Genomic Sequence and Virulence of Clonal Isolates of Vaccinia Virus Tiantan, the Chinese Smallpox Vaccine Strain

Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector.


Introduction
Poxviruses are cytoplasm-replicating large double-stranded DNA viruses. Two of the best-known members of the family of Poxviridae are variola virus, the causative agent of smallpox, and vaccinia viruses (VACV), the smallpox vaccine strain. Smallpox was declared eradicated in 1979 by the World Health Organization (WHO) following a long, global vaccination campaign [1]. Despite smallpox eradication, studies on VACV remain relevant today. There is a continuing need for surveillance for potential smallpox outbreaks and bioterrorism acts using variola virus or other orthopoxviruses (OPVs) [2,3,4]. Furthermore, VACV has been employed to develop effective monkeypox vaccines [5,6,7]. The most widely used smallpox vaccine strain in China is the Temple of Heaven strain (also known as the Tiantan strain or VACV-TT). Its comparative strains are EM-63 in Russia, Lister/ Elstree in Europe, New York City Board of Health (NYCBH)/ Dryvax/Wyeth in the United States [8]. The most frequently studied VACV strain is Western Reserve (WR), a mouse brain passaged derivative of NYCBH [9,10]. Virulence attenuation and immunogenicity improvement are important aspects of VACV based vaccine development [7,11,12].
Currently, there are 30 VACV genomes stored in the Viral Bioinformatics Resource Center (http://www.virology.ca). Several reports have illustrated the presence of genomic diversity in these earlier generations of smallpox vaccines. Qin et al., sequenced 11 clones isolated from Dryvax and revealed the presence of genetic diversity, with deletions in the right-hand inverted terminal repeat (ITR) [13]. Esposito et al., reported the presence of 573 single base polymorphisms and 53 insertions and deletions between the NYCBH/Dryvax clone Acam2000 and a more neurovirulent sister clone [14]. Garcel et al., showed that VACV-Lister also contained genotypic and phenotypic diversity [15] and shotgun sequencing of the genome of an unpurified Lister stock yielded more than 1,200 polymorphic sites [16].
The original TT strain was previously reported to have been isolated from skin lesions of a Chinese individual with smallpox in 1926 and had an attenuation history of passages in monkey, rabbit, bovine skin and rabbit testis [17]. TT had similar vaccination reaction characteristics as the contemporary Japanese cowpox strain and was used in the production of smallpox vaccine in China [17,18,19]. Originally, the TT vaccine was prepared from skin lesion materials after intracutaneous inoculations in calves. From 1969, it was produced in chicken embryo fibroblasts (CEF) cells and the TT (752-1) was a viral batch produced in 1975. The genomic sequence of a TT vaccine stock was determined and submitted to GenBank (accession number AF095689.1) in 1998. It was subsequently recognized that several sequencing errors existed [20]. Currently, an HIV candidate vaccine, rTV, uses TT as its vector [21]. It had completed a Phase I clinical trial and began a Phase II trial in 2012 (www.IAVI.org). In this study, we report the results from sequencing and virulence characterization of TT clones derived from TT (752-1).

Cells, Viruses, and Viral DNA Isolation
TT (752-1) was provided by the National Vaccine and Serum Institute, Beijing. NYCBH strain was provided by the AIDS Research and Reference Reagent Program (Catalog number 3929) of the U.S. National Institutes of Health. Both viruses were grown in CEF prepared from 9 to 11 day-old embryonic chicken eggs and cultured in Eagle's medium supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% antibiotic at 37uC in a 5% CO 2 atmosphere. TT clones were isolated from TT (752-1) by plaque-purifications at terminal dilutions. Seven viral plaques were isolated and amplified by sequential four rounds of passages to produce enough material for sequencing [13]. Plaque images (Fig. S1) were processed with ImageJ (National Institutes of Health) [22]. Genomic DNA of each clone was extracted and purified using proteinase K digestion followed by phenol-chloroform extraction as described previously [23].

Genome Sequencing, Assembly, and Annotation
The genomes of TT clones were sequenced by Illumina Hiseq2000 Sequencer (Illumina Inc, CA) using shotgun sequencing of 500 bp paired-end sequencing library. Contigs were assembled and scaffolds were constructed using raw data with SOAP denovo and SOAP aligner (Beijing Genomics Institute) [24]. GenBank BLAST searches were performed for all scaffolds and sequences derived from the host genome were removed. The resulting alignments were analyzed by BioEdit (Ibis Bioscience, CA) and Contigexpress (Informax Inc, MD) to identify gaps between scaffolds. The average size of sequencing reads was 90 bp. Sequence gaps were present in the genome especially in ITRs. They were subsequently filled by polymerase chain reaction and Sanger sequencing.
Genome Annotation Transfer Utility (GATU) [25] was used to analyze ORFs in TT genomes using VACV-Copenhagen (Cop) as the reference. Orthologs and unassigned ORFs (i.e., ORFs that had no ortholog in Cop) were identified. ORFs shorter than 50 amino acids (aa) or without orthologous gene were excluded from the analysis. Unassigned ORFs of 50 aa or longer was categorized as OPVs genes according to the GenBank BLAST result with the highest similarity [26]. Viral Genome Organizer [27] was used to analyze the positions of individual genes. Sequences of ORFs with polymorphisms including frame-shift insertion or deletion among TT clones were further confirmed by Sanger sequencing. In cases where an ORF contained more than one start codon (ATG) near the 59 terminal, BLAST was conducted to compare the orthologous sequences in other VACVs. The most frequently used ATG was selected as the tentative ORF in TT.

Phylogenetic Analysis
BioEdit was used to produce alignments of multiple genomes by Clustal W method [28], and Base-By-Base software (http:// athena.bioc.uvic.ca/virology-ca-tools/base-by-base/) [29,30] was used to trim the alignments and produce a visual summary of whole genome alignments. Phylogenetic analysis was carried out using the alignments of the conserved central region of 160.4 kb DNA sequences between the two ITRs starting from TT_025 and ending with TT_255. This region in TT corresponded with the orthologous region between C7L and B16R in Cop. Global gaps (the gap regions present in more than half of these aligned genomes) [31] were removed from the alignments. Phylogenetic analysis was performed using a maximum-likelihood analysis with general time-reversible (GTR) substitution model, subtree-pruneand-regraft (SPR) improvement, and 1000 bootstrap replicates (http://www.atgc-montpellier.fr/phyml/). The phylogenetic tree was visualized using Figtree (http://tree.bio.ed.ac.uk/software/ figtree/). MEGA 5 [32] was used to calculate pairwise distances between genomes by a Kimura 2-parameter substitution model including transition and transversion substitutions and pairwisedeletion treatment. The average values of pairwise distances between TT or Dryvax clones were calculated to illustrate the sequence diversity within these two groups of viruses. The standard deviation (s) of each group was computed.

Genomic Sequence Orthology
To analyze the genomic sequence orthology among TT clones, a consensus sequence was first derived. TT11 had the highest similarity to the consensus sequence and was therefore chosen as the reference. ORFs of TT11 were compared with orthologous ORFs of four other TT clones and other VACV genomes to calculate the full-length nucleotide sequence similarity using GATU and DNAMan (Lynnon Corporation, Quebec, Canada).

Nucleotide Sequence Accession Numbers
The full genomic sequences of five TT clones were deposited in GenBank with accession numbers from JX489135 to JX489139. The OPVs used in this report and their GenBank accession numbers are listed in Table S1. TT-TW, a variant of TT (752-1) without plaque-purification was sequenced in this laboratory in 2010 (unpublished).

Mouse Brain Based Neurovirulence Determination
TT viruses and the NYCBH strain were serially diluted in phosphate buffered saline (PBS) and then 3 x 10 2 plaque forming units (PFU) in 30 ml PBS were inoculated intracranially to groups of five 3-week old female BALB/c mice anesthetized with 1.25% 2,2,2-tribromoethyl alcohol. Body weights and mortality were recorded daily during the 12-day observation period.

Rabbit Skin Virulence Determination
TT clones and the NYCBH strain were serially diluted from 10 6 to 10 2 PFU/ml in PBS. For each viral dilution, two rabbits were tested. Each rabbit received two dorsal skin intradermal inoculations with 0.1 ml viral preparation per injection site. At day 4 postinfection (p.i.), the diameters of lesions were measured and the incidence of putrescence was recorded.

Ethics Statement
All experiments were conducted in accordance with the guidelines of the Laboratory Animal Center of Chinese Center for Disease Control and Prevention and NCAIDS. All procedures involving animal use and care were approved by the Institutional Committee on Laboratory Animals of NCAIDS.

Clone Isolation and Genome Assembly
Seven randomly selected TT clones were isolated by plaque picking at the terminal dilution on CEF cells and amplified. The sizes of plaques varied after initial runs of plaque picking of each clone. No obvious difference was observed between the plaque sizes of the clones following passage and those of the original vaccine pool, although the average plaque sizes of TT9 and TT11 were significantly smaller (p,0.05) than those of TT7 and TT8 (Fig. S1).
All seven viral genomic DNAs were sequenced and their contigs were assembled. All the assemblies were aligned with Cop and ListerV107 to accurately identify any gaps, particularly in highly repeated elements such as the ITRs. Five TT clones (TT8 to  Table S1. The scale bar of branch length was shown under the phylogenetic tree. doi:10.1371/journal.pone.0060557.g001 Figure 2. Distribution of polymorphic sites in TT genomes. The genomes were aligned by BioEdit and edited using Base-by-Base software. A consensus sequence was generated from TT8 to TT12 by Base-By-Base software. Sites in TT clones different from the consensus sequence are displayed in blue for nucleotide substitutions, green for insertions, and red for deletions. The extent of similarity (%) of each clone with the consensus sequence is displayed. doi:10.1371/journal.pone.0060557.g002 Table 1. Gross differences in ORF size (nt) and complement.

Phylogenetic Analysis
We examined the phylogenetic relationship of various OPVs by maximum-likelihood method using alignments of the conserved 160.4 kb central region (i.e., the ITR sequences were excluded) (Fig.1). These sequences included five TT plaque-purified clones, TT-TW, and 25 other commonly known OPVs including 11 Dryvax clones DPPs (Table S1). Although TT6 and TT7 clones contained incomplete ITRs, the regions used in phylogenetic analysis were complete and thus were included in this analysis.
Notably, TT clones clustered with a bootstrap value of 988 and shared a common ancestry with the TT genomic strain (AF095689.1) published in 1998 and another TT (752-1) derived TT-TW sequenced in 2010. Among the other VACVs, Cop was most similar to the TT cluster and HSPV-MNR76, NYCBH/ Dryvax-derived DPP clones, Acam2000 and Duke exhibited least similarity. Among the analyzed OPV sequences, ectromelia virus strain Moscow (ECTV-Mos) was most distant from the TT cluster.
All the Dryvax derived strains clustered as one group, including the Dryvax clones (DPPs), Acam2000 that were independently isolated from another stock of the same vaccine, and Duke that isolated from a patient experiencing a Dryvax vaccine-associated complication. The TT clones exhibited higher sequence diversity than the Dryvax clones. The average value of pairwise distances between TT clones was 0.00401 (s = 0.00026) and the average value between Dryvax clones was 0.00294 (s = 0.00017).

Gene Annotations and Polymorphisms in Nucleotide Sequence
ORFs longer than 50 aa were initially annotated as genes and those without comparable orthologs in other OPVs were removed from annotation. Small ORFs that embedded in larger ORFs in the genomes of other poxviruses and those found to be truncated or split into two or more portions were removed from the ORF list and reclassified as ''fragments''. Six such fragments were identified: TT_005.1, TT_028.1, TT_191.1, TT_210.1, TT_252.1, and TT_265.1. The genomic organizations of TT clones were identical to other sequenced VACVs. There were 255 unique ORFs in the genome and nine duplicated ORFs (TT_001 to TT_009 and TT_261 to TT_269) in ITRs. There are 273 ORFs found in each of the TT clones (Table S2), including orthologs of four additional conserved ORFs which were recently recognized in Cop and WR. These conserved ORFs have a ''.50 in their names.
The orthology between TT clones was further assessed through the distribution of polymorphic sites. We first generated a consensus sequence using the most frequent nucleotide from each position. The similarities of each TT clone varied from 97.36% to 98.30% and TT11 had the highest similarity with the consensus (Fig. 2). The ORFs derived from TT11 were thus used as the reference for full-length ORF comparisons among TT clones (Table S3). Among the 273 annotated ORFs, the DNA sequences of 241 ORFs in four other TT clones had .95% identity with those in TT11. Conserved ORFs were located in the middle of the viral genome with gene functions related to viral replication and structure. For the remaining 32 ORFs, there was at least one clone displaying ,95% identity with the reference ORF. More than 95% of the polymorphic ORFs exhibited length variations due to the acquisition of one or more point mutations, in-frame insertions or deletions. ORFs with relatively low similarity were located near Table 1. Cont. ITRs where many virulence or immunomodulation-associated proteins reside. Table 1 summarizes the major ORF differences among TT clones and four reference VACVs including Cop, ListerV107, WR, and Acam2000. Four ORFs (TT_147, TT_185, TT_188, and TT_190) in the TT genomic sequences had orthologs in TT genome (AF095689.1) and thus were annotated, but corresponding sequences in the reference genomes were not annotated. Whether these ORFs code for functional proteins remains to be investigated. Two ,4 kb regions in TT strains (ORF TT_011 to TT_019 and TT_184 to TT_190, encoding host defense modulators, ankyrin-like proteins, and A-type inclusion protein) had orthologous regions in Acam2000 and WR, but were absent in Cop. Conversely, the regions coding three ORFs (C19L, C20L and C21L) in the ITRs of Cop were absent in TT genomes.
Several genes had polymorphic lengths because of the presence of substitution mutations. As shown in Fig. 3A, the length of ORF TT_210/210.1 (A39R, also known as semaphorin) in TT8 was the same as that in Cop and ListerV107 (1212 bp). In TT9 and TT10, a C685T mutation truncated this ORF to 687 bp and a T774A mutation truncated this ORF to 774 bp in TT11 and TT12 or 888 bp in WR. In another example, shown in Fig. 3B, the start codons for ORF TT_180 (A22R, Holliday junction resolvase) in TT11 and Cop were altered by a T-A mutation near the 59 terminal and the next available start codon was 33 bp downstream. This change shortens the size of this ORF to 531 bp. Several polymorphisms were generated by insertions or deletions. One interesting example was the addition of copies of 6 bp repeats (ACAGAT) in TT_250 (B11R, function unknown) (Fig. 3C). In TT11, TT12, Acam2000, ListerV107, and WR, there was one copy of ACAGAT and in TT8, TT9, and TT10 there were three copies. Nine copies were present in Cop. In addition, in-frame insertions near the 39 terminal of TT_162 (A9L, intracellular mature virion membrane protein) resulted in ORFs of 327 bp in TT11, TT9, TT12, Acam2000, and WR; 312 bp in TT10; and 300 bp in TT8, Cop, and ListerV107 (Fig. 3D). TT_230 (A53R, TNF receptor CrmC) in TT8, TT12, Cop, and WR consisted of a 16 bp frame-shift deletion near the 59 end and a shift of start codon 16 bp upstream from the ones in TT11, TT9, TT10, Acam2000, and ListerV107 (Fig. 3E).

Polymorphisms in Virulence Reaction Associated Proteins
VACV contains more than 200 genes, some of which have been previously reported to be associated with viral virulence and immunomodulation. We selected seven virulence reaction-associated genes from TT clones and Acam2000 for amino acid  (Table 2). Acam2000 was a clonal Vero cell culture vaccinia virus derived from NYCBH/Dryvax, and its relative gene sequences were selected to represent those of the NYCBH strain used in the our study. TT_213 (A41L) was a secreted virulence factor and its deletion was reported to result in severe lesions and increased viral clearance in the mouse skin model [33]. Six amino acid polymorphic sites were found in TT clones and Acam2000. TT clones displayed a substitution mutation, K122E, located in the chemokine binding protein superfamily conserved domain. The change from alkaline lysine (K) to glutamic acid (E) residue might be associated with the attenuation of TT. Seven polymorphic sites were found in TT_242 (B5R) among TT clones. B5R was a type I integral membrane glycoprotein and extracellular enveloped virion (EEV) complement control protein which blocked complement activation and associated inflammatory responses. Destruction of B5R was reported to contribute to attenuation of VACV [16,34]. There were three substitution mutations among TT clones and Acam2000, L55S, V82I and I153M. L55S changed the polarity of the amino acid.
Six immunomodulation-associated genes were also selected for closer examination (Fig. 4). TT_210 (A39R) encoded a semaphorin influencing the outcome of dermal infection and, when truncated, the protein product could not be exported extracellularly [35]. Presence of a stop codon in the central sema domain yielded a truncated product of 228 aa in TT9 and TT10; and 257 aa in TT11 and TT12 (Fig. 4A). Truncation of the protein was also observed in Acam2000, with a product of 134 aa. TT_255 (B16R) encoded an IL-1 beta receptor. It was 290 aa in length in Cop and all five TT strains. It was longer in Acam2000 due to the presence of additional N-terminal 36 aa (Fig. 4B). TT_259 (B19R) encoded interferon-a/b receptor. In Cop, TT9, and TT12, it was 352 aa in length. There was a 2-aa deletion (Ser-Leu) in TT8, TT9, and TT10 near the N-terminus (Fig. 4C). In Acam2000, B19R was 265 aa and lost the immunoglobulin domain near the C-terminal. TT_028 (C4L) encoded IL-1 receptor antagonist and was 316 aa in  Acam2000  TT8  TT9  TT10  TT11  TT12   TT_025  150 Host range virulence factor 31 TT_170.5 53 IMV membrane protein, virulence factor (Cop-A14.5L) V82I  V82I  V82I  V82I   140  C  -C140Y  -C140Y  C140Y   153  I  I153M  I153M  I153M  I153M  I153M   216  I  ---I216T c  I216T   TT_245  182  Virulence, ER resident  length in Cop, TT8, and TT12 (Fig. 4D). It was shorter in TT9, TT10 and TT11 due to the deletion of 120 aa in N-terminal. In Acam2000, C4L was split into three fragments. TT_230 (A53R) encoded TNF receptor (CrmC) and its length differed greatly among these genomes (Fig. 4E). It was 102 aa in Cop and was 30 aa longer in TT8, TT9, TT12, and Acam2000. In TT10 and TT11, there was a 84 aa elongation in the C-terminal. Two forms of Nterminal sequences were present in A53R as described earlier. TT_252 (B13R) encoded serine protease inhibitors (Serpin 1, 2, 3) and was 116 aa in length in both Cop and TT9 (Fig. 4F). It was 335 aa in four other TT clones due to the presence of additional C terminal 219 aa. In Acam2000 it was 126 aa due to the presence of an additional N-terminal 10 aa.

Virulence of TT Clones and NYCBH in Animal Models
We compared the neurovirulence of parental TT (752-1), TT clones, and NYCBH. Groups of three-week-old BALB/c mice were intracranially inoculated with 3 x 10 2 PFU viral preparations or PBS and observed daily for weight change and mortality. In the control group (injected with PBS), all mice gained weight over the 12-day observation period. Mice that received NYCBH showed severe signs of illness, weight loss (Fig. 5A), and decreased physical activity starting on day 3 p.i. One mouse died on day 4, three more died on day 5 (Fig. 5B). By day 5, the remaining mouse had lost 39.7% of its initial body weight and died at day 6. For the TT (752-1) group, there was an initial weight gain, but on day 3 the mice began showing weight loss that persisted to day 11. One mouse died on day 7, another on day 8. The remaining three mice survived and gained body weight. The weight change among mice infected with TT (752-1) was less than those infected with NYCBH (P,0.05).
We examined the virulence of five TT clones in the same manner. The patterns of weight loss (Fig. 5C) and mortality (Fig. 5D) were similar to their parental strain, but differences among clones were observed. Mice injected with TT8 or TT10 lost 33% of their body weight by day 8 and all of them died on day 9. One of the mice injected with TT12 lost 32% of its body weight, but survived to day 12. Three and four of the mice injected with TT9 and TT11, respectively, initially lost weight but survived and regained body weight up to the original level.
We further characterized the skin virulence of the parental and cloned strains by inoculating four viral preparations, 10 2 to 10 5 PFU, into rabbit dorsal skin. Viral virulence was quantified by the diameter of skin lesion on day 4 p.i. As shown in Table 3, among the viral strains tested, NYCBH produced the largest lesions with the largest lesion measuring 9.8 mm in diameter at 10 5 PFU. Among TT strains, the parental strain yielded the largest lesions at the dilution of 10 5 PFU (4.6 mm) followed by TT8 (4.2 mm), TT9 (3.4 mm), TT10 (3.4 mm), TT12 (1.2 mm), and TT11 (1.0 mm). NYCBH yielded 100% putrescence at all dilutions. TT clones yield 100% putrescence at the highest 10 5 PFU inoculation, much less putrescence at the 10 4 PFU inoculation, and no putrescence at the 10 3 PFU inoculation.

Discussion
Similar to most vaccinia viruses used in the last century, TT has a vague origin and passage history. The earliest record shows its ancestral strain was isolated from skin lesions of a Chinese individual with smallpox in 1926 [17,18,19]. Vaccine stocks were established after monkey-rabbit-bovine passages and were later widely used in China during the smallpox eradication campaign. After 1969, TT vaccines were produced in CEF cell cultures but were never plaque-cloned. The lack or loss of well-organized records for vaccine production further add to the confusion of the identity and lineage of the virus [36]. Here we successfully characterized full genomes of five TT clones isolated from a CEFderived stock, TT (752-1). Sequences from two other clones with incomplete terminal ITRs were also obtained. Our study, analogous to an earlier genetic analysis [37], shows that TT was a vaccinia virus, not a variola virus. Phylogenetic tree analysis shows that all TT clones were a mixture of closely clustered quasi-species with an inter-clone diversity slightly higher than that observed in clones derived from NYCBH/Dryvax. Presence of quasi-species was also reported in Lister/Elstree vaccine strains [12,16,38].
The genome structure of TT is the same as other vaccinia viruses. The genome length of TT clones varied from 189,366 in TT8 to 191,144 in TT11 with 273 identifiable ORFs. Insertions, deletions, and point mutations were present throughout the genome resulting in protein alterations. ORFs located in the middle of the genome were more conserved than those located at the two ITRs. We also examined biological activities of TT strains in mice using intracranial injections. TT (752-1) and its progeny clones behaved different in the induction of weight loss and mortality. These biological variations among TT clones may not be due to the replication abilities since they were similar in our study (data not show). The severity of the weight loss and mortality was less than that produced by the NYCBH strain. Our finding differs from Marennikova et al. who reported that TT displayed higher pathogenicity than NYCBH in mice [39,40]. The TT stock used by Marennikova et al., was calf skin-derived, not one of the CEF-derived viral strains generated after 1969. We did not examine a calf skin-derived strain in our study and thus direct comparison between the calf skin and CEF derived TT viruses could not be made. Using NYCBH as the common denominator in both studies, it appears passage in CEF might have attenuated the virulence of the calf skin-derived TT. Likewise, we found TT strains displayed less virulence than NYCBH in mice and rabbits. This finding is corroborated by historic data. The frequency of post vaccination encephalitis from CEF-derived TT was 2.1 cases/ million for primary vaccinations and 0 for progressive vaccinia; the corresponding data from calf skin-derived NYCBH were 2.9,12.3 and 0.9,1.5, respectively [8,41]. Further systemic genotypic and phenotypic comparison might reveal underlying mechanisms related to viral virulence and immunomodulation.
Our present genomic and phenotypic characterization of TT strains yields valuable information for continuing TT-based research in China or elsewhere to produce safer and more effective smallpox vaccines. It is also useful for the current vaccine research using TT-based vectors for HIV and other pathogenic viruses. Figure S1 Plaque properties of TT (752-1) and seven TT clones. Each clone of the TT (752-1) was plaque-purified and ,50 PFU of each clone was plated on a monolayer of CEF cells. The infected cells were cultured for 3 days and then stained with crystal violet and imaged. The plaques were visualized using ImageJ and their sizes were determined. (TIF)  Table 3. Viral skin virulence in rabbits. and WR were used as the reference. When no ortholog was present in these two reference strains the gene with the highest similarity in OPVs was used. b: orthologs of "X_ORF_X" in Cop were annotated in TT clones. These genes were predicted not to express. c: TT_005.1, TT_028.1, TT_191.1, TT_210.1, TT_252.1, and TT_265.1 were identified as fragments.

Supporting Information
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