.624 l
W n
q
489.77 0 0 371.622 59.981 360.624 cm
/Im1 Do
Q
Q
BT
/F5 1 Tf
9.845 0 0 9.845 59.7543 343.8424 Tm
[(FIG.)-250.4(1.)]TJ
/F4 1 Tf
4.0252 0 TD
[(The)-247.2(chick)-249.3(embryo)-244.4(is)-243.8(permissive)-249.5(to)-244(persistent)-248.5(ZIKV)-242.9(infection)-247.9(in)-244(all)-246.4(organs)-243(and)-246(causes)-251(embryo)-244.4(death.)]TJ
/F5 1 Tf
40.3156 0 TD
(\(A\))Tj
/F4 1 Tf
1.6297 0 TD
[(At)-244.8(3)-242.9(days)]TJ
-45.9705 -1.0135 TD
[(postinfection)-401.6(\(dpi\),)-394.3(chick)-393.3(embryos)-394.9(display)-394.1(a)-399.5(proportional)-393.6(increase)-398.4(in)-393.7(infectivity)-399.9(to)-393.7(ZIKV)-392.6(infection.)-401(As)-394.2(few)-396.6(as)-394.6(0.2)-391.2(viral)]TJ
T*
[(particles)-329.5(were)-323.8(sufcient)-328(to)-324.6(infect)-325.4(embryos,)-323.4(but)-319.9(infection)-328.5(rates)-324(increase)-329.3(with)-321.9(greater)-327.3(numbers)-325.8(of)-318.7(viral)-329(particles.)]TJ
/F5 1 Tf
45.4176 0 TD
(\(B\))Tj
/F4 1 Tf
1.6527 0 TD
(ZIKV-)Tj
-47.0703 -1.0077 TD
[(positive)-262.8(\(ZIKV)]TJ
/F7 1 Tf
7.3837 0 0 6.5625 120.359 318.3306 Tm
(+)Tj
/F4 1 Tf
9.845 0 0 9.845 124.4409 313.9653 Tm
[(\))-260.1(embryos)-262.5(display)-261.7(active)-263.2(viral)-259.9(replication.)-260.3(At)-262.1(3)-168(dpi,)-259.9(the)-257.7(viral)-259.9(load)-264.4(in)-255.5(ZIKV)]TJ
/F7 1 Tf
7.3837 0 0 6.5625 417.3731 318.3306 Tm
(+)Tj
/F4 1 Tf
9.845 0 0 9.845 424.0629 313.9653 Tm
[(embryos)-256.7(dramatically)-266(increased)]TJ
-37.0044 -1.0135 TD
[(from)-310.3(the)-303.8(initial)-308.4(injected)-305.6(dose.)]TJ
/F8 1 Tf
12.329 0 TD
[(Open)-305.4(circles)]TJ
/F4 1 Tf
5.4533 0 TD
[(indicate)-305.6(individual)-305.4(observations,)-311.3(and)]TJ
/F8 1 Tf
15.2371 0 TD
(line)Tj
/F4 1 Tf
1.8197 0 TD
[(indicates)-306.5(the)-303.8(mean)-306.9(of)-307.2(each)-306.9(dose.)]TJ
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13.5671 0 TD
(\(C\))Tj
/F4 1 Tf
-48.4063 -1.0135 TD
(ZIKV)Tj
/F7 1 Tf
7.3837 0 0 6.5625 83.2819 298.3748 Tm
(+)Tj
/F4 1 Tf
9.845 0 0 9.845 90.5386 294.0094 Tm
[(chick)-329.9(embryos)-331.6(demonstrate)-332.5(dose-dependent)-329.5(survival)-326(with)-333.4(high)-326.6(mortality)-328.7(within)-329.8(the)-326.8(rst)-326.3(3)-168(dpi.)]TJ
/F5 1 Tf
38.7723 0 TD
(\(D\))Tj
/F4 1 Tf
1.7103 0 TD
[(Infectious)-329.1(viral)]TJ
-43.6095 -1.0135 TD
[(particles)-271.9(were)-266.3(isolated)-269.8(from)-264.3(E20)-269(chick)-266.6(embryos)-268.2(after)-266.6(infection-substantial)-274(viral)-265.6(loads)-265.3(were)-272(identied)-265(within)-266.4(the)-269.2(brain,)-268.8(eyes,)]TJ
0 -1.0077 TD
[(and)-338.1(all)-332.7(other)-334.5(tissues)-336.2(evaluated)-336.4(relative)-340.2(to)-330.4(0)-335(particles)-335.3(\(sham\))-336.3(injection.)-337.8(ZIKV,)-332.6(Zika)-334.7(virus.)]TJ
/F1 1 Tf
8.9663 0 0 8.9663 59.7543 749.7069 Tm
[(ZIKV)-338.7(IN)-335.1(CHICK)-330.9(EMBRYOS)]TJ
52.4612 0 TD
(1693)Tj
ET
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