Genetic Diversity Characterization of Porcine Reproductive and Respiratory Syndrome Virus Isolates in Romania, Based on Phylogenetic Analysis

Porcine reproductive and respiratory syndrome (PRRS) is a disease produced by the (PRRS) virus, characterized by endemic evolution in the majority of countries, which remains in actuality being a permanent threat to health and economic free farms, as well as for those infected. The aim of this study was to evaluate the genetic diversity of Romanian PRRSV isolates from the four most important pig farms in Romania by comparing the nucleotide sequences obtained for ORF5 and ORF7 with a wide range of sequences from GenBank belonging to the main types of PRRSV; the type 1. Eighteen different sequences were obtained for ORF5 gene and 10 for ORF7 gene. One Romanian isolate (Rom3) was found in three of the four different investigated farms. The phylogenetic analysis revealed that the Romanian PRRSV nucleotide sequences clustered in three groups within the subtype 1 of the virus. The analysis of amino acid sequences evidenced for GP5 and N-nucleocapsid proteins confirmed that the Romanian virus belonged to type 1.


Introduction
Porcine reproductive and respiratory syndrome (PRRS) is the pathological dominant of the 90 decade for pigs.PRRS is a viral disease, characterized by endemic evolution in the majority of countries, which remains in actuality being a permanent threat to health and economic free farms, as well as for those infected.
In March 1991, a group of researchers from the Institute of Lelystad, Netherlands, was able to isolate and identify the etiologic agent and experimental reproduction of the disease [1,2].It has been established that PRRS is caused by a virus that belongs to a new group of RNA viruses.Genome organization, structure and biology are similar to those of increased lactate dehydrogenase virus (dairy Dehydrogenase Elevating virus LDV), virus infectious equine arteritis (equine arteritis virus EAV) and the Ebola virus simian apes (simian hemorrhagic fever SHFV) [3].Comparison of PRRS virus, lactate dehydrogenase (LD) and equine arthritis (AE), allows for the possibility of a common phylogenetic tree, in which the PRRS virus is closer to the LD virus than the AE virus.Hence, the hypothesis that PRRS virus is a variant of LD virus adapted to pigs.
PRRSV is divided into two genotypes: type 1 and type 2. Comparison of antigenic different strains demonstrated that European and American strains are approximately 60% similar to each other [8].
The purpose of the present study was to investigate the PRRSV genetic variability based on sequence transcription, amplification and sequencing of genes of ORF7 and ORF5, using different tissues homogenates, collected from four pig farms in Romania.To confirm the occurrence of type 1 of PRRSV in Romania, both genes of ORF5 and ORF7 were sequenced.
The studied PRRS outbreaks have appeared in four important counties in different geographical areas in Romania.

Results and Discussion
In order to investigate the PRRSV genetic variability in four pig farms in Romania, 605 samples were collected from different individuals.From the total amount of samples, 33 were positive for PRRSV by PCR.We were able to conduct the investigations on both ORF5 and ORF7 genes on 23 samples but for the other 10 samples due to the small quantity of initial tissue, we were only able to fulfill the investigations on the ORF5 gene (Table 1).In our study, we divided Romania into four different areas (Figure 1).
In Table 2, we described the areas, the total number of swine and the number of samples collected and analyzed from four geographical areas.The ORF5 and ORF7 specific PCR products were sequenced to confirm the occurrence of type 1 of PRRSV in Romania.The obtained nucleotide sequences were aligned using CLUSTAL W program [9], resulting in a 606 nucleotides alignment for ORF5 gene and a 387 nucleotides alignment for ORF7.In our study, we indentified 18 sequences for ORF5 and 10 sequences for ORF7 with at least one nucleotide point mutation compared with data already available in the GenBank.
Interestingly, based on ORF5 gene analysis, among the 18 different Romanian isolates, Rom3 PRRSV strain was detected in three of the four pig farms investigated (Braila, Iasi, Arad counties), while Rom5 PRRSV strain was found in 2 different pig farms (Braila and Arad counties).Taking into account the number of identical nucleotide sequences obtained for gene ORF5, PRRSV strain Rom3 was isolated from 14 different individuals (10 pigs from Braila farm, three pigs from Iasi farm and one from Arad farm).Based on the analysis of ORF7 nucleotide sequence, the new PRRSV strains are different among all Romanian pig farms investigated, but the Rom4 isolate is the most prevalent in Braila farm (found in ten different individuals, Table 1).[10].Two phylogenetic trees were constructed, based on the complete sequences obtained for ORF5 gene and ORF7 gene of PRRSV Romanian isolates, together with a wide range of sequences selected from GenBank (Table 3).The evolutionary history was inferred using the Maximum Likelihood method [11].The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed [12].Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed.The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.The evolutionary distances were computed using the Tamura-Nei method [13] and are in the units of the number of base substitutions per site.The rate variation among sites was modeled with a gamma distribution (shape parameter = 1) with Invariant.The differences in the composition bias among sequences were considered in evolutionary comparisons [14].The analysis involved 72 nucleotide sequences for ORF5 and 99 nulcleotide sequences for ORF7.Codon positions included were 1st + 2nd + 3rd + Noncoding.All ambiguous positions were removed for each sequence pair.Evolutionary analyses were conducted in MEGA4 [15] (Figures 1 and 2).The type 1 PRRSV clade is clearly divided into three clusters corresponding to subtypes 1, 2 and 3 [10].The deduced amino acid sequences encoded by both ORF5 and ORF7 genes were aligned using CLUSTAL W program [9], resulting in a 201 amino acids alignment, corresponding to ORF5 gene (Figure 4) and a 128 amino acid alignment corresponding to ORF7 gene (Figure 5).A single exception can be noticed: the amino acid sequence for Rom22 isolate from Braila farm has an asparagine inserted at position 12 of the sequence.It is known that GP5 protein is very polymorphic [16,17] being under the permanent pressure of selection forces due to its exposed position at the exterior of the virion [18].This is why GP5 is highly informative regarding the evolution and origin of different PRRSV isolates.In particular, some known functional domains of GP5, such as the signal peptide, mature chain with transmembrane regions, some motifs in GP5 like primary neutralizing epitope (PNE) and decoy epitope were also analyzed according to a previous report [19].Our aim was to investigate the amino acid difference among the subtype 1 of Romanian isolates.The GP5 amino acid sequences of 17 PRRSV isolates were aligned, together with the Lelystad virus sequence.Multiple alignments of GP5 sequences of Romanian PRRSV isolates indicated that all 17 isolates encode a GP5 protein of 201 amino acid residues (Figure 4).
Nucleocapsid protein (N) is encoded by ORF7 gene [18,20] and has 128 amino acids in type 1 PRRSV.The N-terminus of the protein interacts with the viral genomic RNA [21,22] and the C-terminus has the role of maintaining the tertiary structure of N protein [23].The deduced amino acid sequences for all 10 different Romanian isolates aligned with CLUSTAL W program [21] reveal no extended hypervariable regions, as expected due to the fact that nucleocapsid protein is a very conserved molecule.The distribution of sequence diversity across the ORF7 protein was investigated for all 10 sequences analyzed in this study.The analysis sequences which contains 128 amino acids demonstrate that Romanian isolates have some amino acid substitutions compared with Lelystad virus: Rom22 isolate with an asparagine inserted between positions 11 and 12 of the alignment, and one substitution in position 42, Rom26 with three amino acid substitutions in positions 4, 8 and 16 and Rom30 with one amino acid substitution in position 124 (Figure 5).

Sampling and RNA Extraction
The biological samples were supplied from four pig farms contaminated with PRRSV, with clinical signs of disease.The pig farms were from different geographical areas of Romania: Braila, Arad, Cluj-Napoca and Iasi counties.
Samples used in this study were collected post mortem from all selected pigs from 2010 to 2012.Also, all the samples were collected as early as possible after exitus, deposited in RNA, later buffer, for transportation, and then stored at −80 °C.
Numbers of samples totaled 605 were collected: 260 from pigs from Braila farm, 85 from pigs from Iasi farm, 200 from pigs belonging to Arad farm and 60 from pigs from Cluj-Napoca farm.The tissues used for PRRSV-RNA extraction were tonsil, lung, mediastinal lymph node, liver, spleen, and kidney.
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Austin, TX, USA).200µL tissue homogenate was processed in accordance with the manufacturer's instructions.

PCR Analysis
Two genes specific to PRRSV were analyzed in our study: ORF7 (open reading frame 7) and ORF5 (open reading frame 5).
The PCR products were visualized on 2% agarose gels stained with ethidium bromide.

Sequencing and Phylogenetic Analysis
The PCR reaction products were purified using Wizard ® PCR Preps DNA Purification System (Promega, Madison, WI, USA), and the concentration and purity of the products were evaluated by spectrophotometry (Eppendorf BioPhotometer, Hamburg, Germany).The DNA sequencing reactions were performed for both forward and reverse strands using BigDye Terminator Kit v3.1 (Applied Biosystems, Foster City, CA, USA).
The sequencing was performed on a 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).The sequences obtained were proofread manually, truncated to the real dimensions of the genes (606 bp for ORF5 gene and 387 bp for ORF7 gene) using BioEdit version 7.1.3.0 [24] and aligned using CLUSTAL W application from MegAlign program (DNASTAR, Intelligenetics, Madison, WI, Wisconsin).ORF5 and ORF7 corresponding sequences were compared with a set of reference sequences selected from GenBank to cover a wide range of genetic and geographic diversity from Europe.All the Romanian isolates received GenBank accession numbers (Table 1).The set of reference sequences used to construct the phylogenetic tree is presented in Table 2.The two phylogenetic trees were generated from the aligned sequences in MEGA4 program [25] using a Maximum Likelihood method [15].The evolutionary distances were computed using the Tamura-Nei + gamma + I model [26] and are in the units of the number of base substitutions per site.Percentage reliability values at each internal node of the trees were obtained by performing 1000 bootstrap analyses.

Conclusions
This is the first extensive study on Romanian PRRSV isolates that provides information about the genetic diversity of this virus in the four most important pig farms in Romania.This study completes our early data on the first two Romanian strains analyzed [27].The results obtained from the phylogenetic trees together with the pairwise nucleotide sequence identity confirm the affiliation of all Romanian isolates to the subtype 1 of the virus.The evolution of PRRSV in the Romanian pig farms follows four distinct directions.
In the case of ORF7, we observed the existence of two monophyletic groups.The Romanian sequences Rom 26, 4, 22 and 23 belong to a different monophyletic group compared to sequences of Porcilis vaccine (DQ324710), Lelystad virus (M96262) and sequences from Spain (DQ324698 and DQ324712).
For the amino acid sequences for ORF5, we identified two hypervariable regions, one in the signal peptide and one in the beginning of the mature chain.Regarding N protein, Romanian isolates have some amino acid substitutions compared with Lelystad virus: Rom22 isolate with an asparagine inserted between position 11 and 12 of the alignment, and one substitution in position 42, Rom26 with three amino acid substitutions in positions 4, 8 and 16, Rom30 with one amino acid substitution in position 124.

Figure 1 .
Figure 1.Political map of Romania showing the number of pigs from different areas.

Figure 2 .
Figure 2. Maximum Likelihood tree based on ORF5 for the Romanian sequences, together with similar sequences from GenBank Database.The evolutionary distances were computed using the Tamura-Nei + G + I method.The bootstrap values adjacent to the main nodes represent the probabilities based on 1000 replicates.

Figure 3 .
Figure 3. Maximum Likelihood tree based on ORF7 for the Romanian sequences, together with similar sequences from GenBank Database.The evolutionary distances were computed using the Tamura-Nei + G + I method.The bootstrap values adjacent to the main nodes represent the probabilities based on 1000 replicates.

Figure 5 .
Figure 5. Alignment of the deduced amino acid sequences of ORF7 protein of 10 Romanian isolates and Lelystad virus (M 96262).Substitutions are indicated by the amino acid letter codes.

Table 1 .
Romanian PRRSV isolates and their GenBank accession numbers used in this study.

Table 3 .
Type 1 PRRSV strains used in this study.