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To investigate the viral infection in the mass lymph node samples collected from pigs, the viral library was constructed with pooled lymph samples of porcine and subsequently sequenced by Illumina platform. The sequencing generated 8, 613, 350 reads with 150 nt length, with an approximate total coverage of 32 ×. After debarcoding and trimming, a total of 6, 673, 308 clean reads were obtained. By de novo assembly, a long contig was obtained from 6, 831 cleaned paired reads. The contig contains genes and encoded proteins that are significantly related to homologues of AdVs in the GenBank. This virus was designated tentatively as PAdV-B-HNU1.
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To reveal the prevalence of PAdV-B-HNU1 in clinical porcine samples, a set of primers targeting a 349-bp region within the penton base gene, were used to detect PAdV-BHNU1 in 295 porcine samples. The result showed that 108 out of 295 samples (36.6%) were positive for PAdV-BHNU1, including 93 of 180 (51.7%) lymph nodes, 6 of 59 (10.2%) oral swabs and 9 of 56 (16.1%) rectal swabs (Table 1).
Sample types No. of test samples/no. of positive samples (% positive) Lymph 180/93 (51.7) Oral swab 59/6 (10.2) Rectal swab 56/9 (16.1) Total 295/108 (36.6) Table 1. Prevalence of PAdVB-HNU1 in porcine samples collected in China.
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The full-length genome of PAdV-B-HNU1 was reconstructed by PCR amplification and replicon sequencing using primers designed according to the viral metagenomic contig sequence. The terminal sequences of the viral genome were determined by 5' and 3' RACE amplification and sequencing. After assembly, a 31, 743 bp genome of PAdVB-HNU1 (GenBank no. MK774519) with 55% GC content was obtained.
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Totally, 116 ORFs (open reading frames) were predicted in both strands of the PAdV-B-HNU1 genome using a searching strategy defining ATG as the start codon and a cutoff size of 50 amino acids (aa). After annotation of the ORFs, the genomic organization of PAdV-B-HNU1 was similar to that of most known members in the genus Mastadenovirus (Fig. 2). The GC contents vary from 46 to 65% in different genes, among which genes in the center of the genome, stretching from IVa2 to pVⅢ, showed higher GC contents than the genes at the terminal regions. Among all the putative proteins encoded by PAdV-B-HNU1, the aa sequences of 31 products were homologous to those of other AdVs, of which 21 were located at the positive strand and 10 were located at the complementary strand (Table 2, Fig. 2). Next, genes of PAdV-B-HNU1 were classified into two sets, homologous genes in all genera as 'genus-common genes' and other genes as 'genus-specific genes' (Davison et al. 2003). Similar to other members of the genus Mastadenovirus, the middle part of the PAdV-BHNU1 genome was predicted to contain 18 genus-common genes. These include genes coding for protein related to DNA replication (Pol, pTP, and DBP), DNA encapsidation (52 K and Iva2), virion formation and capsid structure (pⅢa, penton base, pVⅡ, pX, pVI, hexon, protease, 100 K, 33 K, pVⅢ, and fiber); 22 K, which originates from a lack of splicing in 33 K; and U exon which has been lost in PAdV-C. And PAdV-B-HNU1 had the longest inverted terminal repeat compared to other porcine adenoviruses (Fig. 3).
Figure 2. Genome organization of PAdV-B-HNU1 and other adenoviruses. The viral genome is represented by the thick line in the center marked with 10-kb intervals. Rectangles represent genome regions of PAdV-B published online and the dashed line indicates unavailable region of PAdV-B. Arrows underneath the genome line predicted ORFs of adenoviruses and the thin lines indicate the introns.
Gene product Location(s) (nt)a Size (aa) Amino acid identity (%) with: GC content (%) PAdVNADC1 C.sea lion AdV1 PAdVA PAdVC E1 ORF 1 Termini 485–1039 186 94 29 22 23 55 E1B 19 kDa 1762–2220 152 NI 26 14 NI 54 E1B 55 K 2127–3368 413 95 38 24 28 64 IX 3466–3801 111 100 29 24 26 56 184R 3981–4280 99 NA NI 50 NI 48 ORF6 26, 760–27, 041 93 96 NI NI NI 46 ORF7 26, 242–26, 760 172 97 NI NI NI 56 ORF4 27, 022–27, 561 119 99 NI NI NI 49 U exon 27, 560–27393c 55 100 53 38 NI 48 Fiber 27, 575–29, 764 729 94 28 14 14 55 E434kDa 30, 753–30004c 249 NA 36 28 26 51 dUTPase 31, 592–31164c 145 NA 50 NI 40 62 E415kDa 31, 151–30750c 133 NA 30 NI NI 53 IVa2 Center 3891–5218, 5298–5310c 373 NA 69 58 65 50 pol 4982–8410, 12, 937–12966c 1037 NA 69 65 55 55 11.5 kDa 6087–6407 106 NA NI NI NI 54 p52k 10, 128–11, 339 403 NA 61 47 48 56 Penton base 13, 035–14, 483 482 NA 76 60 63 53 pV 15, 234–16, 175 313 NA 28 14 23 61 pX 16, 197–16, 391 64 NA 70 48 59 57 pVI 16, 428–17, 159 244 NA 48 34 44 61 pTP 8263–10, 095, 13, 097–13162c 595 NA 72 55 58 59 pⅢa 11, 266–12, 927 554 NA 62 43 48 58 p33K 24, 249–24, 423, 24, 690–25, 000 161 NA 52 29 NI 64 p22K 24, 625–24, 912 95 NA 48 29 NI 55 pVⅡ 14, 486–15, 178 230 NA 48 27 24 65 Hexon 17, 201–20, 161 986 NA 77 63 66 57 protease 20, 165–20, 791 208 NA 64 63 57 54 DBP 22, 379–20853c 508 NA 55 41 39 65 p100K 22, 394–24, 403 669 NA 64 50 54 58 pVⅢ 25, 031–25, 696 221 94 57 50 52 62 NA not applicable due to the lack of detectable similarity, NI homologous gene not identified.
aThe letter "c" in this column indicates that those genes are encoded by the complementary strand.Table 2. Putative proteins encoded by PAdV-B-HNU1.
Figure 3. Comparison of nucleotide sequences of 5'UTR between PAdV-B-HNU1, PAdV-C, PAdV-A and C. sea lion AdV-1. Pairwise alignments were calculated using DNAMAN. Dots indicate gaps.
A summary of the sequence identity among proteins of PAdV-B-HNU1 and other porcine adenoviruses including California sea lion AdV-1 is presented in Table 2. The amino acid sequences (94–100%) of PAdV-B-HNU1 showed the highest similarity to PAdV-B strain NADC-1 compared to other porcine adenoviruses including PAdV-A and PAdV-C, suggesting that PAdV-B-HNU1 belongs to PAdV-B.
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Several PAdV strains, such as PAdV-SVN1 and PAdV-WI, have not been classified into any species, because only part of their genome has been sequenced for which the speciesreference sequence is lacking. The PAdV-B-HNU1 provides the first representative complete genomic sequence of PAdV-B, which can be used to classify related PAdV strains. The classification of the PAdV-SVN1 and PAdVWI is made by comparing the aa sequence identities. According to multiple alignment of partial DNA polymerase (Fig. S1A) and hexon (Fig. S1B) aa sequence, partial DNA polymerase of PAdV-SVN1 shows 99%, 76%, and 74% identity to PAdV-B-HNU1, PAdV-A, and PAdVC, respectively, while partial hexon of PAdV-WI shows 93%, 93%, 56%, and 56% identity to PAdV-SVN1, PAdVB-HNU1, PAdV-A, and PAdV-C, respectively. This suggests that PAdV-SVN1 and PAdV-WI, the two PAdVs whose taxonomic position was uncertain, belong to PAdVB.
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Phylogenetic analysis was conducted to reveal the genetic and evolutionary status of PAdV-B-HNU1. Phylogenetic trees were constructed based on the amino acid sequences of DNA polymerase, hexon, penton base and pre-terminal protein. In these trees, the PAdV-B-HNU1 strain was clustered with C.sea lion AdV-1 with high bootstrap value, indicating that PAdV-B-HNU1 is a member of the genus Mastadenovirus (Fig. 4, Fig. S2). And the genome of PAdV-B-HNU1 shared highest nucleotide sequence identity (54.1%) with C. sea lion AdV-1 (Table S1).
Figure 4. The evolutionary position of PAdV-B-HNU1 in the phylogenetic tree of adenoviruses. The phylogenetic trees were constructed based on the amino acid sequences of DNA polymerase (A), hexon (B), penton base (C) and pTP (D) of porcine adenoviruses by using the maximum likehood (ML) method with rtREV + F + I, LG + F + R5, LG + I + G + F, and LG + G as the model of protein evolution, respectively. PAdV-B-HNU1 detected in this study was indicated with solid triangle.
A dUTPase was identified at the 3' end of PAdV-BHNU1 genome, which was more similar to dUTPases of bacteria and fungi than that of mastadenoviruses except C. sea lion AdV-1. Phylogenetic analysis further confirmed that PAdV-B-HNU1 dUTPase does not cluster with other mastadenoviral dUTPases and was more related to dUTPases of baculovirus, poxvirus and eukaryotes (Fig. 5). This suggested that PAdV-B-HNU1 is more closed to C. sea lion AdV-1 than other PAdVs in evolutionary positions.
Figure 5. Phylogenetic tree constructed on the amino acid sequences of dUTPase. Phylogenetic analysis of PAdV-B-HNU1 is based on the partial conserved amino acid sequences of dUTPase from selected adenoviruses, bacteria, fungi, prasinovirus and poxviruses by using the Maximum Likelihood method based on the JTT matrix-based model. PAdV-B-HNU1 detected in this study was indicated with solid triangle.