Retractiondownloads.hindawi.com/journals/isrn/2014/624535.pdfISRNVirology VR-2332 TATGACGTATAGGTGTTGGCTCTATGCCTTGGCATTTGTATTGTCAGGAGCTGTGACCATTGGCACAGCCCAAAACTTGCTGCACAGAAACACCCTTCTG

RetractionRetracted The Genomic and Pathogenic Characteristicsof the Highly Pathogenic Porcine Reproductive and RespiratorySyndrome Virus Isolate WUH2

ISRN Virology

Received 25 February 2014 Accepted 25 February 2014 Published 1 June 2014

Copyright copy 2014 ISRN VirologyThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The paper titled ldquoThe Genomic and Pathogenic Character-istics of the Highly Pathogenic Porcine Reproductive andRespiratory Syndrome Virus Isolate WUH2rdquo [1] publishedin ISRN Virology has been retracted as it was submitted forpublication by the author Bin Li without the knowledge andapproval of the coauthor Shaobo Xiao

References

[1] B Li L Fang S Liu Y Jiang H Chen and S XiaoldquoThe genomic and pathogenic characteristics of the highlypathogenic porcine reproductive and respiratory syndromevirus isolate WUH2rdquo ISRN Virology vol 2014 Article ID624535 15 pages 2014

Hindawi Publishing CorporationISRN VirologyVolume 2014 Article ID 965431 1 pagehttpdxdoiorg1011552014965431

Research ArticleThe Genomic and Pathogenic Characteristics of the HighlyPathogenic Porcine Reproductive and Respiratory SyndromeVirus Isolate WUH2

Bin Li12 Liurong Fang1 Suyan Liu1 Yunbo Jiang1 Huanchun Chen1 and Shaobo Xiao13

1 Division of Animal Infectious Diseases State Key Laboratory of Agricultural MicrobiologyCollege of Veterinary Medicine Huazhong Agricultural University Wuhan 430070 China

2 Key Laboratory of Veterinary Biological Engineering and Technology Institute of Veterinary MedicineJiangsu Academy of Agricultural Sciences Ministry of Agriculture Jiangsu Province Nanjing 210014 China

3 Laboratory of Animal Virology College of Veterinary Medicine Huazhong Agricultural University 1 Shi-zi-shan StreetWuhan 430070 China

Correspondence should be addressed to Shaobo Xiao vetmailhzaueducn

Received 18 November 2013 Accepted 24 December 2013 Published 23 February 2014

Academic Editors L Darwich and D J Jackwood

Copyright copy 2014 Bin Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

To fully understand the extent of genetic diversity and pathogenesis of the highly pathogenic PRRSV found inChina we determinedthe genomic sequence of PRRSV WUH2 the pathogenicity of WUH2 was compared to the classical PRRSV isolate CH-1a Ourresults showed that the WUH2 genome had a discontinuous deletion of 30 aa in Nsp2 a 1 nucleotide deletion located in boththe 51015840 and 31015840 UTRs and point mutations within GP5 Experimental infection demonstrated that PRRSV WUH2 reproduced thephenotype and symptoms of porcine high fever syndrome Importantly we found that there were differences in viral burden in theserum and tissues when comparing infections of the pathogenic isolate WUH2 to those of the classical isolate CH-1a These dataprovide insight into the genomic diversity and altered pathogenicity of Chinese PRRSV isolates and help elucidate the evolutionand potential pathogenic mechanisms of PRRSV

1 Introduction

Porcine reproductive and respiratory syndrome (PRRS) is arelatively new viral disease of swine that is characterized bysevere reproductive failure in sows and respiratory distressin piglets and growing pigs Since PRRS was first reportedin the United States in 1987 and Europe in 1990 [1 2]the disease has devastated the swine industry by causingtremendous economic losses around the world it is nowconsidered one of the most important diseases in countrieswith intensive swine industries [3ndash5] PRRS is caused by thePRRS virus (PRRSV) a member of a group of envelopedpositive-sense RNA viruses in the Arteriviridae family [6]The genome of PRRSV is approximately 15 kb containing nineopen reading frames (ORFs) [7] ORF1a and ORF1b encodefor the RNA-dependent RNA polymerase and nonstructuralproteins (Nsps Nsp1120572 Nsp1120573 and Nsp2ndash12) respectively

[8ndash10] ORF2a ORF2b and ORFs 3 through 7 encode theviral structural proteins GP2 E GP3 GP4 GP5 M and Nrespectively [7 11]

PRRSV can be divided into European and North Amer-ican genotypes which share only about 60 nucleotideidentity at the genomic sequence level [12] Isolates from eachgenotype exhibit significant sequence variation particularlyin Nsp2 and ORF5 which represent the most geneticallyvariable regions in the PRRSV genome [4 13 14]

PRRSV was first reported in 1995 and the first isolate wasidentified in mainland China in 1996 Since then PRRS hasbecome one of themost important swine diseases worldwideMany isolates have been obtainedwithinChina fromdifferentregions and times A porcine high fever syndrome causedby a highly pathogenic PRRSV broke out in the south ofChina in 2006 and rapidly spread across the country affectingmore than 20 million pigs [15ndash17] This syndrome was

Hindawi Publishing CorporationISRN VirologyVolume 2014 Article ID 624535 15 pageshttpdxdoiorg1011552014624535

2 ISRN Virology

characterized by a high continuous fever anorexia red discol-orations on the body and blue ears The morbidity rate was50ndash100 and the mortality rate was 20ndash100 Genomicanalysis showed that the etiologic virus was significantlydifferent from previous isolates [15 17ndash19]

In this study we sequenced and analyzed the genomeof the PRRSV isolate WUH2 a highly pathogenic PRRSVisolated in central China during the pandemic period of theporcine high fever syndrome We then compared the patho-genic characteristics of WUH2 to the classical PRRSV isolateCH-1a The reemergence of a highly pathogenic PRRSVand our study of its genome and pathogenic characteristicsdemonstrated the differences between highly pathogenic andclassical isolates

2 Materials and Methods

21 Virus and Cells PRRSV strain WUH2 was isolated inMARC-145 from the brains of pigs suffering from the highfever syndrome in China at the end of 2006 The 5th passageof virus was purified and other pathogens or microbialcontaminants PRRSV strainCH-1a the first isolate identifiedin mainland China in 1996 was used as the classical PRRSVreference strain MARC-145 cells were grown in DulbeccorsquosModified Eagle Medium (DMEM) supplemented with 10fetal bovine serum (FBS) 100 units of penicillin and 20 unitsof streptomycin Cells were maintained in a 37∘C humidifiedchamber with 5 CO

2

22 Primer Design Preparation of RNA RT-PCR andQuanti-tative PCR Oligonucleotide primers amplifying the WUH2genome were designed based on sequence information fromGenBank for PRRSV strains VR-2332 and CH-1a as wellas sequences from highly pathogenic PRRSV isolates [20]Tissue samples were weighed and an equal volume of PBSwas added to the sample before theywere homogenized RNAwas extracted from infected cells serum and tissue usingthe QIAamp viral RNA kit (Qiagen Shanghai China) ViralcDNA was synthesized by reverse transcription reactionsusing random hexanucleotide primers and SuperScript IIreverse transcriptase (Gibco BRL) following the manufac-turerrsquos instructions cDNA was then used as a template insubsequent PCR reactions with a final volume of 50120583Lcontaining 25 pmol of each PRRSV-specific primer describedabove 1x Pfu polymerase buffer 200 120583MdNTPs and 25 unitsof Pfu DNA polymerase (Stratagene)

Viral load was detected by quantitative PCR Primersand probes were designed targeting the ORF7 gene withthe Primer Express software package The probe (51015840-TCC-CGGTCCCTTGCCTCTGGA-31015840) was labeled with FAMat its 51015840 terminus (reporter) and TAMRA at its 31015840 end(quencher) Primer sequences were 51015840-TCAGCTGTGCCA-AATGCTGG-31015840 and 51015840-AAATGGGGCTTCTCCGGGTTT-T-31015840 Amplification was performed using TaqMan mixtures(TaKaRa) The thermal cycler program was 60∘C for 10min95∘C for 15 s and 40 PCR cycles (90∘C for 15 s and 60∘C for60 s) Known amounts of serially diluted in vitro transcribed

RNA (10e12 copies120583L) were used to generate a standardcurve for each plate run

23 DNA Sequencing and Sequence Analysis The 51015840 terminalsequences of viral genomes were amplified and cloned aspreviously described [19] PCR products were purified usingthe QIAquick gel extraction kit (QIAquick) cloned intothe pMD18-T vector (TaKaRa) and confirmed by sequenc-ing For each amplified genomic region three clones weresequenced from each of the three independent amplificationsin both directionsThus nine determinations of the sequenceof each region were performed

Sequence data were assembled and analyzed using Laser-gene software (DNASTAR) Multiple sequence alignmentswere done using Clustal W A phylogenetic tree was con-structed by the neighbor-joining method using MEGA 31[21]The reliability of the neighbor-joining tree was estimatedby bootstrap analysis (1000 replicates)

24 Experimental Infection We performed experimental in-fections in pigs to compare the virulence and pathogenesisof isolate WUH2 to CH-1a Thirty piglets weaned at 5-6weeks of age were obtained from a PRRS-free farm inHubei China The piglets were randomly separated intothree groups that were housed in separate rooms Pigletswere inoculated intramuscularly with 1050 TCID

502mL of

either CH-1a orWUH2 (10 piglets per group) Control pigletswere inoculated with DMEM Experimental piglets wereclinically examined daily Rectal temperatures and mortalitywere recorded from 0 to 21 days after inoculation (dpi)blood was collected at 0 3 7 10 14 and 21 dpi to measureviral load by quantitative PCR Two piglets from each groupwere euthanized and necropsied at 7 10 and 21 dpi Organswere collected including lung brain spleen kidney heartliver stomach intestines and lymph nodes for viral loaddetermination by quantitative PCR and histopathologicalexamination [22]The remaining 6 piglets in each group wereobserved for 21 days to determine death rates Viral loadsof the piglets which were naturally death and two of live at21 dpi were detected too

25 Statistical Analysis Studentrsquos 119905-tests were used to com-pare viremia and viral load between the different groups 119875values lt 005 were considered statistically significant

3 Results

31 The Complete Genomic Sequence of the PRRSV StrainWUH2 The complete WUH2 genome was found to be15350 nucleotides long excluding the poly(A) tail (GenBankaccession number EU678352) with a 189 nucleotide long51015840 UTR and a 150 nucleotide long 31015840 UTR The genomealso contained nine ORFs two large overlapping ORFs(ORF1a and ORF1b) followed by the 51015840 UTR and sevenORFs encoding structural proteins (ORF2a ORFs 2b to 7)Comparison of this genome to 6 representative genomicsequences from other PRRSV isolates showed that WUH2shared 889 sequence identity with the representativeNorth

ISRN Virology 3

Table 1 The amino acid identity of the proteins of PRRSVWUH2 with other strains ()

ORF Cleavage product VR-2332 LV RespPRRS MLV CH-1a HB-2(sh)2002 JXA1Genome 889 579 888 944 919 984

1a

Nsp1120572 958 669 958 970 982 1000Nsp1120573 834 401 825 871 834 972Nsp2 749 291 748 847 809 973Nsp3 870 536 872 901 892 915Nsp4 887 593 887 907 902 941Nsp5 918 712 924 947 929 994Nsp6 938 750 938 1000 1000 1000Nsp7 892 423 896 965 950 996Nsp8 978 667 978 978 956 978

1b

Nsp9 972 745 972 983 980 994Nsp10 964 647 966 982 975 991Nsp11 951 763 951 973 964 996Nsp12 941 360 941 961 954 987

2a GP2 930 606 926 965 938 10002b E 904 645 890 945 945 10003 GP3 858 545 858 929 909 9804 GP4 888 678 888 961 938 9665 GP5 885 566 875 935 915 9956 M 966 799 960 966 971 9897 N 951 580 951 951 919 1000

American strain VR-2332 579 sequence identity with LVand 944 nucleotide identity with CH-1a WUH2 alsohad high sequence similarity to strain JXA1 (984) therepresentative isolate from the highly pathogenic PRRSV inChina in 2006 particularly notable was the presence of adiscontinuous deletion of 30 aa (residues 481 and 533ndash561)in Nsp2 coding regions [16] The detailed identities for eachcoding region of WUH2 compared to representative PRRSVisolates were summarized in Table 1

32 Sequence Analysis of the UTRs The 51015840 UTR of WUH2was 189 nts long and shared 911ndash995 nucleotide identitywith VR-2332 and other representative North Americanisolates Sequence comparison revealed that the eleven strainscontained a conserved 51015840-leader startmotif of 12 nt the leadertranscription regulatory sequence (TRS) and the CACCCmotifsThe string of 8ndash11ndash9 conserved nucleotides were iden-tical between VR-2332 and the Chinese isolates There was aunique 1 nt deletion at position 119 in the 51015840 UTR of WUH2compared to VR-2332 This was a common characteristicof representative highly pathogenic PRRSV isolates (JXA1JXwn06 HUN4 SY0608 XL2008 and SX2009) (Figure 1(a))

The 31015840 UTR of WUH2 was 150 nucleotides long witha unique 1 nt deletion at position 19 compared to VR-2332 (Figure 1(b)) Similar to the 51015840 UTR the deletion wasobserved in all isolates of the highly pathogenic PRRSVGenomic sequences were conserved at positions 82ndash104 inWUH2 and the representative isolates a previous studysimilarly reported that a stretch of 23 bases (positions 82ndash104)was highly conserved between Lelystad and other PRRSVs

[23] The other nucleotide substitutions identified in WUH2were also found in Chinese isolates

33 Sequence Analysis of Nsp2 and GP5 The Nsp2 gene ofPRRSV had the highest genetic diversity of all genes in theviral genome In our study analysis of sequence homologyindicated that Nsp2 from WUH2 shared only 749 and291 amino acid identity with VR-2332 and LV respectivelythe highest degree of sequence identity (973) was foundwith JXA1 Further sequence analysis revealed that the Nsp2gene from WUH2 was 2850 bps long encoding 950 aminoacids with a discontinuous deletion of 1 and 29 amino acidsat positions 481 and 533ndash561 compared toVR-2332 (Figure 2)These deletions in the Nsp2 region were identical and foundat the same locations in the representative highly pathogenicPRRSV [16 17]

The ORF5-encoded major envelope glycoprotein (GP5)was the least conserved structural protein [13 24ndash26] GP5from WUH2 shared 885 and 566 amino acid identitywith VR-2332 and LV respectively The GP5 gene shared915ndash995 sequence identity with representative Chineseisolates The hypervariable region observed between WUH2and other isolates includes two regions at the amino terminus(the signal peptide sequence and amino acid positions 30ndash36) Amino acid substitution tended to affect the positionand number of potential N-glycosylation sites (Figure 3)There were five N-glycosylation sites inWUH2 and the otherhighly pathogenic PRRSV except SX2009 The variable ofthe primary neutralizing epitope (PNE aa 37ndash44) was foundat amino acid position 39 The L39I mutation was foundin WUH2 and HP-PRRSV and was distinct from the L39F

4 ISRN Virology

VR-2332 TATGACGTATAGGTGTTGGCTCTATGCCTTGGCATTTGTATTGTCAGGAGCTGTGACCATTGGCACAGCCCAAAACTTGCTGCACAGAAACACCCTTCTG 100RespPRRSMLV ----------------------------AC-A---------------------------C--------------G------------------------- 99CH-1a ---------------------------AC-A-----------------------------------------------------G-------------- 99HB-2(sh) ---------------------C--A--AC-A---------------------------------------------------G-G------------C- 99JXA1 ---------------------------AC-------------------------------------------------------G-G--------C--- 99JXwn06 ---------------------------AC-------------------------------------------------------G-G--------C--- 99HUN4 ---------------------------AC-------------------------------------------------------G-G--------C--- 99SY0608 ---------------------------AC-------------------------------------------------------G-G--------C--- 99XL2008 ---------------------------AC-------------------------------------------------------G-G--------C--- 99SX2009 ---------------------------AC-------------------------------------------------------G-G--------C--- 99WUH2

VR-2332RespPRRSMLV CH-1aHB-2(sh)JXA1JXwn06HUN4SY0608XL2008SX2009WUH2

---------------------------AC--------------------------------------T----------------G-G--------C--- 99

12 nt start motif

TGATAGCCTCCTTCAGGGGAGCTTAGGGTTTGTCCCTAGCACCTTGCTTCCGGAGTTGCACTGCTTTACGGTCTCTCCACCCCTTTAACC 190------------------------------------------------------------------------------------------ 190---C----CT-----------A------G-C------------------------------------------------------------ 190---C----CT-----------A------G-C------------------------------------------------------------ 190---C----CT----------A------G-C--------A--------------------------------------------------- 189---C----CT----------A------G-C--------A--------------------------------------------------- 189---C----CT----------A------G-C--------A--------------------------------------------------- 189---C----CT----------A------G-C--------A--------------------------------------------------- 189---C----CT--------C-A------G-C--------A--------------------------------------------------- 189---C----CT----------A------G-C--------A--------------------------------------------------- 189---C----CT----------A------G-C--------A--------------------------------------------------- 189

TRS

(a)

TGGGCTGGCATTCTTGAGGCATCTCAGTGTTTGAATTGGAAGAATGTGTGGTTAACGGCACTGATTGACATTGTG 75----------------------------------------------------G--T------------------- 75--------T------TG----C---------A-------G------------G--T--------------C---- 75--A------------TG----C---G-----ATG-----G------------G--T--------------C---- 75---------------TG---C---------A-------G------------G--T--------------C---- 74------------T--TG--CC---------A-------G------------G--T---C---------CC---- 74---------------TG---C---------A-------G------------G--T--------------C---- 74---------------TG---C---------AT------G------------G--T--------------C---- 74---------------TG---C---------A-------G------------G--T--------------C---- 74---------------TG---C---------AT------G------------G--T--------------C---- 74------A--------TG---C---------A-------G------------G--T--------------C---- 74

CCTCTAAGTCACCTATTCAATTAGGGCGACCGTGTGGGGGTGAGATTTAATTGGCGAGAACCATGCGGCCGAAATT 151---------------------------------------------------------------------------- 150---------------------------------------------------------------------------- 151-----------------------------------------A-AG--------------------------C---- 151-----------------------------------------A-AG--------------------------T---- 150---T----------------------G-G-----G------A-AG--------------------------T---- 150-----------------------------------------A-AG--------------------------T---- 150-----------------------------------------A-AG--------------------------T---- 150--------------------------------------C--A-AG--------------------------T---- 150-----------------------------------------A-AG--------------------------T---- 150-----------------------------------------A-AG--------------------------T--C- 150



GGC

---

---

---

--

--

--

--

--

--

--

(b)

Figure 1 Alignment of the 51015840 UTR (a) and 31015840 UTR (b) of PRRSV isolates WUH2 VR-2332 RespPRRS MLV CH-1a HB-2(sh)2002 JXA1JXwn06 HuN4 SY0608 XL2008 and SX2009 (a)The 12 nt start motif and the leader TRS (black box) were absolutely conservedThe dashedregion indicates the string of 8ndash11ndash9 The CACCC motifs were shown by the red boxes (b) The dashed box indicates a stretch of 23 bases atpositions 82ndash104 The two deleted regions are represented in red

ISRN Virology 5

----------467-----------480----------------500-----------------520-----------------540----------------560--------571--PVSLGGDVPNSWEDLAVSSPFDLPTPPEPATPSSELVIVSSPQCIFRPATPLSEPAPIPAPRGTVSRPVTPLSEPIPVPAPRRKFQQVKRLSSAAAIPPYQDEP -----------------------------------------------------------------------------------------------------N-- -ILM-DN---G---F--GG-L-F---S--M--L--P-LMPAS-H-P--V----G---V----R-----M-------F-S---H-----EEANP--TTLT-----SILA-GG-LNFS--S-LV--LG-P-LMPAS-HVS--V--------V----RI----M------TF-F--W--S---EEANP---TLTC------LM-DN---GS-ET-GG-LNF---S--M--M--P-L-PASRRVPKLM----GS--V----R--TTTLTH---- --LM-DN---GS-ET-GG-LNF---S--M--M--P-L-PASRRVPKLM----GS--V----R--TTTLTH---- --LM-DN---GS-ET-GG-LNF---S--M--M--P-LMPASRRVPKLM----GS--V----R--TTTLTH------LM-DN---GS-ET-GG-LNF---S--M--M--P-L-PASRRVPKLM---GGS--V----R--TTTLTH------LI-DN---GS-ET-GGLLNF---S--M--M--P-L-PASRRVPKLM----GS--V----R--TTTLTH------LM-DN---GS-ET-GGSLNF---S--M-LM--P-L-PASRRVPKLM----GS--V----R--TTTLTH---- --LM-DN---GS-ET-GG-LNF---S--MI-M--P-L-PASRFVPKLM---IRS--V----R--TTTLTH----

LA--F-TTTTTTT

RPVTPLSEPIPVPAPRRKFQQVKRLSSAA-------------------------------M-------F-S---H-----EEANP----M------TF-F--W--S---EEANP--

VR-2332RespPRRSMLVCH-1aHB-2(sh)JXA1JXwn06HUN4SY0608XL2008SX2009WUH2

HB-139 --LM-DN---GS---T-GG-LNF---S--M--M--P-LTPAL-R

VR-2332 IFRPATPLSEPAPIPAPRGTVSRPVTPLSEPIPVPAPRRKFQQVKRLSSAAAIPPYQDEPAPRRS S---VK-----V-V----K-----A-------------------EKVNP---TLGC---FPRRSV01 ---------------------------------------------------------n--PRRSV02 ------------------------------------------------------------PRRSV03 ------------------------------------------------------------GS2002 ---------------------------------------------------------n--GS2003 ---------------------------------------------------------n--GS2004 ---------------------------------------------------------n--BJ-4 ---------------------------------------------------------n--S1 ---------------------------------------------------------n--CC-1 ---------------------------------------------------------n--HN1 ---------------------------------------------------------n--CH-1a -P--V----G---V----R-----M-------F-S---H-----EEANP--TTLT-----CH2002 -P--V----G--RV----R-----M-------F-S---H-----EEANP--TTLT-----CH2003 -P--V----G--RV----R-----M-------F-S---H-----EEANP--TTLT-----CH2004 -P--V----G--RV----R-----M-------F-S---H-----EEANP--TTLT-----HB-2(sh)

HB-1(sh)

VS--V--------V----RI----M------TF-F--W--S---EEANP---TLTC----f----VPKLM---DGS--V----R-----M-------FLS---H-----EEANP-TTTLTH-N--VPKLM--S-GS--V----R-----M------FF-S---Y-----EEANP-TTTLTH----SHB

NB04 VPKLM----GS--V----R-----M-------F-S--QH-----EEANP-TTTLTH----WUH1 VPKLI----GS--V----R--TTTLTH----CG VPKLM----GSmdashV----R--TTTLTH----JSyx VPKLM----GS--V----R--TTTLTH----XH-GD VSKLM----GS--V----R--TTTLTH----

VPKLM----GS--V----R--TTTLTH----BJHenan-1 VPKLM----GS--V----R--TTTLTH----

VPKLM---GGS--V----R--TTTLTH----SY060807BJ VPKLMK---GS--V----RP-TTTLTH----

VPKLMK---GS--V----R--TTTLTH----GDGD2 VPKLM----GS--V----R--TTTLTH----

VPKLM----GS--V----R--TTTLTH----GD2007VPKLM----GS--V----R--TTTLTH----HUN4VPKLM----GS--V----R--TTTLTH----TPVPKLM----GS--V----R--TTTLTH----07QNVPKLM----GS--V----R--TTTLTH----HPBEDV

Em2007

VPKLM----GS--V----R--TTTLTH----JXA1VPKLM----GS--V----R--TTTLTH----HN2007VPKLM----GS--V----R--TTTLTH----LNVPKLM----GS--V----R--TTTLTH----SHH

JXwn06 VPKLM----GS--V----R--TTTLTH----VPKLM----GS--V----R--TTTLTH----TJVPKLM----GS--V----R--TTTLTH----HEB1VPKLM----GS--V----R--TTTLTH----NM1VPKLMA---GS--V----R--TTTLTH----07NMVPKLM----GS--V----R--TTTLTH----07HENVPKLM----GS--V----R--TTTLTH----07HEBTJVPKLM----GS--V----R--TTTLTH----HUB1VPKLM----GS--V----R--TTTLTH----HUB2

HuN VPKLM----GS--V----R--TTTLTH----VPKLM----GS--V----R--TTTLTH----JX143

Jiangxi-3 VPKLM----GS--V----R--TTTLTH----VPKLM----GS--V----R--TTTLTH----NX06VPKLM----GS--V----R--TTTLTH----BJSY06VPKLM----GS--V----R--TTTLTH----JX2006VPKLM----GS--V----R--TTTLTH----XL2008

(a)

Figure 2 Continued

6 ISRN Virology

VPKLM----GS--V----R--TTTLTH----SX2007VPKLM----GS--V----R--TTTLTH----YN2008

BJPG VPKLM----GS--V----R--TTTLTH----BJSD VPKLM----GS--V----R--TTTLTH----CBB-1-F3CWZ-1-F3

VPKLM----GS--V----R--TTTLTH----VPKLM----GS--V----R--TTTLTH----

GDQJ VPKLM----GS--V----R--TTTLTH----GDBY1 VPKLM----GS--V----R--TTTLTH----SD-CXA08 VPKLM----GS--V----R--TTTLTH----SX-1 VPKLM----GS--V-T--R--TTTLTH----SX2009 VPKLM----GS--V----R--TTTLTH----

HB-139 VPKLM---NGS--V----R-----M-------FLS---H-----EEANP-TTTLTH-N--

VPKLM----GS--V----R--TTTLTH----GS2008

(b)

Figure 2 Alignment of partial Nsp2 amino acid sequences of WUH2 with the North American prototype VR-2332 and the representativeChinese isolates The deletions are shown in colors

MLEKCLTAGCCSRLLSLWCIVPFCFAVLANASNDSSSHLQLIYNLTLCELNGTDWLANKFDWAVESFVIFPVLTHIVSYGALTTSHFLDTVALVTVSTAG 100------------Q--------------------------------------------------------------------------------------- 100--G----T--------------------V--NSN----F--------------------------T-------------------------G-------- 100--G-------Y-Q---------------V---SN--P-F------------------ER------T-------------------------G-I------ 100--G-----C------F-------YL---V----NN---I------------------Q-------T-------------------------G-A------ 100--G-----C------F-------YL---V----NN---I------------------Q-------T-------------------------G-A------ 100--G-----C------F-------YL---V----NN---I------------------Q-------T-------------------------G-A------ 100--G-----C------F-------YL--------NN---I------------------Q-------T-------------------------G-A------ 100--G-----C------F-------YL---V----NN---I------------------Q-------T--------------P----------G-A------ 100--G-----C------F-------YL--------SN---I------------------Q-------T-------------------------G-A------ 100--G-----C------F-------YL---V----NN---I------------------Q-------T-------------------------G-A------ 100

FVHGRYVLSSIYAVCALAALTCFVIRFAKNCMSWRYACTRYTNFLLDTKGRLYRWRSPVIIEKRGKVEVEGHLIDLKRVVLDGSVATPITRVSAEQWGRP 200--------------------------------------------------G------------------------------------------------- 200-Y------------------I-----L---------S-----------------------V--G------------------------L----------L 200-Y-R----------------I-----L---------S-------------------P---V--G------------------------L----------L 200YY------------------I-----L---------S-----------------------V--G--------------------A---L------L---L 200YY------------------I-----L---------S-----------------------V--G--------------------A---L----------L 200YY------------------I-----L---------S-----------------------V--G--------------------A---L----------L 200YY------------------I-----L---------S-----------------------V--G--------------------A---L----------L 200YY------------------I-----L---------S-----------------------V--GR-------------------A---L--------A-L 200YY------------------I-----L---------S-----------------------V--G--------------------A---L----------L 200YY------------------I-----L---------S-----------------------V--G--------------------A---L----------L 200

HL

--

-F

-F

-I

-I

-I

-I

-I

-I

-I

SRL

-Q

--

Q-

--

--

--

--

--

--

--

GRL

-G

--

--

--

--

--

--

----

--

YAC

--

-S

-S

-S

-S

-S

-S

-S-S

-S



Figure 3 Amino acid sequence alignment of GP5 ofWUH2 and the representative PRRSV isolatesThe dash box identifies the signal peptidesequence and the hypervariable region The key amino acid mutations were indicated with different colors

mutation in CH-1a The two amino acids R13 and R151 werealtered during virus attenuation [7] and identified in ourstudy inVR-2332These data indicated thatWUH2possessedsequence characteristics of other pathogenic isolates TheA137S mutation was previously hypothesized to differentiatebetween vaccine strains [27] and was found in WUH2 andChinese isolates

34 Phylogenetic Analyses To better understand the geneticcharacteristics ofWUH2 a phylogenetic treewas constructedby the neighbor-joining method based on the representativegenomic sequences of Chinese PRRSV isolated from 1996to 2010 As shown in Figure 4 all Chinese PRRSV isolatesbelonged to the North American genotype and were fur-ther divided into four sub genotypes WUH2 was definedin a group of the highly pathogenic PRRSVs that also

contained JXA1WUH1 JXwn06 SY0608 HUN4HEB1 andHUB2 these strains have all been demonstrated to be highlypathogenic in artificial infection experiments [17 20 28ndash31]

35 Clinical Signs and Mortality The negative control groupshowed no clinical signs of illness at any time duringthe experiment Rectal temperatures of the control groupwere within normal range Piglets inoculated with CH-1ashowed only temporary fever mild respiratory andmoderateanorexia Death was not observed in this group Obviousclinical signs including inappetence lethargy and high con-tinuous fever (405ndash420∘C) were observed in piglets inoc-ulated with WUH2 beginning at 3 dpi (Figure 5(a)) Thesesymptoms showed the rapid evolution of the disease causedby this isolate A few areas of red discoloration on the bodyand blue ears were observed at 5 dpi and spread to the limbs

ISRN Virology 7

HN2007JXA1

HPBEDVGDHUN4GDQJLNTJJXwn06XL2008JX2006YN2008SX2007SD-CXA2008HEB107HEBTJJX143HuNHUB2HUB1JSyxXH-GDCGSY0608

WUH2Henan-1SX-1SX2009WUH1NB04SHBHB-1(sh)2002Em2007HB-2(sh)2002

CH-1aCH2004CH2003CH2002NVSL 97-7895P129SPLMY16244BHN1PA8

VR-2332PRRSV02PRRSV03PL97-1LP1PRRSV01S1MLV RespPRRSReproRespPRRS MLVBJ-4MN184AMN184BLV421LV

100100

100

100

98

4193

82

82

100100

100100

100

100

99

100

100 54

100

100100

100

100

99

100

100

95

76

74

72

58

49

38

46

11

97

40

000005010015020025030035

Figure 4 Phylogenetic tree of WUH2 and Chinese PRRSV isolates based on the complete genomic sequences The phylogenetic tree wasgenerated by the distance-based neighbor-joining method using the MEGA31 software Bootstrap values were calculated on 1000 replicatesof the alignment The different subgenotypes are indicated by different colors The different representative PRRSV isolates were marked withthe triangle

8 ISRN Virology

38539

39540

40541

41542

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Days after infection (DPI)

ControlCH-1aWUH2

(∘C)

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

0

1

2

3

4

5

6

Surv

ival

(pig

s)


ControlCH-1aWUH2

(b)

Figure 5 Pathogenicity comparison among the strainsWUH2 andCH-1a Forty-day-old PRRSV-free piglets were inoculated intramuscularlywith 1050 TCID

502mL of CH-1a andWUH2 respectively Mean rectal temperature (a) and survival rate (b) of each group were recorded for

21 days after inoculation

and belly at 7 dpi The first death occurred at 12 dpi Four ofsix piglets died within 21 dpi with WUH2 (Figure 5(b))

36 Comparison of Gross Lesions and Histopathology Twopiglets from each group were euthanized and necropsied at7 10 and 21 dpi to compare gross lesions and histopathologyNo gross lesions were observed at any time in the controlgroup Piglets in the CH-1a group showed mild lesionsin organs lungs had a typical pulmonary consolidationand mild tumescence while intumescences were found inthe submaxillary and inguinal lymph nodes No obviouspathological changes were observed in the brain or spleenIn contrast there were severe gross lesions in piglets inocu-lated with WUH2 including type and severity of pulmonaryconsolidation severe intumescences and hemorrhages in thelungs brain kidneys and all lymph nodes (Figure 6(a))Histopathology showed that piglets infected with WUH2had severe interstitial pneumonia thickening of the alveolarsepta increased infiltration of mononuclear cells in the lungand brain and increased amounts of alveolar exudate andnecrotic debris compared to piglets inoculated with CH-1a (Figure 6(b)) Nonsuppurative encephalitis also appearedin the brains of piglets inoculated with WUH2 Obviousperivascular cuffing and hemorrhages were seen in the vas-cular wall cells of the hippocampus (Figure 6(c)) Multifocalnecrotic and histopathologic depletion were also found inthe spleen kidney liver and lymph follicles There areobviously lymphoproliferative in the lymph nodes infectedwith WUH2

37 Evaluation ofViremia Bloodwas collected at 0 3 7 10 14and 21 dpi tomeasure viremia by quantitative PCRThemeanPRRSV load in serum after challenge is shown in Table 2PRRSV could not be detected in serum from animals in thecontrol group at any time during the postchallenge periodAll animals were PRRSV negative before inoculation (0 dpi)No significant differences (119875 gt 005) were observed betweenthe level of viremia for CH-1a andWUH2 inoculation groups

before 7 dpi Viral loads in the serum were approximately10e3-10e4 RNA copiesmL Viral loads in the serum werehighest in all animals challenged at 10 dpi equivalent to10e4 RNA copiesmL in piglets inoculated with CH-1a Thisdifference was not statistically significant (119875 gt 005) at 3 7 or10 dpi Piglets from groups inoculated with WUH2 showedthe highest viral loads at 10 dpi (10e7-10e9 RNA copiesmL)In piglets inoculated with WUH2 viral loads at 10 dpi weresignificantly higher (119875 lt 001) than any other time as well asall timepoints for piglets inoculated with CH-1a There was agradual reduction in the viral load from 10 dpi and viral loadsat 21 dpi were similar to those at 3 dpi

38 Viral Burden in Tissues Two piglets from each groupwere euthanized and necropsied at 7 10 and 21 dpi organswere collected for analysis of viral load by quantitative PCRTable 3 represents individual values for viral load in selectedtissues for each animal PRRSV was undetectable in anytissues from piglets in the control group throughout theexperimental period (data not shown) A low viral load forPRRSV was observed in selected organs of piglets inoculatedwith CH-1a throughout the duration of the experiment Inthese same animals a high viral load for PRRSVwas found inthe lungs (10e5 RNA copiesg) while the brain and stomachhad relatively low viral loads (10e2 RNA copiesg)

A significant difference (119875 lt 005) in viral load wasobserved at 10 dpi when comparing CH-1a toWUH2 groupsThe viral load in select organs from WUH2-infected pigletswas measured between 10e6-10e9 RNA copiesg Viral loadwas the highest in the lungs (greater than 10e9 RNA copiesg)followed by the brain and lymph nodes (approximately 10e9RNA copiesg) The stomach and intestines had viral loadsbetween 10e6-10e7 RNA copiesg There was a reduction inthe viral load of PRRSV at 21 dpi in surviving piglets similarto viremia

We also measured the viral load in organs from pigletsthat died naturally during the experimental period (Table 4)

ISRN Virology 9

Lung

Brain

Kidney

Control CH-1a WUH2

(a)

Control CH-1a WUH2

(b)

Control CH-1a WUH2

(c)

Figure 6 Gross lesions and histopathology in pigs experimentally infected with PRRSV CH-1a andWUH2 (a) Gross lesions of lung brainand kidney of pigs experimentally infected at 10 days after inoculation Histopathological findings of HE stained lungs (b) and brains (c) 10days after inoculation Representative sections are shown

10 ISRN Virology

Table 2 Viremia in serum measured by quantitative PCR

Treatment Animal ID number ViremiaDay 0 Day 3 Day 7 Day 10 Day 14 Day 21

Control

1 0 0 0 0 0 02 0 0 0 0 0 03 0 0 0 0 0 04 0 0 0 0 0 05 0 0 0 0 0 0

CH-1a

6 0 343119864 + 03 423119864 + 03 940119864 + 03 Killed7 0 258119864 + 03 594119864 + 03 271119864 + 04 Killed8 0 238119864 + 03 751119864 + 03 321119864 + 04 298119864 + 04 610119864 + 02

9 0 363119864 + 02 868119864 + 03 780119864 + 04 147119864 + 04 259119864 + 03

10 0 142119864 + 03 748119864 + 03 129119864 + 04 525119864 + 03 432119864 + 03

11 0 821119864 + 02 420119864 + 03 218119864 + 04 384119864 + 03 303119864 + 03

12 0 225119864 + 03 332119864 + 03 840119864 + 04 245119864 + 04 117119864 + 03

13 0 222119864 + 03 129119864 + 04 519119864 + 04 746119864 + 04 146119864 + 03

14 0 251119864 + 03 132119864 + 04 Killed15 0 263119864 + 03 179119864 + 04 Killed

WUH2

16 0 726119864 + 03 450119864 + 04 151119864 + 09 Death17 0 294119864 + 03 721119864 + 03 873119864 + 07 Killed18 0 442119864 + 03 252119864 + 04 351119864 + 08 502119864 + 06 Death19 0 271119864 + 04 718119864 + 05 293119864 + 08 Killed20 0 405119864 + 03 329119864 + 04 386119864 + 07 323119864 + 04 584119864 + 03

21 0 449119864 + 03 261119864 + 04 409119864 + 07 304119864 + 04 210119864 + 03

22 0 283119864 + 03 480119864 + 03 334119864 + 08 Death23 0 525119864 + 03 837119864 + 03 154119864 + 09 Death24 0 303119864 + 03 113119864 + 04 Killed25 0 354119864 + 03 956119864 + 03 Killed

The viral load in all organs at natural death was higher thanviral loads in organs from piglets euthanized at 7 10 and21 dpi Terminal viral loads were nearly 10e8 RNA copiesgand the viral load in the lungs was greater than 10e9 RNAcopiesg

4 Discussion

In 2006 an unknown ldquohigh feverrdquo syndrome emergedamong swine herds in the Jiangxi Province of China Thissyndrome rapidly spread to neighboring provinces over-whelming most of the swine herds in mainland China in halfa year The disease affected more than 2000000 pigs withabout 400000 fatal cases causing enormous economic lossesfor the pork-producing community Studies considered thehighly pathogenic PRRSV to be the causative agent of theldquohigh feverrdquo syndrome [16 29] The most remarkable geneticmarkers of the new isolates were two discontinuous deletionsof 30 amino acids in the gene Nsp2 In a recent study wealso found two 1 nt deletions in the 51015840 and 31015840 UTRs [20] Thehighly pathogenic PRRSV exhibits unusually high mortalityin affected pigs of all ages that was different from the classicalPRRSV Therefore it was necessary to perform a detailedcomparison of the genome and pathogenesis of the highlypathogenic PRRSV to the classical strain

In this study we determined the complete nucleotidesequence ofWUH2 characterizing its genome at a molecularlevel Phylogenetic analyses based on the full-length genomesequence indicated that WUH2 belonged to a group ofhighly pathogenic PRRSVs Analysis of genomic sequencedata showed that WUH2 had a discontinuous deletion of 30aa in Nsp2 a 1 nt deletion located in both the 51015840 and 31015840 UTRand some important mutations in GP5

The 51015840 and 31015840 UTRs are known important regulatoryelements in the PRRSV genome that play critical rolesin replication transcription and translation [19 32 33]Interestingly in addition to a discontinuous deletion inNsp2 there was a 1 nucleotide deletion located in the 51015840and 31015840 UTRs in WUH2 that was also found in nearly allother highly pathogenic PRRSV isolates The significance ofthese mutations for PRRSV replication transcription andvirulence remains to be determined

The Nsp2 gene has been shown to be highly variableamong arteriviruses [12 13 34 35] In particular the largemiddle section of Nsp2 (VR-2332 Nsp2 amino acids 148ndash880)is highly variable in length Previously studies noted allinstances of insertion or deletion in this hypervariable mid-dle region [12 26 36ndash38] These studies also showed thatdifferent amino acid insertions or deletions did not affectthe survivorship of the virus but their data were ambiguouson whether other biological characteristics of the virus were

ISRN Virology 11

Table3Viralloadin

selected

tissues

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

7CH

-1a

14203119864+03358119864+03712119864+03343119864+04101119864+04398119864+02143119864+02438119864+02826119864+03

955119864+03

15159119864+03702119864+03188119864+04924119864+04396119864+04221119864+03786119864+02562119864+02274119864+04

258119864+04

WUH2

34319119864+05541119864+05543119864+06105119864+08773119864+05639119864+06532119864+06144119864+05531119864+06

617119864+06

35352119864+05291119864+05219119864+06692119864+07437119864+05866119864+06218119864+06384119864+05459119864+06

431119864+06

10CH

-1a

6532119864+04555119864+04257119864+04102119864+05744119864+04262119864+03561119864+03203119864+04383119864+04

236119864+04

7125119864+04133119864+04585119864+04361119864+05118119864+04126119864+03201119864+03151119864+04579119864+04

712119864+04

WUH2

27576119864+06139119864+07617119864+07194119864+09107119864+08243119864+08788119864+06591119864+06385119864+08

941119864+08

29892119864+06319119864+07121119864+07992119864+08964119864+07631119864+08124119864+07422119864+06105119864+07

269119864+07

21CH

-1a

9568119864+04130119864+04369119864+05361119864+05463119864+04263119864+03124119864+03271119864+03566119864+04

793119864+04

12197119864+03226119864+05176119864+05359119864+05552119864+03298119864+03105119864+04193119864+04332119864+04

203119864+04

WUH2

30386119864+05545119864+05575119864+05846119864+06424119864+04263119864+05393119864+04323119864+04756119864+05

252119864+05

31988119864+05148119864+05167119864+05125119864+07495119864+03111119864+05841119864+04362119864+04122119864+06

966119864+05

12 ISRN Virology

Table4Viralloadin

selected

tissues

ofpigletthatdied

naturally

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

12WUH2

33707119864+08203119864+08391119864+09495119864+09272119864+08656119864+08154119864+08181119864+08216119864+08

185119864+08

14WUH2

26937119864+07146119864+08201119864+08215119864+09103119864+08187119864+08181119864+07986119864+06287119864+08

142119864+08

32697119864+08459119864+07125119864+08231119864+09505119864+07138119864+08181119864+07760119864+07101119864+08

102119864+08

17WUH2

25411119864+07419119864+07176119864+08761119864+09897119864+07147119864+07134119864+07642119864+07521119864+07

110119864+07

ISRN Virology 13

altered [18 35] The variable regions of Nsp2 may also beassociated with cell and tissue tropism and may be involvedin species-specific functions for arteriviruses [6]

The highly pathogenic PRRSV isolated in the 2006 Chi-nese outbreak contained a discontinuous deletion of 1 and 29amino acids at positions 481 and 533ndash561 in Nsp2 respec-tively The deletions were used as a defining characteristic ofthe highly pathogenic strain of PRRSV at the incipient stageof porcine ldquohigh fever syndromerdquo However Zhou et al [39]demonstrated that the discontinuous deletion of 30 aminoacids in Nsp2 of the emerging highly pathogenic PRRSV isnot related to its virulence by utilizing a reverse genetics assaysystem Regardless the Nsp2 gene may be an ideal markerfor monitoring the genetic variation of PRRSV facilitatingresearch on its molecular epidemiology and the developmentof diagnostic tests [34 36]

In addition to deletions inNsp2 and bothUTRs extensivesequence changes were found in GP5 The ORF5-encodedmajor envelope glycoprotein (GP5) is essential for viralinfectivity and contains the primary neutralization epitopeGP5 is also one of the most genetically variable structuralproteins of PRRSVDue to its immunological significance andpolymorphic nature GP5 has also been a target for analysisof the genetic diversity of PRRSV [40ndash42] Residues 13 and151 of GP5 were believed to be associated with virulence R13and R151 were altered with Q13 and G151 respectively whenVR-2332 was attenuated to the RespPRRSRepro vaccinestrain [43] The A

137

Smutation was assumed to differentiate

between the two vaccine strains [27] The key amino acidsfound in GP5 of WUH2 (R13 R151 and S137) were identicalto those found for VR-2332 In our analysis the appearanceof these characterized amino acids (which were hypothesizedto be involved in PRRSVrsquos virulence) suggested that the viru-lence of WUH2 could be higher when compared to classicalisolates Furthermore the substitution of a critical amino acidin the PNE may allow these field isolates to escape antibodyneutralization induced by the attenuated RespPRRSReprovaccine strain resulting in incomplete immune protection byvaccines These results suggest that a more effective vaccineagainst the Chinese PRRSV is urgently required especially toneutralize the emerging highly pathogenic PRRSV

In experimental infection of piglets we observed thatPRRSV WUH2 reproduced a disease state where the clinicalsigns and mortality were virtually identical to what wasseen in the field Mortality was approximately 70 and thehigh continuous fever (over 405∘C) was observed from 3 dpiuntil death Cyanopathy in the ears internal hemorrhag-ing and petechiae were common and significant Typicalinterstitial pneumonia and nonsuppurative encephalitis wereidentified by histopathology Clinical signs gross lesionsand histopathology were more severe in the WUH2 groupcompared to the CH-1a group

We identified important pathogenic characteristics ofhighly pathogenic PRRSV including viremia and viral load intissues by comparing experimental infection with differentisolates to infection with CH-1a by quantitative PCR Theviral load in serum and tissues of animals inoculated withWUH2 exhibited peak levels (10e8 RNA copiesmL) at 10 dpi

and these viral loads were significantly higher (119875 lt 001)than viral loads on other days and in piglets inoculatedwith CH-1a The peak viral load at 10 dpi also correlatedwith high mortality Comparison of viral burden in tissuesindicated that the tissue tropism of PRRSV may have beenchanging [30] The brain typically the representative organfor identifying a change in viral tropism was rarely infectedby CH-1a as determined by the lack of gross lesions andhistopathology In contrast the brains of piglets inoculatedwith WUH2 presented nonsuppurative encephalitis includ-ing obvious perivascular cuffing and hemorrhages At 10 dpithe viral load in the brains of piglets inoculated with WUH2(10e8 RNA copiesmL) was significantly higher comparedto levels in the brains from the CH-1a group (10e2 copiesRNAmL 119875 lt 001) It was also notable that there was achange in the viral burden in the stomach and intestines

In addition to the change in viral burden in tissuesthe highly pathogenic PRRSV emerging in Chinese isolatesseems to adapt to MARC-145 cells more easily than the clas-sical isolates Further exploration of virulence-determiningregions or points in the PRRSV genome is essential toimprove our understanding of pathogenic determinants

5 Conclusion

We analyzed the genomic and pathogenic characteristicsof the highly pathogenic Chinese PRRSV isolates in thisstudy Our data clearly showed that important variationsweredetected in the 51015840 and 31015840 UTRs as well as the Nsp2 andGP5 sequences There were apparent differences in the viralburden in serum and tissues of animals infected with Chineseisolates compared to classical isolates These results provideinsight into the genomic diversity and changes involved inthe pathogenicity of Chinese PRRSV isolates and help toelucidate the evolutionary and pathogenic mechanisms ofPRRSV

Conflict of Interests

The authors declare no potential conflict of interests withrespect to the research authorship andor publication of thispaper

Acknowledgments

This work was supported by the ldquo863rdquo Project (2011A-A10A208) theNational Natural Science Foundation of China(31225027 and 31001066) and the Jiangsu province NaturalSciences Foundation (BK20131330)

References

[1] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoAmerican Association of Swine Practitioners vol 1 no 2 pp 1ndash10 1989

[2] G Wensvoort C Terpstra J M Pol et al ldquoMystery swine dis-ease in The Netherlands the isolation of Lelystad virusrdquoVeterinary Quarterly vol 13 no 3 pp 121ndash130 1991

14 ISRN Virology

[3] L Darwich I Dıaz and E Mateu ldquoCertainties doubts andhypotheses in porcine reproductive and respiratory syndromevirus immunobiologyrdquoVirus Research vol 154 no 1-2 pp 123ndash132 2010

[4] E Mateu M Martın and D Vidal ldquoGenetic diversity and phy-logenetic analysis of glycoprotein 5 of European-type porcinereproductive and respiratory virus strains in Spainrdquo Journal ofGeneral Virology vol 84 no 3 pp 529ndash534 2003

[5] J J Meulenberg ldquoPRRSV the virusrdquo Veterinary Research vol31 no 3 pp 11ndash21 2000

[6] S Dea C A Gagnon H Mardassi B Pirzadeh and D RoganldquoCurrent knowledge on the structural proteins of porcine repro-ductive and respiratory syndrome (PRRS) virus comparisonof the North American and European isolatesrdquo Archives ofVirology vol 145 no 4 pp 659ndash688 2000

[7] T StadejekM BOleksiewicz D Potapchuk andK PodgorskaldquoPorcine reproductive and respiratory syndrome virus strains ofexceptional diversity in eastern Europe support the definition ofnew genetic subtypesrdquo Journal of General Virology vol 87 no 7pp 1835ndash1841 2006

[8] J J Meulenberg E J Meijer and R J Moormann ldquoSubgenomicRNAs of Lelystad virus contain a conserved leader-body junc-tion sequencerdquo Journal of General Virology vol 74 no 8 pp1697ndash1701 1993

[9] S L Ropp C E Wees Y Fang et al ldquoCharacterization ofemerging European-like porcine reproductive and respiratorysyndrome virus isolates in the United Statesrdquo Journal of Virol-ogy vol 78 no 7 pp 3684ndash3703 2004

[10] L C Van Dinten A L Wassenaar A E Gorbalenya W J MSpaan and E J Snijder ldquoProcessing of the equine arteritis virusreplicase ORF1b protein identification of cleavage productscontaining the putative viral polymerase and helicase domainsrdquoJournal of Virology vol 70 no 10 pp 6625ndash6633 1996

[11] T Stadejek A Stankevicius T Storgaard et al ldquoIdentifica-tion of radically different variants of porcine reproductiveand respiratory syndrome virus in Eastern Europe towards acommon ancestor for European and American virusesrdquo Journalof General Virology vol 83 no 8 pp 1861ndash1873 2002

[12] C J Nelsen M P Murtaugh and K S Faaberg ldquoPorcine repro-ductive and respiratory syndrome virus comparison divergentevolution on two continentsrdquo Journal of Virology vol 73 no 1pp 270ndash280 1999

[13] R Forsberg T Storgaard H S Nielsen et al ldquoThe geneticdiversity of European type PRRSV is similar to that of theNorthAmerican type but is geographically skewed within EuroperdquoVirology vol 299 no 1 pp 38ndash47 2002

[14] L Darwich M Gimeno M Sibila et al ldquoGenetic and immuno-biological diversities of porcine reproductive and respiratorysyndrome genotype I strainsrdquo Veterinary Microbiology vol 150no 1-2 pp 49ndash62 2011

[15] Y Feng T Zhao T Nguyen et al ldquoPorcine respiratory andreproductive syndrome virus variants Vietnam and ChinardquoEmerging Infectious Diseases vol 14 no 11 pp 1774ndash1776 2007

[16] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PloS ONE vol 2no 6 article e526 2007

[17] G-Z Tong Y-J Zhou X-F Hao Z-J Tian T-Q An and H-JQiu ldquoHighly pathogenic porcine reproductive and respiratorysyndrome Chinardquo Emerging Infectious Diseases vol 12 no 9pp 1434ndash1436 2007

[18] Z Q Gao X Guo and H C Yang ldquoGenomic characterizationof two Chinese isolates of porcine respiratory and reproductivesyndrome virusrdquo Archives of Virology vol 149 no 7 pp 1341ndash1351 2004

[19] C Tan L Chang S Shen D X Liu and J Kwang ldquoComparisonof the 51015840 leader sequences of North American isolates of refer-ence and field strains of porcine reproductive and respiratorysyndrome virus (PRRSV)rdquo Virus Genes vol 22 no 2 pp 209ndash217 2001

[20] B Li L Fang S Liu et al ldquoThe genomic diversity of Chineseporcine reproductive and respiratory syndrome virus isolatesfrom 1996 to 2009rdquo Veterinary Microbiology vol 146 no 3-4pp 226ndash237 2010

[21] S Kumar K Tamura andMNei ldquoMEGA3 integrated softwarefor molecular evolutionary genetics analysis and sequencealignmentrdquo Briefings in Bioinformatics vol 5 no 2 pp 150ndash1632004

[22] A Z Federico A G Esther D L Ivan et al ldquoAssessment ofthe efficacy of commercial porcine reproductive and respiratorysyndrome virus (PRRSV) vaccines based on measurementof serologic response frequency of gamma-IFN-producingcells and virological parameters of protection upon challengerdquoVeterinary Microbiology vol 123 no 3 pp 69ndash85 2007

[23] S-Y Kang S-I Yun H-S Park C-K Park H-S Choi and Y-M Lee ldquoMolecular characterization of PL97-1 the first Koreanisolate of the porcine reproductive and respiratory syndromevirusrdquo Virus Research vol 104 no 2 pp 165ndash179 2004

[24] I H Ansari B Kwon F A Osorio and A K PattnaikldquoInfluence of N-linked glycosylation of porcine reproductiveand respiratory syndrome virus GP5 on virus infectivity anti-genicity and ability to induce neutralizing antibodiesrdquo Journalof Virology vol 80 no 8 pp 3994ndash4004 2006

[25] M Matrosovich N Zhou Y Kawaoka and R Webster ldquoThesurface glycoproteins of H5 influenza viruses isolated fromhumans chickens and wild aquatic birds have distinguishablepropertiesrdquo Journal of Virology vol 73 no 2 pp 1146ndash1155 1999

[26] R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[27] R D Wesley W L Mengeling K M Lager A C Vorwald andM B Roof ldquoEvidence for divergence of restriction fragmentlength polymorphism patterns following in vivo replication ofporcine reproductive and respiratory syndrome virusrdquo Ameri-can Journal of Veterinary Research vol 60 no 4 pp 463ndash4671999

[28] B Li L Fang Z Xu et al ldquoRecombination in vaccine andcirculating strains of porcine reproductive and respiratorysyndrome virusesrdquo Emerging Infectious Diseases vol 15 no 12pp 2032ndash2035 2009

[29] B Li L Fang X Guo et al ldquoEpidemiology and evolutionarycharacteristics of the porcine reproductive and respiratorysyndrome virus in China between 2006 and 2010rdquo Journal ofClinical Microbiology vol 49 no 9 pp 3175ndash3183 2011

[30] L M Li Q Zhao X N Ge et al ldquoChinese highly pathogenicporcine reproductive and respiratory syndrome virus exhibitsmore extensive tissue tropism for pigsrdquo Virology Journal vol 9article 203 2012

[31] L Zhou S X Chen J L Zhang et al ldquoMolecular variationanalysis of porcine reproductive and respiratory syndromevirus in Chinardquo Virus Research vol 145 no 1 pp 97ndash105 2009

ISRN Virology 15

[32] Y-K Hwang and M A Brinton ldquoA 68-nucleotide sequencewithin the 31015840 noncoding region of simian hemorrhagic fevervirus negative-strand RNA binds to four MA104 cell proteinsrdquoJournal of Virology vol 72 no 5 pp 4341ndash4351 1998

[33] M H Verheije R C Olsthoorn M V Kroese P J MRottier and J J M Meulenberg ldquoKissing interaction between31015840 noncoding and coding sequences is essential for porcinearterivirus RNA replicationrdquo Journal of Virology vol 76 no 3pp 1521ndash1526 2002

[34] R Allende T L Lewis Z Lu et al ldquoNorth American and Euro-pean porcine reproductive and respiratory syndrome virusesdiffer in non-structural protein coding regionsrdquo Journal ofGeneral Virology vol 80 no 2 pp 307ndash315 1999

[35] S Shen J Kwang W Liu and D X Liu ldquoDetermination ofthe complete nucleotide sequence of a vaccine strain of porcinereproductive and respiratory syndrome virus and identificationof the Nsp2 gene with a unique insertionrdquo Archives of Virologyvol 145 no 5 pp 871ndash883 2000

[36] Y Fang D-Y Kim S Ropp et al ldquoHeterogeneity in Nsp2 ofEuropean-like porcine reproductive and respiratory syndromeviruses isolated in the United Statesrdquo Virus Research vol 100no 2 pp 229ndash235 2004

[37] J Han G Liu Y Wang and K S Faaberg ldquoIdentification ofnonessential regions of the nsp2 replicase protein of porcinereproductive and respiratory syndrome virus strainVR-2332 forreplication in cell culturerdquo Journal of Virology vol 81 no 18 pp9878ndash9890 2007

[38] J Han Y Wang and K S Faaberg ldquoComplete genome analysisof RFLP 184 isolates of porcine reproductive and respiratorysyndrome virusrdquo Virus Research vol 122 no 1-2 pp 175ndash1822006

[39] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletionin the Nsp2 of highly pathogenic porcine reproductive andrespiratory syndrome virus emerging in China is not related toits virulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[40] T-Q An Y-J Zhou G-Q Liu et al ldquoGenetic diversity andphylogenetic analysis of glycoprotein 5 of PRRSV isolates inmainland China from 1996 to 2006 coexistence of two NA-subgenotypes with great diversityrdquoVeterinaryMicrobiology vol123 no 3 pp 43ndash52 2007

[41] V G Andreyev R D Wesley W L Mengeling A C Vorwaldand K M Lager ldquoGenetic variation and phylogenetic rela-tionships of 22 porcine reproductive and respiratory syndromevirus (PRRSV) field strains based on sequence analysis of openreading frame 5rdquo Archives of Virology vol 142 no 5 pp 993ndash1001 1997

[42] S A Dee M Torremorell K Rossow C Mahlum S Otakeand K Faaberg ldquoIdentification of genetically diverse sequences(ORF 5) of porcine reproductive and respiratory syndromevirus in a swine herdrdquo Canadian Journal of Veterinary Researchvol 65 no 4 pp 254ndash260 2001

[43] R Allende G F Kutish W Laegreid et al ldquoMutations inthe genome of porcine reproductive and respiratory syndromevirus responsible for the attenuation phenotyperdquo Archives ofVirology vol 145 no 6 pp 1149ndash1161 2000

Research ArticleThe Genomic and Pathogenic Characteristics of the HighlyPathogenic Porcine Reproductive and Respiratory SyndromeVirus Isolate WUH2

Bin Li12 Liurong Fang1 Suyan Liu1 Yunbo Jiang1 Huanchun Chen1 and Shaobo Xiao13

1 Division of Animal Infectious Diseases State Key Laboratory of Agricultural MicrobiologyCollege of Veterinary Medicine Huazhong Agricultural University Wuhan 430070 China

2 Key Laboratory of Veterinary Biological Engineering and Technology Institute of Veterinary MedicineJiangsu Academy of Agricultural Sciences Ministry of Agriculture Jiangsu Province Nanjing 210014 China

3 Laboratory of Animal Virology College of Veterinary Medicine Huazhong Agricultural University 1 Shi-zi-shan StreetWuhan 430070 China

Correspondence should be addressed to Shaobo Xiao vetmailhzaueducn

Received 18 November 2013 Accepted 24 December 2013 Published 23 February 2014

Academic Editors L Darwich and D J Jackwood

Copyright copy 2014 Bin Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

To fully understand the extent of genetic diversity and pathogenesis of the highly pathogenic PRRSV found inChina we determinedthe genomic sequence of PRRSV WUH2 the pathogenicity of WUH2 was compared to the classical PRRSV isolate CH-1a Ourresults showed that the WUH2 genome had a discontinuous deletion of 30 aa in Nsp2 a 1 nucleotide deletion located in boththe 51015840 and 31015840 UTRs and point mutations within GP5 Experimental infection demonstrated that PRRSV WUH2 reproduced thephenotype and symptoms of porcine high fever syndrome Importantly we found that there were differences in viral burden in theserum and tissues when comparing infections of the pathogenic isolate WUH2 to those of the classical isolate CH-1a These dataprovide insight into the genomic diversity and altered pathogenicity of Chinese PRRSV isolates and help elucidate the evolutionand potential pathogenic mechanisms of PRRSV

1 Introduction

Porcine reproductive and respiratory syndrome (PRRS) is arelatively new viral disease of swine that is characterized bysevere reproductive failure in sows and respiratory distressin piglets and growing pigs Since PRRS was first reportedin the United States in 1987 and Europe in 1990 [1 2]the disease has devastated the swine industry by causingtremendous economic losses around the world it is nowconsidered one of the most important diseases in countrieswith intensive swine industries [3ndash5] PRRS is caused by thePRRS virus (PRRSV) a member of a group of envelopedpositive-sense RNA viruses in the Arteriviridae family [6]The genome of PRRSV is approximately 15 kb containing nineopen reading frames (ORFs) [7] ORF1a and ORF1b encodefor the RNA-dependent RNA polymerase and nonstructuralproteins (Nsps Nsp1120572 Nsp1120573 and Nsp2ndash12) respectively

[8ndash10] ORF2a ORF2b and ORFs 3 through 7 encode theviral structural proteins GP2 E GP3 GP4 GP5 M and Nrespectively [7 11]

PRRSV can be divided into European and North Amer-ican genotypes which share only about 60 nucleotideidentity at the genomic sequence level [12] Isolates from eachgenotype exhibit significant sequence variation particularlyin Nsp2 and ORF5 which represent the most geneticallyvariable regions in the PRRSV genome [4 13 14]

PRRSV was first reported in 1995 and the first isolate wasidentified in mainland China in 1996 Since then PRRS hasbecome one of themost important swine diseases worldwideMany isolates have been obtainedwithinChina fromdifferentregions and times A porcine high fever syndrome causedby a highly pathogenic PRRSV broke out in the south ofChina in 2006 and rapidly spread across the country affectingmore than 20 million pigs [15ndash17] This syndrome was

Hindawi Publishing CorporationISRN VirologyVolume 2014 Article ID 624535 15 pageshttpdxdoiorg1011552014624535

2 ISRN Virology





2







502mL of



3 Results


ISRN Virology 3



1a


1b

Nsp9 972 745 972 983 980 994Nsp10 964 647 966 982 975 991Nsp11 951 763 951 973 964 996Nsp12 941 360 941 961 954 987

2a GP2 930 606 926 965 938 10002b E 904 645 890 945 945 10003 GP3 858 545 858 929 909 9804 GP4 888 678 888 961 938 9665 GP5 885 566 875 935 915 9956 M 966 799 960 966 971 9897 N 951 580 951 951 919 1000







4 ISRN Virology



---------------------------AC--------------------------------------T----------------G-G--------C--- 99

12 nt start motif


TRS

(a)





GGC

---

---

---

--

--

--

--

--

--

--

(b)


ISRN Virology 5


LA--F-TTTTTTT





HB-1(sh)







Em2007





(a)

Figure 2 Continued

6 ISRN Virology







(b)




HL

--

-F

-F

-I

-I

-I

-I

-I

-I

-I

SRL

-Q

--

Q-

--

--

--

--

--

--

--

GRL

-G

--

--

--

--

--

--

----

--

YAC

--

-S

-S

-S

-S

-S

-S

-S-S

-S








ISRN Virology 7

HN2007JXA1





100100

100

100

98

4193

82

82

100100

100100

100

100

99

100

100 54

100

100100

100

100

99

100

100

95

76

74

72

58

49

38

46

11

97

40

000005010015020025030035


8 ISRN Virology

38539

39540

40541

41542

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21


ControlCH-1aWUH2

(∘C)

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

0

1

2

3

4

5

6

Surv

ival

(pig

s)


ControlCH-1aWUH2

(b)











ISRN Virology 9

Lung

Brain

Kidney

Control CH-1a WUH2

(a)

Control CH-1a WUH2

(b)

Control CH-1a WUH2

(c)


10 ISRN Virology



Control

1 0 0 0 0 0 02 0 0 0 0 0 03 0 0 0 0 0 04 0 0 0 0 0 05 0 0 0 0 0 0

CH-1a

6 0 343119864 + 03 423119864 + 03 940119864 + 03 Killed7 0 258119864 + 03 594119864 + 03 271119864 + 04 Killed8 0 238119864 + 03 751119864 + 03 321119864 + 04 298119864 + 04 610119864 + 02

9 0 363119864 + 02 868119864 + 03 780119864 + 04 147119864 + 04 259119864 + 03

10 0 142119864 + 03 748119864 + 03 129119864 + 04 525119864 + 03 432119864 + 03

11 0 821119864 + 02 420119864 + 03 218119864 + 04 384119864 + 03 303119864 + 03

12 0 225119864 + 03 332119864 + 03 840119864 + 04 245119864 + 04 117119864 + 03

13 0 222119864 + 03 129119864 + 04 519119864 + 04 746119864 + 04 146119864 + 03

14 0 251119864 + 03 132119864 + 04 Killed15 0 263119864 + 03 179119864 + 04 Killed

WUH2


21 0 449119864 + 03 261119864 + 04 409119864 + 07 304119864 + 04 210119864 + 03



4 Discussion





ISRN Virology 11

Table3Viralloadin

selected

tissues

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

7CH

-1a

14203119864+03358119864+03712119864+03343119864+04101119864+04398119864+02143119864+02438119864+02826119864+03

955119864+03

15159119864+03702119864+03188119864+04924119864+04396119864+04221119864+03786119864+02562119864+02274119864+04

258119864+04

WUH2

34319119864+05541119864+05543119864+06105119864+08773119864+05639119864+06532119864+06144119864+05531119864+06

617119864+06

35352119864+05291119864+05219119864+06692119864+07437119864+05866119864+06218119864+06384119864+05459119864+06

431119864+06

10CH

-1a

6532119864+04555119864+04257119864+04102119864+05744119864+04262119864+03561119864+03203119864+04383119864+04

236119864+04

7125119864+04133119864+04585119864+04361119864+05118119864+04126119864+03201119864+03151119864+04579119864+04

712119864+04

WUH2

27576119864+06139119864+07617119864+07194119864+09107119864+08243119864+08788119864+06591119864+06385119864+08

941119864+08

29892119864+06319119864+07121119864+07992119864+08964119864+07631119864+08124119864+07422119864+06105119864+07

269119864+07

21CH

-1a

9568119864+04130119864+04369119864+05361119864+05463119864+04263119864+03124119864+03271119864+03566119864+04

793119864+04

12197119864+03226119864+05176119864+05359119864+05552119864+03298119864+03105119864+04193119864+04332119864+04

203119864+04

WUH2

30386119864+05545119864+05575119864+05846119864+06424119864+04263119864+05393119864+04323119864+04756119864+05

252119864+05

31988119864+05148119864+05167119864+05125119864+07495119864+03111119864+05841119864+04362119864+04122119864+06

966119864+05

12 ISRN Virology

Table4Viralloadin

selected

tissues

ofpigletthatdied

naturally

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

12WUH2

33707119864+08203119864+08391119864+09495119864+09272119864+08656119864+08154119864+08181119864+08216119864+08

185119864+08

14WUH2

26937119864+07146119864+08201119864+08215119864+09103119864+08187119864+08181119864+07986119864+06287119864+08

142119864+08

32697119864+08459119864+07125119864+08231119864+09505119864+07138119864+08181119864+07760119864+07101119864+08

102119864+08

17WUH2

25411119864+07419119864+07176119864+08761119864+09897119864+07147119864+07134119864+07642119864+07521119864+07

110119864+07

ISRN Virology 13




137







5 Conclusion




Acknowledgments


References



14 ISRN Virology






























ISRN Virology 15













2 ISRN Virology





2







502mL of



3 Results


ISRN Virology 3



1a


1b

Nsp9 972 745 972 983 980 994Nsp10 964 647 966 982 975 991Nsp11 951 763 951 973 964 996Nsp12 941 360 941 961 954 987

2a GP2 930 606 926 965 938 10002b E 904 645 890 945 945 10003 GP3 858 545 858 929 909 9804 GP4 888 678 888 961 938 9665 GP5 885 566 875 935 915 9956 M 966 799 960 966 971 9897 N 951 580 951 951 919 1000







4 ISRN Virology



---------------------------AC--------------------------------------T----------------G-G--------C--- 99

12 nt start motif


TRS

(a)





GGC

---

---

---

--

--

--

--

--

--

--

(b)


ISRN Virology 5


LA--F-TTTTTTT





HB-1(sh)







Em2007





(a)

Figure 2 Continued

6 ISRN Virology







(b)




HL

--

-F

-F

-I

-I

-I

-I

-I

-I

-I

SRL

-Q

--

Q-

--

--

--

--

--

--

--

GRL

-G

--

--

--

--

--

--

----

--

YAC

--

-S

-S

-S

-S

-S

-S

-S-S

-S








ISRN Virology 7

HN2007JXA1





100100

100

100

98

4193

82

82

100100

100100

100

100

99

100

100 54

100

100100

100

100

99

100

100

95

76

74

72

58

49

38

46

11

97

40

000005010015020025030035


8 ISRN Virology

38539

39540

40541

41542

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21


ControlCH-1aWUH2

(∘C)

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

0

1

2

3

4

5

6

Surv

ival

(pig

s)


ControlCH-1aWUH2

(b)











ISRN Virology 9

Lung

Brain

Kidney

Control CH-1a WUH2

(a)

Control CH-1a WUH2

(b)

Control CH-1a WUH2

(c)


10 ISRN Virology



Control

1 0 0 0 0 0 02 0 0 0 0 0 03 0 0 0 0 0 04 0 0 0 0 0 05 0 0 0 0 0 0

CH-1a

6 0 343119864 + 03 423119864 + 03 940119864 + 03 Killed7 0 258119864 + 03 594119864 + 03 271119864 + 04 Killed8 0 238119864 + 03 751119864 + 03 321119864 + 04 298119864 + 04 610119864 + 02

9 0 363119864 + 02 868119864 + 03 780119864 + 04 147119864 + 04 259119864 + 03

10 0 142119864 + 03 748119864 + 03 129119864 + 04 525119864 + 03 432119864 + 03

11 0 821119864 + 02 420119864 + 03 218119864 + 04 384119864 + 03 303119864 + 03

12 0 225119864 + 03 332119864 + 03 840119864 + 04 245119864 + 04 117119864 + 03

13 0 222119864 + 03 129119864 + 04 519119864 + 04 746119864 + 04 146119864 + 03

14 0 251119864 + 03 132119864 + 04 Killed15 0 263119864 + 03 179119864 + 04 Killed

WUH2


21 0 449119864 + 03 261119864 + 04 409119864 + 07 304119864 + 04 210119864 + 03



4 Discussion





ISRN Virology 11

Table3Viralloadin

selected

tissues

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

7CH

-1a

14203119864+03358119864+03712119864+03343119864+04101119864+04398119864+02143119864+02438119864+02826119864+03

955119864+03

15159119864+03702119864+03188119864+04924119864+04396119864+04221119864+03786119864+02562119864+02274119864+04

258119864+04

WUH2

34319119864+05541119864+05543119864+06105119864+08773119864+05639119864+06532119864+06144119864+05531119864+06

617119864+06

35352119864+05291119864+05219119864+06692119864+07437119864+05866119864+06218119864+06384119864+05459119864+06

431119864+06

10CH

-1a

6532119864+04555119864+04257119864+04102119864+05744119864+04262119864+03561119864+03203119864+04383119864+04

236119864+04

7125119864+04133119864+04585119864+04361119864+05118119864+04126119864+03201119864+03151119864+04579119864+04

712119864+04

WUH2

27576119864+06139119864+07617119864+07194119864+09107119864+08243119864+08788119864+06591119864+06385119864+08

941119864+08

29892119864+06319119864+07121119864+07992119864+08964119864+07631119864+08124119864+07422119864+06105119864+07

269119864+07

21CH

-1a

9568119864+04130119864+04369119864+05361119864+05463119864+04263119864+03124119864+03271119864+03566119864+04

793119864+04

12197119864+03226119864+05176119864+05359119864+05552119864+03298119864+03105119864+04193119864+04332119864+04

203119864+04

WUH2

30386119864+05545119864+05575119864+05846119864+06424119864+04263119864+05393119864+04323119864+04756119864+05

252119864+05

31988119864+05148119864+05167119864+05125119864+07495119864+03111119864+05841119864+04362119864+04122119864+06

966119864+05

12 ISRN Virology

Table4Viralloadin

selected

tissues

ofpigletthatdied

naturally

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

12WUH2

33707119864+08203119864+08391119864+09495119864+09272119864+08656119864+08154119864+08181119864+08216119864+08

185119864+08

14WUH2

26937119864+07146119864+08201119864+08215119864+09103119864+08187119864+08181119864+07986119864+06287119864+08

142119864+08

32697119864+08459119864+07125119864+08231119864+09505119864+07138119864+08181119864+07760119864+07101119864+08

102119864+08

17WUH2

25411119864+07419119864+07176119864+08761119864+09897119864+07147119864+07134119864+07642119864+07521119864+07

110119864+07

ISRN Virology 13




137







5 Conclusion




Acknowledgments


References



14 ISRN Virology






























ISRN Virology 15













ISRN Virology 3



1a


1b

Nsp9 972 745 972 983 980 994Nsp10 964 647 966 982 975 991Nsp11 951 763 951 973 964 996Nsp12 941 360 941 961 954 987

2a GP2 930 606 926 965 938 10002b E 904 645 890 945 945 10003 GP3 858 545 858 929 909 9804 GP4 888 678 888 961 938 9665 GP5 885 566 875 935 915 9956 M 966 799 960 966 971 9897 N 951 580 951 951 919 1000







4 ISRN Virology



---------------------------AC--------------------------------------T----------------G-G--------C--- 99

12 nt start motif


TRS

(a)





GGC

---

---

---

--

--

--

--

--

--

--

(b)


ISRN Virology 5


LA--F-TTTTTTT





HB-1(sh)







Em2007





(a)

Figure 2 Continued

6 ISRN Virology







(b)




HL

--

-F

-F

-I

-I

-I

-I

-I

-I

-I

SRL

-Q

--

Q-

--

--

--

--

--

--

--

GRL

-G

--

--

--

--

--

--

----

--

YAC

--

-S

-S

-S

-S

-S

-S

-S-S

-S








ISRN Virology 7

HN2007JXA1





100100

100

100

98

4193

82

82

100100

100100

100

100

99

100

100 54

100

100100

100

100

99

100

100

95

76

74

72

58

49

38

46

11

97

40

000005010015020025030035


8 ISRN Virology

38539

39540

40541

41542

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21


ControlCH-1aWUH2

(∘C)

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

0

1

2

3

4

5

6

Surv

ival

(pig

s)


ControlCH-1aWUH2

(b)











ISRN Virology 9

Lung

Brain

Kidney

Control CH-1a WUH2

(a)

Control CH-1a WUH2

(b)

Control CH-1a WUH2

(c)


10 ISRN Virology



Control

1 0 0 0 0 0 02 0 0 0 0 0 03 0 0 0 0 0 04 0 0 0 0 0 05 0 0 0 0 0 0

CH-1a

6 0 343119864 + 03 423119864 + 03 940119864 + 03 Killed7 0 258119864 + 03 594119864 + 03 271119864 + 04 Killed8 0 238119864 + 03 751119864 + 03 321119864 + 04 298119864 + 04 610119864 + 02

9 0 363119864 + 02 868119864 + 03 780119864 + 04 147119864 + 04 259119864 + 03

10 0 142119864 + 03 748119864 + 03 129119864 + 04 525119864 + 03 432119864 + 03

11 0 821119864 + 02 420119864 + 03 218119864 + 04 384119864 + 03 303119864 + 03

12 0 225119864 + 03 332119864 + 03 840119864 + 04 245119864 + 04 117119864 + 03

13 0 222119864 + 03 129119864 + 04 519119864 + 04 746119864 + 04 146119864 + 03

14 0 251119864 + 03 132119864 + 04 Killed15 0 263119864 + 03 179119864 + 04 Killed

WUH2


21 0 449119864 + 03 261119864 + 04 409119864 + 07 304119864 + 04 210119864 + 03



4 Discussion





ISRN Virology 11

Table3Viralloadin

selected

tissues

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

7CH

-1a

14203119864+03358119864+03712119864+03343119864+04101119864+04398119864+02143119864+02438119864+02826119864+03

955119864+03

15159119864+03702119864+03188119864+04924119864+04396119864+04221119864+03786119864+02562119864+02274119864+04

258119864+04

WUH2

34319119864+05541119864+05543119864+06105119864+08773119864+05639119864+06532119864+06144119864+05531119864+06

617119864+06

35352119864+05291119864+05219119864+06692119864+07437119864+05866119864+06218119864+06384119864+05459119864+06

431119864+06

10CH

-1a

6532119864+04555119864+04257119864+04102119864+05744119864+04262119864+03561119864+03203119864+04383119864+04

236119864+04

7125119864+04133119864+04585119864+04361119864+05118119864+04126119864+03201119864+03151119864+04579119864+04

712119864+04

WUH2

27576119864+06139119864+07617119864+07194119864+09107119864+08243119864+08788119864+06591119864+06385119864+08

941119864+08

29892119864+06319119864+07121119864+07992119864+08964119864+07631119864+08124119864+07422119864+06105119864+07

269119864+07

21CH

-1a

9568119864+04130119864+04369119864+05361119864+05463119864+04263119864+03124119864+03271119864+03566119864+04

793119864+04

12197119864+03226119864+05176119864+05359119864+05552119864+03298119864+03105119864+04193119864+04332119864+04

203119864+04

WUH2

30386119864+05545119864+05575119864+05846119864+06424119864+04263119864+05393119864+04323119864+04756119864+05

252119864+05

31988119864+05148119864+05167119864+05125119864+07495119864+03111119864+05841119864+04362119864+04122119864+06

966119864+05

12 ISRN Virology

Table4Viralloadin

selected

tissues

ofpigletthatdied

naturally

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

12WUH2

33707119864+08203119864+08391119864+09495119864+09272119864+08656119864+08154119864+08181119864+08216119864+08

185119864+08

14WUH2

26937119864+07146119864+08201119864+08215119864+09103119864+08187119864+08181119864+07986119864+06287119864+08

142119864+08

32697119864+08459119864+07125119864+08231119864+09505119864+07138119864+08181119864+07760119864+07101119864+08

102119864+08

17WUH2

25411119864+07419119864+07176119864+08761119864+09897119864+07147119864+07134119864+07642119864+07521119864+07

110119864+07

ISRN Virology 13




137







5 Conclusion




Acknowledgments


References



14 ISRN Virology






























ISRN Virology 15













4 ISRN Virology



---------------------------AC--------------------------------------T----------------G-G--------C--- 99

12 nt start motif


TRS

(a)





GGC

---

---

---

--

--

--

--

--

--

--

(b)


ISRN Virology 5


LA--F-TTTTTTT





HB-1(sh)







Em2007





(a)

Figure 2 Continued

6 ISRN Virology







(b)




HL

--

-F

-F

-I

-I

-I

-I

-I

-I

-I

SRL

-Q

--

Q-

--

--

--

--

--

--

--

GRL

-G

--

--

--

--

--

--

----

--

YAC

--

-S

-S

-S

-S

-S

-S

-S-S

-S








ISRN Virology 7

HN2007JXA1





100100

100

100

98

4193

82

82

100100

100100

100

100

99

100

100 54

100

100100

100

100

99

100

100

95

76

74

72

58

49

38

46

11

97

40

000005010015020025030035


8 ISRN Virology

38539

39540

40541

41542

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21


ControlCH-1aWUH2

(∘C)

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

0

1

2

3

4

5

6

Surv

ival

(pig

s)


ControlCH-1aWUH2

(b)











ISRN Virology 9

Lung

Brain

Kidney

Control CH-1a WUH2

(a)

Control CH-1a WUH2

(b)

Control CH-1a WUH2

(c)


10 ISRN Virology



Control

1 0 0 0 0 0 02 0 0 0 0 0 03 0 0 0 0 0 04 0 0 0 0 0 05 0 0 0 0 0 0

CH-1a

6 0 343119864 + 03 423119864 + 03 940119864 + 03 Killed7 0 258119864 + 03 594119864 + 03 271119864 + 04 Killed8 0 238119864 + 03 751119864 + 03 321119864 + 04 298119864 + 04 610119864 + 02

9 0 363119864 + 02 868119864 + 03 780119864 + 04 147119864 + 04 259119864 + 03

10 0 142119864 + 03 748119864 + 03 129119864 + 04 525119864 + 03 432119864 + 03

11 0 821119864 + 02 420119864 + 03 218119864 + 04 384119864 + 03 303119864 + 03

12 0 225119864 + 03 332119864 + 03 840119864 + 04 245119864 + 04 117119864 + 03

13 0 222119864 + 03 129119864 + 04 519119864 + 04 746119864 + 04 146119864 + 03

14 0 251119864 + 03 132119864 + 04 Killed15 0 263119864 + 03 179119864 + 04 Killed

WUH2


21 0 449119864 + 03 261119864 + 04 409119864 + 07 304119864 + 04 210119864 + 03



4 Discussion





ISRN Virology 11

Table3Viralloadin

selected

tissues

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

7CH

-1a

14203119864+03358119864+03712119864+03343119864+04101119864+04398119864+02143119864+02438119864+02826119864+03

955119864+03

15159119864+03702119864+03188119864+04924119864+04396119864+04221119864+03786119864+02562119864+02274119864+04

258119864+04

WUH2

34319119864+05541119864+05543119864+06105119864+08773119864+05639119864+06532119864+06144119864+05531119864+06

617119864+06

35352119864+05291119864+05219119864+06692119864+07437119864+05866119864+06218119864+06384119864+05459119864+06

431119864+06

10CH

-1a

6532119864+04555119864+04257119864+04102119864+05744119864+04262119864+03561119864+03203119864+04383119864+04

236119864+04

7125119864+04133119864+04585119864+04361119864+05118119864+04126119864+03201119864+03151119864+04579119864+04

712119864+04

WUH2

27576119864+06139119864+07617119864+07194119864+09107119864+08243119864+08788119864+06591119864+06385119864+08

941119864+08

29892119864+06319119864+07121119864+07992119864+08964119864+07631119864+08124119864+07422119864+06105119864+07

269119864+07

21CH

-1a

9568119864+04130119864+04369119864+05361119864+05463119864+04263119864+03124119864+03271119864+03566119864+04

793119864+04

12197119864+03226119864+05176119864+05359119864+05552119864+03298119864+03105119864+04193119864+04332119864+04

203119864+04

WUH2

30386119864+05545119864+05575119864+05846119864+06424119864+04263119864+05393119864+04323119864+04756119864+05

252119864+05

31988119864+05148119864+05167119864+05125119864+07495119864+03111119864+05841119864+04362119864+04122119864+06

966119864+05

12 ISRN Virology

Table4Viralloadin

selected

tissues

ofpigletthatdied

naturally

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

12WUH2

33707119864+08203119864+08391119864+09495119864+09272119864+08656119864+08154119864+08181119864+08216119864+08

185119864+08

14WUH2

26937119864+07146119864+08201119864+08215119864+09103119864+08187119864+08181119864+07986119864+06287119864+08

142119864+08

32697119864+08459119864+07125119864+08231119864+09505119864+07138119864+08181119864+07760119864+07101119864+08

102119864+08

17WUH2

25411119864+07419119864+07176119864+08761119864+09897119864+07147119864+07134119864+07642119864+07521119864+07

110119864+07

ISRN Virology 13




137







5 Conclusion




Acknowledgments


References



14 ISRN Virology






























ISRN Virology 15













ISRN Virology 5


LA--F-TTTTTTT





HB-1(sh)







Em2007





(a)

Figure 2 Continued

6 ISRN Virology







(b)




HL

--

-F

-F

-I

-I

-I

-I

-I

-I

-I

SRL

-Q

--

Q-

--

--

--

--

--

--

--

GRL

-G

--

--

--

--

--

--

----

--

YAC

--

-S

-S

-S

-S

-S

-S

-S-S

-S








ISRN Virology 7

HN2007JXA1





100100

100

100

98

4193

82

82

100100

100100

100

100

99

100

100 54

100

100100

100

100

99

100

100

95

76

74

72

58

49

38

46

11

97

40

000005010015020025030035


8 ISRN Virology

38539

39540

40541

41542

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21


ControlCH-1aWUH2

(∘C)

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

0

1

2

3

4

5

6

Surv

ival

(pig

s)


ControlCH-1aWUH2

(b)











ISRN Virology 9

Lung

Brain

Kidney

Control CH-1a WUH2

(a)

Control CH-1a WUH2

(b)

Control CH-1a WUH2

(c)


10 ISRN Virology



Control

1 0 0 0 0 0 02 0 0 0 0 0 03 0 0 0 0 0 04 0 0 0 0 0 05 0 0 0 0 0 0

CH-1a

6 0 343119864 + 03 423119864 + 03 940119864 + 03 Killed7 0 258119864 + 03 594119864 + 03 271119864 + 04 Killed8 0 238119864 + 03 751119864 + 03 321119864 + 04 298119864 + 04 610119864 + 02

9 0 363119864 + 02 868119864 + 03 780119864 + 04 147119864 + 04 259119864 + 03

10 0 142119864 + 03 748119864 + 03 129119864 + 04 525119864 + 03 432119864 + 03

11 0 821119864 + 02 420119864 + 03 218119864 + 04 384119864 + 03 303119864 + 03

12 0 225119864 + 03 332119864 + 03 840119864 + 04 245119864 + 04 117119864 + 03

13 0 222119864 + 03 129119864 + 04 519119864 + 04 746119864 + 04 146119864 + 03

14 0 251119864 + 03 132119864 + 04 Killed15 0 263119864 + 03 179119864 + 04 Killed

WUH2


21 0 449119864 + 03 261119864 + 04 409119864 + 07 304119864 + 04 210119864 + 03



4 Discussion





ISRN Virology 11

Table3Viralloadin

selected

tissues

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

7CH

-1a

14203119864+03358119864+03712119864+03343119864+04101119864+04398119864+02143119864+02438119864+02826119864+03

955119864+03

15159119864+03702119864+03188119864+04924119864+04396119864+04221119864+03786119864+02562119864+02274119864+04

258119864+04

WUH2

34319119864+05541119864+05543119864+06105119864+08773119864+05639119864+06532119864+06144119864+05531119864+06

617119864+06

35352119864+05291119864+05219119864+06692119864+07437119864+05866119864+06218119864+06384119864+05459119864+06

431119864+06

10CH

-1a

6532119864+04555119864+04257119864+04102119864+05744119864+04262119864+03561119864+03203119864+04383119864+04

236119864+04

7125119864+04133119864+04585119864+04361119864+05118119864+04126119864+03201119864+03151119864+04579119864+04

712119864+04

WUH2

27576119864+06139119864+07617119864+07194119864+09107119864+08243119864+08788119864+06591119864+06385119864+08

941119864+08

29892119864+06319119864+07121119864+07992119864+08964119864+07631119864+08124119864+07422119864+06105119864+07

269119864+07

21CH

-1a

9568119864+04130119864+04369119864+05361119864+05463119864+04263119864+03124119864+03271119864+03566119864+04

793119864+04

12197119864+03226119864+05176119864+05359119864+05552119864+03298119864+03105119864+04193119864+04332119864+04

203119864+04

WUH2

30386119864+05545119864+05575119864+05846119864+06424119864+04263119864+05393119864+04323119864+04756119864+05

252119864+05

31988119864+05148119864+05167119864+05125119864+07495119864+03111119864+05841119864+04362119864+04122119864+06

966119864+05

12 ISRN Virology

Table4Viralloadin

selected

tissues

ofpigletthatdied

naturally

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

12WUH2

33707119864+08203119864+08391119864+09495119864+09272119864+08656119864+08154119864+08181119864+08216119864+08

185119864+08

14WUH2

26937119864+07146119864+08201119864+08215119864+09103119864+08187119864+08181119864+07986119864+06287119864+08

142119864+08

32697119864+08459119864+07125119864+08231119864+09505119864+07138119864+08181119864+07760119864+07101119864+08

102119864+08

17WUH2

25411119864+07419119864+07176119864+08761119864+09897119864+07147119864+07134119864+07642119864+07521119864+07

110119864+07

ISRN Virology 13




137







5 Conclusion




Acknowledgments


References



14 ISRN Virology






























ISRN Virology 15













6 ISRN Virology







(b)




HL

--

-F

-F

-I

-I

-I

-I

-I

-I

-I

SRL

-Q

--

Q-

--

--

--

--

--

--

--

GRL

-G

--

--

--

--

--

--

----

--

YAC

--

-S

-S

-S

-S

-S

-S

-S-S

-S








ISRN Virology 7

HN2007JXA1





100100

100

100

98

4193

82

82

100100

100100

100

100

99

100

100 54

100

100100

100

100

99

100

100

95

76

74

72

58

49

38

46

11

97

40

000005010015020025030035


8 ISRN Virology

38539

39540

40541

41542

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21


ControlCH-1aWUH2

(∘C)

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

0

1

2

3

4

5

6

Surv

ival

(pig

s)


ControlCH-1aWUH2

(b)











ISRN Virology 9

Lung

Brain

Kidney

Control CH-1a WUH2

(a)

Control CH-1a WUH2

(b)

Control CH-1a WUH2

(c)


10 ISRN Virology



Control

1 0 0 0 0 0 02 0 0 0 0 0 03 0 0 0 0 0 04 0 0 0 0 0 05 0 0 0 0 0 0

CH-1a

6 0 343119864 + 03 423119864 + 03 940119864 + 03 Killed7 0 258119864 + 03 594119864 + 03 271119864 + 04 Killed8 0 238119864 + 03 751119864 + 03 321119864 + 04 298119864 + 04 610119864 + 02

9 0 363119864 + 02 868119864 + 03 780119864 + 04 147119864 + 04 259119864 + 03

10 0 142119864 + 03 748119864 + 03 129119864 + 04 525119864 + 03 432119864 + 03

11 0 821119864 + 02 420119864 + 03 218119864 + 04 384119864 + 03 303119864 + 03

12 0 225119864 + 03 332119864 + 03 840119864 + 04 245119864 + 04 117119864 + 03

13 0 222119864 + 03 129119864 + 04 519119864 + 04 746119864 + 04 146119864 + 03

14 0 251119864 + 03 132119864 + 04 Killed15 0 263119864 + 03 179119864 + 04 Killed

WUH2


21 0 449119864 + 03 261119864 + 04 409119864 + 07 304119864 + 04 210119864 + 03



4 Discussion





ISRN Virology 11

Table3Viralloadin

selected

tissues

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

7CH

-1a

14203119864+03358119864+03712119864+03343119864+04101119864+04398119864+02143119864+02438119864+02826119864+03

955119864+03

15159119864+03702119864+03188119864+04924119864+04396119864+04221119864+03786119864+02562119864+02274119864+04

258119864+04

WUH2

34319119864+05541119864+05543119864+06105119864+08773119864+05639119864+06532119864+06144119864+05531119864+06

617119864+06

35352119864+05291119864+05219119864+06692119864+07437119864+05866119864+06218119864+06384119864+05459119864+06

431119864+06

10CH

-1a

6532119864+04555119864+04257119864+04102119864+05744119864+04262119864+03561119864+03203119864+04383119864+04

236119864+04

7125119864+04133119864+04585119864+04361119864+05118119864+04126119864+03201119864+03151119864+04579119864+04

712119864+04

WUH2

27576119864+06139119864+07617119864+07194119864+09107119864+08243119864+08788119864+06591119864+06385119864+08

941119864+08

29892119864+06319119864+07121119864+07992119864+08964119864+07631119864+08124119864+07422119864+06105119864+07

269119864+07

21CH

-1a

9568119864+04130119864+04369119864+05361119864+05463119864+04263119864+03124119864+03271119864+03566119864+04

793119864+04

12197119864+03226119864+05176119864+05359119864+05552119864+03298119864+03105119864+04193119864+04332119864+04

203119864+04

WUH2

30386119864+05545119864+05575119864+05846119864+06424119864+04263119864+05393119864+04323119864+04756119864+05

252119864+05

31988119864+05148119864+05167119864+05125119864+07495119864+03111119864+05841119864+04362119864+04122119864+06

966119864+05

12 ISRN Virology

Table4Viralloadin

selected

tissues

ofpigletthatdied

naturally

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

12WUH2

33707119864+08203119864+08391119864+09495119864+09272119864+08656119864+08154119864+08181119864+08216119864+08

185119864+08

14WUH2

26937119864+07146119864+08201119864+08215119864+09103119864+08187119864+08181119864+07986119864+06287119864+08

142119864+08

32697119864+08459119864+07125119864+08231119864+09505119864+07138119864+08181119864+07760119864+07101119864+08

102119864+08

17WUH2

25411119864+07419119864+07176119864+08761119864+09897119864+07147119864+07134119864+07642119864+07521119864+07

110119864+07

ISRN Virology 13




137







5 Conclusion




Acknowledgments


References



14 ISRN Virology






























ISRN Virology 15













ISRN Virology 7

HN2007JXA1





100100

100

100

98

4193

82

82

100100

100100

100

100

99

100

100 54

100

100100

100

100

99

100

100

95

76

74

72

58

49

38

46

11

97

40

000005010015020025030035


8 ISRN Virology

38539

39540

40541

41542

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21


ControlCH-1aWUH2

(∘C)

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

0

1

2

3

4

5

6

Surv

ival

(pig

s)


ControlCH-1aWUH2

(b)











ISRN Virology 9

Lung

Brain

Kidney

Control CH-1a WUH2

(a)

Control CH-1a WUH2

(b)

Control CH-1a WUH2

(c)


10 ISRN Virology



Control

1 0 0 0 0 0 02 0 0 0 0 0 03 0 0 0 0 0 04 0 0 0 0 0 05 0 0 0 0 0 0

CH-1a

6 0 343119864 + 03 423119864 + 03 940119864 + 03 Killed7 0 258119864 + 03 594119864 + 03 271119864 + 04 Killed8 0 238119864 + 03 751119864 + 03 321119864 + 04 298119864 + 04 610119864 + 02

9 0 363119864 + 02 868119864 + 03 780119864 + 04 147119864 + 04 259119864 + 03

10 0 142119864 + 03 748119864 + 03 129119864 + 04 525119864 + 03 432119864 + 03

11 0 821119864 + 02 420119864 + 03 218119864 + 04 384119864 + 03 303119864 + 03

12 0 225119864 + 03 332119864 + 03 840119864 + 04 245119864 + 04 117119864 + 03

13 0 222119864 + 03 129119864 + 04 519119864 + 04 746119864 + 04 146119864 + 03

14 0 251119864 + 03 132119864 + 04 Killed15 0 263119864 + 03 179119864 + 04 Killed

WUH2


21 0 449119864 + 03 261119864 + 04 409119864 + 07 304119864 + 04 210119864 + 03



4 Discussion





ISRN Virology 11

Table3Viralloadin

selected

tissues

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

7CH

-1a

14203119864+03358119864+03712119864+03343119864+04101119864+04398119864+02143119864+02438119864+02826119864+03

955119864+03

15159119864+03702119864+03188119864+04924119864+04396119864+04221119864+03786119864+02562119864+02274119864+04

258119864+04

WUH2

34319119864+05541119864+05543119864+06105119864+08773119864+05639119864+06532119864+06144119864+05531119864+06

617119864+06

35352119864+05291119864+05219119864+06692119864+07437119864+05866119864+06218119864+06384119864+05459119864+06

431119864+06

10CH

-1a

6532119864+04555119864+04257119864+04102119864+05744119864+04262119864+03561119864+03203119864+04383119864+04

236119864+04

7125119864+04133119864+04585119864+04361119864+05118119864+04126119864+03201119864+03151119864+04579119864+04

712119864+04

WUH2

27576119864+06139119864+07617119864+07194119864+09107119864+08243119864+08788119864+06591119864+06385119864+08

941119864+08

29892119864+06319119864+07121119864+07992119864+08964119864+07631119864+08124119864+07422119864+06105119864+07

269119864+07

21CH

-1a

9568119864+04130119864+04369119864+05361119864+05463119864+04263119864+03124119864+03271119864+03566119864+04

793119864+04

12197119864+03226119864+05176119864+05359119864+05552119864+03298119864+03105119864+04193119864+04332119864+04

203119864+04

WUH2

30386119864+05545119864+05575119864+05846119864+06424119864+04263119864+05393119864+04323119864+04756119864+05

252119864+05

31988119864+05148119864+05167119864+05125119864+07495119864+03111119864+05841119864+04362119864+04122119864+06

966119864+05

12 ISRN Virology

Table4Viralloadin

selected

tissues

ofpigletthatdied

naturally

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

12WUH2

33707119864+08203119864+08391119864+09495119864+09272119864+08656119864+08154119864+08181119864+08216119864+08

185119864+08

14WUH2

26937119864+07146119864+08201119864+08215119864+09103119864+08187119864+08181119864+07986119864+06287119864+08

142119864+08

32697119864+08459119864+07125119864+08231119864+09505119864+07138119864+08181119864+07760119864+07101119864+08

102119864+08

17WUH2

25411119864+07419119864+07176119864+08761119864+09897119864+07147119864+07134119864+07642119864+07521119864+07

110119864+07

ISRN Virology 13




137







5 Conclusion




Acknowledgments


References



14 ISRN Virology






























ISRN Virology 15













8 ISRN Virology

38539

39540

40541

41542

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21


ControlCH-1aWUH2

(∘C)

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

0

1

2

3

4

5

6

Surv

ival

(pig

s)


ControlCH-1aWUH2

(b)











ISRN Virology 9

Lung

Brain

Kidney

Control CH-1a WUH2

(a)

Control CH-1a WUH2

(b)

Control CH-1a WUH2

(c)


10 ISRN Virology



Control

1 0 0 0 0 0 02 0 0 0 0 0 03 0 0 0 0 0 04 0 0 0 0 0 05 0 0 0 0 0 0

CH-1a

6 0 343119864 + 03 423119864 + 03 940119864 + 03 Killed7 0 258119864 + 03 594119864 + 03 271119864 + 04 Killed8 0 238119864 + 03 751119864 + 03 321119864 + 04 298119864 + 04 610119864 + 02

9 0 363119864 + 02 868119864 + 03 780119864 + 04 147119864 + 04 259119864 + 03

10 0 142119864 + 03 748119864 + 03 129119864 + 04 525119864 + 03 432119864 + 03

11 0 821119864 + 02 420119864 + 03 218119864 + 04 384119864 + 03 303119864 + 03

12 0 225119864 + 03 332119864 + 03 840119864 + 04 245119864 + 04 117119864 + 03

13 0 222119864 + 03 129119864 + 04 519119864 + 04 746119864 + 04 146119864 + 03

14 0 251119864 + 03 132119864 + 04 Killed15 0 263119864 + 03 179119864 + 04 Killed

WUH2


21 0 449119864 + 03 261119864 + 04 409119864 + 07 304119864 + 04 210119864 + 03



4 Discussion





ISRN Virology 11

Table3Viralloadin

selected

tissues

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

7CH

-1a

14203119864+03358119864+03712119864+03343119864+04101119864+04398119864+02143119864+02438119864+02826119864+03

955119864+03

15159119864+03702119864+03188119864+04924119864+04396119864+04221119864+03786119864+02562119864+02274119864+04

258119864+04

WUH2

34319119864+05541119864+05543119864+06105119864+08773119864+05639119864+06532119864+06144119864+05531119864+06

617119864+06

35352119864+05291119864+05219119864+06692119864+07437119864+05866119864+06218119864+06384119864+05459119864+06

431119864+06

10CH

-1a

6532119864+04555119864+04257119864+04102119864+05744119864+04262119864+03561119864+03203119864+04383119864+04

236119864+04

7125119864+04133119864+04585119864+04361119864+05118119864+04126119864+03201119864+03151119864+04579119864+04

712119864+04

WUH2

27576119864+06139119864+07617119864+07194119864+09107119864+08243119864+08788119864+06591119864+06385119864+08

941119864+08

29892119864+06319119864+07121119864+07992119864+08964119864+07631119864+08124119864+07422119864+06105119864+07

269119864+07

21CH

-1a

9568119864+04130119864+04369119864+05361119864+05463119864+04263119864+03124119864+03271119864+03566119864+04

793119864+04

12197119864+03226119864+05176119864+05359119864+05552119864+03298119864+03105119864+04193119864+04332119864+04

203119864+04

WUH2

30386119864+05545119864+05575119864+05846119864+06424119864+04263119864+05393119864+04323119864+04756119864+05

252119864+05

31988119864+05148119864+05167119864+05125119864+07495119864+03111119864+05841119864+04362119864+04122119864+06

966119864+05

12 ISRN Virology

Table4Viralloadin

selected

tissues

ofpigletthatdied

naturally

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

12WUH2

33707119864+08203119864+08391119864+09495119864+09272119864+08656119864+08154119864+08181119864+08216119864+08

185119864+08

14WUH2

26937119864+07146119864+08201119864+08215119864+09103119864+08187119864+08181119864+07986119864+06287119864+08

142119864+08

32697119864+08459119864+07125119864+08231119864+09505119864+07138119864+08181119864+07760119864+07101119864+08

102119864+08

17WUH2

25411119864+07419119864+07176119864+08761119864+09897119864+07147119864+07134119864+07642119864+07521119864+07

110119864+07

ISRN Virology 13




137







5 Conclusion




Acknowledgments


References



14 ISRN Virology






























ISRN Virology 15













ISRN Virology 9

Lung

Brain

Kidney

Control CH-1a WUH2

(a)

Control CH-1a WUH2

(b)

Control CH-1a WUH2

(c)


10 ISRN Virology



Control

1 0 0 0 0 0 02 0 0 0 0 0 03 0 0 0 0 0 04 0 0 0 0 0 05 0 0 0 0 0 0

CH-1a

6 0 343119864 + 03 423119864 + 03 940119864 + 03 Killed7 0 258119864 + 03 594119864 + 03 271119864 + 04 Killed8 0 238119864 + 03 751119864 + 03 321119864 + 04 298119864 + 04 610119864 + 02

9 0 363119864 + 02 868119864 + 03 780119864 + 04 147119864 + 04 259119864 + 03

10 0 142119864 + 03 748119864 + 03 129119864 + 04 525119864 + 03 432119864 + 03

11 0 821119864 + 02 420119864 + 03 218119864 + 04 384119864 + 03 303119864 + 03

12 0 225119864 + 03 332119864 + 03 840119864 + 04 245119864 + 04 117119864 + 03

13 0 222119864 + 03 129119864 + 04 519119864 + 04 746119864 + 04 146119864 + 03

14 0 251119864 + 03 132119864 + 04 Killed15 0 263119864 + 03 179119864 + 04 Killed

WUH2


21 0 449119864 + 03 261119864 + 04 409119864 + 07 304119864 + 04 210119864 + 03



4 Discussion





ISRN Virology 11

Table3Viralloadin

selected

tissues

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

7CH

-1a

14203119864+03358119864+03712119864+03343119864+04101119864+04398119864+02143119864+02438119864+02826119864+03

955119864+03

15159119864+03702119864+03188119864+04924119864+04396119864+04221119864+03786119864+02562119864+02274119864+04

258119864+04

WUH2

34319119864+05541119864+05543119864+06105119864+08773119864+05639119864+06532119864+06144119864+05531119864+06

617119864+06

35352119864+05291119864+05219119864+06692119864+07437119864+05866119864+06218119864+06384119864+05459119864+06

431119864+06

10CH

-1a

6532119864+04555119864+04257119864+04102119864+05744119864+04262119864+03561119864+03203119864+04383119864+04

236119864+04

7125119864+04133119864+04585119864+04361119864+05118119864+04126119864+03201119864+03151119864+04579119864+04

712119864+04

WUH2

27576119864+06139119864+07617119864+07194119864+09107119864+08243119864+08788119864+06591119864+06385119864+08

941119864+08

29892119864+06319119864+07121119864+07992119864+08964119864+07631119864+08124119864+07422119864+06105119864+07

269119864+07

21CH

-1a

9568119864+04130119864+04369119864+05361119864+05463119864+04263119864+03124119864+03271119864+03566119864+04

793119864+04

12197119864+03226119864+05176119864+05359119864+05552119864+03298119864+03105119864+04193119864+04332119864+04

203119864+04

WUH2

30386119864+05545119864+05575119864+05846119864+06424119864+04263119864+05393119864+04323119864+04756119864+05

252119864+05

31988119864+05148119864+05167119864+05125119864+07495119864+03111119864+05841119864+04362119864+04122119864+06

966119864+05

12 ISRN Virology

Table4Viralloadin

selected

tissues

ofpigletthatdied

naturally

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

12WUH2

33707119864+08203119864+08391119864+09495119864+09272119864+08656119864+08154119864+08181119864+08216119864+08

185119864+08

14WUH2

26937119864+07146119864+08201119864+08215119864+09103119864+08187119864+08181119864+07986119864+06287119864+08

142119864+08

32697119864+08459119864+07125119864+08231119864+09505119864+07138119864+08181119864+07760119864+07101119864+08

102119864+08

17WUH2

25411119864+07419119864+07176119864+08761119864+09897119864+07147119864+07134119864+07642119864+07521119864+07

110119864+07

ISRN Virology 13




137







5 Conclusion




Acknowledgments


References



14 ISRN Virology






























ISRN Virology 15













10 ISRN Virology



Control

1 0 0 0 0 0 02 0 0 0 0 0 03 0 0 0 0 0 04 0 0 0 0 0 05 0 0 0 0 0 0

CH-1a

6 0 343119864 + 03 423119864 + 03 940119864 + 03 Killed7 0 258119864 + 03 594119864 + 03 271119864 + 04 Killed8 0 238119864 + 03 751119864 + 03 321119864 + 04 298119864 + 04 610119864 + 02

9 0 363119864 + 02 868119864 + 03 780119864 + 04 147119864 + 04 259119864 + 03

10 0 142119864 + 03 748119864 + 03 129119864 + 04 525119864 + 03 432119864 + 03

11 0 821119864 + 02 420119864 + 03 218119864 + 04 384119864 + 03 303119864 + 03

12 0 225119864 + 03 332119864 + 03 840119864 + 04 245119864 + 04 117119864 + 03

13 0 222119864 + 03 129119864 + 04 519119864 + 04 746119864 + 04 146119864 + 03

14 0 251119864 + 03 132119864 + 04 Killed15 0 263119864 + 03 179119864 + 04 Killed

WUH2


21 0 449119864 + 03 261119864 + 04 409119864 + 07 304119864 + 04 210119864 + 03



4 Discussion





ISRN Virology 11

Table3Viralloadin

selected

tissues

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

7CH

-1a

14203119864+03358119864+03712119864+03343119864+04101119864+04398119864+02143119864+02438119864+02826119864+03

955119864+03

15159119864+03702119864+03188119864+04924119864+04396119864+04221119864+03786119864+02562119864+02274119864+04

258119864+04

WUH2

34319119864+05541119864+05543119864+06105119864+08773119864+05639119864+06532119864+06144119864+05531119864+06

617119864+06

35352119864+05291119864+05219119864+06692119864+07437119864+05866119864+06218119864+06384119864+05459119864+06

431119864+06

10CH

-1a

6532119864+04555119864+04257119864+04102119864+05744119864+04262119864+03561119864+03203119864+04383119864+04

236119864+04

7125119864+04133119864+04585119864+04361119864+05118119864+04126119864+03201119864+03151119864+04579119864+04

712119864+04

WUH2

27576119864+06139119864+07617119864+07194119864+09107119864+08243119864+08788119864+06591119864+06385119864+08

941119864+08

29892119864+06319119864+07121119864+07992119864+08964119864+07631119864+08124119864+07422119864+06105119864+07

269119864+07

21CH

-1a

9568119864+04130119864+04369119864+05361119864+05463119864+04263119864+03124119864+03271119864+03566119864+04

793119864+04

12197119864+03226119864+05176119864+05359119864+05552119864+03298119864+03105119864+04193119864+04332119864+04

203119864+04

WUH2

30386119864+05545119864+05575119864+05846119864+06424119864+04263119864+05393119864+04323119864+04756119864+05

252119864+05

31988119864+05148119864+05167119864+05125119864+07495119864+03111119864+05841119864+04362119864+04122119864+06

966119864+05

12 ISRN Virology

Table4Viralloadin

selected

tissues

ofpigletthatdied

naturally

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

12WUH2

33707119864+08203119864+08391119864+09495119864+09272119864+08656119864+08154119864+08181119864+08216119864+08

185119864+08

14WUH2

26937119864+07146119864+08201119864+08215119864+09103119864+08187119864+08181119864+07986119864+06287119864+08

142119864+08

32697119864+08459119864+07125119864+08231119864+09505119864+07138119864+08181119864+07760119864+07101119864+08

102119864+08

17WUH2

25411119864+07419119864+07176119864+08761119864+09897119864+07147119864+07134119864+07642119864+07521119864+07

110119864+07

ISRN Virology 13




137







5 Conclusion




Acknowledgments


References



14 ISRN Virology






























ISRN Virology 15













ISRN Virology 11

Table3Viralloadin

selected

tissues

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

7CH

-1a

14203119864+03358119864+03712119864+03343119864+04101119864+04398119864+02143119864+02438119864+02826119864+03

955119864+03

15159119864+03702119864+03188119864+04924119864+04396119864+04221119864+03786119864+02562119864+02274119864+04

258119864+04

WUH2

34319119864+05541119864+05543119864+06105119864+08773119864+05639119864+06532119864+06144119864+05531119864+06

617119864+06

35352119864+05291119864+05219119864+06692119864+07437119864+05866119864+06218119864+06384119864+05459119864+06

431119864+06

10CH

-1a

6532119864+04555119864+04257119864+04102119864+05744119864+04262119864+03561119864+03203119864+04383119864+04

236119864+04

7125119864+04133119864+04585119864+04361119864+05118119864+04126119864+03201119864+03151119864+04579119864+04

712119864+04

WUH2

27576119864+06139119864+07617119864+07194119864+09107119864+08243119864+08788119864+06591119864+06385119864+08

941119864+08

29892119864+06319119864+07121119864+07992119864+08964119864+07631119864+08124119864+07422119864+06105119864+07

269119864+07

21CH

-1a

9568119864+04130119864+04369119864+05361119864+05463119864+04263119864+03124119864+03271119864+03566119864+04

793119864+04

12197119864+03226119864+05176119864+05359119864+05552119864+03298119864+03105119864+04193119864+04332119864+04

203119864+04

WUH2

30386119864+05545119864+05575119864+05846119864+06424119864+04263119864+05393119864+04323119864+04756119864+05

252119864+05

31988119864+05148119864+05167119864+05125119864+07495119864+03111119864+05841119864+04362119864+04122119864+06

966119864+05

12 ISRN Virology

Table4Viralloadin

selected

tissues

ofpigletthatdied

naturally

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

12WUH2

33707119864+08203119864+08391119864+09495119864+09272119864+08656119864+08154119864+08181119864+08216119864+08

185119864+08

14WUH2

26937119864+07146119864+08201119864+08215119864+09103119864+08187119864+08181119864+07986119864+06287119864+08

142119864+08

32697119864+08459119864+07125119864+08231119864+09505119864+07138119864+08181119864+07760119864+07101119864+08

102119864+08

17WUH2

25411119864+07419119864+07176119864+08761119864+09897119864+07147119864+07134119864+07642119864+07521119864+07

110119864+07

ISRN Virology 13




137







5 Conclusion




Acknowledgments


References



14 ISRN Virology






























ISRN Virology 15













12 ISRN Virology

Table4Viralloadin

selected

tissues

ofpigletthatdied

naturally

measuredby

quantitativeP

CR

DPI

Treatm

ent

AnimalID

number

Viralburdenin

tissues

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Stom

ach

Intestines

Subm

axillary

lymph

nodes

Ingu

inallymph

nodes

12WUH2

33707119864+08203119864+08391119864+09495119864+09272119864+08656119864+08154119864+08181119864+08216119864+08

185119864+08

14WUH2

26937119864+07146119864+08201119864+08215119864+09103119864+08187119864+08181119864+07986119864+06287119864+08

142119864+08

32697119864+08459119864+07125119864+08231119864+09505119864+07138119864+08181119864+07760119864+07101119864+08

102119864+08

17WUH2

25411119864+07419119864+07176119864+08761119864+09897119864+07147119864+07134119864+07642119864+07521119864+07

110119864+07

ISRN Virology 13




137







5 Conclusion




Acknowledgments


References



14 ISRN Virology






























ISRN Virology 15













ISRN Virology 13




137







5 Conclusion




Acknowledgments


References



14 ISRN Virology






























ISRN Virology 15













14 ISRN Virology






























ISRN Virology 15













ISRN Virology 15













Documents

Retractiondownloads.hindawi.com/journals/isrn/2014/624535.pdfISRNVirology VR-2332 TATGACGTATAGGTGTTGGCTCTATGCCTTGGCATTTGTATTGTCAGGAGCTGTGACCATTGGCACAGCCCAAAACTTGCTGCACAGAAACACCCTTCTG