First Nearly Complete Genome Sequence of Porcine Bocavirus Strain from Malaysia

| This paper reports the first nearly complete genome sequence of a Malaysian Porcine bocavirus (PBoV) detected in Malaysia from a porcine organ sample. In Malaysia, both Human bocavirus (HBoV) and PBoV have been reported, at prevalence of 5.6% and 90.9% respectively. Most recently, in 2018, PBoV emerged as a zoonosis when a human case of PBoV-related respiratory infection is reported in a child with history of contact with porcine secretion. With this evidence of human infection with PBoV, considering close contact between humans and pigs in the swine industry, further study of PBoV genetic characterization and epidemiology is warranted. This study attempts to obtain the complete genome sequence of a Malaysian PBoV isolate detected in a pig farm in the state of Selangor. A nearly complete sequence was obtained through primer walking and touchdown PCR method. Based on phylogenetic analysis for the nearly complete genome, the Malaysian PBoV strain is genetically characterized as PBoV G3B, sharing a close relationship with a reference PBoV strain from U.S.A. with 98% homology. This finding may facilitate further epidemiological studies and diagnostic development of PBoV in Malaysia.


Advances in Animal and Veterinary Sciences
September 2019 | Volume 7 | Issue 9 | Page 777 of contact with porcine secretion (Safamanesh et al., 2018). With this evidence of human infection with PBoV, considering close contact between humans and pigs in the swine industry, further study of PBoV genetic characterization and epidemiology is warranted.
Porcine bocavirus was first identified in lymph nodes of pigs in Sweden, co-detected with Porcine circovirus 2 (PCV2) (Blomström et al., 2009). Detection was reported in wide range of samples including saliva, sera, nasopharyngeal swab, feces, lymph nodes, tonsil, lung, kidney, spleen and liver (Choi et al., 2014;Zhou et al., 2014;Gunn et al., 2015), suggestive of systemic infections. It was linked to gastrointestinal and respiratory signs as well as encephalomyelitis (Pfankuche et al., 2016). The pathogenic pathways of PBoV are rather obscured, with multiple reports of co-infections (Blomström et al., 2009;Ndze et al., 2013). It was suggested to be indirectly related to porcine post-weaning multisystemic wasting syndrome (PMWS) and other swine diseases as a triggering factor (Blomström et al., 2009). In Malaysia, PBoV was first detected in 2017 in clinically compromised pigs ( Jacob et al., 2018). As our continual effort in genetic characterization of Malaysian PBoV isolate, this study attempts to obtain the complete genome sequence by primer walking. In summary, here we described the nearly complete genome of one PBoV field strain isolated from Selangor, Malaysia; and its phylogenetic relationship compared to reference bocavirus strains.

saMPLe, Dna eXTraCTion anD PCr assaY
From the tissue samples that were detected positive of PBoV previously ( Jacob et al., 2018), total DNA was isolated using Patho Gene-spin DNA/RNA Extraction Kit (iNtRON Biotechnology, South Korea). A series of spe-cific PCR primers were designed (synthesized by Bioneer, South Korea) to obtain the initial target sequence to be used as a template to 'walk' the length of the target ( Table  1). The primers were paired in various combinations resulting in fragments of varying sizes (Table 2). Real-time PCR was performed using NexQ-9300 NEXpro™ qPCR 2x MasterMix Solution (EvaGreen, U.S.A.) with touchdown cycling conditions to increase annealing specificity and yield of the target fragments (Table 3).

Advances in Animal and Veterinary Sciences
September 2019 | Volume 7 | Issue 9 | Page 778 sequenCing anD PhYLogeneTiC anaLYsis Due to multiple signals being generated from direct sequencing of the fragments, TA cloning using pLUG-Prime TA-Cloning Kit (iNtRON Biotechnology, South Korea) was employed to obtain clean sequences and ascertain whether multiple variants of the PBoV target were present. The near complete genome of PBoV was deduced from the best overlaps spanning the entire sequence of the target PBoV. Phylogenetic analysis of the nucleotide sequences of complete or near complete genome of PBoV and other bocaviruses available in NCBI GenBank database was conducted using MEGA7.0.26 software (Kumar, Stecher and Tamura, 2018) using Maximum Likelihood method based on Tamura-Nei model with 1000 bootstrap replicates (Saitou and Nei, 1987).

RESULTS
A previous selected positive PBoV sample ( Jacob et al., 2018) was subjected to real-time PCR and a nearly complete genome sequence comprising 4836 nucleotide with G+C content of 52% was obtained. Sequence fragments generated from PCR of the primer combinations (Table  2) are shown in Figure 1. Nucleotide BLAST analysis of the initial sequencing data indicated that the target had a high homology of 98% with PBoV Group 3 lineage. The near complete genome of Malaysian PBoV had been submitted to GenBank under accession number MK467456. The nucleotide sequence of the near complete genome is shown in Figure 2.
From the Figure 3, the Malaysian PBoV isolate MK467456 (indicated by round bullet) showed a strong relationship with the both United States PBoV strains KF025382 and KF025386 (indicated by triangle bullets). This relationship could be linked to the importation source of Malaysian

Advances in Animal and Veterinary Sciences
September 2019 | Volume 7 | Issue 9 | Page 779 breeding herd, given that majority of the breeder pigs used in Malaysian swine farms are imported from U.S.A (Singh and Fong, 2014). Considering the possibility of introduction of this PBoV G3 isolate through commercial trading by importation of semen and breeder stock, this virus could be expected to be circulating in Malaysian commercial swine herd, as shown by a pilot molecular detection of PBoV in Malaysia reporting a very high prevalence of 90.9% ( Jacob et al., 2018). Therefore, understanding the genetic characteristics of the Malaysian isolate of PBoV could contribute to the management of bocavirus infection in the local scenario. Screening of breeder animals and semen for bocavirus prior importation and strict biosecurity measures upon arrival may help to prevent any further introduction of new bocavirus strains into the country. In terms of NS1 gene sequence, these 3 strains share 98% of their nucleotide identity. Thus, this Malaysian PBoV isolate can be classified as same species with reference strains KF025382 and KF025386 as Porcine bocavirus G3B. Bocaviruses from G2 and G3 are more distantly related to the HBoV, as compared to G1 which shares a large clade with HBoV. Although the Malaysian PBoV is only distinctly related to HBoV, possible zoonotic risk still cannot be ruled out. Furthermore, strain HM053693 (indicated by square bullet) reported to be closest to the strain in the first human case of PBoV (Safamanesh et al., 2018), is classified as PBoV G2 that shares a clade with G3. This might suggest potential zoonotic risk. Thus, this genetic characterization of Malaysian PBoV G3B isolate could be useful for the start of epidemiology studies of PBoV in Malaysia: further studies to determine whether PBoV G1 and G3 is present in Malaysia, as well as whether the Malaysian PBoV strain poses zoonotic risk.

CONCLUSION
Publication of near complete genome sequences allows researchers to trace the presence of new, important emerging viruses and determine their genetic distribution to plan relevant control programs. With the characterization of Malaysian Porcine bocavirus isolate as strain PBoV G3B having close relationship with U.S.A. reference strains KF025382 and KF025386, we suggest that routine screening of imported breeders could be helpful to monitor the status of new emerging disease in Malaysian swine farms. Further studies are required to establish the clinical impact of the current Malaysian PBoV G3B strain as well as to determine presence of other PBoV strains in Malaysia. In conclusion, PBoV is present in Malaysia and the Malaysian isolate is confirmed to be of PBoV G3B strain.

STATEMENT OF ETHICAL STANDARDS
This research project was approved by the Institutional Animal Care and Use Committee (IACUC), with reference number UPM/IACUC/AUP-R063/2014.