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Keywords:

  • genetic variation of ORF5;
  • North American genotype;
  • phylogenic analysis;
  • porcine reproductive and respiratory syndrome. Author: This phrase has been reworded for clarity. Has the meaning been retained?

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. 1 MATERIALS AND METHODS
  4. 2 RESULTS
  5. 3 DISCUSSION
  6. ACKNOWLEDGMENTS
  7. 4 DISCLOSURE
  8. REFERENCES

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens because it is highly infectious and causes economic losses due to decreased pig productivity. In this study, the 603 bp complete major envelope protein encoding gene (ORF5) of 32 field PRRSV isolates from Vietnam collected during 2008–2012 were sequenced and analyzed. Multiple nucleotide (nt) and deduced amino acid (aa) alignments of ORF5 were performed on the 32 isolates: the representative strains (European and North American genotypes), Chinese strains available in GenBank and vaccine strains licensed for use in Vietnam. The results showed 94.8–100.0% nt identity and 94.0–100% aa similarity among the 32 isolates. These isolates shared similarities with the prototype of the North American PRRSV strain (VR-2332; nt 87.8–89.3%, aa 87.5–90.0%), and Lelystat virus, the prototype of the European PRRSV strain (LV; nt 61.1–61.9%, aa 55.1-57.0%). There was greater similarity with QN07 (nt 96.5-98.5%, aa 96.0-99.0%) from the 2007 PRRS outbreak in QuangNam Province, CH-1a (nt 93.2–95.1%, 91.5–93.5%) isolated in China in 1995 and JXA1 (nt 96.5–98.6%, aa 95.0–98.0%), the highly pathogenic strain from China isolated in 2006. The Vietnamese isolates were more similar to JXA1-R (nt 96.5–98.6%, aa 95.0–98.0%), the strain used in Chinese vaccines, than to Ingelvac MLV/BSL-PS (nt 87.2–89.0%, aa 86.0–89.0%). Phylogenetic analysis showed that the 32 isolates were of the North American genotype and classified into sub-lineage 8.7. This sub-lineage contains highly pathogenic Chinese PRRSV strains. This study documents genetic variation in circulating PRRSV strains and could assist more effective use of PRRS vaccines in Vietnam.

List of Abbreviations
aa

amino acid

EU-type

European genotype (type I)

GP

glycoprotein

LV

Lelystat virus, prototype of European PRRSV strain

NA-type

North American genotype (type II)

nt

nucleotide

ORF

open reading frame

PRRS

porcine reproductive and respiratory syndrome

PRRSV

porcine reproductive and respiratory syndrome virus

VR-2332

prototype of North American PRRSV strain

Porcine reproductive and respiratory syndrome, one of the most serious infectious diseases of pigs, causes reproductive problems and respiratory distress. Because of its economic impact, PRRS is considered one of the most severe viral diseases that can affect pig farms. The causative agent, PRRSV, is a small, enveloped, single-stranded positive-sense RNA virus. PRRSV is a member of the Arteriviridae family, within the order Nidovirales [1]. PRRSV has been divided into two distinct genotypes: the European type (type I) and the North American-type (type II) [2, 3]; these share only about 60% identity at the nt level [4]. There is reportedly much genetic variation among field viruses of the same genotype in different geographical locations. The viral genome is about 15 kb and encodes nine overlapping ORFs [2]. ORF5 encodes the major viral envelope glycoprotein (GP5), which is located on the surface of the virion. GP5 plays an important role in viral infectivity and contains important immunological domains associated with viral neutralization [5, 6]. Several peptide/protein motifs, such as signal peptides, trans-membrane regions, antigenic determinants and glycosylation sites have been recognized in GP5 [7]. Therefore, ORF5 has been widely used for analyzing genetic variation and the molecular epidemiology of PRRSV.

Since at least 1994, when 50 piglets were imported from the USA, PRRSV has been present in Vietnam; however, up until 2007 there were no reports of PRRS disease (popularly known as “blue ear”) in this country [8]. The first known outbreak of PRRS in Vietnam occurred in Hai Duong Province in March 2007 and was recognized as highly pathogenic PRRS. A subsequent epidemic of highly pathogenic PRRS also started in northern Vietnam in April 2010 and rapidly spread southward (July 2010). Outbreaks affecting about 70,577 pigs then occurred in other regions of the country; more than 20,366 pigs had to be killed [9]. Despite efforts to control outbreaks by containment, slaughter and vaccination, the disease has continued to spread throughout Vietnam. Genetic analysis of PRRSV strains collected from outbreaks in China and Vietnam in 2007 indicated high nt identity (99%) among isolates [10]. The aim of this study was to sequence and analyze ORF5 in Vietnamese PRRSV isolates obtained from 2008–2012, thus documenting genetic variation in the circulating PRRSV strains and assisting the effective use of PRRS vaccines in Vietnam.

1 MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. 1 MATERIALS AND METHODS
  4. 2 RESULTS
  5. 3 DISCUSSION
  6. ACKNOWLEDGMENTS
  7. 4 DISCLOSURE
  8. REFERENCES

Serum samples (n = 32) from PRRSV-infected pigs displaying clear clinical signs were collected during 2008–2012 from farms in six different Vietnamese provinces; DienBien is the northern part of Vietnam and other provinces located in the southern part of the country. A summary of the samples is presented in Table 1. The virus used in the current vaccine against PRRS (JAX1-R), originating from China, was also sequenced.

Table 1. PRRSV isolates (n = 32) from Vietnam and representative PRRSV strains used for sequencing and phylogenic analyses
No.Strain*Location - yearReferenceClassificationHaplotype**
  • *

    , strains in bold were isolated and used in this study;

  • **

    , within Vietnamese field strains isolated in this study only.

1DB2012.1Dien Bien - 2012Sublineage 8.77
2DB2012.2Dien Bien - 2012Sublineage 8.77
3DB2012.9Dien Bien - 2012Sublineage 8.77
4HG2012.RV2Hau Giang - 2012JQ860391Sublineage 8.74
5CT2012.HS1Can Tho - 2012JQ860384Sublineage 8.76
6CT2012.HS2Can Tho - 2012JQ860385Sublineage 8.76
7CT2012.HS3Can Tho - 2012JQ860386Sublineage 8.75
8CT2012.C1Can Tho - 2012JQ860382Sublineage 8.75
9CT2012.C2Can Tho - 2012JQ860383Sublineage 8.76
10DT2012.DT7Dong Thap - 2012JQ860387Sublineage 8.73
11DT2012.DT8Dong Thap - 2012JQ860388Sublineage 8.73
12DT2012.DT9Dong Thap - 2012JQ860389Sublineage 8.73
13BD2010.R1Binh Duong - 2010JQ860381Sublineage 8.711
14HCM2010.CC3Ho Chi Minh - 2010JQ860379Sublineage 8.710
15HCM2010.D06Ho Chi Minh - 2010JQ860380Sublineage 8.712
16DN2010.1Dong Nai - 2010JQ860375Sublineage 8.78
17DN2010.4Dong Nai - 2010JQ860376Sublineage 8.71
18DN2010.5.2Dong Nai - 2010JQ860377Sublineage 8.71
19DN2010.11Dong Nai - 2010JQ860378Sublineage 8.72
20DN2009.42Dong Nai - 2009JQ860367Sublineage 8.71
21DN2009.44Dong Nai - 2009JQ860368Sublineage 8.71
22DN2009.59Dong Nai - 2009JQ860369Sublineage 8.71
23DN2009.88Dong Nai - 2009JQ860370Sublineage 8.71
24DN2009.292Dong Nai - 2009JQ860372Sublineage 8.79
25DN2009.1107Dong Nai - 2009JQ860373Sublineage 8.71
26DN2009.1155Dong Nai - 2009JQ860374Sublineage 8.71
27DN2008.153Dong Nai - 2008JQ860371Sublineage 8.79
28DN2008.444Dong Nai - 2008JQ860362Sublineage 8.71
29DN2008.452Dong Nai - 2008JQ860363Sublineage 8.71
30DN2008.460Dong Nai - 2008JQ860364Sublineage 8.71
31DN2008.499Dong Nai - 2008JQ860365Sublineage 8.71
32DN2008.694Dong Nai - 2008JQ860366Sublineage 8.71
33JAX1-R Vaccine ChinaChinese vaccineSublineage 8.7
34HCMC-3341 2010 South VNHo Chi Minh - 2010HQ540647Sublineage 8.7
355402BD 2010 South VNHo Chi Minh - 2010HQ700886Sublineage 8.7
36HCMC-3867 2010 South VNTay Ninh - 2010HQ540654Sublineage 8.7
3707QN 2007 Center VNQuang Nam - 2007FJ394029Sublineage 8.7
38347-T-KS 2010 North VNNorth Vietnam - 2010AB588636Sublineage 8.7
39CH-1a 1995 ChinaChina - 1995AY032626Sublineage 8.7
40NB/04 2004 ChinaChina - 2004FJ536165Sublineage 8.7
41JXA1 2006 ChinaChina - 2006EF112445Sublineage 8.7
42BJ0706 2007 ChinaChina - 2007FJ800759Sublineage 8.7
43XL2008 ChinaChina - 2008EU880436Sublineage 8.7
44YD 2009 ChinaChina - 2009JF748717Sublineage 8.7
45DC 2010 ChinaChina - 2010JF748718Sublineage 8.7
46JilinTN1 2011 ChinaChina - 2011JN157760Sublineage 8.7
47PRRSV55USA - 2000AF176476Sublineage 8.1
4898-31701-1USA - 2001AF339494Sublineage 8.3
49SDSU73USA - 2005AY656993Sublineage 8.5
50Ingelvac ATPUSA - 2004EF532801Sublineage 8.9
5102SP3Thailand - 2002AY297118Lineage 1
52CP07South Korea - 2007FJ972727Lineage 1
53MN184USA - 2002EF484031Lineage 1
54PRRRSV0000008973USA - 2007EU758940Lineage 2
55MD-001Taiwan - 1997AF121131Lineage 3
56Miyagi08-2Japan - 2008AB546105Lineage 4
57VR-2332USA - 1990U87392Sublineage 5.1
58Ksg1South Korea - 2003DQ473455Sublineage 5.1
59NADC-8USA - 1992U66394Sublineage 5.2
60BCL-PS 100Singapore - 2009GU187014Sublineage 5.2
61Ingelvac PRRS MLVUSA - 2005AF066183Sublineage 5.2
62RespPRRS MLVUSA - 2000AF159149Sublineage 5.2
6334075-NEUSA - 1996U66380Lineage 6
64Prime-PacUSA - 1998DQ779791Lineage 7
652000-5424USA - 2008EU556160Lineage 9
66LVNetherlands-1991M96262EU-type

Total RNA was extracted using TRizol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions and then used for synthesis of cDNA with random hexamers (Fermentas, Glen Burnie, MD, USA). Specific primer pairs [11] were used to amplify 720 bp amplicons encoding the complete ORF5. PCR products were purified using a QIAquick Extraction Kit (Qiagen, Hilden, Germany) and directly sequenced (Macrogen, Seoul, Korea). Obtained nt sequences were identified with BLAST [12]. Multiple nt and aa alignments were carried out with BioEdit version 7.0.9.0 using published PRRSV sequences as references [13]. A phylogenetic tree was constructed with Mega 4.1 [14] using the neighbor-joining method. A boot-strap value of 1000 replicates was applied for robustness of phylogeny. Amino acid sequences deduced from ORF5 of 32 PRRSV isolates were aligned and analyzed for changes in the functional domains.

2 RESULTS

  1. Top of page
  2. ABSTRACT
  3. 1 MATERIALS AND METHODS
  4. 2 RESULTS
  5. 3 DISCUSSION
  6. ACKNOWLEDGMENTS
  7. 4 DISCLOSURE
  8. REFERENCES

2.1 Sequence and phylogenetic analysis of open reading frame 5

Nucleotide and amino acid sequences of the 32 PRRSV isolates were compared with those of VR-2332, LV, 07QN and CH-1a and vaccine viruses JXA1-R, Ingelvac MLV and BSL-PS (Table 2). This analysis showed that the 32 isolates shared 94.8–100% nt identity with each other and could be separated into 12 haplotypes. All strains collected from 2008–2009 in Dong Nai province belonged to two haplotypes only (1 and 9); the remaining isolates of 2010–2012 included all 12 haplotypes. There was high nt identity (87.8–90.0%) with VR-2332 but only 61.1–61.9% identity with LV. Compared with 07QN (a highly pathogenic Vietnamese isolate), CH-1a (Chinese isolates) and JXA1-R, nt identities were 96.5–98.5%, 93.2–95.1% and 96.5–98.6%, respectively. Among the 32 Vietnamese isolates two groups of samples, collected in Dong Thap and Dien Bien in 2012, were identified as having mutations at positions 166 (C [RIGHTWARDS ARROW] T), 327 (T [RIGHTWARDS ARROW] C), 417 (C [RIGHTWARDS ARROW] T), 423 ( [RIGHTWARDS ARROW] C) and 509 (A [RIGHTWARDS ARROW] G; data not shown).

Table 2. Nucleotide and amino acid identities (%) for ORF5 among 32 Vietnamese PRRSV isolates and comparison with LV, VR-2332, 07QN, JXA1, CH-1a and Ingelvac PRRS MLV/BSL-PS/JXA1-R isolates
Strain* (Number) 200820092010201207QN347-T-KSHCMC-3341VR-2332LVJXA1CH-1aJXA1-RIngelvac MLV/BSL-PS
  • *

    2008, 2009, 2010, 2012: Vietnamese PRRSV field strains collected from 2008–2012.

2008 (6)nt98.1–10098.1–10096.1–99.196.5–99.598.3–98.598.3–99.596.1–97.388.861.1–61.397.8∼98.694.1–95.097.8–98.688.2–88.5
 aa98.5–10098.5–10096.0–10096.5–99.598.5–99.098.5–99.096.0–97.588.556.096.5∼98.092.0–93.596.5–98.087.0–87.5
2009 (7)nt98.1–10096.1–99.196.5–99.598.3–98.598.3–99.596.1–97.388.861.1–61.397.8∼98.694.1–95.097.8–98.688.2–88.5
 aa98.5–10096.0–10096.5–99.598.5–99.098.5–99.096.0–97.588.556.096.5∼98.092.0–93.596.5–98.087.0–87.5
2010 (7)nt95.8–10094.8–99.196.5–98.396.3–99.595.8–10087.8–89.361.1–61.996.6∼97.893.5–95.196.6–97.887.2–89.0
 aa95.5–10094.0–99.596.0–98.596.0–99.095.5–10087.5–90.055.1–57.095.0∼96.591.5–93.595.0–96.586.0–89.0
2012 (12)nt97.3–10096.6–98.597.8–99.694.8–96.687.8–89.061.1–61.396.5∼98.093.2–94.596.5–98.087.2–88.7
 aa97.0–10096.5–99.097.0–99.594.0–96.587.5–89.056.0–56.595.5∼97.092.0–92.595.5–97.086.0–88.0

The constructed phylogenetic tree based on the sequence of ORF5 is presented in Figure 1. The 32 Vietnamese PRRSV isolates belonged to the NA-type genotype and were grouped as sub-lineage 8.7. This sub-lineage also contains highly pathogenic Chinese strains and the JXA1-R strain used in vaccines. Three isolates collected in Dien Bien in 2012 and JilinTN1 collected in China in 2011 showed a close genetic relationship and were clustered together in one branch.

image

Figure 1. Phylogenetic tree containing 32 Vietnamese isolates from 2008 to 2012 and other representative virus strains. The comparison was based upon on the nucleotide sequence of PRRSV ORF5. The phylogenetic tree was generated by the neighbor-joining method using MEGA 4.1 with bootstrap values of 1000 replicates. Different lineages and sub-lineages are indicated. Our isolates are marked (▴). Vaccines licensed for use in Vietnam are denoted (●).

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2.2 Analysis of deduced type II porcine reproductive and respiratory syndrome virus open reading frame 5 amino acid sequences

The deduced aa sequences for ORF5 of the 32 Vietnamese isolates were aligned with representative PRRSV strains from Vietnam and China and with vaccines (Fig. 2). Pair-wise comparison showed that the 32 strains shared 94.0–100% aa identity with each other, aa identities of 87.5–90.0% with VR-2332, 55.1–57.0% with LV, 96.0–99.0% with 07QN, 91.5–93.5% with CH-1a and 95.0–98.0% with JXA1. The 32 Vietnamese isolates had a higher aa similarity with JXA1-R (95.0–98.0%), the Chinese isolate used in PRRS vaccines, than with Ingelvac MLV and BSL-PS (86.0–89.0%; Table 2).

image

Figure 2. Analysis and comparison of amino acid mutations in GP5 of PRRSV. Functional domains are indicated by black boxes.

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In the extra virion domain, three isolated strains from Dong Nai (DN2008.153, DN2009.292 and DN2010.1), two from Ho Chi Minh (HCM2010.CC3 and HCM2010.D06) and one from Binh Duong (BD2010.R1) shared a mutation at position 29 (A [RIGHTWARDS ARROW] V). This mutation was also seen in the JXA1-R strain and Chinese strains. At a glycosylation site, an aa change was detected in 29 samples (position 34, N [RIGHTWARDS ARROW] S). This was also seen in Chinese strains isolated in 2010 and 2011 (DC and JilinTN1). Two isolates sampled at Ho Chi Minh in 2010 contained a mutation at position 58 (Q [RIGHTWARDS ARROW] E). An aa change at position 104 (G [RIGHTWARDS ARROW] R) was observed in all isolates from 2008, six from 2009, two from 2010 and a Chinese strain from 2004 (NB/04). Three isolates collected from Dong Thap contained an aa mutation at position 10 (C [RIGHTWARDS ARROW] Y) in the peptide signal, three mutations in the extra virion domain (32, S [RIGHTWARDS ARROW] N; 35, N [RIGHTWARDS ARROW] S; 59, K [RIGHTWARDS ARROW] N) and another mutation at position 104 (G [RIGHTWARDS ARROW] E). Three samples from Dien Bien province had a mutation at position 170 (E [RIGHTWARDS ARROW] G).

3 DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. 1 MATERIALS AND METHODS
  4. 2 RESULTS
  5. 3 DISCUSSION
  6. ACKNOWLEDGMENTS
  7. 4 DISCLOSURE
  8. REFERENCES

The first Vietnamese PRRS outbreak was confirmed in the northern province of Hai Duong in March 2007; since then PRRS has continued to spread to other regions of the country. The PRRSV isolates in Vietnam are of the NA-type. ORF5 of the NA-type (603 nt, 201 aa) covers nt 13,788–14,390 of the viral genome and encodes the major envelope glycoprotein (GP5). High nucleotide similarities among Vietnamese and highly pathogenic Chinese PRRSV isolates (Table 2) are consistent with the findings of Feng et al. [10]. They observed 99% identity at the genomic level for Vietnamese and Chinese PRRSV isolates. Compared with isolates from 2010 and later, we detected additional mutations in our isolates. Some nt changes in Vietnamese PRRSV isolates at positions 166, 207, 213, 246, 327, 360, 375, 378, 417 and 423 (data not shown) are also found in isolates from other Asian countries such as Taiwan, Thailand, Japan and Korea. The same nt mutations observed in PRRSV strains from different Asian countries would be the result of transportation of infected pigs, especially between the neighboring countries of Vietnam and China.

Several genotyping studies based on ORF5 sequences have been conducted on type II PRRSVs. Because of the lack of a reference sequence set, no satisfactory classification system is available. Based on analyses of 8624 ORF5 sequences available in GenBank, nine well-defined lineages for type II PRRSV have been fully characterized, intra-lineage diversity ranging from 10.1%–17.4% [15]. Within lineage 8, there are about 1400 selected samples that have been further divided into nine sub-lineages, which show 8.7% nt diversity. For sub-lineage 8.7, 330 selected sequences, with CH-1a as a historical sample, showed 3.9% diversity. Therefore this strain can be used for future genotyping studies and phylogenetic analyses. This phylogeny was also applied in other studies of Thai and Korean PRRSV strains [16, 17]. Studies involving the phylogeny of type II PRRSV ORF5 sequences have used varying classification systems with numerous subgroups or subgenotypes [18-20]. The 32 Vietnamese isolates we focused on were type II PRRSVs and were classified into sub-lineage 8.7. Representative highly pathogenic Chinese isolates from 2006 to 2011 also clustered within this sub-lineage. Nucleotide diversity among the 32 Vietnamese PRRSV strains and the highly pathogenic Chinese strains in our analysis was in the range of 0.5–3.0%. These findings are consistent with a previous classification in which PRRSV isolates from Vietnam and China in 2007 were clustered into a subgroup of type II PRRSVs, which are distantly related to a sub-clade of VR-2332 [10].

Glycoprotein 5, one of the most genetically variable structural proteins of PRRSV, is essential for virus infectivity. The functional domains of GP5, such as signal peptides, ectodomains, transmembrane regions and endodomains, were identified based on previous reports [5, 7]. Amino acid variation in GP5 mainly occurs in the signal peptide and ectodomain [21]. This affects the number of potential N-glycosylation sites where a putative neutralization epitope is located [5, 22]. Potential N-glycosylation sites in GP5 are reportedly involved in viral immune evasion and virus-neutralizing antibody responses [23, 24]. The putative neutralization epitope plays an important role in viral neutralization, residues H38 and L39/F39 being considered the critical amino acids [25]. Five common variable sites (aa positions 32–34, 38–39, 57–59, 137, and 151) have been identified in susceptible and resistant viral isolates [17]. Changes in the aa sequence at three of these sites (32–34,38–39, and 57–59) significantly influence the susceptibility of mutant viruses to virus neutralizing antibodies. Compared to the prototype VR-2332 strain, we found a mutation at position 34 (D [RIGHTWARDS ARROW] N/S) in all 32 isolates and at position 35 (S [RIGHTWARDS ARROW] N) in 27 isolates. These mutations led to the deletion of potential N-glycosylation sites in GP5. Substitution of the aa residue at position 34 with an N resulted in the addition of a new N-glycosylation site to this region. All 32 isolates contained mutations at aa positions 39 (L [RIGHTWARDS ARROW] I) and 58 (N [RIGHTWARDS ARROW] Q/E/K); these have also been observed in highly pathogenic Chinese strains. This mutation was absent from the viruses used in the Ingelvac MLV and Besta BSL-PS vaccines, which are licensed for use in Vietnam. The N [RIGHTWARDS ARROW] Q/E/K change eliminates an N-glycosylation site at position 58 and may confer upon viruses the ability to evade the host immune response.

Additionally, there was a mutation at position 29 (A [RIGHTWARDS ARROW] V) in five isolates. This was considered a decoy epitope, which has been described for JXA1 viral strain [26]. We detected additional aa changes, but only in strains collected in 2012 in Dong Thap and Dien Bien provinces. Three Dong Thap strains showed mutations at positions 10 (C [RIGHTWARDS ARROW] Y), 32 (S [RIGHTWARDS ARROW] N), 59 (K [RIGHTWARDS ARROW] N) and 104 (G [RIGHTWARDS ARROW] E). We identified a mutation at position 170 (E [RIGHTWARDS ARROW] G) in all isolates from Dien Bien. We found no amino acid mutations at amino acid position 151, which is considered to be a highly variable site of mutation. With respect to GP5, PRRSV strains circulating in Vietnam showed greater aa identity to JAX1-R than to virus strains used in other vaccines (Ingelvac PRRS MLV and Besta BSL-PS). This would suggest that the JAX1-R vaccine is more appropriate for use against PRRSV strains circulating in Vietnam.

Taken together, our result shows that the 32 Vietnamese isolates in this study were of the North American genotype and clustered into the unique sub-lineage 8.7, in which high pathogenic Chinese PRRSV strains are also clustered. Several vaccines are being developed to better protect against PRRS. Our work provides genetic information related to PRRSV ORF5 circulating in the field and will assist the effective use of PRRS vaccine in Vietnam.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. 1 MATERIALS AND METHODS
  4. 2 RESULTS
  5. 3 DISCUSSION
  6. ACKNOWLEDGMENTS
  7. 4 DISCLOSURE
  8. REFERENCES

This work was financially supported by Vietnam's National Foundation for Science and Technology (NAFOSTED; grant 106.12-2010.05). The authors sincerely thank Dr E. J. Choi from the National Veterinary Research and Quarantine Service, South Korea for providing the VR-2332 and LV strains.

4 DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. 1 MATERIALS AND METHODS
  4. 2 RESULTS
  5. 3 DISCUSSION
  6. ACKNOWLEDGMENTS
  7. 4 DISCLOSURE
  8. REFERENCES

All funding, support, or sponsorship for the work presented in the manuscript is clearly stated in the acknowledgement section of the manuscript. All authors certify that there is no conflict of interest relevant to financial arrangements.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. 1 MATERIALS AND METHODS
  4. 2 RESULTS
  5. 3 DISCUSSION
  6. ACKNOWLEDGMENTS
  7. 4 DISCLOSURE
  8. REFERENCES
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