design of primers for rt : we used clustalw   to align the orfs and utrs of the 124 na - type prrsv strains for which complete 
 genomic sequences were registered in public databases ( ddbj / embl / genbank ) ( table s1 and fig . 1.phylogenetic relationships of 124 na - type prrsv strains obtained from public 
 nucleotide databases ( ddbj / embl / genbank ) . the phylogenetic tree was constructed 
 according to the neighbor - joining method after using clustalw   to analyze the nucleotide sequences of orf5 of these 124 strains . strains were isolated from japan ( closed circle ) , china ( open 
 triangles ) , the united states ( open boxes ) and south korea ( open diamond ) . bootstrap trials were conducted 1,000 
 times , and values exceeding 90% are shown at nodes . ) . we then designed degenerate consensus rt primers within the conserved regions thus 
 identified ( table 1table 1.oligonucleotide primers used in this studyuseprimer namesequence ( 53)position ( nt)cort - mdaorf1a_r1cgtccaccggagyggctcttc368  348orf1a_r2tgccarccrcarttcccktc1,507  1,488orf1a_r3gcgartcaaacycacaagca4,152  4,133orf1a_r4ggcgtccargcatgycccat6,292  6,273orf1a_r5tcytcrtaraacmcrtcacc7,366  7,347orf1b_r1gccatrgarttcytrgcvgggta8,550  8,528orf1b_r2atgtcaaadacrtarcaatg10,407  10,388orf1b_r3tacacrtcygtyartgtrca11,475  11,456gp2_rgcggtacrtyckrcgcgacaccat12,413  12,390gp5_rtcbgcygaaactytggtta14,361  14,343m_rtgccacccaacacgaggc14,839  14,822rt - pcr5utr_fatgwcgtataggtgtkggctctatg1 253utr_racggycvccctaattgaatagg15,361  15,340primer positions are described according to the genomic sequence of the reference 
 strain edrd-1 ( ab288356.1 ) . ) . phylogenetic relationships of 124 na - type prrsv strains obtained from public 
 nucleotide databases ( ddbj / embl / genbank ) . the phylogenetic tree was constructed 
 according to the neighbor - joining method after using clustalw   to analyze the nucleotide sequences of orf5 of these 124 strains . strains were isolated from japan ( closed circle ) , china ( open 
 triangles ) , the united states ( open boxes ) and south korea ( open diamond ) . bootstrap trials were conducted 1,000 
 times , and values exceeding 90% are shown at nodes . primer positions are described according to the genomic sequence of the reference 
 strain edrd-1 ( ab288356.1 ) . preparation of genomic rna and cdna of viruses : viruses sequenced in the 
 current study were strains edrd-1 ( an na - type reference strain in japan [ 15 , 29 ] , cluster 
 iii ) , nagasaki 11 - 14 ( isolated in 2011 , cluster i ) , jam2 ( aomori 00 ; isolated in 2000 , 
 cluster ii ) , yamagata 10 - 7 ( isolated in 2010 , cluster iii ) and aomori 10 - 5 ( isolated in 
 2010 , cluster iii ) ( fig . 2fig . the 
 phylogenetic analysis was performed according to the neighbor - joining method by using 
 the nucleotide sequences of orf5 
 determined according to clustalw . bootstrap trials were conducted 1,000 
 times , and values exceeding 80% are shown at nodes . closed circles indicate the 
 strains sequenced by the cort - mda method in this study . ) . briefly , porcine alveolar macrophages and the monkey kidney cell 
 line marc-145 were used for virus propagation . propagated prrsv strains in supernatants of 
 the infected cell cultures were concentrated by sucrose density - gradient centrifugation 
 , and viral genomic rna was extracted with 
 trizol reagent ( life technologies , tokyo , japan ) . approximately 1 g of the 
 resulting rna was reverse - transcribed into first - strand cdna by using the consensus 
 oligonucleotide primers ( table 1 ) and 
 superscript iii kit ( life technologies ) according to the manufacturer s instructions . the 
 phylogenetic analysis was performed according to the neighbor - joining method by using 
 the nucleotide sequences of orf5 
 determined according to clustalw . bootstrap trials were conducted 1,000 
 times , and values exceeding 80% are shown at nodes . closed circles indicate the 
 strains sequenced by the cort - mda method in this study . amplification and fragmentation of viral cdna : a 4-l 
 aliquot of the first - strand viral cdna underwent multiple displacement amplification   by using a repli - g ultrafast mini kit ( qiagen , tokyo , 
 japan ) according to the manufacturer s instructions . a 10-l aliquot of the 
 20 l amplified dna product was digested with 1 unit s1 nuclease ( promega , 
 tokyo , japan ) in 50 l of 1 s1 nuclease buffer at 37c for 30 min . the 
 reaction was terminated by adding 1 l 0.3 m tris0.05 m edta ( ph 8.0 ) and 
 incubating for 10 min at 70c . the resultant dna was fragmented in a sonicator ( duty cycle , 
 constant ; output control , 4 ; sonifier 250 , branson ultrasonics , danbury , ct , u.s.a . ) for 5 
 sec . the fragmented dna solution then was desalted ( amicon ultra-0.5 ml 
 50k , merck millipore , billerica , ma , u.s.a . ) . the ends of the fragmented dna were repaired and blunted by using 5 l 10 
 t4 dna polymerase buffer ( life technologies ) , 12.5 l 2 mm dntp mixture and 
 10 units escherichia coli dna polymerase ( life technologies ) at room 
 temperature for 1 hr , followed by treatment with 5 units of t4 dna polymerase ( life 
 technologies ) at 37c for 5 min and at 75c for 20 min . a 25-l aliquot of 
 the blunted dna was treated with 1 unit of calf intestine alkaline phosphatase ( promega ) and 
 1 unit of shrimp alkaline phosphatase ( promega ) at 37c for 1 hr . approximately 1 to 2 kb of 
 the dephosphorylated fragments were size - selected by using 1% seakem gtg agarose ( cambrex , 
 rockland , me , u.s.a . ) and then extracted from the gel by using ultrafree da ( merck 
 millipore ) . chloroform extraction , precipitated 
 with ethanol   and resuspended in 10 
 l of nuclease - free h2o . shotgun sequencing : purified dna fragments were cloned into pcr4 
 blunt - topo ( life technologies ) according to the manufacturer s instructions , and 1 
 l of the resulting plasmid was introduced into dh10b ( life 
 technologies ) . details regarding electroporation conditions , plasmid extraction , sequencing 
 conditions and the sequence assembly method have been described previously [ 14 , 22 ] . briefly , 
 plasmid dna from 192 clones cultured in terrific broth   containing ampicillin was extracted and sequenced by using m13 - 40 ( 5- 
 gttttcccagtcacgacgttg 3 ) and m13-reverse ( 5- ggaaacagctatgaccatg 3 ) primers . the 
 chromatograms were basecalled by using phred [ 5 , 6 ] and assembled by using cap3 . evaluation of accuracy of the cort - mda method : we first evaluated the 
 cort - mda method by sequencing edrd-1 [ 15 , 29 ] . we obtained a contiguous sequence of 13,331 bp in 
 length , comprising 257 shotgun reads and covering more than 86% of the entire reported 
 edrd-1 genomic sequence ( genbank accession number , ab288356.1 ) . because the sequence thus 
 obtained lacked the 5- and 3-ends of the viral genome , we conducted pcr amplification 
 using the consensus rt primers and edrd-1-specific primers ( table s2 ) and sequenced the amplification products . 
 combining the data obtained by cort - mda shotgun sequencing with those from the pcr analysis 
 yielded 15,311 bp of genomic sequence ( excluding nucleotides corresponding to the consensus 
 rt primers at both ends of the genomic sequence ) ( ab811785.1 ) . the sequence of edrd-1 
 obtained by our cort - mda method was perfectly identical with that obtained by rt - pcr , which 
 required 43 primer pairs ( table 
 s3 ) . the edrd-1 virus sequenced in this study had 33 nucleotide 
 substitutions in comparison with the sequence registered in ddbj / embl / genbank ( ab288356.1 ) , 
 because of mutations acquired during in vitro passage ( table 3table 3.mutations revealed through cort - mda sequencing of the edrd-1 strain relative to 
 the reference sequence ( ab288356.1 ) registered in public databases 
 ( ddbj / embl / genbank)nucleotideregionamino acidpositionreferencecort - mdareferencecort - mda158tc5-utr248gaorf1aee856acorf1ada864ctorf1ahy1,866ctorf1all1,879tcorf1ava2,165tcorf1aaa2,682gaorf1aek3,246tcorf1asp3,850ctorf1asf3,994caorf1asy4,998gaorf1ags5,822ctorf1add6,043tgorf1alr6,983tcorf1all7,897tcorf1bss7,962atorf1bhl8,033gaorf1bvi8,368gaorf1bkk8,650ctorf1bvv9,886ctorf1bgg11,104tcorf1bhh11,305gaorf1baa11,308ctorf1bss12,594taorf2yn13,368*ctorf3vvorf4sf13,746ctorf4ff13,837ctorf5ii13,874gaorf5dn13,996gaorf5ll14,598gaorf6vv15,059tgorf7ydpositions are indicated according to those in the reference sequence . * position 
 13,368 belongs to both orf3 and orf4 ; the corresponding respective amino acids are 
 indicated . ) . in addition , we found 2 subpopulations that 
 had a and g , respectively , at nucleotide position 13,850 ( ab811785.1 ) in the edrd-1 virus 
 sequenced here . the mutated locations and nucleotides , including position 13,850 , were 
 identical between those obtained by the cort - mda method and rt - pcr ( data not shown ) , 
 demonstrating the accuracy of the sequence by the cort - mda method and its potential 
 application to detecting ongoing mutations in the viral genome . * position 
 13,368 belongs to both orf3 and orf4 ; the corresponding respective amino acids are 
 indicated . applicability of the cort - mda method to various prrsv strains : next , we 
 evaluated the applicability of the cort - mda method by sequencing 4 prrsv field strains 
 belonging to various clusters classified according to their orf5 sequences [ 11 , 28 ] . viruses 
 nagasaki 11 - 14 ( isolated in 2011 , cluster i ) , jam2 ( aomori 00 isolated in 2000 , cluster ii ) , 
 yamagata 10 - 7 ( isolated in 2010 , cluster iii ) and aomori 10 - 5 ( isolated in 2010 , cluster 
 iii ) underwent sequencing by the cort - mda method ( fig . 
 2 ) . more than 78% of the 384 shotgun sequencing reads were available for assembly 
 ( data not shown ) ; this assembly generated either 1 ( jam2 and yamagata 10 - 7 ) or 2 ( aomori 
 10 - 5 and nagasaki 11 - 14 ) major contigs for each isolate and covered an estimated 80% to 94% 
 of the entire prrsv genome ( table 2table 2.summary of the assemblies of shotgun sequencing reads generated by the cort - mda 
 methodstrainnumber of readslength ( bp)position ( nt)*edrd-125713,3311,831  15,161nagasaki 11 - 14877,081524  7,6051216,4178,712  15,128jam223312,3582,319  14,677yamagata 10 - 727714,458597  15,054aomori 10 - 51849,400964 10,370272,98810,616  13,606*positions of the contigs are indicated relative to the reference sequence edrd-1 
 ( ab288356.1 ) . ) . sequencing 
 of gaps between contigs and unread regions in the 5- and 3-utrs by pcr provided contiguous 
 genomic sequences of 15,313 bp for nagasaki 11 - 14 ( ab811786.1 ) , 15,306 bp for jam2 
 ( ab811787.1 ) , 15,308 bp for yamagata 10 - 7 ( ab811788.1 ) and 15,286 bp for aomori 10 - 5 
 ( ab811789.1 ) ( excluding pcr primer sequences at both ends of the genomic sequences ) . dot - plot analyses by pipmaker   of the 4 viral 
 genome sequences thus obtained against the edrd-1 genome indicated that there were no 
 inversions or low - similarity regions between the compared sequences ( fig . s1 ) . the data shown here demonstrate the robustness and 
 applicability of the method for sequencing of na - type prrsvs in various clusters . * positions of the contigs are indicated relative to the reference sequence edrd-1 
 ( ab288356.1 ) . the cort - mda method for determining the genomic sequences of na - type prrsv isolates had 
 several advantages in comparison with other sequencing methods . the cort - mda method requires 
 far fewer oligonucleotide primers for amplification and sequencing of the prrsv genomes . previous methods require numerous strain - specific primers for step - by - step primer walking 
 [ 16 , 19 , 
 29 ] . the cort - mda method provides a simplified 
 sequencing procedure involving only 11 consensus primers , which can be used for strains from 
 multiple clusters . if unread regions remain after sequencing by the cort - mda method , only a 
 few additional strain - specific primers are required to obtain the entire genomic sequence . laborious iteration of sequencing is not required . the prrsv genomic sequence that we obtained by using the cort - mda method was highly 
 accurate . our method precisely detected mutations in the edrd-1 genome that were generated 
 during passage , and these mutations were confirmed by the conventional pcr - based sequencing 
 method . in this regard , our cort - mda method was able to detect ongoing mutations in the 
 prrsv genome , indicating that the method can be applicable to tracing prrsv genomes , which 
 are unstable and accumulate numerous mutations during a limited number of generations [ 8 , 26 , 30 ] . most importantly , the cort - mda method is robust and versatile regardless of the genetic 
 background of the na - type prrsv to be sequenced . coding sequences of the prrsv genomes 
 determined by the cort - mda method in this study showed identities from 84.0% ( jam2 vs. 
 yamagata 10 - 7 ) to 88.8% ( edrd-1 vs. aomori 10 - 5 ) , demonstrating that the method is 
 applicable for sequencing viral genomes that are more than 10% divergent at the nucleotide 
 level . the consensus primers for our method were designed by using the 124 full - length 
 genomic sequences of na - type viruses , including isolates derived from the united states , 
 china , korea and japan and several strains known to be hp - prrsvs . therefore , our method 
 likely can be used to determine the genomic sequence of any na - type prrsv virus strain , 
 regardless of its country of origin and even if the strain is newly emerged . our rapid , 
 convenient and robust method for the sequencing of prrsv genomes will help greatly to 
 elucidate the genetic factors that affect the pathogenicity of prrsvs by enabling detailed 
 comparisons between the genomic sequences and virulence of different strains . the cort - mda method presented here includes a step for purification by ultracentrifugation 
 through a sucrose gradient . however , if the ultracentrifugation step is dispensable , the 
 method will obtain broader applicability , particularly in the case of samples derived from 
 the field . samples collected in the field or from piggeries under epidemic conditions of 
 prrs  such as viremic plasma from pigs reared in conventional environments  are often 
 contaminated with rna derived from other microorganisms and host animals . in the cort - mda 
 method , the prrsv genome is converted into cdna , which is much more stable during 
 transportation to laboratories than is the rna genome and can be amplified to the amount 
 appropriate for shotgun sequencing . furthermore , because of selective amplification of viral 
 cdna by using prrsv - specific consensus primers , the cort - mda method can be modified for rna 
 samples that are not highly purified . further improvement of the cort - mda method may reduce 
 the need for complicated sample - handling measures at the often insufficiently equipped 
 facilities near epidemic areas . we conducted shotgun sequencing of the amplified viral cdnas according to the sanger 
 method . the method thus presented can be conducted with traditional sequencers and computers 
 without high specification . however , it is worth considering the use of next - generation 
 sequencing technology , which is becoming popular and is potentially more cost - effective than 
 is our current method . in addition , establishment of a cort - mda  based sequencing method for 
 eu - type prrsv strains is worth considering . a random pcr cloning approach that generated 
 partial genomic sequences for an eu - type prrsv strain with an unknown genetic background was 
 reported recently . however , the genomic sequence 
 ( before filling of gaps ) obtained by using the random pcr cloning method was less than half 
 of the entire prrsv genome , and the method s robustness in regard to viruses derived from 
 various clusters has not been well confirmed . development of the cort - mda - based sequencing 
 method for eu - type strains will be beneficial for dissecting prrs outbreaks , because na- and 
 eu - type strains cause analogous clinical signs   and 
 can be epidemic simultaneously in the same location . regardless of the type or cluster to 
 which the strain belongs , cort - mda  based methodology will facilitate rapid identification of 
 the prevailing prrsv isolate on an affected farm .
abstractwe developed a concise and 
 broadly applicable method for accurate genomic sequencing of north american genotype 
 ( na - type ) porcine reproductive and respiratory syndrome viruses ( prrsvs ) that overcomes 
 high genetic variability of the viruses . 
 the method , designated 
 combination of consensus 
 oligonucleotide reverse transcription and multiple displacement amplification 
 ( cort - mda ) , 
 involves reverse - transcription of viral rna followed by shotgun sequencing after 
 amplification using only 11 degenerate oligonucleotide primers ; these primers were 
 designed against consensus regions within the open reading frames of the 124 na - type prrsv 
 strains with reported full - length genomic sequences . 
 sequencing of the 192 shotgun clones 
 generated per virus showed 80% to 94% coverage on the reported prrsv genomic sequence , 
 such that only 2 or 3 unread regions had to be resequenced after pcr amplification using 
 custom primers . 
 direct sequencing of rt - pcr products confirmed absolute consistency 
 between sequences determined by the cort - mda method and those from rt - pcr . 
 these results 
 suggest that our method is applicable to diverse na - type viruses .