A Wide Extent of Inter-Strain Diversity in Virulent and Vaccine Strains of Alphaherpesviruses
[['Alphaherpesviruses are widespread in the human population , with herpes simplex virus 1 ( HSV1 ) and 2 causing oral and genital lesions , respectively , while varicella zoster virus ( VZV ) causes chicken pox and shingles [1]–[3] .', 'In the agricultural industry , a related veterinary alphaherpesvirus , pseudorabies virus ( PRV ) , causes similar disease in swine and significant economic cost due to weight loss in infected adults and reproductive losses during pregnancy and suckling [4] , [5] .', 'As occurs with HSV and VZV , PRV infection has higher morbidity and mortality rates for neonates , with decreasing severity of disease as the age at onset of infection increases [2] , [4] , [6] .', 'PRV and VZV primarily infect via the respiratory mucosa , while HSV-1 primarily infects at the oral mucosa .', 'VZV infection includes a viremic phase that yields widespread vesicular lesions , while PRV and HSV are usually non-viremic and spread predominantly by mucosal infection and neuronal innervation .', 'These alphaherpesviruses are widespread in the population because of their tendency to infect neurons: they establish lifelong latency in the host peripheral nervous system .', 'These latent neuronal infections may occasionally reactivate and spread back the mucosal surfaces where the infection initiated .', 'After further replication , the viruses can spread to new hosts .', 'Among alphaherpesviruses , vaccines are available for VZV and PRV , but not HSV [7] , [8] .', 'Despite considerable effort and recent progress , no broadly effective vaccine candidates have yet emerged for HSV infection [9]–[11] .', 'The co-morbidities of HSV-1 and HSV-2 with human immunodeficiency virus ( HIV ) , which include increased acquisition of HIV due to the inflammation and lesions caused by HSV infection , have added impetus to the search for a vaccine [10]–[13] .', 'PRV serves as a useful model for HSV pathogenesis and vaccine development , because of their similar infectious cycle and ability to infect a variety of animal models [4] , [5] , [8] , [14]–[17] .', 'In contrast , VZV has a more restricted tropism for human cells that complicates its study in animal models [18]–[20] .', 'The agricultural importance of PRV and relative ease of vaccine testing has led to the development of several PRV vaccine strains , whose genetic characteristics have been determined by mapping isolated genomic fragments and sequencing of select regions [8] , [21]–[23] .', 'Of note , the vaccine strain Bartha has a well-characterized deletion of several viral proteins that attenuates its virulence and also limits its spread in neurons , which led to its subsequent development as a tool for trans-neuronal tracing [21] , [24]–[27] .', 'Like several other early vaccine strains , PRV Bartha was attenuated by extensive passage in the laboratory , thus making the full discovery of its genome-wide mutations a priority [22] , [23] , [28] , [29] .', 'Because the only available PRV genome sequence to date is a mosaic of six strains [30] , it has been difficult to discern whether mutations detected in PRV Bartha and other vaccine strains are unique or represent ordinary sequence diversity , i . e . are found in other wild-type genomes [31]–[35] .', 'We therefore applied our recent success in using Illumina high-throughput sequencing ( HTS ) to obtain HSV-1 strain genomes to determining the sequence diversity in the PRV vaccine strain Bartha .', 'In addition to sequence polymorphisms , insertions , and deletions , another major class of variation between nucleic acid sequences lies in copy number variation , either of coding sequences or of repeated structural elements .', 'Herpesvirus genomes have long been known to contain several sites with tandem short sequence repeats ( SSRs ) or reiterations [36]–[40] .', 'Variation in these elements has been described both within and between herpesvirus strains , but their functions were largely unexplored [22] , [35] , [41]–[43] .', 'SSRs can be transcription factor binding sites , chromatin insulators , protein folding motifs , or other regulatory elements [44] , [45] .', 'Recent studies have shown that SSR expansion and contraction , most likely through recombination or polymerase slippage , can generate phenotypic variation [46]–[49] .', "A range of human diseases result from SSR expansion or contraction , including the transcriptional silencing of the gene FMR1 via an upstream SSR , which causes Fragile X syndrome , and the poly-glutamine tract expansion in huntingtin protein , which causes Huntington's disease [50]–[53] .", 'Limited explorations of repetitive elements in viral genomes suggest that SSRs in viral genomes likewise play functional roles [54]–[57] .', 'To explore SSR prevalence and function in herpesviruses , we initiated a global SSR assessment and comparison across viral species , as was recently done for a variety of fungal and bacterial pathogens [49] , [58] .', 'These data highlight the contribution of SSRs to overall sequence diversity in viruses , and through the presence of these elements in both coding and non-coding regions , suggest that viral SSRs may likewise have the potential to affect gene expression and protein functions .', 'We sequenced three widely-studied PRV isolates by HTS: the attenuated vaccine strain Bartha and the virulent strains Kaplan and Becker .', 'This analysis reveals genome-wide sequence diversity between strains , both in the PRV proteome and also in many SSRs .', 'Our comparison of protein coding sequences revealed that 46 of 67 PRV proteins have changes in the vaccine strain Bartha which are not found in the virulent Kaplan or Becker strains .', 'We mapped homologous SSRs in all three strains and provide a comprehensive overview of inter-strain variation in SSR length .', "We compared the proportion of SSRs in PRV to those found in HSV-1 , VZV , the human betaherpesvirus cytomegalovirus ( HCMV ) and gammaherpesviruses Epstein-Barr virus ( EBV ) and Kaposi's sarcoma-associated herpesvirus ( KSHV ) , and Mimivirus .", 'We find that SSRs are likely to be a common property of these large DNA viruses .', 'Finally , we examined the limited number of polymorphic bases detected in these plaque-purified virus stocks , and tested the rate of polymorphism occurrence in purified and non-purified virus populations .', 'These data on sequence variation in PRV strains expand our understanding of viral genome diversity and how attenuated strains lead to successful anti-viral vaccines .'], ['We used Illumina deep sequencing and bioinformatic analyses to assemble millions of sequence reads into three completed genomes of PRV Kaplan , Becker , and Bartha .', 'To produce genetically homogeneous stocks for sequencing , we purified a single plaque from each virus stock , plated it out , selected a progeny plaque , and repeated the process .', 'These plaque-purified stocks were then used to produce viral nucleocapsid DNA for Illumina genomic DNA libraries .', 'Over 15 million Illumina sequence reads were combined for each strain ( details of HTS sequence reads for each strain are listed in Table S1 in Text S1 ) .', 'High quality viral sequence data were used for a 3-phase de novo assembly process ( see Methods for details ) :', '1 ) the automated generation of large blocks of continuous sequence , or contigs , from Illumina sequence data ( usually 0 . 1–30 kilobase pairs ( kb ) in length ) ,', '2 ) the automated generation of super-contigs ( 1–60 kb ) using a long-read assembler , and', '3 ) the manual curation of gaps , joins , and annotations .', 'Assembly quality was checked by BLAST-based alignment of each new genome versus the prior mosaic reference .', 'PCR-validation confirmed regions of the assembly with greatest divergence from the mosaic strain , and guided genome correction in selected regions of the assembly ( Figure S1 and Table S2 in Text S1 ) .', 'The resulting genomes resembled the original mosaic genome in overall size and gene content ( Figure 1A ) .', 'The PRV genome is organized into a unique long ( UL ) region and a unique short ( US ) region , with large inverted and terminal repeats ( IR , TR ) flanking the US region .', 'Overall , DNA sequences are largely conserved between PRV Kaplan , Becker , and Bartha , with the greatest foci of divergence occurring in IR/TR and noncoding regions ( Figure 1B ) .', 'Phylogenetic comparison of the three full-length genomes revealed a closer relationship between PRV strains Kaplan and Bartha than PRV Becker ( Figure 1D ) .', 'To ascertain the quality and depth of coverage of these new genomes , sequence reads were aligned back to the assembled genomes .', 'Median coverage was very high: 3 , 704 sequence reads per base for PRV Kaplan , 4 , 145 reads/base for Becker , and 4 , 137 reads/base for Bartha ( see also Table S1 in Text S1 ) .', 'This coverage was reduced in genome regions with extremely high or low G/C content , as has been observed for both eukaryotic and bacterial genomes ( Figure S2A , B in Text S1 ) [59] , [60] .', 'In addition to analyzing coverage depth , the resulting genomes were used to predict restriction digest patterns , which were compared to actual restriction fragment length polymorphism ( RFLP ) patterns ( Figure 2 ) .', 'Digest patterns match the predicted fragment sizes , with the exception of two classically variable fragments ( BamHI 10 and 12; Figure', '2 ) that have been observed to differ even between repeated passages of the same strain [22] , [41] , [42] .', 'PRV Bartha displays the most divergent phenotype of the PRV strains sequenced here , with severe attenuation of virulence in vivo conferring its suitability for use as a vaccine strain .', 'We compared all protein coding regions of PRV Bartha and the two wild-type strains PRV Kaplan and Becker , to search for novel sequence differences corresponding to potential effects on pathogenicity and attenuation of the vaccine strain ( Tables 1–3 ) .', 'Prior studies mapped a deletion in the Bartha US region that removes all of gE ( US8 ) and US9 and creates an fusion of gI ( US7 ) and US2 , as well as subtle variations in gC ( UL44 ) , gM ( UL10 ) , and UL21 [21] , [28] , [31] , [61]–[65] .', 'Our de novo assembled Bartha genome confirms the boundary of the US region deletion ( position 120 , 927 on the Bartha genome ) as originally mapped by Maxam-Gilbert sequencing [66]; this region spans 3 , 482 bases on the reference PRV Kaplan genome ( positions 120 , 363–123 , 845; see also Figure 1B ) .', 'Adding to these previously reported findings , we identified a total of 46 proteins with coding differences that are unique to PRV Bartha and not found in either wild-type strain ( Table 1 and Figure 3 ) .', "Several of these amino acid ( AA ) changes are conservative , such as a minor Ala13Val change in Bartha's VP18 . 8 ( UL13 ) , or represent expansions or contractions associated with AA repeats ( e . g . VP1/2/UL36 , ICP4/IE180 , AN/UL12 ) .", "Many mutations affect loosely mapped functional protein domains , for instance two differences in the 300 AA chemokine-binding domain of Bartha's gG [67] .", 'Further studies will be necessary to define any functional effects in these regions .', "Several unique Bartha mutations are located within functional domains of proteins not previously considered to affect Bartha's virulence and spread phenotypes , including gH ( UL22 ) , gB ( UL27 ) , and gN ( UL40 . 5 ) .", 'The core fusion process of most alphaherpesviruses consists of receptor binding via gD ( US6 ) , followed by fusion mediated by gB ( UL27 ) and the gH-gL ( UL1 ) heterodimer .', 'PRV gH has recently been crystallized , as have the homologous gH proteins of HSV-2 and Epstein-Barr virus ( EBV ) [68]-[70] .', 'PRV Bartha has a Pro438Ser change in gH .', 'In the recent crystal structure of PRV gH , this proline was highlighted as a key residue , because it mediates a bend at the end of an alpha helix in the gH core ( domain III ) , which is necessary to allow one of four disulfide bonds in the protein [70] .', 'This proline and the neighboring disulfide-bonded cysteine are absolutely conserved across all known herpesvirus sequences , including the evolutionarily distant beta- and gamma -herpesviruses [70] .', 'In Western blot analysis of infected cell lysates ( Figure 4 ) , PRV Bartha produces two bands of gH protein that are comparable to those of the PRV Kaplan and Becker strains .', 'There is no obvious difference in gH produced by these PRV strains .', "We also detected three changes to the key fusion protein gB ( UL27 ) coding sequence in PRV Bartha , which affect several residues immediately adjacent to gB's furin cleavage site ( Ser506Ala , Pro507Ala , and Pro509Gln ) .", 'Furin cleavage of gB has been shown to affect cell-cell spread of PRV and in vivo virulence of VZV [71] , [72] .', 'Transfer of just 11 AAs surrounding this furin cleavage site , corresponding to residues 497–507 of the PRV Kaplan gB sequence ( PAAARRARRSP ) , are sufficient to confer protease-cleavage when inserted into PRV gC [73] .', 'As noted previously [31] , gB is still cleaved in PRV Bartha-infected cells in vitro ( Figure S3 in Text S1 ) , but it is unknown whether these changes in gB affect cleavage efficiency or other aspects of gB function in specialized cell types such as neurons .', 'Finally , PRV Bartha has a Leu7Pro alteration in the signal sequence of gN ( UL49 . 5 ) that may affect glycoprotein processing and/or packaging [62] , [74] .', "A previously detected Leu14Pro difference in Bartha's gC also affects the signal sequence , leading to inefficient maturation of gC , and reduced incorporation of gC into virions [62] .", 'PRV gN is normally packaged into virions and affects the rate of virion penetration into cells [74] , [75] .', 'If this signal sequence mutation affects gN maturation or virion inclusion in a parallel way to that of the gC signal sequence mutation , it may well contribute to the delayed penetration kinetics and cell-to-cell spread phenotype of the attenuated PRV Bartha vaccine strain .', 'The genomes of alphaherpesviruses have long been thought to be quite stable with limited sequence variation among strains [76] , [77] .', 'This idea was well supported when the genome-wide comparison of 18 VZV strains revealed inter-strain coding variation of 1% or less [78] , [79] .', 'The four HSV-1 genome sequences available show modestly increased inter-strain protein-coding variation [80]-[83] .', 'Surprisingly , we find that protein coding variation between PRV strains is higher than that observed for either HSV-1 or VZV ( average of 1 . 6% for PRV , vs . 1 . 3% for HSV-1 or 0 . 2% for VZV; Figure 5 and Table S6 ) [78] , [81] .', 'When the coding sequences for each protein of these three new PRV genomes are compared , the inter-strain variation in AA sequence ( number of AA residues varying between strains , normalized for protein length ) reaches as high as 13% .', 'Starting on the low end of variation , we found eight invariant proteins across these PRV strains ( Figure 3 ) , including the viral DNA polymerase UL30 , the minor capsid proteins VP19c ( UL38 ) and VP23 ( UL18 ) , the nuclear egress components UL20 , UL31 , and UL37 , and the functionally uncharacterized proteins UL24 and UL56 ( ORF-1 ) .', 'In contrast , ICP22 ( US1 ) displays 13% inter-strain variation; this protein has transactivating and regulatory functions in related alphaherpesviruses [84] , [85] , but has only been studied at the level of transcript expression in PRV [86] , [87] .', 'In a similar comparison of AA sequence differences between 3 strains of HSV-1 , the inter-strain variation peaked at 7% ( for ICP34 . 5 ( RL1 ) and US11; Table S6 ) [81] .', 'VZV strains show even less variation in protein coding sequences , with a maximum of 1 . 2% AA variation ( in ORF-1 ) between strains , and just two additional proteins with variation greater than 0 . 5% [78] .', 'One of these two VZV proteins is ORF 62/71 , which is homologous to PRV IE180 and HSV-1 ICP4; this protein is among the most variable across all known strains of these alphaherpesviruses .', 'IE180 is the sole gene expressed with immediate-early kinetics in PRV , and is a key transactivator of viral gene expression [88]–[90] .', 'In contrast , the nuclear egress proteins UL20 and UL31 thus far shows no inter-strain variation in all known genomes of PRV and HSV-1 , while UL31 shows zero coding variation in VZV as well .', 'A comparison of the inter-strain variation in homologous proteins of PRV and HSV-1 ( Figure 5 and Table S6 ) highlights several proteins that appear to vary more substantially in one virus than the other .', 'Although ICP22 is the most variable protein in PRV , it is completely invariant among HSV-1 strains 17 , F , and H129 , as well among the previously described 18 strains of VZV [78] , [79] , [81] .', 'Likewise , the viral egress protein VP13/14 ( UL47 ) is among the most variant in PRV , but it is well-conserved in HSV-1 , while the opposite is true for HSV-1 proteins uracil-DNA glycosylase UNG ( UL2 ) and the ubiquitin E3 ligase ICP0 ( EP0 ) ( Figure 5 , orange vs . green highlighting ) .', "Several proteins , which do not have homologs between HSV-1 and PRV , are also highly variable; these include PRV's viral egress protein UL3 . 5 , which has the third-highest variability of PRV proteins after ICP22 and the tegument protein VP22 ( UL49 ) , and the two most variable HSV-specific proteins , which are the neurovirulence-associated protein ICP34 . 5 ( RL1 ) and the PKR-antagonist US11 .", 'SSRs are widespread in eukaryotic genomes , and mediate functional effects by serving as DNA-binding domains in promoters , protein folding motifs in coding sequences , and sites of inter-molecular recombination [44]–[47] .', 'Since AA repeats generated several examples of inter-strain coding diversity above ( Tables 1–3 ) , we investigated the prevalence of SSRs in the PRV genome .', 'SSRs are generally grouped into three main categories: homopolymers , which include a short run of the same base; microsatellites , where the repeating unit is less than 10 bases; and minisatellites , which have a repeating unit of 10–500 bases [44] , [45] .', 'The initial description of the PRV genome mapped 26 minisatellite SSRs using a DNA identity scoring matrix [30] .', 'Using software designed to identify all size classes of SSRs and include both perfect and imperfect repeats ( see Methods for details ) , we detected a significantly larger number of repeats , a total of 953 distributed across the PRV Kaplan genome ( Table 4 and Table S7; minimum homopolymer length 6 ) .', 'SSRs in PRV occur in both coding and non-coding regions , promoters and open intergenic space , with similar proportions in all three PRV strains ( Table 5 and Figure 6A ) .', 'SSRs of all size classes are distributed throughout the genome , with a slightly higher accumulation of all types in the IR-US-TR region ( Figure 1C and Figure S4 in Text S1 ) .', 'The majority of all SSRs in PRV ( 62% ) contain triplet-based repeats ( e . g . the repeat unit is a 3-mer , 9-mer , 21-mer , etc . ) .', 'Likewise , 69% of homopolymers have a triplet-based length .', 'Half of all SSRs are in coding sequences ( 474/953 ) , and these are largely triplet-based ( 72% ) .', 'Triplet-based repeats , as well as insertions or deletions ( indels ) and partial repeat units of non-triplet-SSRs , help preserve the coding content in the SSR-laden PRV genome because variation in these repeats ( addition or removal of repeat units ) does not change the reading frame of the downstream sequence .', 'All coding sequences , except the small UL11 gene , contain SSRs ( Figure 1C ) .', 'However it is interesting to note that nineteen genes are free of homopolymers , a size class where expansion or contraction of the SSR is likely to disrupt the reading frame ( Table S7 ) .', 'Likewise another 20 genes have regions of at least 1 kb that are homopolymer-free .', 'For instance , the large tegument protein VP1/2 ( UL36; 9 . 2 kb in length ) has no homopolymers in its initial 5 . 5 kb ( Figure 1A , C ) , which contains several domains affecting capsid transport , replication , and neuroinvasion [91]–[95] .', "In contrast , VP1/2's homopolymer-rich C-terminal region has been previously shown to be dispensable for viral replication [96] .", 'Of the 25 core genes found across multiple families of Herpesviridae that are essential for growth in cell culture [76] , 18 have no homopolymers or regions >1 kb that are homopolymer-free .', 'As additional sequences become available for phylogenetic comparison , it may be possible to determine whether this is a chance occurrence or the result of purifying selection .', 'Since SSRs have not been comprehensively examined in other DNA virus families , we extended these analyses to include the genomes of a wide variety of human herpesviruses , including HSV-1 , VZV , HCMV , EBV , and KSHV ( Table 4 and Figure 6B–D ) .', 'To ascertain if these results hold for non-nuclear , non-mammalian viruses , we selected as an outgroup for comparison the nucleocytoplasmic large DNA virus Mimivirus , which infects pathogenic amoebae ( Figure 6E ) [97] .', 'PRV has the highest overall SSR burden , with short repeats encompassing 18% of the genome , which is roughly double the proportion found in HSV-1 , EBV , and Mimivirus , and 5–6 times that of VZV , HCMV or KSHV .', 'In all of these viruses , more than half the SSRs fall into coding regions ( Figure 6 ) , creating potential effects on protein structure if these SSRs vary in length between strains .', 'SSRs also occupy a noticeable fraction of the intergenic and promoter regions in PRV and other genomes ( Figure 6 ) .', 'For those genomes with a biased nucleotide content , the bias is exaggerated in SSRs ( Table 4 ) .', "PRV's overall genome is 74% G/C , but this level is 79% when all SSR sequences are pooled together .", 'This is similar in HSV-1 ( 68% G/C overall; 84% in SSRs ) and EBV ( 59% G/C overall; 77% in SSRs ) , and mirrored in reverse in the A/T-rich genome of Mimivirus ( 72% A/T overall; 80% in SSRs ) .', 'PRV thus provides a rich set of SSRs for analysis of a phenomenon that extends to many other viruses .', 'Previous work in yeast , humans , and other organisms has demonstrated that variation in SSR length , either between individuals or during evolutionary adaptation , can result in phenotypic effects [47]–[50] .', 'Although the overall proportions of SSRs are similar in the PRV Kaplan , Becker , and Bartha genomes ( Figure 6A ) , a comparison across PRV strains revealed that homologous SSRs vary in length between strains ( Table 5 ) .', 'Previously , variation in a selection of microsatellites ( ≤6 bases in length ) has been shown for HSV-1 , HCMV , and HIV [98]–[100] , but the genome-wide complement of all SSR types has not been analyzed .', 'The comparison of homologous SSRs reveals that not all SSRs can be recognized in all three strains ( e . g . SSRKa151 , SSRKa2093 , and SSRKa62103 in Table 5 ) .', 'However the majority of those that do occur in all strains vary in the number of repeating units ( of 861 SSRs found in all three strains , 539 vary in number of repeating units ) .', 'If these SSRs contain transcription factor binding sites or occur in protein coding regions , then these inter-strain differences in SSR copy number may influence gene expression or protein folding domains , and thus lead to phenotypic differences between strains .', 'One of the best characterized biological roles for SSRs in herpesviruses are the CCCTC-binding factor ( CTCF ) binding sites that flank latency-associated transcripts in the genomes of HSV-1 and the gammaherpesviruses EBV and KSHV [101]–[108] .', 'In each of these cases , CTCF binds to motifs within SSRs found near loci that are transcriptionally active during latency; this interaction is proposed to have chromatin insulating and/or silencing effects that maintain a repressed state in flanking genes .', 'CTCF-binding sites occur in several additional conserved locations throughout alphaherpesvirus genomes , as shown by Amelio et al . in a comparison that included HSV-1 , VZV , and PRV [104] .', 'Because many PRV SSRs showed inter-strain variation in copy number or length , we investigated CTCF-binding sites in PRV Kaplan , Becker , and Bartha .', 'Of the 17 CTCF binding sites mapped by Amelio et al , 12 were mapped as falling into SSRs in our inter-strain comparison ( Table 5; CTCF-binding sites in the repeat-unit consensus are underlined and in bold ) .', 'All of these vary in repeat-unit length between strains ( e . g . Table 5: SSRKa31884 , SSRKa115550 ) .', 'Although several have diverged enough to be listed as separate SSRs , their overall location and CTCF-binding ability are preserved ( e . g . Table 5: SSRKa115377 and SSRBe115911; see Table S7 for orthologous SSRBa115943 ) .', 'The greatest inter-strain variation in SSR length occurs at SSRKa15795 , between UL46 and gB ( UL27 ) , where PRV Becker has three times as many repeating-units as either PRV Kaplan or Bartha .', 'This SSR contains both CTCF-binding sites and a non-canonical Egr1/2 binding site , both of which have repressive effects on expression of nearby genes in HSV-1 [57] , [104] , [109]–[111] .', 'Initial studies show that gB levels in PRV Becker-infected lysates do not appear significantly lower than those in PRV Kaplan or Bartha ( Figure S3 in Text S1 ) .', 'Further work will be required to determine if the flanking SSR length affects gB expression and function .', 'In the only previous publication comparing full-length genomes of HSV-1 ( strains 17 , H129 , and F ) , the length of fourteen major SSRs throughout the genome were not determined and were instead set to match the reference genome length [81] .', 'These fourteen SSRs , classically termed reiterations in the HSV literature [37] , [38] , [82] , [83] , correspond to the fourteen CTCCC-domain-containing SSRs defined by Amelio et al . [104] .', 'To discern if inter-strain variation such as that observed in the PRV genomes is found in HSV-1 as well , we PCR amplified and sequenced two of these SSRs from the HSV-1 strains F and H129 .', 'Both SSRs displayed inter-strain variation in copy number , with the reference strain 17 ( GenBank Accession NC_001806 ) having more SSR units at both sites than either the clinical isolate H129 or the laboratory strain F ( IRS reiteration 3 [CTRS3 in Amelio et al . ]: 6 . 5 copies in strain 17 , 4 . 7 copies in H129 , 1 . 7 copies in F; US reiteration 1 [CTUS1 in Amelio et al . ]: 10 copies in strain 17 , 2 copies in H129 , 2 copies in F ) .', 'These data suggest that inter-strain variation in SSR length may affect CTCF-binding efficiency in HSV-1 and could contribute to inter-strain differences in related phenotypes .', 'Annotation of SSRs in the draft PRV genome assemblies had revealed several discrete areas in each genome where peaks of very high coverage coincided exactly with perfect SSRs: for example a peak of over 100 , 000-fold coverage around an SSR at position 15 , 600 in the PRV Becker genome ( Figure S1 in Text S1 and Table 5 ) .', 'This very high coverage ( >2 standard deviations above the median ) occurred at three SSR sites in PRV Kaplan , three SSRs in PRV Becker , and four SSRs in PRV Bartha .', '( Figure S1 and Table S3 in Text S1 , also noted in Table 5 ) .', 'De novo assembly methods cannot distinguish whether repeated sequence reads originate from perfect , extended copies of an SSR unit , or from additional coverage depth of a single unit , and the software therefore creates a final assembly with the minimal number of repeating units supported by the data [112] .', 'In fact , the high coverage peak in PRV Becker coincides with the largest SSR array of perfect repeats in the original mosaic PRV genome , which had 39 copies of a 15-mer at this site [30] , suggesting that this peak might result from de novo-assembly compression of the homologous SSR in PRV Becker .', 'The short unit size of this SSR ( 15-mer ) meant that its copy number could only be estimated by RFLP and Southern blotting , and the likely amount of perfect repeating units could lead to laddering and polymerase slippage errors in PCR analysis .', 'We therefore devised an approach to computationally estimate the length of these perfect tandem repeats that demonstrate potential compaction during assembly , in order to facilitate future HTS-genome assemblies and preserve coverage-based information on inter-strain variation in SSR length .', 'Coverage-Adjusted Perfect Repeat Expansion ( CAPRE ) is based on methods used for copy number variant estimation in HTS data [113] , [114] , which is used in larger genomes to detect duplications of chromosome regions or individual genes .', 'As in copy number estimations , CAPRE takes into account the observed coverage depth and estimates the length of intergenic SSRs based on the expected sequence depth for its G/C nucleotide content ( Figure S2A in Text S1 ) .', 'In order to estimate SSR length conservatively , CAPRE predicts SSR length based on the median coverage expected for a given G/C content , and can also be used to predict potential upper- and lower-range estimates based on the upper and lower quartile ranges of this coverage ( Figure S2A in Text S1 ) .', 'Because it is imprecise , we applied this method sparingly , and used it only at intergenic sites where coverage depth exceeded two standard deviations from the median and coincided with a perfect SSR .', 'We used CAPRE to expand the lengths of three SSRs in PRV Becker , three in Kaplan , and four in Bartha ( Figure S1 and Table S3 in Text S1 ) .', 'This did not affect the overall count of SSRs in Table 4 , but did affect the length of several SSRs included in Table 5 ( e . g . SSRKa15795; these are marked ) .', 'We incorporated these CAPRE-expanded SSRs into the overall assembly of each genome before final annotation and comparisons .', 'The CAPRE method provided a means to estimate the length of these repeats and yielded a more even distribution of sequence read coverage at these sites in the final genome ( Figure S1 in Text S1 ) .', 'To test whether the CAPRE script provides a reasonable estimation of SSR length , we compared the CAPRE-expanded SSRs to alternative sources of data on actual SSR length .', 'First , we compared the three CAPRE-expanded SSRs of PRV Kaplan ( Table S3 in Text S1 ) to their counterparts in the original PRV mosaic genomes .', 'Each of these SSRs falls into areas of the mosaic genome that were originally derived from the Kaplan strain , facilitating comparison of our estimated lengths to SSR lengths that were determined in strain Kaplan by traditional Sanger sequencing .', 'For SSRKa107138 , the CAPRE-estimated length nearly matches that of the Sanger-sequenced Kaplan isolate ( 12 . 5 copies here vs . 10 . 5 copies in the mosaic ) , while for the other two it provides a conservative under-estimate ( SSRKa2093 is 8 . 3 copies here but was 17 . 3 in the mosaic; SSRKa17595 is 13 . 7 copies here , but was 39 copies in the mosaic ) .', 'Next , we used RFLP and Southern blot analysis to estimate the length of the most divergent SSR between strains ( Table 5 , SSRKa15795 ) ; this SSR is also the only one expanded by CAPRE for all three strains ( Table S3 in Text S1; SSRKa15795 , SSRBe15739 , SSRBa15751 ) .', 'We hybridized a probe to this SSR against SalI-digested DNA from PRV Kaplan , Becker , and Bartha ( Figure 7 ) .', 'The size of the SalI fragment reflects a much larger size in PRV Becker than in Kaplan and Bartha , and further reveals that this SSR varies in length even within the purified PRV-Becker stock .', 'A prior Southern blot analysis by Simon et al . showed that this same SSR varied in length between strains and within plaque isolates of a given PRV strain [115] .', 'As occurs here with strain Becker , those authors found that the strain Phylaxia had a wide and blurry band of probe hybridization , while other PRV strains ( Kaplan and Dessau ) had tight bands [115] , suggesting strain-specific differences in SSR length stability .', 'To investigate the stability of this SSR , we serially passaged the plaque-purified PRV Becker stock ten times in culture ( potentially 20–30 cycles of replication at low multiplicity of infection ( MOI ) ; see Methods for details ) .', 'RFLP analysis of this stock , termed Becker p10 , differed from the parental PRV Becker only in the classically variable BamHI fragments 10 and 12 ( Figure 2B and 7A ) , which have been shown to vary with repeated passages [22] , [35] , [41] , 
['Introduction', 'Results/Discussion', 'Methods']
['Alphaherpesviruses are widespread in the human population , and include herpes simplex virus 1 ( HSV-1 ) and 2 , and varicella zoster virus ( VZV ) .', 'These viral pathogens cause epithelial lesions , and then infect the nervous system to cause lifelong latency , reactivation , and spread .', 'A related veterinary herpesvirus , pseudorabies ( PRV ) , causes similar disease in livestock that result in significant economic losses .', 'Vaccines developed for VZV and PRV serve as useful models for the development of an HSV-1 vaccine .', 'We present full genome sequence comparisons of the PRV vaccine strain Bartha , and two virulent PRV isolates , Kaplan and Becker .', 'These genome sequences were determined by high-throughput sequencing and assembly , and present new insights into the attenuation of a mammalian alphaherpesvirus vaccine strain .', 'We find many previously unknown coding differences between PRV Bartha and the virulent strains , including changes to the fusion proteins gH and gB , and over forty other viral proteins .', 'Inter-strain variation in PRV protein sequences is much closer to levels previously observed for HSV-1 than for the highly stable VZV proteome .', 'Almost 20% of the PRV genome contains tandem short sequence repeats ( SSRs ) , a class of nucleic acids motifs whose length-variation has been associated with changes in DNA binding site efficiency , transcriptional regulation , and protein interactions .', 'We find SSRs throughout the herpesvirus family , and provide the first global characterization of SSRs in viruses , both within and between strains .', 'We find SSR length variation between different isolates of PRV and HSV-1 , which may provide a new mechanism for phenotypic variation between strains .', 'Finally , we detected a small number of polymorphic bases within each plaque-purified PRV strain , and we characterize the effect of passage and plaque-purification on these polymorphisms .', 'These data add to growing evidence that even plaque-purified stocks of stable DNA viruses exhibit limited sequence heterogeneity , which likely seeds future strain evolution .']
['Alphaherpesviruses such as herpes simplex virus ( HSV ) are ubiquitous in the human population .', 'HSV causes oral and genital lesions , and has co-morbidities in acquisition and spread of human immunodeficiency virus ( HIV ) .', 'The lack of a vaccine for HSV hinders medical progress for both of these infections .', 'A related veterinary alphaherpesvirus , pseudorabies virus ( PRV ) , has long served as a model for HSV vaccine development , because of their similar pathogenesis , neuronal spread , and infectious cycle .', 'We present here the first full genome characterization of a live PRV vaccine strain , Bartha , and reveal a spectrum of unique mutations that are absent from two divergent wild-type PRV strains .', 'These mutations can now be examined individually for their contribution to vaccine strain attenuation and for potential use in HSV vaccine development .', 'These inter-strain comparisons also revealed an abundance of short repetitive elements in the PRV genome , a pattern which is repeated in other herpesvirus genomes and even the unrelated Mimivirus .', 'We provide the first global characterization of repeats in viruses , comparing both their presence and their variation among different viral strains and species .', 'Repetitive elements such as these have been shown to serve as hotspots of variation between individuals or strains of other organisms , generating adaptations or even disease states through changes in length of DNA-binding sites , protein folding motifs , and other structural elements .', 'These data suggest for the first time that similar mechanisms could be widely distributed in viral biology as well .']
['medicine', 'genome evolution', 'viral classification', 'immunology', 'dna-binding proteins', 'microbiology', 'vaccines', 'genome sequencing', 'dna viruses', 'microbial evolution', 'veterinary science', 'vaccination', 'infectious diseases', 'genome complexity', 'veterinary diseases', 'proteins', 'biology', 'pathogenesis', 'viral evolution', 'biochemistry', 'clinical immunology', 'immunity', 'virology', 'veterinary virology', 'vaccine development', 'viral diseases', 'genomics', 'evolutionary biology', 'genomic evolution', 'computational biology']