Rescue of infectious chimeric viruses from shuffled infectious clones and <i>in vitro</i> growth kinetics of the shuffled chimeric viruses.

<p>Immunofluorescence assay (IFA) (<b>Panel A</b>) was performed with anti-PRRSV N protein monoclonal antibody (SDOW17) to confirm that the chimeric viruses were successfully rescued in MARC-145 cells infected with the supernatant of BHK-21 cells transfected with eight individual clones generated by DNA shuffling (GP4TS14, GP4TS19, GP4TS29, MTS1, MTS5, MTS8, MTS11 and MTS57). Parental backbone strain VR2385 and mock infection were included as positive and negative controls, respectively. The growth kinetics of GP4-shuffled chimeric viruses (<b>Panel B</b>) and M-shuffled chimeric viruses (<b>Panel C</b>) in MARC-145 cells were determined by measuring the infectious viral titers (TCID₅₀/ml) at indicated time points post-infection using the microtitration infectivity assay. The experiments were done in triplicate, and the bars indicated standard errors.</p

Nucleotide sequence alignment of the three GP4-shuffled chimeric viruses and their parents by clustalW method.

<p>The sequence of the backbone virus VR2385 was shown on top, and only differences were indicated for other viruses.</p

Nucleotide sequence alignment of the five M-shuffled chimeric viruses and their parents by clustalW method.

<p>The sequence of the backbone virus VR2385 was shown on top, and only differences were indicated for other viruses.</p

Kinetics of neutralizing antibodies in serum samples collected at 28, 35 and 43 days post-inoculation (DPI) from pigs experimentally infected with chimeric viruses GP4TS14 and MTS57 or with the backbone virus VR2385, respectively.

<p>The NA titers in serum samples at 28, 35 and 43 DPI from pigs experimentally infected with chimeras GP4TS14 and MTS57 or with VR2385 were examined by an <i>in vitro</i> serum virus neutralization test against four parental virus strains VR2385, MN184B, FL-12 and NADC20, respectively (<b>A–E</b>). Each test was performed in triplicate, the average NA titers of 3 or 4 pigs in each group are shown in the figure and the bars indicate standard errors. The asterisk (*) signs indicate a significant difference between the chimeric virus and the parental strain VR2385. One asterisk (*) signs indicate <i>p</i><0.05 and two asterisk (**) signs indicate <i>p</i><0.01. The <i>p</i> values are shown above the asterisk signs.</p

Schematic diagrams of the GP4 (Panel A) or M (Panel B) genes sequences in eight infectious chimeras (GP4TS14, GP4TS19, GP4TS29, MTS1, MTS5, MTS8, MTS11 and MTS57) generated by DNA shuffling.

<p>Each pattern in the shuffled genes represents one of the six different individual parental virus strains, which are shown at the bottom. The crossovers are delineated by the diversity of nucleotides because of the incorporation of the sequences from different parental virus strains. Multiple patterns that are displayed at the same region of the shuffled genes indicate that the sequence in this particular region was conserved at the corresponding parental virus strains. The numbers under the arrows indicate the nucleotide position.</p

Two phylogenetic trees based on the sequence of GP4 (Panel A) or M (Panel B) genes of selected type 2 PRRSV strains.

<p>The six parental viruses (VR2385 JX044140, VR2430 JX050225, MN184B DQ176020, FL-12 AY545985, JXA1 EF112445, and NADC20 JX069953) used in the DNA shuffling are indicated with boldface in the trees. The phylogenetic trees were constructed using the neighbor-joining method with bootstraps of 100 replicates. The numbers above each branch indicate the bootstrap values (percentage of consensus support in bootstrap).</p

Sequence comparison and analyses of GP4 (Panel A) and M (Panel B) amino acid sequences of chimeric viruses and their parents.

<p>Multiple sequence alignments of GP4 and M amino acid sequences of six parental virus strains and shuffled chimeric viruses were performed. The sequence of the backbone virus VR2385 is shown on top, and only differences for other strains are indicated in the alignment.</p

Neutralizing antibody titers against homologous and heterologous strains of PRRSV in serum samples of pigs experimentally infected with eight chimeric viruses or with the backbone virus VR2385, respectively: (A-E): Neutralizing antibody (NA) titers in serum samples collected at 43 DPI from pigs infected with indicated chimeric viruses or VR2385.

<p><i>In vitro</i> cross-neutralization SVN test of respective serum samples was performed against the five available parental virus strains used in the DNA shuffling including VR2385, VR2430, MN184B, FL-12 and NADC20, respectively. <b>F</b>: The average NA titers against all four heterologous strains (VR2430, MN184, FL-12 and NADC20). The NA titers against the heterologous strains in pigs infected with chimeric viruses were averaged and summarized individually, and compared with that in pigs infected with VR2385. The NA titers were calculated as the highest 2-fold dilution (2ⁿ) of the serum sample that showed a 90% or greater reduction in the number of positive fluorescent foci, compared to that of the serum samples from the negative control group in the same dilution. Three independent experiments were performed for each test, and the average NA titer for each group of pigs (3 pigs in test groups and 4 pigs in control groups) was shown in the figure. The error bars indicate standard errors. The asterisk (*) signs indicate a significant difference between the group of chimera pigs and the backbone parental strain VR2385. One asterisk (*) signs indicate <i>p</i><0.05 and two asterisk (**) signs indicate <i>p</i><0.01. The <i>p</i> values are shown above the asterisk signs.</p