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Dear Editor,
Parechoviruses (PeVs) are non-enveloped, spherical viruses of genus Parechovirus and family Picornaviridae. Within the capsid is a naked monopartite, linear, singlestranded positive-sense RNA genome of 7.3 kb, comprising a single long open reading frame (ORF) encoding a polyprotein containing regions P1, P2, and P3. The P1 region encodes three structural proteins (VP0, VP3, and VP1); P2 and P3, non-structural proteins (P2 encoding proteins 2A, 2B, and 2C, and P3 encoding proteins 3A, 3B, 3C, and 3D) (ICTV 2018). PeVs are classified into four species: Parechovirus A, is composed of human parechoviruses (HPeVs) identified worldwide, causing gastrointestinal or respiratory diseases and being implicated in myocarditis and encephalitis (Harvala and Simmonds 2009); Parechovirus B and C have been reported in rodents, including Ljungan viruses (LVs) in bank voles and Sebokele virus (SEBV) in African wood mice (Niklasson et al. 1999; Joffret et al. 2013); Parechovirus D comprises a single virus, ferret parechovirus (FPeV), reported through metagenomics studies on healthy household ferrets in the Netherlands (Smits et al. 2013).
As important hosts for several pathogenic viruses, bats harbor viruses from almost all families of vertebrate viruses, including several genera of family Picornaviridae, including Hepatovirus, Kobuvirus, Crohivirus, and Sapelovirus (Drexler et al. 2015; Wu et al. 2016; Yinda et al. 2017). However, PeVs were not reported in bats until our recent viral metagenomic analysis of 122 adult healthy bats (Pipistrellus pipistrellus) obtained from two locations in Xinjiang (Xinyuan, n = 46; Qapqal, n = 76) in 2016, revealing thousands of reads related to PeV (Zhang et al. 2018). PCR-based screening revealed that 6.5% and 10.5% bats from Xinyuan and Qapqal, respectively, harbored this virus, and preliminary phylogenetic analysis of 396-nt-long amplicons targeting the VP1 region (GenBank accession numbers: MH921430–MH921443) revealed > 91.5% identities among each other and 63.7%–64.2% identity with their closest phylogenetic neighbor, FPeV (Smits et al. 2013; Zhang et al. 2018). This study reports the complete genomic characterization of the first bat PeV to better understand its evolutionary history.
One of the bats harboring the PeV from Qapqal was selected for whole-genome amplification and sequencing, wherein the gut sample contained a single, not multiple PeVs, and this viral isolate was named bat PeV QAPp32 (BtPeV QAPp32). PCR primer pairs were designed using Primer Premier 5.0 from the consensus sequences of contigs and FPeV (Supplementary Table S1). The gut was homogenized with SM buffer (50 mmol/L Tris, 10 mmol/L MgSO4, and 0.1 mol/L NaCl; pH 7.5). After centrifugation at 8000 ×g for 10 min at 4 ℃, 200 lL of the supernatant was subjected to RNA extraction using the QIAamp RNA Mini Kit (Qiagen, Hilden, Germany) and reverse-transcribed using Reverse Transcription Kit (TaKaRa, Dalian, China) in accordance with the manufacturer's instructions. cDNA thus obtained was amplified using the LA PCR kit (TaKaRa) under the following cycling conditions: 35 cycles (outer PCR) or 40 cycles (inner PCR) of denaturation at 94 ℃ for 30 s, annealing at 56 ℃ (or adjusted in accordance with primer pairs) for 30 s, and extension at 72 ℃ for 1 min, with double-distilled water replacing cDNA as the negative control. Expected products of ~ 1500 nt were directly sequenced using an ABI 3730 Sanger sequencer (Comate, Changchun, China), and their 50 and 30 terminal sequences were determined using Rapid Amplification of cDNA Ends (RACE) Kits (TaKaRa) in accordance with the manufacturer's instructions.
The genome of BtPeV QAPp32 (GenBank accession number: MK348056) was determined to be 7174-nt-long, organized as a typical PeV genome (Fig. 1A). It contains a 343-nt-long 5′ untranslated region (UTR), followed by a 6624-nt-long ORF and a 178-nt-long 3′ UTR (Fig. 1A). This ORF encodes a 2207-aa polyprotein further divided into a 730-aa-long P1, 676-aa-long P2, and 801-aa-long P3 regions. Genomic differences between BtPeV QAPp32 and other PeV prototypes are summarized in Table 1. The BtPeV QAPp32 ORF has the same size as that of FPeV; however, it is larger than that of HPeV and smaller than that of LV and SEBV (Table 1), displaying the highest identity with FPeV (72.4% in nt and 79.3% in aa) and ≤ 51.3% with other PeVs (Table 1). To determine its phylogenetic associations, the polyprotein aa sequence of BtPeV QAPp32 was aligned with that of its other counterparts, using ClustalW, available in MEGA6, and a phylogenetic tree was constructed using the maximumlikelihood method with 1000 bootstrap replicates with the best substitution model. The phylogenetic tree showed that those PeVs constituted an independent branch from other picornaviruses such as pasivirus and hepatovirus and further clustered into 4 clades, corresponding to 4 species, and BtPeV QAPp32 clustered nearly together with FPeV within the clade of Parechovirus D (Fig. 1B).
Figure 1. A A schematic representation of the genome structure of QAPp32. Boxes represent the open reading frames encoding structural proteins (P1) and nonstructural proteins (P2, P3). B The maximum-likelihood phylogenetic tree for QAPp32 (filled black triangle) with other prototypical members of family Picornaviridae, based on entire polyprotein sequences, wherein other bat viruses are indicated by filled black circles.
ORF/region QAPp32 HPeVNII561-2000
(AB252582)LV 87-012G
(EF202833)SEBV
(NC021482)FPeVNED 2010
(KF006989)nt aa nt % aa % nt % aa % nt % aa % nt % aa % VP0 726 242 867 42.2 289 34.3 777 48.7 259 39.9 777 49.9 259 39.0 726 70.0 242 77.3 VP3 693 231 756 46.4 252 38.3 732 51.2 244 44.3 729 48.5 243 41.0 693 68.9 231 81.0 VP1 771 257 702 44.0 234 34.6 951 41.3 317 28.7 930 41.8 310 31.5 771 70.8 257 74.0 P1 2190 730 2325 45.6 775 35.3 2460 46.6 820 36.9 2436 46.3 812 36.5 2190 69.9 730 77.3 2A 627 209 450 37.4 150 28.0 405 35.6 135 31.1 402 34.0 134 30.6 627 75.6 209 68.6 2B 408 136 366 47.9 122 36.0 414 54.5 138 41.8 411 48.1 137 40.1 408 72.5 136 80.3 2C 993 331 987 53.2 329 41.7 999 55.1 333 44.7 999 52.7 333 43.7 993 74.0 331 83.7 P2 2028 676 1803 47.7 601 36.8 1818 49.7 606 40.3 1812 46.3 604 39.4 2028 74.5 676 78.7 3A 330 110 351 40.8 117 22.0 390 39.5 130 15.4 372 43.0 124 21.0 330 66.4 110 71.8 3B 90 30 60 34.4 20 37.5 87 49.5 29 34.4 87 53.3 29 43.3 90 74.4 30 90.0 3C 582 194 600 48.8 200 36.3 594 50.4 198 38.9 582 50.9 194 36.6 582 75.4 194 86.6 3D 1401 467 1407 51.5 469 41.7 1410 52.9 470 44.4 1410 52.3 470 43.7 1401 72.9 467 80.7 P3 2403 801 2418 49.4 806 37.0 2481 50.1 827 38.6 2451 50.5 817 38.8 2403 72.6 801 81.3 Polyprotein 6624 2207 6549 47.8 2182 36.5 6762 51.3 2253 39.6 6702 50.3 2233 39.4 6624 72.4 2207 79.3 The highest identities are indicated in bold.
nt, nt length; %, identity; aa, aa length.Table 1. Comparison of amino acid and nucleotide sequence identity between QAPp32 and other representative parechoviruses.
Homogenates of 4 PeV-positive gut tissues were inoculated onto Vero E6, MDCK, and PK-WRL cells in an attempt to culture the viruses; however, after five passages, all cells were normal and RT-PCR analysis did not detect the virus.
BtPeV QAPp32 is a variant and a new member of species Parechovirus D, since its aa identity with FPeV was significantly higher than the species criterion (70%) proposed by International Committee on Taxonomy of Viruses (ICTV 2018). We previously reported that this virus is prevalent in bats sampled from Xinyuan and Qapqal, which are 170 km apart, and this virus was detected in the lung and gut tissues, suggesting that this virus replicates in these organs in P. pipistrellus and is hence unlikely to be transmitted from other animals to bats (Zhang et al. 2018).
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This work was supported by the NSFC-Xinjiang joint fund (U1503283).
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The authors declare that they have no conflict of interest.
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The sampling of bats was approved by the Administrative Committee on Animal Welfare of the Military Veterinary Institute, Academy of Military Medical Sciences, China (Laboratory Animal Care and Use Committee Authorization, Permit No. JSY-DW-2015-01).