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Elizabeth A. Falendysz, Juan G. Lopera, Jeffrey B. Doty, Yoshinori Nakazawa, Colleen Crill, Faye Lorenzsonn, Lem’s N. Kalemba, Monica D. Ronderos, Andres Mejia, Jean M. Malekani, Kevin Karem, Darin S. Carroll, Jorge E. Osorio, Tonie E. Rocke
Characterization of Monkeypox virus infection in African rope squirrels (Funisciurus sp.)
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1.0261 0 Td
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2.863 0 Td
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-34.2649 -1.3039 Td
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3.1748 0 Td
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1.9899 0 Td
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Q
q
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BT
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Q
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3.1748 0 Td
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3.6 0 Td
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3.4186 0 Td
(EP090616A6.)Tj
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3.3732 0 Td
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ET
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[()25()12()18(\n)-4(\r)-3()1(\n)12()12()-9()-4(\r)]TJ
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[()16()6()2()-20()-15()10()18()-9()-4(\r)]TJ
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(the)Tj
1.4684 0 Td
(slurry)Tj
2.5512 0 Td
(to)Tj
1.0261 0 Td
(make)Tj
2.3924 0 Td
(a)Tj
0.652 0 Td
(final)Tj
-34.2196 -1.2983 Td
(concentration)Tj
5.8166 0 Td
(of)Tj
1.0148 0 Td
(10%)Tj
1.9275 0 Td
(\(wgt/vol\).)Tj
4.1103 0 Td
(200)Tj
/F13 1 Tf
1.6497 0 Td
[()]TJ
/F8 1 Tf
(L)Tj
1.2643 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(tissue)Tj
2.4604 0 Td
(slurry)Tj
2.5512 0 Td
(was)Tj
1.7008 0 Td
(used)Tj
2.0579 0 Td
(for)Tj
1.389 0 Td
(DNA)Tj
2.3754 0 Td
(extraction)Tj
4.2576 0 Td
(with)Tj
-35.0586 -1.3039 Td
(the)Tj
1.4683 0 Td
(Zymo)Tj
2.6022 0 Td
(tissue)Tj
2.4604 0 Td
(g-DNA)Tj
3.1918 0 Td
(kit)Tj
1.2813 0 Td
(\(Zymo)Tj
2.9423 0 Td
(Research,)Tj
4.0309 0 Td
(Irvine,)Tj
2.8516 0 Td
(CA\).)Tj
2.1316 0 Td
(200)Tj
/F13 1 Tf
1.6441 0 Td
[()]TJ
/F8 1 Tf
(L)Tj
1.2699 0 Td
(of)Tj
1.0148 0 Td
(whole)Tj
2.6079 0 Td
(blood)Tj
2.5115 0 Td
(was)Tj
1.7007 0 Td
(used)Tj
2.058 0 Td
(for)Tj
-35.7673 -1.2983 Td
(DNA)Tj
2.3754 0 Td
(extraction)Tj
4.2576 0 Td
(with)Tj
1.9956 0 Td
(the)Tj
1.4683 0 Td
(Zymo)Tj
2.6022 0 Td
(g-DNA)Tj
3.1918 0 Td
(kit)Tj
1.2869 0 Td
(\(Zymo)Tj
2.9423 0 Td
(Research,)Tj
4.0309 0 Td
(Irvine,)Tj
2.8459 0 Td
(CA\).)Tj
2.1374 0 Td
(The)Tj
1.7801 0 Td
(quality)Tj
2.9537 0 Td
(of)Tj
1.0148 0 Td
(each)Tj
-34.8829 -1.2982 Td
(DNA)Tj
2.3754 0 Td
(extraction)Tj
4.2576 0 Td
(was)Tj
1.7008 0 Td
(confirmed)Tj
4.3936 0 Td
(using)Tj
2.3868 0 Td
(a)Tj
0.6576 0 Td
(Nanodrop)Tj
4.3653 0 Td
(2000)Tj
2.126 0 Td
(spectrophotometer)Tj
7.8123 0 Td
(\(Nanodrop)Tj
4.7111 0 Td
(prod-)Tj
-34.7865 -1.304 Td
(ucts,)Tj
2.0522 0 Td
(Wilmington,)Tj
5.3801 0 Td
(DE\).)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 569.0267 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Quantitative)Tj
5.4992 0 Td
(real-time)Tj
4.0583 0 Td
(PCR)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 552.0755 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Viral)Tj
2.2336 0 Td
(DNA)Tj
2.3755 0 Td
(in)Tj
1.0374 0 Td
(blood)Tj
2.5115 0 Td
(and)Tj
1.7291 0 Td
(tissues)Tj
2.8233 0 Td
(was)Tj
1.6951 0 Td
(quantified)Tj
4.3087 0 Td
(using)Tj
2.3924 0 Td
(real-time)Tj
3.8551 0 Td
(PCR)Tj
2.0636 0 Td
(to)Tj
1.0261 0 Td
(detect)Tj
2.6136 0 Td
(the)Tj
1.4683 0 Td
(E9L)Tj
1.8028 0 Td
(gene)Tj
2.0806 0 Td
(of)Tj
-36.0167 -1.3039 Td
(orthopoxviruses)Tj
6.684 0 Td
(using)Tj
2.3924 0 Td
(primers)Tj
3.3506 0 Td
(described)Tj
4.0535 0 Td
(by)Tj
1.1792 0 Td
(Li)Tj
1.0261 0 Td
(et)Tj
0.9411 0 Td
(al.)Tj
1.1282 0 Td
([)Tj
0.83 0.64 0.02 0 k
(32)Tj
0 g
(].)Tj
2.0749 0 Td
(SYBR)Tj
2.5285 0 Td
(Green)Tj
2.7043 0 Td
(PCR)Tj
2.0636 0 Td
(Master)Tj
2.999 0 Td
(Mix)Tj
-33.1254 -1.2982 Td
(\(Applied)Tj
3.736 0 Td
(Biosystems,)Tj
4.8755 0 Td
(Carlsbad,)Tj
3.9855 0 Td
(California,)Tj
4.4674 0 Td
(USA\))Tj
2.4491 0 Td
(was)Tj
1.6951 0 Td
(used)Tj
2.0523 0 Td
(in)Tj
1.0318 0 Td
(an)Tj
1.2019 0 Td
(iQ5)Tj
1.7008 0 Td
(Real-Time)Tj
4.4163 0 Td
(PCR)Tj
2.058 0 Td
(Detection)Tj
-33.6697 -1.2983 Td
(System)Tj
3.05 0 Td
(\(Bio-Rad,)Tj
4.0762 0 Td
(Hercules,)Tj
3.9855 0 Td
(California,)Tj
4.473 0 Td
(USA\).)Tj
2.6816 0 Td
(DNA)Tj
2.3754 0 Td
(standards)Tj
4.0875 0 Td
(containing)Tj
4.5241 0 Td
(4.15)Tj
1.8765 0 Td
(ng)Tj
1.2303 0 Td
(to)Tj
1.0261 0 Td
(4.15)Tj
1.8709 0 Td
(x10)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 566.759 517.4929 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(-)Tj
-48.9001 -1.9476 Td
(7)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 203.5842 500.0314 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(ng)Tj
1.2302 0 Td
(were)Tj
2.1259 0 Td
(made)Tj
2.4208 0 Td
(by)Tj
1.1849 0 Td
(extracting)Tj
4.2123 0 Td
(total)Tj
2.0069 0 Td
(DNA)Tj
2.3754 0 Td
(from)Tj
2.2053 0 Td
(purified)Tj
3.4073 0 Td
(MPXV-Congo/Luc+)Tj
8.4641 0 Td
(as)Tj
1.0148 0 Td
(described)Tj
-31.005 -1.2982 Td
(above,)Tj
2.7722 0 Td
(and)Tj
1.7291 0 Td
(creating)Tj
3.4526 0 Td
(eight)Tj
2.1996 0 Td
(10-fold)Tj
3.0898 0 Td
(serial)Tj
2.3244 0 Td
(dilutions)Tj
3.77 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4684 0 Td
(purified)Tj
3.4128 0 Td
(DNA)Tj
2.3755 0 Td
(in)Tj
1.0374 0 Td
(molecular)Tj
4.218 0 Td
(grade)Tj
-32.8646 -1.2983 Td
(water.)Tj
2.6531 0 Td
(Standards)Tj
4.2009 0 Td
(were)Tj
2.1203 0 Td
(aliquoted,)Tj
4.1839 0 Td
(and)Tj
1.7292 0 Td
(each)Tj
2.0295 0 Td
(aliquot)Tj
3.0104 0 Td
(was)Tj
1.6951 0 Td
(used)Tj
2.0636 0 Td
(no)Tj
1.2756 0 Td
(more)Tj
2.3414 0 Td
(than)Tj
2.0296 0 Td
(5)Tj
0.6917 0 Td
(times)Tj
2.3924 0 Td
(to)Tj
1.0261 0 Td
(prevent)Tj
-33.4428 -1.3039 Td
(degradation)Tj
5.0228 0 Td
(of)Tj
1.0148 0 Td
(standards)Tj
4.0819 0 Td
(from)Tj
2.2053 0 Td
(repeated)Tj
3.6397 0 Td
(freeze-thaw)Tj
4.8586 0 Td
(cycles.)Tj
2.7779 0 Td
(Using)Tj
2.5965 0 Td
(these)Tj
2.2564 0 Td
(standards,)Tj
4.3086 0 Td
(assays)Tj
2.6249 0 Td
(were)Tj
-35.3874 -1.2983 Td
(sensitive)Tj
3.6225 0 Td
(enough)Tj
3.2202 0 Td
(to)Tj
1.0261 0 Td
(detect)Tj
2.6135 0 Td
(approximately)Tj
5.9754 0 Td
(2042.49)Tj
3.2995 0 Td
(viral)Tj
1.9956 0 Td
(genomes)Tj
3.7644 0 Td
(in)Tj
1.0375 0 Td
(0.1)Tj
1.3946 0 Td
(mL.)Tj
1.8085 0 Td
(Standard)Tj
3.8324 0 Td
(curves,)Tj
-33.5902 -1.2982 Td
(DNA)Tj
2.3753 0 Td
(concentration,)Tj
6.0434 0 Td
(and)Tj
1.7292 0 Td
(Ct)Tj
1.1848 0 Td
(value)Tj
2.3074 0 Td
(were)Tj
2.1203 0 Td
(calculated)Tj
4.2066 0 Td
(using)Tj
2.3868 0 Td
(the)Tj
1.4683 0 Td
(iQ5)Tj
1.7121 0 Td
(Optical)Tj
3.1521 0 Td
(System)Tj
3.0557 0 Td
(Software,)Tj
3.9232 0 Td
(Ver-)Tj
-35.6652 -1.2983 Td
(sion)Tj
1.9048 0 Td
(2.1.97.1001)Tj
4.6998 0 Td
(\(Bio-Rad\).)Tj
4.4107 0 Td
(The)Tj
1.7858 0 Td
(cutoff)Tj
2.5511 0 Td
(for)Tj
1.3833 0 Td
(fluorescence)Tj
5.1874 0 Td
(signal)Tj
2.5455 0 Td
(was)Tj
1.6951 0 Td
(determined)Tj
4.8529 0 Td
(by)Tj
1.1792 0 Td
(calculating)Tj
-32.1956 -1.3039 Td
(the)Tj
1.4682 0 Td
(average)Tj
3.2258 0 Td
(and)Tj
1.7292 0 Td
(standard)Tj
3.7247 0 Td
(deviation)Tj
3.9458 0 Td
(of)Tj
1.0148 0 Td
(Ct)Tj
1.1848 0 Td
(values)Tj
2.6646 0 Td
(for)Tj
1.3889 0 Td
(standards)Tj
4.0819 0 Td
(across)Tj
2.6759 0 Td
(all)Tj
1.1452 0 Td
(PCRs.)Tj
2.6532 0 Td
(Samples)Tj
3.4979 0 Td
(with)Tj
1.9956 0 Td
(Ct)Tj
-36.3965 -1.2983 Td
(values)Tj
2.6644 0 Td
(that)Tj
1.7802 0 Td
(were)Tj
2.1203 0 Td
(less)Tj
1.6157 0 Td
(than)Tj
2.0239 0 Td
(or)Tj
1.0999 0 Td
(equal)Tj
2.3584 0 Td
(to)Tj
1.0261 0 Td
(2)Tj
0.6973 0 Td
(standard)Tj
3.7191 0 Td
(deviations)Tj
4.3086 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.474 0 Td
(mean)Tj
2.4435 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(lowest)Tj
2.7439 0 Td
(concen-)Tj
-33.5732 -1.2982 Td
(tration)Tj
2.9422 0 Td
(detectable)Tj
4.2293 0 Td
(within)Tj
2.8119 0 Td
(40)Tj
1.1679 0 Td
(cycles)Tj
2.5568 0 Td
(were)Tj
2.1203 0 Td
(considered)Tj
4.5978 0 Td
(positive.)Tj
3.5433 0 Td
(DNA)Tj
2.3754 0 Td
(quantities)Tj
4.1556 0 Td
(were)Tj
2.1203 0 Td
(used)Tj
2.0636 0 Td
(to)Tj
1.0205 0 Td
(cal-)Tj
-35.7049 -1.304 Td
(culate)Tj
2.5737 0 Td
(the)Tj
1.4684 0 Td
(number)Tj
3.4185 0 Td
(of)Tj
1.0148 0 Td
(viral)Tj
1.9899 0 Td
(genomes)Tj
3.7701 0 Td
(in)Tj
1.0375 0 Td
(the)Tj
1.4683 0 Td
(samples.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 341.0078 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Viral)Tj
2.1921 0 Td
(titration)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 324 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Virus)Tj
2.4377 0 Td
(in)Tj
1.0375 0 Td
(swabs)Tj
2.5682 0 Td
(was)Tj
1.7008 0 Td
(detected)Tj
3.5659 0 Td
(and)Tj
1.7291 0 Td
(measured)Tj
4.1102 0 Td
(using)Tj
2.3868 0 Td
(a)Tj
0.652 0 Td
(TCID)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 425.4236 322.3559 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(50)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 434.7212 324 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(assay)Tj
2.2677 0 Td
(and)Tj
1.7291 0 Td
(converted)Tj
4.1952 0 Td
(to)Tj
1.0262 0 Td
(PFU/mL)Tj
3.7303 0 Td
(as)Tj
-36.4192 -1.2982 Td
(described)Tj
4.0535 0 Td
(elsewhere)Tj
4.1046 0 Td
([)Tj
0.83 0.64 0.02 0 k
(21)Tj
0 g
(,)Tj
0.83 0.64 0.02 0 k
(29)Tj
0 g
(].)Tj
3.2485 0 Td
(Briefly,)Tj
3.0784 0 Td
(96)Tj
1.1735 0 Td
(well-plates)Tj
4.4504 0 Td
(were)Tj
2.1203 0 Td
(seeded)Tj
2.9082 0 Td
(with)Tj
1.9956 0 Td
(Vero)Tj
2.2167 0 Td
(cells)Tj
1.9218 0 Td
(\(ATCC)Tj
3.1805 0 Td
(CCL-)Tj
-34.452 -1.2983 Td
(81,)Tj
1.3947 0 Td
(Manassas,)Tj
4.2746 0 Td
(VA\))Tj
1.9389 0 Td
(to)Tj
1.0204 0 Td
(approximately)Tj
5.9754 0 Td
(90%)Tj
1.9333 0 Td
(confluence.)Tj
4.8018 0 Td
(Swabs)Tj
2.6815 0 Td
(were)Tj
2.1259 0 Td
(sonicated)Tj
4.0082 0 Td
(and)Tj
1.7291 0 Td
(vortexed)Tj
3.6907 0 Td
(in)Tj
-35.5745 -1.2982 Td
(the)Tj
1.4684 0 Td
(viral)Tj
1.9955 0 Td
(transport)Tj
3.9232 0 Td
(medium)Tj
3.6056 0 Td
(\(described)Tj
4.3937 0 Td
(in)Tj
1.0375 0 Td
(sample)Tj
3.0217 0 Td
(collection\))Tj
4.4502 0 Td
(after)Tj
2.0353 0 Td
(thawing.)Tj
3.668 0 Td
(Swab)Tj
2.3244 0 Td
(medium)Tj
3.6 0 Td
(was)Tj
-35.5235 -1.304 Td
(diluted)Tj
3.0388 0 Td
(in)Tj
1.0318 0 Td
(DMEM)Tj
3.3108 0 Td
(with)Tj
2.0012 0 Td
(1%)Tj
1.4514 0 Td
(FBS)Tj
1.8028 0 Td
(and)Tj
1.7291 0 Td
(antibiotics)Tj
4.3994 0 Td
(\(1)Tj
/F13 1 Tf
1.0374 0 Td
[()]TJ
/F8 1 Tf
(g/L)Tj
2.058 0 Td
(amphotericin)Tj
5.6238 0 Td
(B,)Tj
1.0261 0 Td
(100)Tj
1.6498 0 Td
(U/mL)Tj
2.6418 0 Td
(penicillin,)Tj
-32.8022 -1.2982 Td
(100)Tj
/F13 1 Tf
1.6441 0 Td
[()]TJ
/F8 1 Tf
(g/mL)Tj
2.88 0 Td
(streptomycin,)Tj
5.7373 0 Td
(and)Tj
1.7291 0 Td
(0.05)Tj
1.8709 0 Td
(mg/mL)Tj
3.1918 0 Td
(gentamycin)Tj
4.9039 0 Td
(\(Life)Tj
2.0805 0 Td
(Technologies,)Tj
5.7543 0 Td
(Grand)Tj
2.8176 0 Td
(Island,)Tj
2.9026 0 Td
(NY,)Tj
-35.5121 -1.2983 Td
(USA\)\).)Tj
3.0217 0 Td
(100)Tj
/F13 1 Tf
1.6441 0 Td
[()]TJ
/F8 1 Tf
(L)Tj
1.2643 0 Td
(of)Tj
1.0148 0 Td
(eight)Tj
2.2053 0 Td
(10-fold)Tj
3.0898 0 Td
(serial)Tj
2.3243 0 Td
(dilutions)Tj
3.7701 0 Td
(were)Tj
2.1203 0 Td
(added)Tj
2.6589 0 Td
(to)Tj
1.026 0 Td
(each)Tj
2.0296 0 Td
(well)Tj
1.8198 0 Td
(in)Tj
1.0375 0 Td
(a)Tj
0.652 0 Td
(96)Tj
1.1735 0 Td
(well)Tj
1.8199 0 Td
(plate.)Tj
2.364 0 Td
(Plates)Tj
-35.0359 -1.3039 Td
(were)Tj
2.1203 0 Td
(incubated)Tj
4.1783 0 Td
(at)Tj
0.9524 0 Td
(37C)Tj
2.228 0 Td
(with)Tj
1.9956 0 Td
(5%)Tj
1.4513 0 Td
(CO)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 343.3889 231.3637 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 349.1716 233.0078 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(for)Tj
1.3832 0 Td
(3)Tj
0.6974 0 Td
(days)Tj
1.9955 0 Td
(before)Tj
2.7383 0 Td
(being)Tj
2.4321 0 Td
(fixed)Tj
2.194 0 Td
(with)Tj
1.9956 0 Td
(0.1%)Tj
2.1543 0 Td
(crystal)Tj
2.812 0 Td
(violet)Tj
2.4151 0 Td
(in)Tj
-35.7333 -1.2982 Td
(10%)Tj
1.9276 0 Td
(formalin.)Tj
3.9288 0 Td
(After)Tj
2.2847 0 Td
(fixation)Tj
3.2938 0 Td
(and)Tj
1.7292 0 Td
(staining,)Tj
3.6338 0 Td
(viral)Tj
1.9956 0 Td
(plaques)Tj
3.2372 0 Td
(were)Tj
2.1203 0 Td
(counted)Tj
3.4752 0 Td
(and)Tj
1.7348 0 Td
(titer)Tj
1.8765 0 Td
(was)Tj
1.7008 0 Td
(determined)Tj
-32.9383 -1.2983 Td
(with)Tj
1.9956 0 Td
(a)Tj
0.652 0 Td
(Reed)Tj
2.2167 0 Td
(and)Tj
1.7348 0 Td
(Muench)Tj
3.5602 0 Td
(calculator)Tj
4.1329 0 Td
([)Tj
0.83 0.64 0.02 0 k
(21)Tj
0 g
(,)Tj
0.83 0.64 0.02 0 k
(29)Tj
0 g
(].)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 178.0157 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Antibody)Tj
4.063 0 Td
(detection)Tj
4.2284 0 Td
(and)Tj
1.8661 0 Td
(serum)Tj
2.9717 0 Td
(neutralization)Tj
6.0851 0 Td
(assay)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 161.0078 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
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