A Bat-Derived Putative Cross-Family Recombinant Coronavirus with a Reovirus Gene


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A Bat-Derived Putative Cross-Family Recombinant Coronavirus with a Reovirus Gene

Material Information

Title:
A Bat-Derived Putative Cross-Family Recombinant Coronavirus with a Reovirus Gene
Series Title:
PLoS Pathogens
Creator:
Huang, Canping
Liu, William J.
Xu, Wen
Jin, Tao
Zhao, Yingze
Song, Jingdong
Shi, Yi
Ji, Wei
Jia, Hao
Zhou, Yongming
Wen, Honghua
Zhao, Honglan
Liu, Huaxing
Li, Hong
Wang, Qihui
Wu, Ying
Wang, Liang
Liu, Di
Liu, Guang
Yu, Hongjie
Holmes, Edward C.
Lu, Lin
Gao, George F.
Publisher:
PLOS ONE
Publication Date:
Language:
English

Subjects

Subjects / Keywords:
Bat-Derived ( local )
Bats ( local )
Coronavirus ( local )
Reovirus Gene ( local )
Genre:
serial ( sobekcm )

Notes

Abstract:
The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 has generated enormous interest in the biodiversity, genomics and cross-species transmission potential of coronaviruses, especially those from bats, the second most speciose order of mammals. Herein, we identified a novel coronavirus, provisionally designated Rousettus bat coronavirus GCCDC1 (Ro-BatCoV GCCDC1), in the rectal swab samples of Rousettus leschenaulti bats by using pan-coronavirus RT-PCR and next-generation sequencing. Although the virus is similar to Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9) in genome characteristics, it is sufficiently distinct to be classified as a new species according to the criteria defined by the International Committee of Taxonomy of Viruses (ICTV). More striking was that Ro-BatCoV GCCDC1 contained a unique gene integrated into the 3’-end of the genome that has no homologs in any known coronavirus, but which sequence and phylogeny analyses indicated most likely originated from the p10 gene of a bat orthoreovirus. Subgenomic mRNA and cellular-level observations demonstrated that the p10 gene is functional and induces the formation of cell syncytia. Therefore, here we report a putative heterologous inter-family recombination event between a single-stranded, positive-sense RNA virus and a double-stranded segmented RNA virus, providing insights into the fundamental mechanisms of viral evolution.
Original Version:
PLoS Pathogens, Vol. 12, no. 9 (2016-09-27).

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Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
This object is protected by copyright, and is made available here for research and educational purposes. Permission to reuse, publish, or reproduce the object beyond the bounds of Fair Use or other exemptions to copyright law must be obtained from the copyright holder.
Resource Identifier:
K26-05156 ( USFLDC: LOCAL DOI )
k26.5156 ( USFLDC: LOCAL Handle )

USFLDC Membership

Aggregations:
University of South Florida
Karst Information Portal

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serial

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n rn nr nr n n r nn n n n n n n n n n n n r n n n n n ! " n n n # n$ n % n & ' n ()* r+ , n rn %* % nnnnn r nr r r r r r r r r r r! r ! " # $r%r r %r% &' $r% (rr# &' r ) r%r r r *+r! ) r r nr ," -.' ,.' ' r *# " # ! r% $r%"r !# ++!# r $r%"r !# r %+# %r% r r! r r r $! # $! ) r " *# " # $r%"r $"r% !rr! r $r%"r! # r %+# %r% r r! r $ n/+ r r $r%"r!# r %+# %r% r r! r ' rrr %r r *# " # r% #./ r! % r r r r r r r! r , $r r r %r r r% r# -r % r!r% %r% ## $r% % (rr# ## ## n/ & r "# r !r r! %%r r r r r r% !# r %+# %r% r r r % n/++r# n r r! r r %r% r %+# %r% r r! r 0r% r%., r r r r r r! r n %r" 1# r /-2 r3#%%2% 4 !3%r %%2% ,5, +!% % 6r# #+ %r . r 7887 $r 6r# #+ %r $. r 7897 ! + r r "rrr# !+r% %.6%r +rr 6r %r 6%r# + " % + 6%r +++2 r / rrr %r 6rr# r! " %r. ,9 . ,9 r % /" +6 2OUSETTUS LESC HENAULTI " "# r! 6.%r . :.! r 1%r!2 ! r r r+r " %r *(; . *(; r !+ %%. rr% r r r%r# rr% " %rr / 6%r %%r! %rr r "# r ++r :+# r 2 $ r-r! / . ,9 %r r1 ! r! r <=. !+ +! r # -/ %r " /r% 1% 6#!. # # rr% + r-# r!r + 698 ! " r2 ". !+r% +n %. "r + 698 ! r %r r% +r % #%#r2 / 6 6r ! r.+r# %+"rr "/ r!. 6rr. 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 9 A 7B 23(1 $&&(66 &LWDWLRQ +XDQJ & /LX :;X : -LQ 7 =KDR < 6RQJ HW DO $ %DW'HULYHG 3XWDWLYH &URVV )DPLO\ 5HFRPELQDQW &RURQDYLUXV ZLWK D 5HRYLUXV *HQH 3/R6 3DWKRJ H GRL MRXUQDOSSDW (GLWRU 6XVDQ %DNHU /R\ROD 81,7(' 67 $ 7(6 5HFHLYHG 0D\ $FFHSWHG $XJXVW 3XEOLVKHG 6HSWHPE HU &RS\ULJKW ‹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n r ". !+ n r 6rr! rr! r . + +%r+ r r2 ++# nn nr n n n n n n n n n n n nr r n n r nr n n nnr n rr n n n nr nnnr nn r nn n n rr nn !"#$ n n n n nr nr nn n nrn n %" n rn n %" nn n n r n n r rn n rr n n r nn ./*0/ &n r nr nrn n nn n %" n '( ) n n nnr *nnnr & +n , *&,$ n r n r n nnr r r n n '. /( )n n n rn '.( &n r n n r n rn n %" n rn n n n n nn '0( nn rr n n n rn '1( n n r n '2( nn n /3n n rn r n n n r n n n n n '1( " n r nn nr r rrn n n n 4 nn r r n 5 n n n #"#&,$ '678( n n n r n nr n n n rn nr n nr n nn n '( r 8 n n r nn n n n r nrn nr r n ) rr nr n nn n n n nn n n ..89 '( n #"#&, '.( n rr r :9#&, '/ 0( " nn n ; rrn n ;n '176( *n n nr n r n &n n nr n n <&&=& )&, <&&=&$ :rr rn n n )&, <&&=& n nn n n n nr n n n r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 7 A 7B 1DWLRQDO *UDQG 67 6SHFLDO 3URMHFW *UDQW 1R =; WR <: DQG *)* LV D OHDGLQJ SULQFLSOH LQYHVWLJDWRU RI WKH 16)& ,QQRYDWLY H 5HVHDUFK *URXS *UDQW 1R 7KH IXQGHUV KDG QR UROH LQ VWXG\ GHVLJQ GDWD FROOHFWLRQ DQG DQDO\VLV GHFLVLRQ WR SXEOLVK RU SUHSDUDWL RQ RI WKH PDQXVFULS W &RPSHWLQJ ,QWHUHVWV 7KH DXWKRUV KDYH GHFODUHG WKDW QR FRPSHWLQJ LQWHUHVWV H[LVW

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1+/+ %r /r 6r %+"r r ! " r > r r nn r n ?nnn n &n n n n %" @ 06 0A$ r n n B& n n )C"# r n n n n n r n 5DE8 )&, 5DE8$ '>( n r =$ 5 r r n n rr rn nrn , 92 )5D. :=&D "108 59. &&. n rr rn : n nr rr rn nr r n rn r % n n n n n r rn r nn n rr r r r n n n n n r n /02 n /12$ n n+nn nn %<#$ "nr n r >2 n$ n %"nn %" r $ n n nr n rr nr n n r '8( nr n n <&&=& )&, <&&=&$ < n n )&, <&&=& r n r n n n n #n nn !nrr 13 n /3n )&, <&&=& n n n 13 n /3 "&9 ! $ %r */ 2 /0 +/*2 3 145/6 %# ,+ !r'r . , 92 n% ! 6r + % 6r % ! B=. <=.( r /r #/ " r! " % 6%r #2 +-" 698 ! r / r 2 6r r!r 698 ! r rr% "# / 1r +-2 1 % %r6r ! # 1% r%# / /r % "2 " 2OUSETTUS LESC HENAULTI r / 6%r . , 9 / r%2 %+r% rr %r r / r rrr r 6 #2 %+ r% rr r !+ 9 !+ r %r + 6r" r!r 698 !2 GRLMRXUQ DOSSDW J r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D < A 7B

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9+rn rnr r /3n n )&, <&&=& /.8 n n rn ( 222A 260A nr n #rr )&, <&&=& r rn n )&, 5DE8 + n n n /3 n n r n n+ n$ 4 n n @!$ nr nr 13 r @!G #$G%#/Gnr 9$Gn :$Gnr %$ rr n nn nnr n %#B$ ! n r $ r % n n "n n nr @! # %#/ 9 : n % n )&, <&&=& n )&, 5DE8 n n n r $ "r n /3n )&, <&&=& n 4 n % n r n n n &rr %#Bnn @! "n nnn n @! r n %#6 %#6 n %#6 r n .8A 1/A n n n )&, 5DE8 n n r n # r$ B n )&, <&&=& n n rr n @! .62 n % n %#6 n "r @! n r n nn n n n + /A 108A n n n n @! n # n n n '.( rn n %" rnn r @! nrr n r n n ! $ r n nn r n #$ )&, <&&=& n n rr;n n n nn n # n r $ # n 13"&<""&/3 nn #"#&, )&, 5DE8 n n ! rn r # r n n n n nn n n <$ 6> &$ ! n r $ %r n # n n nr n nn n r $ nnr n %#B$ n @! nn r rr n r n nn n /&r n /&C$ n nr n BC$ &n nn n ; n n rn n n2 n r n n n r . B n B n /&C r +rr rn H$ '.. ./( 5 nr .20. n )&, <&&=& n nr nn rn H$ n 5$ : nnr n rn n n n .6/ I8.$ n r n nn n n )&, 5DE8 C5J"< n n nr rn r /&C r n %#B8 n %#B " r nnn r n %#B n %#B. )&, <&&=& &"< n &"& n nn H 5 n n n r .$ !rrn n n n *&, ' /( n n r n )&, <&&=& r nr r /$ n n n nn n n )&, <&&=& r >00A >0>A n )&, 5DE8 r 8A r n r /$ 5n r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D E A 7B

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nr n )&, <&&=& n nr n n n n rn n )&, <&&=& # % n n 4 rn nr Brn # n % n rr )&, <&&=& 9rn nFDnFD?.0F.2 9 )&, Dn$ nFDnFD?2F.2 )&, Dn$ n )&, 5DE8 n rr rn = n ! .$ n 7 //n /*+0*/ */*2 +80+ +80 0r*+ + 3 145/6 %# )/ 145/6 9:' +/*+n ;14 6n 5/6 9: +/+n <(146 n 5/6 9: +/*+ 5/6 9: +/*+ =1-+ >0/ +/+ ?+/*/ / @ A*0/ + ?@ 3 */ A*)+ r 0 //2 ?B@ 145/ 6 %# VS * 1 * /*0 1 7 #+/0 ?/@ 3*r 1 / ;% 9:'+ 0 ;14 6 9:+ <(146 3 9:+ 9:+ =17 7 7E;;8F7B D@D 77C EC2;F B82@ .! .! .! .! .! ,;%; ;,, < ( 7BD@DF7B ;8D @D D92CF D;2@ <72; <72; 7<2@ 7D2< 7D2< ,,%;; ,, E < 7B;9EF7D B@; 779 @B2DF C82B E<27 E72 7DD7@F7@ ;BC EE< DC28F @92@ <;29 <;2BF E828 E92@ E92EF E92@ " C 7C7C@F7C CB8 9C@ E828 .! .! .! .! .! ;%; ; B >"7 7CCD"0 r 7;EEEF7; C;< 9E; <828F B<28 .! .! .! .! .! ,,;%;; , E D % /r $,D G79H r! 6r/r r 6r G9

0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D B A 7B

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r )&, <&&=& n /02 n /12$ n rn )&, Dn n )&, 5DE8 n + 7 A*0/ 3 / //E F 7 0E +/+ 3 145/6 %# 7+ 0r *+ )/ *//2 0*E *++ >A r 0+ +/+ =1-F =1-7 A*0/ + ?@ 3 */ 4 //E 0E +/ A//E 30/ r?+ @ 7 >A $ 9 ., 9@E 9@E ,>,n (-/ >A , 9@B ., @@7 B;C ,,>,* (-/ >A , @@< ., 7DB< 9CC9 ,>,n 7 6 >A , 7DBE .J <9E@ E;E J>, #6 "r% +r >A <9EC .J < 6 >A n #6 "r% +r >A" <@E7 .J (-/ >A$ nn r+ >A' n E87B . E9A, E9<@ . E7@B 9<; G;> (-/ >A E7@D . E7C; 9E (-/ 66r 059 >A E7@D .J B78@ ;<7 J> 6 >A B78@ .J BC8C D89 J> n >A BC8; .J D< :n n$ >A D<<; .J DDC8 * n ( >A DDC9 . D;@< 7;< 0.$ ? ,%%r +" r r r #r ? . nI88E@ 9C4 *(9 nI88DB@@ 4 nI889E B94 nI898C8 84 nI88<8E B4 $ nI889C ED4 nI88< EA ? *@ C<2A ?11@ C;2CF;82< @72< @72A ?n >A+@ ;82CF;92B @<2@ @<2@F@E28 @72@ @<29 @72;F@<29 >A ?( >n ><@ C729FC72C D92E D92CFD729 D929 D829 D82@ >A ?>:@ @82;F@<2< E;2@ E;2EFE;2@ ED2D ED28 E@2BFE@2C >A ?=<@ C829FCE27 D<2C D<29FD<2B D92C D729 D72B 0/ / r+ CE2EFCE2C DE2E DE2EFDE2D D72E D72 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D D A 7B

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r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D @ A 7B

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r A$ 5 nr n rn n n n n ! /$ n )&, <&&=& n rr r Bn$ n "r n rn )&, <&&=& n rn rr rr r n n nn ! /$ n )&, <&&=& r r% ! %+"rr r 698 ! %+"rr r . ,9 +r r rn =%" r n r %<# %<# nr; n n 4nn nn n$ n % n n n n 4nn n n %#6 n # ! n # !r$ *n n n n nn n+ n nn n n " r r B& n n + rn rn rn n n " n n ! 0" n n nn r % n r n n r %#6 n n #. !r$ 5n r n nn n r n n )&, <&&=& n nn #nn nn n # n r n nn n % n n 13"&"""&/3 + nr nr n nn n 13"&<""&/3$ ! 0)$ r 86 nr n n # n nn n ! 0)$ rn n nnn n n +n n r # n @! r $ " n n ! 0) n #. ! n n r r rn # n n r n n 4n n n % n < = %r @! )&, <&&=& % n nn K+L n n nn n /3n n n rn ! 1$ "!+r% % . ,9 "n nn r rn r n # $ n n n &, <&&=& ! 2"$ ) n # n nn n n nn nr n %" )&, <&&=& nrn # %#/ 9 : % %#6 %#6 n %#6 ! 2)$ *n n n nr 13 r n r n %" /3n n /M nr n n n n %" n n n rn n n rr n r rr rr nr )&, <&&=& n %" n r A2 /0 2+ + 3 **+/ /E 0*E *++n 0 145/ 6 %# ? 64 ? ? n / r r! +:r++ r-r + r" r #$2 6 / 2 ,%% r +" r r #r r r 7 "2 GRL MRXUQDOSSDW J r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D C A 7B

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n B& n n n r " r n ! 2& r n n rn r n n %" n n ! 2) 5 nr r n r r nn " n n +r n rn 9 n n n n %" %#/ rnr r n n @n rn r n rn nn r n n n r #n rn n %" %#6 r rn n B& r n n n B& n n n n rn %#6. n ! 2& n n rn nn r ! 2= n #/ !$ n n r n # n r n n nn n %" nn n n nn n ! 2) rr n n n %" +n n %" n r n n n n n n n )&, <&&=& 698 ! . ,9 r %r = n n + > n n nr n . n rn$ n .> Krr nL n A2 /0 2+ + 3 , 3*r **+/ /E *E*+ + 145/6 %# / r r! +:r++ r-r + r" r #$2 6 / 2 , %% r +" r r #r r r < "2 GRL MRXUQDOSSDW J r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D ; A 7B

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r ! 6$ 5n n n n )&, <&&=& r r r r ! n )&, <&&=& nnr + n )5D. rr rr n Br nr < n nrn nn n nn n n rr n ! >" n >) n #0" !$ rn n n n n nn n )&, <&&=& n %" n n r n n r n n )&, <&&=& 5 r rn $ n 4 n r ./30 / 3 / *0r7 / , /+ 1 r+ r M:r%N 698 !2 1% % 6 + %r r "/ n 698 ! / + %r r "/ 698 n@ ! r /r 1%r! 6 2 ! r!r % 1 % "/ n 698 ! r rr% /r +"2 6%r ! n@ ! r r!r% 1% r +/r /2 rr%r 698 !2 698 ! r n ! r r /r 1%r ! 62 r% + (, % 698 ! r rr% /r +" 2 ! r! r% 1% "/ n ! ! / + n ! rr% /r +" 2 ! / + n ! +/r /2 GRL MRXUQDOSSDW J r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 98 A 7B

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+ n rn r n r r n nnn n n %" n nnr +n ! >&$ n r n n )5D. rr n n rr n < n nrn nn ! >= n #0) !$ n n n nn n rn r )&, <&&=& *nrn nn r rrnr n )&, <&&=& n n Br ! >) n ! >=$ *n n n )&, <&&=& n n " n )&, <&&=& !"# n n n nr n n n )&, <&&=& n r r r n rrrr n n n n n n #1" !$ '.0 .1( %r n rr n rr n n n n r n n n #1) !$ n nnr n )&, <&&=& r n n nr n n rn nn !"# n '.0 .1( n +n )&, <&&=& n rn )$ n n n n nn r n r r +n )&, <&&=& r #2 !$ #+0++ n nr n )&, <&&=& nn rn = n n r )&, 5DE8 '>( "n n *&, '( )&, <&&=& nr n n nr n : n )&, <&&=& r* + 3 / H4/*r + 3 / > 3 145/6 %# )/ /+ 3 145/6 9:' +/*+ 145/6 92 r!+ % r +r %r 1 % 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 99 A 7B

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nn n r /3n n n r nn 7 r0 +/*0/* + 3 145/ 6 %# %+ r% . ,9 !+2 !+ r 6 "# "%r4 05 B=.( <=.( rr% "# #/ !# / 6%r #2 +/r + r!2 !+r% %r "# / /r " / 6%r #2 %+r% % 6r %r" "!+ r% +n2 "!+ 6 "# "%%! %++ 1% r "# " ":2 ! r / 6r+ +rr% /r 5 6%r #2 / +" / r % "! +r% +n2 "%+" r! rr% 6r +" !+ % " +6rr r! r 6r+ /r 6 % +" !+ "r r r :6r+ /r% % 6 +" " % +/r / ! !2 ! ! %6 r 6% "!+ r% +n2 / " +/r / % r 6%rr% +6r% % "!+ r% +n2 0 +" +6r% 66 "!+r% +n / r 5r! D2 +n %r % "!+ r% +n2 r r / r "%+2 "r 698 ! r r!r! /r #/ "%-2 1% rr%2 ! r! r% 1 % / /r +"2 GRL MRXUQDOSSDW J r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 97 A 7B

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5r nn n nr n r %" rn n n n rn ' .67.8( 5 r r n n r n +n r nn ! +r " n 59 n '/ /( nr nr nrn; & nn %" n n '/ /( n r n n nr nn r r n 59 n %" '/.( Enn nr nn n r n n n n n '//( n rr n '/0( n nn n :3 r+ n n '/1 /2( *n n n rn n nnr nr n n )&, <&&=& rr n nr r n n n rn n nn n r 5n r n " r* + 3 / , */ 3 145/ 6 %# )/ /+ 3 E 7/ * */*E *+ " # r!# + # .% +r %r 698 6r r 6r # G7DH r /r " ! "%% 6%r #2 +r +r r 698 +% 6 6r # 6 G7DH2 $r 6 r %+r #6 "r% 6%4 $ % +r + r 6#"r% % + +" +r $ 6r% /r #/ %!2 % %#r r /2 / %#r r %+ r + r.+ % rr "2 +6r 698 6r . ,9 /r r " r!r r C r +r %r r%r! 7 +r %r r r 7C " # % +r %r #+"r' /r 2 GRL MRXUQDOSSDW J r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 9< A 7B

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nr nn n nrn n %" n rn n %" n nn nr r nn r nn n n n n nnn nF n rr n n n # n n rr nn !"#$ n !"# n nr nn nnn r n n rrrr n rr n n n n n '.( n nn n rr '/6( !"# n n nn n !"# r + nrn n n n n n / 0 n 1 n rn n n n r n 2 n .. n *nnr n n n )&, <&&=& !"# n n n nr n n r n n !"# r $ 202/r 3*r/ 30/ 2+ + 3 145/6 %# , %%r r :6r 6+r 698 ! " 6,, %2 r :6r 698 ! #%#r + +r2 D "r #%#r + +r /r &r!., r+ rr! +# *.79 % % /r %+"r 6+r r 698 ! %+"r 6+r . , 9 698 ! +6# 6, , %4 5//rD " r #%#r + +r /r rr% r++ % % rr! % %r" "2 %% r "!+ r% 6+r 698 !2 6r "!+ 698 / % r 6%n< 28.r %2 r :6r 698 ! #%#r+ +r /r %+"r "!+ r% 698 6+r2 D " r #%#r+ +r /r &r!.,r+ rr! +# *.79 % % /r %+ "r 6+r r 698 ! %+"r 6+r 698 "!+ . ,9 +6# 6%n<28 %4 5//rD " r #%#r+ +r /r rr% r++ %% rr! % %r" "2 &r! .,r+ rr! ? r +# / r+! r! 0#+6 PB95 Q@8 +r%% 6 988R +!rr% r % " S 788 +4 rr% r++ %% rr! ? r +# / r+! r! nr0 .$ +r%% 6 788R +!rr% r % " S B8 +2 GRL MRXUQDOSSDW J r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 9E A 7B

PAGE 15

Ennr rn )&, <&&=& r n rr r n n r n r n n r r )&, <&&=& 5 nnr nr n n r n n n n n rr n nn nr rrrr * r n nn nn nr )&, <&&=& B nr nn n n n nrn; & r r r 59 n n " '/>( *nnr n n 5DE @&0/ n n n r 59 n n n; r n nn r r r n r n n nn '/8( & nnn n n nn n nn n '0 0( rn n n n nn nn n n ) &, <&&=& nr nr n n r r n n n rn nn *n n r n nr #"#&, n :9#&, nn n n '0. 0/( ) n n n n :r :r,$ '00( r r nn n n nr n r n @ r n nn n nr r nn n n n n *n n n @! n r )&, <&&=& r B n /&C r %#B8F%#B n %#BF%#B. nn H 5 n r n r %#B8 %#B n %#B. " %#B. r %" r n rnnn r+ &$ n %#B rr rr n n rr %#B r+ rn r )&, <&&=& r r rn * r nn n r n %#B/F%#B0 r r rn n n 5DE n n n %C2/ &rr nn rr n n rn nn n rn n rn n rn r )&, <&&=& nrn n n nF n r Brnrr n rn n rn n n rn '01 02( En n nn nr rrG n nnG r ) r n nr r nn n r n n )&, <&&=& nn B +n n n rn '0 06708( nn n n n nn n rr r *n n rn nnn r #"#&, n rn n n ./ '1( r r n nn n rr rn n n r )&, <&&=& )&, 5DE8 n rn rr r r nrn r n nn r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 9B A 7B

PAGE 16

QC %" r B& r R>O& nr .%r . r %" + r nn n n n %" n nn B& nr n n r n n n n n #0 r$ " nn n n;n =%" ## *** n *nn En #$ n B& + rn n .88 n n&,@! n n&, @$ n ..> n n&,*! n n&,@$ n rn r "rr r n n %" + nr r nn B =%" rn n$ n nrr nn nn #0 r$ n n ")* B /6/ r r n "r ) En #$ r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 9D A 7B

PAGE 17

+6 !+ 1%r! ! %" + r n /02 n /12 n n %" 4 %+ 5:.88 5:. n 5:. r$ nn # %#/ 9 : n % @! r " n @! /3n n nn r r r+$ n %&S2166 %&S01 %&S> %&S/01 %&S>02 n %&S/0/2 r$ n r n nn n &r n n n r n 2 nnr n *n n n 13 nn r n 13E$ n /3 nnr n /3E$ n n r n r n # rr n n #"#&, r+r 698 ! rn r r rn =%" r n r %<# %<# n; n + n nn n rr 4nn n % n n n n 4nn n n %#6 n *n n n n n n nn n nn n n n n #1 r$ rn nr rn n :=> n nn r 4 #n nn #!r% # n rn n nr n n n nn n n n # n % n rn nr 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 9@ A 7B

PAGE 18

rn n n rnn n n + rr rr n B:C '11( r r 9 n n C< r n n r n n B '12( !nrr rr r n r n nn ! r % +6 r rr #r n r n , 92 )5D. :=&D "108 59. &&. rr rr n n r; n rr rn rr rn nr r n rn r r nn n rr rr n r rn n n n n n "!+ rrr%r 1%r! % n %" n n rn r n 5n nn n %" n r n n rn n nr; r rn n nr n n n n n r n %" n n r r ! n n r n 13n r n n n %" r n n @! n nn n n n n #2 r$ # rn + rn n n rn n :=> nn r nr n n r + n n 4 nn #n rn n %" %#6 r rn n B& r n n n B& n n r r n n rn nn 698 r+ 6%r n r n nr; n B!": '16( n *nB# n '1>( Bn nn n n :5:: '18( : n BBn '2( B nn n n n .7/6$ n r n r // n $ n n #6 r$ n n; Pn )rr nr & C= )4n &n$ " n4n r r nn DC:$ n ; n; n n $ n4 nrr n r . Q n4 + 4n rr nr 0 n4 #n rr n r rr r "n n n n;rn nn 9C*#"$ #%#r #r % rr! *n rr n n n n n rr n n n n n @! n n # n * r n n n r n rr r r n nnn n '.2( r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 9C A 7B

PAGE 19

#n n rn nr n n )&, <&&=& rr n n n & n nr r r 4 n n )&, <&&=& n n > n n nrn . rn$ n .> rr Urr n3 n ; !"# r ! 6$ 5n n +r nr r n n r r )&, <&&=& n n r r r r n n n n rn n &"<<# n nn r ! >"$ n n n )5D. rr n Brrn B9* Brn *n$ n n3 r " nn rrrr n n n n r n < nn n n n nr n r n rrnr n n Br '08( r rn n &"<<# nr &rr n n &"<<# nr n+ n n n nr n rn r )&, <&&=& n n r n n n rn r )&, <&&=& n %" nn n nr ! rn n n =%"/ n n r r ! >&$ r n n %" nn n %" n n rr nn r n n )5D. rr n rr n nn r Br n rr nr &rr n =%"/ nr $r #r 698 6r . ,9 " n )&, <&&=& n n nr n n n n )&, <&&=& r n '.0 .1( ! nr n+n + n n rr )5D. nr rr n 2rr r n . Q r =%" n Brrn B9* Brn *n$ n n 1 rn nn + =:9: *nn$ rn A r n <*)&@$ n rr r+ n n < n n n n n V nn n n @r *P1!CI=B6 & "r! & nr n r n +n )5D. rr nn +n r n &"<<#$ nr )r n n )5D. rr r 4 #=#B"<9 n n B,=! n n r 1A nn r rn n n n n 0O& rr + n4 n n *< 5IC$ Nnn 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 9; A 7B

PAGE 20

%%r +" r n n )&, <&&=& n /02 n /12 n n r r n n r .0 n r */ .3*r/ nrn n 9"= + %<# n n r n )&, <&&=& n 5:.88 5:. 5:.$W )&, rn) n BrnF=rn1.1<.F .> ")10/12$W 9)&, Dn9rn n FDnFD?.0F.2 5H6.>0>.$W )&, DnnFDnF D?2F.2 5H6.>0>/$ *!$ !nrn "nr #$ n nrn %# "rn n %" nrn # %#/ 9 : % %#6 %#6 n %#6 n n rr nn n r n # n &=# r n rr r # n rr r *!$ & '' (nr( n $) n "$ nn +n n n n n * rr n ="B* r n * ! n n nr n %n =*"B5@:= n .V nn #r X 1 Q$ )$ nn +n n n n n nn n r * rr n ="B* rn * ! n n nr n %n =*"B5@:= n .V nn #r X 1 Q$ *!$ + nrn'( n #,n n n' rn n 'n "n n "$ # nn n n n n n n n rr n n r r : n n n 5B W &: n $ nn B) r$ n nr nn n :=$ rr nr n n &$ n n n n n nrr r n )$ nn + n n n n n n nr )5D. rr n r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 78 A 7B

PAGE 21

nn r r )&, <&&=& n n n n &"<<# < nnn nr n n @r *P1!CI=B6 n V nn r X . Q$ *!$ . )('r''( /n "( #,+n n n r r r n rn r # >n r n )&, <&&=& %" )5D.)5D. rr n nn +n r n &"<<#$ nr ! /0 n /0>! r n r nn r )&, <&&=& %" &n #nn r 06 ) &, <&&=& %" *!$ %"' n rr( n 0n #,n #,1234 n n ' " rn =@&P$ %"' % 'r rn n ' n,n5 rrn n" n rnrn ('nr ##6 5 n r'r n =@&P$ %"' % 'r n ' n,n5 rrn n" n rnrn ('nr n =@&P$ & %"' 3n' n nrn #%)# n !nrn =@&P$ + %"' ) rnn n( n rnn( rn/ !nr nn n =@&P$ . %"' ) rn ' "nr #$ =@&P$ 7 %"' ) n n"( rn =@&P$ 8 %"' % !nr n rrn n" n ( =@&P$ ' % n "$ ' nn n "( r'nn "$ ;0C)r/ + r = P4n < n = P N %nr *n ,r = &nr n Bnn &n &n = &nr n Bnn n n n n n n = ?n ?n n B r .5+r # %+"r r 0 ! > 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 79 A 7B

PAGE 22

n4 n %nr *n ,r = &nr n Bnn &n &n = &nr n Bnn n =%"/ n rr rr n rr &=& n Pnnn ?nnn Bn &n n n r rrn n )4n 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 77 A 7B

PAGE 23

, P) r U )! P + V , 6r U 2 r %% r'r " .r% r 7 %62 n2 789<4 B8<@E @@?B 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 7< A 7B

PAGE 24

, # & " U n 5 'r$ 6 &U2 1% + 6rr r B; +n 7? rr%r n %+ "rr "/ %r r' r2 r. !#2 9;CC4 9DD7?E9B F772 $? 7CEBDBB2 V! P$ * *, ' U2 +!! rrA ! + %r r 0E 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 7E A 7B

PAGE 25

$ V 5 ) $! * Ur! ) V ) 2 Pr r r / " r r r r2 % r2 78984 9BBC?97; BF;2 6" 7898A8BA7B2 r? 982988@A8 8@8B.898.8 D;8.E $? 78E;BC 0?9829<@ 9A 26 62988BCC< 6 +" 7@ 789D 7B A 7B


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