Arch Virol DOI 10.1007/s00705-015-2373-7
BRIEF REPORT
Prevalence of emerging porcine parvoviruses and their co-infections with porcine circovirus type 2 in China Jianhui Sun • Liping Huang • Yanwu Wei • Yiping Wang • Dongjie Chen • Wenjuan Du Hongli Wu • Changming Liu
•
Received: 23 December 2014 / Accepted: 15 February 2015 Ó Springer-Verlag Wien 2015
Abstract A total of 450 samples from domestic pigs in China were tested for porcine parvoviruses (PPVs) and coinfections with porcine circovirus type 2 (PCV2), and their complete capsid genes were sequenced. The prevalence of PPV1, PPV2, PPV3, PPV4, and PCV2 was 5.56 %, 39.56 %, 45.11 %, 21.56 %, and 47.33 %, respectively, and co-infection with PCV2 occurred in 4 % (PPV1), 22.44 % (PPV2), 24 % (PPV3), and 12 % (PPV4) of the samples. Phylogenetic analysis revealed two main lineages for each virus, and residues that differentiated these viruses were identified. The co-infections of emerging PPVs and PCV2 were prevalent, indicating their cooperative roles in porcine circovirus-associated diseases.
Parvoviruses are small, single-stranded DNA viruses that cause severe clinical diseases, as well as mild and subclinical infections. They belong to the family Parvoviridae, which is divided into two subfamilies (Parvovirinae and Densovirinae) based on distinct host tropisms. Additionally, the subfamily Parvovirinae is further subdivided into eight genera. The classical porcine parvovirus type 1 (PPV1) belongs to the genus Protoparvovirus and is J. Sun and L. Huang contributed equally to this article.
Electronic supplementary material The online version of this article (doi:10.1007/s00705-015-2373-7) contains supplementary material, which is available to authorized users. J. Sun L. Huang Y. Wei Y. Wang D. Chen W. Du H. Wu C. Liu (&) Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Harbin 150001, China e-mail: [email protected]
responsible for causing reproductive failure in swine, usually described as stillbirth, mummification, embryonic death, and infertility [1]. Porcine circovirus type 2 (PCV2), another small DNA virus with a circular genome, acts as the dominant causative agent of post-weaning multisystemic wasting syndrome (PMWS) [12] and is associated with other porcine circovirus-associated diseases (PCVAD), which include porcine reproductive disorders, porcine dermatitis, nephropathy syndrome, necrotic lymphadenitis, and porcine respiratory disease [5]. Additionally, enhanced PCVAD- and PCV2-related lesions have been found in pigs co-infected with PPV1 and PCV2 [13, 16]. As a result of the widespread use of molecular detection technologies, several novel PPVs have been detected in pigs. Porcine parvovirus type 2 (PPV2) was first found in swine sera in Myanmar [8], and it shares high genetic similarity with subsequently discovered parvoviruses in China, which were found in pigs with clinical symptoms of ‘‘high fever’’ and PMWS [18]. Porcine parvovirus type 3 (PPV3) was identified in Hong Kong in 2008, and initially termed porcine hokovirus. Recent diagnostic investigations have suggested a widespread distribution of PPV3 in wild boars (50.54 %) [3] and domestic pigs (51.3 %) [11]. Porcine parvovirus type 4 (PPV4) was originally reported in North Carolina (USA) and found in swine herds that were co-infected with PCV2 and afflicted with PCVAD [6]. Until now, the specific pathogenic mechanisms of novel PPVs have not been well elucidated, but their prevalence has been reported to be higher in PCVAD cases than in non-PCVAD cases [15], indicating their active roles in PCVAD [11, 15]. Additionally, the high detection rate of PPV2 in the hearts (55 %) and tonsils (78 %) of healthy fattening pigs in Germany is striking [17]. As there have been no reports simultaneously describing emerging PPVs and their relationships with PCV2
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J. Sun et al. Table 1 Primers used for the detection of porcine parvovirus type 1 (PPV1) and amplification of complete major capsid genes of porcine parvoviruses (PPVs) and porcine circovirus type 2 (PCV2) Primer name
Primer sequence
Locationa
Lengthb
PPV1-DF
5’-AATAGAAACCGACATAAGAGCCTGC-3’
2217-2679
463
PPV1-DR
5’-TGGTTGTTGAGATGTAGTTGGTGAG-3’
PCV2-AF
5’-CGTAATTGTTTTTATTATT-3’
1012-1755
744
PCV2-AR
5’-CACTTCTTTCGCTTTCAGCT-3’
PPV1-AF
5’-GCTCTAGAGCCACCATGGGTGAAAATGTGGAACAACACAA-3’
2810-4549
1740
PPV1-AR
5’-AAAACTGCAGTCAATGGTGATGGTGATGATGGTATAATTTTCTTGGTATAAGTTG-3’
PPV2-AF PPV2-AR
5’-GGACGTCGAGGAGGAACCAA-3’ 5’-TATACACGATGAGCGCGTCC-3’
1922-53548
3427
PPV3-AF
5’-ATGGCTGCGCCGGTAACGGGCGCCACCGGTGGG-3’
3349-5016
1668
PPV3-AR
5’-TTACAATTTGCGGGAGAAATTCTGGGCAACCCACTGATCAGAAGGAA-3’
PPV4-AF
5’-ATGCAGAAACACGGTCACTGGCCTCATTTATGGGC-3’
3681-5867
2187
PPV4-AR
5’-TCATCTGCGGTGTCTGGGTCTCTGTTTTGCGGT-3’
a
Nucleotide positions for PPV1, PPV2, PPV3, PPV4 and PCV2 are according to those of NADL-2 (GenBank no.: NC001718), JH13 (GenBank no.: GU938300), HK7 (GenBank no.: EU200677), GQ387499 (GenBank no.: GQ387499), and AF055392 (GenBank no.: AF055392), respectively. F, forward orientation; R, reverse orientation. b Amplicon size is indicated as base pairs
published to date in China, the objective of this study is to determine the prevalence of classical PPV1 and emerging PPV2–4, as well as their co-infections with PCV2, in archived and recently collected domestic pig samples. The clinical tissue samples (lungs, livers, kidneys, brains, lymph nodes, or spleens) from suspected PCVAD pigs were collected and stored at -80 °C. A total of 450 specimens, collected from 2004 to 2014 in different areas of China, were grouped according to their date of collection and availability. Tissue samples of approximately 0.1 g were ground in minimal essential medium (Gibco, USA) using a TissueLyser (Qiagen, Hilden, Germany). Total nucleic acids were extracted from sample homogenates using a TIANamp Genomic DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was used to test all the samples for the presence of PPVs and PCV2 using an Ex Taq DNA polymerase kit (Takara, Dalian, China). Primers used to detect PCV2, PPV2, PPV3, and PPV4 were described previously [4, 11, 15]. Primers for the detection of PPV1 were designed using Primer Premier 5.0 software (www.premierbiosoft. com/primerdesign/index.html) according to the conserved regions in the NS1 gene of the reference PPV1 sequence (accession no.: NC_001718) (Table 1). Our results showed that among the 450 tissue samples tested, 25 (5.56 %) were PPV1 positive, 178 (39.56 %) were PPV2 positive, 203 (45.11 %) were PPV3 positive, 97 (21.56 %) were PPV4 positive, and 213 (47.33 %) were PCV2 positive. The PPV3- and PPV4-positive rates were significantly higher than those in North America (9.4 % for PPV3 and 2.9 % for PPV4) [15] and in Hungary (9.7 % for PPV3 and 6.4 % for PPV4) [7], while the frequency of PPV2 resembled that in North America (35.2 %) but
123
considerably exceeded that in Hungary (6.4 %). Furthermore, the concurrent infection rates with PCV2 of novel PPVs (22.44 % for PPV2, 24 % for PPV3, and 12 % for PPV4) far exceeded that of classical PPV1 (4 %) (Table 2). Above all, PPV2, PPV3, and PPV4 were all detected in tissues from 2004 onward, and this was the first report that PPV4 could be detected in China prior to 2009 [9]. The genomes of the PPVs and PCV2 mainly include two open reading frames (ORFs), ORF1 and ORF2. ORF1 encodes non-structural proteins, whereas ORF2 encodes viral capsid proteins [6, 8, 10]. Recently, a relatively high evolutionary rate, similar to that of RNA viruses, is observed for emerging PPVs [4]. To investigate the possibility of genetic changes among the different strains, further sequencing of the full-length major capsid genes was conducted. Based on the time of sampling and availability, we selected ten PPV1-, eight PPV2-, five PPV3-, one PPV4-, and 12 PCV2-positive samples, and the target genes were amplified using the amplification primers listed in Table 1. PCRs were performed using a KOD FX Neo kit (Toyobo, Shanghai, China) according to the manufacturer’s instructions. The PCR conditions included an initial denaturation step at 94 °C for 2 min, followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 63 °C (PPV1), 60 °C (PPV2), 68 °C (PPV3), 60 °C (PPV4), or 55 °C (PCV2) for 30 s, an extension at 68 °C for 2 min, and a final elongation step at 68 °C for 5 min. The PCR products were purified using an AxyPrey DNA Gel Extraction Kit (Axygen Scientific, Inc., Union City, CA, USA) according to the manufacturer’s instructions. Then, the purified PCR fragments were sequenced by the Beijing Genomics Institute (Beijing, China). Assembly of the obtained sequences was performed using the SeqMan program of the
5 (9.80 %)
4 (18.18 %) 54 (12 %) 21 (95.45 %) 108 (24 %)
12 (23.53 %) 20 (39.22 %)
19 (86.36 %) 101 (22.44 %) 5 (22.73 %) 18 (4 %)
2 (3.92 %) 42 (82.35 %)
21 (95.45 %) 213 (47.33 %) 5 (22.73 %) 97 (21.56 %)
5 (9.80 %) 14 (27.45 %)
22 (100 %) 203 (45.11 %) 20 (90.91 %) 178 (39.56 %)
21 (41.18 %) 51
22 450
2013
2014 Total
5 (22.72 %) 25 (5.56 %)
40
2 (3.92 %)
0 (0 %)
5 (12.5 %) 5 (12.5 %) 6 (15 %) 1 (2.5 %) 24 (60 %) 6 (15 %) 9 (22.5 %) 10 (25 %)
0 (0 %)
2012
1 (2.5 %)
7 (38.89 %)
0 (0 %) 0 (0 %)
6 (33.33 %) 0 (0 %)
0 (0 %) 0 (0 %)
12 (66.67 %) 1 (5.56 %)
16.67 (0 %) 4 (66.67 %)
9 (50 %) 9 (50 %)
3 (50 %) 0 (0 %)
0 (0 %)
6
18 2011
70 2009
2010
6 (10.91 %)
9 (12.86 %) 6 (8.57 %)
15 (27.27 %) 12 (21.82 %)
9 (12.86 %) 5 (7.14 %)
0 (0 %) 18 (32.73 %)
15 (21.43 %) 23 (32.86 %)
12 (21.82 %) 39 (70.91 %)
19 (27.14 %) 24 (34.29 %)
27 (49.09 %) 55 2008
7 (10 %)
70
2 (3.64 %)
9 (25 %)
6 (8.57 %) 5 (7.14 %) 6 (8.57 %) 0 (0 %) 15 (21.43 %) 15 (21.43 %) 29 (41.43 %) 24 (34.29 %)
9 (12 %)
2007
1 (1.43 %)
14 (38.89 %) 10 (27.78 %) 3 (8.33 %) 22 (61.11 %) 11 (30.56 %) 22 (61.11 %) 16 (44.4 %) 36 2006
4 (11.1 %)
1 (14.29 %) 2 (28.57 %)
21 (28 %) 12 (16 %)
1 (14.29 %) 0 (0 %)
2 (2.67 %) 40 (53.33 %)
4 (57.14 %) 1 (14.29 %)
17 (22.67 %) 33 (44 %)
3 (42.86 %) 1 (14.29 %)
23 (30.67 %) 2005
0 (0 %) 7
75
2004
PPV4 PPV3 PPV2 PPV1
3 (4 %)
PPV4 PPV3 PPV2 PPV1
Prevalence of concurrent PPVs/PCV2 infection Prevalence of PCV2 Prevalence of PPVs Number Year
Table 2 Frequency of porcine parvoviruses (PPVs) and porcine circovirus type 2 (PCV2) and their co-infections occurrence detected by polymerase chain reaction (PCR) in tissues collected from 2004 to 2014 in China
Porcine parvoviruses and porcine circovirus type 2 in China
Lasergene software (DNASTAR, USA). Phylogenetic trees were constructed by the neighbor-joining (NJ) method, with 1000 bootstrap replicates, using Molecular Evolutionary Genetics Analysis v5.1 software (www.megasoft ware.net/). Mutations based on amino acid sequences were analyzed using the Quick Alignment method, with a gap penalty of 3 using DNAMAN v6.0 software (http://www. lynnon.com/dnaman.html). Out of the ten amplified PPV1 VP2 sequences, only KP245936 joined Cluster A, which included two representative isolates of PPV1, NADL-2 (non-virulent) and Kresse (virulent). The remaining nine sequences (KP245930–KP245935 and KP245937–KP245939), together with that of one Chinese strain (EU790642) retrieved from GenBank, formed a separate cluster (Cluster B), indicating a large phylogenetic distance between Chinese strains and those foreign ones. The phylogenetic trees were shown in Fig. 1A. Homology analysis based on amino acid sequences was carried out, and the mutated residues were listed in the Supplementary Table 1A. Notably, there was an amino acid insertion at residue 366 in KP245939 and KP245937 that originated in 2008 and 2014, respectively. Additionally, among the three most important amino acid residues (379, 384, and 437) that are responsible for the distinct biological properties of the NADL-2 and Kresse strains [2], residue 437 might be the main factor critical to the formation of Cluster B. Whether the insertion of a tyrosine at residue 366 would affect the antigenic characteristics of PPV1 VP2, and whether strains of Cluster B have different virulence properties compared with those of Cluster A, needs further investigation. As for PPV2, one nearly full-length PPV2 genome (KP245947), together with another seven PPV2 capsid genes (KP245940–KP245946), were sequenced. Only KP245946 isolated from 2013 grouped with the originally identified PPV2 sequences from Myanmar (AB076669), while the others grouped with the sequences from China (Fig. 1B). Amino acid analysis indicated that residues 349, 437, 598, 689, 784,795/796, and 990 might be critical for differentiating the lineages of PPV2 (Supplementary Table 1B). Regarding the phylogenetic tree of PPV3 (Fig. 1C), the five sequences identified in this study (KP245948– KP245952) only clustered with those from Hong Kong, which further illustrated that the Chinese mainland and Hong Kong strains shared a certain degree of homology [11]. An analysis based on amino acid sequences showed that there were only four mutated residues (Supplementary Table 1C) in the complete major capsid genes compared with the initial isolates from Hong Kong, indicating the high conservation of the PPV3 capsid protein. Strangely, the only PPV4 capsid gene identified in this study (KP245953) did not cluster with either the Chinese strains archived in GenBank or the American strains, but
123
J. Sun et al. PPV1-7, 2009, KP245931
67 98
A
100 76
100 92
97 PPV1-5, 2009, KP245938 China, 2009, EU790642 PPV1-74, 2009, KP245930
62 48
Cluster B
77 81 100
99
100
China, 2012, JN860197 USA, 1996, U44978 100
98
China, 2009, AY583318
Cluster A
100
USA, 2012, NC001718 Korea, 2003, AY390557
42
PPV2-11G, 2012, KP245943 PPV2-10C, 2012, KP245942 China, 2006-2007, GU938299
PPV2-L, 2013, KP245945 China, 2006-2007, GU938300 PPV2-215, 2007, KP245947 China, 2006-2007, GU938301 PPV2-K, 2013, KP245946 Myanmar, 2001, AB076669
PPV1-77, 2014, KP245936
94
Cluster B
Cluster A
0.005
Germany, 2006, AY684872
59
PPV2-15A, 2012, KP245940 PPV2-20B, 2012, KP245941 PPV2-8H, 2012, KP245944
93
PPV1-10, 2005, KP245932 PPV1-59, 2006, KP245933
50
39
B
PPV1-HJ, 2004, KP245934
PPV1-LH, 2005, KP245935 PPV1-HB, 2014, KP245937 PPV1-249, 2008, KP245939
0.001
HongKong, 2005-2007, EU200677
93
C
78
99
D
PPV3-26, 2009, KP245949
61 95 88
94
PPV3-208, 2007, KP245951 PPV3-211, 2007, KP245952
56
HongKong, 2005-2007, EU200675 HongKong, 2005-2007, EU200676
93
Cluster A
HongKong, 2005-2007, EU200672 PPV3-205, 2007, KP245950 HongKong, 2005-2007, EU200674 HongKong, 2005-2007, EU200673 PPV3-7, 2014, KP245948
0.008
0.006
0.004
0.002
Cluster A
75 China, 2010, GU978968 China, 2010, GU978964 92 China, 2010, GU978965 USA, 2009-2010, GQ387500 USA, 2009-2010, GQ387499 Cluster B 94 USA, 2009-2010, NC014665 PPV4-HB3, 2012, KP245953
0.001
0.000
Cluster B
0.004
Romania, 2006-2007, JF738351
59
China, 2010, GU978966 China, 2010, GU978967
100
Romania, 2010-201, 1JF738368 HongKong, 2005-2007, EU200671
China, 2010, HM031134 China, 2010, HM031135
0.003
0.002
0.000
E
0.035
PCV2-F9, 2014, KP245919
61
PCV2-8C3, 2012, KP245922 PCV2-L4, 2014, KP245918 PCV2-C6, 2013, KP245925 China, 2012, HQ395061 China, 2010, HM038017 China, 2012, HQ395032
100
USA, 2012, JX535296 99
USA, 2012, JX535297 PCV2-C2, 2012, KP245921 China, 2009, AY181946
100
PCV2b
PCV2-C7, 2013, KP245926
64
PCV2-C9, 2012, KP245928 PCV2-C4, 2013, KP245923 PCV2-C1, 2012, KP245920 PCV2-C5, 2013, KP245924 U.K., 2009, AF055394 2B
100 60
PCV2-C8, 2013, KP245927
63
PCV2-C10, 2013, KP245929 China, 2010, HM038034 U.K., 2009, AF055392 2A
72 100 80 75 0.04
0.03
0.02
0.01
China, 2012, HQ395054
PCV2a
SOUTH KOREA, 2012, FR823451 Canada, 2012, JQ994269 0.00
Fig. 1 (A-E) Phylogenetic trees based on the complete major capsid genes of porcine parvoviruses (PPVs) and porcine circovirus type 2 (PCV2). A: PPV1, B: PPV2, C: PPV3, D: PPV4, E: PCV2. The newly identified PPVs and PCV2 strains in the present study are indicated
with ‘‘d’’. Other known sequences retrieved from GenBank are indicated by their origins, years of isolation, and accession numbers. The phylogenetic trees were constructed by the neighbor-joining method, with 1,000 bootstrap replicates, using MEGA5.1 software
instead formed a separate branch (Fig. 1D). Amino acid sequence analysis suggested that the predominant residues responsible for phylogenetic division were at positions 455
and 469 (Supplementary Table 2D). As 3D structural models for novel PPVs had not been established, comparisons based on amino acid sequences might provide some
123
Porcine parvoviruses and porcine circovirus type 2 in China
clues regarding viral evolution, although further studies are required to examine this. Among the 12 amplified PCV2 capsid genes, four sequences (KP245918, KP245919, KP245922 and KP245925) grouped with mutant PCV2b strains discovered in China (HM038017) and in the USA (JX535296 and JX535297), which were reported to be more virulent than classical PCV2a/2b strains [14]. Another six sequences (KP245920, KP245923, KP245924 and KP245926– KP245928) grouped with the traditional PCV2b strains (AF055394) isolated in France in 2009, whereas KP245921 was located separately between these two branches (Fig. 1E). Furthermore, there was one particular sequence, KP245929, that possessed amino acid characteristics of PCV2a as well as PCV2b and formed a separate branch between the two genotype clusters. The virulence properties of KP245929 and whether it represents a different genotype remain to be explored. Further analysis based on the amino acid sequences revealed that ten residues (Y-8-F, F-53-I, A-68-N, R-89-L, S-90-T, S-121-T, T-134-N, S-169-R, E-210-D, and V-215-I) were responsible for the distinction between traditional PCV2b and mPCV2b (Supplementary Table 1E). Additionally, it is likely that these identified residues are related to the virulence of PCV2b. We think it is necessary to note that PCV2b is playing a predominant role in clinical infections compared with PCV2a, and the high prevalence of PCV2 described in this study further confirmed that the current PCV2a-based commercial vaccines are not fully protective against PCV2b [15]. However, in consideration of the complexity of field conditions, this hypothesis requires more investigations. In conclusion, we report the prevalence of PPVs and their co-infection rates with PCV2 in domestic pigs, and the availability of full-length sequences of major capsid genes has allowed us to conduct a comprehensive phylogenetic analysis and amino acid sequence comparison of different strains. To the best of our knowledge, this is the first analysis of the complete capsid genes of novel PPVs and classical PPV1 and their co-infections with PCV2 in China. We believe that our data will provide valuable information for later diagnostic investigations of diseases in pigs, and it will support future molecular analyses. As novel PPVs are highly prevalent and their isolation in vitro is difficult [17], we suggest the preparation of subunit vaccines as an effective way of inducing specific antibodies to protect swine from continuous infections. Additionally, the great economic losses caused by PCVAD suggest that monitoring of the pathogenic potential of emerging, highly prevalent PPVs is needed. Acknowledgments This study was supported by the National High Technology R&D Program (863) of China (No. 2011AA10A208), the
National Natural Science Foundation of China (No. 31302110), the Public Welfare Special Funds for Agricultural Scientific Research (No. 201203039), and the State Key Laboratory of Veterinary Biotechnology (No. SKLVBP201411).
References 1. Adlhoch C, Kaiser M, Ellerbrok H, Pauli G (2010) High prevalence of porcine Hokovirus in German wild boar populations. Virol J 7:171 2. Bergeron J, Hebert B, Tijssen P (1996) Genome organization of the Kresse strain of porcine parvovirus: identification of the allotropic determinant and comparison with those of NADL-2 and field isolates. J Virol 70(4):2508–2515 3. Cadar D, Csagola A, Lorincz M, Tombacz K, Spinu M, Tuboly T (2011) Distribution and genetic diversity of porcine hokovirus in wild boars. Arch Virol 156(12):2233–2239 4. Cadar D, Csagola A, Kiss T, Tuboly T (2013) Capsid protein evolution and comparative phylogeny of novel porcine parvoviruses. Mol Phylogenet Evol 66(1):243–253 5. Chae C (2005) A review of porcine circovirus 2-associated syndromes and diseases. Vet J 169(3):326–336 6. Cheung AK, Wu G, Wang D, Bayles DO, Lager KM, Vincent AL (2010) Identification and molecular cloning of a novel porcine parvovirus. Arch Virol 155(5):801–806 7. Csagola A, Lorincz M, Cadar D, Tombacz K, Biksi I, Tuboly T (2012) Detection, prevalence and analysis of emerging porcine parvovirus infections. Arch Virol 157(6):1003–1010 8. Hijikata M, Abe K, Win KM, Shimizu YK, Keicho N, Yoshikura H (2001) Identification of new parvovirus DNA sequence in swine sera from Myanmar. Jpn J Infect Dis 54(6):244–245 9. Huang L, Zhai SL, Cheung AK, Zhang HB, Long JX, Yuan SS (2010) Detection of a novel porcine parvovirus, PPV4, in Chinese swine herds. Virol J 7:333 10. Lau SK, Woo PC, Tse H, Fu CT, Au WK, Chen XC, Tsoi HW, Tsang TH, Chan JS, Tsang DN, Li KS, Tse CW, Ng TK, Tsang OT, Zheng BJ, Tam S, Chan KH, Zhou B, Yuen KY (2008) Identification of novel porcine and bovine parvoviruses closely related to human parvovirus 4. J Gen Virol 89:1840–1848 11. Li S, Wei Y, Liu J, Tang Q, Liu C (2013) Prevalence of porcine hokovirus and its co-infection with porcine circovirus 2 in China. Arch Virol 158(9):1987–1991 12. Meehan BM, McNeilly F, Todd D, Kennedy S, Jewhurst VA, Ellis JA, Hassard LE, Clark EG, Haines DM, Allan GM (1998) Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. J Gen Virol 79:2171–2179 13. Opriessnig T, Fenaux M, Yu S, Evans RB, Cavanaugh D, Gallup JM, Pallares FJ, Thacker EL, Lager KM, Meng XJ, Halbur PG (2004) Effect of porcine parvovirus vaccination on the development of PMWS in segregated early weaned pigs coinfected with type 2 porcine circovirus and porcine parvovirus. Vet Microbiol 98(3–4):209–220 14. Opriessnig T, Xiao CT, Gerber PF, Halbur PG (2013) Emergence of a novel mutant PCV2b variant associated with clinical PCVAD in two vaccinated pig farms in the U.S. concurrently infected with PPV2. Vet Microbiol 163(1–2):177–183 15. Opriessnig T, Xiao CT, Gerber PF, Halbur PG (2014) Identification of recently described porcine parvoviruses in archived North American samples from 1996 and association with porcine circovirus associated disease. Vet Microbiol 173(1–2):9–16 16. Ostanello F, Caprioli A, Di Francesco A, Battilani M, Sala G, Sarli G, Mandrioli L, McNeilly F, Allan GM, Prosperi S (2005)
123
J. Sun et al. Experimental infection of 3-week-old conventional colostrum-fed pigs with porcine circovirus type 2 and porcine parvovirus. Vet Microbiol 108(3–4):179–186 17. Streck AF, Homeier T, Foerster T, Fischer S, Truyen U (2013) Analysis of porcine parvoviruses in tonsils and hearts from healthy pigs reveals high prevalence and genetic diversity in Germany. Arch Virol 158(8):1173–1180
123
18. Wang F, Wei Y, Zhu C, Huang X, Xu Y, Yu L, Yu X (2010) Novel parvovirus sublineage in the family of Parvoviridae. Virus Genes 41(2):305–308
BRIEF REPORT
Prevalence of emerging porcine parvoviruses and their co-infections with porcine circovirus type 2 in China Jianhui Sun • Liping Huang • Yanwu Wei • Yiping Wang • Dongjie Chen • Wenjuan Du Hongli Wu • Changming Liu
•
Received: 23 December 2014 / Accepted: 15 February 2015 Ó Springer-Verlag Wien 2015
Abstract A total of 450 samples from domestic pigs in China were tested for porcine parvoviruses (PPVs) and coinfections with porcine circovirus type 2 (PCV2), and their complete capsid genes were sequenced. The prevalence of PPV1, PPV2, PPV3, PPV4, and PCV2 was 5.56 %, 39.56 %, 45.11 %, 21.56 %, and 47.33 %, respectively, and co-infection with PCV2 occurred in 4 % (PPV1), 22.44 % (PPV2), 24 % (PPV3), and 12 % (PPV4) of the samples. Phylogenetic analysis revealed two main lineages for each virus, and residues that differentiated these viruses were identified. The co-infections of emerging PPVs and PCV2 were prevalent, indicating their cooperative roles in porcine circovirus-associated diseases.
Parvoviruses are small, single-stranded DNA viruses that cause severe clinical diseases, as well as mild and subclinical infections. They belong to the family Parvoviridae, which is divided into two subfamilies (Parvovirinae and Densovirinae) based on distinct host tropisms. Additionally, the subfamily Parvovirinae is further subdivided into eight genera. The classical porcine parvovirus type 1 (PPV1) belongs to the genus Protoparvovirus and is J. Sun and L. Huang contributed equally to this article.
Electronic supplementary material The online version of this article (doi:10.1007/s00705-015-2373-7) contains supplementary material, which is available to authorized users. J. Sun L. Huang Y. Wei Y. Wang D. Chen W. Du H. Wu C. Liu (&) Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Harbin 150001, China e-mail: [email protected]
responsible for causing reproductive failure in swine, usually described as stillbirth, mummification, embryonic death, and infertility [1]. Porcine circovirus type 2 (PCV2), another small DNA virus with a circular genome, acts as the dominant causative agent of post-weaning multisystemic wasting syndrome (PMWS) [12] and is associated with other porcine circovirus-associated diseases (PCVAD), which include porcine reproductive disorders, porcine dermatitis, nephropathy syndrome, necrotic lymphadenitis, and porcine respiratory disease [5]. Additionally, enhanced PCVAD- and PCV2-related lesions have been found in pigs co-infected with PPV1 and PCV2 [13, 16]. As a result of the widespread use of molecular detection technologies, several novel PPVs have been detected in pigs. Porcine parvovirus type 2 (PPV2) was first found in swine sera in Myanmar [8], and it shares high genetic similarity with subsequently discovered parvoviruses in China, which were found in pigs with clinical symptoms of ‘‘high fever’’ and PMWS [18]. Porcine parvovirus type 3 (PPV3) was identified in Hong Kong in 2008, and initially termed porcine hokovirus. Recent diagnostic investigations have suggested a widespread distribution of PPV3 in wild boars (50.54 %) [3] and domestic pigs (51.3 %) [11]. Porcine parvovirus type 4 (PPV4) was originally reported in North Carolina (USA) and found in swine herds that were co-infected with PCV2 and afflicted with PCVAD [6]. Until now, the specific pathogenic mechanisms of novel PPVs have not been well elucidated, but their prevalence has been reported to be higher in PCVAD cases than in non-PCVAD cases [15], indicating their active roles in PCVAD [11, 15]. Additionally, the high detection rate of PPV2 in the hearts (55 %) and tonsils (78 %) of healthy fattening pigs in Germany is striking [17]. As there have been no reports simultaneously describing emerging PPVs and their relationships with PCV2
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J. Sun et al. Table 1 Primers used for the detection of porcine parvovirus type 1 (PPV1) and amplification of complete major capsid genes of porcine parvoviruses (PPVs) and porcine circovirus type 2 (PCV2) Primer name
Primer sequence
Locationa
Lengthb
PPV1-DF
5’-AATAGAAACCGACATAAGAGCCTGC-3’
2217-2679
463
PPV1-DR
5’-TGGTTGTTGAGATGTAGTTGGTGAG-3’
PCV2-AF
5’-CGTAATTGTTTTTATTATT-3’
1012-1755
744
PCV2-AR
5’-CACTTCTTTCGCTTTCAGCT-3’
PPV1-AF
5’-GCTCTAGAGCCACCATGGGTGAAAATGTGGAACAACACAA-3’
2810-4549
1740
PPV1-AR
5’-AAAACTGCAGTCAATGGTGATGGTGATGATGGTATAATTTTCTTGGTATAAGTTG-3’
PPV2-AF PPV2-AR
5’-GGACGTCGAGGAGGAACCAA-3’ 5’-TATACACGATGAGCGCGTCC-3’
1922-53548
3427
PPV3-AF
5’-ATGGCTGCGCCGGTAACGGGCGCCACCGGTGGG-3’
3349-5016
1668
PPV3-AR
5’-TTACAATTTGCGGGAGAAATTCTGGGCAACCCACTGATCAGAAGGAA-3’
PPV4-AF
5’-ATGCAGAAACACGGTCACTGGCCTCATTTATGGGC-3’
3681-5867
2187
PPV4-AR
5’-TCATCTGCGGTGTCTGGGTCTCTGTTTTGCGGT-3’
a
Nucleotide positions for PPV1, PPV2, PPV3, PPV4 and PCV2 are according to those of NADL-2 (GenBank no.: NC001718), JH13 (GenBank no.: GU938300), HK7 (GenBank no.: EU200677), GQ387499 (GenBank no.: GQ387499), and AF055392 (GenBank no.: AF055392), respectively. F, forward orientation; R, reverse orientation. b Amplicon size is indicated as base pairs
published to date in China, the objective of this study is to determine the prevalence of classical PPV1 and emerging PPV2–4, as well as their co-infections with PCV2, in archived and recently collected domestic pig samples. The clinical tissue samples (lungs, livers, kidneys, brains, lymph nodes, or spleens) from suspected PCVAD pigs were collected and stored at -80 °C. A total of 450 specimens, collected from 2004 to 2014 in different areas of China, were grouped according to their date of collection and availability. Tissue samples of approximately 0.1 g were ground in minimal essential medium (Gibco, USA) using a TissueLyser (Qiagen, Hilden, Germany). Total nucleic acids were extracted from sample homogenates using a TIANamp Genomic DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was used to test all the samples for the presence of PPVs and PCV2 using an Ex Taq DNA polymerase kit (Takara, Dalian, China). Primers used to detect PCV2, PPV2, PPV3, and PPV4 were described previously [4, 11, 15]. Primers for the detection of PPV1 were designed using Primer Premier 5.0 software (www.premierbiosoft. com/primerdesign/index.html) according to the conserved regions in the NS1 gene of the reference PPV1 sequence (accession no.: NC_001718) (Table 1). Our results showed that among the 450 tissue samples tested, 25 (5.56 %) were PPV1 positive, 178 (39.56 %) were PPV2 positive, 203 (45.11 %) were PPV3 positive, 97 (21.56 %) were PPV4 positive, and 213 (47.33 %) were PCV2 positive. The PPV3- and PPV4-positive rates were significantly higher than those in North America (9.4 % for PPV3 and 2.9 % for PPV4) [15] and in Hungary (9.7 % for PPV3 and 6.4 % for PPV4) [7], while the frequency of PPV2 resembled that in North America (35.2 %) but
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considerably exceeded that in Hungary (6.4 %). Furthermore, the concurrent infection rates with PCV2 of novel PPVs (22.44 % for PPV2, 24 % for PPV3, and 12 % for PPV4) far exceeded that of classical PPV1 (4 %) (Table 2). Above all, PPV2, PPV3, and PPV4 were all detected in tissues from 2004 onward, and this was the first report that PPV4 could be detected in China prior to 2009 [9]. The genomes of the PPVs and PCV2 mainly include two open reading frames (ORFs), ORF1 and ORF2. ORF1 encodes non-structural proteins, whereas ORF2 encodes viral capsid proteins [6, 8, 10]. Recently, a relatively high evolutionary rate, similar to that of RNA viruses, is observed for emerging PPVs [4]. To investigate the possibility of genetic changes among the different strains, further sequencing of the full-length major capsid genes was conducted. Based on the time of sampling and availability, we selected ten PPV1-, eight PPV2-, five PPV3-, one PPV4-, and 12 PCV2-positive samples, and the target genes were amplified using the amplification primers listed in Table 1. PCRs were performed using a KOD FX Neo kit (Toyobo, Shanghai, China) according to the manufacturer’s instructions. The PCR conditions included an initial denaturation step at 94 °C for 2 min, followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 63 °C (PPV1), 60 °C (PPV2), 68 °C (PPV3), 60 °C (PPV4), or 55 °C (PCV2) for 30 s, an extension at 68 °C for 2 min, and a final elongation step at 68 °C for 5 min. The PCR products were purified using an AxyPrey DNA Gel Extraction Kit (Axygen Scientific, Inc., Union City, CA, USA) according to the manufacturer’s instructions. Then, the purified PCR fragments were sequenced by the Beijing Genomics Institute (Beijing, China). Assembly of the obtained sequences was performed using the SeqMan program of the
5 (9.80 %)
4 (18.18 %) 54 (12 %) 21 (95.45 %) 108 (24 %)
12 (23.53 %) 20 (39.22 %)
19 (86.36 %) 101 (22.44 %) 5 (22.73 %) 18 (4 %)
2 (3.92 %) 42 (82.35 %)
21 (95.45 %) 213 (47.33 %) 5 (22.73 %) 97 (21.56 %)
5 (9.80 %) 14 (27.45 %)
22 (100 %) 203 (45.11 %) 20 (90.91 %) 178 (39.56 %)
21 (41.18 %) 51
22 450
2013
2014 Total
5 (22.72 %) 25 (5.56 %)
40
2 (3.92 %)
0 (0 %)
5 (12.5 %) 5 (12.5 %) 6 (15 %) 1 (2.5 %) 24 (60 %) 6 (15 %) 9 (22.5 %) 10 (25 %)
0 (0 %)
2012
1 (2.5 %)
7 (38.89 %)
0 (0 %) 0 (0 %)
6 (33.33 %) 0 (0 %)
0 (0 %) 0 (0 %)
12 (66.67 %) 1 (5.56 %)
16.67 (0 %) 4 (66.67 %)
9 (50 %) 9 (50 %)
3 (50 %) 0 (0 %)
0 (0 %)
6
18 2011
70 2009
2010
6 (10.91 %)
9 (12.86 %) 6 (8.57 %)
15 (27.27 %) 12 (21.82 %)
9 (12.86 %) 5 (7.14 %)
0 (0 %) 18 (32.73 %)
15 (21.43 %) 23 (32.86 %)
12 (21.82 %) 39 (70.91 %)
19 (27.14 %) 24 (34.29 %)
27 (49.09 %) 55 2008
7 (10 %)
70
2 (3.64 %)
9 (25 %)
6 (8.57 %) 5 (7.14 %) 6 (8.57 %) 0 (0 %) 15 (21.43 %) 15 (21.43 %) 29 (41.43 %) 24 (34.29 %)
9 (12 %)
2007
1 (1.43 %)
14 (38.89 %) 10 (27.78 %) 3 (8.33 %) 22 (61.11 %) 11 (30.56 %) 22 (61.11 %) 16 (44.4 %) 36 2006
4 (11.1 %)
1 (14.29 %) 2 (28.57 %)
21 (28 %) 12 (16 %)
1 (14.29 %) 0 (0 %)
2 (2.67 %) 40 (53.33 %)
4 (57.14 %) 1 (14.29 %)
17 (22.67 %) 33 (44 %)
3 (42.86 %) 1 (14.29 %)
23 (30.67 %) 2005
0 (0 %) 7
75
2004
PPV4 PPV3 PPV2 PPV1
3 (4 %)
PPV4 PPV3 PPV2 PPV1
Prevalence of concurrent PPVs/PCV2 infection Prevalence of PCV2 Prevalence of PPVs Number Year
Table 2 Frequency of porcine parvoviruses (PPVs) and porcine circovirus type 2 (PCV2) and their co-infections occurrence detected by polymerase chain reaction (PCR) in tissues collected from 2004 to 2014 in China
Porcine parvoviruses and porcine circovirus type 2 in China
Lasergene software (DNASTAR, USA). Phylogenetic trees were constructed by the neighbor-joining (NJ) method, with 1000 bootstrap replicates, using Molecular Evolutionary Genetics Analysis v5.1 software (www.megasoft ware.net/). Mutations based on amino acid sequences were analyzed using the Quick Alignment method, with a gap penalty of 3 using DNAMAN v6.0 software (http://www. lynnon.com/dnaman.html). Out of the ten amplified PPV1 VP2 sequences, only KP245936 joined Cluster A, which included two representative isolates of PPV1, NADL-2 (non-virulent) and Kresse (virulent). The remaining nine sequences (KP245930–KP245935 and KP245937–KP245939), together with that of one Chinese strain (EU790642) retrieved from GenBank, formed a separate cluster (Cluster B), indicating a large phylogenetic distance between Chinese strains and those foreign ones. The phylogenetic trees were shown in Fig. 1A. Homology analysis based on amino acid sequences was carried out, and the mutated residues were listed in the Supplementary Table 1A. Notably, there was an amino acid insertion at residue 366 in KP245939 and KP245937 that originated in 2008 and 2014, respectively. Additionally, among the three most important amino acid residues (379, 384, and 437) that are responsible for the distinct biological properties of the NADL-2 and Kresse strains [2], residue 437 might be the main factor critical to the formation of Cluster B. Whether the insertion of a tyrosine at residue 366 would affect the antigenic characteristics of PPV1 VP2, and whether strains of Cluster B have different virulence properties compared with those of Cluster A, needs further investigation. As for PPV2, one nearly full-length PPV2 genome (KP245947), together with another seven PPV2 capsid genes (KP245940–KP245946), were sequenced. Only KP245946 isolated from 2013 grouped with the originally identified PPV2 sequences from Myanmar (AB076669), while the others grouped with the sequences from China (Fig. 1B). Amino acid analysis indicated that residues 349, 437, 598, 689, 784,795/796, and 990 might be critical for differentiating the lineages of PPV2 (Supplementary Table 1B). Regarding the phylogenetic tree of PPV3 (Fig. 1C), the five sequences identified in this study (KP245948– KP245952) only clustered with those from Hong Kong, which further illustrated that the Chinese mainland and Hong Kong strains shared a certain degree of homology [11]. An analysis based on amino acid sequences showed that there were only four mutated residues (Supplementary Table 1C) in the complete major capsid genes compared with the initial isolates from Hong Kong, indicating the high conservation of the PPV3 capsid protein. Strangely, the only PPV4 capsid gene identified in this study (KP245953) did not cluster with either the Chinese strains archived in GenBank or the American strains, but
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J. Sun et al. PPV1-7, 2009, KP245931
67 98
A
100 76
100 92
97 PPV1-5, 2009, KP245938 China, 2009, EU790642 PPV1-74, 2009, KP245930
62 48
Cluster B
77 81 100
99
100
China, 2012, JN860197 USA, 1996, U44978 100
98
China, 2009, AY583318
Cluster A
100
USA, 2012, NC001718 Korea, 2003, AY390557
42
PPV2-11G, 2012, KP245943 PPV2-10C, 2012, KP245942 China, 2006-2007, GU938299
PPV2-L, 2013, KP245945 China, 2006-2007, GU938300 PPV2-215, 2007, KP245947 China, 2006-2007, GU938301 PPV2-K, 2013, KP245946 Myanmar, 2001, AB076669
PPV1-77, 2014, KP245936
94
Cluster B
Cluster A
0.005
Germany, 2006, AY684872
59
PPV2-15A, 2012, KP245940 PPV2-20B, 2012, KP245941 PPV2-8H, 2012, KP245944
93
PPV1-10, 2005, KP245932 PPV1-59, 2006, KP245933
50
39
B
PPV1-HJ, 2004, KP245934
PPV1-LH, 2005, KP245935 PPV1-HB, 2014, KP245937 PPV1-249, 2008, KP245939
0.001
HongKong, 2005-2007, EU200677
93
C
78
99
D
PPV3-26, 2009, KP245949
61 95 88
94
PPV3-208, 2007, KP245951 PPV3-211, 2007, KP245952
56
HongKong, 2005-2007, EU200675 HongKong, 2005-2007, EU200676
93
Cluster A
HongKong, 2005-2007, EU200672 PPV3-205, 2007, KP245950 HongKong, 2005-2007, EU200674 HongKong, 2005-2007, EU200673 PPV3-7, 2014, KP245948
0.008
0.006
0.004
0.002
Cluster A
75 China, 2010, GU978968 China, 2010, GU978964 92 China, 2010, GU978965 USA, 2009-2010, GQ387500 USA, 2009-2010, GQ387499 Cluster B 94 USA, 2009-2010, NC014665 PPV4-HB3, 2012, KP245953
0.001
0.000
Cluster B
0.004
Romania, 2006-2007, JF738351
59
China, 2010, GU978966 China, 2010, GU978967
100
Romania, 2010-201, 1JF738368 HongKong, 2005-2007, EU200671
China, 2010, HM031134 China, 2010, HM031135
0.003
0.002
0.000
E
0.035
PCV2-F9, 2014, KP245919
61
PCV2-8C3, 2012, KP245922 PCV2-L4, 2014, KP245918 PCV2-C6, 2013, KP245925 China, 2012, HQ395061 China, 2010, HM038017 China, 2012, HQ395032
100
USA, 2012, JX535296 99
USA, 2012, JX535297 PCV2-C2, 2012, KP245921 China, 2009, AY181946
100
PCV2b
PCV2-C7, 2013, KP245926
64
PCV2-C9, 2012, KP245928 PCV2-C4, 2013, KP245923 PCV2-C1, 2012, KP245920 PCV2-C5, 2013, KP245924 U.K., 2009, AF055394 2B
100 60
PCV2-C8, 2013, KP245927
63
PCV2-C10, 2013, KP245929 China, 2010, HM038034 U.K., 2009, AF055392 2A
72 100 80 75 0.04
0.03
0.02
0.01
China, 2012, HQ395054
PCV2a
SOUTH KOREA, 2012, FR823451 Canada, 2012, JQ994269 0.00
Fig. 1 (A-E) Phylogenetic trees based on the complete major capsid genes of porcine parvoviruses (PPVs) and porcine circovirus type 2 (PCV2). A: PPV1, B: PPV2, C: PPV3, D: PPV4, E: PCV2. The newly identified PPVs and PCV2 strains in the present study are indicated
with ‘‘d’’. Other known sequences retrieved from GenBank are indicated by their origins, years of isolation, and accession numbers. The phylogenetic trees were constructed by the neighbor-joining method, with 1,000 bootstrap replicates, using MEGA5.1 software
instead formed a separate branch (Fig. 1D). Amino acid sequence analysis suggested that the predominant residues responsible for phylogenetic division were at positions 455
and 469 (Supplementary Table 2D). As 3D structural models for novel PPVs had not been established, comparisons based on amino acid sequences might provide some
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Porcine parvoviruses and porcine circovirus type 2 in China
clues regarding viral evolution, although further studies are required to examine this. Among the 12 amplified PCV2 capsid genes, four sequences (KP245918, KP245919, KP245922 and KP245925) grouped with mutant PCV2b strains discovered in China (HM038017) and in the USA (JX535296 and JX535297), which were reported to be more virulent than classical PCV2a/2b strains [14]. Another six sequences (KP245920, KP245923, KP245924 and KP245926– KP245928) grouped with the traditional PCV2b strains (AF055394) isolated in France in 2009, whereas KP245921 was located separately between these two branches (Fig. 1E). Furthermore, there was one particular sequence, KP245929, that possessed amino acid characteristics of PCV2a as well as PCV2b and formed a separate branch between the two genotype clusters. The virulence properties of KP245929 and whether it represents a different genotype remain to be explored. Further analysis based on the amino acid sequences revealed that ten residues (Y-8-F, F-53-I, A-68-N, R-89-L, S-90-T, S-121-T, T-134-N, S-169-R, E-210-D, and V-215-I) were responsible for the distinction between traditional PCV2b and mPCV2b (Supplementary Table 1E). Additionally, it is likely that these identified residues are related to the virulence of PCV2b. We think it is necessary to note that PCV2b is playing a predominant role in clinical infections compared with PCV2a, and the high prevalence of PCV2 described in this study further confirmed that the current PCV2a-based commercial vaccines are not fully protective against PCV2b [15]. However, in consideration of the complexity of field conditions, this hypothesis requires more investigations. In conclusion, we report the prevalence of PPVs and their co-infection rates with PCV2 in domestic pigs, and the availability of full-length sequences of major capsid genes has allowed us to conduct a comprehensive phylogenetic analysis and amino acid sequence comparison of different strains. To the best of our knowledge, this is the first analysis of the complete capsid genes of novel PPVs and classical PPV1 and their co-infections with PCV2 in China. We believe that our data will provide valuable information for later diagnostic investigations of diseases in pigs, and it will support future molecular analyses. As novel PPVs are highly prevalent and their isolation in vitro is difficult [17], we suggest the preparation of subunit vaccines as an effective way of inducing specific antibodies to protect swine from continuous infections. Additionally, the great economic losses caused by PCVAD suggest that monitoring of the pathogenic potential of emerging, highly prevalent PPVs is needed. Acknowledgments This study was supported by the National High Technology R&D Program (863) of China (No. 2011AA10A208), the
National Natural Science Foundation of China (No. 31302110), the Public Welfare Special Funds for Agricultural Scientific Research (No. 201203039), and the State Key Laboratory of Veterinary Biotechnology (No. SKLVBP201411).
References 1. Adlhoch C, Kaiser M, Ellerbrok H, Pauli G (2010) High prevalence of porcine Hokovirus in German wild boar populations. Virol J 7:171 2. Bergeron J, Hebert B, Tijssen P (1996) Genome organization of the Kresse strain of porcine parvovirus: identification of the allotropic determinant and comparison with those of NADL-2 and field isolates. J Virol 70(4):2508–2515 3. Cadar D, Csagola A, Lorincz M, Tombacz K, Spinu M, Tuboly T (2011) Distribution and genetic diversity of porcine hokovirus in wild boars. Arch Virol 156(12):2233–2239 4. Cadar D, Csagola A, Kiss T, Tuboly T (2013) Capsid protein evolution and comparative phylogeny of novel porcine parvoviruses. Mol Phylogenet Evol 66(1):243–253 5. Chae C (2005) A review of porcine circovirus 2-associated syndromes and diseases. Vet J 169(3):326–336 6. Cheung AK, Wu G, Wang D, Bayles DO, Lager KM, Vincent AL (2010) Identification and molecular cloning of a novel porcine parvovirus. Arch Virol 155(5):801–806 7. Csagola A, Lorincz M, Cadar D, Tombacz K, Biksi I, Tuboly T (2012) Detection, prevalence and analysis of emerging porcine parvovirus infections. Arch Virol 157(6):1003–1010 8. Hijikata M, Abe K, Win KM, Shimizu YK, Keicho N, Yoshikura H (2001) Identification of new parvovirus DNA sequence in swine sera from Myanmar. Jpn J Infect Dis 54(6):244–245 9. Huang L, Zhai SL, Cheung AK, Zhang HB, Long JX, Yuan SS (2010) Detection of a novel porcine parvovirus, PPV4, in Chinese swine herds. Virol J 7:333 10. Lau SK, Woo PC, Tse H, Fu CT, Au WK, Chen XC, Tsoi HW, Tsang TH, Chan JS, Tsang DN, Li KS, Tse CW, Ng TK, Tsang OT, Zheng BJ, Tam S, Chan KH, Zhou B, Yuen KY (2008) Identification of novel porcine and bovine parvoviruses closely related to human parvovirus 4. J Gen Virol 89:1840–1848 11. Li S, Wei Y, Liu J, Tang Q, Liu C (2013) Prevalence of porcine hokovirus and its co-infection with porcine circovirus 2 in China. Arch Virol 158(9):1987–1991 12. Meehan BM, McNeilly F, Todd D, Kennedy S, Jewhurst VA, Ellis JA, Hassard LE, Clark EG, Haines DM, Allan GM (1998) Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. J Gen Virol 79:2171–2179 13. Opriessnig T, Fenaux M, Yu S, Evans RB, Cavanaugh D, Gallup JM, Pallares FJ, Thacker EL, Lager KM, Meng XJ, Halbur PG (2004) Effect of porcine parvovirus vaccination on the development of PMWS in segregated early weaned pigs coinfected with type 2 porcine circovirus and porcine parvovirus. Vet Microbiol 98(3–4):209–220 14. Opriessnig T, Xiao CT, Gerber PF, Halbur PG (2013) Emergence of a novel mutant PCV2b variant associated with clinical PCVAD in two vaccinated pig farms in the U.S. concurrently infected with PPV2. Vet Microbiol 163(1–2):177–183 15. Opriessnig T, Xiao CT, Gerber PF, Halbur PG (2014) Identification of recently described porcine parvoviruses in archived North American samples from 1996 and association with porcine circovirus associated disease. Vet Microbiol 173(1–2):9–16 16. Ostanello F, Caprioli A, Di Francesco A, Battilani M, Sala G, Sarli G, Mandrioli L, McNeilly F, Allan GM, Prosperi S (2005)
123
J. Sun et al. Experimental infection of 3-week-old conventional colostrum-fed pigs with porcine circovirus type 2 and porcine parvovirus. Vet Microbiol 108(3–4):179–186 17. Streck AF, Homeier T, Foerster T, Fischer S, Truyen U (2013) Analysis of porcine parvoviruses in tonsils and hearts from healthy pigs reveals high prevalence and genetic diversity in Germany. Arch Virol 158(8):1173–1180
123
18. Wang F, Wei Y, Zhu C, Huang X, Xu Y, Yu L, Yu X (2010) Novel parvovirus sublineage in the family of Parvoviridae. Virus Genes 41(2):305–308
