Virus Genes DOI 10.1007/s11262-015-1262-1
Bufavirus Protoparvovirus in feces of wild rats in China Shixing Yang1 • Dawei Liu2 • Yan Wang1 • Fanyong Qu3 • Yilin He4 Zixuan Sun1 • Quan Shen1 • Wang Li1 • Xingli Fu1 • Xutao Deng5 • Wen Zhang1 • Eric Delwart5
•
Received: 5 October 2015 / Accepted: 8 November 2015 Ó Springer Science+Business Media New York 2015
Abstract Bufavirus (BuV) was first discovered from feces of children with acute diarrhea. It was subsequently detected from several animal species including shrews, bats, and nonhuman primates. In this study, we identified a novel Protoparvovirus, designated RatBuV, from the intestinal contents of wild rats using viral metagenomics. The near complete genome was 4643 nt encoding NS1, VP1, and VP2 proteins. Phylogenetic analysis over the complete genome showed that RatBuV clustered with Mpulungu BuV from shrews. Sequence analysis indicated that the putative protein sequences of NS1, VP1, and VP2 of RatBuV shared identities of 50.6–77.2, 48.3–77.3, and 47.1–78.3 %, respectively, with those of human BuVs, MpBuV, and WUHARV parvovirus, suggesting RatBuV belongs to a new species of Protoparvovirus. Our
Edited by Juergen A. Richt. Shixing Yang and Dawei Liu have contributed equally to this work. & Wen Zhang
[email protected] 1
School of Medicine, Jiangsu University, Zhenjiang, People’s Republic of China
2
College of Biology and the Environment, Nanjing Forestry University, Nanjing, People’s Republic of China
3
Department of Interventional Medicine, Yantai Affiliated Hospital of Binzhou Medical University, Yantai, People’s Republic of China
4
Division of Acute Infectious Disease Prevention and Control, Taizhou Center for Disease Control and Prevention, Taizhou, People’s Republic of China
5
Department of Laboratory Medicine, Blood Systems Research Institute, University of California San Francisco, San Francisco, CA, USA
epidemiologic study indicated that the prevalence rate of RatBuV in the cohort of 40 wild rats is 12.5 % (5/40), which is higher than that of BuV in humans in a previous study. Keywords Bufavirus Wild rats Phylogentic analysis Genome structure Bufavirus, a novel parvovirus, assigned the Protoparvovirus in the subfamily parvovirinae [1], was originally genetically characterized from diarrheic human feces in Burkina Faso in 2012 [2]. At present, three genotypes of human BuVs have been identified from different countries including Burkina Faso [2], Bhutan [3], Finland [4], Thailand [5], Netherlands [6], and China [7]. Parvoviruses related to human BuVs were also found in wild animals such as shrews, bats, and nonhuman primates [8, 9]. The significant divergence of encoding protein sequences between known animal BuVs and human BuVs suggested different species of BuVs was prevalent in mammals. China’s 1.3 billion people, many from rural areas, still often live in poor sanitary conditions. Wild rats, which are widely present in the rural area, are known as reservoirs and vector for many zoonotic pathogens [10]. In the present study, using viral metagenomics method, we identified a new BuV from the intestinal contents of wild rats and characterized the near complete genome. In order to investigate their intestinal virome, 40 intestinal content samples were collected from wild rats captured by the Chinese Center for Disease Control and Prevention in Taizhou City from three districts of Taizhou City including Taixing (n = 15), Gaogang (n = 15), and Hailing (n = 10) from June to August in 2004. All of the wild rats were adults and the exact age was unknown.
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Virus Genes Fig. 1 New species of BuV genome and phylogeny. a Organization of the BuV genome. The predicted splicing for expression of VP1 is shown. The conserved ATP-binding Walker loop motif and the PLA2 similarity region, including the calcium-binding region and catalytic resides, is also shown. b Phylogenetic analyses of the near complete genome of bufavirus of different genotypes and species. Strain name and GenBank accession numbers of BuVs are shown. Scale bar indicates nucleotide substitutions per site. Bootstrap values (based on 1000 replicates) for each node are given
Viral metagenomics method was used to identify viral sequences in these samples. Four separate pools were randomly generated, each of which contained 10 fecal specimens. After low-speed centrifugation and filtration the samples were treated with DNase and RNase [11], to reduce levels of rat nucleic acids while viral genome are protected from digestion within viral capsid. Four libraries were then constructed using Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using the Miseq Illumina platform with 250 bases paired ends with a distinct molecular tag for each pool. Bioinformatics analysis was performed according to a previous study [12]. Sixty one sequence reads showed the best BLASTx matches to WUHARV parvovirus which was identified in the rhesus monkeys and closely related to human BuVs
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[13]. PCR to bridge sequence gaps were used to acquire the nearly complete parvovirus genome. The BuV-like virus was then named RatBuV and submitted to GenBank with accession no. KT716186. The near complete genome of RatBuV consists of 4643 nt and encodes three proteins including the nonstructural protein (NS) 1, the viral capsid protein (VP) 1 and VP2. The length of NS1 is 644 aa, including an ATP- or GTP-binding Walker loop motif (403GPASTGKS410) and two conserved replication initiator motifs (129GLHIHLILH137) and (193VLQYTHQTTR202) (Fig. 1a). Previous reports revealed that the VP1 gene of human BuVs was discontinuous gene encoded by two exons [2], therefore, we predicted the putative intron in the VP1 gene of RatBuV by aligning it with the known BuVs genome sequences and analyzing the putative intron
Virus Genes Table 1 Pairwise percent amino acid sequence identities among NS1, VP1, and VP2 regions of bufavirus of this study with the genotype of different bufavirus reference strains; ND, no data: sequence could not be determined
Protein and virus
% Amino acid identity This study
BuV1
BuV2
BuV3
MpBuV
WUHARV
NS1 This study
100
BuV1
50.6
100
BuV2
50.8
94.9
100
BuV3
50.6
95.6
94.4
100
MpBuV
77.2
53.1
53.7
53.5
100
WUHARV
58.1
58.9
59.4
59.2
56.3
100
VP1 This study
100
BuV1
52.5
100
BuV2 BuV3
49.8 51.8
71.1 78.1
100 71.4
100
MpBuV
77.3
51.9
49.2
51.7
100
WUHARV
48.3
67.9
72.3
67.2
49.6
100
VP2 This study
100
BuV1
51.1
100
BuV2
47.1
64.5
BuV3
49.7
73.1
64.9
100
MpBuV
78.3
50.8
47.6
50.8
100
WUHARV
ND
ND
ND
ND
ND
100
ND
Bold symbol in each panel indicates the highest amino acid identity
sequence using the NetGene2 Server (http://www.cbs.dtu. dk/services/NetGene2/). Results indicated that the VP1 of RatBuV was encoded by an interrupted gene consisting two potential splicing sites, where the donor site (CG/GT) was at nucleotide 2110 and the receptor site (AG/GC) was at nucleotide 2368 (Fig. 1a). In the N-termini of VP1, protein presents a phospholipase A2 (PLA2) motif with the conserved calcium-binding site and the phospholipase catalytic resides which is similar to the BuVs of human and shrews (Fig. 1a). VP2 gene overlapped completely with the ORF of VP1, encoding 585 aa, of which a glycine-rich sequence was present in the N terminus. Phylogenetic analysis based on the whole genome sequences showed that RatBuV clustered with Mpulungu bufavirus (MpBuV), forming a separate clade (Fig. 1b). Comparing RatBuV with human BuVs, MpBuV, and WUHARV parvovirus, the amino acid sequence identities of NS1, VP1, and VP2 were 50.6–77.2 %, 48.3–77.3 %, and 47.1–78.3 %, respectively (Table 1). The NS1 of RatBuV shared the highest identity (77.2 %) with that of MpBuV which was a novel bufavirus isolated from shrews’ feces, while only 50.8 % identity with that of Human BuVs. According to the International Committee on Taxonomy of Viruses, a virus being divided into a new parvovirus species should have\85 % identity of NS1 protein comparing with the other known parvovirus species,
RatBuV can therefore be classified into a novel species within the Protoparvovirus genus based on our data. To investigate the prevalence of RatBuV, viral DNA genome was extracted from all 40 individual samples using TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (TaKaRa, Japan). A set of nested primers designed basing on the VP2 nucleotide sequence of RatBuV was used to perform PCR screening in the 40 rat’s fecal samples. Primer RatBuV F1 (50 -ACCGGGTGCGTT TCCTTCACA-30 ) and RatBuV R1 (50 -ACCTAACACAGT TGGTGGACTTGGT-30 ) were used for the first round of PCR, and RatBuV F2 (50 -TTCATTACTCTTCTGGAGG AC-30 ) and RatBuV R2 (50 -CAGATTTCTCCCCATG GGTAA-30 ) for the second round; the expected length of amplified fragment was 379 bp. Our results indicated 5 samples were positive with the positive rate of 12.5 % (5/40), higher than that reported for bufaviruses in humans [2–5]. The specific PCR products were sequenced by Sanger method. Sequence analysis indicated that the five 379 bp sequences shared [98 % nucleotide identities, indicating a single strain was present in the wild rats in this area. In summary, we identified a novel parvovirus in wild rats and characterized its near complete genome. Phylogenetic analysis indicated that the RatBuV clustered with MpBuV. The NS1 amino acid sequence of RatBuV shared
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Virus Genes
the highest identity (77.2 %) with MpBuV, suggesting RatBuV represents a novel species within the Protoparvovirus genus. Epidemiologic study suggested a single strain was prevalent in the wild rats in this area and the prevalence rate of RatBuV was higher than that of its close relatives. There is currently no evidence that BuV can transmit between humans and rats but a macaque bufavirus showed evidence of past recombination with human bufavirus possibly reflecting their potential for cross-species transmission [9]. Acknowledgments This work was partly supported by the Natural Science Foundation of Jiangsu Province No. BK20130502 and BK20140537, the National Natural Science Foundation of China No. 31302107, the Professional Research Foundation for Advanced Talents of Jiangsu University Nos. 12JDG085, 13JDG025, and 13JDG087, the Postdoctoral Foundation of Jiangsu Province No. 1302058C, and China Postdoctoral Special Foundation No. 2015T80503.
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