Introduction

Tetraparvovirus (previously proposed name, “Partetravirus”) is a newly established genus in the family Parvoviridae [1, 2] that includes primate tetraparvovirus 1 (human parvovirus 4, PARV4) and five novel PARV4-related animal parvoviruses described in the last decade: chiropteran tetraparvovirus 1 (Eidolon helvum [bat] parvovirus, Ba-PARV4), chimpanzee parvovirus 4 (Ch-PARV4), ungulate tetraparvovirus 1 (bovine hokovirus 1, B-PARV4), ungulate tetraparvovirus 2 (porcine hokovirus, P-PARV4), ungulate tetraparvovirus 3 (porcine Cn virus, CnP-PARV4), and ungulate tetraparvovirus 4 (ovine hokovirus, O-PARV4). Members of the family Parvoviridae are small, non-enveloped, single-stranded DNA viruses with an icosahedral capsid. They infect a wide range of hosts and are divided into two subfamilies: Parvovirinae, whose members infect vertebrates, and Densovirinae, whose members infect insects and other arthropods [1, 2]. Many novel parvoviruses have been discovered in recent years, although, historically, they were considered genetically stable [1–8]. Therefore, the subfamily Parvovirinae has been expanded to include, in addition to the previous five genera, three new genera, Aveparvovirus (Ave, bird), Copiparvoviru (Copi, cow and pig) and Tetraparvovirus [1, 2]. There have been only two reports describing members of the species Ungulate tetraparvovirus 1, B-PARV4-1 and B-PARV4-2, which were discovered in Hong Kong by the same research group in 2008 and 2011, respectively [3, 4]. To date, there has been no other report of this novel parvovirus anywhere in the world.

Since their domestication at least 5000 years ago by ancestors of the present Chinese Tibetan people, yaks (Bos grunniens) have lived exclusively in the cold highlands surrounding the Qinghai-Tibet Plateau (altitudes > 3000 m, average annual temperature < 0 °C), including Qinghai and Gansu Provinces in northwestern China. Because they live in a relatively secluded and very cold geographic region, yaks were never suspected of hosting any pathogens prior to recent discoveries of yaks carrying hepatitis E virus and Brucella spp. [9, 10]. We have been involved in research on P-PARV4 and the surveillance of yak infections [8, 9]. The aim of this study was to determine whether yaks host the recently discovered virus B-PARV4. We detected B-PARV4 in domestic yaks in China for the first time and determined its nearly full-length genomic sequence by PCR.

In this study, 370 blood samples (195 from Qinghai Province and 175 from Gansu Province) were collected from apparently healthy domestic yaks between May and October 2014 (Table 1). DNA was extracted from all of the samples, using an E-Z 96® Blood DNA Kit (Omega) according to the manufacturer’s instructions, and it was used as the template for PCR amplification of full-length sequences. To detect PARV4-related sequences, the conserved primers B-PARV4-F and B-PARV4-R (Table 2) were designed, targeting a 629-bp fragment of the conserved VP2 region based on a multiple alignment of the reference PARV4 genomic sequences available in the National Center for Biotechnology Information (NCBI) GenBank database. Precautions were taken to avoid PCR contamination, and no false positives were permitted in the negative controls. All positive samples were subjected to nearly full-length sequencing. (Sequencing the full-length genome was hampered by the two terminal hairpin structures in the linear DNA genomes of parvoviruses.)

Table 1 Frequency of B-PARV4s detected by PCR assay in blood from domestic yaks of different ages in two provinces of northwestern China
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Table 2 Primers used for detection and full-length genome amplification of B-PARV4
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The nearly full-length genomic sequences were determined (two replicates for each sample) by multiplex PCR amplification using four pairs of primers (Table 2) that target overlapping fragments spanning the complete coding region of the PARV4 genome. The PCR products were purified using a QIAquick Gel Extraction Kit (QIAGEN, Germany), sequenced (TaKaRa, Dalian, China), assembled, and edited manually to produce the final sequences of the viral genomes. The sequence divergence of the genomes detected in this study was determined by multiple alignment and phylogenetic analysis of the genomic sequences of all of the B-PARV4s, O-PARV4s, and P-PARV4s available in the NCBI GenBank database, using the MEGA version 5 software and 1000 bootstrap replications.

PCR detected 22 positive samples among the 370 samples tested, with an overall prevalence of 5.95 %. The prevalence was similar in Qinghai (13/195, 6.67 %) and Gansu Provinces (9/175, 5.14 %) but varied significantly between the two age groups tested: 8.47 % (15/177) in yaks ≤ 1 year old and 3.6 % in yaks > 1 year old (7/193). The incidence in the younger group (15/22, 68.18 %) was more than twice that in the older group (7/22, 31.82 %) (p < 0.05; Table 1). Six different nearly full-length genomic sequences were identified using a multiple alignment of all of the genome sequences identified in the 22 positive samples, with no sequence discrepancies between the two replicates of each sample. The sequences were deposited in GenBank (accession numbers KT225725–KT225730), designated Yak/GS1–2 (two from Gansu Province) and Yak/QH1–4 (four from Qinghai Province). A phylogenetic tree clearly showed three main branches, encompassing B-PARV4s, O-PARV4s, and P-PARV4s. The six new yak genomes clustered on the same branch as all known B-PARV4s, including B-PARV4-1 (EU200669/bovine/HK1) and B-PARV4-2 (JF504698/bovine/HK4-B38) from Hong Kong, indicating that yaks host B-PARV4s, but not P-PARV4s or O-PARV4s. The new yak B-PARV4s shared 61.9–65.3 % and 64.7–68.3 % sequence identity with P-PARV4s and O-PARV4s, respectively, and 99.0–99.7 % identity with each other and other known B-PARV4s except JF504698/bovine/HK4-B38, with which they shared only 88.6 % sequence identity (Fig. 1).

Fig. 1
figure 1

Neighbor-joining tree showing the phylogenetic relationship among yak B-PARV4 strains based on nearly full-length gene sequences and comparison with previously identified bovine, porcine and ovine hokoviruses available in the GenBank database. Bootstrap values, expressed as percentages of 1,000 replications, are given at the branch points. GenBank accession numbers and animal species and strains are shown at each branch point. The two newly identified yak B-PARV4s strains in Gansu province described in the present study (GenBank accession numbers KT225725-KT225726) are indicated by ‘‘●’’, and four identified yak B-PARV4s strains in Qinghai province (GenBank accession numbers: KT225727-KT225730) are indicated by ‘‘♦’’. The scale bar indicates nucleotide substitutions per site

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On the phylogenetic tree, the main branch of B-PARV4s was divided into two further branches. JF504698/bovine/HK-B38 was the only strain on one branch [11], representing a separate genotype. The six new yak B-PARV4 strains were closely related to each other and formed one branch with EU200669/bovine/HK1, EU200668/bovine/HK3, and JF504697/bovine/HK-B15, demonstrating that only one B-PARV4 genotype is circulating in the yak populations in China.

The detection and phylogenetic analysis of this virus demonstrates for the first time the presence of B-PARV4s (but no other PARV4-related viruses) in a previously unsuspected host, domesticated highland yaks, extending our knowledge of the host range of B-PARV4. The yak B-PARV4s whose sequences are presented here are not associated with any disease, because the samples were collected from apparently healthy yaks. In previous studies of B-PARV4s, and also O-PARV4s and P-PARV4s, the viruses were identified in samples collected from both healthy and diseased animals, and no association was found with any disease when their prevalence was compared in the healthy and diseased animals. Therefore, the pathogenicity of B-PARV4s requires further detailed study. The overall prevalence (5.95 %) of yak B-PARV4 was much lower than that of B-PARV4s in bovines in Hong Kong (13 %) [3], and even lower than that of the closely related P-PARV4s in pigs, which generally ranges from 44.4 % to 71 % [3, 5–8], and that of O-PARV4s in ovine species (~70 %) [4]. The data for yak B-PARV4s reported here show a unique and significant negative correlation between prevalence and age, which is contrary to the phenomenon observed for PARV4 infections in other animals, such as B-PARV4 infections in bovine species, O-PARV4 infections in ovine species, and P-PARV4 infections in pigs [3–8].

Significantly, in this study, we have demonstrated the presence of B-PARV4s in yaks. There is as yet no sign of cross-species infection or transmission, and the detection and further analysis of B-PARV4 in wild yaks and other bovine species will be very important for clarifying the source, circulation pattern, zoonotic potential, and public-health risk of this virus. The mechanisms of its pathogenicity, transmission, evolution, and persistence also require urgent clarification.