Prevalence and structural protein encoding gene sequence (VP) of porcine parvovirus 2 (PPV2) in slaughtered pigs in Central provinces of Vietnam

. Porcine parvovirus (PPV) is a DNA virus and causative agent of several reproductive problems in sows. This study was conducted to determine the prevalence and analyze the DNA sequence of structural protein encoding gene (VP) of PPV2 genotype in pigs. A total of 146 samples (lung and blood samples) were collected from slaughtered pigs of seven provinces in Central Vietnam during 2018-2019. The overall prevalence of PPV2 was 56.2% (82/146). PPV2 positive rate in each province ranged from 37.5% for Quang Nam to 100% for Quang Binh, with the exception of Da Nang, where no PPV2 positive samples were detected. Nearly complete PPV2-VP gene sequences of three strains were identified with the length of 2,493 nucleotides and deposited in GenBank with accession numbers of OL913365-OL913367 . Four nucleotide substitutions were detected in Vietnamese PPV2 isolates and were not observed in PPV2 reference strains. Multiple alignment and comparison of nucleotide and deduced amino acid sequences showed the high similarity within Vietnamese PPV2 strains (95.6-96.5% and 94.7-96.9%, respectively). The PPV2 strains from this study clustered together with the "primitive" PPV2 strains from Myanmar, and strains from China in a main clade in the phylogenetic tree (Cluster A). This is the first report on the prevalence of PPV2 genotype and its VP gene sequence in pigs in Vietnam. This also provides the valuable information on the molecular evolution of locally circulating PPV2 and contributes to the control of PPV-induced SMEDI syndrome in sows, especially in the central provinces of Vietnam.


Introduction
To date, the clinical significance of porcine parvovirus (PPV) has been well-described with the predominant effect being impaired fertility in sows [1].PPV are small, non-enveloped, isometric, 18-26 nm in diameter viruses, with a genome structure that resembles a linear, nonsegmented molecule of ssDNA which is approximately 4-6.3 kb in size [2] that causes porcine reproductive failure in swine [3].PPV genome is characterized by a hairpin structure at two 5'-3' ends [4,5] and two open reading frames (ORF) coding for non-structural protein (NSP) and viral coat and capsid protein (VP).PPV is stable to environmental factors, living in a pH range of 3.0-8.0and is heat resistant for hours at 80°C [6].
Analysis of the genetic relationship between PPV1 and PPV2 revealed that PPV2 is "distantly related" to PPV1 with 32.2-34.5% genomic identity and amino acid identity of 20.2-21.5% for ORF1 and 16.5-16.9%for ORF2 [17].The efforts to propagate PPV2 in vitro have so far been unsuccessful, so studies on the pathogenicity of PPV2 have not been possible [18].However, recent evidence shows that PPV2 may be a co-factor or triggering agent associated with PCVAD [17] or 'high fever disease' [19].
Further epidemiological studies of PPV2 have been reported differently worldwide [20].About a decade after it was first detected, PPV2 was identified in 8.8% of serum samples obtained from commercial pig farms in Southeastern China [19].
Subsequently, PPV2 has been discovered in other samples, including hearts, blood, faeces and lungs in many other countries including Hungary, the USA, Germany, Japan, and Thailand with prevalence of 6.4%, 20.7%, 55%, 58% and 83%, respectively [18,21,22,23,24].Meanwhile, using tonsil samples in PPV2 screening has reported a higher prevalence, ranging from 78% (Germany) to 100% (Japan), leading to the hypothesis that viral prevalence may depend on the organ type examined, although the routes of infection and viral loci are yet to be elucidated experimentally [22,23,24].  1 The number of samples collected in each abattoir was less than five.
2 From each pig, only a single sample was collected (either lung or blood).

DNA extraction and PCR analysis
Total DNA was extracted and purified according to the methods of Sambrook and Russell [25] with small modifications: blood samples were washed several times with PBS buffer until they became white (try to remove all the hemoglobin) before using for DNA extraction.DNA was diluted in TE buffer, and stored at 4°C for analysis.
To detect PPV2, specific PCR assays were carried out in a single reaction with 50-100 ng of DNA template, 2× PCR master mix (Thermo Scientifc, Lithuania), and 5 pmol of each primer.
The PPV2 primers for detection were followed Streck et al. [22].Primers for the PPV2 sequencing (Table 2) were designed using the program Primer3 based on reference sequences listed below (Table 3).Information on primer sequence, annealing temperature (Ta) and product size are shown in

PPV2-VP2 gene sequencing and sequence analysis
Five primer pairs, designed based on PPV2 gene sequence published on GenBank, were used to amplify structural protein encoding genes (VP genes) of three PPV2 positive strains (Table 2).
The PCR products were sent to Macrogen Inc., South Korea for sequencing using an ABI-3100 Avant Genetic Analyzer automated sequencer according to the standard Sanger method [26].
The published PPV2 sequence information used as a reference is presented in Table 3. Obtained nucleotide sequences were identified with the Basic Local Alignment Search Tool (BLAST, NCBI) [28].
The phylogenetic tree was constructed with Mega X software using the neighbor-joining (NJ) method [29] and computed with the Kimura 2parameter method [30].Bootstrap values were calculated using 1.000 replicates of the alignment.prevalence rates for each province ranged from 37.5% for Quang Nam to 100% for Quang Binh, with the exception of Da Nang, where no PPV2positive samples were detected (Table 4).[15,33,1,34].In contrast, a high prevalence of PPV2 in pig herds was observed in Germany (78.0%) and Thailand (82%) [22,24].
In Vietnam, PPV-induced fertility decline syndrome has been of concern since the early 90s of the last century and has caused considerable damage to the pig industry.Therefore, the detection of PPV genotypes in pigs, including PPV2, is not an exception.In a previous survey of sows in Long An province by serological testing, Nguyen and Tran [35] reported that the positive rate for PPV was quite high at 69%.In addition, analysis of 52 stillbirth samples detected PPV virus and PPV antibodies simultaneously.
Recently, surveys in some localities have initially confirmed the prevalence of the PPV2 genotype in pig farms.In a study on pig lung samples collected from abattoirs in four northern provinces of Vietnam, Cuong [36] reported a PPV2 infection rate of 17.6%.Most recently, Thuy et al. [37] found PPV2 prevalence up to 28% in finishing pig from 13 provinces in three regions of Vietnam, including North, Central and South Vietnam.
Taken together, the above data have shown that our results on PPV2 prevalence are in the average range compared with other previous studies.It could be explained by the type of sample collected (mainly lung sample) and the status of investigated animal (slaughtered pig) in our study.This result supports the hypothesis of Saekhow and Ikeda [24]: viral prevalence may depend on the type of sample collected and screened, although the routes of infection and the viral tissue tropism are yet to be elucidated.

2 (
PPV2) (formally name is Ungulate tetraparvovirus 3), belongs to the Tetraparvovirus genus, was discovered in 2001 during a survey for Hepatitis E virus (HEV) in swine sera collected in Myanmar [9]; PPV3 (Ungulate tetraparvovirus 2) was discovered in Hong Kong in 2008; PPV4 (Ungulate copiparvovirus 2) and PPV5 (unclassifed) were identified in the USA in 2010 and 2013, respectively; PPV6 (Ungulate copiparvovirus 4) was discovered in China in 2014 and most recently, PPV7 (proposed genus Chappaparvovirus) was identified in the USA in 2016

Fig. 2 ,
Fig.2, the BLAST analyses also revealed a high nucleotide identity (98%) between PPV2 sequence and reference PPV2 strain (MG345019).In the present study, among the 146 lung and blood samples from 32 different abattoirs in seven provinces of Central Vietnam, PPV2 DNA was detected with 56.2% (82/146).PPV2

Fig. 2 .
Fig. 2. BLAST result revealed the positive sample with 98% nucleotide identity compared with reference PPV2 strain (MG345019) proteins of three PPV2 strains collected from Quang Binh, Hue and Quang Ngai were identified and analyzed.The length of the partial VP gene of three PPV2 isolates in this study is 2,493 nucleotides, including VP1 and VP2 sequences, without insertions and deletions in the coding regions.The sequences were deposited in the GenBank database with the accession numbers OL913365-OL913367.Comparing many reference strains found that there are four base substitutions (444: T→A, 734: C→T, 820: A→C, 1974: T→A) in PPV2 QN03-VN strain, completely different compared with the reference strains; two of them (734: C→T, 820: A→C) resulted in amino acid changes in the VP gene sequence (245: S→F, 274: K→Q).No variation was detected in the deduced amino acid sequence of the two strains, PPV2 HU10-VN and PPV2 QB05-VN.For the PPV1 genotype, nucleotide changes leading to amino acid changes at several potential sites on the VP2 gene and responsible for antigenicity were observed in

4 ConclusionsFig. 3 .
Fig. 3. Phylogenetic tree based on the VP nucleotide sequence of PPV2 (2,493 nucleotides) obtained from three provinces of Vietnam (in this study) and reference sequences from GenBank database.The black square box indicates the Vietnamese PPV2 sequence.The number indicated the bootstrap value of 1.000 replicates

Table 1 .
Sample information collected in this study

Table 2
. The PCR consisted of an initial activation step at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 56/60/50/54°C for 30 sec, extension at 72°C for 30 sec, final synthesis at 72°C for 5 minutes and holding at 14°C.The resulting PCR products were analyzed by electrophoresis on a 2% agarose gel using a 100 bp DNA ladder (Thermo Fisher Scientific).Appropriate positive and negative extraction and PCR controls (obtained from the University of Edinburgh and confirmed by sequencing) were used for each extraction and PCR run.

Table 2 .
Primer information used in this study for genotyping and sequencing.The virus target was U. tetraparvovirus 3 (PPV2)

Table 3 .
List of PPV2 strains used in this study

Table 4 .
Number of DNA-positive samples (percentage) in this study